Loss of OXGR1 function uncouples intrarenal RAS activation from pendrin and αENaC regulation and blunts angiotensin II-induced hypertension

FIGURE 1. Systolic (A) and diastolic (B) arterial blood pressure in mice infused with angiotensin II (Ang II) with or without montelukast (ML) for 14 days. Ang II augments systolic, diastolic and mean arterial blood pressure, while ML impairs this effect. **p < 0.01, ***p < 0.001 versus sham.

FIGURE 2. Natriuretic responses to a saline load. Na+ excretion (% of injected Na+) was significantly reduced in mice infused with Ang II compared with sham controls. Pharmacological blockade of OXGR1 with montelukast (Ang II + ML) attenuated the antinatriuretic effect of Ang II, restoring Na+ excretion toward control values, while ML alone did not alter Na+ excretion. Data are presented as mean ± SEM with individual values shown; *P < 0.05 versus sham.

FIGURE 3. Effect of Ang II infusion and OXGR1 inhibition on pendrin, NDBCE and αENaC protein abundances. Representative immunoblots (left panels) and densitometric analyses (right panels) showing renal protein abundance of key intrarenal on pendrin, NDBCE and the cleaved active form of αENaC in the experimental groups. Chronic Ang II infusion increased the abundance of pendrin and αENaC compared with sham-treated controls. Pharmacological inhibition of OXGR1 with montelukast (ML) attenuated these responses, preventing the Ang II-induced increases in protein expression. Quantitative data are presented as mean ± SEM, normalized to the corresponding loading control β-actin and expressed relative to the control group. Individual data points are shown for each animal. *p < 0.05, n = 6.

FIGURE 4. Effects of Ang II infusion and OXGR1 blockade on full-length (membrane-bound) and soluble form of the PRR protein abundance in the kidney. Representative immunoblots (left) and densitometric analyses (right) of full-length (FL)-PRR and soluble PRR (sPRR) protein abundance in control mice, Ang II-infused mice, and Ang II-infused mice treated with montelukast (ML). Ang II infusion significantly increased FL-PRR abundance compared with controls. In contrast, ML treatment prevented the Ang II-induced increase in FL-PRR, maintaining protein levels near control values. No significant differences were observed in sPRR abundance among experimental groups. *p < 0.05, n = 6.

FIGURE 5. Immunohistochemical localization of pendrin and αENaC in renal collecting ducts. Representative immunohistochemical images showing pendrin and αENaC distribution in kidney sections from the indicated experimental groups. (A) High-magnification images (100X) illustrating prominent apical membrane staining of pendrin and αENaC in collecting duct epithelial cells from control mice. (B) Positive immunoreactivity is visualized as a brown diaminobenzidine (DAB) precipitate, whereas nuclei are counterstained with hematoxylin. In control kidneys, staining was localized in the cytoplasm and apical, however in Ang II-infused mice the DAB-positive pixels were augmented along with more prominent apical membrane intensity. (C) DAB-positive pixels were less intense in Ang II plus ML treatment. Images are representative of 4-6 fields per animal of each group. *p < 0.05 vs. sham. Scale bar 20 μm.

FIGURE 6. Systolic (A) and diastolic (B) blood pressure overview in wild type or OXGR1−/− with or without Ang II infusion. Systolic and diastolic blood pressure was augmented in Ang II infused wild type mice but not in OXGR1−/− mice t days 7, 9 and 13. *p < 0.05; **p < 0.01; ***p < 0.001 versus sham.

FIGURE 7. Natriuretic responses to a saline load in wild type and OXGR1−/− mice after 6 h. Chronic Ang II infusion significantly reduced Na+ excretion compared with sham mice, indicating an antinatriuretic response. Genetic deletion of OXGR1 prevented the Ang II-induced reduction in Na+ excretion, restoring values toward those observed in sham controls, whereas OXGR1−/− mice alone showed no differences from sham. Data are presented as mean ± SEM with individual values shown; *P < 0.05 versus sham.

FIGURE 8. Effect of Ang II infusion and OXGR1 deletion on pendrin, NDBCE and αENaC protein abundances. Representative immunoblots (left) and densitometric analyses (right) showing renal protein abundance of pendrin, NDBCE, and the cleaved active form of αENaC in wild-type Sham, wild type Ang II-infused, OXGR1−/− + Ang II-infusion and OXGR1−/− mice. Compared with sham animals, Ang II infusion increased the abundance of pendrin and the cleaved αENaC, as indicated by the arrow in the left panel. In contrast, Ang II infusion did not modify pendrin abundance in OXGR1-deficient mice. Data are presented as mean ± SEM, with individual values shown for each animal. *p < 0,05 versus sham, n = 6.

FIGURE 9. Representative immunoblots (left) and densitometric analyses (right) of the membrane-bound full-length (FL)-prorenin receptor (PRR) and soluble PRR (sPRR) protein abundance in renal tissues from WT and OXGR1−/− mice following 14 days of Ang II infusion. Ang II significantly increased FL-PRR abundance in WT mice, whereas this response was absent in OXGR1−/− mice. In contrast, sPRR levels remained unchanged across all experimental groups. Data are presented as mean ± SEM, and representative immunoblots are shown adjacent to their corresponding quantification panels. *p < 0.05 versus sham, n = 6.

FIGURE 10. Representative immunohistochemical staining of αENaC and pendrin and in renal collecting ducts from WT and OXGR1−/− mice after 14 days of Ang II infusion. (A) High-magnification images reveal distinct cell-specific localization of αENaC cytoplasmic and apical aspects in principal cells (PC), while pendrin was mainly localized in the apical side of intercalated cells (IC). (B) Pendrin and αENaC immunoreactivity were augmented in the collecting ducts of wild-type mice infused with Ang II. OXGR1−/− infused with Ang II showed less intensity of DAB-positive pixels. (C) The right panels show quantification of DAB-positive pixels. Images are representative of 4-6 fields per animal of each group. *p < 0.05 vs. sham. Scale bar 20 μm.