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 in vivo, 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+/HCO3- 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.
Project description:Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder caused by impaired distal acidification due to a failure of type A intercalated cells (A-ICs) in the collecting tubule. dRTA is characterized by persistent hyperchloremia, a normal plasma anion gap, and the inability to maximally lower urinary pH in the presence of systemic metabolic acidosis. Common clinical features of dRTA include vomiting, failure to thrive, polyuria, hypercalciuria, hypocitraturia, nephrocalcinosis, nephrolithiasis, growth delay, and rickets. Mutations in genes encoding three distinct transport proteins in A-ICs have been identified as causes of dRTA, including the B1/ATP6V1B1 and a4/ATP6V0A4 subunits of the vacuolar-type H+-ATPase (H+-ATPase) and the chloride-bicarbonate exchanger AE1/SLC4A1. Homozygous or compound heterozygous mutations in ATP6V1B1 and ATP6V0A4 lead to autosomal recessive (AR) dRTA. dRTA caused by SLC4A1 mutations can occur with either autosomal dominant or AR transmission. Red blood cell abnormalities have been associated with AR dRTA due to SLC4A1 mutations, including hereditary spherocytosis, Southeast Asia ovalocytosis, and others. Some patients with dRTA exhibit atypical clinical features, including transient and reversible proximal tubular dysfunction and hyperammonemia. Incomplete dRTA presents with inadequate urinary acidification, but without spontaneous metabolic acidosis and recurrent urinary stones. Heterozygous mutations in the AE1 or H+-ATPase genes have recently been reported in patients with incomplete dRTA. Early and sufficient doses of alkali treatment are needed for patients with dRTA. Normalized serum bicarbonate, urinary calcium excretion, urinary low-molecular-weight protein levels, and growth rate are good markers of adherence to and/or efficacy of treatment. The prognosis of dRTA is generally good in patients with appropriate treatment. However, recent studies showed an increased frequency of chronic kidney disease (CKD) in patients with dRTA during long-term follow-up. The precise pathogenic mechanisms of CKD in patients with dRTA are unknown.
Project description:Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the ?-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.
Project description:The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.
Project description:We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.
Project description:Autosomal dominant distal renal tubular acidosis (dRTA) has been associated with several mutations in the anion exchanger AE1 gene. The effect of an 11-amino-acid C-terminal dRTA truncation mutation (901 stop) on the expression of kidney AE1 (kAE1) and erythroid AE1 was examined in transiently transfected HEK-293 cells. Unlike the wild-type proteins, kAE1 901 stop and AE1 901 stop mutants exhibited impaired trafficking from the endoplasmic reticulum to the plasma membrane as determined by immunolocalization, cell-surface biotinylation, oligosaccharide processing and pulse-chase experiments. The 901 stop mutants were able to bind to an inhibitor affinity resin, suggesting that these mutant membrane proteins were not grossly misfolded. Co-expression of wild-type and mutant kAE1 or AE1 resulted in intracellular retention of the wild-type proteins in a pre-medial Golgi compartment. This dominant negative effect was due to hetero-oligomer formation of the mutant and wild-type proteins. Intracellular retention of kAE1 in the alpha-intercalated cells of the kidney would account for the impaired acid secretion into the urine characteristic of dRTA.
Project description:Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.
Project description:Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of a-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400-Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin-Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of a-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.
Project description:It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in ?-intercalated cells and the basolateral anion exchanger (AE1) in ?-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and ?-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6-20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base.
Project description:Acid-base transport in the renal collecting tubule is mediated by two canonical cell types: the ?-intercalated cell secretes HCO(3) by an apical Cl:HCO(3) named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the ?-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HCO(3) exchanger (kAE1). We previously suggested that the ?-cell converts to the ?-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical ?-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical ?-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical ?-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of ?1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of ?- to ?-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HCO(3) secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM.
Project description:Autosomal dominant distal renal tubular acidosis (dRTA) is a rare disorder caused by a mutation in the AE1 gene encoding the chloride-bicarbonate (Cl-/HCO3-) anion exchanger 1 (AE1). Most patients with this disorder present with clinical symptoms in adulthood and their phenotype is milder than that of those with autosomal recessive dRTA. In this report, we describe a Japanese family with autosomal dominant dRTA in which the mother and her daughter presented with severe symptoms caused by hypokalemia at 2 years of age. The heterozygous AE1 mutation G609R, which is a known causative mutation of dRTA, was identified in both patients. To our knowledge, this is the first report of a Japanese family with autosomal dominant type dRTA caused by an AE1 mutation. We, therefore, propose that alterations of AE1 should be considered causative of autosomal dominant dRTA even if typical symptoms appear during early childhood and the clinical features are severe.