RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal

The conditionally immortalized mouse podocyte cell line (MPC) was a kind gift from Dr. Jochen Reiser (Rush University Medical Center, Chicago, IL, USA), and cultured as described previously [38]. Cells were cultured at 33 °C in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco BRL, USA) and recombinant IFN-γ (growth permissive conditions; CYT-358, ProSpec, Tany Technogene Ltd, Israel). To induce differentiation, podocytes were reseeded and cultured at 37 °C in 100 cm² culture dish coated with 12 mg/ml type-I collagen (BD Bioscience, Bedford, MA, USA) and in RPMI-1640 medium supplemented with 5 % FBS, deprived of IFN-γ(growth restrictive conditions) for 10 to 13 days. After differentiation, podocytes was confirmed by the identification of synaptopodin, a podocyte differentiation marker, 10⁶cells were synchronized into quiescence by growing cells in serum-free RPMI-1640 medium for 24 h, and then treated with LPS (100 μg/ml), ADR (0.125 μg/ml) or small interfering RNA (siRNA, 50nM) for 48 h. Each reaction was repeated in triplicates.

The siRNAs against RhoA, mDia, YAP, dendrin and control were designed and synthesized by RIBOBIO CO., LTD. (Guangzhou, China), and were transfect into podocytes using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The sequences of siRNAs used in this study were as follows: RhoA-siRNA 5′-GGUAAGACAUGCUUGCUCA dTdT-3′, mDia-siRNA 5′-GCAAGUUCUUGUCCUCUUU dTdT-3′, YAP-siRNA 5′-CGAGAUG AGAGCACAGACA dTdT-3′, Dendrin-siRNA 5′-GGCUCCACCUUCUUAUGAA dTdT-3′.

Podocytes were planted on cover slides in six-well plates. After subjected to various treatments, differentiated podocytes were fixed with 4 % paraformaldehyde at room temperature for 10 min, and then treated with 0.1 % Triton X-100 for 10 min, blocked with 1 % bovine serum albumin for 20 min at room temperature, and incubated with rabbit anti-YAP (Santa Cruz, USA, 1:250) overnight at 4 °C. After incubated with the goat anti-rabbit Alexa Fluor 488 (Cell Signaling Technology, USA, 1:1000) for 1 h at room temperature, podocytes were stained with phalloidin (Cytoskeleton, USA, 1:500) for 30 min and then with DAPI (Sigma, St Louis, MO) for 5 min at room temperature. Photomicrographs were taken with laser confocal microscopy (LCSM, Zeiss KS 400, Postfach, Germany). All images were analyzed by two investigators blinded to the identity of the samples.

As described previously [6], RNA samples were prepared using Trizol RNA isolation system (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNAs using the PrimeScriptTM RT regent Kit following the instructions provided by the manufacturer ((Takara Bio Inc, Japan), and then cDNAs were used for real-time PCR analysis by a Plantinum SYBR Green SuperMix-UDG kit (Takara Bio Inc, Japan). The primers used are listed as follows: RhoA, forward 5′-AGCTT GTGGTAAGACATGCTTG-3′, reverse 5′-GTGTCCCATAAAGCCAACTCTAC-3′, Bax, forward 5′-CTGGACCATAGGTCGGAGTG-3′, reverse 5′-AATTCGCCGGAGACACTCG-3′, GAPDH, forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGT CA-3′.

Whole and nuclear proteins were prepared as described previously [6]. An aliquot of cell lysates containing 30 μg of protein was separated on 10 % sodium dodecyl sulfate–polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes (Amersham Biosciences). After blocked by 5 % skim milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-YAP (Cell Signaling Technology, USA, 1:1000), rabbit anti-Histone (Cell Signaling Technology, USA, 1:3000), rabbit anti-RhoA (Santa Cruz, USA, 1:500), rabbit anti-Bax (Santa Cruz, USA, 1:500), rabbit anti-GAPDH (Bioworld Technology, China, 1:10000), rabbit anti-Dia (Santa Cruz, USA, 1:500), rabbit anti-Dendrin (Santa Cruz, USA, 1:500). The membranes were then incubated with anti-rabbit IgG (Jackson Immuno Research, USA, 1: 4000) at room temperature for 1 h. Finally, membranes were treated with ECL reagents (Pierce Chemical, IN, USA), followed by exposing to X-ray film (Kodak, USA). The bands of the resulting autoradiographs were quantified densitometrically using Bandscan software. Protein expression was quantified as the ratio of specific band to Histone (nuclear fractions) or GAPDH.

Apoptotic cells in different groups were determined using an Annexin V/PI apoptosis detection kit according to manufacturer’s protocol (Nanjing KeyGEN Biotech, China). Briefly, podocytes were resuspended with binding buffer followed by incubation with 5 ml of Annexin V (conjugated with FITC) and 5 ml of PI in the dark for 10 min. Cell fluorescence was then analyzed using a Cell Lab Quanta™ SC Flow cytometer (Beckman Colter, Inc, USA). Cells positive for Annexin V-FITC were considered as apoptosis.

Active RhoA was measured by G-LISA RhoA Activation Assay Biochem kit (colorimetric assay, Cytoskeleton, USA) following the manufacturer’s instruction. And the signal was measured at 490 nm with a microplate reader (MRX, Dynatech Laboratories). Total RhoA protein expression of each group was measured by WB, as described above. Results were expressed as fold activity of stimulated in relation to non-stimulated controls normalized to total RhoA protein content.

All values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 19.0 (SPSS, Inc., Chicago, IL, USA). Statistical analysis of the data from multiple groups was performed by analysis of variance followed by LSD or Bonferonni tests. Comparisons between two groups were conducted by Student’s t test. P-values <0.05 were considered significant.