Fig 2: B1R expression is increased and correlates with collagen expression in human kidney sections from hypertensive chronic kidney disease. Representative immunohistochemistry photomicrographs showing B1R specific immunoreactivity in kidney sections of healthy controls (A), specifically in glomerulus (B), tubules (C) and arteries (D). The B1R specific immunoreactivity is increased in kidney sections from renal disease (E), specifically in glomerulus (F), tubules (G) and arteries (H). Representative photomicrographs showing Masson Trichrome staining for collagen deposition in health control kidney section (I) with in glomerulus (J), tubules (K) and arteries (L). The collagen deposition was increased in kidney sections from the kidney disease subjects (M), specifically in glomerulus (N), tubules (O) and arteries (P). Quantification data showing increased B1R (Q) and collagen (R) expression in kidney sections from renal disease subjects (n = 5, Unpaired, 2-tailed t-test, *P < 0.01 vs. Control). B1R immunoreactivity is positively correlated with collagen deposition (S). Pearson correlation and least-squares linear regression were used to determine correlation coefficients between variables.

Fig 3: Immunohistochemical results. (A) Representative immunohistochemical images of B1R, IL-6 and IL-10 in plaques of asymptomatic, TIA and stroke patients of <30, 30–90, 90–180 and >180 days. (B) Quantification of immunohistochemistry analysis of B1R, IL-6 and IL-10 staining of section from asymptomatic, TIA and stroke patients of <30, 30–90, 90–180 and >180 days. Percentage of positive staining area in each microscopic view (at 200x) showed that stroke plaques experienced initial increases of B1R and IL-6 contents at <30 days, and subsequent reduction from 30–90 to >180 days. However, a noticeable increase in IL-10 contents was found in stroke plaques from <30 to >180 days. (***P<0.001 vs. asymptomatic or TIA group). Scale bar: 100 µm.

Fig 4: B1R gene deletion prevents gene expression of renal fibrosis markers in DOCA-salt hypertension. Gene expression measured by real time RT-PCR showing significantly increased mRNA of renal fibrosis markers in the kidney cortex of DOCA-salt treated hypertensive mice compared with sham mice. B1R knockout mice did not show this DOCA-salt-induced increase in renal fibrosis markers. Data was normalized to ß-actin and presented as mean ± SEM (n = 6, Two-way ANOVA, *P < 0.01 vs. Sham, †P < 0.05 vs. WT + DOCA).

Fig 5: B1R expression in human kidney proximal tubular epithelial (HK-2) cells. (A) Representative photomicrographs showing immunofluorescence of B1R in HK-2 cells treated vehicle or B1R specific agonist DAKD for 24 h. Control HK-2 cells without primary antibody (No 1°Ab control) incubation and with only secondary antibody incubation shows no staining. (B) The quantification of B1R immunoreactivity shows that the DAKD treatment of HK-2 cells for 24 h significantly upregulated the B1R expression (n =7–8, Unpaired, 2-tailed t-test, *P < 0.01 vs. Vehicle). (C) Representative western blot image and quantification data showing significantly increased B1R protein expression in HK-2 cells following stimulation with DAKD for 24 h (n = 5, Unpaired, 2-tailed t-test, *P < 0.01 vs. Vehicle).