Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloridesensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na + and Cl -in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na + -driven Cl -/HCO 3 -exchanger (NDCBE/SLC4A8) and the Na + -independent Cl -/HCO 3 -exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na + reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt-and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1 -/-mice) displayed renal loss of NaCl, K + , and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na + -driven chloride/ bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E 2 (PGE 2 ) and ATP levels in Atp6v1b1 -/-mice. Inhibition of PGE 2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calciumactivated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in β-ICs induced release of PGE 2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE 2 paracrine signaling originating from β-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.

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The Na + concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na + along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na + has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na + /K + -ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H + vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na + /K + -ATPase had no effects. Basolateral NaCl exit from β-intercalated cells was independent of the Na + /K + -ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H + V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H + V-ATPase.

NH(4)(+) transport by the distal nephron and NH(4)(+) detoxification by the liver are critical for achieving regulation of acid-base balance and to avoid hyperammonemic hepatic encephalopathy, respectively. Therefore, it has been proposed that rhesus type B glycoprotein (Rhbg), a member of the Mep/Amt/Rh NH(3) channel superfamily, may be involved in some forms of distal tubular acidosis and congenital hyperammonemia. We have tested this hypothesis by inactivating the RHbg gene in the mouse by insertional mutagenesis. Histochemical studies analyses confirmed that RHbg knockout (KO) mice did not express Rhbg protein. Under basal conditions, the KO mice did not exhibit encephalopathy and survived well. They did not exhibit hallmarks of distal tubular acidosis because neither acid-base status, serum potassium concentration, nor bone mineral density was altered by RHbg disruption. They did not have hyperammonemia or disturbed hepatic NH(3) metabolism. Moreover, the KO mice adapted to a chronic acid-loading challenge by increasing urinary NH(4)(+) excretion as well as their wild-type controls. Finally, transepithelial NH(3) diffusive permeability, or NH(3) and NH(4)(+) entry across the basolateral membrane of cortical collecting duct cells, measured by in vitro microperfusion of collecting duct from KO and wild-type mice, was identical with no apparent effect of the absence of Rhbg protein. We conclude that Rhbg is not a critical determinant of NH(4)(+) excretion by the kidney and of NH(4)(+) detoxification by the liver in vivo.

Acid–base transport in the renal collecting tubule is mediated by two canonical cell types: the β-intercalated cell secretes HCO 3 by an apical Cl:HCO 3 named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the α-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HCO 3 exchanger (kAE1). We previously suggested that the β-cell converts to the α-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical α-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical β-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical β-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of β1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of β- to α-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HCO 3 secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K + intake, we hypothesized that this enzyme might regulate plasma K + concentration ([K + ]). We showed in wild-type mice that renal K + and TK excretion increase in parallel after a single meal, representing an acute K + load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption ( TK −/− ) were used to assess the impact of the enzyme on plasma [K + ]. A single large feeding did not lead to any significant change in plasma [K + ] in TK +/+ , whereas TK −/− mice became hyperkalemic. We next examined the impact of TK disruption on K + transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK −/− mice exhibit net transepithelial K + absorption because of abnormal activation of the colonic H + ,K + -ATPase in the intercalated cells. Finally, in CCDs isolated from TK −/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H + ,K + -ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K + load.

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