Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model

Abstract

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis , we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na/HCO cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H secretion and possibly lysosomal acidification.

Genotype–Phenotype Analysis in Pediatric Patients with Distal Renal Tubular Acidosis

“…One study [19] showed that retention of heterologous AE1 p.R589H in the endoplasmic reticulum of polarized Madin-Darby canine kidney cells, along with co-retention of co-expressed wild-type AE1, suggesting dominant negative trafficking defects. However, a recent study [20] with an Ae1 p.R607H knock-in mouse model (which corresponds to human AE1 p.R589H) showed that both heterozygous and homozygous knock-in mice exhibited incomplete dRTA (less severe in heterozygotes than in homozygotes). Interestingly, the targeting of Ae1 p.R607H to the basolateral plasma membrane of the α-intercalated cells was normal, as was transport activity in vivo .…”

New Findings on the Pathogenesis of Distal Renal Tubular Acidosis

“…Hence, NH 4 + becomes the molecular readout of efficient distal acid secretion. This mechanism is maximized during acidosis, where not only the abundance of vH + ATPase, but also the number of A-IC is increased in the inner stripe of the outer medulla [8] . This is the rationale for using NH 4 Cl to evocate a maximal urinary acidification.…”

“…To recapitulate a mouse model of AE1-dependent dRTA, we recently generated a transgenic mouse expressing one of the most common mutations of the AE1 gene, namely R589H AE1, corresponding to the R607H mutation in mouse AE1 [8] . As humans, these mice do not present RBC abnormalities, but they exhibit incomplete unmasked dRTA when they are challenged with an NH 4 Cl load.…”

“…AE1 is expressed at the basolateral membrane of A-type intercalated cells (A-IC) in the distal nephron, where it is required to transport bicarbonate generated during acid secretion from the cytosol to the renal interstitium [7] . Loss of function mutations of AE1 are not always associated with both renal and hematological phenotype simultaneously, even though band 3 is the most abundant protein of the membrane of RBC [8] , revealing a different functional setting in these two cell types. Indeed, AE1 dysfunction leading to hereditary ovalocytosis or spherocytosis is commonly associated with the autosomal recessive form of dRTA, but not with autosomal dominant dRTA [6] .…”

Renal tubular acidosis in pediatrics: A review article

“…It is associated with gene mutations in kidney anion exchanger 1 (kAE1) 5,6 caused by decrease in anion exchange thus hinder trafficking and regulation of V-type ATPase, venturing acidification of lysosomes. 7 In a case report, down syndrome was associated hypothyroidism and renal acidosis, with antithyroid peroxidase antibodies positive and with treatment of hypothyroidism, symptoms of acidosis resolved. 8 A case series of periodic paralysis, otosclerosis and fluorosis, result from a mutation in anion exchanger 1 gene (AE1) codes band 3.…”