identification of promising urinary microrna biomarkers in two rat

Identification of Promising Urinary MicroRNA

Biomarkers in Two Rat Models of Glomerular InjuryRounak Nassirpour1 Bruce L Homer Sachin Mathurdagger Yizheng Lidagger

Zhonghan Li Tom BrownDagger Deborah Carraher James WarnekeSteven Bailey Karen Percival Shawn P OrsquoNeil and Laurence O Whiteley

Drug Safety Pfizer Worldwide Research and Development daggerBusiness Technology Pfizer Research andDevelopment Andover Massachusetts 01810 and DaggerDrug Safety Pfizer Research and Development GrotonConnecticut 063401To whom correspondence should be addressed at Business Technology Pfizer Research and Development 1 Burtt Rd Andover MA 01810Fax thorn18454744065 E-mail rounaknassirpourpfizercom

ABSTRACT

MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein levels posttranscriptionally miRNAs play importantregulatory roles in many cellular processes and have been implicated in several diseases Recent studies have reportedsignificant levels of miRNAs in a variety of body fluids raising the possibility that miRNAs could serve as useful biomarkersHere changes in miRNA expression patterns are described in 2 different rodent models of glomerular injury (acutepuromycin aminonucleoside nephropathy and passive Heymann nephritis) By employing 2 different modes of glomerularinsult oxidative stress and immune-mediated toxicity miRNA changes in both isolated glomeruli as well as urinespecimens allow for identification of urinary miRNA biomarkers that are suggestive of drug-induced injury specifically tothe glomerulus Subsets of glomerular urinary miRNAs associated with these different modes of glomerular toxicity seem tobe dependent on the mechanism of the induced injury while 9 miRNAs that changed early in both glomerular and urinespecimens were common to both studies We further show that the miRNAs identified as mechanism-specific earlyglomerular injury biomarkers target key pathways and transcripts relevant to the type of insult while the insult-independent changes might serve as ideal glomerular injury biomarkers

Key words microRNA puromycin Heymann nephritis glomerular injury biomarker urine

Despite potentially beneficial therapeutic effects a growingnumber of molecules fail in early nonclinical development dueto drug-induced renal toxicity Although there are a number ofbiomarkers available for detection of renal tubular injury glo-merular injury often goes undetected (ANON 2010) The currentdiagnostic gold standard is histological evaluation of renal bi-opsy specimens Biomarkers such as serum creatinine (Cr) esti-mated glomerular filtration rate andor urinary protein areroutinely used to monitor renal function (Bonventre et al 2010Dieterle et al 2010) However by the time urinary albumin ortotal protein are elevated glomerular damage may already beirreversible or unresponsive to therapy Thus sensitivity andspecificity of currently available biomarkers of glomerulopathy

are mostly considered inadequate for nonclinical screening orclinical monitoring of glomerular injury Therefore sensitiveaccurate and early biomarkers of glomerular injury are greatlyneeded

There is a growing interest in the role of microRNAs(miRNAs) in the pathogenesis of renal diseases (Li et al 2010)and investigations into the role miRNAs may serve as potentialbiomarkers of glomerulopathy have increased miRNAs arehighly conserved endogenous small (19ndash25 nucleotides)noncoding RNAs that play a primary role in posttranscriptionalsilencing (Khella et al 2013) Due to their stability miRNAs arereadily quantifiable in serum plasma urine and other body flu-ids (Scian et al 2013) Specific miRNAs identified to date as

VC The Author 2015 Published by Oxford University Press on behalf of the Society of ToxicologyAll rights reserved For Permissions please e-mail journalspermissionsoupcom

35

TOXICOLOGICAL SCIENCES 148(1) 2015 35ndash47

doi 101093toxscikfv167Advance Access Publication Date August 7 2015Research Article

Downloaded from httpsacademicoupcomtoxsciarticle-abstract1481351659715by gueston 14 April 2018

biomarkers are able to distinguish tissue of origin with in-creased diagnostic accuracy (De Guire et al 2013) miRNA ex-pression has been shown to be distinct among differentanatomical regions of the kidney and alterations in miRNA ex-pression have been associated with a variety of kidney diseasesincluding diabetic nephritis hypertension glomerulonephritisand cancer (Alvarez and DiStefano 2013 Khella et al 2013Scian et al 2013 Wang et al 2009) A critical role of miRNA reg-ulation in the progression of glomerular damage and the devel-opment of proteinuria has been suggested by studies in micewith podocyte-specific deletion of Dicer (Harvey et al 2008 Hoet al 2008 Ho and Marsden 2008 Shi 2008) and more recentlyDrosha (Zhdanova et al 2011) critical enzymes involved inmiRNA biogenesis

In order to assess the utility of miRNA measurements inurine for detection of site-specific renal toxicity the Health andEnvironmental Sciences Institute (HESI) Biomarkers ofNephrotoxicity Committee is conducting a collaborative pro-gram using toxicants or models that are selective for specificnephron segment injury This study describes the effects on uri-nary induction of miRNAs in 2 different rat models of glomeru-lar injury The objective of the rodent glomerular injury studiesreported here was to characterize the miRNA expression andexcretion profile following the induction of glomerular injuryAccordingly we performed global miRNA expression profilingfrom isolated glomeruli and urine specimens following induc-tion of injury by 2 different mechanisms oxidative stress andimmune-mediated complement activation The aim was to de-termine if urinary miRNA changes would correlate with glomer-ular injury and severity in longitudinal time-course studies in 2well-established models of glomerulopathy passive Heymannnephritis (HN a rat model of immune-mediated glomerulop-athy) and rat puromycin aminonucleoside (PAN) podocytop-athy Although glomerular injury was induced in rats by 2 verydifferent mechanisms podocyte injury played a critical role inthe pathogenesis and progression of both immune and nonndashimmune mediated injury in these models (Patrakka andTryggvason 2009) Podocytes highly specialized terminally dif-ferentiated cells and integral components of the glomerular fil-tration apparatus are highly susceptible to the injurious effectsinduced by a variety of molecules including complement reac-tive oxygen species (ROS) and nephrotoxicants (Leeuwis et al2010) In passive HN a rat model of human membranous ne-phritis intravenous injection of an antibody against the alpha-3beta-1 integrin matrix receptor results in the formation of glo-merular subepithelial immune deposits and activation of thecomplement cascade leading to podocyte foot process efface-ment and detachment with proteinuria (Pippin et al 2009) Thecombined insults of sublytic amounts of complement ldquomem-brane attack complexrdquo fragment C5b-9 and ROS induce podo-cyte injury and proteinuria (Salant et al 1980) Similarly inPAN-induced nephrosis a model of idiopathic nephrotic syn-drome direct oxidative stress induces podocyte foot process ef-facement and apoptosis leading to proteinuria (Rincon et al2004) PAN is used to induce podocyte foot process effacementapoptosis and detachment leading to massive proteinuriasimilar to those described in human nephrosis (Grond et al1988) PAN-induced glomerular injury is mediated by directDNA damage via production of ROS Changes in cytoskeletaland slit diaphragm proteins lead to potentially deleterious cel-lular consequences in podocytes and proteinuria (Gwinner et al1997) miRNA expression profiles were investigated in isolatedglomeruli and urine specimens The ability of a subset of uri-nary miRNAs to distinguish rats with or without glomerular

injury was evaluated by correlating miRNA changes with histo-pathologic lesions (the diagnostic benchmark) and urinary al-bumin and total protein levels (Alousi et al 1969) To confirmthat the miRNA changes observed were not specific to theimmune-mediated injury model (HN) an additional well-established glomerular injury rat model (PAN) with a differentmode of injury was conducted

PAN- and HN-induced glomerular injury in both rat modelswas associated with the modulation of glomerular and urinarymiRNAs These investigations provided a means to identify glo-merular miRNA changes in urine samples that were induced bydistinct mechanisms of injury as well as a subset of glomerularmiRNAs that changed expression in urine specimens indepen-dent of the mode of glomerular injury Therefore these miRNAsmay not only be useful biomarkers of glomerular injury but mayalso distinguish the mechanism of induced injury Finally genetargets associated with urinary glomerular miRNA changescommon to both modes of injury were predicted and reported

MATERIALS AND METHODS

Animals Studies were conducted in accordance with the currentguidelines for animal welfare (National Research Council Guidefor the Care and Use of Laboratory Animals 2011 AnimalWelfare Act [AWA] 1966 as amended in 1970 1976 1985 and1990 and the AWA implementing regulations in Title 9 Code ofFederal Regulations Chapter 1 Subchapter A Parts 1ndash3)Procedures used in these studies were reviewed and approvedby the Institutional Animal Care and Use Committee (AnimalCare and Use Protocol AND-2011-00380) Male Sprague Dawleyrats (225ndash70 g Charles River Laboratories) were maintained in acentral animal facility housed individually in polycarbonatecages with autoclaved woodchip bedding (Lillico) enriched withLillico paperwool (nesting material) The room environmentwas maintained at 21C 6 2C and 55 6 10 relative humidityat all times in an alternating 12-h lightndashdark cycle Rats wereremoved from cages during injection of PAN or HN reagent andblood and urine collection Animals were acclimated to the lab-oratory environment for a minimum of 5 days prior to initiationof dosing Water (purified by reverse osmosis) and CertifiedRodent Diet 5002 (PMI Feeds Inc St Louis Missouri) were pro-vided ad libitum Animals were euthanized by isoflurane gasanesthesia followed by exsanguination

Dosing In the PAN study rats were injected once intraperito-neally (ip) with 150 mgkg of PAN (Sigma-Aldrich St LouisMissouri) on study day 1 Dose and time points were selectedbased on our pilot study (data not shown) and previous publica-tions (Yu et al 2005) Control animals were injected once ipwith sterile solution of 09 saline pH 7 (adjusted with 1MNaOH) In the HN study sheep anti-Fx1A serum (Probetex SanAntonio Texas) was injected once intravenously (iv) at a dose of1 ml200 g body weight into rats on study day 1 Dose and timepoints were selected based on our pilot study (data not shown)and previous publications (Pippin et al 2009) Control rats wereinjected once iv with a sterile solution of 09 saline pH 7(adjusted with 1M NaOH)

Sample collection Animals were placed into metabolic cages for16-h overnight urine collection into chilled (4C) containers onstudy days 3 and 6 (PAN) and days 3 9 and 16 (HN) All animalswere fasted during urine collection but provided free access towater Total urine volume for each animal was recorded Urinewas centrifuged (1500 rpm for 5 min at 4C) to remove any

36 | TOXICOLOGICAL SCIENCES 2015 Vol 148 No 1


contaminants and cellular debris and aliquots were frozen at70C Urine albumin and total protein were normalized tothe total amount excreted in the volume of urine collected andto the urine Cr concentration (mgdl) to control for the effect ofdilution There was no change in the amount of Cr excreted inthe urine of control or treated animals over the time course ofthe studies (data not shown) Urine albumin total protein andCr were analyzed using Siemens Advia 1800 automated technol-ogy Data are expressed as mean 6 standard deviationStatistical differences (assumed for Plt 05) were assessed usingone-way ANOVA (represented by a single asterisk where appli-cable) All graphs were generated by Prism software (GraphPadSoftware 60)

Light microscopic examination Kidneys were weighed at schedulednecropsy and sections were fixed overnight in 10 neutral buf-fered formalin then dehydrated through graded alcoholscleared in xylene and infiltrated and embedded in paraffinSerial 5 micron coronal sections at the hilus were stained withhematoxylin and eosin (HampE) and Periodic Acid Schiff (PAS)reagent with a hematoxylin counterstain Histological sectionsfor both kidneys included cortex medulla and pelvisHistopathological evaluation was performed by a board certifiedveterinary pathologist who had knowledge of the treatmentgroups and necropsy data (organ weights and macroscopicobservations) but who was blinded as to clinical pathologydatasets including results from biomarker evaluationsBackground findings in vehicle control animals were identifiedto differentiate disease model-related glomerular findingsfrom incidental background spontaneous lesions and theseverity of glomerular injury was scored using a grading scaleof 0ndash5 with histopathological changes described as 0(no lesions) 1 (minimal) 2 (slight) 3 (moderate) 4 (marked) or 5(severe)

Electron microscopic examination After kidneys were collected atnecropsy a tissue slice through the cortex was immersed in 1glutaraldehyde and 4 formaldehyde at a volume ratio of 101fixative to tissue and held at 4C until processed for ultrastruc-tural assessment For processing tissues were trimmed trans-ferred to 01 M phosphate buffer postfixed (1 bufferedosmium tetroxide 2 h 4C) and rinsed in deionized waterSamples were then dehydrated in graded ethanols containingpropylene oxide infiltrated and embedded in resin that wasallowed to polymerize at 60C for 24 h Resin-embedded sam-ples were sectioned at one-half micron stained with toluidineblue and glomerulus-containing areas were identified Thin(75ndash90 nm) sections were cut from these areas mounted on 200mesh copper palladium grids counter-stained with uranyl ace-tate and lead citrate and examined with a Hitachi 7100 trans-mission electron microscope at 75 kV and digital images werecaptured (Advanced Microscopy Techniques DanversMassachusetts) The control electron microscopy (EM) image inFigure 2d was obtained from a vehicle control rat that wasinjected ip with saline and was sacrificed at study day 11 of oneof our pilot studies

Laser capture microdissection Glomeruli were identified by phasecontrast microscopy and isolated from 9 micron cryosections ofkidney cortex from PAN D3 and D6 and HN D3 and D16 rats andrespective controls using a Molecular Devices Arcturus XT lasercapture microdissection (LCM) instrument Dissected glomeruliwere captured onto Arcturus HS caps and immersed in RNAextraction buffer

RNA extraction and quantitative PCR Total RNA was isolated from200 ll of rat urine or isolated LCM specimens from PAN D3 andD6 and HN D3 and D16 time points using a modified protocol(miRNeasy Serumplasma RNA extraction kit Qiagen RedwoodCity CA) according to the manufacturerrsquos instructions Briefly700 ll of QIAzol reagent was added to 200 ll of urine After vor-texing vigorously with chloroform the samples were then cen-trifuged at 12 000 g for 15 min at 4C The upper aqueousphase was transferred to a new tube and 15 volume of ethanolwas added The sample was then applied to the column andwashed and the immobilized RNA was collected from themembrane with 10 ll of RNase free water Total RNA concentra-tion was measured at 260 nm using a NanoDrop 2000c spectro-photometer (Thermo Scientific Waltham Massachusetts)Quantitative miRNA analysis was performed using TaqManmiRNA assays from Applied Biosystems RNA was reverse tran-scribed into complementary DNA (cDNA) using megaplex pri-mers (16C for 30 min 42C for 30 min 85C for 5 min) ThecDNA product was then used in a preamplification stepQuantitative PCR (qPCR) was performed using TaqMan low-density array (TLDA-A Life Technologies) in a Viia 7 thermocy-cler instrument (Life Technologies Grand Island New York)with the following temperature profile 95C for 10 min followedby 40 cycles of 95C for 15 s and 60C for 1 min Threshold cycle(Ct) values of 10 biological replicates from each experimentalcondition (treatment model time point and respective controls)were extracted using GeneData Expressionist software and athreshold of 32 was chosen (Ct values gt32 were consideredunreliable) Because samples for each treatment time pointalong with the corresponding control samples were processedin the same batch (same day same set of arrays) 20 sampleswere analyzed at a time and not merged with other time pointsThis avoided any batch effects that could be introduced by ana-lyzing multiple time points in the same assay Unless statedotherwise all analyses were performed using R (Bioconductor)

Each time point consisted of 20 samples (10 controlthorn 10treated) miRNAs that were not reliably detected (Ctgt 32) in atleast 33 of the samples were discarded from the dataset Urinesamples showed higher variability (correlation 01ndash09) com-pared with LCM samples (correlation 08ndash097) Consequentlyhierarchical clustering was employed and heat maps were gen-erated using the Spearman correlation coefficient to detect out-liers which were subsequently removed

Because currently no single normalization technique is com-monly accepted we compared 3 methods in our datasets Twomethods (NormFinder and GENorm) use invariant miRNAswhich iterate variance and ranking of Ct values through thedataset to find invariant miRNAs (those that do not changeacross samples) and use them to normalize the entire datasetThe third method loess-based normalization constructs areference array using the mean of all arrays and normalizeseach array to the reference array Hierarchical clustering andheat maps were generated for each group per time point usingthe normalization methods described above to determine thebest segregation between the control and treated samplesAdditionally the criterion of coefficient of variance (CV) wasused in comparing normalized values in individual groups (con-trol treated) and the combined set (Supplementary Table S9)Because the CV scores within each group should be less thanthe combined groups (controlthorn treated) calculating theincrease in CV for the combined groups as a percentage of aver-age individual grouprsquos CVs was used to determine which nor-malization method had the best separation between the controland treated groups (ie Increase CVfrac14 100CV (of the dataset

NASSIRPOUR ET AL | 37


(controlthorn treated)mean (CV (controls)thornCV (treated)) A higherIncrease CV value indicates that the normalization methodhad better separation between the control and treatmentgroups Loess provided the highest Increase CV and hencewas used for the reported data here

To increase our confidence regarding the normalizationstrategy chosen deltandashdelta Ct (ddCt) values were calculated formiRNAs using all 3 normalization methods Invariant miRNAswere found by NormFinder and GeNorm to calculate deltandashCtvalues (data not shown) To generate deltandashCt values in loesswe used an artificial invariant miRNA that had 0 as Ct valuesand subtracted it from Ct values of other miRNAs yielding theoriginal values as deltandashCt values (reported here for both LCMand urine data sets) Thereafter ddCt values were calculatedbetween the control and treated samples A Welch t test wasapplied to assess statistical significance and P values were cor-rected using BenjaminindashHochberg for multiple testing Eventhough adjusted P valuelt 05 is a well-accepted standard itmay introduce false negatives and can be overly restrictive Werelaxed the adjusted P value cutoff tolt 15 while using miRNAswith P valueslt 05 Hence miRNAs with adjusted P valuelt 15were deemed significantly altered in each dataset Fold changesare reported as 2(ddCt) The adjusted P value False DiscoveryRate (FDR) lt15 with a fold change cutoff of 6135 was used forthe reported analysis throughout the manuscript

Prediction of toxicity effects for significantly altered miRNAs ThemiRNAs identified as significant were analyzed separately byQIAGENrsquos Ingenuity Pathway Analysis (IPA QIAGEN RedwoodCity wwwqiagencomingenuity) Analysis was performed oneach set and the most significant ldquoTox Functionsrdquo are reported

RESULTS

Histopathology Electron Microscopy and Urinary Protein BiomarkerData Confirm Induction of Glomerular Injury in Both Rat ModelsHN male Sprague Dawley rats were injected with Sheep anti-Fx1A serum and urine and kidney specimens were collected at 3time points (days 3 9 and 16 of the study) as depicted by theschematic in Figure 1a Urine microalbumin and total protein(Fig 1b) histopathology (Fig 1c) and ultrastructural analysis byelectron microscopy (EM) (Fig 1d) all confirmed injury to glo-meruli Urine total protein and albumin assessed at each timepoint (D3 D9 and D16) revealed that proteinuria was inducedby D9 (Fig 1b) No morphological evidence of tubule injury wasobserved for this model Histopathologic lesions were notdetected in D3 glomeruli (Fig 1c) On D16 there was foamylightly positive PAS-positive material in glomerular tufts(arrows) in all 10 rats examined EM analysis revealed ultra-structural injury by D3 consisting of small subepithelial elec-tron dense deposits consistent with immune complexes(arrows) and minimal podocyte foot process swelling (Fig 1d)On D16 changes were similar but more severe with moreprominent subepithelial dense deposits (arrows) and associatedfoot process effacement (stars)

In the PAN study urine albumin levels increased by D3 andthere were significantly increased levels of urinary total proteinby D3 (Fig 2b) consistent with glomerular injury These urinebiomarker levels correlated well with the histopathological find-ings The earliest glomerular lesion identified by light micro-scopy in PAN dosed rats was observed 48 h after dosing (D3of the study) and was characterized by increased numbers ofPAS-positive granules within glomerular podocytes (Fig 2c)

The incidence and severity including numbers of PAS-positivegranules and numbers of affected glomeruli increased fromstudy day 3 (incidencefrac14 4 of the 10 rats dosed with PAN) to D6(incidencefrac14 9 of the 10 rats dosed with PAN) At study termina-tion on study day 6 minimal to mild hypertrophy of podocytes(arrows) andor parietal epithelial cells (stars) was observed inseveral glomeruli (Fig 2c) of PAN dosed rats Tubular epithelialchanges were not observed until study day 6 At day 6 intratub-ular protein and hyaline casts were observed in 8 of the 10 ratkidneys mostly in proximal tubules Minimal tubular epithelialcell changes included flattening associated with cytoplasmicattenuation cytoplasmic basophilia and very occasionalmitotic figures In addition there was evidence of ultrastruc-tural glomerular injury at D3 (Fig 2d) which consisted of mini-mal to mild podocyte foot process effacement well-circumscribed electron-dense cytoplasmic bodies (arrows) con-sistent with protein and microvillus extensions from podocytesinto the urinary space (stars) consistent with early podocytehypertrophy

Levels of miRNAs in Isolated Glomeruli Were Significantly AlteredFollowing Induced Glomerular InjuryAfter establishing that both the HN and the PAN rat models suc-cessfully induced glomerular injury (Figs 1 and 2) we investi-gated the changes in patterns of miRNA expression within thesite of injury Frozen kidney specimens were embedded inoptimum cutting temperature media from the control (nfrac14 10)and treated (nfrac14 10) rats at D3 and at the last time point for eachstudy LCM was employed to isolate a pure population of glo-meruli from sections collected at D3 and D6 (PAN study) as wellas D3 and D16 (HN study) Glomerular RNA was isolated at eachof these time points and profiles of 376 rodent miRNAs weregenerated by using a low-density array qPCR platform (TLDA-A)(Fig 3a) There was a measurable signal below the arbitrary cut-off set at 32 qPCR cycles (Ct) for 171 6 84 miRNAs for both thecontrol and treated samples at D3 (Supplementary Table S1)Sixty miRNAs changed in LCM-isolated glomeruli from theHN rats at D3 of which 28 were decreased and 32 increased(Table 1) At D16 235 6 45 miRNAs were detected (Table 1) outof which 119 changed significantly in the HN rats 64 miRNAswere decreased and 55 were significantly increased(Supplementary Table S2) Of these 45 miRNAs matchedbetween the 2 time points

In the PAN study 220 6 103 miRNAs were detected at D3compared with 248 6 62 at D6 (Table 1) The administration ofPAN led to alterations in 85 miRNAs at D3 of which 43 increasedand 42 decreased (Plt 05) (Supplementary Table S3) At D6changes were observed in 85 miRNAs 41 decreased while 44significantly increased (Supplementary Table S4) Among thesethere were 36 miRNA matches between the 2 time pointsNotably 84 miRNAs were consistently modulated in both setsof glomeruli isolated from the PAN and HN rat glomerular injurymodels

Levels of miRNAs in Urine Were Significantly Changed FollowingInduced Glomerular InjuryTo investigate the possibility that certain miRNAs could act assensitive and specific biomarkers of glomerular injury weassessed changes in urinary miRNA expression in rat models ofglomerular injury The transcript levels of 376 miRNAs weremeasured in the urine specimens that correlated with each ofthe LCM-measured time points in the HN (D3 and D16) and thePAN (D3 and D6) studies (Fig 4a)



FIG 1 Passive Heymann nephritis (HN) rat model of nephrotoxicity induces injury to glomeruli a Schematic depicting study design Briefly sheep anti-Fx1A serum

was injected intravenously into rats daily and urine and kidney specimens were collected at days 3 9 and 16 post injections b Urinary protein and microalbumin (nor-

malized to urine Cr) indicate renal injury at day 9 (D9 mean 6 standard deviation Plt 05 Plt 01) c Compared with vehicle-dosed rats (control) day 16 (HN-D16) but

not day 3 (HN-D3) rats had foamy lightly positive Periodic Acid Schiff (PAS)-positive material in glomerular tufts (arrows) PAS stain scale barsfrac14100 mm d Compared

with vehicle-dosed rats (control) anti-Fx1A-related ultrastructural findings were multifocal subepithelial electron dense deposits (arrows) consistent with immune

complexes at D3 By D16 more prominent multifocal subepithelial electron dense deposits (arrows) and effacement of podocyte foot processes adjacent to these

deposits (stars) were observed Original negative magnifications5000 (control) and10 000 (HN-D3 and HN-D16) Scale barfrac14500 nm



FIG 2 Puromycin-induced glomerulopathy in rats a Study design depicting vehicle control male Sprague Dawley and rats dosed with 150 mgkg puromycin aminonu-

cleoside (PAN) b Urinary protein and microalbumin (normalized to urine creatinine [Cr]) indicate renal injury at day 3 (D3) and 6 (D6 mean 6 standard deviation

Plt 05 Plt 01) c Puromycin-induced glomerulopathy in rats Study day 3 (control rat glomerulus) with Periodic Acid Schiff (PAS) stain Study day 3 (PurondashD3) PAN-

dosed rat glomerulus with prominent PAS-positive granules in podocytes (arrows) Study day 6 (PurondashD6) PAN-dosed glomerulus with several hypertrophied podocytes

(arrows) and parietal epithelial cells (stars) characterized by minimally enlarged nuclei and increased amount of cytoplasm HampE stain Scale barsfrac1450mm d Study

day 3 puromycin-associated ultrastructural changes included electron dense material in the cytoplasm of podocytes (arrows) and microvillus podocyte cytoplasmic

extensions into the urinary space (stars) Scale bar (control)frac14500 nm Scale bar (PAN)frac14 1060 nm Original negative magnifications 5000 (control) and6000

(PurondashD3)



At D3 130 6 135 miRNAs were detected by qRT-PCR with Ctvalues 32 in at least 20 of the urine specimens analyzed fromthe HN study (Table 1) A total of 62 miRNAs changed in urinespecimens collected at D3 in the HN group 33 increased and 30

decreased while 1 was absent in the control group and 1 miRNAwas not detected in the HN group (Supplementary Table S5)At D16 a total of 114 6 176 miRNAs were detected in urine(Table 1) 25 of these changed significantly of which 14

FIG 3 Relative quantification of microRNA (miRNA) responders in rat glomeruli with nephrotoxicity a Workflow of quantitative RT-PCR analysis for the laser capture

microdissection-isolated glomeruli b Volcano plots show miRNAs that are significantly regulated at day 3 and 16 in the immune-mediated Heymann nephritis rat

model of glomerular injury (FDRlt015 and FCgt135 in either direction) c Volcano plots show miRNAs that are significantly regulated at day 3 and 6 post Puromycin-

induced glomerular injury (FDRlt015 and FCgt135 in either direction) Glomerular miRNA changes that were also detected in urine specimen are highlighted

Horizontal line Pfrac14 05 vertical lines FC at 13 and 13 Data are normalized using loess normalization



decreased and 11 increased (Supplementary Table S6) In thePAN model of glomerular injury 101 6 98 miRNAs weredetected in D3 urine specimens Significant changes weredetected in 21 of these miRNAs including 10 miRNAs withdecreased expression and 11 with increased expression levels(Supplementary Table S7) At D6 however 140 6 143 miRNAswere detected in urine specimens from PAN rats (Table 1) withsignificant changes in 38 miRNAs of which 17 decreased and 21increased Two miRNAs detected in the PAN rats were absent inthe control samples (Supplementary Table S8) Fourteen of themiRNAs detected in urine specimens changed in both rat mod-els of glomerular injury at D3

Cross-referencing urine miRNA changes with those observedin the LCM specimens provided further characterization of theglomerular miRNAs that not only changed at the site of injurybut were also detected in the earliest urine specimens and thusmay be potential biomarkers In the HN study significantchanges were measured in 45 miRNAs that were detected inboth D3 urine and D3 or D16 glomeruli In the PAN model 14miRNAs that changed significantly in urine were also altered inthe glomeruli at D3 or D6 Cross-study comparison revealedchanges in 9 miRNAs (miR-106a 125a-5p 17 218 223 27b 30c574-3p and 196c) that changed in both models at D3 urine sam-ples as well as LCM specimens (highlighted in Figs 3 and 4Table 2) Interestingly significant changes in miR-574-3p weredetected in every dataset analyzed

Prediction of miRNA Targets and Pathway Analysis Relevant toRenal InjuryIngenuity modeling of the differentially expressed miRNAsidentified in the PAN and HN specimens was undertaken tobetter understand the targets and pathways regulated Theexperimentally verified and predicted miRNA targets from theqRT-PCR analysis were analyzed through Ingenuityrsquos ToxFunction prediction Figure 5a shows the significant (Plt 01) dis-eases molecular and cellular functions and nephrotoxicityfunctions in the cross-referenced miRNAs in HN (44) and PAN(14) studies The nephrotoxicity functions were obtained byusing the gene targets of miRNAs Interestingly cross-referenceanalysis identified 35 miRNAs that were altered significantly inHN but not in PAN D3 urine specimens In contrast 5 miRNAswere significantly altered in PAN but not in HN D3 urine speci-mens (Figs 5b and c)

The 9 common miRNAs targeted 261 genes and cellularpathways such as ldquoRenal Necrosisrdquo and ldquoGlomerular Injuryrdquo(Plt 019) The signaling pathways and renal toxicity relatedfunctions are illustrated in Figure 5d along with the miRNAsand their targets

To investigate if there was any difference in HN and PANmechanisms the HN-only (Fig 5b) and PAN-only miRNAs(Fig 5c) were analyzed The HN-only (35) miRNAs targeted 588(7284 predicted targets) genes and PAN-only (5) targeted 208(4691 predicted targets) genes out of which 118 (2935 predicted)were in common This suggests that these seemingly differentsets of miRNAs might be functionally related Some of the bio-logical functions enriched in HN-only targets were immunemediated (labeled by IPA as Organismal Injury) however B-cellreceptor signaling was among the top 5 hits (Plt 1 1010) forPAN-only targets

The analysis also revealed an interesting trend in the datasetwhich we tried to capture in Figure 5e and Table 2 The directionof change in tissue versus urine was inverse for most of the 9miRNAs that changed in both models of glomerular injury Forthe expression pairing analysis the urinary miRNA changes atD3 were cross-referenced to the LCM changes that occurredeither at D3 or D6D16 Thus miRNAs that were decreased inglomeruli were highly increased in corresponding urine speci-mens possibly implying a link between induced tissue injuryand release of the miRNAs in the biofluid

DISCUSSION

We have described glomerular miRNA changes that wereinduced by 2 different modes of insult and were altered in earlyurine specimens and may thus potentially serve as early site-specific and mechanism-related biomarkers of glomerularinjury In the immune-mediated HN model highly reproducibleprogressive concordant changes in 45 miRNAs were observedin the earliest urine specimens analyzed as well as isolated D3and D16 glomeruli Similarly treatment with a single high doseof puromycin resulted in 14 miRNA changes in D3 urine thatwere associated with altered expression patterns in the isolatedglomeruli collected at D3 and D6 These miRNA alterations cor-related with histologic findings of glomerular injury and withincreases in concentrations of benchmark protein biomarkers

miRNAs are short (22 nucleotides) noncoding RNAs thatregulate translational repression of their target transcriptsmiRNAs are transcribed as much longer precursors which aresequentially processed by 2 different RNase 3 enzymes Droshaand Dicer to their mature forms Mice with podocyte-specificdeletions of these miRNA processing enzymes suffer from pro-gressive glomerular and tubular defects and hence miRNAs arebelieved to play pivotal roles in normal renal physiology as wellas various pathological processes in the kidney Due to specific-ity in patterns of cell and tissue expression and remarkablestability in various biofluids miRNAs have gained popularity asbiomarkers of various diseases including renal disordersmiRNA changes within the tissue of origin must also be detect-able in a biofluid before the miRNA may be proposed as a bio-marker of injury Employing 2 established rat models thatinduce glomerular injury by different mechanisms we identi-fied several miRNA changes in LCM-isolated glomeruli as wellas urine specimens collected during early stages of glomerularinjury by PCR-based genomic profiling

We had reported previously interplatform differencesbetween qPCR and next-generation sequencing (NGS) platformsfor profiling urinary miRNAs namely that although NGS mightbe a more accurate platform due to its ability to identify miRNAisoforms qPCR seems to be a more sensitive profiling technique(Nassirpour et al 2014) We therefore employed low-densityarrays for quantitative real-time PCR (TLDA) for miRNA expres-sion investigations reported here with the caveat that miRNAs

TABLE 1 Summary of miRNA Analysis Observed WithNephrotoxicants

Condition Detected Increased Decreased

LCM HN-D3 171 32 28HN-16 241 55 65Puromycin-D3 220 43 42Puromycin-D6 252 44 41

Urine HN-D3 133 33 30HN-16 116 11 14Puromycin-D3 104 11 10Puromycin-D6 140 21 17

LCM laser capture microdissection miRNA microRNA



FIG 4 Relative quantification of microRNA (miRNA) responders in urine with nephrotoxicity a Workflow of quantitative RT-PCR analysis for the urine specimen b

Volcano plots show urinary miRNAs that are significantly regulated at day 3 and 16 in the immune-mediated Heymann nephritis rat model of glomerular injury

(FDRlt 015 and FCgt135 in either direction) c Volcano plots show urinary miRNAs that are significantly regulated at day 3 and 6 post Puromycin-induced glomerular

injury (FDRlt015 and FCgt135 in either direction) Glomerular miRNA changes that were also detected in urine specimen are highlighted Horizontal line Pfrac14 05 verti-

cal lines FC at13 and 13 Data are normalized using loess normalization



expressed in their isomeric forms are probably unfortunatelymissed As with any other gene expression profiling effectiveanalysis methods to produce reliable and high-quality resultsinclude normalization (Deo et al 2011) Adequate normalizationminimizes the effects of systematic as well as measurementand technical errors and variations and is critical for properbiological interpretations However due to the nature of in vivostudies low concentrations of ribonucleic acids in urine col-lected from metabolic cages incomplete understanding of thesource and biology of miRNAs in biofluids and low mean con-cordance in miRNA normalization platforms additional studieswill be necessary to identify the most suitable techniques formiRNA normalization (Mohammadian et al 2013) Previouslywe investigated the impact of different normalization methodson intra- and interplatform performance of 2 distinct and com-monly used miRNA profiling platforms namely qPCR and NGS(Nassirpour et al 2014) In this study we compared the per-formance of 3 different normalization methods NormFinder(Andersen et al 2004) and geNorm (Vandesompele et al 2002)which are commonly used and reported in various profilingstudies employ the variance and ranking of Ct values to iden-tify nonvariant miRNAs against which to normalize the datasetThe third method locally weighted scatterplot smoothing(loess Cleveland and Devlin 1988) is a nonparametric localregression model that constructs a reference array using themean of all arrays and normalizes each array to this referencearray This method showed the best performance in hierarchi-cal clustering and heat map generation for each treatment pertime point measuring segregation among samples andincreasing the correlation among replicate samples better thanthe other 2 nonvariant methods Thus as reported previously inother miRNA tissue profiling studies (Meyer et al 2012) we pro-pose loess normalization for identification of differentiallyexpressed miRNAs in urine as it seemed to minimize standarddeviations and increased the area under the ROC curve both ofwhich are established measures of statistical performance Inaddition we found that locally weighted scatterplot smoothingalso increased interplatform concordance of differential expres-sion further endorsing loess as our choice of normalizationmethod employed in this study (Nassirpour et al 2014)

Although each study revealed a subset of miRNAs that seemto be dependent on the induced mechanism of injury 9miRNAs including miR-106a 125a-5p 17 218 223 27b 30c

574-3p and 196c changed in an insult-independent mannerTherefore these miRNAs are proposed as candidate urinary glo-merular injury biomarkers Furthermore although the transportmechanisms or mechanisms of excretion of glomerular or circu-lating miRNAs into the urinary space are not well elucidatedthe direction of change for these miRNAs was noteworthy asthe miRNAs that were increased in the glomeruli weredecreased in urine specimens and vice versa It is thus plausi-ble that the increase in miRNA expression in urine may be theresult of cell death in glomeruli (eg podocyte) or active secre-tion from the surviving cells Additional studies are needed tolocalize the cellular source of the miRNAs that were identifiedin urine in these rat models of glomerular injury

Among the miRNA biomarkers that were common to bothstudies miR-574-3p was unique in that its expression wasaltered in the glomeruli and urine specimens collected at everytime point Interestingly miR-574-3p was also shown by Kontaet al (2014) to be the only miRNA that was significantly changedin clinical urine samples collected from patients with 4 differentrenal diseases miRNAs 30a-c 194 197 and 200c whichchanged in both glomeruli and urine specimens in these ratmodels were also proposed as potential urinary biomarkers ofdiabetic nephropathy (DN) in the Konta study thus the glomer-ular biomarkers we identified in these preclinical rat modelsmight have clinical implications and warrant further inves-tigation Although we know very little about the role of miR-574-3p in renal diseases it has been implicated as an inhibitorof differentiation of multipotent mesenchymal stromal cellsinto chondrocytes (Guerit et al 2013) has been proposed as atumor suppressor in gastric (Su et al 2012) and bladder cancercells (Tatarano et al 2012) and has been shown to negativelyregulate the proliferation of keratinocytes (Chikh et al 2011)Therefore it is possible that this miRNA also plays a role inrenal function and may influence podocyte differentiationHowever this miRNA was also reported as altered in urine sam-ples analyzed after gentamicin-induced tubule injury(Nassirpour et al 2014) Therefore we cannot propose thismiRNA as glomerular specific Comparing the 9 miRNAs identi-fied in this manuscript against other published tubule injury ratmodels will also shed light on their utility as glomerular-specificbiomarkers For example miR-17 and 218 were also altered inurine analyzed from rats with cisplatin-induced tubular injury(Kanki et al 2014) Similarly Pavkovic et al (2014) reported uri-nary changes in miR-196c and 223 with cisplatin However inboth cases their analysis focused on later time points and afterextensive tubular injury Unfortunately because glomerulartoxicity was not assessed in these studies we are unable toassess their site specificity Interestingly Pavkovic et al (2015)recently identified 5 miRNAs in a glomerular rat injury modelinduced by nephrotoxic serum All 5 were also detected at mul-tiple time points in our glomerular injury models Howeverbecause our objective was to identify early biomarkers of injurywe focused our efforts on early time points (day 3) before mor-phologically significant induced glomerular injury occurred

The majority of the differentially expressed glomerular andurine miRNA biomarkers identified here have also been shownto be highly enriched in the kidney and conserved across spe-cies (Saal and Harvey 2009) A recent review (Khella et al 2013)summarized the association of several miRNA families andtranscripts with renal physiology and disease including themiR-30 family shown to be crucial for podocyte functions rolesfor the miR-200 family and miR-17 in polycystic kidney diseaseroles for the miR-29 family and miR-192 in renal fibrosis andthe involvement of miR-192 and miR-27 in the pathogenesis of

TABLE 2 Summary of the Significantly Altered Glomerular miRNAUrinary Changes Observed in Both Glomerular Injury Rat Models

microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine

mmu-miR-106a-5p 107 330 170 127 128 139mmu-miR-223-3p 101 207 662 145 114 241mmu-miR-574-3p 158 182 155 132 180 343rno-miR-125a-5p 112 222 332 119 121 251rno-miR-17-5p 100 370 474 121 119 160rno-miR-196c-5p 103 152 509 118 124 313rno-miR-218a-5p 152 113 183 156 168 157rno-miR-27b-3p 116 142 245 110 121 151rno-miR-30c-5p 117 150 150 150 125 154

LCM and urinary miRNA changes are highlighted according to their direction

(increases are highlighted in bold)

HN Heymann nephritis LCM laser capture microdissection miRNA microRNA

PAN puromycin aminonucleoside



lupus nephritis Additionally miR-223 appears to regulate keypathways in IgA nephropathy (Bao et al 2014) and may serve asa biomarker for human allograft rejection (Anglicheau et al2009) Lai et al (2015) have also demonstrated that glomerularmiR-21 expression is positively associated with albumin-to-Crratios in patients with DN and that loss of miR-21 is associatedwith accelerated glomerular damage and podocyte apoptosis ina murine model of DN and Tgfb1-TG mice Similarly Ichii et al(2014) have shown that miR-26a regulates podocyte differentia-tion and cytoskeletal integrity and its altered levels in glomeru-lus and urine may serve as a marker of injured podocytes inautoimmune glomerulonephritis Therefore the miRNA bio-markers proposed here may play important roles in renal physi-ology and in the development of renal diseases as well asserving as novel and site-specific biomarker candidates of

glomerular injury In fact several global consortia such as HESIare comparing site-specific induced preclinical toxicologicalstudies such as this one against studies reported by othernephrotoxicants and in different animal models to evaluate theutility of these proposed biomarkers in drug-induced toxicologi-cal evaluations Methodical comparisons with respect to thedegree of induced toxicity as well as characterizations of thesite of induced injury within a nephron and technical and ana-lytical strategies used are critical in obtaining a better under-standing of applicability of the proposed miRNA biomarkers

In this study we compared the timing of onset for biomarkeralterations of the urinary miRNAs that we identified versusestablished protein urine biomarkers (eg microalbumin andtotal protein) and against benchmark histopathology In thePAN model the rapid progression of the injury that was induced

FIG 5 Prediction of renal functions for microRNA (miRNA) biomarkers of glomerular injury a Diseases molecular and cellular functions as well as nephrotoxicity

functions in the cross-referenced miRNAs in Heymann nephritis (HN) (44) and puromycin aminonucleoside (PAN) (14) studies were analyzed through Ingenuityrsquos Tox

Function prediction (P lt 01) b The nephrotoxicity functions were obtained by using the gene targets of the 35 miRNAs that changed only in HN D3 urine c The corre-

sponding number in PAN but not in HN D3 urine was 5 and their targets and nephrotoxicity functions depict glomerular injury d The 9 common miRNAs targeted

261 genes and cellular pathways such as Renal Necrosis and Glomerular Injury (Plt 019) e Directionality of change in miRNA expression patterns in tissue versus

urine for the 9 common miRNAs as observed in the isolated glomeruli and D3 urine specimen



did not provide sufficient time to assess whether urine miRNAbiomarkers would change prior to increases in urine proteinbiomarkers However in the more slowly developing immune-mediated HN model miRNA changes at D3 preceded significantincrease in urine albumin or total protein Therefore the differ-entially expressed miRNAs that we identified during the earlystages of passive HN may hold promise for improving earlyidentification of drug-induced immune injury Furthermorepathway analysis revealed close association between the 9miRNA biomarkers of glomerular injury and target genes linkedto glomerular injury inflammation and apoptosis Thereforedue to their high translatability and conservation across spe-cies miRNA transcripts that respond very early after drug-induced injury in rodents might have higher probability ofhuman translation in addition to being more specific for podo-cyte or glomerular injury than the functional glomerular injurybiomarkers that are currently in use

SUPPLEMENTARY DATA

Supplementary data are available online at httptoxscioxfordjournalsorg

ACKNOWLEDGMENTS

This study was conducted as part of Pfizerrsquos GlomerularInjury Biomarker team efforts and we would like toacknowledge all present and past members for their variouscontributions to this project with special thanks to ShashiRamaiah Zaher Radi Patrick Lappin Eva Nagiec andDeborah Burt We would like to extend our gratitude to DrDale Morris Dr Denise Robinson-Gravatt and PfizerrsquosScience and Technology Board for their generous help sup-port and commitment This study supports the efforts ofthe HESI Biomarkers of Nephrotoxicity Committee The HESIis a nonprofit institution whose mission is to engage scien-tists from academia government and industry to identifyand resolve global health and environmental issues Wewould like to especially acknowledge Dr Jean-CharlesGautier for his critical reading of our manuscriptAdditionally we would like to thank Mr Edward Germondwho as a talented summer intern significantly contributedto data acquisition from a pilot puromycin miRNA study(data not shown)

REFERENCESAlousi M A Post R S and Heymann W (1969) Experimental

autoimmune nephrosis in rats Morphogenesis of the glo-merular lesion Immunohistochemical and electron micro-scopic studies Am J Pathol 54 47ndash71

Alvarez M L and DiStefano J K (2013) The role of non-codingRNAs in diabetic nephropathy Potential applications as bio-markers for disease development and progression DiabetesRes Clin Pract 99 1ndash11

Andersen C L Jensen J L and Oslashrntoft T F (2004)Normalization of real-time quantitative reverse transcrip-tion-PCR data A model-based variance estimation approachto identify genes suited for normalization applied to bladderand colon cancer data sets Cancer Res 64 5245ndash5250

Anglicheau D Sharma V K Ding R Hummel A SnopkowskiC Dadhania D Seshan S V and Suthanthiran M (2009)

MicroRNA expression profiles predictive of human renal allo-graft status Proc Natl Acad Sci USA 106 5330ndash5335

ANON (2010) Biomarkers on a roll Nat Biotech 28 431Bao H Chen H Zhu X Zhang M Yao G Yu Y Qin W

Zeng C Zen K and Liu Z (2014) MiR-223 downregulationpromotes glomerular endothelial cell activation by upregu-lating importin [alpha]4 and [alpha]5 in IgA nephropathyKidney Int 85 624ndash635

Bonventre J V Vaidya V S Schmouder R Feig P andDieterle F (2010) Next-generation biomarkers for detectingkidney toxicity Nat Biotech 28 436ndash44

Chikh A Matin R N H Senatore V Hufbauer M Lavery DRaimondi C Ostano P Mello-Grand M Ghimenti CBahta A et al (2011) iASPPp63 autoregulatory feedbackloop is required for the homeostasis of stratified epitheliaEMBO J 30 4261ndash4273

Cleveland W S and Devlin S J (1988) Locally weighted regres-sion An approach to regression analysis by local fitting JAm Stat Assoc 83 596ndash610

De Guire V Robitaille R Tetreault N Guerin R Menard CBambace N and Sapieha P (2013) Circulating miRNAs assensitive and specific biomarkers for the diagnosis and mon-itoring of human diseases Promises and challenges ClinBiochem 46 846ndash860

Deo A Carlsson J and Lindlof A (2011) How to choose a nor-malization strategy for miRNA quantitative real-time (qPCR)arrays J Bioinform Comput Biol 9 795ndash812

Dieterle F Perentes E Cordier A Roth D R Verdes PGrenet O Pantano S Moulin P Wahl D Mahl A et al(2010) Urinary clusterin cystatin C [beta]2-microglobulinand total protein as markers to detect drug-induced kidneyinjury Nat Biotech 28 463ndash469

Grond J Muller E W van Goor H Weening J J and Elema JD (1988) Differences in puromycin aminonucleoside ne-phrosis in two rat strains Kidney Int 33 524ndash529

Guerit D Philipot D Chuchana P Toupet K Brondello J MMathieu M Jorgensen C and Noel D (2013) Sox9-regulated miRNA-574-3p inhibits chondrogenic differentia-tion of mesenchymal stem cells PLoS One 8 e62582

Gwinner W Landmesser U Brandes R P Kubat B Plasger JEberhard O Koch K M and Olbricht C J (1997) Reactiveoxygen species and antioxidant defense in puromycinaminonucleoside glomerulopathy J Am Soc Nephrol 81722ndash1731

Harvey S J Jarad G and Cunningham J (2008) Podocyte-specific deletion of dicer alters cytoskeletal dynamics andcauses glomerular disease J Am Soc Nephrol 19 2150ndash2158

Ho J Ng K H Rosen S Dostal A Gregory R I and KreidbergJ A (2008) Podocyte-specific loss of functional microRNAsleads to rapid glomerular and tubular injury J Am SocNephrol 19 2069ndash2075

Ho J J and Marsden P A (2008) Dicer cuts the kidney J AmSoc Nephrol 19 2043ndash2046

Ichii O Otsuka-Kanazawa S Horino T Kimura J NakamuraT Matsumoto M Toi M and Kon Y (2014) DecreasedmiR-26a expression correlates with the progression of podo-cyte injury in autoimmune glomerulonephritis PLoS One 9e110383

Kanki M Moriguchi A Sasaki D Mitori H Yamada AUnami A and Miyamae Y (2014) Identification of urinarymiRNA biomarkers for detecting cisplatin-induced proximaltubular injury in rats Toxicology 324 158ndash168

Khella H W Z Bakhet M Lichner Z Romaschin A D JewettM A S and Yousef G M (2013) MicroRNAs in kidney



disease An emerging understanding Am J Kidney Dis 61798ndash808

Konta T Ichikawa K Suzuki K Kudo K Satoh H Kamei KNishidate E and Kubota I (2014) A microarray analysis ofurinary microRNAs in renal diseases Clin Exp Nephrol 18711ndash717

Lai J Y Luo J OrsquoConnor C Jing X Nair V Ju W RandolphA Ben-Dov I Z Matar R N Briskin D et al (2015)MicroRNA-21 in glomerular injury J Am Soc Nephrol 26805ndash816

Leeuwis J W Nguyen T Q Dendooven A Kok R J andGoldschmeding R (2010) Targeting podocyte-associateddiseases Adv Drug Deliv Rev 62 1325ndash1336

Li J Y Yong T Y Michael M Z and Gleadle J M (2010)Review The role of microRNAs in kidney disease Nephrology15 599ndash608

Meyer S U Kaiser S Wagner C Thirion C and Pfaffl M W(2012) Profound effect of profiling platform and normaliza-tion strategy on detection of differentially expressedmicroRNAsmdashA comparative study PLoS One 7 e38946

Mohammadian A Mowla S Elahi E Tavallaei M NouraniM and Liang Y (2013) Normalization of miRNA qPCR high-throughput data A comparison of methods Biotechnol Lett35 843ndash851

Nassirpour R Mathur S Gosink M Li Y Shoieb A Wood JOrsquoNeil S Homer B and Whiteley L (2014) Identification oftubular injury microRNA biomarkers in urine Comparison ofnext-generation sequencing and qPCR-based profiling plat-forms BMC Genomics 15 485

Patrakka J and Tryggvason K (2009) New insights into the roleof podocytes in proteinuria Nat Rev Nephrol 5 463ndash468

Pavkovic M Riefke B and Ellinger-Ziegelbauer H (2014)Urinary microRNA profiling for identification of biomarkersafter cisplatin-induced kidney injury Toxicology 324 147ndash157

Pavkovic M Riefke B Frisk A L Groticke I and Ellinger-Ziegelbauer H (2015) Glomerulonephritis-induced changesin urinary and kidney microRNA profiles in rats Toxicol Sci145 348ndash359

Pippin J W Brinkkoetter P T Cormack-Aboud F CDurvasula R V Hauser P V Kowalewska J Krofft R DLogar C M Marshall C B Ohse T and Shankland S J(2009) Inducible rodent models of acquired podocyte dis-eases Am J Physiol Renal Physiol 296 F213ndashF229

Rincon J Romero M Viera N Pedreanez A and Mosquera J(2004) Increased oxidative stress and apoptosis in acute pu-romycin aminonucleoside nephrosis Int J Exp Pathol 8525ndash33

Saal S and Harvey S (2009) MicroRNAs and the kidneyComing of age Curr Opin Nephrol Hypertens 18 317ndash323

Salant D J Belok S Madaio M P and Couser W G (1980) Anew role for complement in experimental membranous ne-phropathy in rats J Clin Invest 66 1339ndash1350

Scian M J Maluf D G and Mas V R (2013) MiRNAs in kidneytransplantation Potential role as new biomarkers Expert RevMol Diagn 13 93ndash104

Shi S (2008) Podocyte-selective deletion of dicer induces pro-teinuria and glomerulosclerosis J Am Soc Nephrol 19 2159ndash2169

Su Y Ni Z Wang G Cui J Wei C Wang J Yang Q Xu Yand Li F (2012) Aberrant expression of microRNAs in gastriccancer and biological significance of miR-574-3p IntImmunopharmacol 13 468ndash475

Tatarano S Chiyomaru T Kawakami K Enokida H YoshinoH Hidaka H Nohata N Yamasaki T Gotanda TTachiwada T et al (2012) Novel oncogenic function of me-soderm development candidate 1 and its regulation by MiR-574-3p in bladder cancer cell lines Int J Oncol 40 951ndash959

Vandesompele J De Preter K Pattyn F Poppe B Van Roy NDe Paepe A and Speleman F (2002) Accurate normaliza-tion of real-time quantitative RT-PCR data by geometric aver-aging of multiple internal control genes Genome Biol 3RESEARCH0034

Wang G Kwan B C H Lai F M M Choi P C L Chow K-MLi P K T and Szeto C-C (2009) Intrarenal expression ofmicroRNAs in patients with IgA nephropathy Lab Invest 9098ndash103

Yu D Petermann A Kunter U Rong S Shankland SJ andFloege J (2005) Pathophysiology of renal disease and pro-gression Urinary podocyte loss is a more specific marker ofongoing glomerular damage than proteinuria JASN 16 1733ndash1741

Zhdanova O Srivastava S Di L Li Z Tchelebi L DworkinS Johnstone D B Zavadil J Chong M M Littman D Ret al (2011) The inducible deletion of Drosha and microRNAsin mature podocytes results in a collapsing glomerulopathyKidney Int 80 719ndash730



kfv167-TF1

kfv167-TF2

kfv167-TF3

biomarkers are able to distinguish tissue of origin with in-creased diagnostic accuracy (De Guire et al 2013) miRNA ex-pression has been shown to be distinct among differentanatomical regions of the kidney and alterations in miRNA ex-pression have been associated with a variety of kidney diseasesincluding diabetic nephritis hypertension glomerulonephritisand cancer (Alvarez and DiStefano 2013 Khella et al 2013Scian et al 2013 Wang et al 2009) A critical role of miRNA reg-ulation in the progression of glomerular damage and the devel-opment of proteinuria has been suggested by studies in micewith podocyte-specific deletion of Dicer (Harvey et al 2008 Hoet al 2008 Ho and Marsden 2008 Shi 2008) and more recentlyDrosha (Zhdanova et al 2011) critical enzymes involved inmiRNA biogenesis

In order to assess the utility of miRNA measurements inurine for detection of site-specific renal toxicity the Health andEnvironmental Sciences Institute (HESI) Biomarkers ofNephrotoxicity Committee is conducting a collaborative pro-gram using toxicants or models that are selective for specificnephron segment injury This study describes the effects on uri-nary induction of miRNAs in 2 different rat models of glomeru-lar injury The objective of the rodent glomerular injury studiesreported here was to characterize the miRNA expression andexcretion profile following the induction of glomerular injuryAccordingly we performed global miRNA expression profilingfrom isolated glomeruli and urine specimens following induc-tion of injury by 2 different mechanisms oxidative stress andimmune-mediated complement activation The aim was to de-termine if urinary miRNA changes would correlate with glomer-ular injury and severity in longitudinal time-course studies in 2well-established models of glomerulopathy passive Heymannnephritis (HN a rat model of immune-mediated glomerulop-athy) and rat puromycin aminonucleoside (PAN) podocytop-athy Although glomerular injury was induced in rats by 2 verydifferent mechanisms podocyte injury played a critical role inthe pathogenesis and progression of both immune and nonndashimmune mediated injury in these models (Patrakka andTryggvason 2009) Podocytes highly specialized terminally dif-ferentiated cells and integral components of the glomerular fil-tration apparatus are highly susceptible to the injurious effectsinduced by a variety of molecules including complement reac-tive oxygen species (ROS) and nephrotoxicants (Leeuwis et al2010) In passive HN a rat model of human membranous ne-phritis intravenous injection of an antibody against the alpha-3beta-1 integrin matrix receptor results in the formation of glo-merular subepithelial immune deposits and activation of thecomplement cascade leading to podocyte foot process efface-ment and detachment with proteinuria (Pippin et al 2009) Thecombined insults of sublytic amounts of complement ldquomem-brane attack complexrdquo fragment C5b-9 and ROS induce podo-cyte injury and proteinuria (Salant et al 1980) Similarly inPAN-induced nephrosis a model of idiopathic nephrotic syn-drome direct oxidative stress induces podocyte foot process ef-facement and apoptosis leading to proteinuria (Rincon et al2004) PAN is used to induce podocyte foot process effacementapoptosis and detachment leading to massive proteinuriasimilar to those described in human nephrosis (Grond et al1988) PAN-induced glomerular injury is mediated by directDNA damage via production of ROS Changes in cytoskeletaland slit diaphragm proteins lead to potentially deleterious cel-lular consequences in podocytes and proteinuria (Gwinner et al1997) miRNA expression profiles were investigated in isolatedglomeruli and urine specimens The ability of a subset of uri-nary miRNAs to distinguish rats with or without glomerular

injury was evaluated by correlating miRNA changes with histo-pathologic lesions (the diagnostic benchmark) and urinary al-bumin and total protein levels (Alousi et al 1969) To confirmthat the miRNA changes observed were not specific to theimmune-mediated injury model (HN) an additional well-established glomerular injury rat model (PAN) with a differentmode of injury was conducted

PAN- and HN-induced glomerular injury in both rat modelswas associated with the modulation of glomerular and urinarymiRNAs These investigations provided a means to identify glo-merular miRNA changes in urine samples that were induced bydistinct mechanisms of injury as well as a subset of glomerularmiRNAs that changed expression in urine specimens indepen-dent of the mode of glomerular injury Therefore these miRNAsmay not only be useful biomarkers of glomerular injury but mayalso distinguish the mechanism of induced injury Finally genetargets associated with urinary glomerular miRNA changescommon to both modes of injury were predicted and reported

MATERIALS AND METHODS

Animals Studies were conducted in accordance with the currentguidelines for animal welfare (National Research Council Guidefor the Care and Use of Laboratory Animals 2011 AnimalWelfare Act [AWA] 1966 as amended in 1970 1976 1985 and1990 and the AWA implementing regulations in Title 9 Code ofFederal Regulations Chapter 1 Subchapter A Parts 1ndash3)Procedures used in these studies were reviewed and approvedby the Institutional Animal Care and Use Committee (AnimalCare and Use Protocol AND-2011-00380) Male Sprague Dawleyrats (225ndash70 g Charles River Laboratories) were maintained in acentral animal facility housed individually in polycarbonatecages with autoclaved woodchip bedding (Lillico) enriched withLillico paperwool (nesting material) The room environmentwas maintained at 21C 6 2C and 55 6 10 relative humidityat all times in an alternating 12-h lightndashdark cycle Rats wereremoved from cages during injection of PAN or HN reagent andblood and urine collection Animals were acclimated to the lab-oratory environment for a minimum of 5 days prior to initiationof dosing Water (purified by reverse osmosis) and CertifiedRodent Diet 5002 (PMI Feeds Inc St Louis Missouri) were pro-vided ad libitum Animals were euthanized by isoflurane gasanesthesia followed by exsanguination

Dosing In the PAN study rats were injected once intraperito-neally (ip) with 150 mgkg of PAN (Sigma-Aldrich St LouisMissouri) on study day 1 Dose and time points were selectedbased on our pilot study (data not shown) and previous publica-tions (Yu et al 2005) Control animals were injected once ipwith sterile solution of 09 saline pH 7 (adjusted with 1MNaOH) In the HN study sheep anti-Fx1A serum (Probetex SanAntonio Texas) was injected once intravenously (iv) at a dose of1 ml200 g body weight into rats on study day 1 Dose and timepoints were selected based on our pilot study (data not shown)and previous publications (Pippin et al 2009) Control rats wereinjected once iv with a sterile solution of 09 saline pH 7(adjusted with 1M NaOH)

Sample collection Animals were placed into metabolic cages for16-h overnight urine collection into chilled (4C) containers onstudy days 3 and 6 (PAN) and days 3 9 and 16 (HN) All animalswere fasted during urine collection but provided free access towater Total urine volume for each animal was recorded Urinewas centrifuged (1500 rpm for 5 min at 4C) to remove any















RESULTS

























(PurondashD3)


















DISCUSSION
























microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine




















SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3













RESULTS

























(PurondashD3)


















DISCUSSION
























microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine




















SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3

















(PurondashD3)


















DISCUSSION
























microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine




















SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3
















DISCUSSION
























microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine




















SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3














microRNA HND3LCM

HND16LCM

HND3Urine

PAND3LCM

PAND6LCM

PAND3Urine




















SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3













SUPPLEMENTARY DATA


ACKNOWLEDGMENTS




















































kfv167-TF1

kfv167-TF2

kfv167-TF3


























kfv167-TF1

kfv167-TF2

kfv167-TF3

identification of promising urinary microrna biomarkers in two rat

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