The effect of donor pretreatment with the heat shock protein-inducer geranylgeranylacetone on brain death-associated inflammation in the kidney

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Donation of kidneys after brain death van Dullemen, Leon

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van Dullemen, L. (2017). Donation of kidneys after brain death: Protective proteins, profiles, and treatment strategies. Rijksuniversiteit Groningen.

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512316-L-bw-Dullemen 512316-L-bw-Dullemen 512316-L-bw-Dullemen 512316-L-bw-Dullemen Processed on: 9-8-2017 Processed on: 9-8-2017 Processed on: 9-8-2017

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The effect of donor pretreatment with the heat shock protein-inducer geranylgeranylacetone on brain death-associated inflammation in the kidney

Leon F.A. van Dullemen Henri G.D. Leuvenink

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ABBREVIATIONS DBD: Deceased Brain Dead HO1: Heme Oxygenase 1 HSP: Heat Shock Protein HSPA1A: Heat Shock Protein 70 HSPB1: Heat Shock Protein 27 HSPC1: Heat Shock Protein 90a GGA: Geranylgeranylacetone LD: Living Donor

PMN: Polymorphonuclear Cell UPS: Ubiquitin Proteasome System

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ABSTRACT

Background. Deceased brain dead (DBD)-derived kidney grafts have inferior transplantation outcomes compared to living donated kidneys. Factors affecting the graft quality are diminished organ perfusion, metabolic changes, and a systemic inflammatory reaction during the pathologic process of brain death. To reduce DBD-related kidney injury, we aim to increase the HSPA1A (HSP70) expression prior to the process of brain death. In this study we investigated whether geranylgeranylacetone (GGA), an HSPA1A inducer, can reduce the pro- inflammatory changes and improve kidney donor quality in an in vivo brain death rat model.

Method. Male F344 rats (275-300g, n=15) underwent slow induction of brain death and were kept brain dead for 4 hours. We administered GGA (400 mg/kg orally) or a saline vehicle 20h and 1h prior to brain death induction. Sham-operated animals (n=14) received the same treatment.

Results. At the moment of organ retrieval, the expressions of HSPA1A and other HSPs are not increased with GGA-treatment. However, kidney interleukin-6 (IL-6) mRNA levels in GGA pretreated DBD rats were lower compared to saline-treated controls. Systemic ASAT levels were also reduced by GGA, indicating decreased systemic injury.

Conclusion. GGA reduces some markers for injury and inflammation during the brain death period, despite the unchanged expression of HSPA1A and kidney function at the time of organ retrieval. Thus, oral administrations of GGA appears not to be an effective treatment in DBD donors.

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INTRODUCTION

Kidney transplantation is the treatment of choice for most people with end-stage renal failure.

Worldwide the majority of donor kidneys are retrieved from deceased brain dead (DBD) donors due to cerebrovascular haemorrhage or trauma. Pathophysiological changes in the DBD donor affect the organ quality, reflected by higher rates of delayed graft function (DGF) and increased risk of acute rejection, and lower survival rates.(1-3) The injury mechanism in DBD donors is not fully elucidated but it is known that it initiates a systemic stress reaction with progressive upregulation of pro-inflammatory mediators, as well as cause oxidative stress to the donor organs with subsequent induction of metabolic changes.(2,4-8) Pharmacological

‘pretreatment’ of the DBD donor to improve organ quality by reducing the exposure to stress and inflammation would an elegant approach to improve transplantation outcomes.

Upregulation of the cytoprotective Heat Shock Proteins (HSPs) is such a treatment option that could benefit potential donor organs.

Stressed cells induce the expression of protective HSP genes, named after the discovery that thermal stress is a strong inducer of these genes.(9) Other stressors to induce HSPs are ischaemia, anoxia, surgical stress, heavy metal ions, and viral agents. Following cellular stress the expression of particularly HSP72 (or HSPA1A) is rapidly increased.(10) The intracellular function of this protein is that of a chaperone, assisting in the folding or degradation of native and damaged proteins. In this way, HSPA1A inhibits the formation of protein aggregates and prevents ubiquitin proteasome system (UPS)-related stress, thus removing the stimuli for apoptosis, cell necrosis, and release of pro-inflammatory cytokines via NFkB activation.

(11,12) Upregulation of HSPA1A has shown promising results in ischaemia reperfusion injury (IRI) in in vivo animal experiments in kidney(13-17), liver (12,18,19), heart (20-22), lung (23), and small intestine (24).

Unfortunately, thermal stress is not a suitable procedure to induce HSP expression, and many drugs known to upregulate HSPA1A expression have toxic side effects. Geranylgeranylacetone (GGA) is a drug known to rapidly enhance HSPA1A expression through dissociation of Heat shock factor-1 (HSF1) from HSPA1A, which then activates transcriptional expression of the HSPA1A-gene. The optimal oral GGA dosage for up-regulating HSPA1A expression in rat kidney was found to be 400mg/kg, 24h and 1h prior to inflicting injury to the kidney.(15,22,25-29) The effects of GGA appear to be systemic, since HSPA1A upregulation was also found in liver, heart, lung, intestine, and gastric mucosa.(15,18,22,25-29) Therefore, GGA seems a suitable candidate drug for HSPA1A upregulation and protection of multiple organs against the DBD-related organ injury.

The aim of this study was to investigate whether administration of Geranylgeranylacetone (GGA) to deceased brain dead (DBD) rats can improve the quality of donor kidneys by increasing the expression of heat shock proteins.

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MATERIAL AND METHODS Animals

Adult male Fischer F344 rats (N=32), weighing 278-310 gram, were used (Harlan, Horst, the Netherlands). Animals were housed in cages at 22˚C with a light-dark cycle of 12/12h and were allowed free access to food and water. Acclimatization period was 1 week before starting experiments. The experiments were approved by the local animal care committee and performed according to the guidelines of the Institutional Animal Care and Use Committee following National Institutes of Health guidelines.

Experimental groups

To study the effects of oral GGA administration in the brain dead (BD) donor, 4 groups (n=8) were studied (Figure 1). Group 1: sham-operated treated with saline, group 2: sham-operated treated with GGA, group 3: 4h BD treated with saline, group 4: 4h BD treated with GGA. GGA (400 mg/kg) was administered 20h and 1h before the BD induction or the sham operation (group 2 and 4). GGA was purchased at Eisai CO., Ltd (Tokyo, Japan), and emulsified in 5% arabic gum (51198-250G, Sigma Aldrich, Saint Louis, US). Control rats received saline with 5% arabic gum 20h and 1h before the BD induction or the sham operation (group 1 and 3).

Brain death model

Brain death was induced as described previously.(chapter 2)(30) The procedure was as follows.

Rats were anesthetised using isoflorane (Pharmachemie BV, Haarlem, the Netherlands) with 100% O2. One cannula was inserted in the femoral artery to monitor blood pressure (BP), a second cannula was inserted in the femoral vein to administer drugs. Animals were intubated via a tracheostomy and ventilated throughout the experiment. A no. 4 Fogarty catheter (Edwards Lifesciences Co., Irvine, US) was placed intracranially via a frontolateral drillhole in the skull. The catheter was slowly inflated (16µl/min) with saline using a syringe pump (Terufusion, Termo Co., Tokyo, Japan). This slow inflation model simulates an epidural hematoma leading to brain death. During balloon inflation, a hypotensive period occurred.

When BP returned to 80 mmHg, inflation of the balloon and anaesthesia were stopped. The balloon was kept inflated throughout the experiment. BD was confirmed by the absence of corneal and pupillary reflexes, and an apnoea test. After 30 min of BD the ventilated air was switched from 100% O2 to 50% O2 in air. Temperature was monitored rectally and kept constant at 37˚C. Animals were kept BD for 4 hours. If BP 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 lung pressure, decreasing ventilation rate, administering HAES 10% (Fresenius Kabi AG, Bad Homburg, Germany), administrating noradrenalin 0.1mg/ml (Centrafarm services BV, Nieuwe Donk, the Netherlands). Exclusion criteria were a BP below 80 mmHg for more than 10 min, or a maximal administration of 10 ml of liquids. After 4 hours of brain death, 500 IU heparin (Leo Pharma BV, Breda, the Netherlands) was used 5 min before the end of the BD period. Recuronium bromide 0.6mg/kg (D50162.A, Sandoz BV, Almere, the Netherlands) was used 10 min before the end of the BD period to achieve full muscle relaxation and allow abdominal surgery. Blood and urine were collected from the aorta and bladder. Organs were

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flushed with ice-cold saline via the aorta. Left kidneys were removed and snap frozen in liquid nitrogen or fixated in 4% paraformaldehyde. Blood was centrifuged for 10 min at 960g and 4˚C.

Blood plasma was collected and stored at -80˚C. Sham operated rats (same operation except insertion of the balloon catheter) served as controls. Anaesthesia continued for 30 minutes after the sham operation to mimic the BD induction period, blood, urine, and organs were collection directly hereafter.

Histology and immunohistochemistry

Staining for polymorphonuclear cells (PMNs) was performed on kidney cryosections (5µm).

Sections were fixated using acetone. Endogenous peroxidise was blocked using 0.01% H2O2 in phosphate-buffered saline (PBS) for 30 min. Sections were stained with primary antibody His- 48 mAB (supernatant, UMCG, Groningen, the Netherlands). Staining for HSPA1A (HSP72) and ED-1 (monocytes) were performed on kidney paraffin sections (4μm). Sections were de-waxed, rehydrated and subjected to heat-induced antigen retrieval by microwave heating in 1 mM EDTA (pH=8.0, HSPA1A), or 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 antibodies included HSPA1A (SPA-810, Enzo Life Sciences, Farmingdale, US) and ED-1 (MCA341R, Serotech, Raleigh, US). Incubation of the primary antibodies on paraffin- and cryosections lasted for 60 min at room temperature (RT), binding of the antibody was detected by incubation with appropriate peroxidase-labelled secondary and tertiary antibodies (DAKO, Glostrup, Denmark) for 30 min at RT. Antibody dilutions were made in PBS 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, Glostrup, Denmark) for paraffin sections. Sections were counterstained with haematoxylin. Negative antibody controls were performed. In order to assess morphologic changes, kidney paraffin sections (4µm) were de-waxed, rehydrated and subjected to periodic acid-Schiff (PAS) reagent.

Morphometric analysis of histology and immunohistochemistry

PAS-stained kidney sections were assessed by a pathologist in a blind fashion. 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). Infiltration of PMNs and monocytes in renal tissue was assessed in His-48-stained cryosections and ED-1-stained paraffin sections. Kidney sections were scored by two observers in a blindly fashion. For each tissue section, His-48 and ED-1 positive cells were counted in 10 microscopic fields of the cortex at 200x magnification. Positive staining in renal sections were also calculated with Aperio Imagescope software including a cytoplasmic IHC algorithm (Supplementary figure 1). This algorithm is based on the spectral differentiation between red (positive) and blue (counter) staining, and validated by comparing the results to those scored in a blind fashion (Supplementary figure 2). Values are expressed as percentage strong positive surface area. HSPA1A expression in renal cortex tissue was assessed in HSPA1A- stained paraffin sections. Positive staining was assessed with Aperio Imagescope software using the same cytoplasmic IHC algorithm. Values are expressed as percentage strong positive surface area.

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79 Biochemical determinations

At the Laboratory Centre of the University Medical Centre Groningen, the following measurements were determined in a routine fashion: Creatinine in plasma and urine (31), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) enzyme activity in plasma and urine (32), lactate dehydrogenase (LDH) activity in plasma (based on the conversion rate of lactate to pyruvate), and urea concentration in plasma. Sodium and potassium in plasma and urine was determined with flow photometry. To estimate tubular damage, N-acetyl-b-D- glucosaminidase (NAG) activity in urine was measured using a method based on enzymatic hydrolysis of p-Nitrophenyl N-acetyl-b-D-glucosaminidase to p-Nitrophenyl and N-acetyl-b- D-glucosaminidase. Enzymatic activity was expressed as the amount of enzyme required to release 1µmol of product per minute. NAG levels were normalized for urine creatinine levels and expressed as U/mmol UCr. Interleukin-6 (IL-6) levels in plasma were measured using a rat IL-6 duoset ELISA (DY506, R&D systems, Abingdon, the UK) and expressed in ng/mL.

Westernblot

Per sample, six 20μm cryosections were lysed in 200µL RIPA buffer (1% NP₄₀, 0.1% SDS, 10 mM β-mercaptoethanol) containing protease inhibitors (Roche, Basel, Switzerland). 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, Hercules, US). Equal amounts of protein were loaded on to SDS/PAGE (10% polyacrylamide gels). Proteins were transferred on to nitrocellulose membranes and incubated with HSPA1A antibody (SPA-810, Enzo Life Sciences, Farmingdale, US). The house keeping-gene GAPDH (glyceraldehyde-3- phosphate dehydrogenase) was used as loading control and was detected with a secondary 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 qRT-PCR

Total RNA was isolated from kidney cryosections using the TRIzol (15504-020, Invitrogen, Waltham, US) method and using a DNase treatment step with deoxyribonuclease I (AMP-D1, Sigma Aldrich, Saint Louis, US). RNA quality was verified for absence of DNA contamination by performing real-time polymerase chain reactions (RT-PCR) in which reverse transcriptase was omitted using glyceraldehyde-3-phosphate dehydrogenase primers. Gene-specific primers were designed using Primer express 2.0 (Applied Biosystems, Foster city, US) and published gene-sequences. Primers are shown in (Table 1). Amplification and detection of the PCR products were performed with 7900 HT real-time polymerase chain reaction systems (Applied Biosystems, Carlsbad, US) using SYBR Green (SYBR Green master mix; Applied Biosystems, Carlsbad, US). 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.

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Statistical analyses

The distribution of data was assessed with Q-Q plots and the Kolmogorov-Smirnov test for normality. Data is expressed as mean ± standard deviation. Non-normally distributed data was tested with Mann-Whitney U test for difference in mean. Correlations between variables were assessed with one-way ANOVA, after logarithmic transformation if required. A p value < 0.05 was considered significant. Statistical analyses were performed using SPSS version 18.0 (SPSS Inc, Chicago, US).

RESULTS

Brain death induction

Induction of brain death showed a consistent blood pressure pattern as described before (30). The induction period took approximately 30 minutes. Blood pressure was kept at a mean arterial pressure of at least 80 mmHg during the 4h BD period. Brain dead groups had an average infusion of 3.2 mL HAES 10% and 1.6 mL noradrenalin (NA) to maintain stable blood pressure. There was no difference in administration of HAES (P=0.34) or NA (P=0.54) between brain dead groups.

Heat shock protein expression in the brain dead donor kidney

HSPA1A (HSP72) quantities in the kidney were measured using Westernblot analysis (Figure 2) and normalized for GAPDH expression on the same blot. HSPA1A levels were also assessed using immunohistochemistry and quantified using Aperio Imagescope software. No difference in HSPA1A expression was found between DBD rat pretreated with or without GGA. Heat shock protein expression was also measured by quantifying the mRNA levels using qRT- PCR for HSPB1 (HSP27), HO-1, HSPA1A, and HSPC1 (HSP90a) (Figure 3). DBD donor kidneys had increased mRNA levels of HSPB1 (P=0.013), HO-1 (P<0.01), and HSPC1 (P<0.01) compared to sham-operated controls. HSPA1A mRNA levels did not increase in the DBD donor kidney measured at the time-point of 4h brain death. GGA pretreatment had no effect on kidney HSPA1A mRNA levels in the DBD rat.

The effect of GGA on inflammation in the kidney

Brain death induced an evident inflammatory response in the donor kidney with elevated mRNA levels for the (Interleukin-6) IL-6 (P<0.01), and E-selectin (P<0.01) genes (Figure 4). In order to assess the amount of apoptosis, the ratio for the mRNA levels for Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) was calculated. DBD donor kidneys had higher Bax/Bcl-2 levels, reflecting an higher degree of activated pro-apoptotic genes (Figure 4). GGA pretreatment had a significant effect on downregulating the gene-expression for IL-6 in the DBD donor kidney (Figure 4A). A similar effect was seen in the samples analysed from DBD livers, there the relative IL-6 mRNA expression was decreased by 2-fold (P=0.011) in GGA-treated animals (data not shown).

Systemic inflammation was assessed using an Elisa-kit for IL-6 (Figure 5A). Systemic IL-6 levels in sham-operated controls were too low to measure. GGA pretreatment decreased the

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81 amount of systemic circulating IL-6, although it did not reach significance (P0.075). To evaluate the effect of HSPA1A on injury and inflammation we performed a univariate linear regression.

Renal protein expression of HSPA1A was correlated with a lower systemic IL-6 concentration (R²=0.368, P=0.01), renal IL-6 mRNA levels (R²=0.266, P=0.05), and renal influx of PMNs (R²=0.368, P=0.01).

Biochemical analysis and histological findings

Blood and urine was collected after the end of the animal experiments. After brain death, creatinine levels in plasma were 88.7µL±6.9, compared to 40.6 µL±2.9 in sham-operated rats (P<0.01). In urine, no differences were found in creatinine levels between brain dead and sham groups (5.8 mmol/L±0.52, P=0.71). After correcting urine samples for urine creatinine levels, no difference was found between sodium (10±1.56, p=0.32) and potassium (21.6±2.1, P=0.09) concentrations between BD and sham-operated rats.

To evaluate cellular damage, plasma lactate dehydrogenase (LDH), plasma and urine aspartate aminotransferase (ASAT), plasma alanine aminotransferase (ALAT), plasma urea concentration, and urine N-acetyl-b-D-glucosaminidase (NAG) were measured (Table 2). ASAT plasma levels were increased after BD, and GGA pretreatment significantly reduces ASAT plasma levels (P=0.04). The differences found in plasma LDH (P=0.19), and ALAT (P=0.23) levels were not significant between BD control and GGA pretreated rats. No effect of GGA administration was seen on the other injury parameters.

To assess the effect of brain death and GGA pretreatment on kidney injury we used a Periodic Acid-Schiff stain. The renal cortex showed no morphologic changes between sham-operated and brain dead rats. As expected, GGA pretreatment did not affect the renal morphology in DBD pretreated donors (Figure 6).

DISCUSSION

Geranylgeranylacetone (GGA) has been shown to boost HSP expression in kidneys of orally pretreated rats (15,17). Enhanced intracellular expression of HSPA1A may reduce the harmful effects of ischaemia reperfusion injury (IRI) (17) which is inevitably related to donation and transplantation. This protective effect is also documented in other organs (18,27,29). In this study we assessed the effects of oral GGA pretreatment on the organ quality of deceased brain dead (DBD) kidney donors in a rat model. Here we show that GGA pretreatment reduces renal IL-6 mRNA levels, which also could be confirmed in the livers from the same animals.

However, systemic IL-6 plasma levels from GGA pretreated brain dead rats were not found to be significantly different from untreated subjects. During brain death, the severely damaged brain could have leaked IL-6 into the systemic circulation via the destructed blood brain barrier, thus increasing the systemic IL-6 concentrations. This could possibly mask the decreased release of IL-6 from other transplantable organs, where GGA pretreatment affects the IL-6 gene expression. GGA also reduced systemic ASAT levels, an injury marker, released by the mitochondria of several organs like the liver, kidney, heart, muscles, and the brain.

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Remarkably, GGA pretreatment did not increase the expression of HSPs in renal or liver tissue in sham-operated animals (data not shown). We hypothesized that the GGA should be enhanced following a simultaneous stress stimulus at the time of administration due to the proposed working mechanism, as GGA competes with HSF1 for its binding to HSPA1A and thus facilitating HSF1 dissociation, trimerisation, and translocation to the nucleus for binding to the heat shock element (HSE) promoter region.(25,33,34) We expect that GGA functions as a HSP booster instead of an inducer, however, the stress stimulus of brain death did not boost HSPA1A protein expression in DBD pretreated rats, nor did it increase the expression of the other HSF1 induced genes like HSPB1 (HSP27) and HSPC1 (HSP90a). HSPB1, HSPC1, and HO-1 mRNA expression were increased in the DBD donor kidney compared to the sham-operated controls, but HSPA1A mRNA levels did not change in DBD donor kidneys compared to shams, illustrating the difficulties with the window of expression and time-dependant changes during brain death. Schuurs et al. have shown in the DBD rat model that HSPA1A mRNA levels are increased after 0.5 and one hour, but already normalises at four hours of brain death.(35) One explanation for the unchanged expression of HSPA1A and other HSF1 related genes in GGA pretreated brain dead rats at four hours in this present study is that the combination of GGA with the brain death stress stimulus is not severe enough to maintain upregulation of HSPA1A in this window of four hours. Suzuki et al. (15) have shown up regulation of HSPA1A after GGA pretreatment in rats subjected to 15 min of IRI. In that study, IRI produced massive injury to the kidney, reflected in a great amount of morphological changes in the renal cortex.

Brain death-related stress did not produce any morphological changes like necrosis or swollen cytoplasm in our experiment. The timing and dosage of this compound was adopted from several studies where GGA was administered once or twice 24 - 18 hours and two - one hours prior to the stress stimulus with a dosage ranging from 200mg/kg to 600mg/kg.(15,16,18,22,25- 28,36) In those studies from Suzuki et al and Wang et al the dosage, method of administration, and timing were similar to that performed in this experiment, however the injury subjected to the kidneys in those studies were IRI instead of DBD-related stress.(15,36) We suspect that the small window of four hours and the low degree of histological injury could explain why GGA pretreatment was ineffective in enhancing the HSP expression in this study. A study that did show protective properties of the heat shock proteins in DBD donors is that of Kotsch et al where authors treated rats with a single dosage of cobalt protoporphyrin (CoPP) one hour after brain death induction and were transplanted after six hours.(37) CoPP enhanced HO-1 expression and improved the survival rate after kidney transplantation in this severe kidney injury model.

A drawback of our study is that the pretreatment of the rat had to occur prior to brain death induction, in a clinical setting this would be difficult to realise. Also, the oral treatment of the DBD donor is not a practical approach, however, for GGA to function properly, it has to maintain its hydrophobic regions, which makes solubilisation of this compound extremely difficult. We have experimented with intraperitoneal administration of this compound in three rats, but this

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83 inflicted a sterile inflammatory response within 20 hours of administration. Another limitation of this study is the brain death injury model where large intra- and inter study variations are observed, as reflected by the difference in HSP mRNA expression between sham-operated and BD-induced rats in this study and that of a previous study of ours.(38)

In conclusion, our study was not able to show an increased renal expression of HSPA1A after pretreatment in donor animals prior to brain death induction. GGA pretreatment decreased renal IL-6 mRNA and systemic ASAT levels in DBD donors rats, but the effects were minimal and oral administration of GGA does not appear to be a very effective treatment option to improve the graft quality of the DBD donor. Interestingly, higher HSPA1A expression did correlate with lower systemic and renal IL-6 levels, indicating that enhancing HSPA1A expression in the DBD donor may be an interesting approach in treating and improving graft quality.

DISCLOSURE

The authors declare no conflict of interests.

ACKNOWLEDGEMENTS

The authors would like to thanks R. Mencke, D. Hoeksma, P.J. Suichies-Ottens, Z. Veldhuis, J.

Zwaagstra, and H.H. Kampinga for their help, advice, and technical support.

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TABLES AND FIGURES

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

Gene Primer sequences

β-actin 5’-GGAAATCGTGCGTGACATTAA-3’,

5’-GCGGCAGTGGCCATCTC-3’

IL-6 5’-CCAACTTCCAATGCTCTCCTAATG-3’,

5’-TTCAAGTGCTTTCAAGAGTTGGAT-3’

E-selectin 5’-GTCTGCGATGCTGCCTACTTG-3’,

5’-CTGCCACAGAAAGTGCCACTAC-3’

Bax 5’-CTGGGATGCCTTTGTGGAA-3’,

5’-TCAGAGACAGCCAGGAGAAATCA-3’

Bcl-2 5’-CGGCGGCTGGTGGTATAA-3’,

5’-CTGTAAAGGCCACCCCAGTAGTAT-3’

HSPB1 (HSP27) 5’-AAGGCTTCTACTTGGCTCCAG-3’,

5’-ACATGGCTACATCTCTCGGTG-3’

HO-1 5’-ACTTTCAGAAGGGTCAGGTGTCC-3’,

5’-TTGAGCAGGAAGGCGGTCTTAG-3’

HSPA1A (HSP72) 5’-CTGACAAGAAGAAGGTGCTGG-3’,

5’-AGCAGCCATCAAGAGTCTGTC-3’

HSPC1 (HSP90a) 5’-CATGCCACACAGATGTTTTAAATGTT-3’,

5’-GATGCTTACCTTCATTCCTTCTGATAATAT-3’

Table 2. Cellular injury parameters in plasma and urine Sham (n=7)

Sham + GGA (n=7)

BD (n=7)

BD + GGA (n=8) Plasma Urea (mmol/L) 12.1 ± 0.4 11.4 ± 0.3 20.6 ± 0.8 * 20.0 ± 0.6 Plasma LDH (U/L) 120.4 ± 8.4 148.1 ± 15.2 342.1 ± 50.1 * 251.1 ± 38.2 Plasma ASAT (U/L) 79.6 ± 5.2 74.7 ± 3.4 166.3 ± 15.0 * 120.1 ± 9.4 ^† Plasma ALAT (U/L) 66.3 ± 10.1 58.0 ± 3.2 114.3 ± 14.6 * 84.6 ± 5.6 Urine ASAT (U/mmol UCr) ^‡ 3.3 ± 0.5 6.1 ± 1.7 9.4 ± 1.8 * 9.6 ± 1.3 Urine NAG (U/mmol UCr) ^‡ 0.11 ± 0.03 0.08 ± 0.02 0.09 ± 0.02 0.13 ± 0.03 Results are presented as mean±sd. BD: brain dead, GGA: geranylgeranylacetone, LDH: lactate dehydrogenase, ALAT: alanine aminotransferase, ASAT: aspartate aminotransferase, NAG: N-acetyl-b-D- glucosaminidase

* significant at P<0.05 as compared to sham-operated controls

† significant at P<0.05 as compared to BD

‡ corrected for urine creatinine levels

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85 Figure 1. Experimental design.

Sham-operated and deceased brain dead donor rats were treated with either Geranylgeranylacetone (GGA) or a vehicle 20 hours and 1 hour prior to the operation. Organs were flushed and collected after the brain dead (BD) period of four hours. Organs from sham-operated controls were flushed and harvested after the sham-induction period of 30 minutes.

Figure 2. HSPA1A (HSP72) protein expression in the DBD donor kidney.

The degree of HSPA1A protein expression in the DBD donor kidney was not affected by GGA pretreatment measured by Westernblot analysis (A) or the amount of positive staining (B) measured in paraffin sections of vehicle-treated (C) or GGA-treated (D) DBD donor kidneys

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Figure 3. Renal HSP mRNA expression.

qRT-PCR showed an increase in (A) HSPB1 (HSP27) (P=0.013), (B) HO-1 (P<0.01), and (D) HSPC1 (HSP90a) (P<0.01) mRNA levels in DBD donor kidneys. There was no change in (C) HSPA1A (HSP72) mRNA expression between DBD and sham-operated controls. GGA pretreatment did not increase renal HSP mRNA expression levels.

Figure 4. Inflammatory and apoptosis related gene-expression.

qRT-PCR shows an increased mRNA levels of the inflammatory proteins (A) IL-6 (P<0.01) and (B) E-selectin (P<0.01) in DBD donor kidneys. Bax/Bcl2 ratio (C), which is a measure for apoptosis, also increased significantly. GGA pretreatment decreased IL-6 mRNA levels in brain dead rats (P=0.015), but had no effect on E-selectin or Bax/Bcl2 expression.

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87 Figure 5. Inflammatory status of the DBD donor kidney.

Pretreatment of DBD donor rat with GGA did not significantly decrease (A) systemic IL-6 levels (P=0.075) measured with Elisa in plasma. Systemic IL-6 levels in sham-operated rats were too low to measure. Influx of mononuclear cells (ED-1) (B) and polymorphonuclear cells (HIS48) (C) were measured on cryo- and paraffin sections respectively. In DBD donor kidneys there was an increased influx of HIS48-positive cells (P<0.01), however, GGA pretreatment had no effect on the amount of influx.

Figure 6. PAS-staining of the renal cortex.

Renal tissue from sham-operated (A), brain dead (B), sham-operated pretreated with GGA (C), and brain dead rats pretreated with GGA (D) were stained with PAS. There were no morphological changes between sham-operated and brain dead rats. Images were taken at 200x magnification.

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SUPPLEMENTARY TABLES AND FIGURES

Supplementary figure 1. Aperio Imagescope software algorithm.

(A) Infiltrating polymorphonuclear cells (PMNs) positive for His48 in the renal cortex. (B) Positive staining was calculated with Aperio Imagescope software using an algorithm based on the spectral differentiation between red (positive) and blue (counter) staining. In the algorithm, yellow represents negative staining, orange represents positive staining, and red represents strong positive staining. Images were taken at 200x magnification.

Supplementary figure 2. Validation of Aperio Imagescope software algorithm.

(A) Infiltrating polymorphonuclear cells (PMNs) in the renal cortex, stained positive for His48. The amount of infiltration was expressed as percentage positive per area (%) calculated with Aperio Imagescope software. (B) Average number of His48-positive cells in the renal cortex scored in random 10 fields at 200x magnification. The results of the quantitative analysis with the Aperio Imagescope software are comparable to the results scored in a blindly fashion.

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