Fig 2: Renal interstitial renin is induced by renal fibrosis and related to the increase in systemic blood pressure. (a) Ren1 mRNA levels in injured (I) and contralateral (C) kidneys of mice subjected to UUO for 3, 14, and 21 days. The average expression level (red bar) in non-treated kidneys (N) was set as 1. (b) Scatter plots of the relationship between systemic blood pressures (DBP, green; MBP, red; SBP, blue) and Ren1 mRNA levels in injured kidneys. R2 values for the relation are shown. (c) Representative images of serial sections stained with Ren1 ISH (upper, brown) and αSMA IHC (lower, red) demonstrate that interstitial cells positive for Ren1 mRNA expression (white arrowheads) are αSMA-positive myofibroblasts in injured kidneys 21 days after UUO surgery. Ren1+ JG cells (red arrows) next to glomeruli (dotted circles) and renal arterioles (A) are also positive for αSMA. The scale bar represents 100 μm. (d) Quantification of Ren1 ISH in injured kidney sections from mice after UUO surgery. n = 3–4 per group. Green and brown lines indicate P<0.01 and P<0.05, respectively, in the Tukey-Kramer HSD test (a and d).

Fig 3: Renal interstitial renin is induced by antihypertensive agents. (a) Vasodilator (hydralazine, Hyd) administration causes hypotension and renal renin induction in wild-type C56BL/6 mice. The experimental scheme, blood pressure, Ren1 mRNA levels in kidney, and Agt mRNA levels in liver are shown. (b) Representative images and quantification of ISH of Ren1 mRNA in kidney sections of mice freely drinking Hyd for 4 days. (c) Injection of an AngII receptor blocker reagent (losartan, LST) into wild-type DBA/2 mice resulted in hypotension and the induction of the genes encoding renin (Ren1 and Ren2). The experimental scheme, blood pressure, and Ren1 and Ren2 mRNA levels in the kidney are shown. (d) Representative images and quantification of ISH of Ren1 mRNA in kidney sections of mice after LST administration. The average expression levels (red bars) in the control mice were set as 1 for each gene expression analysis (a and c). n = 4–7 per group. Dotted circles and red arrows indicate glomeruli and JG cells, respectively (b and d). White arrowheads indicate interstitial fibroblasts positive for Ren1 (blue in b and d). Scale bars represent 100 μm (b and d). Green and brown lines indicate P<0.01 and P<0.05, respectively, in the Wilcoxon-Mann-Whitney test. .

Fig 4: Chronic anaemia induces renal renin expression in mice. (a) RT-qPCR analyses were performed to determine the relative mRNA expression of the Ren1, Agt, and Ace genes in the kidney, liver, and lung, respectively, which were obtained from ISAM and control mice at 11 weeks of age. (b) Immunoblotting of control and ISAM plasma with an anti-renin antibody detected renin, its precursor (prorenin), and nonspecific bands (ns), which were probably immunoglobulin. (c) Blood haemoglobin concentrations and renal Ren1 mRNA expression levels in control and ISAM mice treated with C.E.R.A. or vehicle (V). Two schemes of C.E.R.A. administration, e.g., 6H and 28D, are shown in the top panel. The average expression levels (red bars) in the control mice were set as 1 for each gene expression analysis (a and c). Green lines indicate P<0.01 in the Wilcoxon-Mann-Whitney test (a) or Tukey-Kramer HSD test (c). n = 5–7 per group (a and c).

Fig 5: Acute anaemia induces Ren1 mRNA expression in the renal interstitium. (a) Epo and Ren1 mRNA expression levels were elevated in kidneys from wild-type C57BL/6 mice by phlebotomy-induced haemorrhagic anaemia. The experimental scheme, erythrocyte counts, and Epo and Ren1 mRNA levels are shown. (b) Representative images of ISH of Epo (red) and Ren1 (blue) mRNA in kidney sections from control and haemorrhagic mice. (c) Epo and Ren1 mRNA expression levels were elevated in kidneys from wild-type C57BL/6 mice by phenylhydrazine (PHZ)-induced haemolytic anaemia. The experimental scheme, erythrocyte counts, and Epo and Ren1 mRNA levels are shown. (d) Representative images of ISH of Epo (red) and Ren1 (blue) mRNA in kidney sections from control and haemolytic mice. (e) Epo and Ren1 mRNA expression levels were elevated in kidneys from wild-type Slc:SD rats by PHZ-induced haemolytic anaemia. The experimental scheme, erythrocyte counts, and Epo and Ren1 mRNA levels are shown. (f) Representative images of ISH of Ren1 mRNA (brown) in kidney sections from control and haemolytic rats. The average expression levels (red bars) in the control mice were set as 1 for each gene expression analysis (a, c, and e). Green and brown lines indicate P<0.01 and P<0.05, respectively, in the Wilcoxon-Mann-Whitney tests (a, c, and e). n = 4–6 per group. Dotted circles and red arrows indicate glomeruli and JG cells, respectively (b, d, and f). White, black, and yellow arrowheads indicate interstitial fibroblasts positive for Ren1 (b, d, and f), Epo (b and d), and both (b and d), respectively. Scale bars represent 100 μm (b, d, and f).