Human tissue samples
Renal biopsy samples were obtained from diagnostic renal biopsies performed at Juntendo University Hospital after the approval of the Ethics Committee on Human Research of Juntendo University Faculty of Medicine. Samples from human subjects with diabetic nephropathy (DN, n = 5), minor glomerular abnormality (n = 1), focal segmental glomerular sclerosis (FSGS, n = 4), anti-neutrophil myeloperoxidase cytoplasmic antigen-antibody-related nephritis (ANCA-RN, n = 4), and nephrosclerosis (n = 1) were evaluated.
Assessment Of Dpp-4 Activity In Renal Biopsy Specimens
Frozen kidney sections (3 µm) were fixed with formalin, phosphate-buffered saline (PBS), and acetone (1:35:15) and washed with water. The samples were incubated with a coloring solution (1.76 mol/L glycyl-prolyl-4-methoxy-β-naphthylamide, 2.52 mol/L Fast Blue B, 3.71 vol% N,N-dimethyl formamide, 95.7 mmol/L phosphate buffer) [5]. After rinsing with water, images were acquired with a BX43 Microscope (Olympus, Tokyo, Japan).
Cell Cultures And Measurement Of Dpp4 Activity
Conditionally immortalized human podocytes were kindly provided by Dr. Moin A. Saleem (Bristol Royal Hospital for Children Bristol, Bristol, UK) and cultured as previously described [16]. Cultured podocytes were treated with 0.15 µg/mL of ADR (ADR group) or normal saline (control) for 10 days. At 2 and 4 days after ADR treatment, podocytes were treated with saxagliptin (1, 10, and 100 nM, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) (ADR + saxagliptin group). After treatment, podocytes were collected using a scraper, pelleted by centrifugation, and washed twice with ice-cold PBS. DPP-4 activities of cultured podocytes were measured using DPP4 Activity Assay Kit (Abcam, Cambridge, UK) and FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) according to the manufacturer’s protocol.
Immunostaining For Cytoskeleton Protein
Differentiated podocytes were cultured on collagen type I-coated cover slips and then treated with saline (control), ADR alone (ADR group), or ADR with saxagliptin (ADR + saxagliptin group) for 8 days. The cells were fixed with 2% paraformaldehyde and incubated with blocking solution (2% fetal calf serum, 2% bovine serum albumin, 0.2% fish gelatin in PBS). The primary antibody against synaptopodin (Progen Biotechnik GmbH, Heidelberg, Germany) was used at a 1:10 dilution. The primary antibody against Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) as a marker of F-actin, a stress fiber, was used at 1:250 dilution. As a secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific) was used at a 1:300 dilution. Images were acquired using FV1000 Confocal Microscope (Olympus). The areas of synaptopodin and F-actin were measured with Image J software (National Institutes of Health, Bethesda, MD, USA).
Assessment Of Podocyte Detachment
Podocytes were cultured in 6-well plates at a concentration of 1.5 × 105 cells/well. On day 0, the number of cells per field were counted as a baseline number. The average number of cells in 5 fields in 3 independent sets of experiments was determined. After treatment with ADR with or without 100 nM saxagliptin for 2 days, podocytes were counted in the same 5 fields of each well. The ratio of detachment was also evaluated.
Western Blotting
The cell pellet was re-suspended in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate buffer and incubated on ice for 30 min. The cell lysate was cleared by centrifugation for 10 min. Samples were separated by sodium dodecyl sulfate–polyacrylamide gels and then proteins were transferred to membranes and blocked with Block-ACE (DS Pharma Biomedical Co., Ltd., Osaka, Japan). The membranes were incubated with the appropriate primary antibodies. The antibodies against synaptopodin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and RhoA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used at 1:500 and 1:300 dilutions, respectively. Peroxidase-conjugated goat anti-mouse IgG was used as a secondary antibody at a 1:10,000 dilution (Jackson Immunoresearch, West Grove, PA, USA). Equal protein loading was confirmed by reprobing the membrane with GAPDH at 1: 20,000 (Sigma-Aldrich, St. Louis, MO, USA). Images were scanned with a C-Digit chemiluminescent western blot scanner, and densitometry analysis was performed using Image Studio Digits software (LI-COR Biosciences, Lincoln, NE, USA).
Statistics
All values are shown as the means ± standard deviation. Statistical significance (defined as P < 0.05) was calculated using Prism 6.0 software (GraphPad, Inc., La Jolla, CA, USA) followed by t-test.