常染色体隐性遗传性远端肾小管酸中毒患儿ATP6V0A4和ATP6V1B1基因突变分析

目的 分析常染色体隐性遗传性远端肾小管酸中毒(rdRTA)患儿ATP6V0A4和ATP6V1B1基因的突变,进行基因型和表型的相关性研究.方法 PCR扩增基因组DNA,直接测序分析来自3个家系3例患儿的ATP6V0A4和ATP6V1B1基因的突变位点,选取不相关的100例健康人作为对照.结果 1例患儿携带ATP6V0A4基因的1个新的纯合无义突变(p.R194X);1例患儿携带ATP6V1B1基因1个新的杂合无义突变(p.R114X)和1个已经报道过的杂合突变p.I386fsX441;第3例患儿未发现以上2个基因的突变.结论 对中国rdRTA患者基因突变分析有利于了解该类疾病的基因型和表型的相关性,增强临床医生对该类疾病的认识和治疗.     

The effect of kidney transplantation on distal tubular vacuolar H+-ATPase

BACKGROUND: The presence of vacuolar (v)H+-ATPase in distal tubule alpha-intercalated cells is essential for hydrogen excretion and maintenance of acid-base homeostasis. Loss of vH+-ATPase after kidney transplantation could cause posttransplant distal renal tubular acidosis (dRTA). METHOD: Immunostaining of the kidney specific vH+-ATPase of cortical collecting duct cells (CCT) was performed in 37 kidney biopsies taken immediately prior to transplantation and after engraftment (median [range]: 10 [1-181] months). Apical or intracytoplasmatic staining intensity was classified as grade 0 (absent), grade 1 (weak), or grade 2 (strong), and positive cells expressed as percentage of all CCT cells. In addition, kidney biopsies were scored for damage by the Banff schema. Serum and urinary pH, anion gap, and serum potassium were obtained for the diagnoses of dRTA. RESULTS: Fourteen transplant recipients had dRTA type I, 5 had rate-limited RTA, six had type IV dRTA, and 12 had no RTA. In pretransplant biopsies, 40% [3-77%] of CCT cells were positive for vH+-ATPase but only 17% [0-39%] after transplantation (P<0.0001). The loss of vH+-ATPase expression was similar in patients with dRTA type I (-21%), type IV (-25%), rate limited RTA (-21%), or no RTA (-29%). The decrease affected predominantly the apical proton pump expression. The individual loss of vH+-ATPase expression was not related to the time elapsed since transplantation, immunosuppressive drugs, acute transplant rejection, or tubulointerstitial changes. CONCLUSION: Kidney transplantation leads to a general decrease of distal tubular vH+-ATPase expression. Loss of proton pump activity occurs unrelated to immunosuppressive therapy or transplant related histologic changes.     

Vacuolar H+ -ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells

Point mutations in the B1 subunit of vacuolar H+ -ATPase are associated with impaired ability of the distal nephron to secrete acid (distal renal tubular acidosis). For testing of the hypothesis that these mutations interfere with assembly and trafficking of the H+ -ATPase, constructs that mimic seven known point mutations in inherited distal renal tubular acidosis (M) or wild-type (WT) B1 were transfected into a rat inner medullary collecting duct cell line to express green fluorescence protein (GFP)-B1WT or GFP-B1M fusion proteins. In co-immunoprecipitation studies, GFP-B1WT formed complexes with other H+ -ATPase subunits (c, H, and E), whereas GFP-B1M did not. Proteins that were immunoprecipitated with anti-GFP antibody from GFP-B1WT cells had ATPase activity, whereas proteins from GFP-B1M cells did not. Proton pump-mediated intracellular pH transport was inhibited in GFP-B1M-transfected cells but not in GFP-B1WT cells. GFP-B1WT and GFP-B1M are present in the apical membrane and increased with cellular acidification. In GFP-B1WT cells, the apical membrane fraction of GFP-B1, endogenous B1, and the 31-kD subunits of the H+ -ATPase increased with cell acidification. In GFP-B1M cells, the endogenous B1 and 31-kD subunits did not increase with acidification. B1 point mutations prevent normal assembly of the H+ -ATPase and also may act as an inhibitor of H+ -ATPase function by competing with endogenous intact H+ -ATPase for trafficking in inner medullary collecting duct cells.     

Genetic investigation of autosomal recessive distal renal tubular acidosis: evidence for early sensorineural hearing loss associated with mutations in the ATP6V0A4 gene

Mutations in the ATP6V1B1 and ATP6V0A4 genes, encoding subunits B1 and 4 of apical H(+) ATPase, cause recessive forms of distal renal tubular acidosis (dRTA). ATP6V1B mutations have been associated with early sensorineural hearing loss (SNHL), whereas ATP6V0A4 mutations are classically associated with either late-onset SNHL or normal hearing. The phenotype and genotype of 39 new kindreds with recessive dRTA, 18 of whom were consanguineous, were assessed. Novel and known loss-of-function mutations were identified in 31 kindreds. Fourteen new and five recurrent mutations of the ATP6V0A4 gene were identified in 21 families. For the ATP6V1B1 gene, two new and two previously described mutations were identified in 10 families. Surprisingly, seven probands with ATP6V0A4 gene mutations developed severe early SNHL between the ages of 2 mo and 10 yr. No mutation was detected in eight families. These data extend the spectrum of disease-causing mutations and provide evidence for genetic heterogeneity in SNHL. The data also demonstrate that mutations in either of these genes may cause early deafness, and they highlight the importance of genetic screening for recessive forms of dRTA independent of hearing status.     

The urine-blood PCO gradient as a diagnostic index of H(+)-ATPase defect distal renal tubular acidosis

BACKGROUND: Urine pH during acidemia and urine PCO2 upon alkalization both may be useful to indicate H+ secretion from collecting ducts. The urine anion gap has been used to detect urinary NH4+ for differential diagnosis of hyperchloremic metabolic acidosis. We have previously demonstrated that the lack of normal H(+)-ATPase may underlie secretory defect distal renal tubular acidosis (dRTA). In this study we evaluated the diagnostic value of the urine-blood (U-B) PCO2 in H(+)-ATPase defect dRTA, and compared it with that of urine pH and urine anion gap during acidemia. METHODS: In H(+)-ATPase defect dRTA, the diagnostic values of three urinary parameters were evaluated: (1) urine pH measured after acid (NH4Cl) loading; (2) urine-to-blood carbon dioxide tension gradient (U-B PCO2) during alkali (NaHCO3) loading; and (3) urine anion gap during acidemia. Seventeen patients were diagnosed as having H(+)-ATPase defect dRTA based on reduced urinary NH4+ and an absolute decrease in H(+)-ATPase immunostaining in intercalated cells on renal biopsy. Eight patients with non-dRTA renal disease served as control patients. RESULTS: Upon NaHCO3 loading, U-B PCO2 was < or =30 mm Hg in all 17 dRTA patients and >30 mm Hg in all 8 control patients. With NH4Cl loading, urine pH was >5.4 in 15 of 17 dRTA patients and < or =5.4 in 7 of 8 control patients, and the urine anion gap was >5 mmol/L in 13 of 17 dRTA patients and< or =5 mmol/L in 6 of 8 control patients. Therefore, the sensitivity and specificity of U-B PCO2 < or =30 mm Hg during NaHCO3 loading were both 100%, whereas those of urine pH >5.4 or urine anion gap >5 mmol/L during NH4Cl loading were below 90%. In control patients, the U-B PCO2 was found to be well correlated with the urinary NH4+ (r= 0.79, P < 0.05). CONCLUSION: The U-B PCO2 during NaHCO3 loading is an excellent diagnostic index of H(+)-ATPase defect dRTA.     

Confirmation of the ATP6B1 gene as responsible for distal renal tubular acidosis

Primary distal renal tubular acidosis (dRTA) type I is a hereditary renal tubular disorder, which is characterized by impaired renal acid secretion resulting in metabolic acidosis. Clinical symptoms are nephrocalcinosis, nephrolithiasis, osteomalacia, and growth retardation. Biochemical alterations consist of hyperchloremic metabolic acidosis, hypokalemia with muscle weakness, hypercalciuria, and inappropriately raised urinary pH. Autosomal dominant and rare forms of recessive dRTA are known to be caused by mutations in the gene for the anion exchanger AE1. In order to identify a gene responsible for recessive dRTA, we performed a total genome scan with 303 polymorphic microsatellite markers in six consanguineous families with recessive dRTA from Turkey. In four of these there was an association with sensorineural deafness. The total genome scan yielded regions of homozygosity by descent in all six families on chromosomes 1, 2, and 10 as positional candidate region. In one of these regions the gene ATP6B1for the ss1 subunit of the vacuolar H(+)-ATPase is localized, which has recently been identified as causative for recessive dRTA with sensorineural deafness. Therefore, we conducted mutational analysis in 15 families and identified potential loss-of-function mutations in ATP6B1in 8. We thus confirmed that defects in this gene are responsible for recessive dRTA with sensorineural deafness.     

A distal (type 1) renal tubular acidosis case that mimic coronary ischemia

Abstract

Introduction: Type 1 (distal) renal tubular acidosis (RTA) is a rare clinical condition characterized with defect of urinary acidification in distal tubulus. If diagnosis delays, RTA may cause metabolic and clinical complications and comorbidities. We describe here a type 1 distal RTA case with symptoms mimicking coronary ischemia. Case report: A 46-year-old woman admitted with complaints of chest pain, palpitation, walking disability, fatigue and nausea. On physical examination muscles were weaken 3/5 in four extremities. An electrocardiogram revealed supraventricular tachycardia and ST depression on precordial V2-6 derivations. An acute coronary syndrome diagnosis made based on anginal symptoms, supraventricular tachycardia, ST depression on V2-6 derivations and elevated cardiac enzymes. Urgent coronary angiography was normal except a 30% narrowing in LAD. She had recurrent nephrolithiasis and had operated because of hydronephrosis. She had two episodes of fatigue and walking disability previously. Hyperchloremic metabolic acidosis with normal anion gap determined in blood gas analyze. Patient diagnosed with type I RTA with the signs and symptoms of recurrent nephrolithiasis, fatigue, severe hypokalemia (1.8?mmol/L), hyperchloremic metabolic acidosis with normal anionic gap, alkaline urine (pH 8) and positive urinary anionic gap (13.7?mmol/L). Sodium bicarbonate infusion and potassium replacement therapy administered. Clinical and laboratory signs of the patient dissolved during treatment. Conclusion: Type 1 RTA should be considered in acidotic patients admitted with hypokalemia and coronary symptoms. Urinary and blood gas analyses should be done beside cardiac tests initially. Therefore, a precise diagnosis may be possible without the possible complications of unnecessary coronary interventions.     

Absence of H(+)-ATPase in cortical collecting tubules of a patient with Sjogren's syndrome and distal renal tubular acidosis.

Distal urinary acidification abnormalities may arise from transepithelial voltage defects, permeability defects, or proton-secretory defects, but tests to determine the cellular mechanisms underlying secretory abnormalities have not previously been reported. A patient with Sjogren's syndrome and distal renal tubular acidosis due to a secretory defect is described, whose kidney biopsy was examined by fluorescent immunocytochemistry with an antibody to the M(r) 31,000 subunit of the mammalian kidney vacuolar H(+)-ATPase and was compared with normal human kidney. Staining with the anti-H(+)-ATPase antibody in normal human kidney was detected in the brush border microvilli and subvillar invaginations of the proximal tubule and in intercalated cells in the collecting duct. A biopsy sample from the patient was devoid of any anti-H+-ATPase staining in the intercalated cells. Staining was also absent from the proximal tubule brush border microvilli but was present in the subvillar invaginations. Although autoantibodies to normal human kidney membrane proteins were detected in the serum by immunoblot analysis, no immunocytochemical evidence for anti-intercalated cell autoantibodies was observed in the patient's serum. This report demonstrates that the basis for the proton secretory defect in some patients with distal renal tubular acidosis is likely the absence of H(+)-ATPase in the intercalated cells. It also illustrates the potential diagnostic utility of anti-H(+)-ATPase antibodies in the classification of distal renal tubular acidoses.     

ATP6V1B1 mutations in distal renal tubular acidosis and sensorineural hearing loss: clinical and genetic spectrum of five families

Abstract

Distal renal tubular acidosis (DRTA) is characterized by tubular defects in urinary acidification and hyperchloremic metabolic acidosis, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis and nephrolithiasis. Mutations in ATP6V1B1 cause DRTA associated with sensorineural hearing loss. The objective of this multicenter study is to screen DRTA patients with sensorineural hearing loss for ATP6V1B1 gene mutations and present genotype/phenotype correlation. Clinical data in five unrelated consanguineous families with DRTA and hearing loss were obtained in Turkey. For mutation screening, all coding exons of ATP6V1B1 were PCR-amplified and sequenced from genomic DNA. In our cohort of five families, there were four different homozygous ATP6V1B1 mutations in affected individuals: c.91C>T (p.R31X), c.232G>A (p.G78R), c.497delC (p.T166RfsX9) and c.1155dupC (p.I386HfsX56). Our study shows that rare and family-specific variants in ATP6V1B1 are responsible for DRTA and sensorineural hearing loss syndrome in Turkey. While firm genotype–phenotype correlations are not available, detailed clinical and molecular analyses provide data to be used in genetic counseling.     

常染色体显性遗传性远端肾小管酸中毒一家系SLC4A1基因突变分析

目的 研究肾小管酸中毒（RTA）一家系的遗传方式和临床特征及分析致病基因SLC4A1突变情况。 方法 通过家系调查和检测肾小管酸化功能,研究分析遗传特点和肾小管缺陷的定位。通过直接测序法分析SLC4A1基因。100例健康正常人作为对照。 结果 该家系共有6例患者确诊为远端肾小管酸中毒（dRTA）,6例均有严重的临床表型（发育障碍、肾脏钙质沉着和肾功能不全）;遗传特点符合常染色体显性遗传。SLC4A1基因分析发现位于20号外显子的一个新突变位点（c.2713dupG,band 3 Qingdao）,该突变导致第905位密码子起始的移码突变（p.Asp905Glyfs15）。 结论 常染色体显性遗传性dRTA可表现严重的临床表型。1个新的SLC4A1突变位点被确定与显性遗传性dRTA有关。这是国内首次对遗传性RTA的报道。     

特发性高草酸尿症并不完全性远端肾小管酸中毒1例报告及文献复习

目的:探讨特发性高草酸尿症并不完全性远端肾小管酸中毒的临床特点.方法:回顾性分析1例特发性高草酸尿症并不完全性远端肾小管酸中毒患者的临床资料,并结合文献复习予以讨论.结果:连续两次24 h尿草酸检测结果分别为89.90 mg和93.83 mg,提示为高草酸尿症,结合临床特点诊断为特发性高草酸尿症.血气分析和血电解质未见异常,肾小管酸化功能检测结果:可滴定酸(TA)为0 mmol/L,NH4 为12.93 mmol/L,净酸排泄量(NAE)为12.93 mmol/L,氯化铵负荷试验阳性,提示为不完全性远端肾小管酸中毒.结论:特发性高草酸尿症与远端肾小管酸中毒均为尿路结石形成和复发的原因之一,对尿路结石患者应积极寻找结石成因,以期进行合理的治疗和有效的预防.     

Incomplete distal renal tubular acidosis coinherited with a mutation in the band 3 (AE1) gene

Background. Band 3 (anion exchanger 1, AE1) is one of the most abundant proteins of the erythrocyte membrane. We have previously characterized twenty AE1 gene defects underlying spherocytic haemolytic anaemia with band 3 deficiency. Since AE1 is also expressed in the intercalated cells of renal cortical collecting ducts where it is thought to participate in urine acidification, we asked whether the spherocytogenic AE1 mutations also affect the regulation of urine acidity. Methods. We examined 10 patients from seven unrelated families with hereditary spherocytosis with band 3 deficiency using the short urine acidification test with CaCl2 administration at a dose of 0.2 g/g b.w. To assess the ability of the nephron to secrete protons, 400 mol of NaHCO3 were infused over a period of 2 h. Results. While we detected no significant abnormalities in eight patients, we have diagnosed incomplete distal renal tubular acidosis (dRTA) in two patients from one family whose urinary pH 5 h after a CaCl2 administration were 6.65 and 6.89. Administration of bicarbonate in these two patients resulted in high urinary HCO3^- concentration. The patients carry the previously characterized mutation band 3^PRIBRAM that encodes a C-terminally truncated band 3 containing only the cytoplasmic domain and the first three putative transmembrane segments. Conclusions. This finding shows an association of a band 3 defect with abnormal urinary acidification perhaps secondary to Cl^-/HCO3 exchange in the basolateral membrane of &agr;-intercalated cells of cortical collecting ducts.     

Case of hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome associated with nephrocalcinosis and distal renal tubular acidosis

Hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder characterized by hypoparathyroidism, sensorineural deafness and renal dysplasia. Herein, we report a case of HDR syndrome associated with nephrocalcinosis and distal renal tubular acidosis. A 34-year-old woman was admitted to investigate recurrent stone formation and bilateral nephrocalcinosis. As a 3-year-old child, she had been diagnosed with HDR syndrome without chromosome evaluation. She had spontaneous stone passages on several occasions. On laboratory examination, serum calcium and intact parathyroid hormone at lower levels. Urinary citrate excretion was extremely low at 51.6 mg/day. On an ammonium chloride loading test, complete distal renal tubular acidosis was proved. To prevent the nephrocalcinosis from deteriorating, she was given potassium-sodium citrate. Since administration, she has not experienced spontaneous stone passage or renal colic. Nephrocalcinosis and recurrent urolithiasis will strongly affect renal prognosis in this case and we consider that citrate medication is an effective therapy in avoiding progress of her nephrocalcinosis.     

Val2Ala mutation in the Atp6v0a4 gene causes early-onset sensorineural hearing loss in children with recessive distal renal tubular acidosis: a case report

A young female patient born to consanguineous parents was admitted to our clinic at the age of 3 years with a 5-month history of weight loss and recurrent urinary tract infections. Based on clinical findings (delayed growth and O-bein deformity) and laboratory tests (hypokalemia, hyperchloremia, partially compensated metabolic acidosis, alkaline urine and nephrocalsinosis), a diagnosis of distal renal tubular acidosis (dRTA) was made. Then, the audiogram revealed a bilateral sensorineural hearing loss (SNHL). On follow-up, bilateral SNHL progressively worsened requiring the need for hearing aid. The ATP6V0A4 gene mutation analysis showed homozygote Val2Ala mutation. To the best of our knowledge, this is the first report describing a Turkish girl with dRTA who suffered from early-onset SNHL caused by Val2Ala mutation in the ATP6V0A4 gene.     

Hypokalaemic muscle paralysis secondary to a sporadic form of type I renal tubular acidosis. A case report

A patient presenting with hypokalaemic muscle weakness is described. An associated hyperchloraemic metabolic acidosis was noted, which gave the clue to the final diagnosis of type I ('classic' or 'distal') renal tubular acidosis. Treatment resulted in clinical and biochemical recovery.     

Characterization of a highly polymorphic marker adjacent to the SLC4A1 gene and of kidney immunostaining in a family with distal renal tubular acidosis.

BACKGROUND: Mutations in the human SLC4A1 (AE1/band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (dRTA). The molecular diagnosis of AE1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some dRTA-associated AE1 mutations remain unclear. Here, we characterized a polymorphic dinucleotide repeat close to the human AE1 gene and performed an immunocytochemical study of kidney tissue from a patient with inherited dRTA with a defined AE1 mutation. METHODS: One CA repeat region was identified in a phage P1-derived artificial chromosome (PAC) clone containing most of the human AE1 gene and the upstream flanking region. We determined its heterozygosity value in multiple populations by PCR analysis. Genotyping of one family with dominant dRTA identified the AE1 R589H mutation, and family member genotypes were compared with the CA repeat length. AE1 and vH(+)-ATPase polypeptides in kidney tissue from an AE1 R589H patient were examined by immunocytochemistry for the first time. RESULTS: This CA repeat, previously reported as D17S1183, is approximately 90 kb upstream of the AE1 gene and displayed considerable length polymorphism, with small racial differences, and a heterozygosity value of 0.56. The allele-specific length of this repeat confirmed co-segregation of the AE1 R589H mutation with the disease phenotype in a family with dominant dRTA. Immunostaining of the kidney cortex from one affected member with superimposed chronic pyelonephritis revealed vH(+)-ATPase-positive intercalated cells in which AE1 was undetectable, and proximal tubular epithelial cells with apparently enhanced apical vH(+)-ATPase staining. CONCLUSIONS: The highly polymorphic dinucleotide repeat adjacent to the human AE1 gene may be useful for future studies of disease association and haplotype analysis. Intercalated cells persist in the end-stage kidney of a patient with familial autosomal dominant dRTA associated with the AE1 R589H mutation. The absence of detectable AE1 polypeptide in those intercalated cells supports the genetic prediction that the AE1 R589H mutation indeed causes dominant dRTA.     

Distal renal tubular acidosis in mice lacking the AE1 (band3) Cl-/HCO3- exchanger (slc4a1)

Mutations in the human gene that encodes the AE1 Cl(-)/HCO(3)(-) exchanger (SLC4A1) cause autosomal recessive and dominant forms of distal renal tubular acidosis (dRTA). A mouse model that lacks AE1/slc4a1 (slc4a1-/-) exhibited dRTA characterized by spontaneous hyperchloremic metabolic acidosis with low net acid excretion and, inappropriately, alkaline urine without bicarbonaturia. Basolateral Cl(-)/HCO(3)(-) exchange activity in acid-secretory intercalated cells of isolated superfused slc4a1-/- medullary collecting duct was reduced, but alternate bicarbonate transport pathways were upregulated. Homozygous mice had nephrocalcinosis associated with hypercalciuria, hyperphosphaturia, and hypocitraturia. A severe urinary concentration defect in slc4a1-/- mice was accompanied by dysregulated expression and localization of the aquaporin-2 water channel. Mice that were heterozygous for the AE1-deficient allele had no apparent defect. Thus, the slc4a1-/- mouse is the first genetic model of complete dRTA and demonstrates that the AE1/slc4a1 Cl(-)/HCO(3)(-) exchanger is required for maintenance of normal acid-base homeostasis by distal renal regeneration of bicarbonate in the mouse as well as in humans.     

Immunocytochemical localization of vacuolar H+-ATPase and Cl−-HCO 3 − anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: Effect of calcium-deficient diet on polar expression of the H+-ATPase pump

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H⁺-ATPase, and a functional Cl⁻-HCO ₃ ⁻ anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H⁺-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl⁻-HCO ₃ ⁻ anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H⁺-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H⁺-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P =0.09). These changes were not accompanied by a significant increase in the intensity of staining for H⁺-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H⁺-ATPase polarization. Parts of this work have been presented in the American Society of Nephrology in November 1992