A novel I551F variant of the Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity.

Homozygous mutations in SLC4A4, which encodes the electrogenic Na+/[Formula: see text] cotransporter (NBCe1), cause proximal renal tubular acidosis associated with extrarenal symptoms. Although 17` mutated sites in SLC4A4 have thus far been identified among patients with proximal renal tubular acidosis, the physiological significance of other nonsynonymous single-nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From the National Center for Biotechnology Information dbSNP database, we identified 13 SNVs that have not previously been characterized in the highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that the I551F variant was present predominantly in the cytoplasm in human embryonic kidney (HEK)-293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK-293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69% reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants in both nonpolarized HEK-293 cells and polarized Madin-Darby canine kidney cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39% of that of the wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence experiments in HEK-293 cells indicated that the I551F variant retained wild-type NBCe1-A in the cytoplasm. These data demonstrate that the I551F variant of NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention and suggest that the variant can have a dominant negative effect by forming complexes with wild-type NBCe1-A.NEW & NOTEWORTHY Electrogenic Na+/[Formula: see text] cotransporter 1-A (NBCe1-A) in the proximal tubule regulates the acid/base balance and fluid volume homeostasis. From the National Center for Biotechnology Information dbSNP database, we identified the I551F variant of NBCe1-A, which showed reduced glycosylation, cell surface expression, and transport activity. We also found that the I551F variant can exert a dominant negative effect on wild-type NBCe1-A, suggesting its physiological significance.

ENaCγ in Urinary Extracellular Vesicles as an Indicator of MR Signaling in PA.

BACKGROUND: Aldosterone and the MR (mineralocorticoid receptor) are important therapeutic targets for hypertension and cardiovascular diseases. However, biomarkers of tissue MR signaling are not fully established. Extracellular vesicles released from eukaryotic cells can provide information on tissue signaling. Using samples from patients with primary aldosteronism (PA), we explored the potential of urinary extracellular vesicles (uEVs) as a noninvasive indicator of MR signaling to guide treatment. METHODS: We analyzed proteins contained in PA uEVs by liquid chromatography tandem mass spectrometry. We narrowed down candidate biomarkers by referring to an existing database of urinary exosomes. The results were validated through Western blot analysis involving 63 patients with PA and 11 healthy volunteers. RESULTS: We identified a total of 1940 proteins in PA uEVs. Comparative analysis with the existing database narrowed down the pathways enriched in PA uEVs, which were related to diabetic complications, Rac1 signaling, and aldosterone-regulated sodium reabsorption. A closer look at the identified proteins revealed ENaCγ (epithelial Na+ channel γ) peptides near the proteolytic cleavage sites, and Western blot analysis confirmed the predominant presence of cleaved ENaCγ, a marker of aldosterone signaling in renal tubules. In PA uEVs, cleaved ENaCγ showed a 4.8-fold increase compared with healthy volunteers and was significantly correlated with the aldosterone-to-renin ratio, aldosterone levels, and fractional excretion of K+. Targeted treatment in PA reduced the abundance of cleaved ENaCγ, suggesting a causal role for MR in its induction. CONCLUSIONS: This study provides a list of proteins contained in PA uEVs and suggests that ENaCγ in uEVs is a promising biomarker for renal MR signaling.

Characterization of pendrin in urinary extracellular vesicles in a rat model of aldosterone excess and in human primary aldosteronism.

Pendrin is a Cl-/HCO3- exchanger selectively present in the intercalated cells of the kidney. Although experimental studies have demonstrated that pendrin regulates blood pressure downstream of the renin-angiotensin-aldosterone system, its role in human hypertension remains unclear. Here, we analyzed the quantitative changes in pendrin in urinary extracellular vesicles (uEVs) isolated from a total of 30 patients with primary aldosteronism (PA) and from a rat model of aldosterone excess. Western blot analysis revealed that pendrin is present in dimeric and monomeric forms in uEVs in humans and rats. In a rodent model that received continuous infusion of aldosterone with or without concomitant administration of the selective mineralocorticoid receptor (MR) antagonist esaxerenone, pendrin levels in uEVs, as well as those of epithelial Na+ channel (ENaC) and Na-Cl-cotransporter (NCC), were highly correlated with renal abundance. In patients with PA, pendrin levels in uEVs were reduced by 49% from baseline by adrenalectomy or pharmacological MR blockade. Correlation analysis revealed that the magnitude of pendrin reduction after treatment significantly correlated with the baseline aldosterone-renin ratio (ARR). Finally, a cross-sectional analysis of patients with PA confirmed a significant correlation between the ARR and pendrin levels in uEVs. These data are consistent with experimental studies showing the role of pendrin in aldosterone excess and suggest that pendrin abundance is attenuated by therapeutic interventions in human PA. Our study also indicates that pendrin analysis in uEVs, along with other proteins, can be useful to understand the pathophysiology of hypertensive disorders.