Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca 2+ , the production of NO and prostaglandin (PGE 2 ), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na ϩ excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel-dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption. 20: 172420: -173220: , 200920: . doi: 10.1681 It is well established that renal tubular epithelial cells release ATP, 1-4 which then binds to purinergic receptors along the nephron and collecting ducts to regulate salt and water reabsorption [5][6][7][8][9][10][11][12] ; however, the molecular mechanism of ATP release and the identity of ATP channels are less clear. The possible mechanisms include vesicular release, 1,2 pannexin or connexin hemichannels, 13-16 ATP-permeable anion channels such as the cystic fibrosis transmembrane conductance regulator, 17 or the maxi anion channel. 18 Previous work also suggested that the release of ATP in epithelia may be triggered by mechanical stimulation, 19 -21 including elevations in tubular fluid flow rates. For example, tubular flow-dependent ATP release resulting in purinergic calcium signaling has been well characterized in the cortical collecting duct (CCD) [21][22][23] ; however, the ATP release mechanism has not been identified. J Am Soc Nephrol

Connexins (Cxs) are widely-expressed proteins that form gap junctions in most organs, including the kidney. In the renal vasculature, Cx37, Cx40, Cx43, and Cx45 are expressed, with predominant expression of Cx40 in the endothelial cells and Cx45 in the vascular smooth muscle cells. In the tubules, there is morphological evidence for the presence of gap junction plaques only in the proximal tubules. In the distal nephron, Cx30, Cx30.3, and Cx37 are expressed, but it is not known whether they form gap junctions connecting neighboring cells or whether they primarily act as hemichannels. As in other systems, the major function of Cxs in the kidney appears to be intercellular communication, although they may also form hemichannels that allow cellular secretion of large signaling molecules. Renal Cxs facilitate vascular conduction, juxtaglomerular apparatus calcium signaling, and tubular purinergic signaling. Accordingly, current evidence points to roles for these Cxs in several important regulatory mechanisms in the kidney, including the renin angiotensin system, tubuloglomerular feedback, and salt and water reabsorption. At the systemic level, renal Cxs may help regulate blood pressure and may be involved in hypertension and diabetes.

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1 Ϫ/Ϫ mice. Urinary ATP levels were reduced by 30% in Panx1 Ϫ/Ϫ compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. ATP release; channel; immunofluorescence; kidney PURINERGIC SIGNALING IS INVOLVED in several major regulatory mechanisms in the kidney, including tubuloglomerular feedback, control of renin release, and tubular fluid and electrolyte transport (17). ATP concentrations consistent with P2 receptor activation have been found in the tubular lumen (40). Additionally, the identity of several P2X and P2Y receptors along the renal epithelium, as well as their effect on ion transport, has already been determined (17). However, the mechanisms by which ATP, the ligand, is released into the tubular lumen remain contentious.In the renal tubules, ATP may be released by both vesicular and channel-based release mechanisms. Several different transporters have been proposed as ATP conduits, including the cystic fibrosis transmembrane conductance regulator and the P2X7 receptor, but subsequently their ATP permeability has been challenged (24). It has also been suggested that connexin (Cx) proteins can form hemichannels, or connexons, which allow ATP release to the extracellular fluid (8,10,12,24). Recently, a study from our laboratory provided evidence that Cx30 is required for luminal ATP release in the cortical collecting duct and that this mechanism is involved in pressurenatriuresis (35). However, the issue of functional hemichannels formed solely by connexins under physiological conditions is controversial (37).A recently identified plasma membrane ATP channel is the pannexin channel (3-7). Pannexins are a class of proteins that also form hexameric transmembrane channels, similar to those formed by connexins (4, 5, 7). Despite having similar structures, connexins and pannexins have no sequence homology. ...