Materials and Methods
Transgenic Mice Creation
All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Harvard University. Gα12 (Q229L) EE tagged was cloned into the CMV-floxed LacZ cassette kindly provided by Dr Larry Holzman. C57/B6 mice were injected at the Brigham and Women's Hospital Transgenic Mouse Facility and were then crossed with Nphs2/Cre mice on the same C57/BL6 background.
Urine Microalbumin, Serum Creatinine, LPS and Tissue Harvesting
Male and female mice were analyzed for urine microalbumin/creatinine ratio at specified ages using Bayer DCA 200+ Analyzer with software version E3.11/01.04. Mice were defined as proteinuric when microalbumin/creatinine ratio was ≥34 owing to the detection limits of the analyzer. LPS (Invivogen, San Diego, CA, USA) (10 μg/g, intraperitoneally) was administered to 2- to 6-month-old mice, and urine was collected 18 h post-injection and microalbumin/creatinine ratio was determined. Serum was collected and BUN and creatinine were analyzed using an iStat System with CHEM8+ cartridges. Mice were anesthetized using inhaled isofluorane (Phoenix Pharmaceuticals, St Louis, MO, USA). For whole kidney collection, organs were perfused with cold PBS. Kidneys were removed and processed according to methods below.
GST-TPR Pull-down Assay
The GST-TPR construct was kindly provided by Dr M Negishi (Kyoto University, Kyoto, Japan). GST-TPR was prepared as described previously. Harvested kidneys were homogenized in lysis buffer with eComplete (Roche, Indianapolis, IN, USA) protease inhibitors, lysed through 22 G needle and normalized for protein concentration. GST-TPR (1 μg) coupled to glutathione-agarose beads (Amersham Biosciences) was added to 800 μg of total protein and rocked overnight at 4°C. Beads were pelleted with low-speed centrifugation and washed three times with PBS, 0.1% Triton X-100, resuspended in Laemmli sample buffer and analyzed by SDS-PAGE and western blot using a Gα12 antibody (Santa Cruz, Santa Cruz, CA, USA).
Immunogold Electron Microscopy
Immunogold labeling and electron microscopy (EM) was performed at the Membrane Biology Program, Massachusetts General Hospital. Normal mouse kidney was fixed in 2% freshly made paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and processed according to standard conditions. Grids were incubated with rabbit anti-Gα12 (Santa Cruz) overnight at 4°C, followed by a 25 μl droplet of anti-goat IgG conjugated with gold (10 nm). Grids were washed and imaged using Philips CM10 electron microscope (Philips Electronics, Mahwah, NJ, USA).
Fixed kidney tissue was paraffin processed and 4 μm sections were stained with PAS. Light microscopic evaluation included quantification of the total number of glomeruli present in one microscopic section as well as quantification of glomeruli with lesions (global sclerosis, segmental sclerosis, glomerular collapse and glomerular hypertrophy).
Fixed specimens were trimmed, post-fixed with osmium tetroxide, dehydrated in serial ethanols and embedded in epoxy resin. Ultrathin sections were cut at 80 nm, mounted on 200 mesh copper grids, treated with uranyl acetate and lead citrate, and examined in a JEOL 1010 transmission electron microscope (Tokyo, Japan). Electron micrographs showing glomerular ultrastructure were analyzed in a blinded manner. Podocytes, endothelium, glomerular basement membrane (GBM) and mesangium were examined and scored from 0 (no abnormality) to 4 (severe abnormality). Podocytes were scored for foot process (FP) effacement and irregularity, microvillous degeneration, vacuolization and subepithelial deposits. Endothelium was scored for: double contours, subendothelial deposits and swelling. GBM abnormalities were scored for thickness and irregularity, and the mesangium was scored for deposits, expansion by matrix and cellular expansion.
LacZ, TUNEL and Immunostaining
Fixed kidneys were washed in PBS and rehydrated overnight in 30% sucrose. Kidneys were embedded in OCT Compound (TissueTek, Sakura Finetek, Torrence, CA, USA) and frozen in liquid nitrogen. For Wilms's tumor-1 (WT-1) and TUNEL staining, sections were labeled using the 'In Situ Cell Death Detection Kit' (Roche) according to the manufacturer's protocol. Kidneys were incubated in methanol, blocked and incubated in anti-WT-1 antibody (sc-192; Santa Cruz) at 4°C overnight. Sections were washed, incubated with appropriate secondary antibody and mounted using Prolong Gold (Invitrogen, Carlsbad, CA, USA). Kidneys were photographed on a Nikon E-1000 equipped with a SPOT digital camera. For collagen staining, kidneys were blocked in 1% BSA and incubated with primary antibodies (generous gift of DB Borga, Vanderbilt, Nashville, TN, USA) or (Rockland Gilbertsville, PA, USA). All sections were incubated with appropriate secondary antibody for 1 h at room temperature, and mounted using Prolong Gold (Invitrogen). Images were taken on a Nikon C1 D-Eclipse confocal microscope.
Glomerular isolation was performed using magnetic beads. A measure of 200 μl Dynabeads (M450 Tosylactivated; Invitrogen) were washed with 0.1% PBS and incubated overnight in 0.2 M Tris (pH 8.5) with 0.1% BSA. Beads were washed and resuspended in 30 ml HBSS. Mice were anesthetized and organs perfused with HBSS. Kidneys were digested in 1 mg/ml collagenase A (Roche) and 100 U/ml DNaseI (New England Biolabs, Ipswich, MA, USA) at 37°C for 30 min. Digests were strained though a 100 μm cell strainer, centrifuged and resuspended in HBSS. Glomeruli were obtained using magnetic separator (New England Bio Labs).
RhoA Activation ELISA and Western Blot
Isolated glomeruli were resuspended in lysis buffer with eComplete (Roche) protease inhibitors and lysed through 22 G needle. Rho activity was determined using G-LISA™ RhoA Activation Assay Kit (Cytoskeleton, Denver, CO, USA) and absorbance at 490 nm of an HRP-active RhoA antibody. In addition, samples were analyzed by SDS-PAGE and western blot analysis using an anti-RhoA antibody (Cytoskeleton) as described previously.
Purified glomeruli were lysed in TRIzol reagent (Invitrogen). RNA concentrations were determined, and RNA was incubated in DNAseI (New England Biolabs) with RNAse inhibitor (Roche). DNAse-treated RNA was reverse transcribed using Transcriptor Reverse Transcriptase (Roche). Negative control without enzyme was included in each analysis. The cDNA template was treated with RNaseH (Invitrogen). TaqMan Gene Expression assays (Applied Biosystems, Foster City, CA, USA) were performed using an ABI 7300 (Applied Biosystems) with the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Melting-curve analysis and gel electrophoresis of PCR products verified that a single product of the expected size was generated with each primer set. Data analysis used the ΔΔCt method. Ct was normalized to 18S ribosomal subunit.
Data are expressed as medians or means±s.e.m. as indicated. Statistical analysis was performed using Prism 4 for Macintosh (GraphPad, La Jolla, CA, USA) using two-way ANOVA, followed by Bonferroni's post-hoc test. Statistical significance was identified at P<0.05.
Lab Invest. 2012;92(5):662-675. © 2012 Nature Publishing Group