Hydrogen Sulfide Protects Hyperhomocysteinemia-Induced Renal Damage by Modulation of Caveolin and eNOS Interaction

Abstract

The accumulation of homocysteine (Hcy) during chronic kidney failure (CKD) can exert toxic effects on the glomeruli and tubulo-interstitial region. Among the potential mechanisms, the formation of highly reactive metabolite, Hcy thiolactone, is known to modify proteins by N-homocysteinylation, leading to protein degradation, stress and impaired function. Previous studies documented impaired nitric oxide production and altered caveolin expression in hyperhomocysteinemia (HHcy), leading to endothelial dysfunction. The aim of this study was to determine whether Hhcy homocysteinylates endothelial nitric oxide synthase (eNOS) and alters caveolin-1 expression to decrease nitric oxide bioavailability, causing hypertension and renal dysfunction. We also examined whether hydrogen sulfide (H₂S) could dehomocysteinylate eNOS to protect the kidney. WT and Cystathionine β-Synthase deficient (CBS+/-) mice representing HHcy were treated without or with sodium hydrogen sulfide (NaHS), a H₂S donor (30 µM), in drinking water for 8 weeks. Hhcy mice (CBS+/-) showed low levels of plasma H₂S, elevated systolic blood pressure (SBP) and renal dysfunction. H₂S treatment reduced SBP and improved renal function. Hhcy was associated with homocysteinylation of eNOS, reduced enzyme activity and upregulation of caveolin-1 expression. Further, Hhcy increased extracellular matrix (ECM) protein deposition and disruption of gap junction proteins, connexins. H₂S treatment reversed the changes above and transfection of triple genes producing H₂S (CBS, CSE and 3MST) showed reduction of vascular smooth muscle cell proliferation. We conclude that during Hhcy, homocysteinylation of eNOS and disruption of caveolin-mediated regulation leads to ECM remodeling and hypertension, and H₂S treatment attenuates renovascular damage.

Figure 1

Hhcy decreases plasma H₂S level and glomerular filtration rate (GFR) and H₂S treatment improves GFR. Plasma Hcy was measured by HPLC and GFR by FITC Inulin method. (A) Plasma Hcy levels, (B) Plasma H₂S levels and (C) Glomerular filtration rate (GFR), (D) H₂S reduces systolic BP in hyperhomocysteinemic (CBS+/−) mice. Values are mean ± SEM, n = 7/group. *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups, ^‡p < 0.05 vs. WT (control).

Figure 2

Hhcy reduces renal cortical blood flow. (A) Laser Doppler flowmetry line trace showing renal blood flow (blue line, black arrow) in the kidney. Black trace, is from aorta, red trace from renal artery and pink trace from renal vein. (B) Flux unit change from WT. Flux units = No. of RBCs x velocity. It is used as a surrogate for blood flow. n = 6/group, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups.

Figure 3

H₂S restores Hhcy induced reduced renal cortical vascularity. (A) Representative barium angiogram of left kidneys showing total vascularity. Interlobar arteries are shown by white arrows, arcuate arteries by red arrows and interlobular arteries are shown by yellow arrows, (B) Analysis of renal vessels by Vessel Segmentation and analysis software, (C) Change in total vascular density as % from control mice (WT). n = 6/group, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups. TV, total vascularity; ILA, interlobular artery; AA, arcuate artery.

Figure 4

H₂S increases eNOS activity by caveolin-1 modulation. (A) Immunoprecipitation of endothelial nitric oxide synthase (eNOS, conc.: 2 µg/200 µg of protein) and immunoblotting for eNOS (MW: 140 kDa) and Hcy (MW: 150–160 kDa) in the kidney. Caveolin-1 (Cav-1), (MW: 20 kDa) was quantified in the non-immunoprecipitated sample (cropped immunoblot image), (B) Fold change of proteins eNOS and Hcy, (C) Fold change of Cav-1, (D) Representative images of DAF-2DA fluorescence in MGECs treated without and with Hcy and H₂S, and (E) Bar graph showing mean fluorescence ± SEM of DAF-2DA in MGECs. Magnification × 60, scale bar: 20 µm. MW: Molecular weight. n = 5/group, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups, in vitro experiments, *p < 0.05 vs. Hcy, ^†p < 0.05 vs. Ctr and H₂S.

Figure 5

The expressions of MMP/TIMP is altered during Hhcy and H₂S restores normal MMP/TIMP balance and reduces collagen deposition. (A) Representative cropped immunoblot images of protein expression of MMP-2, -9 and -13 (MW: 72, 82, and 45 kDa resp.) and TIMP-1, -2 and -4 (MW: 23, 21 and 26 kDa resp.); (B,C) MMP and TIMP fold change, (D,E) Representative gelatin zymogram in MGECs and media and bar graph, (F,G) Zymography of kidney lysate and bar graph. Analysis was done using ImageJ. Values are expressed as fold change. MW: Molecular weight. n = 6/group for Western blot and n = 4/group for zymography, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups, in vitro experiments, *p < 0.05 vs. Hcy, ^†p < 0.05 vs. Ctr and H₂S.

Figure 6

Hhcy increased collagen deposition in the glomeruli, tubulointerstitium and renal cortical arteries. (A) Representative images of Masson Trichrome staining for collagen (blue arrows), (B) Interlobular arteries show increased collagen type I (yellow arrow) with pricrosirius red stain. Magnification ×20 for Masson Trichrome, scale bar: 60 µm, and magnification ×100 for picrosirius red stain, scale bar: 20 µm. n = 5, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups.

Figure 7

H₂S reduces Hhcy-induced vascular smooth muscle cell proliferation. (A) Fluorescence images of Ki-67 in VSMCs treated without or with Hcy (75 µM) and H₂S (30 µM), (B) Quantification of Ki-67, C) MTT assay showing relative VSMCs proliferation to control over 48 h. Magnification ×60, scale bar: 20 µm. n = 4, *p < 0.05 vs. Hcy, ^†p < 0.05 vs. Ctr and H₂S.

Figure 8

Triple gene transfection reduces BrdU incorporation in Hhcy mice. Fluorescence image of renal arteries. (A) Triple gene transfection (CBS, CSE and 3MST) and H₂S reduces BrdU incorporation in renal artery explants in Hhcy, (B) The number of strong fluorescence signals representing BrdU were analyzed using ImageJ software. Renal artery triple gene transfection: n = 4/group, magnification ×100, scale bar: 100 µm. *p < 0.05 vs. Hcy, ^†p < 0.05 vs. other groups.

Figure 9

H₂S downregulates Hhcy-induced increased expression of connexin 40 and 43. (A) Representative images of Cx40 in glomeruli and tubular regions (yellow arrows) and Cx43 in the tubules (white arrows), (B) Mean fluorescence analysis, (C,D) mRNA expression and fold change. Quantification was done using ImageJ software. Magnification × 60, scale bar: 20 µm, n = 6/group, *p < 0.05 vs. CBS+/− mice without H₂S treatment, ^†p < 0.05 vs. WT groups, ^‡p < 0.05 vs. WT (control).