Original Contribution
Protection of podocytes from hyperhomocysteinemia-induced injury by deletion of the gp91phox gene

https://doi.org/10.1016/j.freeradbiomed.2010.01.029Get rights and content

Abstract

In this study, mice lacking the gp91phox gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that a folate-free diet increased plasma homocysteine levels, but failed to increase O2radical dot− production in the glomeruli from gp91phox gene knockout (gp91−/−) mice, compared with wild-type (gp91+/+) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, whereas the glomerular filtration rate (GFR) was higher in gp91−/− than in gp91+/+ mice when they were on the folate-free diet (urine albumin excretion, 21.23 ± 1.88 vs 32.86 ± 4.03 μg/24 h; GDI, 1.17 ± 0.18 vs 2.59 ± 0.49; and GFR, 53.01 ± 4.69 vs 40.98 ± 1.44 μl/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91+/+ mice were not observed in gp91−/− mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91−/− mice. In in vitro studies of podocytes, homocysteine was found to increase gp91phox expression and O2radical dot− generation, which was substantially inhibited by gp91phox siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A production was abolished by gp91phox siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis.

Section snippets

Animal procedures

C57BL/6J wild-type (WT; 6 weeks of age, male) and gp91phox knockout (KO) mice (6 weeks of age, male; The Jackson Laboratory, Bar Harbor, ME, USA) were used. All protocols were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University. Given the difficulty of the development of hyperhomocysteinemia in mice with both kidneys because of treatment that requires a long period of time, all gp91phox KO mice and WT mice were subjected to a uninephrectomy, which

Reduction of hHcys-induced O2radical dot− production in gp91phox KO mice

As shown by HPLC analysis, plasma Hcys levels were similar in WT and gp91phox KO mice on the normal diet. FF diet treatment significantly increased plasma Hcys levels in both WT and gp91phox KO mice (Fig. 1A). ESR analysis showed that glomerular O2radical dot− production was lower in KO mice than in WT mice on the normal diet. On the FF diet, glomerular O2radical dot− production increased by 2.4-fold in WT mice. However, FF diet-induced O2radical dot− production was much less in KO mice compared with WT mice (Figs. 1B and 1C).

Alleviation of glomerular injury in gp91phox KO mice on the FF diet

Discussion

The goals of this study were to determine whether podocytes are a direct target of hHcys and to investigate the role of NADPH oxidase-dependent O2radical dot− production in hHcys-induced podocyte injury. Using an FF diet-induced hHcys animal model in gp91phox gene-deficient mice and in vitro cultured podocytes, we clearly demonstrated that hHcys directly induces podocyte injury and depletion through gp91phox-containing NADPH oxidase activation and O2radical dot− production.

There is considerable evidence supporting

Acknowledgments

This study was supported by Grants DK54927, HL075316, and HL57244 from the National Institutes of Health.

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