Podocin

Protein expression in cells and renal tissues was detected by Western blotting as described previously44 (link). An equal amount of protein (50 μg) from each sample was separated by SDS-PAGE and transferred to PVDF membranes. After blocking in blocking buffer containing 5% skim milk for 30 min, the membranes were incubated with primary antibodies for HDAC9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-STAT3 (ab76315, Abcam, Cambridge, MA, USA), STAT3 (Abcam), Bcl-2 (Santa Cruz Biotechnology), Bax (Santa Cruz Biotechnology), cleaved Caspase 3 (Abcam), Nephrin (Abcam), Podocin (Abcam) and GAPDH (Cell signaling Technology, Danvers, MA, USA) separately at 4 °C overnight. The membranes were incubated with appropriate HRP-conjugated secondary antibody at room temperature for 1 h and signals were detected by using enhanced chemiluminescence (BioRad, Richmond, CA, USA). The blotting bands were quantified by densitometric analysis using Image J software (National Institutes of Health, Bethesda, MD, USA).

The primary antibodies used in this study targeted SDF-1α (Novus Biologicals, Littleton, CO, United States), DPP-4 (Abcam, Cambridge, MA, United States), p-PKA (clone EP2606Y, Abcam, Cambridge, MA, United States), PKA (Cell Signaling, Danvers, MA, United States), NOX2 (clone EPR6991, Abcam, Cambridge, MA, United States), p-ERK1/2 (clone D13.14.4E, Cell Signaling, Danvers, MA, United States), ERK1/2 (Cell Signaling, Danvers, MA, United States), transforming growth factor-β1 (TGF-β1) (clone EPR18163, Abcam, Cambridge, MA, United States), podocin (Abcam, Cambridge, MA, United States), desmin (Abcam, Cambridge, MA, United States), nephrin (clone B-12, Santa Cruz Biotechnology Inc., Dallas, TX, United States), fibronectin (Proteintech, Rosemont, IL, United States), Fsp1 (clone EPR14639(2), Abcam, Cambridge, MA, United States), and α-SMA (clone 1A4, Abcam, Cambridge, MA, United States). ROS levels in renal tissue were tested with a dihydroethidium (DHE) assay kit (Beyotime, Jiangsu, China), and ROS levels in cells were examined with a DCFH-DA assay kit (Beyotime, Jiangsu, China). DPP-4 enzyme activity was tested using a DPP-4 assay kit (Enzo Life Sciences, Farmingdale, NY, United States).

Kidney tissues were fixed in 4% paraformaldehyde in PBS at 4°C. Tissues were incubated in 30% sucrose in PBS overnight at 4°C and mounted in OCT (Tissue-Tek, Tokyo, Japan) for sectioning [34 (link)]. After blocking, tissue sections were stained with the indicated primary antibodies and affinity-purified secondary antibodies-conjugated HRP (DAKO, Glostrup, Denmark). Primary antibodies used included antibodies against αSMA (Sigma), desmin (DAKO, Glostrup, Denmark), podocin (Abcam, Cambridge, MA, USA), and 8-hydroxy-2′-deoxyguanosine (8OHdG) (JaICA, Shizuoka, Japan). Quantitative analysis of afferent arterioles was performed as previously described [16 (link)]. For afferent arterioles, vessels with internal elastic lamina adjacent to glomeruli were selected. Arcuate arteries were identified by the location at the border of renal cortex and medulla. Areas positive for αSMA in the cross section of these vessels were quantitated using NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan) and Aperio ImageScope (Leica, Buffalo Grove, IL, USA). For quantification of desmin, podocin, and 8OHdG in the glomeruli, the percentage of positive area was determined as positive pixels per total pixels in a glomerulus using Image Scope software. For each rat, 20 glomeruli were randomly analyzed.