The B1-subunit of the H(+) ATPase is required for maximal urinary acidification

Abstract

The multisubunit vacuolar-type H(+)ATPases mediate acidification of various intracellular organelles and in some tissues mediate H(+) secretion across the plasma membrane. Mutations in the B1-subunit of the apical H(+)ATPase that secretes protons in the distal nephron cause distal renal tubular acidosis in humans, a condition characterized by metabolic acidosis with an inappropriately alkaline urine. To examine the detailed cellular and organismal physiology resulting from this mutation, we have generated mice deficient in the B1-subunit (Atp6v1b1(-/-) mice). Urine pH is more alkaline and metabolic acidosis is more severe in Atp6v1b1(-/-) mice after oral acid challenge, demonstrating a failure of normal urinary acidification. In Atp6v1b1(-/-) mice, the normal urinary acidification induced by a lumen-negative potential in response to furosemide infusion is abolished. After an acute intracellular acidification, Na(+)-independent pH recovery rates of individual Atp6v1b1(-/-) intercalated cells of the cortical collecting duct are markedly reduced and show no further decrease after treatment with the selective H(+)ATPase inhibitor concanamycin. Apical expression of the alternative B-subunit isoform, B2, is increased in Atp6v1b1(-/-) medulla and colocalizes with the H(+)ATPase E-subunit; however, the greater severity of metabolic acidosis in Atp6v1b1(-/-) mice after oral acid challenge indicates that the B2-subunit cannot fully functionally compensate for the loss of B1. Our results indicate that the B1 isoform is the major B-subunit isoform that incorporates into functional, plasma membrane H(+)ATPases in intercalated cells of the cortical collecting duct and is required for maximal urinary acidification.

Fig. 1.

Generation of H⁺ ATPase B1-subunit deficient mice. (a) Structure of the endogenous locus, targeting construct and targeted allele are shown. The segments not endogenous to the WT allele are shown in gray. Numbered boxes represent exons of Atp6v1b1. The sizes of restriction fragments resulting from HindIII (H) and EcoRI (E) digestion of the WT and targeted alleles, and the locations of probes (P1, P2) used for Southern blotting are shown. “Neo” denotes neomycin resistance gene; “TK” denotes thymidine kinase gene. (b and c) Confirmation of Atp6v1b1 gene targeting. Genomic DNA of WT (+/+) or heterozygous (+/-) targeted deletion ES cell lines were digested with indicated enzymes, fractionated on 0.8% agarose gels, and subjected to Southern blotting. (b) Hybridization of probe P1 to HindIII-digested DNA is shown. (c) Hybridization of probe P2 to EcoRI-digested DNA is shown. Fragments corresponding to the targeted (-) alleles are seen only in targeted clones. (d) Genotyping of Atp6v1b1 in +/+, +/-, and -/- mice. PCR of genomic DNA was performed by using primer pairs specific for the WT (intron/exon 9) and deleted alleles (Neo cassette) and genomic DNA as template (see Methods). Products were resolved on a 2% agarose gel. The inferred genotypes are indicated. (e) Immunoblotting of total kidney homogenates with antibodies to the B1-subunit was performed (see Methods). Expression is seen in Atp6v1b1^+/+ and Atp6v1b1^+/- mice, but is absent in Atp6v1b1^-/- mice.

Fig. 2.

Immunolocalization of CD cell type-specific markers in WT and Atp6v1b1^-/- kidney. Renal sections were prepared, stained, and imaged as described in Methods. (a) Distribution of Atp6v1b1 in WT outer medullary collecting duct (OMCD). CD segments are identified by staining of principal cells with aquaporin-2 (AQP-2, in green); Atp6v1b1 is stained in red, and localizes to intercalated cells (ICs). (b) Staining of OMCD from Atp6v1b1^-/- mouse was performed as in a. Architecture of the nephron segment appears normal, but Atp6v1b1 staining is absent. (c) Presence of A-ICs in Atp6v1b1^-/- kidney. Sections were stained for AQP2 (green) and AE1 (red). AE1 staining is present and localizes to the basolateral membrane of ICs of the OMCD, a normal distribution like that seen in WT (not shown). (d) Presence of B-ICs in Atp6v1b1^-/- kidney. Pendrin immunoreactivity (a B-IC marker, red) is detected in an apical distribution in subset of cortical CD cells. This intracellular distribution is similar to that seen in WT (not shown). (Scale bars, 16 μm.)

Fig. 3.

Defective renal H⁺ secretion in Atp6v1b1^-/- mice. Values in Atp6v1b1^+/+ and Atp6v1b1^-/- littermates are compared and represented as means ± SE. (a-c) Venous pH, plasma HCO^-₃ and urine pH were measured on a standard rodent diet with free access to either tap water (standard diet) or a 1.5% NH₄Cl/1% sucrose solution (acid load) (see Methods). n = 6-12 mice per genotype. ^**, P < 0.001 standard diet vs. acid load. (a) Venous pH. The -/- mice have significantly lower venous pH after acid loading. (b) Venous plasma HCO^-₃. The -/- mice have significantly lower plasma HCO^-₃ after acid loading. (c) Urine pH. Despite lower plasma pH, the -/- mice have higher urine pH, indicating a defect in renal acidification. (d-f) Sodium excretion, potassium excretion, and urinary pH were measured in Atp6v1b1^+/+ (n = 9) and Atp6v1b1^-/- (n = 9) mice before and after i.v. administration of furosemide as described in Methods. ^**, P < 0.01, without furosemide vs. with furosemide. (d) Sodium excretion. Furosemide dramatically increased natriuresis to a similar degree (P = 0.10) in both +/+ and -/- mice. (e) Potassium excretion. Furosemide induced potent kaliuresis to a similar degree in both +/+ and -/- mice (P = 0.337). (f) Urine pH. Furosemide induced a marked reduction in urine pH in +/+ mice but no significant change in -/- mice.

Fig. 4.

Impaired proton secretion from single ICs of Atp6v1b1^-/- mice after acute acidification. CD fragments were isolated and acidified as described in Methods, and the recovery of pH was measured in the presence or absence of concanamycin (CON). (a) ICs were identified on morphologic criteria; the ability to distinguish ICs was shown by staining for AE1 as in Fig. 2. (b) Measurement of recovery of intracellular pH after acute acidification. Cells were acidified with NH₄Cl, and recovery of intracellular pH was monitored with the pH-sensitive dye BCECF in the absence of Na⁺ as described in Methods. The rate of recovery was determined from the slope of the increase in pH with time (bracket). (c) Distribution of rates of pH recovery in +/+ and -/- mice in the absence and presence of CON. Each data point represents the rate of recovery in a single cell. +/+ ICs show a markedly greater mean rate of recovery than cells from -/- mice (P < 0.001). CON inhibited recovery of pH in +/+ cells (P < 0.001) to a rate indistinguishable from that seen in -/- cells in the absence or presence of the drug.

Fig. 5.

Increased apical expression of the H⁺ ATPase B2-subunit in Atp6v1b1^-/- kidney. Kidney sections were stained for the B2-subunit and visualized as described in Methods.(a) B2 is predominantly cytoplasmic in +/- inner medullary collecting duct (IMCD). (b) B2 shows significant enhancement at the apical membrane in -/- IMCD. (c-e) Colocalization of B2 (green) with the H⁺ ATPase E subunit (red) at the apical membrane of IMCD of -/- mice. (Insets) A single A-IC at higher magnification. (Scale bars, 15 μm in a and b, 30 μm in c-e, and 5 μm in Insets.)