Acid Loading Unmasks Glucose Homeostatic Instability in Proximal-Tubule-Targeted Insulin/Insulin-Like-Growth-Factor-1 Receptor Dual Knockout Mice.

BACKGROUND/AIMS: Metabolic syndrome and type 2 diabetes are associated with some degree of acidosis. Acidosis has also been shown to upregulate renal gluconeogenesis. Whether impaired insulin or insulin-like-growth factor 1 receptor (IGF1) signaling alter this relationship is not known. Our aim was to determine the effects of deletion of insulin and IGF1 receptors (Insr and Igf1r) from renal proximal tubule (PT) on the gluconeogenic response to acidosis. METHODS: We developed a mouse model with PT-targeted dual knockout (KO) of the Insr/Igf1r by driving Cre-recombinase with the gamma-glutamyl transferase (gGT) promoter. Male and female mice were maintained as control or acidotic by treatment with NH4Cl in the drinking water for 1-week. RESULTS: Acidosis in both genotypes increased renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1-bisphosphatase (FBP1), but not glucose-6-phosphatase catalytic subunit (G6PC), which showed significantly lower expression in the KO regardless of treatment. Several differences between KO and WT suggested a protective role for insulin/IGF1 receptor signaling in maintaining relative euglycemia in the face of acidosis. First, the increase in FBP1 with acid was greater in the KO (significant interactive term). Secondly, proximal-tubule-associated FOXO1 and AKT overall protein levels were suppressed by acid loading in the KO, but not in the WT. Robust intact insulin signaling would be needed to reduce gluconeogenesis in PT. Third, phosphorylated FOXO1 (pS256) levels were markedly reduced by acid loading in the KO PT, but not in the WT. This reduction would support greater gluconeogenesis. Fourth, the sodium-glucose cotransporter (SGLT1) was increased by acid loading in the KO kidney, but not the WT. While this would not necessarily affect gluconeogenesis, it could result in increased circulatory glucose via renal reabsorption. Reduced susceptibility to glucose-homeostatic dysregulation in the WT could potentially relate to the sharp (over 50%) reduction in renal levels of sirtuin-1 (SIRT1), which deacetylates and regulates transcription of a number of genes. This reduction was absent in the KO. CONCLUSION: Insulin resistance of the kidney may increase whole-body glucose instability a major risk factor for morbidity in diabetes. High dietary acid loads provide a dilemma for the kidney, as ammoniagenesis liberates α-ketoglutarate, which is a substrate for gluconeogenesis. We demonstrate an important role for insulin and/or IGF1 receptor signaling in the PT to facilitate this process and reduce excursions in blood glucose. Thus, medications and lifestyle changes that improve renal insulin sensitivity may also provide added benefit in type 2 diabetes especially when coupled with metabolic acidosis.

Changes in V-ATPase subunits of human urinary exosomes reflect the renal response to acute acid/alkali loading and the defects in distal renal tubular acidosis.

In the kidney, final urinary acidification is achieved by V-ATPases expressed in type A intercalated cells. The B1 subunit of the V-ATPase is required for maximal urinary acidification, while the role of the homologous B2 subunit is less clear. Here we examined the effect of acute acid/alkali loading in humans on B1 and B2 subunit abundance in urinary exosomes in normal individuals and of acid loading in patients with distal renal tubular acidosis (dRTA). Specificities of B1 and B2 subunit antibodies were verified by yeast heterologously expressing human B1 and B2 subunits, and murine wild-type and B1-deleted kidney lysates. Acute ammonium chloride loading elicited systemic acidemia, a drop in urinary pH, and increased urinary ammonium excretion. Nadir urinary pH was achieved at four to five hours, and exosomal B1 abundance was significantly increased at two through six hours after ammonium chloride loading. After acute equimolar sodium bicarbonate loading, blood and urinary pH rose rapidly, with a concomitant reduction of exosomal B1 abundance within two hours, which remained lower throughout the test. In contrast, no change in exosomal B2 abundance was found following acid or alkali loading. In patients with inherited or acquired distal RTA, the urinary B1 subunit was extremely low or undetectable and did not respond to acid loading in urine, whereas no change in B2 subunit was found. Thus, both B1 and B2 subunits of the V-ATPase are detectable in human urinary exosomes, and acid and alkali loading or distal RTA cause changes in the B1 but not B2 subunit abundance in urinary exosomes.

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

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.

Regulation of luminal acidification by the V-ATPase.

Specialized cells in the body express high levels of V-ATPase in their plasma membrane and respond to hormonal and nonhormonal cues to regulate extracellular acidification. Mutations in or loss of some V-ATPase subunits cause several disorders, including renal distal tubular acidosis and male infertility. This review focuses on the regulation of V-ATPase-dependent luminal acidification in renal intercalated cells and epididymal clear cells, which are key players in these physiological processes.

Novel ATP6V0A4 mutation described in a Tunisian patient with distal renal tubular acidosis.

UNLABELLED: Few data regarding molecular diagnosis of primary distal renal tubular acidosis (DRTA) in Tunisian population are available. CASE REPORT: 25-day-old male patient from consanguineous parents of Tunisian origin diagnosed with DRTA and without hearing impairment observed later in life. ATP6V0A4 gene sequencing demonstrated a novel homozygous G deletion in exon 13 (c.1221delG, p.Met408CysfsX10), leading to a premature stop codon. CONCLUSION: A novel ATP6V0A4 gene mutation confirmed autosomal recessive DRTA with normal hearing in the patient. Molecular analysis may help to rapidly diagnose autosomal recessive DRTA in Tunisian population.

Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness.

H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis. Patients with ATP6B1 mutations also have sensorineural hearing loss; consistent with this finding, we demonstrate expression of ATP6B1 in cochlea and endolymphatic sac. Our data, together with the known requirement for active proton secretion to maintain proper endolymph pH, implicate ATP6B1 in endolymph pH homeostasis and in normal auditory function. ATP6B1 is the first member of the H+-ATPase gene family in which mutations are shown to cause human disease.

Novel carbonic anhydrase autoantibodies and renal manifestations in patients with primary Sjogren's syndrome.

OBJECTIVE: Anti-carbonic anhydrase II (anti-CA II) antibodies have been related to renal manifestations of primary SS (pSS), and animal studies have even suggested a pathogenic role for them. However, not all pSS patients with renal tubular acidosis (RTA) present with anti-CA II antibodies. Recently, several novel CA isoenzymes have been recognized and we aimed to investigate whether antibodies to these are associated with renal manifestations of pSS. METHODS: We examined anti-CA II antibodies as well as anti-CA I, VI, VII and XIII antibodies by ELISA tests in 74 pSS patients on whom detailed nephrological examinations had been performed and, as controls, in 56 subjects with sicca symptoms, but no pSS. RESULTS: The levels of anti-CA I, II, VI and VII antibodies were significantly higher in patients with pSS compared with subjects with sicca symptoms but no pSS. None of the anti-CA antibodies was associated with the presence of complete or incomplete RTA or proteinuria or urinary α1m excretion in patients with pSS. However, levels of anti-CA II, VI and XIII antibodies correlated significantly with urinary pH, and inversely with serum sodium concentrations. The degree of 24-h urinary protein excretion correlated weakly with levels of anti-CA VII antibodies. CONCLUSION: Not only antibodies to CA II, but also anti-CA VI and XIII antibodies seem to be associated with renal acidification capacity in patients with pSS.

Identification of a novel mutation in an Indian patient with CAII deficiency syndrome.

Carbonic anhydrase II (CAII) deficiency syndrome characterized by osteopetrosis (OP), renal tubular acidosis (RTA), and cerebral calcifications is caused by mutations in the carbonic anhydrase 2 (CA2) gene. Severity of this disorder varies depending on the nature of the mutation and its effect on the protein. We present here, the clinical and radiographic details along with, results of mutational analysis of the CA2 gene in an individual clinically diagnosed with renal tubular acidosis, osteopetrosis and mental retardation and his family members to establish genotype-phenotype correlation. A novel homozygous deletion mutation c.251delT was seen in the patient resulting in a frameshift and a premature stop codon at amino acid position 90 generating a truncated protein leading to a complete loss of function and a consequential deficiency of the enzyme making this a pathogenic mutation. Confirmation of clinical diagnosis by molecular methods is essential as the clinical features of the CAII deficiency syndrome are similar to other forms of OP but the treatment modalities are different. Genetic confirmation of the diagnosis at an early age leads to the timely institution of therapy improving the growth potential, reduces other complications like fractures, and aids in providing prenatal testing and genetic counseling to the parents planning a pregnancy.

The calcineurin inhibitor FK506 (tacrolimus) is associated with transient metabolic acidosis and altered expression of renal acid-base transport proteins.

Calcineurin inhibitors like FK506 (tacrolimus) are routinely used for immunosuppression following transplantation. Its use is limited by many side effects, including renal tubular acidosis (RTA), mainly of the distal type. In this study, rats were treated with FK506 and at baseline (after 9 days) systemic acid-base status was similar to that in control animals. However, FK506-treated rats given NH(4)Cl in the drinking water for 2 days developed a more severe metabolic acidosis than control animals. Urine pH was more alkaline, but net acid excretion was normal. After 7 days of acid load, all differences related to acid-base homeostasis were equalized in both groups. Protein abundance of type IIa Na-P(i) cotransporter, type 3 Na(+)/H(+) exchanger, and electrogenic Na(+)-bicarbonate cotransporter, and both a4 and B2 subunits of the vacuolar H(+)-ATPase were reduced under baseline conditions, while induction of metabolic acidosis enhanced protein abundance of these transporters in FK506-treated animals. In parallel, protein expression of AE1 was reduced at baseline and increased together with pendrin during NH(4)Cl loading in FK506 rats. Protein abundance of the Na(+)-bicarbonate cotransporter NBCn1 was reduced under baseline conditions but remained downregulated during metabolic acidosis. Morphological analysis revealed an increase in the relative number of non-type A intercalated cells in the connecting tubule and cortical collecting duct at the expense of principal cells. Additionally, subcellular distribution of the a4 subunit of the vacuolar H(+)-ATPase was affected by FK506 with less luminal localization in the connecting tubule and outer medullary collecting duct. These data suggest that FK506 impacts on several major acid-base transport proteins in the kidney, and its use is associated with transient metabolic acidosis and altered expression of key renal acid-base transport proteins.

Medullary sponge kidney associated with primary distal renal tubular acidosis and mutations of the H+-ATPase genes.

BACKGROUND: Medullary sponge kidney (MSK) is a rare congenital disease characterized by diffuse ectasia or dilation of precalyceal collecting tubules. Although its pathogenesis is unknown, the association with various congenital diseases suggests that it could be a developmental disorder. In addition to the typical clinical features of nephrocalcinosis and urolithiasis, patients with MSK show tubular function defects of acidification and concentration. These are considered to be secondary to morphological changes of collecting tubules. Primary distal renal tubular acidosis (dRTA) is a rare genetic disease caused by mutations in different genes involved in the secretion of H(+) ions in the intercalated cells of the collecting duct required for final excretion of fixed acids. Both autosomal dominant and autosomal recessive forms have been described, the latter is also associated with sensorineural hearing loss. METHODS AND RESULTS: We report two patients presenting with dRTA, late sensorineural hearing loss and MSK, in whom molecular investigations demonstrated the presence of mutations of the H(+) proton pump ATP6V1B1 and ATP6V0A4 genes. CONCLUSIONS: These observations, including a previous description of a similar case in the literature, indicate that MSK could be a consequence of the proton pump defect, thus can potentially provide new insights into the pathogenesis of MSK.

Carbonic anhydrase II deficiency a novel mutation.

Carbonic anhydrase II (CA II) deficiency is an extremely rare autosomal recessive disorder, characterised by a triad of osteopetrosis, renal tubular acidosis and cerebral calcifications. A 12 year old boy with classical features of CA II deficiency is reported who was found to be homozygous for the mutation in CA II gene and parents were heterozygous for the same mutation .To the best of our knowledge this is the first case report of mutation proven CA II deficiency from India.

Presence of serum autoantibodies to vacuolar H+ -ATPase in patients with renal tubular acidosis.

OBJECTIVE: Concomitant presence of renal tubular acidosis (RTA) and autoimmune diseases is indicative of the potential role of immune factors in the pathogenesis of RTA. Our study aimed to detect the serum antibodies to renal tubular epithelial cells in RTA patients. METHODS: We enrolled 11 RTA patients, eight primary Sjögren's syndrome (pSS) patients and eight healthy controls (HC). Serum biochemical test, urinary regular test, and 24 hours urinary protein quantification were measured using a fully automated analyzer. Immunofluorescence assay was performed to detect the antibodies to subunit B1 and subunit B2 of v-H+-ATPases (adenosine triphosphatases) in the serum of the participants. RESULTS: Clinically, RTA patients showed hyperchloremia, acidosis and paradoxical alkaline urine. We detected the serum antibodies to renal tubular epithelial cells and there were 6/11 positive in RTA patients, much higher than that in the pSS group (0/8) and the HC group (0/8). Subsequently, we demonstrated that in normal renal tissue, the B1 subunit of v-H+-ATPase specifically expressed in intercalated cells, while the B2 subunit continuously expressed along the lumen of renal tubular epithelial cells. Moreover, the antibody to subunit B1/B2 of v-H+-ATPase was positive in the sera of 6 RTA patients (54%), while it was negative in both the pSS and HC group. CONCLUSIONS: We detected the presence of serum autoantibodies to subunit B1 and subunit B2 of v-H+ -ATPase in RTA patients. Our findings may provide novel mechanism insights into the pathogenesis of RTA and the potential diagnostic utility of antibodies to v-H+ -ATPase in the classification of RTA.

Molecular cloning and characterization of a novel form of the human vacuolar H+-ATPase e-subunit: an essential proton pump component.

Several of the 13 subunits comprising mammalian H(+)-ATPases have multiple alternative forms, encoded by separate genes and with differing tissue expression patterns. These may play an important role in the intracellular localization and activity of H(+)-ATPases. Here we report the cloning of a previously uncharacterized human gene, ATP6V0E2, encoding a novel H(+)-ATPase e-subunit designated e2. We demonstrate that in contrast to the ubiquitously expressed gene encoding the e1 subunit (previously called e), this novel gene is expressed in a more restricted tissue distribution, particularly kidney and brain. We show by complementation studies in a yeast strain deficient for the ortholog of this subunit, that either form of the e-subunit is essential for proper proton pump function. The identification of this novel form of the e-subunit lends further support to the hypothesis that subunit differences may play a key role in the structure, site and function of H(+)-ATPases within the cell.

Enzymatically inactive red cell carbonic anhydrase B in a family with renal tubular acidosis.

An inactive mutant form of red cell carbonic anhydrase B is described in three members of a large kindred who manifest infantile renal tubular acidosis and nerve deafness. A combination of enzymatic and immunologic investigations permitted its detection, despite the fact that both antigenic and electrophoretic properties of the mutant were identical to those of the normal form.

Inactive form of erythrocyte carbonic anhydrase B in patients with primary renal tubular acidosis.

Evidence was found for an inactive form of carbonic anhydrase type B in the erythrocytes of two children with primary renal tubular acidosis. The addition of zinc chloride to hemolysates from these patients resulted in a marked increase in the activity of this enzyme. No such effect was noted with hemolysates of control subjects. No significant differences were observed in the zinc levels of hemolysates of these patients and of normal individuals. However, the level of zinc in the carbonic anhydrase B isolated from one of these patients was low, suggesting a modified form of the enzyme. The restoration of activity upon the addition of zinc was reversed by ethylenediamine tetraacetate, but no such effects were noted for the carbonic anhydrase B of normal individuals. Thus the abnormal carbonic anhydrase B has decreased zinc binding. The ultraviolet difference spectrum of the carbonic anhydrase B of normal individuals and that of a patient showed a peak at 305 nm which decreased upon the addition of zinc. The abnormal form of carbonic anhydrase B was not distinguishable from that of normal individuals by either immunological or electrophoretic criteria.

A phenocopy of CAII deficiency: a novel genetic explanation for inherited infantile osteopetrosis with distal renal tubular acidosis.

The rare bone thickening disease osteopetrosis occurs in various forms, one of which is accompanied by renal tubular acidosis (RTA), and is known as Guibaud-Vainsel syndrome or marble brain disease. Clinical manifestations of this autosomal recessive syndrome comprise increased bone density, growth failure, intracerebral calcification, facial dysmorphism, mental retardation, and conductive hearing impairment. The most common cause is carbonic anhydrase II (CAII) deficiency. Several different loss of function mutations in CA2, the gene encoding CAII, have been described. To date, there have been no exceptions to the finding of CAII deficiency in patients with coexistent osteopetrosis and RTA. Most often, the RTA is of mixed proximal and distal type, but kindreds are reported in which either distal or proximal RTA predominates. We report the molecular genetic investigation of two consanguineous kindreds where osteopetrosis and distal RTA (dRTA) were both manifest. One kindred harbours a novel homozygous frameshift alteration in CA2. In the other, CAII levels were normal despite a similar clinical picture, and we excluded defects in CA2. In this kindred, two separate recessive disorders are penetrant, each affecting a different, tissue specific subunit of the vacuolar proton pump (H(+)-ATPase), providing a highly unusual, novel genetic explanation for the coexistence of osteopetrosis and dRTA. The osteopetrosis is the result of a homozygous deletion in TCIRG1, which encodes an osteoclast specific isoform of subunit a of the H(+)-ATPase, while the dRTA is associated with a homozygous mutation in ATP6V1B1, encoding the kidney specific B1 subunit of H(+)-ATPase. This kindred is exceptional firstly because the coinheritance of two rare recessive disorders has created a phenocopy of CAII deficiency, and secondly because these disorders affect two different subunits of the H(+)-ATPase that have opposite effects on bone density, but which have only recently been determined to possess tissue specific isoforms.

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss.

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

ATP6B1 gene mutations associated with distal renal tubular acidosis and deafness in a child.

A large proportion of autosomal recessive distal renal tubular acidosis (RTA) is associated with mutations in the ATP6B1 gene encoding the B1 subunit of H+-ATPase. H+-ATPase is one of the key membrane transporters for net acid excretion in the alpha-intercalated cells of the medullary collecting duct. Sensorineural hearing loss frequently accompanies this type of distal RTA. Mutational analysis of the ATP6B1 gene in a 9-year-old Korean boy with distal RTA and sensorineural hearing loss found 2 heterozygous missense point mutations. Although a single case report, this is the second report documenting ATP6B1 mutations in recessive distal RTA with sensorineural hearing loss after the original report by Karet et al and confirms the novelty of these mutations.

Unusual renal features of Lowe syndrome in a mildly affected boy.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by congenital cataracts, mental retardation, and renal tubular dysfunction. The gene responsible for OCRL was identified by positional cloning and encodes a lipid phosphatase, phosphatidylinositol 4,5, bisphosphate [PtdIns(4,5)P2]5-phosphatase, which localizes to the Golgi apparatus and is suspected to play a role in Golgi vesicular transport [Suchy et al., 1995]. In addition to the ocular and renal manifestations, most boys with OCRL have cognitive problems and maladaptive behaviors including tantrums and stereotypies. We report a boy with a history of congenital cataracts and mild developmental delay who was also found to have hematuria with proteinuria but minimal signs of renal tubular dysfunction. Subsequent renal biopsy was compatible with a diagnosis of a noncomplement fixating chronic glomerulonephritis. Despite the atypical renal findings, skin fibroblast analysis for PtdIns (4,5)P2 5-phosphatase was performed, and enzyme activity was low, consistent with the diagnosis of OCRL. Western blot analysis from cell lysates showed the ocrl protein was decreased in size and amount. Our report shows atypical renal features of OCRL in a mildly affected boy. The possibility of OCRL should be considered in boys with cataracts and glomerular disease, even in the absence of renal tubular defects and frank mental retardation usually associated with the syndrome. Am. J. Med. Genet. 95:461-466, 2000. Published Wiley-Liss, Inc.

Transport enzymes and renal tubular acidosis.

By analogy to the findings with other transport disorders such as Bartter's or Liddle's syndrome, it might be expected that the various forms of renal tubular acidosis (RTA) could result from defects in H-ATPase or H-K-ATPase. However, the available data do not yet support such a simple explanation. With regard to distal RTA, inhibition of H-K-ATPase with inhibitors such as vanadate blocks the increase in enzyme activity observed with potassium depletion, but does not produce distal RTA. H-K-ATPase does not increase with metabolic acidosis, and inhibition of its activity does not decrease ammonium or total acid excretion unless K depletion is also present. Maleic acid administration produces proximal RTA along with other proximal tubular dysfunction in experimental animals. However, it acts by reducing Na,K-ATPase activity rather than by affecting specific H+ ion transporters. This is pertinent to the findings that Na,K-ATPase activity is reduced in obstructive uropathy. Although the acidification defect in this disorder has been ascribed to a defect in H-ATPase, Na-K-ATPase function is also impaired. Thus, the role of isolated defects in H+ transporters in the development of clinical acidification disorders remains to be elucidated.