Connexin40 regulates renin production and blood pressure N. Krattinger, A. Capponi, L. Mazzolai, J.-F. Aubert, D. Caille, P. Nicod, G. Waeber, P. Meda, J.-A. Haefliger  Kidney International  Volume 72, Issue 7, Pages 814-822 (October 2007) DOI: 10.1038/sj.ki.5002423 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Loss of Cx40 alters the organization of the RSC. (a) In control mice, in situ hybridization shows that Cx40 mRNA is expressed along the endothelial cell (e) that line the lumen of the afferent arteriole (aa), as well as between the RSC (rc) that form the media of the juxtaglomerular (gl) portion of this vessel. (b) Immunocytochemistry shows a similar localization of the cognate protein. Bar=20 μm. (c) Immunofluorescence reveals the alignment of RSC in three afferent arterioles of a Cx40-/- mouse. (d and e) Mice lacking Cx40 had more renin-containing cells per glomerule than WT controls. (f–i) When compared to controls (f), the RSC of Cx40-/- mice (g) were smaller (e) and contained fewer (h), and more round secretory granules (i). Data are mean±s.e.m. values. ***P<0.001 Cx40-/- (solid bars) vs WT values (open bars). Bar=30 μm in (c) and 2 μm in (f) and (g). Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Cx40-/- mice are hypertensive due to increased levels of circulating renin. (a) Mean blood pressure was higher in Cx40-/- mice (solid bar) than in heterozygous (+/-; shaded bar) and WT littermates (open bar). (b) Plasma renin activity (PRA) was markedly increased in the hypertensive Cx40-/- mice. Values are mean±s.e.m. ***P<0.001 Cx40-/- vs WT levels. Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 High blood pressure and renin levels of Cx40-/- mice are caused by an activation of the RAS. (a) Inhibitors of the RAS lowered blood pressure of Cx40-/- mice, but did not affect that of WT controls. (b) Circulating renin was not significantly affected by these treatments in Cx40-/- mice, but was increased in WT controls (WT). (c) Basal expression of renin mRNA was higher in Cx40-/- than in WT mice. Candesartan and ramipril significantly increased the expression of renin in WT animals. (d) Expression of the angiotensin 1 receptor subtype (AT1A) mRNA was lower in Cx40-/- than in WT mice. After induction of hypertension in the 2K1C model, the AT1A levels of WT mice decreased to the values observed in Cx40-/- mice. Values are mean±s.e.m. **P<0.01, ***P<0.001 Cx40-/- (solid bars) vs WT levels (open bars). §P<0.05 vs levels in untreated Cx40-/- mice. Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 The regulation of renin secretion by salt is altered in Cx40-/- mice. (a) Mean blood pressure of WT (WT) and Cx40-/- mice was not altered after feeding a normal (NS), a low (LS), or a high-salt diet (HS). (b) Under the latter diet, the circulating levels of renin decreased in both WT and Cx40-/- mice. In contrast, a low salt diet increased renin secretion in WT but not Cx40-/- mice. (c) Northern blots showed that the low and the high-salt diets had opposite effects on the expression of the renin gene. Data are mean±s.e.m. values. *P<0.05, **P<0.01, ***P<0.001 vs the NS value of WT mice (open bars); §P<0.05 vs. NS values of Cx40-/- mice (solid bars). Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Renin secretion and hypertension depend on the expression of Cx40. (a) The 2K1C procedure increased the blood pressure of WT but not of Cx40-/- mice. (b) Plasma renin followed the same pattern model. (c) Changes of renin expression took place in both the hypoperfused (LK) and the controlateral (RK) kidneys of both WT and Cx40-/- mice. However, the effects on renin mRNA levels were smaller in Cx40-/- than in WT animals. (d) In situ hybridization showed that the levels of this transcript in the afferent arterioles (aa) also decreased much less in the kidneys of 2K1C Cx40-/- mice than in those of WT controls (right panels). G, glomerulus. Bar=80 μm. Data are mean±s.e.m. values of the number of mice indicated in Table 2. *P<0.05, **P<0.01, ***P<0.001 vs the cognate sham value in WT mice (open bars); §P<0.05 vs sham value of Cx40-/- mice (solid bars); ##P<0.001 vs the value in the right kidney (RK) of WT mice. Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 6 Cx37 and Cx43 are not over expressed in the RSC of the hypertensive Cx40-/- mice. (a) Analysis of total kidney RNA revealed different changes of the transcripts of Cx40, Cx43 and Cx37 in both the hypoperfused (LK) and the controlateral (RK) kidneys of WT (open bars) and Cx40-/- mice (solid bars) submitted to the 2K1C procedure. Data are mean±s.e.m. values of the number of mice indicated in Table 2. *P<0.05, vs sham value in WT mice. °°P<0.01. (b) Antibodies detected Cx37 in the intra-glomerular vessels of WT mice (top panels), as well as in the media of the juxta-glomerular portion of the afferent arterioles, where RSC are located (arrows in middle top panel), and between the endothelial cells of interlobular arteries (arrows in left bottom panel). A similar distribution was observed in Cx40-/- mice, even though the levels of Cx37 appeared decreased in both glomeruli (middle bottom panel) and interlobular vessels (right bottom panel). (c) Cx43 was expressed by the endothelial cells of renal vessels, including the afferent arteriole (left top panel). However, and at variance with Cx37, Cx43 was not found in the RSC of this vessel (arrow in left top panel). A similar distribution and expression levels were observed in the afferent arterioles (aa) of Cx40-/- mice (middle and right top panels; arrows point to putative RSC). In these mice, Cx43 was also observed between the endothelial cells of interlobular arteries (ila, arrows in left bottom panel), at the low levels observed in controls. Cx43 appeared increased between the smooth muscle cells of the media of large arcuate arteries (arrows in right bottom panel). gl, glomerulus; aa, afferent arteriole; ila, interlobular artery; md, macula densa; ara, arcuate artery. Bar=25 μm. Kidney International 2007 72, 814-822DOI: (10.1038/sj.ki.5002423) Copyright © 2007 International Society of Nephrology Terms and Conditions