Donation of kidneys after brain death van Dullemen, Leon

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University of Groningen

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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Protective Proteins, Profiles, and Treatment Strategies

Leon Frederik Albert van Dullemen

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ISBN: 978-94-028-0729-5 Cover design: Jan Willem Deiman

Lay-out: Tara Kinneging, Persoonlijk Proefschrift Printed by: Ipskamp Printing (www.proefschriften.net)

All rights reserved. No part of this thesis may be reproduced or transmitted in any form of by any means, electronically or mechanically, including photocopy, recording or any other information storage or retrieval system, without permission in writing from the author, or, when appropriate, of the publishers of the publications. Financial support by the Dutch Kidney Foundation, Junior Scientific Masterclass, and University Medical Center Groningen for the publication of this thesis is gratefully acknowledged.

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Donation of Kidneys after Brain Death

Protective Proteins, Profiles, and Treatment Strategies

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op maandag 18 september 2017 om 09.00 uur

door

Leon Frederik Albert van Dullemen

geboren op 29 juni 1988

te Tilburg

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Promotores

Prof. dr. H.G.D. Leuvenink Prof. dr. R.J. Ploeg

Beoordelingscommissie

Prof. dr. J.L. Hillebrands

Prof. dr. B. Jespersen

Prof. dr. R.P.H. Bischoff

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drs. M.H.W. van Dullemen

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Chapter 1.

Introduction and rationale 9

Chapter 2.

Heat shock proteins and their protective role in organ transplantation 21

Chapter 3.

Brain death induces renal expression of heme oxygenase-1 and heat shock

protein-A1A 55

Chapter 4.

The effect of donor pre-treatment with the heat shock protein-inducer

geranylgeranylacetone on brain death-associated inflammation in the kidney 73

Chapter 5.

Donor pre-treatment with Nyk9354, a geranylgeranylacetone derivate, reduces brain death-associated inflammation in the kidney at organ retrieval 93

Chapter 6.

Systematic review for the treatment of deceased organ donors 115

Chapter 7.

Lipid catabolism provides an alternative energy source and compensates for

mitochondrial dysfunction in reperfused rat kidneys after ischaemia 155

Chapter 8.

Deceased donor kidney proteomic profiles correlate with 12-month allograft

function after transplantation 221

Chapter 9.

Impact of pre-analytical factors on the proteome and degradome in human blood 253

Chapter 10.

Discussion and future perspectives 293

Nederlandse samenvatting 301

Author affiliations 305

Dankwoord 307

About the author 311

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Introduction and rationale

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Chapter 1

ABBREVIATIONS

BD: Brain Death

CAN: Chronic Allograft Nephropathy CKD: Chronic Kidney Disease DBD: Donation after Brain Death ESRD: End-stage Renal Disease GGA: GeranylGeranylAcetone HO1: Heme Oxygenase-1 HSPA1A: Heat Shock Protein 70 ICU: Intensive Care Unit

IRI: Ischaemia Reperfusion Injury

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RENAL FAILURE AND KIDNEY TRANSPLANTATION

Chronic kidney disease (CKD) is the progressive loss of kidney function and is recognised as a public health problem, affecting 10% of the Dutch population.(1) Major risk factors for developing CKD are diabetes mellitus, elevated blood pressure, and atherosclerosis, explaining the higher prevalence of CKD with age. Every year about 2.000 people develop end-stage renal disease (ESRD) in The Netherlands.(2) ESRD is a great burden to the patient, as it is associated with many complications e.g. cardiovascular disease, and has to be eventually treated with renal replacement therapies. The most common treatment for ESRD is dialysis with currently 6.500 people subjected to this type of treatment in The Netherlands.(2) Although patients with ESRD obviously can survive whilst on dialysis compared to no treatment, the 5-year patient survival rate is still on average only 50%.(2) To date the preferred choice of treatment for ESRD is kidney transplantation for those in good enough health, which is life prolonging, quality of life enhancing, and becomes cost-effective within two years after transplantation.(3) The availability of kidney transplantation is however limited since there is a persistent shortage of viable donor organs. The average time a patient has to wait for an organ in The Netherlands is 3.2 years, and as a consequence 10-12% of patients waiting for a kidney will die or become too ill to receive a transplant.(4) Therefore, it is of crucial importance to be able to optimise all potential donor kidneys and utilise most of them once they have been procured. Unfortunately, in a deceased donor the sequence of cerebral injury, followed by donor management, procurement and preservation will expose the graft-to-be to a series of inevitable injury mechanisms that negatively affect the graft quality and outcome after transplantation. After the period of donor management in the intensive care unit (ICU) and during procurement and cold preservation, the kidney graft becomes subjected to ischaemic injury followed by reperfusion injury at time of transplantation in the recipient. The transplanted organ will find itself in a rather hostile immune system of the recipient that is activated even more when recognising the pre-existent injury due to donation and after transplantation. Common strategies to reduce injury and increase the chance of immediate function are to improve organ storage conditions by using machine perfusion at either hypo- or normothermic temperatures, as well as administering dedicated immunosuppressive therapies to the recipient.

It is important to realise that donor organ injury does not start with preservation as viable organs can also be injured in the donor even prior to organ procurement and cold storage.

Different type of organ donors are associated with different short- and long-term results, and kidneys from deceased donors after brain death (DBD) have been reported to result in inferior transplantation outcomes when compared to those derived from living donors.

(5) Due to the donor organ shortage in The Netherlands, living kidney donation has been stimulated significantly in the past decade, resulting nowadays in that approximately half of the kidneys transplanted are derived from living related or unrelated donors whilst the other half are procured from deceased donors(4). This situation is quite unique to The Netherlands as in most other developed countries the majority of kidneys comes from DBD donors.

(2) Unfortunately, the physiological state of brain death itself predisposes for considerable metabolic- and pro-inflammatory changes in donor kidneys prior to procurement and with the

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ischaemia and reperfusion injury (IRI) still to be added after preservation.(6-8) Prevention or at least reduction of this type of early donor injury could ameliorate early graft function as well as render grafts less susceptible to chronic allograft nephropathy (CAN) with better long-term graft survival.

BRAIN DEATH AND ITS IMPACT ON THE DONOR ORGAN

Brain death is defined as an irreversible coma with absence of brain stem reflexes, and apnoea but with a functioning systemic circulation. The major causes of brain death (BD) are cerebrovascular injury, anoxia, or traumatic injury, and the clinical diagnosis of brain death should only be considered in patients that suffer from severe irreversible brain injury of an identifiable origin. Patients that are diagnosed with BD are considered dead and organs donated from these patients for transplantation are referred to as deceased BD donor organs.

Although the patient remains ventilated during their stay in the ICU and a stable blood pressure is maintained, the organs procured from these donors are more susceptible to IRI, cold storage and rewarming injury, and immune mediated graft injury. During the onset of BD a cascade of events occur due to the cerebral ischaemia, cerebral oedema, and cerebral hypertension.

The increased intracranial pressure results in a strong parasympathic response followed by a sympathic response with severe vasoconstriction due to endogenous catecholamine release.

This haemodynamic response is named the Cushing reflex that aims to maintain adequate cerebral perfusion pressure.(9) After the onset of BD there is an increased systemic circulation of cytokines, chemokines, and adhesion molecules.(6,10-13) Eventually BD results in organ dysfunction due to a series of detrimental responses including; microthrombus formation, decreased peripheral perfusion, generation of reactive oxygen species (ROS), increased vascular permeability, leukocyte mobilisation and infiltration, and pro-inflammatory changes in the transplantable organs (Figure 1).(6,7,11,13,14)

The trigger for the inflammatory response is not fully understood, but cerebral injury itself is associated with a systemic inflammatory response syndrome (SIRS).(15,16) In addition, the integrity of the blood-brain barrier is lost during brain death, resulting in release of central nervous system-derived cytokines, like matrix metalloproteinases, that may induce inflammation in the peripheral organs.(17-19) The pathological process of brain death including its systemic changes has a detrimental effect on organ quality. Kidneys having suffered from BD induced haemodynamic instability and inflammation, have been reported to have higher rates of primary non-function and poorer short- and long-term outcomes when compared to optimal living donors after transplantation.(5,20,21) Several studies have shown increased plasma creatinine levels in brain dead animals and humans, increased leukocyte influx, enhanced expression of vascular adhesion molecules, and activation of the innate immune system.(10,11,22) Also, other donor organs such as liver, heart, and lungs are affected by this pathological process of brain death demonstrating significant pro-inflammatory changes.

(13,17,23) Therefore, early intervention in the donor after diagnosis of brain death could prevent the detrimental changes, potentially better preserving organ quality, and improving

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13 transplantation outcomes.

At the same time, it should be noted that not only harmful molecules become upregulated during BD. There is also an enhanced simultaneous expression of cytoprotective proteins, especially those of the family of heat shock proteins.(10,11,24) In particular heat shock protein-72 (HSP72 or HSPA1A) and heme oxygenase-1 (HO1 or HSP32) appear to be rapidly upregulated after cellular stress and during BD. In tissue culture and animal models, elevated levels of HSPs can substantially reduce the level of cell death and has led to the insight that HSPs can protect cells or even influence the course of disease.(25-27) Enhanced expression of HSPA1A and HO1 are able to provide protection against the detrimental effects of ischaemia and reperfusion injury (IRI) in the kidney(28,29), liver(30,31), and heart(32,33). In the context of brain death it is conceivable that the balance between cytoprotective- and inflammatory proteins eventually will determines the graft quality, function, and survival.

RATIONALE

The aim of this thesis is to explore the expression and properties of protective proteins and pathways in the deceased donor kidney after brain death to improve donor quality and outcomes after kidney transplantation. It is conceivable that enhancing the expression of protective proteins may improve the capacity of the donor organ to better cope with injury during donation and transplantation, and therefore provide benefit to the recipient with improved graft quality. This approach is investigated in the first part of this thesis by exploring the expression profile and protective properties of heat shock proteins in the setting of the deceased brain dead donor. The goal of this thesis is to improve the outcome after kidney transplantation. Since donor management affects all organs, the effects of protective strategies was also considered for other transplantable-organs in chapter 2 and 6.

In Chapter 2 the protective working mechanism of the family of heat shock proteins is explained, including HSPA1A and HO1. Chapter 2 also contains a detailed overview of the immune-active properties of heat shock proteins, since their release in the extracellular space can elicit an immune response. Especially donor organs from deceased donors are susceptible to inflammation. Despite the ability of heat shock proteins to induce inflammation, it appears that upregulation of these proteins is protective during ischaemia reperfusion injury and organ transplantation. The evidence for these protective effects is reviewed in the final section of Chapter 2.

In Chapter 3 the effect of brain death-related kidney stress on heat shock protein expression is assessed in an animal model of brain death that has been developed by our group. The expression levels of several heat shock proteins are objectified using different techniques after a period of four hours of brain death. In particular, expression levels of HO1 and HSPA1A are analysed whether they appear to be upregulated.

Not only cellular stress, but also circulating compounds can enhance the expression of

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cytoprotective proteins. In Chapter 4 we aimed to stimulate heat shock protein levels in the donor kidney to protect the renal tissue from the hostile environment during brain death. To accomplish this, animals are treated orally with the drug GeranylGeranylAcetone (GGA) at 24 hours prior to the induction of brain death. Protective effects are evaluated.

As oral administration of drugs is not the preferred route for deceased brain dead donors, since the absorption is not optimal during this pathophysiological state, in addition, a derivate of GGA with improved pharmacochemical properties will be used to investigate whether the expression of HSPA1A increases in the deceased brain dead donor kidney. In Chapter 5 we describe the effect after intravenous administration of this compound prior to brain death induction on the inflammatory and cytoprotective response. Treating the donor prior to organ procurement could be an elegant and also clinically relevant strategy to improve the donor quality.

In Chapter 6 the results of a systematic review and meta-analyses are reported for different treatment strategies for deceased donors tested in randomised controlled trials.

In the latter part of this thesis the injury mechanisms leading to inferior graft quality in kidney transplantation are explored on a more molecular level. The goal of these studies is to reveal injury mechanisms or pathways that may either be affected after intervention, or could assist in better assessment of the donor organ quality.

In Chapter 7 the effect of ischaemia and reperfusion injury is investigated on the molecular level in a rat model by applying 45 minutes of ischaemia to the kidney followed by subsequent four or 24 hours of reperfusion. IRI is especially relevant to donor management since the organs from deceased circulatory dead donors endure prolonged periods of ischaemia, after which reperfusion injury occurs. Since IRI is inevitable in solid organ transplantation, a better understanding of the molecular mechanisms leading to injury after reperfusion could identify new treatment strategies to prevent detrimental effects. Mass spectrometry is used to identify the changes occurring after IRI on the protein and metabolite level.

In Chapter 8 we analyse kidney biopsies from deceased brain dead donors with either a good or suboptimal kidney function after transplantation. Using an -Omics approach, the goal is to see whether a difference can be observed in the donor proteomic profile at time of procurement of those transplanted kidneys that were found to have either a good or a suboptimal transplant function at the long-term. With the current need to accept more older and higher risk donor kidneys to meet the demand of patients on the waiting list, clinicians often have the dilemma of uncertainty when they have the choice to accept or decline a kidney. To date, the transplant community lacks more sophisticated diagnostic tools to support evidence-based decisions whether to accept or discard a kidney for transplantation. The use of a pre-transplantation biopsy scoring combined with donor age, cold ischaemia time, donor hypertension, and latest donor creatinine plasma levels are assumed to provide the best clinical assessment predicting graft function. However, such a scoring system has been shown not be able to discriminate between good and suboptimal kidney donors as outlined in Chapter 8.

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15 Identification of biomarkers in blood of the deceased donor that could help with the clinical decision-making whether to accept or decline a kidney graft for transplantation could be of significant impact for the clinician burdened with this difficult decision. An -Omics approach would be the first logical step towards identification of biomarkers in blood of deceased brain dead donors. Biobanks that store blood or urine from deceased donors are of great value and importance to answer this question. However, sample collection during transplantation usually occurs during out-of-office hours and can result in prolonged room temperature storage of samples prior to correct storage, potentially inducing pre-analytical variability with artefacts.

In Chapter 9 an -Omics approach was used to investigate the effect of sample storage at room temperature for different periods of time. In this chapter we evaluate the results and how a protocol can be developed allowing sufficiently high quality proteomics data to support identification and validation of molecular profiles and biomarkers associated with lower or higher risk kidneys and their outcomes after transplantation.

In Chapter 10 the findings in this thesis are summarised and discussed with a brief outlook into future developments and some recommendations are provided.

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Figure 1. Brain injury results in loss of the blood brain barrier (BBB) with increased systemic circulation of central nervous system (CSN)-derived or activated cytokines, chemokines, and adhesion molecules.

(6,10-13). Progression of brain injury leading to brain stem death results in a strong sympathic response with cathecholamine release, known as the Cushing reflex. Eventually, brain injury will compromise organ quality due to detrimental responses leading to reactive oxygen species (ROS) formation and pro- inflammatory changes in the graft.(6,7,11,13,14)

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REFERENCES

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3. Tonelli M, Wiebe N, Knoll G, Bello A, Browne S, Jadhav D, et al. Systematic review:

kidney transplantation compared with dialysis in clinically relevant outcomes. Am J Transplant. 2011 Oct;11(10):2093–109.

4. 2015 year report Dutch Transplant Society, http://www.transplantatiestichting.nl.

5. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival rates of kidney transplants from spousal and living unrelated donors. N Engl J Med. 1995 Aug 10;333(6):333–6.

6. Nijboer WN, Schuurs TA, van der Hoeven JAB, Fekken S, Wiersema-Buist J, Leuvenink HGD, et al. Effect of Brain Death on Gene Expression and Tissue Activation in Human Donor Kidneys. Transplantation. 2004 Oct;78(7):978–86.

7. Akhtar MZ, Huang H, Kaisar M, Faro Lo ML, Rebolledo R, Morten K, et al. Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death. Am J Transplant.

2016 May;16(5):1421–40.

8. Damman J, Bloks VW, Daha MR, van der Most P, Sanjabi B, van der Vlies P, et al.

Hypoxia and Complement-and-Coagulation Pathways in the Deceased Organ Donor as the Major Target for Intervention to Improve Renal Allograft Outcome. Transplantation.

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Relationship between sympathetic nervous activity and inflammatory response after subarachnoid hemorrhage in a perforating canine model. Auton Neurosci. 2009 May 11;147(1-2):70–4.

10. Schuurs TA, Gerbens F, van der Hoeven JAB, Ottens PJ, Kooi KA, Leuvenink HGD, et al.

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Insights in the Processes of Brain Death. Am J Transplant. 2004 Dec;4(12):1972–81.

11. Schuurs TA, Morariu AM, Ottens PJ, ‘t Hart NA, Popma SH, Leuvenink HGD, et al. Time- Dependent Changes in Donor Brain Death Related Processes. Am J Transplant. 2006 Dec;6(12):2903–11.

12. Hoeger S, Bergstraesser C, Selhorst J, Fontana J, Birck R, Waldherr R, et al. Modulation of brain dead induced inflammation by vagus nerve stimulation.

Am J Transplant. 2010 Mar;10(3):477–89.

13. Avlonitis VS, Wigfield CH, Kirby JA, Dark JH.

The Hemodynamic Mechanisms of Lung Injury and Systemic Inflammatory Response Following Brain Death in the Transplant Donor. Am J Transplant. 2005 Apr;5(4):

684–93.

14. Hoeksma D, Rebolledo RA, Hottenrott CMV, Bodar YS, Wiersema-Buist JJ, van Goor H, et al. Inadequate anti-oxidative responses in kidneys of brain-dead rats. Transplantation.

2016 Aug 5.

15. Campbell SJ, Perry VH, Pitossi FJ, Butchart AG, Chertoff M, Waters S, et al. Central nervous system injury triggers hepatic CC and CXC chemokine expression that is associated with leukocyte mobilization and recruitment to both the central nervous system and the liver. Am J Pathol. 2005 May;166(5):1487–97.

16. Campbell SJ, Jiang Y, Davis AEM, Farrands R, Holbrook J, Leppert D, et al.

Immunomodulatory effects of etanercept in a model of brain injury act through attenuation of the acute-phase response. J Neurochem. 2007 Dec;103(6):2245–55.

17. Skrabal CA, Thompson LO, Potapov EV, Southard RE, Joyce DL, Youker KA, et al. Organ-specific regulation of pro- inflammatory molecules in heart, lung, and kidney following brain death. J Surg Res.

2005 Jan;123(1):118–25.

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18. Koudstaal LG, ‘t Hart NA, Ottens PJ, van den Berg A, Ploeg RJ, van Goor H, et al.

Brain death induces inflammation in the donor intestine. Transplantation. 2008 Jul 15;86(1):148–54.

19. Higashida T, Kreipke CW, Rafols JA, Peng C, Schafer S, Schafer P, et al. The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood- brain barrier disruption and brain edema after traumatic brain injury. J Neurosurg.

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20. Bos EM, Leuvenink HGD, van Goor H, Ploeg RJ. Kidney grafts from brain dead donors: Inferior quality or opportunity for improvement? Kidney International. 2007 Jul 25;72(7):797–805.

21. Roodnat JI, van Riemsdijk IC, Mulder PGH, Doxiadis I, Claas FHJ, IJzermans JNM, et al. The superior results of living-donor renal transplantation are not completely caused by selection or short cold ischemia time: a single-center, multivariate analysis.

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22. Nijboer WN, Ottens PJ, van Dijk A, van Goor H, Ploeg RJ, Leuvenink HGD.

Donor pretreatment with carbamylated erythropoietin in a brain death model reduces inflammation more effectively than erythropoietin while preserving renal function*. Critical Care Medicine. 2010 Apr;38(4):1155–61.

23. Dziodzio T, Biebl M, Pratschke J. Impact of brain death on ischemia/reperfusion injury in liver transplantation. Current Opinion in Organ Transplantation. 2014 Apr;19(2):

108–14.

24. van Dullemen LFA, Bos EM, Schuurs TA, Kampinga HH, Ploeg RJ, van Goor H, et al.

Brain death induces renal expression of heme oxygenase-1 and heat shock protein 70. J Transl Med. 2013;11:22.

25. Jäättelä M, Wissing D, Kokholm K, Kallunki T, Egeblad M. Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 1998 Nov 2;17(21):

6124–34.

26. Mosser DD, Caron AW, Bourget L, Denis- Larose C, Massie B. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Molecular and Cellular Biology. 1997 Sep;17(9):5317–27.

27. Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance:

degradation and reactivation of damaged proteins. Annu Rev Genet. 1993;27:437–96.

28. Suzuki S, Maruyama S, Sato W, Morita Y, Sato F, Miki Y, et al. Geranylgeranylacetone ameliorates ischemic acute renal failure via induction of Hsp70. Kidney International.

2005 Jun;67(6):2210–20.

29. Wang Z, Gall JM, Bonegio RGB, Havasi A, Hunt CR, Sherman MY, et al. A3 Induction of HSP70 inhibits renal injury (knockout).

Kidney International. Nature Publishing Group; 2011 Jan 26;79(8):861–70.

30. Kuboki S, Schuster R, Blanchard J, Pritts TA, Wong HR, Lentsch AB. Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice. Am J Physiol Gastrointest Liver Physiol. 2007 Apr;292(4):G1141–9.

31. Fudaba Y, Ohdan H, Tashiro H, Ito H, Fukuda Y, Dohi K, et al. Geranylgeranylacetone, a heat shock protein inducer, prevents primary graft nonfunction in rat liver transplantation.

Transplantation. 2001 Jul 27;72(2):184–9.

32. Hutter JJ, Mestril R, Tam EK, Sievers RE, Dillmann WH, Wolfe CL. Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo. Circulation.

1996 Sep 15;94(6):1408–11.

33. Plumier JC, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, et al. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest. 1995 Apr;95(4):1854–60.

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Heat shock proteins and their protective role in organ transplantation

Leon F.A. van Dullemen Bianca J.J.M. Brundel Henri G.D. Leuvenink

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Chapter 2

ABBREVIATIONS

APC: Antigen Presenting Cell BD: Brain Death

CO: Carbon Monoxide

CMA: Chaperone Mediated Autophagy DAF: Decay-Accelerating Factor

DAMP: Damage-Associated Molecular Pattern DBD: Donation after Brain Death

DCD: Donation after Circulatory Death DNAJB1: Heat Shock Protein-40 GGA: Geranylgeranylacetone HO1: Heme Oxygenase-1 or HSP32 HSF-1: Heat Shock Factor-1 HSP: Heat Shock Protein

HSPA1A: Heat Shock Protein-70/-72 HSPB6: Heat Shock Protein-25/-27 HSPC1: Heat Shock Protein-90 HSPD1: Heat Shock Protein-60 ICP: Ischaemic Preconditioning IRI: Ischaemia Reperfusion Injury MHC: Major Histocompatibility Complex

LOX-1: Lectin-like Oxidized Low Density Lipoprotein Receptor-1 NEF: Nucleotide Exchange Factor

NFkB: Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells PAMP: Pathogen-Associated Molecular Pattern

PQC: Protein Quality Control System PRR: Pattern Recognition Receptor ROS: Reactive Oxygen Species TLR: Toll-Like Receptor

UPS: Ubiquitin Proteasome System

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ABSTRACT

Heat shock proteins (HSPs) are a conserved family of chaperones that become rapidly expressed upon cellular stress to protect the cell. During transplantation, donor organs are exposed to multiple types of injury varying from deceased brain death-related injury to hypoxia, ischaemia, reperfusion, and activation of the innate- and adaptive immune system.

The upregulation of cytoprotective HSPs could preserve or increase graft quality and thereby improve transplantation outcome. In this review we consider in depth the intra- and extracellular working mechanism of heat shock proteins in organ transplantation and based on experimental studies we present evidence of organ protection in transplantation by heat shock protein upregulation.

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Chapter 2

INTRODUCTION

Organ transplantation is inevitably linked with hypoxia and ischaemia during organ procurement, followed by reperfusion injury. Ischaemia/reperfusion injury (IRI) can be very detrimental for organ function and may negatively affects the short- and long-term graft survival. Compared to living donors, organs derived from deceased donors after brain death (DBD) or after circulatory death (DCD) are more susceptible to IRI.(1,2) Interestingly, although the DBD patient remains ventilated on the intensive care unit and a stable blood pressure is maintained, the organs procureded from these donors show an inflammatory response that could explain their high susceptibility to IRI and transplantation related graft injury. The inflammatory response in the DBD donor is not fully understood yet, but it originates from the cerebral injury and increased intracranial pressure, resulting in a strong sympathic response with severe vasoconstriction due to endogenous catecholamine release. After the onset of BD there is an increased systemic circulation of cytokines, chemokines, and adhesion molecules.

(3-7) Eventually, BD will lead to decreased peripheral perfusion, generation of reactive oxygen species (ROS), leukocyte mobilisation and infiltration, and pro-inflammatory changes in the graft-to-be.(4,5,7-10) The pathological process of brain death with its systemic changes negatively affects the organ quality and is associated with a higher rate of primary non-function following transplantation as well as poorer short- and long-term outcomes when compared to optimal living donors.(1,11,12)

However, not only detrimental molecules are upregulated during BD and after IRI. At the same time an enhanced expression of cytoprotective proteins, in particular heat shock proteins, can be seen.(3,4,13-15) Heat shock protein-72 (HSP72 or HSPA1A), heat shock protein-25 (HSPB6 or HSP25), and heme oxygenase-1 (HO1 or HSP32) are proteins that are upregulated after stress.

In tissue culture and animal models, elevated levels of HSPA1A, HSPB6, and HO-1 will protect or may repair damage thus preventing cell death.(16-18) Increased expression of HSPA1A and HO1 is able to provide protection against the detrimental effects of IRI in kidney(19-21), liver(22-24), and heart(25-27), and in the context of organ transplantation it is conceivable that enhancing the cellular contents of these proteins will benefit the donor organ quality. Furthermore, HSPs are especially interesting target molecules for transplantation since the expression can be boosted with non-toxic compounds, like GernalyGeranylAcetone (GGA).(28)

Although many articles in the literature focus on HSPs in transplantation or IRI, an overview how HSPs will exert their protective properties in transplantation is lacking. The aim of this review is to explain on a molecular level how HSPs protect against certain damage leading to reduced apoptosis and necrosis during transplantation-related injury in solid organ transplantation. The first part of this review will consider the mechanism how HSPs become upregulated and how these chaperones assist in the refolding or disposal of damaged proteins. Two common and extensively used pathways are highlighted in this review that dispose of proteins and protein aggregates and are facilitated by HSPS: the proteasome and autophagy pathway.

HSPs also exert extracellular functions that can trigger the innate- and adaptive immune system, which could be detrimental in allograft transplantation when provoking immune activation in

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25 the graft. Therefore, the second part of this review focuses on the mechanism how HSPs are released into the extracellular space and in what manner they may initiate innate- and adaptive immune responses. The latter part of this review will summarise the evidence of the protective effects of HSP upregulation in deceased donors, IRI, and organ transplantation.

HEAT SHOCK PROTEINS AND THEIR INTRACELLULAR FUNCTIONS

The viability of cells depends on synthesis and the functionality of its proteins. Proteins depend on their secondary and tertiary structure to fulfil their specific function. However, proteins are sensitive to extra- and intracellular stressors that influence their stability and conformation by exposing hydrophobic regions. Depending on the severity of injury and the conformational changes, proteins can no longer fulfil their specific function or are even no longer soluble and may form protein clusters in the cell, also known as protein aggregates. The accumulation of misfolded proteins and insoluble aggregates can be toxic to the cell and eventually lead to loss of function and induction of apoptosis. Therefore, it is important that the cell has a healthy homeostasis of protein synthesis, folding, assembly, and clearance, also referred to as proteostasis.(29) All cells are equipped with a protein quality control system (PQC) that assists in protein folding and protein degradation. The need for a correct functioning PQC is evident as cytoplasmic aggregates can affect membrane integrity, damage mitochondria, interfere with endoplasmic reticulum-associated degradation, and impair autophagy and vesicle transport. During transplantation a functioning PQC is important, since IRI is very potent in damaging proteins.(30)

Central to the PQC are HSPs.(31) HSPs were originally categorised into several families according to their molecular weight; HSP110, HSP90, HSP70, HSP60, HSP40, and small HSPs (e.g. HSP25).

To date a new nomenclature has been proposed that is based on the function and similarity of domain functions; HSPH (HSP105=HSPH1), HSPC (HSP90=HSPC1), HSPA (HSP70=HSPA1A), HSPD (HSP60=HSPD1), DNAJ (HSP40=DNAJB1), and small HSPs (HSP27=HSPB1.(32) Several members of the HSP family are constitutively expressed proteins, but for some members their intracellular concentration can rapidly be enhanced upon cell stress, referred to as a heat shock response (HSR); hence the name of heat shock proteins (Figure 1). Most HSPs function as chaperones, taking part in the assembly, stabilisation, or folding of proteins. The protective mechanism of a heat shock relates to its preservation of cellular protein homeostasis and prevention of aggregation.

The heat shock response is regulated by interaction of the heat shock factor (HSF) transcription factors with the heat shock elements (HSE) in the promoter regions of the heat shock genes, resulting in enhanced HSP expression (Figure 2). There are four HSFs identified of which HSF1 is the main transcription factor in response to cell stress.(33,34) HSF1 is present in the cytoplasm as a monomeric molecule, unable to bind DNA or activate transcription. Upon proteotoxic stress, monomeric HSF1 becomes converted to a trimer that translocates to the nucleus and binds to the HSE.(33,35) Activation of HSF1 is further fine-tuned with posttranslational

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modifications such as phospohoryaltion and controlled by binding of HSPA1A and heat shock protein binding factor 1 (HSBP1) to the transactivation domain of HSF1.(33) HSPC1 (HSP90) and CCT are a negative regulator of this process (36) and active inhibition of HSPC1 enhances HSF1 related gene expression, a strategy often applied to increase cellular HSPA1A levels.(37) The most prominent HSPs of the cytosolic HSF1-dependent stress response are HSPB1 (HSP27), HSPB5 (alfaB crystalline), DNAJB1 (HSP40), HSPA1A (HSP70), HSPA6, and HSPA8 (HSC70).(38) Proteins central to the PQC system are chaperonins (HSP60, TCP-1 ring complex [TRiC]), the HSP90 (HSPC) family, the HSP70 (HSPA) family, the small HSPs (HSPB), and a set of co-chaperones, including the HSP40 (DNAJ) family and nucleotide exchange factor (NEF) (Figure 2).(39,40) Heat shock protein 70 (HSPA1A) is involved in protein refolding and rapidly upregulated upon stress. The HSPAs contain an N-terminal ATPase domain and a C-terminal peptide-binding domain,(41) and the function of HSPA1A is ATP-dependent and regulated by co-chaperones such as HSP40 (DNAJ) and nucleotide exchange factors (NEFs).(42) DNAJs recognise the unfolded or damaged protein and subsequently bind to the ATP-bound form of HSPA, after binding the ATP molecule is hydrolysed and DNAJ is released. Upon ATP-hydrolysis, HSPA undergoes a conformational change and binds the unfolded protein. The NEF binds to the ADP-bound HSPA and mediates the exchange of ADP for ATP, after which the target protein can be folded and released.(43,44)

The recognition of HSPs for misfolded proteins is through their affinity for hydrophobic regions, binding to these regions prevents the initiation of aggregate formation. In circumstances when aggregates are formed, HSPs can also prevent further nucleation of these aggregates. HSPA and HSPB family members are found to accumulate on the surface of aggregates, potentially reducing the toxic effects of these aggregates by preventing the trapping of other proteins to it.(45) Failure of damaged proteins to be refolded can lead to HSP-directed degradation through the proteasomal degradation machinery, or disposal of damaged proteins by exosomes, or autophagy (Figure 2).(46-50) How HSPA select substrates to be reused and refolded or disposed and degradated is not fully elucidated. DNAJs and NEFs contain ubiquitin interacting motifs or ubiquitin-like domains that could influence the protein folding machinery and direct it to degradation through the ubiquitin proteasome system (UPS).(40,51,52) Ubiquitin proteasome system and HSPs

It is acknowledged that cytokines and metabolic dysfunction, as a consequence of e.g. DBD- related injury, IRI, or an activated immune system, can result in the production of ROS and damage proteins. When HSPs fail to refold a damaged protein, the protein can still be targeted for degradation by ubiquitylation (Figure 2). The UPS is an important pathway that can degrade these oxidative damaged proteins(53), and the PQC that efficiently degrades these proteins protects the cell from accumulating toxic aggregates.

Target protein ubiquitylation is a three-step enzymatic cascade; an E1 enzyme activates ubiquitin and transfers it onto an E2 ubiquitin-conjugating enzyme, subsequently, ubiquitin is shifted to a substrate-specific E3 ligases. The E3 ligases facilitate convalent binding of ubiquitin to the target protein, which targets it for degradation by the 26S-proteasome.(54) There are multiple

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27 genes encoded for E3 ligases, and one of such E3-ligase is C-terminus of Hsc70 Interacting Protein (CHIP or STUB1) which interacts mainly with DNAJ and HSPA chaperones.(55) In this way HSPs act as recognition factors of misfolded proteins and delivers those to the E3-ligase CHIP. Furthermore, HSPA1A and DNAJs are also essential to keep substrates soluble and make it accessible for the 26S-proteasome.(56,57) HSPA1A depletion has an inhibitory effect on the aggregation prevention of partially misfolded proteins that are otherwise soluble.

Interfering with the UPS system seems feasible in transplantation, although the protective mechanism seems counter intuitive. During the cold preservation of organs, intracellular proteins become massively ubiquitylated(58,59) resulting in an overloaded UPS. When inhibiting the UPS during organ preservation, the ubiquitin conjugate pool is preserved and the proteasome function is maintained at reperfusion(58). Two studies show that inhibition of the UPS during heart IRI is cytoprotective and prevents inflammation.(60,61) The protective effect of UPS inhibition in IRI is through the inhibition of NFkB. NFkB becomes activated after degradation of its inhibitor IkB, and UPS inhibitor administration prior to heart IRI prevents NFkB activation, cardiac dysfunction, and limits the infarct size.(60,61)

HSP-assisted autophagy

As mentioned above, besides targeting proteins for degradation, HSPs are also involved in protein degradation through autophagy on several levels. Autophagy is a pathway for the cell to degrade and recycle damaged organelles and proteins. The importance of autophagy in kidney transplantion has recently been reviewed by Pallet et al.(62) When autophagy is increased, the graft is more protected against transplant-related injury, while inhibition of autophagy enhances the cellular injury.(63)

During the process of autophagy a double membrane-vesicle is formed, containing the substrates targeted for degradation. The vesicle will fuse with a lysosome after which hydrolysis can take place. Soluble proteins can be chaperoned toward lysosomes by a pathway called chaperone-mediated autophagy (CMA).(64) The mediater of CMA is HSPA8 (HSC70), a constitutively expressed protein that recognises a specific peptide motif in the target protein. This allows it to bind the target protein and chaperone it to the lysosomes, where the protein will eventually be degraded. Besides CMA, HSPs can also target large growing aggregates for invagination by autophagosomes.(65) As described above, HSPs facilitate the polyubiquitination of damaged proteins, which allows recognition by ubiquitin-binding complexes leading to autophagy, also known as macroautophagy.(66) In addition, it is also believed that HSPs can re-route proteins to autophagosomes in the case that the proteasomal pathways are overloaded. (67,68)

Heme oxygenase

Another protective protein related with cell stress and extensively researched in IRI and transplantation is heme oxygenase-1 (HO1) or Heat shock protein-32 (HSP32). Heme oxygenase (HO) has three isoforms, of which HO1 is the inducible form that is upregulated in the deceased organ donor and proves to be protective in IRI.(13,21,69) HO1 is highly stress inducible but not

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by heat shock via HSF1. HO1 upregulation is mediated by inhibition of BACH1 and upregulation of NRF2.(70), A wide variety of stimuli can increase HO1 expression, possibly through oxidative stress. Stimuli that increase HO1 expression are e.g.; renal ischaemia, interleukins, TNF, IFN, TGF-beta, HGF, BMP-7, VEGF, heme, hemin, iron, angiotensin II, nitric oxide, carbon monoxide, gentamycin, cyclosporine, proteosomal inhibition, NGAL, and osmotic stress.(21,70,71) HO1 is often referred to as a HSP or stress protein although it lacks conventional HSP functions like refolding and stabilization of denatured proteins. HO1 is an enzyme that is involved in the breakdown of heme. Heme is a molecule constructed in many proteins like haemoglobin, mitochondrial cytochromes, NADPH oxidase, cyclo-oxygenase, and catalase. When cells become damaged, heme becomes released and inflicts cell injury.(72) In large quantities heme is very toxic and affects the plasma membrane, cytoskeleton, mitochondria, DNA, cytosolic enzymes, and induce renal injury.(73) Breakdown products of HO1-mediated heme metabolism are carbon monoxide (CO), free iron (Fe²⁺), and biliverdin that subsequently is converted to bilirubin by the enzyme biliverdin reductase. The cytoprotective effects of HO1 are in part derived from the downstream products; 1. CO, 2. bile salts, and 3. through it’s effects on heme- and iron homeostasis.

Carbon monoxide (CO) is notorious for its toxic effects in large quantities, however, in low concentrations CO has cytoprotective, anti-apoptotic, vaso-relaxant and anti-inflammatory effects.(74,75) CO attenuates the expression of pro-inflammatory cytokines like TNF-alfa, and promotes the expression of anti-inflammatory cytokines like IL-10.(76) In animal experiments, exposure of rats to a CO-containing atmosphere protects the kidney from ischaemia reperfusion injury.(77) In this context, inhibition of HO1 also results in reduced glomerular filtration rate (GFR), renal blood flow (RBF) and these effects are reversible by administration of CO-releasing molecules.(78) Consistent with this, the protective effects of CO are not attenuated by inhibition of HO1 after administration of CO donor compounds.(79) Secondly, HO1 is responsible for the conversion of heme into biliverdin. While accumulation of bile salts are associated with cell injury, they exert anti-inflammatory and anti-oxidant properties also when present in low quantities.(80,81) Low elevated levels of bile salts are associated with lower NADPH activity, reduced ERK1/2 signalling, and reduced TNF-alfa induced endothelial activation.(82) The third downstream product of HO1 is iron (Fe²⁺). Iron is an oxidative reagent that can induce the expression of ferritin and activate iron-transporters. HO1 upregulation is associated with an improved iron homeostasis and decreased free intracellular iron and iron induced cytotoxicity.(73,83-86) A potential fourth protective effect of HO1 is thought to be mediated by decreased expression of the pro-inflammatory protein: inducible nitric oxide synthase (iNOS).(87,88)

HSPS AND THEIR IMMUNOACTIVE PROPERTIES

Eukaryotic HSPs are considered intracellular proteins that are released upon cell necrosis and subsequently can initiating a range of pro-inflammatory processes in the extracellular environment (Figure 3).(89,90) It has been acknowledged that extracellular HSPs can function

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29 as inter-cellular signalling ligands and activate dendritic cells, monocytes, and other cells related to the innate- and adaptive immune system.(91-94) The exact receptor- and binding mechanism is not yet fully elucidated but potential receptors for HSPs include Toll-like receptors (TLRs)(95-97), scavenger receptors (SR)(98-100), CD40(101,102), and CD91(103). There is still no concensus if HSPs are able to initiate an immune response alone, or if the immune response is through HSP-bound peptides.(91) In skin transplantation the latter option seems to be the case, since HSP inhibition prevents allograft rejection.(104)

It has become apparent that HSPs have more than merely a passive role in activating the immunological processes upon cell decay, for instance, HSPs have also been detected in the serum of healthy individuals.(105-108) Apart from necrosis, the release of HSPs into the extracellular space is regulated through multiple pathways.

Extracellular release of HSPs

In the process of transplantation it is likely that the increase in extracellular HSPs is the result of ROS- and pro-inflammatory related cell damage and necrosis. The content of a cell is very high with HSPA1A, which can be up to 200µg of protein per 1g of necrotic tissue.(91) It is conceivable that irreversible injury to a cell, leading to necrosis, can easily enhance the extracellular HSP content measured in blood. An illustrative ischaemia reperfusion experiment in rats, where the blood flow to the middle lobe of the liver was blocked for 30 min, showed that after reperfusion there was a marked increase of HSPA1A in the plasma of 60.3ng/ml while the sham controls had levels of 0.02ng/ml.(109) A similar increase in HSPA1A was observed after treating animals with a heat shock of 42°C for 10 min, where plasma HSPA1A elevated from 0.04ng/ml to 99.4ng/ml.(110) Whereas, in the latter experiment one would only expect minimal if not any cell necrosis. However, it should be noted that HSPA1A expression is measured with ELISA in these studies that could bias the numbers due to aspecific binding for other HSPs.

Although two studies have found that HSPs secretion is an active and ATP-dependent process in multiple cell types (Figure 2), more evidence is needed.(111,112) One such excretion pathway is through secretory lysosomes. Extracellular HSP levels correlate with the appearance of the lysosomal marker, LAMP-1, on the cell surface, and HSP release can be inhibited with lysosomotropic agents.(112,113) The ABC-transporterappears to be a potential mechanism to translocate HSPs across the vesicle membrane, enriching the lysosome with these proteins.

(112) Another way of excreting protein aggregates is by microvesicle formation and release via the exosomal pathway, or release through membrane blebbing.(47-50,114) It also appears that HSPs can become translocated across the plasma membrane, the exact mechanism is not understood but the mentioned ABC-transporter could be involved in this mechanism.

The function of this HSP transport mechanism seems to be Mg²⁺-mediated, elevated levels of extracellular Mg²⁺ inhibit Hsp70 secretion, while decreased Mg²⁺-levels enhances HSPA1A secretion.(112) The in the end released extracellular HSPs are immonuactive and can initiate and activate the immune cascade by interacting with antigen presenting cells (APS).

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HSPs and the innate immune system

Molecular characteristics of prokaryote cell products are known as pathogen-associated molecular patterns (PAMPs) that can activate the immune response by interacting with specific receptors known as patterns recognition receptors (PRR), which triggers the innate immunity. One of those PRRs is the family of Toll-like receptors (TLRs) that can activate pro- inflammatory signalling. Similar to PAMPs, the release of intracellular components will also trigger the innate immunity in a sterile manner by molecules referred to as damage-associated molecular patterns (DAMPs).(115)

When cells die by apoptosis, their intracellular content remains enveloped with an external membrane, preventing extracellular release that could trigger inflammation. The apoptotic cells express ‘eat me’ signals on the surface, which induces phagocytosis by macrophages. The roll of HSPs in phagocytosis is unclear, but scavenger receptors associated with endothelial cells (SREC-I)(99,100) and CD91 posses sequence similarities to HSPs, suggesting a role in the process of phagocytosis.(98) Upon stress, cells are able of expressing HSPs on their extracellular plasma membrane that has shown to activate macrophages, and upregulation of HSPA1A (HSP72) in macrophages increases their phagocytotic capacity.(116-118) Furthermore, increased levels of extracellular HSPB1 (HSP27) can trigger the NF-kB pathway in macrophages.

(119) Interaction of HSPA1A with CD40 also activates macrophages and enhances interleukin secretion (Figure 3).(120) Consistent with this, extracellular HSPD1 (HSP60) could increase the influx of macrophages since it induces the expression of adhesion molecules E-selectin, intracellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)- 1. In addition, HSPD1 also increases the secretion of IL-6 from several cell types and macrophages.(121,122)

Many reports suggest that Toll-like receptors (TLRs) can become activated by HSPs, however, there is much controversy around this topic. HSPs have been produced in bacteria and lipopolysacharide (LPS) derived from those bacteria can become bound to the HSP. LPS triggers NF-kB and MAP-kinase signalling via the activation of TLR4 and therefore could be a biased activation mechanism of TLRs in in vitro studies with purified HSPs. The mechanism how HSPs trigger TLRs is not fully understood but it is likely to be through the interaction of the chaperoned protein. However, several studies have shown that extracellular HSPA1A function in a LPS independent manner and can elicit the production of cytokines, even after adding a LPS inhibitor.(89,90)

The extracellular function of HSPA1A as a DAMP seems to be mediated through TLR2 and -4, enhancing the expression of NF-kB, ICAM-1, MCP-1, IL-6, -8, TNF-alfa, and IL-1beta.(123-128) Also, HSPA1A and its bound peptides isolated from tumour-dendritic cell can induce an anti-tumour immunity reaction, while knock-out of the TLR2 and -4 genes decreases the inflammatory response of an HSPA1A vaccine.(99) The exact mechanism is not known but some studies show that HSPD1 and HSPA activate TLR2 and -4 pathways through SREC-1, LOX-1, and CD14 receptors.(90,95,99,121)

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31 Besides the TRL-system, HSPs might also interact with the complement system, a major arm of the innate immune system. The inhibition of extracellular HSPA1A in tumour cells also has an inhibitory effect on the expression of complement components C3, C5, and C9.(129) The opposite appears for overexpression of HO1, which enhances the expression of decay- accelerating factor (DAF) through increased bilirubin levels, and improves protection against C3-deposition and complement-mediated cell lysis.(130)

HSPs and the adaptive immune system

The adaptive immune system can be triggered by major histocompatibility complex (MHC) class I or –II receptors. MHC-I molecules are present on all nucleus-containing cells and activate CD8+ T-lymphocytes, while MHC-II molecules are only present on antigen-presenting cells (APCs) and activate CD4+ T-lymphocytes. Extracellular antigens are internalized into an endosome or phagosome and those peptides are presented on MHC-II molecules, activating CD4+ T-lymphocytes upon appropriate co-stimulation. A second pathway to present antigens involves translocation of the antigen into the cytosol and degradation via the ubiquitin- proteasome pathway where it can subsequently be presented by MHC-I molecules to CD8+

T-lymphocytes.(131) This process is called cross-presentation and is important for an effective host defence against viral infections and malignancies.(132) The molecule CD91 present on APCs interacts with HSP GP96 and becomes internalized upon interaction (Figure 3).(103) The chaperone with the bound peptide either becomes degraded and loaded to a MHC class-I or –II molecule, or transferred to the cytosol, degraded by a proteasome and subsequently presented on MHC class-I molecules.(133-136) Also, HSPC1 associates with proteins destined for proteosomal degradation and HSPC1 inhibition affects protein loading on MHC-I molecules.

(137) Furthermore, HSPC1 also plays a role in chaperoned antigen presentation on MHC-II molecules, and increased levels of extracellular HSPC1 enhances cross presentation of these antigens.(137,138) Downregulation of HSF1 and HSPC1 seems to decrease antigen presentation of APCs on both MHC-I and -II molecules, and also affect T-lymphocyte activation via MHC-I molecules on target cells.(139-141) Therefore, it seems that chaperoning of proteins and peptides is important for effective cross-presentation and activation of the adaptive immune system. Furthermore, HSPA1A can also capture antigenic substrates that are released, and after binding to receptors on APCs, they subsequently trigger CD8+ T-lymphocytes by antigen presentation on MHC-I molecules.(93,99,142)

Another function of extracellular HSPs is activation of APCs after interacting with CD40.

(120,124,143) It appears that binding of HSPA1A to APCs generates a pro-inflammatory response through the activation of various immune processes, such as cytokine production (IL1b, TNF- alfa, and IL6,), co-stimulatory molecule expression (MHC class II and CD86), and nitric oxide (NO) release. Another candidate receptor for HSPs is the scavenger receptor (SR) family, which are expressed on the surface of APCs. It has been shown that HSPA1A can interact with at least three of those members, including SREC-1, FEEL-1/CLEVER-1, and LOX1.(100,144) The latter, LOX-1, is involved in the homing and adhesion of APCs to endothelial cells, mediating cell influx that could initiate an immune response.

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Although HSPs have not been extensively investigated in the context of transplantation and immune activation, it is probable that HSP-mediated activation of the innate- or adaptive immune system will be detrimental for the graft. However, as outlined in the next paragraph, it appears that for some HSP-members upregulation results in a better functioning graft post IRI, also after allogeneic transplantation. This was illustrated in a mouse heart transplantation model where enhanced expression of HSPB1 resulted in lower rates of acute graft rejection and apoptosis.(145)

PROTECTIVE EFFECTS OF HEAT SHOCK PROTEINS IN TRANSPLANTATION

The precise protective mechanism of HSPs in inflammation- and ischaemia reperfusion related injury is not very well understood, but may be mediated through their biological function as molecular chaperones. HSPs became of interest in the setting of transplantation since they first appeared to be protective in ischaemia reperfusion injury in hearts.(146) Upregulation of HSPs also appears to be protective for IRI and DBD-associated injury in in vivo animal experiments of several organs, including the kidney(19,20,147,148), liver (23,149,150), heart (25,26,151,152), lung (153), and small intestine (154) (Table 1). Upregulation of HSPA1A protects cells from TNF-alfa and monocyte induced inflammation and cytotoxicity.(155-157) In a sepsis model HSPA1A upregulation is associated with protection of the endovasculature and the lung.(158- 161) Also, cold shock, a common injury mechanism in the preservation of organs, is associated with decreased expression of HSPA1A, HSPB1, and HSPC1. Enhancing the expression of these depleted HSPs by heat shock provides protection against cold storage and rewarming injury.

(162) Similar protective properties against IRI have also been observed after enhancing HO1 expression in kidney and liver.(21,163-165) Pre-treatment of the donor by upregulating HO1 appears to be a useful strategy for improving the graft quality in a rat transplantation model.

Cobalt-protophyrin administration is a commonly used method to enhance HO-1 expression, although it should be noted that this compound is not solely specific for HO-1. A single dosis of cobalt-protoporphyrin improved the kidney graft in an allogeneic- and also in a brain dead transplant model.(166,167) HO1 upregulation improved the survival rate, ameliorated the amount of proteinuria, and inhibited NFkB activation and cytokine expression. Just this year Thomas et al. showed very promising results on the possibility to enhance HO1 expression in deceased human donor kidneys.(168) The clinical importance of HO1 is emphasized by the observation that donors with a polymorphism in the HO1 gene, resulting in higher HO1 expression levels, have a superior graft function and survival.(169,170) In addition, older age is associated with inferior transplantation outcomes. This could partly be mediated via HSP expression since both HO1 and HSPA1A expression levels appear to be age-associated.

(170-172) Also with increasing age the PQC system fails to prevent and eliminate misfolded proteins, lysosomal function decreases, and autophagy becomes more impaired.(173-176) In a rat IRI model, older rats have an impaired ability to enhance HSPA1A expression and are more susceptible for ischaemic injury.(172) Consistent with this data, the ability of HSF1 to bind to the HSP-promoter regions also diminishes with ageing.(177) A possible contributing cause could be that SIRT1, a molecule that affect HSF1 acetylation and expression, shows an age- dependent decline.(178)