1. Introduction

2. Current and new animal

models of AKI

3. Need for new animal models of

AKI

4. Advances in AKI animals

models

5. New potential therapeutic

approaches to AKI

characterized in experimental

models

6. Clinical therapies for AKI

treatment

7. Conclusions

8. Expert opinion

Review

Progress in the development ofanimal models of acute kidneyinjury and its impact on drugdiscoveryAna B Sanz, Marıa Dolores Sanchez-Nino, Catalina Martın-Cleary,Alberto Ortiz† & Adrian M Ramos†Fundacion Renal Inigo Alvarez de Toledo (FRIAT), Madrid, Spain

Introduction: Acute kidney injury (AKI) is a clinical syndrome characterized

by the acute loss of kidney function. AKI is increasingly frequent and is associated

with impaired survival and chronic kidney disease progression. Experimental AKI

models have contributed to a better understanding of pathophysiological mech-

anisms but they have not yet resulted in routine clinical application of novel

therapeutic approaches.

Areas covered: The authors present the advances in experimental AKI models

over the last decade. Furthermore, the authors review their current and

expected impact on novel drug discovery.

Expert opinion: New AKI models have been developed in rodents and non-

rodents. Non-rodents allow the evaluation of specific aspects of AKI in both

bigger animals and simpler organisms such as drosophila and zebrafish. New

rodent models have recently reproduced described clinical entities, such as

aristolochic and warfarin nephropathies, and have also provided better mod-

els for old entities such as thrombotic microangiopathy-induced AKI. Several

therapies identified in animal models are now undergoing clinical trials in

human AKI, including p53 RNAi and bone-marrow derived mesenchymal

stem cells. It is conceivable that further refinement of animal models in com-

bination with ongoing trials and novel trials based on already identified

potential targets will eventually yield effective therapies for clinical AKI.

Keywords: apoptosis, fibrosis, inflammation, kidney, necroptosis, nephrotoxicity, transgenic

mice, TWEAK

Expert Opin. Drug Discov. [Early Online]

1. Introduction

Acute kidney injury (AKI) is a clinical syndrome characterized by the acute loss ofkidney function that leads to increased serum creatinine or oliguria [1]. AKI mayresult in the need for renal replacement therapy (RRT). However, even when notneeding RRT, AKI is still a serious disorder, conferring an increased risk of deaththat persists for over a year following the AKI episode and also increasing the riskof chronic kidney disease (CKD) progression [2-5]. AKI and CKD have been recentlyrecognized as an integrated clinical syndrome and much research is being carried outon the interface of both clinical conditions [6]. Indeed, AKI and CKD share key bio-logical processes such as cell death, cell proliferation, cell dedifferentiation, inflam-mation and fibrosis and also share biomarkers [7-10]. In general, the magnitude ofthese processes is greater in AKI than in CKD, except for fibrosis which is associatedto chronicity. The short time-course and the severity of histological and functionalchanges have fueled the study of experimental animal models of AKI to characterizemediators involved in kidney injury in order to identify novel therapeutic

10.1517/17460441.2013.793667 © 2013 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 1All rights reserved: reproduction in whole or in part not permitted

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approaches and biomarkers. However, despite many success-ful reports in animal models, no novel biomarker resultingfrom these studies is currently in use in routine clinical prac-tice, and there is no routine therapeutic approach to AKIbased on experimental advances on the molecular and cellularmechanisms of kidney injury. This lack of tools to preventinjury or accelerate recovery reflects a still incomplete under-standing of molecular and cellular events, the deficiencies ofcurrently available animal models and probably a lack of accu-rate tools for clinical staging of AKI. In this regard, the cur-rent clinical definition of AKI based on functional criteriaidentifies late events in the course of kidney injury. A noveldefinition that allows an earlier diagnosis and therapeuticintervention is required and a search for novel biomarkers isunderway. Animal models of AKI will play a decisive role inthe quest for biomarkers and novel therapeutic strategies.We now review current and upcoming animal models ofAKI, illustrate some emerging therapeutic approaches identi-fied in recent years by the use of these models, critically reviewongoing and recent clinical trials based on experimentalmodel data and provide expert opinion on the current stateof the field and unmet medical needs.

2. Current and new animal models of AKI

AKI is a syndrome that recognizes many triggering events inclinical situations, including nephrotoxic therapeutic drugs,environmental contaminants and poisons, urinary tractobstructions, bacterial toxins, ischemic episodes and, fre-quently in clinical practice, a sequential or concomitant pres-ence of several of these factors. Thus, several experimentalanimal models have been developed to mimic these differentpotential clinical settings (Table 1). Comprehensive reviewsof the available models, their advantages and limitations,

were performed more than a decade ago [11]. More recentlytechnical details for a wide range of ischemia-reperfusion,toxic and septic models have been collected [12]. We referthe readers to these manuscripts for an in-depth descriptionof traditional models of AKI, while we will concentrate, inthe present review, on emerging models of AKI and theimpact of experimental models on drug discovery.

3. Need for new animal models of AKI

Animal models of AKI have proved to be valuable in under-standing molecular mechanisms underlying AKI initiationand progression, but they also have limitations that haveprecluded widespread clinical translation. Thus, AKIremains an entity devoid of specific therapy, beyond symp-tomatic therapy, and all clinical trials completed to datebased on preclinical data have failed. Several factors contrib-ute to this failure including animal models that do not fullyreproduce the clinical situation, such as the classic ischemia/reperfusion model, relative timing of insult and therapy(most successful preclinical interventional approaches areprophylactic), the insufficient biomarker resources inhumans to stage the disease and, potentially, interspeciesdifferences. The translational gap between experimentaltherapies for AKI and their effective application in the clin-ical setting remains a concern in the scientific community.In order to overcome drawbacks detracting reliability fromusually employed in vivo models for AKI, researchers con-tinue to develop novel models that try to best fit clinicalconditions or better dissect the role of different moleculesor cells or allow a more detailed follow-up of molecular orcellular events. New proposed in vivo models focus on tar-geting specific kidney areas and epithelial subtypes as wellas pathological events previously identified as the startingpoint to a further widespread renal damage, a purpose diffi-cult to achieve with traditional models. Many new animalmodels deal with genetically modified mice. These animalsmay express proteins that under specific conditions damagethe cellular type where they are expressed, explore the roleplayed by specific molecules, molecular pathways and bio-chemical processes underlying experimental AKI in tradi-tional models or are designed to closely follow-up cell fateor molecular events (Table 1).

4. Advances in AKI animals models

Recent advances in AKI animal models have focused onimproving previous models or developing new models tomeet previously unmet needs usually in rodent animal modelsbut sometimes expanded into non-rodents or employinggenetically modified animals. Specific attention has beendevoted to address the AKI to CKD transition and to repro-duce special high-risk clinical situations that predispose toAKI initiation and progression.

Article highlights.

. Experimental AKI models have contributed to a betterunderstanding of pathophysiological mechanisms buthave not resulted yet in routine clinical therapy for AKI.

. New AKI models have been developed in rodents andnon-rodents.

. Non-rodents allow the evaluation of specific aspects ofAKI in both bigger animals and simpler organisms.

. New rodent models have reproduced recently describedclinical entities, have provided better models for oldentities and have allowed exploration of factorspredisposing to AKI.

. Genetically modified animals allow the functionalevaluation of specific molecules or tubular segments,lineage tracing and real-time monitoring oftubular injury.

. In recent years a spate of novel druggable targets hasbeen identified and clinical trials are underway for someof them.

This box summarizes key points contained in the article.

A. Sanz et al.

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Table 1. Traditional and novel AKI animal models.

Model Characteristics

RodentsToxic or drug-induced AKI*Therapeutic drugs:

Cisplatin [11,12]

Aminoglycosides [11,12]

Nonsteroidal anti-inflammatory drugs [128]

Acetaminophen (Paracetamol) [12]5/6 nephrectomy + warfarin [30]

Non-therapeutic drugs:Folic acid [12]

z

Diagnostic drugs:Radiocontrast [12]

Poisons/ambient contaminants:Ethylene glycol [140]z

Aristolochic acid [129]

Mercury compounds and derivatives [36]

Pigment-inducedGlycerol-induced rhabdomyolysis [12]

OtherMaleate nephrotoxicity [23]

AdvantagesSimplicity and reproducibilityClinical correlate: may simulate renal adverse effects of pharmacologicaltreatments and common intoxications or poisoning affecting the kidneyShort time to AKI production (1 -- 6 days) and achieved with single orrepetitive dosePredisposing factors for human AKI can be reproduced in animal modelsEvolution of damage and renal function, including the recovery phase, parallelhuman AKIUseful to study long-term consequences of AKI, including transition to CKDand fibrosis in some modelsCan be performed in small animals

DisadvantagesDose to produce AKI can be much higher than those for human use, raisingquestions of clinical significanceWidespread kidney cell damage beyond tubular cellsNo adequate mouse models of radiocontrast-induced AKINo clinical correlate of maleate nephrotoxicity, single case report of accidentalfolic acid nephrotoxicity

Observations5/6 nephrectomy + warfarin is the only model available to study AKI caused byanticoagulants. Only available in ratsThere are alternative methods to study rhabdomyolysis including myoglobin orhemoglobin infusion but they poorly reproduce human physiopathologicalaspectsMaleate causes proximal tubular cell specific coenzyme A depletion, ATP andGSH depletion simulating ischemiaAristolochic acid induces rapidly progressive CKD and cancer in aristolochicacid nephropathy and has been implicated in Balkan nephropathy, allowing theexploration of the AKI/CKD/cancer interface

Obstructive AKI§

UUO [11]

Adenine ingestion [130]

Cast nephropathy following immunoglobulinlight chain injection [112]

Folic acid [12]z

Ethylene glycol [87]z

AdvantagesUUO: accelerated model of AKI leading to renal fibrosisAllows variations in the timing, severity and duration. Reversal of theobstruction permits the study of recovery. It presents with tubular damage(apoptosis and necrosis) (UUO)Technically simple and reproducible (adenine ingestion, cast nephropathy)Clinical relevance of UUO and cast nephropathy, the latter in the setting ofoverproduction of monoclonal immunoglobulin free light chains, clinicallyassociated to multiple myelomaCan be performed in small animals

DisadvantagesUUO requires surgery. Not widely used as AKI modelOwing to their novelty, adenine ingestion and cast nephropathy need furthervalidationUUO: renal function cannot be measured since it is compensated by the non-ligated kidney

Ischemia-reperfusion injury (IRI)§

Time-dependent ischemia [11]

Warm/cold [11,12]

AdvantagesClinically very relevant, including for kidney transplantsThe most employed model to study AKI which give a high knowledgebackground

*A more comprehensive list is provided in Refs [11] and [12].zBoth toxicity and intratubular precipitation contribute to AKI.§Additional details in reference to these models are given in Ref. [11].{Experimental AKI has been performed in genetically modified mice in order to explore the role of specific molecules in AKI. Here, we reflect examples of the use

of genetically modified mice to answer more general questions about AKI, such as the role of specific cell types, the sensitization to a particular form of kidney

injury or cell tagging for better assessment of specific aspects of AKI.

Progress in the development of animal models of AKI and its impact on drug discovery

Expert Opin. Drug Discov. [Early Online] 3

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Table 1. Traditional and novel AKI animal models (continued).

Model Characteristics

Unilateral or bilateral [11,12]Renal artery occlusion + E. coli injection [131]

The variable ischemia time allows different forms of injuryCan be performed in small animals

DisadvantagesMurine model is less reproducible than the rat modelTechnical and surgical procedures require carefully monitoring and surgeonsmust be well trained. Close post-surgery care is also neededMost common clinical situation is prolonged low level ischemia with potentialrepeat episodes, while the experimental setting implies complete short-term occlusion

ObservationsBilateral ischemia is more relevant to human pathology where abnormal bloodsupply is usually bilateral

Endotoxic AKI§

LPS injection or infusion [11]

Cecal puncture/ligation [11]

Bacterial infusion [11]

Uterine obstruction + bacterial inoculation [72]

AdvantagesLPS injection is simple and inexpensiveThe dose of endotoxin and bacteria can be standardizedLPS mimic systemic inflammatory reaction occurring in sepsisLeaking of cecal bacteria avoid transient inflammatory response of toxininjectionAppropriate for small animals

DisadvantagesVariable hemodynamic response between modelsMultiorgan dysfunction is dependent on the modelThe extent of renal damage is variable and the acute tubular necrosis is notalways achievedThere is a variable outcome with bacterial strainSurgical methods are not well standardizedLPS does not reproduce the presence of live bacteria occurring in the clinicalsituationCecal puncture/ligation involves surgery. Use of antibiotics in the clinicalsituation differs from animal model

Thrombotic microangiopathy-induced AKIStx injection to different strains of mice [26]

LPS + Stx [29]

Genetically modified Stx-expressingbacteria [27]

AdvantagesClinically relevant for HUS, the most common cause of AKI in childrenStx injection simple and inexpensiveThe dose of toxin and bacteria can be standardizedGenetically modified Stx-expressing bacteria allow reproduction of theinfectious agent-driven clinical situationAppropriate for small animals

DisadvantagesStx does not reproduce the presence of live bacteria occurring in the clinicalsituation

ObservationsCAST/Ei mice are more susceptible to thrombotic diseases than C57BL/6

Selected examples of genetically modified mice{

Ischemia-reperfusion in PKD KO mice [132] Allows study of mechanisms of cystogenesisSelective expression of human HB-EGF (thediphtheria toxin receptor, hDTR) to sensitize todiphtheria toxin [48,49]

Selective expression of HSV1-tk transgene tosensitize to ganciclovir [52]

Allows selective killing of cells expressing these proteins

Genetically labeled cells [52] Allows lineage tracingNgal reporter mice [55] Allows in vivo monitoring and localization of kidney injury

*A more comprehensive list is provided in Refs [11] and [12].zBoth toxicity and intratubular precipitation contribute to AKI.§Additional details in reference to these models are given in Ref. [11].{Experimental AKI has been performed in genetically modified mice in order to explore the role of specific molecules in AKI. Here, we reflect examples of the use

of genetically modified mice to answer more general questions about AKI, such as the role of specific cell types, the sensitization to a particular form of kidney

injury or cell tagging for better assessment of specific aspects of AKI.

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4.1 Novel rodent AKI modelsMouse and rat rodent models are by far the most used tostudy AKI pathological events, potential druggable targetsand select specific biomarkers of disease. Mice and rats arerelatively inexpensive to grow and maintain, they are fastbreeders and their genetics and physiology have been widelystudied and can be easily compared to humans. Indeed,mice, rats and humans share 99% of genes [13,14]. Rats arelarger than mice and this may be an advantage for techni-cally demanding models. However, mice constitute anextraordinary platform to get genetically engineered animalscarrying induced mutations and there are more gene tar-geted mice than rats. However, tools currently available tocreate genetically modified mice are not applicable to rats.Instead, genetically modified rats can be obtained frompoint mutations induced in master genes by treating maleswith usual chemical mutagens and further mating tountreated females, then followed by selection of disease phe-notypes of interest carrying a traceable mutation. In addi-tion, other genetic tools are currently being developed [15].Recently, a novel approach based on chromosome substitu-tion illustrates the way that genetic approaches in the ratmay help to identify protective genes against AKI. Genesexpressed in intrinsic AKI-resistant strains were transferredto other AKIs susceptible to create a consomic strain, thusconferring this later with the same natural component ofresistance [16].

Novel rodent AKI models have been developed in the pastdecade that address novel clinical entities or try to improvemodeling of clinical conditions. These add to already availableAKI models (Table 1). We will now briefly review some selectedexamples of experimental AKI induced by specific exogenoustoxins, thrombotic microangiopathy and warfarin-inducedhematuria that try to more closely reproduce relatively novelclinical situations (aristolochic acid or warfarin nephropathies)or clinical situations requiring improved modeling (thromboticmicroangiopathy and ischemia-reperfusion injury).

4.1.1 Exogenous toxinsAristolochic acid was long known to induce AKI in rats andmice [17]. However, it was not until the 1994 description ofChinese herbs nephropathy (hereafter named as aristolochicacid nephropathy to avoid referring in totality all Chinese herbsas causal of nephropathy), a rapidly progressive interstitial renalfibrosis initially reported in young women on a slimming regi-men including Chinese herbs, that interest in understanding thepathophysiogical mechanism of kidney injury boomed [18]. Ini-tially, rabbit and rat models focused on reproducing the humanCKD and helped to establish a link between this toxin and Bal-kan endemic nephropathy [19-21], but more recent mice modelshave been successfully used to identify new druggable targets inAKI and its transition to CKD, such as Jun N-terminal kinases(JNK) signaling role in G2/M arrest during AKI leading toresidual fibrosis [22].

Table 1. Traditional and novel AKI animal models (continued).

Model Characteristics

Whole mouse or cell specific KO oroverexpression of potential mediators of AKI

Allows characterization of specific molecular pathway or functionsAllows functional exploration of gene products in vivoAdvantagesThe main advantages have been specified for each model

DisadvantagesGenetically engineered mice are technically laborious to obtain and requirespecific knowledge on geneticsMay not be freely available

Non-rodentPigs and AKI involving surgical procedures,including kidney transplantation andcardiovascular surgery [83,133]

AdvantagesSize and physiology closer to humanMay one day be a source of xenografts

DisadvantagesBig size requires complex logistics and breeding facilities

Drosophila: malpighian tubules and adult kidneystem cells [92]

Zebrafish: gentamicin and cisplatinnephrotoxicity [91]

AdvantagesLow technical demands for breedingPossibility of high-throughput chemical or mutagenesis screensZebrafish kidneys contain self-renewing nephron stem/progenitor cells: studymolecular pathways for reactivation of this program in mammals [93]

DisadvantagesSimpler kidneys with less physiological similarity to humans

*A more comprehensive list is provided in Refs [11] and [12].zBoth toxicity and intratubular precipitation contribute to AKI.§Additional details in reference to these models are given in Ref. [11].{Experimental AKI has been performed in genetically modified mice in order to explore the role of specific molecules in AKI. Here, we reflect examples of the use

of genetically modified mice to answer more general questions about AKI, such as the role of specific cell types, the sensitization to a particular form of kidney

injury or cell tagging for better assessment of specific aspects of AKI.

Progress in the development of animal models of AKI and its impact on drug discovery

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Table

2.Someexamplesofmolecu

lartargets

exploredin

recentexperimentalAKImodels.

Candidate

target

Anim

almodel

Species

Targetingprocedure

Phenotypic

resp

onses

Renalfunction

Refs.

JNK,p53

UUO,IRI,aristolochic

acid

Mouse

PIF-a

(p53inhibitor)

SP600125(pan-JNKinhibitor)

#G2/M

arrest

#Fibrosis

Notassessed

[22]

CXCR4,CXCR7

Stx-m

ediated

Mouse

CXCR4antagonist

(AMD3100/plerixafor)

"Survival

Inpart

restoredBUN,sC

r[26]

Oat1

Mercury(II)chloride

Rat

Mouse

Oat1-KO

mice

#Histopathologicaldamage

NotchangeBUN

[36]

Smad3

IRI

Mouse

Smad-/-mice

#Histopathologicalinjury,renalIL-6

andendothelin,bloodIL-6

#BUN,sC

r[37]

MMP9

FA,IRI

Mouse

MPP9-/-mice

"Tubulardilationandapoptosis

Delayedrecovery

ofsC

r[38]

Dicer

IRI

Mouse

Dicer(flox/flox) X

CREY(PT-Dicer-/-)mice

#Tissuedamageandapoptosis

"Survival

#BUN,sC

r[39]

CCR1

IRI

Mouse

CCR1antagonism

(BX471)

CCR1-/-mice

#Leukocyte

infiltration#r

enal

CCL3,CCL5

NotchangesC

r[40]

Tweak

FAMouse

TweakKO

mice

#Tubularapoptosisandproliferation

#sCr

[41]

Fn14

IRI

Mouse

Fn14blockade(ITEM-2

blockingantibody)

#Inflammation

#Apoptosisandfibrosis

#BUN,sC

r[42]

ATF3

IRI

Mouse

ATF3-KO

mice

"Inflammation,apoptosis

"BUN,sC

r[43]

ATF3

genetransfer

#Apoptosis

"Survival

#BUN,sC

r

HO-1

Glycerol,cisplatin

Mouse

HO-1

transgenic

"Survival

#sCr

[44]

CSF-1

IRIDT-induced

Mouse

GW2580(c-fmsinhibitor)

Csf1-/-mice

Persistentrenalinjury

"BUN

[48]

TGFb

/Smadpathway

IRI

Mouse

THR-123(Alk3peptideagonist)

BMP7

#Tubulardamage

#Fibrosis

NochangeBUN

[66]

Dnmt1/Rasal1

pathway

FAMouse

5-Azacytidine

Dnmt1

+/-mice

#Progressionto

fibrosis

#sCr

[67]

Renalprogenitors

cells

(RMPs)

expressingCXCR7/CXCR4

Glycerol

Mouse

RMPswith/w

ithoutanti-CXCR4,

anti-CXCR7antibodies

CXCR4antagonist(AMD3100)

#Severity,fibrosis

(CXCR4orCXCR7antagonism

prevents

beneficialeffects)

Recovered

[110]

BUN:Bloodureanitrogen,FA

:Folic

acid,IRI:Ischemia-reperfusioninjury,sC

r:Serum

creatinine,UUO:Unilateralureteralobstruction.

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Maleate-induced proximal tubular cell toxicity and AKI inmice had received little attention because of a lack of a clinicalcorrelate. However, recent studies have ‘rediscovered’ thisform of renal injury as having similarities to metabolic eventsin ischemic/hypoxic tubular cell death, such as proximal tubu-lar cell specific coenzyme A, ATP and glutathione (GSH)depletion-simulating ischemia [23].

4.1.2 Thrombotic microangiopathyThere have also been advances in thrombotic microangiop-athy-induced AKI, as can be observed during human hemo-lytic uremic syndrome (HUS), the most common cause ofAKI in children. This is a rare disease in need of adequate ani-mal models and although great advances have been made inthe pathogenesis and treatment with eculizumab of atypical,genetic complement defect-related HUS [24], there is stillmuch confusion about therapy of typical HUS, as evidencedby the recent 2011 German outbreak where none of the tradi-tional forms of therapy (plasmapheresis and corticosteroids)proved beneficial and the long-discarded antibiotic therapyappeared to have a role [25]. An adequate choice of mousestrain and specific Shiga toxin (Stx) appears to be important.Shiga toxin 2 (Stx2) injection to CAST/Ei mice reproducedthe disease better than C57BL/6 mice, which are less suscep-tible to thrombotic diseases [26]. In CAST/Ei mice, a singlei.v. dose-dependent injection of Stx2 resulted in increasedmortality and, after 4 days, AKI with severe defects in corticalperfusion of the functional vasculature and increased urinealbumin/creatinine ratios [26]. Stx2 is the isoform associatedwith the most severe forms of human disease. The 2011German outbreak was caused by Stx2-producing Escherichiacoli. However, toxin injection does not reproduce the infec-tious nature of typical human HUS, usually the result ofenteric infection by toxin-expressing enterohemorrhagicE. coli (EHEC). However, EHEC infection in conventionalmice does not manifest key features of the disease, such asattaching and effacing lesions, intestinal damage and systemicillness. Genetic modification of the murine pathogen Citro-bacter rodentium has been used to express Stx-reproducedintestinal epithelium and Stx-dependent intestinal inflamma-tory damage and kidney damage [27]. Additional modelsinclude murine AKI induced by infusion of concanavalin Aand anti-concanavalin A antibodies [28] and lipopolysaccha-ride (LPS) plus Stx administration [29]. A transgenic murinemodel of atypical HUS is described in the next section.

4.1.3 Warfarin nephropathyA new rat model of warfarin-induced hematuric AKI couldonly be reproduced in five of six nephrectomized animalswith prior CKD [30]. This model provided a biological basisfor the recently described warfarin nephropathy syndrome ofAKI in humans overcoagulated with oral agents and will allowthe development of therapeutic approaches [31]. In this regard,hematuria results in kidney recruitment of macrophagesexpressing the CD163 hemoglobin scavenger receptor, and

hemoglobin toxicity is probably the key element in macro-scopic hematuria-associated AKI in IgA nephropathy [32-35].The need for prior five of six nephrectomies makes thebigger-sized rat a better animal model than the mouse andunderscores the clinical observation that prior CKD is a riskfactor for this form of AKI [31].

In summary, the AKI researcher is presented with anexpanding choice of potential models. Potential risk factors(e.g., diabetes and aging) in the targeted clinical entity aswell as method for inducing AKI (toxin, drug, route, dosing,animal strain and genomic background) are all aspects to beconsidered when choosing a model to identify and test drugcandidates.

4.2 Genetically modified miceMice are well suited for genetic modification involving tar-geted knockout (KO) or knock-in genes or able to condition-ally express proteins of interest. A growing body of AKIresearch is carried out in genetically modified mice, exploitingsome of these possibilities. Genetically modified mice havebeen used to perform functional studies of the role of specificgenes in AKI, to induce targeted injury of specific tubular seg-ments, for lineage tracing and for imaging of injured tubularsegments.

4.2.1 Functional studies of the role of specific genesGenetically modified mice have been used to address the func-tional role of specific AKI mediators (Table 2). Approximately15% of the mouse genome genes have been successfully targetedby means of genetic engineering tools. As the number of poten-tial AKI mediators amply exceeds those studied in geneticallymodified mice, further use of this technology in drug discoveryis expected. Already studied KO mice include, but are notlimited to, renal transporters (Oat1/Slc22a6), molecular effec-tors of relevant intracellular pathways (Smad3), metalloprotei-nases (MMP9), RNA regulators (Dicer), cytokines orreceptors (CCR1, TWEAK and Fn14) and transcription factors(ATF3) [36-43]. KO mice have been subjected to different mod-els of AKI to evaluate modification of the injury as a result ofthe gene manipulation and, thus, the role of the targeted genein AKI. These modifiers were manipulated, for example, inmercury- [36], folic acid- [38,41] and ischemia-induced AKI back-grounds [37,39,40,43]. Humanized transgenic mice were used toevaluate beneficial HO-1 induction during AKI [44]. TransgenicCfh-/-.FHD16-20 mice express a genetically modified comple-ment H protein that mimics the human mutations reportedin atypical HUS [45]. These animals develop spontaneous micro-angiopathy and AKI, thus providing a model to investigate therole of C5 complement factor in atypical HUS.

4.2.2 Targeted injury of specific tubular segmentsThe kidney is a complex organ composed of several epitheliaand other metabolically active cell types that interact withone another. Tubular epithelial cells are considered key targetsin AKI and may undergo death, dedifferentiaton and

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activation [9]. However, most classic models of AKI are notselective and promote diffuse tubular cell injury, frequentlyin several tubular types, in addition to vascular and glomeru-lar injury; the relative role of tubular cell toxicity should beclarified. Proximal tubules are the main targets of injury forseveral nephrotoxins, including tenofovir and cidofovir thataccumulate into these cells because of their particular set oftransporters [46,47]. These transporters may be used in target-ing therapeutic agents to proximal tubules as a strategy totreat AKI.

4.2.2.1 Targeted injury of proximal tubulesSeveral genetically modified systems have been used foraddressing the specific role of proximal tubules in AKI. Trans-genic mice are used to induce selective tubular lesions and tospecifically study how injury primarily affects specific tubularcells and how this lesion expands to other cell types. Takingadvantage of the fact that rodents are 103 -- 106 times moreresistant to diphtheria toxin than other mammals, becausethe toxin does not bind to their heparin-binding epidermalgrowth factor (HB-EGF), selective expression of humanHB-EGF (the diphtheria toxin receptor, hDTR) in cells ofinterest allows induction of targeted injury. Genetically mod-ified mice that express hDTR under diverse promoters allowsensitization of proximal tubular cells, S3 segment proximaltubular cells or kidney epithelial cells to diphtheria toxinallowing selective killing of these cells upon diphtheria toxinadministration [48-50]. In mice expressing hDTR in proximaltubules, diphtheria toxin-induced AKI was characterized bymarked renal proximal tubular cell apoptosis with tubule dila-tion, loss of brush border, sloughing of individual epithelialcells and distal cast formation. The rate of increase in bloodurea nitrogen (BUN) and creatinine was slower than that ofischemia-reperfusion: it was observed at day 2 and peaked at5 -- 6 days. Apoptosis and regeneration were observed within2 days. Apoptosis had decreased by 5 days and evidence ofregeneration peaked at 8 days and decreased by 12 days.Complete recovery of proximal tubule function took longerand albuminuria persisted for up to 4 weeks [48].The S3 segment of the proximal tubule was selectively

targeted in transgenic mice expressing hDTR under the tran-scriptional control of the S3-specific promoter Gls5(Gsl5-hDTR) [49]. Systemic diphtheria toxin resulted in adose-dependent AKI characterized by injury mainly to theS3 segment but also to S1 and S2 segments, AQP7 expressionloss and distal cast formation.

4.2.2.2 Simultaneous targeted injury of different tubular

segmentsAnother model to selectively target tubular cells wasobtained by Cre-LoxP recombinant technology [50]. Cre-LoxP bigenic mice (Six2-GFPCre+-LoxP,iDTR+/DTRrec)expressing the diphtheria toxin receptor (iDTR+) inmetanephric-derived cells (Six2+), including proximal anddistal tubules, podocytes and the loop of Henle, suffer AKI

with diffuse tubular injury but recover completely followinga single challenge with diphtheria toxin. However, repeateddoses resulted in maladaptive repair with interstitial capillaryloss, fibrosis, and glomerulosclerosis, showing that selectiveepithelial injury can drive CKD. This may be especially rel-evant to the clinical situation in which repeated tubularinsults may occur in the course of hospitalization. Condi-tional gene expression systems, such as the tetracycline-dependent (Tet) on system, have also been employed to tar-get tubular epithelia. The Pax8/rtTA transgenic mouse is amodel of conditional gene expression created to study renaldamage that allow targeting genes to all proximal and distaltubules and the entire collecting duct system of both em-bryonic and adult kidneys [51]. On the basis of the exclusiverenal Pax8 promoter stimulation, tubular epithelial cellsexpress the reverse tetracycline-dependent transactivator(rtTA). Mice of this genetic background were crossed withTetO-X transgenic to generate double transgenicPax8-rtTA/TetO-X, where X represents different target pro-teins of interest, the expression of which is activated undertetracycline dosage. This mouse was used to model renalcancer, cystic and fibrotic lesions and it is potentially usefulin testing molecules mediators that could play a role in AKI.

4.2.2.3 Targeted injury of the thick ascending limb of the

loop of HenleThe contribution of the thick ascending limb to AKI wasinvestigated in transgenic mice expressing a herpes simplexvirus I thymidine kinase gene (HSV1-tk) commanded bythe Tamm--Horsfall promoter THP (THP-HSV1-tk), a spe-cific protein synthesized in this nephron portion [52]. Micecarrying the THP-HSV1-tk transgene are susceptible to gan-ciclovir apoptotic action whereby they showed in thickascending limbs, apoptosis, flattening of cells, dilation oftubular lumen along with cast formation, decreased renalfunction established by serum urea and creatinine rise andloss of ability to concentrate urine, all of which defined theAKI occurrence. Conditional tubular expression or deletionmay be obtained. As an example, mice with specific tubulardeletion of the focal adhesion kinase were designed to studycontributions of this signaling pathway in mediating tubulardamage in AKI [53].

4.2.3 Lineage tracingGenetically modified mice can also be used to trace cell line-age in the course of AKI or to image tubular cell stress.Genetic labeling and fate mapping of renal epithelial cells inAKI models evolving to chronicity and kidney fibrosis, suchas ischemia-reperfusion or unilateral ureteral obstruction(UUO), by Cre/Lox techniques showed no direct transitionof epithelial cells to fibroblasts [54].

4.2.4 Imaging of injured tubular segmentsNgal is a biomarker of AKI that may be assessed in humanurine. Ngal reporter mice allowed illumination of injured

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tubules in vivo in real time. Specific cells of the distal nephronwere the source of Ngal. This system allowed characterizingthe action of injurious and therapeutic drugs [55].

4.3 Use of AKI models to better understand the AKI

to CKD transitionThere is a growing concern about long-term consequences ofAKI that clinically may result in CKD evolving to end-stage renal disease (ESRD) [56-58]. Animal models haveallowed the exploration of the impact of AKI on the develop-ment and progression of CKD [59-62]. Modifications of thetraditional ischemia-reperfusion, folic acid or aristolochicacid-induced AKI models are commonly used to explore thetransition to chronicity [22,63-65]. UUO induces AKI, althoughit is not classically considered as a model of AKI, because earlycompensatory mechanisms of the non-ligated kidney maskloss of function of the obstructed kidney. This precludesassessment of the impact of therapy in renal function. How-ever, the extended early damage produced in the course ofan UUO is found especially useful in investigating the transi-tion to chronicity [21,66]. Fibrosis can also occur when renalinjury is mild and renal function loss is not evident [65].Depending on the dose of a unique insult or on the repetitivenature of the insult, ischemia-reperfusion, folic acid or aristo-lochic acid-induced AKI may lead to either recovery of kidneyfunction preceded by a rapid regenerative stage after the acuteinsult or chronic inflammation and fibrosis. Unraveling thecellular and molecular mechanisms may be useful in prevent-ing both the AKI to CKD transition and progression of CKDitself. A single dose of folic acid in mice resulted in fibrosisthat increased from 7 to 147 days [67]. Repeated diphtheriatoxin administration to genetically modified mice expressingthe diphtheria toxin receptor in proximal tubules also resultedin tubulointerstitial scarring associated with glomerulosclero-sis and areas of inflammatory infiltrates, 5 weeks afterAKI [50].

4.4 Novel rodent models that reproduce special

high-risk situations for AKI initiation and

progressionAKI incidence, severity and progression to CKD are more fre-quent in patients with diabetes and in the elderly [68-70]. Thus,several models of AKI have been developed in mice or ratsthat are either diabetic or aged in order to better understandthe molecular mechanisms of this predisposition and whetherthese patients require specific therapeutic approaches. Sus-ceptibility to AKI was observed in streptozotocin-induceddiabetic rats and mice [71] and septic aged mice [72]. High glu-cose and glucose degradation products promote tubular cellapoptosis and inflammation and thus may contribute to thehigher incidence of AKI in diabetics [73-76]. In mice, the con-tribution of aging-related cumulative telomere shortening tosusceptibility to apoptosis and reduced proliferative kidneyresponses to extrinsic stress have also been observed in

telomerase-deficient mice subjected to ischemic AKI. Thesemice showed increased histopathological damage and reducedproliferation of tubular, glomerular and interstitial cells com-pared to mice with mild or no telomere deficiency [77]. A roleof the antiproliferative Zag protein in preventing regenerationafter ischemia/reperfusion-induced AKI in aged mice was alsoestablished [78].

4.5 Non-rodent AKI modelsAlthough rodent models have constituted a breakthrough inunderstanding the basic processes characterizing importantgenetic as well as acquired kidney diseases, the results obtainedwith them are not exempt from misinterpretation. This is espe-cially true for conclusions from preclinical therapies that ulti-mately failed in clinical trials. Non-rodent animal models ofAKI are available and involve both larger animals that presentsome technical and physiological advantages and smaller ani-mals that better suit for conducting high-throughput chemicalor mutagenesis screens as recently reviewed [79]. Large animals,such as pigs, have similarities in size, physiology and anatomywith humans and may offer advantages to develop and validatetherapeutic approaches and transfer them into clinical trials.The growing knowledge about pig genetics has also allowedthe development of genetically modified pigs with potentialuse in research and clinical application [80]. However, applica-tion of genetically modified pig models to study AKI is stillnascent. As an example, the design of strategies blocking renalgenes identified as responsible for rejecting or inducing theexpression of other protective may potentially be useful inavoiding graft AKI in renal xenotransplants [81]. Transgenicpigs expressing the human protective HO-1 protein that mayhelp withstand ischemic insults were recently reported [82].Pigs are well suited to explore ischemia-reperfusion-inducedAKI in the context of major surgery such as renal transplanta-tion and cardiovascular surgery involving cardiopulmonarybypass [83]. Swine have also been used as a surrogate model toreproduce ischemia through maneuvers different from cardio-pulmonary bypass [84,85]. Swine is also suitable to reproduceseptic AKI and is proposed as having advantages over otheranimal models to test preclinical therapies. Production of mul-tiorgan failure with kidney compromise is not always observedin septic AKI models. A pig model combining surgical proce-dures to produce sepsis and gut ischemia/reperfusion was suc-cessful in achieving both systemic inflammation anddysfunction of the major organ systems that is typically seenin human sepsis [86]. Another model of septic AKI was alsosuitable to reproduce and study the impact of the decreasedrenal blood flow and inflammation, which have been sug-gested as mechanisms of kidney dysfunction [87]. Pig AKImodels may be used to search for molecular mediators of dis-tinctive pathological processes of AKI, such as tubular apopto-sis, as well as to study early damage biomarkers and theregeneration process accompanying recovery from AKI [88-90].

Smaller animals have also been used to model kidney dis-ease, including the worm (Caenorhabditis elegans), the fruit

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fly (Drosophila melanogaster), the zebrafish (Danio rerio) andthe little skate (Leucoraja erinacea) [91]. In particular, malpigh-ian tubules of Drosophila are used to learn about adult kidneystem cells [92]. Gentamicin and cisplatin nephrotoxicity andAKI have been reproduced in larval zebrafish and have beenshown to respond to therapeutic intervention with a specificinhibitor of the apoptosis-related protease Omi/HtrA2 [91].Contrary to mammals, zebrafish add nephrons throughouttheir lifespan and regenerate nephrons de novo after injury,providing a model for understanding how mammalian renalregeneration may be therapeutically activated. Transplanta-tion of single aggregates comprising 10 -- 30 cells is sufficientto engraft adults and generate multiple nephrons, demonstrat-ing the presence of self-renewing nephron stem/progenitorcells in zebrafish kidneys [93].

5. New potential therapeutic approaches toAKI characterized in experimental models

The review of all potential therapeutic targets identified inrecent years in animal models of AKI is beyond the scope ofthis review. In our opinion the greatest advances are relatedto the modes of tubular cell death, the role of TWEAK inkidney inflammation and tubular cell death, the transitionto fibrosis and the recruitment and role of macrophages andmarrow cells.Tubular cell death is a key feature of AKI. Both apoptosis

and necrosis contribute to tubular cell injury and the relativecontribution may depend on the severity of injury and thespecific trigger [7,9]. The role of apoptosis has been extensivelystudied but has not yet resulted in any medication in clinicaluse. By contrast, necrosis was neglected because it was thoughtto be a passive process not susceptible to therapeutic manipu-lation. This view has changed in recent years and there is hopethat a particular form of regulated necrosis, necroptosis,

which is controlled by the kinases RIPK1 and RIPK3, mayprovide novel therapeutic tools [94]. Necroptosis may beinhibited by necrostatin-1, a highly specific inhibitor ofRIPK1. Necrostatin-1 reduced kidney damage and renal fail-ure, even when administered after reperfusion, resulting in asignificant survival benefit in a model of lethal renal ische-mia/reperfusion injury in which the pan-caspase inhibitorzVAD, an inhibitor of apoptosis, was not protective [95].

Two novel therapeutic targets encompass the cell death--inflammation interface. A polymeric nanomedicine, whichwas previously shown to inhibit cell death induced by theproapoptotic molecule Apaf-1, was recently shown to preventtubular cell death and inflammatory responses both in cultureand during folic acid-induced AKI in mice [96,97]. TWEAK isa TNF superfamily cytokine that promotes tubular cellinflammatory responses and tubular cell death through activa-tion of the Fn14 receptor [8,98]. TWEAK or Fn14 targeting byantibodies or in KO mice prevents folic acid and ischemia-reperfusion-induced AKI, tubular cell apoptosis andinflammation [8,41,42,99-101]. Interestingly TWEAK targetingpreserved the kidney expression of Klotho during AKI [102].Klotho behaves both as an FGF23 receptor and a soluble hor-mone with anti-aging, anti-inflammatory and anti-fibroticproperties [103,104].

Fibrosis is a general feature of CKD and a determinant ofprogression to ESRD [105]. The repair process after AKI canbe incomplete, with persisting tubulointerstitial inflammationand fibrosis characterized by proliferation of fibroblasts andexcessive deposition of extracellular matrix [106]. The molecu-lar mechanisms that mediate renal fibrosis after AKI are notwell understood. AKI animal models have been used to dissectthe molecular mechanisms and find new therapeutic tar-get [107]. Recent studies have shown that fibrosis is promotedby tubular cell G2/M cell cycle arrest during AKI or even dur-ing milder renal injury not leading to serum creatinine

Table 3. Molecular and biological pathways targeted in recent or ongoing AKI clinical trials.

Candidate target Model Species Targeting procedure Phenotypic responses Renal

function

Clinical

trial

Refs.

Multipotent MSCs IRI Rat AC607 # Tubular apoptosisand inflammation

Improved Ongoing [124]

Oxidative stress IRI Rat Administration of N-AC(inhibition of endothelialnitric oxide synthase)

# Infiltrating leukocytes Non-significanttrend towardsimprovement

Completedand ongoing

[134]

Apoptosis IRI Rat Administration of EPO # Tubular damage,apoptosis and NF-kBimmunostaining

Improved Completedand ongoing

[135]

Vessel vasoconstriction IRI Rat Administration of ANP # Acute tubular necrosis Improved Completed [136]

Dopamine (D-1)receptor

IRI Rat Administration offenoldopam, D-1 agonist

# Activation of NF-kB Not assessed Completedand ongoing

[137]

Dopamine (D-1)receptor

IRI Pig Administration offenoldopam, D-1 agonist

# Acute tubular necrosis Improved Completedand ongoing

[138]

p53 IRI andcisplatin

Rat I5NP: p53 siRNA # Tubular apoptosis Improved Ongoing [139]

IRI: Ischemia-reperfusion injury.

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elevations [22]. G2/M-arrested proximal tubular cells activateJNK signaling, leading to the release of profibrotic cytokines,such as TGF-b1 and CTGF, which in turn stimulated prolif-eration and collagen synthesis by fibroblasts. These studiesidentified two potential druggable targets: JNK inhibition orbypassing the G2/M arrest by administration of a p53inhibitor prevented fibrosis. The process was observed afterkidney injury induced by severe or unilateral ischemia/re-per-fusion injury, aristolochic acid nephropathy and UUO [22].Indeed, p53 targeting is already undergoing clinical trials inAKI (see below) and the oral JNK inhibitor is undergoingclinical trials for inflammatory endometriosis (clinicaltrials.gov/ct2/show/NCT01630252). Demonstration of safetymay facilitate its assessment in AKI o renal fibrosis.

UUO and ischemia-reperfusion AKI illustrated the poten-tial therapeutic interest of THR-123 to prevent progressionto fibrosis. THR-123 is a peptide agonist of the activin-like kinase 3 (Alk3) receptor. BMP7 antagonizes the TGFb/Smad pathway by binding to Alk3 [66,108]. THR-123 repro-duced BMP7 actions and decreased tubular damage andfibrosis, but did not prevent the increase in BUN followingischemia-reperfusion [66].

HIPK2 is a protein kinase identified as a kidney fibrosispromoter by a systems biology approach in the fibrotic kid-neys of Tg26 mice transgenic for HIV [109]. The role ofHIPK2 in kidney fibrosis was validated in murine UUOand folic acid-induced renal fibrosis. Absence of HIPK2 pre-vented both fibrosis and loss of renal function in these mod-els [109]. As other kinases, HIPK2 is a therapeutic targetpotentially druggable by oral small molecules.

The contribution of methylation epigenetic modificationsto kidney fibrosis was established in kidney injury inducedby a single dose of folic acid [67]. Fibrosis was reduced in het-erozygous mice by the expression of the methyltransferaseDnmt, suggesting that this enzyme is a potential therapeutictarget. Cell culture indicated that methylation of the Rasregulatory molecule Rasal1 played a key role.

Several studies have addressed the role of CXCR4 andCXCR7, the two receptors for the chemokine stromal-derivedfactor-1 (SDF-1), in AKI. In SCID mice, rhabdomyolysis-induced AKI following glycerol injection CXCR4 andCXCR7 played an essential role in the therapeutic homingof human renal progenitor cells, tissue regeneration and renalfunction improvement [110]. This has important implicationsfor the development of stem cell-based therapies. A note ofcaution is needed since in a model of Stx2-induced AKI inCAST/Ei mice, inhibition of the CXCR4--SDF-1 interactiondecreased endothelial activation and organ injury andimproved animal survival [26]. Thus, CXCR4/SDF-1 targetingprotects from experimental HUS-induced AKI but mayimpair homing of renal progenitor cells.

Marrow cells also contribute to AKI recovery. A role of res-ident renal macrophages and dendritic cells in AKI recoverywas observed after ischemia/reperfusion injury or followingdiphtheria toxin selective injury to proximal tubules [48].

Macrophage/dendritic cell depletion during the recoveryphase increased functional and histological injury and delayedregeneration. Genetic or pharmacological inhibition of mac-rophage colony-stimulating factor (CSF-1) signaling blockedmacrophage/dendritic cell proliferation, decreased M2 polari-zation and inhibited recovery. These findings demonstratedthat CSF-1-mediated expansion and polarization of residentrenal macrophages/dendritic cells is an important mechanismmediating renal tubule epithelial regeneration after AKI [48].

Multiple myeloma is a plasma cell dyscrasia in which over-production of light chains may result in AKI and CKD causeda light chain precipitation in association with Tamm--Horsfallprotein into the tubular lumen (cast nephropathy). The mainprognostic factor for cast nephropathy is the response of thetumor to chemotherapy. An unmet medical need is the treat-ment of non-responsive patients. Cast nephropathy and AKIwere reproduced in rats 72 h after a single intraperitonealinjection of monoclonal light chains (k3 or l2) obtainedfrom myeloma patients with cast nephropathy [111]. Thismodel was used to illustrate protection from casts and AKIprovided by a cyclized competitor peptide that inhibited thebinding of light chains to Tamm--Horsfall protein, thusproviding the first therapy for cast nephropathy that isindependent from the tumor response to treatment [112].

6. Clinical therapies for AKI treatment

Current clinical strategies for AKI prevention are limited torestriction of nephrotoxic agents and avoiding hypoperfusionand volume depletion. However, there are no established ther-apies for AKI beyond symptomatic management. The currentstatus of clinical trials in AKI has been recently reviewed [113].As of November 2011, there were 126 trials registered onclinicalTrials.org studying AKI: 75 addressed prevention ofAKI and 51 addressed management of established AKI. Themajority of the prevention trials are conducted for cardiacsurgery-associated AKI and contrast-induced AKI [113-115].N-acetylcysteine (NAC), erythropoietin (EPO), natriureticpeptides, fenoldopam, I5NP and AC607 are some of theagents undergoing clinical testing in AKI based on resultsfrom experimental animals (Table 3). Neutralizing anti-TWEAK antibodies are not undergoing clinical trials inAKI, but there is a first human clinical trial addressing itsnephroprotective action in lupus nephritis, and if this trialsucceeds, we might expect trials in AKI to follow.

NAC has been studied for AKI prevention, since 2000.However, results have been mixed [116]. The PRESERVEstudy (Prevention of Serious Adverse Events FollowingAngiography) is enrolling 8,680 patients to settle theissue [117].

EPO is an erythropoietic hormone produced by the kidney.Accumulating evidence suggests that EPO has additionalorgan protective effects [118]. A randomized, pilot study ofpreoperative EPO in 71 patients who underwent elective cor-onary artery bypass graft surgery reported renoprotective

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effects [119]. In a different setting, randomization of 162 ICUpatients at risk of AKI based on urinary biomarkers (urinaryGGT and alkaline phosphatase) to placebo or two doses ofEPO did not result in differences in the incidence ofAKI [118]. There are currently three randomized controlledtrials using preoperative EPO.Although there are currently no clinical trials testing car-

peritide (atrial natriuretic peptide) and nesiritide (brain natri-uretic peptide), they are worth mentioning as examples ofpromising preclinical data not mirrored in the clinical setting.A recent meta-analysis comprising data from 15 randomized,controlled trials in cardiovascular surgery patients reported anincreased urine output and a reduction in serum creatininelevels compared with controls. However, the effect isdescribed in the immediate perioperative period and noconclusions can be drawn on outcomes [120].Fenoldopam is a dopaminergic DA1 agonist that selectively

increases renal medullary blood flow and natriuresis and hasbeen tested in clinical trials as prophylaxis for septic, postop-erative and contrast-induced AKI with no clear benefits [121].Fenoldopam is currently being tested as a prophylactic agentfor AKI in cardiopulmonary bypass surgery.I5NP is a synthetic RNAi that temporarily inhibits the

expression of p53. I5NP is being developed to protect cellsfrom acute ischemia/reperfusion injuries and AKI followingmajor cardiac surgery and renal transplantation [122]. Thep53 is a proapoptotic protein that additionally promotes G2/M cell cycle arrest and kidney fibrosis in murine AKI mod-els [7,22]. I5NP was tested in a Phase I clinical trial in 16 patientsat risk of AKI who had undergone cardiovascular surgery. In aPhase II trial, I5NP is being tested in patients receivingdeceased donor kidney transplantation with > 24 h of coldischemia time, to evaluate efficacy in preventing delayed graftfunction. However, a dose escalation and safety study ofI5NP to prevent AKI in high-risk major cardiovascular surgerypatients was terminated [123].AC607 is composed of human bone marrow-derived

mesenchymal stem cells (MSCs). In preclinical research, itdecreased inflammation and cell death in anesthetized ratswith ischemia-reperfusion-induced AKI [124]. An ongoingclinical trial is testing effects of AC607 in AKI patients within48 h of cardiac surgery. Once enrolled, subjects will receive asingle administration of AC607 or placebo. Kidney recoverywill be evaluated over the subsequent 30 days and death orthe need for dialysis will be evaluated within 90 days of dos-ing. After 90 days (evaluation period), subjects will enter a3-year extension phase of the study to monitor safety andlong-term outcomes (follow-up period) [125].

7. Conclusions

In conclusion, while the yield to date of animal models hasbeen poor as assessed by the number of drugs approved forthe treatment of human AKI, there is a new crop of optimizedmodels and it is conceivable that further refinement of animal

models will eventually result in effective therapies for clinicalAKI. It should be kept in mind, however, that these modelsshould be used to test the therapeutic rather than prophylacticuse of agents, given that in the clinic, many patients presentwith already established AKI. In addition, advances in bio-marker science should be incorporated to adequately stageAKI and individualize therapy according to AKI stage.

8. Expert opinion

The number of experimental AKI models has increased inrecent years trying to address concerns resulting from thepoor performance of prior therapeutic targets identified in con-ventional AKI models when translated to clinical practice andalso trying to keep pace with newly described entities. In addi-tion, the number of species used for AKI models has expandedto include non-rodents. Non-rodents allow the evaluation ofspecific aspects of AKI in bigger animals, such as complex sur-gical interventions and xenograft protection from AKI in pigsand large-scale genetic screenings in simpler organisms suchas drosophila and zebrafish. New rodent models have repro-duced recently described clinical entities, such as aristolochicand warfarin nephropathies, have provided better models forold entities such as thrombotic microangiopathy-induced AKIor cast nephropathy, have optimized models to explore factorsthat predispose AKI in clinical practice such as prior CKD,old age or diabetes and have modified the model to answer spe-cific questions such as the factors driving the AKI to CKD tran-sition. Furthermore, genetically modified animals have beendesigned to explore functional contributors to AKI, to help tar-get specific cell types and to track the contribution of specificcell types. A combination of traditional and new models hasallowed the identification of novel druggable targets that mod-ulate the development of AKI or try to correct the failure torecover from injury. These new targets include, but are not lim-ited to, modulators of cell death, such as RIP1/RIP3 regulatingnecroptosis, inflammatory cytokines, such as TWEAK, media-tors of the AKI to CKD transition, such as JNK, Alk3,HIPK2 and Dnmt and regulators of bone marrow cells andstem cell biology, such as CXCR4/CXCR7/SDF-1 andCSF-1. Agents targeting some of these molecules are alreadyundergoing clinical trials. Thus, the deep understanding ofthe role of TWEAK/Fn14 in kidney disease justifies the factthat the first clinical trial addressing the efficacy of neutralizinganti-TWEAK antibodies in humans studies nephroprotectionin lupus nephritis. An eventual success of this trial may openthe doors for studies in AKI. JNK, Alk3, HIPK2 and Dnmtrepresent receptors and enzymes which can be inhibited bysmall molecules, some of which, such as JNK inhibitors, areundergoing clinical trials for inflammatory diseases [126]. Atleast 17 trials are studying or have studied CXCR4 inhibitorsor antagonists for diverse clinical indications [127]. Furthermore,several therapies identified in AKI animal models are undergo-ing clinical trials in human AKI, including p53 RNAi andbone-marrow derived MSCs.

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Acknowledgements

The authors have received support from the Instituto de SaludCarlos III through the grants FIS PS09/00447, ISCIII-RETIC, REDinREN/RD06/0016. Furthermore, they havereceived support via the European Regional DevelopmentFund (FEDER), RD12/0021 and the Comunidad deMadrid/CIFRA S2010/BMD-2378. The authors have alsoreceived support via the Instituto de Salud Carlos III in theform of: MD Sanchez-Nino has received a FIS-Sara Borrell

post-doctoral fellowship; AM Ramos and A Sanz have receiveda post-doctoral Miguel Servet fellowship, while A Ortiz hasreceived salary support through the Programa IntensificacionActividad Investigadora (ISCIII/Agencia Laın-Entralgo/CM).Finally C Martin-Cleary is the recipient of a ‘Rio Hortega’fellowship grant from the Instituto de Salud Carlos III.

Declaration of Interest

The authors have no conflict of Interest

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AffiliationAna B Sanz1,2 PhD,

Marıa Dolores Sanchez-Nino3 PhD,

Catalina Martın-Cleary1,2 MD,

Alberto Ortiz†1,2,4 MD PhD &

Adrian M Ramos1,2 PhD†Author for correspondence1Renal and Vascular Pathology Laboratory,

Instituto de Investigacion Sanitaria-Fundacio

Jimenez Dıaz/Universidad Autonoma de Madrid

(IIS-FJD-UAM), Madrid, Spain2Red de Investigacion Renal (REDINREN),

Madrid, Spain3IDIPAZ, Madrid, Spain4Fundacion Renal Inigo Alvarez de Toledo

(FRIAT), C/Jose Abascal, 42,

28003, Madrid, Spain

E-mail: aortiz@fjd.es

Progress in the development of animal models of AKI and its impact on drug discovery

Expert Opin. Drug Discov. [Early Online] 17

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