1

Name student: R.G.M. Lammerts

Student number: S1836005

Faculty supervisor: H.G.D. Leuvenink

External supervisor: Prof. R.J. Ploeg

Daily supervisor: Z.M. Akhtar

Preventing brain death induced kidney injury through

exploiting the hypoxia inducible factor pathway

Oxford Transplant Centre, Department of Surgical Sciences. University of Oxford

2

Abstract in English

Background and aims: Brain death-derived kidney allografts have inferior short and

long-term outcomes when compared to the living donated allograft, even when HLA

mismatches and cold ischemia times are taken into consideration. Brain death leads to a

physiologically abnormal state resulting in dramatic hemodynamic, hormonal, coagulation

and inflammatory disturbances culminating in kidney injury prior to organ procurement.

Two promising therapies evaluated in the brain death setting include heme-oxygenase 1

(HO-1) and erythropoietin (EPO) administration. These are both downstream effectors of the

hypoxia inducible factor (HIF) pathway. HIF is the universal cellular oxygen sensing

mechanism and mediates over 100 hypoxia responsive genes. The HIF pathway is

responsible for the early and delayed response of ischaemic preconditioning, a pathway in

what exposure of an organ to a short period of ischemia protects the organ against exposure

to subsequent periods of ischemia. We hypothesized that up regulation of HIF in brain death

donors could protect against brain death induced kidney injury and improve the outcomes of

kidney transplantation. To begin to evaluate this hypothesis I characterized the effects of

brain death on HIF1α expression in comparison to other models of hypoxic/ischemic injury.

Methods: the expression of HIF1α was characterized in four models of kidney

ischemic injury. Adult male Fischer rats were used in the described animal model.

(n=8, 230-350g). Brain death (DBD) was induced by a gradual inflation of a subdurally

placed balloon catheter. In addition an ischemia reperfusion model(n=2) and a deceased after

circulatory death model (n=2) was developed. Cell culture NRK-49 cells were grown in

DMEM medium. Western blotting was performed to characterize the differential expression

of HIF and HO-1, a downstream effector of HIF.

Results: A brain death animal model was developed. Induction of brain death showed

a drop in blood pressure after approximately 40 min. It was not possible to increase the blood

pressure to 80 mmHg after brain death induction. Western blotting showed HIF1α expression

in the 4 models of kidney injury. HO-1 expression was seen in 3 models.

Conclusions: The BD model needs optimization to maintain the animals stable for a

longer period of time. These results suggest that HIF1α is activated in the 4 different models

of kidney injury. Western blotting for kidney samples showed more HIF1α and HO-1

expression in the IRI experimental group than in the control group. In the DCD model HIF1α

is less expressed in the experimental group than in the control group, whereas HO-1 is more

expressed in the DCD experimental group. As for the DBD group, both HIF1α and HO-1 are

more expressed in the experimental group than in the control group. Western blotting for

liver samples showed more HIF1α expression in the IRI control group than in the IRI

experimental group. In the liver DCD model HIF1α is more expressed in the control group

than in the DCD experimental group, whereas in the DBD model both HIF1α and HO-1 are

more expressed in the experimental group than in the control group. However, to confirm this

further research needs to be performed.

Expression of the prolyl hydroxylases 1,2 and 3 and expression of HIF2 α and HIF 3α are

questions yet to be answered before administration of a prolyl hydroxylase inhibitor and

translation to the clinical setting.

Keywords:HIF, hypoxia inducible factor, DBD, DCD, brain death, deceased after

circulatory death, transplantation, ischemia reperfusion, IRI, animals, heme oxygenase-1,

HO-1, EPO,1, erythropoietin

3

Samenvatting in het Nederlands

Achtergrond en doelstellingen: Nieren verkregen van hersendode orgaan donoren

hebben een slechtere korte en lange termijn overleving wanneer dit wordt vergeleken met de

levende donor, zelfs wanneer HLA mismatches en koude ischemie tijden in beschouwing

worden genomen. Hersendood leidt tot een fysiologische abnormale staat die resulteert in

dramatische hemodynamische-, hormonale-, coagulatie-, en ontstekings- verstoringen die

samenklonteren in de nier voordat orgaan donatie begint. Twee veel belovende therapieën in

de hersendood setting zijn de toediening van heme-oxygenase-1 en erythropoietin (EPO). Dit

zijn beide eindproducten van de hypoxia inducible factor(HIF) pathway. HIF is een cellulair

zuurstof gevoelig mechanisme en medieert meer dan 100 genen die reageren op hypoxie. HIF

is verantwoordelijk voor de vroege en de late fase van preconditie, een pathway waarin de

blootstelling van een orgaan aan een korte periode van ischemie, het orgaan beschermd tegen

de volgende langere periode van ischemie. De hypothese is dat de activatie van HIF in

hersendode orgaandonoren bescherming biedt aan nierschade door hersendood en hiermee de

uitkomsten van niertransplantatie verbeteren. Om te beginnen met het testen van deze

hypothese, werden de effecten van hersendood op HIF1α expressie gekarakteriseerd in

vergelijking met andere modellen van hypoxie/ischemie schade.

Methoden: De expressie van HIF1α werd gekarakteriseerd in vier modellen van nier

ischemie schade. Volwassen mannelijke Fischer ratten werden gebruikt in het beschreven

diermodel. (n=8, 230-350g). Hersendood (DBD) werd geïnduceerd door een langzaam

opgeblazen dubduraal geplaatste ballon katheter. Ook werd ischemie reperfusie model (IRI)

(n=2) en een overleden na circulatie model (DCD) (n=2) ontwikkeld. Het was niet mogelijk

om de bloeddruk weer naar 80 mmHg te laten stijgen na hersendood inductie. NRK-49 cellen

werden gekweekt in DMEM medium. Western blotting werd gebruikt om de verschillen in

expressie van HIFα en HO-1, een eindproduct van HIF1α, te karakteriseren.

Resultaten: Een hersendood dier model werd ontwikkeld. Inductie van hersendood liet

een daling in bloeddruk zien na ongeveer 40 minuten. Western blotting liet HIF1α expressie

zien in de 4 modellen van nier schade. HO-1 expressie werd gezien in 3 modellen.

Conclusie: Het hersendood model heeft optimalisatie nodig om de dieren stabiel te houden

voor een langere tijd. Deze resultaten suggereren dat HIF1α is geactiveerd in de 4

verschillende modellen van nier schade. Western blotting voor de nier liet zien dat HIF1α en

HO-1 meer tot expressive komen in de experimentele IRI groep dan in de IRI controle groep.

In de DCD setting HIF1α komt minder tot expressie in de experimentele groep dan in de

controle groep, terwijl HO-1 meer tot expressie komt in de experimentele groep. In de DBD

groep, komen beide HIF1α en HO-1 meer tot expressie in de DCD experimentele groep dan

in de controle groep. Western blotting voor de lever liet zien dat HIF1α meer tot expressie

komt in de controle groep dan in de experimentele groep. In het lever DCD model komt

HIF1α meer tot expressie in de controle groep dan in de DCD experimentele groep terwijl in

het DBD model beide HIF1α en HO-1 meer tot expressie komen in de experimentele groep

dan in de controle groep. Echter, om dit te bevestigen is er meer onderzoek nodig. Expressie

van de prolyl hydroxylases 1,2 en 3 en expressie van HIF2α en HIF3α zijn vragen die nog

beantwoord dienen te worden voordat administratie van een prolyl hydroxylase inhibitor en

vertaling naar de klinische setting kan plaatsvinden.

Sleutel woorden: HIF, hypoxia inducible factor, DBD, DCD, IRI, hersendood, overladen

naar circulatie stop, transplantatie, ischemie reperfusie, dieren, heme oxygenase-1, HO-1,

EPO, erythropoietin

4

Preface

This is the final report of the scientific clerkship for the Master in Medicine. The emphasis of

this project lies on 3 major aims;

- The development of a slow induction brain death animal model in rodents;

- The exploitation of the hypoxia inducible factor pathway;

- The establishment of the QUality in Organ Donation (QUOD) bio-bank.

Once this clerkship is finished, research and collaboration between the University of Oxford

and the University of Groningen will continue. Experience gained from previous research

projects plus this clerkship in Oxford encourages my ambition to keep on working within this

research field.

The main goal of the scientific internship was to improve different skills: performing animal

experiments, performing laboratory work, scientific writing, scientific thinking, to take part

in a management team and of course the experience of living abroad. This research is based

on developing a brain death animal model, deceased after circulatory death model and

ischemia reperfusion model. While analyzing the samples creative scientific thinking was

required to develop and proceed to the next step.

To develop a parallel translational strategy the Quality in Organ Donation (QUOD) initiative

was established. The key of QUOD is to increase the number and quality of organs procured

from deceased donors for transplantation by evaluating pathways of injury and repair in

organ donors and establishing a platform for the investigation of interventional strategies in

organ donors. In fact comparable to the animal work but QUOD is the translation to the

human setting. During my stay in Oxford a management team was constructed for the

establishment of the QUOD initiative. The management team was constructed to establish the

collection of biological materials and clinical data from organ donors.

It is important for optimizing donor organ quality to bring together all the participants in

organ retrieval, including clinical and scientific experts. The QUOD initiative is a beginning

project providing opportunities for clinicians and researchers to aid and perform research in

the optimization of donation and transplantation.

Working in the QUOD management team gave me the chance to gain experience in writing

protocols, understanding the UK transplant system and work with people from different

fields.

Whilst living abroad during my scientific internship and working in the QUOD national

management team I gained a lot of life and-, research experience and developed my

communication skills.

Rosa Lammerts

5

Table of contents

Abstract in English .................................................................................................................................. 2

Samenvatting in het Nederlands ............................................................................................................. 3

Preface .................................................................................................................................................... 4

Table of contents ..................................................................................................................................... 5

Introduction ............................................................................................................................................. 6

Internship in Oxford ............................................................................................................................ 6

Organ Donation and transplantation .................................................................................................. 7

Donation after brain death (DBD) ....................................................................................................... 7

Donation after circulatory death (DCD) .............................................................................................. 8

Phases of organ injury during donation .............................................................................................. 8

Ischemia/reperfusion injury (IRI) ........................................................................................................ 9

Hypoxia inducible factor ................................................................................................................... 12

Rationale ............................................................................................................................................... 14

Methods ................................................................................................................................................ 15

Results ................................................................................................................................................... 20

Conclusion and Discussion ................................................................................................................... 28

Translational strategy; relevancy for the human setting .................................................................. 32

Conflict of interest ................................................................................................................................ 35

Acknowledgements ............................................................................................................................... 35

References ............................................................................................................................................. 36

Supplementary protocols ...................................................................................................................... 40

6

Introduction

Internship in Oxford

Following participation in the Transplantation summer school I was inspired to carry out my

scientific internship at the Oxford Transplant Centre, University of Oxford under the

guidance of Prof Rutger J Ploeg. Prof Ploeg had recently been appointed as Professor of

Transplant Biology and Honorary Consultant Transplant Surgeon in Oxford and was

establishing a number of exciting initiatives.

I planned to join Prof Ploeg and his team in Oxford for 6 months to work on two of the

major initiatives:

- To work with a DPhil student, Zeeshan Akhtar, to establish a rodent model of brain

death in Oxford. This model had been developed in Groningen by the Surgical

Research Laboratory and has been used as a basic science model to investigate the

pathways responsible for organ injury following brain death and the effects of

therapeutic interventions. (1)We aimed to collaborate on a project looking at novel

interventions in the brain dead organ donor. In preparation for this aspect of the

project I spent several weeks during the summer becoming familiar with the model in

Groningen. I learned the western blotting laboratory technique in Oxford, which was

used to characterize the hypoxia inducible factor pathway.

- To aid in the establishment of a national organ donor bio-repository to identify

biomarkers in the donor that can be used to predict the outcomes of transplantation. A

project termed Quality in Organ Donation (QUOD) and sponsored by NHS Blood and

Transplant.

Both of these initiatives concern addressing the most important issue in transplantation; the

lack of good quality organs. It is estimated in the UK that one in three patients a day will

either die or become too unwell to receive a transplant whilst on the waiting list. Thus there is

an urgent need to protect, preserve and repair donor organs whilst also identifying markers to

predict the outcomes of transplantation. It is the translational prospect of these projects and

their clinical relevance that appeared scientifically and clinically interesting to me. My

previous experience as President of the Prometheus kidney team would aid me in assisting in

the establishment of the QUOD bio-repository. I participated as part of this team collecting

biopsies during my Bachelor and as a President responsible for the operational aspects of the

team.

One of the trends observed across Europe in the last decade has been that increasingly

“marginal donors” are being used to address the organ deficit. These are organs obtained

from older donors with additional co-morbities, termed extended criteria donors (ECDs) and

even donors after cardiocirculatory arrest (DCDs). The vast majority of organs obtained from

deceased individuals still come from brain dead organ donors (DBDs), although record

numbers of donors were achieved from DCD donors last year in the UK accounting for 40%

of all deceased donation.

The application I wrote 7 months ago concerned the effect of antithymocyte globulin (ATG)

on renal injury in brain stem dead rats. Since submission of the application a different

approach to addressing kidney injury following brain death has been pursued, by attempting

to exploit the hypoxia inducible factor (HIF) pathway. The rationale for this change was

because of an increasing body of literature supporting the exploitation of the HIF pathway as

an ischemic pre-conditioning and post-conditioning strategy. In addition work performed by

another group in Argentina and presented at the American Transplant Congress demonstrated

7

only a modest improvement in terms of organ injury when ATG was administered to brain

dead rats(2).

Organ Donation and transplantation

Every year organ transplantation saves the lives of thousands of patients. Despite increasing

numbers of allografts obtained from living donors, DCD donors and DBDs, a significant gap

exists between the supply and demand of

organs. (See figure 1(3)) This trend is

observed not only in Europe but also

throughout the world. It has become apparent

that organs obtained from brain dead organ

donors, even when HLA mismatches and

ischemia times are taken into consideration,

have poorer short and long term outcomes

compared to the living donor. By attempting

to improve the outcomes of kidney

transplantation from brain dead organ donors, fig. 1 Eurotransplant kidney transplant.

especially ECDs we can increase the number

of good quality allografts available for transplantation and also improve graft survival and

function.

Donation after brain death (DBD)

Brain dead donor kidneys are affected profoundly by the brain death process. From the point

of herniation of the brain stem a cerebral injury cascade ensues which results in

hemodynamic compromise, disturbance of metabolic and hormonal processes that leads to a

profoundly unphysiological state and diminished perfusion of peripheral organs. (4)This

stress reaction results in a progressive up-regulation of pro-inflammatory mediators,

increased cellular infiltration, and other inflammatory changes in the donor organs. (5)(6).

These changes may enhance the immunogenicity of the graft and increase the risk of acute

graft rejection and also chronic allograft dysfunction.(7) Cerebral injury and brain death in

DBD donors may be a risk factor for the development of vascular rejection after

transplantation, risk factors which don’t exist in living donors. Authors have attempted to

prevent brain death induced organ injury through hemodynamic resuscitation and hormone

replacement. These strategies have only had a modest effect on improving the outcomes of

kidney transplantation. An increasing body of literature supports the theory that ischemic

injury to the donor organ whilst they remain in-situ, in addition to the effects of warm and

cold ischemia during the preservation period can significantly damage donor organs. This

injury is accelerated following reperfusion after engraftment into the recipient. This injury

process is termed ischemia reperfusion injury.

8

Donation after circulatory death (DCD)

In order to enlarge the donor pool, many centres are revisiting donation after circulatory

death (DCD). In DCD donors the organs are subjected to a substantial period of oxygen

deprivation and ischaemic injury prior to retrieval. In addition, in this donor pool the organs

suffer from warm and cold ischemia. The amount of injury differs for the various donor

categories. (table 1) (25) (8)

Category 3 donors are most widely

used, since the duration of WI is

known, usually short and organ

recovery can be planned in advance. In

the potential donor the time interval

between withdrawal of therapy and

circulatory stop may account for

additional WI injury due to low oxygen

and organ hypoperfusion. Uncontrolled

DCD may have an advantage over

controlled DCD,

because serious brain damage is

associated with a pro-inflammatory and

pro-coagulatory state in the donor,

which has a negative effect table 1. Maastricht classification

on the organ quality. (26) On the other hand the profound warm ischemia time needs to be

taken into account. The result of this is that these organs suffer more from coagulatory and

negative immunological effects compared to organs from uncontrolled DCD donors because

the primary medical condition of these donors is mostly not neurologic. Apparently, the

detrimental effect of delayed graft function on the graft survival is more pronounced in

kidneys obtained from brain injured donors compared to organs obtained from uncontrolled

DCD donors. (27)

Phases of organ injury during donation

The organs from older and less healthy donors, from the DBD donors and those that have

been removed from the deceased donor after a period of circulatory arrest usually have

normal or near normal function before the process of death. During the process of death,

hypotension, diabetes insipidus, natremia and relative hypothermia are common in the donor,

this all affects the potential organs to donate. (9)In addition, progressive injury takes place at

every point along the donation process; during the retrieval, storage (preservation) and

transplantation. Longer cold and warm ischemic periodes correlates with higher rates of

organ dysfunction following transplantation. To prevent injury to organs during this sequence

researchers have been devising methods to optimize the condition of donor organs before

retrieval, to repair sub-optimal donor organs after retrieval, to preserve these organs and to

minimize the injury that occurs when an organ is transplanted and exposed to the recipient’s

circulation. Figure 2 shows the different phases a potential organ to transplant goes through

prior to retrieval, after organ retrieval and during implantation. It is important to assess and

monitor the organ in the donor during this procurement, storage, transplantation and

implantation phase. In the DBD donor maintaining physiological parameters, counteract brain

death induced inflammatory changes already before the declaration of death and during the

Table 1. Maastricht classification of donors after

circulatory death

1 Dead on arrival Uncontrolled

2 Unsuccessful

resuscitation

Uncontrolled

3 Awaiting cardiac arrest

(withdrawal of therapy)

Controlled

4 Cardiac arrest while

brain death

uncontrolled

5 Unexpected cardiac

arrest in a critically ill

patient

uncontrolled

9

agonal phase is of a great value, whereas in the DCD donor it is important to reduce the warm

ischemia time in this period. In both the DCD and DBD donors it is important to reduce

damage to the organ during organ procurement, ex-vivo storage and transportation. By

cooling the procured organs as soon as possible warm ischemia injury could be prevented. In

addition, preserving the organs through in-vivo and ex-vivo perfusion with nutrients, oxygen

and other additives is an intervention which provides benefit in reducing the injury that

occurs in donor organs. In the donation and transplantation process ischaemia reperfusion

injury is a central mechanism resulting in organ injury. It is important to begin to address this

injury mechanism in the donor, through understanding the pathways of injury.

Figure 2

Ischemia/reperfusion injury (IRI)

Ischemia

The definition of ischemic injury is cessation of arterial blood flow with immediate oxygen

deprivation of cells (ie, hypoxia with accumulation of metabolic products.)

Ischemia/reperfusion injury (IRI) is unfortunately unavoidable in transplantation and it can be

a serious complication of kidney transplantation.

Different organs have different degrees of tolerance to ischemic injury. The brain for example

suffers permanent loss of function when exposed to a few minutes of ischemia as illustrated

in rodent models of ischemia reperfusion injury. (10)In kidney transplantation allograft

damage is reversible if the warm ischemia time of the kidney is less than 30 minutes,

prolonging the warm ischemia time results in delayed graft function (DGF) and diminished

allograft survival. (11)

Ischemia involves oxygen and nutrient deprivation, switch to anaerobic metabolism and the

accumulation of catabolites with the capacity to induce cell death and attract erythrocytes,

leukocytes and platelets. The renal cortex has an especially high requirement for oxygen.

Under normal physiological conditions in normoxia, intracellular potassium and magnesium

10

levels are kept relatively high, whereas sodium and calcium concentrations are kept low. This

physiological state is maintained due to the activity of the ATP dependent sodium-potassium

pump and calcium-magnesium pump. In an hypoxic/ischemic cell, ATP is no longer

produced and thus these pumps are no longer able to maintain the concentration gradients.

Sodium, chloride and water diffuse into the cell and potassium and magnesium diffuse out.

Cell swelling ensues and cell lysis pathways are activated.(10).

During organ donation an organ is damaged during recovery, preservation and transplantation

and this occurs primarily as a result of ischemia. Techniques for organ preservation, like

cooling of the organs to decrease cell metabolism, serve to minimize this damage. In a brain

death donor the brain death process itself leads to a physiologically abnormal state resulting

in dramatic hemodynamic, hormonal, coagulatory and inflammatory disturbances

culminating in kidney injury prior to organ procurement. This injury is exacerbated during

the preservation phases and worsened on reperfusion in the repecient..(12)

Repefusion injury is irreversible damage after ischemia. Figure 3 illustrates the effects of

hypoxia and ischemia on renal cell metabolism. During warm ischemia, adenosine tri-

phosphate is degraded to adenosine monophosphate. Because of calcium influx the cytosolic

proteases are activated. They produce xanthin with the formation of superoxide free radical

and hydrogen peroxide.

Also, after ischemia the cell no longer has its usual reserve of free radical scavengers. This

increases the vulnerability of cellular and subcellular membranes to injury by lipid

peroxidation. The subcellular and cellular membranes are damaged, so after reperfusion the

capacity of restoration of homeostasis in the cell is decreased. Erythrocytes, leukocytes and

platelets adhere to damaged vasculature, because production of oxygen free radicals also

initiates production of prostaglandins, including leukotriene B4, which is an chemoattractant.

These neutrophils can cause secondary hypoxia in the kidney, followed by further

preservation injury. (10)

Figure 3. Results of hypoxia and warm ischemia on renal cell metabolism.

11

Protection of the kidney from ischemia injury

In recent years authors have investigated strategies to tackle ischemia reperfusion injury.

Examples of these treatments include; calcium channel blockers, allopurinol, free radical

scavengers, steroids, vasoactive drugs energy replenishment therapies and several others. But

despite the plethora of therapies the efficacy of these agents to reduce ischemic damage is

limited.

Two promising therapies have emerged showing an ability to ameliorate brain death induced

kidney injury in an experimental setting. This has been the application of heme-oxygenase 1

(HO1) upregulators and carbamylated erythtopoietin (cEPO). (13)These are both downstream

effectors of the hypoxia inducible factor (HIF) pathway, and are cytoprotective molecules

that are thought to protect against ischemic injury. They decrease the expression of several

proinflammatory genes, including vascular adhesion molecule-1, an adhesion molecule that

regulates leukocyte migration from the blood into tissues in addition to p-selectin and e-

selectin. These are both mediating the attachment of leukocytes to endothelial cells and are

early adhesion molecules. cEPO also impairs IL-1 and IL-6 expression, which are important

proinflammatory cytokines. HO-1 and EPO also decrease the polymorphonuclear (PMN) cell

infiltration (leukocyte migration) in the kidney. (14)

In addition, HO-1 is responsible for the breakdown of heme proteins, resulting in the

generation of biliverdin, carbon monoxide (CO) and iron. These products play a critical role

in the normal function of the kidney as well as protecting the kidney from ischemic insults

and exposure to nephrotoxins, so they have an important anti-oxidant, anti-apoptotic,

cytoprotective and anti-inflammatory property. (15)

EPO has, in addition to its ability to decrease PMN cell infiltration more complex actions.

EPO rescues cells from apoptosis (programmed cell death) to increase their survival. Other

effects include stimulation of angiogenesis, stimulation of endothelial and vascular smooth

muscle cell proliferation, increasing endothelin production, up-regulation of tissue renin and

change in vascular tissue prostaglandins production. (16)

In the recent years authors have shown that activation of HIF and its downstream effectors

prior to renal injury protects the kidney from ischemia reperfusion injury. (17)This is called

ischemic preconditioning (IPC) and it is a therapeutic intervention which aims at protecting

against subsequent exposure to ischemic injury. This was first described by Murry et al. (18).

They showed that brief periods of coronary occlusion followed by a short period of

reperfusion reduced the infarct size caused by an ischemic insult in a canine model. After this

discovery it has been found to be a near-universal phenomenon in all organs. IPC is a

biphasic phenomenon. It has a short lasting phase of about 2 hours of protection within

minutes of the initial ischaemic insult and a delayed phase of protection which becomes

apparent around 24 hours later and lasts for about 3 days.(19) IPC is a potent renoprotective

strategy which has not yet been translated successfully into clinical practice, despite

impressive preclinical results. However, it remains interesting to induce genes that are

normally activated during the early phase of an ischemic insult. Small molecule inhibition of

the oxygen-sensing HIF-prolyl hydroxylases have been identified. This offers the possibility

to mimic the hypoxic response, by pharmacological stabilization of HIF in order to achieve

organ protection. Therefore, a promising therapeutic strategy for the prevention of organ

failure and organ injury is oxygen-independent activation of HIF.

12

Hypoxia inducible factor

Hypoxia inducible factor (HIF) is a DNA-binding transcription factor that associates with

specific nuclear cofactors under hypoxic conditions. HIF consists of two subunits subunit

HIFα and an abundantly expressed β-subunit, HIFβ. Both subunits are part of the basic Helix-

Loop-Helix PER-ARNT-SIM (bHLH-PAS) family of transcription factors. (20) HIFα is

constitutively transcribed and translated in cells, but under normoxia it has a short half-life of

less than 5 min. There are three HIFα isoforms; HIF1α, HIF2α and HIF3α, these isoforms

are all oxygen dependent.

The isoforms consist of an oxygen

dependent degradation domain (ODD)

and an N-terminal transactivation

domain (NAD), HIF1α and HIF2α also

have a C-terminal transactivation

domain (CAD). The N-terminal

transactivation domains are essential

for targeting gene specificity, CAD on

the other hand contributes to the

regulation of most HIF target genes.

(21)HIF1α and HIF2α have a similar

architecture and are regulated in the

same

manner, whereas HIF3α is less closely

related and its regulation is less well

understood. The HIF1α and HIF2α oxygen dependent degradation domains are located in

the central region of the molecule. HIF1α is the subject to a further control that involves a

nuclear localization, figure 4. HIF and its downstream effectors

which is mediated by the active exclusion of

HIF1α from the nucleus in the presence of oxygen. (22) To date, far more than 100 HIF target

genes have been identified, including the previous described EPO and HO-1, further

downstream effectors are VEGF and glucose transporters. These downstream effectors have

all the potential to protect the kidney under different conditions. (23) (See figure 4)

13

HIFα regulation by prolyl hydroxylation and factor inhibiting HIF

HIFα protein levels are regulated by several mechanisms. The most important one is the

degradation pathway. Under aerobic conditions, HIFα undergoes proteasomal degradation.

This happens via the ubiquitin-dependent pathway and involves hydroxylation of specific

proline residues within the ODD domain. This degradation is done by prolyl hydroxylases

(PHDs), non-heme, oxygen-, Fe(II)- and 2-oxoglutarate-dependent dioxygenases. The PHD

proteins act as oxygen sensors and so far three of them have been identified: PHD1, 2 and 3.

(24) Hydroxylated HIFα is bound by the von Hippel-Lindau protein (pVHL). This protein

targets HIFα for degradation by the 26S proteasome. Binding of pVHL to HIFα is prevented

when PHDs are inactive, this is during hypoxia or in case of lack of the cofactors Fe(II) or 2-

oxoglutarate. In this case HIFα is able to escape ubiquitination and proteasomal degradation

and is transported to the nucleus where it binds with HIF β. After recruitment of co-activators

they bind together to the hypoxia responsive element (HRE) at the target gene loci. (figure 5)

Figure 5. HIF1α regulation by prolyl hydroxylation

14

Rationale

Ischemia related injury plays a central role in both the pathophysiology of organ injury from

both brain dead and deceased circulatory death donors. Following brain dead dramatic

physiological disturbances occur, resulting in non-function of the central nervous system,

hemodynamic instability, systemic hormonal changes and diminished perfusion of peripheral

organs. This is worsened by exposure to periods of ischemia during the preservation period

followed by reperfusion in the recipient, this activates toxic reactive oxygen species

production and cell death via apoptosis. These changes may enhance the immunogenicity of

the grafts and increase the risk of acute graft rejection.

In the DCD setting the organs are subjected to a substantial period of oxygen deprivation and

ischemic injury, prior to the retrieval. Concluded from the data from the previous described in

the DCD chapter, is reasonable to suggest that in the controlled DCD group warm ischemia

plus profound cerebral injury could account for a different form of delayed graft function

than observed in uncontrolled DCD group that only have suffered warm ischemia. It would

be interesting to see whether and on what level the hypoxia inducible factor is up-regulated in

a rat model which mimics uncontrolled DCD.

After promising studies in the experimental brain dead setting of application of HO-1, one of

the most important downstream effectors of HIF, and HIF being negatively regulated by

PHD1,2 and 3, we reasoned that the induction of protective mechanisms before the initiation

of the acute injury associated with transplantation of BD and DCD kidneys might be an

effective way to improve early and possibly late graft function. Our hypothesis is that the

induction of genes that have become inducible by hypoxia could confer such protection. For

testing this hypothesis we first aimed to characterize the expression of HIF and its

downstream effector HO-1 following both brain death and also deceased after circulatory

death.

Main objectives

- To develop a gradual onset brain death model in rats.

- To determine the effect of 4 methods of renal ischemia on HIF1α and HO-1

expression. The models include an ischemia reperfusion model (IRI),donated after

circulatory death (DCD), donated after brain death (DBD) and NRK-49 cell culture.

- To aid in the development of a national transplant donor bio-bank.

15

Methods Animal justification

This animal research was carried out in accordance with the Home Office guidance on

welfare for animals and the experiments were approved by the animal committee.

We have employed the principles of reduction, replacement and refinement on the animal

study. The study has been reviewed by the Oxford veterinary services. Experiments were

performed in the Animal facility in the South Parks road, Oxford. The number of animals for

the experiments have been based on the following question: What are the effects of BD and

DCD on HIF and HIF downstream effector production in the donor kidney?

For the establishment of the BD model 30 animals are going to be used because the model

needs to be set up and optimized. 8 animals were used during my stay in Oxford for the pilot

experiments to begin to characterize the HIF pathway in 4 different types of kidney injury.

Two animals were used per group.

.

Animals

Adult male Fischer rats where used weighing 230- 350g were used. 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 of at least 1 week before starting experiments.

Experimental group brain death model

To establish the brain death animal model one group were used.(n=8)

Experimental groups DBD, DCD, IRI and NRK-49 cells

To study the amount of HIF1α upregulation and its downstream effector HO-1 in different

models of kidney injury, 3 groups were used.(Figure 7) The samples from group 2 were

historical samples taken from previous BD experiments done in the University medical centre

Groningen. Rat kidney cells exposed to 24 hours of hypoxia were used as a positive control.

Group 1: deceased brain death group. (n=2)

Group 2: donor after circulatory death. (n=2)

Group 3: ischaemia reperfusion group. (n=2)

Group 4: rat kidney cells 24 hours hypoxia.

Fig. 7 Experimental protocol for the DBD, DCD and IRI experimental groups.

16

Brain death model

Experimental preparation

Protocols for the animal experiments were written and worksheets were designed for

recording the physiological parameters e.g. temperature, mean arterial pressure, heart rate and

oxygen saturations. (See supplementary protocols and worksheets.)

To set up the brain death model we had to monitor several parameters, and investigate which

set up and anesthesia settings were optimal. This is done by monitoring parameters including

peak pressures in the lungs, the tidal volume (the volume of gas inhaled and exhaled during

one respiratory cycle), the oxygen flow and % isoflurane administered. We calibrated the

optimal settings for rodents of different weights. The normal lung pressure of a male rat is

10-12 mmHg.

Anaesthesia and surgical preparation

Rats were anesthetised using 3-5% isoflurane with 100% oxygen (1L/minute) and during the

procedure maintained at 2% isoflurane on average. The rats were weighed and placed in

supine position. After shaving and cleaning the operation field with 70% alcohol, the

operation was commenced.

Brain death animal model

For the brain death rat experiments we used a gradual onset brain death model. This model

has been described previously (1) (figure 8) The aim was to simulate cerebral haemorrhage

by slowly increasing the intracranial pressure. Anaesthesia and preparation was performed

like described previously. Via a tracheostomy the animals were intubated and ventilated

throughout the experiment with a respiratory rate of 50-70 per minute. In this model we

mimicked intracranial hemorrhage, which finally led to brain death, with increasing a

subdurally placed no.4 Fogarty catheter (Edwards Lifesciences Co., Irvine, CA). The catheter

was slowly inflated (16 μl/min) with saline using a syringe pump. During the brain death

induction the percentage of isoflurane was reduced to 2 %. To monitor the blood pressure a

polyethylene catheter was inserted into a femoral artery. After brain death establishment the

anesthesia was turned off and the rats were ventilated with oxygen (1L/min), this was

continued during the whole experiment.

Fig. 8. Brain death model

17

Brain death procedure (DBD)

Using a microdrill a hole ( 2mm) was drilled through the skull frontolateral to the bregma.

Into the extradural space a balloon catheter was inserted. By gradually increasing the

intracranial pressure by inflating the balloon with 16μL saline per min, induction of brain

death was started. Stopping Inflation of the balloon and withdrawal of anaesthesia was done

after a hypotensive period followed by a short peak and a subsequent drop in blood pressure

and after this returning of the blood pressure to its basal level again. Until the end of the

experiment the balloon was kept inflated. Confirmation of brain death was done 30 -40 min

after the onset of brain death, by the absence of corneal reflexes and a positive apnoea test.

Donor management

A mean arterial pressure (MAP) above 80 mmHg was considered to be normotensive during

brain death. Blood pressure was recorded by the Labview program nerve Chart version 1.02

on a device provided by the Evans group. When blood pressure decreased below 80 mmHg,

venous return was increased by repositioning the rodent. If this failed to increase the MAP

colloid infusion were administered in boluses of 0.2 ml (Voluven). By using a heating pad

and a rewarming lamp we attempted to maintain the body temperature at 37°C.

Collecting tissues

A midline laparotomy was performed 10 minutes before the end of the experiment. Urine was

collected just before the organs were flushed with 40 mL ice-cold saline via aortic infusion.

The kidneys were collected after complete blood flush out. The tissue samples were fixated in

4% paraformaldehyde and frozen on dry ice (+/- ethanol).

DBD samples, DCD model, IRI model and NRK-49 cells

Deceased brain death samples (DBD)

Historical kidney and liver samples were taken from previous BD experiments done in the

University of Groningen. There the BD procedure described by Kolkert et al. was performed.

(1)

Donation after circulatory death model (DCD)

Anaesthesia and preparation was performed in a similar manner to that described previously.

The left and right kidney were randomized by flipping a coin. The control kidney was taken

out at time point 0 by ligating the renal pedicle and dissection of the kidney. After tying the

kidney was retrieved, dissected longitudinally and frozen on dry ice (+/- ethanol) or fixated in

4% paraformaldehyde. The rat was kept anesthetized and the wound was closed.

Subsequently cervical dislocation was performed. Cessation of cardiac death was monitored

by palpation. After 30 minutes following cervical dislocation the abdomen was reopened and

the second kidney retrieved, dissected longitudinally and frozen on dry ice (+/- ethanol) or

fixated in 4% paraformaldehyde.

Ischaemia reperfusion model (IRI)

Anaesthesia and preparation was performed in a similar manner to that described previously.

The left and right kidney were randomized to IRI vs control and the renal artery and vein

were dissected. After dissecting a non crushing bulldog arterial clamp was applied for a

period of 45 min. 10 U / ml heparin was administered subcutaneously 10min prior to

removal of the clamp. After 45 min the abdomen was reopened again and the clamp removed.

Bupivicaine was administered as pain relief (10 mg/ml) and the wound closed. The rat was

recovered using 100% O2 and placed in a heating room for slowly waking up. After 4 hours

18

the experiment was terminated. Anestheasia was induced with 3-5% isoflurane and the

abdomen opened again. The aorta was cannulated and flushed with normal saline. Both

kidneys were retrieved, dissected longitudinally into halfs and frozen on dry ice (+/- ethanol)

or fixated in 4% paraformaldehyde. Following the first anesthesia period the animals woke

up and were alert after approximately 30 min, while being kept in a heating room.

Preparation of slices

Laboratory experiments were performed in the welcome Wellcome Trust Centre for Human

Genetics.

Precision-cut kidney slices of 50 mg were prepared on dry ice. For the homogenisation 1 ml

of urea /SDS lysis buffer was used for 100 mg of tissue. The ultraturrax T25 homogenizer

was used and samples were homogenized for one minute. After homogenizing the tissue

homogenate were kept on urea SDS buffer for proper lysising and vortexed every minute.

The cells were centrifuged twice at 13k rpm for 10 minutes for separation with the

supernatant. The supernatant was piptetted into fresh Eppindorfs. After a last spin and an

additional equal volume of lysis buffer the samples were stored in a -20°C freezer.

Cell culture

As a positive control, NRK-49 cells were exposed to 1% of hypoxia for 24 hours in a hypoxia

station.

Initial culture

For the initial culture NRK-49 cells were the cells of interest. The cells were defrosted by

placing them in a 37 °C bath until defrosted. The cells were prepared in the culture hood. 5

mL of DMEM culture medium, 5 mL of a 5% glutamine dilution, 5 mL of a 5% Pen and

Strep dilution and 50 mL of a 10% Bovine serum dilution was added. The supernatant was

removed and the pellet was resuspend in 18 mL of culture medium. After assessment of cell

roundness, fullness and quality the box with cells was placed into the incubator at 37 °C with

5% CO2.

Splitting cells

Cells were daily checked under the microscope. Confluency of the cells was an indicator that

splitting needed to take place. The medium was removed from the cells and disposed.

Phosphate buffered saline (PBS) was added to the non cell side of the flask to wash the cells

and the PBS was removed after washing. Then 1 mL of trypsin is added to make the cells

detach from the bottom of the plate. Trypsin works best in a warm surrounding, so the plate is

incubated for 1-2 minutes in 37 °C. The cells are tapped to free them up and examined under

the microscope to see whether they move or not. In order to stop the reaction and resuspend

the cells 10 mL of medium is added. To make up the right dilution (1:10) 9 mL of the

medium was discard. To make up to the volume started with 17 mL of medium was added.

Seeding cells

For splitting the cells into the right concentration that will be the most useful in the

experiment the cells were seeded. The confluences needed were 60% and 50% to allow them

to be approximately 80% confluent after the 24 hypoxia period.

The cells were washed in the culture bottle. First the DMEM medium was removed and the

cells were washed using 10 mL phosphate buffered saline (PBS). After washing the PBS was

removed. 1 mL of trypsin was added and incubated for 1 minute at 37 °C with 5% CO2. The

cells were rescued by adding 14 mL of the DMEM medium. To make a confluence of 60%,

355 μL was added in 9 separate wells. The wells were further filled with DMEM until a

19

volume of 1mL in each well. After seeding the cells were stored in the incubator at 37 °C

with 5% CO2.

Exposing to hypoxia

To expose the cells to hypoxia, the wells with a 50% and 60% confluence were placed in the

hypoxia chamber for 24 hours. The oxygen tension maintained was 1%.

Harvesting cells

To harvest the cells, the control cells (normoxia cells) were harvested first. 8 μL of 4x SDS

sample buffer (2.0 mL 1M tris-HCl pH6.8, 0.8 g SDS, 4.0 mL 100% glycerol, 0.4 mL 14.7 M

β-mercaptoeethanol, 1.0 mL 0.5 M EDTA, 8mg bromophenol Blue) was added to prelabeled

eppindorfs. The DMEM medium was removed from the wells containing the samples. 34 μL

USDS lysis medium was added to the wells. The samples were aspirated and placed into the

prelabeled eppindorfs containing the 4x SDS sample buffer.The eppindorfs were centrifuged

for one minute and vortexed. The same steps were followed for the positive control hypoxia

cells. Once all samples were obtained the were heated at 95°C for 5 minutes and virgorously

vortexed for 15 sec per sample. After centrifugation the cells were checked for siffucient

liquification and stored at -20 °C.

Western blotting

We evaluated whether exposing cells to 24 hours of hypoxia could induce HIF1α response

using Western blotting. For this equal amounts of protein were loaded on to SDS/PAGE gels.

(stacking solution; Tris/HCL/SDS pH 8.8, 10% acrylamide, dH20, TEMED, 10%APS.

Resolving solution; Tris/HCL/SDS pH6.8, acrylamide, dH20, TEMED, 10%APS) The first

well was filled by a page marker and the gels were run at 180V and 60-80 mA for 1 hour.

The proteins were transferred on to nitrocellulose membranes by running at 100V for 1 hour

and incubated with Novus Biologicals NB100-479 antibody for 1 hour, an antibody used in

the laboratory which shows cross-reactivity with rat HIF1α. The blots were subsequently

incubated with a rabbit anti- IgG antibody a as an secondary antibody for 1 hour. Β-actin was

used as a loading control and was detected with conjugated antibody. Visualization was

peformed by incubation of the membranes with SSWD/SSWS solutions (super signal west

dura peroxide buffer/super signal west dura luminal/enhancer solution) and exposing films on

the membrane to infrared light. Detected signal was quantified an normalized. After a

positive result, confirmation of hypoxic cells, detected by the Novus biological antibody we

compared 3 different types of antibodies on the hypoxic and normoxic cells, to see which

antibody gives the strongest signal. After detection of the best antibody (Novus Biological

NB100-479 in a 1:1000 solution) we tested whether this antibody was cross-reactive with

our tissue samples.

Statistical analyses

Using the photography software program image J for scientific image processing the western

blots were converted raw data.

The statistical program Instat 3 was used to perform the statistical analyses.The distribution

of data was assessed with Q-Q plots and the Kolmogorov-Smirnov test for normality.

Normally distributed data are going to be expressed as mean ± standard error of the mean.

Non-normally distributed data are going to be tested with the Mann-Witney U test.

20

Results

Brain death experiments

To optimize the ventilation settings and to set a start point for each following experiment, the

pressure in the lungs, tidal volume and flow was measured. (figure 9 and 10) The tidal

volume could be adjusted by turning a button on the ventilator. A sample bag, from what the

ventilator could sample oxygenated air mixed with isoflurane was used in the brain death

model from the University of Groningen. In our brain death model set up a comparison was

made whether optimal anaesthesia was with or without the sample bag. The normal pressure

in the lungs of a rat is 10 -12 mmHg, so without the sample bag, a tidal volume of 8 turns and

a flow of 0.4 L/min the optimal starting settings are achieved.

21

Induction of brain death showed a drop in blood pressure after approximately 40 minutes.

(figure 11) Following the drop the blood pressure didn’t raise above 80 mmHg. The brain

death group had an average infusion of 3.2 mL heparinised saline through the femoral artery

to maintain stable blood pressure. Experiments were terminated after 30 minutes, 60 minutes

and 120 minutes.

22

Deceased after circulatory death experiments

2 DCD experiments were performed. Cervical dislocation resulted in immediate cessation of

breathing and cessation of cardiac contractility at 5 min after cervical dislocation. After 30

minutes of warm ischemia the second kidney was retrieved.

Ischemia reperfusion injury experiments

The renal artery of the randomized kidney was clamped to induce ischemia, which was

verified by the change in renal color. During ischemia the abdomen was closed. Time

between start of the experiment and a clamp on the randomized kidney was the same in all

experiments and took 25 minutes. After 45 minutes of WI the abdomen was reopened, the

clamp was removed and reperfusion started. The pain relief bupivicaine was administered

before closure of the wound. After 4 hours of reperfusion the kidneys were retrieved.

Cell culture

The cells were harvested at a confluence of 50% and 60%. (figure 12)

23

Detection of HIF1α in the normoxia(Nx) and hypoxia(Hx) cells.

Western blotting was used to detect HIF1α in the grown cells with a confluence of 50% and

60%. (figure 13) The antibody Novus Biologicals NB100-479, known to be cross reactive

with kidney epithelial cells, was used to incubate and detect the proteins with. Β-actin was

used as a control. Figure 14 shows a significant upregulation of HIF1α in the hypoxic cells.

Figure 13. Detection of HIF-1α in normoxic (Nx) vs hypoxic (Hx, 1% O2 for 24 hours) NRK-49 cells using

anti-rat HIF1a antibody (Novus Biologicals NB100-479) comparing cells harvested from 50% and 60%

confluence at seeding. Β- actin as a control.

Figure 14. Detection of HIF 1 α expression in Nx and Hx cell culture. 24 hours of 1% hypoxia significantly

induced HIF 1 α in the hypoxic cells.

Antibody determination

To find the antibody which detects HIF1α the best in the samples of interest using western

blots, two different antibodies were tested one of them in a 1:1000 and a 1:3000 dilution,

using the Nx and Hx cells. The first one was anti-rat HIF1α Novus Biological (NB100-479),

raised in rabbit, in a 1:1000 dilution and in a 1:3000 dilution. The second one was anti-rat

HIF1α Transduction Laboratories (610959) raised in mouse, in a 1:1000 dilution. As shown

in figure 15, the anti-rat HIF1α Novus Biological (NB100-479), raised in rabbit, in a 1:1000

dilution is the best antibody to use.

Figure 15. Comparison of

antibodies detecting HIF1α using

normoxic (Nx) and hypoxic (Hx)

NRK-49 cells, B-actin as control.

24

Comparison of HIF1a expression in 4 models of kidney ischemic injury

After finding and a detection of the HIF1α by the antibody Novus Biological (NB100-479),

raised in rabbit, in a 1:1000 dilution, 4 models of kidney ischemic injury were compared. As

shown in fig. 16 and figure 19 HIF1α is more expressed after 45 min of WI in the IRI group

than in the IRI control group.

30 min post cervical dislocation there is less HIF1α expression than in the DCD control

group. Also, after 4 hours of brain death, the HIF1α expression is less HIF1α upregulation

than in the control group.

Figure 16. Comparison of HIF1α

expression in 4 models of kidney

ischemic injury: cell hypoxia (NRK-

49 cells 1%O2 for 24 hours), IRI

model (45 min WI 4 hours

reperfusion), DCD model (30min

WI following cervical dislocation),

DBD model (4 hours of BD). Β-

actin as a control.

25

Comparison of HIF1a and HO-1 expression in 4 models of kidney ischemic injury

To characterize the expression of HIF1α and its downstream effectors following brain death,

ischemia reperfusion and deceased after circulatory death, the expression of HO-1, a

downstream effector of HIF was detected using western blot. (figure 17 and 19 )Westerns

blotting shows a difference in the HO-1 expression between the IRI control and experimental

group, but figure 19 shows this is not significant. The DCD experimental group shows a

higher HO-1 expression when compared to the control group, with the reverse seen with

regards to HIF1α expression. In the DBD control group HIF1α is stronger expressed than in

the experimental group, with an adverse effect when looking at HO-1.Westerns blotting

demonstrates no HO-1 protein expression in the Hx and Nx cells.

Figure 17. Comparison of HIF1α and HO-

1 expression in 4 models of kidney

ischemic injury: cell hypoxia (NRK-49

cells 1%O2 for 24 hours), IRI model (45

min WI 4 hours reperfusion), DCD model

(30min WI following cervical dislocation),

DBD model (4 hours of BD) β-actin as a

control

To control for the previous experiment same experiments with different samples were

performed.(figure 18) Again western blotting showed no HO-1 protein expression in the Hx

and Nx cells. The expression of HIF1α and HO-1 was similar as the expression of the same

proteins in the previous western blot for the IRI and DCD groups. Figure 18 shows a stronger

HIF1α expression in the DBD experimental group than in the DBD control group. HO-1

protein expression in the DBD control and experimental group showed the same result in this

western as in the previous western blots. Figure 19 and 20 show that the differences in

expression between HIF1α and HO-1 in all experimental groups are not significant.

Figure 18. Comparison of HIF1a and

HO-1 expression in 4 models of kidney

ischemic injury: cell hypoxia (NRK-49

cells 1%O2 for 24 hours), IRI model (45

min WI 4 hours reperfusion), DCD

model (30min WI following cervical

dislocation), DBD model (4 hours of

BD) β-actin as a control.

26

27

Comparison of HIF1a expression in 4 models of liver ischemic injury

To characterise and explore the hypoxia inducible pathway and its downstream effectors

furthermore it is interesting to see whether the same expression could be shown in other

organs like the liver. Figure 21 shows western blotting for liver samples, using the antibody

Novus Biologicals NB100-479. HIF1α expression is slightly higher in the control IRI group

than in the experimental IRI group. HIF1α is more expressed in the DCD control group than

in the DCD experimental group. Whereas HIF1α is more expressed in the DBD experimental

group than in the DBD control group. Figure 22 shows that these differences are not

significant.

Figure 21. Comparison of HIF1α

expression in 4 models of liver

ischemic injury: cell hypoxia (NRK-

49 cells 1%O2 for 24 hours), IRI

model (45 min WI 4 hours

reperfusion), DCD model (30min WI

following cervical dislocation), DBD

model (4 hours of BD). Β-actin as a

control.

28

Comparison of HIF1a expression in 2 models of liver ischemic injury

To explore the HIF1α pathway in the DBD group western blotting was performed on liver

samples and HIF1α and HO-1 expression was detected. Figure 23 shows a stronger

expression of HO-1 in the DBD experimental group than in the control group. The same

conclusion can be made regarding to HIF1α expression. Figure 24 shows a significant

difference in expression of HO-1 in de DBD experimental group versus the DBD control

group. Figure 24 shows no significant difference between the HIF1α expression in the DBD

control and DBD experimental group.

Figure 23. Comparison of HIF1α

and HO-1 expression in 2 models of

kidney ischemic injury: cell hypoxia

(NRK-49 cells 1 % O2 for 24 hours)

and the DBD model (4 hours of BD)

β-actin as a control.

29

Conclusion and Discussion

This study was designed to develop a brain death model which was the same as described

before(1), but then on a different location. This preliminary study aimed to characterise the

effect of brain death on HIF1α expression and HO-1 in comparison to other models of kidney

ischemic injury.

The importance of this study lies in the fact that the donor and pre-donation management and

state of the organs determines post-transplant function and graft survival.

Brain death model

A brain death animal model was established. Figure 11 shows that the results in this model

are extreme variable. There is a large variation in the parameters within the same group. This

variation could be due to technical problems and/or physiological problems. During the

performance of the experiments we faced several technical problems with the ventilator and

blood pressure measurements. On the other hand, the technical failures reflect an ‘unstable’

BD organ donor on the ITU and therefore are representative for the clinical setting.

Concluding from the fact that the lack of reflexes, a positive apnea test and the pressure

change pattern are similar to that of previous experiments,(1) it can be stated that the animals

were brain death after the brain death induction.

We observed after the initial decrease in blood pressure, a gradual increase in blood pressure

just before a peak was observed. This is due to the physiological response to increasing

intracranial pressure and is followed by a decrease in cerebral perfusion pressure. Normally

this event leads to ischemia in the brainstem, which results in peripheral vasoconstriction due

to excitation of different neurons and a subsequent increase in arterial pressure.(25) The

arterial pressure exceeds the intracranial pressure and blood flow to the brain stem is ensured.

But in our experiments the MAP of the peak wasn’t as high as described before by Kolkert et

al. (1)This effect could be because of hemodynamic effects due to brain death and this was

not corrected properly enough. Also, it could be due to reduced release in catecholamines and

pulmonary changes. In most studies using a sudden inflammation animal brain death model

(a model which simulates acute and significant cerebral trauma by increasing the intracranial

pressure within 30-60 sec.) a hypotensive period after the catecholamine-induced peak has

been described.(26) (27) Our results show a decrease in blood pressure after the peak

occurred, which is similar to the observations described in the sudden onset brain death

model. Kolkert et al. described the gradual onset brain death model and they showed a

plateau at levels of 100 mmHg after the peak in blood pressure. In our model optimization

and clarification needs to be performed to make sure we induce brain death on a gradual

onset instead of a sudden onset, which is what the blood pressure curve suggests.

In our brain dead model we didn't have a venous cannula. So blood pressure couldn’t be kept

stable by administration of HAES or noradrenalin, as described before. (1)A next step would

be to insert a venous cannula.

In our setting we used mechanical ventilation. In the past researchers looked at how to apply

mechanical ventilation and it has become evident that a number of ventilator strategies can

produce or worsen lung injury. The use of large tidal volumes (VT)(28), high inspiratory

flows,(29) high peak airway pressures, (30) and high respiratory rates (RR)(31)play a role in

the pathogenesis of ventilator induced injury. The epithelial and endothelial barrier is

damaged and compartments of alveolar cytokines can be lost into the vascular system during

acute lung injury. A process that can initiate a systemic response. (32). Tremblay et al. have

shown that ventilation with zero positive end-expiratory pressure (PEEP) and/or excessive

30

end-inspiratory lung volume increased the concentration of lung cytokines and ‘leak’ into the

systemic circulation. (33) During the optimization process we started doing the animal

experiments with a respiration frequency of the animal around 80 breaths per minute and with

a PEEP of zero. We corrected for this ventilation settings during the optimization of the brain

death model.

The occurrence of ischemia was one potential important primary trigger which was analyzed

in relation to the brain death process. Like described before, (34)periods in the slow-

induction model of BD and the IRI model are associated with a blood pressure drop followed

by a period of hypotension. The vasoconstriction and increase in peripheral vascular

resistance due to catecholamines release after the sharp increase in blood pressure can result

in a period of hypotension and a decrease in peripheral flow to the abdominal organs. This is

something which results in the occurrence of ischemic episodes and the occurrence of HIF1α

activation due to ischemia followed by hypotension instead of hypoxia.

Eckhardt. Et al. described an ischemia reperfusion model in which the control kidney was

removed before the ischemia/reperfusion of the contralateral kidney, to obtain information

about the efficacy of precondition.(19) in our experimental setting the control kidney of the

DCD is not the same as the control kidney of the IRI group. This is due to a different

procedure and therefore the 2 control groups are not comparable. The IRI control kidney was

removed after two periods of anaesthesia, opening the abdomen and placing a clamp on the

ischemia/reperfusion kidney. After this procedure western blotting showed a different

accumulation of HIF 1α in the DCD versus the IRI model.(figure 16, 17, 18, 21, 23)

Benedikt et al. have shown that anaesthetics induce early and late preconditioning in several

organs by activation of the transcription factor HIF 1 α and its downstream effectors. (35)

This might be an explanation of the difference between the HIF 1 α accumulation in our DCD

and IRI model.

Collection, preparation and analysing of slices

HIF1α and HIF2α have been shown to be differentially expressed in the kidney following

systemic hypoxia. HIF1 α is predominantly found in the tubular cells and in particular in the

medullary collecting ducts. HIF2 α is expressed in the glomerular cells, peritubular

endothelial cells and interstitial fibroblasts. (36) In our models we dissected the explanted

kidneys longitudinally into halfs and snap froze the pieces. 50 mg of these were prepared on

ice for analysis. Since we were first looking at HIF1 α we tried to cut the 50 mg pieces

longitudinally, so that we got the region were HIF1 α was expressed and we could detect it. It

was possible to control the way the tissues were collected and prepared in the experiments

performed by ourselves , however, the possibility we only received the cortex of the organs

taken from the BD experiments done in the Medical Centre in Groningen and therefore have

less tissue with a HIF1 α response is not excluded.

Interestingly, figure 18 shows a stronger HIF1α expression in the DBD experimental group

than in the DBD control group. This is in contrast with the western blot shown in 17 and 16.

We can’t explain this observation other than only having prepared the cortex instead of the

medulla where HIF1α is strongly expressed. Wrong loading during western blotting is not a

possible reason, because HO-1 is in both figure 17and 18 stongly expressed in the DBD

control group. For further research it is essential to develop a strategy to prepare the slices so

that the localization of HIF1α is not missed. For example through homogenezing whole

kidneys.

31

Figures 16, 17 and 18 of the kidney samples show a low HIF1α protein expression in the

experimental DCD group. Testing for HO-1 in figure 17 and 18 shows the reverse effect.

This phenomenon could be explained because of the short half-life of HIF1 α (37) and the

longer half-life of HO-1. HIF1α could already be disappeared when left on the bench for 30

minutes in the DCD group, whereas HO-1 is still detectable. So we could ‘miss’ seeing

HIF1α and we need to look at other time points during the experiments.

Figure 21, the western blot of liver samples, shows a low HO-1 protein expression in the

DCD experimental group when compared to the control group. As seen in the β-actin control

this effect could be due to technical reasons and another western blot needs to be performed

before making any conclusions.

Another explanation of the high expression of HO-1 where there is low expression of HIF1α

could be the fact that HO-1 is a heat shock protein, because of its responsiveness to thermal

stress.(38) Thermal stress is stress caused by temperature change. Because the thermometer

used during the animal experiments was not accurate there might be a possibility HO-1 is

highly expressed because of the temperature change instead of being a downstream effect of

HIF1α.

HO-1 could also be activated by a pathway independent from HIF1α. HO-1 is an enzyme that

has a transcription which is regulated by a large variety of stimuli. These include its substrate,

heme, signaling proteins nerve growth factor, TNFα, IL1β and interferon γ.(39) These are all

enzymes expressed as an adaptive response that increases cell resistance to oxidative injury.

However, HO-1 still maintains one of the major downstream effectors of HIF (40) so further

research needs to be performed to determine whether HO-1 is upregulated by HIF1α in these

models of kidney injury.

As shown in the results, in none of the groups the difference in HIF1α is significantly

different expressed. This is probably due to the size of the n and/or the constructed models.

More experiments with different samples need to be performed to reduce large variations in

the outcomes within the same groups and to make conclusions which could be significant.

The assumption that HIF activation protects tissues against hypoxic damage so far mainly has

been indirect, all on the basis of the characeristics of HIF and its downstream effectors. For

example it has been shown that EPO expression is predominantly HIF 2 α and not HIF 1 α

dependent. (41) So for the next step in our approach on the HIF pathway it is interesting to

clarify what happens with HIF downstream effectors like HO-1, EPO and VEGF.

Furthermore, a clarification of the prolyl hydroxylase 1,2 and 3 and of HIF 1 α and HIF2 α

expression in the different models would be of a great value. In addition a time course effect

of the HIF accumulation at 0.5, 1, 2, and 4 hours is interesting to see.

32

Recommendations for further research

After a complete clarification of the hypoxia inducible factor pathway it is interesting to

investigate whether the pharmacological induction of HIF and its protective downstream

effectors target ischaemia related injury in the brain death setting and deceased after

circulatory death setting. In the clinical context of organ donation, the opportunity to

‘pretreat’ organs is limited. In an IRI study performed by Bernhardt et al. animals were

treated with prolyl hydroxylase inhibitors prior to ischaemia.

In normoxia the prolyl hydroxylases are continuously inactivating HIFα. In this way they

prevent the dimerisation with HIFβ and consequently preventing the transcription of hypoxia

responsive genes. In addition to oxygen, prolyl hydroxylases also require Fe2+, 2-

oxoglutarate, and ascorbate. Knowing this, there is a possibility to pharmacologically activate

PHDs in the BD and DCD setting by for example the administration of DMOG

(dimethyloxalylglycine) a 2-oxoglutrate analogue, or Fe2+ and cobaltous ions (42).

Oxyglutarate analogues act as competitive inhibitors of PHDs and other HIF stabilizers. (17)

No data exists on whether treatment can be administered after ischaemic kidney injury. (43)

However, a rodent study found that administration of the prolyl hydroxylase inhibitor

dimenthyloxaylglycine (DMOG) 30-60 min following middle cerebral artery occlusion

reduced ischaemic injury (44) and in cardiac postconditioning HIF plays a critical role (45).

So after completing to assess the effect BD and DCD have on HIF and HIF target genes in

the kidney, investigating whether DMOG can activate HIF following deceased after

circulatory death and brain death is of great value. Activating HIF following an ischaemic

period by DMOG administration might reduce the pro inflammatory state of the donor and

subsequent effects of IRI, this improves the outcome of kidney transplantation.

Figure 16, 17, 18 and 21 showed HIF1α expression in an model which mimics uncontrolled

DCD. After further exploitation and confirmation of the HIF1α expression in an uncontrolled

DCD model it will be interesting to see whether and on what level the hypoxia inducible

factor is up-regulated in a rat model which mimics controlled DCD. At a later state it would

be interesting to investigate whether it is possible to use the hypoxia inducible factor to

precondition DCD donors.

Translational strategy; relevancy for the human setting

Quality in Organ donation

In order to attempt the issue that fewer optimal organ donors will be available, because of

reasons like changes in neurosurgical practice as well as improvements in the

management of cardiovascular disease and a fall in road accident deaths, the

transplantation community has been turning to organs preciously considered

unsuitable for donation. These are organs from older donors following brain

death, the extended criteria donors (ECD) and donors following circulatory

arrest (DCD). The abnormal physiological state in these donors results in

significant organ damage even prior to procurement. Investigating these injury

mechanisms in the donor, by understanding the pathways of injury and

applying therapeutics, we think that organ injury can be prevented and short and long-term

organ survival as well as the function of the graft can be improved. We propose that it helps

transplant teams in deciding the suitability of specific organs for individual patients if we are

able to identify markers in the donor to predict the short and long-term outcome of

transplantation. This would also help to improve or alter the post-operative management in

the specific recipient.

33

Success in transplantation starts in the organ donor. There is a number of points at which

there is a possibility to intervene to prevent organ damage, from the moment of the initial life

threatening event on. (See figure) These potential points include interventions during the

ICU, management of the brain dead and dying patient, strategies to prevent ischemia

reperfusion injury and optimizing retrieval and organ preservation techniques.

The key aims of the QUality in Organ Donation (QUOD) project are:

To increase the number and quality of organs procured from DBD and DCD donors for

transplantation by optimizing donor management and resuscitating and preserving marginal

organs.

- To make previously unusable organs transplantable and increase the ‘donor pool’.

- To identify pathways of injury and apply targeted interventions to repair donor organ

injury.

- To translate validated experimental methods and technologies into clinical use and

best practice protocols.

- To identify bio-markers and functional parameters that predict outcome following

transplantation.

- To streamline collaboration and dissemination between scientific and clinical experts

in academic institutions across the United Kingdom.

The team which worked on this project consisted of Prof RJ Ploeg, who was the co-

ordinating principle investigator, Zeeshan Akhtar and Maria Kaisar, the research students,

Luke Marsden and Christian Brailsford the expert advisors, Sandrine Rendel, the national

operational coordinator and myself, also a research student with the capacity to aid in

establishing the national donor bio-bank because of my previous experience as President of

the Prometheus kidney team in the University medical Centre in Groningen. Together we are

the QUOD National management team and we propose to create a national Consortium on

donor organ quality. We want to bring together all the participants in the National Organ

34

Retrieval System (NORS), these include clinical and scientific experts from the ICUs and

transplant centres in the United Kingdom. The centres to start the project include Cambridge,

Edingburgh, Newcastle-upon-Tyne, Kings College from London and Oxford. The idea is to

collect demographic and clinical information from organ donors following informed consent.

Also, blood, urine and tissue biopsies will be collected at certain timepoints during

procurement. Tissue biopsies include biopsies from the left and right kidney, from the liver,

the ureter and the spleen. In addition, information about ex-vivo preservation techniques and

procurement related injury will be recorded. We will use the samples to analyse pathways

involved in organ injury and repair and to identify novel bio-markers. This will be correlated

with the organ outcomes in the recipient. The samples are going to be stored in a central

biobank and this biobank will be accessible to researchers.

The outcome measures are;

Assessment of donor organ injury using clinical (donor profile and organ function) and

biological parameters.

Clinical functional assessment in the recipient (primary non-function, delayed graft function,

immediate function) and expression of relevant biological parameters for injury and/or repair.

Identification of donor risk factors

Evaluation of specific post-post transplant related complications related to donor organ

quality.

My role in the QUOD national management team was to aid in the establishment of this

organ donor database and bio-bank using my expertise, gained as being part and president of

the Prometheus Kidney team in the University medical centre in Groningen. I used this

expertise to establish, implement, check and control laboratory and procedure protocols. I

wrote protocols for the specialized nurses in organ donation (SNODs) which we provided to

NHSBT. To check protocols before implementation into the QUOD standards I went with the

retrieval surgeons on organ retrieval and followed and revised the protocols. Furthermore I

wrote the worksheets on what the SNODs and perfusionists have to record the time, places,

facts and data of the collection of biological materials and clinical data.

I communicated between the members of the management team and encouraged

collaboration of the different participants of organ retrieval; clinical and scientific experts

from all over England and Scotland as well. I wrote patient leaflets and worked on ethics

documents which had to be submitted and approved before starting the collection of samples

and biopsies. All together this became the QUOD booklet to which participants of the QUOD

team are able to refer to and adjust documents when required.

35

Conflict of interest

The author declares that she has no conflict of interest.

Acknowledgements

First of all I want to thank prof. Rutger Ploeg. Thank you for giving me the opportunity to

come to Oxford and work with you and your team. Secondly Henri Leuvenink, thank you for

all your visits and the support in hard times. Zeeshan Akhtar, Zee, thank you for all the hours

we worked together and for becoming such a good friend. Petra Ottens, thank you for coming

over to Oxford and helping us with the brain death model. Furthermore I want to thank

Michiel Voskuil, Sandrine Rendel, Maria Kaisar and Beverly Burcham. It has been a

pleasure to work with you!

36

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40

Supplementary protocols

Brain Death

1.0 Brain death induction

In most brain death models, an increase in intracranial pressure is generated by an

expanding intracranial balloon, finally leading to brain death. Our model simulates

cerebral hemorrhage by slowly increasing the intracranial pressure, resulting in less

hemodynamic instability and maintenance of normotension during brain death for several

hours.

2.0 Equipment

O2/isoflurane anesthesia

Compressed air

Ventilator: Harvard apparatus, model 683

Trachea canula/”tube”: shortened 16G i.v. cathether ( 2cm) (BD insyte)

Equipment for bloodpressure measurements (Meetcase, Organ Assist)

A. femoralis canula: PE canula 0.40x0,80 mm, 6 cm long with a blunted end 26G

needle.

V. femoralis canule: silicone canule (0.5x1.0 mm, 8 cm long)with a blunt ended 25G

Ligatures 6/0 (Braun: 11340119) and 3/0 (Braun: 01134043)

Suture 4/0 mersilene (R-683H, Ethicon)

Heparin 10 U / ml in saline. (Pharmacy, 5000 IU / ml)

Arterial embolectomy catheter 3F (UMCG, inkoop)

Shaver

10% HAES (37oC). (Pharmacy)

70% alcohol.

papaverine sulphate (50 mg / ml), lidocaine (10 mg / ml), nor-epinephrine (1 mg/ml)

(Pharmacy)

Syringe Pump (Terufusion Syringe Pump, model STC-521)

3.0 Procedure

1. Start surgery:

Anestethise the rat using O2/isoflurane 5%,

Anesthesia is maintained with O2/isoflurane 2%

Turn on the heating mat (stand 2)

Weigh the rat

Shaving: head, throat and one of the legs.

Place the rat in supine position

Clean the operation areas with 70% alcohol.

2. Femoral artery en vene canulation

41

Start with a medial incision in the leg of the rat.

The femoral artery and vein are directly visible under the skin.

Dissection of the femoral artery and vein.

Put two ligatures (6/0) around the femoral vein.

Ligate the distal ligature.

Cut a small hole in the vein and insert the vene canula into the vein.

Fixate the canula with the rostral ligature. Infuse some saline with heparin into the

femoral vein.

Place a drop of papaverine on the artery.

Put two ligatures (6/0) around the femoral artery

Ligate the distal ligature.

Bring a blunt curved forceps rostral under the artery to lift and close the artery.

Cut a hole in the artery and insert the canula.

Fixate the canula with the rostral ligature. Infuse some heparinised saline into the canula

Suture the wound (4/0).

3. Intubation of the rat

Prepare a medial incision through the skin of the throat.

Pull the salivary glands laterally from each other.

Watch out for the blood vessels at the rostral side of the salivary glands that may not be

damaged.

Separate the m. sternohyoideus (muscles)

Dissect the blood vessels from the trachea

Place two ligatures around the trachea (3/0 suture)

Just before intubation, the ventilator has to be switched on.

Slightly lift the trachea with the rostral trachea ligature, make a little incision between 2

cartilage rings below the thyroid glands and the tube can be inserted inside the trachea.

Don’t insert the tube more than 1 cm, because otherwise one of the two lungs will be

ventilated.

Connect the tube to the ventilator and fixate the tube with the caudal ligature.

Ventilator:

Breathing frequency: 50 breaths/min, and a peak of 13-15 (acceptable until 100

breaths/min)

Tidal volume: 3-4 ml/stroke

The EtCO2 will be measured.

If the EtCO2 > 22 mmHg increase the breathing frequency

If the EtCO2 < 20 mmHg decrease the breathing frequency

Suture the wound (4/0 suture) and fixate the tube.

4. Turn rat to prone position

Disconnect the blood pressure measurements.

Disconnect the ventilator and change the rat form supine position to prone position.

Connect the ventilator and the blood pressure measurements.

Infuse some heparinised saline through the a. femoralis to avoid blood clotting.

Place a temperature probe into the rectum of the rat. Control the temperature by turning

on the lamp.

Connect the pulse oximeter.

42

5. Craniotomie + insertion of the catheter

Prepare an incision in the skin above the skull.

Place some drops of lidocaïne / marcaïne on the skull. Wait for several minutes.

Drill a hole through the skull frontolateral to the bregma, using a microdrill. Don’t hit the

dura.

Drill also a line rostral from the hole to gently infuse the catheter.

Using a blunt forceps, prepare some extradural space between the dura and the skull.

Check whether the catheter is intact (with some fluid)

Fill the balloon catheter with 1 ml demi water without air bubbles in the syringe or catheter.

Place the catheter with syringe in an infusion pump and set the infusion rate to 16 ml/h

(equal to 0.5 ml fluid in the catheter during 30 minutes)

Gently insert the balloon catheter until the end of the balloon is just underneath the skull.

Wait till the blood pressure is about 80-100 mmHg

Start the brain death induction.

During balloon inflation, a hypotensive period occurred followed by a short peak and a

subsequent drop in blood pressure.

When the blood pressure returned to it’s basal level during an increasing peak, inflation

of the balloon is stopped and anesthesia is withdrawn.

The first 30 minutes of brain death the rat will be ventilated with 100% O2 (1000 ml/min).

Confirm the state of brain death 30 minutes after onset of brain death:

The absence of corneal reflexes

A positive apnoea test

Disconnect the ventilator for approximately 1 minute.

if the rat isn’t brain death, he will start breathing, otherwise the test will be

positive.

6. Donor managment:

After 30 minutes of brain death, the ventilator will be switched to O2 / air (1:1).

Blood pressure:

A mean arterial pressure (MAP) above 80 mmHg is considered to be normotensive.

o When MAP decreased below 80 mmHg, a drip with norepinephrine

(0.01 mg/ml) is connected to the femoral vene canula.

Start slowly with the infusion untill the MAP will be 100 mmHg. If the blood

pressure is stable at 100 mmHg, slowly decrease the infusion rate of the drip

to zero.

o With the use of a norepinephrine drip we can manage the MAP till 80-100

mmHg

Temperature:

o The temperature of the rat will be controlled with a lamp in combination with

a heating mat

Ventilation:

The EtCO2 will be maintained to 20-22 mmHg.

o If the EtCO2 > 22 mmHg increase the breathing frequency

o If the EtCO2 < 20 mmHg decrease the breathing frequency

7. 4 hrs of Brain Death

43

15 min before 4 hrs of BD, administration of Esmeron (0.6 mg/kg)

o 0,2 ml/100 gram i.a (333 ul stock + 9667 saline)

10 min before 4 hrs of BD, turn the rat in suprine position and open the abdomen

5 min before 4 hrs of BD, administration of heparin i.a

At 4 hrs of BD, collect blood, urine and organs.

44

DCD model

1.0 the model

the model is relevant to non-recovery of rodents

2.0 Equipment

O2/isoflurane anesthesia

Compressed air

Ligatures 6/0 (Braun: 11340119) and 3/0 (Braun: 01134043)

Suture 4/0 mersilene (R-683H, Ethicon)

Heparin 10 U / ml in saline. (Pharmacy, 5000 IU / ml)

Shaver

70% alcohol.

papaverine sulphate (50 mg / ml), lidocaine (10 mg / ml), nor-epinephrine (1

mg/ml) (Pharmacy)

Scale

3.0 Procedure

1. Start surgery

Anesthetise the rat using O2/isoflurane 5%,

Anesthesia is maintained with O2/isoflurane 2%

Turn on the heating mat (stand 2)

Weigh the rat

Place the rat in supine position

Shave the operation field

Clean the operation areas with 70% alcohol.

2. Retrieving first kidney

Midline abdominal incision

Randomize the kidneys

Tie the renal artery

Retrieve first kidney

Dissect longitudinally into halfs- ½ snap freeze, ½ in formalin

Close wound

3. Cervical dislocation

Keep the rat anesthetised

Dislocate the cervix of the rat, using a suffle

Check the heart rate and breathing

Wait for the determined time period

4. Retrieving second kidney

Open abdomen

Retrieve the second kidney

Retrieve other organs as controls

Dissect longitudinally into halfs- ½ snap freeze, ½ in formalin

Close wound

45

Renal ischemia reperfusion injury model

1.0 The model

This model is relevant to non-recovery of rodents.

2.0 Equipment

O2/isoflurane anesthesia

Compressed air

Equipment for blood-pressure measurements (Meetcase, Organ Assist)

A. femoralis canula: PE canula 0.40x0,80 mm, 6 cm long with a blunted end 26G

needle.

Ligatures 6/0 (Braun: 11340119) and 3/0 (Braun: 01134043)

Suture 4/0 mersilene (R-683H, Ethicon)

Heparin 10 U / ml in saline. (Pharmacy, 5000 IU / ml)

Shaver

70% alcohol.

papaverine sulphate (50 mg / ml), lidocaine (10 mg / ml), nor-epinephrine (1 mg/ml)

(Pharmacy) #

scale

3.0 Procedure

1. Start surgery:

Anesthetise the rat using O2/isoflurane 5%,

Anesthesia is maintained with O2/isoflurane 2%

Turn on the heating mat (stand 2)

Weigh the rat

Place the rat in supine position

Shaving: operation field

Clean the operation areas with 70% alcohol.

3. Clamping of the renal artery

Midline abdominal incision

Randomize the kidneys to IRI vs control

Dissect renal artery and vein and apply non crushing clamp for period e.g. 30-45min

Administer heparin through femoral arterial access (check if required)

Close wound

Reopen following ischemic period and remove the clamp

Administer pain relief

Close wound

4. Terminating experiment following ischemic period

Open abdomen

Cannulate aorta

Flush with normal saline

Explant kidneys

Dissect longitudinally into halfs- ½ snap freeze, ½ in formalin

46

DBD experiment

Date:

Ref e.g. DBD001

Type rat:

Male/female:

Weight:

Time start operation:

Time arterial cannulation:

Time intubation:

End of experiment:

Event time temp Heart rate %SpO2 MAP ETCO2

Brain death induction

Time:

5’

10’

15’

20’

25’

30’

45’

1hr

1hr 15’

1hr 30’

1hr 45’

2hrs

2hrs 15’

2hrs 30’

2hrs 45’

3 hrs

3hrs 15’

3hrs 30’

3hrs 45’

4hrs

Time perfusion of abdominal organs:

Volume of urine:

Volume of blood:

Number Eppindorfs kidney left:

Number Eppindorfs kidney right:

Number Eppindorfs pancreas:

Number Eppindorfs liver:

47

DCD experiment

Date:

Ref e.g. DCD001

Type rat:

Male/female:

Weight:

Time of anaesthesia:

Cervical dislocation

Time to cessation of cardiac contractility

Time start operation:

Administration of heparin:

Length of warm ischemia kidney left:

Length of warm ischemia kidney right:

Time perfusion of abdominal organs

End of experiment:

Time perfusion of abdominal organs:

Volume of urine

Volume of blood

Number Eppindorfs kidney left:

Number Eppindorfs kidney right:

Number Eppindorfs pancreas:

Number Eppindorfs liver:

48

Renal IRI experiment

Date:

Ref e.g. RIS001

Type rat:

Male/female:

Weight:

Time of anaesthesia:

Time start operation:

Time re-open the abdomen after 4 hours:

Clamp on left/ right :

Clamp off (reperfusion):

Time ischemic period:

Administration of heparin time & amount:

Time arterial cannulation & flush:

End of experiment:

Length of warm ischemia:

Event time temp Heart rate %SpO2 MAP ETCO2

Application of clamp

Time:

05’

10’

15’

20’

25’

30’

35’

40’

45’

1hr

1hr 15’

Time administration pain relief:

Abdomen closed:

Amount administration normal saline:

Event time temp Heart rate %SpO2 MAP ETCO2

Reperfusion:

15’

30’

Time animal in oxygen/ recovery cabin:

Time waking up:

Time normal behaviour:

Time perfusion of abdominal organs:

Volume of urine:

Volume of blood:

Number Eppindorfs kidney left:

Number Eppindorfs right:

Number Eppindorfs pancreas:

Number Eppindorfs liver: