Sam68 mediates high glucose‑induced podocyte apoptosis through modulation of Bax/Bcl‑2

Abstract

Hyperglycemia promotes podocyte apoptosis and contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms of hyperglycemia‑induced podocyte apoptosis remain unknown. Recent studies have implicated Src‑associated substrate during mitosis of 68 kDa (Sam68) in various cellular processes including RNA metabolism, apoptosis, signal transduction. This study sought to examine the effect of Sam68 on high glucose (HG)‑induced podocytes apoptosis, and the mechanism underlying this effect. Immortalized mouse podocytes were exposed to medium containing normal glucose, or HG and Sam68 siRNA, respectively. The expression of Sam68 in podocytes was determined by fluorescence quantitative PCR (qPCR), immunofluorescence and immunoblotting. The role of Sam68 in HG‑induced podocyte apoptosis was further evaluated by inhibiting Sam68 expression by Sam68 siRNA and performing flow cytometry. The mRNA and protein expression of pro‑apoptosis gene Bax and anti‑apoptotic gene Bcl‑2 were assessed by qRCR and immunoblotting. In the present study, it was first demonstrated that Sam68 was upregulated in a time and dose‑dependent manner in in vitro HG‑treated podocytes. Pretreatment with Sam68 siRNA markedly decreased nuclear Sam68 expression. Moreover, the effects of HG‑induced apoptosis were also abrogated by Sam68 knockdown in cultured podocytes. Furthermore, HG increased Bax and decreased Bcl‑2 protein expression in cultured podocytes, and this effect was blocked by Sam68 knockdown. The results of the present study revealed that Sam68 mediated HG‑induced podocyte apoptosis, probably through the Bax/Bcl‑2 signaling pathway, and thus may be a potential therapeutic target for DN.

Figure 1.

Sam68 is increased in HG-treated podocytes in vitro. (A) The effects of HG with different concentrations on the mRNA expression of Sam68. (B) The effects of HG at different time-points on the mRNA expression of Sam68. The mRNA level of Sam68 was detected using real-time-quantitative PCR, and was calculated with the 2^−ΔΔCq method. (C) The protein level of Sam68 was detected by immunoblotting in the nuclear extract of podocytes treated with different concentrations of glucose. Histone H3 was used as a nuclear protein loading control. (D) Densitometric analysis of three independent experiments displayed in C. Data are expressed as the mean ± SEM. *P<0.05 HG (20 mM, 30 mM, 40 mM) vs. NG. (E) The protein expression of Sam68 was detected by immunoblotting in the nuclear extract of podocytes incubated with HG at different time-points. Histone H3 was used as the nuclear protein loading control. (F) Densitometric analysis of three repetitions of the experiment displayed in E. Data are expressed as the mean ± SEM. *P<0.05, 24 HG (30 mM) or 48 h HG (30 mM) or 72 h HG (30 mM) vs. NG. Sam68, Src-associated substrate during mitosis of 68 kDa; HG, high glucose; PCR, polymerase chain reaction.

Figure 2.

Increased Sam68 expression in cultured podocytes with HG is inhibited by Sam68 knockdown. (A) The effects of siRNA sequences targeting Sam68 on the mRNA expression of Sam68 in in vitro cultured podocytes. Three pairs of siRNA sequences that targeted Sam68 and one pair of control-siRNA were designed and synthesized. The third pair of siRNA sequence was selected for further experiments based on the interference effect. NG (5.3 mM) group. Values are expressed as the mean ± SEM. *P<0.05 NG+Sam68-siRNA1 or NG+Sam68-siRNA2 vs. NG; ^#P<0.01, NG+Sam68-siRNA1-3 vs. NG+Con-siRNA. (B) The effects of Sam68 siRNA on the mRNA expression of Sam68 in HG-cultured podocytes. The mRNA level of Sam68 was examined with real-time-PCR, and was calculated with the 2^−ΔΔCq method. (C) Double immunofluorescence staining of Sam68 (red), synaptopodin-identified podocytes (green), DAPI-stained nuclei (blue) and merged images (purple) in cultured podocytes treated with HG for 48 h. (D) The protein expression of Sam68 was detected by immunoblotting in the nuclear extract of podocytes incubated with HG (30 mM) for 48 h. Histone H3 was used as the nuclear protein loading control. (E) Densitometric analysis of three independent experiments displayed in D. NG (5.3 mM) group; HG (30 mM) group; MA, NG (5.3 mM)+MA (24.7 mM) group, as an osmolality control; HG+Sam68-siRNA, HG (30 mM)+Sam68-siRNA (50 nM) group; HG+Con-siRNA, HG (30 mM)+non target-siRNA (50 nM) group. Values are expressed as the mean ± SEM. *P<0.05, HG or HG+Con-siRNA vs. NG; ^#P<0.05, HG+Sam68-siRNA vs. HG. Sam68, Src-associated substrate during mitosis of 68 kDa; HG, high glucose; PCR, polymerase chain reaction; NG, normal glucose; HG, high glucose; MA, mannitol.

Figure 3.

Sam68 mediates HG-induced podocyte apoptosis. (A) Podocytes cultured under different conditions were stained with Annexin V-FITC/PI for flow cytometric analysis. Cells that stained positive for Annexin V-FITC and negative for PI were considered apoptotic. (B) Histogram revealing changes in the percentage of apoptotic cells. NG (5.3 mM) group; HG group (30 mM); MA, NG (5.3 mM)+MA (24.7 mM) group, as an osmolality control; HG+Sam68-siRNA, HG (30 mM)+Sam68-siRNA (50 nM) group; HG+Con-siRNA, HG (30 mM)+non target-siRNA (50 nM) group. Data are expressed as the mean ± SEM. The experiments were performed in triplicate. *P<0.01, HG or HG+Con-siRNA vs. NG; ^#P<0.01, HG+Sam68-siRNA vs. HG. Src-associated substrate during mitosis of 68 kDa; HG, high glucose; NG, normal glucose; HG, high glucose; MA, mannitol.

Figure 4.

Sam68 regulates Bax and Bcl-2 expression in HG-cultured podocytes. (A) The protein expression of Bax and Bcl-2 were determined by western blotting analysis (n=3). (B and C) Densitometric analysis of three independent experiments presented in A. NG (5.3 mM) group; HG, (30 mM); MA, NG (5.3 mM) + MA (24.7 mM) group, as an osmolality control; HG+Sam68-siRNA, HG (30 mM)+Sam68-siRNA (50 nM) group; HG+Con-siRNA, HG (30 mM)+non target-siRNA (50 nM) group. All data are expressed as the mean ± SEM. *P<0.05 HG or HG+Con-siRNA vs. NG; ^#P<0.05 HG+Sam68-siRNA vs. HG. Src-associated substrate during mitosis of 68 kDa; HG, high glucose; NG, normal glucose; HG, high glucose; MA, mannitol.