Yonsei Med J.  2016 Sep;57(5):1252-1259. 10.3349/ymj.2016.57.5.1252.

Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells

• Wu Y
• Zhang M
• Liu R
• Zhao C

• KMID: 2374173
• DOI: http://doi.org/10.3349/ymj.2016.57.5.1252

Abstract

PURPOSE
Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells.
MATERIALS AND METHODS
Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis.
RESULTS
HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions.
CONCLUSION
Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.

Keyword

NHE1; diabetic nephropathy; oxidative stress; PKG

MeSH Terms

Antioxidants/metabolism
Apoptosis/*drug effects
Cation Transport Proteins/*metabolism
Cell Cycle/drug effects
Cell Line
Dose-Response Relationship, Drug
Epithelial Cells/*cytology/drug effects/*metabolism
Glucose/*pharmacology
Glutathione/metabolism
Humans
Kidney Tubules/*cytology
Oxidative Stress/*drug effects
Reactive Oxygen Species/metabolism
Signal Transduction/drug effects
Sodium-Hydrogen Antiporter/*metabolism
Antioxidants
Cation Transport Proteins
Glucose
Glutathione
Reactive Oxygen Species
Sodium-Hydrogen Antiporter

Figure

• 

  Fig. 1 High glucose (HG) induced apoptosis and oxidative stress in HK-2 cells. (A) Flow cytometry analysis and quantification of cell apoptosis in HK-2 cells incubated in 5 mM (NG) and 30 mM glucose (HG) at 48 h. (B) Apoptosis associated proteins Bax, Bcl-2, and caspase 3 were detected by western blotting after 48 h incubation with normal and HG. The proteins expressed in NG-treated HK-2 cells were set as "1." (C) ROS production in HG-stimulated cells was monitored by DCFH-DA and relative ROS was indicated. (D) DNA oxidative product 8-OHdG was stained using its specific antibody and cell nucleus were stained with DAPI. n=3. *p<0.05, †p<0.01, compared with NG treated cells. ROS, reactive oxygen species; DCFH-DA, dichlorofluorescein-diacetate; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; DAPI, 4',6-diamidino-2-phenylindole.

• 

  Fig. 2 High glucose (HG) upregulated NHE1 expression and increased pHi. (A) Expression of NHE1 in NG and HG-treated HK-2 cells at 0, 12, 24, and 48 h. (B) Intracellular pH changes in NG and HG-treated HK-2 cells at 0, 12, 24, and 48 h. n=3. *p<0.05, compared with NG treated cells. NHE1, Na+/H+ exchanger isoform 1.

• 

  Fig. 3 NHE inhibition repressed high glucose (HG)-induced HK-2 apoptosis. (A) Inhibitory efficiency of NHE1 siRNA on the expression of NHE1 as determined by western blotting. †p<0.01, compared control. (B) The negative control siRNA (NC) and NHE1 siRNA transfected cells were cultured in HG for 48 h, and the cell apoptosis was analyzed using flow cytometry. (C) HK-2 cells were pretreated with 20 nM NHE1 inhibitor DMA, then exposed to HG, and cell apoptosis was evaluated after 48 h. *p<0.05, compared with HG treated cells. (D) Intracellular pH change in HG-stimulated HK-2 cells in response to NHE1 inhibition by siRNA and DMA at 48 h. *p<0.05. Each experiment was performed in triplicates. NHE1, Na+/H+ exchanger isoform 1; DMA, 5-(N,N-dimethyl)-amiloride.

• 

  Fig. 4 Roles of PKG in high glucose (HG) induced NHE1 dysfunction. HK-2 cells were incubated with 5 mM GSH, 1 mM N-acetyl cysteine (N-Cys) or 1 mM exogenous H2O2 in the presence or absence of HG for 48 h. Then ROS (A) and NHE1 expression (B) were evaluated. (C) PKG level after stimulation with HG. (D) PKG inhibition with 20 nM KT-5823 on apoptosis of HG stressed HK-2 cells. (E) HK-2 cells were exposed to 3 µM exogenous cGMP and 20 nM KT-5823 in the presence or absence of HG for 24 h, and then ROS content was determined. (F) PKG signaling activation by cGMP or inhibition by KT-5823 on the expression of NHE1. *p<0.05, †p<0.01, compared with NG, ‡p<0.05, compared with HG. Each experiment was performed in triplicate. GSH, glutathione; NHE1, Na+/H+ exchanger isoform 1; ROS, reactive oxygen species.

• 

  Fig. 5 of the roles of NHE1 in high glucose induced tubular cell death. High glucose elevates cellular cGMP levels and leads to the activation of PKG, which enhances oxidative stress. NHE1 is induced in response to oxidative response, and cellular pHi increases by extruding H+. The elevated NHE1 triggers cell apoptosis by activating pro-apoptotic proteins. NHE1, Na+/H+ exchanger isoform 1; pHi, intracellular pH; ROS, reactive oxygen species.

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