Cdc42 upregulation under high glucose induces podocyte apoptosis and impairs β-cell insulin secretion

Abstract

Objectives: The progressive impairment of β-cell function results in prolonged deterioration in patients with type 2 diabetes mellitus (T2DM). Interestingly, the finding on pancreatitis secondary to renal injury suggests that potential communication exists between kidney and pancreas. Therefore, we aimed to investigate cell division cycle 42 (Cdc42)-mediated podocyte apoptosis and its effect on insulin secretion in islet β-cells.

Methods: Type 2 diabetic nephropathy mouse models were established to identify the expression of Cdc42 in podocytes by immunohistochemistry. An in vitro co-culture of mouse podocyte MPC5 and β-TC6 cells was preliminarily established. Subsequently, podocyte apoptosis induced by high glucose and Cdc42 was detected by TUNEL staining and western blotting. In addition, the JNK pathway was examined to determine the mechanism of apoptosis in MPC5 cells. Finally, insulin secretion and expression in β-TC6 cells as well as malondialdehyde (MDA) and superoxide dismutase (SOD) levels in both cell types were examined after the regulation of Cdc42 in MPC5 cells.

Results: Cdc42 was highly expressed in the podocytes of diabetic nephropathy mice. Exposure to 25 mM glucose for 48 h induced a significant upregulation of Cdc42, Bax, and cleaved caspase-3 as well as a decreased Bcl-2 expression. In addition, marked apoptosis of MPC5 cells was observed compared to normal glucose treatment. After transfection with Cdc42 plasmid, apoptosis of MPC5 cells was enhanced with an increased expression of p-JNK, whereas inhibition of Cdc42 significantly alleviated podocyte apoptosis accompanied by a downregulation of p-JNK. The glucose-stimulated insulin secretion level of β-TC6 cells decreased after the upregulation of Cdc42 in MPC5 cells. Immunofluorescence staining for insulin showed that co-culture with MPC5 cells carrying the Cdc42 plasmid significantly reduced insulin expression, whereas inhibition of Cdc42 in MPC5 cells alleviated the above-mentioned abnormality of β-TC6 cells. The expression of Cdc42 and p-p38 in β-TC6 cells increased following the upregulation of Cdc42 in MPC5 cells; this was concurrent with augmented MDA levels and decreased SOD activity. The opposite result was observed for Cdc42 knockdown in MPC5 cells.

Conclusions: Cdc42 in podocytes plays a crucial role in insulin secretion by β-cells, which may provide a new therapeutic target to prevent the vicious cycle of β-cell dysfunction in T2DM.

Keywords: Cdc42; apoptosis; co-culture; podocyte; β-cells.

Figure 1

Examination of Cdc42 and Bax when treated with various glucose concentrations at different timepoints. (A–E) The protein expression levels of Cdc42, Bax, and cleaved caspase-3 increased, while those of Bcl-2 decreased, when treated with 25 mM glucose for 48 h. Data are presented as mean ± SEM; *P < 0.05.

Figure 2

Podocyte apoptosis was detected by TUNEL staining with different glucose concentrations. (A) Podocyte apoptosis rate increased significantly in response to high glucose (25 mM). White arrows point to TUNEL-positive podocytes (apoptotic podocytes); magnification: ×200; scale bar = 25 μm. (B) Absolute count of TUNEL-positive podocytes. Data were expressed as the mean numbers of apoptotic podocytes from 20 randomly selected fields and were from at least three independent experiments. Data are presented as mean ± SEM; *P < 0.05; ns, not significant.

Figure 3

Upregulation of Cdc42 increased the Bax levels in MPC5 cells. (A, B, D) The protein expression levels of Cdc42 and Bax were detected. When transfected with a plasmid carrying the Cdc42 gene, the Cdc42 and Bax proteins were significantly increased compared to the negative control, whereas transfection with Cdc42-siRNA reduced the Cdc42 and Bax protein levels. (C, E) The mRNA expression of Cdc42 and Bax increased in plasmid-transfected podocytes compared to the control; conversely, the mRNA expression of Cdc42 and Bax decreased in siCdc42-transfected podocytes. The data were from at least three independent experiments and are presented as mean ± SEM; *P < 0.05.

Figure 4

Upregulation of Cdc42-induced podocyte apoptosis. (A) The control podocytes, podocytes transfected with the plasmid carrying the Cdc42 gene, podocytes transfected with Cdc42-siRNA, and podocytes transfected with negative control siRNA were cultured in a medium containing glucose (5.5 mM). White arrows point to TUNEL-positive podocytes (apoptotic podocytes); magnification: ×200; scale bar = 25 μm. (B) The apoptosis rate (%) was calculated by dividing the number of TUNEL-positive cells by a population of 100 counted cells per condition. The podocyte apoptosis rate increased significantly after Cdc42 upregulation. The data were from at least three independent experiments. (C, D) The protein expression levels of JNK and p-JNK were detected. When transfected with the plasmid carrying the Cdc42 gene, the p-JNK/JNK ratio decreased significantly compared to the negative control, whereas transfection with Cdc42-siRNA increased the p-JNK/JNK ratio. The data are means ± SEM; *P < 0.05.

Figure 5

Upregulation of Cdc42 in podocytes impaired glucose-stimulated insulin secretion (GSIS) and insulin expression in β-TC6 cells. (A) A co-culture with MPC5 cells resulted in impaired insulin secretion from β-TC6 cells stimulated with 16.7 mM glucose compared with the monocultured group. The upregulation of Cdc42 in MPC5 cells diminished the GSIS of β-TC6 cells compared to the siNC group without a change in basal insulin secretion, while the impaired GSIS of β-TC6 cells was reversed by co-culture with MPC5 cells transfected with siCdc42. However, insulin secretion was partially restored when cotransfected with siCdc42 and Cdc42-res in MPC5 cells. (B) Immunofluorescence staining for insulin (red dots) showed a lower expression in the β-TC6 cells when co-cultured with MPC5 cells compared with monocultured β-TC6 cells. The upregulation of Cdc42in MPC5 cells significantly reduced the insulin expression in β-TC6 cells compared to the siNC group. When Cdc42 was suppressed in MPC5 cells, the insulin level was significantly upregulated in β-TC6 cells. Nevertheless, this effect was counteracted when co-cultured with MPC5 cells transfected with siCdc42 and Cdc42-res; original magnification: ×400. (C) The quantitative expression of insulin was examined, and no significant difference was observed. The data are presented as mean ± SEM; *P < 0.05.

Figure 6

The upregulation of Cdc42 in podocytes increased the Cdc42 expression and the p-p38/p38 ratio in β-TC6 cells, which were accompanied by an increase in malondialdehyde (MDA) levels and a decrease in SOD activity. (A–C) The protein expression levels of Cdc42, p-38, and p-p38 in β-TC6 cells were detected. (D–G) The levels of MDA and SOD in MPC5 cells and β-TC6 cells were measured. Data are presented as mean ± SEM; *P < 0.05.