University of Groningen Donation of kidneys after brain death van Dullemen, Leon

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Donation of kidneys after brain death

van Dullemen, Leon

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2017

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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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Donor pretreatment with a

HSP-inducing compound

reduces brain death-

associated inflammation in

the kidney at organ retrieval

Leon F.A. van Dullemen* Jurian H. Kloeze* Bianca J.J.M. Brundel Henri G.D. Leuvenink

*Authors contributed equally

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ABBREVIATIONS

BD: Brain Death BP: Blood Pressure CIT: Cold Ischaemia Time DBD: Deceased Brain Dead ECD: Extended Criteria Donor ESRF: End-Stage Renal Failure GGA: Geranylgeranylacetone HAES: Hydroxyethyl Starch HLA: Human Leucocyte Antigen HSF1: Heat Shock Factor-1 HSP: Heat Shock Protein

HSPA1A: Heat Shock Protein 70/-72 IRI: Ischaemia Reperfusion Injury LD: Living Donor

Nyk9354: Derivate of Geranylgeranylacetone PMN: Polymorphonuclear Cell

ROS: Reactive Oxygen Species UPS: Ubiquitin Proteasome System WBC: White Blood Cells

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ABSTRACT

Background. Brain dead-derived kidney grafts have inferior transplantation outcomes compared to living donated kidneys. This is due to increased inflammation during the brain death (BD) period. Therefore, research is directed at identification of druggable targets to enhance the expression of cytoprotective proteins and limit inflammation and injury of donated kidneys. We investigated whether intravenous (i.v.) treatment with HSP-inducing compounds, Nyk9354 or geranylgeranylacetone (GGA), can increase HSPA1A expression, provide cytoprotection and reduce the pro-inflammatory changes associated with brain death. Method. Male F344 rats (n=24) underwent slow induction of brain death and were ventilated for 4 hours. Nyk9354 (0.56 mg/kg i.v.), GGA (0.56 mg/kg i.v.) or a vehicle was administered at 20h and 0h prior to brain death induction. Kidneys, blood, and urine were collected directly after the 4h BD period.

Results. Renal HSPA1A protein expression was significantly upregulated in Nyk9354- (2.14±0.54) compared to GGA- (1.04±0.14), and saline-treated groups (0.92±0.04). Renal mRNA expression of the adhesion molecules E-selectin and ICAM-1 were significantly lower in Nyk9354- (0.56±0.08 and 0.48±0.07) compared to saline- (1.13±0.14 and 0.76±0.09), or GGA-treated groups (0.89±0.20 and 0.71±0.06). In accordance with downregulation of these adhesion molecules, renal granulocyte infiltration was significantly decreased in Nyk9354- (2.81±0.29) compared to saline- (5.78±0.94) and GGA-treated (4.40±1.27) rats. In addition, renal mRNA IL-6 expression was lower in Nyk9354- (1.08±0.46) compared to saline- (2.70±0.74), and GGA-treated (1.84±0.39) groups.

Conclusion. These results indicate that the HSP-inducer Nyk9354 is a suitable compound for enhancing HSPA1A expression and reducing pro-inflammatory changes during the brain death period.

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INTRODUCTION

Kidney transplantation is the treatment of choice for patients with end-stage renal failure (ESRF), since it provides superior quality of life and increased long-term survival compared to dialysis.(1,2) However, there is a structural shortage of transplantable donor organs resulting in a still growing number of patients waiting for a suitable graft.(3) The growing population of elderly recipients is in part the cause for the expanding waiting list since age-related comorbidity such as hypertension, diabetes, and cardiovascular disease are pertinent risk factors for developing ESRF. To date, there is no real age restriction for kidney transplantation and older individuals will qualify for an alllograft as it offers a better quality of life and survival compared to dialysis treatment, even when donor kidneys are transplantated retrieved from higher risk older extended criteria donors (ECD).(4-6) To increase the donor pool of acceptable quality and transplantable organs is essential to decrease the waiting list. The gold standard quality donor organs are obtained from young age living donors.(7) However, donor kidneys from deceased brain death (DBD) donors represent the majority of grafts used in transplantation. When compared to living donor kidneys, these are associated with a higher chance of delayed graft function (DGF) and inferior long-term outcomes, which are independent risk factors from cold ischaemia time (CIT) or human leukocyte antigen (HLA) mismatches. (7-9) As previously described, the onset of brain death causes a release of catecholamines in response to cerebral oedema and increased intracranial pressure, in order to maintain cerebral perfusion pressure. As a consequence decreased peripheral organ perfusion will occur with a production of reactive oxygen species (ROS), hormonal dysregulation, an altered metabolic state, and an increased secretion of systemic pro-inflammatory cytokines, followed by an influx of monocytes and neutrophils.(10-16) During brain death the cells in the kidney will also enhance the expression of cytoprotective proteins, in particular of heat shock proteins (HSPs). (16,17) Heat shock protein-72 (HSP72 or HSPA1A) is a cytoprotective chaperone that is rapidly upregulated after stress and during brain death. It is conceivable that the balance between cytoprotective and inflammatory proteins eventually determines graft quality. Enhanced expression of HSPA1A may provide protection against the detrimental effects of ischaemia reperfusion injury (IRI) in the kidney, liver, and heart.(18) HSPA1A is an especially interesting agent as the protein can be targeted with the non-toxic drug GeranylGeranylAcetone (GGA). (19) However, GGA is a very hydrophobic compound and difficult to solubilise (Log P value of approximately 9), limiting the concentration that can be administered intravenously. Oral administration of GGA has shown to be protective in renal ischaemia reperfusion injury (IRI), but oral ingestion is not feasible in a DBD donor.(18,20) Several derivates of GGA have been synthesised with improved pharmacochemical properties and shown to express a more potent ability to provoke a heat shock response in cardiomyocytes and Drosophila melanogaster.(21) The possibility of intravenous administration and the need of lower concentrations to elicit a heat shock response are clinically relevant benefits of this compound.

The aim of this study was to reduce the inflammatory response in the DBD donor. Therefore, we tested the effects of GGA and of the promising derivate Nyk9354 in DBD donor rats, assessing the effect on kidney organ quality and inflammation after a period of four hours of brain death.

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MATERIAL AND METHODS

Animals

Adult male Fischer F344 rats (N=24), weighing 281-308 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 one 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 HSPA1A (HSP72) upregulation on DBD-related kidney injury, three groups were studied (Figure 1). Group 1: BD treated with saline 20h and 0h before BD induction, group

2: BD treated with geranylgeranylacetone (GGA) (0.56mg/kg) 20h and 0h before BD induction, group 3: BD treated with Nyk9354 (0.56mg/kg) 20h and 0h before BD induction. Nyk9354 was

kindly provided by Nyken BV (Groningen, The Netherlands) and solubilised in 0.9% NaCl for infusion (Baxter BV, Utrecht, The Netherlands). GGA was purchased at Eisai Co., Ltd (Tokyo, Japan), and solubilised 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%. Saline with 10% kleptose HPB and 0.5% DMSO was administered as a vehicle control.

Brain death model

Brain death was induced as described previously (Figure 2).(22) 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 four 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 10% hydroxyethyl starch (HAES) (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

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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 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.

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 monocytes was performed on kidney paraffin sections (4μm) with a monoclonal antibody directed against ED-1 (also known as CD68) (MCA341R, Serotech, Raleigh, US). 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). Endogenous peroxidase was blocked with 0.03% H2O2 in PBS for 30 min. 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-labeled 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. Analysis of Immunohistochemistry

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 who were blinded for the group allocation of each section. For each tissue section, His-48 and ED-1 positive cells were counted in 10 microscopic fields of the cortex at 200x magnification. Biochemical measurements

At the clinical laboratory facility of the UMCG, the following measurements were determined in a routine fashion: creatinine in plasma and urine (23), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) enzyme activity in plasma and urine (24), 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 were determined with flow photometry.

Western blotting

Per sample, six 20μm cryosections were lysed in 200 µL RIPA buffer (1% NP₄₀, 0.1% SDS, 10 mM β-mercaptoethanol) containing protease inhibitors (Roche, Basel, Switserland). 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

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amounts of protein were loaded on to SDS/PAGE (10% polyacrylamide gels). Proteins were transferred 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 anti-mouse 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 series. The samples were amplified as follows, first an activation step at 50°C for two 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.

Cell culture and RNAi transfection

Cell culture experiments were performed with a human kidney cell-line (HK-2)(American Type Culture Collection (ATCC), Rockville, US). HK-2 cells were cultured in a monolayer in a DMEM (Lonza, Basel, Switserland) supplemented with 1% penicillin, 1% streptomycin, supplemented with L-glutamin and 10% fetal bovine serum in humidified air with 5% CO2 at 37°C. HK-2 cells (1 x 105_{) were seeded in 6-wells plates and transfected using HSPA1A and HSF1 targeted silencer}

select RNAi. RNAi for HSPA1A and HSF1 are shown in Table 2 (Thermofisher, Waltham, US). All controls in the RNAi-experiment were performed using scramble RNAi. Transfection was performed using Lipofectamine-2000 (Thermofisher, Waltham, US) according to manufacturer’s protocol. The amount of siRNA was optimized to achieve a knockdown of 80% after five days. To increase HSPA1A expression, cells were serum starved for 12 h before stimulation with a heat shock of 42°C for 30 min. After stimulation, cells were trypsinised and used for either measuring phagocytosis or analysed for HSPA1A expression.

Phagocytosis assay

Blood from healthy volunteers was collected and red blood cells were lysed at 4°C using ammonium chloride (UMCG, The Netherlands) and centrifuged for five min at 1000g. The pellet

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of white blood cells (WBC) was resuspended in RPMI (Lonza, Basel, Switserland) and stored at 37°C until use. HK-2 cells were labelled using CFSE (Thermofisher, Waltham, US) in serum-free medium and incubated for 20 minutes at RT, after incubation the cells were washed in medium with 10% serum and added to the WBC. A total of 3 x105_{WBC and 3 x10}5_{HK-2 cells were added}

in one vial and incubated for two hours prior evaluation with flow cytometry a FACSCalibur (Beckton Dickinson, Germany) and analysed using Cytoflog software (Cytoflog Ltd, Finland). The FACS was gated for granulocytes and the amount of apoptosis was calculated according to the amount of CFSE-positive granulocytes. Incubation of HK-2 cells with an antibody directed against CD47 functioned as a positive control.

Statistical analyses

The distribution of data was assessed with Q-Q plots and the Kolmogorov-Smirnov test for normality. Normally distributed data are expressed as mean ± standard deviation. Non-normally distributed data was tested with Mann-Whitney U tests for differences of the mean. A p value < 0.05 was considered significant. Statistical analyses were performed using SPSS version 22.0 (SPSS Inc, Chicago, US).

RESULTS

Brain death induction

Induction of brain death (BD) took approximately 30 minutes and showed a consistent blood pressure pattern as described before.(22) Blood pressure was kept at a mean arterial pressure of at least 80 mmHg during the four hour BD period. BD groups had an average infusion of 1.1 (±0.45) mL HAES 10% and 1.0 (±1.03) mL noradrenalin (NA) to maintain a stable blood pressure profile (Table 3). There was no difference in administration of HAES or NA, mean arterial pressure (MAP), and blood pressure profile between pretreated brain dead groups (Figure 3). Nyk9354 induced HSPA1A expression in the brain dead donor kidney

HSPA1A levels in the kidney were measured using Western blot analysis (Figure 4 and S-figure 1) and normalized for GAPDH expression on the same blot. HSP expression was also measured by quantifying the mRNA levels using qRT-PCR for HSPA1A and HO-1 (Figure 4). Nyk9354-treated brain dead rats had higher protein levels of HSPA1A (P=0.042) compared to saline-treated controls. The mRNA levels of HSPA1A and HO-1 from Nyk9354- or GGA-saline-treated rats were comparable to saline-treated rats at the time-point of 4h brain death.

Nyk9354-treatment protects against renal inflammation

To assess the effect of Nyk9354-treatment on BD-induced renal inflammation, the mRNA levels for interleukin-6 (IL-6), E-selectin (also known as CD62E or ELAM-1), and intracellular adhesion molecule 1(ICAM-1) were determined (Figure 5). Nyk9354-treated DBD rat kidneys showed a significant reduction in expression levels of IL-6, E-selectin, and ICAM-1 compared to saline-treated controls. GGA-treatment showed no difference in renal mRNA expression of IL-6, E-selectin, or ICAM-1 compared to saline-treated controls. To evaluate the effect of adhesion

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molecule downregulation by Nyk9354 treatment, we assessed the amount of granulocyte and monocyte infiltration in the renal cortex. Nyk9354- and GGA treatment both significantly inhibited the influx of His48 positive-stained granulocytes (Figure 6) however, no effect of either treatment was found on the influx of ED-1-positive monocytes (data not shown). In order to analyse the amount of apoptosis, the ratio for the mRNA levels for Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) was calculated. Nyk9354- and GGA- treatment of the DBD donor kidneys had no effect on Bax/Bcl-2 levels (Figure 5). Interestingly, the mRNA levels of Bcl-2 were significantly lower (P=0.02) in Nyk9354- (0.59±0.04) compared to GGA- (0.69±0.04) or saline treated rats (0.78±0.08), while the mRNA levels of BAX in Nyk9354- (1.89±0.61), GGA- (1.99±0.17), or saline-treated rats (1.81±0.17) were not changed.

Nyk9354 treatment does not prevent kidney dysfunction or cellular damage

Blood and urine was collected after the end of the animal experiments. After BD, creatinine levels in plasma and urine did not show improvement after Nyk9354- or GGA treatment (Table 3). To evaluate cellular damage and kidney function, plasma lactate dehydrogenase (LDH), plasma aspartate aminotransferase (ASAT), and fractional sodium excretion were calculated (Table 3). No effect was seen of Nyk9354- or GGA treatment on plasma LDH- and ASAT levels, or fractional sodium excretion.

HSPA1A does not modulate phagocytosis in human kidney cells

To assess whether high levels of intracellular HSPA1A could affect the activation of granulocytes we designed a phagocytosis assay. HK-2 cells were treated with a heat shock (HS) and/ or with RNAi for HSPA1A and HSF1. HSPA1A increased 5-10 fold after a HS. Treatment with RNAi for HSPA1A and HSF1 resulted in an 80% knockdown (Figure 7). The amount of base-line phagocytosis was around 5% and upregulation or knockdown of HSPA1A and HSF1 did not affect the amount of phagocytosis of HK-2 cells by healthy human granulocytes (Figure 8).

DISCUSSION

In this series of experiments we have assessed the effects of intravenous administration of the GGA derivate Nyk9354 prior to brain death induction in a rat model. We show that it is possible to prevent pro-inflammatory changes in the kidney of a deceased brain dead donor by upregulation of heat shock protein HSPA1A. Nyk9354 pretreatment prior to brain death induction resulted in lower gene-expression of the pro-inflammatory cytokine Interleukin-6, and the adhesion molecules E-selectin and Intercellular Adhesion Molecule-1 (ICAM-1). Nyk9354 pretreatment also resulted in decreased influx of polymorphonuclear cells, which is consistent with a lower expression level of adhesion molecules. HSP upregulation has been previously shown to prevent cell death. In our study, however, we obsereved downregulation of the anti-apoptotic protein Bcl2, whilst the ratio between Bax (pro-apoptotic) and Bcl2 was not significantly altered. The mechanism leading to this change was not further explored, but since Bcl2 also has anti-autophagic properties (25), the decreased expression of Bcl2 could reflect a cellular switch to autophagy. Combining these results, we suggest that this study shows that

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DBD donor pretreatment with Nyk9354 can prevent the development of pro-inflammatory changes in the renal graft prior to transplantation.

Our group and those of others have previously demonstrated that brain death induces a profound pro-inflammatory and pro-coagulatory systemic response that increases the immunogenicity of the donor organ.(15,16,26-28) Graft quality and long-term allograft dysfunction are to some extent defined early on in the donor and certainly prior to organ retrieval.(8,29,30) The hostile pro-inflammatory environment renders the graft more susceptable to the damage associated with prolonged CIT and warm IRI, resulting in delayed graft function (DGF) and reduced graft survival.(7,31) Currently, the management of the deceased donor focuses on three aspects; haemodynamic stability, ventilatory support, and hormonal replacement therapy. However, to date no target specific treatments have been adopted in the donor prior to organ procurement.(32) Nyk9354 pretreatment could potentially be a suitable drug to better condition DBD donors. As mentioned above, Nyk9354 is a derivate of the non-toxic anti-ulcer drug GGA, but with a superior HSPA1A upregulating potential in cardiomyocytes and in the DBD rat kidney.(21) GGA facilitates the dissociation of heat shock factor-1 (HSF1) from HSPA1A, which is then able to translocate to the nucleus and bind to the promotor region of HSPA1A, enhancing HSPA1A expression.(33) HSPA1A is a cytoprotective chaperone that assists in the refolding or degradation of denaturated proteins.(34-37) HSPA1A has shown to be rapidly induced after cellular stress and is also an accurate biomarker for renal IRI.(38) The rapid induction properties of HSPA1A make it a suitable candidate for targeted treatment in kidney donation and transplantation. GGA treatment and HSPA1A upregulation has shown to be protective in several animal kidney IRI studies.(18,20,39,40) The studies conducted with GGA have used oral administration of this compound, which is an impractical administration route in deceased donors. Nyk9354 has improved pharmacochemical properties, allowing to solubilise in water achieving concentrations of up to 31mg/ml, whilst GGA can only be solubilised up to 0.5mg/ml.

The anti-inflammatory effects of HSPA1A upregulation can be explained by its assisting in protein (re)folding and targeting proteins in the ubiquitin proteasome system (UPS). Brain death- and inflammatory-related oxidative stress results in protein modification and damage. Consequently these damaged proteins tend to aggregate and form aggregate-like induced structures. Irreversibly damaged proteins are potentially toxic and require elimination which is mediated by UPS or autophagy.(41) Removal of these cell-toxic aggregates upon UPS upregulation by HSPA1A prevents NFkB-activation, cytokine release, and innate immune system activation in the cell.(42) This is important as the activation of the innate immune system will lead to complement formation of factor C3a, C5a, and C5b release, which is known to enhance influx and activation of neutrophils.(43) In our study we showed that up- or downregulation of HSPA1A did not affect the activation and extent of phagocytosis by granulocytes added

in vitro to HK-2 kidney cells. This is consistent with the literature describing that HSPA1A is

an intracellular cytoprotective molecule and suggesting that enhancing the expression of this protein does not increase the kidney graft’s more immunogenicity. The inhibiting effect of HSPA1A on cytokine production and granulocyte influx is most likely related to lower levels of

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NFkB activation and cell necrosis. Such inhibitory effects of HSPA1A have also been reported in a sepsis and an acute respiratory distress rat-model.(44,45)

In this brain death model we did not see any effect of Nyk9354 pretreatment on the kidney function reflected by fractional sodium excretion, and systemic or urinal creatinine and urea levels. However, donor kidney function does not seem to be a very reliable parameter for posttransplantat graft survival. Damman et al. showed that pretreatment of brain dead rats with the soluble complement receptor inhibitor (sCR1), did reduce the inflammatory response significantly but not affect plasma creatinine levels in the donor. In contrast, the same donor kidney pretreatment was effective in lowering creatinine levels in the recipient after allogeneic kidney transplantation.(46) To date, there is no reliable parameter to predict kidney function of the deceased donor after transplantation. The use of pretransplantation biopsy scoring in combination with donor age, CIT, donor hypertension, and premortem creatinine plasma levels may provide the best clinical assessment to predict short-term graft function. However, no scoring system based on clinical parameters has yet been able to accurately define which organs should be discarded due to a high risk of graft failure.(47,48)

This study shows that upregulation of HSPA1A with Nyk9354 in the organ donor could be a potential therapeutic approach to improve the kidney graft quality. It is conceivable that targeting multiple injury mechanisms during transplantation is probably the most successful approach, aiming at preventing the accumulation of reactive oxygen species, enhancing the expression of cytoprotective proteins, and of autophagy, as well as inhibition of the innate immune system. Several of these target mechanisms have been tested in transplantation-related animal models and were reviewed by Mundt et al.(32)

In conclusion, we show that pretreatment of the DBD donor with Nyk9354 is a feasible approach to enhance HSPA1A expression and reduce the expression of pro-inflammatory genes and influx of polymorphonuclear cells, suggesting that treatment of the DBD donor with NYK9354 could improve kidney graft quality.

DISCLOSURE

The authors declare no conflict of interests.

ACKNOWLEDGEMENTS

We would like to thank P.J. Ottens and Z. Veldhuis for their technical assistance, furthermore we would like to thank dr. H. Steen and dr. M. de Haan for providing Nyk9354 and W.T. van Haaften for critically reviewing this manuscript.

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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’ ICAM-1 5’-CCAGACCCTGGAGATGGAGAA-3’ 5’-AAGCGTCGTTTGTGATCCTCC-3’ Bax 5’-CTGGGATGCCTTTGTGGAA-3’, 5’-TCAGAGACAGCCAGGAGAAATCA-3’ Bcl-2 5’-CGGCGGCTGGTGGTATAA-3’, 5’-CTGTAAAGGCCACCCCAGTAGTAT-3’ HO-1 5’-ACTTTCAGAAGGGTCAGGTGTCC-3’, 5’-TTGAGCAGGAAGGCGGTCTTAG-3’ HSPA1A (HSP72) 5’-CTGACAAGAAGAAGGTGCTGG-3’, 5’-AGCAGCCATCAAGAGTCTGTC-3’

Table 2. RNAi sequences for the genes (sense, antisense)

Gene Primer sequences

HSPA1A (HSP72) 5’-CGAUAUGUUCAUUAGAAUUtt-3’ 5’-AAUUCUAAUGAACAUAUCGgt-3’

HSF1 5’-GGACAAGAAUGAGCUCAGUtt-3’ 5’-ACUGAGCUCAUUCUUGUCCag-3’

Table 3. Brain death-related parameters

Saline GGA Nyk9354 P-value

Brain death induction time (min) 32.8±1.8 32.6±1.4 32.9±2.1 0.87

Mean arterial pressure (mmHg) 105±9 101±12 101±11 0.92

Weight (gram) 295±13 292±5 281±32 0.93

HAES administration (ml) 1.5±0.2 1.0±0.2 1.0±0.3 0.825

Noradrenalin administration (ml) 1.1±0.3 1.0±0.5 1.1±0.5 0.79 Data is presented as mean±sd

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Table 4. Biochemical parameters in plasma and urine

Saline GGA Nyk9354 P-value

Plasma sodium (mmol/L) 142.5±1.0 141.4±1.2 141.5±2.0 0.58

Plasma creatinine (µmol/L) 73±18 81±24 84±27 0.71

Plasma LDH (U/L) 461±218 497±231 371±299 0.42

Plasma ASAT (U/L) 130±24 148±39 119±27 0.56

Urine sodium (mmol/mmol Ucr) 38±25 44±28 57±44 0.64

Urine creatinine (µmol/L) 5.3±1.6 5.7±1.9 5.3±2.0 0.62

Fractional sodium excretion 0.36±0.18 0.43±0.30 0.66±0.33 0.38

Data is presented as mean±sd

Figure 1. Experimental design.

Deceased brain dead donor rats were treated with a bolus of either Nyk9354 (0.5mg/kg), geranyl-geranylacetone (GGA) (0.5mg/kg) or a vehicle 20 hours before and at the start of the operation. Organs were flushed and collected after the brain dead (BD) period of four hours.

Figure 2. Blood pressure profile.

Mean arterial blood pressure (MAP) was recorded at the start of brain death induction and considered as time 0 at the start of the brain death period. MAP was maintained above 80mmHg by administration of either noradrenalin or HAES. There was no difference in the blood pressure profile between the different treatment groups.

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Figure 3. Renal heat shock protein expression.

Pretreatment of DBD donors with Nyk9354 resulted in higher upregulation of HSPA1A (HSP72) protein expression (P=0.042). Renal mRNA levels for HSPA1A (HSP72) and HO-1 were not changed between different treated groups at the time point of 4 hours brain dead.

Figure 4. Renal inflammation and apoptosis related gene-expression.

qRT-PCR analysis showed lower levels of IL-6 (P=0.049), E-selectin (P<0.01), and ICAM-1 (P=0.037) in Nyk9354-treated DBD donors compared to saline-treated controls. Bax/Bcl2 ratio was not significantly changed between different treatment groups.

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Figure 5. Influx of polymorphonuclear cells.

Influx of HIS48-positive polymorphonuclear cells (PMNs) was counted in 10 microscopic fields at 200x magnification for DBD rats treated with a vehicle (B), GGA, or Nyk9354 (C). Pretreatment with Nyk9354 (P=0.001) and GGA (P=0.008) resulted in a decreased influx of PMNs (A).

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Figure 6. Effect of heat shock on HSPA1A (HSP72) protein expression.

A heat shock (HS) at 42°C for 30min, 12 hours prior to supernatant collection showed an 10-fold upregulation. Transfection of HK-2 cells with RNAi against HSPA1A and HSF1 resulted in HSPA1A knockdown of 80% measured with Western blot.

Figure 7. Phagocytosis of HK-2 cells.

Phagocytosis HK-2 cells by granulocytes was measured using FACS. The FACS was gated for granulocytes

(A), subsequently the outliers were selected and removed using a negative control as reference (B),

resulting in a percentage of CFSE-positive granulocytes as shown in a positive control (C). D. Phagocytosis of HK-2 cells was increased after incubating the cells with a CD47 antibody (positive control), upregulation of HSPA1A with a heat shock (HS) or downregualtion with RNAi for HSPA1A and HSF1 did not the affect phagocytosis of HK-2 cells. Incubation of granulocytes with CFSE-stained HK-2 cells at 4°C served as a negative control.

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SUPPLEMENTARY DATA

Supplementary figure 1. Original western blot data for HSPA1A and GAPDH on kidney tissue.

Equal amounts of tissue from rats treated with vehicle (V), Nyk9354 (N2), or geranylgeranylacetone (G) was loaded on a gel and transferred to a blot. On every blot a control (C) was loaded to correct for inter-blot variability. Some animals were treated with a single injection of Nyk9354 (N1), however this group was not included in this article.

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