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Renal perfusion and oxygenation during acute kidney injury
Aksu, U.
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Download date: 06 Sep 2018
RENAL PERFUSION AND OXYGENATION DURING ACUTE KIDNEY INJURY
Uğur Aksu
ISBN: 978-94-91715-08-2
Printing by: NetzoDruk Groningen bv
Cover: Resuscitation rain to kidneyCover design: İlker Traş
Copyright © Uğur AKSU, Amsterdam, 2015. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means mechanically, by photocopy, by recording, or otherwise, without the prior written permission of the holder of copyright.
Renal perfusion and oxygenation during acute kidney injury
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het College voor Promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel
op dinsdag 17 november 2015 om 12.00 uur
door Uğur Aksu geboren te Üsküdar, Turkije
Promotiecommissie
Promotor: prof. dr. ir. C. Ince Universiteit van Amsterdam
Copromotor: dr. R. Bezemer Universiteit van Amsterdam
Overige leden: prof. dr. F. Toraman Acibadem University prof. dr. J.H. Ravesloot Universiteit van Amsterdam prof. dr. S. Florquin Universiteit van Amsterdam prof. dr. E.T. van Bavel Universiteit van Amsterdam dr. E.G. Mik Erasmus Universiteit Rotterdam dr. C.T.P. Krediet Academisch Medisch Centrum
Faculteit der Geneeskunde
“As far as we can discern, the sole purpose of human existence is to kindle a light in the darkness of mere being”
Carl Gustav Jung
In memory of my dear father…
Contents • General Introduction: The pathogenesis of acute kidney injury and the toxic triangle
of oxygen, reactive oxygen species and nitric oxide 7
• Outline of the thesis 19
• Chapter 1: Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and
inflammation 23
• Chapter 2: The acute effects of acetate-balanced colloid and crystalloid resuscitation
on renal oxygenation in a rat model of hemorrhagic shock 45
• Chapter 3: Acute effects of balanced versus unbalanced colloid resuscitation on renal
macrocirculatory and microcirculatory perfusion during endotoxemic shock 67
• Chapter 4: Effect of tempol on redox homeostasis and stress tolerance in mimetically
aged Drosophila 83
• Chapter 5: Scavenging ROS in the acute phase of renal I/R injury also protects
kidney oxygenation and NO levels 101
• Summary and conclusions 117
• Samenvatting en conclusies 121
• References list 125
• Acknowledgments 143
• Curriculum vitae and portfolio 145
• List of publications 149
7
GENERAL INTRODUCTION
THE PATHOGENESIS OF ACUTE KIDNEY INJURY AND THE TOXIC
TRIANGLE OF OXYGEN, REACTIVE OXYGEN SPECIES AND NITRIC OXIDE
Aksu U1,3, Demirci C3, Ince C1,2
1Department of Translational Physiology, Academic Medical Center, University of
Amsterdam, Amsterdam The Netherlands 2Department of Intensive Care, Erasmus MC University Hospital Rotterdam, Rotterdam,
The Netherlands 3Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey
Published in: Contrib Nephrol. 2011; 174:119-28. Review.
8
Abstract
Despite the identification of several of the cellular mechanisms thought to underlie the
development of acute kidney injury (AKI), the pathophysiology of AKI is still poorly
understood. It is clear, however, that instead of a single mechanism being responsible for its
etiology, AKI is associated with an entire orchestra of failing cellular mechanisms. Renal
microcirculation is the physiological compartment where these mechanisms come together
and exert their integrated deleterious action. Therefore, the study of renal microcirculation
and the identification of the determinants of its function in models of AKI can be expected to
provide insight into the pathogenesis and resolution of AKI. A major determinant of adequate
organ function is the adequate oxygen (O2) supply at the microcirculatory level and utilization
at mitochondrial levels for ATP production needed for performing organ function. The highly
complex architecture of the renal microvasculature, the need to meet a high energy demand
and the borderline hypoxemic nature of the kidney makes it an organ that is highly vulnerable
to injury. Under normal, steady-state conditions, the oxygen supply to the renal tissues is well
regulated and utilized not only for mitochondrial production of ATP (mainly for Na+
reabsorption), but also for the production of nitric oxide and the reactive oxygen species
needed for physiological control of renal function. Under pathological conditions, such as
inflammation, shock or sepsis, however, the renal microcirculation becomes compromised,
which results in a disruption of the homeostasis of nitric oxide, reactive oxygen species, and
oxygen supply and utilization. This imbalance results in these compounds exerting pathogenic
effects, such as hypoxemia and oxidative stress, resulting in further deterioration of renal
microcirculatory function. Our hypothesis is that this sequence of events underlies the
development of AKI and that integrated therapeutic modalities targeting these pathogenic
mechanisms will be effective therapeutic strategies in the clinical environment.
9
Introduction
Despite the advances made in unravelling the pathogenesis and improving the treatment of
acute kidney injury (AKI), current therapeutic modalities have been ineffective for adequate
treatment. Consequently, AKI remains a condition with a poor prognosis in hospitals today.
Important factors leading to AKI are renal ischemia and hypoxia that can occur as a result of
kidney transplantation, treatment of suprarenal aneurysms, cardiac surgery, renal artery
reconstructions, contrast agent-induced nephropathy, cardiac arrest, sepsis, and shock
[Lameire et al., 2005]. AKI is associated with higher early and late mortality rates [Lameire et
al., 2005] and with higher costs, especially related to the demand for retransplantation and/or
hemodialysis. Among critical care patients who have AKI and survive, up to 30% will require
long-term dialysis [Bagshaw, 2006].
Understanding of the acute kidney injury
AKI, classified according to the RIFLE criteria, is characterized by the sudden loss of
glomerular filtration rate (GFR). Current views concerning the pathophysiology of AKI
implicate a reduction in renal blood flow and consequent renal ischemia as the cause of
depressed GFR which in turn causes disturbances in fluid, electrolyte, and acid-base balances
[Kellum, 2008]. Inflammatory processes can be triggered by ischemic insults and lead to
increased expression of adhesion molecules and impaired tubular sodium reabsorption due to
intraluminal debris from tubular cells. Endothelial injury directly affects afferent arterioles
and causes endothelin release and further vasoconstriction [Abuelo, 2007], which together
results in renal microcirculatory dysfunction.
Measurements of biomarkers in blood and/or urine have recently been developed for the
diagnosis of AKI at an early stage, which can, potentially, be used to prevent progression.
Hence early warnings (i.e., before GFR falls) are important in determining the therapeutic
strategies. According to AKI definition, serum creatinine and/or blood urea nitrogen increase.
However, traditional blood and urine biomarkers (such as the fractional excretion of sodium)
are nonspecific and not sensitive. New biomarkers have been discovered by using advanced
molecular techniques. These biomarkers have been assessed primarily after a specific insult,
such as cardiac surgery, kidney transplantation, contrast administration and sepsis. Currently,
some urine biomarkers, such as neutrophil gelatinase lipocalin (NGAL), cystatin C, kidney
injury molecule, interleukin-18 have been tested for early diagnosis of AKI and was discussed
to be AKI-spesific biomarker [Coca et al., 2008; Geuss et al., 2011]. Despite these findings in
10
the diagnosis of AKI, however, the pathophysiology and progress of AKI to renal failure in
the respect of molecular basis remain poorly understood [Wan et al., 2008].
Shock, fluid therapy and acute kidney injury
Currently, hemorrhagic and septic shock is one of the main contributors to the development of
AKI and one of the leading causes of death in intensive care [Kellum, 2008]. Shock
constitutes a major hit to renal function as it induces a massive increase in inflammatory
mediators and activated leukocytes, which together cause severe microcirculatory dysfunction
and disruption of oxygen homeostasis that leads to oxidative stress and hypoxemia [Legrand
et al., 2008]. In the early stages of sepsis, impairment of the renal microcirculation is a key
complication potentially leading to AKI through hypoxia-induced tubular epithelial cell injury
and acute tubular necrosis.
Current treatment strategies for hemorrhagic and septic shock involve rapid and aggressive
fluid resuscitation to restore blood pressure and tissue perfusion prior to blood transfusion.
Fluid resuscitation is a cornerstone of the treatment of sepsis because it is considered crucial
for the preservation of adequate intravascular volume and the maintenance of blood pressure
[Vincent and Gerlach, 2004]. Such fluid therapy is expected to promote microvascular
perfusion and thereby renal oxygenation. However, the extent to which fluid therapy is
effective in promoting renal oxygenation has recently been questioned [Legrand et al., 2010].
Fluid resuscitation can have severe deleterious effects on the microcirculation [Boldt and
Ince, 2010] and hemodilution in a range of therapeutic scenarios have been found to lead to
renal failure [Habib et al., 2005]. Excessive fluid administration in sepsis has been found to be
associated with renal failure [Payen et al., 2008], although restrictions in fluid use can lead to
hypovolemia. Therefore, determining the optimal fluid volume to administer during sepsis to
deal with hypovolemia remains a source of controversy [Boldt and Ince, 2010]. In addition,
the type of fluid that yields the best renal outcome when used for resuscitation in sepsis is also
currently a source of uncertainty. This controversy not only includes the use of crystalloid
versus colloid solutions, but also encompasses the use of balanced versus unbalanced colloid
solutions. Because most colloid preparations are saline-based, liberal fluid resuscitation
regimes may lead to non-physiologically high sodium and chloride concentrations and may be
associated with the development of (hyperchloremic) metabolic acidosis that can affect
inflammatory and coagulation homeostasis, thereby contributing to the deterioration of renal
function. This insight has led to development of modern hydroxyl-ethylated starch (HES)
11
preparations, based on balanced plasma-adapted crystalloid solutions, and to the notion of
developing a totally balanced fluid resuscitation concept, including balanced crystalloids and
balanced colloids. Studies have also found that HES solutions have anti-inflammatory
properties [Hoffmann et al., 2002]. In contrast, however, various investigations have
identified potential adverse effects of HES solutions on renal function [Winkelmayer et al.,
2003, Schortgen, et al., 2001, Cittanova et al., 1996].
Despite much literature showing its deleterious effects, 0.9% NaCl is widely used as a
resuscitation solution in emergency departments and intensive care units today. However, it is
known that crystalloids have a poor plasma expanding effect since they rapidly leave the
intravascular space. Compared to colloids in this respect, about three times more volume of
crystalloids needs to be given to reach a similar systemic hemodynamic endpoint [Dubin et
al., 2010]. Therefore, colloid fluids are more effective as plasma expanders because less is
needed. However, colloids have also been shown to have adverse affects on coagulation
pathways and are often dissolved in high chloride solutions (e.g. 0.9% NaCl). Excessive
chloride levels can have deleterious effects on renal function. For example, intrarenal
administration of a chloride solution provokes renal vasoconstriction and reduces GFR
[Wilcox, 1983]. Solutions containing high amounts of chloride can cause hyperchloremic
acidosis, while solutions with buffers, such as acetate, and more physiological concentrations
of strong ions, do not. Proinflammatory mechanisms involving acidosis have been elaborately
described elsewhere [Kellum et al., 2004].
Relation between oxygen, reactive oxygen species and nitric oxide
In addition to the vasodilatory effect of NO, when it is produced by endothelial nitric oxide
synthase (NOS), NO is thought to prevent vascular dysfunction by inhibiting platelet
aggregation and preventing leukocyte activation and infiltration via endogenous anti-
inflammatory properties. The depletion of suitable cofactors of the NO-producing enzyme
(e.g. BH4), as occurs during reperfusion injury and sepsis, can enhance the production of
reactive oxygen species (ROS) by uncoupling endothelial NOS [Rabelink and Zonneveld,
2006]. Excessive NO produced by cells (occurring, for example, as a result of inflammation
from inducible NOS activation) can inhibit mitochondrial respiration by competing with
oxygen mitochondrial cytochrome oxidase in a dose-dependent manner [Cooper and Giulivi,
2007]. Thus, the production and utilization of oxygen, NO, and ROS are intrinsically
dependent on each other and a proper balance is required for ensuring adequate renal function.
12
This delicate homeostasis is pathogenically altered during inflammation and hypoxemia, and
leads to oxidative stress and tissue damage. Oxidative stress is an imbalance between oxidants
and antioxidants that favors oxidants and causes a disruption of redox signaling and control,
leading to damage of cellular molecular structures [Clanton, 2007]. Oxygen radicals can be
released after the reduction of oxygen, and the outcome is cell injury and dysfunction. ROS is
a common term that is used for both oxygen radicals (O2– and OH–) and nonradical (H2O2,
HOCl, O3) compounds. Another commonly used term is ‘oxidant’. O2– and H2O2 can function
as both oxidizing and reducing agents.
Under normal circumstances, ROS are released at low concentrations and are neutralized by
endogenous antioxidant compounds, which can be both enzymatic, such as superoxide
dismutase, catalase and glutathione peroxidase, and nonenzymatic, such as glutathione and
vitamins C and E. Both high and low levels of oxygen promote oxidative stress, making the
need for keeping levels of tissue oxygen tensions at physiological levels imperative [Clanton,
2007].
Several studies have promoted the idea that targeting the ROS associated with cellular injury
in acute or chronic kidney disease may aid the design of future therapeutic approaches.
Oxidative stress commonly results in the degeneration of cells via apoptotic pathways.
Apoptotic-induced oxidative stress in conjunction with processes of mitochondrial
dysfunction forms the corner stone of triggered mechanisms in nephropathic conditions. The
dependency of ROS activity on oxygen availability was recently shown in a model of
oxidative stress in spontaneously hypertensive rats in which a loss of bioactive NO by high
ROS production was found to interfere with normal oxygen usage in the kidney. In addition, it
was shown that superoxide produced by NADPH oxidase was inhibited when oxygen tensions
dropped below 20 mmHg [Adler and Huang, 2004].
The main sources of ROS in the microcirculation are mitochondria, NADPH oxidase, NO
synthase and xanthine oxidase. Moreover, cytochrome P450 and cyclooxygenase are capable
of producing O2–. In mitochondria, there is continuous production of ROS during cellular
respiration. A percentage of the oxygen used in mitochondria is reduced to superoxide. This
process occurs by blockade of the electron transfer chain at the flavin mononucleotide group
of complex I or at the ubiquinone site of complex III. This free radical generation is under the
13
control of the endogenous antioxidant defense system, and Mn-superoxide dismutase in
mitochondria converts the superoxide to H2O2. Superoxide generates much of its biological
effects by scavenging the NO produced by three isoforms of NOS, each expressed in the
kidney: neuronal NOS, inducible NOS, and endothelial NOS. In their pathogenic action, ROS
mostly cause their deleterious effects by inducing lipid peroxidation, activation of apoptotic
pathways, alteration of intracellular calcium concentrations, and inducement of adhesion
molecule expression. Oxidative stress can also increase mitochondrial membrane
permeability, resulting in loss of mitochondrial NAD+ residues and subsequent radical
generation [Maiese and Chong, 2003]. In addition to their pathogenic action, superoxide and
NO are involved in normal kidney and vascular functions. Both may mediate the maintenance
of vascular tone (especially in the afferent arterioles) and tubular function. Angiotensin II,
mediated by superoxide together with NO, is also responsible for maintaining afferent
arteriolar tone in perfused isolated mouse afferent arterioles. Renal oxygen consumption, in
contrast, has been found to be increased by l-NG-monomethyl-arginine, a nonselective NOS
inhibitor, and S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor [Deng et al.,
2005], which emphasizes the roles of the different isoforms of NOS in modulating oxygen
utilization. These and the above-mentioned studies illustrate the complicated interdependency
between oxygen and NO species; their homeostasis becomes severely disrupted during
conditions of renal inflammation and ischemia-reperfusion (I/R), resulting in oxidative stress
and loss of renal function.
Inflammation and ischemia can severely disrupt the balance between oxygen transport and
utilization, reactive oxygen and NO metabolism, resulting in oxidative stress and regional
hypoxemia that lead to renal failure. Sepsis and reperfusion injury are the most severe
manifestations of such an inflammatory insult attacking microcirculatory function at all
levels; they can result in a viscous, self-perpetuating spiral of pathogenic events that lead to
renal failure. In this sequence of events, activated leukocytes and inflammatory mediators
disrupt the homeostasis of renal oxygenation, which leads to functional deterioration. This
model of the pathogenesis of AKI can be seen in Figure 1. In the view of the progress of AKI,
therapeutic compounds need to correct all the elements of this pathology (e.g. inflammation,
microcirculatory oxygenation, NO and oxidative stress) in an integrated manner to effectively
alter the course of AKI. We performed empirical studies on compounds that have multiple
correcting effects on these pathogenic mechanisms (e.g. dexamethasone, l-NIL, iloprost, and
14
APC;), and we found them successful in reverting AKI in rat models [Johannes et al., 2009;
Legrand et al., 2009; Johannes et al., 2009].
Conclusion
It is clear that physiological function of the kidney relies on a delicate balance between
oxygen transport and utilization, reactive oxygen and NO metabolism, and that this balance
affects the renal microcirculation and is essential for renal function. The question now is
whether the approach based on this integrative model provides a strategic therapeutic
rationale for the treatment of AKI in experimental scenarios.
Fig.1. An integrated model of the pathogenesis of AKI. Inflammation-induced leukocyte-endothelium interactions lead to a distortion of the homeostatic balance between O2, NO and ROS. This imbalance perpetuates the distorted leukocyte endothelium interaction, and a spiral of pathogenic events will follow. It is hypothesized that, taken together, the imbalances will fuel microcirculatory dysfunction which will lead to AKI and ultimately renal failure.
Acknowledgements The author is grateful to Rick Bezemer who drew Figure 1.
15
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19
OUTLINE OF THE THESIS
20
OUTLINE OF THE THESIS
The current study presented in this thesis was conducted at Department of Translational
Physiology of the Academic Medical Center of the University of Amsterdam. As most fluid
preparations are saline based, liberal fluid resuscitation regimens might lead to non-
physiologically high sodium and chloride concentrations and may be associated with the
development of (hyperchloremic) metabolic acidosis, which could affect inflammatory and
coagulation homeostasis and thereby deteriorated organ function. The ultimate aim of this
thesis is to test if fluid resuscitation regimens, besides antioxidant therapies, effect on kidney
oxygenation and redox homeostasis by using different rat models of kidney injury.
Current treatment strategies for hemorrhagic shock involve rapid and aggressive fluid
resuscitation to restore blood pressure and tissue perfusion prior to blood transfusion. In
Chapter 1, we aimed to test the hypothesis that balanced crystalloid resuscitation would be
better for the kidney than unbalanced crystalloid resuscitation in a rat hemorrhagic shock
model. For integrative investigation of the effects of hemorrhage and fluid resuscitation on the
complex interrelation between oxygen and reactive oxygen species, we measured renal
microvascular oxygen tension and plasma levels of malondialdehyde (oxidative stress
marker). Furthermore, glycocalyx degradation was assessed by measuring plasma levels of
hyaluronan (glycocalyx compartment) and plasma levels of TNF-alpha and interleukin-6 were
measured as markers of inflammation.
The use of conventional crystalloid solutions as initial resuscitation fluids is still implemented
in emergency departments even though it is known that crystalloid solutions have poor plasma
expander capacities and just 20% of the given volume remains contained in the intravascular
space. In Chapter 2, we aimed to investigate the acute effects of acetate-balanced colloid and
crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. To this
end, we examined the effects of resuscitation with different fluids: (1) 0.9% NaCl; (2)
acetated Ringer’s solution; (3) 6% HES with a molecular weight of 130 kDa and molar
substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution; and (4) 6% HES with a molecular
weight of 130 kDa and molar substitution of 0.42 (HES 130/0.42) in acetate-balanced
Ringer’s solution. We hypothesized that acetated solutions would have superior resuscitation
capacities compared to the other solutions with respect to improving renal oxygenation after
21
severe hemorrhage. In this line; we measured renal oxygen consumption and cortical-
medullary oxygenation in a rat model of hemorrhagic shock.
The aim of Chapter 3 was therefore to investigate the acute effects of balanced versus
unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in
a rat model of lipopolysaccharide-induced endotoxemia. We tested the hypothesis that
balanced colloid resuscitation would be better for the kidney than unbalanced colloid
resuscitation. The acute effects of two clinically applied resuscitation regimens were
investigated: 6% 130/0.4 HES in NaCl as an unbalanced colloid solution and 6% 130/0.4 HES
in Ringer’s acetate solution as a balanced colloid solution. Renal perfusion was assessed at the
macrocirculatory level using Doppler ultrasound on the renal artery and at the
microcirculatory level using laser speckle imaging on the renal cortex.
According to free radical theory, reactive oxygen species (ROS) may cause oxidative injury to
living organisms through their lifetime. Prolonged oxidative stress may cause functional
cellular decline and various age-related disorders in humans and experimental animals. The
aim of Chapter 4 was to test the hypothesis whether tempol, a tempol scavenger, restores
impaired redox homeostasis and increases stress tolerance in a mimetic aging model of
Drosophila. To this end, we investigated the extent of oxidative stress and, specifically,
oxidative protein damage in mimetically aged flies following tempol administration.
Therefore, oxidative stress parameters and, sialic acid (SA) as cell surface glycocprotein
levels were determined.
It is well known that reactive oxygen species are fundamentally implicated as primary culprits
in the pathophysiology of renal I/R injury and consequent acute kidney injury. The excess
generation of reactive oxygen species and decreases in antioxidant defenses are known to
contribute to I/R injury. In a series of recent reviews, we have described that our hypothesis
that a disturbed balance between oxygen, nitric oxide, and reactive oxygen species might form
an important component of the pathogenesis of I/R-induced acute kidney injury. In Chapter
5, we aimed to test whether the proven protective effects of tempol are indeed associated with
improved renal oxygenation and nitric oxide levels in a short-term rat model of renal I/R.
Therefore, kidney oxygenation and consumption were determined beside nitric oxide levels of
kidney tissues in a rat model of renal I/R.
22
23
CHAPTER 1
BALANCED VS UNBALANCED CRYSTALLOID RESUSCITATION IN A NEAR-
FATAL MODEL OF HEMORRHAGIC SHOCK AND THE EFFECTS ON RENAL
OXYGENATION, OXIDATIVE STRESS, AND INFLAMMATION
Aksu U1,2, Bezemer R1, Yavuz B3, Kandil A2, Demirci C2, Ince C1
1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, The Netherlands
2Department of Biology, Faculty of Science, University of Istanbul, 3Department of Biochemistry, Cerrahpasa Medical School, University of Istanbul, Istanbul,
Turkey
Published in: Resuscitation. 2012 Jun;83(6):767-73.
24
Chapter 1
Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic
shock and the effects on renal oxygenation, oxidative stress, and inflammation
Running title: Crystalloid resuscitation in hemorrhagic shock
Abstract
Background: The aim of the present study was to test the hypothesis that balanced crystalloid
resuscitation would be better for the kidney than unbalanced crystalloid resuscitation in a rat
hemorrhagic shock model.Methods: Male Wistar rats were randomly assigned to four groups
(n = 6/group): (1) time control; (2) hemorrhagic shock control; (3) hemorrhagic shock
followed by unbalanced crystalloid resuscitation (0.9% NaCl); and (4) hemorrhagic shock
followed by acetate and gluconate-balanced crystalloid resuscitation (Plasma Lyte). We tested
the solutions for their effects on renal hemodynamics and microvascular oxygenation,
strongion difference, systemic and renal markers of inflammation and oxidative stress
including glycocalyx degradation as well as their effects on renal function. Results: The main
findings of our study were that: (1) both the balanced and unbalanced crystalloid solutions
successfully restored the blood pressure, but renal blood flow was only recovered by the
balanced solution although this did not lead to improved renal microvascular oxygenation; (2)
while unbalanced crystalloid resuscitation induced hyperchloremia and worsened metabolic
acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevented hyperchloremia,
restored the acid–base balance, and preserved the anion gap and strong ion difference in these
animals; (3) in addition balanced crystalloid resuscitation significantly improved renal oxygen
consumption (increased VO2, decreased EFNa+); and (4) however neither balanced nor
unbalanced crystalloid resuscitation could normalize systemic inflammation or oxidative
stress. Functional immunohistochemistry biomarkers showed improvement in L-FABP in
favor of balanced solutions in comparison to the hemorrhagic group although no such benefit
was seen for renal tubular injury (measured by NGAL) by giving either unbalanced or
balanced solutions. Conclusions: Although balanced crystalloid resuscitation seems superior
to balanced crystalloid resuscitation in protecting the kidney after hemorrhagic shock and is
certainly better than not applying fluid resuscitation, these solutions were not able to correct
systemic inflammation or oxidative stress associated with hemorrhagic shock.
25
Introduction
Hemorrhagic shock is one of the leading causes of death among trauma patients. Current
treatment strategies for hemorrhagic shock involve rapid and aggressive fluid resuscitation to
restore blood pressure and tissue perfusion prior to blood transfusion.
However, even when systemic vital parameters are restored, tissue oxygenation in some
organs such as the kidney remains inadequate due to disturbed (micro) vascular regulatory
mechanisms as a result of hemorrhage-induced coagulation and inflammation [Legrand et al.,
2010; Cai et al., 2009]. In this line, it has been shown that an important step during
microvascular inflammation is the loss of the endothelial cell glycocalyx [Pries and Kuebler,
2006; Taylor and Gallo, 2006; Vink and Duling, 1996; Rubio-Gayosso et al., 2006;
Nieuwdorp et al., 2006]. This loss of the glycocalyx significantly affects capillary wall
permeability, coagulation, and leukocyte adhesion and thereby disturbs microvascular
function [Constantinescu et al., 2003]. Additionally, dysfunctional nitric oxide metabolism
during oxidative stress has been proposed to cause inefficient utilization of oxygen for sodium
reabsorption [Welch et al., 2001]. Hence, there is a complex interrelation between oxygen,
nitric oxide, and reactive oxygen species in the tissues [Freeman et al., 1982; Demoncheaux et
al., 2005].
In rat models of hemorrhagic shock, it has been shown that excessive saline (0.9% NaCl)
administration for resuscitation can lead to metabolic acidosis, which has been associated with
microvascular dysfunction and consequent tissue hypoxia in sensitive organ systems such as
the kidney. Hyperchloremia, in addition, has been shown to depress renal blood flow and
glomerular filtration rate and has been shown to disturb nitric oxide production and
utilization. Therefore, using balanced crystalloids to avoid acid–base disturbances and
hyperchloremia could be beneficial. The type of buffer that should be used for balancing
fluids, however, is still subject of debate [Parekh, 2002; Pedoto et al., 1999; Wilcox,1983;
Dorje et al., 2000; Zander, 2002]. Attempts to prevent metabolic acidosis include partial
replacement of chloride by rapidly metabolized anions such as l-lactate, acetate, and
gluconate. The aim of the present study was to test the hypothesis that balanced crystalloid
resuscitation would be better for the kidney than unbalanced crystalloid resuscitation in a rat
hemorrhagic shock model. For integrative investigation of the effects of hemorrhage and fluid
resuscitation on the complex interrelation between oxygen and reactive oxygen species, we
measured renal microvascular oxygen tension and plasma levels of malondialdehyde
26
(oxidative stress marker). Furthermore, glycocalyx degradation was assessed by measuring
plasma levels of hyaluronan (glycocalyx compartment) and plasma levels of TNF-α and
interleukin 6 were measured as markers of inflammation.
Materials and methods
Animals
All experiments in this study were reviewed and approved by the institutional animal
experimentation committee of the Academic Medical Center of the University of Amsterdam.
Care and handling of the animals were in accordance with the guidelines for Institutional and
Animal Care and Use Committees. Experiments were performed on 24 male Wistar rats
(Harlan, the Netherlands) with body weight of 335 ± 15 g.
Surgical preparation
The rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg
ketamine (Nimatek®; Eurovet, Bladel, the Netherlands), 0.5 mg/kg medetomidine (Domitor;
Pfizer, New York, NY), and 0.05 mg/kg atropine-sulfate (Centrafarm, Etten-Leur, the
Netherlands). After tracheotomy the animals were mechanically ventilated with a FiO2 of 0.4.
Body temperature was maintained at 37 ± 0.5 "C during the entire experiment by external
warming. Ventilator settings were adjusted to maintain end-tidal PCO2 between 30 and 35
mmHg and arterial PCO2 between 35 and 40 mmHg.
For drug and fluid administration and hemodynamic monitoring, vessels were cannulated with
polyethylene catheters (outer diameter = 0.9 mm; Braun, Melsungen, Germany). A catheter in
the right carotid artery was connected to a pressure transducer to monitor arterial blood
pressure and heart rate. The right jugular vein was cannulated for continuous infusion of
Ringer Lactate (Baxter, Utrecht, the Netherlands) at a rate of 15 ml/kg/h and maintenance of
anesthesia. The right femoral artery was cannulated for blood shedding and the right femoral
vein for fluid resuscitation. The left kidney was exposed, decapsulated, and immobilized in a
Lucite kidney cup (K. Effenberger, Pfaffingen, Germany) via a ~4 cm incision in the left
flank. Decapsulating the kidney allowed low-noise phosphorimetric measurements of renal
microvascular oxygenation (see below). Renal vessels were carefully separated under
preservation of nerves and adrenal gland. A perivascular ultrasonic transient time flow probe
was placed around the left renal artery (type 0.7 RB; Transonic Systems Inc., Ithaca, NY,
USA) and connected to a flow meter (T206; Transonic Systems Inc.) to continuously measure
27
renal blood flow (RBF). The left urethra was isolated, ligated, and cannulated with a
polyethylene catheter for urine collection. A small piece of aluminum foil was placed on the
dorsal site of the renal vein to prevent contribution of underlying tissue to the
phosphorescence signal in the venous PO2 measurement (as described below).
After the surgical protocol, one optical fiber was placed 1 mm above the decapsulated kidney
and another optical fiber was placed 1 mm above the renal vein to measure renal
microvascular and venous oxygenation, respectively, using phosphorimetry. Oxyphor G2 (a
two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin, Oxygen
Enterprises Ltd., Philadelphia, PA, USA) was subsequently infused (i.e. 6 mg/kg i.v. over 5
min), followed by 30 min stabilization time. The surgical field was covered with a humidified
gauze compress throughout the entire experiment to prevent drying of the exposed tissue. A
short description of the phosphorimetric method is given below and an extensive description
of the technology can be found elsewhere [Johannes et al., 2006].
Experimental protocol
After stabilization, the animals were bled through the left femoral artery catheter at a rate of 1
ml/min using a syringe pump (Harvard 33 syringe pump; Harvard Apparatus, South Natick,
MA) till reaching a MAP of 30 mmHg. Coagulation of the shed blood was prevented by
adding 200 UI of heparin in the syringe. This MAP was maintained for 1 h by re-infusing or
withdrawing blood. At the end of this phase, the animals were randomized into: (1) balanced
fluid resuscitation with Plasma Lyte (Lyte) (n = 6) (Na+ 140 mmol L−1, Cl− 98 mmol L−1, K+
5 mmol L−1, Mg2+ 1.5 mmol L−1, acetate 27 mmol L−1, gluconate 23 mmol L−1; Plasma Lyte
148®, Baxter, Valencia, Spain); and (2) unbalanced fluid resuscitation for 90 min with saline
(NaCl) (n = 6) (0.9% NaCl). Resuscitation continued until a target MAP of 80 mmHg was
reached or for 90 min. Additionally, time control (Control) and hemorrhagic shock control
(HS) experiments were performed (n = 6 per group). The experiments were terminated by
infusion of 1 ml of 3 M potassium chloride (KCl) after which the kidney was removed and
weighed.
Hemodynamic, blood gas parameters, lactate, and acid base balance
Mean arterial pressure (MAP), heart rate (HR), and renal blood flow (RBF) were measured
continuously. Arterial blood samples (0.5 ml) were taken from the femoral artery at three time
points: (1) baseline (BL, t = 0 min); (2) hemorrhagic shock (HS, t = 60 min); and (3) 90 min
28
after starting resuscitation (RS, t = 150 min). The blood samples were replaced by the same
volume of balanced crystalloid (Plasma Lyte). The samples were used to determine of blood
gas parameters (ABL505 Blood Gas Analyzer; Radiometer, Copenhagen, Denmark), the
hemoglobin concentration, and the hemoglobin oxygen saturation. Renal oxygen delivery was
calculated as: DO2ren (ml/min) = RBF × arterial oxygen content (1.31 × hemoglobin × SaO2) +
(0.003 × PaO2), where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure
of oxygen.
Renal oxygen consumption was calculated as: VO2ren (ml/min/g) = RBF × (CaO2 − CvO2),
where renal venous oxygen content (CvO2) was calculated as (1.31 × hemoglobin × SrvO2) +
(0.003 × PrvO2). An estimation of the renal vascular resistance (RVR) was made as: RVR
(dynes s cm−5) = (MAP/RBF) × 100.
Plasma lactate levels were measured by an enzymatic colorimetric method using the Roche
Modular P800 automatic analyzer (Roche Diagnostics) from samples taken at t = 0 min
(baseline) and t = 150 min (end of experiment). The anion gap (AG) was calculated as the
sum of sodium and potassium concentrations minus the sum of bicarbonate and chloride
concentrations. For calculation of strong ion difference (SID), the sum of chloride and lactate
concentrations was subtracted from the sum of sodium and potassium concentrations.
Renal function and plasma osmolality
Creatinine clearance (Clearcrea (ml/min)) was assessed as an index of the glomerular filtration
rate. Calculation of the clearance was done using: Clearcrea = (Ucrea × V)/Pcrea, where Ucrea is
the concentration of creatinine in urine, V is the urine volume per unit time and Pcrea is the
concentration of creatinine in plasma. Additionally, excretion fraction of Na+ [EFNa (%)] was
calculated to use as a marker of tubular function as: EFNa+ = (UNa × Pcrea)/(PNa+ × Ucrea) × 100,
where UNa is Na+ concentration in urine and PNa is the Na+ concentration in plasma. Clearcrea
and EFNa+ were determined at t = 0 min and t = 150 min. Furthermore, the renal energy
efficiency for sodium transport (VO2ren/TNa+ ) was assessed using the ratio of the total amount
of VO2ren over the total amount of sodium reabsorbed (TNa+ , mmol/min). The osmolality of
the plasma and urine were determined using the freezing point method using an osmotic
pressure meter (Osmostation, OM-6050; Arkray) from a sample taken at the end of the
experiment.
29
Renal microvascular oxygenation and renal venous PO2
Renal microvascular PO2 (µPO2) and renal venous PO2 (PrvO2) were measured by oxygen
dependent quenching of phosphorescence lifetimes of the systemically infused albumin
targeted (and therefore circulation confined) phosphorescent dye Oxyphor G2 [Vinogradov et
al., 2002]. A linear relationship between reciprocal phosphorescence lifetime and oxygen
tension (given by the Stern–Volmer relation) allows quantitative measurement of µPO2.
Oxyphor G2 (a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin)
has two excitation peaks (λexcitation1 = 440 nm, λexcitation2 = 632 nm) and one emission peak
(λemission = 800 nm) [ Dunphy et al., 2002].These optical properties allow (near) simultaneous
lifetime measurements in microcirculation of the kidney cortex (CµPO2) and the outer
medulla (MµPO2) due to different optical penetration depths of the two excitation
wavelengths [Johannes et al., 2006]. For the measurement of renal venous PO2 (PrvO2), a
mono-wavelength (λexcitation = 632 nm) phosphorimeter was used [Mik et al., 2008].
Oxidative stress and inflammation markers
Determination of malondialdehyde (MDA) levels was used to quantify the lipid peroxidation
in tissues and plasmas. Tissues were homogenized in 5 mM (cold) Na-phosphate buffer. The
homogenates were centrifuged at 12,000 × g for 15 min at 4 oC and supernatants were used
for MDA determination. The level of lipid peroxides was expressed as micromoles of MDA
per miligram of protein (Bradford assay). Plasma levels of TNF-α and IL-6 were measured by
ELISA as markers of systemic inflammation.
Glycocalyx degradation
Hyaluronan is the main component of endothelial glycocalyx, and alterations in its
concentration can be attributed to glycocalyx volume loss [Nieuwdorp et al., 2006]. Plasma
hyaluronan concentrations were determined using the Corgenix hyaluronic acid test kit
(Corgenix Inc., Westminster, Colo) that is based on an enzyme-linked hyaluronic acid binding
protein assay.
Immunohistochemical analysis
Kidney tissues were fixed in 4% formalin and embedded in paraffin. Kidney sections (5 µm)
were deparaffinized with xylene and rehydrated with decreasing percentages of ethanol and
finally with water. Antigen retrieval was accomplished by microwaving slides in citrate buffer
30
(pH 6.0) (Thermo Scientific, AP-9003-500) for 10 min. Slides were left to cool for 20 min at
room temperature and then rinsed with distilled water. Surroundings of the sections were
marked with a PAP pen. The endogenous peroxidase activity was blocked with 3% H2O2 for
10 min at room temperature and later rinsed with distilled water and PBS. Blocking reagent
(LabVision, TA-125-UB) was applied to each slide followed by 5 min incubation at room
temperature in a humid chamber. Kidney sections were incubated for overnight at 4 oC with
Lipocalin 2 antibody (NGAL) (abcam 41105) and polyclonal antibody to rat L-FABP (Hycult
Biotect HP8010). Antibodies were diluted in a large volume of UltrAb Diluent (Thermo
Scientific, TA-125-UD). The sections were washed in PBS three times for 5 min each time
and then incubated for 30 min at room temperature with biotinylated goat anti-rabbit
antibodies (LabVision, TP-125-BN). After slides were washed in PBS, the streptavidin
peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room
temperature in a humid chamber. The colored product was developed by incubation with
AEC. The slides were counterstained with Mayer’s hematoxylin (LabVision, TA- 125-MH)
and mounted in vision mount (LabVision, TA-060-UG) after being washed in distilled water.
Both the intensity and the distribution of specific L-FABP and NGAL staining were scored.
For each sample, a histological score (HSCORE) value was derived by summing the
percentages of cells that stained at each intensity multiplied by the weighted intensity of the
staining [HSCORE = S Pi (i + 1), where i is the intensity score and Pi is the corresponding
percentage of the cells] [Senturk et al., 1996].
Statistical analysis
Values are reported as mean ± SEM. The decay curves of phosphorescence intensity were
analyzed using software programmed in Labview 6.1 (National Instruments, Austin, TX,
USA). Statistical analysis was performed using GraphPad Prism version 4.0 for Windows
(GraphPad Software, San Diego, CA, USA). Two-way ANOVA for repeated measurements
with a Bonferroni post hoc test was used for comparisons; a p-value of <0.05 was considered
statistically significant.
Results
Fluid resuscitation and plasma osmolality
For restoration of the MAP from 30 mmHg to 80 mmHg, 56.8 ± 3.8 ml of saline was required
and only 30.4 ± 4.7 ml of Plasma Lyte was required (p < 0.01 vs HS + NaCl group). The
plasma osmolality at the end of resuscitation time point was 237 ± 4 mOsm/kg in the control
31
group, 253 ± 2 mOsm/kg in the HS group (p < 0.01 vs control), 244 ± 2 mOsm/kg in the HS +
NaCl group (p < 0.01 vs HS), and 247 ± 2 mOsm/kg in HS + Lyte group.
Plasma ions
Anion gap values, the negative strong ion difference, and plasma ion levels are presented in
Table 1. Hemorrhagic shock did not affect the anion gap, the SID, and the sodium and
chloride levels. Hemorrhagic shock did induce metabolic acidosis as reflected by a decreased
pH (p < 0.05 vs control) and plasma HCO3− level (p < 0.01 vs control). The metabolic
acidosis could be restored by Plasma Lyte resuscitation, but not by NaCl resuscitation. NaCl
resuscitation, moreover, increased the plasma chloride levels (p < 0.001 vs control), which
were decreased by Plasma Lyte resuscitation (p < 0.05 vs HS). Sodium concentration was
similar in all groups. The NaCl resuscitation group had the lowest anion gap (p < 0.01) and
negative SID was also significantly lower in the NaCl group compared to the other groups
(p < 0.05). The highest value of negative SID was found in the HS + Lyte group.
Systemic and renal hemodynamics
Systemic and renal hemodynamics are presented in Table 2. Baseline values were found
similar in each group. Hemorrhage caused marked effects on hemodynamics without
significant differences among groups (p > 0.05). In all groups, MAP and RBF decreased
during hemorrhage but RVR and HR did not change. During resuscitation, MAP was
increased in all groups and the target MAP of 80 mmHg was successfully achieved and
maintained throughout resuscitation in all groups. Resuscitation did not affect HR (p > 0.05).
During hemorrhagic shock, RBF dropped ∼74% (p < 0.01) without differences among groups.
The most effective fluid to restore RBF was the Plasma-Lyte preparation. None of the fluids
significantly affected RVR during resuscitation
32
Table 1. Anion gap, negative strong ion difference and pH values and plasma ion levels at baseline (t= 0 min) and at the end of resuscitation (t = 150 min). Cp< 0.05 vs control, Hp<0.05 vs HS, Np< 0.05 vs HS+NaCl.
Baseline Resuscitation Anion gap
Control 20.8 ± 0.5 22.3 ± 3.2 HS 18.6 ± 2.5 30.2 ± 1.6 HS+NaCl 16.3 ± 1.2 0.6 ± 4.0C,H HS+Lyte 22.6 ± 3.3 29.8 ± 6.0 Strong ion difference (negative)
Control 39.7 ± 1.8 34.3 ± 6.0 HS 37.1 ± 2.6 26.2 ± 3.1 HS+NaCl 36.0 ± 0.8 12.6 ± 5.0C HS+Lyte 39.2 ± 2.6 47.6 ± 5.3H,N pH
Control 7.38 ± 0.01 7.38 ± 0.01 HS 7.37 ± 0.01 7.18 ± 0.00C HS+NaCl 7.39 ± 0.03 7.18 ± 0.00C HS+Lyte 7.38 ± 0.03 7.36 ± 0.04H,N HCO3
-(mmol/l) Control 20.9 ± 1.9 20.6 ± 2.2
HS 20.3 ± 0.4 6.9 ± 1.0C HS+NaCl 21.7 ± 0.7 15.2 ± 1.4 HS+Lyte 21.0 ± 0.6 21.3 ± 0.9H Cl- (mmol/l)
Control 107.7 ± 3.4 110.7 ± 2.6 HS 106.4 ± 2.8 113.8 ± 1.9 HS+NaCl 109.2 ± 1.1 133.8 ± 4.0C,H HS+Lyte 105.5 ± 3.2 95.3 ± 5.6H,N Na+ (mmol/l)
Control 144.7 ± 0.3 146.0 ± 1.5 HS 140.8 ± 1.9 144.2 ± 1.0 HS+NaCl 142.8 ± 1.1 145.2 ± 0.7 HS+Lyte 143.5 ± 2.1 141.5 ± 0.5
33
Table 2. Hemodynamic parameters at baseline (t=0 min), during shock (t=60 min) and resuscitation (t=150 min). Cp < 0.05 vs. control, Hp < 0.05 vs. HS
Baseline Shock Resuscitation Mean arterial pressure (mmHg)
Control 103 ± 2 102 ± 1 102 ± 3 HS 102 ± 2 34 ± 1 C 35 ± 1C HS+NaCl 99 ± 4 35 ± 2 C 76 ± 6 C,H HS+Lyte 100 ± 5 34 ± 3 C 75 ± 6 C,H Heart rate (bpm)
Control 245 ± 6 253 ± 3 254 ± 11 HS 229 ± 7 256 ± 6 247 ± 12 HS+NaCl 226 ± 8 237 ± 16 247 ± 13 HS+Lyte 260 ± 13 260 ± 15 281 ± 12 Renal blood flow (ml/min)
Control 5.1 ± 0.7 5.9 ± 0.8 6 ± 0.9 HS 5.1 ± 0.2 1.4 ± 0.1 C 1.2 ± 0.1 C HS+NaCl 5.2 ± 0.8 1.8 ± 0.3 C 3.3 ± 1.1 HS+Lyte 5.4 ± 0.2 1.7 ± 0.2 C 3.9 ± 0.2 Renal vascular resistance (dyn s cm-5)
Control 1435 ± 114 1505 ± 164 1299 ± 171 HS 1584 ± 47 1927 ± 260 2518 ± 465 HS+NaCl 1393 ± 170 1704 ± 235 3117 ± 1443 HS+Lyte 1916 ± 15 1937 ± 195 1516 ± 106
Renal oxygenation and function
Renal oxygenation and function parameters are presented in Figs. 1 and 2, respectively. DO2,
VO2, CµPO2, and MµPO2 all decreased during the hemorrhage. CµPO2 and MµPO2 could not
be improved by fluid resuscitation using NaCl or Plasma Lyte. Both NaCl and Plasma Lyte
resuscitation increased DO2 and VO2 although the increase in VO2 was more pronounced in
the HS + Lyte group. VO2/TNa+ was increased during hemorrhage and could not be improved
by fluid resuscitation (0.68 ± 0.20 in the control group vs 1.67 ± 0.62 and 1.69 ± 0.32 in the
HS + NaCl and HS + Lyte groups, respectively, p < 0.05). Creatinine clearance was decreased
in all groups compared to the control group. NaCl resuscitation significantly increased the
EFNa+ group (p < 0.05 vs all groups) (Fig. 2).
34
Fig.1. Renal oxygenation at baseline (t=0 min) and during shock (t=60 min) and resuscitation (t=150 min). µPO2: microvascular oxygen tension; DO2: renal oxygen delivery; VO2: renal oxygen consumption. Cp< 0.05 vs control group, Hp< 0.05 vs HS group.
Fig.2. Renal function parameters at baseline (t=0 min) and the end of resuscitation (t=150min).Cp<0.05 vs controlgroup, Np<0.05 vs HS+NaCl group.
35
Oxidative stress and inflammation
All types of resuscitation decreased the plasma lactate levels. Additionally, lactate levels in
resuscitated groups were similar with control group (Table 3). The plasma MDA levels in the
resuscitated groups were lower than in the control group, reflecting the dilution of plasma by
resuscitation. Additionally, tissue MDA levels in all groups were higher than in the control
group (p < 0.05) (Table 3). This was accompanied by increase in plasma levels of hyaluronan,
an endothelial glycocalyx component (p < 0.05 vs control group) (Table 3). Plasma cytokines
levels TNF-α and IL-6 increased during shock (p < 0.01 vs control) and resuscitation did not
restore these parameters (Table 3).
Immunohistochemical analysis
Lipocalin 2 (NGAL) and L-FABP reactivity were both increased during hemorrhagic shock
(p < 0.01). Fluid resuscitation could only slightly reduce the NGAL levels (p > 0.05 in the HS
+ Lyte group and p < 0.05 in the HS + NaCl group). NaCl resuscitation could not correct the
L-FABP levels while Plasma Lyte resuscitation could significantly reduce the L-FABP levels
(p < 0.01; Fig. 3).
36
Table 3. Plasma and tissue biochemistry parameters. Cp < 0.05 vs. control, Hp < 0.05 vs.HS Resuscitation Plasma lactate (mmol/l)
Control 2.8 ± 0.2 HS 11.2 ± 1.8 C HS+NaCl 3.3 ± 0.7 H HS+Lyte 3.6 ± 0.5 H Plasma NO (µmol)
Control 76.9 ± 14.1 HS 81.2 ± 18.5 HS+NaCl 147.9 ± 23.5 HS+Lyte 102.2 ± 21.6 Tissue NO /protein content (µmol/g)
Control 1.2 ± 0.1 HS 1.9 ± 0.2 HS+NaCl 1.6 ± 0.6 HS+Lyte 0.7 ± 0.3 Plasma TNF-α (pg/ml)
Control 68 ± 13 HS 291 ± 16.26 C HS+NaCl 368 ± 47.99 C HS+Lyte 247 ± 48.7 C Plasma IL-6 (pg/ml)
Control 170 ± 25 HS 14132 ± 4347 C HS+NaCl 21258 ± 5312 C HS+Lyte 12808 ± 6146 C Plasma MDA (µmol/l)
Control 20.7 ± 2 HS 21.6 ± 3.1 HS+NaCl 15.9 ± 1.6 HS+Lyte 16.6 ± 1.8 Tissue MDA/protein content (umol/g)
Control 1.7 ± 0.2 HS 3.4 ± 0.6 C HS+NaCl 3.5 ± 0.6 C HS+Lyte 3.1 ± 0.3 C Plasma hyaluronan (ng/ml)
Control 7.9 ± 2.7 HS 158.1 ± 20.8 C HS+NaCl 97.1 ± 13.4 C HS+Lyte 152.7 ± 56.9 C
37
Fig.3. Renal tissue NGAL and L-FABP levels at the end of resuscitation (t=150 min). Cp<0.05 vs control group, Hp<0.05 vs HS group.
Discussion
In the present study, an acetate and gluconate-balanced crystalloid solution was tested for its
effects on the plasma ion levels and acid–base balance; renal oxygenation, oxidative stres
status, glycocalyx integrity; and systemic cytokine levels in a rat model of hemorrhagic shock.
The main findings of our study were that: (1) both the balanced and unbalanced crystalloid
solutions successfully restored the blood pressure, but renal blood flow was only recovered by
the balanced solution although this did not lead to improved renal oxygenation; (2) less
balanced fluid was required to restore blood pressure; (3) while unbalanced crystalloid
resuscitation induced hyperchloremia and worsened metabolic acidosis in hemorrhaged rats,
balanced crystalloid resuscitation prevented hyperchloremia, restored the acid–base balance,
and preserved the anion gap and strong ion difference in these animals; (4) neither balanced
nor unbalanced crystalloid resuscitation could normalize systemic inflammation (TNF-α and
IL-6); (5) only balanced crystalloid resuscitation significantly reduced renal oxidative stres as
reflected by reduced L-FABP reactivity, but none of the fluids could restore the increased
NGAL, MDA, and hyaluronan levels; and (6) balanced crystalloid resuscitation significantly
improved renal oxygen consumption (increased VO2, decreased EFNa+ ), but none of the fluids
was able to restore creatinine clearance rate in this shortterm protocol.
The results of our study show that renal microcirculatory hypoxia occurs during hemorrhage
and remains after crystalloid resuscitation. In addition, this appears to be relatively
independent from systemic and renal macrohemodynamics, but arises from intrarenal
mechanisms that may be associated with hypoxia in the microcirculation. A mechanism
38
potentially responsible for the disassociation between macro- and microcirculatory parameters
could be the inadequate resolution of inflammatory activation by just improving systemic
hemodynamics [Ulloa and Tracey, 2005]. Indeed, crystalloid resuscitation was unable to
prevent systemic inflammation. Combined with a suboptimal fluid composition, this possibly
leads to disturbed plasma ion levels (e.g., hyperchloremia), reduced oxygen carrying capacity,
and consequent microvascular dysfunction and hypoxia.
As renal dysfunction is a common complication following major hemorrhage and fluid
resuscitation, there is a continuing research on the efficacy of fluid resuscitation strategies to
protect the kidney. However, the type of fluid that should be used for resuscitation to yield the
best renal outcome remains controversial today and the composition of resuscitation fluids is
still subject of debate. Buffers such as acetate, gluconate, and lactate are commonly used in
resuscitation fluids. These buffers are converted to bicarbonate in the liver, raising the pH of
the solution toward normal blood pH of 7.4 [Stewart 1983; Richards et al., 1982].
Furthermore, recent investigations have shown that unbalanced solutions containing high
amounts of chloride (e.g., 0.9% NaCl) might cause hyperchloremic acidosis [Scheingraber et
al., 1999; McFarlane and Lee, 1994; Prough and Bidani, 1999] whereas solutions with buffers
and more physiological concentrations of strong ions do not [Kellum et al., 1998; Kellum,
1998]. As demonstrated by the present work, unbalanced crystalloid resuscitation leads to
hyperchloremia since a reduction in the strong ion difference is required to match the diluted
non-volatile weak acid. In this line, the decrease in pH as observed following NaCl
resuscitation in the present study appears to have been caused by chloride loading.
Resuscitation with the balanced crystalloid preparation, in contrast, completely restored
metabolic acidosis and improved renal blood flow. Clinical studies have also shown that
balanced solutions alter the acid–base status significantly less compared to saline solutions
[Khajavi et al., 2008]. The role of chloride in modulating vasoconstrictor responses to
vasoactive agents has previously been investigated in isolated rat kidneys [Quilley et al.,
1993]. Hyperchloremic fluids induced intrarenal vasoconstriction as indicated by increased
RVR and decreased RBF and glomerular filtration rate [Pedoto et al., 1999; Wilcox, 1983;
Naylor and Forsyth, 1986]. This is in agreement with the findings of increased RVR and
decreased microcirculatory perfusion in the present study. Whether these perturbations are
caused by the high levels of chloride directly or from the consequent disturbances in the acid–
base balance remains unknown.
39
Acidemia might affect a variety of vasoregulatory mechanisms. First, acidemia increases
endogenous catecholamine release, which induces the release of both pro and anti-
inflammatory cytokines [Le Tulzo et al., 1997; Liskaser et al., 2000] and nitric oxide [Haque
et al., 2003; Celotto et al., 2008]. Moreover, studies have shown that fluid resuscitation
following hemorrhage can exacerbate the systemic inflammatory responses which may be
even more harmful than the initial hemorrhage [Le Tulzo et al., 1997]. Jensen et al. have
shown that as a result of acid loading, macrophages increase their tumor necrosis factor
secretion [Jensen et al., 1990]. Increased levels of tumor necrosis factor might have influenced
microvascular perfusion either by the direct vasoactive properties of these molecules, which
could have contributed to the worsening of shock, or via direct tissue injury.
To test the effects of fluid resuscitation on systemic inflammation and oxidative stress in our
model, we measured plasma TNF-α and IL-6 levels as markers of systemic inflammation,
MDA and L-FABP as markers of oxidative stress, hyaluronan levels as markers of glycocalyx
degradation, and NGAL as a marker of renal tubular injury. We found that neither balanced
nor unbalanced crystalloid resuscitation could restore systemic inflammation, oxidative stress,
glycocalyx, or renal injury. The balanced crystalloid resuscitation, however, could
significantly reduce L-FABP reactivity. Nonetheless, our results suggest that even balanced
crystalloid resuscitation fails to prevent harmful systemic inflammatory responses and
oxidative stress following hemorrhagic shock.
In this study we found that hemorrhage severely disturbed the renal oxygen
consumption/sodium reabsortion balance and that this could not be restored by resuscitation
with balanced or unbalanced crystalloid solutions. Under normal physiological conditions,
there is a positive correlation between renal oxygen consumption (VO2) and tubular sodium
reabsorption and glomerular filtration rate [Kiil, 1977; Adler and Huang 2002; Lassen and
Thaysen, 1961]. Excessive nitric oxide (NO) production by cells as a result of inducible NO
synthase activation consequent to inflammation can inhibit mitochondrial respiration by
competing with oxygen mitochondrial cytochrome oxidase [Terada et al., 1992; Cooper and
Giulivi, 2007]. Thus, the production and utilization of oxygen, NO, and reactive oxygen
species are directly dependent on each other and together determine the adequacy of renal
function. In this line, renal oxygen consumption has been found to be increased by
nonselective NOS inhibition and selective neuronal NOS inhibition [Deng et al., 2005], which
demonstrates that different isoforms of NOS are involved in modulating renal oxygen
40
utilization. Hence, there is a complex interdependency between oxygen, reactive oxygen
species, and NO; their homeostasis becomes severely altered during conditions of renal
inflammation and ischemia/reperfusion, resulting in oxidative stress and loss of renal
function.
Conclusions
While unbalanced crystalloid resuscitation induces hyperchloremia and worsens metabolic
acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevents hyperchloremia,
restores the acid–base balance, and preserves the anion gap and strong ion difference in these
animals. Balanced crystalloid resuscitation prevents renal hypoperfusion better than
unbalanced crystalloid resuscitation. However, although the balanced preparation improves
some parameters, it does not improve oxidative stress and systemic inflammation.
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44
45
Chapter 2
THE ACUTE EFFECTS OF ACETATE-BALANCED COLLOID AND
CRYSTALLOID RESUSCITATION ON RENAL OXYGENATION IN A RAT
MODEL OF HEMORRHAGIC SHOCK.
Almac E1,2, Aksu U1,3, Bezemer R1, Jong W1, Kandil A3, Yuruk K1, Demirci-Tansel C3,
Ince C1
1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
2Department of Anesthesiology, St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands
3Department of Biology, Faculty of Science, University of Istanbul, Istanbul, Turkey
Published in: Resuscitation. 2012 Sep;83(9):1166-72.
46
Chapter 2
The acute effects of acetate-balanced colloid and crystalloid resuscitation on renal
oxygenation in a rat model of hemorrhagic shock
Running title: Colloid vs crystalloid resuscitation in hemorrhagic shock
Abstract
Introduction: Fluid resuscitation therapy is the initial step of treatment for hemorrhagic shock.
In the present study we aimed to investigate the acute effects of acetate-balanced colloid and
crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We
hypothesized that acetate-balanced solutions would be superior in correcting impaired renal
perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions.
Methods: In anesthetized, mechanically ventilated rats, hemorrhagic shock was induced by
withdrawing blood from the femoral artery until mean arterial pressure (MAP) was reduced to
30 mmHg. One hour later, animals were resuscitated with either hydroxyethyl starch (HES,
130/0.42 kDa) dissolved in saline (HES-NaCl; n = 6) or a acetate-balanced Ringer’s solution
(HES-RA; n = 6), as well as with acetated Ringer’s solution (RA; n = 6) or 0.9% NaCl alone
(NaCl; n = 6) until a target MAP of 80 mmHg was reached. Oxygen tension in the renal
cortex (CµPO2), outer medulla (MµPO2), and renal vein were measured using
phosphorimetry. Results: Hemorrhagic shock (MAP = 30 mmHg) significantly decreased
renal oxygenation and oxygen consumption. Restoring the MAP to 80 mmHg required 24.8 ±
1.7 ml of NaCl, 21.7 ± 1.4 ml of RA, 5.9 ± 0.5 ml of HES-NaCl (p < 0.05 vs. NaCl and RA),
and 6.0 ± 0.4 ml of HES-RA (p < 0.05 vs. NaCl and RA). NaCl, RA, and HES-NaCl
resuscitation led to hyperchloremic acidosis, while HES-RA resuscitation did not. Only HES-
RA resuscitation could restore renal blood flow back to ∼85% of baseline level (from 1.9 ±
0.1 ml/min during shock to 5.1 ml ± 0.2 ml/min 60min after HES-RA resuscitation) which
was associated with an improved renal oxygenation (CµPO2 increased from 24 ± 2 mmHg
during shock to 50 ± 2 mmHg 60 min after HES-RA resuscitation) albeit not to baseline level.
At the end of the protocol, creatinine clearance was decreased in all groups with no
differences between the different resuscitation groups. Conclusion: While resuscitation with
the NaCl and RA (crystalloid solutions) and the HES-NaCl (unbalanced colloid solution) led
to hyperchloremic acidosis, resuscitation with the HES-RA (acetate-balanced colloid solution)
47
did not. The HES-RA was furthermore the only fluid restoring renal blood flow back to ∼85%
of baseline level and most prominently improved renal microvascular oxygenation.
48
Introduction
Hemorrhagic shock is the major cause of mortality after major trauma and aggressive fluid
resuscitation is often the initial step to restore the circulating intravascular volume to prevent
organ hypoperfusion, organ failure, and eventually death [Coimbra et al., 2006; Liu et al.,
2003]. Acute renal failure (ARF) is a serious complication contributing to the high mortality
in these patients [Morris et al., 1991]. The use of conventional crystalloid solutions (e.g.,
isotonic saline) as initial resuscitation fluids is still implemented in emergency departments
even though it is known that crystalloid solutions have poor plasma expander capacities and
just 20% of the given volume remains contained in the intravascular space [Svensen and
Hahn, 1997]. Hence, to restore perfusion, large volumes of crystalloid solutions are required.
Additionally, hyperchloremic acidosis is a known risk in patients treated with isotonic saline.
Hyperchloremia is suggested to cause afferent renal artery vasoconstriction in animal models,
possibly leading to kidney dysfunction [Wilcox, 1983; Bullivant et al., 1989; Wilcox and
Peart, 1987]. Hydroxyethyl starch (HES) solutions have been used clinically as a colloid
solution, and have been shown to have superior plasma expanding capacities compared to
traditional crystalloid solutions [Haisch et al., 2001]. However, these HES solutions, in turn,
have been suggested to have adverse effects on systemic coagulation properties and are
potentially harmful for the kidney [Warren and Durieux, 1997]. Consequently, new HES
solutions (mean molecular weight: 130 kDa, degree of substitution: 0.4; HES 130/0.4) have
been developed and have been shown to improve microvascular perfusion and reduce
macromolecular leakage [Boldt et al., 1996; Nohe et al., 2005; Hoffmann et al., 2002].
Although effective in restoring systemic hemodynamic parameters, aggressive (i.e., large
volume) fluid resuscitation introduces non-physiologic levels of plasma ions which depress
microvascular function and organ perfusion [Legrand et al., 2010]. The kidney is especially
susceptible for this type of injury due to its complex microvascular structure and high oxygen
dependency [Evans et al., 2008]. Over the past few years, research has therefore been focused
on balancing fluids to optimally match physiological conditions and thereby prevent
microvascular dysfunction and organ hypoperfusion [Spahn et al., 2007; Stern, 2001; Horstick
et al., 2002]. Balanced fluids are suggested to have a more physiological electrolyte
composition than conventional saline-based fluids.
In the present study we aimed to investigate the acute effects of acetate-balanced colloid and
crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. To this
49
end, we examined the effects of resuscitation with different fluids: (1) 0.9% NaCl; (2)
acetated Ringer’s solution; (3) 6% HES with a molecular weight of 130 kDa and molar
substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution (HES-NaCl); and (4) 6% HES with a
molecular weight of 130 kDa and molar substitution of 0.42 (HES 130/0.42) in acetate-
balanced Ringer’s solution (HES-RA). We hypothesized that acetated solutions would have
superior resuscitation capacities compared to the other solutions with respect to improving
renal oxygenation after severe hemorrhage.
Materials and methods
Animals
All experiments in this study were approved by the institutional Animal Experimentation
Committee of the Academic Medical Center of the University of Amsterdam. Care and
handling of the animals were in accordance with the guidelines for Institutional and Animal
Care and Use Committees. Experiments were performed on 30 Sprague-Dawley rats (Harlan,
the Netherlands) with mean ± SD body weight of 350 ± 20 g.
Surgical preparation
The rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg
ketamine (Nimatek®; Eurovet, Bladel, the Netherlands), 0.5 mg/kg medetomidine (Domitor;
Pfizer, New York, NY), and 0.05 mg/kg atropine-sulfate (Centrafarm, Etten-Leur, the
Netherlands). After tracheotomy, the animals were mechanically ventilated with an FiO2 of
0.4. Body temperature was maintained at 37 ± 0.5 ◦C during the entire experiment by external
warming. The ventilator settings were adjusted to maintain end-tidal PCO2 between 30 and 35
mmHg and arterial PCO2 between 35 and 40 mmHg. Vessels were cannulated with
polyethylene catheters (outer diameter = 0.9 mm; Braun, Melsungen, Germany) for drug and
fluid administration and hemodynamic monitoring. A catheter in the right carotid artery was
connected to a pressure transducer to monitor mean arterial blood pressure (MAP) and heart
rate. The right jugular vein was cannulated for continuous infusion of Ringer Lactate (Baxter,
Utrecht, the Netherlands) at a rate of 15 ml/kg/h. The right femoral artery was cannulated for
blood shedding and the right femoral vein for fluid resuscitation. The left kidney was
exposed, decapsulated, and immobilized in a Lucite kidney cup (K. Effenberger, Pfaffingen,
Germany) via a 4 cm incision in the left flank. Renal vessels were carefully separated under
preservation of nerves and adrenal gland. A perivascular ultrasonic transient time flow probe
was placed around the left renal artery (type 0.7 RB; Transonic Systems Inc., Ithaca, NY,
50
USA) and connected to a flow meter (T206; Transonic Systems Inc.) to continuously measure
renal blood flow (RBF). An estimation of the renal vascular resistance (RVR) was made as
RVR [dynes s cm−5] = (MAP/RBF) × 100. The left ureter was isolated, ligated and cannulated
with a polyethylene catheter for urine collection. The surgical field was covered with a
humidified gauze compress throughout the entire experiment to prevent drying of the exposed
tissue.
After the surgical protocol (approximately 60 min) one optical fiber was placed 1 mm above
the decapsulated kidney and another optical fiber 1 mm above the renal vein to measure
oxygenation using a phosphorescence lifetime technique. A small piece of aluminum foil was
placed on the dorsal site of the renal vein to prevent contribution of underlying tissue to the
phosphorescence signal in the venous PO2 measurement. Oxyphor G2 (a two-layer glutamate
dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin; Oxygen Enterprises Ltd.,
Philadelphia, PA, USA) was subsequently infused (6 mg/kg IV over 5 min) followed by a 30
min stabilization period. A short description of the phosphorescence quenching method is
given below and a more detailed description of the technology has been previously described
[Johannes et al., 2006].
Experimental protocol
After stabilization, the animals in experimental groups were bled by the left femoral artery
catheter at a rate of 1 ml/min using a syringe pump (Harvard 33 syringe pump; Harvard
Apparatus, South Natick, MA) until a MAP of 30 mmHg was reached which was maintained
for 1 h by re-infusing or withdrawing blood. Coagulation of the shed blood was prevented by
adding 200 UI of heparin in the syringe.
At the end of the hemorrhage phase, the animals were randomized into 5 groups for
resuscitation until a target MAP of 80 mmHg was reached with: (1) 0.9% NaCl (NaCl; Na+
154 mmol l−1, Cl− 154 mmol l−1; pH 5.5; n = 6); (2) Ringer’s Acetate (RA; Na+ 130 mmol l−1,
Cl− 112 mmol l−1, K+ 5.4 mmol l−1, Ca+2 0.9 mmol l−1, Mg+2 1.0 mmol l−1, acetate− 27 mmol
l−1; pH = 5.0–7.0; n = 6); (3) 6% HES with a molecular weight of 130 kDa and molar
substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution (HES-NaCl; Voluven®, Fresenius
Kabi, Bad Homburg, Germany; n = 6); or (4) 6% HES with a molecular weight of 130 kDa
and molar substitution of 0.42 (HES 130/0.42) in acetate-balanced Ringer’s solution (HES-
51
RA; Plasma Volume®, Baxter, Germany; n = 6). In addition, sham operated control
experiments were performed (n = 6).
The experiments were terminated by infusion of 1 ml of 3 M potassium chloride (KCl).
Blood gas parameters
Arterial blood samples (0.5 ml) were taken from the femoral artery at time points: (1) baseline
(BL, t = 0 min); (2) after hemorrhagic shock (HS, t = 60 min); (3) 15 min after starting
resuscitation (R15, t = 75 min), and (4) at the end of the protocol (R60, t = 120 min).
The blood samples were replaced by the same volume of test solution. The samples were used
to determine blood gas parameters (ABL505 blood gas analyzer; Radiometer, Copenhagen,
Denmark), hemoglobin concentration, and hemoglobin oxygen saturation (OSM 3,
Radiometer).
Renal microvascular and venous oxygenation
Microvascular oxygen tension in the renal cortex (CµPO2), outer medulla (MµPO2), and renal
venous oxygen tension (PrvO2) were measured by oxygen-dependent quenching of
phosphorescence lifetimes of the systemically infused albumin-targeted (and therefore
circulation-confined) phosphorescent dye Oxyphor G2 [Johannes et al., 2006; Mik et al.,
2004; Vinogradov et al., 2002; Dunphy et al., 2002]. Oxyphor G2 (a two-layer glutamate
dendrimer of tetra- (4-carboxy-phenyl) benzoporphyrin) has two excitation peaks (λexcitation1 =
440 nm, λexcitation2 = 632 nm) and one emission peak (λemission = 800 nm) [Dunphy et al.,
2002]. These optical properties allow (near) simultaneous lifetime measurements in
microcirculation of the kidney cortex and the outer medulla due to different optical
penetration depths of the excitation light [Johannes et al., 2006]. For the measurement of renal
venous PO2 (PrvO2), a mono-wavelength phosphorimeter was used [Mik et al., 2008]. Oxygen
measurements based on phosphorescence lifetime techniques rely on the principle that
phosphorescence can be quenched by energy transfer to oxygen resulting in shortening of the
phosphorescence lifetime. A linear relationship between reciprocal phosphorescence lifetime
and oxygen tension (given by the Stern–Volmer relation) allows quantitative measurement of
PO2. Details of the technique have previously been published [Johannes et al., 2006].
Renal oxygen delivery and consumption
52
Arterial oxygen content (AOC) was calculated by (1.31 ×hemoglobin × SaO2) + (0.003 ×
PaO2), where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure of oxygen.
Renal venous oxygen (RVOC) content was calculated as (1.31 × hemoglobin × SrvO2) +
(0.003 × PrvO2), where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial
pressure of oxygen. Renal oxygen delivery was calculated as DO2 (ml/min) = RBF × AOC.
Renal oxygen consumption is calculated as VO2ren (ml/min/g) = RBF × (AOC − RVOC). The
renal oxygen extraction ratio was calculated as O2ERren (%) = VO2ren/DO2 × 100.
Assessment of kidney function
Creatinine clearance (Clearcrea, [ml/min]) was assessed as an index of the glomerular filtration
rate. Calculation of the clearance was done using the standard formula: Clearcrea = (Ucrea ×
V)/Pcrea, where Ucrea is the concentration of creatinine in urine, V is the urine volume per unit
time and Pcrea is the concentration of creatinine in plasma.
Furthermore, all urine samples were analyzed for sodium (Na+) concentration. The renal
energy efficiency for sodium transport (VO2ren/TNa+ ) was assessed using the ratio of the total
amount of VO2ren over the total amount of sodium reabsorbed (TNa+ , [mmol/min]).
Statistical analysis
Values are reported as the mean ± SEM. The decay curves of phosphorescence intensity were
analyzed using software programed in Labview 6.1 (National Instruments, Austin, TX, USA).
Statistical analysis was performed using GraphPad Prism version 4.0 for Windows (GraphPad
Software, San Diego, CA, USA). Twoway ANOVA with a Bonferroni post hoc test was used
and a p-value of <0.05 was considered statistically significant.
Results
Fluid and electrolyte balance
The amount of fluids given during resuscitation and the plasma chloride and sodium levels
and plasma pH are presented in Table 1. Restoring the MAP from 30 mmHg (shock) to 80
mmHg required 24.8 ± 1.7 ml of NaCl, 21.7 ± 1.4 ml of RA, 5.9 ± 0.5 ml of HES-NaCl (p <
0.05 vs. NaCl and RA), and 6.0 ± 0.4 ml of HES-RA (p < 0.05 vs. NaCl and RA). Plasma
chloride levels were significantly increased (p < 0.05 vs. time control) after NaCl (119.6 ± 6.1
mmol l−1), RA (110.2 ± 1.7 mmol l−1), and HES-NaCl (112.4 ± 3.5 mmol l−1) resuscitation,
53
but not after HES-RA (106.0 ± 3.5 mmol l−1) resuscitation. Similarly, plasma pH was
significantly decreased (p < 0.05 vs. time control) after NaCl (7.10 ± 0.03), RA (7.15 ± 0.01),
and HESNaCl (7.20 ± 0.02) resuscitation, but not after HES-RA (7.26 ± 0.02) resuscitation.
Hence, NaCl, RA, and HES-NaCl resuscitation led to hyperchloremic acidosis, while HES-
RA resuscitation did not.
Table 1: Amount of resuscitation fluid required to increase the mean arterial pressure from 30 to 80 mmHg and the plasma sodium (Na+ ) and chloride (Cl−) concentrations and plasma pH at baseline (BL) and after 60 min of resuscitation (R60). Tp<0.05 vs. time control, Np<0.05 vs. 0.9% NaCl, Rp<0.05 vs. Ringer’s Acetate.
BL R60 Amount of fluid required (ml)
Time control HS control
0.9 % NaCl
24.8 ± 1.7 Ringer's Acetate
21.7 ± 1.4
HES-NaCl
5.9 ± 0.5N,R HES-RA
6.0 ± 0.4N,R
Cl- (mmol/l) Time control 104.0 ± 1.0 105.0 ± 2.0
HS control 110.4 ± 1.1 119.5 ± 3.2 0.9 % NaCl 87.2 ± 5.4 119.6 ± 6.1T Ringer's Acetate 105.6 ± 3.7 110.2 ± 1.7T HES-NaCl 97.6 ± 1.2 112.4 ± 3.5T HES-RA 102.4 ± 4.1 106.0 ± 3.5 Na+ (mmol/l)
Time control 142.0 ± 2.0 143.0 ± 2.0 HS control 148.4 ± 2.0 149.0 ± 3.1 0.9 % NaCl 141.8 ± 1.0 143.2 ± 0.8 Ringer's Acetate 142.3 ± 1.2 144.2 ± 0.5 HES-NaCl 143.2 ± 1.0 143.8 ± 0.6 HES-RA 142.5 ± 0.9 143.8 ± 0.5 pH
Time control 7.31 ± 0.10 7.30 ± 0.10 HS control 7.35 ± 0.01 7.11 ± 0.02 0.9 % NaCl 7.27 ± 0.01 7.10 ± 0.03T Ringer's Acetate 7.31 ± 0.01 7.15 ± 0.01T HES-NaCl 7.29 ± 0.03 7.20 ± 0.02T HES-RA 7.31 ± 0.01 7.26 ± 0.02
54
Systemic and renal hemodynamics
Systemic and renal hemodynamic variables are presented in Table 2. The baseline values
measured in each group were found to be similar (p > 0.05). In all groups, MAP, RBF
decreased during hemorrhage without significant differences between groups.
During resuscitation, MAP was consistently increased in all groups, though the target MAP of
80 mmHg was not successfully maintained after 60 min of resuscitation. In crystalloid treated
groups, NaCl and RA, MAP was lower at the end of the protocol (44 ± 4 and 48 ± 3 mmHg,
respectively) compared to in the colloid treated groups, HES-NaCl and HES RA (58 ± 5 and
52 ± 3 mmHg, respectively).
Resuscitation improved RBF in all groups starting in the early phase of resuscitation (p <
0.05). Improvement of RBF after 60 min of resuscitation was most pronounced in the HES-
RA group (5.1 ± 0.2 ml/min; 85% of baseline value) and least in 0.9% NaCl group (2.4 ± 0.5
ml/min; 42% of baseline value).
Renal oxygenation
Renal DO2, VO2, CµPO2, and MµPO2 are presented in Table 3. All these parameters
decreased during hemorrhage without significant differences between groups. At the end of
resuscitation, DO2 was improved compared to hemorrhagic shock. This increase was
significant in the RA group (0.41 ± 0.07 ml O2/min) and HES-RA group (0.39 ± 0.06 ml
O2/min) compared to HS control (p < 0.05). VO2, however, could not be increased by fluid
resuscitation (p > 0.05 vs. HS control).
Resuscitation improved CµPO2 and MµPO2 albeit not to baseline level. At R60, CµPO2 was
higher in the HES-RA group compared to other groups and significantly different comparing
to the NaCl group (p < 0.05).
55
Table 2: Mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) at baseline (BL), during hemorrhagic shock (HS), and after 15 and 60 min of resuscitation (R15 and R60, respectively). Hp < 0.05 vs. HS control, Np < 0.05 vs. 0.9 % NaCl. BL HS R15 R60 MAP (mmHg) Time control 102 ± 1.0 104 ± 2.0 99 ± 3.0 105 ± 4.0 HS Control 95 ± 9.0 30 ± 2.0 30 ± 3.0 30 ± 2.0 0.9% NaCl 102 ± 2.0 31 ± 1.0 73 ± 8.0 44 ± 4.0 Ringer's Acetate 101 ± 2.0 33 ± 1.0 57 ± 2.0H 48 ± 3.0 HES-NaCl 102 ± 3.0 30 ± 1.0 67 ± 6.0 58 ± 5.0 HES-RA 101 ± 3.0 32 ± 2.0 62 ± 3.0 52 ± 3.0 RBF (ml.min-1) Time control 5.6 ± 0.4 5.6 ± 0.1 5.8 ± 0.6 5.5 ± 1.0 HS Control 5.4 ± 0.2 1.2 ± 0.2 1.1 ± 0.3 0.9 ± 0.2 0.9% NaCl 5.6 ± 0.3 1.4 ± 0.1 2.9 ± 0.6H 2.4 ± 0.5 Ringer's Acetate 5.6 ± 0.4 1.4 ± 0.2 3.8 ± 0.6H 3.6 ± 0.4H HES-NaCl 5.5 ± 0.2 1.7 ± 0.3 3.1 ± 0.6H 3.4 ± 0.4H HES-RA 5.9 ± 0.2 1.9 ± 0.1 4.9 ± 0.6H 5.1 ± 0.2H,N RVR (dyn.s.sec-5) Time control 16.4 ± 1.1 15.1 ± 1.5 17.2 ± 1.2 16.9 ± 1.5 HS Control 17.5 ± 1.8 24.5 ± 5.2 27.6 ± 5.4 37.7 ± 5.6 0.9% NaCl 18.6 ± 1.5 22.3 ± 1.8 29.3 ± 4.6 21.4 ± 3.5 Ringer's Acetate 18.5 ± 1.5 20.8 ± 1.2 15.8 ± 2.6 14.2 ± 2.0 HES-NaCl 18.6 ± 1.3 19.9 ± 3.0 21.4 ± 3.5 18.7 ± 2.7 HES-RA 17.1 ± 1.0 17.6 ± 0.9 13.1 ± 1.1 10.2 ± 0.5H,N
Renal function
Creatinine clearance and VO2/TNa+ are presented in Fig. 1. There were no differences at
baseline in creatinine clearance (not shown). During hemorrhagic shock urine production
decreased dramatically. In the HS control group, all animals suffered from anuria at the end of
the protocol. All groups had a lower creatinine clearance at the end of resuscitation (p < 0.05
vs. time control). The NaCl resuscitated group had the lowest creatinine clearance rate at R60.
The VO2/TNa+ was found to be unaffected by fluid resuscitation.
56
Table 3: Renal oxygen delivery (DO2), oxygen consumption (VO2) and microvascular oxygen tension in the renal cortex (CµpO2) and medulla (MµpO2) at baseline (BL), during hemorrhagic shock (HS), and after 15 and 60 min of resuscitation (R15 and R60, respectively). Hp < 0.05 vs. HS control, Np < 0.05 vs. 0.9% NaCl.Rp < 0.05 vs. Ringer’s Acetate.
BL HS R15 R60
DO2 (ml O2/min) Time control 1.30 ± 0.10 1.32 ± 0.15 1.27 ± 0.08 1.4 ± 0.01
HS control 1.42 ± 0.11 0.19 ± 0.05 0.16 ± 0.05 0.13 ± 0.05 0.9 % NaCl 1.36 ± 0.09 0.22 ± 0.01 0.33 ± 0.06 0.27 ± 0.06
Ringer's Acetate 1.24 ± 0.03 0.22 ± 0.04 0.48 ± 0.08H 0.41 ± 0.07H HES-NaCl 1.39 ± 0.04 0.29 ± 0.09 0.4 ± 0.09H 0.35 ± 0.05H
HES-RA 1.32 ± 0.08 0.2 ± 0.02 0.53 ± 0.09H 0.39 ± 0.06H VO2 (ml O2/min/g)
Time control 0.20 ± 0.02 0.25 ± 0.01 0.24 ± 0.01 0.28 ± 0.02 HS control 0.15 ± 0.08 0.07 ± 0.02 0.07 ± 0.02 0.06 ± 0.02
0.9 % NaCl 0.19 ± 0.06 0.07 ± 0.01 0.08 ± 0.02 0.05 ± 0.01 Ringer's Acetate 0.10 ± 0.03 0.06 ± 0.02 0.10 ± 0.04 0.07 ± 0.03
HES-NaCl 0.21 ± 0.03 0.11 ± 0.04 0.09 ± 0.03 0.07 ± 0.02 HES-RA 0.15 ± 0.02 0.07 ± 0.01 0.11 ± 0.03 0.11 ± 0.02
CµpO2 (mmHg) Time control 80.0 ± 2.0 78.0 ± 2.0 78.0 ± 2.0 76.0 ± 2.0
HS control 83.0 ± 2.0 28.0 ± 6.0 22.0 ± 4.0 19.0 ± 5.0 0.9 % NaCl 85.0 ± 4.1 19.7 ± 2.5 42.9 ± 2.9 33.0 ± 2.5
Ringer's Acetate 75.0 ± 6.0 21.0 ± 3.8 43.6 ± 3.1 41.0 ± 2.0 HES-NaCl 81.0 ± 7.0 27.0 ± 4.0 39.8 ± 3.5 45.0 ± 5.0
HES-RA 85.0 ± 4.0 24.1 ± 1.5 53.1 ± 3.5R 49.8 ± 2.4N MµpO2 (mmHg)
Time control 67.0 ± 2.0 66.0 ± 1.0 64.0 ± 1.0 64.0 ± 2.0 HS control 62.0 ± 2.0 19.0 ± 6.0 15.0 ± 2.0 14.0 ± 2.0
0.9 % NaCl 67.0 ± 3.0 9.1 ± 1.2 41.4 ± 1.6 30.4 ± 1.4 Ringer's Acetate 61.0 ± 4.5 11.2 ± 3.1 30.7 ± 2.8 29.6 ± 1.0
HES-NaCl 73.0 ± 1.3 15.6 ± 2.7 34.9 ± 2.3 29.9 ± 2.6 HES-RA 67.0 ± 3.3 22.2 ± 2.7 39.2 ± 5.8 30.5 ± 3.5
57
Fig.1. Creatinine clearance and the ratio of the renal oxygen consumption (VO2) over the total amount of sodium reabsorbed (TNa+) after 60 min of resuscitation. Tp < 0.05 vs. time control, Np < 0.05 vs. 0.9% NaCl.
Discussion
In the present study, we examined the acute effects of acetatebalanced colloid and crystalloid
resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We tested the
hypothesis that acetate-balanced solutions would be superior in correcting impaired renal
perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions. Our
main findings were that: (1) hemorrhagic shock was associated with acute decreases in blood
pressure, renal perfusion and oxygenation, and urine production; (2) volume replacement
therapy with balanced and unbalanced crystalloid and colloid solutions partially corrected
these parameters; and (3) the acetate-balanced colloid solution HES-RA was the only
resuscitation fluid that could restore renal blood flow back to ∼85% of baseline level which
was associated with the most prominently improved renal oxygenation.
Hemorrhagic shock is one of the major causes of acute renal failure due to decreased blood
pressure and consequent hypoperfusion of the kidney. The presence of acute renal failure
significantly increases morbidity and mortality [Lindseth et al., 1975]. The first step in the
correction of hemorrhage-induced hypotension is aggressive volume replacement therapy
[Spahn et al., 2007] which aims to increase the circulating intravascular volume, blood
pressure, and organ perfusion [Hoffmann et al., 2002; Kemming et al., 2005]. However, in
contrast to blood, resuscitation fluids have poor oxygen transporting capacity and rheological
properties. In addition, the fluids used for volume replacement therapy have been suggested to
58
increase inflammation and disturb homeostasis and the acid–base balance [Xiao et al., 2004;
Crimi et al., 2006; Santibanez-Gallerani et al., 2001; Yada-Langui et al., 2004]. Over time, a
variety of colloid and crystalloid solutions has been used, including isotonic saline and saline-
based colloid solutions. Although saline-based solutions have been associated with disturbed
acid–base balance due to non-physiological electrolyte composition and pH, these yet remain
the most popular solutions for volume replacement therapy in peri-operative care
[Scheingraber et al., 1999; Waters et al., 2001; Waters et al., 1999; Juca et al., 2005; Wilcox,
1983; Bullivant et al., 1989; Wilcox and Peart 1987]. With respect to the kidney, saline-based
solutions are known to be more frequently associated with hyperchloremic acidosis, due to
their high levels of chloride, resulting in renal vascular constriction and decreased renal
perfusion [Kellum, 2002; Brill et al., 2002; Williams et al., 1999]. This we have confirmed in
the present study.
Balanced solutions, in contrast, provide an alternative with optimized physiological
composition in terms of sodium, potassium, calcium, magnesium, and chloride levels, and
their relative contributions regarding osmolality. Buffers such as acetate, gluconate, pyruvate,
and lactate can be used in resuscitation fluids and are converted to bicarbonate in liver and
raise the pH of the solution to normal blood pH (7.4). These solutions achieve a physiological
acid–base balance with either bicarbonate or metabolizable anions and reduce of the risk of
iatrogenic disruptions. In animal models of sepsis it has also been demonstrated that balanced
solutions lead to less metabolic acidosis, reduced inflammatory cytokine levels, and longer
survival compared to resuscitation with normal saline [Kellum, 2002; Kellum et al., 2006].
Infusion of solutions containing lactate, however, has multiple side effects and, aside from
those, lactate buffers require high levels of liver metabolism and oxygen consumption
[Zander, 2002].
In our model, as shown by others, hyperchloremia led to progressive renal vasoconstriction
(increased RVR and decreased RBF) and a fall in glomerular filtration rate (decreased
creatinine clearance). These phenomena have been shown to be independent of the renal
nerve system and to be related to tubular chloride reabsorption and chloride-induced renal
vasoconstriction [Wilcox, 1983]. Increased RVR and decreased creatinine clearance were
most pronounced following NaCl resuscitation and were less pronounced in the HES-RA
resuscitated group. Furthermore, HES-RA resuscitation was the only regime that could
significantly increase renal DO2. This can be explained by the composition of the different
59
fluids: where 0.9% NaCl has a chloride content of 154 mmol l−1, HES-RA has a chloride
content of 112 mmol l−1. It should be pointed out, however, that the improved renal
oxygenation in the HES-RA group compared to the other groups is not directly associated
with acetate-balancing, per se; rather, it is probably due to less chloride infused in HES-RA in
this MAP-targeted resuscitation protocol. Acetate itself does not correct hyperchloremic
acidosis, lactic acidosis, and does not protect renal function. However, as HES-RA
resuscitation prevented hyperchloremic acidosis, it also led to avoiding microvascular
constriction in renal cortex and medulla by which renal oxygenation was improved.
Therefore, in essence, this study provided evidence that the excess chloride in resuscitation is
toxic and disturbs both the acid–base balance and the organ function.
In this line, metabolic acidosis has been shown to be a common complication in critically ill
patients and has been shown to serve as an independent predictor of outcome [Smith et al.,
2001; Gunnerson et al., 2006]. Furthermore, restricting chloride-rich fluids in intensive care
have been shown significantly improve the acid–base status in critically ill patients [Yunos et
al., 2011]. However, although several animal studies, including the present study, suggest that
hyperchloremic metabolic acidosis leads to renal vasoconstriction and potentially to kidney
dysfunction, whether this also occurs in patients remains to be verified.
The results from our study demonstrated once more the need for larger volumes of
crystalloids to achieve similar systemic and microcirculatory goals, compared to colloids.
Blood pressure increased the first 15–20 min of resuscitation and then gradually declined even
though fluid infusion continued. Hence, the volume expansion effect of both crystalloids and
colloids were temporary. Nonetheless, significantly lower volumes of colloids were required
and the colloid solutions were also more effective in maintaining blood pressure after 1 h of
resuscitation. The low efficacy of the crystalloid solutions can be explained by the fact that
only 20% of their volume remains in the vascular lumen and 80% leaks out, leading to tissue
edema and consequent impaired tissue oxygenation.
Excessive fluid overload leads to hemodilution which eventually may impair tissue
oxygenation. In experimental studies it has been demonstrated that acute isovolemic
hemodilution is associated with increases in red blood cell aggregation which triggers
endothelium-dependent thrombogenic and pro-inflammatory responses [Morariu et al., 2006].
Animal studies have demonstrated the direct influence of hemodilution on microvascular flow
60
and renal oxygen supply [Johannes et al., 2007]. Johannes et al. have found that the renal
microvascular oxygenation drops at very early stages of isovolemic hemodilution. It was also
shown that the kidney is particularly vulnerable to decreases in oxygen delivery and that the
critical hematocrit associated with a decrease in microvascular oxygenation is much higher
for the kidney than for the heart or intestines [van Bommel et al., 2008]. This was underscored
by a study demonstrating an increased risk of acute kidney injury in cardiopulmonary bypass-
associated hemodilution [Habib et al., 2005]. The reasons for such a high sensitivity to
hemodilution could involve endothelial dysfunction with an inflammatory component leading
to tissue edema and increase of diffusion distance from microcirculation to the tissue cells.
Although earlier studies suggested negative effects of colloids on microcirculation, there is
increasing evidence supporting the opposite [Krieter et al., 1995]. Compared to crystalloid
solutions, colloid solutions increase plasma viscosity. Elevating plasma viscosity in extreme
hemodilution has been shown to increase microvascular flow through nitric oxide-mediated
vasodilation [Tsai et al., 2005]. Others have demonstrated the importance of sufficient blood
viscosity with respect to functional capillary density and tissue oxygenation. Hence, during
acute hemodilution as occur during aggressive fluid resuscitation, increasing plasma viscosity
by administration of colloids may be beneficial for the microcirculation [Tsai and Intaglietta,
2001]. Indeed, the administration of hyperoncotic and hyperviscous solutions has been shown
to be advantageous in hemorrhagic shock due to normalization of colloid osmotic pressure
which leads to the recovery of microcirculatory perfusion and oxygenation [Wettstein et al.,
2006]. Furthermore, Lang et al. described that colloids improved microvascular perfusion and
reduced endothelial tissue edema. In contrast, the authors showed that crystalloids leak
rapidly into the interstitium, causing endothelial tissue swelling and consequently reducing
capillary perfusion and increasing the oxygen diffusion distance [Lang et al., 2001]. The
results from the present study confirm this as microvascular oxygenation in the renal cortex
was lower in the crystalloid resuscitated groups compared to the colloid resuscitated groups.
This was most marked when comparing the unbalanced crystalloid olution (NaCl) to the
acetate-balanced colloid solution (HES-RA) and was also translated into a significantly higher
creatinine clearance rate in the HES-RA group compared to the NaCl group.
Our study has, however, some limitations which should be acknowledged. First, translation of
the findings in our animal model to clinical scenarios should be done with utmost care. Here,
we imitated major hemorrhage by withdrawing blood until mean arterial pressure was
61
decreased to 30 mmHg. Most trauma patients, however, suffer from multiple injuries which
may influence their inflammatory state, potentially interfering with the hemorrhageinduced
hypovolemia and subsequent treatment. Moreover, this model does not reflect the challenges
in treatment of a neurological trauma patient. Nonetheless, our model does demonstrate the
efficacy of volume replacement therapy using different types of fluids on renal perfusion and
oxygenation after severe hemorrhage. Second, the rather short follow-up period after of
hemorrhagic shock and resuscitation does not allow assessment of renal (dys)function and
injury in the long-term. Third, blood lactate and base excess levels were not monitored in the
experiments so the effects of the tested solutions on these parameters remain to be elucidated.
Conclusions
In conclusion, while resuscitation with the NaCl and RA (crystalloid solutions) and the HES-
NaCl (unbalanced colloid solution) led to hyperchloremic acidosis, resuscitation with the
HES-RA (acetatebalanced colloid solution) did not. The acetate-balanced colloid solution
HES-RA was furthermore the only fluid restoring renal blood flow back to ∼85% of baseline
level and most prominently improved renal microvascular oxygenation. However, the
longterm effects of HES-RA resuscitation on renal function warrants further study.
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66
67
Chapter 3
ACUTE EFFECTS OF BALANCED VERSUS UNBALANCED COLLOID
RESUSCITATION ON RENAL MACROCIRCULATORY AND
MICROCIRCULATORY PERFUSION DURING ENDOTOXEMIC SHOCK
Aksu U1,2, Bezemer R1, Demirci C2, Ince C1.
1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
2Department of Biology, Faculty of Science, University of Istanbul, Istanbul, Turkey
Published in: Shock. 2012 Feb;37(2):205-9.
68
Chapter 3
Acute effects of balanced versus unbalanced colloid resuscitation on renal
macrocirculatory and microcirculatory perfusion during endotoxemic shock
Running title: Balanced vs unbalanced colloid resuscitation in sepsis
This study was designed to investigate the acute effects of balanced versus unbalanced colloid
resuscitation on renal macrocirculatory and microcirculatory perfusions during
lipopolysaccharide-induced endotoxemic shock in rats. We tested the hypothesis that balanced
colloid resuscitation would be better for the kidney than unbalanced colloid resuscitation.
Shock was induced by lipopolysaccharide (10 mg/kg i.v. over 30 min). When mean arterial
pressure (MAP) was decreased to 40 mmHg, fluid resuscitation was started with either
hydroxyethyl starch (HES130/0.42) dissolved in saline (HES-NaCl) as an unbalanced colloid
solution or HES130/0.42 dissolved in Ringer’s acetate (HES-RA) as a balanced colloid
solution. Microvascular perfusion in the renal cortex was monitored using laser speckle
imaging, and in addition, systemic hemodynamics, renal artery blood flow (RBF), and plasma
ion levels were measured. Shock decreased MAP, led to anuria, and worsened all other
parameters. Hydroxyethyl starch-NaCl improved MAP (p > 0.05) but did not improve RBF (p
> 0.05), metabolic acidosis (p > 0.05), and plasma ion levels (p > 0.05). Hydroxyethyl starch-
RA improved MAP (p < 0.05), RBF (p < 0.05), and renal microvascular perfusion (p < 0.05),
but did not improve metabolic acidosis (p > 0.05) and plasma ion levels (p > 0.05). Both
HES-NaCl and HES-RA treatment could normalize creatinine clearance but not fractional
sodium excretion. In endotoxemic rats, balanced colloid (HES) resuscitation was shown to be
superior to un- balanced colloid resuscitation in terms of improvement of renal macrovascular
and microvascular perfusions. However, whether this results in improved renal function in the
long term warrants further study.
69
Introduction
In the early stage of sepsis, impairment of the renal microcirculation is a key complication
potentially leading to renal failure through hypoxia-induced tubular epithelial cell injury and
acute tubular necrosis [Weinberg et al., 1991; De Backer et al., 2002; Sakr et al., 2004;
Klenzak and Himmelfarb, 2005]. Fluid resuscitation during sepsis is considered crucial for the
preservation of adequate intravascular volume and blood pressure and thereby promotion of
microvascular perfusion and renal oxygenation [Rivers et al., 2001]. It has been shown,
furthermore, that hypoxic microvascular areas might arise in the renal cortex in untreated
endotoxemia [Johannes et al., 2009].
As renal dysfunction is a key complication in intensive care units, there is a continuing
concern about the efficacy of fluid resuscitation. However, the type of fluid that should be
used for resuscitation in sepsis to yield the best renal outcome remains controversial today
[Marx, 2003]. This controversy includes not only the use of crystalloid versus colloid
solutions but also the use of balanced versus unbalanced colloid solutions. It is known that
crystalloid solutions have poor plasma expander capacities, and just 20% of the given volume
remains contained in the intravascular space [Svensen and Hahn, 1997]. Colloid solutions,
because of their high colloid osmotic pressure, are known to have superior plasma expanding
capacities compared with traditional crystalloid solutions. As most colloid preparations are
saline based, liberal fluid resuscitation regimens might lead to nonphysiologically high
sodium and chloride concentrations and may be associated with the development of
(hyperchloremic) metabolic acidosis, which could affect inflammatory and coagulation
homeostasis and thereby deteriorated organ function [Morgan, 2005; Schindler, 2004]. This
insight has led to development of modern hydroxyethyl starch (HES) preparations based on
balanced, plasma-adapted, crystalloid solutions and to the idea of developing a totally
balanced fluid resuscitation concept, including balanced crystalloids and balanced colloids
[Schindler, 2004]. However, whether these new, balanced fluids are able to improve renal
microcirculatory perfusion and renal function under septic conditions remains to be
elucidated.
The aim of this study was therefore to investigate the acute effects of balanced versus
unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in
a rat model of lipopolysaccharide (LPS)-induced endotoxemia. We tested the hypothesis that
balanced colloid resuscitation would be better for the kidney than unbalanced colloid
70
resuscitation. The acute effects of two clinically applied resuscitation regimens were
investigated: 6% 130/0.4 HES in NaCl (HES-NaCl) as an unbalanced colloid solution and 6%
130/0.4 HES in Ringer’s acetate solution (HES-RA) as a balanced colloid solution. Renal
perfusion was assessed at the macrocirculatory level using Doppler ultrasound on the renal
artery and at the microcirculatory level using laser speckle imaging (LSI) on the renal cortex.
Materials and methods
Animals
The protocol of the present study was approved by the Animal Research Committee of the
Academic Medical Center at the University of Amsterdam. Animal care and handling were
performed in accordance with the national guidelines for the care of laboratory animals. The
experiments were performed in 20 male Wistar rats with a mean +/- SEM body weight of 385
+/- 1 g.
Preparation
The rats were anesthetized with an intraperitoneal injection of a mixture of 90 mg/kg
ketamine, 0.5 mg/kg medetomidine, and 0.05 mg/kg atropine. Anesthesia was maintained
with ketamine 50 mg/kg per hour administered intravenously. A tracheotomy was performed,
and a polyvinylchloride tube was inserted into the trachea to enable mechanical ventilation
with oxygen of FiO2 = 0.4. A capnometer (Capstar-100; CWE, Inc, Ardmore, Pa) was used to
measure end-tidal carbon dioxide partial pressure (EtCO2), which was used to adjust
ventilator settings to maintain an arterial PCO2 between 35 and 40 mmHg. Body temperature
was measured with a thermocouple placed in the rectum and was maintained at 37.0 ± 0.5 oC
with a heating pad below and a warming lamp above the animal.
A catheter was placed into the right carotid artery and connected to a pressure transducer for
continuous monitoring of arterial blood pressure and heart rate. A polyethylene catheter (outer
diameter, 0.9 mm) was inserted into the right jugular vein for intravenous administration of
fluid. To compensate for fluid loss, saline was infused continuously at a rate of 15 mL/kg per
hour. Catheters were advanced into both the right femoral artery and vein for withdrawal of
blood for blood gas measurements and administration of drugs, respectively.
After a left-sided laparotomy of lower abdomen, the left kidney was exposed from adipose
tissues, and a catheter was placed in the ureter to collect urine during the experiment. A 0.5-
71
mm perivascular flow probe (Transonic Systems Inc, Ithaca, NY) was placed around the renal
artery and connected to a flow meter (T206; Transonic Systems Inc).
Study design
After the surgical preparation, the animals were randomly assigned to one of four groups. In
three groups (n = 5/group), endotoxemic shock was induced by infusion of 10 mg/kg LPS
over 30 min (Escherichia coli 055:B5; Sigma, Paris, France). When mean arterial pressure
(MAP) was decreased to 40 mmHg (after approximately 4 h), fluid resuscitation was started
in two groups of rats (unbalanced colloid and balanced colloid resuscitation), and in one
group, no resuscitation fluids were given as an LPS control group. An additional group (n = 5)
of animals was included as a sham-operated control group receiving no LPS and no additional
fluids.
Unbalanced colloid resuscitation (LPS + HES-NaCl) was done with 15 mL/h 6% HES
130/0.4 prepared in saline solution (Na+ 154 mmol/L, Cl- 154 mmol/L; Voluven, Fresenius
Kabi, Bad Homburg, Germany). Balanced colloid resuscitation (LPS + HES-RA) was done
with 15 mL/h balanced 6% HES 130/0.4 dissolved in RA preparation (Na+ 130 mmol/L, Cl-
112 mmol/L, K+ 5.36 mmol/L, Ca+2 0.912 mmol/L, Mg+2 0.984 mmol/L, acetate- 27.2
mmol/L; Plasma Volume; Baxter, Melsungen, Germany). The infusion rates were determined
in previous studies by our group [Johannes et al., 2006; Legrand et al., 2010]. Time points
were (a) baseline; (b) during endotoxemic shock at a MAP of 40 mmHg; and (c) after 30 min
of resuscitation.
Systemic hemodynamics
Arterial pressure was measured in the carotid artery. Mean arterial pressure (in mmHg) was
calculated as MAP = 2/3 x diastolic pressure + 1/3 x systolic pressure. Heart rate (in beats/min;
HR) was determined by derivation of arterial pressure signal. Blood samples (0.2 mL) were
taken from the femoral artery at each time point and replaced with the same volume of saline.
The samples were used to determine blood gas values, plasma ion levels, and pH (ABL505;
Radiometer, Copenhagen, Denmark) as well as hematocrit, hemoglobin concentration, and
hemoglobin oxygen saturation (OSM 3; Radiometer).
72
Renal macrovascular and microvascular perfusions
Blood flow in the renal artery (RBF [in mL/min]) was measured continuously and normalized
to body weight. Renal vascular resistance (RVR [dyn/s per cm-5]) was estimated as
(MAP / RBF) x 100.
Laser speckle imaging was used to visualize the spatiotemporal characteristic of renal cortical
perfusion changes during endotoxemia and fluid resuscitation as described previously
[Bezemer et al., 2010; Legrand et al., 2011]. Typical laser speckle images of the renal
microcirculation before and during endotoxemic shock are provided elsewhere [Legrand et
al., 2011]. For LSI measurements, a commercially available system was used (Moor
Instruments, Devon, UK) in which a 785-nm class 1 laser diode was used for illumination of
the tissue to a depth of approximately 1 mm. Laser speckle images were acquired using a 576
x 768-pixel grayscale CCD camera at a frame rate of 25 Hz and converted to pseudo-color
speckle contrast images where the perfusion was scaled from blue (low perfusion) to red (high
perfusion). For LSI of the rat kidney, the field of view was set to ~1.8 x 2.4 cm
(corresponding to ~30 µm/pixel). Using a 5 x 5-pixel window to calculate speckle contrast,
the maximal image resolution was ~150 µm/pixel area. The laser speckle images were
analyzed for mean renal microvascular perfusion (arbitrary unit [AU]) and perfusion
heterogeneity (AU), calculated as the range in perfusion values divided by the mean perfusion
value.
Renal function
Creatinine clearance (clearcrea [mL/min]) was assessed as an index of the glomerular filtration
rate. The concentrations of creatinine in urine and plasma were determined by colorimetric
methods. Calculation of the clearance was done using the standard formula: clearcrea = (Ucrea x
V)\ Pcrea, where Ucrea is the concentration of creatinine in urine, V is the urine volume per unit
time, and Pcrea is the concentration of creatinine in plasma. In addition, excretion fraction of
Na+ (EFNa+ [%]) was calculated to use as a marker of tubular function as: EFNa+ = (UNa+ x
Pcrea) / (PNa+ x Ucrea) x 100, where UNa+ is Na+ concentration in urine, and PNa+ is the Na+
concentration in plasma.
Statistical analysis
Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, Calif) and
presented as mean ± SEM. Results obtained in different groups were compared using two-
73
way analysis of variance and, when appropriate, post hoc analyses with Bonferroni tests.
Differences were considered statistically significant at p < 0.05.
Results
Although LPS infusion induced significant effects on systemic and renal hemodynamics and
renal function, all animals survived for the duration of the experiment.
Systemic hemodynamics
Systemic hemodynamic parameters and blood acidic state parameters are presented in Tables
1 and 2, respectively, and plasma ion levels are presented in Table 3. Lipopolysaccharide
infusion decreased MAP in all animals but did not affect the heart rate. Hydroxyethyl
starch-NaCl and HES-RA resuscitation improved the MAP (p < 0.05 vs. LPS) but could not
restore MAP back to baseline (p < 0.05 vs. control).
Table 1. MAP and HR. *p < 0.05 vs. control group. †p < 0.001 vs. LPS group.
Baseline Shock Resuscitation MAP (mmHg) Time-Control 99 + 2 99 + 2 93 + 1 LPS 96 + 4 45 + 4 39 + 2* LPS+HES-NaCl 93 + 1 48 + 2 56 + 4*
† LPS+HES-RA 96 + 2 49 + 2 59 + 5*
† HR (BPM) Time-Control 263 + 3 268 + 2 277 + 7 LPS 264 + 7 257 + 26 253 + 26 LPS+HES-NaCl 260 + 11 239 + 11 285 + 27 LPS+HES-RA 278 + 11 275 + 19 256 + 19
Lipopolysaccharide infusion induced metabolic acidosis and increased plasma potassium
levels (p < 0.05 vs. control), which could not be restored by HES-NaCl or HES-RA
resuscitation (p > 0.05 vs. LPS and control). HES-NaCl resuscitation, moreover, slightly
increased the plasma chloride level (p > 0.05 vs. LPS and control), which was slightly
reduced by HES-RA resuscitation (p > 0.05 vs. LPS and control). Plasma chloride levels were
significantly lower after HES-RA resuscitation compared with after HES-NaCl resuscitation.
74
Table 2. Arterial pH, base excess, and HCO3- levels. *p < 0.05 vs. control group.
Baseline Shock Resuscitation pH
Time-control 7.37 + 0.02 7.49 + 0.05 7.50 + 0.03 LPS 7.41 + 0.01 7.19 + 0.07 7.11 + 0.06* LPS + HES-NaCl 7.40 + 0.02 7.16 + 0.05 7.06 + 0.03* LPS + HES-RA 7.39 + 0.02 7.15 + 0.04 7.07 + 0.05* Base excess (mmol/L)
Time-control -1.4 + 0.5 1.6 + 1.1 2.5 + 0.6 LPS -0.9 + 0.9 -12.3 + 0.5 -11.4 + 1.0* LPS + HES-NaCl 0.5 + 0.7 -13.4 + 1.9 -13.4 + 1.0* LPS + HES-RA -2.6 + 0.2 -12.4 + 2.1 -13.3 + 1.6* HCO3
-(mmol/L) Time-control 21.8 + 1.1 22.6 + 1.0 23.1 + 0.4
LPS 21.7 + 0.8 16.3 + 2.2 13.2 + 2.1* LPS + HES-NaCl 21.7 + 1.2 15.9 + 0.8 14.9 + 0.9* LPS + HES-RA 19.5 + 1.4 15.6 + 1.2 19.6 + 1.0*
Renal hemodynamics
Renal macrocirculatory and microcirculatory perfusions are shown in Figure 1. Endotoxemia
was associated with a decreased RBF (p < 0.05 vs. control) and increased RVR (p < 0.05 vs.
control). Whereas HES-NaCl resuscitation was ineffective in restoring these parameters
(p > 0.05 vs. LPS), HES-RA resuscitation could significantly decrease RVR and increase
RBF (p < 0.05 vs. LPS). The reduction in RBF consequent to LPS infusion was accompanied
by a decrease in cortical microvascular perfusion, which could be partially restored by HES-
NaCl (p > 0.05 vs. LPS) and HES-RA (p < 0.05 vs. LPS). In addition, the increased cortical
perfusion heterogeneity that arose during endotoxemia could be countered by HES-NaCl and
HES-RA resuscitation (p < 0.05 vs. LPS).
Table 3. Plasma ion levels after resuscitation. *p < 0.05 vs. control group, †p < 0.05 vs. HES-NaCl group.
Na+ (mmol/L) K+ (mmol/L) Cl- (mmol/L) Time-control 141.3 + 1.1 4.0 + 0.3 109.3 + 1.3 LPS 145.5 + 1.3 7.2 + 0.4* 110.3 + 0.8 LPS + HES-NaCl 147.6 + 1.0* 6.1 + 0.4* 113.5 + 1.3 LPS + HES-RA 141.3 + 1.4 6.9 + 0.3* 107.7 + 1.4†
75
Table 4. Clearcrea and fractional Na+ excretion after resuscitation *p < 0.05 vs. control group.
Clearcrea(ml/min) Fractional Na+ excretion
Time-control 0.34 + 0.09 0.6 + 0.1 LPS Anuric Anuric
LPS + HES-NaCl 0.33 + 0.02 3.9 + 0.2* LPS + HES-RA 0.31 + 0.09 3.5 + 0.5*
Renal function
Parameters of renal function are reported in Table 4. As LPS infusion resulted in anuria,
fractional sodium excretion and clearcrea rate could not be determined for the endotoxemic
time point. Both resuscitation regimens resulted in an increased fractional sodium excretion
with respect to the time control group (p < 0.05), and none of the regimens affected clearcrea
rate.
Fig.1. Renal blood flow (A), RVR (B), cortical microvascular perfusion (C), and cortical perfusion
heterogeneity (D) at baseline (BL) and during shock and resuscitation (RS). *p < 0.05 vs. control
group, †p < 0.001 vs. LPS group, #p < 0.01 vs. HES-NaCl group.
76
Discussion
The aim of this study was to investigate the acute effects of balanced versus unbalanced
colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in a rat model
of LPS-induced endotoxemia. To test the hypothesis that balanced colloid resuscitation would
be better for the kidney than unbalanced colloid resuscitation, we resuscitated with HES-NaCl
as an unbalanced colloid solution and HES-RA as a balanced colloid solution. The main
findings were that (a) LPS-induced endotoxemia was associated with deteriorated systemic
and renal hemodynamics, acid-base balance, mean cortical microvascular perfusion, and
perfusion heterogeneity and caused anuria; (b) both HES-NaCl and HES-RA resuscitation
improved systemic blood pressure, but only HES-RA resuscitation improved renal
macrovascular and microvascular perfusions; (c) neither HES-NaCl nor HES-RA
resuscitation could restore the metabolic acidosis or fractional sodium excretion; and (d)
plasma chloride levels were significantly lower after HES-RA resuscitation compared with
after HES-NaCl resuscitation.
As renal dysfunction is a common complication in intensive care patients, there is a
continuing research on the efficacy of fluid resuscitation strategies to protect the kidney
[Kellum, 2002; Thijs and Thijs, 1998]. As such, many recent studies underscore the
importance of early fluid resuscitation in severe sepsis and sepsis-induced tissue
hypoperfusion [Kortgen et al., 2006; Sebat et al., 2005; Dellinger et al., 2008]. However, the
type of fluid that should be used for resuscitation in sepsis to yield the best renal outcome
remains controversial today [Natanson et al., 1986; Mythen et al., 1993; Lacy and Wright,
1992; Khajavi et al., 2008]. This controversy includes both the use of crystalloid versus
colloid solutions and balanced versus unbalanced colloid solutions [Brunkhorst and Oppert,
2008; Mills, 2007; Wiedermann, 2004]. In pigs with severe sepsis, colloid solutions were
shown to have superior resuscitation capacity compared with saline solutions [Marx et al.,
2002]. Moreover, HES solutions have been shown to have antiinflammatory properties, and a
new HES solution (mean molecular weight, 130 kd; degree of substitution, 0.4; HES 130/0.4)
has been shown to improve microvascular perfusion and reduce macromolecular leakage
[Nohe et al., 2005; Hoffman et al., 2002]. However, contrastingly, various investigations have
concluded that HES solutions have potential adverse effects on renal function [Legendre et
al., 1993].
77
The present study was designed to assess the effects of a balanced volume replacement
regimen including a new balanced HES preparation (HES-RA) on renal macrocirculatory and
microcirculatory perfusions in comparison to an unbalanced fluid regimen. To this end, we
measured renal macrocirculatory blood flow using a transit-time ultrasound flow probe
around the renal artery, and we monitored cortical microvascular perfusion and perfusion
heterogeneity using LSI. Laser speckle imaging is a noninvasive technique for macroscopic
mapping of tissue perfusion, but sensitive to microcirculatory flow alterations, and allows
quantitative assessment of mean microcirculatory perfusion and microcirculatory perfusion
heterogeneity. In the present study, all rats suffered from LPS-induced endotoxemic shock as
indicated by reduced MAP and increased RVR and decreased RBF. The increased RVR was
caused by severe intrarenal vasoconstriction during septic acute renal failure as has been
shown previously [Kellum et al., 2004]. Using LSI, we showed acute beneficial effects of the
two HES solutions on cortical microvascular perfusion, which was most pronounced in the
HES-RA group.
The observed differences between HES-NaCl and HES-RA resuscitation with respect to their
effects on RVR, RBF, and cortical microvascular perfusion could be attributed to the
differential effects on the plasma chloride levels. Where HES-NaCl resuscitation slightly
increased the plasma chloride level, this was slightly decreased by HES-RA resuscitation,
which led to a significantly lower plasma chloride level in the HES-RA group. The role of
chloride in modulating vasoconstrictor responses to vasoactive agents has previously been
investigated in isolated rat kidneys [Quilley et al., 1993]. Hyperchloremic fluids induced
intrarenal vasoconstriction as indicated by increased RVR and decreased glomerular filtration
rate [Wilcox, 1983]. Furthermore, the observed acidemia affects a variety of vasoregulatory
mechanisms such as by increasing endogenous catecholamine release, which induces the
release of both proinflammatory and antiinflammatory cytokines [Le Tulzo et al., 1997] and
nitric oxide [Celotto et al., 2008], and by direct effects on the renal microvasculature [Quilley
et al., 1993]. Jensen and coworkers [Jensen et al., 1990] have shown that as a result of acid
loading, macrophages increase their tumor necrosis factor secretion, which affects
microvascular perfusion by the direct vasoactive properties and by direct tissue injury.
However, in the present study, we did not measure plasma tumor necrosis factor levels, and
the present study therefore does not permit testing of contribution of these molecules to the
observation of decreased renal microcirculatory perfusion.
78
The clearcrea rate, as an index of kidney function, could be restored to baseline levels
regardless of the type using HES-NaCl and HES-RA resuscitation, but fractional sodium
excretion, in contrast, remained elevated after HES resuscitation, which could indicate tubular
dysfunction. However, changes in creatinin clearance rate and fractional sodium excretion
should be regarded only as gross indicators of renal function in sepsis, although both are
accepted indicators of renal function as described by the AKIN and RIFLE criteria. As more
sensitive markers would be desirable, novel biomarkers are currently under investigation (e.g.,
neutrophil gelatinase-associated lipocalin and fatty acid binding protein), but the significance
of their detection in the context of endotoxemia is a matter of concern.
We are aware that our study suffers from several limitations inherent to the use of an animal
model of endotoxemia. First, LPS-induced endotoxemia may not reflect all the situations
encountered in human sepsis and may lack relevance in grampositive sepsis. Second, the
present study allows only assessment of the LPS-related effects in this short-term rat model of
acute endotoxemia. Third, LSI allows only assessment of cortical microvascular perfusion and
does not take changes in the medullar microcirculation into account. Furthermore, imaging
renal microcirculatory perfusion using LSI has been performed only in rats [Legrand et al.,
2011; Holstein-Rathlou et al., 2011], and clinical application of this technique would be
limited to surgical scenarios with exposed kidney (e.g., during renal transplantation). Fourth,
HES-based resuscitation strategies are controversial. However, only the old generation of
high-molecular-weight HES molecules has been reported to be associated with acute renal
failure in a dosedependent fashion. There is no evidence for such an association with the low-
molecular-weight (130/0.4) HES we used in this study, which actually has been shown to
have protective effects on the microcirculation. In line with this, we showed that fluid
resuscitation with HES-based solutions led to an improvement of renal macrocirculatory and
microcirculatory perfusion. Fifth, as vehicle control experiments (NaCl only, RA only) have
not been performed, it is difficult to determine what proportion of the improvement in renal
microvascular perfusion is due to the inherent properties of RA. However, the specific aim of
the present study was to investigate the potential beneficial effect of resuscitation with a
balanced colloid solution (HES-RA) compared with resuscitation with an unbalanced colloid
solution (HES-NaCl) on renal microvascular perfusion in endotoxemic rats. We have clearly
demonstrated this.
79
Conclusions
In endotoxemic rats, balanced colloid (HES) resuscitation was shown to be superior to
unbalanced colloid resuscitation in terms of improvement of renal macrovascular and
microvascular perfusions. However, whether this results in improved renal function in the
long-term warrants further study.
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83
Chapter 4
EFFECT OF TEMPOL ON REDOX HOMEOSTASIS AND STRESS TOLERANCE IN
MIMETICALLY AGED DROSOPHILA
Aksu U1, Yanar K2, Terzioglu D2, Erkol T3, Ece E3, Aydin S2, Uslu E2, Cakatay U2
1Department of Biology, Science Faculty, Zoology Division, Istanbul University, 2Department of Medical Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, 3Department of Biology, Science Faculty, General Biology Division, Istanbul University,
Istanbul, Turkey
Published in: Arch Insect Biochem Physiol. 2014 Sep;87(1):13-25
84
Chapter 4
Effect Of tempol on redox homeostasis and stress tolerance in mimetically aged
Drosophila
Running title: Archives of Insect Biochemistry and Physiology.
We aimed to test our hypothesis that scavenging reactive oxygen species (ROS) with tempol,
a membrane permeable antioxidant, affects the type and magnitude of oxidative damage and
stress tolerance through mimetic aging process in Drosophila. Drosophila colonies were
randomly divided into three groups: (1) no D-galactose, no tempol; (2) D-galactose without
tempol; (3) D-galactose, but with tempol. Mimetic aging was induced by D-galactose
administration. The tempol-administered flies received tempol at the concentration of 0.2% in
addition to D-galactose. Thiobarbituric acid reacting substance (TBARS) concentrations,
advanced oxidation protein products (AOPPs), Cu,Zn-superoxide dismutase (Cu,Zn-SOD),
sialic acid (SA) were determined. Additionally, stress tolerances were tested. Mimetically
aged group without tempol led to a significant decrease in tolerance to heat, cold, and
starvation (p < 0.05), but tempol restored these parameters to control levels. The Cu,Zn-SOD
activity and SA concentrations were lower in both mimetically aged and tempol-administered
Drosophila groups compared to control (p < 0.05), whereas there were no significantly
difference between mimetically aged and tempol-administered groups. Mimetically aged
group without tempol led to a significant increase in tissue TBARS and AOPPs
concentrations (p < 0.05). Coadministration of tempol could prevent these alterations.
Scavenging ROS using tempol also restored redox homeostasis in mimetically aged group.
Tempol partly restored age-related oxidative injury and increased stress tolerance.
85
Introduction
Free radical theory of aging is one of the widely accepted theories set forth in relation to
cellular effects of both natural and mimetic aging [Yanar et al., 2011; Aydin et al., 2012].
According to this theory, reactive oxygen species (ROS) is the cause of oxidative injury that a
living organism undergoes throughout its lifetime. Increased oxidative stress may cause
functional decline and various age-related disorders in humans and experimental animals
[Cakatay, 2011].
Aerobic organisms continuously produce ROS through their lifespan. Free radicals are
molecules with unpaired electron in the outermost molecular orbitals and these molecules
cause oxidative damage to cellular macromolecules such as DNA, proteins, and lipids
[Cakatay, 2011]. To protect itself against the harmful toxic effects of ROS and modulate the
physiological effects of ROS, the cell has developed endogenous antioxidant systems. Under
normal circumstances, ROS are metabolically formed but are removed efficiently by
antioxidant systems virtually instantly, so that no macromolecular damage occurs in the cell.
However, this homeostatic process becomes less efficient in aging favoring ROS formation
[Cakatay, 2011]. Impaired redox homeostasis originates both by the inefficiency of
antioxidant systems and by increased ROS formation due to the aging process. The ability of
amphipathic antioxidants to penetrate into cellular lipid bilayers is crucial to the protection
against macromolecular oxidation [Cakatay, 2006; Zhou et al., 2010].
Several routes of superoxide dismutase administration have been described, however Cu,Zn-
superoxide dismutase (Cu,Zn-SOD) cannot easily penetrate biological membranes to
attenuate the effects of intracellular production of superoxide radical anion [Fridovich, 1995].
Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl), a low molecular weight
piperidine nitroxide, can effectively penetrate biological membranes and scavenge superoxide
radicals. Mitochondrial ROS are known to be the main sources of all oxygen-related free
radicals, and antioxidant derivatives of tempol are accumulated in the mitochondria. The
possible proposed biochemical mechanism whereby tempol controls mitochondrial oxidative
stress is attributed to hydroxylamine reduction of tempol as well as nitroxide formation
[Wang et al., 2003; Wilcox, 2010]. Moreover, tempol has been reported to improve chronic
high salt intake induced kidney injury [Carlstrom et al., 2013], and to be effective in
preventing several of the adverse consequences of oxidative stress [Wilcox, 2010], and type 1
diabetes induced organ injury [Zheng et al., 2013] in animal models. Here, we demonstrated
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the beneficial effects on alleviation of oxidative protein damage by tempol and stress
tolerance in a mimetic aging model of Drosophila.
Biomarkers of oxidative protein damage are often measured to assess the status for study of
oxidative stress. Several oxidative protein modifications such as advanced oxidation protein
products (AOPPs) formation may result from ROS oxidative stress and lead to the formation
of the high molecular weight insoluble aggregates that are common in aging and age-related
disorders [Cakatay, 2011]. AOPPs contain a variety of protein oxidation products such as
protein carbonyl groups, dityrosine, and advanced glycation end products [AGEs; Selmeci,
2011]. Besides protein oxidation marker, other oxidative damage markers of lipid
peroxidation include malondialdehyde, lipid hydroperoxides, isoprostanes, and thiobarbituric
acid reacting substances [TBARS; Buege and Aust, 1978; Hanasand et al., 2012]. TBARS are
a group of reactive aldehydes resulting from ROS-induced degradation of polyunsaturated
membrane lipids [Buege and Aust, 1978; Hanasand et al., 2012].
Increase in oxidative stress may be one of the reasons for the decrease in the stress tolerance,
which develops through natural and mimetic aging [Yanar et al., 2011; Aydin et al., 2012].
Research on D-galactose has shown that the optimum doses for establishing a mimetic aging
model of D-galactose can affect the redox homeostasis by increasing the formation of
hydrogen peroxide, galactitol, and AGEs [Yanar et al., 2011; Aydin et al., 2012]. Although
majority of the mimetic aging studies related to D-galactose administration were performed
by using rodents, D-galactose-induced aging model has also been applied to Drosophila [Cui
et al., 2004] where Cui and co-workers showed that D-galactose administration shortens the
lifespan of Drosophila. Although use of synthetic antioxidants has recently become
widespread, their effects on protecting and restoring cellular redox homeostasis is not entirely
known [Augustyniak et al., 2010].
Organisms such as Drosophila are mostly composed of postmitotic cells where studies from
this invertebrate support the free radical theory of aging much more so than results from
vertebrates. Additionally, postmitotic cells in vertebrate such as neurons and muscles are
more sensitive than other type of cells with regard to oxidative stress mediators [Cakatay,
2011].
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The aim of this study was to test the hypothesis whether tempol restores impaired redox
homeostasis and increases stress tolerance in a mimetic aging model of Drosophila. For this
reason, we investigated the extent of general oxidative stress and, specifically, oxidative
protein damage in mimetically aged flies following tempol administration. To this end
TBARS, AOPPs, Cu,Zn-SOD, and sialic acid (SA) were determined.
Materials and Methods
Chemicals and Apparatuses
Chemicals and solvents used in the experiments were of the highest purity and analytical
grade. All chemicals and reagents were purchased from Merck (Darmstadt, Germany) or
Sigma-Aldrich (St Louis, MO). Deionized water was used in the analytical procedures.
Reagents were stored at +4°C. The reagents were maintained in equilibrium at room
temperature for 0.5 h before use. All centrifugation procedures were performed with a Sigma
3–18 KS centrifuge (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany).
Oxidative stress parameters were run in duplicate by using the Biotek SynergyTM H1 Hybrid
Multi-Mode Microplate Reader (BioTek US, Winooski, VT).
Animals
In this study Drosophila melanogaster (Diptera: Drosophilidae; fruit fly) of the Oregon-R
strain, was used. All the individuals making up the experimental groups were exposed to 60%
relative humidity and ambient temperature of 25°C during the experimental period. Animals
were kept in 25 × 100 mm glass bottles containing 2 ml standard nutrient.
Experimental Groups
_Control group: In standard feeding environment. Standard feedlot: contained 8.5 g corn
flour, 0.75 g agar, 0.75 g dry yeast, 6.5 g sucrose, 0.5 ml 100% propionic acid, and 90 ml
distilled water.
_ Mimetically aged Drosophilas: By adding same amount of D-galactose instead of sucrose to
the standard feeding environment.
_ Mimetically aged Drosophilas + Tempol administration: By adding tempol in the
concentration of 0.2% D-galactose [Izmalylov and Obukhova, 1996]. Two male and two
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female individuals were allowed to live for 4 weeks in organized environment described
above, and the experimental process was terminated. In this period, the flies were middle
aged. At the end of the fourth week, male flies were isolated at which time both stress
tolerance tests and biochemical analyzes were carried out. Female flies were discarded due to
possible antioxidant effect of estrogens [Altun et al., 2011].
Stress Tolerance Tests (Cold, Heat, and Starvation)
The stress response tests need to be run precisely and carefully so that no other physical
factors can contribute to the reason the flies are dying. Flies exposed to the different diets
were assayed simultaneously and percentage of survival ratio was calculated as Ns/Nt × 100,
where Ns is the number of survived flies and Nt is the number of total flies.
Cold Stress. Flies in put in an empty bottle, in groups of 10–15 individuals, and exposed to a
temperature of 0 °C for 2 h. At the end of the period, flies were taken into standard diet
medium. After 24 h, the flies that survived were counted and the percentage of ratio
calculated by taking the average of three repeated processes.
Heat Stress. Flies in groups of 10–15 individuals were exposed to 38.3°C and 60% relative
humidity for 2 h, and the survivors counted after 24 h and the percentage of ratio calculated
by taking the average of three repeated processes into account.
Starvation Stress. The flies in groups of 10–15 individuals were kept in bottles with no food.
Dehydration was prevented by putting water absorbed Whatman filter papers in the bottles.
After 24 h, the dead and surviving flies were counted and the percentage of ratio calculated by
taking the average of three repeated processes into account.
Biochemical Methods
Flies (25 in each group) were sacrificed by exposure to −80 °C for 15 min for three times. For
the frozen 25 flies, homogenization was performed in ice-cold phosphate buffer solution by a
glass homogenizer (Potter-Elvehjem). Afterwards, the resulting homogenates were
centrifuged at the rate of 7,000 rcf for 10 min and the resulting supernatants stored at −80 °C
until they were analyzed. The by-product of the centrifugation, supernatants of homogenates
was used for the biochemical assays.
89
Measurement of Advanced Oxidative Protein Products (AOPPs). Modified method of
Hanasand et al. [2012] was performed for spectrophotometric determination of AOPPs
concentrations. According to the procedure, homogenates were diluted with citric acid, 10 µl
of 1.16 M KI was added to the diluted solution, 2 min later followed by 20 µl acetic acid. The
absorbance of the reaction mixture was immediately read at 340 nm against the blank
solution. AOPPs concentrations were expressed as micromoles per liter of chloramine-T
equivalents.
Measurement of Protein-Bound Sialic Acid. SA concentrations were determined by the
thiobarbituric acid (TBA) method of Tram et al. [1997], who have made some modifications
to previous methods [Aminoff, 1961] resulting in improved sensitivity and higher
reproducibility. Homogenate proteins (30 µl) were precipitated with 500 µl trichloroacetic
acid with the volume of 20% (w/v). The upper layer was removed and discarded. The
precipitated proteins were dissolved in 280 µl H2SO4 and then incubated in 80°C for 1 h for
hydrolysis. N-acetylneuraminic acid was used as a standard. The samples, standards, and
blank were treated with 70 µl of periodate reagent (25 mM periodic acid in 0.125N sulfuric
acid) and incubated at 37°C for 30 min. The reaction was terminated by adding 70 µl of
sodium arsenite (2% sodium arsenite in 0.22 M hydrochloric acid). Once the yellow color of
liberated iodine had disappeared, 140 µl of TBA (0.1 M, pH 9.0) was added and the solution
heated in temperature-controlled water bath for 7.5 min, and then cooled in icy water.
Dimethyl sulfoxide (560 µl) was added and corresponding absorbances were measured at 549
nm.
Assay of Thiobarbituric Acid Reacting Substances. The rate of lipid peroxidation was
determined by the procedure of Buege and Aust [1978]. One of the major secondary products
of lipid peroxidation is reactive aldehydes. TBARS, along with other by-products, react with
TBA to generate a colored product that absorbs maximally at 535 nm wavelength,
representing the color produced by all the TBARS. The coefficients of intra- and
interassayvariations for TBARS assay were 3.4 and 5.4%, respectively.
Assay of Superoxide Dismutase Activity (Cu,Zn-SOD). Determination of Cu,Zn-SOD (EC
1.15.1.1) activity was assayed in supernatant fractions based on the method developed by Sun
90
et al. [1988]. This assay involves the inhibition of nitroblue tetrazolium reduction, with
xanthine oxidase used as a superoxide radical generator. One unit of Cu,Zn-SOD is defined
as the amount of enzyme needed to exhibit a 50% dismutation of superoxide radical. The
coefficients of intra- and interassay variations were 2.4 and 2.7%, respectively.
Total Protein Assay. Supernatants were stored at −70°C for protein measurement. Total
protein was determined by the Folin phenol procedure [Lowry et al., 1951].
Statistical Analyses
Data sets are shown as mean ± SE. While the results were statistically evaluated, one-way
ANOVA and post hoc Bonferroni tests were performed. The significance level of p < 0.05
was considered as significant for the statistical evaluations.
Results
Stress Tolerance Test Results
Test results are shown in Figures 1–3. After the exposure to heat and cold, more flies in the
mimetic aging group died compared to the respective control group (p < 0.05). Although the
ratio of those that died of starvation was not much, it is statistically significant l (p < 0.05 vs.
control). Resluts showed that tempol administration caused the percentage of survival in all
three tests to increase to a level close to that of the control group (p > 0.05 vs. control).
Fig.1. Survival percentage of groups after exposure to heat. (The bars represent mean of 25 animals ±
SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)
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Fig.2. Survival percentage of groups after exposure to cold. (The bars represent mean of 25 animals ±
SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)
Fig.3. Survival percentage after the starvation stress. (The bars represent mean of 25 animals ± SE. *p < 0.05 vs. control group.)
Biochemical Results
Cu,Zn-SOD activities and TBARS concentrations for the experimental groups are shown,
respectively, in Figures 4 and 5. In comparison to the control group, the Cu,Zn-SOD activity
decreased (p < 0.05 vs. control) and the TBARS concentration significantly increased (p <
0.05 vs. control) by D-galactose administration. On the other hand, no significant variation
was observed in Cu,Zn-SOD activity by tempol administration compared to D-galactose
administration (p > 0.05), whereas TBARS concentration dropped to control group
concentration level (p < 0.05 in comparison to control group, p < 0.05 in comparison to
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mimetically aged group). SA and AOPPs concentrations for the experiment groups are
presented, respectively, in Figures 6 and 7. The SA concentrations showed no significant
difference between the mimetically aged and -administered groups (p >0.05), whereas SA
concentrations were found to be lower in both mimetically aged and tempol-administered
groups compared to the control group (p < 0.05). AOPPs concentrations in the mimetically
aged group male flies were significantly higher than those in the male control group (p <
0.05), and additionally tempol administration decreased AOPPs concentrations (p < 0.05 vs.
mimetically aged group.)
Fig.4. Superoxide dismutase (Cu,Zn-SOD) activity values of the groups. (The bars represent mean of
25 animals ± SE. *p < 0.05 vs. control group.)
Fig.5. Thiobarbituric acid reacting substances (TBARS) concentrations of the groups. (The bars
represent mean of 25 animals ± SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)
93
Fig.6. Protein-bound sialic acid (SA) values of the groups. (The bars represent mean of 25 animals ±
SE. *p < 0.05 vs. control group.)
Fig.7. Advanced oxidative protein products (AOPPs) concentrations of the groups. (The bars represent
mean of 25 animals ± SE. *p < 0.05 vs. control; #p < 0.05 vs. Mimetically aged group.)
Discussion
The idea that oxidative damage underlies mimetic aging is mainly supported by studies in
rodents [Yanar et al., 2011; Aydin et al., 2012; Cakatay et al., 2013]. Increased oxidative
protein damage and free radical mediated desialylation of cellular proteins is another
important mechanism thought to underlie cellular aging in rodents [Cakatay et al., 2013]. On
the other hand, Drosophila is used widely to examine the relationship between oxidative
stress and aging [Cui et al., 2004; Lushchak et al., 2013; Yamamato et al., 2013] because
Drosophila genetic systems are well known in postmitotic tissues [Clancy and Birdsall,
2013].
94
When D-galactose is present at high levels, it can be converted to aldose hydroperoxides with
the catalysis of galactose oxidase, and generate superoxide radical anion and other ROS
[Zhong et al., 2009]. Previous studies showed that mitochondrial dysfunction maybe be a key
componenet in the mechanism of accelerated aging caused by D-galactose [Long et al., 2007;
Kumar et al., 2010]. Additionally, it has been demonstrated that D-galactose causes damage
on the integrity of the mitochondria and disturbs the efficiency of ATP production, which in
turn contributes to more ROS generation in mitochondria [Long et al., 2007].
Prooxidant–antioxidant homeostasis was determined to investigate prooxidation due to D-
galactose-induced mimetic aging, although there are some differences in results obtained from
various mitotic and postmitotic tissues [Yanar et al., 2011; Aydin et al., 2012; Cakatay et al.,
2013]. In this study, the effect of tempol on an aging model in terms of stress tolerance,
general oxidative stress, and specifically oxidative protein damage has been to our knowledge
investigated for the first time. Our results support the idea that tempol has a positive impact
on the increase of stress tolerance as well as the detrimental effects of oxidative damage in
mimetically aged flies.
The present study has shown that that stress tolerance levels of older individuals increases
with tempol application. Increase in this resistance l ensures survival and therefore extend
lifespan. In the current study, the lifespan of the fly colony was not been determined.
However, increases in the amount of flies resistant to cold, heat, and hunger could also be
explained by an increase in lifespan. Tempol administration improved stress tolerance
response against heat and cold. Although, the effect of tempol on starvation response was not
statistically significant, there was an increased trend on mean values. In fact, starvation is
considered favorable for organisms due to the lesser mitochondrial electron leakage during
decreased glucose uptake and utilization [Gredilla and Barja, 2005].
At high concentrations, free radicals and radical-derived, nonradical reactive species are
hazardous for living organisms and damage all major cellular constituents. At moderate
concentrations, however, nitric oxide (NO), superoxide anion, and related ROS play an
important role as regulatory mediators in nuclear signaling processes. Many of the ROS-
mediated responses actually protect the cells against oxidative stress and reestablish “redox
homeostasis.” Higher organisms, however, have evolved the use of NO and ROS also as
signaling molecules for other physiological functions [Dröge, 2002].
95
There are many irreversible degenerative molecular processes in proteome of aging cells
[Cakatay, 2011]. The most important process is the formation of ROS and the fact that they
cause damage to lipid, protein, and DNA in cells [Huangfu et al., 2013; Na et al., 2013]. In
addition, more reactive secondary derived metabolites from such macromolecules form in
time due to disruption in redox homeostasis in aging cells. In our study, this case has been
observed by means of the increase in TBARS concentration, which is the indicator of cellular
membrane damage. Moreover, it has been originally shown in our study that mimetic aging
significantly accelerates free radical mediated deterioration of redox homeostasis in various
stress conditions.
Cu,Zn-SOD is an essential antioxidant enzyme in the front line defense system that converts
dismutates superoxide radical anion to hydrogen peroxide and molecular oxygen within the
mitochondrial matrix [Maiese and Chong, 2004]. It is well known that during the aging
process, there is a reduced Cu,Zn-SOD activity [Lawler et al., 2009] and antioxidant enzyme
expression [Fleenor et al., 2012; Ramesh et al., 2012]. In our study, tempol administration in
the mimetically aged flies had no significant effect on the activity of Cu,Zn-SOD, which was
already at lower levels compared to the untreated galactose administered rodents. Since it was
shown that free radicals have an impact on the aging process, the notion that it increases the
lifespan by the inhibition of these molecules is proposed. Therefore, the studies with synthetic
antioxidants are being undertaken. Although the use of antioxidants seems to be beneficial,
care has to be taken that the endogenic defense system becomes of secondary importance.
This was observed in our study as well since no significant change was found in Cu,Zn- SOD
activity in the tempol application. This could have been caused by a decreased Cu,Zn-SOD
activity and/or expression level through aging process [Uzun et al., 2013].
The spectral characteristics of AOPPs correspond to several chromophores, including
dityrosine, carbonyls, and pentosidine, although nitrotyrosine is not in this group [Breusing
and Grune, 2010]. Oxidative modifications of cellular proteins, as in AOPPs formation,
usually results in a loss of protein function. When mimetically aged flies are compared to the
tempol-administered group, the impaired redox homeostasis was reversed by tempol by
means of decreased AOPPs concentrations. Impaired protein redox homeostasis, which
appears to occur in mimetically aged group, may be an enhancing factor in the propagation of
protein oxidation, as indicated by the AOPPs concentrations.
96
Protein-bound SA residues play a significant role in various biological functions [Li and
Chen, 2012]. Desialylation shows its effect not only by altering the structure of glycoforms
and also function of glycoproteins, but also by increasing the concentration of SA, which
leads to the emergence of pathologies in tissues [Goswami and Koner, 2002]. SA residues
occupying terminal positions in N-linked oligosaccharides of glycoproteins have been shown
to play an important role in a variety of biological functions [Aminoff, 1961]. In our study,
there was no significant change in protein-bound SA concentrations in mimetically aged flies
compared to tempol-administered groups. To maintain the critical functions of SA groups
mentioned above, this finding might be explained by the importance of the strict maintenance
of the redox homeostasis of glycoproteins in proteomes of flies. In other words, the current
results of our study suggest that in D-galactose induced mimetic aging, there is an association
between desialylation of protein-bound SA and increased protein oxidation that leads to a
clustering of age-related disorder results obtained in these aged flies.
Conclusion
In conclusion, our study has demonstrated that scavenging ROS using tempol not only partly
reduced organism oxidative damage during aging, but also directly scavenged the mediators
related to oxidative stress rather than improving the reduced endogenous defense system,
thereby causing an improved endurance against environmental stress. Taken together, these
effects led to a modest improvement of aging-related frailty.
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Chapter5
SCAVENGING ROS IN THE ACUTE PHASE OF RENAL I/R INJURY ALSO
PROTECTS KIDNEY OXYGENATION AND NO LEVELS
Aksu U1,2, Ergin B1,2, Bezemer R1, Kandil A2, Milstein D.M. J.1, Demirci-Tansel C2, Ince C1
1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, The Netherlands
2Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey
Published in: Intensive Care Medicine Experimental 2015;3:21:1:10
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Chapter 5
Scavenging ROS in the acute phase of renal I/R injury also protects kidney oxygenation
and NO levels
Running title: ROS, O2, and NO in I/R-induced AKI
Background: We aimed to test our hypothesis that scavenging ROS with tempol would also
protect renal oxygenation and nitric oxide (NO) levels in the acute phase of renal I/R.
Methods: Rats were randomly divided into four groups of six: 1) no I/R, no tempol; 2) no I/R,
but with tempol; 3) I/R without tempol; and 4) I/R with tempol. I/R was induced by 30-min
clamping of the renal artery. The tempol-treated animals received 200 µmol/kg/h tempol
intravenously 15 min prior to I/R. Results: I/R without tempol led to a significant decrease in
renal DO2 and microvascular oxygenation, but tempol was able to these parameters. At R90,
the creatinine clearance rate was lower in the I/R-subjected group that did not receive tempol
compared to that in the other groups. I/R injury without tempol led to a significant increase in
tissue malondialdehyde levels (marker of oxidative stress) and a significant decrease in tissue
NO levels. Tempol administration before I/R could prevent these alterations. Conclusions:
Scavenging ROS using tempol also protects renal oxygenation and NO levels in the acute
phase of renal I/R. This demonstrates that renal oxygen, ROS, and NO levels are strongly
related in conditions of I/R.
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Introduction
Acute kidney injury (AKI) and is a complex clinical complication and is associated with a
high incidence of morbidity and mortality [Bagshaw, 2006; Bell and Martling, 2007]. One of
the most common causes of AKI is renal ischemia/reperfusion (I/R) injury as can occur in
numerous scenarios such as during surgery and also as a result of shock and resuscitation
[Lameire et al., 2005; Hoste and Kellum, 2006]. Despite the identification of several
mechanisms underlying the development of AKI, the pathophysiology of AKI is still
incompletely understood. It is clear, however, that instead of a single mechanism being
responsible for its etiology, AKI is associated with an entire orchestra of failing cellular
mechanisms [Welch et al., 2001; Adler and Huang, 2002; Welch et al., 2003; Aksu et al.,
2011].
It is well known that reactive oxygen species (ROS) are fundamentally implicated as primary
culprits in the pathophysiology of renal I/R injury and consequent AKI. The excess generation
of ROS and decreases in antioxidant defenses are known to contribute to I/R injury.
Superoxide dismutase (SOD), a ubiquitous intrinsic biological antioxidant, catalyzes the
dismutation of superoxide anions into oxygen and hydrogen peroxide. Tempol (4-hydroxy-
2,2,6,6-tetramethyl piperidinoxyl) is a membrane-permeable, metal-independent SOD
mimetic specific for superoxide anions (O2-). Several studies have demonstrated that tempol
may reduce renal I/R injury through its free radical scavenging activity [Chatterjee et al.,
2000; Fujii et al., 2005].
In a series of recent reviews, we have described that our hypothesis that a disturbed balance
between oxygen, nitric oxide (NO), and ROS might form an important component of the
pathogenesis of I/R-induced AKI [Legrand et al., 2008; Le Dorze et al., 2009; Aksu et al.,
2011]. In the present study we aimed to test whether the proven protective effects of tempol
are indeed associated with improved renal oxygenation and NO levels in a short-term rat
model of renal I/R.
Materials and methods
Animals
All experiments in this study were approved by the institutional Animal Experimentation
Committee of the Academic Medical Center of the University of Amsterdam. Care and
handling of the animals were in accordance with the guidelines for Institutional and Animal
104
Care and Use Committees. The study has been carried out in accordance with the Declaration
of Helsinki. Experiments were performed on 24 Sprague-Dawley rats (Harlan Netherlands
BV, Horst, The Netherlands) with a mean ± SD body weight of 348 ± 21 g.
Surgical preparation
All animals were anesthetized with an intraperitoneal injection of a mixture of 75 mg/kg
ketamine (Nimatek®, Eurovet, Bladel, The Netherlands), 0.5 mg/kg dexmedetomidine
(Dexdomitor, Pfizer Animal Health BV, Capelle aan den IJssel, The Netherlands), and 0.05
mg/kg atropine-sulfate (Centrafarm Pharmaceuticals BV, Etten-Leur, The Netherlands). After
preparing a tracheotomy the animals were mechanically ventilated with a FiO2 of 0.4. Body
temperature was maintained at 37±0.5 °C during the entire experiment by an external thermal
heating pad. Ventilator settings were adjusted to maintain end-tidal pCO2 between 30 and 35
mmHg and arterial pCO2 between 35 and 40 mmHg.
For drug and fluid administration and hemodynamic monitoring, vessels were cannulated with
polyethylene catheters with an outer diameter of 0.9 mm (Braun, Melsungen, Germany). A
catheter in the right carotid artery was connected to a pressure transducer to monitor mean
arterial blood pressure (MAP) and heart rate. The right jugular vein was cannulated for
continuous infusion of Ringer’s Lactate (Baxter, Utrecht, The Netherlands) at a rate of 15
mL/kg/hour and maintenance of anesthesia. The right femoral artery was cannulated for
drawing blood samples and the right femoral vein for fluid resuscitation.
The left kidney was exposed, decapsulated, and immobilized in a Lucite kidney cup (K.
Effenberger, Pfaffingen, Germany) via ~4 cm incision in the left flank in each animal. Renal
vessels were carefully separated under preservation of nerves and the adrenal gland. A
perivascular ultrasonic transient time flow probe was placed around the left renal artery (type
0.7 RB Transonic Systems Inc., Ithaca, NY, USA) and connected to a flow meter (T206,
Transonic Systems Inc., Ithaca, NY, USA) to continuously measure renal blood flow (RBF).
An estimation of the renal vascular resistance (RVR) was made as: RVR (dynes.sec.cm-5) =
(MAP/RBF) × 80. The left ureter was isolated, ligated, and cannulated with a polyethylene
catheter for urine collection.
After the surgical preparation one optical fiber was placed 1 mm above the decapsulated
kidney and another optical fiber was placed 1 mm above the renal vein to measure renal
105
microvascular and venous oxygenation using phosphorimetry (explained in more detail
below). A small piece of aluminum foil was placed on the dorsal side of the renal vein to
prevent contribution of the underlying tissues to the phosphorescence signal in the venous pO2
measurements. Oxyphor G2, a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl)
benzoporphyrin (Oxygen Enterprises Ltd., Philadelphia, PA, USA) was subsequently infused
(i.e. 6 mg/kg IV over 5 min), followed by 30 min of stabilization time. The surgical field was
covered with a humidified gauze compress throughout the entire experiment to prevent drying
of the exposed tissues.
Experimental protocol
After a stabilization period of 30 min, the animals were randomly divided into four groups of
six: 1) no I/R, no tempol (CTRL); 2) no I/R, but with tempol (TMPL); 3) I/R without tempol
(I/R); and 4) I/R with tempol (I/R+TMPL). Ischemia/reperfusion was induced by 30-min non-
destructive clamping of the renal artery. The tempol-treated animals received 200 µmol/kg/h
of 4-hydroxy-TEMPO (tempol) intravenously 15 min prior to initiation of I/R. Measurements
were performed up to 90 min post-ischemia and after the experiments, the kidneys were
isolated and renal tissue malondialdehyde and nitric oxide levels were measured.
Blood variables
Arterial blood samples (0.5 ml) were taken from the femoral artery at baseline (BSLN) and
after 15 and 90 min of reperfusion (R15 and R90, respectively). The blood samples were
replaced by the same volume of Ringer’s Lactate. Samples were analyzed for blood gas
values (ABL505 blood gas analyzer; Radiometer, Copenhagen, Denmark), hemoglobin
concentration, and hemoglobin oxygen saturation (OSM3; Radiometer, Copenhagen,
Denmark). Additionally, plasma creatinine and sodium concentrations were determined in all
samples.
Renal microvascular and venous oxygenation
Microvascular oxygen tension in the renal cortex (CµPO2), outer medulla (MµPO2), and renal
venous oxygen tension (PrvO2) were measured by oxygen-dependent quenching of
phosphorescence lifetimes of the systemically infused albumin-targeted (and therefore
circulation-confined) phosphorescent dye Oxyphor G2 [Johannes et al., 2006]. Oxyphor G2
has two excitation peaks (λexcitation1 =440 nm, λexcitation2 =632 nm) and one emission peak
(λemission =800 nm). These optical properties allow (near) simultaneous lifetime measurements
106
in microcirculation of the kidney cortex and the outer medulla due to different optical
penetration depths of the excitation light [Johannes et al., 2006]. For the measurement of renal
venous PO2 (PrvO2), a mono-wavelength phosphorimeter was used [Mik et al., 2008]. Oxygen
measurements based on phosphorescence lifetime techniques rely on the principle that
phosphorescence can be quenched by energy transfer to oxygen resulting in shortening of the
phosphorescence lifetime. A linear relationship between reciprocal phosphorescence lifetime
and oxygen tension (i.e., the Stern-Volmer relation) allows quantitative measurement of PO2
[Bezemer et al., 2010].
Renal oxygen delivery and consumption
Arterial oxygen content (AOC) was calculated by (1.31×hemoglobin×SaO2)+(0.003×PaO2),
where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure of oxygen. Renal
venous oxygen content (RVOC) was calculated as (1.31×hemoglobin×SrvO2)+(0.003×PrvO2),
where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial pressure of oxygen
(measured using phosphorimetry). Renal oxygen delivery was calculated as DO2
(mL/min)=RBF×AOC. Renal oxygen consumption was calculated as VO2 (mL/min)
=RBF×(AOC–RVOC).
Renal function
For analysis of urine volume, creatinine concentration, and sodium (Na+) concentration at the
the end of the protocol, urine samples from the left ureter were collected for 10 min.
Creatinine clearance rate (CCR) per gram of renal tissue was calculated with standard
formula: CCR [mL/min] = (UC×V)/PC, where UC is the urine creatinine concentration, V is
the urine volume per unit time, and PC is the plasma creatinine concentration. Renal sodium
reabsorption (TNa+, [mmol/min]) was calculated as TNa+ = (PNa+×CCR)-(UNa+×V) , where UNa+
is the urine sodium concentration and PNa+ is the plasma sodium concentration.
Renal tissue oxidative stress
Renal tissue malondialdehyde (MDA) levels were determined to assess lipid peroxidation as a
measure of renal oxidative stress. All kidneys were homogenized in cold 5 mM sodium
phosphate buffer. The homogenates were centrifuged at 12,000 g for 15 min at 4 ºC and
supernatants were used for MDA determination. The level of lipid peroxides was expressed as
micromoles of MDA per milligram of protein (Bradford assay).
107
Renal tissue NO levels
NO undergoes a series of reactions in biological tissues leading to the accumulation of the
final products nitrite and nitrate. Thus, the index of the total NO accumulation is the sum of
both nitrite and nitrate levels in the tissue samples. To reduce the nitrate and nitrate pressnet
in the tissue samples to NO, the samples were put in the reducing agent vanadium (III)
chloride (VCl3) in 1 mol/L HCl at 90 oC. The VCl3 reagent converts nitrite, nitrate, and S-
nitroso compounds to NO gas which is guided towards an NO chemiluminescence signal
analyzer (Sievers 280i analyzer, GE Analytical Instruments) allowing the direct detection of
NO [Yang et al., 1997]. Within the reaction vessel, NO reacted with ozone to generate oxygen
and excited-state NO species, of which the decay is associated with the emission of weak
near-infrared chemiluminescence. This signal is detected by a sensitive photodetector and
converted to millivolts (mV). The area under the curve of the detected chemiluminescence
(mV·s) represents the amount of NO-ozone reactions in time and thus the amount of
bioavailable NO in the tested samples. The ratio of tissue NO to tissue protein content was
used to for standardization of the NO measurements.
Data analysis
Data analysis and presentation were performed using GraphPad Prism (GraphPad Software,
San Diego, CA, USA). Values are reported as the mean ± SD. Two-way ANOVA for repeated
measurements with a Bonferroni post hoc test were used for comparative analysis between
groups. A p-value of <0.05 was considered statistically significant.
Results
Systemic and renal hemodynamics and oxygenation
All systemic and renal hemodynamic and oxygenation variables are presented in Tables 1 and
2. MAP and renal VO2 remained stable throughout the entire protocol in all groups. Tempol
administration in the sham-operated animals (i.e., without I/R) did not affect any of the
systemic and renal hemodynamic and oxygenation variables. I/R without tempol
administration led to a significant decrease in RBF (2.5 ± 0.6 mL/min at R15 and 2.4 ± 0.3
mL/min at R90) and DO2 (1.05 ± 0.28 mL O2/min at R15 and 0.90 ± 0.22 mL O2/min at R90)
and a significant increase in RVR (3298 ± 955 dyn·s·cm-5 at R15 and 3352 ± 426 dyn·s·cm-5
at R90). Tempol administration prior to I/R was able to preserve RBF (4.0 ± 0.9 mL/min at
R15 and 4.1 ± 1.6 mL/min at R90), DO2 (1.61 ± 0.46 mL O2/min at R15 and 1.75 ± 0.70 mL
108
O2/min at R90), and RVR (1999 ± 471 dyn·s·cm-5 at R15 and 2200 ± 1046 dyn·s·cm-5 at
R90).
Table 1: Mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), renal oxygen delivery (DO2), and renal oxygen consumption (VO2) at baseline (Bsln) and after 15 and 90 min of reperfusion (R15 and R90, respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R.
Bsln R15 R90 MAP [mmHg] CTRL 103 ± 7 103 ± 5 96 ± 6 TMPL 103 ± 8 96 ± 8 93 ± 4 I/R 101 ± 10 96 ± 6 98 ± 6 I/R+TMPL 105 ± 11 95 ± 16 96 ± 16 RBF [mL/min] CTRL 4.3 ± 1.3 4.1 ± 1.4 3.8 ± 0.5 TMPL 4.2 ± 0.7 3.8 ± 1 3.7 ± 1.3 I/R 4.0 ± 0.6 2.5 ± 0.6 CT 2.4 ± 0.3 CT I/R+TMPL 4.4 ± 1 4 ± 0.9 I 4.1 ± 1.6 I RVR [dyn.s.cm-5] CTRL 2060 ± 583 2143 ± 542 2070 ± 240 TMPL 1989 ± 379 2189 ± 712 2223 ± 733 I/R 2064 ± 414 3298 ± 955 CT 3352 ± 426 CT I/R+TMPL 1968 ± 454 1999 ± 471 I 2200 ± 1046 I DO2 [mL O2/min] CTRL 1.77 ± 0.53 1.65 ± 0.52 1.52 ± 0.22 TMPL 1.75 ± 0.20 1.54 ± 0.18 1.45 ± 0.21 I/R 1.62 ± 0.33 1.05 ± 0.28 CT 0.9 ± 0.22 CT I/R+TMPL 1.88 ± 0.42 1.61 ± 0.46 I 1.75 ± 0.70 I VO2 [mL O2/min/g] CTRL 0.12 ± 0.04 0.11 ± 0.02 0.12 ± 0.02 TMPL 0.13 ± 0.07 0.13 ± 0.03 0.11 ± 0.03 I/R 0.13 ± 0.04 0.1 ± 0.03 0.1 ± 0.03 I/R+TMPL 0.14 ± 0.04 0.13 ± 0.05 0.13 ± 0.04
Renal microvascular oxygenation in the cortex and medulla were decreased quickly during
ischemia but normalized immediately upon reperfusion. However, at R90, microvascular
oxygenation was significantly decreased in the I/R-subjected group that did not receive
tempol (44 ± 11 mmHg in the cortex and 41 ± 5 mmHg in the medulla) while this was
maintained in the I/R-subjected group that did receive tempol (57 ± 4 mmHg in the cortex and
51±2 mmHg in the medulla).
109
Table 2: Microvascular oxygen tension in renal cortex (CµpO2) and medulla (MµpO2) at baseline
(Bsln), at the end of 30 min of ischemia (Isch), and after 15 and 90 min of reperfusion (R15 and R90,
respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R.
Bsln Isch R15 R90 CµpO2 [mmHg] CTRL 76 ± 2 70 ± 4 70 ± 4 62 ± 7 TMPL 76 ± 3 71 ± 8 73 ± 6 58 ± 6 I/R 79 ± 4 11 ± 4 CT 59 ± 4 44 ± 11 CT I/R+TMPL 77 ± 6 10 ± 4 CT 66 ± 9 57 ± 4 I MµpO2 [mmHg] CTRL 61 ± 5 57 ± 6 54 ± 4 51 ± 4 TMPL 57 ± 9 56 ± 10 55 ± 7 50 ± 7 I/R 59 ± 6 7 ± 1 CT 50 ± 3 C 41 ± 5 CT I/R+TMPL 59 ± 5 7 ± 1 CT 59 ± 7 I 51 ± 2 I
Renal function
The renal function variables are presented in Table 3. Tempol administration in the sham-
operated animals (i.e., without I/R) did not affect renal function. I/R without tempol
administration led to a significant decrease in CCR (0.3 ± 0.1 mL/min at R15) and TNa+ (0.04
± 0.01 mmol/min at R15) and tempol administration prior to I/R could not prevent these
reductions in CCR (0.4 ± 0.2 mL/min at R15) and TNa+ (0.06 ± 0.03 mmol/min at R15). At
R90 these decreases were mostly normalized except for the CCR in the I/R-subjected group
that did not receive tempol.
Table 3: Creatinine clearance rate (CCR) and sodium reabsoption (TNa+) at baseline (Bsln) and after 15 and 90 min of reperfusion (R15 and R90, respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R. Bsln R15 R90 CCR [mL/min] CTRL 1.2 ± 0.7 1.3 ± 0.3 1.5 ± 0.7 TMPL 1.1 ± 0.3 1.1 ± 0.3 1.2 ± 0.4 I/R 1.2 ± 0.4 0.3 ± 0.1 CT 0.7 ± 0.4 C I/R+TMPL 1.4 ± 0.6 0.4 ± 0.2 CT 1.0 ± 0.3 TNa+ [mmol/min] CTRL 0.18 ± 0.09 0.18 ± 0.09 0.14 ± 0.07 TMPL 0.15 ± 0.04 0.14 ± 0.04 0.13 ± 0.03 I/R 0.16 ± 0.06 0.04 ± 0.01 CT 0.09 ± 0.04 I/R+TMPL 0.20 ± 0.09 0.06 ± 0.03 CT 0.14 ± 0.05
110
Renal oxidative stress and NO levels
The renal microvascular oxygenation, oxidative stress, and NO levels at the end of the
protocol are presented in Figure 1. Tempol administration without I/R injury led to a
significant decrease in tissue MDA levels (1.6 ± 0.17) and I/R injury in the absence of tempol
led to a significant increase in tissue MDA levels (3.8 ± 0.9). Tempol administration before
I/R could partially prevent this increase in MDA levels (2.4 ± 0.7). Tissue NO levels were not
affected by tempol administration without I/R injury (240 ± 100), but were significantly
decreased after I/R in the absence of tempol (72 ± 21). Tempol administration before I/R
could completely normalize the tissue NO levels (265 ± 143). Hence, tempol administration
prior to I/R injury reduced renal oxidative stress and normalized renal oxygenation and tissue
NO levels.
Fig.1. Renal oxygenation, oxidative stress, and nitric oxide (NO) levels at the end of the protocol. (A) Microvascular oxygen tensions (µpO2) in the renal cortex; (B) Microvascular oxygen tensions (µpO2) in the renal medulla; (C) renal tissue malondialdehyde (MDA) levels normalized to the tissue protein content; and (D) tissue NO levels normalized to the tissue protein content. *p<0.05 versus all other groups; Cp<0.05 versus the CTRL group; Tp<0.05 versus the TMPL group.
111
Discussion
In the present study we aimed to test the hypothesis scavenging ROS using tempol would be
associated with improved renal oxygenation and NO levels in a short-term rat model of renal
I/R. We have found that I/R was associated with a significant increased in tissue MDA
(marker of oxidative stress) and a significant decrease in tissue NO. The decrease in tissue
NO was followed by an increase in RVR and consequent decrease in RBF, renal DO2, and
renal microvascular oxygenation. These disturbances were associated with reduced renal
function in terms of sodium reabsorption and creatinine clearance. Pre-ischemic
administration of tempol, a known superoxide scavenger, was able to decrease oxidative
stress and increase renal tissue NO and microvascular oxygenation and thereby improve renal
function. Furthermore, we have shown that administration of tempol in the absence of I/R
leads to a reduction in the renal MDA levels normally present in renal tissue, but did not
affect any of the other parameters.
I/R injury is a multi-pathway process in which decreased ROS scavenging and increased ROS
generation are particularly important mediators leading to tissue injury [Nath and Norby,
2000; Aksu et al., 2011]. ROS are created in mitochondria [Yoshikawa et al., 2012], and
excess ROS injure the mitochondria themselves, impair cellular function, and promote
apoptosis [Huttemann et al., 2012]. It has previously been shown that antioxidants can
decrease cellular and tissue damage by decreasing intracellular ROS levels and suppressing
oxidative stress [Patel et al., 2002; Chatterjee, 2007; Guz et al., 2007; Roth et al., 2011;
Gomes et al., 2012; Riccioni et al., 2012]. In this study, we showed that tempol reduced renal
lipid peroxidation in renal tissue after renal I/R as reflected by decreased tissue MDA levels
[Michel et al., 2008]. In line, Patel et al. have previously shown that administration of
tempone, an unmetabolized form of tempol, reduced I/R-induced injury to peritubular cells by
thereby reduced renal dysfunction [Patel et al., 2002]. They showed, moreover, that this was
without the adverse cardiovascular effects observed when using other nitroxyl radical
scavenging agents. Noiri et al. also demonstrated that both L-NIL (i.e., a selective iNOS
inhibitor) and lecithinized SOD administration improve renal function due to scavenging of
peroxynitrite and thereby preventing lipid peroxidation and oxidative damage to DNA [Noiri
et al., 2001].
In this study tempol effectively inhibited an I/R-induced decrease in tissue NO concentration.
Decreased NO production via eNOS during renal I/R contributes to renal hypoperfusion and
112
renal injury. This has been confirmed by studies showing that L-arginine (i.e., a precursor of
NO) and NO donors improve renal function after I/R [Chatterjee et al., 2007; Kucuk, et al.,
2006; Jeong et al., 2004]. On the other hand, also the administration of iNOS inhibitors has
been shown protect the kidney against I/R injury [Chatterjee et a., 2002; Mark et al., 2005;
Vinas et al., 2006; Noiri et al., 2001]. In the present study, however, the protocol was too
short for iNOS expression to occur. Nonetheless, the administration of tempol did scavenge
the excess ROS generated during I/R and thereby prevented the interaction of eNOS-derived
NO and ROS forming peroxynitrite and leaving the NO available for maintenance of
microvascular perfusion. Hence, scavenging ROS has a double beneficial effect.
Our study has of course a number of limitations. First, this study was performed in rats and
the effects of tempol could be different in humans. Second, the duration of renal ischemia was
30 min and measurements were performed up to 90 min post-ischemia and thus long-term
effects of I/R and tempol were not studied. Additionally, a longer duration of ischemia might
have caused more severe renal dysfunction. Third, we did not measure ROS directly but
instead measured MDA as a marker of lipid peroxidation as a result of oxidative stress.
Conclusions
In conclusion, our study clearly demonstrated that scavenging ROS using tempol not only
reduced renal oxidative stress following I/R, but also normalized renal tissue NO levels and
thereby reduced RVR and improves RBF, renal DO2, and renal microvascular oxygenation.
Taken together, these effects led to a modest (albeit not statistically significant) improvement
of renal function after I/R.
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117
Summary and conclusions
In Chapter 1, an acetate- and gluconate-balanced crystalloid solution was tested for its effects
on the plasma ion levels and acid–base balance, renal oxygenation, oxidative stress status,
glycocalyx integrity, and systemic cytokine levels in a rat model of hemorrhagic shock. The
main findings of our study were that: (1) both the balanced and unbalanced crystalloid
solutions successfully restored the blood pressure, but renal blood flow was only recovered by
the balanced solution although this did not lead to improved renal oxygenation; (2) less
balanced fluid was required to restore blood pressure; (3) while unbalanced crystalloid
resuscitation induced hyperchloremia and worsened metabolic acidosis in hemorrhaged rats,
balanced crystalloid resuscitation prevented hyperchloremia, restored the acid–base balance,
and preserved the anion gap and strong ion difference in these animals; (4) neither balanced
nor unbalanced crystalloid resuscitation could normalize systemic inflammation (TNF-a and
IL-6); (5) only balanced crystalloid resuscitation significantly reduced renal oxidative stress,
as reflected by reduced L-FABP reactivity, but none of the fluids could restore the increased
NGAL, MDA, and hyaluronan levels; and (6) balanced crystalloid resuscitation significantly
improved renal oxygen consumption (increased VO2 , decreased EFNa+), but none of the
fluids was able to restore creatinine clearance rate in this short-term protocol. In conclusion,
while unbalanced crystalloid resuscitation induces hyperchloremia and worsens metabolic
acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevents hyperchloremia,
restores the acid–base balance, and preserves the anion gap and strong ion difference in these
animals. Balanced crystalloid resuscitation prevents renal hypoperfusion better than
unbalanced crystalloid resuscitation. However, although the balanced preparation improves
some parameters, it does not improve oxidative stress and systemic inflammation.
In Chapter 2, we examined the acute effects of acetate-balanced colloid and crystalloid
resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We tested the
hypothesis that acetate-balanced solutions would be superior in correcting impaired renal
perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions. Our
main findings were that: (1) hemorrhagic shock was associated with acute decreases in blood
pressure, renal perfusion and oxygenation, and urine production; (2) volume replacement
therapy with balanced and unbalanced crystalloid and colloid solutions partially corrected
these parameters; and (3) the acetate-balanced colloid solution HES-RA was the only
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resuscitation fluid that could restore renal blood flow back to ∼85% of baseline level which
was associated with the most prominently improved renal oxygenation. While resuscitation
with the NaCl and RA (crystalloid solutions) and the HES-NaCl (unbalanced colloid solution)
led to hyperchloremic acidosis, resuscitation with the HES-RA (acetate-balanced colloid
solution) did not. In conclusion, the acetate-balanced colloid solution, HES-RA, was
furthermore the only fluid restoring renal blood flow back to ∼85% of baseline level and most
prominently improved renal microvascular oxygenation.
The aim of Chapter 3 was to investigate the acute effects of balanced versus unbalanced
colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in a rat model
of LPS-induced endotoxemia. To test the hypothesis that balanced colloid resuscitation would
be better for the kidney than unbalanced colloid resuscitation, we resuscitated with HES-NaCl
as an unbalanced colloid solution and HES-RA as a balanced colloid solution. The main
findings were that (1) LPS-induced endotoxemia was associated with deteriorated systemic
and renal hemodynamics, acid-base balance, mean cortical microvascular perfusion, and
perfusion heterogeneity and caused anuria; (2) both HES-NaCl and HES-RA resuscitation
improved systemic blood pressure, but only HES-RA resuscitation improved renal
macrovascular and microvascular perfusion; (3) neither HES-NaCl nor HES-RA resuscitation
could restore the metabolic acidosis or fractional sodium excretion; and (4) plasma chloride
levels were significantly lower after HES-RA resuscitation compared with after HES-NaCl
resuscitation. In conclusion, this confirmed our hypothesis that balanced colloid resuscitation
is superior to unbalanced colloid resuscitation in terms of improvement of renal
macrovascular and microvascular perfusions. However, whether this results in improved renal
function in the long-term warrants further study.
A role for oxidative damage in mimetic aging is mainly supported by studies in rodents.
Increased oxidative protein damage and free radical mediated desialylation of cellular proteins
is another important mechanism for cellular aging in rodents. On the other hand, the
Drosophila is used widely to examine the relationship between oxidative stress and aging,
because the Drosophila genetic systems are well-known and postmitotic tissues. When D-
galactose is present at high levels, it can be converted to aldose hydroperoxides with the
catalysis of galactose oxidase, resulting in the generation of a superoxide radical anion and
other ROS. Previous studies showed that mitochondrial dysfunction maybe a key issue in the
mechanism of accelerated aging caused by D-galactose. Additionally, it was demonstrated
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that D-galactose causes damage to the integrity of the mitochondria and disturbs the
efficiency of ATP production, which in turn contributes to more ROS generation in the
mitochondria. In Chapter 4, the aim of the study was to test the hypothesis whether tempol
restores impaired redox homeostasis and increases stress tolerance in a mimetic aging model
of Drosophila. For this reason, we investigated the extent of general oxidative stress and,
specifically, oxidative protein damage in D-galactose administered mimetically aged flies
following tempol administration. Our study demonstrated that scavenging ROS using tempol
not only (1) reduced oxidative damage during aging, but also (2) directly scavenged the
mediators related to oxidative stress, rather than only improving the reduced endogenous
defense systems. This led to (3) an improved endurance against environmental stress. In
conclusion, the restoration of redox homeostasis led to a modest improvement of aging-
related frailty upon tempol administration.
In Chapter 5, we aimed to test the hypothesis that scavenging ROS using tempol would be
associated with improved renal oxygenation and NO levels in a short-term rat model of renal
I/R. We have found that (1) I/R was associated with a significant increase in tissue MDA
(marker of oxidative stress) and (2) a significant decrease in tissue NO. The decrease in tissue
NO was followed by (3) an increase in RVR and consequent decrease in RBF, renal DO2, and
renal microvascular oxygenation. (4) These disturbances were associated with reduced renal
function in terms of sodium reabsorption and creatinine clearance. Pre-ischemic
administration of tempol, a known superoxide scavenger, was able to decrease oxidative
stress and increase renal tissue NO and microvascular oxygenation and thereby improve renal
function. Furthermore, we have shown that administration of tempol in the absence of I/R
leads to a reduction in the renal MDA levels normally present in healthy renal tissue, but did
not affect any of the other parameters. In conclusion, our study clearly demonstrated that
scavenging ROS using tempol not only reduced renal oxidative stress following I/R, but also
normalized renal tissue NO levels and thereby reduced RVR and improves RBF, renal DO2,
and renal microvascular oxygenation. Taken together, these effects led to a modest (albeit not
statistically significant) improvement of renal function after I/R.
In conclusion, this thesis presents the findings of various experimental therapatic approaches
on in the treatment of acute kidney injury in different experimental models. The findings
indicate that the resuscitation fluids commonly used with the idea of protecting the kidney
actually do not correct systemic inflammation or oxidative stress, and therefore do not prevent
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renal ischemia and hypoxia. Nonetheless, eventhough fluid resuscitation does not have any
effects on renal oxygenation, it is of course better than not resuscitating at all. Therefore,
optimization of fluid therapy, such as balancing fluids, and other therapeutic approaches
aimed to protect the kidney, is utmost important. Taking into account our studies on
antioxidants, a new generation of fluids could be developed, incorporating antioxidant
properties. However, the long-term effects of balanced and antioxidant-enriched resuscitation
on renal function warrants further study.
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Samenvatting en conclusies
In hoofdstuk 1 werd een acetaat- en gluconaat-gebalanceerde kristalloïdoplossing getest op de
effecten op de plasma-ion niveaus, het zuur-base-evenwicht, the renale oxygenatie, oxidatieve
stress status, glycocalyx integriteit, en systemische cytokine niveaus in een rat model van
hemorragische shock. De belangrijkste bevindingen van ons onderzoek waren dat: (1) zowel
de gebalanceerde en ongebalanceerde kristalloïde oplossingen met succes de bloeddruk
herstelde, maar de doorbloeding van de nier werd alleen hersteld door de gebalanceerde
oplossing, hoewel dit niet leidde tot een betere renale oxygenatie; (2) minder gebalanceerde
vloeistof nodig was om de bloeddruk te herstellen vergeleken met ongebalanceerde vloeistof;
(3) terwijl ongebalanceerde kristalloïde therapie hyperchloremie veroorzaakte en metabole
acidose verslechterde in hemorrhagische ratten, gebalanceerde kristalloïde therapie voorkwam
hyperchloremie, herstelde het zuur-base-evenwicht, en bewaarde de anion gap en het sterke
ion verschil in deze dieren; (4) zowel niet-gebalanceerde als gebalanceerde kristalloïdtherapie
konden niet de systemische inflammatie (TNF-a en IL-6) normaliseren; (5) gebalanceerde
kristalloïde oplossing verminderde aanzienlijk de renale oxidatieve stress, zoals weerspiegeld
door de gereduceerde L-FABP reactiviteit, maar geen van de vloeistoffen kon de verhoogde
NGAL, MDA en hyaluronzuur niveaus herstellen; en (6) gebalanceerde kristalloïde therapie
verbeterde aanzienlijk het renale zuurstofverbruik (gestegen VO2, gedaalde EFNa+), maar
geen van de vloeistoffen in staat was om de creatinineklaring te herstellen in dit kortduurende
protocol. In conclusie, terwijl ongebalanceerde kristalloïde therapie hyperchloremie
induceerde en metabole acidose verslechterde in ratten met hemorrhagische shock,
gebalanceerde kristalloïd therapie voorkwam hyperchloremie, herstelde het zuur-base-
evenwicht en bewaarde het anion gap en sterke-ionen verschil in deze dieren. Gebalanceerde
kristalloïde therapie voorkwam renale hypoperfusie beter dan ongebalanceerde kristalloïde
therapie. Hoewel de gebalanceerde oplossingen een aantal parameters verbeterde, geen een
oplossing kon de systemische oxidatieve stress en inflammatie verbeteren.
In hoofdstuk 2 hebben we onderzocht wat de acute effecten van acetate-gebalanceerde
colloïde en kristalloïde therapie op de renale oxygenatie zijn, in een ratmodel van
hemorragische shock. We hebben de hypothese getest dat acetaat-gebalanceerde oplossingen
superieur zijn in het corrigeren van insufficiënte nierperfusie en -oxygenatie na ernstige
bloeding, in vergelijking met ongebalanceerde oplossingen. De belangrijkste bevindingen
waren dat: (1) hemorrhagische shock was geassocieerd met acute verlaging van de bloeddruk,
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renale perfusie en oxygenatie en urineproductie; (2) volume-therapie met gebalanceerde en
ongebalanceerde kristalloïde en colloïdale oplossingen gedeeltelijk deze parameters
cirrigeerde; en (3) acetaat-gebalanceerde colloïdale oplossing (HES-RA) de enige
therapievloeistof is die de nierperfusie tot ~85% van het baseline-niveau kon brengen, wat
geassocieerd was met de sterkst verbeterde renale oxygenatie. Terwijl therapie met de NaCl
en RA (kristalloïde oplossingen) en de HES-NaCl (ongebalanceerde colloïdale oplossing)
leidde tot hyperchloremische acidose, therapie met de HES-RA (acetate- gebalanceerde
colloïdale oplossing) deed dat niet. De acetaat-gebalanceerde colloïdale oplossing HES-RA
was bovendien de enige vloeistof die de renale perfusie terug naar ~85% van het baseline
niveau kon herstellen en het sterkst de renale microvasculaire oxygenatie verbeterde. Echter,
de lange-termijn effecten van HES-RA therapie op de nierfunctie dient verder bestudeerd te
worden.
Het doel van het hoofdstuk 3 was om de acute effecten van gebalanceerde versus
ongebalanceerde colloïde therapie op de renale macro- en microcirculatie te testen in een
ratmodel van LPS-geïnduceerde endotoxemie. Om de hypothese te testen dat gebalanceerde
colloïde therapie beter is voor de nieren dan ongebalanceerde colloïde therapie, hebben we de
dieren behandeld met HES-NaCl als een ongebalancerde colloïde oplossing en HES-RA als
gebalanceerde colloïde oplossing. De belangrijkste bevindingen waren dat (1) LPS-
geïnduceerde endotoxemie was geassocieerd met verslechterde systemische en renale
hemodynamiek, zuur-base-evenwicht, gemiddelde corticale microvasculaire perfusie en
perfusie-heterogeniteit, en urineproductie; (2) zowel HES-NaCl als HES-RA therapie de
systemische bloeddruk verbeterde, maar alleen HES-RA therapie de renale macro- en
microcirculatie verbeterde; (3) noch HES-NaCl noch HES-RA therapie de metabole acidose
of de fractionele excretie van natrium kon herstellen; en (4) plasma-chloride niveaus
significant lager waren na HES-RA therapie opzichte van na HES-NaCl therapie.
In hoofdstuk 4 was het doel van de studie om de hypothese te testen dat TEMPOL de
verstoorde redox homeostase kan herstellen en de stresstolerantie kan verhogen in een
mimetische-veroudering model van Drosophila. Daarom onderzochten we de omvang van de
algemene oxidatieve stress en met name oxidatieve schade aan eiwitten in deze vliegen na
TEMPOL toediening. Een rol voor oxidatieve schade in mimetische veroudering wordt
voornamelijk ondersteund door studies met knaagdieren. Verhoogde oxidatieve schade eiwit
en vrije radicalen gemedieerde desialylation van cellulaire eiwitten is een ander belangrijk
123
mechanisme voor cellulaire veroudering bij knaagdieren. Aan de andere kant worden
Drosophila veel gebruikt om de relatie tussen oxidatieve stress en veroudering te
ouderzoeken, omdat de genetische systemen van Drosophila bekend zijn. Wanneer D-
galactose aanwezig is in hoge concentratie kan het omgezet worden naar aldose
hydroperoxiden met katalyse galactoseoxidase, resulterend in de vorming van een superoxide
radicaal anion en andere ROS. Eerdere studies toonden aan dat mitochondriale dysfunctie een
essentieel onderdeel is van het mechanisme van versnelde huidveroudering door D-galactose
kan zijn. Bovendien werd aangetoond dat D-galactose schade veroorzaakt aan de integriteit
van de mitochondriën en de efficiëntie van ATP-productie verstoort, wat weer bijdraagt aan
ROS generatie in de mitochondria. Tot slot, onze studie toonde aan dat het wegvangen van
ROS met behulp van TEMPOL niet slechts gedeeltelijk oxidatieve schade vermindert tijdens
veroudering, maar ook direct de factoren gerelateerd aan oxidatieve stress verminderen in
plaats van alleen het verbeteren van het verminderde endogene afweersysteem, waardoor een
verbeterde bescherming tegen de omgeving bewerkstelligd wordt. Samengevat, deze effecten
leiden tot een bescheiden verbetering van de verouderings-gerelateerde kwetsbaarheid van
cellen.
In hoofdstuk 5 hebben we geprobeerd om de hypothese te testen dat het wegvangen van ROS
met behulp van TEMPOL geassocieerd zou zijn met een verbeterde nierfunctie,
zuurstoftransport, en NO productie in een kortdurend ratmodel van renale ischemie/reperfusie
(I/R) schade. We hebben gevonden dat I/R geassocieerd was met een significante toename in
weefsel MDA (marker van oxidatieve stress) en een significante afname in weefsel NO. De
afname in weefsel NO werd gevolgd door een toename in de RVR en daaruit voortvloeiende
verlaging van de RBF, renale DO2, en renale microvasculaire oxygenatie. Deze verstoringen
waren geassocieerd met een verminderde nierfunctie in termen van natriumreabsorptie en
creatinineklaring. Pre-ischemische toediening van TEMPOL, een bekende superoxide
scavenger, kon oxidatieve stress verlagen en nierweefsel NO en microvasculaire oxygenatie
beschermen en daardoor de nierfunctie verbeteren na I/R. Verder hebben we aangetoond dat
toediening van TEMPOL in afwezigheid van I/R leidt tot een verlaging van de renale MDA-
niveaus normaal aanwezig in nierweefsel, maar dit had geen effect op de andere parameters.
Onze studie heeft duidelijk aangetoond dat het wegvangen van ROS met behulp van
TEMPOL niet alleen renale oxidatieve stress verminderde na I/R, maar ook nierweefsel NO
niveaus genormaliseerde alsmede de RVR verminderde en de RBF, nier-DO2, en renale
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microvasculaire oxygenatie verbeterde. Samengevat, de effecten van TEMPOL leiden tot een
bescheiden (maar niet statistisch significante) verbetering van de nierfunctie na I/R.
125
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Acknowledgement
I am grateful for this moment in my life while I am writing this page of my thesis and reminisce moments of this wonderful chapter of my life. I am thankful to all the incredible people who played a vital role in helping to bring this project to finalize.
First of all, I would like to express my sincere gratitude to my supervisor, değerli hocam, Prof. Dr. Can Ince, for all motivation and supports, for all discussions, suggestions and feedbacks during last 14 years, especially my PhD study. Also, thank you for the patience during the whole period, despite of problems that I believe normally is not part of a supervisor responsibility. Thank you for trusting and giving me the possibility to work in an international research center and allowed me to develop my scientific career in a high standard and pleasant environment and you introduced me to clinical world and you made my dream came true. For me, hocam, you are a true mentor.
My thanks are also for my co-promoter Dr. Rick Bezemer who guided me through the process. Thank you for suggestions and all the comments, you put in the drafts, really helpful in improving my skills. I wish you all the best in your career!
I would like to thank to all PhD committee members for their willingness to join the committee: Prof. dr. F. Toraman, Prof. dr. J.H. Ravesloot, Prof. dr. S. Florquin, Prof. dr. E.T. van Bavel, Dr. E.G. Mik, Dr. C.T.P. Krediet.
Thanks to Dr. Jesse Ashruf. You came at the most trouble time of mine. Thank you very much for the contribution of the thesis to be published.
My sincere gratitude is also for Prof. Dr. Fevzi Toraman. Thank you for all opportunities me to add the clinical insight and you have always been a role model. It is honor for me to be a member of your team.
I also owe a debt of gratitude to Dr. Mathieu Legrand and Dr. Emre Almac for teaching a very hard surgery technique. Open abdominal surgery was really hard to perform, especially in rats.
Special thanks to Floris De Vries, Late Bas Bartels, Sema Aydin, Roos Koopman, Koray Yuruk and Bulent Ergin for warm friendship and fun work environment. Without you, my job would have undoubtedly been more difficult. I wish you lots of success with your further careers and rest in peace Bas!
Dear Peter, it was a great pleasure to know a person like you. I will never forget you and the period when we shared the room in AMC. Thanks for the heritage of NO analyzer. Rest in peace!
Special thanks for Prof. Dr. Gulderen Sahin. I will always remember your belief and motivation to me, which gave hope and strength.
I would also like to thank my dear friend Umut Naci. You always became a good example for me. I hope our friendship will last forever.
Finally, I would like to thank my family that in any way supports my PhD journey during all these years. Especially thank to my dear father. All successes in my life have something from you. Surely, you were with me from the beginning. Rest in peace!
Thanks to my dear sister Çiğdem. Maybe I could not show you my gratitude but surely I will need your support and constant love forever. Thanks to my dear mother. Great woman with great patience at all times.
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And special thanks to my dear wife Berna. Living with you in Amsterdam was amazing. Your personal support and motivation, especially during my stay in Amsterdam for months is one of the spines of this thesis.
Last but not least, I would like to express my gratitude to the Dutch culture as well as the research systems. Since 2001, the continuous interaction between us has shaped me and transformed me into the person that I am today.
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Curriculum vitae Ugur Aksu was born on February 1, 1979 in Istanbul, Turkey. He attended high school at
Haydarpasa Lycee in Istanbul. In 1996, he started his bachelor studies at the University of
Istanbul. During this time he developed an interest in physiology and participated in several
projects. The first project such project was “The effects of TNF-alpha on leukocyte
endothelial cell interactions during sepsis” project in which he was responsible for building a
research system in his department besides doing the experiments. This project was partly
supported by Prof. Ince from Academic Medical Centre in the Netherlands. He developed
skills in experimentation, enhanced his theoretical knowledge in cardiovascular system and
microcirculation and was oriented to scientific research environment. In 2003, he received his
master degree wıth the thesis titled “The effects of different nitric oxide synthase inhibitors
upon hemodynamic of rats received lipopolysaccaride“. In the same year, he started his PhD
studies at the Department of Biology, University of Istanbul. Besides his academic activities,
between November 2000 and January 2010, he was employed at the University of Istanbul as
a research assistant. In this position, his primary responsibility was to participate in teaching
activities and also to supervise laboratory work. In June 2009, he receveid PhD degree in
Biology with the thesis titled “Effects of β-3-agonists on cardiovascular system and adhesion
molecules in hyperglycemic rats.“ His thesis was about the investigation of β3-ARs’ effects
on the cardiovascular system and immunologic state during hyperglycemia. From July 2009
to August 2010, he worked on the basis of a research grant in the Department of Translational
Physiology at the Academic Medical Center, University of Amsterdam, The Netherlands.
Until present his research has focused lies on kidney perfusion and oxygenation changes in
various rat models of acute kidney injury. His research was supported by Dutch Kidney
Foundation and published in numerous international journals. Currently he is employed as an
associated professor in Istanbul University.
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Portfolio
Name PhD student: Uğur AKSU
PhD period: September 2009-November 2015
Name PhD supervisor: Prof.Dr. Can Ince
1) PhD Training
General courses
- Project management
- Biotek Epoch
- Biotek Synergy H1-M
- Course on Laboratory Animal Science
- Course on Neurobiology
- Course on Biostatistics
- Course on Cognitive Electrophysiology: ERP in the Evaluation of Cognitive Disorders
- PowerLab Training Course
- III. Ege Biennial Int. Neuroscience Grd. Summer School
- Symposium of Evolution on Biology Education
- Flow Cytometry Training VII
- National Student Scientific Session with International Participation
Specific courses
- CELL AND TISSUE PATOLOGY
- INTRACELLULAR TRAFFIC OF PROTEINS
- INTRO. TO CANCER BIOLOGY
- HORMONES OF VERTEBRATES
- ADVANCED PHYSIOLOGY I
- MEMBRANE PHYSIOLOGY
- ADVANCED PHYSIOLOGY II
(in doctorate)
- THE USE OF ANIMALS FOR EXPERIMENTS
- TISSUE CULTURES AND APPLICATION FIELDS
- TRACE ELEMENTS
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- ANTIOXIDATIONS AND DETOXIFICATIONS IN BIOLOGICAL SYSTEMS
- IN VIVO TECHNIQUES
- CYTOLOGICAL TECHNIQUES
- NEUROPHYSIOLOGY
Presentations
- "Fluoxetine Reduces The Lung Injury Induced By Infrarenal Abdominal Aortic Ischemia-Reperfusion In Rats.", 37th International Union of Physiological Sciences (IUPS) Congress,England
- "The effects of balanced and unbalanced colloid and crystalloid solutions on renal microvascular perfusion in endotoxemic rats", 31st ISICEM (International Symposium on Intensive Care and Emergency Medicine) 168 pp., Belgium 2010
- "The effects of balanced and unbalanced colloid and crystalloid solutions on renal oxygenation in a rat model of hemorrhagic shock and resuscitation" 31st ISICEM (International Symposium on Intensive Care and Emergency Medicine) 168 pp., Belgium, 2010
- "Atorvastatin improves development of pentylentetrzol-induced kindling, learning and memory disorders in rats" The 36th Congress of the International Union of Physiological Sciences P3PM-6-6 pp., Kyoto-Japan, 2009
- "Evaluation of the effects of α-lipoic acid and pycnogenol supplementation on NO release with ONOO-, 3-NTyr and total nitrite/nitrate levels in experimental cerebral ischemia-reperfusion subjected to diabetic rats", HSSR/AIST-NIEHS/NIH Joint International Symposium "Biomarkers of Oxidative Stress in Health and Disease" P4-6-1 pp., Osaka-Japan, 2008
- "Effects of lipoic acid on oxidative and nitrosative in cerebral ischemia reperfusion exposed diabetic rats" 17th IFCC-FESCC European Congress of Clinical Chemistry and Laboratory Medicine-Euromedlab, T128 pp., Amsterdam-Holland 2007
- "Exploring the recovering effects of pycnogenol on cerebral ischemia reperfusion in experimental diabetes model", 17th IFCC-FESCC European Congress of Clinical Chemistry and Laboratory Medicine-Euromedlab T127 pp., Amsterdam-Holland 2007
- "Effects of glucocorticoids on alpha adrenergic response during sepsis", 11th Annual Meeting of the European-Council- for Cardiovascular Research 769 pp., Nice-France, 2006
- "Lipoic acid attenuates oxidative and nitrosative stress, simultaneously sialic acid content in liver tissues of diabetic rats" 31st Congress of the Federation-of-European-Biochemical-Societies (FEBS), 179 pp., İstanbul-Türkiye, 2006
- "Comparative effects of nitric oxide inhibition by aminoguanidine before and after dopamine infusion on intestinal perfusion during endotoxemia", 24th Conference of the European-Society for Microcirculation 50 pp., Amsterdam-Holland, 2006
- "Alpha-Lipoic acid prevents oxidative injury in diabetic rats subjected to cerebral ischemia-reperfusion" 16th European Congress of Clinical Biochemistry and Laboratory Medicine (EUROMEDLAB 2005, 174 pp., Glasgow, 2005
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- "Mesenteric blood flow can be affected by aminoguanidine during sepsis", XXXV. International Congress of Physiological Sciences 11 pp., San Diego-USA, 2005
- "Oxidative injury in cerebral ischemia reperfusion exposed to diabetic rats", 13th Balkan Biochemical Biophysical Days & Meeting on Metabolic Disorders, 158 pp., Kuşadası-Türkiye, 2003.
Invited Oral presentations:
- Microcirculation in health and disease, Cerrahpasa Med. School, Istanbul, Turkiye, 2015.
- Perfusion heterogeneity in sepsis, İzmir Inovation meeting, Izmir, Turkiye, 2014.
- A biological mask: Glycocalyx, Acibadem University, Turkiye, 2013.
- Oxidative stress in disease, Acibadem University, Turkiye, 2013.
2. Teaching
Lecturing
- Animal Physiology (2015- )
- Selected Topics in Nervous System (2014- )
- The Modelling in Experimental Animals (2014- )
- Professional English (2010) (2013- )
- Supervising Student laboratory (2000-2013)
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List of publications
• Aksu U., Ergin B., Bezemer R., Milstein D., Ince C. Scavenging Reactive Oxygen Species Using Tempol In The Acute Phase Of Renal Ischemia/Reperfusion And Its Effects On Kidney Oxygenation And Nitric Oxide Levels, Intensive Care Medicine Experimental, vol.3 , pp.1-10, 2015
• Ischemia Modified Albumin; Does it Change During Pneumoperitoneum in Robotic
Prostatectomies? Accepted in International Braz J Urol, 2015
• Arıtürk C, Ozgen ZS, Kilercik M, Ulugöl H, Ökten EM, Aksu U, Karabulut H, Toraman F. Comparative Effects of Hemodilutional Anemia and Transfusion during Cardiopulmonary Bypass on Acute Kidney Injury: A Prospective Randomized Study. Arıtürk C, Ozgen ZS, Kilercik M, Ulugöl H, Ökten EM, Aksu U, Karabulut H, Toraman F. Heart Surg Forum. 2015 Aug 30;18(4):E154-E160.
• Erman H., Guner I., Yaman M.O., Uzun D.D., Gelisgen R., Aksu U., Yelmen N.,
Sahin G., Uzun H. The Effects Of Fluoxetine On Circulating Oxidative Damage Parameters In Rats Exposed To Aortic Ischemia-Reperfusion, European Journal of Pharmacology, vol.749, pp.56-61, 2015
• Toraman F., Aksu U. Monitoring of tissue oxygenation and perfusion. Turkiye
Klinikleri J Anest Reani, vol.8, pp.8-14, 2015
• Almac E., Bezemer R., Kandil A., Aksu U., Milstein D.M.J., Bakker J., Demirci-Tansel C., Ince C. Bis Maltolato Oxovanadium (Bmov) And Ischemia/Reperfusion-Induced Acute Kidney Injury In Rats. Intensive Care Medicine Experimental, vol.2, pp.1-9, 2014
• Aksu U., Guner I., Yaman O.M., Erman H., Uzun D., Sengezer-Inceli M., Sahin A.,
Yelmen N., Gelisgen R., Uzun H., Sahin G. Fluoxetine ameliorates imbalance of redox homeostasis and inflammation in an acute kidney injury model. J Physiol Biochem. 2014 Dec;70(4):925-34.
• Aksu U., Yanar K., Terzioglu D., Erkol T., Ece E., Aydin S., Uslu E., Cakatay U.
Effect of tempol on redox homeostasis and stress tolerance in mimetically aged Drosophila. Arch Insect Biochem Physiol. 2014 Sep;87(1):13-25.
• Guner I., Yaman M.O., Aksu U., Uzun D., Erman H., Inceli M., Gelisgen R., Yelmen
N., Uzun H., Sahin G. The effect of fluoxetine on ischemia-reperfusion after aortic surgery in a rat model. J Surg Res. 2014 Jun 1;189(1):96-105.
• Aksu U., Bezemer R., Ince C. Reply to: crystalloid resuscitation in hemorrhagic
shock. Resuscitation. 2012 Aug;83(8):e173. Epub 2012 May 4. No abstract available.
• Almac E., Aksu U., Bezemer R., Jong W., Kandil A., Yuruk K., Demirci-Tansel C., Ince C. The acute effects of acetate-balanced colloid and crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. Resuscitation. 2012 Sep;83(9):1166-72. Epub 2012 Feb 19.
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• Aksu U., Bezemer R., Yavuz B., Kandil A., Demirci C., Ince C. Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and inflammation. Resuscitation. 2012 Jun;83(6):767-73.
• Aksu U., Bezemer R., Demirci C., Ince C. Acute effects of balanced versus
unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusion during endotoxemic shock. Shock. 2012 Feb;37(2):205-9.
• Aksu U., Demirci C., Ince C. The pathogenesis of acute kidney injury and the toxic
triangle of oxygen, reactive oxygen species and nitric oxide. Contrib Nephrol. 2011;174:119-28. Epub 2011 Sep 9. Review.
• Uzum G., Akgun-Dar K., Aksu U. The effects of atorvastatin on memory deficit and
seizure susceptibility in pentylentetrazole-kindled rats. Epilepsy Behav. 2010 Nov;19(3):284-9.
• Guner I., Sahin G., Yelmen N.K., Aksu U., Oruc T., Yildirim Z.
Intracerebroventricular serotonin reduces the degree of acute hypoxic ventilatory depression in peripherally chemodenervated rabbits. Chin J Physiol. 2008 Jun 30;51(3):136-45. Erratum in: Chin J Physiol. 2008 Aug 31;51(4):261.
• Diler A.S., Uzüm G., Akgün Dar K., Aksu U., Atukeren P., Ziylan Y.Z. Sex
differences in modulating blood brain barrier permeability by NO in pentylenetetrazol-induced epileptic seizures. Life Sci. 2007 Mar 13;80(14):1274-81. Epub 2007 Jan 25.
• Guner I., Sahin G., Karaturan-Yelmen N., Aksu U., Oruc T., Yildirim Z. The Role of
Central Serotonin on Respiratory Regulation in Anaesthetized Rabbits. Cerrahpasa J Med 2006; 37: 98 – 102.