Connexin43-dependent mechanism modulates renin secretion and hypertension

Abstract

To investigate the function of Cx43 during hypertension, we studied the mouse line Cx43KI32 (KI32), in which the coding region of Cx32 replaces that of Cx43. Within the kidneys of homozygous KI32 mice, Cx32 was expressed in cortical and medullary tubules, as well as in some extra- and intraglomerular vessels, i.e., at sites where Cx32 and Cx43 are found in WT mice. Under such conditions, renin expression was much reduced compared with that observed in the kidneys of WT and heterozygous KI32 littermates. After exposure to a high-salt diet, all mice retained a normal blood pressure. However, whereas the levels of renin were significantly reduced in the kidneys of WT and heterozygous KI32 mice, reaching levels comparable to those observed in homozygous littermates, they were not further affected in the latter animals. Four weeks after the clipping of a renal artery (the 2-kidney, 1-clip [2K1C] model), 2K1C WT and heterozygous mice showed an increase in blood pressure and in the circulating levels of renin, whereas 2K1C homozygous littermates remained normotensive and showed unchanged plasma renin activity. Hypertensive, but not normotensive, mice also developed cardiac hypertrophy. The data indicate that replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin, thus preventing the renin-dependent hypertension that is normally induced in the 2K1C model.

Figure 1

Cx43 and Cx32 were differentially distributed in the kidneys of KI32 mice. (A–D) Antibodies (A, C, and D) and a lacZ reporter gene construct (B) revealed the presence of Cx43 between the ECs of both interlobular renal arterioles (D) and afferent arterioles (B and C). Cx43 was also expressed by the ECs of the glomeruli (A and B). (E) In contrast, antibodies against Cx32 showed no staining of glomeruli and arterioles, but abundant levels of Cx32 in cortical proximal tubules. (F) In kidneys of homozygous KI32 mice, Cx32 was immunolocalized in both proximal and distal convoluted tubules, as well as in ECs of glomeruli. (G) The protein was also immunolabeled at gap junctions of ECs in interlobular arteries. In these vessels, the protein A–coated gold particles that were reacted with the Cx32 antibodies selectively decorated minute areas of apposition between EC membranes characteristic of gap junctions. g, glomerulus; aa, afferent arteriole; md, macula densa; ila, interlobular arteriole; pct, proximal convoluted tubule; dct, distal convoluted tubule; ec, endothelial cells. Scale bar: 30 μm (A, C, D, and G); 50 μm (B and E); 20 μm (F); 215 nm (H).

Figure 2

Renin expression was reduced after replacement of Cx43 by Cx32. (A) Northern blots revealed that the levels of the renin transcript were decreased by about half in the kidneys of homozygous KI32 mice, relative to those of GAPDH. *P < 0.05 versus WT mice. (B) Consistent with this finding, in situ hybridization (top panels) and immunofluorescence labeling (bottom panels) of the afferent arteriole of homozygous KI32 mice indicated reduced levels of the hormone transcript and of the cognate protein, respectively. Scale bar: 30 μm.

Figure 3

Transcription of the renin gene was differentially regulated after replacement of Cx43 by Cx32. (A) Mean blood pressure measurements remained normal in KI32 mice fed a high-salt (6%) diet. (B) Under these conditions, Northern blots revealed that the expression of the renin gene was decreased by about 40% in the kidneys of WT mice but was not altered in the kidneys of homozygous KI32 animals. ^#P < 0.01 versus value observed under a control NaCl diet (0.3%); *P < 0.05 versus WT mice. (C) A similar difference was found by quantitative RT-PCR. This approach further revealed that the renin expression of heterozygous KI32 mice was decreased by the high-salt diet, as seen in WT. ^§P < 0.05 versus controls fed a normal-salt diet; *P < 0.05 versus WT mice. Data are mean ± SEM values of 5 mice per group.

Figure 4

Renal artery clipping increased blood pressure in WT and heterozygous KI32 mice, but not in homozygous littermates. Four weeks after clipping of a renal artery, mean intra-arterial blood pressure of WT, 2K1C mice (white bar) was higher than that of sham-operated controls that remained normotensive (black bar). Similar observations were made in heterozygous KI32 mice. In contrast, no change in blood pressure was observed in homozygous KI32 littermates. Data are mean ± SEM values of the number of mice indicated in Table 1. White bars, 2K1C mice; black bars, sham-operated mice. *P < 0.05; **P < 0.01.

Figure 5

Heart weight increased in control and heterozygous KI32 mice, but not in homozygous littermates. (A) Immunofluorescence labeling for Cx43 and Cx32 showed the exclusive presence of the former and latter connexins in sections of ventricular myocytes of WT and homozygous KI32 mice, respectively. In the myocardium of heterozygous littermates, the 2 connexins colocalized at intercalated discs. Bar: 120 μm. (B) Four weeks after clipping of a renal artery, the cardiac weight index of WT, 2K1C mice that were hypertensive (white bar) was higher than that of sham-operated controls that remained normotensive (black bar), reflecting cardiac hypertrophy. Similar observations were made in heterozygous 2K1C mice. In contrast, no change in cardiac index was observed in homozygous 2K1C littermates. Data are mean ± SEM values of the number of mice indicated in Table 1. White bars, 2K1C mice; black bars, sham-operated mice. *P < 0.05; ***P < 0.001.

Figure 6

Plasma renin activity was increased in hypertensive but not in normotensive KI32 mice. Four weeks after clipping of a renal artery, plasma renin activity of the hypertensive, WT 2K1C mice (white bar) was higher than that of the normotensive sham-operated controls (black bar). Similar observations were made in heterozygous 2K1C mice. However, after clipping of the renal artery, these animals showed lower renin levels than WT littermates. In contrast, homozygous KI32 mice showed a much lower plasma renin activity (black bar), which was not affected by clipping of the renal artery (white bar). Data are mean ± SEM values of the number of mice indicated in Table 1. White bars, 2K1C mice; black bars, sham-operated mice. **P < 0.01; ***P < 0.001.

Figure 7

Expression of renin mRNA increased in the clipped kidneys of hypertensive, but not of normotensive, KI32 mice. (A) Northern blots revealed that, 4 weeks after surgery, the expression of the renin gene was similar in the left kidney (LK) and right kidney (RK) of WT, sham-operated control mice. In contrast, the renin transcript was markedly increased in the clipped kidney (LK), and decreased in the contralateral kidney (RK), of 2K1C animals. No significant change in the expression of the renin gene was observed in 2K1C homozygous KI32 littermates. As a result of these changes, the ratio of renin mRNA levels between the left (clipped) and the right kidney, as quantified in 4 independent experiments, was increased approximately 10-fold in WT 2K1C mice, and unchanged in homozygous KI32 2K1C littermates. (B) Similar changes were evaluated using quantitative RT-PCR. This approach further revealed that the renin expression of heterozygous KI32 2K1C mice was also increased in the clipped kidney, though to a level that was about half that evaluated in WT littermates. ***P < 0.001.

Figure 8

Relative expression of connexin mRNA was unchanged in kidneys of 2K1C mice. Four weeks after surgery, the expression of Cx32 and Cx43, as evaluated by the ratio of the cognate transcripts in the left (clipped) and right kidneys, was unchanged in the 3 mouse genotypes that we compared.