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Sepsis-induced acute kidney injury
“The role of the P2X7 receptor (P2X7R) in renal injury and sepsis through the local
production of cytokines and chemokines”
Groningen International Program of Science in Medicine (GIPS-M)
Marije (ML) Sixma
Supervisor London, United Kingdom Supervisor Groningen, Netherlands
Professor Mervyn Singer Professor C.G.M. Kallenberg,
Professor of Intensive Care Medicine, Head Professor Department of Rheumatology
Research Department of Clinical Physiology and Clinical Immunology
University College London University Medical Centre Groningen
Division of Medicine, NIHR Senior Investigator ’09 Chairman of the GIPS-M Committee
[email protected] [email protected]
Co-supervisor
Dr. Nish Arulkumaran, PhD at the division of
Intensive Care Medicine, UCL
London, January – June 2013
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Table of contents
ABBREVIATIONS 4
ENGLISH SUMMARY 5
INTRODUCTION
Sepsis and acute kidney injury 7
Current tools and limitations 7
Haemodynamic and histopathological findings 8
Inflammation and the inflammasome 9
The NLRP3 pathway 10
P2X7 receptor 11
Macrophages and monocyte chemoattractant protein 1 12
Aim of study 12
MATERIAL AND METHODS
1. In vivo methods 13
(i). Rat model of acute sepsis
(ii). Rat model of sepsis and recovery
2. Homogenization of Frozen Kidney Tissues 14
3. Bicinchoninic acid (BCA) protein estimation assay 14
4. Western blotting 14
5. Immunohistochemistry 15
6. Enzyme-linked immunosorbent assays (ELISAs) 15
7. Statistics 16
RESULTS
Polymicrobial model of acute sepsis
1. Analysis of renal dysfunction by use of serum creatinine 17
2. Immunohistochemistry
(i) Assessment of histological damage 17
(ii) P2X7 expression in kidney tissue 18
(iii) Detection of macrophages 20
3. Enzyme-linked immunosorbent assay’s
(i) Expression of serum and renal IL-1β 20
(ii) Expression of serum and renal MCP-1 21
4. Western Blotting 22
Recovery model of sepsis
1. Immunohistochemistry 23
3. ELISA; expression of renal MCP-1 24
DISCUSSION 25
CONCLUSIONS 27
FUTURE WORK 27
3
DUTCH SUMMARY 29
ACKNOWLEDGMENTS 31
REFERENCES 32
APPENDICES
I. Buffers and solutions 36
ABSTRACTS 37
1. “P2X7 Receptor and Haemorrhage-reperfusion- Induced Acute Tubular Injury”
2. “Renal macrophage infiltration in a rat model of sepsis and recovery”
3. “Temporal changes in renal haemodynamics and oxygenation in a rat model of sepsis”
4
Abbreviations
MOF multi organ failure
AKI acute kidney injury
ICU intensive care unit
RRT renal replacement therapy
ADQI Acute Dialysis Quality Initiative
RIFLE Risk- Injury- Failure- Loss and End stage renal disease criteria
sCr serum creatinine
AKIN Acute Kidney Injury Network
KDIGO Kidney Disease: Improving Global Outcomes guidelines
GFR glomerular filtration rate
ARF acute renal failure
RBF renal blood flow
MAP mean arterial pressure
ATN acute tubular necrosis
TECs tubular epithelial cell
LPS lipopolysaccharide
PRR pattern recognition receptor
PAMPs pathogen-associated molecular patterns
DAMPs danger-associated molecular patterns
ATP adenosine 5’-triphosphate
TLR toll-like receptor
NFkB nuclear factor kappa
IL-1β interleukin-1β
TNF-α tumor necrosis factor-α
COX-2 cyclooxygenase-2
NLRP-3 nod-like receptor protein 3
P2X7R P2X7 receptor
IFN-γ interferon-γ
mRNA messenger ribonucleic acid
NTN nephrotoxic nephritis
MCP-1 monocyte chemoattractant protein 1
CKD chronic kidney disease
NGAL neutrophil gelatinase-associated lipocalin
KIM-1 kidney injury molecule-1
SDS-PAGE Sodium dodecylsulphate-Polyacrylamide gel electrophoresis
PVDF Polyvinylidene difluoride
ELISA Enzyme-linked immunosorbent assay
PAS Periodic acid–Schiff (PAS)
ICE interleukin-1β converting enzyme
MAP kinase mitogen-activated protein kinase
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Summary
Introduction
Sepsis is an exaggerated inflammatory response to infection that may lead to multiple organ
failure and death. The kidney is commonly affected in the septic process leading to
dysfunction or complete failure. Of note, survivors of sepsis-induced acute kidney injury
(AKI) have an increased risk of developing end-stage chronic kidney disease. Release of pro-
inflammatory cytokines such as IL-1β and IL-18 are associated with the pro-inflammatory
cascade and immunological factors are likely to contribute to the development of sepsis-
induced AKI. However, initial release of these mediators is, in itself, insufficient. A
considerable role for the post-transcriptional processing and release of mature cytokines is via
the formation of a large multiprotein complex, the NLRP3 inflammasome. The cell surface
P2X7 receptor (P2X7R) facilitates assembly of NLRP3. As well as mediating inflammatory
responses, the P2X7R is also involved in apoptotic cell death. Upregulation of this receptor is
associated with renal injury. Moreover, A-438079, a selective P2X7 antagonist, attenuated this
response in a rat model of glomerulonephritis. These findings suggest that the P2X7R could
function as a potential therapeutic target. P2X7 expression is best known in immune cells such
as macrophages which play a critical role in the initiation, maintenance, and resolution of
inflammation. Macrophages both release and are influenced by chemokines such as MCP-1
that play a prominent role in the acute inflammatory response in several models of kidney
disease. Moreover, IL-1 β (after maturation and release by the P2X7/inflammasome) induces
expression of MCP-1 by epithelial cells.
Aims
With a working hypothesis that P2X7R upregulation causes renal injury through local
production of cytokines and chemokines, I sought to determine whether renal P2X7R
expression increases in a fluid-resuscitated three-day rat model of faecal peritonitis, and
specifically to define its relationship and co-localization to histopathological changes and
macrophage infiltration. In a separate longer term (2-week) rat model of zymosan-induced
peritonitis, I sought to describe renal P2X7R expression and pathological changes from the
early stages into the recovery phase.
Methods
For the acute model, sepsis was induced in instrumented, awake male Wistar rats by
intraperitoneal injection of faecal slurry followed by fluid resuscitation at 10 ml/kg/hr
commencing 2 hours post-induction of sepsis. Sham animals received a similar fluid regimen
but no i.p. injection. Septic and sham-operated animals were sacrificed post-sepsis at either 6
hours (n = 6 per group) or 24 hours (n= 8 per group). Kidneys were harvested and blood
analyzed for serum creatinine, histological evidence of renal injury, P2X7 expression,
macrophage infiltration, and cytokine and chemokine expression. Comparison was made
against naïve, non-instrumented animals (n=6).
In the long-term model, non-instrumented male Wistar rats were given i.p. zymosan (a yeast
cell wall product) as a more prolonged septic insult. Kidneys were harvested at day 2 (4 sham,
4 sepsis animals) and day 14 (4 sham, 8 sepsis), and analyzed for renal injury, macrophage
infiltration and chemokine expression.
6
Continuous variables are presented as mean ± SD. Parametric and non-parametric data were
compared by unpaired t-test and Mann Whitney U tests, respectively. One-way ANOVA
assessed differences between more than two groups of continuous variables. Post-hoc Tukey’s
test ascertained between-group differences with P values <0.05 being taken as statistically
significant.
Results
In the acute septic AKI model, serum creatinine was significantly higher at 24 hours (p<0.05).
However, no significant renal histological damage was seen at either 6 or 24 hour timepoints.
In septic kidneys, P2X7R expression increased at 24 hours (p< 0.05) though a trend was
apparent even at 6 hours. Severely septic animals (with a 3-fold increase in serum creatinine)
showed evidence of intraluminal debris that stained intensely for P2X7. Increased expression
of P2X7 correlated with elevated serum and renal tissue IL-1β levels. Serum IL-1β were
elevated in septic animals, though levels were falling by 24 hours (p<0.05). By contrast, renal
tissue IL-1β levels were elevated at 6 hours after sepsis (p=0.05) but further increased at 24
hours (p<0.01). Consistent with these findings, septic AKI was associated with increased
renal expression of caspase-1 and pro-IL-18. No macrophage infiltration was seen at either
timepoint and there was only minimal elevation in renal MCP-1 levels.
In the long-term model of sepsis, there was also minimal evidence of acute renal injury at
Days 2 or 14. P2X7 staining has yet to be analyzed; however, macrophage infiltration was
evident in both glomeruli and interstitium in all Day 14 septic animals but in none of the sham
controls. Renal MCP-1 levels were also significantly elevated in septic animals (p<0.01) at
this timepoint. By contrast, macrophage infiltration was only seen in one of the four septic
animals on Day 2 and renal MCP-1 levels were similar to sham animals.
Conclusions
A significant increase in renal P2X7 expression was seen in septic animals after 24 hours,
correlating with elevated levels of IL-1β. In severely septic animals more renal injury was
apparent with a distinct pattern in staining, suggestive of a role in cell death. This finding,
together with an absence of early renal macrophage infiltration, suggests renal P2X7 is likely
to be expressed by intrinsic kidney cells. While significant histological damage was not seen,
reflecting findings in septic patients, there was biochemical evidence of renal dysfunction.
Further research is needed to evaluate whether P2X7 antagonism offers protection. By
contrast, the longer term model (in which P2X7 staining has not yet been analyzed) did show
significant renal macrophage infiltration within the glomeruli and interstitium with a
corresponding increase in renal MCP-1 at day 14. Whether this contributes to long-term renal
damage requires further study.
7
Introduction
Sepsis is a life-threatening disorder and a leading cause of mortality among critically ill
patients. With an associated mortality rate exceeding 20%, sepsis is a major global health
problem (1-4). Sepsis represents an excessive systemic inflammatory host response to an
infectious agent that may result in multiple organ dysfunction (5), of which acute kidney
injury (AKI) is a common manifestation. The reported incidence of AKI in intensive care
units (ICU) may be as high as 70%, of which sepsis is associated with half the cases (1, 6, 7).
Overall mortality rates of patients with AKI remain high. Approximately one-third of patients
who require renal replacement therapy (RRT) die while still in the ICU (8-11). However, as
the biochemical abnormalities and fluid balance can be managed by renal replacement
therapy, it is uncertain whether or not AKI directly contributes to mortality. There is no
specific treatment for AKI once it occurs; current management is largely preventive or
supportive. Despite septic AKI being a commonplace problem, relatively little is known about
its pathogenesis. Clearly, outcomes should be improved with a better understanding of the
pathophysiology leading to more directed therapies.
Current tools and limitations
Although AKI is common, and even mild forms of AKI portend worse clinical outcomes, it is
often recognized late (7, 12). AKI defines a broad spectrum of renal impairment, ranging from
minimal elevations in serum creatinine to anuric renal failure. Because of great variations in
this clinical syndrome, a uniform definition was lacking until relatively recently. The Acute
Dialysis Quality Initiative (ADQI) proposed the RIFLE criteria (Risk, Injury, Failure, Loss
and End-stage renal disease) (13). This classification system for diagnosing and classifying
AKI is defined by criteria for serum creatinine (sCr) and urine output (Figure 1).
Subsequently, these criteria were refined by the Acute Kidney Injury Network (AKIN)
(Figure 1) (14). Both have been recently amalgamated into the ‘Kidney Disease: Improving
Global Outcomes (KDIGO)’ guidelines for Acute Kidney Injury (15), that offer a means of
standardizing epidemiological data and clinical studies.
Both AKIN and RIFLE criteria have been validated by a number of epidemiological studies.
Despite their merits, the criteria must be interpreted within their limitations. They lack detail
on the mechanisms and the precise timing of injury. Furthermore, knowledge of the patient’s
baseline serum creatinine is required, and monitoring of urine output is highly variable
depending on their hydration status and the use of diuretics.
The use of serum creatinine as a biomarker has several limitations. There may be a significant
loss of renal functional reserve (up to 70%) before serum creatinine rises. It may also not
accurately reflect the glomerular filtration rate (GFR) in acute illness due to fluctuations in
intravascular volume status (16). Alterations in creatinine production that accompany critical
illness, due in part to loss of muscle mass, further confound the use of serum creatinine as a
renal biomarker in acute illness. The need for more sensitive biomarkers of renal dysfunction
is therefore required, which may allow earlier initiation of appropriate management.
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Figure 1. RIFLE and AKIN classifications for acute kidney injury. Adapted from (13)
RIFLE: Risk–Injury–Failure–Loss–Endstage renal disease; AKIN: Acute Kidney Injury
Network; ARF: acute renal failure; Cr: creatinine; GFR: glomerular filtration rate.
Haemodynamic and histopathological findings
Knowledge of the pathophysiology of septic AKI is still in its infancy. It was generally
believed that, like other causes of shock, AKI was mainly due to renal hypoperfusion. Sepsis
results in hypovolaemia due to increased capillary leak, and hypotension due to arterial
vasodilatation and a loss in vascular tone. The resulting drop in renal blood flow (RBF) was
felt to cause renal ischaemia and, consequently, renal failure. However, Bellomo et al
systematically reviewed changes in RBF in sepsis and found that RBF was actually elevated
in human studies (17). Thereafter, they analyzed 159 animal studies and also found RBF was
either preserved or elevated during sepsis, provided the animals were fluid-resuscitated. They
confirmed these findings in a sheep model of septic shock which showed prominent renal
vasodilatation, accompanied by a marked increase in RBF, despite a three-fold increase in
serum creatinine and progressive oliguria (18, 19).
Another long-held paradigm in septic AKI was that acute tubular necrosis (ATN) would be
the predominant histological finding. Tubular injury evolves as a result of generalized or
localized impairment of oxygen and nutrient delivery to, and waste product removal from,
tubular epithelial cells (TECs) (20). TECs are metabolically very active and therefore
particularly sensitive to anoxia and vulnerable to toxins. This susceptibility partly derives
from the arrangement of the vascular supply to the outer medulla. In health, the pars recta of
the proximal tubule and the thick ascending limb are on the verge of hypoxia due to the
countercurrent exchange properties of the vasa recta, what increases by alterations of flow to
these areas (21). Other factors that predispose tubules to toxic injury include a vast
electrically-charged surface for tubular reabsorption, active transport systems for ions and
organic acids, and the capability for effective concentration. This potentially leads to
reabsorption of substances from the tubular lumen that may cause toxicity to epithelial cells
(22). Toxins may include drugs or high concentrations of endogenous substances such as
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myoglobin, potassium or urea (22). Tubular ischaemia initially leads to damage of TECs
followed by shedding. The resulting cellular debris is thought to exacerbate excretory failure
by blockage of the tubules. Despite this cascade being conceivable for septic AKI it does not
correspond with the histopathological findings normally found in septic AKI. While a local
inflammatory response with leukocyte infiltration and tubular apoptotic bodies has been
described in the kidney (23); acute tubular necrosis only occurs in 22% of renal
histopathological specimens, though usually to minimal degrees only, and fails to explain the
degree of functional impairment seen in vivo (24).
Other haemodynamic changes within the kidney such as efferent arteriolar vasodilatation, re-
distribution of blood flow due to periglomerular shunting, and late vasoconstriction may all
occur at different timepoints during the course of sepsis. However, the development of septic
AKI is likely not due to renal haemodynamic changes alone, particularly in view of the lack
of correlation between histopathological changes and functional abnormality. This has led to
other hypotheses including a role for immunological factors and other sequelae of
inflammation including endothelial dysfunction, coagulation abnormalities, endothelial
dysfunction, and oxidative stress (15).
Inflammation and the inflammasome
In response to inflammatory stimuli, a sequence of host-microbial interactions occur which
activates the innate immune response. Innate immunity coordinates the first-line defence
response against pathogens and involves both humoral and cellular components. Besides
being involved in the initiation of inflammation, it also has a warning function by alerting the
adaptive immune system. The components of innate immunity recognizes structures shared by
various classes of antigens, so-called pattern recognition receptors (PRRs) (25). PRRs
recognize pathogen-associated molecular patterns (PAMPs) on invading organisms, as well as
endogenous stress signals termed danger-associated molecular patterns (DAMPs). To date,
various DAMPs and PAMPs have been recognized. Examples of PAMPs include LPS,
peptidoglycan, bacterial DNA, viral RNA and fungi. Many DAMPs are nuclear or cytosolic
proteins that get released outside the cell or are exposed on the cell surface following stress,
tissue injury or cell death (26). Non-protein DAMPs include purines such as adenosine 5’-
triphosphate (ATP) (27), uric acid (28) and adenosine (29).
Toll-like receptors (TLRs), a class of PRRs, are transmembrane proteins primarily expressed
by innate immune cells, but also by endothelial and epithelial cells (30). Neutrophils and
macrophages recognize microbes via these surface receptors. During sepsis, there is a
significant upregulation in TLR-2 and TLR-4 expression (31). TLR-4 has been discovered as
a key mediator in renal inflammation (32). TLRs activate pro-inflammatory transcription
factors such as nuclear factor kappa B (NFkB) with subsequent production of cytokines (e.g.
interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and IL-6, enzymes such as
cyclooxygenase-2 (COX-2), and a variety of chemokines, cellular adhesion molecules,
receptors and other proteins (33, 34) (Figure 2, i and ii).
IL-1β is a key initiator of the acute inflammatory response and plays a pivotal role in the
pathogenesis of septic shock (35, 36). However, initial IL-1β processing and release is
insufficient and a secondary stimulus is necessary (36). A considerable role for post-
transcriptional processing and release of mature cytokines is via formation of a large multi-
protein complex, the NLRP3 inflammasome. Initial activation of PRRs not only results in
NFκB-dependent transcription of pro-IL-1β but also upregulates NLRP3 (37, 38).The cell
surface P2X7 receptor (P2X7R) facilitates assembly of the NLRP3 inflammasome (39-41).
10
ATP released into the extracellular milieu during inflammation is a potent stimulus for P2X7R
activation (38, 42, 43), leading to IL-1β processing from activated monocytes, macrophages
and microglia (44). Monocytes from transgenic mice lacking the P2X7R were unable to
release IL-1β in response to ATP (45). Pro-inflammatory molecules such as interferon-γ,
(IFN-γ), TNF-α and endotoxin can induce upregulation in P2X7R synthesis (46). ATP-driven
P2X7R activation results in formation of an ion pore causing potassium (K+) efflux (figure 2
iii). The following endogenous depletion of K+ is a key step in inflammasome activation (47,
48). The NLRP3 inflammasome recruits pro-caspase-1 which, by proteolysis to caspase-1, is
the final step in the processing and release of bioactive IL-1β (figure 2 iv).
Figure 2. Schematic figure of the inflammasome pathway.
i) Innate immunity is activated via
DAMP signalling with initial
activation of TLR-4.
ii) TLR-4 then activates pro-
inflammatory transcription factors
such as NFkB, leading to production
of pro-IL-1β and pro-IL-18 and
upregulation of NLRP3.
iii) ATP released into the extracellular
milieu during inflammation stimulates
P2X7R activation, a key step in
inflammasome activation.
iv) NLRP3 recruits pro-caspase-1
which is converted to caspase-1, the
final step in the processing and
release of bioactive IL-1β and IL-18.
Stimulation mediated through P2X7R/ NLRP3 rapidly releases large amounts of mature IL-1β
at rates up to 100x faster than with PAMP exposure alone (49, 50). Extended stimulation of
the P2X7 inflammasome cascade induces caspase-1-mediated death of monocytes and
macrophages (51-53). IL-1β may also be activated by P2X7-independent pathways. This
includes shedding via microvesicles (54), processing to biological active IL-1β by neutrophil-
derived serine proteases (cathepsin G, neutrophil elastase, and proteinase-3) (55, 56), or by
mast cell–derived serine proteases (granzyme A and chymase) (57, 58).
IL-18 is also an important inflammatory mediator. Excessive levels are associated with septic
shock and autoimmune syndromes (59). IL-18 shares structural features with IL-1, but is not
an endogenous pyrogen (60, 61). IL-18 binds to a receptor inducing synthesis of other pro-
inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, IFN-γ) and chemokines (60). IL-18 is also
a substrate for the caspase-1 inflammasome, functioning via the P2X7R (37). IL-18 excretion
is higher in septic compared to non-septic AKI; increased levels predict deterioration of
kidney function 24-48 hours before AKI becomes clinically apparent (62, 63).
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NLRP3 pathway
Activation of the innate immunity occurs via DAMP and PAMP signalling, resulting in
release of the pro-inflammatory cytokines, IL-1β and IL-18. An additional stimulus is
necessary for extracellular release of active IL-1β and IL-18. Inflammasome-mediated post-
translational processing of IL-1β (and other inflammasome-inducing stimuli such as IL-18) is
linked to efficient export of these molecules to the extracellular compartments. Extracellular
ATP, acting on the P2X7R, is one of the best established stimuli for this pathway (59, 64).
How the inflammasome exactly mediates these series of events has yet to be fully resolved
since it can be triggered by different mechanisms in different cell types.
P2X7 receptor
The purine ATP is an important extracellular messenger in various physiological processes,
including synaptic transmission and inflammation (65, 66). Its effects are mediated through
activation of purinergic receptors, e.g. P1 adenosine and P2 nucleotide receptors (67). P2
receptors are classified into two subfamilies, the P2X ligand-gated ion channels and the P2Y
G protein-coupled receptors. P2 receptors are expressed differentially throughout the kidney.
Purinoceptor signaling has important roles in water and sodium transport, control of the renal
microcirculation and in autoregulatory responses (68-70). By its structural differences from
the other P2X members and the requirement of high concentrations of ATP for activation, the
P2X7 receptor (P2X7R) has several unique properties among the P2X family.
The P2X7R, first characterized in immune cells including monocytes, macrophages,
lymphocytes and bone-marrow derived cells, has a wide expression pattern, particularly in
epithelial cells of the gastrointestinal tract, skin, salivary glands and urinary tract (71, 72).
Demonstration of the extracellular release of ATP and the presence of P2X7R in the renal tract
are consistent with a functional role for nucleotide signaling. Attempts to define the functional
importance of this potential signaling system have not yet led to complete understanding.
The P2X7 membrane receptor functions as a ligand-gated ion channel; it activates cell
membrane permeabilization and various downstream-signaling pathways, including
inflammatory responses and modulation of cell turnover (72). This pro-inflammatory response
results in release of inflammatory cytokines such as IL-1β and IL-18 from macrophages,
changes in plasma membrane lipid distribution, and can lead to cell death by necrosis or
apoptosis (54, 70, 73). As discussed, the P2X7 subtype is the receptor responsible for ATP-
driven maturation and release of IL-β (44, 45, 74-76). Pro-inflammatory cytokines and
bacterial products (e.g. LPS) can also upregulate P2X7R expression and increase its sensitivity
to extracellular ATP (64). As the P2X7R is non-desensitizing, the pore stays open as long it is
bound by ATP. Sustained activation by extracellular ATP confers the ability of the molecule
to form a large plasma membrane ‘pore’, enabling the receptor to function not only as a non-
selective cation channel, but to also mediate cell membrane permeabilization by allowing
passage of large molecules up to 900Da (42). Should ATP stimulation be prolonged, cells
become irreversibly injured and death occurs (42, 70, 77).
The ability of the P2X7R to mediate inflammatory responses and its dual role in cell turnover
(by potentially effecting both apoptosis and proliferation) suggest its importance in situations
in which these processes are prominent. These include both normal and abnormal renal
function (78). In vitro studies where rat mesangial cells were stimulated by LPS, IFN-γ or
TNF-α showed significant upregulation in mRNA expression of P2X7. Exposure of these cells
to external ATP resulted in apoptotic cell death (79, 80). In vivo studies of healthy rat kidneys
however yielded very low levels of P2X7R expression (80, 81). A key role for P2X7R has
been suggested in glomerulonephritis in experiments using a rodent model of nephrotoxic
12
nephritis (NTN) (70). A three-fold upregulation of glomerular P2X7R mRNA was seen by day
4, correlating with the onset of significant proteinuria and peak macrophage infiltration (71).
In the same model, P2X7-knockout mice were used, showing P2X7 deficiency was
significantly renoprotective as evidenced by better renal function, a striking reduction in
proteinuria and decreased histological glomerular injury (82). Other experiments with A-
438079, a selective P2X7 antagonist, prevented the development of antibody-mediated
glomerulonephritis in rats (82). These data support a pro-inflammatory role for P2X7 in
immune-mediated renal injury and suggest that the P2X7R is a potential therapeutic target.
Macrophages and monocyte chemoattractant protein 1 (MCP-1)
Macrophages, as major components of the mononuclear phagocyte system, play a significant
role in inflammation. A normal inflammatory response involves signals that initiate, maintain
and terminate this process. An imbalance leaves inflammation unchecked, resulting in cellular
and tissue damage. In inflammation, macrophages have three major functions, namely antigen
presentation, phagocytosis and immunomodulation via production of cytokines and growth
factors. Therefore, macrophages play a critical role in the initiation, maintenance, and
resolution of inflammation. Different cytokines can influence this inflammatory process by
activating and deactivating macrophages.
Monocyte chemoattractant protein 1 (MCP-1) belongs to a family of small cytokines called
chemokines. These play a major role in selectively recruiting monocytes, neutrophils and
lymphocytes, and in inducing chemotaxis. MCP-1 is a key chemokine that regulates migration
and infiltration of macrophages; it is not only expressed by macrophages but also by
endothelial, mesangial and epithelial cells (83). By eliciting and activating leukocytes, this
regulatory mediator plays an important role in the inflammatory cascade and, perhaps, the
pathogenesis of sepsis. P2X7R has a central role in macrophage IL-1β secretion via the
NLRP3 inflammasome (82). In an experimental model of glomerulonephritis a link was found
between P2X7, MCP-1 and macrophage infiltration (82). Moreover, MCP-1 was shown to be
important in glomerular recruitment of macrophages and crescent formation in NTN (84, 85);
it has been studied as a biomarker for the mononuclear inflammatory processes that occur
following ischaemia-induced AKI (86). Aim of study
My fellowship is aimed at researching the expression of renal P2X7R and any relationship
with the course and pathogenesis of septic AKI. I aimed to define (an early) role for the
P2X7R in a fluid-resuscitated three-day rat model of faecal peritonitis and, specifically, to
define its relationship and co-localization to histopathological changes seen in septic AKI. I
hypothesized that upregulation of the ATP-sensitive P2X7R causes renal injury in sepsis
through local production of cytokines and chemokines and mediation of cell death.
Despite minimal histological damage (87), survivors of acute kidney injury who require acute
renal replacement therapy have an up to 28-fold increased risk of developing stage 4 or 5
chronic kidney disease (CKD) (88, 89). Most of the focus of AKI has been on the acute injury
phase but little attention to date has been paid to recovery. Mechanisms underlying resolution
of AKI in sepsis are poorly understood and may be potentially injurious. Therefore, I also
aimed to describe temporal changes in renal inflammation and recovery following sepsis and
to establish the involvement of renal macrophages and cytokines in a long-term rat model of
zymosan-induced peritonitis.
13
Materials & Methods
For buffers and solutions, see Appendices I
1. In vivo methods
Rat model of acute –polymicrobial- sepsis
Male Wistar rats weighing 300-350g were used. All invasive procedures and imaging
techniques were performed under isoflurane general anaesthesia. Twenty-four hours
following insertion of a tunnelled central venous catheter, sepsis was induced by
intraperitoneal injection of faecal slurry. Sham-operated animals had lines inserted but no
intraperitoneal injection. All animals were inspected four times daily and given a clinical
score based on their physical appearance (90). Fluid resuscitation with crystalloid was
initiated at 10 ml/kg/hr 2 hours following induction of sepsis. Sham animals received a similar
fluid regimen. At either 6 or 24 hours, animals were anaesthetized and an echocardiography
and renal ultrasound scan performed, followed by arterial blood gas analysis.
Subsequently, a laparotomy was performed. The urinary bladder was directly punctured with
a 22 gauge needle to drain and measure urine output. The left kidney was then resected. The
upper pole of the kidney was placed in 20% formalin and the remaining kidney was snap-
frozen into liquid nitrogen (LN2). A thoracotomy was performed followed by cardiac puncture
to obtain blood. This was centrifuged at 6500 rpm for 10 minutes, with subsequent snap
freezing in LN2. Sections of liver, spleen, lung, heart and aorta were collected and stored in
formalin and the remaining segments of these organs were snap-frozen in LN2. There were six
animals for each of the 6 hour timepoint groups and 8 animals for each of the 24 hour
timepoint groups. Each timepoint had a sham-operated and sepsis group. Separately, samples
were taken from non-instrumented naïve animals under anaesthesia.
Rat model of sepsis and recovery
Male Wistar rats weighing 300-350g were used. Rats were randomly divided into two groups:
sham or sepsis with timepoints at days 2 and 14. Animals in the septic group received
intraperitoneal zymosan* while sham animals received intraperitoneal saline. To assess their
clinical course, all animals were inspected daily and given a clinical score based on their
physical appearance. Body weight and food intake were also measured daily. Animals were
culled at days 2 or 14 post-zymosan injection and the left kidney harvested within 2 minutes
of the animal being culled. Sections of liver, spleen, lung, heart and aorta were collected and
stored in formalin and the remaining tissues were snap-frozen in LN2.
* Zymosan is a yeast-derived compound used to induce sterile inflammation. An advantage of
the zymosan-induced peritonitis (ZIP) model is that it generates a more prolonged
inflammatory insult than that seen in bacterial models. By adjusting the dose of zymosan,
insult severity can be varied. Clinical recovery in zymosan-treated animals commences from
48-72 hours, mimicking that seen in many immunocompetent individuals.
2. Homogenization of Frozen Kidney Tissues
Snap-frozen kidney sections (approx 2x3x2 mm) were put into a sterilin container containing
2 ml ice-cold radioimmunoprecipitation assay (RIPA) buffer. Tissue was homogenized on ice
using an electric homogenizer. Blades were rinsed in between with dH2O, 70% IMS and
14
dH2O, respectively. Following thorough lysis, the homogenate was centrifuged for 5 minutes
at 16000g at 4°C to remove tissue debris. The supernatant was aliquotted and stored at -80°C.
3. Bicinchoninic acid (BCA) protein estimation assay
Following homogenization of the frozen kidney tissue, protein concentration was estimated
using a BCA Protein Assay kit (Novagen). A standard curve was prepared by diluting bovine
serum albumin (2 mg/ml) to achieve final concentrations of 2, 1.5, 1, 0.5, 0.25, 0.125 and
0.025 mg/ml using dH2O as a diluent. Tissue homogenate was diluted 1:10 in dH2O. To
prepare the BCA assay working reagent, 50 parts of reagent A were combined with 1 part of
reagent B. A blank 96-well plate was used and 25 μl of the standards or diluted samples were
added in duplicates. Two hundred μL of BCA working reagent were added to each well, and
placed on a shaker for 30 seconds. The plate was sealed and incubated at 37°C for 30 minutes.
Afterwards, the plate was left to cool to room temperature and the absorbance at 562 nm was
determined using a spectrophotometer (Synergy 2, Biotek). The recorded absorbance was
corrected for the dilution and the protein concentrations of the samples were extrapolated
from the standard curve.
4. Western blotting
Homogenization was performed as described previously, and protein concentrations estimated
using the BCA Protein Assay kit (Novagen). Kidney tissue homogenate was corrected for an
equal amount of 4 μg/μL protein per sample. SDS-sample buffer was added in a 1:1 ratio. To
reduce and denature the proteins, the cell lysate in sample buffer was heated at 90°C for 5
minutes, then quickly centrifuged at 16000g in a microcentrifuge for 1 minute and kept on ice
before use.
Equal amounts of protein were electrophoresed on 12% SDS-PAGE Tris-glycine minigels in
an electrode tank containing running buffer at a current of 80-120 Volt (for 2 gels: 1-1.5
hours). Subsequently, the gels were transferred onto pre-treated polyvinylidene difluoride
PVDF membrane (GE Healthcare). Semi-dry electroblotting was performed on a Bio-Rad
Trans-blot SD semi-dry transfer cell where the electrode plates are in direct contact with filter
paper as buffer reservoirs. Filter paper and membranes were trimmed to match the dimensions
of the gel and equilibrated in transfer buffer. The blotting assembly from anode to cathode
contained a filter paper sheet, the gel, PVDF membrane and a second filter paper. The
electroblotting was performed at a constant current of 15V for 45 minutes. Non-specific
antibody binding was minimized by blocking the membrane in TBST buffer (TBS with 0.1%
Tween 20) supplemented with 5% non-fat dry milk for 1 hour at room temperature. The
membranes were then incubated with the primary antibodies overnight at 4°C with gentle
agitation. The following antibodies were used; IL-18 (rabbit polyclonal, Santa Cruz) diluted
1:1000 in 5% milk/TBST and Caspase-1 (rabbit polyclonal, Santa Cruz), diluted 1:1000 in 5%
BSA in TBST. Membranes were then incubated with appropriate secondary antibodies
(Polyclonal Rabbit Anti-Goat IgG/HRP, DAKO or anti-rabbit IgG peroxidise labelled
polymer, DAKO), respectively, both diluted 1:3000 in 5% milk/TBST. Afterwards, the
membranes were washed three times for 8-10 minutes. Bands were visualized by enhanced
chemiluminescence (ECL) detection kit (GE-Amersham Biosciences). X-ray films were
manually developed at several incubation timepoints.
15
5. Immunohistochemistry
To assess the amount and localization of P2X7, slides were stained with rabbit anti-rat P2X7
(Alamone). Signal development was performed using an anti-rabbit Envision kit (Dako
EnVision+ HRP-systems, DAB). Rat anti-mouse macrophage CD68 (AbD Serotec Inc.) was
used to assess the amount and localization of macrophages, visualization of these antibody
was obtained using an anti-mouse kit (Dako EnVision+ HRP-systems, DAB). Periodic acid-
Schiff (PAS) and Hematoxylin and eosin (H&E) staining are not described as they were
processed through the lab at Imperial College London, South Kensington.
Rat renal and spleen tissue samples were harvested and kept in 20% formalin prior to fixation.
After 24 hours, tissue was rinsed in 70% ethanol and embedded in paraffin wax. Tissue
sections, 5μm thick, were cut with a microtome and mounted onto coated glass slides.
Tissue sections were deparaffinized in xylene and rehydrated in decreasing concentrations of
ethanol to water. The slides were then heated in 0.01M citrate buffer [pH 6.0] at 90°C to
retrieve antigen exposure and allowed to cool for 5 minutes. Slides were washed three times
for 5 minutes each with excess PBS. In case of P2X7 staining, PBST (0.05% Tween 20 in
PBS) instead of PBS was used for all washes to reduce the amount of background staining.
To quench any endogenous peroxidase activity, sections were blocked with 3% H2O2 for 10-
30 minutes, followed by 3x5 min washes with excess PBS(T). Non-specific binding was
blocked by incubation with 10% milk in PBS (for P2X7) or 20% normal goat serum (NGS)
diluted in PBS (for CD68). Subsequently, the sections were incubated with the following
primary antibodies; P2X7 (rabbit anti-rat, 1:100, 004 Alamone) diluted in 1% BSA/0.05M
Tris-HCL [pH 7.2] or CD68 (mouse anti-rat, 1:500, AbD Serotec) diluted in PBS. The slides
were incubated for 1 hour at room temperature. One section from each sample was incubated
with antibody diluent instead of primary antibody and served as control. After washing, the
slides were incubated with the appropriate peroxidase-labelled polymer for 30 minutes at
room temperature. For P2X7 this was biotinylated goat anti-rabbit immunoglobulins in Tris-
HCL buffer (Dako), and for CD68 biotinylated goat anti-mouse immunoglobulins in Tris-
HCL buffer (Dako). After washing in PBS(T) to remove excessive unbound antibody, nickel
intensified 3-3′-diaminbenzidine (DAB, Dako) was used reveal antibody binding. Sections
were counterstained with Harris‘s haematoxylin solution, then dehydrated and mounted with
Eukitt (VWR International) and left to dry overnight.
The slides were examined with a Zeiss Axioplan light microscope (Carl Zeiss International).
Images were taken with a Leica DC200 digital camera (Leica Microsystems). Quantification
of staining was assessed by using Image Pro Plus software (Media Cybernetics) and each
slide was given an arbitrary score based on this method. For each section, 10 random fields of
view (x20 magnification) within the outer cortex were taken and the mean intensity of
staining was calculated.
6. Enzyme-linked immunosorbent assays (ELISAs)
IL-1β and MCP-1 estimations were performed to assess the presence of rat IL-1β and MCP-1
in renal tissue homogenate and serum. For the IL-1β and MCP-1 Sandwich ELISAs, rat IL-1β
DuoSet ELISA kit (R&D Systems) and rat MCP-1 ELISA set (BD Biosciences) were used.
16
A 96 blank plate was coated with 100 μL of capture antibody, sealed and left to incubate
overnight. The following capture antibodies were used; goat anti-rat IL-1β (1:180 in PBS,
incubation at RT) and goat anti-rat MCP-1 (1:250 diluted in 0.1M Sodium Carbonate [pH
9.5], incubation at 4°C). Each well was aspirated and washed with wash buffer for three times
each. The plate was blocked with the appropriate assay diluent. For IL-1β this was 1% BSA in
PBS [pH 7.2-7.4], 300 μL/well. For MCP-1 this was 10% FBS in PBS, 200 μL/well. Both
plates were incubated for 1 hour at room temperature. After washing of the wells, again for 3
times each, 100 μL of the standards or samples were added in duplicates and incubated for 2
hours at room temperature, followed by 5x aspirating and washing of the wells.
For IL-1β, the method of antibody detection occurred in two subsequent steps:
(1) 100 μL of detection antibody (biotinylated goat anti-rat IL-1β, 1:180 in assay diluent) was
added to each well; the plate was then sealed and incubated for 2 hours at room temperature
(2) After washing (5x), 100μL of working dilution of Streptavidin-horseradish peroxidase
conjugate (SAv-HRP, 1:200 in assay diluent) was added to each well with an incubation
period of 20 minutes at room temperature.
For MCP-1, working detector was made containing biotinylated anti-rat MCP-1 (1:500
diluted in assay diluent) supplemented with SAv-HRP (1:250). One hundred μL of this
solution was added to each well with an incubation period of 1 hour at room temperature.
Afterwards, both plates were washed 7 times with soaking of the wells in wash buffer for 1
minute per wash. One hundred μL of substrate solution (1:1 mixture of Colour Reagent A
(H2O2) and Colour Reagent B (Tetramethylbenzidine) was added and incubated for 20
minutes in the dark at room temperature. Fifty μL of stop Solution (2N H2SO4) was added to
each well and absorbance was read at 450 nm using a spectrophotometric ELISA plate reader.
Using BCA protein estimation, kidney tissue homogenate was corrected for an equal amount
of protein per sample. Samples that were out of range were diluted in a 1:2-1:100 manner with
re-estimation of the protein concentration.
7. Statistics
All statistical analyses were performed using SPSS (IBM. Version 20) and graphs were drawn
using Graphpad Prism (GraphPad Software, Version 5.0d). Normality of continuous data was
assessed using the Shapiro-Wilk test. Continuous variables are presented as means (standard
deviation). Parametric data were compared using unpaired t-test, whereas non-parametric data
were compared using the Mann Whitney U test. One-way ANOVA (analysis of variance) was
used to assess difference between more than two groups of continuous variables. Post-hoc
Tukey’s test was performed to ascertain differences between individual groups. Pearson’s
correlation was used to assess linear association between two groups of continuous variables.
A p value <0.05 was taken as statistically significant.
17
Results
I. Polymicrobial model of acute sepsis
1. Analysis of renal dysfunction by use of serum creatinine
Serum creatinine levels were analyzed as a measure of kidney dysfunction. At 6 hours, sham
and septic animals demonstrated similar, non-significantly raised, serum creatinine levels
(26±3 μmol/L sham vs 24±3 μmol/L septic; p = 0.399). In sepsis, a significant rise in serum
creatinine was seen at 24 hours (24±3 sham vs 30±5 μmol/L septic; p = 0.012) (Figure 1).
Serum creatinine levels in naives were within the normal range (20-25 μmol/L).
Figure 1. Creatinine
values expressed in
μmol/L; significantly
increased serum
creatinine levels were
seen in septic rats at 24 hours compared
to controls (sham) (indicated by asterisk,
p <0.05). Serum creatinine in naive and
sham-operated rats, are within normal
ranges (20-25 μmol/L). Data represent
mean±SD. N= 6 for naive and 6 hour
groups (sham and septic), and n=8 for
both 24 hour groups. sCR: serum
creatinine.
2. Immunohistochemistry
2a. Assessment of histological damage
To assess histopathological damage in kidneys during sepsis, I examined the Periodic acid–
Schiff (PAS) stained kidney sections. Acute renal histological injury was evaluated by a pre-
defined scoring system (82). This quantifies for tubular cell necrosis, loss of luminal brush
border, tubular cast formation and tubular dilatation by ascribing the following scales: mild-
moderate-severe. For each group, i.e. naive, sham (6 and 24 hr) and sepsis (6 and 24 hr), a
minimum of 6 slides were scored. Perimortem animals were excluded from analysis as such
animals had significant global haemodynamic compromise that may affect the histological
appearances.
No significant difference in overall acute renal histological injury was seen between sham and
septic kidneys at either 6 or 24 hours (figure 2). Both sham and septic animals had contained
areas with minimal dilated tubules and minimal loss of brush border. There was no evident
presence of necrosis or cast formation throughout the kidney. Looking at the time of sepsis,
there were no major histological changes at either 6 or 24 hours after initiation of sepsis.
Despite clinical, biochemical, and renal haemodynamic changes of septic AKI, histology
remains relatively preserved.
Creatinine (umol/L)
Naive
6hr s
ham
6hr s
epsis
24hr
sha
m
24hr
sep
sis
0
10
20
30
40
6 hrs 24 hrsNaive
*
Naive
Sham
Sepsis
18
Figure 2: Histological assessment of rat kidneys for renal damage. A. Naive renal tissue
without any significant damage. B. Renal tissue obtained from a haemorrhage-reperfusion
model (A. Dyson, UCL), showing several characteristics of ATN such as dilated tubules,
ischaemic glomeruli and tubular casts. C. Kidney from a sham animal without any significant
damage. D. Kidney section from a 24 hour septic rat showing a similar pattern (no significant
damage) compared to shams. (PAS-staining, magnification x 20)
2b. P2X7 expression in kidney tissue
I then assessed P2X7 expression to see whether P2X7 correlates with severe sepsis at either 6
or 24 hour timepoints. In septic animals, P2X7-dense areas were seen in a patchy, discreet
manner with relatively more proximal than distal tubular P2X7 staining. As shown in picture
3A there was minimal P2X7 expression in naive kidneys. Picture 3b shows the minimal P2X7
expression seen in sham animals, and mainly located within the capillary loops of the
glomeruli and at the basolateral regions of the tubules. P2X7 expression was evident at both
timepoints in sham and septic animals (Figures 3B and 3C, illustrating P2X7 expression in 24
hour sham and septic animals respectively). In severely septic animals (with a serum
creatinine elevated at least three-fold compared to sham animals), the pattern of P2X7 staining
differed with evidence of intraluminal debris that stained intensely for P2X7 (3D). This pattern
of tubular P2X7 is suggestive of a role in cell death. No P2X7 staining was observed in naive,
sham or septic renal tissue when the primary antibody was replaced with non-immune serum.
For each group (naive, sham and sepsis) and timepoints (6 and 24 hours), ≥6 animals were
scored. Scoring was based on the area and intensity of staining as described previously.
Briefly, quantification of staining was assessed by using Image Pro Plus software, and each
slide was given an arbitrary score based on this method. For each section, 10 random fields of
view (x20 magnification) within the outer cortex were taken and the mean intensity of
staining was calculated.
19
Figure 3; Histological assessment of rat kidney for P2X7. A: Naive kidney with minimal
P2X7; B: Kidney from a 24hour sham animal with minimal P2X7 mainly within the capillary
loops of the glomeruli and at the basolateral regions of tubules; C: Kidney from a 24 hour
septic animal showing a similar pattern as the 24 hour shams; D: Kidney from a severe septic
animal at 24 hours with overwhelming intraluminal, intraluminal debris, with intense staining
for P2X7. (P2X7-staining, magnification x 20)
P2X7 was visible in both tubules and glomeruli of naïve kidney tissue (Figure 3). At 6 hours,
there was a wide variation in the level of P2X7 expression. There was no significant difference
in the expression of renal P2X7 at 6 hours between sham and septic rats (0.011 vs 0.013, p =
0.31). At 24 hours, there was a significant difference in P2X7 in sham compared to septic rats
(0.0067 vs. 0.0138, p= 0.0025) (Figure 4).
Figure 4. Semi-quantitative analysis
of P2X7; a significant increase in
P2X7 was seen in septic rats at 24
hours compared to controls
(indicated by asterisk, p <0.05). Data
represent mean±SD. N= 6 for naive
and 6 hour groups (sham and septic),
and n=8 for both 24 hour groups.
P2X7 score
Naive
6hr S
ham
6hr S
epsis
24hr
Sha
m
24hr
Sep
sis
0.000
0.005
0.010
0.015
0.020
0.025
His
tolo
gy s
co
re
*
Naive 6 Hrs 24 Hrs
Naive
Sham
Sepsis
20
2c. Detection of macrophages
P2X7 is predominantly expressed by macrophages. Macrophages are a major component of
the mononuclear phagocyte system and play a critical role in the initiation, maintenance, and
resolution of inflammation. Therefore, I assessed the level of macrophage expression
throughout the kidney to seek any correlation between macrophage expression and sepsis-
induced AKI. Furthermore, I wanted to determine the relative contribution of infiltrating
macrophages to renal P2X7 staining. The presence of macrophages was sought using CD68
staining as described. At least six animals were included in each group.
Naive kidneys showed no or minimal staining of macrophages. When present, macrophages
were found within the interstitium. A similar pattern was seen in sham and septic animals at 6
and 24 hours (Figure 5). The minimal presence of macrophages in the kidney tissue and the
pattern of staining of P2X7 suggest P2X7 is likely to be expressed by intrinsic renal cells.
Figure 5; Histological assessment of rat kidneys for presence of macrophages. A: Naive
kidney with no visible macrophage staining; B: Kidney from a 24 septic animal with minimal
staining for macrophages, as indicated by the arrow (CD68-staining, magnification x 20).
3. Enzyme-linked immunosorbent assays (ELISA)
3a. Expression of serum and renal IL-1β
Interleukin-1β (IL-1β) is a key initiator in acute inflammation and plays a pivotal role in the
pathogenesis of sepsis (35, 36). A pro-inflammatory response progressing to a “cytokine
storm” causes inflammatory cytokines such as IL-1β to be released from macrophages (54,
70, 73). IL-1β then binds to receptor target cells, eliciting a signalling cascade that enhances
the inflammatory response which may, in turn, lead to cell death (91). The P2X7 subtype is
identified as the receptor responsible for ATP-driven maturation and release of IL-β.
Therefore I sought to evaluate the role of IL-1β in this model of septic AKI. Serum and renal
tissue IL-1β levels were quantified by ELISA. Experiments were repeated in duplicate using
six animals for naïve studies and 6 hour timepoints, and 8 animals for the 24 hour timepoints.
Serum IL-1β is expressed as pg/mL and renal IL-1β as a ratio of μM/μg of protein.
There was no detectable serum IL-1β in naive animals, with minimal to none in sham animals
at both 6 and 24 hour timepoints (Figure 6A). There was a significant rise in serum IL-1β in
septic compared to sham animals at both 6 and 24 hour timepoints (Table 1). There was a
non-significant fall in serum IL-1β levels from 6 to 24 hrs in septic animals (p = 0.206). For
21
renal IL-1β levels, a significant rise was seen in the septic animals at both 6 and 24 hours
(Figure 6b). In contrast to serum levels, renal IL-1β revealed a consistent rise in sepsis from 6
to 24 hours (p=0.0009).
Figure 6; Serum and renal expression of the pro-inflammatory cytokine IL-1β. Left-hand
graph shows a significant increase in serum IL-1β in the septic group at both 6 and 24 hours
(p<0.05), with a non-significant fall at 24 hours. Right-hand panel: renal IL-1β protein ratio
increases in septic animals at 6 hours and further at 24 hours (p<0.05). Significance is
indicated by the asterisk. Data represent mean ± SD. Six rats were used for naive and 6 hour
groups (sham and septic), and 8 for both 24 hour groups.
3b. Expression of serum and renal MCP-1
In view of the lack of renal macrophage infiltration, I measured renal MCP-1 levels. Renal
MCP-1 levels were not elevated at 6 hours (97±23 (sham) vs 125±37 (septic) vs 88±13 pg/μL
(naive); p = 0.3359). (Figure 7). At 24 hours, the difference in renal MCP-1 levels in septic
compared to sham animals neared significance (178±78 vs 92±41 pg/μL; p = 0.055).
Figure 7. Renal MCP-1 levels.
These were not elevated in sham or septic
animals at 6 hours. At 24 hours, a near
significant increase was seen in septic
animals compared to controls (p=0.0553).
Data represent mean ± SD. Six rats were used
naive and 6 hour groups (sham and septic),
and eight for both 24 hour groups.
Renal IL-1beta (uMol/ug Protein)
Naive
6hr s
ham
6hr s
epsis
24hr
sha
m
24hr
sep
sis
0
100
200
300
400
500
6hrs 24hrsNaive
*
*
Serum IL-1beta (pg/mL)
Naive
6hr s
ham
6hr s
epsis
24hr
sha
m
24hr
sep
sis
0
500
1000
6hrs 24hrsNaive
*
*
Renal MCP-1 (pg/uL)
Naive
6hr s
ham
6hr s
epsis
24hr
sha
m
24hr
sep
sis
0
50
100
150
200
250
Naive
Sham
Sepsis
Naive
Sham
Sepsis
22
Table 1. Overview of cytokines and creatinine measured in this model of acute sepsis.
Significant differences are indicated by an asterisk.
4. Western Blotting
Formation of the NLRP3 inflammasome converts pro- to active caspase-1. Inactive caspase-1
(Pro-C1) has a weight of 45 kDa in rats, whereas the active fragment of caspase-1 (p10)
weights 10 kDa. Caspase-1, or interleukin-1β converting enzyme (ICE), then promotes
maturation of interleukin IL-1β and IL-18 by proteolytic cleavage of precursor forms into
biologically active pro-inflammatory cytokines. I determined the relative abundance of
precursors and active forms of caspase-1 and IL-18 in kidney tissue.
Similar quantity of total protein was loaded into each well (20 μg). Ten μL of pre-stained
protein ladder, Broad Range (10-230 kDa) (New England Biolabs) was added once and
served as a reference indicator. This ladder consists of 12 bands, namely; 10, 15, 20, 25, 30,
40, 50, 60, 80, 100, 150, 230.
Immunoblotting of renal tissue homogenate revealed a major band at 45 kDa, the predicted
size of pro-caspase-1 in all groups (Fig 8). There was no visible difference in the presence of
the precursor caspase-1 between naïve, 24 hours sham and septic animals. For active caspase-
1, with a predicted weight of 10kDa, no band was detected in naive renal tissue. Compared to
naives, there was a slight increase in active caspase-1 in septic compared to sham animals.
Figure 8. Western blot analysis of kidney homogenate obtained from naive, control and septic
rats. Migration of the 45 kDa active caspase-1 and the 10 kDa pro-caspase-1 molecular forms
are indicated (N=3 for naives and N=4 for sham and septic rats).
Naive
6 hours 24 hours
Sham Septic P Sham Septic P
Renal
IL-1β
0 (0-0) 22 ± 55 834 ± 451 0.005* 0 (0-0) 544 ± 534 <0.001*
Serum
IL-1β
20 ± 20 36 ± 21 117 ± 22 0.004* 92 ± 62 360 ± 101 0.002*
Renal
MCP-1
88 ± 13 97 ± 23 125 ± 37 0.339 92 ± 41 178 ± 78 0.055
Creatinine 23 ± 3 26 ± 3 24 ± 3 0.399 24 ± 3 30 ± 5 0.012*
23
Immunoblotting of kidney homogenate for IL-18 was performed in naïve rats and 24 hour
sham and septic rats. Minimal levels of pro-IL-18 were detected in renal tissue, with a relative
increase of pro-IL-18 in septic compared to sham animals (Fig 9). Active IL-18 protein was
detected at very low levels in all groups with no visible difference between groups.
Figure 9. Western blot analysis of
kidney homogenate obtained from
naive, control and septic rats.
Migration of the 24 kDa pro-IL-18 and
the 18 kDa active IL-18 molecular
forms are indicated (N=3 for naïve rats
and N=4 for sham and septic rats).
II. Recovery model of sepsis: Results
1. Immunohistochemistry
Periodic acid–Schiff (PAS)-stained kidney sections were examined for histopathological
damage at days 2 and 14 in sham and septic animals. Acute renal injury was evaluated by a
pre-defined scoring system as described earlier. For each group, at least 4 slides were scored.
Minimal, if any, evidence of renal injury was seen in sham and septic kidneys at Days 2 or 14.
To evaluate the role of macrophages in this recovery model, I examined kidney sections for
CD68 in 4 sham and 4 septic at Day 2, and 4 sham and 8 septic kidneys at Day 14.
Macrophage infiltration was evident in glomeruli and interstitium in all Day 14 septic animals
but in no sham animal (Fig 10). At Day 2, 1 septic animal had renal macrophage infiltration
and 1 sham animal had minimal glomerular macrophage infiltration (Fig 11).
Figure 10; Histological assessment of rat kidney for presence of macrophages. On the left:
No presence of macrophages in sham animals. On the right: Evident staining for macrophages
in the glomeruli and interstitium (CD68-staining, magnification x 20).
24
Figure 11; Kidneys showing macrophage
infiltration.
A significant difference was seen in septic
animals at day 14 (p=<0.05). N= 4 in all
groups except for sepsis day 14, N=8.
2. Enzyme-linked immunosorbent assays (ELISA)
At day 2 similar renal MCP-1 levels were seen in sham and septic animals (72±15 vs 78±14
pg/μL, p=0.875). By day 14, renal MCP-1 levels were significantly elevated in septic
compared to sham animals (187±55 vs. 86±18 pg/μL, p=0.009). A significant elevation in
renal MCP-1 was seen at day 14 in septic animals compared to sham animals. This coincides
with increased macrophage infiltration and clinical recovery seen at day 14.
Figure 12; Renal MCP-1 levels in long-
term model.
A significant increase was seen in septic
animals at day 14 as indicated by asterisk
(p<0.05). Data represent mean ± SD.
N= 4 in all groups except for sepsis day 14;
N=8.
Renal MCP-1 (pg/uL)
MC
P-1
(p
g/u
L)
Sham
Day
2
Sepsis
Day
2
Sham
day
14
Sepsis
day
14
0
50
100
150
200
250*
Day 2 Day 14
Clinical Score
6hrs
24hr
s
0
1
2
3
4Sham
Sepsis* *
25
Discussion
Despite clinical, haemodynamic (data not shown) and biochemical alterations, no abundant
characteristic pathological features were found on renal histology. This finding is similar to
that predominantly reported in the literature (92). There is still no defined consensus on a
scoring system for histopathological damages seen in AKI. This is partly due to a wide
variation in histological features (varying from no damage, to minimal loss of brush border
and a few dilated tubules, to severe acute tubular necrosis) but also as a result of a poor
correlation between histopathological findings and in vivo functional impairment. To
determine AKI severity, I used a scoring system that grossly divides animals into mild-
moderate and severe groups. I received confirmation of a lack in difference from a blinded
histopathologist (Dr. Paul Bass, UCL). Despite minimal, if any histological damage, my
results revealed overall levels of increased P2X7 expression in this model of AKI.
I found increased P2X7 expression on both glomeruli and tubules, and showed a pattern in
P2X7 expression suggestive of a role in cell death. The physiological function of the P2X7R is
still under investigation but a number of roles have been proposed. Expression in shedding
epithelia such as skin (93), duodenum (94), vagina and uterus (95) suggest it may have a role
in normal cell turnover. There are several reports of P2X7R activation causing either necrosis
or apoptosis of cells of haemopoietic origin such as macrophages and lymphocytes (43, 77,
96). Furthermore, the ability of extracellular ATP to trigger apoptosis via the P2X7R has been
reported in other cell types including thymocytes (97), dendritic cells (98) and mesangial cells
(80). In HEK-293 cells, dramatic membrane blebbing and micro-vesciculation was observed
within seconds to minutes of receptor activation, a phenomenon in which large membrane-
bound vesicles protrude rapidly from the cell surface, associated with cells undergoing
apoptosis (96). Normally, apoptosis is a tightly regulated mechanism for maintaining normal
and healthy cell numbers; in kidney it occurs at a low level (99). However, apoptosis
increases following several forms of glomerular injury including ischaemia (100).
Besides P2X7, I also assessed the role of other components of the NLRP3 inflammasome
pathway including caspase-1, IL-1β and IL-18. Western blotting revealed a slight increase in
active caspase-1 and pro IL-18, but this needs re-evaluation with inclusion of a positive
control (spleen), and quantified in relation to a housekeeping protein’ (β-actin). Furthermore,
differences between bands should be formally assessed using relative densitometry.
As expected, serum IL-1β levels showed a clear rise during the acute phase of sepsis (35, 36).
Moreover, renal IL-1β showed a further rise at 24 hours compared to 6 hours in this model of
septic AKI. Glomerular mesangial cells and podocytes are capable of producing IL-1β (101,
102); this cytokine is considered largely responsible for leukocyte infiltration in anti-GBM
glomerulonephritis (103). IL-1β binds to receptor target cells and elicits a signalling cascade
that enhances the inflammatory response leading to cell death (91). Target cells include
endothelial cells which, when exposed to IL-1β, are induced to secrete chemokines such as
monocyte chemotactic peptide-1 (MCP-1) (104).
MCP-1 is not only expressed by macrophages but also by endothelial cells, mesangial cells
and epithelial cells (83). Macrophages play a critical role in the initiation, maintenance, and
resolution of inflammation. Therefore, they mediate both the pro and anti-inflammatory
cascades. MCP-1 is a key chemokine that regulates migration and infiltration of macrophages.
As it is produced by, and acts on macrophages, it has a dual role in eliciting and activating
26
leukocytes. Chemokines play a prominent role in the acute inflammatory response in several
models of kidney disease, and a specific role has been revealed for MCP-1.
MCP-1 provides a stimulus for chemotaxis in glomerulonephritis by facilitating glomerular
recruitment of macrophages and crescent formation in both rats and mice (85, 105, 106). In a
rat model of ischaemia-induced AKI, increased levels of kidney MCP-1 mRNA, protein and
an increase in urinary MCP-1 excretion were reported (86). In addition, a correlation was seen
between areas of increased MCP-1 expression and infiltration of mononuclear cells into the
kidney. In cultured astrocytes P2X7R activation increased MCP-1 expression via a MAP
kinase-dependent mechanism which includes P38-MAP kinase. This pathway plays a role in
the cascade of programmed cell death (107) and may provide a link with the apoptotic
function of the P2X7. In an experimental glomerulonephritis model a link was seen between
P2X7, MCP-1 and macrophage infiltration. P2X7 knockout mice showed a 96% reduction in
urine MCP-1 levels correlating with a reduction of macrophage infiltration into the glomeruli,
but not the interstitium. Treatment with A-438079, a selective P2X7 antagonist, caused a
significant reduction in macrophage infiltration in renal tissue, and of urinary MCP-1 levels.
Although renal MCP-1 will not differentiate between different causes of AKI, it may be a
biomarker for differentiation between active inflammation and resolution. The correlation
between renal MCP-1 levels and the amount of macrophage infiltration in renal injury, as
described in the literature, corresponds with findings in our long-term model of sepsis and
recovery. However, in other models of renal injury, both MCP-1 and macrophages were
detected at early stages. At the very start of clinical recovery (day 2 in our long term model), a
minimal presence of macrophages was seen with correspondingly low basal levels of renal
MCP-1. Similarly, in our model of sepsis, no infiltration of macrophages was seen at either 6
or 24 hour timepoints. This may suggest that in sepsis, renal macrophages and MCP-1 are
likely to play more of a role in (late) resolution of inflammation rather than in the acute phase.
Activated macrophages can be classified into M1 and M2 phenotypes (108, 109). The M1
macrophages, or classically-activated macrophages, are immune effector cells with an acute
inflammatory phenotype. They are highly aggressive against microbes and can produce large
amounts of cytokines. The M2 macrophages, or alternatively-activated macrophages, possess
anti-inflammatory properties and can be divided into at least three subgroups. These subtypes
have various functions including regulation of immunity, maintenance of tolerance and tissue
repair. I propose that the upregulation of macrophages seen at day 14 in our model of
recovery of sepsis belongs to the M2 subtype. The exact pathophysiological role for
macrophages and MCP-1 at different timepoints in this rat model of sepsis remains to be
determined. Whether there is initial MCP-1 expression in the kidney that attracts
macrophages, or whether macrophages mainly induce expression of renal MCP-1 needs to be
elucidated. Most likely they are synergic partners that both play an important role in the
pathogenesis and recovery of sepsis-induced renal injury.
27
Conclusions
A significant increase in renal P2X7 expression (in both glomeruli and tubuli) was seen in
septic animals after 24 hours, correlating with elevated levels of IL-1β. Consistent with these
findings, septic AKI was associated with increased renal expression of caspase-1 protein and
pro-IL-18. In peri-mortem animals, there was evidence of intraluminal cellular debris, which
stained intensely for P2X7. This pattern of tubular P2X7 suggests a possible role in cell death.
Despite upregulation of P2X7, there was absence of macrophage infiltration. This, in
combination with the pattern of staining of P2X7, suggests renal P2X7 is likely to be
expressed by intrinsic kidney cells. While significant histological damage was not seen,
reflecting findings in septic patients, there was biochemical evidence of renal dysfunction.
Further research is needed to evaluate whether P2X7 antagonism offers protection.
By contrast, the longer-term sepsis model that also incorporated a recovery phase did shown
significant renal macrophage infiltration within the glomeruli and interstitium with a
corresponding increase in renal MCP-1 at day 14. Whether this contributes to long-term renal
damage requires further study.
Future work
P2X7 antagonism
An important reason for looking at the P2X7 in septic AKI is its possible role in inflammation,
particularly as the P2X7R is considered a possible therapeutic target in inflammation.
Antagonists are currently in Phase II clinical trials. Therefore, our next step would be to
evaluate the effects of P2X7 antagonism on renal function and survival.
Define renal histological features in sepsis in detail and attempt to score injury
Because there were clear differences not only in the amount but also in the pattern and areas
of P2X7 expression, I have attempted to define a scoring system to differentiate between the
functional areas within the kidney and the pattern of expression. This scoring is based on 1:
the location of staining, which was subdivided into (i) glomerular, tubular, or interstitial, (ii)
cortical or medullary (iii) cytoplasmic, basolateral or apical expression (in tubules) 2: the
pattern of staining (discrete or diffuse) and 3: the amount of staining. We are awaiting
validation of this scoring system by a certified histopathologist. If possible, this could also be
used to score immunohistochemistry on renal tissue for NLRP3, macrophages and α-SMA.
The inflammasome and the role of other immunological mediators
Although different components of the inflammasome pathway have been assessed, these
results should be correlated against upregulation and/or presence of the NLRP3
inflammasome itself. We are currently performing immunohistochemistry for NLRP3. As
discussed, P2X7 is associated with cell apoptosis, so we would like to further investigate this
association with immunohistochemistry using Terminal deoxynucleotidyl transferase dUTP
nick end labelling (TUNEL)-staining. Furthermore, we propose to further investigate the role
of other immune cells in septic AKI.
28
Temporal changes in renal biomarkers
As previously discussed, creatinine is a poor biomarker in early septic AKI. Although several
other biomarkers have been proposed (including neutrophil gelatinase-associated lipocalin
[NGAL], kidney injury molecule-1 [KIM-1], Cystatin C and IL-18), none have convincingly
proven reliability (110). There is a need for (new) biomarkers of renal cell injury that may
identify patients with AKI at an earlier stage, and may contribute to the prognosis of septic
AKI. A potential biomarker could be MCP-1. The kinetics and location of MCP-1 and the
early detectability of MCP-1 protein suggests MCP-1 could function as a potential early
biomarker for the mononuclear inflammatory processes that occurs in acute renal injury. In
mouse models with pre-, intra- and post-renal injury, its role as a biomarker was assessed
(111). During intrarenal injury, both urinary MCP-1 protein and MCP-1 gene activation
increased to a greater extent than NGAL. Moreover, uraemia in the absence of renal injury
induced the NGAL gene but not the MCP-1 gene. This suggests a possible higher specificity
for MCP-1 in AKI. Clinical findings in patients have endorsed this idea by discriminating
patients with and without AKI by use of urinary MCP-1 concentrations. It would therefore be
interesting to evaluate whether urine MCP-1 levels in our septic AKI models could also be an
early detector of septic AKI.
Determine inflammation and resolution at different time points in the long-term model
The source of MCP-1 and the role of the macrophage in renal injury and recovery require
further elucidation. To determine inflammation and resolution at different time points in our
long-term model, earlier and later timepoints (e.g. days 7 and 28) should be included.
Furthermore, it needs to be established if the macrophages belong to the M1 or M2
phenotypes. We propose to perform histological assessment of kidney sections and to confirm
macrophage specificity by double labelling with macrophage and immunofluorescent-labelled
M1 and M2 markers. To evaluate whether P2X7 correlates with inflammation or resolution in
this long-term model of sepsis and to assess fibrosis we are currently performing
immunohistochemistry with P2X7 and α-SMA-staining, and are awaiting formal
quantification.
29
Dutch Summary
Sepsis is een overwelmende inflammatoire reactie op een infectie, wat kan leiden tot multi-
orgaan falen en de dood. Gedurende het septische proces raken de nieren vaak aangetast, wat
leidt tot dysfunctie of compleet nierfalen. Daarnaast hebben overlevers van sepsis-
geïnduceerd acuut nierfalen (AKI - acute kidney injury) een sterk verhoogd risico op het
ontwikkelen van eindstadium nierfalen. Het vrijkomen van pro-inflammatoire cytokines, zoals
IL-1β en IL-18, is geassocieerd met de pro-inflammatoire cascade die sepsis met zich
meebrengt en immunologische factoren lijken mee te dragen aan de ontwikkeling van
septisch-AKI. De mediatoren die aanvankelijk vrijkomen (in pro-vorm) zijn opzichzelf
insufficient. Een aanzienlijke rol voor de post-transcriptionele verwerking en het vrijlaten van
de actieve cytokines is via de formatie van een groot multi eiwitcomplex, het NLRP3
inflammasoom. De transmembrane cel receptor P2X7 (P2X7R) faciliteert de vorming van
NLRP3. Naast een belangrijke rol in de inflammatoire respons, speelt de P2X7R ook een rol
in apoptotische celdood. Opregulatie van deze receptor is geassocieerd met renale schade.
Daarentegen heeft A-438079, een selectieve P2X7 antagonist een reno-protectief effect in een
rat model met glomerulonefritis. Deze bevindingen wekken de suggestie dat P2X7R als een
potentieel therapeutisch doelwit kan worden gezien. P2X7 expressie is het best bekend in
immuuncellen zoals macrofagen, die een cruciale rol spelen in de initiatie, het onderhouden
van en de resolutie van inflammatie. Macrofagen komen vrij en worden beïnvloed door
chemokines zoals MCP-1. MCP-1 speelt een prominente rol in de acute inflammatoire
respons in meerdere modelen van nierziekten. Bovendien, induceert IL-1β (na maturatie en
vrijlating door de P2X7/inflammasome) expressie van MCP-1 door epitheelcellen.
Doel
Met de volgende werk hypothese; “P2X7R opregulatie veroorzaakt renale schade door lokale
productie van cytokines en chemokines”, heb ik onderzocht of renale P2X7 expressie
verhoogd is in een 3-daags rat-model met faecale peritonitis en vloeistofresuscitatie. Specifiek
heb ik gekeken naar een relatie met histopathologische veranderingen tijdens septisch-AKI en
naar de co-lokalisatie van macrofaag infiltratie.
In een ander lange termijn model (2 weken), met Zymosan-geinduceerde peritonitis, heb ik
onderzoek gedaan naar renale P2X7 expressie en naar de pathologische veranderingen vanuit
de vroege naar de ‘recovery’ fase.
Methoden
In het acute model werd sepsis geïnduceerd in geïnstrumenteerde, wakkere, mannelijke
Wistar ratten door middel van een intraperitoneale injectie van faecale ‘slurry’. Dit werd
gevolgd door vloeistofresuscitatie (10ml ml/kg/uur) vanaf 2 uur na het induceren van sepsis.
Sham (controle groep) ratten kregen hetzelfde vloeistof regime, maar geen i.p. injectie.
Septische en sham geopereerde ratten werden post-sepsis geofferd na 6 uur (n= 6 per groep)
of na 24 uur (n = 8 per groep). Bloed en nieren werden verkregen en geanalyseerd voor serum
creatinine, histologisch beoordeling van renale schade, P2X7 expressie, macrofaag infiltratie
en cytokine en chemokine expressie. Dit werd vergeleken met naïve, niet-geïnstrumenteerde
ratten (n=6).
In het lange termijn model, kregen niet-geinstrumenteerde mannelijke Wistar ratten een .i.p.
injectie met Zymosan (een glycaan afkomstig uit de celwand van een gist) voor een verlengd
septisch insult. Nieren werden verkregen op dag 2 (4 sham, 4 septische ratten) en dag 14 (4
sham, 8 sepsis) en geanalyseerd voor renale schade, macrofaag infiltratie en chemokine
expressie.
30
Continue variabelen zijn weergegeven als gemiddelde ± SD. Parametrische data werd
geanalyseerd door middel van een unpaired T-test en non-parametrische data door middel van
de Mann Whitney U test. Verschillen tussen meer dan twee groepen continue variabelen werd
beoordeeld met one-way ANOVA. Door middel van de Post-hoc Tukey’s test werden
verschillen tussen groepen vastgesteld. Een p-waarde <0.05 werd als statistisch significant
beschouwd.
Resultaten
In het acute model werd een significant hoger serum creatinine gevonden in septisch-AKI na
24 uur (p<0.05). Echter, na zowel 6 en 24 uur werd geen significante histologische renale
schade gezien. In septische nieren was P2X7 expressie verhoogd na 24uur (p<0.05), een trend
was echter al zichtbaar na 6 uur. In ratten met een ernstige sepsis (met een 3x verhoogd serum
creatinine) was intraluminaal debris zichtbaar wat intens kleurde voor P2X7. Verhoogde
expressie van P2X7 correleerde met verhoogde niveau’s van IL-1β in zowel serum als in de
nieren. Serum IL-1β was verhoogd in septische dieren, deze niveaus daalden na 24 uur
(P<0.05). Daarentegen was renaal IL-1β verhoogd 6uur na sepsis, wat doorsteeg op 24 uur
(p<0.01). Tevens is septisch-AKI geassocieerd met verhoogde niveaus van renale expressie in
caspase-1 en pro IL-18. Na zowel 6 als 24 uur was er geen macrofaag infiltratie zichtbaar en
was er sprake van slechts minimale stijging in renale MCP-1 niveaus.
In het lange termijn model van sepsis werden tevens minimale aanwijzingen gevonden van
acuut renale schade op dag 2 en dag 14. P2X7 kleuring moet nog geanalyseerd worden, echter
macrofaag infiltratie was evident in zowel glomeruli en interstitium in alle septische dieren op
dag 14, terwijl macrofaag infiltratie afwezig was in alle dieren van de controlegroep.
De renale MCP-1 niveaus waren tevens significant verhoogd in de septische dieren (p<0.01)
op dit tijdpunt. Daarentegen, werd macrofaag infiltratie slechts in één van de 4 septische
dieren gezien op dag 2. Renale MCP-1 niveaus waren soortgelijk in shams.
Conclusies
Een significante verhoging in renale P2X7 expressie werd gezien in septische dieren na 24
uur, correlerend met verhoogde niveaus van IL-1β. In ernstig septische dieren werd een
distinctief kleurings patroon gevonden, suggestief voor een rol in celdood. Deze bevinding
tezamen met de afwezigheid van vroege macrofaag infiltratie, wekt de suggestie dat renaal
P2X7 hoogstwaarschijnlijk tot expressie gebracht wordt door intrinsieke cellen in de nier.
Ondanks biochemische afwijkingen van renale dysfunctie werd er geen significante
histologische renale schade gezien, dit reflecteert bevindingen in septische patienten. Verder
onderzoek is nodig om te evalueren of P2X7 antagonisme bescherming kan bieden.
In tegenstelling tot bevindingen in het acute model, werd in het lange termijn model van
sepsis significant macrofaag infiltratie gezien op dag 14, wat in zowel de glomeruli als het
interstitium zichtbaar was. Dit correspondeert met verhoogde niveaus van renaal MCP-1. Of
dit bijdraagt aan lange-termijn schade in de nieren behoeft verder onderzoek.
31
Acknowledgments
I sincerely would like to thank all those who have given me their generous assistance, support and
guidance during the realization of my thesis. Not only could I not have performed my experiments
without their contributions but their uplifting feedback and energy gave me the opportunity to
start each day with renewed energy and kept me on my feet during the ups and downs of the entire
process.
I would particularly like to thank:
Prof. Mervyn Singer, for your guidance and support. It has been a great honour to be given the
opportunity to participate in the research with your group. You are a true role model of how to
lead a team; despite your tedious rushed schedules you would always make sure your door was
open for any questions or help in other sorts of ways. With your everlasting enthusiasm and
cheerful comments (even despite your awful taste for picking nicknames), I felt part of ‘the Singer
family’ straight away.
A special thanks to Dr. Nish Arulkumaran who provided me with great opportunities, both in and
outside the lab. I could not have succeeded in finishing this project without your generous
assistance. Your contagious ardour about science and uplifting motto kept me motivated, while
keeping my expectations right. It has been a real pleasure working with you!
I would also like to thank Dr. Frederick Tam and Prof. Charles Pusey for their hospitality in the
lab, Dr. Paul Bass, for his assistance and feedback regarding the histopathology and Prof. Robert
Unwin. Also a warm thanks to all the other members of the Research Department of Clinical
Physiology, UCL and the Renal Research Department at Hammersmith Hospital, Imperial for the
interesting and useful laboratory meetings and happy times working in the lab. Specifically I
would like to thank Gurtjeet Bangal and Clare Turner for their generous help in teaching my all
the ins and outs about immunohistochemistry.
I thank the GIPS-M committee who gave me the opportunity to participate in the GIPS-M
program, in specific Prof. Cees Kallenberg for his time and effort during my preparations and for
reviewing this thesis.
In particular, I would like to thank Maarten for his everlasting support, motivation and believe.
Your hardworking and positive mindset always 'boost' me to achieve my goals. Last but definitely
not least, I would like to thank my family for encouraging me throughout my life and education,
and my friends for the very much appreciated distraction and laughter in my spare time and for
always being there for me. I hope I can return you all with the same amount of love and laughter
on our continuing journey.
Sponsors I would like to thank the following sponsors for their financial support:
Nierstichting Nederland,
Fundatie van Vrijvrouwe van Renswoude,
Marco Polo Fonds,
JO Kolk studiefonds voor vrouwen,
Jan Kornelis de Cock Stichting,
Groninger Universiteisfonds.
32
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36
Appendices I – Buffers and solutions
1. RadioImmunoPrecipitation Assay (RIPA) buffer: 50 mM Tris [pH 7.4], 150 mM
NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% , 1mM EDTA supplemented
with 10% protease inhibitors (Sigma-Aldrich Co)
2. SDS-sample buffer: 0.125M Tris-HCL [pH6.8], 4% SDS, 20% (v/v) glycerol, 0.6M
β-mercaptoethanol and 0.02% Bromophenol blue
3. 12% SDS-PAGE Tris-glycine minigels. The SDS-page gel consisted of a stacking
gel (i) overlaid onto a resolving gel (ii)
(i) 5% stacking gel: 30% Acrylamide/bis solution (ratio 37.5:1) in a buffer of 1.5M
Tric-HCL [pH 6.8] containing 0.1% (w/v) SDS . Polymerisation is initiated by
addition of 0.1% (w/v) ammonium persulphate and 0.05% (v/v)
tetramethylethylenediamine (TEMED).
(ii) 12% Resolving gel: 10% of a 30% Acrylamide/bis solution (ratio 37.5:1) in a
buffer of 0.375M Tris-HCL [pH 8.8] containing 0.1% SDS. Polymerisation is
initiated by addition of 0.1% (w/v) ammonium persulphate and 0.016% (v/v)
tetramethylethylenediamine (TEMED).
4. Running Buffer: 3,03g Tris base, 14,4g Glycine, 1g SDS make up to 1L with dH2O
5. Transfer (Towbin) buffer 3g 25mM Tris, 14,4g 192mM Glycine, 10% methanol
make up to 1L with dH2O
6. Tris-buffered saline (TBS): 2,42g 20mMTris, 8,2g 140mM NaCl, make up to 1L
with dH2O and adjust for [pH 7.6] with 1.0M HCL
7. 0.01M citrate buffer [pH 6.0]: 2.94g Tri-sodium citrate to 1L dH20, adjust for [pH
6.0] with 1.0M HCL
8. PBS: 8g NaCl + 1,15g Na2NPO4 + 0,2g KCL + 0,2g KH2PO4 make up to 1L with
dH2O
9. 0.05M Tris-HCL [pH 7.2]: 1.576g Trizma HCL to 200mL dH20 and adjust for [pH
7.2] with NaOH
10. 0.1M Sodium Carbonate [pH 9.5]: 7,13g NaHCO3 + 1,59g Na2CO3, make up to 1L
with dH20 and adjust for pH with 10N NaOH
37
P2X7 Receptor and Haemorrhage-reperfusion- Induced Acute Tubular Injury
1M Sixma,
1A Dyson,
1, 2, 3N Arulkumaran,
2P Bass,
2RJ Unwin,
3F Tam,
1M Singer
1Bloomsbury
Institute of Intensive Care Medicine and
2Dept of Nephrology, University College London, UK and
3Imperial College Kidney and Transplant Institute, Hammersmith Hospital, London UK
INTRODUCTION
The P2X7 purinoreceptor (P2X7R) triggers activation of the inflammasome with release of pro-inflammatory
cytokines (e.g. IL-1β, IL-18) and the pro-apoptotic caspase-1. Constitutive expression of the P2X7R in the
kidney is minimal. However, in a rat glomerulonephritis model (1), expression, mainly localized to the
glomeruli, was upregulated, with selective P2X7R receptor antagonism being protective.
OBJECTIVES To determine the pattern of renal P2X7R expression in a rat model of severe haemorrhage-reperfusion injury.
METHODS
Anaesthetized male Wistar rats underwent insertion of carotid arterial and jugular venous lines. After 30 min
stabilization, 50% estimated circulating blood volume was removed from the arterial line over 15 min. Animals
were monitored for a further 90 min prior to resuscitation. This was immediately followed by administration of
shed blood over 15 min followed by a background infusion of n-saline (10 ml/kg/hr). Animals were culled at 6
hours post-reperfusion with kidneys taken for analysis. Paraffin-embedded kidney sections were stained for
periodic acid Schiff (PAS) and P2X7 (anti- P2X7 primary antibody, Alamone,).
RESULTS Major histological damage was seen, including loss of brush border from tubular epithelial cells, tubular casts,
dilated tubules and tubular cell death (Figure 1). Immunohistochemistry demonstrated widespread upregulation
of renal P2X7R in tubules of rats that underwent haemorrhage-reperfusion injury but none in naïve rats. P2X7R
expression was localized to areas of tubular damage (Figure 1). However, the glomeruli were negative for P2X7.
CONCLUSIONS
In this rat model of severe haemorrhage-reperfusion injury, there was histological evidence of significant tubular
injury. Coexistence of tubular injury and P2X7R upregulation suggests that P2X7R is implicated in the
pathophysiology of AKI. Further work is required to determine the functional significance of tubular P2X7R, and
the potential benefit of P2X7 receptor antagonism in haemorrhage-reperfusion injury-induced AKI.
References:
(1). Taylor SR et al. P2X7 deficiency attenuates renal injury in experimental glomerulonephritis. J Am Soc Neph
2009; 20:1275-81
Grant acknowledgement:
NA Wellcome, MS Dutch Kidney Foundation
Figure 1: Immunohistochemistry demonstrating severe renal tubular injury (left panel), with P2X7R
(stained brown) in kidneys taken from naïve (middle panel), and haemorrhage reperfusion rats (right
panel)
38
Renal macrophage infiltration in a rat model of sepsis and recovery
N. Arulkumaran, M. Sixma, S Saeed, G Bangal, P Bass, F Tam, M. Singer
INTRODUCTION
Despite minimal histological damage (1), survivors of acute kidney injury (AKI) are at risk of developing
chronic kidney disease (2). Most of the focus of AKI has been on the acute injury phase but little attention to
date has been paid to recovery. Mechanisms underlying resolution of AKI in sepsis are poorly understood and
may be potentially injurious.
OBJECTIVES
To describe temporal changes in renal inflammation and recovery following sepsis in a long-term rat model of
zymosan-induced peritonitis
METHODS
Intraperitoneal zymosan was injected into male Wistar rats under isoflurane anaesthesia. Sham animals received
intraperitoneal saline. At day 2 or day 14, animals were culled and kidneys taken for analysis. Renal tissue was
homogenized and MCP-1 (monocyte chemoattractant protein-1) levels were measured by ELISA (R&D
-1 is a key chemokine regulating
monocyte/macrophage migration and infiltration. Paraffin-embedded kidney sections were stained for
macrophages (anti-ED-1 primary antibody, Abcam). Statistics were performed using independent t-tests. A p-
value of <0.05 was taken as being statistically significant. Data are presented as mean ± standard deviation, p-
value.
RESULTS In this model the severity of illness (clinical severity, organ dysfunction, weight loss, food intake) peaks at day 2
with gradual recovery over the subsequent 12 days. At Day 2 there was minimal renal cell damage with similar
renal MCP-1 -1
Macrophage infiltration was evident in glomeruli and interstitium in all day 14 septic animals but in none of the
sham animals (Figure 1). At day 2, just 1 septic animal demonstrated renal macrophage infiltration while 1 sham
animal had minimal glomerular macrophage infiltration.
CONCLUSIONS
In the recovery phase of sepsis in this long-term rodent model, significant renal macrophage infiltration was
present within the glomeruli and interstitium with a corresponding increase in renal MCP-1. These changes were
not seen during the acute injury phase. The source of MCP-1 and the role of the macrophage in renal injury and
recovery require further elucidation.
References
(1) Takasu O, et al. Mechanisms of cardiac and renal dysfunction in patients dying of sepsis. Am J Respir Crit
Care Med 2013;187:509–17. (2) Chawla LS, et al. The severity of acute kidney injury predicts progression to
chronic kidney disease. Kidney Int 2011; 79:1361-9.
Grant acknowledgment
SS is supported by a Medical Research Council (UK) training fellowship. NA is supported by a Wellcome Trust
training fellowship. MS is supported by the Dutch Kidney Foundation.
Figure 1: Immunohistochemistry for ED-1 (stained brown) in septic (Day 14) rat in the glomerulus (left
panel) and interstitium (right panel).
39
Temporal changes in renal haemodynamics and oxygenation in a rat model of sepsis
1,2,3
N Arulkumaran, 1M Sixma,
2P Bass,
3F Tam,
2RJ Unwin,
1M Singer
1Bloomsbury
Institute of Intensive Care Medicine and
2Dept of Nephrology, University College London, UK and
3Imperial College Kidney and Transplant Institute, Hammersmith Hospital, London UK
INTRODUCTION
Postulated mechanisms for sepsis-induced acute kidney injury (AKI) include altered global and intra-renal
haemodynamics and bioenergetic dysfunction. However, temporal changes are not well elucidated.
OBJECTIVES
To characterize temporal changes in renal haemodynamics and tissue oxygenation in a long-term, fluid-
resuscitated rat model of faecal peritonitis.
METHODS
Tunnelled central venous lines were inserted into male Wistar rats under isoflurane anaesthesia. Twenty-four
hours later, sepsis was induced by intraperitoneal injection of faecal slurry (n=14). Sixteen animals served as
sham-operated controls. Fluid resuscitation (10 ml/kg/hr) was commenced at 2h post-slurry. At either 6hr or 24hr
animals were terminally anaesthetized and instrumented for measurement of cardiac output (echocardiography),
renal blood flow (ultrasonic flow probe), renal cortical tissue oxygen tension (Oxylite sensor), and renal oxygen
extraction ratio (from the renal arterio-venous oxygen difference). Statistics were performed using independent t-
tests. P values <0.05 were taken as statistically significant. Data are presented as mean ± standard deviation.
RESULTS
At 6 hours, septic animals had a lower renal vascular resistance (MAP/RBF) (0.072±0.001 vs. 0.084±0.011
sham, p<0.05) and a lower renal resistive index (peak systolic velocity/(peak systolic velocity-diastolic velocity))
(0.42±0.02 vs. 0.52±0.0.9 sham, p<0.05). No significant differences were seen between groups in global oxygen
delivery, renal blood flow, renal oxygen delivery, renal cortical oxygen tension or renal oxygen extraction.
By 24 hours, global oxygen delivery, renal vascular resistance and resistive index were similar between septic
and sham animals, whereas renal oxygen delivery was lower (285±68 vs. 371±41mL/min, p<0.05) in the septic
animals. Renal oxygen extraction ratio was however similar (43±7 vs 43±9% sham, p=0.876) and this was
associated with a significantly lower renal cortical oxygen tension (10.8 ± 3.2 septic vs 14.9±4.2 mmHg sham,
p<0.05).
CONCLUSIONS
In early sepsis (6h), renal autoregulation (fall in renal vascular resistance) maintains renal oxygenation.
However, at 24h, despite a maintained global DO2, renal autoregulation may be impaired. This results in reduced
renal O2 delivery and renal cortical hypoxia.
GRANT ACKNOWLEDGMENT.
NA is supported by a Welcome trust training fellowship. MS is supported by the Dutch Kidney Foundation.
40
“If we knew what it was we were doing, it would not be called research, would it?”
― Albert Einstein