ToxSci Advance Access published August 7, 2015

IDENTIFICATION OF PROMISING URINARY MICRORNA BIOMARKERS IN TWO RAT

MODELS OF GLOMERULAR INJURY

Rounak Nassirpour*1, Bruce L Homer1, Sachin Mathur2, Yizheng Li2, Zhonghan Li1, Tom

Brown3, Deborah Carraher1, James Warneke1, Steven Bailey1, Karen Percival1, Shawn P

O’Neil1, and Laurence O Whiteley1

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1
Drug Safety, Pfizer Worldwide Research and Development, 1 Burtt Rd, Andover, MA 01810

2
Business Technology, Pfizer Research and Development, Burtt Rd, Andover, MA 01810

3
Drug Safety, Pfizer Research and Development, MS 8274 E. Point Road, Groton, CT 06340

*Corresponding author: Rounak Nassirpour

1 Burtt Rd, Andover, MA 02140

Tel: 1.978.247.2904

Email: Rounak.nassirpour@pfizer.com

Running title: urinary microRNA biomarkers of glomerular injury rat models

Keywords: microRNA; Puromycin; Heymann Nephritis; glomerular injury; biomarker; urine

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ABSTRACT

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-

transcriptionally. miRNAs play important regulatory roles in many cellular processes and have

been implicated in several diseases. Recent studies have reported significant levels of miRNAs

in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers.

Here, changes in miRNA expression patterns are described in two different rodent models of

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glomerular injury (acute puromycin aminonucleoside nephropathy and passive Heymann

nephritis). By employing two different modes of glomerular insult, oxidative stress and immune

mediated toxicity, miRNA changes in both isolated glomeruli as well as urine specimens allow

for identification of urinary miRNA biomarkers that are suggestive of drug-induced injury

specifically to the glomerulus. Subsets of glomerular urinary miRNAs associated with these

different modes of glomerular toxicity seem to be dependent on the mechanism of the induced

injury, while nine miRNAs that changed early in both glomerular and urine specimens were

common to both studies. We further show that the miRNAs identified as mechanism specific

early glomerular 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.

INTRODUCTION

Despite potentially beneficial therapeutic effects, a growing number of molecules fail in

early nonclinical development due to drug induced renal toxicity. Although, there are a number

of biomarkers available for detection of renal tubular injury, glomerular injury often goes

undetected (ANON, 2010). The current diagnostic gold standard is histological evaluation of

renal biopsy specimens. Biomarkers such as serum creatinine (Cr), estimated glomerular

filtration rate (eGFR), and/or urinary protein are routinely used to monitor renal function

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(Bonventre et al., 2010; Dieterle et al., 2010). However, by the time urinary albumin or total

protein are elevated, glomerular damage may already be irreversible or unresponsive to

therapy. Thus, sensitivity and specificity of currently available biomarkers of glomerulopathy

are mostly considered inadequate for nonclinical screening or clinical monitoring of glomerular

injury. Therefore, sensitive, accurate and early biomarkers of glomerular injury are greatly

needed.

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There is a growing interest in the role of micro-ribonucleic acids (miRNAs) in the

pathogenesis of renal diseases (Li et al., 2010), and investigations into the role miRNAs may

serve as potential biomarkers of glomerulopathy have increased. miRNAs are highly conserved,

endogenous, small (19–25 nucleotides), non-coding RNAs, that play a primary role in post-

transcriptional silencing (Khella et al., 2013). Due to their stability, miRNAs are readily

quantifiable in serum, plasma, urine and other body fluids (Scian et al., 2013). Specific miRNAs

identified to date as biomarkers are able to distinguish tissue of origin with increased diagnostic

accuracy (De Guire et al., 2013). miRNA expression has been shown to be distinct among

different anatomical regions of the kidney, and alterations in miRNA expression have been

associated with a variety of kidney diseases, including diabetic nephritis, hypertension,

glomerulonephritis, and cancer (Alvarez and DiStefano, 2013; Khella et al., 2013; Scian et al.,

2013; Wang et al., 2009). A critical role of miRNA regulation in the progression of glomerular

damage and the development of proteinuria has been suggested by studies in mice with

podocyte-specific deletion of Dicer (Harvey et al., 2008; Ho et al., 2008; Ho and Marsden,

2008; Shi, 2008), and more recently Drosha (Zhdanova et al., 2011), critical enzymes involved

in miRNA biogenesis.

In order to assess the utility of miRNA measurements in urine for detection of site-

specific renal toxicity, the Health and Environmental Sciences Institute (HESI) Biomarkers of

Nephrotoxicity Committee is conducting a collaborative program using toxicants or models that

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are selective for specific nephron segment injury. This study describes the effects on urinary

induction of miRNAs in two different rat models of glomerular injury. The objective of the rodent

glomerular injury studies reported here was to characterize the miRNA expression and excretion

profile following the induction of glomerular injury. Accordingly, we performed global miRNA

expression profiling from isolated glomeruli and urine specimens following induction of injury by

two different mechanisms: oxidative stress and immune mediated complement activation. The

aim was to determine if urinary miRNA changes would correlate with glomerular injury and

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severity in longitudinal time-course studies in two well-established models of glomerulopathy:

passive Heymann nephritis (HN; a rat model of immune-mediated glomerulopathy) and rat

puromycin aminonucleoside (PAN) podocytopathy. Although glomerular injury was induced in

rats by two very different mechanisms, podocyte injury played a critical role in the pathogenesis

and progression of both immune and non-immune mediated injury in these models (Patrakka

and Tryggvason, 2009). Podocytes, highly specialized terminally differentiated cells, and

integral components of the glomerular filtration apparatus, are highly susceptible to the injurious

effects induced by a variety of molecules, including complement, reactive oxygen species

(ROS), and nephrotoxicants (Leeuwis et al., 2010). In passive Heymann nephritis, a rat model

of human membranous nephritis, intravenous injection of an antibody against the alpha 3 beta

1-integrin matrix receptor results in formation of glomerular sub-epithelial immune deposits and

activation of the complement cascade, leading to podocyte foot process effacement and

detachment with proteinuria (Pippin et al., 2009). The combined insults of sub-lytic amounts of

complement “membrane attack complex” fragment C5b-9 and ROS induce podocyte injury and

proteinuria (Salant et al., 1980). Similarly, in PAN–induced nephrosis, a model of idiopathic

nephrotic syndrome, direct oxidative stress induces podocyte foot process effacement and

apoptosis leading to proteinuria (Rincon et al., 2004). PAN is used to induce podocyte foot

process effacement, apoptosis and detachment, leading to massive proteinuria, similar to those

described in human nephrosis (Grond et al., 1988). PAN-induced glomerular injury is mediated

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by direct DNA damage via production of reactive oxygen species (ROS). Changes in

cytoskeletal and slit diaphragm proteins lead to potentially deleterious cellular consequences in

podocytes and proteinuria (Gwinner et al., 1997). miRNA expression profiles were investigated

in isolated glomeruli and urine specimens. The ability of a subset of urinary miRNAs to

distinguish rats with or without glomerular injury was evaluated by correlating miRNA changes

with histopathologic lesions (the diagnostic benchmark), and urinary albumin and total protein

levels (Alousi et al., 1969). To confirm that the miRNA changes observed were not specific to

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the immune mediated injury model (HN), an additional well-established glomerular injury rat

model (PAN) with a different mode of injury was conducted.

PAN- and HN-induced glomerular injury in both rat models was associated with the

modulation of glomerular and urinary miRNAs. These investigations provided a means to

identify glomerular miRNA changes in urine samples that were induced by distinct mechanisms

of injury as well as a subset of glomerular miRNAs that changed expression in urine specimens

independent of the mode of glomerular injury. Therefore, these miRNAs may not only be useful

biomarkers of glomerular injury but may also distinguish the mechanism of induced injury.

Lastly, gene targets associated with urinary glomerular miRNA changes common to both modes

of injury were predicted and reported.

MATERIALS AND METHODS

Animals. Studies were conducted in accordance with the current guidelines for animal welfare

(National Research Council Guide for the Care and Use of Laboratory Animals, 2011; Animal

Welfare Act (AWA), 1966, as amended in 1970, 1976, 1985, and 1990, and the AWA

implementing regulations in Title 9, Code of Federal Regulations, Chapter 1, Subchapter A,

Parts 1-3). Procedures used in these studies were reviewed and approved by the Institutional

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Animal Care and Use Committee (Animal Care and Use Protocol AND-2011-00380). Male

Sprague Dawley rats (225-270 g, Charles River Laboratories) were maintained in a central

animal facility housed individually in polycarbonate cages with autoclaved woodchip bedding

(Lillico) enriched with Lillico paperwool (nesting material). The room environment was

maintained at 21°C ± 2°C and 55 ± 10% relative humidity at all times in an alternating 12-h

light–dark cycle. Rats were removed from cages during injection of PAN or HN reagent, and

blood and urine collection. Animals were acclimated to the laboratory environment for a

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minimum of 5 days prior to initiation of dosing. Water (purified by reverse osmosis) and Certified

Rodent Diet 5002 (PMI Feeds, Inc., St. Louis, MO) were provided ad libitum. Animals were

euthanized by isoflurane gas anesthesia followed by exsanguination.

Dosing. In the PAN study, rats were injected once intraperitoneally (ip) with 150 mg/kg of

puromycin aminonucleoside (PAN, Sigma-Aldrich, St. Louis, MO) on study day one. Dose and

time points were selected based on our pilot study (data not shown) and previous publications

(e.g. Yu et al., 2005). Control animals were injected once ip with sterile solution of 0.9% saline,

pH to 7 (adjusted with 1M NaOH). In the HN study, sheep anti-Fx1A serum (Probetex, San

Antonio, TX) was injected once intravenously (iv) at a dose of 1 ml / 200 g body weight into rats

on study day one. Dose and time points were selected based on our pilot study (data not

shown) and previous publications (e.g. Pippin et al., 2009). Control rats were injected once iv

with a sterile solution of 0.9% saline, pH to 7 (adjusted with 1M NaOH).

Sample Collection. Animals were placed into metabolic cages for 16-hour overnight urine

collection into chilled (4˚C) containers on study days 3, and 6 (PAN) and days 3, 9, and 16

(HN). All animals were fasted during urine collection, but provided free access to water. Total

urine volume for each animal was recorded. Urine was centrifuged (1500 rpm for 5 min at 4˚C)

to remove any contaminants and cellular debris, and aliquots were frozen at ≤-70˚C. Urine

albumin and total protein were normalized to the total amount excreted in the volume of urine

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collected, and to the urine creatinine concentration (mg/dl) to control for the effect of

dilution. There was no change in the amount of creatinine excreted in the urine of control or

treated animals over the time course of the studies (data not shown). Urine albumin, total

protein and creatinine were analyzed using Siemens Advia 1800 automated technology. Data

are expressed as mean ± standard deviation. Statistical differences (assumed for p < 0.05) were

assessed by one-way ANOVA (represented by a single asterisk (*) where applicable). All

graphs were generated by Prism software (GraphPad Software 6.0).

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Light Microscopic Examination. Kidneys were weighed at scheduled necropsy, and sections

were fixed overnight in 10% neutral buffered formalin, then dehydrated through graded alcohols,

cleared in xylene and infiltrated and embedded in paraffin. Serial five micron coronal sections at

the hilus were stained with hematoxylin and eosin (H&E) and Periodic Acid Schiff (PAS) reagent

with a hematoxylin counterstain. Histological sections for both kidneys included cortex, medulla

and pelvis. Histopathological evaluation was performed by a board certified veterinary

pathologist who had knowledge of the treatment groups and necropsy data (organ weights and

macroscopic observations) but who was blinded as to clinical pathology datasets, including

results from biomarker evaluations. Background findings in vehicle control animals were

identified to differentiate disease model-related glomerular findings from incidental background

spontaneous lesions, and the severity of glomerular injury was scored using a grading scale of

0-5, 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 at necropsy, a tissue slice

through the cortex was immersed in 1% glutaraldehyde and 4% formaldehyde at a volume ratio

of 10:1, fixative to tissue, and held at 4°C until processed for ultrastructural assessment. For

processing, tissues were trimmed, transferred to 0.1M phosphate buffer, post-fixed (1% buffered

osmium tetroxide, 2 hours, 4°C), and rinsed in deionized water. Samples were then dehydrated

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in graded ethanols containing propylene oxide, infiltrated and embedded in resin that was

allowed to polymerize at 60°C for 24 hours. Resin-embedded samples were sectioned at one-

half micron, stained with toluidine blue, and glomerulus-containing areas were identified. Thin

(75-90 nm) sections were cut from these areas, mounted on 200 mesh copper palladium grids,

counter-stained with uranyl acetate and lead citrate, and examined with a Hitachi 7100

transmission electron microscope (TEM) at 75kV, and digital images were captured (Advanced

Microscopy Techniques, Danvers MA). The control EM image in Figure 2D was obtained from a

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vehicle control rat that was injected ip with saline and was sacrificed at study day 11 of one of

our pilot studies.

Laser Capture Microdissection (LCM). Glomeruli were identified by phase contrast

microscopy and isolated from seven micron cryosections of kidney cortex from PAN D3 and D6

and HN D3 and D16 rats and respective controls using a Molecular Devices Arcturus XT LCM

instrument. Dissected glomeruli were captured onto Arcturus HS caps and immersed in RNA

extraction buffer.

RNA Extraction and Quantitative Polymerase Chain Reaction (qPCR). Total RNA was

isolated from 200 µL of rat urine or isolated LCM specimens from PAN D3 and D6 and HN D3

and D16 time points using a modified protocol (miRNeasy Serum/plasma RNA extraction kit,

Qiagen, Redwood City, CA) according to the manufacturer’s instructions. Briefly, 700 µL of

QIAzol reagent was added to 200 µL of urine. After vortexing vigorously with chloroform, the

samples were then centrifuged at 12,000 g for 15 min at 4˚ C. The upper aqueous phase was

transferred to a new tube and 1.5 volume of ethanol was added. The sample was then applied

to the column and washed, and the immobilized RNA was collected from the membrane with 10

µL of RNase free water. Total RNA concentration was measured at 260 nm using a NanoDrop

2000c spectrophotometer (Thermo Scientific, Waltham, MA). Quantitative miRNA analysis was

performed using TaqMan miRNA assays from Applied Biosystems. RNA was reverse

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transcribed into complementary DNA (cDNA) using megaplex primers (16°C for 30 min; 42°C

for 30 min; 85°C for 5 min). The cDNA product was then used in a pre-amplification step.

Quantitative PCR was performed using TaqMan Low Density Array (TLDA-A, Life Technologies)

in a Viia 7 thermocycler instrument (Life Technologies, Grand Island, NY) with the following

temperature profile: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Threshold cycle (Ct) values of 10 biological replicates from each experimental condition

(treatment model time point and respective controls) were extracted using GeneData

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Expressionist software, and a threshold of 32 was chosen (Ct values >32 were considered

unreliable). Since samples for each treatment time point along with the corresponding control

samples were processed in the same batch (same day, same set of arrays), 20 samples were

analyzed at a time and not merged with other time points. This avoided any batch effects that

could be introduced by analyzing multiple time points in the same assay. Unless stated

otherwise, all analyses were performed using R (Bioconductor).

Each time point consisted of 20 samples (10 control + 10 treated). miRNAs that were

not reliably detected (Ct>32) in at least 33% of the samples were discarded from the dataset.

Urine samples showed higher variability (correlation 0.1-0.9) compared to LCM samples

(correlation 0.8-0.97). Consequently, hierarchical clustering was employed, and heat maps were

generated using the Spearman correlation coefficient to detect outliers which were subsequently

removed.

Since currently no single normalization technique is commonly accepted, we compared three

methods in our datasets. Two methods (NormFinder and GENorm) use invariant miRNAs,

which iterate variance and ranking of Ct values through the dataset to find invariant miRNAs

(those that do not change across samples) and use them to normalize the entire dataset. The

third method, loess-based normalization, constructs a reference array using the mean of all

arrays and normalizes each array to the reference array. Hierarchical clustering and heat maps

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were generated for each group per time point using the normalization methods described above

to determine the best segregation between the control and treated samples. Additionally, the

criterion of coefficient of variance (CV) was used in comparing normalized values in individual

groups (control, treated) and the combined set (Supplementary Table 9). Since the CV scores

within each group should be less than the combined groups (control + treated), calculating the

increase in CV for the combined groups as a percentage of average individual group’s CVs was

used to determine which normalization method had the best separation between the control and

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treated groups (i.e. % Increase CV = 100 * CV (of the dataset (control + treated) / mean (CV

(controls) + CV (treated)). A higher %Increase CV value indicates that the normalization method

had better separation between the control and treatment groups. Loess provided the highest %

Increase CV and hence was used for the reported data here.

To increase our confidence regarding the normalization strategy chosen, delta-delta Ct

(ddCt) values were calculated for miRNAs using all three normalization methods. Invariant

miRNAs were found by NormFinder and GeNorm to calculate delta-Ct values (data not shown).

To generate delta-Ct values in loess, we used an artificial invariant miRNA that had 0 as Ct

values, and subtracted it from Ct values of other miRNAs, yielding the original values as delta-

Ct values (reported here for both LCM and urine data sets). Thereafter ddCt values were

calculated between the control and treated samples. A Welch T-test was applied to assess

statistical significance, and p-values were corrected using Benjamini-Hochberg for multiple

testing. Even though adjusted p-value <0.05 is a well-accepted standard, it may introduce false

negatives and can be overly restrictive. We relaxed the adjusted p-value cutoff to < 0.15 while

using miRNAs with p-values < 0.05. Hence miRNAs with adjusted p-value <0.15 were deemed

significantly altered in each data set. Fold changes are reported as 2^(-ddCt). The adjusted p-

value FDR <15% with a fold change cutoff of ±1.35 was used for the reported analysis

throughout the manuscript.

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Prediction of Toxicity Effects for Significantly Altered miRNAs. The miRNAs identified as

significant were analyzed separately by QIAGEN’s Ingenuity® Pathway Analysis (IPA®,

QIAGEN, Redwood City, www.qiagen.com/ingenuity). Analysis was performed on each set and

the most significant “Tox Functions” are reported.

RESULTS

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Histopathology, electron microscopy and urinary protein biomarker data confirm

induction of glomerular injury in both rat models.

HN male Sprague-Dawley rats were injected with Sheep anti-Fx1A serum and urine and

kidney specimens were collected at three time points (days 3, 9 and 16 of the study), as

depicted by the schematic in Figure 1a. Urine microalbumin and total protein (Figure 1b),

histopathology (Figure 1c) and ultrastructural analysis by electron microscopy (EM) (Figure 1d)

all confirmed injury to glomeruli. Urine total protein and albumin, assessed at each time point

(D3, D9 and D16), revealed that proteinuria was induced by D9 (Figure 1b). No morphological

evidence of tubule injury was observed for this model. Histopathologic lesions were not

detected in D3 glomeruli (Figure 1c). On D16, there was foamy lightly positive PAS-positive

material in glomerular tufts (arrows) in all 10 rats examined). EM analysis revealed

ultrastructural injury by D3, consisting of small subepithelial electron dense deposits consistent

with immune complexes (arrows), and minimal podocyte foot process swelling (Figure 1d). On

D16, changes were similar but more severe, with more prominent subepithelial dense deposits

(arrows) and associated foot process effacement (stars).

In the PAN study, urine albumin levels increased by D3, and there were significantly

increased levels of urinary total protein by D3 (Figure 2b), consistent with glomerular injury.

These urine biomarker levels correlated well with the histopathological findings. The earliest

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glomerular lesion identified by light microscopy in PAN dosed rats was observed 48 hours after

dosing (D3 of the study), and was characterized by increased numbers of PAS positive granules

within glomerular podocytes (Figure 2c). The incidence and severity, including numbers of

PAS positive granules and numbers of affected glomeruli increased from study day 3 (incidence

= 4 of 10 rats dosed with PAN) to D6 (incidence = 9 of 10 rats dosed with PAN). At study

termination on study day 6, minimal to mild hypertrophy of podocytes (arrows) and / or parietal

epithelial cells (stars) was observed in several glomeruli (Figure 2c) of PAN dosed rats. Tubular

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epithelial changes were not observed until study day 6. At day 6, intra-tubular protein and

hyaline casts were observed in 8 out of 10 rat kidneys, mostly in proximal tubules. Minimal

tubular epithelial cell changes included flattening associated with cytoplasmic attenuation,

cytoplasmic basophilia, and very occasional mitotic figures. In addition, there was evidence of

ultrastructural glomerular injury at D3 (Figure 2d), which consisted of minimal to mild podocyte

foot process effacement, well-circumscribed electron-dense cytoplasmic bodies (arrows),

consistent with protein, and microvillus extensions from podocytes into the urinary space (stars)

consistent with early podocyte hypertrophy.

Levels of miRNAs in isolated glomeruli were significantly altered following induced

glomerular injury.

After establishing that both the HN and the PAN rat models successfully induced

glomerular injury (Figure 1-2), we investigated the changes in patterns of miRNA expression

within the site of injury. Frozen kidney specimens were embedded in Optimum Cutting

Temperature - (OCT) media from the control (n=10) and treated (n=10) rats at D3 and at the last

time point for each study. LCM was employed to isolate a pure population of glomeruli from

sections collected at D3 and D6 (PAN study), as well as D3 and D16 (HN study). Glomerular

RNA was isolated at each of these time points and profiles of 376 rodent miRNAs were

generated by using a low density array qPCR platform (TLDA-A) (Figure 3a). There was a

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measurable signal below the arbitrary cut-off set at 32 qPCR cycles (Ct) for 171 + 8.4 miRNAs,

for both the control and treated samples at D3 (Supplementary Table 1). Sixty miRNAs changed

in LCM isolated glomeruli from the HN rats at D3, of which 28 were decreased and 32 increased

(Table 1). At D16, 235 + 4.5 miRNAs were detected (Table 1) out of which 119 changed

significantly in the HN rats; 64 miRNAs were decreased and 55 were significantly increased

(Supplementary Table 2). Of these, 45 miRNAs matched between the two time points.

In the PAN study, 220 + 10.3 miRNAs were detected at D3 compared to 248 + 6.2 at D6

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(Table 1). The administration of PAN led to alterations in 85 miRNAs at D3, of which 43

increased and 42 decreased (p-value < 0.05) (Supplementary Table 3). At D6, changes were

observed in 85 miRNAs; 41 were decreased while 44 were significantly increased

(Supplementary Table 4). Among these, there were 36 miRNA matches between the two time

points. Notably, 84 miRNAs were consistently modulated in both sets of glomeruli isolated from

the PAN and HN rat glomerular injury models.

Levels of miRNAs in urine were significantly changed following induced glomerular

injury.

To investigate the possibility that certain miRNAs could act as sensitive and specific

biomarkers of glomerular injury, we assessed changes in urinary miRNA expression in rat

models of glomerular injury. The transcript levels of 376 miRNAs were measured in the urine

specimens that correlated with each of the LCM-measured time points in the HN (D3 and D16)

and the PAN (D3 and D6) studies (Figure 4a).

At D3, 130 + 13.5 miRNAs were detected by qRT-PCR with Ct values < 32 in at least

20% of the urine specimens analyzed from the HN study (Table 1). A total of 62 miRNAs

changed in urine specimens collected at D3 in the HN group; 33 were increased and 30 were

decreased while one was absent in the control group and one miRNA was not detected in the

HN group (Supplementary Table 5). At D16, a total of 114 + 17.6 miRNAs were detected in

urine (Table 1); 25 of these changed significantly, of which 14 were decreased and 11 were

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increased (Supplementary Table 6). In the PAN model of glomerular injury, 101 + 9.8 miRNAs

were detected in D3 urine specimens. Significant changes were detected in 21 of these

miRNAs, including 10 miRNAs with decreased expression and 11 with increased expression

levels (Supplementary Table 7). At D6, however, 140 + 14.3 miRNAs were detected in urine

specimens from PAN rats (Table 1), with significant changes in 38 miRNAs, of which 17 were

decreased and 21 were increased. Two miRNAs detected in the PAN rats were absent in the

control samples (Supplementary Table 8). Fourteen of the miRNAs detected in urine specimens

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changed in both rat models of glomerular injury at D3.

Cross-referencing urine miRNA changes with those observed in the LCM specimens

provided further characterization of the glomerular miRNAs that not only changed at the site of

injury but were also detected in the earliest urine specimens and thus may be potential

biomarkers. In the HN study, significant changes were measured in 45 miRNAs that were

detected in both D3 urine and D3 or D16 glomeruli. In the PAN model, 14 miRNAs that

changed significantly in urine were also altered in the glomeruli at D3 or D6. Cross-study

comparison revealed changes in nine miRNAs (miR-106a, 125a-5p, 17, 218, 223, 27b, 30c,

574-3p, and 196c) that changed in both models at D3 urine samples as well as LCM specimens

(highlighted in Figure 3-4, Table 2). Interestingly, significant changes in miR-574-3p were

detected in every data set analyzed.

Prediction of miRNA targets and pathway analysis relevant to renal injury.

Ingenuity modeling of the differentially expressed miRNAs identified in the PAN and HN

specimens was undertaken to better understand the targets and pathways regulated. The

experimentally verified and predicted miRNA targets from the qRT-PCR analysis were analyzed

through Ingenuity’s Tox Function prediction. Figure 5a shows the significant (p-value <0.01)

diseases, molecular & cellular functions and nephrotoxicity functions in the cross-referenced

miRNAs in HN (44) and PAN (14) studies. The nephrotoxicity functions were obtained by using

the gene targets of miRNAs. Interestingly, cross reference analysis identified 35 miRNAs that

14
were altered significantly in HN but not PAN D3 urine specimens. In contrast, five miRNAs were

significantly altered in PAN but not HN D3 urine specimens (Figure 5b-c).

The nine common miRNAs targeted 261 genes, and cellular pathways such as ‘Renal

Necrosis’ and ’Glomerular Injury’ (p<0.019). The signaling pathways and renal toxicity related

functions are illustrated in Figure 5d, along with the miRNAs and their targets.

To investigate if there was any difference in HN and PAN mechanisms, the HN-only

(Figure 5b) and PAN-only miRNAs (Figure 5c) were analyzed. The HN-only (35) miRNAs

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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 different sets of miRNAs might be functionally related. Some of the biological

functions enriched in HN-only targets were immune mediated (Labeled by IPA as Organismal

Injury), however B-cell receptor signaling was among the top 5 hits (p value <1E-10) for PAN-

only targets.

The analysis also revealed an interesting trend in the data set which we tried to capture

in Figure 5e and Table 2. The direction of change in tissue versus urine was inverse for most of

the nine miRNAs that changed in both models of glomerular injury. For the expression pairing

analysis, the urinary miRNA changes at D3 were cross referenced to the LCM changes that

occurred either at D3 or D6/D16. Thus, miRNAs that were decreased in glomeruli were highly

increased in corresponding urine specimens, possibly implying a link between induced tissue

injury and release of the miRNAs in the biofluid.

DISCUSSION

We have described glomerular miRNA changes that were induced by two different

modes of insult and were altered in early urine specimens, and may thus potentially serve as

early, site-specific, and mechanism-related biomarkers of glomerular injury. In the immune-

15
mediated HN model, highly reproducible, progressive, concordant changes in 45 miRNAs were

observed in the earliest urine specimens analyzed as well as isolated D3 and D16 glomeruli.

Similarly, treatment with a single high dose of puromycin resulted in 14 miRNA changes in D3

urine that were associated with altered expression patterns in the isolated glomeruli collected at

D3 and D6. These miRNA alterations correlated with histologic findings of glomerular injury and

with increases in concentrations of benchmark protein biomarkers.

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miRNAs are short (~22 nucleotides) non-coding RNAs that regulate translational

repression of their target transcripts. miRNAs are transcribed as much longer precursors which

are sequentially processed by two different RNase 3 enzymes, Drosha and Dicer, to their

mature forms. Mice with podocyte-specific deletions of these miRNA processing enzymes

suffer from progressive glomerular and tubular defects and, hence, miRNAs are believed to play

pivotal roles in normal renal physiology as well as various pathological processes in the kidney.

Due to specificity in patterns of cell and tissue expression and remarkable stability in various

biofluids, miRNAs have gained popularity as biomarkers of various diseases including renal

disorders. miRNA changes within the tissue of origin must also be detectable in a biofluid

before the miRNA may be proposed as a biomarker of injury. Employing two established rat

models that induce glomerular injury by different mechanisms, we identified several miRNA

changes in LCM-isolated glomeruli as well as urine specimens collected during early stages of

glomerular injury by PCR-based genomic profiling.

We had reported previously inter-platform differences between qPCR and Next

generation Sequencing (NGS) platforms for profiling urinary miRNAs, namely that although

NGS might be a more accurate platform due to its ability to identify miRNA isoforms, qPCR

seems to be a more sensitive profiling technique (Nassirpour et al., 2014). We therefore

employed low-density arrays for quantitative real-time PCR (TLDA) for miRNA expression

investigations reported here, with the caveat that miRNAs expressed in their isomeric forms are
16
probably unfortunately missed. As with any other gene expression profiling, effective analysis

methods to produce reliable and high-quality results include normalization (Deo et al., 2011).

Adequate normalization minimizes the effects of systematic, as well as measurement and

technical errors and variations, and is critical for proper biological interpretations. However, due

to the nature of in vivo studies, low concentrations of ribonucleic acids in urine collected from

metabolic cages, incomplete understanding of the source and biology of miRNAs in biofluids,

and low mean concordance in miRNA normalization platforms, additional studies will be

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necessary to identify the most suitable techniques for miRNA normalization (Mohammadian et

al., 2013). Previously, we investigated the impact of different normalization methods on intra-

and inter-platform performance of two distinct and commonly used miRNA profiling platforms,

namely qPCR and NGS (Nassirpour et al., 2014). In this study we compared the performance

of three different normalization methods. NormFinder (Andersen et al., 2004) and geNorm

(Vandesompele et al., 2002), which are commonly used and reported in various profiling

studies, employ the variance and ranking of Ct values to identify non-variant miRNAs against

which to normalize the dataset. The third method, locally weighted scatterplot smoothing

(Loess, (Cleveland and Devlin, 1988)), is a nonparametric local regression model that

constructs a reference array using the mean of all arrays and normalizes each array to this

reference array. This method showed the best performance in hierarchical clustering and heat

map generation for each treatment per time point, measuring segregation among samples, and

increasing the correlation among replicate samples better than the other two non-variant

methods. Thus, as reported previously in other miRNA tissue profiling studies (Meyer et al.,

2012), we propose Loess normalization for identification of differentially expressed miRNAs in

urine, as it seemed to minimize standard deviations and increased the area under the ROC

curve, both of which are established measures of statistical performance. In addition, we found

that locally weighted scatterplot smoothing also increased inter-platform concordance of

17
differential expression, further endorsing Loess as our choice of normalization method

employed in this study (Nassirpour et al., 2014).

While each study revealed a subset of miRNAs that seem to be dependent on the

induced mechanism of injury, nine miRNAs, including miR-106a, 125a-5p, 17, 218, 223, 27b,

30c, 574-3p and 196c, changed in an insult-independent manner. Therefore, these miRNAs are

proposed as candidate urinary glomerular injury biomarkers. Furthermore, although the

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transport mechanisms or mechanisms of excretion of glomerular or circulating miRNAs into the

urinary space are not well elucidated, the direction of change for these miRNAs was noteworthy,

as the miRNAs that were increased in the glomeruli were decreased in urine specimens, and

vice versa. It is thus plausible that the increase in miRNA expression in urine may be the result

of cell death in glomeruli (for example, podocyte) or active secretion from the surviving cells.

Additional studies are needed to localize the cellular source of the miRNAs that were identified

in urine in these rat models of glomerular injury.

Among the miRNA biomarkers that were common to both studies, miR-574-3p was

unique in that its expression was altered in the glomeruli and urine specimens collected at every

time point. Interestingly, miR-574-3p was also shown by Konta et al. (2014) to be the only

miRNA that was significantly changed in clinical urine samples collected from patients with four

different renal diseases. miRNAs 30a-c, 194, 197 and 200c, which changed in both glomeruli

and urine specimens in these rat models, were also proposed as potential urinary biomarkers of

diabetic nephropathy in the Konta study; thus, the glomerular biomarkers we identified in these

preclinical rat models might have clinical implications and warrant further investigation.

Although we know very little about the role of miR-574-3p in renal diseases, it has been

implicated as an inhibitor of differentiation of multipotent mesenchymal stromal cells into

chondrocytes (Guérit et al., 2013), has been proposed as a tumor suppressor in gastric (Su et

al., 2012) and bladder cancer cells (Tatarano et al., 2012), and has been shown to negatively
18
regulate the proliferation of keratinocytes (Chikh et al., 2011). Therefore, it is possible that this

miRNA also plays a role in renal function and may influence podocyte differentiation. However,

this miRNA was also reported as altered in urine samples analyzed after gentamicin induced

tubule injury (Nassirpour et al, 2014). Therefore, we cannot propose this miRNA as glomerular

specific. Comparing the nine miRNAs identified in this manuscript against other published

tubule injury rat models will also shed light on their utility as glomerular specific biomarkers. For

example, miR-17 and 218 were also altered in urine analyzed from rats with cisplatin induced

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tubular injury (Kanki et al., 2014). Similarly, Pavkovic et al. (2014) reported urinary changes in

miR-196c and 223 with cisplatin. However, in both cases their analysis focused on later time

points and after extensive tubular injury. Unfortunately, since glomerular toxicity was not

assessed in these studies we are unable to assess their site specificity. Interestingly, Pavkovic

et al. (2015) recently identified five miRNAs in a glomerular rat injury model induced by

nephrotoxic serum. All 5 were also detected at multiple time points in our glomerular injury

models. However, since our objective was to identify early biomarkers of injury, we focused our

efforts on early time points (day 3), before morphologically significant induced glomerular injury

occured.

The majority of the differentially-expressed glomerular and urine miRNA biomarkers

identified here have also been shown to be highly enriched in the kidney and conserved across

species (Saal and Harvey, 2009). A recent review (Khella et al., 2013) summarized the

association of several miRNA families and transcripts with renal physiology and disease,

including: the miR-30 family, shown to be crucial for podocyte functions; roles for the miR-200

family and miR-17 in polycystic kidney disease; roles for the miR-29 family and miR-192 in renal

fibrosis; and the involvement of miR-192 and miR-27 in the pathogenesis of lupus nephritis.

Additionally, miR-223 appears to regulate key pathways in IgA nephropathy (Bao et al., 2014)

and may serve as a biomarker for human allograft rejection (Anglicheau et al., 2009). Lai et al.

(2015) have also demonstrated that glomerular miR-21 expression is positively associated with

19
albumin-to-creatinine ratios in patients with diabetic nephropathy (DN) and that loss of miR-21 is

associated with accelerated glomerular damage and podocyte apoptosis in a murine model of

DN and Tgfb1-TG mice. Similarly, Ichii et al. (2014) have shown that miR-26a regulates

podocyte differentiation and cytoskeletal integrity, and its altered levels in glomerulus and urine

may serve as a marker of injured podocytes in autoimmune glomerulonephritis. Therefore, the

miRNA biomarkers proposed here may play important roles in renal physiology and in the

development of renal diseases, as well as serving as novel and site-specific biomarker

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candidates of glomerular injury. In fact, several global consortia, such as HESI, are comparing

site-specific induced pre-clinical toxicological studies, such as this one, against studies reported

by other nephrotoxicants and in different animal models to evaluate the utility of these proposed

biomarkers in drug-induced toxicological evaluations. Methodical comparisons with respect to

the degree of induced toxicity as well as characterizations of the site of induced injury within a

nephron and technical and analytical strategies used are critical in obtaining a better

understanding of applicability of the proposed miRNA biomarkers.

In the current study, we compared the timing of onset for biomarker alterations of the

urinary miRNAs that we identified versus established protein urine biomarkers (e.g.,

microalbumin and total protein), and against benchmark histopathology. In the PAN model, the

rapid progression of the injury that was induced did not provide sufficient time to assess whether

urine miRNA biomarkers would change prior to increases in urine protein biomarkers. However,

in the more slowly developing, immune-mediated HN model, miRNA changes at D3 preceded

significant increase in urine albumin or total protein. Therefore, the differentially expressed

miRNAs that we identified during the early stages of passive HN may hold promise for

improving early identification of drug-induced immune injury. Furthermore, pathway analysis

revealed close association between the nine miRNA biomarkers of glomerular injury and target

genes linked to glomerular injury, inflammation and apoptosis. Therefore, due to their high

20
translatability and conservation across species, miRNA transcripts that respond very early after

drug-induced injury in rodents might have higher probability of human translation, in addition to

being more specific for podocyte or glomerular injury than the functional glomerular injury

biomarkers that are currently in use.

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Table 1. Summary of miRNA analysis observed with nephrotoxicants.

Condition Detected Increased Decreased

HN-D3 171 32 28

HN-16 241 55 65

LCM Puromycin-D3 220 43 42

Puromycin-D6 252 44 41

HN-D3 133 33 30

HN-16 116 11 14

Urine Puromycin-D3 104 11 10

Puromycin-D6 140 21 17

21
 Table 2. Summary of the significantly altered glomerular miRNA urinary changes observed in

both glomerular injury rat models. LCM and urinary miRNA changes are highlighted according

to their direction (green = positive; red = negative).

HN HN HN PAN PAN PAN

microRNA
D3 LCM D16 LCM D3 Urine D3 LCM D6 LCM D3 Urine

mmu-miR-106a-5p 1.07 -3.30 -1.70 -1.27 -1.28 1.39

mmu-miR-223-3p 1.01 2.07 -6.62 -1.45 -1.14 2.41

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mmu-miR-574-3p -1.58 1.82 1.55 -1.32 -1.80 -3.43

rno-miR-125a-5p -1.12 2.22 3.32 1.19 -1.21 -2.51

rno-miR-17-5p 1.00 -3.70 -4.74 -1.21 -1.19 1.60

rno-miR-196c-5p -1.03 1.52 5.09 -1.18 -1.24 -3.13

rno-miR-218a-5p -1.52 -1.13 1.83 -1.56 -1.68 1.57

rno-miR-27b-3p 1.16 -1.42 2.45 1.10 1.21 -1.51

rno-miR-30c-5p 1.17 -1.50 1.50 1.50 1.25 -1.54

22
FIGURE LEGENDS

Figure 1. Passive Heymann Nephritis 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 (normalized to urine creatinine), indicate renal

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injury at day 9 (D9, Mean +/- SD; * denotes p value < 0.05; ** denotes p value < 0.01). (c)

Compared to vehicle-dosed rats (Control), day 16 (HN-D16) but not day 3 (HN-D3) rats had

foamy lightly positive PAS-positive material in glomerular tufts (arrows). PAS stain, scale bars =

100 um. (d) Compared to 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 magnifications 5,000x (Control) and 10,000x (HN-D3 and HN-D16).

Scale bar = 500 nm.

Figure 2. Puromycin-induced glomerulopathy in rats.

(a) Study design depicting vehicle control male Sprague Dawley and rats dosed with 150 mg/kg

puromycin aminonucleoside (PAN). (b) Urinary protein and microalbumin (normalized to urine

creatinine), indicate renal injury at day 3 (D3) and day 6 (D6; Mean +/- SD; * denotes p value <

0.05; ** denotes p value < 0.01). (c) Puromycin-induced glomerulopathy in rats. Study day 3

(Control rat glomerulus) with PAS stain. Study day 3 (Puro–D3); PAN-dosed rat glomerulus

with prominent PAS positive granules in podocytes (arrows). Study day 6 (Puro–D6); PAN-

dosed glomerulus with several hypertrophied podocytes (arrows) and parietal epithelial cells

(stars), characterized by minimally enlarged nuclei and increased amount of cytoplasm. H&E

23
stain. Scale bars = 50 um. (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) = 500 nm.

Scale bar (PAN) = 1060 nm. Original negative magnifications: 5000x (control) and 6000x (Puro-

D3).

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Figure 3. Relative quantification of miRNA responders in rat glomeruli with

nephrotoxicity.

(a) Workflow of qRT-PCR analysis for the laser capture microdissection isolated glomeruli. (b)

Volcano plots shows miRNAs that are significantly regulated at day 3 and 16 in the immune

mediated Heymann Nephritis (HN) rat model of glomerular injury (FDR < 0.15 and FC > 1.35 in

either direction). (c) Volcano plots shows miRNAs that are significantly regulated at day 3 and 6

post Puromycin (Puro) induced glomerular injury (FDR < 0.15 and FC > 1.35 either direction).

Red denotes glomerular miRNA changes that were also detected in urine specimen. Horizontal

line: P-value 0.05; vertical lines: FC at -1.3 and 1.3. Data are normalized using loess

normalization.

Figure 4. Relative quantification of miRNA responders in urine with nephrotoxicity.

(a) Workflow of qRT-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 (HN) rat model of glomerular injury (FDR < 0.15 and FC > 1.35 either direction). (c)

Volcano plots show urinary miRNAs that are significantly regulated at day 3 and 6 post

Puromycin (Puro) induced glomerular injury (FDR < 0.15 and FC > 1.35 either direction). Red

denotes glomerular miRNA changes that were also detected in urine specimen. Horizontal line:

P-value 0.05; vertical lines: FC at -1.3 and 1.3. Data are normalized using loess normalization.

24
Figure 5. Prediction of renal functions for miRNA biomarkers of glomerular injury.

(a) Diseases, molecular & cellular functions as well as nephrotoxicity functions in the cross-

referenced miRNAs in HN (44) and PAN (14) studies were analyzed through Ingenuity’s Tox

Function prediction (p-value <0.01). (b) The nephrotoxicity functions were obtained by using the

gene targets of the 35 miRNAs that changed only in HN D3 urine. (c) The corresponding

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number in PAN, but not in HN D3 urine was 5 and their targets and nephrotoxicity functions

depict glomerular injury. (d) The nine common miRNAs targeted 261 genes, and cellular

pathways such as ‘Renal Necrosis’ and ’Glomerular Injury’ (p<0.019). (e) Directionality of

change in miRNA expression patterns in tissue vs. urine for the nine common miRNAs as

observed in the isolated glomeruli and D3 urine specimen.

25
SUPPLEMENTARY DATA DESCRIPTION

Tables 1-8 describe glomerular and urinary miRNA alterations observed relative to control at D3

and D6 in rats treated with puromycin and D3 and D16 in the Heymann nephritis model. Table 9

compares the performance of the three normalization methods investigated for the data sets in

tables 1-8.

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ACKNOWLEDGEMENTS

This study was conducted as part of Pfizer’s Glomerular Injury Biomarker team efforts

and we would like to acknowledge all present and past members for their various contributions

to this project, with special thanks to Shashi Ramaiah, Zaher Radi, Patrick Lappin, Eva Nagiec,

and Deborah Burt. We would like to extend our gratitude to Dr. Dale Morris, Dr. Denise

Robinson-Gravatt and Pfizer’s Science and Technology Board, for their generous help, support,

and commitment. This study supports the efforts of the HESI Biomarkers of Nephrotoxicity

Committee. The Health and Environmental Sciences Institute (HESI) is a nonprofit institution

whose mission is to engage scientists from academia, government and industry to identify and

resolve global health and environmental issues. We would like to especially acknowledge Dr.

Jean-Charles Gautier for his critical reading of our manuscript. Additionally, we would like to

thank Mr. Edward Germond, who as a talented summer intern significantly contributed to data

acquisition from a pilot puromycin miRNA study (data not shown).

26
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Figure 1. Passive Heymann Nephritis 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 (normalized to urine creatinine), indicate renal injury at day 9 (D9, Mean +/- SD;
* denotes p value < 0.05; ** denotes p value < 0.01). (c) Compared to vehicle-dosed rats (Control), day
16 (HN-D16) but not day 3 (HN-D3) rats had foamy lightly positive PAS-positive material in glomerular tufts
(arrows). PAS stain, scale bars = 100 um. (d) Compared to 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 magnifications 5,000x (Control) and 10,000x (HN-D3 and HN-D16). Scale bar = 500 nm.

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Figure 2. Puromycin-induced glomerulopathy in rats.
(a) Study design depicting vehicle control male Sprague Dawley and rats dosed with 150 mg/kg puromycin
aminonucleoside (PAN). (b) Urinary protein and microalbumin (normalized to urine creatinine), indicate
renal injury at day 3 (D3) and day 6 (D6; Mean +/- SD; * denotes p value < 0.05; ** denotes p value <
0.01). (c) Puromycin-induced glomerulopathy in rats. Study day 3 (Control rat glomerulus) with PAS
stain. Study day 3 (Puro–D3); PAN-dosed rat glomerulus with prominent PAS positive granules in podocytes
(arrows). Study day 6 (Puro–D6); PAN-dosed glomerulus with several hypertrophied podocytes (arrows) and
parietal epithelial cells (stars), characterized by minimally enlarged nuclei and increased amount of
cytoplasm. H&E stain. Scale bars = 50 um. (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) = 500 nm. Scale bar (PAN) =
1060 nm. Original negative magnifications: 5000x (control) and 6000x (Puro-D3).
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Figure 3. Relative quantification of miRNAs responders in rat glomeruli with nephrotoxicity.
(a) Workflow of qRT-PCR analysis for the laser capture microdissection isolated glomeruli. (b) Volcano plots
shows miRNAs that are significantly regulated at day 3 and 16 in the immune mediated Heymann Nephritis
rat model of glomerular injury (FDR < 0.15 and FC > 1.3 in either direction). (c) Volcano plots shows
miRNAs that are significantly regulated at day 3 and 6 post Puromycin induced glomerular injury (FDR <
0.15 and FC > 1.3 either direction). Red denotes glomerular miRNA changes that were also detected in urine
specimen. Horizontal line: P-value 0.05; vertical lines: FC at -1.3 and 1.3. Data are normalized using loess
normalization.

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Figure 4. Relative quantification of miRNAs responders in urine with nephrotoxicity.
(a) Workflow of qRT-PCR analysis for the urine specimen. (b) Volcano plots shows urinary miRNAs that are
significantly regulated at day 3 and 16 in the immune mediated Heymann Nephritis rat model of glomerular
injury (FDR < 0.15 and FC > 1.3 either direction). (c) Volcano plots shows urinary miRNAs that are
significantly regulated at day 3 and 6 post Puromycin induced glomerular injury (FDR < 0.15 and FC > 1.3
either direction). Red denotes glomerular miRNA changes that were also detected in urine specimen.
Horizontal line: P-value 0.05; vertical lines: FC at -1.3 and 1.3. Data are normalized using loess
normalization.

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Figure 5. Prediction of renal functions for miRNA biomarkers of glomerular injury.
(a) Diseases, molecular & cellular functions as well as nephrotoxicity functions in the cross-referenced
miRNAs in HN (44) and PAN (14) studies were analyzed through Ingenuity’s Tox Function prediction (p-
value <0.01). (b) The nephrotoxicity functions were obtained by using the gene targets of the 35 miRNAs
that changed only in HN D3 urine. (c) The corresponding number in PAN, but not in HN D3 urine was 5 and
their targets and nephrotoxicity functions depict glomerular injury. (d) The nine common miRNAs targeted
261 genes, and cellular pathways such as ‘Renal Necrosis’ and ’Glomerular Injury’ (p<0.019). (e)
Directionality of change in miRNA expression patterns in tissue vs. urine for the nine common miRNAs as
observed in the isolated glomeruli and D3 urine specimen.

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