Supplementary Materials for - Science Translational Medicine · 2012-02-13 · Supplementary...

www.sciencetranslationalmedicine.org/cgi/content/full/4/121/121ra18/DC1

Supplementary Materials for

MicroRNA-21 Promotes Fibrosis of the Kidney by Silencing Metabolic Pathways

B. Nelson Chau,* Cuiyan Xin, Jochen Hartner, Shuyu Ren, Ana P. Castano, Geoffrey

Linn, Jian Li, Phong T. Tran, Vivek Kaimal, Xinqiang Huang, Aaron N. Chang, Shenyang Li, Aarti Kalra, Monica Grafals, Didier Portilla, Deidre A. MacKenna, Stuart

H. Orkin, Jeremy S. Duffield*

*To whom correspondence should be addressed. E-mail: [email protected] (J.S.D.); [email protected] (B.N.C.)

Published 15 February 2012, Sci. Transl. Med. 4, 121ra18 (2012)

DOI: 10.1126/scitranslmed.3003205

This PDF file includes:

Materials and Methods Fig. S1. Up-regulation of miRNAs in kidney injury models. Fig. S2. Purification of kidney cells by flow cytometric sorting. Fig. S3. Characterization of miR-21–deficient mice. Fig. S4. Inhibition of miR-21 by the anti–miR-21 oligonucleotide in vitro in HeLa cells as detected using a miR-21–specific luciferase reporter assay. Fig. S5. ERK phosphorylation in whole kidney of miR-21 knockout and anti–miR-21–treated mice, and examination of miR-21 target genes in whole kidney and in kidney myofibroblasts. Fig. S6. Lipid metabolic genes are up-regulated in Ppara transgenic mice. Fig. S7. Anti–miR-21 administration to primary epithelial cultures or systemic administration to mice derepresses critical genes identified in miR-21−/− mice including PPARα. Table S1. List of up-regulated miRNAs. Table S2 legend Table S3. Primers used to detect genes by qPCR. References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/4/121/121ra18/DC1)

Table S2. List of seed-matched target derepressed mRNA in UUO kidneys from miR-21+/+ compared with miR-21−/− mice. (Excel file)

CHAU ET AL, 2012

1 miR-21 Promotes Fibrosis

Supplementary Data

Supplementary Materials and Methods

Mouse models of fibrosis

Unilateral ureteric obstruction (UUO) was performed in adult (8-12wk) mice as described (S1). Briefly,

under anaesthesia by ketamine/xylazine (100/10 mg/kg i.p), the left ureter was exposed through flank

incision in the prone position. The ureter was ligated twice using 4-0 nylon surgical sutures at the level of

the lower pole of kidney. In some experiments, sham operation was performed by flank incision only.

The unilateral IRI model was performed as described (S2) with a 30-minute ischemic time at 36.8-

37.3°C core temperature. In some experiments the healthy kidney was removed at 7d after IRI. Urine

was collected and quantified for albumin and urine creatinine. In experiments performed on Kap2-

Ppara transgenic female or strain matched control mice a 5mg pellet of testosterone, which lasts 21 days

was placed subcutaneously for the duration of the experiment, five days prior to surgery (S3).

Human Studies

Biopsies were taken from patients undergoing kidney donation for transplantation (normal) or

undergoing a kidney transplant biopsy in the course of their medical management under an IRB

approved protocol (DR-1 LCID-2010-044 at the Lahey Clinic). Biopsies were placed immediately in

RNA later, stored at 4ºC for 24h prior to storage at -80ºC. A second core was scored by a Renal

Pathologist for diagnosis and extent of fibrosis. A diagnosis of chronic allograft dysfunction (CAD) or

acute kidney injury (AKI) was made according to standard criteria.

CHAU ET AL, 2012


Cell purification, culture and in vitro assays

Purification of cells from kidney. Cell purification from normal and diseased kidney was described

previously (S4). Briefly the kidney was decapsulated, diced, then incubated at 37ºC for 30 min with

liberase DL (0.5 mg/ml, Roche) and DNase (100U/ml, Roche) in serum free DMEM. After

centrifugation, cells were resuspended in 5ml of PBS/1% BSA, and filtered (40µm).

Pericytes/myofibroblasts were purified from the single cell suspension isolating Coll-GFP+ cells in Coll-

GFP mice using FACSAria cell sorting, then total RNA was isolated from RLT buffer or Trizol. Other

cells were separated using specific antibodies or lectins added to the single cell suspension: (anti-CD11b-

APC (1:400, Ebioscience) for macrophages, anti-CD31-PE (1:400 EBioscience) for endothelial cells,

lotus lectin-fluorescein (1:200, DAKO) for proximal epithelium and anti-Kim1-biotin (1:200,

EBioscience) followed by streptavidin-APC (1:2000, Jackson Immunoresearch) for injured proximal

epithelium. Negative gates were set to exclude other cell types from the positive selection.

Purification and culture of myofibroblasts from kidney. Purification and culture of kidney

myofibroblasts from kidney d7 after UUO was described previously (S2) The primary cultured cells

used in this study were between passages 4 and 6, cultured in DMEM/F12 with 10% FCS and have been

characterized previously (2) (9). For transcriptional analysis, total RNA was isolated from sorted UUO

kidney myofibroblasts using RNeasy Mini Kit (Qiagen). Cells were transfected with anti-miR21 or

control anti-miR using Lipofectamine RNAiMAX reagent (Invitrogen) and incubated for 48h. Total

RNA was isolated using Trizol reagent and silencing of >90% was confirmed by Q-PCR as described

above.

Primary kidney epithelial cell culture. Digested kidneys were prepared as previously described.

Digested tubules were cultured in RPMI medium supplemented with ITS and hydrocortisone (S5) but no

serum for 7-10 days until confluent monolayers were achieved. Cells were not passaged.

Migration assay. Confluent monolayers of typical cobblestone appearing kidney epithelial cells were

used. A scratch ‘wound’ was performed using a 200µl pipet tip, wells were washed X2 with epithelial

culture medium. At pre-fixed points along the scratches photomicrographs were performed at 0h and

CHAU ET AL, 2012


24h after initialization of the wound. Migration of cells and quantification of the wound area and its

closure after 24h was analyzed using ImageJ software.

Hypoxia & TGFβ stimulation assays. Cells were incubated for 8 or 22h in a hypoxic chamber with

normal CO2 levels. The level of oxygen was <0.3%. Cells were harvested in Trizol. TGFβ stimulation.

Confluent monolayers were washed with fresh medium, then TGFβ (Peprotech) was added at 10ng/ml).

In vitro reporter for anti-miR-21

HeLa cells stably transfected with a luciferase reporter with a perfect-matched miR-21 binding sites

were transfected with indicated concentration of anti-miR-21 or a control anti-miR oligonucleotide

(highest dose). 24 hours post-transfection, normalized luciferase activities were determined.

In situ Hybridization

PFA fixed paraffin embedded 3µm sections were prepared in RNAse free environment. Tissues sections

were stained with LNA-modified miR-21 probes or control probes (Exiquon) and performed according

to protocol previously reported (S6).

Tissue preparation and histology

Mouse tissues were prepared and stained as previously described (S7). Briefly, tissues for cryosectioning

(7µm) were fixed in PLP solution for 2h, then washed in 18% sucrose solution overnight prior to cryo-

preservation. Tissues for paraffin sectioning (3µm) were fixed for 12h in 10% formalin solution prior to

70% ethanol solution. For fluorescence detection, primary antibodies against the following proteins were

used for immunolabeling: αSMA-Cy3 (1:200, clone 1A4, Sigma), (1:200, Abcam, Cambridge, UK),

F4/80 (1:300 Invitrogen), CD31 (1:300, BD), Pparα (1:200, Abcam), Mpv17l (1:200 Gift), Reck (1:200,

Cell Signaling Technology). Fluorescent conjugated affinity purified secondary antibody labeling

CHAU ET AL, 2012


(1:400-1:800, Jackson Immunoresearch), co-labeled with DAPI, mounting with Vectashield/DAPI,

image capture and processing were carried out as previously described (S7). To detect 8-oxo-DG

paraffin sections were re-hydrated, treated with hydrochloric acid 2N, 10min followed by quenching

with sodium borate. Primary antibodies (anti-8-oxo-DG 1:200, Abcam) were incubated 4°C overnight in

BSA buffer followed by secondary Cy3-conjugated Ab (1:500 JacksonImmunoresearch), then washing

and mounting as above. Positive nuclei were identified by red color within blue labeled nuclei. Detection

of apoptotic cells was performed on paraffin sections as previously described and quantification was

performed as previously described (S8). Quantification of specific cells in tissue sections was carried out

as previously described. In brief, sections were co-labeled with DAPI, cells were identified by blue and

green nuclear co-localization; αSMA+, and F4/80+ cells were identified by greater than 75% of the cell

area immediately surrounding nuclei (detected by DAPI) staining positive with Cy3 fluorescence

indicative of the antigen expression; Ki-67+ cells were identified by positive nuclear staining for Cy3

fluorescence. Specific cells were counted in 10 cortical interstitial fields randomly selected at 400X

magnification per mouse. Vessel fluorescence was analyzed in images at 400X magnification captured

from CD31-stained sections of 10 different fields from 6 different animals. Based on fluorescence

intensities ranging from 0 to 255, peritubular capillaries were distinguished from background by

empirically determining threshold values that marked only blood vessels in specimens from control

kidney in sham-operated mice. The threshold was constant for all measurement (S9). Interstitial fibrosis

was quantified in picrosirius red-stained paraffin sections. The morphometry of CD31+ peritubular

capillary and picrosirius red+ collagen was quantified using Photoshop (Adobe) as described previously

(S10). Epithelial injury was quantified in PAS stained kidney sections. Epithelial injury scores were

previously described (S11). Brush border scores were obtained by counting brush-border positive

epithelial cells in sequential images across the entire sagittal section of each kidney.

CHAU ET AL, 2012


Western blot analysis

Kidneys were homogenized in ice-cold lysis buffer (50mM Tris/HCl, pH 7.4, 150mM NaCl, 10%

glycerol, 1% Triton X100, 2mM EDTA, 2mM EGTA, 40mM b-glycerophosphate, 50mM sodium

fluoride, 10ug/ml leupeptin, 10ug/ml aprotinin, 1uM pepstatin A, 1mM phenylmethyl sulphonyl

fluoride) and homogenized by 10 passes through an 18G-needle fitted to a 1ml syringe. Samples were

centrifuged for 10 min at 14000 x g and the supernatant was taken for protein determination. Cell

extracts containing 50µg of protein were prepared in SDS-sample buffer and subjected to SDS-PAGE.

Proteins were transferred on to nitrocellulose paper. After the transfer, immunostaining was performed

as previously described in detail. Antibodies were diluted with 1:1000 in blocking buffer. Bands were

detected by the enhanced chemiluminescence (ECL) method (Pierce) as recommended by the

manufacturer and luminescence captured by FluorChem-Q (ProteinSimple). The following primary

antibodies were used to detect the specific protein: anti-Pparα (Abcam), anti-Mpv17l (Gift), anti-Reck

(Cell Signaling), anti-Sprouty1 (Abcam), anti-NFIA, anti-NFIB, anti-Cyr61 (Abcam), anti-Timp3 (Cell

signaling), anti-Actin (SantaCruz), anti-pERK1/2 and anti-ERK1/2 (Cell signaling), anti-Kim1/Tim1

(EBioscience).

Isoelectric focusing

Whole kidneys were lysed in bicine/CHAPS lysis buffer and mixed with gel containing fluorescent pI

markers. The mix is drawn into capillary tubes and placed across cathode and anode buffers in a

NanoPro machine (ProteinSimple). Current is applied across the tubes until the all proteins have stopped

migrating. After fixation with UV light, anti-ERK antibodies (1:200, Cell Signaling) are applied

followed by anti-IgG-HRP conjugates followed by a chemiluminescence-based detection. The tubes are

imaged by a camera in situ to detect the proteins.

CHAU ET AL, 2012


RT-PCR

Total RNA was extracted using Trizol. Purity was determined by A260 to A280. cDNA was

synthesized using oligo(dT) and random primers (iScript, Biorad). Quantitative PCR was performed

using ABI machine, iTaq SYBR Green supermix with ROX (Biorad) using methods described

previously (S11). The specific primer pairs used in Q-PCR were tested for specificity and are listed in

Table S3. For RT-PCR for microRNAs, RNA was extracted using Trizol. For some experiments an

initial reaction was performed to anneal polyA sequences to all RNA (polyA tailing) then cDNA was

generated using conventional Superscript-II RT and a universal primer (Invitrogen). Q-PCR for miR-21

or the housekeeping small nucleolar RNA Sno234 was performed using the reverse universal primer

specific to the annealed polyA tail and the specific forward primer to the miR-21 or Sno234 unique

sequence (See Table S3). Other primers used are inventoried assays obtained from Applied Biosystems.

Microarray analysis

Total RNA from tissues were extracted as per manufacturer’s instructions (miREASY kit, Qiagen).

mRNA expression profiles were determined using Mouse Genome 430 2.0 array (Affymetrix) per

manufacturer’s instruction. mRNA microarrays were run in triplicates. microRNA arrays were from

Agilent and were performed on total RNA purified from cells or tissues lysed in Trizol.

CHAU ET AL, 2012


Supplementary Figures

CHAU ET AL, 2012


microRNAs that were changed at p<0.01 (FDR corrected) by comparing the day 10 of IRI or

UUO to sham. A common set of 24 miRNAs were regulated in both models. (B) Heat maps

(blue low and red high) showing relative intensity (expression level) of each sample in focusing

on the 24 miRNAs. Note miR-21 standouts as a highly expressed miRNA (pinkish-red color) in

normal and it was further induced). (C) In situ hybridization showing miR-21 expression (purple)

in sham and d7 after UUO kidney. Note highest expression in the interstitium of UUO kidney, but

epithelium is shows positive signal. WT kidneys are shown alongside miR-21-/- kidneys for

specificity. A positive control detecting the nuclear small RNA U6 is shown (g = glomerulus, bar

= 10µm). (D-F) The effect of hypoxia or TGFβ1 stimulation on miR-21 expression in primary

cultures of kidney proximal epithelial cells (PTECs) as detected by Q-PCR.

Fig. S1. Up-regulation of miRNAs in kidney injury models. (A) Venn diagram showing identified

CHAU ET AL, 2012


Fig. S2. Purification of kidney cells by flow cytometric sorting. (A) Total kidney cells from single

cell preparation are selected for viable cells and singularity by initial forward and side scatter

gates (B) Example of fluorescent channels used to select macrophages from d2 after UUO. NK1.1

and Ly6G + cells are selected and excluded from collection. CD11b+ cells are then collected. (C)

Example of endothelial cell collection from normal kidney. CD31 + cells are collected but

negatively selected cells include F4/80+ cells. (D) Proximal epithelial cells in normal kidney are

selected by lotus lectin binding, and the absence of the leukocyte marker CD45, but in injured

kidney injured proximal epithelium is selected by expression of Kim-1. (E) Example of

CHAU ET AL, 2012


myofibroblast collection using Coll-GFP mice from d4 after UUO showing negative gates to

select out leukocytes and endothelial cells. (F) Agilent bioanalyzer graph showing RNA collected

in Trizol. Note the minor peak which represents microRNA. The large peak is an artifact due to

the green marker. (G) Q-PCR of the pericyte marker, PDGFRβ (left) or the endothelial cell

marker, CD31 (right) in purified kidney pericytes (PC) and endothelial cells (EC) purified from

normal Coll-GFP mouse kidney and mRNA isolated in RLT buffer. (H) Agarose gel showing 35

cycle RT-PCR of myofibroblasts purified from d7 after UUO kidney detecting the macrophage

specific, F4/80 antigen transcripts, Col1a1 transcripts of epithelial specific transcripts for Kim1.

CHAU ET AL, 2012


transcripts and Gapdh transcripts in WT, miR21flox/+ mice and miR21-/- mice. (B) Sylarray

(S12) showing de-repression (enrichment) of seed-matched miR21 target mRNA transcripts in

the kidney on d10 after IRI comparing WT gene expression with miR21-/- gene expression.

Fig. S3. Characterization of miR-21-deficient mice. (A) Northern blot showing Tmem49 (Vmp1)

CHAU ET AL, 2012


Fig. S4 Inhibition of miR-21 by the anti-miR-21 oligonucleotide in vitro in HeLa cells as

detected using a miR-21-specific luciferase reporter assay.

CHAU ET AL, 2012


CHAU ET AL, 2012


examination of miR-21 target genes in whole kidney and in kidney myofibroblasts. (A) Western blot of

pERK and ERK levels in sham operated or UUO d10 whole kidneys from mice treated with anti-

miR-21 or controls or vehicle. (B) Western blot of pERK and ERK levels in sham operated or

UUO d10 kidneys from miR-21+/+ vs miR-21-/- mice. (C) IEF for pERK in sham operated or

UUOd10 kidneys from miR-21+/+ vs miR-21-/- mice. Note that pERK1 levels are selectively

reduced in the absence of miR-21. Unphosphrylated ERK1 levels are not different between miR-

21+/+ vs miR-21-/- kidneys. (D-E) Whole kidney Western blots for protein (D) and Q-PCR for

transcripts (E) of candidate seed matched miR-21 target genes that were not regulated in kidney

injury in miR-21+/+ vs miR-21-/- mice. (F-G) Analysis of the impact of miR-21 in myofibroblasts.

(F) Spry1 relative transcript levels in primary myofibroblast cultures transfected with anti-miR21

or control anti-miR for 48h. (G) Sylarray (10) showing no evidence of widespread de-repression

(enrichment) of seed-matched miR-21 target mRNA transcripts in purified myofibroblasts from

d4 after UUO wild type kidneys from mice treated for 10 days with control anti-miR vs anti-

miR21. (H) Sylarray (10) showing no evidence of de-repression (enrichment) of seed-matched

miR-21 target mRNA transcripts in primary cultures of kidney myofibroblasts cultures

transfected with anti-miR21 or control anti-miR for 48h.

Fig. S5. ERK phosphorylation in whole kidney of miR-21 knockout and anti-miR-21-treated mice, and

CHAU ET AL, 2012


Fig. S6. Lipid metabolic genes are upregulated in Ppara transgenic mice. (A) Graph showing gene

ontogeny groups over-expressed in mice transgenic for Ppara in muscle. (B) Venn Diagram

showing lipid metabolic genes upregulated in Ppara transgenic compared with miR21-/- UUO

kidneys.

CHAU ET AL, 2012


Fig. S7. Anti-miR-21 administration to primary epithelial cultures or systemic administration to mice

-/-

primary PTECs showed miR-21 specific de-repression of PPARA 48h after transfection with anti-

miR21 without an effect of control anti-miR. (B) UUO surgery decreases Ppara in mouse kidney

and systemic treatment with anti-miR-21 shows increased levels of Ppara transcripts at d4 after

UUO. (C) Western blots showing whole kidney expression of Pparα, Mpv17l and Reck proteins

in wild type mouse kidneys that underwent sham surgery or UUO 7d previously and were treated

derepresses critical genes identified in miR21 mice including PPARα. (A) TGF-β stimulated human

CHAU ET AL, 2012


with vehicle, control anti-miR or anti-miR-21. Note that anti-miR-21 specifically de-represses

these proteins in UUO kidney. (D) Primary mouse epithelial cells from miR21-/- kidneys show de-

repression of Ppara, and Reck in control conditions and in response to TGFβ1 stimulation.

Mpv17l is transiently de-repressed in miR21-/- kidney epithelial cells in response to TGFβ1

stimulation.

CHAU ET AL, 2012


Supplementary Tables

Table S1

that were regulated in both models).

!" #$%&'()* +',-.'#/* -0' #/ *1!2 +345 ! "#$%&'()* +',-.'#/* -0' #/ * 1! 2+345

66/ 76 89 7:; < == 1> = < 1< : ?7> @ = 1> A? 7> = @1< ; 1;> ?7> B C 1:= ?7> =

66/ 76 89 7;A A '7= D :A 1= < A 1< E ?7> = E 1; >? 7> E C 1: ; B 1E> ?7> @ C 1:= ?7> =66/ 76 89 7;< : 7ED :> 1E < = 1E ; ?7> = : 1: >? 7> E B 1@ = ; 1>> ?7> < E 1:> ?7> E

66/ 76 89 7;A A '7E D ;C 1B ; B 1> E ?7> = : 1C >? 7> E = 1A = : 1@A ?7> B ; 1>> ?7> <

66/ 76 89 7E< : 7ED ;< 1E @ : 1A ; ?7> = ; 1= >? 7> E = 1> E E 1=@ ?7> = ; 1@> ?7> E66/ 76 89 7;@ ' ;E 1A < : 1< A ?7> C < 1> >? 7> < < 1C = E 1A> ?7> = ; 1@> ?7> E

66/ 76 89 7:; ;E 1= A ; 1B B ?7> = ; 1; >? 7> E = 1= : A 1>> ?7> C ; 1;> ?7> E

66/ 76 89 7AE ;E 1< = ; 1> A ?7> = @ 1> >? 7> < : 1; B : 1>> ?7> < E 1=> ?7> E

66/ 76 89 7;@ E ; :1= ; 1: B ?7> C < 1> >? 7> < E 1; A = 1>> ?7> ?7> E66/ 76 89 7AA F ; :1; = 1; C ?7> = : 1: >? 7> E : 1> < C 1>> ?7> < @ 1>> ?7> E

66/ 76 89 7:: E A 1B B < 1C > ?7> C = 1> >? 7> < A 1< @ ; 1>> ?7> < : 1@> ?7> E

66/ 76 89 7;< C ' B1@ E 1: B ?7> C < 1> >? 7> < < 1= = B 1>> ?7> < A 1E> ?7> E66/ 76 89 7;A A FG B1C C 1A A ?7> = : 1C >? 7> E E1A ; 1>= ?7> = ; 1:> ?7> E

66/ 7#*H 7B8 C 1A = A 1< A ?7> = E 1; >? 7> E :1B @ 1:< ?7> = : 1=> ?7> E

66/ 76 89 7;A ' C 1A < ; 1> > ?7> < E 1A >? 7> E : 1> A = 1>> ?7> ?7> E

66/ 76 89 7;= F C 1; E : 1> > ?7> < C 1B >? 7> E : 1= @ : 1>> ?7> < < 1:> ?7> E66/ 76 89 7:> ' = 1C @ E 1> > ?7> ? 7> E : 1: A E 1>> ?7> < = 1=> ?7> E

66/ 76 89 7:= = 1 > ?7> < @ 1A >? 7> E : 1; = < 1>> ?7> < C 1C> ?7> E

66/ 76 89 7;> C F = 1< C E 1> > ?7> ? 7> E : 1; @ : 1>> ?7> < < 1>> ?7> E66/ 76 89 7E= > = 1> = : 1B >? 7> E : 1= B : 1>> ?7> < < 1>> ?7> E

66/ 76 89 7CB < G < 1B E < 1> < ?7> = ; 1A >? 7> E : 1C : = 1>B ?7> = : 1>> ?7> E

66/ 76 89 7;E : < 1C < : 1B A ?7> C < 1> >? 7> < C 1E A : 1>> ?7> < E 1=>?7> E

66/ 76 89 7A: ' < 1> B = 1> > ?7> < A 1C >? 7> E ; 1C A < 1>> ?7> < C 1C> ?7> E66/ 76 89 7;= ' E 1@ E E 1> > ?7> ? 7> E ; 1@ < ; 1>> ?7> < E 1:> ?7> E

!"#$%&&' (#(

List of up-regulated miRNAs. The log2 intensity of each sham and day10 samples (of the 24 miRNAs

CHAU ET AL, 2012


Table S2 +/+ compared with miR21-/-

mice.

See attached spreadsheet (Excel file).

Table S3

Gene Sequences PPARa

Forward 5’-ATGCCAGTACTGCCGTTTTC-3’ Reverse 5’-GGCCTTGACCTTGTTCATGT-3’

MPV17L Forward 5’-GACCTTCCACGGAAACTTCA-3’ Reverse 5’-TCTGATCGCATAGCACCTTG-3’ RECK Forward 5’-CCTGCAATAAGGAGGCAGAG-3’ Reverse 5’-ACTGCTGGAGACCTGCACTT-3’ Spry1 Forward 5’-GGCCTATTAGGACGGTCTCC-3’ Reverse 5’-GGCCGTACACTCTCCACATT-3’ NFIA Forward 5’-GGCCCCAATTTTTCTCTCTC-3’ Reverse 3’-ACAGACTTGAGGCGCTTTGT-3’ NFIB

Forward 5’-ACCCGTGCTGTGTCTTATCC-3’ Reverse 5’-CGCTCTCCATCCGTACTCTC-3’

Cyr61 Forward 5’-CAAGAAATGCAGCAAGACCA-3’ Reverse 5’-TTCTGGTCTGCAGAGGTGTG-3’ Col1a1 Forward 5’-GAGCGGAGAGTACTGGATCG-3’ Reverse 5’-GTTCGGGCTGATGTACCAGT-3’ GAPDH Forward 5’-CTGCAGAAACCTGCCAAGTA-3’

List of seed-matched target derepressed mRNA in UUO kidneys from miR21

Primers used to detect genes by qPCR

CHAU ET AL, 2012


Reverse 5’-AAGAGTGGGAGTTGCTGTTG-3’ 18S Forward 5’-GATTCCGTGGGTGGTGGTG-3’ Reverse 5’-GCCAGAGTCTCGTTCGTTATCG-3’ miR-21 Forward 5’-CGGTAGCTTATCAGACTGATGTTGA-3’ SnoRNA234 Forward 5’-GCGCGGAACTGAATCTAAGTGATTTAACAA-3’ Supplementary References

S1. S.L. Lin, T. Kisseleva, D.A. Brenner & J.S. Duffield. Pericytes and perivascular

fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of

the kidney. Am J Pathol 173, 1617-1627 (2008).

S2. A.P. Castano, et al. Serum amyloid P inhibits fibrosis through Fc gamma R-dependent

monocyte-macrophage regulation in vivo. Sci Transl Med 1, 5ra13 (2009).

S3. S. Li, et al. Transgenic expression of proximal tubule peroxisome proliferator-activated

receptor-alpha in mice confers protection during acute kidney injury. Kidney Int 76,

1049-1062 (2009).

S4. L. Alonso, et al. Effect of ruminally protected Methionine on the productive and

reproductive performance of grazing Bos indicus heifers raised in the humid tropics of

Costa Rica. Trop Anim Health Prod 40, 667-672 (2008).

S5. T. Ichimura, et al. Kidney injury molecule-1 is a phosphatidylserine receptor that confers

a phagocytic phenotype on epithelial cells. J Clin Invest 118, 1657-1668 (2008).

S6. J.T. Pena, et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat

Methods 6, 139-141 (2009).

S7. J.S. Duffield, et al. Restoration of tubular epithelial cells during repair of the

postischemic kidney occurs independently of bone marrow-derived stem cells. J Clin

Invest 115, 1743-1755 (2005).

S8. J.S. Duffield, et al. Selective depletion of macrophages reveals distinct, opposing roles

during liver injury and repair. J Clin Invest 115, 56-65 (2005).

CHAU ET AL, 2012


S9. F. Kuhnert, et al. Soluble receptor-mediated selective inhibition of VEGFR and

PDGFRbeta signaling during physiologic and tumor angiogenesis. Proc Natl Acad Sci U

S A 105, 10185-10190 (2008).

S10. B. Li, et al. Mobilized human hematopoietic stem/progenitor cells promote kidney repair

after ischemia/reperfusion injury. Circulation 121, 2211-2220.

S11. S.L. Lin, et al. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc

Natl Acad Sci U S A 107, 4194-4199 (2010).

S12. http://www.ebi.ac.uk/enright-srv/sylarray/

Supplementary Materials for - Science Translational Medicine · 2012-02-13 · Supplementary...

Documents

Transcript of Supplementary Materials for - Science Translational Medicine · 2012-02-13 · Supplementary...