IQGAP1 regulates actin cytoskeleton organization in podocytes through interaction with nephrin

Abstract

Increasing data has shown that the cytoskeletal reorganization of podocytes is involved in the onset of proteinuria and the progression of glomerular disease. Nephrin behaves as a signal sensor of the slit diaphragm to transmit cytoskeletal signals to maintain the unique structure of podocytes. However, the nephrin signaling cascade deserves further study. IQGAP1 is a scaffolding protein with the ability to regulate cytoskeletal organization. It is hypothesized that IQGAP1 contributes to actin reorganization in podocytes through interaction with nephrin. IQGAP1 expression and IQGAP1-nephrin colocalization in glomeruli were progressively decreased and then gradually recovered in line with the development of foot process fusion and proteinuria in puromycin aminonucleoside-injected rats. In cultured human podocytes, puromycin aminonucleoside-induced disruption of F-actin and disorders of migration and spreading were aggravated by IQGAP1 siRNA, and these effects were partially restored by a wild-type IQGAP1 plasmid. Furthermore, the cytoskeletal disorganization stimulated by cytochalasin D in COS7 cells was recovered by cotransfection with wild-type IQGAP1 and nephrin plasmids but was not recovered either by single transfection of the wild-type IQGAP1 plasmid or by cotransfection of mutant IQGAP1 [△1443(S→A)] and wild-type nephrin plasmids. Co-immunoprecipitation analysis using lysates of COS7 cells overexpressing nephrin and each derivative-domain molecule of IQGAP1 demonstrated that the poly-proline binding domain and RasGAP domain in the carboxyl terminus of IQGAP1 are the target modules that interact with nephrin. Collectively, these findings showed that activated IQGAP1, as an intracellular partner of nephrin, is involved in actin cytoskeleton organization and functional regulation of podocytes.

Keywords: Actin cytoskeleton; IQ domain GTPase-activating protein 1; Nephrin; Podocyte.

Fig. 1

The expression and distribution of IQGAP1 in glomeruli during PAN nephrosis. (A) Representative microscopy images of renal pathological changes in various groups (PAS staining, original magnification ×200). Scale bar = 20 μm. The black arrows indicate protein cast in the renal tubular lumen. (B) Quantitative analysis of urinary protein excretion in various groups (n = 6). (C) Coomassie brilliant blue staining of urinary protein in various groups. (D) Representative transmission electron microscopy images of glomerular capillary walls (original magnification ×10,000) and quantitative analysis of the FP width and FP fusion rate (n = 6). Scale bar = 1900 nm. Podo, podocyte. Cap, capillary lumen. (E) Western blotting analysis of glomerular IQGAP1 in various groups (n = 6). (F) Double immunolabeling of nephrin and IQGAP1 in glomeruli (original magnification ×400). The colocalization area of nephrin and IQGAP1 indicated by the blue dotted line is an enlarged view of the boxed region. Scale bar = 20 μm. *p < 0.05 relative to control.

Fig. 2

The expression of IQGAP1 in podocytes stimulated by PAN. (A) (B) Real-time PCR analysis of IQGAP1 expression in cultured podocytes stimulated by various concentrations of PAN for 6 h or by 50 μg mL⁻¹ PAN for 0–24 h (n = 3). (C) (D) Western blotting analysis of IQGAP1 expression in cultured podocytes treated with PAN (n = 3). *p < 0.05, #p < 0.01 relative to control.

Fig. 3

The interaction between IQGAP1 and nephrin and the phosphorylation of IQGAP1 in PAN-stimulated podocytes. (A) Double-immunolabeling of nephrin and IQGAP1 in cultured podocytes (original magnification ×400). The area indicated by the blue dotted line is the region of colocalized nephrin and IQGAP1. Scale bar = 20 μm. (B) Co-immunoprecipitation analysis between IQGAP1 and nephrin in podocytes treated with PAN (50 μg mL⁻¹ for 6 h) (n = 3). Control IgG is a normal rabbit IgG that replaced the anti-nephrin pAb in the precipitation process. (C) The phosphorylation of IQGAP1 detected by co-immunoprecipitation (n = 3). Control IgG is a normal rabbit IgG that replaced the anti-IQGAP1 pAb in the precipitation process. #p < 0.01 relative to control.

Fig. 4

Effects of altered IQGAP1 expression on nephrin, phosphorylated nephrin, cytoskeletal reorganization, and the interaction between IQGAP1 and nephrin in podocytes. (A) Western blotting analysis of the expression of nephrin, phosphorylated nephrin and IQGAP1 in cultured podocytes stimulated by PAN and pre-transfected with IQGAP1 siRNA or scrambled siRNA (n = 3). (B) Western blotting analysis of the expression of nephrin, phosphorylated nephrin and IQGAP1 in cultured podocytes stimulated by PAN and pre-transfected with the wild-type IQGAP1 plasmid or null vector (n = 3). (C) Co-immunoprecipitation analysis between IQGAP1 and nephrin in podocytes transfected with the relevant siRNA or plasmid in the presence or not in the presence of PAN (n = 3). Lysate indicates the podocyte lysate and was used as a positive control. Control IgG is a normal rabbit IgG and was used as a negative control. (D) FITC-phalloidin staining of the actin cytoskeleton in podocytes with altered IQGAP1 expression in the presence or not in the presence of PAN (original magnification ×400), and the quantitative analysis of CFS (n = 100). The nuclei were stained with DAPI. Scale bar = 20 μm. *p < 0.05 relative to control, #p < 0.05 relative to PAN-treated cells.

Fig. 5

Involvement of IQGAP1 in podocyte migration and spreading. (A) Representative migration results of podocytes stimulated with PAN and pre-transfected with IQGAP1 siRNA or plasmids (original magnification ×100). Scale bar = 100 μm. (B) Quantitative analysis of podocyte migration. (n = 3). (C) Representative spreading images of podocytes stimulated with PAN in the presence of IQGAP1 siRNA or plasmids (original magnification ×400). Scale bar = 30 μm. (D) Quantitative analysis of podocyte spreading (n = 3). *p < 0.05 relative to control, ^#p < 0.05 relative to PAN-treated cells.

Fig. 6

Involvement of the nephrin–IQGAP1 interaction and IQGAP1 phosphorylation in cytoskeletal regulation. (A) Western blotting analysis of the expression of nephrin and IQGAP1 in COS7 cells transfected with wild-type IQGAP1, mutated IQGAP1 (ΔSer1443) or wild-type nephrin plasmids. The podocyte lysate was used as a positive control. (B) The FITC-phalloidin staining of actin cytoskeleton in COS7 (or NCOS7) cells pretreated with CyD and transfected with IQGAP1 wild-type or mutated plasmids (original magnification ×400). Scale bar = 10 μm.

Fig. 7

Identification of the specific domains of IQGAP1 necessary for interacting with nephrin. (A) Schematic diagram depicting the domain organization of human IQGAP1 and various truncated IQGAP1 mutants. The location indicates the amino acid residues. (B) Detection of the nephrin binding sites in IQGAP1. The co-immunoprecipitation study was performed using COS7 cell lysates with (+) or without (−) nephrin overexpression and each derivative molecule of IQGAP1. Input: total protein extract.

Fig. 8

A schematic model for the involvement of IQGAP1 in actin cytoskeleton organization in podocytes.