Lisa Kurtz

Connexin40 is essential for the pressure control of renin synthesis and secretion.

Renin secretion and synthesis in renal juxtaglomerular cells are controlled by short feed back loops involving angiotensin II and the intrarenal blood pressure. The operating mechanisms of these negative feed back regulators are widely unknown, except for the fact that both require calcium to exert their inhibitory action. We here show that in the absence of connexin40 (Cx40), which form gap junctions between juxtaglomerular and endothelial cells, the negative control of renin secretion and synthesis by angiotensin II and by intravasal pressure is abrogated, while the regulation by salt intake and &bgr;-adrenergic stimulation is maintained. Renin secretion from Cx40-deficient kidneys or wild-type kidneys treated with the nonselective gap junction blocker 18&agr;-glycyrrhetinic acid (10 &mgr;mol/L) resembles the situation in wild-type kidneys in the absence of extracellular calcium. This disturbed regulation is reflected by an enhanced plasma renin concentration despite an elevated blood pressure in Cx40-deficient mice. These findings indicate that Cx40 connexins and likely intercellular communication via Cx40-dependent gap junctions mediate the calcium-dependent inhibitor effects of angiotensin II and of intrarenal pressure on renin secretion and synthesis. Because Cx40 gap junctions are also formed between renin producing cells and endothelial cells our finding could provide additional information to suggest that the endothelium may be strongly involved in the control of the renin system.

Lack of Connexin 40 Causes Displacement of Renin-Producing Cells from Afferent Arterioles to the Extraglomerular Mesangium

In the adult kidney, renin-producing cells are typically located in the walls of afferent arterioles at the transition into the glomerular capillary network. The mechanisms that are responsible for restricting renin expression to the juxtaglomerular position are largely unknown. This study showed that in mice that lack connexin 40 (Cx40), the predominant connexin of renin-producing cells, renin-positive cells are absent in the vessel walls and instead are found in cells of the extraglomerular mesangium, glomerular tuft, and periglomerular interstitium. Blocking macula densa transport function by acute administration of loop diuretics strongly enhances renin secretion in vivo and in isolated perfused kidneys of wild-type mice. This effect of loop diuretics is markedly attenuated in vivo and even blunted in vitro in Cx40-deficient mice. Even after prolonged stimulation of renin secretion by severe sodium depletion, renin expression is not seen in juxtaglomerular cells or in cells of more proximal parts of the arterial vessel wall as occurs normally. Instead, renin remains restricted to the extra-/periglomerular interstitium in Cx40-deficient mice. In contrast to the striking displacement of renin-expressing cells in the adult kidney, renin expression in the vessels of the developing kidney was found to be normal. This is the first evidence to indicate that cell-to-cell communication via gap junctions is essential for the correct juxtaglomerular positioning and recruitment of renin-producing cells. Moreover, these findings support the notion that gap junctions are relevant for the macula densa signaling to renin-producing cells.

Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension

Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure.

Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension

Connexins (Cxs) are a family of transmembrane proteins that form gap junctions with unique and redundant biophysical functions. Juxtaglomerular cells express Cx40, which is crucial to the control of renin secretion by blood pressure and angiotensin II, and mice that lack Cx40 have high plasma renin and hypertension. To examine whether normal juxtaglomerular cell function depends on the unique properties of Cx40, we measured renin release in mice where the coding sequence for Cx40 was replaced by that for Cx45, using the knock-in method. We first found that the knock-in strategy indeed resulted in expression of Cx45 but not Cx40 in the juxtaglomerular cells of these mice. The plasma renin concentration of the knock-in mice was similar to that in wild-type mice. The high blood pressure of the Cx40 knockout mice was significantly reduced when Cx45 was knocked into the locus but remained mildly elevated compared to wild-type mice. Blockade of angiotensin II formation by enalapril increased the plasma renin concentration in wild-type and the Cx45 knock-in mice but not in the Cx40 knockout mice. Infusion of angiotensin II into isolated perfused kidneys results in decreased renin release, a phenomenon that was attenuated in the Cx40 knockout mice. However, in the Cx45 knock-in mice, angiotensin II suppressed renin release similar to its effect in wild type mice. Unilateral renal artery stenosis increased the plasma renin concentration and blood pressure in both the wild-type and the Cx45 knock-in mice but not in the Cx40 knockout mice. Since Cx40 can be replaced by Cx45, a connexin with a significantly lower conductivity, we suggest that the regulation of renin release is not dependent on the unique electrical properties of these channel proteins.

The Connexin40 A96S Mutation Causes Renin-Dependent Hypertension

Deletion of the gap-junction-forming protein connexin40 leads to renin-dependent hypertension in mice, but whether observed human variants in connexin40, such as A96S, promote hypertension is unknown. Here, we generated mice with the A96S variant in the mouse connexin40 gene. Although mice homozygous for the A96S mutations had normal expression patterns of connexin40 in the kidney, they were hypertensive, had sixfold higher plasma renin concentrations, and had 40% higher levels of renin mRNA than controls. Renin-expressing cells were aberrantly located outside the media layer of afferent arterioles, and increased renal perfusion pressure did not inhibit renin secretion from kidneys isolated from homozygous A96S mice. Treatment with a low-salt diet in combination with an ACE inhibitor increased renin mRNA levels, plasma renin concentrations, and the number of aberrantly localized renin-producing cells. Taken together, these findings suggest that the A96S mutation in connexin40 leads to renin-dependent hypertension in mice. Modulation of renin secretion by BP critically depends on functional connexin40; with the A96S mutation, the aberrant extravascular localization of renin-secreting cells in the kidney likely impairs the pressure-mediated inhibition of renin secretion.

Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation

Connexins are important in vascular development and function. Connexin 40 (Cx40), which plays a predominant role in the formation of gap junctions in the vasculature, participates in the autoregulation of renal blood flow (RBF), but the underlying mechanisms are unknown. Here, Cx40-deficient mice (Cx40-ko) had impaired steady-state autoregulation to a sudden step increase in renal perfusion pressure. Analysis of the mechanisms underlying this derangement suggested that a marked reduction in tubuloglomerular feedback (TGF) in Cx40-ko mice was responsible. In transgenic mice with Cx40 replaced by Cx45, steady-state autoregulation and TGF were weaker than those in wild-type mice but stronger than those in Cx40-ko mice. N omega-Nitro-L-arginine-methyl-ester (L-NAME) augmented the myogenic response similarly in all genotypes, leaving autoregulation impaired in transgenic animals. The responses of renovascular resistance and arterial pressure to norepinephrine and acetylcholine were similar in all groups before or after L-NAME inhibition. Systemic and renal vasoconstrictor responses to L-NAME were also similar in all genotypes. We conclude that Cx40 contributes to RBF autoregulation by transducing TGF-mediated signals to the afferent arteriole, a function that is independent of nitric oxide (NO). However, Cx40 is not required for the modulation of the renal myogenic response by NO, norepinephrine-induced renal vasoconstriction, and acetylcholine- or NO-induced vasodilation.

Connexin Expression in Renin-Producing Cells

Absence of connexin 40 (Cx40) leads to ectopic juxtaglomerular renin expression and abrogates recruitment of renin-expressing cells in the adult kidney but does not disturb renin expression during kidney development. To find an explanation for these observations, we aimed to analyze the expression pattern of major vascular Cxs in normal juxtaglomerular epithelioid cells, in recruited renin-expressing cells, and in fetal renin-expressing cells. We found that during kidney development, the appearance of renin-producing cells paralleled the expression of Cx40 and, to a lesser extent, Cx45 but not other Cxs. In the adult kidney, juxtaglomerular epithelioid cells expressed Cx40 and lesser amounts of Cx37 and Cx43 but not Cx45, which localized to arteriolar smooth muscle cells. Recruitment of renin-producing cells in adult kidneys in response to long-term salt deprivation of mice correlated with the reappearance of only Cx40. Cx40-null renin-producing cells did not express Cx37, Cx43, or Cx45. These findings suggest that Cx40 expression is a characteristic of renin-producing cells in the kidney, and it seems to be essential in the recruitment of renin-producing cells in the adult but not the fetal kidney.

High-Level Connexin Expression in the Human Juxtaglomerular Apparatus

Recent evidence obtained in rodents indicates that gap junctions in the juxtaglomerular apparatus play an important role in the control of renin-producing cells and in tubuloglomerular signaling. These gap junctions are formed by cell-specific expression patterns of the vascular connexins Cx37, Cx40, Cx43 and Cx45. In order to obtain a first indication if gap junctions might play a similar important functional role in the juxtaglomerular apparatus of human kidneys, this study aimed to characterize the juxtaglomerular localization of Cx40, Cx37, Cx43 and Cx45 in human kidney specimens. We found Cx37, Cx40 and Cx43, but not Cx45 expression in high density in the extraglomerular mesangium. Renin-producing cells displayed strong immunoreactivity for Cx40 and Cx37. Cx37, Cx40 and Cx43 were also seen in the endothelium of arteries/arterioles outside of the glomeruli, whereas Cx45 was located in vascular smooth muscle cells. All four connexins were also expressed within the glomeruli. These findings indicate that the expression pattern of vascular connexins in the human kidney cortex is very similar to that previously found for mouse and rat kidneys, suggesting that the intrarenal expression pattern of vascular connexins is conserved among the mammalian species. Because of this similarity, and in particular in view of the strong expression of Cx37 and Cx40 in the juxtaglomerular area, we infer that those functions of connexins that have already been demonstrated for rodent kidneys, such as a central role of Cx40 for the development and function of renin-producing cells and for tubuloglomerular signal transmission, might hold for human kidneys as well.

Connexin 37 is dispensable for the control of the renin system and for positioning of renin-producing cells in the kidney

Within the juxtaglomerular apparatus, renin-producing cells and endothelial cells of the afferent arterioles express connexin (Cx)37 and Cx40, which form abundant gap junctions among these cells. Deletion of Cx40 leads to strong hyperreninemia and ectopic localization of renin-producing cells; however, the relevance of Cx37 for the renin system in the kidney has not been investigated. We therefore studied renin expression and renin secretion in kidneys from Cx37-deficient mice, both on normal salt diet and during chronic challenge of the renin system by pretreatment of mice with a low-salt diet in combination with an angiotensin I-converting enzyme inhibitor. This treatment procedure strongly enhances renin gene expression and renin secretion. We found that renal renin mRNA abundance and plasma renin concentration did not differ between wild-type and Cx37−/− mice under normal conditions. The stimulation of renin gene expression and renin secretion by salt depletion was even more pronounced in Cx37−/− as compared to wild-type mice. The regulation of renin secretion from isolated perfused kidneys by perfusion pressure and by angiotensin II was normal in Cx37−/− mice. In addition, the localization of renin-expressing cells was also regular in Cx37−/− kidneys. Finally, the expression pattern of other vascular Cxs such as Cx40, Cx43, and Cx45 was not altered in Cx37−/− kidneys. Our findings suggest that Cx37 is not essential for normal development and function of renin-producing cells. As a consequence, it appears unlikely that Cx40 exerts its important function in renin-producing cells via Cx37/Cx40 heteromeric gap junctions.

Replacement of connexin 40 by connexin 45 causes ectopic localization of renin-producing cells in the kidney but maintains in vivo control of renin gene expression.

Deletion of connexin 40 (Cx40) leads to ectopic hyperplasia of renin-producing cells in the kidney, which is associated with dysregulated hyperreninemia and hypertension. The aim of this study was to determine whether Cx45 is able to substitute the function of Cx40 with regard to the localization of renin-producing cells. For this purpose, we have studied the distribution of renin-expressing cells under both normal conditions and during a stimulatory challenge of the renin system by inducing salt deprivation in mice, achieved by replacing the coding sequence of the Cx40 gene with that of Cx45 (Cx40ki45). In both wild-type (WT) mice and Cx40ki45 mice under normal conditions, renin-expressing cells were located at the juxtaglomerular position, whereas in Cx40-deficient mice they were located in the periglomerular interstitium. Upon challenge of the renin system, renin mRNA and the number of renin-expressing cells increased in WT mice in the media layer of afferent arterioles, while neither parameter changed significantly in Cx40-deficient mice. In Cx40ki45 mice, challenge of the renin system markedly increased both renin mRNA and the number of renin-expressing cells. However, the newly recruited renin-expressing cells were localized mainly outside the afferent vessels in the periglomerular interstitium. We found no evidence of cell divisions in renin-expressing cells in any of the genotypes investigated in this study, suggesting that the ectopically localized, renin-expressing cells in Cx40ki45 mice were already preexisting but were not renin-expressing under normal conditions. In summary, we infer from our findings that the function of Cx40 for the localization of potential renin-producing cells cannot be substituted by that of Cx45, although the regulability of renin gene expression can.