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

Abstract

In this study, mice lacking the gp91(phox) 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 O(2)(-) production in the glomeruli from gp91(phox) 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 microg/24 h; GDI, 1.17+/-0.18 vs 2.59+/-0.49; and GFR, 53.01+/-4.69 vs 40.98+/-1.44 microl/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 gp91(phox) expression and O2(*)(-) generation, which was substantially inhibited by gp91(phox) siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A production was abolished by gp91(phox) 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.

Figure 1. Effects of normal and FF diets on plasma Hcys levels and glomerular O₂^.− production in gp91^phox KO and WT mice

A. Plasma Hcys levels measured by HPLC in 4 groups of mice (n=6). B. Representative ESR spectra traces for O₂^.− production in gp91^phox KO and WT mice (n=5). C. Summarized data show the fold changes of O₂^.− production, which are normalized to WT mice on the normal diet (n=5). * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet; & p<0.05 vs. KO mice on the normal diet.

Figure 2. Proteinuria was attenuated in gp91^phox KO mice on the FF diet

A. Urinary total protein levels in 4 groups of mice (n=6). B. Urinary albumin excretion in 4 different groups of mice as indicated (n=6). * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet; & p<0.05 vs. KO mice on the normal diet.

Figure 3. Glomerular damage was alleviated in gp91^phox KO mice on the FF diet

A. PAS staining shows glomerular morphological changes (original magnification, ×400). B. Summarized data of glomerular damage index (GDI) by semi-quantitation of scores in 4 different groups of mice (for each group, n=6). For each kidney section, 50 glomeruli were randomly chosen for the calculation of GDI. * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet.

Figure 4. Creatinine clearance (Ccr) and arterial blood pressure in gp91^phox KO and WT mice

A. Creatinine clearance in WT and KO mice on a normal or FF diet (n=7). B. Mean arterial pressure (MAP) in WT and KO mice with or without FF diet (n=5). * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet.

Figure 5. Expressions of nephrin, podocin, and desmin in gp91^phox KO and WT mice

A. Real-time RT-PCR analysis of the expressions of nephrin, podocin, and desmin in the glomeruli of gp91 KO and WT mice (n=6). B. Immunofluorescent staining of nephrin, podocin, and desmin in the glomeruli of 4 groups of mice (n=4). * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet; & p<0.05 vs. KO mice on the normal diet.

Figure 6. Attenuation of foot process effacement and podocyte loss in the glomeruli of gp91^phox KO mice

A. gp91^phox gene deletion improved podocyte ultrastructure in FF diet-treated mice. Arrow denotes the area of foot process effacement in WT mice on the FF diet. Images are representative of 6 TEM images per kidney from 3 mice per group. Original magnification: ×8,000. B. Typical images of WT1-stained glomeruli from 4 groups of mice (Original magnification, ×400). C. Summarized data showing podocyte numbers per glomerulus in each group (n=6). * p<0.05 vs. WT mice on the normal diet; # p<0.05 vs. WT mice on the FF diet.

Figure 7. L-Hcys increases gp91^phox expression and NADPH oxidase-dependent O₂^.− production in cultured podocytes

A. L-Hcys stimulation for 12 h caused elevated gp91^phox mRNA expression in podocytes as determined by real-time RT-PCR (n=4). B. ESR analysis shows that Hcys induces O₂^.− production in a concentration-dependent manner in podocytes (n=5). C. ESR analysis shows that gp91^phox siRNA transfection or NADPH oxidase inhibition by DPI reduces O₂^.− production in podocytes. Ctrl: Control; Vehl: Vehicle; Scra: scrambled sRNA; siRNA: gp91^phox siRNA. (n=6). * p<0.05 vs.ctrl, # p<0.05 vs. Hcys.

Figure 8. Effects of gp91^phox gene silencing on L-Hcys-induced decrease in VEGF-A production and secretion in podocytes

A. Real-time RT-PCR analysis shows that Hcys stimulation for 12 h decreases VEGF-A mRNA levels in a concentration-dependent manner (n=4). B. Real-time RT-PCR analysis shows the expression of VEGF-A mRNA challenged by Hcys (80 μmol/L) for 12 h with different pretreatments. PAN was used as a positive control (n=4). C. VEGF-A secretion in the culture medium detected by ELISA after pretreatment with different inhibitors and Hcys for 24 h. Ctrl: Control; Vehl: Vehicle; Scra: scrambled sRNA; siRNA: gp91^phox siRNA. (n=5). * p<0.05 vs. ctrl. # p<0.05 vs. Hcys.