Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 31 (5), 442-55

Hydrogen Sulfide Regulates Homocysteine-Mediated Glomerulosclerosis

Affiliations

Hydrogen Sulfide Regulates Homocysteine-Mediated Glomerulosclerosis

Utpal Sen et al. Am J Nephrol.

Abstract

Background/aims: In this study we tested the hypothesis that H(2)S regulates collagen deposition, matrix metalloproteinases (MMP) and inflammatory molecules during hyperhomocysteinemia (HHcy) resulting in attenuation of glomerulosclerosis and improved renal function.

Materials and methods: A genetic model of HHcy, cystathionine beta-synthase heterozygous (CBS+/-) and wild-type (WT) 2-kidney (2K) mice were used in this study and supplemented with or without NaHS (30 micromol/l, H(2)S donor) in drinking water for 8 weeks. To expedite the renal damage associated with HHcy, uninephrectomized (1K) mice of similar groups were also used.

Results: Results demonstrated that NAD(P)H oxidase (p47(phox)subunit) and blood pressure were upregulated in WT 1K, CBS+/- 2K and CBS+/- 1K mice with downregulation of H(2)S production and reduced glomerular filtration rate. These changes were normalized with H(2)S supplementation. Both pro- and active MMP-2 and -9 and collagen protein expressions and glomerular depositions were also upregulated in WT 1K, CBS+/- 2K and CBS+/- 1K mice. Increased expressions of inflammatory molecules, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1, as well as increased macrophage infiltration, were detected in WT 1K, CBS+/- 2K and CBS+/- 1K mice. These changes were ameliorated with H(2)S supplementation.

Conclusion: Together, these results suggest that increased oxidative stress and decreased H(2)S in HHcy causes matrix remodeling and inflammation resulting in glomerulosclerosis and reduced renal function.

Figures

None
Fig. 1
Fig. 1
Attenuated renal cortical tissue H2S production and CSE expressions were normalized by H2S treatment. a In the presence of 10 mmol/l L-cysteine and 2 mmol/l pyridoxol 5′-phosphate, H2S production in the kidney cortex tissue homogenates was measured following the procedure as described in the ‘Materials and Methods’. ∗ p < 0.01 vs. 2K WT; p < 0.05 vs. 2K CBS+/−; p < 0.05 vs. 1K CBS+/−. b Representative Western blot showed CSE expression in the kidney cortex tissue in different experimental animal groups. The bar diagram showed densitometric analyses of CSE expression against β-actin loading control. Data represents the mean ± SEM, n = 4–7. ∗ p < 0.01 vs. respective 2K mice and p < 0.01 vs. respective 1K mice.
Fig. 2
Fig. 2
Increased blood pressure was normalized with H2S supplementation. Blood pressure was measured using radiotelemetry devices (model TA11PA-C10; DSI, St. Paul, Minn., USA). A miniature intra-arterial catheter was surgically implanted in the ventral subcutaneous space with the catheter inserted into the left carotid artery. Mice were allowed to recover for 1 week before they were placed on telemetric matrix while they remained in their cages, and devices were turned on for hemodynamics measurement. Summarized data (n = 4–6 mice/group).
Fig. 3
Fig. 3
Impaired GFR in HHcy mice was ameliorated by H2S supplementation. NaHS (H2S donor, 30 μmol/l) was supplemented with drinking water for 8 weeks in the appropriate groups as shown. GFR was measured using the gold standard method (FITC-inulin) as described in ‘Materials and Methods’. Data represents the mean ± SEM, n = 7 in each group.
Fig. 4
Fig. 4
H2S mitigated NAD(P)H oxidase (p47phox subunit) in the HHcy kidney. Western blot was performed to measure the p47phox subunit of NAD(P)H oxidase in the kidney cortex tissue extract using anti-p47phox antibody. Data represents the mean ± SEM, n = 5–7. ∗ Significant difference (p < 0.05) compared to 2K WT, and ∗∗ significant difference (p < 0.05) compared to 2K CBS+/−.
Fig. 5
Fig. 5
H2S diminished Hcy-induced p47phox upregulation in mesangial cells as well as in the glomerulus. a Kidney mesangial cells were cultured in 8-well chamber slides. Cells were pretreated with H2S (NaHS, 30 μmol/l) 15 min before cells were exposed to Hcy (50 μmol/l) for 48 h (NaHS and Hcy were freshly added after every 12 h). Cells were fixed, permeabilized, blocked with 1% BSA in PBS, and immunostained with anti-p47phox antibody secondarily conjugated with FITC. Also, cells were counterstained with DAPI. Fluorescence images were taken under the laser scanning confocal microscope (FluoView 1000, Olympus). Green fluorescence, as indicated by arrows, indicated p47phox expression. Representative images from independent experiments (n = 5). b Representative images of p47phox immunostained glomerulus (n = 5–7 animals/group) from different experimental animal groups.
Fig. 6
Fig. 6
H2S attenuated MMP-2 and -9 expressions in the kidney. Western blot analyses were performed to measure MMP-2 and -9 in the kidney cortical tissue extract using anti-MMP-2 and anti-MMP-9 antibodies, respectively. The bar diagram indicated densitometric analyses of expressed protein. Data represents the mean ± SEM; n = 5–7 per group. ∗ p < 0.05 vs. respective 2K; ∗∗ significant differences (p <0.05) vs. respective 1K; p < 0.05 vs. 2K WT.
Fig. 7
Fig. 7
Excessive collagen deposition in HHcy mice was ameliorated by H2S supplementation. Histological kidney tissue sections were stained with Masson trichrome (collagen appears as blue, and indicated by arrows). 2K CBS+/− mice showed increased collagen deposition in the glomerular basement membrane, compared to 2K WT. This collagen deposition in the glomerulus of 1K CBS+/− mice was even higher than in 2K CBS+/− mice. When a separate group of 1K CBS+/− mice was supplemented with H2S (NaHS, 30 μmol/l in drinking water) for 8 weeks postsurgery, the collagen appeared much less in the glomerulus. Notably, their 1K WT littermates had very little collagen appearance and this disappeared with NaHS supplementation (n = 7/group; ×200).
Fig. 8
Fig. 8
H2S-attenuated collagen IV protein expression. Kidney cortex tissues were analyzed by Western blot using anticollagen IV antibody. While collagen expression was high in 1K WT and both 2K and 1K CBS+/− mice, this increased expression was ameliorated with H2S supplementation (NaHS, 30 μmol/l) for 8 weeks postsurgery. The bar diagram indicated densitometric analyses of the blots. Data represents the mean ± SEM; n = 5–7/group. ∗ Significant difference (p <0.05) compared to 2K WT.
Fig. 9
Fig. 9
Expression of collagen IV gene in the kidney cortical tissues. Renal cortical collagen protein was analyzed by Western blot. The bar diagram showed densitometric analyses of blots. Data represents the mean ± SEM, n = 5–7/group. ∗ p < 0.01 compared to WT 2K; ∗∗ p < 0.05 compared to CBS+/− 2K.
Fig. 10
Fig. 10
Inflammatory molecule ICAM-1 and VCAM-1 were mitigated by H2S. Representative Western blot showed ICAM-1 and VCAM-1 expressions in the kidney cortex tissue in different experimental animal groups. The bar diagram indicated densitometric analyses of expressed protein in the respective blots. Data represents the mean ± SEM, n = 5–7/group. ∗ Significant difference (p < 0.01) compared to 2K WT littermates; p < 0.01 vs. 1K WT; # p < 0.01 vs. 2K CBS+/−; p < 0.01 vs. 1K CBS+/−.
Fig. 11
Fig. 11
Increased macrophage infiltration was mitigated by H2S. a Kidney tissue cryosections (2-μm) were immunostained with macrophage F4/80 marker and visualized with DAB (3,3′-diaminobenzidine) staining. Increased brown staining, as indicated by arrows, in 1K WT, 2K CBS+/− and 1K CBS+/− mice indicated more macrophage infiltration in the glomerulus. In the similar groups, H2S supplementation prevented this infiltration. Representative images (n = 5/group); data represent the mean ± SEM. b Bar diagram showed intensity changes of DAB staining against 2K WT glomerulus. ∗ p < 0.05 vs. 2K WT; ∗∗ p < 0.05 vs. 1K WT; †,‡ p < 0.05 vs. 2K CBS+/− and ‡‡ p < 0.05 vs. 1K CBS+/−.

Similar articles

See all similar articles

Cited by 31 articles

See all "Cited by" articles

Publication types

Feedback