Folic Acid Mitigates Angiotensin-II-Induced Blood Pressure and Renal Remodeling

Citation: Pushpakumar SB, Kundu S, Metreveli N, Sen U ( Folic Acid Mitigates Angiotensin-II-Induced Blood Pressure and Renal Remodeling Sathnur B. Pushpakumar 0 Sourav Kundu 0 Naira Metreveli 0 Utpal Sen 0 Sebastien Fuchs, Cedars-Sinai Medical Center, United States of America 0 Department of Physiology and Biophysics, University of Louisville School of Medicine , Louisville, Kentucky , United States of America Clinical data suggests an association between systolic hypertension, renal function and hyperhomocysteinemia (HHcy). HHcy is a state of elevated plasma homocysteine (Hcy) levels and is known to cause vascular complications. In this study, we tested the hypothesis whether Ang II-induced hypertension increases plasma Hcy levels and contributes to renovascular remodeling. We also tested whether folic acid (FA) treatment reduces plasma Hcy levels by enhancing Hcy remethylation and thus mitigating renal remodeling. Hypertension was induced in WT mice by infusing Ang II using Alzet mini osmotic pumps. Blood pressure, Hcy level, renal vascular density, oxidative stress, inflammation and fibrosis markers, and angiogenic- and anti-angiogenic factors were measured. Ang II hypertension increased plasma Hcy levels and reduced renal cortical blood flow and microvascular density. Elevated Hcy in Ang II hypertension was associated with decreased 4, 5Diaminofluorescein (DAF-2DA) staining suggesting impaired endothelial function. Increased expression of Nox-2, -4 and dihydroethidium stain revealed oxidative stress. Excess collagen IV deposition in the peri-glomerular area and increased MMP-2, and -9 expression and activity indicated renal remodeling. The mRNA and protein expression of asymmetric dimethylarginine (ADMA) was increased and eNOS protein was decreased suggesting the involvement of this pathway in Hcy mediated hypertension. Decreased expressions of VEGF and increased anti-angiogenic factors, angiostatin and endostatin indicated impaired vasculogenesis. FA treatment partially reduced hypertension by mitigating HHcy in Ang IItreated animals and alleviated pro-inflammatory, pro-fibrotic and anti-angiogenic factors. These results suggest that renovascular remodeling in Ang II-induced hypertension is, in part, due to HHcy. - Funding: This study was supported, in part, by National Institutes of Health grant HL-104103 to US. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Utpal Sen is a PLOS ONE Editorial Board member. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Renovascular injury and fibrosis due to angiotensin II (Ang II) is a leading cause of cardio-renovascular morbidity and mortality. Clinical data suggest an association between elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), and systolic hypertension [1]. In addition, plasma Hcy level has an inverse relation with renal function [2]. Although Ang II has predominant actions on the renal vasculature causing a reduction in renal blood flow, the effect of HHcy and its contribution to renovascular remodeling in Ang II-induced hypertension is unclear. HHcy induces reactive oxygen species (ROS) production by auto-oxidation or by homocysteinylation of lysine residues of other cellular proteins [3]. In addition, HHcy is also known to decrease the antioxidant status [4]. The generation of ROS triggers leukocyte infiltration and cytokine release leading to glomerular inflammation and subsequent injury [5,6]. Chronic HHcy has also been reported to alter ECM components contributing to glomerulosclerosis [7,8]. Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), play a major role in ECM remodeling under physiological and pathological conditions [9,10]. Although the kidney expresses all the currently described TIMPs, (TIMP-1 - 4) their expression and activities are varied [1113]. TIMP-1, -2 and -4 mediate their action by blocking the MMPs catalytic core, whereas TIMP-3 binds to ECM and protects it from MMP mediated injury [14]. Thus, TIMPs regulate ECM by inhibiting MMPs. HHcy induces MMP-2, -9 [13] and also modulates TIMPs [15] to promote matrix accumulation [16]; however, whether a similar mechanism is involved in Ang II-induced kidney remodeling has not been reported. During vascular remodeling, vascular endothelial growth factor (VEGF) plays an important role by promoting endothelial cell proliferation, migration and tube formation [17]. However, during HHcy these processes are inhibited suggesting impairment of vessel growth [18,19]. Additionally, HHcy induced MMP activation can also lead to increased production of anti-angiogenic factors, endostatin and angiostatin, further inhibiting vascular growth by down regulation of VEGF [20]. The anti-angiogenic molecules specifically target endothelial cells to inhibit proliferation, survival, migration, and sprouting [21]. Since VEGF is widely expressed in the kidney, the consequences of VEGF inhibition can result in loss of vascular and glomerular integrity leading to renal dysfunction [22,23]. Folic acid (FA) is a B-vitamin which acts as a co-factor in the Hcy remethylation pathway to reduce plasma Hcy level and thus reducing Hcy-induced oxidative stress and DNA damage [24]. However, the role of FA in hypertension-induced HHcy, glomerular injury, inflammation, and subsequent glomerulosclerosis remains largely unknown. The current study was undertaken to delineate the potential role of Hcy in Ang II-induced hypertension and renovascular remodeling. Additionally, considering its potential effects to reduce Hcy levels, FA was given to mitigate Hcy mediated renal damage. Materials and Methods Animal groups and protocol Wild type (WT, C57BL/6J) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in the animal care facility at University of Louisville. All animal procedures were performed in accordance with the National Institute of Health Guidelines for animal research and were approved by the Institutional Animal Care and Use Committee of the University of Louisville, School of Medicine. Animals were allocated into the following groups: 1) Vehicle (saline), 2) Ang II, 3) Ang II + FA, and 4) FA. Hypertension was created by infusing Ang II (1000 ng/kg/min) using Alzet mini osmotic pump intraperitoneally for 4 weeks. Folic acid was given at 0.015 g/L in drinking water starting from 2 weeks after Ang II pump insertion and continued till the end of the experiment. Water was changed on alternate days. In previous studies, FA has been given by different routes and at varied concentration from 0.1425 to 375 mg/25 g b.w./day [25,26]. Since FA in drinking water changes the taste, we chose a dose based on previous work from our laboratory [27] and also t (...truncated)