Hydrogen sulfide inhibits high glucose-induced NADPH oxidase 4 expression and matrix increase by recruiting inducible nitric oxide synthase in kidney proximal tubular epithelial cells

Hak Joo Lee,Doug Yoon Lee,Meenalakshmi M Mariappan,Denis Feliers,Goutam Ghosh-Choudhury,Hanna E Abboud,Yves Gorin,Balakuntalam S Kasinath

doi:10.1074/jbc.m116.766758

Abstract

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H2S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H2S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H2S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H2S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N(ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H2S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H2S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.

Highlights

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells
Our data show that H2S inhibits high glucose-induced NOX4 expression, subsequent ROS generation, and matrix protein laminin increase in MCT cells by harnessing inducible NOS (iNOS) activation
The serial events are as follows: H2S stimulates an increase in iNOS expression (Fig. 3D), and the increased iNOS expression and NO generation mediate H2S inhibition of high glucoseinduced NOX4 expression (Fig. 3H), ROS generation (Fig. 4H), and increased matrix laminin content (Fig. 7C)

Summary

Results

Because NOX4 is constitutively active, the amount of ROS released by the enzyme is regulated by control of its protein expression [11, 22]. The inhibitory effect of NONOate on high glucose-induced NOX4 was blocked by compound C (Fig. 5E), indicating that it was AMPK-dependent These data show that NaHS promotes iNOS expression (Fig. 3D) and NO generation, leading to AMPK activation (Fig. 5A) and inhibition of high glucoseinduced NOX4 expression (Fig. 2E). That NO was involved in NaHS amelioration of the high glucose-induced increase in laminin ␥1 in an AMPK-dependent manner was confirmed by directly employing NONOate, the NO donor, and compound C, the AMPK inhibitor (Fig. 7, D and E) Together, these observations lead to the conclusion that H2S inhibition of highglucose stimulation of laminin ␥1 is mediated by NO generation by iNOS in MCT cells

Discussion

Cell culture

NADPH oxidase assay