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  • 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: jeremysd@u.washington.edu (J.S.D.); nchau@regulusrx.com (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

    2 miR-21 Promotes Fibrosis

    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

    3 miR-21 Promotes Fibrosis

    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

  • CHAU ET AL, 2012

    4 miR-21 Promotes Fibrosis

    (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

    5 miR-21 Promotes Fibrosis

    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 pass