Knockout of the V-ATPase Interacting Protein Tldc2 Inhibits Renal B-Intercalated Cell Function and Urine Alkalinization

Abstract

The V-ATPase, or proton pump, generates electrochemical gradients across membranes driving cell biological processes. The kidney-specific V-ATPase is expressed in intercalated cells (ICs), which regulate acid-base homeostasis by adjusting plasma membrane V-ATPase via regulated traffcking. In the kidney, A-ICs transport protons into the tubule lumen while B-ICs move protons into the peritubular space and, ultimately, the blood. The V-ATPase is distributed between intracellular vesicles and the apical plasma membrane in A-ICs, whereas B-ICs express V-ATPase not only in intracellular vesicles, but also basolaterally. In general, V-ATPase dysfunction in ICs leads to distal renal tubular acidosis (dRTA) implying a functional dominance of A-IC in acid/base physiology. B-ICs have, however, been implicated in blood pressure regulation due to their role in chloride reabsorption and, indirectly, Na transport. We recently identified a new class of V-ATPase interacting proteins defined by their TLDc domain (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2), all of which interact with the IC-specific B1 subunit of the V-ATPase. Of importance, Ncoa7 knockout mice had decreased V-ATPase expression in ICs and developed dRTA, which led us to hypothesize that TLDc proteins regulate the kidney V-ATPase. While Ncoa7 is specifically expressed in A-ICs, Tldc2 is one of the most differentially expressed proteins in B-ICs, prompting us to ask whether Tldc2 deletion affected V-ATPase function in these cells. Using global Tldc2 knockout mice (KO), we found that urine pH is significantly more acidic in KO mice than in WT mice in both males and females (pH 5.8 in male KOs versus pH 6.4 in male WTs and pH 5.8 in female KOs versus pH 6.2 in female WTs). After an acid challenge with NH4Cl, the urine pH of WT mice was significantly decreased, but urine pH was unchanged in KO mice, since it was already low under baseline conditions (pH 5.8 for male and female WT and KO mice). After base challenging mice with NaHCO3−, urine pH was greatly increased in WT mice (pH 7.8 in male WTs and pH 7.5 in female WTs), while urine pH increased to a lesser, but still significant amount, in KO mice (pH 6.3 in male KOs and 7.2 in female KOs). Interestingly, after the bicarbonate challenge urine pH was significantly lower in KO males versus WT males (pH 6.3 in male KOs versus pH 7.8 in male WTs). In females, urine pH was also lower in KO mice versus WT mice after the base challenge but did not reach statistical significance due to high variability between mice (pH 7.2 in female KOs versus pH 7.5 in female WTs). There was no significant difference in blood pH between Tldc2 KO and Tldc2 WT mice of either sex, with or without treatment, suggesting possible respiratory compensation. The inability of KO mice to alkalinize their urine points to a defect in bicarbonate-secreting B-ICs, in which Tldc2 is specifically expressed. To examine this, localization of the B1 subunit was visualized in B-ICs by immunofluorescence, and membrane accumulation was quantified by line intensity scanning using FIJI. Our preliminary results show that basolateral membrane accumulation of B1 was reduced in KO mice relative to WT mice. In summary, our data suggest that loss of Tldc2 negatively affects B-IC function most likely by impairing basolateral accumulation of the V-ATPase. We conclude that Tldc2 is part of a regulatory mechanism that participates in the accumulation of the V-ATPase holoenzyme at the basolateral surface of B-ICs. Whether this is due to changes in assembly or traffcking of the holoenzyme remains to be determined. NIH/NIDDK R01DK121848. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.