Although it is not clear whether hyperhomocysteinemia (HHcy), an elevated plasma homocysteine (Hcy) level, causes hypertension, recent clinical data suggest an association between systolic hypertension and HHcy. Both angiotensin II (Ang II) and Hcy decrease blood flow;however, the role of Hcy in Ang II mediated decrease in blood flow and vascular density is unclear. Interestingly, preliminary studies of this proposal suggest that Ang II increases plasma Hcy level in mice. Treatment of Ang II animals with folic acid (FA) decreases plasma Hcy and mitigates hypertension. Based on our preliminary studies, in this proposal, we hypothesize that HHcy decreases blood flow in part by decreasing vascular density, increasing oxidative, pro-inflammatory, pro-fibrotic and anti-angiogenic factors in Ang II mediated renovascular remodeling. FA treatment through Hcy clearance mechanism reduces plasma Hcy level that mitigates renal remodeling. The hypothesis will be tested by following their specific aims: (1) To determine whether the Hcy contributes to Ang II hypertension, in part, by inducing oxidative stress (Nox2, gp47phox, Nox4 and mtROS), exacerbating renal inflammation by inducing MCP-1, MIP-2, ICAM-1, VCAM-1, and if FA ameliorates these changes;(2) To determine whether the Hcy instigates Ang II hypertension and renovascular fibrosis, in part, by inducing collagen IV, MMP-2, -9, -13;TIMP-1,-2, -3, -4;and if FA mitigates this renovascular fibrosis;and (3) To determine whether the Hcy promotes Ang II hypertension and decreases renovascular blood flow, in part, by unbalancing of angiogenic (VEGF) and anti-angiogenic factors (angiostatin and endostatin), and if FA increases vascular density and blood flow. WT (C57BL/6J) mouse, genetic mouse model of HHcy (CBS) and mouse deficient with angiotensin receptor I (AT1R-/-) will be used in this study. In WT and CBS mice, hypertension will be created by infusing Ang II (1000 ng/kg/min for 4 weeks) through alzet mini pump. Control animals will receive only vehicle. In a separate group of animals FA (0.015g/L, in drinking water) will be introduced after 2 weeks of Ang II infusion and will be continued until the end of experiments (total 2 weeks). Appropriate FA controls will be used. AT1R-/- mice will be treated with or without Hcy (1.8 g/L for 4 weeks) to determine whether the effect of HHcy is AT1R dependent. Ambulatory blood pressure will be measured by DSI radiotelemetry (model TA11PA- C10). Plasma Hcy will be measured by HPLC. Vascular density will be measured by in vivo soft-tissue Barium sulfate-contrast X-ray angiography and renal cortical blood flow by moorFLPI full-filled laser perfusion imager. Renal function will be determined by measuring glomerular filtration rate (GFR). Histological kidney sections will be used to detect ROS, collagen, mesangial widening and podocyte injury. Plasma MCP-1, MIP-2, VCAM- 1 and ICAM-1 will be measured by ELISA. Tissue collagen, MMPs, TIMPs, Nox2, p47phox, Nox4, ICAM-1, VCAM-1 protein expressions will be measured by Western blot and immunostaining. MMPs activities will be measured by in gel zymography, TIMPs activity by reverse zymography, and mRNA abundance by Q-PCR. In addition to confirming the preliminary studies, the scope of the research will be extended to understand the implications of FA treatment in Ang II-induced hypertension to modulate pro-inflammatory, pro-fibrotic and anti- angiogenic factors. The results of this study will increase our understanding of role of Hcy in Ang II hypertension and renovascular remodeling. Additionally, the research outcome will provide the missing information of HHcy as a potential risk factor of renovascular fibrosis, which exacerbates hypertension, and will lead to develop or modify current therapeutic strategies of renovascular disease.

Public Health Relevance

Angiotensin II (Ang II) is a main player in the pathogenesis of hypertensive renovascular disease. Elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is frequently observed in patients with renal dysfunction. Although it is not well known whether HHcy induces blood pressure;recent clinical data suggest elevation of plasma Hcy level in essential hypertensive patients. The preliminary studies of this proposal suggested that in Ang II-induced hypertensive mouse model, an elevated plasma level of Hcy was also observed. Hcy is a known risk factor of cardio-renovascular disease, however, the role of Hcy in hypertensive individuals in the progression of renovascular remodeling and glomerulosclerosis is unclear. Folic acid (FA) reduces plasma Hcy level, and therefore modulates risks of cardio-renovascular disease caused by HHcy. Herein, this study proposes to investigate the role of Ang II hypertension in inducing HHcy, and whether this HHcy aggravates hypertensive renovascular remodeling by triggering oxidative, pro-inflammatory and pro- fibrotic factors. The contribution of Hcy in reducing vascular density and blood flow by decreasing angiogenic and increasing anti-angiogenic factors will also be investigated through FA treatment. To delineate these issues, inflammatory molecules, such as MCP-1, MIP-2, ICAM-1 and VCAM-1 will be measured in Ang II- induced hypertensive mouse model. The matrix metalloproteinase-2, -9 &-13, and their tissue inhibitors, TIMP- 1, -2, -3 &-4, as well as extracellular matrix protein, collagen will be measured in hypertensive mice. The angiogenic factor, VEGF and anti-angiogenic factors, angiostatin and endostatin will also be measured. The contribution of HHcy in AngII-induced glomerulovascular remodeling and glomerulosclerosis will be determined through FA treatment. This is a pure, elegant, and mechanistic study to investigate the potential role of HHcy in Ang II-induced glomerular extracellular matrix remodeling and sclerosis. The research outcome of this proposal will lead us to better understand the hypertensive renovascular disease and the findings of this study will open new avenues for therapeutic strategies of hypertensive renovascular disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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OH, Youngsuk
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University of Louisville
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Weber, Gregory J; Pushpakumar, Sathnur B; Sen, Utpal (2017) Hydrogen sulfide alleviates hypertensive kidney dysfunction through an epigenetic mechanism. Am J Physiol Heart Circ Physiol 312:H874-H885
Pushpakumar, Sathnur; Ren, Lu; Kundu, Sourav et al. (2017) Toll-like Receptor 4 Deficiency Reduces Oxidative Stress and Macrophage Mediated Inflammation in Hypertensive Kidney. Sci Rep 7:6349
John, A M Sashi Papu; Kundu, Sourav; Pushpakumar, Sathnur et al. (2017) GYY4137, a Hydrogen Sulfide Donor Modulates miR194-Dependent Collagen Realignment in Diabetic Kidney. Sci Rep 7:10924
Amin, Matthew; Pushpakumar, Sathnur; Muradashvili, Nino et al. (2016) Regulation and involvement of matrix metalloproteinases in vascular diseases. Front Biosci (Landmark Ed) 21:89-118
Sen, Utpal; Pushpakumar, Sathnur (2016) Mini-review: diabetic renal complications, a potential stinky remedy. Am J Physiol Renal Physiol 310:F119-22
Weber, Gregory J; Pushpakumar, Sathnur; Tyagi, Suresh C et al. (2016) Homocysteine and hydrogen sulfide in epigenetic, metabolic and microbiota related renovascular hypertension. Pharmacol Res 113:300-312
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Kundu, Sourav; Pushpakumar, Sathnur; Sen, Utpal (2015) MMP-9- and NMDA receptor-mediated mechanism of diabetic renovascular remodeling and kidney dysfunction: hydrogen sulfide is a key modulator. Nitric Oxide 46:172-85
Sen, Utpal; Pushpakumar, Sathnur B; Amin, Matthew A et al. (2014) Homocysteine in renovascular complications: hydrogen sulfide is a modulator and plausible anaerobic ATP generator. Nitric Oxide 41:27-37
Pushpakumar, Sathnur; Kundu, Sourav; Sen, Utpal (2014) Endothelial dysfunction: the link between homocysteine and hydrogen sulfide. Curr Med Chem 21:3662-72

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