Different sulfonylureas induce the apoptosis of proximal tubular epithelial cell differently via closing KATP channel

Abstract

Background: Sulfonylureas (SUs) are widely prescribed for the treatment of type 2 diabetes (T2DM). Sulfonylurea receptors (SURs) are their main functional receptors. These receptors are also found in kidney, especially the tubular cells. However, the effects of SUs on renal proximal tubular epithelial cells (PTECs) were unclear.

Methods: Three commonly used SUs were included in this study to investigate if different SUs have different effects on the apoptosis of PTECs. HK-2 cells were exposed to SUs for 24 h prior to exposure to 30 mM glucose, the apoptosis rate was evaluated by Annexin/PI flow cytometry. Bcl-2, Bax and the ratio of LC3II to LC3I were also studied by western blot in vitro. Diazoxide was used to evaluate the role of K_ATP channel in SUs-induced apoptosis of PTECs. A Student's t-test was used to assess significance for data within two groups.

Results: Treatment with glibenclamide aggravated the apoptosis of HK-2 cells in high-glucose, as indicated by a significant decrease in the expression of Bcl-2 and increase in Bax. Additionally, the decreased LC3II/LC3I reflects that the autophagy was inhibited by glibenclamide. Similar but less pronounced change was found in glimepiride group, however, nearly opposite effects were found in gliclazide group. Further, the effects of glibenclamide on apoptosis promotion and the decreased LC3II/LC3I were ameliorated obviously by treatment with 100uM diazoxide. The potential protection effect of gliclazide was also inhibited after opening the K_ATP channel.

Conclusion: Our results suggest that, the effects of glibenclamide and glimepiride on PTECs apoptosis, especially the former, were achieved in part by closing the K_ATP channel. In contrast to glibenclamide and glimepiride, therapeutic concentrations of gliclazide showed an inhibitory effect on apoptosis of PTECs, which may have a benefit in the preservation of functional PTECs mass.

Keywords: ATP-dependent potassium channel; Diabetes kidney disease; Glibenclamide; Gliclazide; Glimepiride; Proximal tubular epithelial cells.

Fig. 1

Effect of diazoxide on SUs-induced apoptosis was determined with Annexin V-FITC/PI staining by flow cytometry. a Flow cytometry results with Annexin V-FITC/PI staining. Cells were treated with SUs (left) and K_ATP channel opener (right), DZ, as described above. After culture for 24 h, cells were harvested and then apoptosis was analyzed with an Annexin V-FITC Apoptosis Detection Kit by flow cytometry. Cells were classified as healthy cells (Annexin V⁻, PI⁻), early apoptotic cells (Annexin V⁺, PI⁻), late apoptotic cells (Annexin V⁺, PI⁺), and damaged cells (Annexin V⁻, PI⁺). b The ratio of apoptosis among different experiment groups. Apoptosis ratio was early apoptosis percentage plus late apoptosis percentage. The date were presented as the mean ± SD. Columns, mean of three independent experiments; bars, SD; * p < 0.05, ** p < 0.01, n.s. not significant

Fig. 2

Bcl-2 and Bax expression in SUs and SUs + DZ groups. a Representative image of Western Blot of cells extracts. b Quantificaton of Western Blot results. Mean ± SD of three independent experiments *P < 0.05 versus control, ** p < 0.01, n.s. not significant

Fig. 3

. Effect of SUs and SUs + DZ on expression of autophagy-related protein. LC3I and LC3II expression in SUs and SUs + DZ groups. a Western Blot analysis of cells extracts confirmed almost complete loss of LC3I to LC3II conversion after treated with DZ. b Quantificaton of Western Blot results. Mean ± SD of three independent experiments * P < 0.05 versus control, ** p < 0.01, n.s. not significant