Pyruvate alleviates high glucose-induced endoplasmic reticulum stress and apoptosis in HK-2 cells

Abstract

Endoplasmic reticulum (ER) stress plays a critical role in the development of diabetic nephropathy (DN). We previously demonstrated that pyruvate (Pyr)-enriched oral rehydration solution improved glucometabolic disorders and ameliorated DN outcome in db/db mice. Here, we investigated the effects of Pyr on high glucose-induced ER stress and apoptosis in HK-2 cells. Our results suggest that high glucose can induce reactive oxygen species production, apoptosis and ER stress in HK-2 cells, and that Pyr treatment can ameliorate these effects and restore the expression of key proteins involved in ER stress. Thus, Pyr may have potential for the development of novel strategies for the prevention and treatment of clinical DN.

Keywords: HK-2 cells; apoptosis; diabetes; diabetic nephropathy; endoplasmic reticulum stress; pyruvate.

Fig. 1

Pyr effects on cell proliferation as evaluated by CCK‐8. (A) HK‐2 cells were exposed to HG (30 mm) and treated with 0.01–10 mm Pyr for 72 h. (B) HK‐2 cells were exposed to HG (30 mm glucose) and treated with Pyr (0.5 mm) for 4 days. Con: cells were treated in 5 mm glucose in the DMEM. Values were means ± SEM (n = 5). Data were analyzed by one‐way ANOVA. *P < 0.05; **P < 0.01.

Fig. 2

Pyr effects on cell apoptosis by flow cytometry analysis. HK‐2 cells were exposed to HG and treated with Pyr (0.5 mm) for 72 h. Cells were stained with Annexin V/PI for flow cytometry analysis (A). The Q2 (Annexin V–FITC⁺/PI⁺) and Q3 (Annexin V–FITC⁺/PI⁻) were considered as early stage and late stage of apoptotic cells, respectively. Thus, the apoptosis ratio (B) was quantified by Q2 + Q3. The apoptosis ratio was expressed in a histogram. Con: cells were treated with 5 mm glucose in the DMEM; HG: cells were treated with 30 mm glucose in the DMEM; HG + Pyr: cells were treated with 30 mm glucose and 0.5 mm Pyr in the DMEM; Pyr: cells were treated with 5 mm glucose and 0.5 mm Pyr in the DMEM. Values are represented as mean ± SEM (n = 5). Data were analyzed by one‐way ANOVA. *P < 0.05.

Fig. 3

Pyr effects on apoptosis‐related protein expressions. HK‐2 cells were exposed to HG (30 mm glucose) and treated with Pyr (0.5 mm) for 72 h. (A) The protein expressions of Bcl‐2, BAX and Caspase‐3 of HK‐2 cells after exposure to HG in the presence or absence of Pyr were detected by western blot analyses. Con: cells were treated with 5 mm glucose in the DMEM; HG: cells were treated with 30 mm glucose in the DMEM; HG + Pyr: cells were treated with 30 mm glucose and 0.5 mm Pyr in the DMEM; Pyr: cells were treated with 5 mm glucose and 0.5 mm Pyr in the DMEM. The percentages of Bcl‐2 (B), BAX (C) and caspase‐3 (D)/β‐actin in the bar graphs were quantified by imagej software. Values were means ± SEM (n = 5). Data were analyzed by one‐way ANOVA. *P < 0.05; **P < 0.01.

Fig. 4

Pyr effects on ER stress‐related protein and ROS level. HK‐2 cells were treated with Pyr (0.5 mm) and/or HG (30 mm glucose) for 3 days. The protein expressions (A) of GRP78, CHOP, ATF4 and p‐EIF2α of HK‐2 cell were detected by western blot analyses. The percentages of GRP78 (B), CHOP (C), ATF4 (D) and p‐EIF2α (E)/β‐actin in the bar graphs were quantified by imagej software. Expressions of GRP78 (B), CHOP (C), ATF4 (D) and p‐EIF2α (E) were decreased after Pyr treatments, which ascertained that exogenous Pyr ameliorated the ER stress in HK‐2 cells. (F) HK‐2 cells were treated with Pyr (0.5 mm) and/or HG (30 mm glucose) for 3 days. Further, HG‐induced ROS increases were inhibited by Pyr treatments in HK‐2 cells. Con: cells were treated with 5 mm glucose in the DMEM; HG: cells were treated with 30 mm glucose in the DMEM; HG + Pyr: cells were treated with 30 mm glucose and 0.5 mm Pyr in the DMEM; Pyr: cells were treated with 5 mm glucose and 0.5 mm Pyr in the DMEM. Values were means ± SEM (n = 5). Data were analyzed by one‐way ANOVA. *P < 0.05; **P < 0.01.