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. 2017 Apr 7;292(14):5665-5675.
doi: 10.1074/jbc.M116.766758. Epub 2017 Feb 10.

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  1   2 , Doug Yoon Lee  1 , Meenalakshmi M Mariappan  1   2 , Denis Feliers  1   2 , Goutam Ghosh-Choudhury  1   2 , Hanna E Abboud  1   2 , Yves Gorin  1 , Balakuntalam S Kasinath  3   2
Affiliations

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 et al. J Biol Chem. .

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.

Keywords: AMP-activated kinase (AMPK); diabetic nephropathy; laminin; oxidative stress; reactive oxygen species (ROS).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
H2S inhibits high glucose-stimulated NOX4 expression. Quiescent MCT cells were incubated with 5 or 30 mm glucose (Glc) for 24 h with or without NaHS. A and B, immunoblotting was done for NOX4 expression, with actin as a loading control. C and D, NOX4 and GAPDH mRNA were measured by quantitative RT-PCR. E, cells were preincubated with actinomycin D (ACD, 2 μg/ml) for 30 min before incubation with high glucose; immunoblotting was done to detect NOX4 and actin. In A–E, composite data from three to six experiments are shown in scatterplots. F, the NADPH oxidase assay was done as described under “Experimental Procedures;” data from three experiments (mean ± S.D.) are shown in a scatterplot. *, p < 0.05; **, p < 0.01 versus 5 mm glucose; #, p < 0.05; ##, p < 0.01 versus 30 mm glucose by ANOVA.
Figure 2.
Figure 2.
H2S inhibition of high glucose-induced NOX4 expression is AMPK-dependent. A and B, immunoblotting showed that NaHS promoted Thr-172 phosphorylation of AMPK and rectified its reduction induced by high glucose (Glc). Man, mannitol. C and D, NaHS increased Ser-79 phosphorylation of ACC, an AMPK substrate, and corrected its reduction induced by high glucose. E, compound C, an AMPK inhibitor, abolished NaHS inhibition of high glucose-induced NOX4 expression. A–E, composite data from three to four experiments (mean ± S.D.) are shown in scatterplots. A and C, *, p < 0.5; **, p < 0.01 versus no NaHS. B, D, and E, *, p < 0.05 versus 5 mm glucose; #, p < 0.05; ##, p < 0.01 versus 30 mm glucose; §§, p < 0.01 versus 30 mm glucose + NaHS by ANOVA.
Figure 3.
Figure 3.
H2S inhibition of high glucose-induced NOX4 expression is associated with an increase in iNOS. A, immunoblotting showed that NaHS inhibition of high glucose (Glc)-induced NOX4 expression was abolished by l-NAME, a general NOS inhibitor. B, NaHS decreased Ser-1177 phosphorylation of eNOS but did not affect its expression. C, MCT cells did not express nNOS; however, nNOS was expressed by the brain (Br), employed as a positive control. D, NaHS increased iNOS expression in a time-dependent manner. E, NaHS increased the mRNA expression of iNOS as measured by quantitative RT-PCR. F, preincubation with ACD (2 μg/ml) for 30 min abolished the NaHS-induced increase in iNOS expression at 4 h. G, NaHS-induced iNOS expression could be inhibited by compound C, indicating that AMPK activation is required for NaHS stimulation of iNOS. H, cells were transfected with siRNA against iNOS (si-iNOS) or control siRNA (si-con) and incubated with or without high glucose and with or without NaHS. Expression of si-iNOS abolished NaHS inhibition of the high glucose-induced NOX4 increase at 24 h. In A, B, and D–H, composite data from three to four experiments (mean ± S.D.) are shown in scatterplots. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus respective control; #, p < 0.05; ###, p < 0.001 versus 30 mm glucose; †, p < 0.05 versus NaHS, §§§, p < 0.001 versus 30 mm glucose + NaHS by ANOVA.
Figure 4.
Figure 4.
H2S inhibition of high glucose-induced ROS generation is iNOS-dependent. A–H, DCF fluorescence was examined by confocal microscopy as an index of intracellular ROS. Exposure of cells to high glucose (Glc, 30 mm) for 24 h promoted an increase in ROS generation (B) compared with cells incubated with 5 mm glucose (A). NaHS abrogated high glucose-induced ROS generation (D), an effect that was reversed by l-NAME (F) and the iNOS-selective inhibitor 1400W (H). NaHS (C), l-NAME (E), and 1400W (G) did not affect basal ROS expression.
Figure 5.
Figure 5.
The NO donor NONOate inhibits high glucose-induced NOX4 expression by stimulating AMPK phosphorylation. A, immunoblotting showed that NONOate, an NO donor, promoted AMPK phosphorylation in a time-dependent manner. B, NaHS induced a second wave of AMPK phosphorylation at 8 h following an increase in iNOS expression seen at 4 h (Fig. 3D). C, expression of si-iNOS abolished NaHS induced AMPK phosphorylation at 8 h. D, NONOate inhibited high glucose (Glc)-induced NOX4 expression at 24 h. E, NONOate inhibition of high glucose-induced NOX4 expression was abolished by compound C (CC), indicating the requirement for AMPK activation. In A–E, composite data from three to six experiments (mean ± S.D.) are shown in scatterplots. *, p < 0.05; **, p < 0.01 versus respective control; #, p < 0.05; ##, p < 0.01 versus 30 mm glucose; †, p < 0.05 versus NaHS; ♢♢, p < 0.001 versus NONOate + 30 mm glucose by ANOVA.
Figure 6.
Figure 6.
High-glucose-induced laminin γ1 increase is abolished by H2S and is NOX4-dependent. A, immunoblotting showed that NaHS inhibited the high glucose (Glc)-induced increase in laminin γ1 (lamγ1) expression. Man, mannitol. B, cells were transfected with siRNA against NOX4 (si-NOX4) or control siRNA (si-con) and incubated with or without high glucose. Immunoblotting was done with antibodies against the indicated proteins. Composite data from three experiments (mean ± S.D.) are shown in scatterplots. A, *, p < 0.05 versus 5 mm glucose; #, p < 0.05 versus 30 mm glucose by ANOVA. B, *, p < 0.05; **, p < 0.01 versus 5 mm glucose (si-con); ###, p < 0.001 versus 30 mm glucose by ANOVA.
Figure 7.
Figure 7.
H2S inhibition of high glucose-induced laminin γ1 and NOX4 is iNOS-dependent. A and B, immunoblotting showed that NaHS inhibited the high glucose (Glc)-induced increase in laminin γ1 (Lamγ1) expression, and the increase was abolished by l-NAME, a general NOS inhibitor, or by 1400W, a selective inhibitor of iNOS. C, the immunoblot of lysates from cells transfected with si-iNOS or control si-RNA and treated with or without high glucose or NaHS from Fig. 3H was probed with antibody against laminin γ1; thus, the iNOS and actin bands are the same as in Fig. 3H. NaHS inhibition of high glucose-induced laminin γ1 at 24 h could be abrogated by si-iNOS. D, NONOate abolished high glucose-induced laminin γ1 expression. E, compound C (CC) inhibited NONOate reduction of high glucose-induced laminin γ1 expression. Composite data from three to five experiments (mean ± S.D.) are shown in scatterplots. *, p < 0.05; **, p < 0.01 versus 5 mm glucose; #, p < 0.05; ##, p < 0.01 versus 30 mm glucose; ♢, p < 0.05 versus NONOate + 30 mm glucose; §, p < 0.05; §§, p < 0.01 versus 30 mm glucose + NaHS by ANOVA.
Figure 8.
Figure 8.
A schematic showing pathways of interaction between NO and H2S signaling pathways in high glucose-treated MCT cells that were explored in this study.
See this image and copyright information in PMC

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