(B6;129.Cg-Gt(ROSA)26Sor Tm20(CAG-Ctgf-GFP)Jsd) Were Crossed with Female Foxd1cre/+ Heterozygote Mice 1, and Experimental Mice Were Selected As Foxd1cre/+; Rs26cig/+
Total Page：16
File Type：pdf, Size：1020Kb
Supplemental Information SI Methods Animal studies Heterozygote mice (B6;129.Cg-Gt(ROSA)26Sor tm20(CAG-Ctgf-GFP)Jsd) were crossed with female Foxd1Cre/+ heterozygote mice 1, and experimental mice were selected as Foxd1Cre/+; Rs26CIG/+. In some studies Coll-GFPTg or TCF/Lef:H2B-GFPTg mice or Foxd1Cre/+; Rs26tdTomatoR/+ mice were used as described 2; 3. Left kidneys were subjected to ureteral obstruction using a posterior surgical approach as described 2. In some experiments recombinant mouse DKK1 (0.5mg/kg) or an equal volume of vehicle was administered by daily IP injection. In the in vivo ASO experiment, either specific Lrp6 (TACCTCAATGCGATTT) or scrambled negative control ASO (AACACGTCTATACGC) (30mg/kg) (Exiqon, LNA gapmers) was administered by IP injection on d-1, d1, d4, and d7. In other experiments anti-CTGF domain-IV antibodies (5mg/kg) or control IgG were administered d-1, d1 and d6. All animal experiments were performed under approved IACUC protocols held at the University of Washington and Biogen. Recombinant protein and antibody generation and characterization Human CTGF domain I (sequence Met1 CPDEPAPRCPAGVSLVLDGCGCCRVCAKQLGELCTERDPCDPHKGLFC), domain I+II (sequence Met1CPDEPAPRCPAGVSLVLDGCGCCRVCAKQLGELCTERDPCDPHKGLFCCIFGGT VYRSGESFQSSCKYQCTCLDGAVGCMPLCSMDVRLPSPDCPFPRRVKLPGKCCEE) were cloned and expressed in 293 cells, and purified by Chelating SFF(Ni) Column, tested for single band by SEC and PAGE, and tested for absence of contamination. Domain-IV (sequence GKKCIRTPKISKPIKFELSGCTSMKTYRAKFCGVCTDGRCCTPHRTTTLPVEFKCPDGE VMKKNMMFIKTCACHYNCPGDNDIFESLYYRKMY) was purchased from Peprotech. Mouse or human DKK1 was generated from the coding sequence with some modifications and a tag. Secreted protein was harvested from 293 cells, and purified by nickel column, and tested for activity in a supertopflash (STF) assay 4. DKK1 showed EC50 of 0.69nM for WNT3a-induced WNT signaling in STF cells. Single band was confirmed by SEC and non-reducing PAGE, and proteins tested for endotoxin with LAL 1 assay. Rabbits were immunized with CTGF Domain IV to generate a polyclonal antiserum using standard methods. This was affinity purified and tested for activity in a migration activity with an IC50 of approximately 100ng/ml. Human CTGF domain IV or domain I+II or rDKK1 was adhered to assay plates in increasing concentrations from 1nM to 10,000nM. After washing and blocking with BSA, LRP6-Fc or CD109-His (R&D systems) was incubated in blocking solution, washed and detected with either anti-Fc- biotin and avidin-HRP or anti-his tag-HRP followed by enzymatic reaction. Cell purification, culture and assays Purification and culture of pericytes. Pericytes were purified from healthy kidneys and characterized as described 2. Kidney pericytes were purified from C57BL6 mice, Coll-GFPTg or TCF/Lef:H2B-GFPTg or Ctnnb1fl/fl mice by MACS immunoaffinity column purification from kidney single cell preparation of mouse kidney as above, using positive selection by anti-PDGFRβ antibodies as described 2. Purified cells were cultured in DMEM/F12 containing 10%FBS and ITS on gelatin coated plates, and confirmed to lack epithelial or leukocyte contamination. The function and purity of these cells has been previously well characterized 5. Human pericytes were purified from fetal human kidneys obtained following voluntary pregnancy interruptions (d110 to d130 of gestation) performed at the University of Washington Medical Center, (IRB447773EA University of Washington) or from Novogenix, Los Angeles, CA as described 6. Informed consent for the use of fetal tissues were obtained from all patients. Decapsulated fetal kidneys were minced and digested to single cells and filtered as described. The single cell preparation was depleted of epithelial cells by passing down an anti-CD326 magnetic bead Column (Miltenyi Biotech). Remaining cells were sorted for a population PDGFRβ+, NG2+ population using the following antibodies (anti-PDGFRβ APC, anti-NG2 PE). Pericytes were then cultured in pericyte medium as described above on 0.2% gelatin-coated plates. All pericytes were studied functionally between P2 and P5 6; 7. Cells were cultured similarly to mouse pericytes, and confirmed to lack epithelial or leukocyte contamination. In vitro cell culture assays. Cell Migration: This was studied using modifications of a protocol described 2. Briefly, confluent pericytes in 6 well-plates were cultured O/N in serum free medium. Cells were washed again and a scratch placed across the culture with a pipette tip. Cytokines, 2 proteins or vehicle were added to the medium. At T0 the scratches were imaged at marked places and at timepoints after this the same areas were imaged. Migration is expressed as a percentage of the area of culture denuded of cells at T0 that has been re-covered. Each timepoint is an average of 6 separate experiments, and migration assessed at 8, 16 and 24h. Human CTGF domain IV (Peprotech) at concentrations indicated, and domains I, I+II were used as described above. DKK1 was recombinant protein as described above and used at 1-3µg/ml. WNT3a was from conditioned supernatants as previously described 2 with appropriate control. Cell Activation: Confluent pericytes in 12-well plates were cultured overnight in serum medium. Vehicle or CTGF domain IV, I+II, I were added to the medium at 100 or 200 ng/ml. Activation was assessed at 48h by Q-PCR of cDNA from cultured pericytes using primer sequences to detect specific transcripts (Table S1). Cytoskeletal reorganization: Pericytes were cultured on gelatin-coated glass coverslips for 48 hr at 60 – 80% confluence, washed 3 times with PBS and cultured in serum free medium stimulated with CTGF domain IV, I+II, I at 100 or 200 ng/ml for 24h. PFA (1%)- fixed, TX100 (0.5%)-permeablized cells were stained with αSMA (Sigma), visualized and images taken from 10 random fields in a blinded manner. Images were captured by confocal fluorescence microscopy (Nikon A1R Confocal, Nikon, Japan). Cytoskeletal reorganization was assessed by detecting stress fiber positive cells on coverslips. All data points conatained at least 3 independent replicates. Fibrogenesis: Confluent pericytes were cultured in serum free medium for 24 or 48h. Vehicle or CTGF domain IV, I+II, I were added to the medium at 100 or 200 ng/ml for 24h or 48h. DKK1 recombinant protein was added at 3µg/ml as described above. Fibrogenesis was detected by PAI-1, collagen-I, fibronectin protein expression in pericytes cell lysates by Western blot. Cell signaling: Confluent pericytes in 6 well plates were cultured in serum free medium for 24 or 48h and stimulated with CTGF domain IV, I+II, I at concentration of 200 ng/ml with or without 3µg/ml DKK1 recombinant protein. Cells were harvested and lysed on ice at 10min, 2h, and 24h in RIPA buffer (Cell Signaling) with proteinase inhibitors (Roche) and 1mM PMSF as described below. Gene silencing: siRNA for LRP-6, β-catenin and control siRNA (Life technology) were resuspended in RNase free water and stored (−80°C, 10 µM). 24h before transfection, pericytes were seeded into a six-well plate with 300,000 cells/well. siRNA (final concentration of 25 nM/well) was resuspended in Opti-MEM®Medium, lipofectamine® 3 RNAiMAX (life technology) was added, gently mixed, and further incubated for 5 min at RT. Medium containing siRNA was added to the wells and plates were swirled gently. Cells were incubated under normal conditions for 16 hours, followed by substitution of additional culture medium. After 48 hours, cells were evaluated for gene silencing. Lentiviral transduction: Genomic recombination at loxP sites in primary pericyte cultures was performed using Cre-recombinase expressed by lentiviral transduction using Lenti- GFP (control) or Lenti-Cre (provided by the Diabetes Research Center, University of Washington, Seattle, WA). Confluent (70–80%) pericytes in six-well plates were treated with 105 IU of virus per 104 cells and 10 µg/mL Polybrene (Sigma). The medium was changed 24 h later, and GFP expression was confirmed in 100% of cells at 48 h. Experiments were performed from 48–72 h after transduction. Western blot and immunoprecipitation analysis Cells were collected in ice-cold RIPA buffer (Cell signaling), homogenized, centrifuged (10 min, 13000 rpm) and the supernatant was taken for protein concentration. Cell extracts containing 10-20µg of protein were prepared in SDS-sample buffer and subjected to SDS-PAGE, transferred to nitrocellulose paper. After transfer, immunodetection was performed as described 2. Antibodies were diluted at 1:1000 in blocking buffer. Bands were detected by the SuperSignal West Dura Extended Duration Substrate (Pierce) as recommended by the manufacturer and luminescence captured by BIO-RAD ChemiDoc MP Imaging System. Primary antibodies against the following antigens were used: CTGF (L-20, Santa Cruz) p-P42/44, p-JNK, p-P38, p-LRP6, p- Smad2/3, β-catenin, PAI-1, LRP6 (Cell Signaling), αSMA (Sigma), Fibronectin, Laminin (abcam), Collagen I (Novusbio), NG2(R&D), PDGFRb (eBioscience), F480 (Invitrogen), GAPDH (Santa Cruz). Immunoprecipitation was performed as described 2. In brief, cell extracts containing 100µg of protein and 5% of fetal bovine serum in lysis buffer were incubated with either p-LRP6 (Cell Signaling) antibody or isotype control at 1:1000 dilution (4°C O/N). Thereafter, 25µl of ProteinA – Sepharose 4B CL slurry (Invitrogen) was added and incubated (2h RT). After precipitation, sepharose was washed with 0.2M Tris (pH 8.5) then heated (5min, 95°C) in Laemmli buffer prior to SDS-PAGE and western