Bud 2016 dapest, 2 Docto oral SchoSemme ool of Clielweis Un nical Meniversity edicine Raine er Ober rbauer PR REVEN NTION N OF ACI CUTE RINJURY RENALY L ALLO OGRAF FT

(1)

PR

Con Offi Hea Mem

REVEN

nsultant: A icial review ad of the F

mbers of th

NTION

Docto

Attila Szab wers: Atti Balá inal Exam

Zolt he Final E

Fere Zsol

N OF AC I

P

Raine

oral Scho Semme

ó, M.D., D ila Tordai ázs Antus mination C án Szabol Examinatio enc Rényi-

lt Csapó M

Bud

CUTE R INJURY

PhD thesi

er Ober

ool of Cli elweis Un

DSc.

M.D., DS MD., DSc Committee

lcs M.D., P on Comm -Vámos M M.D., Ph.D

dapest, 2

RENAL Y

is

rbauer

nical Me niversity

Sc.

c.

e:

Ph.D.

mittee:

M.D., Ph.D D.

2016

L ALLO

edicine

D.

OGRAF FT

(2)

1

The list of abbreviations

DGF delayed graft function DSA donor specific antibodies

ARF acute renal failure

ICU intensive care unit

OR odds ratio

MI myocardial infarction

ReOp reoperation BUN blood urea nitrogen DOI distant organ injury GFR glomerular filtration rate

CFR chronic renal failure

ADH antidiuretic hormone

RCT randomized controlled trial

LCM laser capture microdissection

QC quality control

OEDTR Österreichisches dialysis and transplant registry

POC proof of concept

BCAR biopsy confirmed acute rejection GEO gene expression omnibus

SNPs single nucleotide polymorphism

SIRS systemic inflammatory response syndrome ITT Intention to treat (analysis)

(5)

4

ET Eurotransplant SAM significance analysis of microarrays FDR false discovery rate

NaPi2a natrium(sodium) phosphate cotransporter HRPTEC human renal proximal tubule epithelial cells MM mismatch

ASO antisense oligonucleotide

IRI ischemia reperfusion injury PCR polymerase chain reaction HCV hepatitis C virus

(6)

5

1 Introduction

Kidney transplantation is clearly the preferred treatment of end stage kidney disease since it allows for a nearly normal life quality and is in the long run considerably cheaper than maintenance dialysis (24).

The incidence rates of patients undergoing kidney transplantation vary considerably among countries and regions reflecting different national regulations and public opinion on organ donation and transplantation in general. The incidence as well as the prevalence of kidney transplantations per million population is in strong correlation with the socioeconomic situation and the Human Development Index (HDI) and income level of countries. Industrialized regions in the world such as USA, Australia and Europe with high HDIs of well above 0.6 show a high incidence (> 30 per million population) of patients undergoing kidney allografting but with a considerable regional variability.

Accordingly, there is also a marked regional and global disparity in the prevalence of kidney transplant patients. The prevalence rates also correlate tight with the socioeconomic status and range from 50 to 300 per million population where the poorest regions have the lowest rates. In countries with a HDI below 0.3 there is virtually no renal transplantation performed. In addition, sound data in these regions are not available and thus an informed discussion on transplantation in these regions is not possible. Lastly, the medical insurance type, religious believes and philosophy are key determinants for access to transplantation.

Given the expenses of this high-end medical procedure requiring the tight collaboration between specialists from different areas such as surgery, immunology, pathology, radiology, tissue typing, infectious disease and nephrology render it intuitive that only countries with working medical infrastructure can offer such regimen. In addition, usually only patients with medical insurance covering such initially expensive

(7)

proce allog affor of th dram have kidne

How one y bette

Figu data

Now dono surgi the

edures can graft need to

rdable to pa ese modern matically. N been achie ey transplan

wever; most year graft fu er pre-transp

ure 1. Ann from the Vi

wadays, mod or HLA ant

ical procedu main achie

undergo t o take life-lo

tients with h n immunosu National and eved over t ntation (25)

of the impr function not plant risk ev

nual attrition ienna Trans

dern transpl tibodies (DS

ure in live d evements i

this form o ong immun

health insur uppressants d internation

the last yea .

rovement th t only due to valuation (fi

n of renal splant Cente

lant medici SAs or don donor transp in mid-term

6 of treatmen nosuppressiv

rance or to v in the mid n nal data un ars in prolo

hat was achi o better imm igure 1).

allografts p er).

ine requires nor specific

plantation, a m graft pa

nt. Lastly, t ve drugs, wh

very wealth ninety eight nambiguous onging patie

ieved can b munosuppre

per period o

s the determ antibodies) appropriate atency, the

the recipien hich are exp hy patients.

ties prolong sly show th ent and gra

e attributed essive drug

of transplan

mination of ), technical postoperati

rate of n

nts of a k pensive and

The introdu ged graft sur hat great su aft survival

d to the incr gs but also d

ntation (per

f preformed l planning o

ive care. De not immed

kidney d only uction rvival uccess after

reased due to

rsonal

anti- of the

espite iately

(8)

funct high

The early resol all re (figu

Figu last o survi DGF

This risk f DGF prim week

tioning tran level.

incidence o y clinical co lves after so ecipients of ure 2) (26).

ure 2. The in one and a h ival. The re F/ARF comp

highly pre factor for a F have a mo mary or imm

ks but recen

nsplants from

of post-tran ondition wit

ome days b f deceased d

ncidence ra half decade elative risk pared to prim

valent cond a reduced al ore than dou mediate gra nt data sugg

m deceased

splantation th the strong but delayed

donor transp

ates of ARF e remained of graft lo mary functi

dition in de lograft surv ubled risk fo ft function gest that the

7 d organ don

acute rena gest negativ

allograft fu plants but v

F/DGF in th stable at 2 oss more th ioning graft

eceased don vival. Studie or graft loss

(1, 27). D ese allograf

ors remaine

l failure (A ve impact o unction (DG virtually nev

he applicant 25% with a

han doubled ts (PF).

nor transpla es showed t s compared DGF eventu fts are prone

ed unchange

ARF) is the on survival.

GF) affects ver in live d

’s home ins strong asso d in those

ntation repr that kidney to matched ally resolve e to a more

ed at a relat

leading sin ARF event roughly 25 donor transp

stitution ove ociation of transplants

resents the y transplants d transplants es over the e accelerated

tively

ngular tually 5% of plants

er the f graft with

main s with s with e first

d rate

(9)

8

of fibrosis, specifically under the current standard immunosuppression of cyclosporine or tacrolimus. These facts clearly show that there is an urgent need for improvement.

1.1 Background and significance

1.1.1 Delayed Graft Function

DGF has been identified as the key target for intervention and great efforts have been undertaken to study the underlying pathophysiology and identify risk factors for ARF as main cause of DGF. There is no uniform definition of post-transplant DGF but most authors use the need of post-transplant dialysis as dichotomous outcome. However, on a more fine-granular basis, DGF/ARF is actually a continuum which ranges from one post-transplant dialysis due to volume management or hyperkalemia to the most extreme other end of the spectrum called primary non-function. The rate of slow or incomplete function is clearly associated with reduced long term patency of the graft and mortality. Longer DGF is also associated with a more pronounced immune response and inflammation in the grafts as evidenced by studies that utilized early protocol biopsies. It is still unclear whether treatment of these early inflammation in these management biopsies with high doses of corticosteroids for example will lead to less fibrosis in the long run and increased graft longevity. Therefore it is of utmost importance to study the signal cascade of DGF/ARF on a molecular level.

It is likely that post-transplant ARF shares some common features of ARF in the native kidney occurring in roughly one out of four to six patients admitted to the medical ICU (2). If the ARF is so severe that a form of renal replacement therapy is required such as hemofiltration or hemodialysis, the risk of mortality increases dramatically. Studies in patients after cardiac surgery for example showed, that ARF is the second strongest predictor of death after cardiac arrest and ranks even before such devastating conditions as perioperative myocardial infraction or prolonged mechanical ventilation (3) (table 1).

(10)

9

Table 1. Risk of mortality expressed as odds ratio (OR) in patients after cardiac surgery developing dialysis dependent ARF compared to equally sick patients without ARF (adapted from Chertow GM et al. (3)).

These results seem initially not intuitive given the fact that renal failure can be treated by hemofiltration, a rather routinely used technique without procedural risk. So why is the risk of dying still so high in patients with ARF?

The likely explanation is that hemofiltration can be used efficiently to remove fluid and low molecular weight ‘toxins’ such as the renal markers creatinine or BUN but cannot normalize the many other malfunctions that are associated and/or caused by ARF. These adverse entities have been recently coined as ‘distant organ injury’ or DOI (4). DOI includes cardiac problems (high output failure, cardiomyopathy, pericarditis), pulmonary comorbidities (edema, alveolitis, pneumonia, hemorrhage), gastrointestinal disease (erosions, ulcerations, hemorrhage, pancreatitis, colitis), neuromuscular pathologies (neuropathy, myopathy, encephalopathy), hematologic/immunologic deficiencies (anemia, thrombopathy, hemorrhage and impaired humoral and cellular host defense) and protein catabolism.

These simple epidemiological analyses which highlight the devastating and essentially poorly treatable condition of the ARF syndrome makes it intuitively clear that prevention of ARF of native kidneys in the ICU and after transplantation is of paramount importance. In order to prevent this condition more insight is required from the initiating and early processes of developing ARF up to the full blown and eventually

OR (95% CI) Cardiac Arrest 23 (19 – 28) ARF pre-post adjusted OR 8 (6 – 10) Perioperative MI 5 (4 – 6) C. Insult or Coma 4 (3 – 6) Low output failure 4 (3.3 – 4.3) ReOp - Hemorrhage 2.7 (2.3 – 3.2) Prolong. mechan. Ventilation 1.3 (1.1 – 1.5)

(11)

10

resolving ARF. Since it is not possible to perform sequential renal biopsies on a daily basis experimental animal studies have been used to study postischemic ARF.

1.1.2 Experimental ARF

One of the most representative experimental models for the human situation is the unilateral nephrectomy and clamping of the contralateral renal vascular pedicles in rats for 40 minutes at normothermia. Dialysis dependent ARF in humans is usually multifactorial and occurs predominantly in kidneys with some form of pathology such as atherosclerosis and reduced GFR. Therefore the single kidney ARF of the rat clamp model is also very representative for the human renal allograft setting.

Within one hour after reperfusion the tubular epithelial cell lining is already damaged and one or the other cell became detached from the basement membrane, the tubules are dilated and the creatinine increased twofold (figure 3). One day after reperfusion a full blown ARF developed on a morphological as well as functional level. Most of the tubuli are filled with detritus and fibrin; epithelial cells are predominantly necrotic as evidenced by pycnotic nuclei, and some apoptotic cells. No open lumen is visible within the tubular cross sections and the serum creatinine increased tenfold.

One week after reperfusion the creatinine has returned to normal but the morphology is by far not normal suggesting that creatinine is not a good kidney injury marker because it underestimates the severity of damage dramatically. Morphology improves over time but the late consequences of the ARF are cystic dilatation of some tubuli and higher propensity of chronic renal failure (CRF) as also overserved in patients with initially resolved ICU-ARF. Reemphasizing again however that no morphological data in humans exist because sequential biopsies are not feasible.

(12)

Figu Time magn reper arrow nucle the conc morp towa of re Cysti

Drill a mo This trans 1.1.3 The accom cellu dama

ure 3. Morp e on the x-a nification 4 rfusion, tub w). One day ei are pykno

remaining entration h phology is ards its base enal injury.

ic dilated tu

ling from th olecular leve

is especial splant ARF

Donor Fa allogenic t mpanied w ular alteratio

age by reac

phology of d axis refer to

00x, adapte bular dilatio y after reper otic and adj tubule lum has increas

still severe eline value s The late co ubuli are ma

he morpholo el shows sur lly true in

studies.

actors transplantat

ith a period ons of the ti ctive oxygen

developing o 1hour, 1 d ed from Hau on can be s rfusion all a jacent to the men (green sed to its ely impaire suggesting t onsequences aking up mo

ogical level rprising som

the reparat

ion of orga d of hypoxi

ssue of the n species, n

11 ARF in a r day, 1 week user P, Obe seen with o avital cell h

e cell memb n arrow).

maximum ed but the that it is not s of ARF ca ost of the re

deeper into me choreog tion phase

ans from o a over hour donor orga nitric oxide

rat kidney i k and 20 we erbauer R (2 occasional have been sh

brane. Fibri At this st of 3.5mg serum crea t a good bio an be seen a enal parench

o the events raphed proc

of ARF as

one to anot rs leading t n. These ch or peroxini

schemia rep eeks after re 28)). Alread

loss of epi hedded into inous detrit tage the s g/dl. After

atinine has omarker to a at 20 weeks hyma (blue

and process cesses in the s has been

ther organis o a series o hanges main itrite as one

perfusion m eperfusion dy one hour

ithelia cells o the lumen tus is obstru serum creat one week almost retu assess the d s after the i

arrow).

ses that occ e ongoing c shown in

sm is inevi of molecula nly include t e of the stro

model.

(print r after s (red n. Cell

uction tinine k, the

urned degree nsult.

cur on chaos.

post-

itably ar and tissue ongest

(13)

12

oxidants that develop in response to hypoxia (5). In addition, a considerable share of the injury observed in the deceased donor kidney is derived from processes occurring in the brain dead donor.

Brain death induces an autonomous storm of cytokines and catecholamines which trigger inflammation followed by profound hypotension after an initial hypertensive crisis and subsequent tissue hypoxemia. This event is perpetuated by the absence of ADH release from the death brain and culminated in the kidneys inability to reabsorb the huge amounts of filtered water eventually culminating in full blown diabetes insipidus. Reactive renal vasoconstriction perpetuates the ischemia/hypoxia of renal tubule epithelial cells as the main target of injury. White and colleagues recently showed that heterotrimeric G-proteins are among the key regulators of renal epithelial cell function during homeostasis and in response to ischemic injury (6). The authors hypothesized that G-protein βγ subunit (Gβγ) dimer activity may be a novel therapeutic target since it is strongly activated during IRI. Eventually, iatrogenic vasopressor use and developing hypernatremia perpetuate these catastrophic processes. A recent study showed that desmopressin use in the donor can ameliorate hyponatremia and was associated with higher propensities of immediate graft function (7).

Ischemic damage of the tissue is further on continued by subsequent reperfusion and flushing with oxygen. Once again, highly reactive and cytotoxic species like the hydroxyl radical lead to further partially irreversible alterations and destruction of the tissue. Even more important, reperfusion of blood into previously damaged tissue is accompanied with a strong immune response resulting in the infiltration of monocytes and neutrophils through the activated endothelium. Taken together, ischemic injury with reperfusion associated inflammatory responses seems to be important factors for the development of ARF.

Additionally, there are certainly post-donor factors such as the duration of cold and likely more important the warm ischemic period that contribute considerably to ARF in

(14)

13

the human renal allograft setting. Studies showed that cold static ischemia above twenty hours gradually increase the risk of ARF (27). In recent years machine perfusion techniques have been developed that showed that the risk of ARF, especially in older donors, could be reduced (8). However the reported benefit may even increase by normothermic perfusion of the donor organs (9).

1.1.4 Recipient Factors

Accordingly, the duration of the suture of the vascular anastomosis takes a skilled surgeon in patients with normal anatomy between 30 and 40 minutes. Longer anastomosis times however increase the risk of ARF unproportionally (10). How much the alloimmune response contributes to ARF is not unambiguously determined. Cellular rejection can be clearly differentiated from DGF but the contribution of preformed HLA and non-HLA antibodies is not so easily discernible and no studies investigated morphological and serological features of antibody mediated rejection in first week management or protocol biopsies. However most of the transplant centres nowadays screen patients on the waiting list for the presence of donor specific HLA-antibodies and thus the incidence is likely very low. The contribution of non-HLA antibodies to DGF has not been evaluated yet. In fact there is plausible evidence coming from clinical observations that non-HLA alloimmunity is contributing to ARF/DGF. Patients with Alport syndrome exhibit a full loss of function of the alpha five chain of the type four collagen. If these recipients receive a donor kidney with an intact collagen structure, recipients will make antibodies and a rapid Goodpasture like syndrome develops in roughly 5% leading to early graft loss after prolonged DGF. It is not clear yet why only a minority of cases develop this devastating rapid immune response and why some of the patients develop anti-collagen antibodies but exhibit early and mid-term graft patency. Recent global consortia are currently evaluation the impact of these genetic mismatches between donor and recipient in large genetic associational studies such as the International Genetics & Translational Research in Transplantation Network (www.igenetrain.org).

(15)

14 1.2 Related Work of other Investigators

Since ARF/DGF represents one of the main risk factors for premature graft loss and mortality and occurs in roughly one in four transplant recipients, the clinical need for prevention and therapy is evident. Thus many research groups tackled this pathology and investigated adjacent processes from the molecular level up to clinical intervention trials. Recent reviews nicely summarized the current understanding and clinical state of DGF/ARF research and prophylaxis/therapy (11-13, 29).

The transfer of organs from one to another organism inevitably causes some decree of hypoxia which leads to a cascade of molecular and cellular choreographed but also anarchic events and in the donor organ. Ischemic damage of the tissue is aggravated by subsequent reperfusion of the kidney with oxygen containing blood and the alloimmune response of the recipient. Even more important, reperfusion of blood into previously damaged tissue is accompanied with a strong immune response resulting in the infiltration of monocytes and neutrophils through the activated endothelium (30). In summary, ischemic injury with reperfusion associated inflammatory responses seems to be important factors for the development of ARF. There is even evidence that severe brain injury might potentiate the immunologic responses to the graft as a variety of cytokines have been demonstrated to be released in deceased donors (14, 15).

Given the overriding risk of ARF for reduced long-term graft survival and the identified risk factor, several studies investigated preventive measures or strategies. Among the modifiable factors are an optimal donor management and a minimum duration of cold and especially warm ischemic times. Counterbalance of the systemic inflammation of the deceased donor by dopamine has been reported to be associated with a reduced risk of ARF (16). The same authors subsequently showed in a RCT that dopamine donor treatment reduced the need for dialysis after transplantation. However, most of the donors will require vasopressor use anyway and thus we were interested in any additional benefit of an anti-inflammatory intervention. Studies in the late 1970s and early 1980s tackled this enigma but none was randomized and all were of small sample

(16)

15

size (17-20). Furthermore, that endpoint was graft loss in this pre-cyclosporine era.

Thus these old studies are not amenable to nowadays clinical practice.

In contrast, three small randomized studies found that corticosteroids plus additional cyclophosphamide in 2 studies did not effect on graft survival within the first year (21- 23). The primary outcome in these trials was short-term graft survival, and their ability to detect important effects of treatment was limited by the low incidence of early graft failure.

The only one adequately powered state RCT on the effect of inhibition of systemic donor inflammation of the incidence and duration of ARF has been conducted by the applicant and will be discussed in detail below (31).

Once ARF has developed in native as well as post-transplant kidneys no causal therapy exists. It is advisable however that those parameters known to contribute to ARF such as arterial hypotension and renal hypoperfusion, infection and inflammation as well as dehydration should be corrected. A scientific proof that these activities actually led to a reduced duration of DGF remains elusive. Nevertheless, the overall ARF rate remains still at a constant high level so that novel approaches for the prevention and treatment of ARF are urgently needed.

(17)

16

2 Objectives

The overall aim of this thesis was to elucidate the molecular pathophysiology and discover prognostic factors for posttransplant ARF. The objective was not only to explain the underlying pathophysiology but rather to invent potential preventive and therapeutic strategies to overcome this unwanted phenomenon. By amelioration of the high DGF rates we speculate that the mid- and long term graft survival will increase and shortage of the scare donor organs will be alleviated due to longer lasting grafts. This utilitarian approach has been introduced also in large transplant networks such as in the United States (UNOS) where the donor organ quality is matched with the expected survival of the recipients to avoid futility because currently most of the allograft recipients die with a functioning graft.

In fact based on the findings of our studies a precise goal was to test in vitro and in vivo strategies in experimental models of ARF. Based on these proofs of concept experiments a series of large animal studies will be ultimately performed to test for the applicability of our findings in the setting of human renal transplantation.

Objectives: The molecular regulation of ARF is highly complex and not all parts of the kidney are equally affected. Most of the injury occurs in the tubular epithelial cells and therefore we set out to elucidate the sequential molecular events in microdissected tubules of deceased donor human kidneys. Live donor kidney biopsy specimens were used as healthy controls. Genome wide studies were utilized as tool to derive candidate features of regulation. Since it was expected that several dozens of regulators will be found and that it will be unlikely to find a single therapeutic intervention that captures all of these mediators, miRNA profiles were obtained as well. The specifically designed bioinformatics suite miRway was applied to search for the candidate miRNA that regulated most of these candidates. The causal inference was tested by designing ASOs (antisense oligonucleotides against the target miRNA) to be used for in vivo experiments of ARF.

(18)

2.1 A ca half conse overv

Figu trans OED conc

To a decea order rough Semm trans the M to ov recor

Work plan arefully desi

in order t equences a view of this

ure 4. Work splant settin DTR … OE

ept)

assess the ased donor r to have a hly 25%

melweis U splant cente Medical Uni

ver 400 ren rded into a w

n

igned work to thorough and potentia s work plan

k plan for ng (LCM Esterreichis

association kidneys, p a sufficient of decease University C

r roughly 2 iversity of V nal transplan

web-based d

plan has be hly study t al prophyla is illustrate

the study

… laser ca ches Dialy

ns of morp pre-implanta t number o ed donor Clinic in B

00 kidney t Vienna and nts per yea database (fi

17 een set up a the epidem actic and th ed in figure

of ARF ex apture mic yse and Tr

phologic an ation biopsi of outcomes kidney tra Budapest w transplants d the Elisabe

ar. The post igure 5).

and followed miology, pat

herapeutic 4.

xperimentall rodissection ansplant R

nd molecul ies were ob s, i.e. cases ansplants. A was initiate

are perform ethinen hosp t-transplant

d over the l thophysiolo intervention

ly and in t n; QC … Registry; PO

lar features btained in l

s of ARF, A collabor ed since in med annually pital in Linz course was

last decade ogy and cl ns. A sche

the human quality co OC … pro

s in the h large amoun

which occ ration with n this exc

y. Together nz, we had a as monitored

and a inical matic

post- ontrol;

of of

uman nts in cur in h the

ellent r with access d and

(19)

Figuure 5. Screennshot of thee web-fronte

18

end of the AARF database

(20)

19

3 Methods

A detailed description of all methodological utilized in this research may be found at the applicants website http://www.meduniwien.ac.at/nephrogene under ‘protocols’.

In brief, the following techniques were used in the research projects:

 Affymetrix mRNA and miRNA arrays

 cDNA Arrays

 Cell culture

 ELISA

 Immunohistochemistry

 Peptide Arrays

 Protein concentration

 qRT - PCR

 Western blotting

A detailed description of the animal experiments may be found at the respective sections of this paper.

3.1 Histogenomics

To account for the fact that the kidney does not consist of homogenous tissue but is rather structured into the functional units of nephrons, many of the biopsy specimens obtained were subjected to microdissection. The genome wide gene expression profile was analysed in donor kidney biopsies which developed ARF/DGF after transplantation and compered to signatures in kidneys with primary graft function. The Venn diagram in figure 6 shows the genomic features that discriminated ARF from the primary functioning grafts by tissue compartments.

(21)

Figu Abbr glom fibro

The webs

Since given ARF clear

ure 6. Histo reviations r merulosclero

osis, ta … tu

detailed pr site http://ww

e most of t n the highl F, we used a

r results of t

ogenomics o refer to h osis, as … a ubular atrop

rotocol of k ww.meduniw

the gene ex ly complex

an integrati these part of

of donor ki istological arterioloscle

hy, ii … int

kidney gene wien.ac.at/nep

xpression pr changes d ive analytic f the studies

20 idneys (n=8

findings i erosis, ati … terstitial inf

e expression phrogene/ind

rofiles follo during initia cal approach

s.

82) (adapted in the kidn

… acute tub flammation.

n profiling dex.php/prot

ow a highly ation, estab h to derive

d from Perc ney compa ular injury,

may be fo tocol/whole-g

y choreogra blishment an

a sound un

co P et al.

artments: g , if … inter

ound on my genome-arra

aphed patte nd resolutio nderstanding

(32)).

gs … rstitial

y lab- ays-2

er and on of g and

(22)

3.2 The colla Emer betw litera

Figu 13,00 biolo

Systems B integrative aboration wi rgentec has ween protein

ature that m

ure 7. Integ 00 vertices ogical data (

Biology e analyses

ith Bernd M s developed n coding f mirror the ph

grated Anal and 800,00 (33).

using a s Mayer and co

an in silico features der hysiological

lyses of a S 00 edges an

21 systems bi oworkers fr o network o rived from

dependenc

Systems Bi nd allows f

ology appr rom emerge of molecula various om ies (figure 7

iology deriv for the integ

roach was entec (www.

r processes mics tracks 7).

ved omicsN grated analy

carried o .emergenetc.

and interac s and publ

NET that c yses of com

ut in com).

ctions lished

covers mplex

(23)

The 8A a

Figu

Figu The and i

donor kidne and 8B).

ure 8A. Sche

ure 8B. Sch ARF specif interpretatio

ey profiles

ematic repr

heme of the fic segment on section.

are superim

esentation o

e disease sp ted network

22 mposed on t

of the molec

pecific segm k is shown

the annotate

cular netwo

mented subg in figures

ed relations

ork ‘omicsN

graphs and 10A and 10

s network (f

NET’

functional 0B of the re

figure

units.

esults

(24)

3.3 After biops unco of flo obtai in the BAC

Figu expre Indep in sil

Discovery r the transci sies in a n over the com

ow chart is in roughly t e first week CR were tak

ure 9. Wor ession in th pendent val lico analysis

y and Valida iptomics stu novel prosp

mplex mole s depicted i

the same re k and a half ken in the sa

rk flow of he same bi lidation of o s and statist

ation Studie udies, cDNA ective coho cular regula in figure 9.

epresentative after transp ame period a

f the simul opsies in a observed fin tical testing

23 es

A and miRN ort of rena ation of AR . The samp e numbers plantation. T

and served

ltaneous de a prospectiv ndings in th compared t

NA arrays w l allograft RF in furthe ple size of 1

of ARF and Ten protoco

as control ti

etermination ve cohort o

he discover to public do

were conduc recipients w r depth. A 122 donors d BCAR fo ol biopsies w

issue.

n of mRN f renal allo ry cohort w omain data (

cted on the with the ai CONSORT s were chos ollow up bio without ARF

NA and mi ograft recip was performe

(GEO).

same im to T type

sen to opsies F and

iRNA pients.

ed by

(25)

4.1 Base defin signi figur the trans

Figu

4 R

ARF Spec ed on the a ned and su

ificant featu re 10B. Mo

gene onto scription fac

ure 10A. AR

Results

cific Molecu available om ubsequently ures and de st of the di logies of ctors.

RF specific

ular Netwo mics profil y superimp ependencies fferentially inflammati

molecular n

24 rk

les (figure posed on t s/interaction y regulated f ion, immun

network of t

10A), an A the omicsN ns between

features in nity and h

the various

ARF specif NET and s the nodes the ARF ki host defen

omics categ

fic network segmented.

are display idneys belo nce, some

gories

k was The yed in ong to were

(26)

Figu post- featu

4.2 The i the k well such to ar inflam expre endo repre that kidne

ure 10B. In -transplant ures (34).

The Inflam interpretatio kidneys occu to the abov as the syst rterial hypo

mmation ca ession of pr othelial adh esentants IC subsequent eys that exh

nflammation ARF. The

mmatory R on of the tra urs in the d ve describe emic inflam otension, di

an not only roteins that hesion mol CAM-1, VC tly develop hibited subs

n network i colour cod

Response ansciptomic deceased don

d events th mmatory res iabetes insi y be seen o t are the ma

ecules. Imm CAM-1 and

ed post-tra equently im

25 in donor ki de refers to

cs findings nor before t hat occur du sponse synd ipidus and on the RNA ain effector

munohistoc ELAM-1 sh ansplant AR mmediate gr

idneys whi the gene o

is unambigu the organs a uring the st drome (SIR vasopresso A level but s of the inf chemical st howed a str RF but was raft function

ch subsequ ontologies o

uously that are harveste ay of the d S), renal hy or use. The t is also tra flammation

taining of ong signal i s virtually n (figure 11)

uently deve of the indiv

inflammati ed. This fits donor in the ypoperfusio e observed ranscribed i

cascade su the three in donor kid

absent in d ).

loped vidual

ion in s very e ICU n due renal in the uch as main dneys donor

(27)

Figu expre but a pane that mole nece was d

The howe assoc thus cond reduc was d

ure 11. In essed in kid are not det ls). (adapte systemic in ecules in th ssary causa designed an

observed i ever were d ciational fin can only b ducted a RC ction of the defined as t

nflammatory dneys with

tectable in d from Sch nflammation he donor ki ally related

nd conducte

inflammator derived from nding. The be conducte CT to test w e incidence the need for

y proteins subsequent

kidneys w hwarz et al ( n in the bra idneys. It i

to the subse ed (see figur

ry signals m observati

proof of c ed in an in whether the of post-tra r more than

26 (adhesion developme with subsequ

(30)). These ain death d is of notice equent risk re 9 in the n

in the don ional studie ausality of nterventiona treatment o ansplant AR n one post-tr

molecules ent of post-t

uent primar e immunohi donor cause e that the o

of ARF an next paragra

nor kidneys es and thus inflammati al study. T of inflamm RF/DGF. DG

ransplant di

s) are hig transplant A ry allograft istochemica es upregulat observed as nd thus the s aph on RCT

with post- can only b ion as deter herefore, w ation in the GF is the p ialysis. A fi

ghly abund ARF (left pa ft function

al stainings tion of adh ssociation i subsequent T).

t-transplant be consider rminant of we designed e donor cau primary end iner resoluti

dantly anels) (right show hesion is not

RCT

ARF red as ARF d and uses a dpoint ion of

(28)

27

the potential effect was the duration of DGF and the number of dialysis session required.

4.3 The RCT – a Logistic Challenge

In this blinded multicentre trial 207 deceased donors were randomized to 1000mg of methylprednislon or placebo single shot injection three to six hours before the organ retrieval. Preimplantation biopsies were obtained to test whether the randomization, timing and dose of the intervention (1000mg methylprednislon) was appropriately chosen to reduce the inflammation cascade on a genome wide level in the kidneys. The biopsies were subjected to microarray testing and the post-transplant course was monitored. A web base case record form (CRF) was used to facilitate the administration of the study and allow for a state of the art online monitoring of the participating centers.

The trial was quite challenging since it is unforeseen where a potential donor will become available and if one is registered, the blinded study medication needed to be carried by the transplant coordinator to the donor site and the verum/placebo injected in the predefined time frame before organ harvest.

The ethical committees of the Medical University of Vienna as well as Eurotransplant approved the study (Ethical Committee of the Medical University of Vienna, Vienna, Austria [study protocol EK-067/2005; to be found at http://ohrp.cit.nih.gov/search]) and the Eurotransplant kidney advisory committee (study protocol 6021KAC06)). A more in depth presentation of the methods and details about the study protocol may be found in the methods section of the publication.

(29)

The DGF calcu

Figu dono comp size f an LFU

trial was p F from a hi ulation in th

ure 12. Pow or kidney tr puted samp for 8% lost ITT an U/NU)2=176

powered to istorical 25 he screensho

wer calculat ransplantati ple size to h to follow u nalysis in 6(1/.92)2=20

detect a 50 5% of all d

ot of figure

tion of the ion. Event half event ra up and non-t ncreased

07]

28 0% reductio deceased do

12).

RCT on t rate in the ate was 176 transplanted the samp

on in the i onor transp

he preventi e control g 6. The adju

d (NU … no ple size

ncidence o lants to 12

ion of DGF group was

stment of th on-used) or

to 207

f post-trans 2.5% (see p

F after dec set at 24%

he donor sa rgan plannin 7 [N*=N

splant power

eased

%, the ample ng for N(1/1-

(30)

The prede partic the c

Figu al. (3

Detai

number of efined excl cipating ce conducted st

ure 13. CON 31)).

ils on the dem

f 207 dono lusion crite enters. The tudy is illus

NSORT flow

mographic p

ors needed eria includi

CONSORT strated in fig

w chart of t

arameters of

29 to be incr ing kidneys T flow cha gure 13.

the steroid

f the study po

reased to 2 s shipped art (http://ww

donor study

opulation is p

274 to acco in the ET ww.consort-s

y (adapted f

provided in t

ount for va region to statement.org

from Kainz

table 2.

arious non- g/) of

z A et

(31)

Tablee 2. Demogrraphic characcteristics of d

30

donors and reecipients by treatment asssignment

(32)

31

The transcriptomics experiments on randomly selected 20 biopsies, 10 of each intervention group (steroid/placebo) were conducted on cDNA microarrays holding 41,421 features (batch SHEO) from the Stanford University Functional Genomics core facility. Data analysis was performed by unsupervised hierarchical clustering of biopsies by gene expression (figure 14). The y-axis shows the grouped gene signatures by ontologies. The colour code below the x-axis indicated the relative up- (red) or downregulation (green) of features compared to a set of reference transcripts (Agilent).

As clearly indicated, the steroid pre-treatment of the donors led to a reduction of many features of the inflammation cascades (top lines) and activation of metabolism genes (bottom lines). Based on these data we were confident that the coordination, timing, dosing and intervention of this blinded and logistically challenging worked fine.

(33)

Figu two indic diffe ampl 4.4 Desp treatm

ure 14. Uns treatment cated on the

rential regu lified RNA

Results on pite the gl

ment, the in

supervised h groups. Th e right side o ulation of fe (Agilent).

n DGF in th obal suppr ncidence of

hierarchical he gene o of the figur features vs a

he RCT ression of

DGF was n

32 l clustering ontologies o re. The colo

a standard

inflammati not different

of biopsies of differen our code at t

Stratagene

ion in the t between g

s by gene e tially regul the bottom s Universal h

donor org roups (table

expression i ulated gene

shows the x human refe

gans by st e 3).

in the s are x-fold erence

teroid

(34)

Tabl 25%

dialy

le 3. The in in the pla ysis was not

ncidence of acebo group t different b

post-transp p respective

etween the

33 plant DGF/A

ely. Also th two groups

ARF in the he number s.

steroid grou of required

up was 22%

d post-trans

% and splant

(35)

Acco unch

Figu value

ordingly, th hanged after

ure 15. Traj e refers to th

he resolutio r donor stero

jectories of he F-test in

on of DGF oid treatmen

f post-transp the mixed l

34 as indicate nt (figure 15

plant creati linear mode

ed by creat 5).

inine by ran el for longitu

tinine trajec

ndomization udinal data.

ctories rem

n group. T .

mained

The p-

(36)

As se graft evalu of inj

Figu rejec The deriv (adap

econdary en t function o uated. Amat jury and gra

ure 16. Kapl ction (log-ra number of ved from lo

pted from A

ndpoint of t of other tra tschek et al aft patency

lan–Meier e ank p = 0.9 f subjects a og-rank test Amatschek A

this labour, ansplant org

. showed th in liver allo

estimates of 6) risks are at risk is p ts. BCAR, A et al. (35)

35 time and co gans of the hat steroid p ografts (figu

f (A) surviv e shown for provided ab biopsy-con )).

ost intensive e enrolled d pre-treatmen ure 16) (35)

val (p = 0.24 r steroid pre bove the x- nfirmed reje

e academic deceased do nt did not re

.

4) and (B) b e-treated liv axis. Given ection; TX,

study, the i onors have educe that d

biopsy confi vers and pla

n p values , transplant

initial been degree

irmed acebo.

were ation.

(37)

4.5 Stero Furth peri- betw fores

Figu treatm (from

4.6 Since suffic were respo In or

Effect of S oid pre-trea hermore, th

transplant l ween treatme st plot in fig

ure 17. Inte ment group m Amatsche

The miRN e it became cient to cha e investigate

onsible for i rder to cov

Steroid Don atment did he trajectori

liver injury ent and base gure 17.

eraction an p. No effect ek A et al. (3

NA Discove e clear that t

ange the ear ed assumin initiation an ver a wider

nor Treatm not effect es of the tr y by steroid eline risk fa

nalysis of g t modificati

35)).

ries the suppres rly outcome ng that othe nd maintena range of r

36 ment on othe

t on the i ransaminase d pre-treatm actors, i.e. d

graft loss ion by the s

ssion of infl es ARF/DG er contribut ance of ARF regulatory t

er Transpla incidence o es showed ment. Post h

donor charac

by various steroid inte

lammation i GF after eng tors than in F leading to target pathw

nted Organ of rejection no impact hoc analysi cteristics is

donor cha rvention co

in the donor graftment, n

nflammatio o DGF after ways than s

ns

ns or mort in the degr s of interac

illustrated i

aracteristics ould be obs

r organs wa novel appro on were cau

r transplant steroid treat

tality.

ree of ctions

in the

s and erved

as not aches usally ation.

tment

(38)

37

could accomplish, the microRNA (miRNA) expression in the organs at risk of ARF were determined.

MicroRNAs (miRNAs) are a class of small non-coding 18 to 24 nucleotide-long RNAs that have been implicated recently in diverse cellular functions. miRNAs interfere in translation of mRNAs for a wide variety of proteins regulating cell proliferation, cell death, cellular morphogenesis and differentiation. Production and function of miRNAs requires a set of proteins summarized in the term miRNA machinery (figure 18). A recent summary of the genesis of miRNA, their contribution to pathologies in the transplant kidney as well as potential therapeutic applications may be found in the review by J. Wilflingseder et al.

(36).

(39)

Figurre 18. Schemme of miRNAA processing

38

g and its effeccts on transcription and trtranslation

(40)

The proto adjus The (37).

Figu accor

simultaneou ocol biopsie stment for m

significant

ure 19. Mol rding to the

us analysis es yielded multiple tes features of

lecular featu eir p-value o

of mRNA several fea sting using f both analy

ures discrim of significan

39 and miRN atures that r

the SAM fa ysis in aggr

minating DG nce.

A transcrip remained s false discove

regate form

GF from pri

pts in the A tatistically ery rate <10 m are display

imary graft

ARF compar significant 0% penaliz yed in figu

t kidney bio red to

after ation.

ure 19

opsies

(41)

4.7 The p and predi comp indiv miRw http:/

Figu and comm

miRNA T prediction o only few ta iction routi putational vidual adjus

way

//mirway.nep

ure 20. Scre the underly munication)

Target Predi of miRNA t arget protei ines yield algorithm stment of th

is a

phrogene.at/M

eenshot of t ying databa ).

iction targets is cu ins have be rather inho that allows he significa accessible MirWayFron

the miRNA ase (right p

40 urrently mai een experim omogeneou s the integ ance thresho

on ntend/faces/s

A target pred panel) (Ivce

inly based o mentally val us results,

gration of old (figure my tart.xhtml

diction fron evic S, Stü

on computa lidated. Sin we designe all availab 20). This a lab-w

tend MIRW ütz C et al

ational predi nce the ava

ed a web-b ble routines

algorithm c website

WAY (left p l. 2014 per

iction ilable based s and

oined at

panel) rsonal

(42)

We i (figu

Figu ARF

identified m ure 21).

ure 21. Stati F vs PF.

miR-182-5p

istically sign

as the best

nificant disc

41 discrimina

criminator m

ator between

mRNA lead

n DGF and

ds of miR-1

primary pa

82-5p targe atency

ets for

(43)

Targ prese cycle FOX

Figu cell c as FO

get candidat ented to be e regulators XO3 (figure

ure 22. Netw cycle regula OXO3 were

te proteins involved in s such as P

22).

work regula ators such a e identified

were ident n ARF regu

CNA, cdc2

ation of pre as PCNA, c as candidat

42 tified and s ulation by c 2, cdk4 or c

edicted miR cdc2, cdk4 tes.

showed man cell cycle re

clock and tr

R-182 target or clock an

ny proteins egulation an ranscription

t proteins. S nd transcript

s that have nd repair su n factors su

Several plau tion factors

been uch as uch as

usible s such

(44)

Valid and s

Figu (PGF

4.8 In ex admi DNA in th cells

dation of th showed sim

ure 23. Vali F) vs those t

In vivo Ki xperimenta inistered sh A) end up in

e proximal for at least

he array exp milar regulat

idation of m that develop

inetics and l rat studie hort (18-22m

n the kidney tubule epith four days (

periments fo ion (figure 2

miR-182 arr ped acute ki

Efficacy of es we foun mers) and y. Specifica helial cells (figure 24) (

43 for the lead

23).

ray data by idney injury

f Antisense nd longer syntheticall ally, about 5

of the kidn (27).

miRNA-18

qPCR for g y (AKI).

Oligonucle time ago, ly derived 50% of the eys and rem

82 was perf

grafts with p

eotides that most oligonucleo injected do main stable

formed by q

primary fun

of system otides (anti ose is reabso

inside the t qPCR

nction

ically isense orbed ubule

(45)

Figu cells

In ur micro and t

This cotra proxi reabs oligo apica reabs

ure 24. Upt four hours

rine, degra oscopic stu thus may be

hypothesis ansporter N imal tubule sorption. W onucleotides

al membran sorption in i

take of the (A) and fou

aded produc dies showed e suitable fo

s was tested NaPi2a. NaP e and is r We showe s was possi ne of the

isolated bru

antisense o ur days (B)

cts appear d that the ol or antisense

d in vivo by Pi-2a was c responsible

d that a ible and cau proximal tu ush border m

44 oligonucleot

respectively

as early a ligonucleot inhibition o

y inhibiting chosen beca for more

specific used a decr

ubule cell membrane v

tides in the y.

as few hour ides are not of target mR

g the mRNA ause this tr

than 90%

inhibition rease in pho

(25). The vesicles is d

e proximal

rs after inj t trapped in RNAs and m

A of the so ransporter i

of the tub of NaPi2a osphate tran kinetics o isplayed in

tubule epit

jection. Ele n endo-lysos miRNAs.

odium-phos is located i ubular phos a by anti nsport acros

f the phos figure 25.

thelial

ectron somes

sphate in the sphate

isense ss the sphate

(46)

Figu antis contr from

4.9

4.9.1 Havi oligo and epith

ure 25. Sele ense oligon rols and the m Oberbauer

Clinical A

In vitro D ing identifi onucleotides

efficacy of helial cells (

ective inhibi nucleotides e unaffected r R et al. (38

Applicability

Data

ed miR-18 s (ASO, an f miR-182 (HRPTECs)

ition of the (AS). Rev d sodium su 8)).

y and Utility

2 as candi natgomiRs) inhibition ).

45 renal sodiu ersed seque ulfate transp

ty

idate interv against mi in cell cul

um phospha ences of th porter were

ventional le iR-182 and ture of hum

ate cotransp e AS (rAS) used as ref

ead, we des d evaluated man renal

porter NaPi ) and scram ference (ad

signed anti their selec proximal t

2a by mbled dapted

isense ctivity ubule

(47)

Studi

Figu 25mM as w expe

ies showed

ure 26. Anta M antagom well as vehi

riments.

a dose depe

agomiR inh miR-182 (AS

icle and sa

endent inhib

hibition of m SO) blocke

line contro

46 bition of the

miR-182 in ed the targe ol did not a

e target miR

n cultured H t miRNA, m affect miRN

RNA in vitro

HRPTEC in mismatched NA abunda

o (figure 26

n vitro. Dos d controls ( ance in the

6).

ses of (MM) PCR

(48)

4.9.2 Acut left r

Figu cross

Rats 25mM expre (figu

In vivo da te renal failu renal artery

ure 27. Rat s clamping o

were pretr M) or equa ession of m ure 28).

ata

ure was ind for 40 minu

model of A of the left re

reated 12 ho al concentra miR-182 re

duced in SD utes (figure

ARF. Anim enal artery f

ours before ations of m mained sel

47 rats by righ

27).

mals were su for 40 minu

e insult with mismatched lectively re

ht uninephr

ubjected to utes.

h either ana antisense duced in th

ectomy and

sided unin

atgomiR-18 oligonucleo he anatagom

d clamping o

nephrectomy

82 (ASO 2.5 otides. Intra

miR treated of the

y and

5 and arenal d rats

(49)

Figu reper antis

The injur insul (crea respe

Figu reper equa

ure 28. Intr rfusion. Ra ense 12 hou

trajectories ry. Histolog lt. Antagom atinine and

ectively.

ure 29. Traj rfusion inju al concentrat

rarenal exp ats were p urs before in

s of serum gical evalua miR-182 (A

BUN) and

ajectories of ury in rats.

tions of mis

pression of pretreated w

nduction of

creatinine ation of kid ASO) treatm

morpholog

f serum cre ASO low smatched co

48 miR-182 a with either f injury.

were deter dney was pe

ment led t gical insult

eatinine ove refers to an ontrol.

at various r antagomi

rmined dail erformed on to an amel

as evidenc

er the first n antimiR c

time points iR (ASO)

ly for seven n days two lioration of ce in figure

seven day concentratio

s after isch or misma

n days afte and seven f the funct es 29, 30 an

ys after isch on of 2.5m

hemia atched

er the n after tional nd 31

hemia mM or

(50)

Figu injur conc

ure 30. Traj ry in rats.

entrations o

jectories of ASO low of mismatch

f BUN ove refers to hed control.

49 er the first

an antimiR

seven days R concentr

after ische ration of 2

emia reperf 2.5mM or

fusion equal

(51)

Figu treate antim phos degre left comp

ure 31. Hist ed as well miR pretrea phatase pro ee of injury lower corn pared to the

tomorpholo as untreate atment as oduction an y. The treat ner. ASO ( e control (pl

ogy of repre ed controls.

indirectly nd thus diff

tment group (lower righ lacebo and A

50 esentative r . Damage c

also evide ferent colou p and time ht) treated ASO-misma

rat kidneys could be dr nce by int ur intensitie after injury rats showe atch) group

of anti-miR ramatically tact brush es are corre y in days is ed dramatic

s.

R and mism y reduced b

border alk esponding t

indicated i cally less i

match by the

kaline to the in the injury

(52)

Morp patho evalu histo

Figu after high

phological e ologist. Inju uated kidn opathology s

ure 32. Sem IRI. Anti-m to 25mmol

evaluation o ury severity

eys per g scores are p

miquantiativ miR (ASO) l.

of the vario y was score groups and provided in f

ve scoring o ), MM (mis

51 ous treatme ed semiquan

time afte figure 32.

of the injury smatched A

nt groups w ntitatively f er ischemia

y severity b ASO), low re

was perform from 0 to 3.

a reperfusi

by treatmen efers to a d

med by a bl . The numb ion as we

nt class and dose of 2.5m

inded ber of ell as

d time mmol,

(53)

The seven the o (figu

Figu selec ASO discr effica

long-term e n day exper ontologies o ure 33).

ure 33. Unsu cted three A O (antimiR-1

riminate at d acy of ASO

effects of th riments but

of fibrosis

upervised cl ASO and thre

182) treated day two bu O (antimiR-1

he ASO int it is interest were mark

lustering of ee control tr d rats (indic ut already at 182) in fibro

52 tervention c

ting that alr kedly suppr

f gene ontol reated rat k cated as t_) t day seven

osis preven

can certainl ready at sev ressed in A

logies of fib kidneys two ) and placeb

suggesting tion.

y not be ev ven days, ge ASO treated

brosis pathw and seven d bo (indicate g also poten

valuated in enes belongi d rats vs co

ways in rand days after in ed as p_) di ntial of long

these ing to ontrol

domly njury.

id not g term

(54)

53

The following 20 genes were selected on the basis of public domain databases such as GEO (table 4).

Table 4. Fibrosis and connective tissue markers represented on the Affymetrix platforms. These ontologies were specifically analysed in order to elucidate whether ASO (antiMir-182) could act on prevention of fibrosis as the long-term consequence of ARF.

Fibrosis marker Collagens

AngII COL4A1 AT1 COL4A2 AT2 COL4A3 AGE COL4A4 RAGE COL4A5 TGFB1 COL4A3bp TGFBR2

TGFBRI IL1B TNFA SMAD2 SMAD3 SMAD4 SMAD7 IKBA P50 P65 ERK P38

(55)

4.9.3 Ther treatm figur http:/

and c

Figu (© n

So fa How apop

Abundanc re are likely

ment given re 18). Ou //mirway.nep cell viability

ure 34. miR ephrogene)

far only few wever, many ptosis. Thus

ce of miR-18 y many effec n the fact th ur custom phrogene.at/M

y/apoptosis

R-182 target .

w of the pr y targets b s it is of n

82 target pro ctor protein hat miRNA mir targ MirWayFron regulatory

features pre

redicted targ belong to note that s

54 oteins in injur ns of the obs As regulate a get predicti

ntend/faces/s proteins an

edicted by o

get proteins protein net suppression

red kidneys served effec a whole ne ion tool m

tart.xhtml p nd adjacent p

our custom

s have been tworks of

of miRs t

ct of anatgo twork of pr mirway, to predicted se pathways (f

mirway bio

n experime cell cycle that inhibit

omiR-182 (A roteins (see o be foun everal cell figure 34).

oinformatic

entally valid regulation t cell cycle

ASO) e also nd at

cycle

s tool

dated.

n and e and

(56)

antia hypo 48 an were

Figu anti-m injec

apoptosis w othesis, mice

nd 96 hours e performed

ure 35. Bcl- miR-182 (A cted with AS

ould lead to e were injec s respectivel d and the res

-2 and Foxo ASO) versu

SO or place

o a more ef cted with A ly. Western sults are disp

o-1 protein s placebo (M ebo.

55 fficient rege ASO or place n blots of th

played in fi

are activate MM-ASO)

eneration of ebo control he candidate

igure 35.

ed (inhibitio . Experimen

f injured tis and kidney e proteins B

on of inhibit nts were pe

ssue. To tes ys harvested cl.2 and FO

tory miRNA erformed in

st this d after OXO1

A) by mice

(57)

56

After investigations from cell culture to whole animal experiments showed that anti- miR-182 selectively inhibited miRNA-182 activity and activation of target proteins as well as amelioration of IRI in the rat models, we proceeded with studies of anti-miR- 182 in ex vivo perfused pig kidneys. Freshly harvested pig kidneys were subjected to ex vivo normotheric machine perfusion with UW solution (University of Wisconsin) solution at a pressure of 80mmHg. The setup of the experiment is illustrated in figure 36.

(58)

Figu 80mm

ure 36. Nor mHg.

rmothermicc machine p

57

perfusion oof a pig kidney with UW solutiion at

(59)

58 The experimental set up is provided as:

• Run for 6.5 hours

• Antisense oligonucleotide was administered after 30 minutes of perfusion and circulated for 6 hours

• Pressure: 75-90 mmHg (depending on position of the cannulas)

• Flow: 0.14-0.188 L/min (increased in the first 2 hours then remained constant)

• O2 consumption 68% after 30 minutes of perfusion, 48% after 3h of perfusion

• Biopsies were taken regularly (every 2 hours) For RNA extraction

For protein extraction

For histology

• Urine samples were taken regularly

• Urine produced: 20-40ml/30min, increased slowly

(60)

Quan miR- deter isola

Figu biops miRN

ntitative PC -182 over t rmined sinc ated perfused

ure 37. Inhi sies of norm NA to test t

CR of the bi the perfusio ce the half-li

d organs lar

ibition kine mothermic e the selectivi

iopsies from on time of

ife of these rgely unkno

etics of miR ex vivo per ity of the an

59 m the pig k

six hours proteins is own.

R-182 by a rfused pig k ntisense con

kidney show (figure 37) considerab

antisense ol kidneys. miR nstruct.

wed a select ). Target pr ly longer an

ligonucleoti R-146b was

tive inhibiti roteins wer nd the kinet

ide in sequ s used as co

ion of re not tics in

ential ontrol