COX-2-derived PGE2 triggers hyperplastic renin expression and hyperreninemia in aldosterone synthase-deficient mice

Figures

• 

  Fig. 1. (A–D) Immunohistochemistry of COX-2 (red) and renin (green) in kidney sections of a wildtype mouse (A) and a mouse lacking aldosterone synthase (B–D). Renin cells in wild-type kidneys were small in number and located in the classical juxtaglomerular position within the media layer of the afferent arteriole. In contrast, kidneys of the AS−/− mouse showed a strong increase in the number of renin-positive cells surrounding afferent arterioles. In direct proximity to the renin producing cells, tubular cells of the AS−/− mice showed a strong expression of COX-2, whereas no COX-2 staining was found in the cortex of wild-type mice. Bars indicate 50 μm. (E–H) Renin and COX-2 expression is increased in kidneys of AS−/− mice. E and F show renin mRNA abundance and COX-2 mRNA abundance in kidneys of WT and AS−/− mice. G shows plasma renin concentration in WT and AS−/− mice. H shows quantitative analysis of renin immunofluorescence in kidney cortices of WT and AS−/− mice. Data are means ± SEM of 6–18 mice of each group. Asterisks indicate p < 0.05

• 

  Fig. 2. Renin mRNA and plasma renin concentration are sensitive to COX-2 inhibition in AS−/− mice. Kidney renin mRNA abundance (upper panel) and plasma renin concentration (lower panel) in AS−/− mice and in wild-type controls were measured under basal conditions and after 1 week of SC-236 treatment. Data are means ± SEM of 6 mice of each genotype. Asterisks indicate p < 0.05

• 

  Fig. 3. Hyperplastic renin cells in AS−/− mice express EP2 and EP4 receptors. In situ hybridization with the RNAscope method showed clear expression of both EP2 receptor mRNA (A; blue dots) and EP4 receptor mRNA (B; blue dots) in hyperplastic renin cells of AS−/− mice. Hyperplastic renin cells (thin dotted line) were identified as PDGFR-β mRNA-expressing (red dots) granular cells, surrounding afferent arterioles (aa). G and thick dotted lines indicate glomeruli. Bars indicate 50 μm

• 

  Fig. 4. Enlarged view of EP2 receptor mRNA in situ hybridization (blue dots) in hyperplastic renin cells of AS−/− mice. Arrowheads highlight selected EP2 receptor mRNA expression signals. Thick dotted line indicates a glomerulus. Thin dotted line indicates hyperplastic renin cells. aa, afferent arteriole; red dots, PDGFR-β mRNA expression. Bar indicates 50 μm

• 

  Fig. 5. Enlarged view of EP4 receptor mRNA in situ hybridization (blue dots) in hyperplastic renin cells of AS−/− mice. Arrowheads highlight selected EP4 receptor mRNA expression signals. Thick dotted line indicates a glomerulus. Thin dotted line indicates hyperplastic renin cells. aa, afferent arteriole; red dots, PDGFR-β mRNA expression. Bar indicates 50 μm

• 

  Fig. 6. Deletion of EP2/EP4 receptors in renin cells lowers renin mRNA and plasma renin concentration. Kidney renin mRNA abundance (upper panel) and plasma renin concentration (lower panel) in wild-type, AS−/−, AS−/−/EP2−/−, AS−/−/mRen-rtTAm2/LC1 Cre/EP4fl/fl, and AS−/−/EP2−/−/mRen-rtTAm2/LC1 Cre/EP4fl/fl mice. Renin mRNA abundance was measured 10 days after 21 days of doxycycline treatment. Plasma renin concentration was measured before and 10 days after 21 days of doxycycline treatment. Data are means ± SEM of 5–20 mice of each genotype. Asterisks indicate p < 0.05

• 

  Fig. 7. Deletion of EP2/EP4 receptors in renin cells reduces renin immunoreactivity in the cortex of AS−/− mice. Immunostaining (A, B; Renin, green; α-SMA, red) and quantitative analysis of renin immunofluorescence (C) showed a clear decrease in renin-positive areas in the cortex of AS−/− mice after deletion of EP2 and EP4 in renin cells. Kidneys of AS−/−/EP2−/−/mRen-rtTAm2/LC1 Cre/EP4fl/fl were analyzed before and 10 days after 21 days of doxycycline treatment. Data are means ± SEM of 4 mice of each group. Asterisks indicate p < 0.05. Bars indicate 100 μm

• 

  Fig. 8. Renin cell lineage labeling in AS−/−/mRen-rtTAm2/LC1 Cre/mT/mG mice. After doxycycline induction, GFP-expression was irreversibly switched on in all vascular and perivascular renin cells. A shows a combined image of GFP (red), α-SMA (cyan), and renin (green) immunoreactivity. B–D shows immunoreactivity for GFP, α-SMA, and renin alone. G and dotted lines indicate glomeruli. Bars indicate 50 μm

• 

  Fig. 9. Renin cell lineage tracing reveals perivascular downregulation of renin expression after EP2 and EP4 deletion in AS−/− mice. Deletion of EP2 and EP4 in AS−/−/EP2−/−/mRen-rtTAm2/LC1 Cre/EP4fl/fl/mT/mG mice results in a clear reduction of the renin signal and a persistence of large areas of perivascular GFP-expressing renin-negative cells 10 days after doxycycline induction. The decrease in renin expression was mainly seen in the perivascular renin cells while in vascular renin cells, expression of renin was not affected. A shows a combined image of GFP (red), α-SMA (cyan), and renin (green) immunoreactivity. B–D shows immunoreactivity for GFP, α-SMA, and renin alone. G and dotted lines indicate glomeruli. Bars indicate 50 μm