Incomplete distal renal tubular acidosis from a heterozygous mutation of the V-ATPase B1 subunit

Abstract

Congenital distal renal tubular acidosis (RTA) from mutations of the B1 subunit of V-ATPase is considered an autosomal recessive disease. We analyzed a distal RTA kindred with a truncation mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of V-ATPase. All heterozygous carriers in this kindred have normal plasma HCO3- concentrations and thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria were present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also had inappropriate urinary acidification with acute ammonium chloride loading and an impaired urine-blood Pco2 gradient during bicarbonaturia, indicating the presence of a H+ gradient and flux defects. In normal human renal papillae, wild-type B1 is located primarily on the plasma membrane, but papilla from one of the heterozygote who had kidney stones but not nephrocalcinosis showed B1 in both the plasma membrane as well as diffuse intracellular staining. Titration of increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+ pump activity of wild-type B1 in mammalian human embryonic kidney-293 cells and in V-ATPase-deficient Saccharomyces cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of a mutant B1 subunit that cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

Keywords: V-ATPase; distal renal tubular acidosis; haploinsufficiency; kidney stones.

Fig. 1.

Top: genotype and phenotype of the pedigree. A GC insertion leads to loss of a phenylalanine and a frame shift, resulting and premature termination (p.Phe468fsX487). The arrow indicates the proband. *The patient without frank renal tubular acidosis (RTA) who presented with kidney stones. dRTA, distal RTA. Bottom: 24-hour and spot urine values. NA, not available.

Fig. 2.

NH₄Cl and HCO₃⁻ loading tests as well as immunohistochemistry of B1. Four members of the family consented to acidification experiments. Homo, homozygous mutant; Hetero, heterozygous carriers; WT, wild type. A: urine pH (UpH) changes with NH₄Cl load. B: urinary NH₄ excretion rate (U_NH4V) with NH₄Cl load. C: urine and blood values from the NaHCO₃ loading test. U-BPco₂, urine minus blood Pco₂. D: staining for B1 in the renal medulla. One heterozygous family member required surgery for nephrolithiasis and had an intraoperative biopsy. B1 was stained with an antibody that reacts with both WT and mutant B1 (green). β-Actin counterstain is shown in red. One normal subject is shown, but independent staining of three normal individuals showed similar results.

Fig. 3.

Mammalian cell expression system. Endogenous B1 and B2 were knocked down in human embryonic kidney (HEK)-293 cells, and V-ATPase function was measured in isolated endosomes. A: design of the small interfering (si)RNAs targeting B1 and B2 and protection of the heterologously transfected B1. B: original tracings of the acridine orange assay. HEK-293 cells were transfected with control siRNA (left), combined B1 + B2 siRNA (middle), or B1 + B2 siRNA along with FLAG-tagged WT human B1 with synonomous mutations as shown in A (right). Tracings show ATP-induced endosomal acidification followed by the collapse of the pH gradient by H⁺ ionophore 1799 (*). C: experimental conditions were similar to those in B. Cell lysates were immunoblotted with anti-B1 or anti-FLAG antibody.

Fig. 4.

Heterologous expression of WT and truncation mutant (TM) B1 in HEK-293 cells. A: V-ATPase function in endosomes was measured as ATP-induced acidification and expressed as a percentage of untransfected control cells. Top, WT and TM B1 were transfected into HEK-293 cells with scrambled control siRNA. Bottom, WT and TM B1 were transfected into HEK-293 cells where endogenous B1 and B2 were knocked down with siRNA. Each bar represents mean ± SE from three independent transfections with triplicate plates per transfection. Statistically significant differences were assessed by ANOVA. *P < 0.05. B: immunoblot of transfected FLAG epitope-tagged WT and TM B1 in conditions identical to those in A, bottom. C: titration of transfected WT (FLAG tagged) and TM B1 [hemagglutinin (HA) tagged] where the amount of cDNA of one was kept constant and the other varied. D: HEK-293 cells were transfected with 4 μg FLAG-tagged WT B1 (top) or 4 μg FLAG-tagged WT B1 with 8 μg HA-tagged TM B1 (bottom) and stained with anti-FLAG antibody. Left, fluorescent images only; right, results superimposed on DIC images.

Fig. 5.

Effect of TM B1 on native B1 in HEK-293 cells. TM B1 was transfected into HEK-293 cells, and its effects on native B1 expression and assembly with other subunits were studied. After cell lysis, transfected heterologous TM B1 was removed by immunodepletion with anti-HA antibody beads, and native B1 was immunoprecipitated with anti-B1. The immunocomplex was resolved by SDS-PAGE and immunoblotted for the antigens stated. A, top: immunoblot of transfected TM B in cell lysates with anti-HA. Middle, immunoblot of lysates after capture of TM B by immunoprecipitation (IP) for TM B HA, native B, A, and E subunits that are part of V₁. Bottom, immunoblot of the native B immunocomplex by anti-B. The complex was immunoblotted for B, A, and E subunits, which are parts of V₁. B: endosomal V-ATPase activity in untransfected, vector-transfected, and TM B-transfected HEK-293 cells.

Fig. 6.

Expression of WT and TM B1 in yeast. Expression plasmids p425TEF and p426TEF are described in methods. P425 and p426 denote plasmid only. WT and various human B1 mutations are shown. A, left: the yeast growth assay is permissive at pH 5.5 and restrictive at pH 7.5. A total of 5 × 10⁴ yeast cells were plated with serial 10-fold dilutions from left to right, and growth was assessed after 4 days. Right, expression of WT and TM COOH-terminally HA-tagged human B1 constructs in yeast. Equal amounts of yeast lysates of 10 optical density units were loaded. Immunoblot analysis was performed with a monoclonal anti-HA antibody. B: 12 other documented naturally occurring human B1 mutations were tested in a similar fashion in null yeast as described above. Left, yeast growth assay. Right, immunoblot.