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

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.

transsulfuration pathway yields cysteine and hydrogen sulfide (H 2 S). H 2 S is a gasotransmitter known to have multiple functions including regulation of vascular tone, neuromodulation, anti-oxidant and as an anti-inflammatory molecule 10,11 . A reduction of H 2 S producing enzymes and thus H 2 S has been implicated in animal model of CKD and clinical study 12,13 . Low plasma H 2 S has also been linked to decreased glomerular function and increased cardiac risk in CKD patients 14 . In contrast, supplementation of H 2 S has been shown to be beneficial in several studies [15][16][17][18] . Some of the beneficial effects of H 2 S are attributed to the activation of K ATP (ATP-sensitive K+) channels or scavenging free radicals 19 . Recently, it has been speculated whether H 2 S is involved in posttranslational protein modification for some of its biological effects 20 . It is possible that H 2 S may modify the course of protein-S-S bridge formation and reverse homocysteinylation of proteins such as eNOS.
The aim of the present study was to investigate whether homocysteinylation of eNOS and disruption of caveolin-mediated eNOS regulation leads to hypertension and renal dysfunction. Further, we investigated whether H 2 S supplementation dehomocysteinylates eNOS and reduces vascular smooth muscle cell proliferation and extracellular matrix protein deposition to protect the kidney from Hhcy mediated injury.

Results
Plasma H 2 S, renal perfusion and glomerular filtration rate (GFR) is reduced during hyperhomocysteinemia. Plasma Hcy was measured by HPLC (Fig. 1A). Plasma Hcy was increased more than two-fold in CBS+/− mice compared to WT mice (Fig. 1A). In addition, CBS+/− mice had low levels of plasma H 2 S compared to WT groups (Fig. 1B). NaHS supplementation reduced plasma Hcy levels in CBS+/− mice and increased plasma H 2 S levels (Fig. 1A,B respectively). Plasma H 2 S increased in WT mice treated with NaHS (Fig. 1B).
FITC-Inulin clearance showed reduction of GFR in CBS+/− mice compared to WT groups without or with H 2 S treatment (Fig. 1C). Following H 2 S treatment, GFR returned to normal in CBS+/− mice suggesting recovery of renal function (Fig. 1C). GFR was not affected by H 2 S in WT mice.
At 12 weeks of age, there was no difference in systolic blood pressure (SBP) between the groups. CBS+/− mice exhibited progressive increase in SBP commencing at 16 weeks until the end-point of the experiment (20 wks) compared to WT mice (Fig. 1D). SBP was attenuated following H 2 S treatment in CBS+/− mice (Fig. 1D). There was no difference in the baseline SBP in WT and CBS+/− mice. H 2 S treatment did not affect the SBP in WT mice.
Laser Doppler flowmetry was used to measure red blood cell flux (No. of RBCs × velocity) as an index of microvascular blood flow in the renal cortex. CBS+/− mice showed lower renal flux units compared to WT groups reflecting reduced perfusion ( Fig. 2A, black arrow). H 2 S supplementation restored normal perfusion in CBS+/− mice ( Fig. 2A,B). There was no difference renal flux units in WT groups without or with H 2 S treatment ( Fig. 2A,B).
Hhcy homocysteinylates eNOS, increases caveolin-1 expression and reduces nitric oxide production. In order to determine whether the protein-amino acid (eNOS-homocysteine) interaction and thus homocysteinylation of eNOS occurred, coimmunoprecipitation experiment was done. In CBS+/− mice, there was a prominent interaction of Hcy with eNOS as observed by eNOS immunoprecipitation followed by Hcy  immunodetection which showed high levels of Hcy and low levels of eNOS (Fig. 4A,B). In CBS+/− mice which received H 2 S, there was significant reduction of Hcy and increased eNOS expression (Fig. 4A,B). In the WT mice without or with H 2 S, Hcy and eNOS levels were not affected by the treatment (Fig. 4A,B).
Normally upon stimulus, endothelial cells produce NO by the activation of eNOS, which diffuses into the smooth muscle cells to further activate soluble guanylyl cyclase resulting in vessel relaxation. We therefore checked for NO production in MGECs by challenging it with acetylcholine as a measure of eNOS activity. MGECs treated with Hcy only, did not increase NO generation via eNOS stimulation as indicated by low fluorescence with DAF-2DA (Fig. 4D,E). In contrast, acetylcholine enhanced the fluorescence of MGECs treated with Hcy + H 2 S  H 2 S antagonizes ECM protein accumulation and smooth muscle cell proliferation. The activation of matrix metalloproteinases (MMPs) and inhibition of their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), lead to excess collagen deposition and allows for vascular smooth muscle cell proliferation in the vasculature. MMP-2 and -9 are gelatinases which cleave denatured collagen and collagen IV in the basement membrane and MMP-13 degrades fibrillar collagen. In CBS+/− mice, MMP-2, -9, and -13 were upregulated compared to the other groups (Fig. 5A,B). Further, CBS+/− mice demonstrated significant decrease in TIMP-1 and -2 compared to WT groups (Fig. 5A,C). TIMP-4 expression was nonexistent in CBS+/− mice compared to WT groups ( Fig. 5A,C). H 2 S treatment to CBS+/− mice reduced the expression of MMP-2, -9, and -13 and upregulated TIMP-1, -2 and -4 ( Fig. 5A-C). In the WT groups, the expression of MMP-2, -9 and -13 and TIMP-1 and -2 was similar without or with H 2 S treatment ( Fig. 5A-C). There was a non-significant decrease in TIMP-4 in WT mice treated with H 2 S (Fig. 5A,C).
MMP-9 activity was determined in in vitro experiments using MGECs and lysate from the kidney by gelatin zymography. Hcy treated cell lysate and media showed 1.3 and 2.03-fold increase in MMP-9 activity respectively compared to cells that did not receive any treatment (Fig. 5D,E). H 2 S treatment reduced MMP-9 activity significantly in cells treated with Hcy but did not affect cells treated with H 2 S only (Fig. 5D,E). Similarly, kidney lysate from CBS+/− mice showed nearly 2-fold increase in MMP-9 activity compared to WT mice (Fig. 5F,G). MMP-9 activity was mitigated by H 2 S supplementation (Fig. 5F,G). There was no difference in MMP-9 activity in WT groups (Fig. 5F,G).
Renal fibrosis can occur in all compartments of the kidney. In CBS+/− mice, Masson Trichrome staining revealed increased collagen accumulation in the glomeruli and tubulointerstitium (Fig. 6A, blue arrows). In CBS+/− kidney sections stained with picrosirius red, there was increased type I collagen in the interlobular arteries suggesting arteriosclerosis (Fig. 6B, yellow arrow). Collagen deposition was reduced in all areas following H 2 S supplementation (Fig. 6A,B). WT mice treated with H 2 S had similar collagen as that of WT control mice (Fig. 6A,B).
In separate experiments, we examined for vascular smooth cell proliferation (VSMCs) using Ki-67 marker. VSMCs treated with Hcy (75 µM) showed increased Ki-67 expression in the nucleus and decreased following H 2 S treatment (Fig. 7A,B). Since increased cell proliferation corresponds to increased metabolic activity, we performed MTT assay to confirm the findings above. There was increased absorbance in the VSMCs treated with Hcy (75 µM) alone compared to VSMCs without or with H 2 S (Fig. 7C) indicating increased metabolic activity. VSMCs treated with Hcy and H 2 S showed significant reduction in metabolism which was comparable to untreated VSMCs (Fig. 7C). The thymidine analog, 5′-bromo-2′-deoxyuridine (BrdU), is incorporated into newly synthesized DNA of replicating cells. Because H 2 S treatment reduced cell proliferation in Hcy treated VSMCs above, we wanted to test whether H 2 S treatment to renal artery explants would inhibit VSMCs proliferation in the tunica media. Renal arteries treated with Hcy alone showed increased BrdU immunostaining (Fig. 8A,B). In response to H 2 S, the arteries that were treated with Hcy showed significant reduction in BrdU fluorescence (Fig. 8A,B) suggesting reduction in VSMCs proliferation.  To confirm whether enhancing the endogenous activity of H 2 S producing enzymes (CBS, CSE and 3MST) would similarly affect VSMCs proliferation in renal arteries, we transfected CBS, CSE and 3MST enzymes ex vivo into renal arteries of WT mice (Fig. 8A,B). As expected, triple gene transfection reduced BrdU incorporation in renal artery rings of WT mice that was treated with Hcy, which was similar to H 2 S treated WT artery explants (Fig. 8A). There was no change in the expression of BrdU to triple gene transfection in WT artery explant (Fig. 8A,B).
Hhcy increases connexin expression. In addition to intercellular communication, connexins have multiple other roles such as cell signaling, mesangial cell proliferation, blood pressure regulation and as mediators of interstitial fibrosis. We therefore checked for changes in the expression of connexin40 and 43 (Cx40 and 43 respectively), as they are widely expressed in the kidney. In CBS+/− mice, both Cx40 and 43 was highly expressed (Fig. 9A,B). Cx40 was seen in the glomerular and tubular regions (Fig. 9A,B, yellow arrows) and Cx43 localized predominantly to the tubules (Fig. 9A,B, white arrows). H 2 S supplementation reduced the expression of Cx40 and 43 in CBS+/− mice similar to that seen in WT groups (Fig. 9A,B). H 2 S treatment did not affect the expression of connexins in WT mice. mRNA quantification for Cx40 and 43 confirmed the findings above (Fig. 9C,D).

Discussion
This study demonstrates that Hhcy reduces hydrogen sulfide (H 2 S) generation in the kidney and homocysteinylation of eNOS and upregulation caveolin-1 expression together reduce eNOS activity resulting in hypertension. Hhcy associated renal vascular changes include reduction in vascular density, blood flow and increased smooth muscle cell proliferation contributing to impaired renal function. Further, Hhcy-induced MMP-2, -9 and -13 activation, decreased TIMP-1, -2 and -4 and upregulation of gap junction Cx40 and 43 led to dysregulation of ECM metabolism and excess collagen deposition in the glomerular and interstitial regions. We found that H 2 S supplementation dehomocysteinylates eNOS and reduces caveolin-1 expression to increase eNOS activity thus decreasing blood pressure. In addition, H 2 S restored normal MMPs/TIMPs axis to reduce glomerular and tubulointerstial sclerosis and improved renal function. Heterozygous CBS+/− mice have approximately 50% reduction in the CBS enzyme activity and twice the normal plasma Hcy levels, which is clinically significant. Although there has been much debate whether Hhcy contributes to the development of hypertension, emerging evidence suggests it has a significant role. Earlier, a case control study demonstrated that an increase in 5 µM of Hcy was associated with an increase of systolic blood pressure by 0.7 and 1.2 mm of Hg in men and women respectively 3 . More recently, Yang et al. demonstrated that high levels of homocysteine is associated with hypertension in the presence of comorbidities such as, obesity, dyslipidemia and family h/o of hypertension suggesting a complex relationship between Hhcy and the development of hypertension 21 . In CKD patients, Hcy and cysteine levels are consistently increased which are also the principal substrates for H 2 S generation in the body 22,23 . However, during Hhcy, the serum levels of H 2 S was found to be significantly decreased in CKD patients 24 . Several studies have shown that H 2 S controls vascular tone and reduces blood pressure 25,26 . Conversely, low levels of H 2 S has been observed in the kidneys of spontaneously hypertensive rats and after targeted deletion of CSE leading to hypertension 25,27 . The findings from the present study adds further credence that Hhcy and low levels of H 2 S contribute to hypertension and renal pathology and supplementation of H 2 S reduces systolic blood pressure. Further, exogenous H 2 S supplementation is reported to increase CSE activity, which may therefore reduce plasma Hcy levels as observed in our study 28 .
Several studies have documented that Hhcy impairs endothelial function in various tissues [29][30][31] . In renal failure, endothelial and kidney production of NO is impaired 32 . As a result, there is loss of vascular relaxation, which over time leads to microvascular disruption, decreased blood flow and hypoxia. Capillary rarefaction is a crucial stage in the development of renal damage and fibrosis 33,34 . In the present study, we observed a reduction in the renal vascularity during Hhcy that was associated with reduction of cortical blood flow and poor renal function. H 2 S is a well-known vasodilator and direct infusion into the renal artery was shown to increase renal blood flow, GFR, and urinary excretion of sodium and potassium 35 . Exogenous supplementation of H 2 S in this study showed increase in the terminal branches of the renal cortex suggesting improved perfusion.
The vascular tone is regulated by the constriction and relaxation of vascular smooth muscle cells which is in part regulated by nitric oxide (NO) 36 . The endothelium-derived eNOS catalyzes the formation of NO from L-arginine. A decrease in the expression/function of eNOS can therefore affect NO production and thus impair vascular function. Protein homocysteinylation is one of the mechanisms proposed that contributes to Hhcy induced pathology in the kidney 37 . Further, homocysteinylation of plasma proteins has been documented in uremic patients undergoing hemodialysis 38 . Several other studies reported homocysteinylation of plasma proteins including albumin, hemoglobin, fibrinogen, and others such as, E-Cadherin, and actin during Hhcy and have been associated with multiple diseases 39,40 . Since endothelial dysfunction is a feature of Hhcy, we queried whether eNOS was a target of Hhcy. Our findings show that during Hhcy, eNOS is homocysteinylated and its activity is diminished in the kidney. The eNOS activity in the endothelial cells is regulated by its interaction with caveolin-1. In an in vitro study on human coronary artery endothelial cells, Hhcy was found to downregulate caveolin-1 expression and translocation of eNOS from the caveolar fraction to non-caveolar fractions in the cytoplasm thereby decreasing eNOS availability 41 . However, in other studies of acute renal insufficiency, a marked increase in caveolin-1 expression was observed in the kidney including aorta and liver 42,43 . The increased caveolin-1 expression seen in the present study during Hhcy are in agreement with the latter studies above as it also explains decreased eNOS availability due to its binding to eNOS.
Nitric oxide and H 2 S have vasoregulatory roles in the body and a significant cross talk exists between the two molecules 44 . In a recent study, Wesseling et al. demonstrated that the gasotransmitters NO, H 2 S and carbon monoxide (CO) have a complex relationship in the development of hypertension and renal injury 45 . A reduction of H 2 S was associated with reduction of NO products but enhanced CO and CO appears to be a mediator between NO and H 2 S molecules 45 . In another study, H 2 S was shown to increase eNOS phosphorylation by activating PI3K/ Akt pathway to increase NO production 44 . In animal models of left ventricle hypertrophy and acute myocardial injury, exogenous H 2 S increased NO by upregulating eNOS activity to offer cardiac protection 46,47 . In the present study, exogenous supplementation enhanced eNOS expression and activity in Hhcy and decreased caveolin-1 expression suggesting increased NO bioavailability. NO has been shown to increase CSE expression, thus H 2 S, in mouse aortic endothelial cells of caveolin1−/− mice to offer protection against atherosclerosis 48 . The decrease in caveolin-1 expression seen in our study could therefore be due to negative regulation of caveolin-1 by H 2 S or caveolin-1 translocation from the membrane into the cytoplasm for degradation to enable increased eNOS activation. These possible mechanisms need further exploration.
The disruption of MMP/TIMP axis and excess accumulation of ECM proteins during Hhcy has been demonstrated in several studies including our own [49][50][51] . The gelatinases, MMP-2 and -9, have increased affinity to collagen IV in the basement membrane. Their inhibitors, TIMPs, are known to have dual role. For example, at low concentration TIMP-2 activates MMP-2 whereas high concentration inhibits MMP-2 52 . TIMP-1 is the main inhibitor of MMP-9 and TIMP-4 has low affinity for MMP-9 53,54 . Therefore, low levels of TIMP-1 and TIMP-4 can upregulate MMP-9 as seen in the present study. Although the gelatinases degrade collagen, because collagen turnover is faster than elastin, oxidatively modified collagen is deposited in the ECM 55 . In this study, the upregulation of MMPs and collagen deposition led to global fibrosis in the kidney.
An essential component of vascular fibrosis involves VSMC proliferation leading to vessel stiffness. Hhcy promotes VSMCs proliferation in a ROS dependent manner and the use of antioxidant/Hcy lowering agent such as, folic acid, has been shown to inhibit proliferation [56][57][58] . Further, H 2 S treatment has been shown to inhibit VSMCs in several in vitro and in vivo studies 59 59 . Our in vitro data supports these earlier findings, further, we confirmed inhibition of VSMCs proliferation in the renal artery explants by triple gene transfection which showed decreased BrdU immunofluorescence.
Connexins are gap junction proteins that play a prominent role in maintaining intercellular communication and cell homeostasis. A disruption in their expression can result from oxidative stress and inflammation both of which are associated with Hhcy. Cx40 upregulation was observed in 2 kidney, 1 clip, rat model of hypertension 63 . Cx43 showed increased expression in the damaged tubular areas of patients suffering from glomerulonephritis 64 . In another study involving rat model of puromycin-induced nephritis, Cx43 was upregulated in glomerular podocytes however, whether this increase worsens renal injury or is beneficial was not known 65 . The present study showed similar increase in Cx43 expression in the tubular areas and in the glomeruli of Hhcy mice and was associated with renal damage. Previously, we demonstrated that reduction of H 2 S in diabetic mice was associated with NMDA-R1 and Cx40 and 43 mediated renal fibrosis 66 . In the present study, H 2 S reduced the expression of Cx40 and 43 to mitigate renal injury.

Conclusion
In this study, we show that during Hhcy, homocysteinylation of eNOS and upregulation of caveolin-1 results in decreased eNOS and NO production and a concurrent decrease in H 2 S leads to impaired vasomotor response, hypertension and poor renal perfusion. In addition, Hhcy disrupts the MMP/TIMP balance and increased Cx40 and 43 expression leading to adverse ECM remodeling. Exogenous H 2 S supplementation and triple gene therapy (CBS/CSE/3MST) dehomocysteinylated eNOS and reduced caveolin-1 to increase eNOS availability. Finally, H 2 S inhibited renovascular fibrosis and improved renal function in Hhcy-induced renal injury. WT and CBS+/− mice (n = 7/group) received sodium hydrogen sulfide (NaHS, 30 µM), a H 2 S donor, in drinking water and their respective controls received plain water for 8 weeks. Because NaHS has short half-life, the water containing NaHS was changed at 3-h interval during the day.
Blood pressure measurement by DSI telemetry. BP was measured continuously in conscious mice using PhysioTel telemetry system (DSI telemetry, St. Paul, MN) as described previously 67 . Under tribromoethanol anesthesia, the pressure transmitter, TA11PA-C10, was surgically introduced via the right carotid artery into the aortic arch. After a one-week recovery period, individual mice in cages were placed on receivers, which captured digital signals and relayed it to a computer via DSI matrix. Data was viewed and analyzed by Ponemah v5.20 software (DSI).
High Performance Liquid Chromatography. Plasma homocysteine levels were measured by HPLC as described previously 51 . Briefly, the following were added to a microcentrifuge tube: 200 µl of plasma, 100 µl of water, 300 µl of 9 M urea (pH 9.0), 50 µl of n-amyl alcohol, and 50 µl 10% NaBH 4 solution (wt/vol in 0.1 N NaOH). The solution was incubated at 50 °C for 30 minutes. After cooling to room temperature, 500 µl of 20% trichloroacetic acid was added to precipitate protein. Samples were centrifuged at 12,000 g for 5 minutes. and the supernatant was collected after passing through 0.22 µm filter. The sample was used for HPLC analysis. The mobile phase solution was a mixture of 0.1 M monochloroacetic acid and 1.8 mM octylsulfate in HPLC grade water at pH 3.2. The constant flow rate of isocratic solvent was 0.8 ml/minute. A Shimadzu Class-VP 5.0 chromatograph (Shimadzu, Columbia, MD) system was used to analyze samples.

Plasma H 2 S measurement.
Plasma H 2 S levels were measured as described before 68 . Briefly, freshly collected plasma (100 µL) was mixed with PBS (350 µL) and Zinc acetate (250 µL) and sealed immediately. To this mixture, N,N-dimethyl-p-phenylenediamine sulfate (20 mM, 133 μL) in 7.2 M HCl, and FeCl3 (30 mM, 133 μL) in 1.2 M HCl was added and incubated at 37 °C for 45 minutes. The reaction was terminated by adding Trichloroacetic acid solution (10% w/v, 250 μL). After centrifugation (×2,700 g for 5 minutes), aliquots were transferred to a 96-well plate and absorbance was read at 670 nm using Spectramax M2e (Molecular devices, CA). The values were calculated by using a standard curve of known concentrations.
Laser Doppler flowmetry and Barium angiography. Renal cortical blood flow was measured using Speckle Contrast Imager (MoorFLPI, Wilmington, DE) as described before 69 . The Moor FLPI speckle contrast imager uses a diverging infrared laser beam to illuminate the tissue of interest to produce an interference pattern called as 'laser speckle' . A charge couple device sensor in the camera captures the light and the inbuilt software processes the high and low contrast areas to yield video and trace lines. The flux units are automatically calculated by the software as No. of RBCs × velocity. The left kidney was exposed posteriorly and positioned approximately 15 cm from the camera which was focused on the kidney, renal vessels and aorta. High-speed images with a display rate of 25 Hz including line traces were obtained for 2 minutes.
Renal angiography was done by infusing Barium sulfate solution in 50 mM of TRIS buffer (100 mg/mL at pH 5.0) through the renal artery and imaged using KODAK in vivo Imaging System FX Pro (Molecular Imaging System; www.Bruker.com) 69 . Analysis was done using Vessel Segmentation and Analysis software (http://www. isip.uni-luebeck.de/?id=150) 69 .

Glomerular filtration rate (GFR) measurement. GFR was measured by FITC-Inulin clearance method 70 .
Mini osmotic pumps (Alzet Model 2001D, Cupertino, CA) were loaded with 200 µL of dialysed 5% FITC-Inulin. Under 2,2,2-Tribromoethanol anesthesia, osmotic pumps were implanted in the abdominal cavity. After 2 h recovery, the mice were placed individually in metabolic cages and urine was collected over 24 h period including blood samples. Since pH affects the FITC fluorescence, urine and plasma samples were buffered to pH 7.4 with 500 mM HEPES. Separate standard curves were prepared for urine and plasma as described before 71 . Samples were transferred to 96-well plate and fluorescence measured using SpectraMax M2 e (San Jose, CA) with excitation set at 485 nm and emission at 538 nm. Inulin clearance (GFR) was calculated as urinary FITC-Inulin excretion rate (Fluorescence counts/24 h) divided by plasma FITC-Inulin concentration (Fluorescence counts/µL). (rabbit anti-mouse IgG-HRP, Cat. no.: SC358920; and goat anti-rabbit IgG-HRP, Cat. no.: SC2004 respectively) were used at 1:1000 dilution and bands were detected using chemiluminescence (Luminata Forte, BioRad  Ki-67 is a nuclear marker for proliferating cells. In a second set of experiments, VSMCs were treated with Hcy and H 2 S as above and stained with anti-Ki-67. In separate experiments, renal arteries from WT mice were transfected ex vivo with triple gene (CBS, CSE and 3MST) as described before 73 . Renal arteries were excised from WT mice (n = 4) and rinsed in ice-cold physiological salt solution. The arteries were cut into 2 mm rings and placed in a 12-well plate containing DMEM F/12 50/50 cell culture media (Mediatech Inc, VA). Arteries were transfected with plasmid containing CBS, CSE and 3MST genes (0.4 μg DNA/cm 2 of growth area) or control plasmid without genes using jetPRIME transfection reagent (Polyplus Transfection) following the manufacturer's instructions. After 6 h, the arteries were mounted on the bottom of plate using Matrigel (BD Biosciences, San Diego, CA) and treated without or with Hcy (75 µM) in fresh growth media. After 48 h, artery rings were fixed with Tissue Tek OCT compound. Sections of 5 μm thickness were cut and stained with 5-bromo-2′-deoxyuridine (BrdU) antibody (Millipore Sigma, MA) and images were captured with Olympus FluoView1000 microscope (B&B Microscope Ltd., PA).
Gelatin Zymography. The proteolytic activity of MMP-9 to HHcy and H 2 S treatment was detected by gelatin zymography as described previously 69 . Statistics. Values are presented as mean ± SEM. Data was analyzed by ANOVA followed by post hoc, Bonferroni correction to identify differences between two groups. Mann-Whitney U test was done for nonparametric data. A 'p' value of <0.05 was considered significant.