Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice. Am J Physiol Renal Physiol 293: F1915-F1926, 2007. First published September 26, 2007; doi:10.1152/ajprenal.00160.2007.-Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar protonpumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1Ϫ/Ϫ mice than in B1ϩ/ϩ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1Ϫ/Ϫ mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28 -40% of normal) V-ATPase function is preserved in medullary ICs from B1Ϫ/Ϫ mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions. proton pump; immunofluorescence; pH homeostasis; urinary acidification; Atp6v1b1Ϫ/Ϫ mice THE MAIN MEDIATOR OF INTRACELLULAR organelle acidification in eukaryotic cells and of proton (H ϩ ) secretion along the distal renal nephron is the ubiquitous vacuolar proton-pumping ATPase (vacuolar, or V-type, H ϩ -ATPase, or V-ATPase). The V-ATPase is a complex enzyme, consisting of two large sectors or domains (V 0 , the transmembrane domain involved in H ϩ translocation, and V 1 , the cytosolic domain, responsible for hydrolyzing ATP), which together contain at least 13 distinct

Final urinary acidification is mediated by the action of vacuolar H ϩ -ATPases expressed in acid-secretory type A intercalated cells (A-IC) in the collecting duct. Angiotensin II (AngII) has profound effects on renal acid-base transport in the proximal tubule, distal tubule, and collecting duct. This study investigated the effects on vacuolar H ϩ -ATPase activity in A-IC in freshly isolated mouse outer medullary collecting ducts.AngII (

Final urinary acidification is achieved by electrogenic vacuolar H(+)-ATPases expressed in acid-secretory intercalated cells (ICs) in the connecting tubule (CNT) and the cortical (CCD) and initial medullary collecting duct (MCD), respectively. Electrogenic Na(+) reabsorption via epithelial Na(+) channels (ENaCs) in the apical membrane of the segment-specific CNT and collecting duct cells may promote H(+)-ATPases-mediated proton secretion by creating a more lumen-negative voltage. The exact localization where this supposed functional interaction takes place is unknown. We used several mouse models performing renal clearance experiments and assessed the furosemide-induced urinary acidification. Increasing Na(+) delivery to the CNT and CCD by blocking Na(+) reabsorption in the thick ascending limb with furosemide enhanced urinary acidification and net acid excretion. This effect of furosemide was abolished with amiloride or benzamil blocking ENaC action. In mice deficient for the IC-specific B1 subunit of the vacuolar H(+)-ATPase, furosemide led to only a small urinary acidification. In contrast, in mice with a kidney-specific inactivation of the alpha subunit of ENaC in the CCD and MCD, but not in the CNT, furosemide alone and in combination with hydrochlorothiazide induced normal urinary acidification. These results suggest that the B1 vacuolar H(+)-ATPase subunit is necessary for the furosemide-induced acute urinary acidification. Loss of ENaC channels in the CCD and MCD does not affect this acidification. Thus, functional expression of ENaC channels in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification.

Hypothyroidism in humans is associated with incomplete distal renal tubular acidosis, presenting as the inability to respond appropriately to an acid challenge by excreting less acid. Here, we induced hypothyroidism in rats with methimazole (HYPO) and in one group substituted with l-thyroxine (EU). After 4 wk, acid-base status was similar in both groups. However, after 24 h acid loading with NH(4)Cl HYPO rats displayed a more pronounced metabolic acidosis. The expression of the Na(+)/H(+) exchanger NHE3, the Na(+)-phosphate cotransporter NaPi-IIa, and the B2 subunit of the vacuolar H(+)-ATPase was reduced in the brush-border membrane of the proximal tubule of the HYPO group, paralleled by a lower abundance of the Na(+)/HCO(3)(-) cotransporter NBCe1 and a higher expression of the acid-secretory type A intercalated cell-specific Cl(-)/HCO(3)(-) exchanger AE1. In contrast to control conditions, the expression of NBCe1 was increased in the HYPO group during metabolic acidosis. In addition, net acid excretion was similar in both groups. The relative number of type A intercalated cells was increased in the connecting tubule and cortical collecting duct of the HYPO group during acidosis. Thus thyroid hormones modulate the renal response to an acid challenge and alter the expression of several key acid-base transporters. Mild hypothyroidism is associated only with a very mild defect in renal acid handling, which appears to be mainly located in the proximal tubule and is compensated by the distal nephron.