Folic acid treatment increased renal cortical blood flow and vascular density in Ang II infused kidney.

<p>(<b>A</b>) Renal cortical blood flow was measured at end-point using Speckle contrast Imager (MoorFLPI, Wilmington, DE). Animals were anesthetized with TBE (Tribromoethanol, 240 mg/kg b.w. i.p.) and the left kidney exposed. All measurements were done under standard conditions of light and temperature control. (<b>B</b>) Data was first analyzed with ANOVA and pairwise comparison was performed using Bonferroni method. Summarized bar diagram represents mean ± SEM, n = 5–6 animals/group. * p<0.05 vs. vehicle and ^†p<0.05 vs. Ang II. (<b>C</b>) Mice were infused with Barium sulfate (100 mg/ml, at pH 5.0) through PE10 catheter (ID -0.28 MM, Franklin Lakes, NJ) inserted in the carotid artery directed towards the aorta and a constant rate of 200 µL/min was injected. Two minute X-ray images were captured with Kodak 4000 MM image station (Molecular Imaging System; Carestream Health Inc., Rochester, NY). Image analyses were done by ImagePro software (a representative analysis image is shown at the bottom right). Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagram indicates percent change of vascular density against the background using vehicle treatment as control, n = 3 mice/group. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II.</p

Folic acid (FA) reduced blood pressure and plasma Hcy levels in Ang II-induced hypertension.

<p>(<b>A</b>) Ang II was infused using alzet mini pump (1000 ng/kg/min) for 4 weeks and blood pressure was measured by radiotelemetry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Folic acid (0.015 g/L) was given in drinking water 14 days after pump insertion and continued till the end of the experiment; n = 8 animals/group. * p<0.05 between Ang II + FA vs. Ang II. (<b>B</b>) Plasma Hcy was measured by high performance liquid chromatography (HPLC) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Data was first analyzed with ANOVA and pairwise comparison was performed using Bonferroni method. Data is presented as mean ± SEM, n = 6 mice/group. * p<0.05 vs. vehicle (saline) and, ^† p<0.05 vs. Ang II (1000 ng/kg/min), ^‡ p = 0.05 vs. Vehicle.</p

mRNA and protein expression of CBS/CSE and MTHFR is decreased in Ang II induced hypertension

<p>Effect of vehicle, Ang II and FA on the mRNA and protein expression of CBS (<b>A</b>); CSE (<b>C</b>) and MTHFR (<b>E</b>) as determined by semiquantitative RT-PCR and Western blotting. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change from n = 6 experiments using GAPDH as control. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

Food and water intake.

<p>Food and water intake was measured for 5 days. Vehicle group represents animals treated with normal saline; Ang II, angiotensin II; FA, folic acid. Data is presented as mean ± SEM. * p<0.01 vs. vehicle control; ^† p<0.05 vs. Ang II + FA.</p

FA mitigated glomerulosclerosis and collagen IV expression in Ang II hypertension.

<p>(<b>A</b>) Histological kidney sections (5 µm thick) were stained with Masson's trichrome. Collagen is shown as deep blue color (yellow arrows); 200× magnification. (<b>B</b>) Collagen IV was detected in the cortical tissue extracted protein by Western blot. (<b>C</b>) Bar diagram represents densitometric analysis of collagen IV expression as fold change against vehicle group, n = 5–6 group. Statistical analyses were performed by Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle and, ^† p<0.05 vs. Ang II.</p

FA treatment reduces ROS production in Ang II hypertension by decreasing Nox-2 and Nox-4 isoforms.

<p>(<b>A</b>) Equal amount of protein from each group was separated on a SDS-PAGE and immunoblotted with anti-Nox-2, and -4 antibodies. (<b>B</b>) The pixel densities of bands from n = 6/group were quantified using ImageJ software (National Institute of Health, NIH) and presented as fold change using β-actin as control. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle; p<0.05 vs. Ang II. (<b>C</b>) Superoxide was detected in the glomerulus by dihydroethidium (DHE) staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Scale 20 µm.</p

HHcy in Ang II hypertension increases expression of ADMA and suppresses eNOS.

<p>Effect of vehicle, Ang II and FA on mRNA and protein expression of ADMA (<b>A</b>) and protein expression of eNOS (<b>C</b>) by RT-PCR and Western blotting. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change for ADMA (<b>B</b>) and eNOS (<b>C</b>) from n = 6 animals/group. GAPDH was used as control. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

FA normalized altered expression of angiogenic and anti-angiogenic factors in Ang II hypertension.

<p>Western blot was performed to measure VEGF, angiostatin and endostatin expression using specific antibodies in the renal cortical tissue extracted protein. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change from n = 5–6 mice/group. * p<0.05 vs. vehicle and ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

mRNA primers.

<p>mRNA primers.</p

mRNA expression of MMP-9 and TIMP-3.

<p>A) mRNAs are amplified using respective primers and the bands were quantified using densitometry. <b>B, C</b>) Bar graphs represent respective mRNA expression over GAPDH expression by RT-PCR and real time PCR assay. Data represents mean ±SE from n = 6 per group; *p<0.05 was considered significant compared to sham and ^#p<0.05 compared to vehicle treated group.</p