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J Biol Chem, Vol. 273, Issue 11, 6380-6388, March 13, 1998

Autosomal Dominant Distal Renal Tubular Acidosis Is Associated in Three Families with Heterozygosity for the R589H Mutation in the AE1 (Band 3) Clminus /HCO3minus Exchanger*

Petr Jarolimabcd, Chairat Shayakuldef, Daniel Prabakarande, Lianwei Jiange, Alan Stuart-Tilleye, Hillard L. Rubina, Sarka Simovac, Jiri Zavadilc, John T. Herringh, John Brouilletteik, Michael J. G. Somersgh, Eva Seemanovac, Carlo Brugnarabj, Lisa M. Guay-Woodfordil, and Seth L. Alperefmno

From the e Molecular Medicine and f Renal Units, Beth Israel Deaconess Medical Center, g Division of Nephrology and j Department of Laboratory Medicine, The Children's Hospital, a Department of Pathology, Brigham and Women's Hospital, and Departments of m Medicine, h Pediatrics, n Cell Biology, and b Pathology, Harvard Medical School, Boston, Massachusetts 02215; the i Division of Nephrology and Departments of k Medicine and l Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294, and c Charles University School of Medicine and Institute of Hematology and Blood Transfusion, 12820 Prague, Czech Republic

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed ~20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To maintain systemic acid/base balance, human kidneys must excrete 40-70 milliequivalents of H+/day. This acid excretion is mediated in substantial part by type A (acid-secreting) intercalated cells (IC)1 of the renal collecting ducts. Failure of this distal acidification mechanism early in life leads to chronic metabolic acidosis (often with hypokalemia) and growth retardation. Later in life, nephrocalcinosis and hypercalciuria are variable concomitants. The clinical diagnosis of distal renal tubular acidosis (dRTA) of the "complete form" is established by less-than-maximal urinary acidification in the presence of pre-existing acidosis. An "incomplete form" of dRTA in individuals without spontaneous acidosis has been defined as less-than-maximal urinary acidification following administration of a standard acid load. Complete dRTA can be successfully treated by chronic oral supplementation with bicarbonate or its metabolic precursor, citrate (1).

Defects in any one of several transporters of the type A IC required for transepithelial acid secretion and bicarbonate reabsorption might cause heritable dRTA (1, 2). Among these components are the multiple gene products that comprise the vacuolar H+-ATPase (vH+-ATPase) thought to mediate most H+ secretion across the lumenal plasma membrane of the type A IC (3, 4); the kAE1 (kidney band 3) Cl-/HCO3- exchanger (5, 6) that extrudes HCO3- across the basolateral membrane of the type A IC (7, 8); cytoplasmic carbonic anhydrase II (CAII) whose activity provides both H+ for lumenal secretion by the vH+-ATPase and HCO3- for basolateral extrusion by kAE1 (1, 9); and, at least in conditions of K+ deprivation, the H+,K+-ATPase(s) (10, 11).

Several autosomal recessive mutations of CAII have been described (9) that result in proximal renal tubular acidosis and metastatic calcinosis. A similar syndrome is present in mice homozygous for loss of CAII activity (12), leading to loss of collecting duct IC (13). Although mutations in the multiple genes encoding the subunits of the vH+-ATPase have not yet been reported in familial dRTA, absence of immunoreactive vH+-ATPase has been noted in kidney biopsy specimens from patients with acquired dRTA secondary to Sjogren's syndrome (14). A recently described autosomal recessive AE1 nonsense mutation in cattle noted the presence of systemic acidosis in addition to severe anemia and red cell fragility (15). In addition, genetically engineered AE1 -/- mice were noted to lack AE1 immunostaining in type A IC (16).

These observations suggest AE1 as a candidate gene for familial dRTA. However, the many heterozygous missense and nonsense mutations in the AE1 gene associated with ~25% (17-19) of hereditary spherocytosis (HS) cohorts have not been associated with metabolic acidosis. In most reported HS cases, heterozygosity for wild-type (wt) AE1 was associated with the presence in patient red cells of immunoreactive AE1 polypeptide and anion transport function at levels 60-70% of those in wt family members (17, 18).

We have examined AE1 as a candidate gene in familial dRTA of an autosomal dominant pattern. In this article, we report the presence in three unrelated families of heterozygosity for a single AE1 missense mutation in all clinically affected individuals. Microsatellite haplotypes within families also showed linkage with disease phenotype and with AE1 genotype, but differed among families. Functional analysis of AE1-mediated sulfate uptake into red cells and of recombinant AE1-mediated Cl-/HCO3- exchange in Xenopus oocytes revealed a mild partial loss-of-function phenotype for the mutant AE1 polypeptide. Co-expression of wt and mutant polypeptides in Xenopus oocytes did not reveal dominant negative properties of the mutant polypeptide. In addition, one apparently unaffected at-risk child also shows heterozygosity for the mutation, but has not undergone acid-load testing for incomplete dRTA.

Thus, heterozygosity for the hypofunctional AE1 R589H allele is associated with dRTA and may contribute to its pathogenesis, whereas heterozygosity for null AE1 alleles in most HS patients has no evident renal phenotype.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Clinical Studies-- Antecubital venipuncture and blood collection into tubes containing heparin or citrate were performed under protocols approved by the Clinical Investigation Committees of The Children's Hospital (Boston, MA) and the Institute of Hematology and Transfusion (Prague, Czech Republic). Red cell indices were measured using the H3 Autoanalyzer (Technicon). Red cell cation content was measured by atomic absorption spectrometry (20).

Red Cell Anion Transport Studies-- [35S]Sulfate influx studies in the presence and absence of the inhibitor, 4,4'-diisothiocyano-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Calbiochem or Sigma) were performed at 37 °C as described previously in the presence of 4 mM sodium sulfate in 84 mM trisodium citrate, 10 mM MOPS, pH 6.4 (16-19, 21). Influx inhibition data as a function of inhibitor concentration were fit to the Michaelis-Menten equation with Ultrafit 2.0 (Elsevier).

pH dependence of [35S]sulfate influx experiments was assessed at 37 °C in the presence of 4 mM sodium sulfate in flux medium containing 140 mM sodium sulfamate, 10 mM MOPS/Tris titrated to the indicated pH values between 8 and 5. Influx data as a function of pH were fit to the equation v = Vmax × 10pH50/(10pH50 + 10pH).

Red Cell Polypeptide Analysis-- Washed red cells were lysed in 5 mM sodium phosphate, pH 8, containing Complete® protease inhibitor mixture (Boehringer Mannheim). Ghost protein was measured by the bicinchoninic acid assay (Pierce). Ghost membrane treatment with peptidyl-N-glycosidase F (New England Biolabs) was as described previously (18). Protein (10 µg) was dissolved in SDS-load buffer and subjected to SDS-polyacrylamide gel electrophoresis as described previously (16-18). Gels were stained with Coomassie Blue R250 or used for immunoblot analysis. Antibodies to mouse AE1, mouse spectrin, human protein 4.1, and human protein 4.2 were previously described (16). Autoradiograms were scanned in transmittance mode or digitally photographed (22).

Genetic Analyses-- Genomic DNA was prepared from whole blood buffy coats using the QiaAmp kit (Qiagen), according to the manufacturer's instructions. All exonic sequences of human AE1 were PCR-amplified using the flanking intronic oligonucleotide primers and cycling conditions described previously (19). Amplified exonic fragments were subjected to single-strand conformational polymorphism analysis (SSCP) (23) as described previously (19, 24). DNA sequencing was performed with ABI 373 and 377 automated sequencers. Restriction digestions were analyzed by agarose gel electrophoresis.

The AE1 locus (SLC4A1) has been previously mapped to human chromosome 17q21-q22 in tight linkage with the gene encoding nerve growth factor receptor (NGFR) (25). Included among the ordered polymorphic markers on chromosome 17q that are tightly linked to NGFR on the WC17.6 YAC contig2 are the following: centromere-D17S1814-D17S800-D17S934-D17S920-D17S1861-telomere. Microsatellite polymorphisms amplified by PCR in the presence of [alpha -32P]dCTP using standard conditions. Amplified products were separated on 6% denaturing polyacrylamide gels and analyzed by autoradiography. Two-point linkage analyses were performed using the LINKAGE package, version 5.1 (26). Gene frequency was set as 0.00001 with a penetrance of 100% for affected individuals and 0% for unaffected family members. In addition, haplotypes were constructed from the genotype data. The most likely haplotypes were inferred by minimizing the number of crossover events in each sibship.

Mutagenesis-- The plasmid encoding human erythroid AE1 (eAE1) has been previously described (27, 28). The eAE1 R589H plasmid was constructed by four primer PCR (29). The flanking primers were F1 (5'-TCCCGCTATACCCAGGAG-3', nt 1660-1677) and F2 (5'-GGATGACCCAGCCCCGGG-3', nt 2062-2045). The internal mutagenic primers were M1 (5'-CATGATGCTGCACAAGTTCAAGA-3', nt 1866-1888) and M2 (5'-TCTTGAACTTGTGCAGCATCATG-3', nt 1888-1866). The 402-base pair PCR fragment was then cleaved at its internal BclI sites, and the internal 230-base pair fragment encompassing the mutant site was cloned into the similarly cleaved human eAE1 pBluescript KS plasmid (Stratagene). Mutant and wild-type subclones were defined by restriction digestion, and their integrity confirmed by DNA sequencing.

Human kAE1 versions of the wild-type and mutant eAE1 cDNAs were constructed by KpnI digestion of the wt and R589H eAE1 cDNAs to remove the 5'-most 311 nt, yielding constructs in which the next downstream ATG corresponded to the kAE1 initiator ATG. kAE1 constructs were inserted into the pXT7 vector (Promega) containing as 5'- and 3'-flanking regions the corresponding untranslated regions from Xenopus beta -globin mRNA.

Functional Expression of eAE1- and kAE1-mediated Anion Exchange in Xenopus Oocytes-- cRNA was transcribed from linearized plasmid template with the Megascript kit (Ambion). cRNA was injected into defolliculated Xenopus laevis oocytes of stages V-VI. Oocytes were maintained in ND-96 buffer at 19 °C for 2-3 days. 36Cl- influx studies were performed as described previously (29, 30) with the following modifications. Isotopic influx periods were 10 or 15 min. Some influx experiments were performed at 37 °C and/or in extracellular solutions of pH 5.5 or 9.0, as indicated. Hypertonic influx medium contained ND-96 (212 mosM) supplemented with NaCl to a final osmolarity of 280 mosM (30). 36Cl- efflux studies were performed as described previously (31). Cl-/HCO3- exchange activity was measured by ratiometric video imaging of oocytes (32) loaded with 2,7-bis(2-carboxyethyl)-5(6)carboxy-fluorescein acetoxymethyl ester.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Clinical Presentation and Diagnosis-- Family pedigrees are shown in Fig. 1 (panels A-C). Propositi (arrows) presented to medical attention with histories of growth retardation, in one case accompanied by repeated urinary tract infections. All affected individuals (filled symbols) were short of stature. The complete form of dRTA was diagnosed by the combined detection of urinary pH values > 5.5; the absence of glucosuria (indicating normal proximal tubular function), and by the presence of metabolic acidosis without concomitant gastrointestinal bicarbonate losses (such as might result from diarrhea). Nephrocalcinosis (radiologically detected renal calcification) was present in all affected individuals except for family L members IV:3 and IV:4, who were diagnosed with incomplete dRTA before 1 year of age and then treated successfully with oral citrate (metabolized to bicarbonate). Unaffected individual IV:2 underwent an acid-challenge test at 9 months of age with normal results. Clinical variability among affected individuals within families was evident. In family L, individual III:2 has had >30 kidney stones without severe hypokalemia, whereas her sibling III:4 has had persistent refractory hypokalemia but few kidney stones. Among propositi, only individual III:2 of family B was hypokalemic at presentation.


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  		Fig. 1.   Pedigrees and genotypes of dRTA families. Individuals are identified by generation (Roman numerals at right margin of each pedigree) and individually (Arabic numerals at upper left of each unaffected (open symbols) or affected individual (filled symbols). The chromosome 17 microsatellites tested are listed with their genetic map positions at the left margin of each pedigree. Haplotypes are presented underneath individual symbols. Propositi are indicated by arrowheads. In insets, genetically affected individuals are indicated by black dots under genotypes. A, top, family L pedigree and haplotypes (horizontal line in haplotype of individual IV:2 indicates recombination compared with parental haplotypes); inset, SSCP profile of PCR-amplified AE1 exon 14. B, top, family B pedigree and haplotypes; inset, HhaI digest of PCR amplified AE1 exon 14 from selected family members. C, family K pedigree and haplotypes; inset, HhaI digest of PCR-amplified AE1 exon 14. D, representative DNA sequences of AE1 exon 14 from PCR-amplified genomic DNA of affected individual II-2 (top) and of unaffected individual III-1 (bottom) of family K, showing the heterozygous AE1 substitution G1766A encoding the missense mutation R589H only in II-2.

Detection of an AE1 Mutation in Genomic DNA from Individuals of Three Unrelated Families with dRTA-- Genomic DNA previously prepared from the available members of family L was subjected to SSCP analysis of exons 11-20, encoding the anion-transporting transmembrane domain of the AE1 polypeptides. Only exon 14 showed reproducible polymorphism among family members. Fig. 1A (inset) shows that of the 12 genomic DNAs tested, an identical exon 14 polymorphism was present in all five members of family L with dRTA. The polymorphism was absent from unaffected individual IV:2, in whom the absence of incomplete dRTA was confirmed by acid-loading test, and in all other apparently unaffected individuals except for one. The polymorphism was present in apparently unaffected individual IV:1, who has been unavailable for an acid loading test that would could diagnose incomplete dRTA. Although not acidotic, individual IV:1 has been and remains short of stature.

DNA sequencing of PCR-amplified exon 14 from all affected individuals in family L revealed the heterozygous mutation G1766A (numbering from the initiator codon ATG), encoding the amino acid substitution R589H. The heterozygous mutation was also present in apparently unaffected individual IV:1, whose exon 14 displayed the conformational polymorphism. Absence of the heterozygous G1766A substitution was confirmed by exon 14 DNA sequencing in three individuals without clinical dRTA: III:3, III:5, and IV:2. The mutation abolished a restriction site recognized by FspI and HhaI. FspI digestion of PCR-amplified DNA confirmed the heterozygous presence of the mutation in all individuals with the conformational polymorphism, and its absence in the DNA of those without the polymorphism.

In family K (Fig. 1B), heterozygous loss of the HhaI restriction site in exon 14 cosegregated strictly with disease (inset). This was confirmed in all individuals by FspI digestion and by exon 14 DNA sequences, portions of which are shown in Fig. 1D for affected individual II:2 and for unaffected individual III:1. Unaffected individual II:1 has had a normal acid challenge as an adult. In family B, heterozygous loss of the HhaI restriction site also cosegregated with disease (inset), and was confirmed by exon 14 DNA sequences in all individuals (data not shown). This mutation was not detected by SSCP analysis among 224 normal and affected inidividuals from families with hereditary ovalocytosis or hereditary spherocytosis (data not shown).

The AE1 gene of individual II:2 of family K was completely sequenced through all coding exons and exon-intron splice junctions, without detection of additional mutations. The DNA sequence of individual II:5 of family L was obtained for exons 4, 5, and 10-20 (encoding the juxtamembrane region and the transmembrane ion transport domain) of the AE1 gene. Except for homozygosity for the previously described (19) silent polymorphism CTG right-arrow CTA in codon 441 (Leu) of exon 12, no other mutations were detected. The AE1 Memphis I polymorphism (33) was absent in all three families.

Intron 3, which includes at least part of the kidney-specific promoter for the kidney band 3 isoform, kAE1, was also sequenced in entirety in affected individuals I:1 and II:2 of family K, and in II:5 of family L. The intron 3 sequence in all these individuals conformed to family L intron 3 sequence, and were identical to the GenBank L35930 AE1 gene sequence (34), except for the homozygous absence of nt 7509-7512. These four nucleotides were similarly absent in the human AE1 (Memphis I variant) gene sequence (GenBank X77738) of Schofield et al. (35).3

Disease Phenotype Linkage with Additional Nearby Genetic Markers-- The uncertain clinical status of short-of-stature, genetically affected individual IV:1 of family L, in whom acid-loading test has not yet been performed, encouraged further testing of the hypothesis of linkage between the AE1 mutation R589H and disease phenotype by examination of linked polymorphic markers. Within each family, R589H heterozygotes shared a common allele of the bimorphic intragenic PstI restriction fragment length polymorphism in AE1 intron 3 (38).

One shared haplotype of microsatellite markers flanking the AE1 locus (Fig. 1) was evident in all R589H heterozygotes within each family as indicated by D17S1814, D17S800, D17S934, D17S920, and D17S1861 in families L and K and by D17S800, D17S934, and D17S1861 in family B. In a set of two-point linkage analyses, the maximum LOD score calculated with the value of theta set to zero was 3.01 in favor of linkage for each of the microsatellites D17S934 and D17S1861. Moreover, the recombination event evident in individual IV:2 of family L (Fig. 1A) refines the genetic locus of the AE1 gene to a position telomeric to D17S800.

Properties of Red Blood Cells from the dRTA Families-- The R589H mutation associated with the presence of dRTA in the three families is predicted to be present in both erythroid AE1 (eAE1) and in kidney AE1 (kAE1) polypeptides. Since multiple heterozygous AE1 loss-of-function mutations associated with Southeast Asian ovalocytosis (SAO; Ref. 21) and with HS (17-19) are associated with reduced DIDS-sensitive sulfate uptake into red cells, this index of erythroid AE1 function was examined in red cells from the dRTA families.

Cells from affected and unaffected family members were normocytic and had normal indices as detected by Technicon H3 autoanalyzer (data not shown). Red cell sulfate influx varied among families. However, dRTA cells showed influx values that were 86% (family L), 81% (family K), and 78% (family B) of the values of cells from related unaffected individuals (Table I). These relative values are somewhat higher than those measured in red cells from families with AE1-deficient HS (17-19) and with SAO (21). The ID50 for inhibition of sulfate influx by DIDS did not differ between cells from normal and dRTA individuals. Similarly, but in contrast to SAO (21) and to HS (16, 18), the DIDS concentration required for (extrapolated) maximal inhibition of sulfate influx did not differ between cells from unaffected and affected individuals (Table I).

                              
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Table I
Red cell sulfate uptake
Table shows characterization of eAE1-mediated [35S]SO4 influx into red cells from members of families L, B, and K, compared with healthy control cells and with AE1-deficient HS red cells. Cells from all individuals were blocked >99.5% by 3 µM DIDS at pH 6.4. pKa and ID50 values are means ± S.E. from the fit curves. ND, not determined.

The IC of the renal collecting duct are exposed to extracellular pH values more acidic than are red cells. Since mutation of the positively charged Arg to the titratable His might be hypothesized to alter the pH dependence of AE1-mediated transport sufficiently to contribute to or cause dRTA, the pH dependence of erythroid sulfate influx was examined (Fig. 2). However, values of pKa varied among healthy individuals between 5.92 and 6.41, and pKa values for dRTA cells fell within that range (Table I).


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  		Fig. 2.   pH dependence of eAE1-mediated [35S]sulfate influx into erythrocytes incubated in sodium sulfamate medium of the indicated pH values. Each set of symbols in each panel represents cells from an individual depicted in the pedigrees of Fig. 1. A, family L: square, III:5; inverted triangle, II:5; upright triangle, III:4; circle, IV:3; diamond, IV:4. B, family K: square, I:1; circle, II:2; diamond, unrelated patient with hereditary spherocytosis. C, family B: triangle, III:2; circle, III:1; diamond, II:3. Relative standard errors of plotted mean values are <5% (except for a single value of 8%).

The polypeptide composition of dRTA red cell ghosts did not differ from that of ghosts from unaffected individuals, as detected by Coomassie Blue profile (Fig. 3). Immunoblot analysis of spectrin, AE1, and the AE1-binding proteins 4.1 and 4.2 did not indicate consistent differences in abundance between affected and unaffected individuals (Fig. 3). Treatment with peptidyl-N-glycosidase F of ghosts from normal and dRTA individuals yielded deglycosylated AE1 polypeptides of indistinguishable apparent abundance and Mr (Fig. 4).


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  		Fig. 3.   Red cell membrane proteins from selected affected and unaffected members of dRTA families. Upper rows, immunoblots of alpha -spectrin, eAE1, protein 4.1, and protein 4.2;. Bottom, Coomassie Blue staining profile of ghost membranes fractionated by SDS-polyacrylamide gel electrophoresis.


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  		Fig. 4.   N-Deglycosylation of red cell eAE1 in family B. Coomassie Blue staining of eAE1 polypeptide from 10 µg of red cell ghost protein after incubation with (left lanes) and without (right lanes) peptidyl-N-glycosidase F (PNGase F). Filled circles above lanes indicate affected individuals.

Functional Analysis of Recombinant AE1 R589H-- Since type A IC were not available for study, recombinant wt and R589H kAE1 polypeptides were expressed in Xenopus oocytes from cRNA as described previously (5, 28-32). Three modes of AE1 function were examined: 36Cl- influx (Fig. 5 (A-C), Table II) and 36Cl- efflux modes of Cl-/Cl- exchange (Table II), and Cl-/HCO3- exchange (Fig. 5D, Table III).


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  		Fig. 5.   kAE1-mediated 36Cl- transport in oocytes expressing wt and R589H kAE1 polypeptides individually (from 1 ng of cRNA) or together (from 0.5 ng of each cRNA), measured 72 h after cRNA injection into oocytes from a single frog. A, kAE1-mediated 36Cl- uptake measured in isotonic medium of pH 7.4 at 20 °C and 37 °C. B, kAE1-mediated 36Cl- uptake measured in isotonic medium of pH 5.5 at 20 °C and 37 °C. C, kAE1-mediated 36Cl- uptake measured in hypertonic medium of pH 5.5 at 20 °C and 37 °C. Means ± S.D. for 6-8 oocytes are shown. D, Cl-/HCO3- exchange activity in individual oocytes expressing wt AE1 (squares) or AE1 R589H (filled triangles) in isotonic and hypertonic media, and in isotonic medium with DIDS (100 µM). *, p < 0.005 compared with wt kAE1; **, p < 0.05 compared with wt kAE1.

                              
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Table II
Cl-fluxes in Xenopus oocytes expressing wt kAE1, kAE1 R589H, or both
Table shows kAE1-mediated 36Cl- influx into oocytes previously injected with 1 ng of cRNA encoding either wt kAE1 or kAE1 R589H, or with 0.5 ng of each. Fluxes were measured at the indicated temperatures and pH values. Hypertonic medium was 280 mosM; isotonic medium was 212 mosM. Number of influx experiments (each testing 6-8 oocytes) is indicated within parentheses. Influx values are expressed relative to those of wt AE1 to allow pooling of results and comparison of different conditions. Each efflux experiment tested a single oocyte. *, p < 0.03 versus wt kAE1-mediated efflux.

                              
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Table III
Cl-/HCO3- exchange activity of kAE1 R589H
Table shows kAE1-mediated Cl-/HCO3- exchange in oocytes previously injected with cRNA encoding wt kAE1, kAE1 R589H, or an equimolar mix of both. Number of individual oocytes studied is indicated in parentheses. *, p < 0.01 compared to wt kAE1; without asterisk, p > 0.05. 

kAE1 R589H was modestly but significantly less active than wt kAE1 at pH 7.4, 20 °C (Fig. 5 (A-C) and Table II), with influx rates 73% of wt values. Other measures of kAE1 R589H function confirmed similar mild loss-of-function phenotypes. Thus, R589H-mediated 36Cl- efflux was 52% that of wt kAE1 (n = 4, p < 0.03) at 20 °C at pHo 7.4 in isotonic conditions (Table II). Rates of kAE1 R589H-mediated Cl-/HCO3- exchange were 50-62% of wild-type values as estimated from measurements of dpHi/dt, and retained DIDS sensitivity (Fig. 5D and Table III).

A more severe loss of kAE1 function might be elicited only "conditionally" at the human body temperature of 37 °C, or in the more extreme conditions of pH and tonicity to which type A intercalated cells of the medullary collecting duct are exposed. Therefore, oocytes expressing wt and R589H variants of kAE1 were subjected to tests of Cl- influx at pH 5.5, and in hypertonic conditions at 20 °C and 37 °C. As shown in Fig. 5 (A-C) and in Table II, elevated temperature increased AE1-mediated 36Cl- influx in every condition, consistent with the temperature dependence of red cell AE1-mediated anion transport (39). However, elevated temperature did not elicit further loss-of-function in kAE1 R589H. As expected, wt kAE1 was not activated by hypertonicity (30) and showed little inhibition by acute extracellular acidification or alkalinization (29). Furthermore, these conditions did not dramatically enhance the loss-of-function phenotype exhibited by kAE1 R589H (Table II, Fig. 5).

A mutant polypeptide with a partial loss-of-function phenotype might produce a clinical loss-of-function in the context of a dominant inheritance pattern by acting as a dominant negative mutant. Were kAE1 R589H to be a dominant negative mutant, it would be predicted to decrease AE1 function when coexpressed with wt kAE1 polypeptide. The oocyte expression system is the most convenient in which to assess the consequences of co-expression of wt and mutant polypeptides, mimicking the heterozygous state. However, oocytes previously co-injected with 0.5 ng each of cRNA encoding wt and mutant kAE1 exhibited 20 °C transport activities intermediate between wt and mutant levels of transport. This was so whether evaluated as AE1-mediated 36Cl- influx (Fig. 5 (A-C), Table II) or as kAE1-mediated Cl-/HCO3- exchange (Fig. 5D, Table III). Neither the higher temperature of 37 °C (Table II) nor increasing by 4-fold the amount of injected R589H cRNA relative to wt cRNA at 20 °C (data not shown) led to the decreased transport activity predicted for a dominant negative mechanism. Moreover, oocyte levels of wt and R589H AE1 polypeptides did not differ as detected by metabolic labeling and immunoprecipitation (n = 4). Accumulation of in vitro-translated wt and mutant polypeptides in pancreatic microsomes also did not differ (data not shown).

Unlike the modest reduction in kAE1 activity produced in Xenopus oocytes by the R589H mutation, eAE1 R589H-mediated Cl-/Cl- exchange activity was indistinguishable from that of wt eAE1 (n = 4; data not shown).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The above results describe three unrelated families with autosomal dominant dRTA in which clinical disease (complete dRTA) is associated with heterozygosity for the AE1 point mutation G1766A (Fig. 1). This mutation is absent in >200 other unrelated individuals, some normal and some with eAE1-deficient HS or SAO (19).4 The mutation encodes the amino acid substitution R589H, changing a positively charged side chain to a titratable group at a position thought to be at the cytoplasmic end of AE1 transmembrane span 6 (27, 40). Because wt eAE1, in contrast to the related protein AE2, is known to be only slightly inhibited by acid pH (29), the mutation of a cytoplasmically disposed Arg to His suggested the possibility that kAE1 R589H might be more susceptible to inhibition by acid pH than is wt kAE1.

AE1 R589H-associated dRTA was not associated with any clinical erythroid abnormality. Presence of the heterozygous mutation was unaccompanied by altered abundance, Mr, or glycosylation of red cell eAE1 polypeptide (Figs. 3 and 4), but was associated with 14-22% reduction in eAE1-mediated sulfate transport without apparent change in pKa or in DIDS sensitivity (Table I, Fig. 2).

Functional comparison in Xenopus oocytes of recombinant wt kAE1 with kAE1 R589H revealed 20-50% reduction in Cl- influx in different conditions (Fig. 5, Table II), and 40-50% reduction in Cl- efflux (Table II) and in Cl-/HCO3- exchange (Fig. 5, Table III). Minimal inhibition of wt kAE1 function at pH 5.5 was not enhanced in kAE1 R589H. Coexpression of wt and R589H kAE1 designed to mimic the heterozygous state revealed no evidence for a dominant negative effect of the R589H mutant. Recombinant wt and R589H kAE1 polypeptides accumulated to equal extents in Xenopus oocytes and in pancreatic microsomes present during in vitro translation reactions.

These findings contrast with those in individuals with the many, distinct AE1 mutations causing HS, whose red cells exhibit up to 40% reduction both in red cell sulfate uptake and in eAE1 polypeptide abundance. AE1 Prague I, the only HS variant polypeptide studied in Xenopus oocytes, was nonfunctional and failed to accumulate to detectable levels (28). However, in six family cohorts with AE1-deficient HS, including the loss-of-function frameshift mutations AE1 Prague I and AE1 Smichov, as well as four families not yet genotyped, no systemic acidosis was found (41), and the urinary acidification response to CaCl2 loading (1) was normal, suggesting the absence of incomplete dRTA. Therefore, the HS mutations suggest that haploinsufficiency of eAE1 (assumed to be accompanied by haploinsufficiency of kAE1) can be compatible with clinically normal urinary acidification.5

How can the absence of dRTA and the presence of 40% reduction in red cell eAE1 abundance and function in the heterozygous AE1 disease HS be reconciled with the presence of dRTA and 14-22% reduction in red cell AE1 function in the heterozygotes for AE1 R589H? The absence of stable cell culture models of type A IC and the difficulty in obtaining human renal tissue from affected individuals restrict the types of data that can be gathered to address this question. However, several explanations can be considered.

The first explanation that must be considered is the unlikely possibility that the R589H mutation is not in fact linked to dRTA. However, all R589H heterozygotes within each of the three families share a common allele of the intragenic PstI restriction fragment length polymorphism in AE1 intron 3 (38). In addition, two nearby polymorphic microsatellite markers display 1000:1 odds in favor of linkage with the AE1 R589 genotype. However, neither intragenic nor flanking haplotypes support a recent common origin of the R589H mutation among the three families. (One individual of uncertain clinical status, IV:1 in family L, is heterozygous for the AE1 R589H mutation and may have incomplete dRTA, a diagnosis that requires acid-challenge testing.) Yet, additional support for linkage comes from four recently described unrelated families with autosomal dominant dRTA in individuals heterozygous for AE1 mutations: two families with the same R589H substitution reported here, one with R589C, and one with S613F (42). Interestingly, these two latter mutations pose the same paradox of mild or absent functional impairment of anion transport.

As a second explanation, the R589H mutation in one allele of the AE1 gene might be necessary but not sufficient to produce dRTA. In this scenario, there must be present a required allele of one or more modifier genes for sufficient reduction of collecting duct acid secretion to result in dRTA. This incremental effect may be entirely mediated by further reduction in Cl-/HCO3- exchange in the type A IC, or (more likely) due to impairment of yet another process required for or contributing to urinary acid excretion (and encoded by a linked gene).

As a third explanation, heterozygosity for kAE1 R589H may indeed suffice to cause dRTA. However, a more extreme loss-of-function phenotype of kAE1 R589H than that exhibited by recombinant protein expressed in Xenopus oocytes might indeed be required to cause dRTA. Manifestation of this hypothesized more extreme (and possibly dominant negative) phenotype, might require conditions or cofactors present in mammalian cells but absent from Xenopus oocytes. An example of such a condition would be temperature sensitivity of the AE1 R589H mutation, such that biosynthesis and/or intracellular trafficking might be impaired at 37 °C, even though transport activity of protein already in the plasma membrane is not impaired at 37 °C (Fig. 5). Such conditions or cofactors might be restricted to epithelial cells, to polarized epithelial cells, or maybe even restricted to type A IC in situ. The latter case would be consistent with the observed mild loss-of-function in red cells and more severe loss-of-function in type A IC.

Fourth, the mild loss-of-function phenotype of recombinant kAE1 R589H measured in Xenopus oocytes may have indeed accurately reflected its relative anion exchange activity in type A IC. However, the more extreme renal loss-of-function phenotype hypothesized to occur in vivo might arise from loss of polarized kAE1 targeting in type A IC. This could lead to nonpolarized secretion of bicarbonate, effectively short-circuiting urinary acid excretion. Such a loss of polarization of bicarbonate secretion could explain AE1-linked dRTA in the presence of less severe impairment of red cell AE1 function than that measured in HS with normal urinary acidification.

Expression of kAE1 R598H in polarized mammalian epithelial cells should allow experimental tests of these hypotheses. Ultimately, the ability and sufficiency of kAE1 mutations to produce dRTA may be tested in mice -/+ and -/- for the AE1 gene (16).

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK43495 and DK34854 (to S. L. A.), and HL15157 (to C. B. and S. L. A.); by a grant from the Alabama Kidney Foundation (to L. G.-W.); and by Grant 4118-3 from the Grant Agency of the Ministry of Health, Czech Republic (to P. J.). Portions of this work were presented in abstract form on November 2-5, 1997, at the 30th Annual Meeting of the American Society of Nephrology, San Antonio, TX, and on December 5-9, 1997, at the 39th Annual Meeting of the American Society of Hematology, San Diego, CA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

d These authors contributed equally to this work.

o Established Investigator of the American Heart Association. To whom correspondence should be addressed: Molecular Medicine Unit, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2930; Fax: 617-667-2913; E-mail: salper{at}bidmc.harvard.edu.

1 The abbreviations used are: IC, intercalated cell(s); dRTA, distal renal tubular acidosis; vH+-ATPase, vacuolar form of the H+-ATPase; kAE1, kidney isoform of AE1 (band 3); eAE1, human erythroid AE1; CAII, carbonic anhydrase II; HS, hereditary spherocytosis; wt, wild-type; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; MOPS, 3-(N-morpolino)propanesulfonic acid; SSCP, single-strand conformational polymorphism analysis; SAO, Southeast Asian ovalocytosis; contig, group of overlapping clones; PCR, polymerase chain reaction; nt, nucleotide(s).

2 Information is available via the World Wide Web (http://www.gdb.org).

3 Additional sequencing of intron 3 in six randomly selected normal individuals showed identity in this region with GenBank X77738 (35) rather than with GenBank L35930 (34). This finding prompted the re-examination of intron 3 in two clonal isolates of intron 3 used in the sequencing of the non-Memphis human AE1 gene. Both clones lacked nt 7508-7511 (numbering from Ref. 34). The portion of intron 3 encompassing this region is not present in kAE1 mRNA in human kidney (36) or mouse kidney (5, 37). GenBank L35930 has been revised to reflect this correction.

4 P. Jarolim, unpublished results.

5 Rysava et al. (41) also described a mother and daughter with HS secondary to the AE1 Pribram mutation, in which a G to A substitution at position +1 of intron 12 disrupts the intron 12 splice acceptor site, leading to retention of intron 12 and premature polypeptide termination 8 neoresidues beyond codon 477. These individuals displayed incomplete dRTA, but also had the unusual accompanying finding of spontaneous bicarbonaturia.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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