The following primary antibodies were used in the current study: Mouse anti-cofilin-1 (ab54532), rabbit anti-p-(Ser3)-cofilin-1 (ab131274), rabbit anti-p-(Tyr1105)-p190RhoGAP (growth regulating factor 1; GRF1; ab182636), mouse anti-LIM kinase 1 (LIMK1; ab119084), mouse anti-Fyn (ab1881), and rabbit anti-p-(Thr508)-LIMK1 antibody (ab194798; all from Abcam, Cambridge, MA, USA); rabbit anti-CMIP (12851–1-AP; ProteinTech Group, Inc., Chicago, IL, USA); rabbit anti-GRF1 polyclonal antibody (PA5-38253; Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA); rabbit anti-Src^Tyr418 antibody (OPA1-03091; Invitrogen; Thermo Fisher Scientific, Inc.); rabbit anti-Src^Tyr527 antibody (2105; Cell Signaling Technology, Inc., Danvers, MA, USA); mouse anti-GAPDH (G8795; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and mouse anti-β-actin (A5441; Sigma-Aldrich; Merck KGaA) antibodies.

A total of 12 male Wistar rats, weighing 100–120 ^g were i.p. injected with Sigma-Aldrich PA (10 mg/100 g body weight; Merck KGaA) or an equal volume of saline control. A 24-h urine sample was collected from each rat, and proteinuria was measured on an automatic biochemical analyzer (7170A, Hitachi, Ltd., Tokyo, Japan) (15). At day 0, 7 and 15 following the injections, all rats were sacrificed under ether anesthesia according to the Guide for the Care and Use of Laboratory Animals at the Kunshan First People's Hospital (Kunshan, China). Rat glomeruli were isolated using a typical sequential sieve (150, 100 and 75 µm) and stored at −80°C for further protein extraction.

The mouse podocyte cell line was provided by Professor Peter Mundel (Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA), and cultured at 33°C for proliferation in Gibco RPMI-1640 media (Thermo Fisher Scientific, Inc.) supplemented with Gibco 10% fetal calf serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin/streptomycin and 10 U/ml recombinant mouse interferon (IFN)-γ (Sigma-Aldrich; Merck KGaA). Cells were subsequently moved to 37°C and cultured for 14 days to induce differentiation by removal of IFN-γ. Podocytes were treated with 75 µg/ml PA for 12, 24 and 48 h. The small GTPase Rho inhibitor I (CT04-A; Cytoskeleton, Inc., Denver, CO, USA) was used at concentrations of 0.5 and 1.0 mg/ml to explore the role of RhoA in PA-induced podocyte damage. Podocytes overexpressing pcDNA-CMIP (a kind gift from Professor Dil Sahali, Inserm U955, Paris, France) were used in the present study.

pSilencer 2.1-U6 (Ambion; Thermo Fisher Scientific, Inc.) was used to express siRNAs that specifically target mouse CMIP and LIMK. The validated siRNA target sequences were obtained from Sigma-Aldrich and were as follows: siCMIP, 5′-gaggatccttaaacataat-3′; si-LIMK, 5′-gctaaacttcatcacagagtac-3′ (Merck KGaA). Mouse podocytes were transfected with the above-mentioned siRNA plasmids using Lipofectamine 2000 (11668027; Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, to establish stable knockdown cells, cells were selected with neomycin (500 µg/ml) until resistant colonies of cells appeared.

Total RNA was extracted from the rat kidney cortex and cultured podocytes using TRIzol reagent (Thermo Fisher Scientific, Inc.). RNA (1.5 µg) was used for cDNA synthesis (Thermo Fisher Scientific, Inc.) and 2 µl cDNA was used to perform qPCR with 1X SYBR Green Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 250 nM of primers on a 7700 real-time PCR Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primer sequences were used: Sense, 5′-gctgcttgggagcgacgtgt-3′ and antisense, 5′-cgttaggtggtgcggctccc-3′ for CMIP; sense, 5′-caaggatgccatcaagaa-3′ and antisense, 5′-gtccttgacctcctcgta-3′ for cofilin-1; and sense, 5′-tgtgtccgtcgtggatctga-3′ and antisense, 5′-cctgcttcaccaccttcttga-3′ for GAPDH. The PCR thermocycling program was started with initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing and extension at 60°C for 30 sec. The normalized mRNA gene level was calculated using 2^−ΔΔCq and standardized to control (16).

Podocytes were cultured on coverslips in 12-well plates. Cells were fixed with 4% paraformaldehyde, prepared in phosphate-buffered saline (PBS) and permeabilized with 0.25% Triton X-100/PBS (Sigma-Aldrich; Merck KGaA). Non-specific staining was reduced by incubation in 3% bovine serum albumin (BSA)/PBS at room temperature for 30 min. Actin cytoskeleton was revealed with Alexa 488-conjugated phalloidin. After three washes with PBS, the coverslips were mounted on glass slides with immunofluorescence anti-fade reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Images were obtained using immunofluorescence microscopy and processed with Photoshop software (Adobe Systems, Inc., San Jose, CA, USA). Actin filaments were analyzed using image thresholding techniques using the ImageJ 1.49p plugin, Analyze Particles (National Institutes of Health, Bethesda, MD, USA). The lengths of actin filaments, defined as Feret's diameter (the longest distance between any two points along the selection boundary) were measured, and the average length (mean ± standard deviation) of actin filaments was compared.

Total glomerular and cellular protein was extracted using RIPA buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 1 mM EGTA (pH 8.0) and 10% glycerol) supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Protein concentration was quantified using a bicinchoninic acid assay kit (Bio-Rad Laboratories, Inc.). In total, 75 µg protein was used for 7.5 and 12.5% SDS polyacrylamide gel electrophoresis (90 V for 1–1.5 h), and the protein was transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The membrane was blocked for 1 h at room temperature in 5% BSA/Tris-buffered saline containing 0.05% Tween-20 (TTBS). The following primary antibodies were diluted in 5% BSA/TTBS and incubated at 4°C overnight: Rabbit anti-CMIP (1:500), rabbit anti-p-cofilin-1 (1:1,000), mouse anti-cofilin-1 (1:1,000), mouse anti-LIMK1 (1:400), rabbit anti-p-LIMK1 (1:600), rabbit anti-p-GRF1 (1:800), rabbit anti-GRF1 (1:500), mouse anti-Fyn (1:1,000), rabbit anti-Src^Tyr418 (1:750), rabbit anti-Src^Tyr527 (1: 1,000 mouse anti-GAPDH (1:5,000), and mouse anti-β-actin (1:10,000). After five washes with TTBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG (1:5,000 in 5% BSA/TTBS; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The blots were developed with an enhanced ECL chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.), and the specific band was scanned and quantified using ImageJ version 1.47 (National Institutes of Health, Bethesda, MD, USA).

To analyze the specific activity of the Src kinase, Fyn, an immunoprecipitation assay was performed. Briefly, a total of 250 µg protein was incubated with 2 µg mouse anti-Fyn antibody overnight at 4°C and 2 µg of normal mouse IgG (sc-2025; Santa Cruz, Shanghai, China) served as a control. Protein G agarose beads (50 µl) were added and incubated for 4 h. Following three washes by centrifugation (3,000 × g, at 4°C for 1 min), 20 µl of 2X loading buffer was added to the beads and boiled for 5 min. Finally, an immunoblot assay as described above was performed using the anti-pSrc418 antibody.

The Rho switch operates by alternating between an active guanosine-5′-triphosphate (GTP)-bound state and an inactive guanosine diphosphate (GDP)-bound state. The Raf-like Ras-binding domain (RBD) motif of the Rho effector protein, Rhotekin, has been demonstrated to bind specifically to the GTP-bound form of RhoA. Therefore, glutathione S-transferase-tagged Rhotekin-RBD protein on colored agarose beads (Cytoskeleton, Inc.) was used to quantify RhoA activation in cells Briefly, 200 µg samples were combined with 20 µl reconstitute Rhotekin-RBD beads, and gently rotated at 4°C for 1 h. Beads were then washed five times in Wash Buffer (25 mM Tris (pH 7.5), 30 mM MgCl₂ and 40 mM NaCl) by centrifugation (3,000 × g, at 4°C for 1 min). Finally, bead pellets were resuspended in 30 µl of 2X SDS Lamelli buffer (1610737; Bio-Rad Laboratories, Inc.) supplemented with 20 mM DTT and boiled for 5 min. GTPγS- and GDP-treated samples served as positive and negative controls, respectively.

Data are presented as the mean ± standard deviation. Statistical evaluation was performed using one- or two-way analysis of variation and P<0.05 was considered to indicate a statistically significant difference.

The alteration of CMIP and cofilin-1 expression levels was assessed in PA-induced podocyte damage in vitro and in vivo. At the indicated time points, no marked changes in total cofilin-1 abundance were observed at the mRNA and protein expression levels following PA treatment in cultured mouse podocytes (Fig. 1A and B; P>0.05). However, the immunoblot assay detected a time-dependent significant increase of p-cofilin-1^Ser3 in PA-treated podocytes compared with untreated cells (Fig. 1B; P<0.01). The mRNA and protein abundance of CMIP significantly increased (P<0.01) in a time-dependent manner in PA-treated podocytes (Fig. 1A and B). Nephrosis was induced in rats by i.p. injection of PA (10 mg/100 g body weight), and 24-h proteinuria was analyzed. Compared with day 0, proteinuria was significantly detected at day 3, peaked at day 7, and then decreased at day 14 following the PA injections (Fig. 1C; P<0.01). Immunoblotting of the isolated glomeruli indicated that CMIP mRNA and protein expression levels were significantly increased at days 3 and 7 in PA-injected rats (Fig. 1D and E). Total cofilin-1 did not change, whereas p-cofilin-1^Ser3 increased significantly (P<0.01) at days 3 and 7 following the PA injections (Fig. 1F). Notably, the increased p-cofilin-1 expression level indicates a reduction of cofilin-1 activity (11). Thus, increased CMIP and reduced cofilin-1 activity may be associated with PA-induced podocytes.

To assess the potential association between CMIP and cofilin-1, knockdown of CMIP was performed using the specific siRNA against the mouse CMIP gene (siCMIP) and the podocyte clone, which stably expresses siCMIP, was obtained by selection with neomycin. As compared with control siRNA (siCTL), the CMIP protein expression level was significantly decreased in non-PA-treated podocytes expressing siCMIP. PA significantly induced CMIP expression in the control siRNA-expressing podocytes, but not in the siCMIP-expressing cells (Fig. 2A). In non-PA-treated podocytes, CMIP knockdown exhibited no obvious effect on the p-cofilin-1^Ser3 expression level. However, PA-induced upregulation of the p-cofilin-1^Ser3 expression level was significantly prevented in siCMIP-expressed podocytes (Fig. 2B). Therefore, the current in vivo and in vitro results demonstrate that stimulation of CMIP expression decreased the activity of cofilin-1. Cofilin is an actin-binding protein that is essential for actin de-polymerization (13). Subsequently, the effect of CMIP on actin remodeling in PA-treated podocytes was evaluated in the current study. CMIP knockdown alone did not alter the architecture of the actin cytoskeleton. Compared with non-PA-treated cells, the application of PA led to a marked re-organization of the actin cytoskeleton, which was displayed as nucleation and loss of actin filaments, which was prevented in the CMIP knockdown podocytes, but not in the control knockdown cells (Fig. 2C). The average length (mean ± standard deviation) of actin filaments was further measured and compared, demonstrating that the length of actin filaments was significantly decreased (P<0.01) in PA-treated podocytes, which was protected by siCMIP, but not by siCTL (Fig. 2D).

Previous studies reported that the small GTPase, RhoA is crucial in regulating the activity of cofilin-1 (17). The level of activated RhoA was evaluated in PA-treated podocytes, and it was identified that, when compared with the control, the activated RhoA level was significantly increased at 12 and 24 h following PA administration (Fig. 3A; P<0.01). Consistently, the p-LIMK^Thr508, a downstream effector of RhoA, increased significantly in the PA-treated podocytes (Fig. 3B; P<0.01). It was demonstrated that RhoA regulates cofilin-1 activity via LIMK (18). In addition, it was found that PA-induced upregulation of p-cofilin-1^Ser3 was reduced significantly by LIMK knockdown (Fig. 3C; P<0.01). The role of RhoA signals was further investigated using RhoA inhibitor, CT04. Two different concentrations (0.5 or 1.0 mg/ml) of CT04 were used in the current study. In cultured podocytes, the two significantly blocked PA-induced RhoA activation (Fig. 3D; P<0.01). The effects of CT04, at a concentration of 1.0 mg/ml, on LIMK and cofilin-1 activities were investigated. The present data indicated that CT04 prevented the increase of p-LIMK^Thr508 and p-cofilin-1^Ser3 in PA-treated podocytes (Fig. 3E).

In CMIP knockdown podocytes, RhoA activity was investigated following PA treatment for 24 h. Firstly, knockdown of CMIP was confirmed in PA-treated podocytes. The immunoblotting results indicate that PA-induced CMIP expression was not detected in cultured podocytes with CMIP knockdown (Fig. 4A). PA-induced RhoA activation was prevented by CMIP knockdown in the cultured podocytes (Fig. 4B), indicating that overproduced CMIP may cause RhoA activation. Furthermore, CMIP knockdown significantly reduced the p-LIMK^Thr508 level in PA-treated podocytes (Fig. 4C; P<0.05). It is well known that Rho GTPase activity is regulated by numerous guanine nucleotide exchange factors and GTPase-activating proteins (19). The current study identified that the phosphorylated level of p190RhoGAP at Tyr1105, also termed GRF1, was significantly decreased in the PA-treated podocytes compared with the untreated cells (Fig. 4D; P<0.05), while PA-induced reduction of p-GRF1^Tyr1105 was significantly prevented by CMIP knockdown (Fig. 4D; P<0.05). In untreated podocytes, CMIP was overexpressed by transfection of pcDNA-CMIP plasmids. In comparison with the control plasmid transfection, decreased p-GRF1^Tyr1105 and increased RhoA activation was detected in CMIP overexpressed podocytes (Fig. 4E). These results indicate that PA-induced overproduction of CMIP leads to RhoA activation potentially by inhibiting p190RhoGAP.

It has been reported that increased CMIP inhibits the activation of the Src kinase, Fyn in CMIP-overexpressed podocytes and in angiotensin II-induced podocyte damage (5,20). The activation status of the Src kinase Fyn is associated with RhoA activity. Src activation leads to the inactivation of RhoA through formation of a complex with p190RhoGAP and mDia interacting protein (21). In PA-treated podocytes, the activation level of Fyn, a predominant Src kinase in podocytes, was analyzed. Overproduction of CMIP was observed in PA-treated podocytes. Therefore, the activity of the Src kinase was investigated in a PA-induced podocyte damage model. The application of PA significantly decreased (P<0.05) the activated level of Src kinase (p-Src^Tyr418), while the inactivated level (p-Src^Tyr527) was increased in the cultured podocytes (Fig. 5A). The specific activity of Fyn was evaluated using an immunoprecipation assay with an anti-Fyn antibody and blotting with anti-pSrc418 antibody. The PA-induced reduction of activated Src kinase (p-Src^Tyr418) was prevented in CMIP knockdown podocytes as compared with the control knockdown cells (Fig. 5B).