Reduced Inflammatory Response of the

Brain Dead Kidney Graft with Nyk pre-

treatment

Naam Jurian Harm Kloeze

Studentennummer 1853597

Supervisoren Dr. H.G.D. Leuvenink

Hoofddocent Chirurgisch Onderzoek

Laboratorium, UMCG

L.F.A. van Dullemen

MD/PhD student Chirurgisch Onderzoek

Laboratorium, UMCG

Afdeling Chirurgisch Onderzoek Laboratorium,

UMCG

2

Abstract Introduction Kidney transplantation is ultimately needed for end-stage renal failure patients to

maintain life. Short-term and long-term outcomes are higher with living kidney donation

compared to heart-beating (HB) kidney donation. The discrepancy cannot be explained with

differences in human leukocyte antigen difference or ischemia times. Comparisons between

living kidney grafts and HB kidney grafts revealed that HB kidney grafts are inflammatory

active at the time of donation. The inflammatory state induces histological damage, which

enhanced ischemia/reperfusion injury in the recipient. Described here is a first attempt with

pre-treatment of geranyl-geranylacetone (GGA)-derivate Nyk, to test if it can reduce the

inflammatory active state in the kidney from HB donors via heat shock protein (HSP)-72 and

heme oxygenaze (HO)-1 expression.

Material and methods A graduate onset brain death model was used with male Fischer rats

(N=36) to analyze the inflammatory and protective response of the kidney to brain death. Rats

were divided in three groups: Nyk, GGA, and Saline. Inflammatory parameters were adhesion

molecules E-selectin, Intracellular adhesion molecule (Icam)-1, and neutrophil and

monocyte/macrophage infiltration.

Results A decreased up-regulation of E-selectin, Icam-1, and subsequent infiltration of

neutrophil was seen after Nyk pre-treatment compared to GGA or Saline pre-treatment. HSP-

72 showed broad expression with Nyk pre-treatment compared to low expression of GGA and

Saline pre-treatment. HO-1 expression was low in all pre-treated HB animals.

Conclusion A reduced renal inflammatory response was seen with Nyk pre-treatment in brain

dead rats compared to GGA or Saline pre-treatment. Nyk did not induce a significant higher

expression of HSP-72 or HO-1. Other HSPs should therefore be analyzed to understand how

Nyk pre-treatment reduces the renal inflammatory response. Short-term outcomes should be

analyzed with ischemia/reperfusion experiments.

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Samenvatting Introductie Patiënten met eindstadium nierfalen hebben uiteindelijk een donornier nodig om

te overleven. Er bestaan grote verschillen tussen de korte en lange termijn uitkomsten van

levende en hersendode nierdonatie. Deze verschillen kunnen niet verklaard worden door

verschillen in humaan leukocytenantigeen of ischemie tijden. Nieren van hersendode donoren

blijken op het moment van donatie inflammatoir actief te zijn vergeleken met nieren van

levende donoren. De inflammatie veroorzaakt histologische schade, wat de

ischemie/reperfusie schade in de ontvanger versterkt. Dit onderzoek is een eerste poging met

geranyl-geranylaceton (GGA)-derivaat Nyk voorbehandeling, om te onderzoeken of het de

inflammatie in de nier van hersendode donoren kan verminderen via heat shock protein

(HSP)-72 en heme oxygenaze (HO)-1 expressie.

Materiaal en methode Een dierenmodel met langzame hersendoodinductie werd gebruikt bij

mannelijke Fisher ratten (N=36) om de inflammatie- en beschermingsrespons te analyseren.

De ratten waren verdeeld in drie groepen: Nyk, GGA en Saline. Inflammatie parameters

waren adhesie moleculen E-selectine, Intracellular adhesie molecuul (Icam)-1 en infiltratie

van neutrofielen en monocyten/macrofagen.

Resultaten Een verminderde expressie van de adhesiemoleculen E-selectin en Icam-1 werd

gemeten, tezamen met een verminderde infiltratie van neutrofielen na voorbehandeling met

Nyk vergeleken met GGA of Saline voorbehandeling. HSP-72 liet een brede expressie zien,

maar de expressie was hoger vergeleken met de andere groepen. HO-1 was in alle groepen

laag.

Conclusie Een verminderde inflammatierespons was gemeten na voorbehandeling met Nyk

vergeleken met GGA of Saline voorbehandeling. Nyk veroorzaakte geen significant hogere

expressie van HSP-72 of HO-1. Andere HSPs zullen onderzocht moeten worden hoe een

voorbehandeling met Nyk de inflammatierespons kan verminderen. Ischemie/reperfusie

experimenten moeten de korte termijnuitkomsten duidelijk maken.

4

Table of contents

Abstract page 2

Samenvatting page 3

Introduction and rationale page 5

- Objectives page 9

Material and methods page 11

Results page 14

Discussion page 20

Conclusion page 23

Acknowledgements page 23

References page 24

Attachments page 28

5

Introduction and rationale

End-stage renal failure (ESRF) is the progressive end result of chronic kidney disease, and is

defined as the need for permanent replacement therapy, i.e. dialysis or transplantation (1). The

former however is not comparable with a functional kidney: it can only adopt some functions

of the kidney and it is associated with a number of complications like bacterial peritonitis,

cardiovascular disease, and sepsis (2). Therefore, renal transplantation is the treatment of

choice for ESRF-patients as quality of life and long-term survival are increased compared to

dialysis (3,4). Due to shortage of organ donors, hundreds of people are each year waiting for a

kidney graft in The Netherlands (5).

Kidney donation and transplantation There are two types of kidney donation: living and cadaveric. The cadaveric kidney donations

are further subdivided in the heart-beating (HB) and non-heart-beating (NHB) donations, and

this subdivision is related to the criteria of brain death. Brain death is defined as the

irreversible loss of all function of the brain, including the brainstem (6). NHB donors do not

meet the criteria for brain death, and therefore the donation is also called donation after

cardiac death.

Short-term transplantation outcomes are defined as delayed graft function (DGF: the need for

dialysis during the first week after transplantation) and primary non function (PNF: the non-

functioning of the kidney graft during the first three months after transplantation ). Long-term

transplantation outcomes are defined as survival rates like one-year or five-year survival. Both

outcomes vary among the donation types. Living has the best outcomes in both the short-term

and the long-term (7-9). HB donation has better short-term outcomes than NHB donation, but

the long-term are almost equal (10).

The better rate in outcomes in living kidney grafts are above all due to the quality of the

kidney grafts, but also due to the circumstances associated with living donation compared to

cadaveric donations. A living donor is uniformly healthy; he is not brain dead, nor is he

injured from trauma or cardiac failure. The donation has minimal warm ischemia time, and

the cold ischemia time is very short as well because transplantation occurs right after

donation.

In HB- and NHB donations, both the quality of the graft and the circumstances are less

optimal. The inferior quality is due to donor conditions and due to the circumstances which

are not optimal for donation. It is almost always in the nighttime, and a long cold ischemia

time exists for transportation to the recipient’s hospital. Furthermore, a long warm ischemia

time occurs with NHB donation.

Besides the donation type, differences exist between recipients as well. Living donations are

more often scheduled. The donor is often a relative, and the donor and recipient are prepared

for the donation and transplantation procedure before the recipient is dialysis-dependent.

Every recipient of a cadaveric kidney graft has been on the waiting list, the largest part for

multiple years. In those years, these patients were dialyzing in the hope to survive the waiting

period. Dialysis itself, and its complications drop the patient’s quality of life and the

possibility to survive surgery (11). Half of the dialyzing patients will not survive a five year

waiting time (12). These conditions associated with dialyzing recipients are also contributing

to the lower outcomes after transplantation.

6

However, many differences between living and HB kidney donation do not explain the graft

quality differences. Donors and recipients in humans are coupled with each other according to

their human leukocyte antigen (HLA) match to prevent rejection. However, even fully

HLA-mismatched living kidney grafts showed higher outcomes compared to reasonable

matched HB kidney grafts (9). Furthermore, the differences in outcomes cannot be fully

attributed to prolonged cold ischemia times for grafts procured from HB donors, because no

significant effect of cold ischemia on kidney outcomes was seen (9). Studies with HB kidney

donation have shown that the process of brain dead has a major contribution to the lower

outcomes compared to living kidney donation (13-16). This effect was seen in histological

differences in kidney grafts. Comparisons between living kidney grafts and HB kidney grafts

revealed that HB kidney grafts are inflammatory active at the time of donation. That is, HB

kidney grafts show a massive increase in leukocytes infiltration and subsequently histological

damage as seen as pronounced expression of histocompatibility antigens, as will be described

below.

After transplantation, these kidneys have a more severe ischemia/reperfusion injury

and subsequently higher chance of DGF and PNF, hence decreasing short-term outcomes

compared to the living kidney grafts (13,17,18). The link between ischemia/reperfusion injury

and short-term outcomes can be explained with the hyperfiltration hypothesis. This postulates

that kidneys with a reduced functioning nephron mass due to profound histological damage

will progress toward failure due to hypertrophy of the remaining nephrons to meet the excess

load, eventually leading to nephron exhaustion. The histological damage is furthermore a

trigger for the recipient’s immune system to actively infiltrate the kidney graft, producing

more damage after transplantation (19-21).

The latter is called the reflow-paradox: reperfusion of the grafts result in severe injury by

neutrophil infiltration and subsequent reactive oxygen substance production, followed by

increasing numbers of infiltrating macrophages and T-cells (22-24). The net effect is a

decreased functioning outcome and/or accelerated acute rejection (13,14).

Brain death

From the moment of a severe brain damaging insult, there is an active process initiated which

may lead to brain death. The local cell death, inflammation, and edema from the insult will

extend globally throughout the brain causing an increase in the intracranial pressure (ICP)

(25,26). An increased ICP suppresses the arterial blood flow to the brain, causing enhanced

brain damage and leads to brain death.

The period of brain damage till brain death is not restricted to the brain. Damage extends early

to the blood brain barrier (BBB) cells as well, resulting in leakage of the produced pro-

inflammatory cytokines into the systemic circulation (Figure 1) (27,28). The leakage into the

systemic circulation will activate peripheral organs, including the kidneys. Pro-inflammatory

cytokines cause endothelial activation (27,29,30), leading to the activation of mitogen-

activated protein (MAP)-kinases: the cell’s regulatory proteins (Figure 6 in attachments).

MAP-kinases include p38, nuclear factor kappa Beta (NFκβ), and extracellulair signal-

regulated kinases (ERK). These will eventually lead to up-regulation and expression of the

adhesion molecules, and further up-regulation of pro-inflammatory cytokines like IL-6

(27,30-38). E-selectin and Icam-1 are important for diapedesis: The release of pro-

inflammatory cytokines activate peripheral tissues like the kidney. This results, besides

adhesion molecules up-regulation and further up-regulation of pro-inflammatory cytokines, in

up-regulation of chemo attractants. Circulating leukocytes like neutrophils and monocytes are

localized to the tissue which produces chemo attractants. E-selectin expressions on the

vascular wall slows the circulating leukocytes down, and initiate leukocyte-rolling over the

7

vessel wall. Icam-1 expression then binds to rolling leukocytes, and assist with tissue-

migration. The final result of these events is neutrophil and monocyte/macrophage infiltration

in the kidney and histological damage, confirming the image of an inflammatory active

kidney graft (16,27,31,33,35,36,39).

ICP>MAP

Global ischemia

brain cellsà brain

death

IL-6 synthesis in

glial cells and

neurons

BBB disruption

IL-6 release in

systemic

circulation

Inflammatory

active kidney graft

Infiltration of

PMNs

Up-regulation of

adhesion

molecules

Endothelial

activation

Necrosis of

hypothalamus

Cushing response

Ischemia

IL-6 release in

systemic

circulation

Intestinal

inflammation

Intestinal

permeability

increases

Bacterial

translocation Endotoxemia

Activation of

pro-

inflammatory

mediators

Intestinal permeability hits the kidney

As well as the kidney, intestinal inflammatory activation is seen in HB donors. As a

consequence of intestinal inflammation, the permeability of the intestine increases, resulting

in bacterial translocation, leading to endotoxemia (39-41). This activates endothelial cells,

followed by the same reaction cascade with leukocytes infiltration and histological damage in

the kidney as pro-inflammatory cytokines (Figure 1) (31,40,41).

Figure 1 Brain damage initiate a systemic inflammation response due to blood-brain barrier dysfunction and

results in inflammatory active kidney grafts. Activation of pro-inflammatory cytokines due to brain death and leakage of

them into the systemic circulation result in peripheral endothelial activation, and subsequently in kidney inflammation via

activation of mitogen-activated protein (MAP) kinase pathways. As well as the kidney, intestinal inflammation occurs, and

results in bacterial translocation leading to endotoxemia. This induces a systemic inflammation response via bacterial toxins

leading to inflammatory active kidneys.

Ischemia is the results of hypothalamus necrosis. Massive catecholamines release induces the Cushing response to maintain

adequate blood pressures to the brain, using severe vasoconstriction. This leads to hypoperfusion or ischemia to the peripheral

organs. Ischemia leads to kidney inflammation via activation MAP kinase pathways.

IL-6 release in systemic circulation has been doubled to show the impact of endotoxemia to the kidney. ICP=

intracranial pressure; MAP= mean arterial pressure; IL-6= interleukin-6

8

Hemodynamic changes alters kidney perfusion

The damage due to brain death include the cells which control the sympathetic nervous

system in the brainstem (25,42). Experiments with brain death induction in rodents showed a

classical blood pressure pattern, with a hypotensive period during brain death induction, and a

hypertensive period when brain dead is reached (Figure 1) (42,43). The net result of the

hemodynamic changes is a reduced perfusion of the peripheral organs, including the kidney,

leading to ischemic periods for the peripheral organs (44).

Besides changes in blood pressure, haemodynamic changes are furthermore related to

inflammatory and hormonal alterations:

- The described events of inflammatory activation and impaired perfusion for the kidney

are also happening to the heart, and induce a diminished cardiac output, thereby

enhances the ischemia in the kidney (45,46).

- Among the damaged brain areas is the hypothalamic-pituitary-axis, which produces

low amount of anti-diuretic hormone (ADH) in brain death donors. This is seen as

diabetes insipidus (DI), thereby reducing the organ perfusion even more (45). DI can

also be caused by the down-regulation of specific proteins, like aquaporin-2, a protein

for urine concentration in the collecting duct cells (36,45).

The resulting ischemia of these effects injures the kidney graft. Ischemia leads to the

disruption of the binding between NFκβ and its inhibitor; Inhibitor kappa β (I-kβ) in renal

cells. Activated NFκβ then induces the same inflammatory actions as explained above

(47,49). Furthermore, ischemia leads to a metabolism shift from aerobic to anaerobic,

reducing the energy levels in the cells, and induces either apoptosis or necrosis (45,50). Both

leads to a decreased functioning mass and necrosis, and furthermore increases the

inflammatory state of the kidney.

Protection mechanisms in the HB donor

The severe cellular stress associated with brain death initiates a protection response in the

kidney, called the stress response, with up-regulation of heat shock proteins (HSPs) (35,51-

53). HSPs were first discovered as a result of heat shock treatment on drosophila, which

induced expression of a specific group of genes afterwards called the heat shock protein genes

(54). Nowadays, it is known that these genes are inducible by a variety of stresses and drugs

but are also conservatively expressed (55,56). Conservative HSPs function in assisting the

folding of newly synthesized polypeptides, the assembly of multiprotein complexes and the

transport of proteins across cellular membranes.

HSPs are divided in families, according to their approximate molecular mass, in which

the HSP-70 family is the most studied and best known family. Within the family, HSP-72 is

the best known stress-inducible variant. HSP-72 has been often studied, and results showed

that it is up-regulated in brain dead animals, and humans (51,52). HSP-72 is a strong anti-

apoptotic protein, and is also known for its anti-inflammatory functions (57,58). Its activation

is dependent on heat shock factor (HSF)-1. In unstressed cells, it is bound to HSF-1 in the

cytoplasm of every cell. In stressed cells, HSP-72 binds to damaged proteins, and is therefore

dissociated from HSF-1. Free activated HSF-1 is then oligomerized, as seen in tripling of their

molecular mass, before entering the nucleus. In the nucleus, HSF-1 binds to the heat shock

responsive element (HSE) in the promoter of HSPs genes inducing transcription and

translation HSPs including HSP-72 (59,60).

Besides HSP-72, another protective protein has been often studied: heme oxygenaze (HO)-1

(also known as HSP-32). HO-1 is the inducible member of the HO family but its protective

9

effects are different. HO-1 has antioxidant capacities and acts as potent anti-inflammatory and

anti-apoptotic protein whenever oxidative injury takes place (61-63). HO-1 is an enzyme that

catalyze the conversion of heme into biliverdin, carbon monoxide and iron. The degraded

heme products act as an antioxidant, a vasodilator, and inhibitor of platelet aggregation. This

prevents tissues from oxidative injury, or reactive oxygen species (ROS). The activation

mechanism of HO-1 has not been unraveled yet but could be related to the HSF-1 associated

HSE-binding in the HSPs genes.

In brain dead human and animal donors however, the up-regulation of both HSP-72 and HO-1

is insufficient to protect renal cells (34,36,51). To increase the protecting abilities of HSP-72

and HO-1 in brain dead donors, their expression should be increased.

Therefore, several attempts have been made to enhance the up-regulation with drugs

administration for a better functioning graft after transplantation. Up-regulation of HSPs

occurs, besides of the effects of brain dead, trough hyperthermia and drug administration.

Hyperthermia is inducing a thermal stress response, and both HSP-72 and HO-1 expression

has been successfully increased by administrating a heat shock to rats and in

ischemia/reperfusion experiments (64,65). However, this has not been studied in humans

because of the clinical problems of dispensing a heat shock to a donor. The idea is simple and

effective, but in order to attempt this with a human donor, with a body hundred times as a rat,

more research has to been done with bigger animals first. Administrating of a locally heat

shock to the human body will probably result in an intolerable dermal pain and is therefore

not a clinical applicable alternative. Therefore, a therapeutic approach prevails to enhance

HSP up-regulation.

Up-regulation of HSP-72 has been successfully attempted in several studies by administrating

Taurine (66), Herbimycin-A (67) , mercury (68), and dexamethasone (69).

HO-1 has, among others, been up-regulated due to cobalt-potoporphyrin (70) and the gene

transfer of the recombinant adenovirus encoding rat HO-1 cDNA (71). Furthermore, HO-1 is

up-regulated in ischemic experiments (72-74).

However, many of the HSP-72 and HO-1 inducers are toxic for humans. Therefore, studies

started to experiment with geranyl-geranylacetone (GGA). GGA is an anti-ulcer drug

developed in Japan which has been used clinically to treat gastritis or gastric ulcers since 1984

without serious adverse reactions. GGA boosts HSP-72 expression in gastric mucosa, small

intestine, hearts, livers, and kidneys, via HSF-1 dissociation (Figure 7 in attachments) (75-

79). However, GGA administration in animal experiments is orally and is therefore dependent

on the metabolic activity of the animal. Intravenous administration therefore prevails, but

GGA is difficult to dissolve. Recently, Nyk has been synthesized as a soluble derivate of

GGA.

In this research, we will study the effects of Nyk pre-treatment on kidney graft quality in a

brain dead rat model. Several inflammatory parameters will be measured in renal tissue after a

four hour brain dead experiment.

Aim

The aim of this experiment is to study whether Nyk pre-treatment to brain dead rats can

reduce the inflammatory active state and of the kidney after donation.

Main objectives First we want to study whether Nyk pre-treatment reduces the adhesion molecules,

10

interleukin-6 expression, and infiltration of neutrophils and monocytes/macrophages in the

kidney graft. Secondly, we want to study if Nyk pre-treatment increases the expression of

HSP-72 and HO-1 in renal tissue. Thirdly, we want to study if the inflammatory expression

with Nyk pre-treatment is lower compared to GGA pre-treatment.

11

Materials and Methods

Animals

Adult male Fischer F344 rats (N=36) (Harlan, Horst, the Netherlands), weighing 278-310

gram (293±1.75 gram) were used in this experiment. The animals were housed in the Central

Animal Facility of the University Medical Centre Groningen (UMCG), and animal care was

according to Experiments on Animals Act (1996) issued by the Dutch Ministry of Public

health, Welfare and Sports. Freedom of access to food and water was accomplished. The

animals were caged in groups in a light-dark cycle of 12h-12h and a temperature-controlled

(22ºC) environment.

Nyk, GGA, and Saline preparation

Nyk was kindly provided by Nyken BV (Groningen, the Netherlands) and solubilized in 0.9%

Sodiumchloride (NaCl) for infusion (Baxter BV, Utrecht, the Netherlands) and administered

in a concentration of 0.56mg Nyk/kg rat weight.

GGA was purchased at Eisai Co., Ltd (Tokyo, Japan), and solubilized in 100% dimethyl

sulfoxide(DMSO) (Merck KGaA, Darmstadt, Germany) and diluted in 10% Kleptose HPB

parenteral grade (Roquette Co., Lestrem, France) with a final DMSO concentration of 0.5%.

GGA pre-treatment was given in the same concentration as Nyk pre-treatment. The

concentration are based on previous experiments with GGA administration in mice.

Standard sterile bags with NaCl were used for Saline administration.

Experimental groups

To study the effects of intravenously (iv.) Nyk pre-treatment in the brain dead donor,

4 groups (n=8-9) were studied. All the groups received their primarily treatment for including

20 hours before brain death induction. The second treatment occurred 0 hours before

induction. Brain death induction was the same in all animals, as described below.

Group 1: iv. Saline at 20h and 0h, group 2: iv. Nyk at 20h and 0h, group 3: iv. Saline at 20h

and iv. Nyk at 0h, group 4: iv. GGA at 20h and 0h.

Brain death model

Brain death was induced as described previously (42). The procedure was as follows. Rats

were anesthetised using isoflorane (Pharmachemie BV, Haarlem, the Netherlands) with 100%

O2. Brain death induction started with placing a balloon-tipped cannula (Edwards

Lifesciences Co., Irvine, CA) through a 1x1 mm frontolateral hole in the epidural space with

the tip pointed towards caudal. Slow inflation of the balloon (16µl/min) simulated an epidural

hematoma, leading to brain death. Blood pressure monitoring occurred with cannula insertion

in the femoral artery. A second cannula in the femoral vein was used for hemodynamic drugs

administering.Animals were intubated via a tracheostomy and mechanically ventilated

throughout the experiment. Ventilation occurred with a small rodent ventilator (683 small

animal ventilator, Harvard Apparatus, UK).

As a result of inflation, hemodynamic changes occurred starting with a hypotensive period

followed by a graduate blood pressure increase. When the blood pressure reached 80 mmHg,

balloon infiltration was stopped. The balloon was kept infiltrated throughout the experiment.

Brain death was confirmed approximately 30 minutes after balloon inflation stop by dilated

and fixed pupils, absence of corneal reflexes and a negative apnea test. After 30 min of brain

death, the ventilated air was switched from 100% O2 to 50% O2 in air. Throughout the

experiment, the temperature was maintained at 37ºC using a heating pad.

12

Animals were kept brain dead for 4 hours. If the blood pressure fell below 80 mmHg, it was

restored with the following order of actions: compressing the rats body, lifting the backside of

the rats body, decreasing post end expiratory pressure, decreasing ventilation rate,

administering hydroxyethyl starch ( Haes) 10% (Fresenius Kabi AG, Bad Homburg,

Germany), administrating noradrenalin 0.01mg/ml (Centrafarm services BV, Nieuwe Donk,

the Netherlands). Exclusion criteria were a blood pressure below 80 mmHg for more than 10

min, or a maximal administration of 10 ml of liquids.

15 min before the end of the 4 hours of brain death, Suxamethoniumchloride (Centrafarm BV,

Etten-Leur, the Netherlands) was given iv. to achieve full muscle relaxation to allow

abdominal surgery. This was followed 10 min later with the administration of 500IU heparin

(Leo Pharma BV, Breda, the Netherlands) iv. After 4 hours of brain death, blood and urine

were collected from the aorta and bladder prior to aorta-flush with ice-cold NaCl. Right

kidneys were removed and sliced in parts. One part was snap frozen in liquid nitrogen and one

part was fixated in 4% paraformaldehyde. Blood was centrifuged for 10 min at 960g at 4˚C.

The resulting plasma were collected and stored at -80ºC.

Histology and immunohistochemistry

Staining for neutrophils was performed on kidney cryosections (4µm) according to protocol.

Sections were fixated using acetone. Endogenous peroxidise was blocked using 0.01%

Hydrogen peroxide (H2O2) in phosphate-buffered saline (PBS) for 30 min. Sections were

stained with primary granulocytes antibody His48 (Stressgen).

Staining for monocytes/macrophages, cleaved caspase-3, and p38 were performed on kidney

paraffin sections (4μm) according to protocol. Sections were de-waxed, rehydrated and

subjected to heat-induced antigen retrieval by overnight incubation in 0.1 M Tris/HCl buffer

at 80°C (pH=9.0, ED-1). Endogenous peroxidase was blocked with 0.03% H2O2 in PBS for

30 min. Primary antibody for p38 was anti-p38 antibody (Stressgen). Monocyte/macrophage

primary antibody was ED-1 (Stressgen). Cleaved caspase-3 primary antibody was Asp175

(Stressgen).

Primary antibody was incubated on paraffin- and cryosections for 60 min at room

temperature. After the initial primary antibody binding, the sections were washed with PBS

for 5 min. Secondary and tertiary antibody (Dakopatts, Glostrup, Denmark) incubation lasted

for 30 min at room temperature to detect binding of the primary antibody. Secondary and

tertiary antibodies were added in PBS, which was supplemented with 1% bovine serum

albumin (BSA) and 1% normal rat serum. Peroxidase activity was visualised using 9-amino-

ethylcarbazole (AEC) for cryosections, and 3,3’-diaminobenzidine tetrahydrochloride

(DAB+, K3468; DAKO) for paraffin sections. Sections were counterstained with

haematoxylin. Negative antibody controls were performed in all staining analysis.

Morphometric analysis of histology and immunohistochemistry

Stained paraffin- and cryosections were scanned with NanoZoomer 2.0-HT (Hamamatsu

Photonics). Digital slides were assessed with the software program Aperio Imagescope

(version 11.1.2.760, Aperio Thechnologies).

Scoring occurred by two observers independently from each other. For each kidney section,

positive cells were counted in 10 microscopic fields of the cortex and medulla at 200x

magnification.

13

Western blotting

Per sample, six 20 μm cryosections were lysed in 200 µL RIPA buffer (1% NP40, 0.1% SDS,

10 mM β-mercaptoethanol) containing protease inhibitors (Complete, Roche). Samples were

lysed on ice, centrifuged for 15 min at 16000g (4°C), and supernatant was collected. Protein

concentrations were measured using the Lowry Protein assay (BioRad).

Equal amounts of protein were loaded on to SDS/PAGE (10% polyacrylamide gels). Proteins

were transferred on to nitrocellulose membranes and incubated with HSP-72 antibody SPA-

810 (StressGen). The house keeping-gene GAPDH (glyceraldehyde-3-phosphate

dehydrogenase) was used as loading control and was detected with mouse antibody (RDI

Research Diagnostics). Blots were subsequently incubated with HRP (horseradish

peroxidase)-conjugated antimouse secondary antibody (Amersham), and visualization was

performed with ECL and hyperfilm. Detected signal was quantified and normalized for the

GAPDH signal on the same blot.

RNA isolation and semi-quantitative RT-PCR Total RNA was isolated from kidney cryosections using TRIzol™ (15504-020, Invitrogen)

method and using a DNase treatment step with deoxyribonuclease I (AMP-D1, sigma

Aldrich). The RNA quantity was measured using a spectrophotometer. RNA quality control

was secured using electrophoresis. Positive samples showed no DNA contamination. Using

Primer express 2.0 (Applied Biosystems, Foster city, CA), gene-specific primers were

designed and gene-sequences were published (Table 1). Amplification and detection of the

PCR products were performed with 7900 HT real-time polymerase chain reaction systems

(Applied Biosystems) using SYBR Green (SYBR Green master mix; Applied Biosystems).

All assays were performed in triplet. The samples were amplified as follows, first an

activation step at 50°C for 2 min and a hot start at 95°C for 10 min. The PCR step consisted

40 cycles at 95°C for 15 sec and 60°C for 60 sec. Specificity of the PCR products was

routinely assessed by performing a dissociation curve at the end of the amplification program.

Gene expression was related to the mean ß-actin gene expression from the same cDNA.

Gene Primer sequences Bp

β-actin 5’-GGAAATCGTGCGTGACATTAA-3’, 5’-GCGGCAGTGGCCATCTC-3’ 74

IL-6 5’-CCAACTTCCAATGCTCTCCTAATG-3’, 5’-TTCAAGTGCTTTCAAGAGTTGGAT-3’ 89

E-selectin 5’-GTCTGCGATGCTGCCTACTTG-3’, 5’-CTGCCACAGAAAGTGCCACTAC-3’ 73

Icam-1 5´-CCAGACCCTGGAGATGGAGAA-3’, 5’-AAGCGTCGTTTGTGATCCTCC-3’ 90

Bax 5’-CTGGGATGCCTTTGTGGAA-3’, 5’-TCAGAGACAGCCAGGAGAAATCA-3’ 70

Bcl-2 5’-CGGCGGCTGGTGGTATAA-3’, 5’-CTGTAAAGGCCACCCCAGTAGTAT-3’ 71

HO-1 5’-ACTTTCAGAAGGGTCAGGTGTCC-3’, 5’-TTGAGCAGGAAGGCGGTCTTAG-3’ 523

Hsp72 5’-CTGACAAGAAGAAGGTGCTGG-3’, 5’-AGCAGCCATCAAGAGTCTGTC-3’ 302

Statistical analyses All data were tested for normality using Q-Q plots and the Kolmogorov-Smirnov test.

Normally distributed data were expressed as mean ± standard deviation of the mean. Non-

normally distributed data are expressed as median± standard error and differences were tested

with Mann-Whitney U tests. Correlations between variables were assessed with bivariate

correlation and Pearson correlation coefficients. A P value < 0.05 was considered significant.

Statistical analyses were performed using SPSS version 21.0 (SPSS Inc, Chicago, US).

Table 1 qRT-PCR primer sequences for the genes (forward, reverse)

IL-6= interleuking-6, Icam-1= Intracellular adhesion molecule-1, HO-1= heme oxygenaze-1, HSP-72= heat

shock protein-72, BAX and Bcl-2 are respectively pro- and anti-apoptotic genes; BP=base-paired.

14

Results

Surgery and brain death induction

The 4 hour brain death experiment succeeded with thirty-four of the thirty-six rats used for

this experiment, resulting in an success rate of 94%. One rat of the Nyk group (group 2) died

during the experiment as the result of a human error, one GGA pre-treated rat experiment

was stopped due to an excessive hypotensive period. All rats received the same surgery and

brain death induction method, performed by one of the two surgeons specialized in rodent-

surgery working in the Surgical Research Laboratory. After anesthesia, the rats were prepared

for brain death induction according to protocol. Group inclusion occurred 20 hours before,

with the administration of either Nyk, GGA, or Saline iv.. The inflammatory response of

group 3 (iv. Saline at 20h and iv. Nyk at 0h), is not included in this report as this group was

not included in the objectives.

Figure 2 shows the mean arterial pressure (MAP) of the groups during the brain death

experiment, grouped as the induction period (-30 till 0 min) and the 4 hour brain death period.

After the initial hypotensive period, an increase is seen in blood pressure. However, we did

not see a sharp peak as seen in humans and animal experiments (42). We see normotensive

levels for the first 1.5 hours after confirmed brain death, followed by hyper-/normotensive

levels for the Saline pre-treated group, and normotensive levels for the Nyk and GGA pre-

treated group. The MAP difference between the groups were not significant (Table 2). The

prominent drop in MAP at the end of the brain death period is due to surgical procedures,

which always started 3.75 hours after confirmed brain death for ureter cannulation, followed

by aorta perfusion at exactly 4 hours of confirmed brain death.

In our experiments, after reaching the threshold MAP level we stopped balloon inflation and

tried to maintain normotensive levels. Hypertensive levels were not corrected. Hypotensive

levels which were not correctable with body changing or ventilator adjustments

were corrected with Haes and/or noradrenalin. The amount of Haes and noradrenalin

administration in the groups were not significant different from each other (Table 2).

Figure 2 Mean arterial pressures of the Nyk, GGA and Saline pre-treated group. No significant difference were

seen in arterial pressure during the brain dead experiment (0 – 240). Also, no hypertensive peak was seen after brain death

induction (-30 – 0 min).

15

We furthermore evaluated if administration of either one or both could influence the

inflammatory response outcome, therefore we tested for correlation (Table 3 and 4).

Table 2 Mann-Whitney U results of independent parameters

(median±standard error) Nyk GGA Saline

Brain death induction time (min) 32,90±0,77 32,84±0,47 32,63±0,80

Mean arterial pressure (mmHg) 105,35±3,56 101,56±4,16 105,36±3,12

Weight (gram) 293,50±2,02 294,00±1,88 295,00±4,60

Haes administration (ml) 1,00±0,25 1,00±0,15 1,50±0,21

Noradrenalin administration (ml) 1,05±0,50 1,00±0,51 1,10±0,32

Table 3 Correlation levels of the hemodynamic drug administration with

inflammatory parameters (median±standard error) Correlation Nyk GGA

Noradrenalin Haes Noradrenalin Haes

E-selectin 0.33 (P=0.42) 0.54 (P=0.17) -0.36 (P=0.38) 0.80 (P=0.02)

Icam-1 0.56 (P=0.15) 0.46 (P=0.25) 0.24 (P=0.57) -0.49 (P=0.22)

WB HSP-72 0.69 (P=0.06) 0.39 (P=0.34) -0.21 (P=0.66) -0.22 (P=0.64)

mRNA HSP-72 0.56 (P=0.15) 0.27 (P=0.52) 0.26 (P=0.54) 0.17 (P=0.68)

mRNA HO-1 0.58 (P=0.13) 0.25 (P=0.56) 0.54 (P=0.17) -0.36 (P=0.38)

mRNA IL-6 0.71 (P=0.05)* 0.46 (P=0.25) 0.59 (P=0.12) -0.00 (P=1.00)

Neutrophil infiltration 0.36 (P=0.38) 0.31 (P=0.46) 0.04 (P=0.93) -0.83 (P=0.02) *

M/M infiltration -0.41(P=0.31) -0.11 (P=0.79) -0.32 (P=0.68) -0.15 (P=0.83)

mRNA BAX 0.28 (P=0.50) -0.03 (P=0.95) -0.47 (P=0.24) 0.12 (P=0.77)

mRNA Bcl-2 0.24 (P=0.56) 0.31 (P=0.46) -0.39 (P=0.35) -0.33 (P=0.43)

mRNA BAX/Bcl-2

ratio

0.13 (P=0.76) -0.20 (P=0.64) -0.05(P=0.91) 0.22 (P=0.60)

Table 4 Correlation levels of the

hemodynamic drug administration

with inflammatory parameters

(median±standard error)

Correlation Saline

Noradrenalin Haes

E-selectin -0.21 (P=0.58) -0.33 (P=0.39)

Icam-1 0.58 (P=0.10) 0.68 (P=0.05)*

WB HSP-72 0.13 (P=0.76) 0.13 (P=0.77)

mRNA HSP-72 0.00 (P=1.00) -0.23 (P=0.55)

mRNA HO-1 0.07 (P=0.85) -0.17 (P=0.66)

mRNA IL-6 0.71 (P=0.03)* 0.33 (P=0.39)

Neutrophil infiltration 0.38 (P=0.31) 0.45 (P=0.22)

M/M infiltration -0.37 (P=0.32) -0.08 (P=0.84)

mRNA BAX -0.67 (P=0.05) -0.25 (P=0.52)

mRNA Bcl-2 0.17 (P=0.66) 0.18 (P=0.64)

mRNA BAX/Bcl-2

ratio

Haes = Hydroxyethyl

starch; GGA=geranyl-

geranylacetone

* P-value is significant (< 0.05) WB=

Western blot; mRNA= messenger

ribonucleic acid; M/M=

monocyte/macrophage; GGA= geranyl-

geranylacetone; Haes= hydroxyethyl

starch

-0.77 (P=0.02) -0.43 (P=0.24)

* P-value is significant

(< 0.05) WB= Western blot;

mRNA= messenger

ribonucleic acid; M/M=

monocyte/macrophage;

GGA= geranyl-

geranylacetone; Haes=

hydroxyethyl starch

16

Real-Time Polymerase Chain Reaction (RT-PCR)

Multiple genes were tested to determine the renal response to brain death in both groups.

Levels of mRNA expression were measured for interleukin (IL)-6, E-selectin, and Icam-1 for

the inflammatory response. HSP-72 and HO-1 were tested for the protective response. Pro-

apoptotic BAX and anti-apoptotic Bcl-2 were measured for the intrinsic apoptotic cascade.

(Figure 4 and Table 5).

Table 5 mRNA expression levels of the inflammatory

and protective response (median±standard error)

Nyk GGA Saline

E-selectin 0,48±0,08 0,76±0,21 0,87±0,14

Icam-1 0,48±0,07 0,67±0,06 0,76±0,08

IL-6 0,54±0,46 1,44±0,39 1,43±0,70

HSP-72 0.92±0,20 0,91±0,27 0,64±0,12

HO-1 0,60±0,09 0,63±0,13 0,61±0,07

BAX 1,89±0,61 1,99±0,17 1,81±0,17

Bcl-2 0,59±0,04 0,69±0,04 0,78±0,08

BAX/Bcl-2 3,28±0,39 2,77±0,34 2,32±0,21

ratio

RT-PCR results showed a significant difference in the renal up-regulation of the genes coded

for E-selectin, Icam-1 and Bcl-2 between Nyk pre-treatment and Saline pre-treatment.

E-selectin was decreased 1.81 fold (P=0.00), Icam-1 1.58 fold (P=0.04) and Bcl-2 1.32 fold

(P=0.02). IL-6 mRNA expression was decreased 2.65 fold but no significant value was

reached. No difference in BAX expression was seen.

Significant difference between Nyk pre-treatment and GGA pre-treatment were seen in Icam-

1 and IL-6 expression. Icam-1 mRNA expression was decreased 1.40 fold (P=0.04), and IL-6

mRNA 2.67 fold (P=0.05) with Nyk pre-treatment. The expression of E-selectin mRNA was

reduced 1.58 fold, but the result was insignificant. No difference was seen in BAX and Bcl-2

expression.

The protective response showed no significant differences between Nyk, GGA, and Saline

pre-treatment. mRNA HSP-72 expression was the highest with Nyk pre-treatment and lowest

with Saline pre-treatment. HO-1 mRNA expression was highest with GGA treatment, and

lowest with Nyk pre-treatment. However, mRNA HO-1 expression were low in all animals.

mRNA= messenger ribonucleic acid;

Icam-1= Intracellulair adhesion

molecule -1; IL-6= interleukin-6;

HSP-72= heat shock protein-72; HO-

1= heme oxygenaze-1; GGA=

geranyl-geranylacetone

17

Western Blot HSP-72 protein expression was measured with Western blotting. One sample of the Saline

pre-treated group was not included in this test due to technical problems. Western blotting

showed an increase of 1.88 fold in HSP-72 expression in the Nyk pre-treated compared to

Saline pre-treatment, and an increase of 1.36 fold compared to GGA pre-treatment, but both

results were not significant (Figure 3). A broad range is seen in the HSP-72 expression in the

Nyk pre-treated group compared to the GGA and Saline pre-treated group.

The protein expression of HSF-1 showed no differences between Nyk, GGA, or Saline pre-

treatment (results not shown).

Immunohistochemistry The inflammatory response was besides RT-PCR and Western blot measured with

immunohistochemistry. Cellular infiltration of the cortical and medullar segments of the right

kidney was measured with staining with granulocytes antibody (HIS48) for neutrophil

infiltration and anti-CD68 antibody (ED-1) for monocytes/macrophages infiltration.

Infiltration of neutrophils was significantly reduced with Nyk pre-treatment compared to

GGA pre-treatment or Saline pre-treatment. The average infiltration per microscopic field for

Nyk pre-treated rats was 3,14±0.23 versus 4,26±0.27 for GGA pre-treated rats (P=0.01) and

5,89±0.35for Saline pre-treated rats (P=0.00) (Figure 5).

Monocytes/macrophages infiltration showed no significant difference between the pre-treated

groups. Nyk pre-treatment showed an average infiltration per microscopic field of 7,73±1,71.

Infiltration with GGA pre-treatment was 8,05±1,56 and Saline pre-treatment levels were

8,52±2,37 (results not shown).

Staining for cleaved caspase-3 and p38 showed no difference in expression, as concluded by

an immunohistochemistry expert (results not shown)

Figure 3 HSP-72 protein

expression between Nyk, GGA,

and Saline pre-treatment.

Differences were not significant.

Broad expression of HSP-72 was seen

in the Nyk pre-treated group.

Nyk

GGA

Sal

ine

0

2

4

6Nyk

GGA

Saline

HSP72 protein

Pre-treatment groups

HS

P-7

2/G

AP

DH

R

ati

o (

Re

lati

ve

to

co

ntr

ol)

18

Figure 4 mRNA expression of E-selectin, Icam-1, IL-6, HSP-72, HO-1, and BAX/Bcl-2 ratio with Nyk,

GGA, and Saline pre-treatment. Significant differences between Nyk and Saline pre-treatment were seen in

E-selectin and Icam-1. Between Nyk and GGA pre-treatment, significant differences were seen in Icam-1 and

IL-6. HSP-72 and HO-1 were both insignificant different between all pre-treated groups.

E-selectin

Nyk

GG

A

Sal

ine

0.0

0.5

1.0

1.5

2.0

2.5Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

n

Icam-1

Nyk

GGA

Salin

e

0.0

0.5

1.0

1.5Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

nInterleukin-6

Nyk

GGA

Salin

e

-2

0

2

4

6

8Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

n

HSP-72

Nyk

GGA

Salin

e

0

1

2

3Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

n

HO-1

Nyk

GGA

Salin

e

0.0

0.5

1.0

1.5Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

n

BAX/Bcl-2 ratio

Nyk

GGA

Salin

e

0

1

2

3

4

5Nyk

GGA

Saline

Pre-treatment groups

Rela

tive m

RN

A e

xp

ressio

n

19

A B

D C

E F

Figure 5 Neutrophil infiltration stained with HIS48 antibody in Nyk (E&F), GGA (C&D), and Saline

(A&B) pre-treatment. Infiltration was significantly reduced with Nyk pre-treatment compared to Saline or GGA pre-

treatment. Median±standard error values infiltration per field for Nyk pre-treatment was 3,14±0.23, for GGA pre-

treatment 4,26±0.27 and for Saline pre-treatment 5,89±0.35. Magnification=200x

20

Discussion

Patients with end-stage renal failure (ESRF) are in need of a kidney graft to maintain life.

There is a significant difference in short- and long-term outcomes between living kidney

donation and heart-beating (HB) kidney donation. The latter provides inflammatory active

kidney grafts with histological damage that results in lower short- and long-term outcomes.

To improve kidney graft outcomes from HB donors, influencing the inflammatory state of the

organ donor has been the focus of experiments the last years.

One option to improve HB kidney grafts is reducing the inflammatory state of the graft.

However, the HB donor cannot be treated before brain death is confirmed and a lot of damage

has already occurred before confirmation. Therefore, increasing the response of the kidney to

the damaging process associated with brain dead is a more clinically relevant option. This

study is based on earlier results with geranyl-geranylacetone (GGA), but elaboration takes

place with the recently developed drug Nyk. As a derivate of GGA, it is better soluble and

should theoretically accomplish improvement in outcomes. Described here is a first attempt

with Nyk pre-treatment, to test if it can reduce the inflammatory active state of the kidney in

HB donors via heat shock protein (HSP)-72 and heme oxygenaze (HO)-1 expression.

Using Nyk pre-treatment in a graduate onset brain death model with rats, we observe a

significant reduction in inflammatory parameters compared to Saline pre-treatment or GGA

pre-treatment.

The significant differences with Saline pre-treatment were seen in the inflammatory

parameters E-selectin, Icam-1, and neutrophil infiltration. The expression of mRNA IL-6 was

more decreased after Nyk pre-treatment, but the result did not reach a significance value.

Significant differences with GGA pre-treatment were seen in the mRNA expression of IL-6

and Icam-1, and neutrophil infiltration. The results of mRNA E-selectin expression were

lower in the Nyk pre-treated group, but the result was insignificant.

Discrepancy was seen in the infiltration of monocytes/macrophages compared with other

studies (15,44,80). This could be the result of a different antigen staining. We have used

ED-1, which is specific for circulating monocytes who have infiltrated into the tissue. The

studies which showed increased monocytes/macrophages infiltration have stained with ED-2,

specific for infiltrating monocytes and resistant macrophages. Both the monocytes and

macrophages are inducing histological damage, but ED-2 was not available in our experiment

period. Therefore, staining for ED-2 has to be performed in the future as we do believe that

the low results are contradictory with the other anti-inflammatory results. Furthermore, one

study found high levels of monocyte chemoattractant protein (MCP)-1. It might help to

measure MCP-1 as well to know whether MCP-1 expression is decreased as well.

Neutrophil granulocytes are known to be increased in several inflammatory diseases,

and induce histological damage by releasing of cytokines and phagocytosis (81-83). As

described in the introduction, inflammation is the main insult in HB kidney donation and

provides the opportunity for neutrophil granulocytes to enter the tissue and induce histological

damage. The result of histological damage is seen as an increased amount of

histocompatibility antigens. These increase the ischemia/reperfusion injury and initiate a more

severe immune response in the recipient after transplantation. Neutrophil granulocytes

infiltration is therefore associated with accelerated graft rejection in several studies (13-

15,84). In theory, the reduced infiltration due to Nyk pre-treatment should therefore reduce

the immunogenicity of the kidney graft, improve short-term and eventually long-term

outcomes. We have tried to measure histological damage with apoptosis and necrosis

21

analyzing. However, necrosis analysis could not be done during the analyzing period.

Apoptotic analyzing showed no differences in BAX/BCL-2 ratio, initiators of the intrinsic

apoptosis cascade (Table 5 and Figure 4). To test if apoptosis was executed we have stained

for cleaved caspase-3. The staining provided no differences between the groups, as concluded

with inspection from an immunohistochemistry expert (results not shown). However, we

believe that only limited histological damage have occurred at the time of donation. As

described, the damage done by brain death leads to a pronounced expression of

histocompatibility antigens. Histological damage itself, by apoptosis and necrosis, is more the

result of ischemia/reperfusion injury than brain dead injury (85). However, this conclusion

cannot be made without necrotic analyzing.

How Nyk induces a reduction in the inflammatory parameters is still unknown. In contrast to

the described hypothesis, we did not find a significant up-regulation of HSP-72 and HO-1 in

the Nyk pre-treated rats. The mRNA expression of HSP-72 was increased 1.33 fold, whereas

HO-1 mRNA expression was even reduced compared to the other groups. However, because

of the possibility of only a short-term up-regulation of mRNA expression, we have measured

HSP-72 protein expression with Western blot. Western blot material for HO-1 was not

available during the analyzing period. HSP-72 protein expression was increased 1.95 fold and

2.16 fold compared to respectively GGA and Saline pre-treatment, but the results were still

insignificant. This could be due to the small size group, but as seen in Figure 3, HSP-72

protein expression has a broad expression in the Nyk pre-treated group. We tried to find an

answer to the broad protein expression in the Nyk pre-treated group. A correlation with one of

the other measured parameters like hemodynamic drug administration, brain death induction

time, mean arterial pressure during the brain death experiment, or the weight of the rats was

not found. None had a correlation level sufficient enough to explain the differences (Table 9

in attachments). Furthermore, we looked at the possibility if gaining experience with the

experiment was a factor, that is, if the HSP-72 expression was highest or lowest at the last rats

compared to the first ones. But the expression was various high and low among the rats so no

explanation could be found there as well. Therefore, it is possible that the insignificant results

are dose-dependent combined with a still unknown other factor. As described, our experiment

was done with one more group, group 3 (Nyk and Saline). Group 3 received half of the

dosage of group 2 (Nyk). In group 3, mRNA expression and Western blot showed a higher

up-regulation of both mRNA and protein expression compared to the Saline group, but the

increase was less compared to group 2. Still, no correlations could be made which explain the

differences in HSP-72 up-regulation but the comparison revealed a dose-dependent up-

regulation of HSP-72. To test if our animals induce a different HSP-72 expression compared

to other studies, a heat shock induction could be valuable. Heat shock is known to increase

HSP-72 up-regulation, and it should be interesting to see if there will still be various HSP-72

up-regulations after heat shock induction (64,65). If so, an independent factor is influencing

the HSP-72 expression and should be ruled out for further experiments. Lastly, the dosage for

each rat has been measured according to their metabolic weight and the metabolic weight of

the GGA mouse treatment. It is possible that the comparison with mice was not sufficient and

therefore lower dosages were administered for the rats who showed low expressions of

HSP-72.

The low expression of mRNA HO-1 is indicating that Nyk pre-treatment does not increase the

up-regulation of mRNA HO-1. In other studies, up-regulation of HO-1 was successfully and

showed to be protective, as described in the introduction. It is possible that lower expressions

are seen due to the ischemic state of our rat kidney grafts. All of the described studies

22

included ischemic periods. It is possible that our low HO-1 expression is associated with

limited ischemia due to maintaining the blood pressure >80 mmHg. In Figure 2 is seen that

after brain death induction, no hypotensive period has occurred in both groups. Also, no

hypertensive period right after induction is seen which is different from the described animal

model (42). We have not applied different animals or material in our method, but the

discrepancy could be explained by a different end induction points and differences in blood

pressure stabilization. One hypothesis is that we were more anticipating for the high peak

pressure and therefore we stopped the induction period sooner. Another (additional)

hypothesis is that we maintained normotensive values with less constricting renal vessels and

subsequently higher renal perfusion by altering the respiratory frequency and –amplitude prior

to hemodynamic drugs administration.

Besides the measured expression of HSP-72 and HO-1, they showed furthermore no

significant correlation with the inflammatory parameters to explain the reduction in E-

selectin, Icam-1, and neutrophil infiltration (Tables 6,7 and 8 in attachments). Therefore, a

possibility for the low HSP-72 and HO-1 outcomes is that other HSPs are functioning instead

with Nyk pre-treatment. Therefore, HSF-1 expression has been measured, as HSF-1 is not

only inducing HSP-72 expression. The HSF-1 expression was indifferent between the groups,

but the 4 hours of brain death could be too long for HSF-1 up-regulation due to Nyk pre-

treatment. Therefore, HSF-1 should be tested with Western blot and other HSPs should be

measured with RT-PCR and Western blot.

The functioning of Nyk pre-treatment on inflammatory reduction could be related to other

HSPs than HSP-72 or HO-1. Plain brain death induction induced only the expression of HSP-

72 and HO-1, and not of HSP-27 and HSP-40 (52). It could be possible that the latter two are

up-regulated due to Nyk pre-treatment and therefore they should be analyzed. Other studies

showed an increased expression of HSP-90, HSP-25, and HSP-27 after (pre-)treatment (86-

89). Therefore, other HSPs should be analyzed prior to other experiments to see how Nyk pre-

treatment reduces the inflammatory response.

The reduction of inflammatory parameters with Nyk pre-treatment could be, regardless the

HSPs expression, due to inhibition of mitogen-activated protein (MAP) kinases. Other studies

have described that several cellular pathways are involved in the brain dead damaged kidneys,

like up-regulation of MAP kinases. In these studies, the produced inflammatory cytokines in

brain death activate the cell’s MAP kinases by binding to the gp130 receptors, which induce

phosphorylation of JNK, p38, and ERK pathways through the JAK/STAT pathways. As a

result of p38 activation, nuclear-factor-kappa-bèta (NFĸß) is activated, and is translocated to

the nucleus where it influence the gene-expression of among others IL-6, E-selectin, and

Icam-1 (27,30-38). Because we observe a reduced expression of E-selectin and Icam-1, the

possibility that the MAP-kinases pathways are inhibited by Nyk pre-treatment arise. We have

not measured all the MAP-kinases, because that would be too expensive and time-taken.

Because NFĸß staining was not available in the analyzing period, we stained for p38, as this is

the main inducer of NFĸß and therefore the pro-inflammatory state in the cell. No differences

were seen between Nyk, GGA, or Saline pre-treatment (results not shown). However, the

indifferent result does not have to be contradictory because p38 is involved in many cellular

processes (37,90). Therefore, more analysis have to be done to analyze the effect of Nyk pre-

treatment on MAP-kinases pathways, because the inflammatory influence of JNK, p38 and

NFĸß inhibition has been observed in other studies (91-95). Because NFĸß is involved in

many cellular processes, an ischemia/reperfusion experiment should be performed to analyze

the anti-inflammatory response to see if NFĸß is directly inhibited by Nyk pre-treatment.

23

Secondly, if NFĸß effects on the inflammatory response is inhibited, the complete pathway

from pro-inflammatory cytokines binding till NFĸß expression should be analyzed to

understand how Nyk pre-treatment reduces the up-regulation of the adhesion molecules and

subsequently neutrophil infiltration. At last, a transplantation model should be done with Nyk

treatment after brain death induction to analyze the inflammatory reduction when the donor is

not pre-treated, as is the clinical manner.

Conclusion We believe that Nyk pre-treatment is a potential suitable drug for the heart-beating donor to

reduce the inflammatory state of the kidney graft. The inflammatory expression is decreased

compared to GGA pre-treatment. At the moment of organ retrieval, Nyk pre-treatment

reduces the expression of adhesion molecules and subsequently infiltration of neutrophil

granulocytes and possible monocyte/macrophages. If the reduction is due to HSP-72

expression or HO-1 expression is unlikely, but more analysis has to be performed to measure

the expression of other HSPs first. The result of Nyk pre-treatment should be further studied

with ischemia/reperfusion experiments to analyze short-term outcomes.

Acknowledgements

Above all, I want to thank Leon van Dullemen and Henri Leuvenink for their supervising,

their approach to introduce me to translational research, and for the discussions and

advisements. Secondly, I am truly thankful for the introduction to the many analyzing

techniques and advice of Petra Ottens, Susanne Veldhuis, Janneke Wiersma, Rik Mencke and

Jacco Zwaagstra.

Lastly, I could not perform the research without the help of Dane Hoeksema and Jelle

Adelmeijer and I want to thank them for their positive influence.

24

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Attachments

Table 6 Correlation levels of Nyk pre-treated HSPs expression with inflammatory

parameters

HSP-72 HO-1 WB HSP-72

E-selectin 0.04 (P=0.92) 0.43 (P=0.40) -0.01 (P=0.98)

Icam-1 -0.05 (P=0.91) 0.60 (P=0.12) 0.08 (P=0.85)

IL-6 -0.20 (P=0.63) 0.55 (P=0.16) 0.08 (P=0.85)

Neutrophil infiltration -0.69 (P=0.06) 0.03 (P=0.94) -0.73 (P=0.04) *

M/M infiltration 0.65 (P=0.08) 0.79 (P=0.02) * 0.58 (P=0.14)

Table 7 Correlation levels of GGA pre-treated HSPs expression with inflammatory

parameters

HSP-72 HO-1 WB HSP-72

E-selectin -0.21 (P=0.62) -0.61 (P=0.11) -0.14 (P=0.77)

Icam-1 -0.49 (P=0.22) 0.31 (P=0.45) 0.39 (P=0.39)

IL-6 0.04 (P=0.94) 0.46 (P=0.31) 0.37 (P=0.47)

Neutrophil infiltration

M/M infiltration 0.36 (P=0.38) 0.07 (P=0.88) -0.30 (P=0.52)

Table 8 Correlation levels of Saline pre-treated HSPs expression with inflammatory

parameters

HSP-72 HO-1 WB HSP-72

E-selectin 0.10 (P=0.80) 0.16 (P=0.65) -0.50 (P=0.21)

Icam-1 -0.09 (P=0.82) 0.12 (P=0.76) 0.05 (P=0.91)

IL-6 -0.40 (P=0.29) -0.36 (P=0.35) 0.05 (P=0.90)

Neutrophil infiltration -0.60 (P=0.09) -0.65 (P=0.06) 0.40 (P=0.33)

M/M infiltration -0.21 (P=0.60) -0.07 (P=0.87) -0.20 (P=0.64)

HSPs= heat shock proteins, HO-1 = heme oxygenaze-1, WB = Western blot, M/M=

monocyte/macrophage, IL-6 = interleukin-6. * P-value is significant (<0.05)

HSP= heat shock proteins, HO-1 = heme oxygenaze-1, WB = Western blot, M/M=

monocytes/macrophage, IL-6 = interleukin-6. * P-value is significant (<0.05)

HSP= heat shock proteins, HO-1 = heme oxygenaze-1, WB = Western blot, M/M=

monocytes/macrophage, IL-6 = interleukin-6. * P-value is significant (<0.05)

29

Table 9 Correlation level between HSP-72 protein expression and parameters

associated with brain death induction and brain death experiment.

WB HSP-72

Noradrenalin 0.69 (P=0.06)

Haes 0.39 (P=0.34)

Weight rats -0.07(P=0.88)

Brain death induction 0.20 (P=0.64)

time

Mean arterial pressure -0.40(P=0.33)

Figure 6. Brain dead associated peripheral inflammation, as described in article reference number

26. Pro-inflammatory cytokine IL-6 produced in the brain bind to IL-6 receptors in the kidney. It activates MAP

kinases pathway, in which NFĸß induce transcription of adhesion molecules genes and further pro-inflammatory

cytokines. IL-6= interleukin-6, MAP= mitogen active protein, NFĸß = nuclear factor kappa ß

* P-value is significant (<0.05). Haes= Hydroxyethyl

starch’WB= Western blot

30

Figure 7. Induction with GGA of HSP-72 (here described as HSP70) via HSF-1 activation.

GGA= geranyl-geranylacetone, HSF-1= heat shock factor-1