Connexin 30 Deficiency Impairs Renal Tubular ATP Release and Pressure Natriuresis

Abstract

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

[Ca2+] Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

“…That said, it is currently not known how HlyA induces ATP release from renal epithelia. One could speculate that ATP leaves through the HlyA pore, but ATP may equally as well be released through one of the established ATP release pathways in renal epithelia; vesicular release (34) or through connexin hemichannels (35).…”

Vesicular and conductive mechanisms of nucleotide release

“…Cx30 −/− mice exhibited saltsensitive elevation in mean arterial pressure. The data suggest that ATP release via Cx30 controls salt and water reabsorption at the collecting ducts, regulating pressure natriuresis and diuresis [129]. In another study, increasing Na + intake resulted in enhanced urinary ATP secretion, which was robust in WT mice but modest in Cx30 −/− animals.…”

“…Microperfused cortical collecting ducts from WT mice displayed flow-and hypotonicity-evoked ATP release (from intercalated cells), which were impaired in Cx30 −/− animals [129]. Urine output and urinary Na + secretion in response to increased arterial pressure (following ligating the distal aorta) were greater in WT relative to Cx30 −/− mice.…”

Renal β-intercalated cells maintain body fluid and electrolyte balance

“…The preceding experiments suggest that PGE 2 reduced ENaC activity in CCD PCs. To get a better insight into the mechanism by which PGE 2 regulates ENaC function in CCD PCs, we measured PGE 2 release into the tubular fluid in freshly isolated microperfused wild-type CCDs in response to pharmacologic inhibition of basolateral H + -ATPases in β-ICs using the previously established PGE 2 biosensor technique (27,28). We used HEK cells overexpressing the calcium-coupled PGE 2 receptor E-prostanoid 1 (EP1) as PGE 2 biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the tubular fluid ( Figure 6A).…”