Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery, and novel therapeutics

Molecular Genetics and Metabolism 80 (2003) 365–376

www.elsevier.com/locate/ymgme

Minireview

Gene expression in early ischemic renal injury: cluestowards pathogenesis, biomarker discovery, and novel therapeutics

Prasad Devarajan,a,b,c,* Jaya Mishra,a Suroj Supavekin,b Larry T. Patterson,a

and S. Steven Potterc

a Department of Nephrology, Cincinnati Children�s Hospital, Medical Center and Research Foundation, 3333 Burnet Avenue, MLC 7022,

Cincinnati, OH 45229-3039, USAb Department of Nephrology, Children�s Hospital at Montefiore, 111 E 210 Street, Bronx, NY 10467, USA

c Department of Developmental Biology, Cincinnati Children�s Hospital, Medical Center and Research Foundation, 3333 Burnet Avenue,

MLC 7022, Cincinnati, OH 45229-3039, USA

Received 8 July 2003; received in revised form 10 September 2003; accepted 10 September 2003

Abstract

Acute renal failure (ARF) represents a common and serious problem in clinical medicine. Renal ischemia–reperfusion injury (IRI) is

the major cause of ARF in the native and transplanted kidney. Several decades of research have provided successful therapeutic

approaches in animal models, but translational efforts in humans have yielded disappointing results. The major reasons for this

include a lack of early markers for ARF (and hence a delay in initiating therapy), and the multi-factorial nature of the disease. This

review focuses on the use of cDNA microarrays to elucidate the molecular genetic mechanisms underlying tubule cell apoptosis, and

to identify novel biomarkers for early renal IRI. Also presented is a comparative temporal analysis of cDNA microarray results

from mature kidneys following IRI and during normal nephrogenesis. Molecular genetic evidence for the notion that regeneration

recapitulates development in the kidney, and that injured tubule cells possess the capacity to de-differentiate to the earliest stages of

development, is presented. The implications of these findings to the ability of the kidney to repair itself and potential strategies for

accelerating recovery are briefly discussed.

� 2003 Elsevier Inc. All rights reserved.

Introduction

Acute renal failure (ARF) represents a significant andpersistent problem in clinical medicine with serious

consequences [1–12]. Renal ischemia–reperfusion injury

(IRI) is the major cause of ARF in the native [1,4], and

transplanted kidney [5]. Several decades of research have

provided successful therapeutic approaches in animal

models, but translational efforts in humans have yielded

disappointing results. An improved understanding of

the molecular mechanisms underlying early renal cellinjury will be critical for innovative and effective ther-

apy. The morphologic and biologic responses of renal

tubular cells to IRI include cell death, de-differentiation

* Corresponding author. Fax: +513-636-7407.

E-mail address: [email protected] (P. Devarajan).

1096-7192/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ymgme.2003.09.012

of viable cells, proliferation, re-differentiation, and res-

titution of a normal epithelium [1,19–22]. Apoptosis has

recently emerged as the major mechanism leading toearly tubule cell death following IRI [23–28] and down-

regulation of apoptosis may offer a novel therapeutic

approach to the amelioration of IRI in both the native

and the transplanted kidney. Attempts at unraveling the

molecular basis of the myriad apoptotic pathways in-

duced by IRI have been facilitated by recent advances in

functional genomics [60–67]. In this review, we will first

outline what is known about the apoptotic mechanismspertinent to renal IRI, and then present our genetic

analysis of this process using cDNA microarrays. We

will then review the current literature on molecular

mechanisms of tubule cell regeneration following IRI,

and present the pertinent cDNA microarray data with

special emphasis on translating this information to the

field of non-invasive biomarker discovery. In the final

mail to: [email protected]

366 P. Devarajan et al. / Molecular Genetics and Metabolism 80 (2003) 365–376

part of this review, we will present a detailed compara-tive analysis of renal gene expression during develop-

ment and following IRI. We will provide molecular

genetic evidence for the hypothesis that injured tubule

cells possess the capacity to de-differentiate to the ear-

liest stages of development, and that the de-differenti-

ated tubule cells can persist in the injured mature kidney

for a relatively long period of time. The implications of

these findings to the ability of the kidney to repair itselfand potential strategies for accelerating recovery will be

briefly discussed.

Background

Acute renal failure

Acute renal failure (ARF) continues to represent a

very significant and potentially devastating problem in

clinical medicine [1–12]. The incidence of ARF is high

(about 200 cases per million adult population per year),

and varies from 5% of hospitalized patients to 30–50%

of patients in intensive care units. Despite significant

technical advances in therapeutics, the mortality and

morbidity associated with ARF remain dismally highand have not appreciably improved during the last four

decades [1–12]. For example, in a recent large study of

1095 patients with ARF [13], the survival rate was 60%

of cases; however, only half of the survivors attained full

recovery of renal function, with the other half displaying

either renal insufficiency or end-stage renal disease

(ESRD). Although this unacceptable prognosis may be

partly attributable to other co-morbid conditions, recentstudies have revealed that several outcome variables are

adversely affected by the presence of ARF, and that

ARF itself is a major risk factor for the development of

non-renal complications. In addition to its implications

on human life and its quality, the treatment of ARF also

represents an enormous financial burden to society [14],

and ARF-associated medical expenses are conserva-

tively estimated at over $8 billion per year in adultsalone. In addition, even sub-clinical episodes of ARF

significantly contribute to the development of ESRD, a

common clinical condition (nearly 400 new cases per

million adult population per year) with a mean survival

rate only slightly better than lung cancer and with

staggering economic implications.

Ischemia–reperfusion injury

Renal ischemia–reperfusion injury (IRI) is the major

cause of ARF in the native kidney [1,4], and an invari-

able occurrence in the transplanted kidney [15]. Com-

mon medical conditions leading to IRI in the native

kidney include cardiovascular disease, stroke, trauma,

dehydration, and surgical procedures. In addition to the

usual complications of ARF, IRI in the transplantedkidney results in delayed graft function [16], which sig-

nificantly increases the risk of graft loss and acute re-

jection [17,18]. Thus, even long-suffering ESRD patients

who finally get a kidney transplant are not spared from

the ravages of IRI. Pioneering work over several de-

cades has illuminated the roles of persistent vasocon-

striction, tubular obstruction, cellular metabolic

alterations, and the inflammatory response in the path-ogenesis of IRI [19–22]. While these studies have paved

the way for successful therapeutic approaches in animal

models, translational research efforts in humans have

yielded disappointing results. One of the major reasons

for this includes a paucity of early biomarkers for hu-

man ARF, which contributes to a delay in initiating

preventive and therapeutic measures that have been ef-

fective in ameliorating ARF in animals. The secondreason pertains to the multi-factorial nature of the dis-

ease. An improved understanding of the molecular

mechanisms underlying early renal cell injury will be

critical for innovative and effective therapy.

Molecular pathogenesis of renal IRI

It is now clear that the morphologic and biologicresponses of renal tubular cells to IRI are multifaceted,

and include cell death, de-differentiation of viable cells,

proliferation, re-differentiation, and restitution of a

normal epithelium [1,19–22], as illustrated in Fig. 1. The

molecular mechanisms underlying each of these re-

sponses are subjects of intense current investigations.

Although the term ‘‘acute tubular necrosis’’ has been

synonymous with ARF, apoptosis has recently emergedas an additional potential mechanism leading to early

tubule cell death following ischemic renal injury [23–28].

Several studies have documented that IRI in animal

models predictably leads to apoptosis of renal tubular

epithelial cells [29–42]. Importantly, this finding has now

been confirmed in biopsy samples from transplanted

cadaveric kidneys, which constitutes a human model for

renal IRI [43–45]. Furthermore, several renal tubule cellculture models of ischemic renal injury have now been

described, which reliably demonstrate the induction of

apoptosis following partial ATP depletion or hypoxia,

and necrotic cell death following more severe grades of

injury [46–52]. These findings have lead to the hypoth-

esis that down-regulation of apoptosis may offer a un-

ique and powerful therapeutic approach to the

amelioration of IRI in both the native and the trans-planted kidney. Considerable attention and efforts have

therefore been directed towards identification of the

molecular pathways involved in the tubule cell apoptosis

following IRI. In recent years, specific proteases be-

longing to the caspase family have surfaced as crucial

effectors of apoptosis [53,54]. Members of this family are

expressed as pro-enzymes and require activation by

Fig. 1. Morphologic events following renal IRI. The four major phases of regeneration are illustrated.

Fig. 2. Apoptotic pathways in renal IRI, as predicted by transcriptome analysis. Pro-apoptotic genes of the extrinsic pathway that are directly

induced by IRI (FADD, DAXX, and p53) are shown in red, whereas those in the intrinsic pathway (Bak and Bad) are in green. The induced anti-

apoptotic gene Bcl2 is in blue.

P. Devarajan et al. / Molecular Genetics and Metabolism 80 (2003) 365–376 367


upstream stimuli in order to commit a cell into the ex-ecution phase of apoptosis. The major intracellular ap-

optotic pathways may be classified according to the type

of pro-caspase that is activated. Activation of the initi-

ator pro-caspase 8 results from signaling via cell surface

death receptors such as Fas (the ‘‘extrinsic’’ pathway)

and their ligands such as FADD and DAXX [55]. On

the other hand, activation of the initiator pro-caspase 9

is dependent primarily on ’’intrinsic’’ mitochondrialsignaling pathways regulated by members of the Bcl-2

family [56]. Activation of pro-apoptotic Bcl-2 family

members such as Bax and Bak can trigger a sequence of

events leading to release of mitochondrial cytochrome c

into the cytosol, and activation of pro-caspase 9 [57–59].

At least three potential levels of ‘‘cross-talk’’ exist be-

tween the extrinsic and intrinsic apoptotic pathways.

First, initial activation of caspase 8 via Fas can inducethe mitochondrial translocation of BID, a pro-apoptotic

member of the Bcl-2 family, with resultant cytochrome c

release and activation of caspase 9 [60]. Second, the p53

gene is a potent transcription factor that regulates ap-

optosis most notably by activating pro-apoptotic Bcl-2

family members as well as the Fas-dependent axis [61].

Third, both pathways culminate in the activation of

caspase 3, with subsequent entry into the ’’execution’’phase of apoptosis, resulting in DNA fragmentation and

cellular morphologic changes characteristic of apoptosis

[53]. The anti-apoptotic Bcl-2 family members such as

Bcl-2 itself play a pivotal protective role by preserving

mitochondrial structure and inhibiting cytochrome c

release [57–59].

Several of these apoptosis-related genes are expressed

and appear to be mechanistically operative in renal tu-bular cells following IRI. Attempts at unraveling the

molecular basis of these myriad apoptotic mechanisms

have been significantly facilitated by recent advances in

functional genomics that have yielded new tools for

genome-wide analysis of complex biologic processes

[60–67]. The DNA microarray methodologies provide

parallel and quantitative expression profiles of thou-

sands of genes, which when combined with bioinfor-matics can identify genes in a biologic pathway,

characterize the function of novel genes, analyze genetic

variation, detect disease subclasses, and identify drug

targets [60–67]. Others [68,69] and we [70] have recently

utilized these genome-wide approaches to analyze the

early renal response to ischemia both in vitro and in

animal models. Our results indicate that apoptosis plays

a major role and that a specific subset of apoptotic genesare induced in tubule cells following early IRI, as de-

tailed below.

Molecular mechanisms of recovery from renal IRI

Renal epithelial cells possess a remarkable ability to

regenerate and proliferate after IRI, a quality that is not

shared by the majority of other tissues. An improvedunderstanding of the molecular mechanisms of repair

may provide clues towards accelerating recovery fol-

lowing renal IRI. In the fully developed kidney, cell

division is minimal but can increase more than 10-fold

after acute injury [71]. Four major phases of this re-

generative process have been described [72–74], as il-

lustrated in Fig. 1. The first phase consists of cell

damage and cell death, during which tubule cells havebeen postulated to generate signals that initiate the re-

generative response. During this very early phase, renal

tubule cells up-regulate the expression of a number of

genes, including stress-activated protein kinases such as

MAP kinases [75,76] and transcription factors such as

c-Fos and Egr-1 [77]. The second phase is characterized

by the appearance of a large pool of de-differentiated

epithelial cells with flattened appearance and poorlydifferentiated brush borders [72–74]. These cells express

vimentin, a marker for multipotent embryonic mesen-

chymal cells [78]. Since the number of resident stem cells

in the native kidney and the number of bone marrow-

derived cells that appear in the post-injury kidney are

both very small, these cells must represent surviving or

sub-lethally injured tubule cells that have de-differenti-

ated [79]. De-differentiation of the tubular epithelial cellmay be a necessary pre-requisite to allow for spreading,

migration and re-population of cells over the denuded

basement membrane. The third phase is exemplified by a

marked increase in the number of proliferating tubule

epithelial cells [78–80] that express genes encoding for a

variety of growth factors such as IGF-1 [81,82], HGF

[83], and FGF [80]. The fourth phase is one of re-dif-

ferentiation, during which the normal tubular epithe-lium is restored with fully differentiated polarized cells.

Tubule epithelial cells in this stage display induction of a

variety of genes including NCAM [84], osteopontin [85],

CD44 [86], and TGF-b1 [87]. Others [68,69] and we [70]

have recently utilized genome-wide approaches to ana-

lyze the early global renal response to ischemia both in

vitro and in animal models. Our results have identified a

number of genes that are induced following early IRIand may play an important role in the recovery process,

as detailed below.

It is interesting to note that during recovery from

IRI, renal tubule cells recapitulate phases and processes

very similar to those during normal kidney develop-

ment. For instance, both the immature developing tu-

bule and the mature injured tubule cells display

apoptotic death as the mechanism for removing dam-aged or unwanted cells [23–27,88,89]. In both situa-

tions, de-differentiated mesenchymal cells undergo

proliferation, differentiation, and establishment of a

polarized epithelium [90]. However, many questions

remain unanswered. For example, it is unknown whe-

ther the inductive interactions between the metanephric

mesenchyme and ureteric bud that are characteristic of


and critical to the developing nephron also play a rolefollowing IRI. Also it is unclear which stage of devel-

opment the injured tubule cells de-differentiate to, and

how the patterns of gene expression resemble or differ

from each other during the two processes. The advent

of cDNA microarrays has, however, revolutionized the

global analysis of gene expression in the developing

kidney. Others [91] and we [92] have recently utilized

these approaches to catalogue gene expression duringkidney development. The results indicate that specific

subsets of genes are induced during various stages of

development. The results are detailed below, along with

an instructive and interesting comparison of the tem-

poral patterns of gene expression during renal devel-

opment and following IRI.

Analysis of gene expression during early recovery from

IRI

Maximally induced transcripts: clues for biomarker

discovery

We have utilized cDNA microarray technology and

stringent statistical analysis to define global changes inrenal gene expression during the early reperfusion peri-

ods following ischemic injury in an established mouse

model [70]. We have screened for changes in expression

of 9000 sequence-verified mouse genes at various early

points (3, 12, and 24 h) following renal IRI. We chose to

examine the immediate and early responses because (a) it

is well known that changes in gene expression occur soon

after an ischemic insult, (b) interventions aimed atameliorating injury and/or accelerating recovery may

need to target these early changes, (c) the temporal pat-

terns of these early morphologic and biologic changes

recapitulate those during renal development, and (d)

genes whose protein products are induced early after

injury may represent biomarkers that have hitherto elu-

ded discovery. We identified several transcripts that have

previously been shown to be over-expressed or repressedfollowing ischemic injury, thereby validating this tech-

nique. For example, our results have confirmed previous

reports documenting by alternative methods the upreg-

ulation of genes such as Bcl-2, Egr-1, c-Fos, HSP-70,

HGF, HB-EGF, IGF-1, TGFb1, p21, heme oxygenase 1,

and a-crystallin following renal IRI [70]. Similarly, pre-

viously described transcripts that are down-regulated

following ischemia that were also identified in our studyinclude NHE3, a-albumin, and members of the cyto-

chrome p450 family [70]. In addition, several genes not

previously associated with IRI were consistently shown

to be differentially expressed, and a detailed analysis of

these transcripts is beyond the scope of this minireview.

Surprisingly, several of the transcripts that were

maximally induced during early IRI were novel to the

field of acute renal failure. In a recent study [93], wehave further characterized one of these previously un-

recognized genes, namely neutrophil gelatinase-associ-

ated lipocalin (NGAL), because it encodes a small

secreted polypeptide that is protease resistant and con-

sequently might be readily detected in the urine. We

confirmed the marked upregulation of NGAL mRNA

by semi-quantitative RT-PCR and protein levels by

Western analysis in the early post-ischemic mouse kid-ney (both greater than 10-fold). NGAL protein expres-

sion was detected predominantly in PCNA-positive

proximal tubule cells that were undergoing proliferation

and regeneration. These findings strongly implicate a

role for this maximally induced gene and protein in the

repair process following IRI. Other recent studies have

also suggested that NGAL enhances the epithelial phe-

notype. During nephrogenesis, NGAL is expressed bythe penetrating ureteric bud, and triggers nephrogenesis

by stimulating the conversion of mesenchymal cells into

kidney epithelia [94]. Another lipocalin, glycodelin, has

been shown to induce an epithelial phenotype when

expressed in human breast carcinoma cells [95]. These

findings are especially pertinent to the mature kidney, in

which one of the well-documented responses to ischemic

injury is the remarkable appearance of de-differentiatedepithelial cells lining the proximal tubules [78]. An im-

portant aspect of renal regeneration and repair after

ischemic injury involves the reacquisition of the epithe-

lial phenotype, a process that recapitulates several as-

pects of normal development [96]. This suggests that

NGAL may be expressed by the damaged tubule in

order to induce re-epithelialization. Support for this

notion derives from the recent identification of NGALas an iron transporting protein that is complementary to

transferrin during nephrogenesis [94]. It is well known

that the delivery of iron into cells is crucial for cell

growth and development, and this is presumably critical

to post-ischemic renal regeneration just as it is during

ontogeny. Since NGAL appears to bind and transport

iron [94], it is also likely that NGAL may serve as a sink

for iron that is shed from damaged proximal tubuleepithelial cells. Because NGAL can be endocytosed by

the proximal tubule, the protein could potentially re-

cycle iron into viable cells. This might stimulate growth

and development, as well as remove iron, a reactive

molecule, from the site of tissue injury, thereby limiting

iron-mediated cytotoxicity.

Importantly, we have found that NGAL is easily

detected in the urine in the very first urine output fol-lowing ischemia in both mouse and rat models of ARF

[93]. The appearance of NGAL in the urine is related to

the dose and duration of renal ischemia, and precedes by

far the appearance of other known urinary markers such

as NAG and b2-microglobulin. The origin of NGAL

was confirmed in cultured human proximal tubule cells

subjected to in vitro ischemic injury, where NGAL


mRNA was rapidly induced in the cells and NGALprotein readily detectable in the culture medium within

one hour of mild ATP depletion. Our results indicate

that NGAL may represent an early, sensitive, non-in-

vasive urinary biomarker for ischemic renal injury that

compares very favorably with other biomarkers that

have been described. One of the best-studied examples is

kidney injury molecule-1 (KIM-1), a putative adhesion

molecule involved in renal regeneration that was alsofirst detected as a result of genomic analysis [103–105].

In a rat model of ischemia–reperfusion injury, KIM-1

was found to be upregulated 24–48 h after the initial

insult, rendering it a reliable but somewhat late marker

of tubular cell damage. Recent elegant studies have

shown that KIM-1 can be detected in the kidney biopsy

and urine of patients with ischemic acute tubular ne-

crosis [105]. However, this detection was documented inpatients with established ischemic renal damage, and the

utility of urinary KIM-1 measurement for the detection

of early subclinical injury has thus far not been vali-

dated. In another recent example, Cyr61 was found to

be a secreted cysteine-rich protein that is detectable in

the urine 3–6 h after ischemic renal injury [106]. How-

ever, this detection required a bioaffinity purification

step with heparin–Sepharose beads, and even after suchpurification several cross-reacting peptides were appar-

ent. In contrast, our study demonstrates that NGAL

was easily and rapidly detected as relatively clean im-

munoreactive peptides in Western blots with as little as

1 ll of the very first unprocessed urine output following

renal ischemia in both mice and rats. In addition, uri-

nary NGAL was evident even after very mild ‘‘subclin-

ical’’ renal ischemia, in spite of normal serum creatininelevels. Translational work in patients who are at risk for

developing sub-clinical forms of ischemic injury is in

progress. It is anticipated that early detection of sub-

clinical renal injury will enable clinicians to begin timely

interventional measures to prevent progression to overt

ARF. It is also anticipated that early detection will al-

low for institution of novel therapeutic approaches that

have thus far been met with limited success due to thelack of early biomarkers.

Induction of apoptosis-related genes: clues for pathogen-

esis and therapy

Our microarray analysis revealed for the first time a

marked upregulation of several pro-apoptotic genes in

the early post-ischemic period [70]. The most prominentchange was noted for FADD, which was upregulated

2.1-fold, 5.4-fold, and 6.1-fold at 3, 12, and 24 h of

reperfusion, respectively. Significant increases in ex-

pression were also noted at all reflow periods for DAXX

(1.9-fold, 2.5-fold, and 2.5-fold at 3, 12, and 24 h of

reperfusion, respectively), p53 (2.2-fold, 2.4-fold, and

2.2-fold at 3, 12, and 24 h of reperfusion, respectively),

and BAD (2.5-fold, 3-fold, and 2.5-fold at 3, 12, and24 h of reperfusion respectively). In contrast, the ex-

pression of BAK was significantly upregulated (3.2-fold)

only at 3 h of reflow. The immediate response to is-

chemic injury was also characterized by increased ex-

pression of the anti-apoptotic genes Bcl-2 and NF-jB.The finding of upregulated pro-apoptotic transcripts by

microarray analysis was confirmed by semi-quantitative

RT-PCR and immunohistochemistry [70]. In serial sec-tions, the majority of tubule epithelial cells that stained

positive for these pro-apoptotic factors also revealed

Tunel positive nuclei, implicating these pathways in the

cell death following IRI.

Products of the induced transcripts FADD and

DAXX are members of the ‘‘extrinsic’’ cell death path-

way, and are known to transduce apoptotic signals

emanating from cell surface receptors such as Fas, withresultant activation of caspase 8 [55]. Expression of Fas

has previously been documented in renal tubule cells

both in vivo [97] and in vitro [52], and upregulation of

Fas protein has been shown to occur in mouse kidney

after a 24 h reflow period following ischemia [97]. Sim-

ilarly, renal expression of FADD has been demonstrated

in vivo and in vitro [70]. Furthermore, we have previ-

ously demonstrated a rapid upregulation of Fas andFADD protein and activation of caspase 8 in cultured

tubule cells following an in vitro ischemic injury [52].

Our previous and present studies suggest that induction

of the Fas-FADD-caspase 8 pathway may play an im-

portant pathogenetic role in the initiation of tubule cell

apoptosis during the early reperfusion period. The pre-

cise role of DAXX in kidney cell apoptotic cascades is

unclear. However, it is worthwhile noting that DAXXmediates both Fas-dependent and TGF-b-induced ap-

optosis, and the marked renal induction of TGF-b fol-

lowing ischemia is well documented in our study [70].

Transcripts belonging to the intrinsic mitochondrial

apoptotic pathway, namely BAD and BAK, were also

consistently induced in our study [70]. These pro-apop-

totic molecules of the Bcl-2 family normally reside in the

cytosol, but can undergo activation and mitochondrialtranslocation following an apoptotic stimulus, leading

to release of cytochrome c and activation of caspase 9

[56–59]. Both molecules are expressed in kidney tubule

cells [70]. BAK was found to be transcriptionally up-

regulated exclusively at the 3 h reflow period, whereas

the induction of BAD was evident at all times examined.

It is interesting to note that the anti-apoptotic genes Bcl-

2 and NF-jB were also upregulated at earlier time pe-riods in our study. Bcl-2 plays a pivotal protective role

by inhibiting mitochondrial cytochrome c release, and

our findings confirm previous reports of enhanced tu-

bule cell Bcl-2 expression following renal ischemia–rep-

erfusion injury [40]. NF-jB comprises a family of

transcription factors that regulate the expression of

several genes involved in inflammation, proliferation,


and anti-apoptosis [98]. Our findings allow us to hy-pothesize that the simultaneous induction of pro- and

anti-apoptotic transcripts may account for the paucity

of apoptotic cells immediately following IRI [70].

However, the continued upregulation of extrinsic and

intrinsic pro-apoptotic pathway transcripts as observed

in the 12 and 24 h reperfusion periods, combined with a

diminished induction of survival factors such as Bcl-2

and NF-jB, may tilt the balance in favor of apoptosis.The p53 gene is a potent transcription factor that

regulates apoptosis most notably by activating pro-ap-

optotic Bcl-2 family members as well as the Fas-FADD

axis [61]. Both the extrinsic and intrinsic pathways cul-

minate in the activation of caspase 3, with subsequent

entry into the ‘‘execution’’ phase of apoptosis [53]. Our

overall findings suggest induction of multiple apoptotic

pathways, as illustrated in Fig. 2, and lend support tothe notion of caspase inhibition as a potential thera-

peutic tool in renal IRI. Indeed, recent animal studies

have revealed a promising role for caspase inhibitors in

ameliorating injury following renal ischemia [101,102].

From a translational perspective, this concept may be

especially pertinent to renal transplantation, wherein the

kidney is perfused with a defined solution and stored

until re-anastomosis. It will be important in futurestudies to explore the efficacy of adding cell-permeant

caspase inhibitors to the solutions used during cold

storage in ameliorating the IRI typical of kidney trans-

plantation.

It is important to recognize that one of the limitations

to using functional genomic approaches is the fact that

alterations in gene expression are not always predictive

of downstream functional and/or pathophysiologicpathways. Although our results have suggested activa-

tion of select pro-apoptotic pathways at the mRNA

level, additional post-transcriptional and post-transla-

tional events may be required to fully implicate these

factors in the programmed cell death following renal

IRI. For example, phosphorylation is a key event that

determines the activity of p53 and BAD, and the role

of FADD is dependent on complex protein–proteininteractions. The cDNA microarray results provide a

stepping stone, and it will be important in future studies

to fully characterize the biology of the identified apop-

totic factors in order to confirm their role in tubule

cell death.

Induction of regeneration-related genes: lessons from the

developing kidney

The advent of microarrays has revolutionized global

studies of gene expression in the developing kidney.

Using microarrays it is now possible to rapidly assay the

expression level of essentially every mouse or human

gene. New powerful target micro-amplification tech-

niques allow the application of this procedure to very

small samples. Valerius et al. [99] generated two clonalcell lines, mK3 and mK4, that by several criteria rep-

resented early metanephric mesenchyme and later in-

duced metanephric mesenchyme, respectively. For

example, the mK3 cells were able to induce branching

morphogenesis of ureteric bud in organ co-culture ex-

periments, and the mK4 cells showed a more cuboidal,

epithelial-like morphology. Microarray analysis of these

cells identified thousands of genes they expressed, pro-viding a detailed molecular fingerprint that confirmed

their developmental stages [99]. It was observed that the

mK3 cells expressed known markers of early meta-

nephric mesenchyme, including collagen I and vimentin,

while the mK4 cells expressed genes such as Pax-8, Pax-

2, Wnt-4, cadherin-6, collagen IV, and LFB3, diagnostic

of a somewhat later developmental stage during which

the metanephric mesenchyme undergoes epithelialtransformation. In addition, over 70 transcription fac-

tors were expressed in the mK3 cells. Six Hox genes were

expressed, Hoxc 9, Hoxa 11, Hoxc 8, Hoxa 9, Hoxa 10,

and Hoxa 5, in order of decreasing abundance. Their

expression in mK3 cells suggested they are expressed in

early, uninduced metanephric mesenchyme.

Stuart et al. [91] used Affymetrix gene chips to ex-

amine kidney development in the rat. Samples werecollected from E13, 15, 17, 19, newborn, one week, and

adult. K-means hierarchical cluster analysis identified

five groups of genes with distinct expression patterns.

The first group consisted of genes with highest expres-

sion in the earliest examined developing kidney and then

diminishing expression with time. The genes in this

group were largely associated with cell proliferation and

morphogenesis, including pleiotrophin, IGF-II, TGF-b2, ganglioside, frizzled, PDGF-R, Wilms tumor 1, and

Bcl2. The genes of group 2 showed highest expression in

the developing kidney at E15 to E19, with lower ex-

pression levels at earlier and later time points. Many of

these genes encoded proteins of the extracellular matrix

or cytoskeleton, including agrin, decorin, and osteo-

nectin. This group also included a number of morpho-

genetic effectors, including jagged, LIM1, HGF, FGF7,and BMP4. Group 3 contained genes that peaked late

during development, and consisted notably of retro-

transposons. The genes in groups 4 and 5 increased in a

linear fashion into adulthood. These included BMP7,

energy-related genes (such as aldolase, fructose bis-

phosphatase 2, hexokinase, and lactate dehydrogenase),

and transporters (such as aquaporin, H+ transporting

ATPase, and organic cation transporter).Affymetrix oligonucleotidemicroarrays have also been

used to study early kidney development in the mouse [92].

In this case the U74A GeneChips were used, with about

12,500 probe set arrays. Kidneys from E11.5, E12.5,

E13.5, E16.5, and adult were examined, as well as micro-

dissected E11.5 ureteric bud and metanephric mesen-

chyme and P1 total mouse as a reference. Stringent


comparison of gene expression profiles found 428 geneswith significantly elevated expression in the E12.5 em-

bryonic kidney versus the adult.About 10%of these genes

are transcription regulators, includingHoxa 11,Hoxd 11,

N-Myc, histone deacetylase 1, nuclear transcription

factor-Yb, and TG interacting factor. Consistent with the

high growth rate of the embryonic kidney, 35 cell cycle

associated genes were found elevated in expression in the

E12.5 kidney. In addition almost 100 genes with highexpression in the developing kidney were involved in

intermediary metabolism, ribosome biogenesis, DNA

synthesis, and other processes connected with rapidly

dividing cells. This investigation also found several

growth factor-related genes with elevated expression

during early kidney embryogenesis, including GDNF,

midkine, Sfrp2, and follistatin-like protein.

We have completed a detailed comparative analysisof renal gene expression during development and fol-

lowing IRI. Table 1 shows the expression profiles of

Table 1

Transcripts induced early during development and at 3, 12, or 24 h followin

Name Description

Cell adhesion and cytoskeleton

Bys1 Bystin-like

Cdh2 Cadherin-2

Cldn6 Claudin 6

Cldn7 Claudin 7

Efs Embryonal Fyn-associated substr

Lamc2 Laminin, c2Ncam1 Neural cell adhesion molecule 1

Ppl Periplakin

Growth-related

Bcl-2 Bcl-2

Cdkn1a Cdk inhibitor 1a (p21)

Cnn2 Calponin 2

Fstl Follistatin-like

Gdnf Glial-derived neurotrophic factor

Hgf Hepatocyte growth factor

Mdk Midkine

Tgfbi Transforming growth factor, b in

Signaling

Cks1 CDC 28 protein kinase 1

Gfra1 GDNF receptor a1Lag Leukemia-associated gene

Sfrs3 Splicing factor, Arg/Ser-rich 3

Tacstd Tumor-associated Ca signal tran

U2af1 U2 small nuclear RNP auxiliary

Transcription

En2 Engrailed 2

Hdac1 Histone deacetylase 1

Nfyb Nuclear transcription factor-Y bNmyc1 N-myc 1

Tgif TG interacting factor

Wt1 Wilm�s tumor homolog 1

Transport

Hba-a1 Hemoglobin a, adult 1

Values represent fold changes in expression compared to control (mean va

2-fold have been left blank.

transcripts that are predominantly induced early duringdevelopment [91,92] and following early IRI in the

mature kidney [70]. It is evident that several of the genes

that have been shown to play a key role during early

renal morphogenesis are also induced within 3 h of renal

IRI, lending strong support to the concept that regen-

eration recapitulates ontogeny in the kidney. Particu-

larly noteworthy in this respect are a number of

transcription and growth factors (such as Nmyc1, Nfyb,Gfra1, HGF, and Bcl-2) that are upregulated during

early nephrogenesis and exclusively in the 3 h reperfu-

sion period. These results provide molecular genetic

evidence for the hypothesis that injured tubule cells

possess the capacity to de-differentiate to the earliest

stages of development. Equally striking is the finding

that the majority of genes whose expression is a signa-

ture of a very de-differentiated stage of early nephro-genesis, that become down-regulated as development

proceeds, however remain induced at the later (12 and

g early IRI

3 h 12 h 24 h

3.2 3.8 3.4

3.0 3.2

4.0 3.6

12.0 9.1 13.2

ate 2.8 2.6 2.6

4.1 6.0 3.4

2.8 5.4 3.4

2.8 2.4 2.4

5.0

14.2 13.0 10.8

3.2 3.0

2.6 3.8 2.8

2.8 2.8 2.8

2.6

3.0 3.0

duced 3.2 3.0 3.6

2.4 4.4 5.0

2.8

3.4 2.6

3.2 4.0 4.0

sducer 2 10.0 6.1 9.0

factor 1 5.0 3.8 3.8

3.2 2.8

2.8 2.8

3.4

3.0

7.6 4.6 4.0

4.0 4.0 3.2

7.1 4.0 11.4

lues from at least 4 mice at each reperfusion period). Changes less than


24 h) reflow periods following IRI in the mature kidney.Examples include Wt1, GDNF, and midkine, all of

which have been extensively investigated during early

development, but the role for their rapid and persistent

induction following IRI in the mature kidney remain

unknown. The results do indicate, however, that

de-differentiated tubule cells can persist in the injured

mature kidney for a relatively long period of time, an

observation that may be pertinent to the ability of thekidney to repair itself during the recovery process and

the temporal patterns with which this occurs.

Table 2 illustrates the expression profiles of tran-

scripts induced at a later stage of nephrogenesis [91,92]

and following IRI in the mature kidney [70]. As ex-

pected, the majority of these genes, including trans-

porters, cell adhesion molecules and energy-related

species whose expression is diagnostic of a terminallydifferentiated state during development, were also found

Table 2

Transcripts induced late during development and at 3, 12, or 24 h following

Name Description

Cell adhesion and cytoskeleton

Gpc4 Glypican 4

Igta6 Integrin a6Muc1 Mucin 1

Timp3 Tissue inhibitor of metalloprotein

Vil Villin

Growth-related

Cish2 Cytokine inducible SH2-containi

Eps8 EGFR pathway substrate 8

Pparbp Ppar binding protein

Umod Uromodulin

Energy-related

Aldo1 Aldolase 1, A isoform

Fbp2 Fructose bisphosphatase 2

Hk1 Hexokinase 1

Ldh1 Lactate dehydrogenase 1, A chai

Signaling

Calml4 Calmodulin-like 4

Cast Calpastatin

Cckar Cholecystokinin type-A receptor

Kdap Kidney-derived Asp protease-like

Mylk Myosin, light polypeptide kinase

Ndrl N-myc downstream regulated

Ppp1cb Protein phosphatase 1, bRhoB Ras homolog gene, AB

Ren1 Renin 1, structural

Transcription

Elk3 ETS oncogene member

Mad4 Max dimerization protein 4

Sp1 Trans-acting transcription factor

Transport

Aqp1 Aquaporin 1

Atp6v1 ATPase, Hþ transporting, A1

Oct1 Organic cationic transporter 1

Snca Synuclein, a

Values represent fold changes in expression compared to control (mean va

2-fold have been left blank.

to be significantly induced in the 24 h reflow periodfollowing IRI. However, a surprising finding is that this

subset of transcripts was also significantly over-ex-

pressed very early following IRI. One interpretation of

this pattern is that it is a reflection of viable cells to

rapidly and appropriately respond to cellular stress

following IRI. An alternative, intriguing possibility is

that the process of de-differentiation and re-differentia-

tion may be significantly accelerated in mature tubulecells following injury. In either case, these findings sug-

gest several potential strategies for accelerating the re-

pair process that may be worthy of exploring.

Accelerating regeneration: clues for novel therapies

Both nephrogenesis and regeneration following renal

IRI are associated with a marked induction of a varietyof growth factors, including HGF, GDNF, TGF-b1,

early IRI

3 h 12 h 24 h

2.4

3.0 3.0 3.0

2.4 2.4 3.1

ases 3 3.2

3.0 2.0

ng 2 2.6 2.6 2.6

4.2

4.4 2.4

4.0 3.4

2.8 2.7 2.6

4.4 3.6 4.1

4.4 2.4 2.8

n 4.0 6.0 5.2

4.0 4.0

4.0 3.8 3.6

2.6 2.8

2.2 3.1 4.2

3.8 4.2 3.4

3.0 2.8

4.0 2.8

7.0

3.6 3.6

3.4 4.4 3.6

3.0 2.4 2.8

1 4.2 3.4 3.4

2.8

3.0 3.0

3.0

2.6 2.4

lues from at least 4 mice at each reperfusion period). Changes less than


IGF-1, IGF-2, and EGF. This has stimulated interro-gation of the effects of exogenously administered growth

factors for enhancing renal repair following IRI. Per-

haps the best studied example is that of IGF-1, which

clearly accelerates functional recovery and regeneration

of damaged tubular epithelium when administered prior

to, concomitant with, or even following renal IRI in

animal models [100]. However, in a prospective ran-

domized trial of IGF-1 in humans with establishedARF, no beneficial effects were discernible [96]. It has

been suggested that a delay in starting IGF-1 therapy

(due to lack of early biomarkers) might have contrib-

uted to the negative outcome [96]. Another explanation

suggested by the available microarray analysis is that the

molecular genetic response to IRI is multifaceted, with

upregulation of a number of growth factors that may act

in concert to accelerate regeneration, and that exoge-nous administration of a single growth factor may be

insufficient. It is likely that a combination of growth

factors (including factors previously not associated with

IRI such as PDGF-a, angiopoietin, and follistatin that

have emerged from our transcriptome analysis) admin-

istered early in the course of ARF (based on novel bi-

omarkers) will provide the effective therapy that has

eluded clinicians thus far.

Acknowledgments

Portions of the work described in this review weresupported by grants from the NIH to P.D. (DK53289,

DK52612), L.T.P. (DK02702), and S.S.P. (DK61916),

and a Fellowship from the Kidney and Urology Foun-

dation to S.S.

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Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery, and novel therapeutics