Most00

A commercial osteopontin (OPN) ELISA kit was purchased (CAT# MOST00, R&D Systems; Minneapolis, MN, USA) and the corresponding protocol was followed. Whole blood serum samples obtained from mouse tail vein bleeds prior to euthanization were added to wells that were pre-coated with OPN polyclonal antibodies. After 1 h of incubation and four washes, OPN conjugate was added to each well. After an additional 2 h of incubation and four additional washes, substrate solution was added. Stop solution was added 30 min later, and the plate was immediately read at 450 nm and 540 nm. Using the standards provided with the assay, OPN concentration (pg/mL) was extrapolated for each sample.

ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions. The concentrations of SPP1 were measured by diluting plasma 300-fold, gWAT-CM 300-fold, BMAds-CM 150-fold and matured 3T3-L1 adipocytes-CM 150-fold.

All reagents were obtained from Sigma-Aldrich unless otherwise stated. Human VEGF-A, PlGF, osteopontin, and EPO were measured in plasma samples with standard enzyme-linked immunosorbent assay (ELISA) kits (refs QVE00B, SPG00, DO ST00, and DEP00, R&D Systems, Minneapolis, MN, respectively). Serum ceruloplasmin was measured by an ELISA (Eagle Biosciences, ref HCP31-K01) adapted on a Beckman Array Protein System (Beckman Instruments Inc, Brea, CA, USA). Rat VEGF-A and osteopontin were quantified in plasma samples using standard ELISAs (refs RRV00 and MOST00, R&D Systems, Minneapolis, MN, respectively). Rat EPO and ceruloplasmin were measured by radioimmunoassay using commercially available antibodies (refs sc-5290 and sc-365205, Santa Cruz Biotechnology).

Blood was harvested into an EDTA-treated tube at the time of euthanasia via intracardiac puncture, spun down at 8,000 rpm for 6 min, and plasma collected and stored at –80°C until use. Enzyme immunoassays were used to measure plasma concentrations of mouse Glu-osteocalcin (MK129;Takara), mouse Gla-osteocalcin (MK127; Takara), mouse osteopontin (MOST00; R&D Systems), and mouse testosterone (55-TESMS-E01; Alpco), according to the manufacturers’ recommendations. Statistical analysis was performed used a linear regression model to test for differences between genotypes via R. We also collected blood (see below) from 10- to 15-week-old males for testosterone measurements analyzed independently by the P30-supported University of Virginia Ligand Assay and Analysis Core Laboratory.

Immunofluorescence staining of the kidney was performed on fixed-frozen sections. Briefly, the tissue sections were activated and labeled with antibodies, including rabbit anti-Ki-67 (Vector, VP-K451, 1 in 200), rabbit anti-KIM-1 (R9²⁵ , 1 in 200), rabbit anti-α-SMA (Sigma, 1 in 400), rat anti-F4/80 (Abcam, ab6640, 1 in 1000), rabbit anti-Hnf1b (Thermo Fisher Scientific, 720259), rabbit anti-GR (Thermo Fisher Scientific, PA1-511A), rabbit anti-Slc34a1 (Novusbio, NBP2-13328), rabbit anti-Spp1 (Abcam, ab8448) and rat anti-Kl (BioLogo, KM2076). The slides were then exposed to FITC or Cy3-labeled secondary antibodies (Jackson ImmunoResearch). The staining was examined with fluorescence microscopes (Nikon TE 1000 and Nikon C1 confocal). At least 7 high-power fields/section for each sample were examined in each evaluation and quantification (positive area or cell count) was performed with an in-house Macro in Image J. Spp1 plasma concentration was measured by ELISA (R&D Systems, MOST00).