Characterization of a highly polymorphic marker adjacent to the SLC4A1 gene and of kidney immunostaining in a family with distal renal tubular acidosis
Chairat Shayakul1,2,3,
Petr Jarolim4,5,6,
Marie Zachlederova4,
Daniel Prabakaran1,3,
Dionisio Cortez-Campeao7,
Dana Kalabova6,
Alan K. Stuart-Tilley1,
Hiroshi Ideguchi8,
Christlieb Haller9,10 and
Seth L. Alper1,2,11
1Molecular Medicine and 2Renal Units, Beth Israel Deaconess Medical Center, 4Division of Clinical Laboratories and Department of Pathology, Brigham and Women's Hospital, Departments of 11Medicine and 5Pathology, Harvard Medical School, Boston, MA, USA, 3Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, 6Institute of Hematology and Blood Transfusion, Charles University School of Medicine, Prague, Czech Republic, 8Department of Laboratory Medicine, Fukuoka University, Fukuoka, Japan, 9Department of Medicine, University of Heidelberg, Heidelberg, 10Hegau-Klinikum, Singen and 7Sinsheim, Germany
Correspondence and offprint requests to: Seth L. Alper, MD, PhD, Molecular and Vascular Medicine and Renal Units, Beth Israel Deaconess Medical Center, RW763 East Campus, 330 Brookline Avenue, Boston, MA 02215, USA. Email: salper{at}bidmc.harvard.edu
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Abstract
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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
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.
Keywords: anion exchanger 1; dinucleotide repeat; intercalated cell; proximal tubule; spherocytosis; vacuolar H+-ATPase
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Introduction
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Mutations in the SLC4A1 (AE1/band 3) gene are associated with inherited distal renal tubular acidosis (dRTA) and disorders of red cell membrane integrity, including hereditary spherocytic anaemia (HS) and Southeast Asian ovalocytosis (SAO). The dRTA mutations are confined to the exons coding for the transmembrane domain and the short C-terminal cytoplasmic tail [1], whereas HS mutations have been found throughout the AE1 gene. Mutant AE1 polypeptides associated with recessive dRTA are characterized by normal erythroid expression and function. Loss of function in Xenopus oocytes is rescued by co-expression of the erythroid AE1-binding protein, glycophorin A [2,3]. In contrast, mutant AE1 polypeptides associated with dominant dRTA are stable and functional both in erythrocytes and in Xenopus oocytes [35]. Abnormalities of mutant trafficking in HEK 293 cells suggest a pathogenic mechanism for dominant dRTA [6,7], but mutant expression in polarized epithelial cells yields a more complex picture [8].
In almost all instances, the renal and erythroid phenotypes appear mutually exclusive. One exception is the homozygous AE1 mutant V488M (Band 3 Coimbra), which presents with severe anaemia and renal acidification defect [9]. Additional exceptions are compound heterozygotes described by Bruce et al. [3] in whom erythroid dyscrasias of varying severity co-exist with dRTA.
Hereditary dRTA is also caused by mutations in at least two genes encoding subunits of the vacuolar H+-ATPase with a restricted spectrum of expression that includes renal intercalated cells [reviewed in 1,10]. Other families have been characterized whose disease cannot be explained by any of the currently defined disease genes. Interestingly, the KCC4 KCl co-transporter (/) mouse also manifests dRTA [11], but linkage of the KCC4 gene to human disease remains unreported.
Mapping disease phenotype with polymorphic genetic markers serves as a useful approach to rule out candidate genes, or to justify their more intensive molecular genetic studies. The mouse AE1 gene has a strain-specific CA repeat length polymorphism within intron 13 [12], but the human AE1 gene lacks such an intragenic polymorphic repeat. Moreover, defined genetic markers near the AE1 gene were sufficiently distant so that linkage analysis was confounded by recombination events [5]. We therefore searched for a candidate polymorphic marker more tightly linked to the human AE1 gene. We report our characterization of a highly polymorphic CA repeat tightly linked to the AE1 gene, its evaluation in previously reported families with dRTA, and its diagnostic evaluation in a newly reported family with dRTA. We also present the first immunohistological analysis of kidney from a patient with dRTA secondary to a defined AE1mutation.
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Subjects and methods
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Study population
The subjects included for heterozygosity determination of the studied CA repeat are 157 normal controls from different ethnic groups (50 Czech, 49 Japanese and 58 Thai) and 65 family members of patients with SAO or with HS. Co-segregation of the repeat with disease phenotype was studied in four dRTA families previously reported by our group [2,5] and a previously unreported German family K with an autosomal dominant inheritance pattern of dRTA (Figure 1). In this kindred, neither I:1 nor I:2 were known to have renal disease or short stature, but a brother of 1:1 had nephrocalcinosis. Among family members with documented dRTA (grey symbols), II:1 and II:2 had short stature, nephrolithiasis and chronic pyelonephritis, the well-known complications of dRTA; III:2 and III:5 have renal insufficiency; and III:3 has short stature and nephrolithiasis. No informative clinical data were available for IV:1.

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Fig. 1. Pedigree of a German K family with dRTA. Grey symbols represent complete dRTA. Current age or age at death are shown underneath each symbol. Codon 589 status (R/R is wild-type, R/H is heterozygous for the disease mutation) is shown inside each symbol. See text for clinical details.
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Characterization of the dinucleotide repeat
Genomic DNA was prepared from peripheral blood sample buffy coats. Phage P1-derived artificial chromosome (PAC) HRPC1067M6 was purchased from the Roswell Park Cancer Institute. PCR primers D17SCS-1 and D17SCS-2 were selected to amplify a 220 bp product encompassing the CA repeat region (Figure 2). Hot start PCR was performed for 3035 cycles consisting of 45 s denaturation at 94°C, 5 min annealing at 57°C, and 2 min extension at 72°C. Final extension was 10 min at 72°C. PCR products were directly sequenced with an ABI 373 DNA sequencer. [32P]dCTP-labelled PCR products were fractionated on 8% polyacrylamide urea gels, with M13 partial reactions as size standards. CA repeats were counted in select genomic DNAs from the DNA sequence, and these were used to calibrate the PAGE autoradiograms, in which the most prominent band of the stutter was considered to represent fragment length.

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Fig. 2. (A) DNA sequence of the PCR amplimer surrounding the CA repeat region cloned from the PAC containing the AE1 gene. The oligonucleotides D17SCS-1 and D17SCS-2 used for PCR amplification studies are underlined at the sequence termini, and enlarged in (B). This clone contains 13 CA repeats. The shaded region in (A) comprises part of STS D17S1183 (GenBank database: 344800).
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Analysis of AE1 gene
Using genomic DNA of members in family K as the template, all coding exons and exonintron boundaries of the AE1 gene were PCR-amplified and analysed for single-strand conformation polymorphism (SSCP) as described previously [5]. The DNA sequence of PCR products with shifted mobility was determined on both strands using the dideoxy termination method on an ABI 373 DNA sequencer.
Immunocytochemistry
Paraffin sections of 8 µm were cut on a Leica Autocut 8800 microtome. One paraffin block from 1992 was from dRTA patient II:2 (Figure 1). A control paraffin block of the same age was of apparently normal kidney adjacent to a resected renal carcinoma. Affinity-purified anti-AE2 C-terminal antibody that recognizes only AE1 in SDS-untreated kidney sections has been described previously [13]. The monoclonal antibody against the 31 kDa common subunit of bovine renal vH+-ATPase (E31 hybridoma supernatant) was the gift of S. Gluck (Universuty of Florida) [13]. Sections were deparaffinized in xylene, and rehydrated in a graded series of ethanol solutions, finishing in phosphate-buffered saline. Primary antibodies were incubated at 20°C for 2 h. Peroxidase-coupled or alkaline phosphatase-coupled secondary anti-rabbit and anti-mouse antibodies (Jackson Immuno-Research, Westport, PA) were incubated at 20°C for 45 min, then developed by enhanced chemiluminescence or with bromochloroindenyl-phosphate and nitro-blue tetrazolium, respectively. Stained sections were viewed with an Olympus BH2 epifluorescence photomicroscope, photographed on 400 ASA Tmax film (Kodak), and push-processed at 1600 ASA. Negatives were scanned in transmission mode for printing of positive images.
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Results
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A CA repeat length polymorphism adjacent to the human AE1 gene
Figure 2 shows the DNA sequence of the PCR amplimer encompassing the CA repeat region noted in the PAC HRPC1067M6 (GenBank accession no. AC003043). This PAC exhibited 13 CA repeats. Figure 3 shows the location of this CA repeat region at nucleotides 30 82930 856 from the telomeric end. It lies
90 kb from the 5' end of the AE1 gene, separated from it by the genes coding for human epithelin/granulin, the mitochondrial carrier protein CGI-69 and the RAP2-interacting protein 8 (RPIP8).

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Fig. 3. Schematic of PAC HRPC1067M6 from the Roswell Park human genomic library. The 139 488 bp PAC contains at its 3' centromeric end most of the AE1 (band 3) gene. Identified genes telomeric to the AE1 gene, denoted within or beneath the bar, include RAP2-interacting protein 8 (RPIP), mitochondrial carrier protein CGI-69, the putative growth factor epithelin, and platelet glycoprotein IIb (gpIIb), and four hypothesized transcripts of unknown function. Distances (in kb) from the telomeric end of the PAC are noted underneath the bar. Distances (in kb) from the centromeric end of the 1.29 Mb contig NT_010755 are shown above the bar. The CA repeat region is located at 30.8 kb from the telomeric end and between GpIIb and epithelin, 90 kb from the 5' end of the AE1 gene.
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We searched for length polymorphism in this CA repeat by PCR amplification of genomic DNA samples. Many individuals are heterozygous, with one allele often containing 13 CA repeats, and a second allele with a larger number of repeats. Figure 4 shows the distribution of CA repeat numbers in normal controls from Czech (Figure 4A), Japanese (Figure 4B) and Thai populations (Figure 4C). Among all groups, the prevalence of heterozygosity for CA repeat numbers is 5056%. The most common allele has 13 CA repeats. The majority of other alleles exhibit between 19 and 27 CA repeats, but a total of 14 allelic variants were detected. The calculated heterozygosity value of this polymorphic (CA) repeat was 0.56 among the unrelated normal individuals from the three tested populations. The distribution of CA repeat lengths among affected and unaffected individuals from SAO families, and HS families with various heterozygous AE1 mutations did not differ from these normal controls (data not shown).

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Fig. 4. Allele frequency determination of the CA dinucleotide repeats and analysis in the German K family. Histograms of repeat numbers found among healthy unrelated residents of (A) the Czech Republic, (B) Thailand and (C) Japan. The percentage of individuals exhibiting heterozygosity for the length polymorphism is noted in the upper portion of each graph.
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CA repeat length polymorphism in distal renal tubular acidosis
In three previously reported families with the AE1 R589H mutation associated with dominant dRTA [5], the mutation co-segregated with the short, common (CA)13 repeat. In a previously reported family with the AE1 G701D mutation associated with recessive dRTA [2], the two affected children were haploidentical for this repeat. The paternal mutant allele co-segregated with the short (CA)13, whereas the maternal mutant allele co-segregated with the long (CA)25 repeat (data not shown).
In the new family K (Figure 1), disease phenotype co-segregated with the (CA)14 repeat, suggesting the AE1 gene as the cause of dominant dRTA in this kindred (Figure 5B). Subsequent SSCP analyses revealed AE1 exon 14 heterozygosity as a potential mutation site in affected individuals (Figure 5A). DNA sequencing confirmed the presence of the G1766A substitution encoding the missense mutation R589H reported previously to be associated with dominant dRTA [4,5].

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Fig. 5. (A) SSCP analysis of AE1 exon 14 in the dRTA family. The autoradiograph illustrates the pattern associated with the R589H mutation. Genotype was verified by DNA sequencing of AE1 exon 14 of all individuals, including IV:1. No additional mutations were found in the complete exonic sequence of the AE1 gene of III:5. (B) CA repeat analysis in the dRTA family. The shorter allele of (CA)14 is present in all members carrying the AE1 R589H mutation, but is absent from family members without the mutation.
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Pathological examination of the kidney from proband II:2
Proband (II:2) of family K underwent a right nephrectomy, then 1 month later a left nephrectomy at the time of a radical resection of highly differentiated, verrucous, squamous cell carcinoma of the bladder infiltrating the bladder wall and perivesical fat, and with epidermoid metaplasia and atypia of the adjacent bladder epithelium. Both kidneys exhibited double collecting systems with chronic, scarring pyelonephritis. The left kidney was also remarkable for pronounced parenchymal atrophy and nephrocalcinosis, areas of pseudostroma, and partly obliterating nephroangiosclerosis with renal arteriosclerosis. The right kidney exhibited in addition acute pyelonephritis and abscess formation with ureteritis, and a highly differentiated renal adenoma 0.9 cm in diameter. A paraffin block of formaldehyde-fixed right kidney cortex was retrieved for immunohistochemical study, along with a similarly fixed paraffin block of diagnostically normal kidney adjacent to a renal cell carcinoma resected from a different patient. The blocks were fixed identically, and were of approximately the same age.
Immunohistochemical study of AE1 R589H dRTA kidney
Human kidney biopsies from patients with sporadic primary dRTA have been reported to be marked frequently by deficiency or absence of vH+-ATPase and of AE1. Figure 6a shows AE1 staining of type A intercalated cell basolateral membranes in initial cortical collecting duct or connecting segment from the normal control tissue block. This staining was competed by excess peptide antigen (Figure 6c). In contrast, the right kidney from dRTA patient II:2, with interstitial thickening and dilated tubules, exhibited heavy staining that did not follow cell boundaries (Figure 6b). This staining was not competed by excess peptide antigen (Figure 6d), and thus represented non-specific trapping of peroxidase product in the areas of nephrocalcinosis. In contrast, erythrocytes exhibited AE1 immunoreactivity (Figure 6a) which was specifically competed by peptide antigen (Figure 6c). Thus, the dRTA kidney lacked the immunohistochemical evidence of kidney AE1.

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Fig. 6. Absence of AE1 from kidney cortex of the dRTA patient II:2. AE1 immunoperoxidase staining of formalin-fixed paraffin-embedded kidney from II:2 (b and d), who underwent kidney resection in 1992 for chronic pyelonephritis and nephrocalcinosis in the setting of bilateral double collecting system, squamous epithelial carcinoma of the bladder and renal adenoma. Control kidney tissue from 1992 (a and c) was treated similarly. Tissue was immunostained with antibody that detects the C-terminal peptide of AE1, in the presence of irrelevant peptide (a and b) or of peptide antigen (c and d). Note the specific staining of intercalated cell basolateral membranes in control tissue (arrows in a). Erythrocytes are also specifically stained (asterisks in a). In contrast, the intense staining in dRTA tissue (b and d) does not follow cellular boundaries, and cannot be competed by peptide antigen.
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This non-specific AE1 staining did not allow identification of intercalated cells in the scarred dRTA kidney sections, in which tubular segment identification was more difficult. Therefore, we examined sections for vH+-ATPase in Figure 7. Figure 7a, c and e shows in representative control sections intense staining of collecting duct intercalated cells either at the apical pole or diffusely throughout the intercalated cells. Much less intense immunostaining was evident in proximal tubular cells at the apical surface. Figure 7b and f shows that vH+-ATPase-positive putative intercalated cells remained detectable (perhaps in lower number) in the dilated tubules of the dRTA kidney. Curiously, most putative collecting ducts of the dRTA kidney exhibited apparent vH+-ATPase staining at the apical surface of nearly all cells. Moreover, vH+-ATPase staining in the dRTA kidney was also greatly enhanced at the apical surface of putative proximal tubules (lacking diffusely stained intercalated cells; Figure 7b and d), compared with control sections. Immunostaining with irrelevant monoclonal antibody of similarly prepared paraffin sections of additional archival blocks from normal and scarred human kidney failed to exhibit this staining (not shown). Specific secondary donkey anti-mouse peroxidase-conjugated Ig in the absence of primary antibody produced no staining.

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Fig. 7. The presence of vH+-ATPase in type A intercalated cells and the increase in proximal tubule in dRTA patient II:2. Anti-vH+-ATPase immunoperoxidase staining in three fields of control kidney (left panels a, c and e), and in three fields of kidney from patient II:2 (right panels b, d and f). In normal kidney, vH+-ATPase is abundant in intercalated cells of collecting duct (CD) and at much lower level in proximal tubule (PT). The dRTA kidney exhibits dilated tubules, luminal debris and apparent interstitial scarring, rendering tubule segment identification more difficult. Putative intercalated cells remain present in apparent CD of dRTA tissue (b and f) but may be less numerous than in control kidney. vH+-ATPase abundance is greatly increased in putative PT cells of dRTA kidney (b, d and f).
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Discussion
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We have described a highly polymorphic CA repeat marker located
90 kb telomeric to the human AE1 gene on chromosome 17. More than half of individuals from various ethnic groups are heterozygous for allele repeat length, and at least 14 allelic variants exist. The CA repeat polymorphism confirmed AE1 linkage to dRTA in four previously published families, and co-segregated with dRTA and the AE1 R589H mutation in the affected family newly reported here. After completion of this work, we learned that the same region of CA repeat had been contributed to the NCBI database previously as STS D17S1183 (GenBank database: 344800) by Miki et al. [14]. They screened 40 unrelated CEPH individuals and reported eight alleles of D17S1183, with a heterozygosity value of 0.50. Our current studies, performed in different ethnic populations in which AE1 mutations have been found, extend their results, and verify the diagnostic utility of D17S1183 for linkage analysis of the AE1 gene.
In one AE1 R589H dRTA patient with nephrocalcinosis and scarring from chronic pyelonephritis, AE1-negative intercalated cells were present (perhaps in lower numbers), and vH+-ATPase exhibited increased abundance in proximal tubule, and in principal or connecting tubular cells. This is the first reported immunohistological analysis of kidney tissue from a patient with familial dRTA caused by a defined mutation.
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Evolution of CA repeats
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The CA repeat allele of highest prevalence was (CA)13. Interestingly, no alleles longer than (CA)29 were found either among 161 normal unrelated individuals of three ethnic backgrounds or among 65 individuals from families with AE1 mutations associated with SAO or with HS anaemia. The evolutionary age of this CA repeat length polymorphism is unclear. The genes present on PAC HRPC1067M6, from human chromosome 17q2122, are arrayed in the same order on mouse chromosome 11. A (CA)25 repeat is present
880 nucleotides centromeric from the 3' end of the mouse epithelin gene. Perfect repeats of a (CA)25 repeat with distinct flanking sequences are found in introns of the adjacent murine LOC217219 gene, but the flanking regions of none of these dinucleotide repeats are homologous with those of the human CA repeat reported here.
The mechanism of repeat generation and amplification must account for the paucity of alleles with 1519 repeats, and the higher prevalence of alleles with
20 repeats. This might suggest one or more ancient duplications of a (CA)13 or (CA)14 allele, followed by sporadic deletions of one or more repeats. In cultured cell transgene models, the rate of insertion or deletion in CA repeats was 100-fold higher than in the rest of the transgene. Dinucleotide repeat mutation rates were also higher than those of tetranucleotide repeats. Single dinucleotide repeat insertion rates exceeded single-repeat deletion rates in engineered tracts of 17 or 30 repeats.
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Utility of the CA repeat in evaluation of familial dRTA
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The CA repeat described in this work is only 90 kb centromeric to the 5' end of the human AE1 gene. It is thus close enough so that recombination is unlikely to diminish its utility. The length variation of this repeat is highly polymorphic. However, the (CA)13 allele represents >50% of all alleles. The utility of the repeat as a diagnostic aid will thus depend, as true for any polymorphic marker, on its informativeness within the family in question. In family K presented here and, retrospectively, in the dRTA families described previously [2,5], the marker was useful to justify further analysis of the AE1 gene. The marker's greatest utility might be to rule out the AE1 gene as a candidate disease gene with a high degree of certainty. The completion of the human genome has lessened the importance of linkage analysis in the consideration of candidate genes. However, the addition of highly polymorphic markers to this region of the genome may nonetheless prove helpful for mapping studies. It remains possible, however, as recently reported for the dinucleotide repeat polymorphism in the murine MMP-9 gene, that repeat length may correlate with disease severity [15].
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Immunohistopathology of dRTA
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The first immunohistochemical case report of dRTA reported the absence of vH+-ATPase staining in intercalated cells of renal biopsies from patients with Sjogren's syndrome, while vH+-ATPase staining was preserved in systemic lupus erythematosus-associated hyperkalaemic dRTA, and only modestly decreased in the dRTA of transplant rejection. Cho et al. [16] also showed immunohistochemical and electron microscopic evidence of diminished vH+-ATPase and AE1 immunoreactivity in another case of dRTA with acute rejection superimposed on chronic allograft nephropathy. Han et al. [17] recently reported immunohistological study of renal biopsies from 11 patients with clinical diagnoses of primary dRTA. Family histories, however, were not mentioned in the study. AE1 immunostaining was undetectable in seven of the 11, and of reduced intensity in three more, while vH+-ATPase immunostaining was characterized as minimal or absent in intercalated cells of three of the 11, and greatly reduced in seven more. Carbonic anhydrase II immunostaining was reduced in all patients.
Immunostaining of the scarred kidney with dilated tubules from patient II:2 of family K revealed undetectable AE1 and reduced vH+-ATPase in putative intercalated cells. These appeared to be reduced in number, reminiscent of the decline in intercalated cell number observed in rats chronically treated with acetazolamide, and of their gradual disappearance in CAR2(/) mice. A novel finding was the increase of vH+-ATPase staining intensity in the lumenal pole of apparent proximal tubular cells. Comparable intensity of proximal staining was not noted in the archival control sample. This finding in hereditary dRTA contrasted with systemic lupus-associated dRTA, in which proximal tubular brush border immunostaining with vH+-ATPase was substantially decreased. We speculate that this increase in apparent proximal tubular vH+-ATPase protein represents a compensatory increase in proximal urinary acidification in response to an AE1 defect of the collecting duct. However, the co-existing extensive, severe, inflammatory and obstructive conditions complicate interpretation of the immunohistological findings in this patient.
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Intercalated cells of human cortical collecting duct
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Kidney AE1 observed by immunocytochemistry was first reported in rabbit kidney by Schuster et al. [18], and thereafter in human, rat and mouse. The rabbit cortical collecting duct (CCD) normally reabsorbs protons, and type A intercalated cells are a small minority of total intercalated cells in rabbit CCD. In contrast, type A intercalated cells comprise
4555% of total intercalated cells in rat and mouse [19]. Curiously, however, Wagner et al. noted that 48% of all human CCD cells were AE1 positive [20]. Since intercalated cells comprised only
50% of total CCD cells, they concluded that nearly all intercalated cells of human CCD may be of type A. Our control cortex sections are consistent with this impression, but Biner et al. warn that considerable inter-individual variation exists among human kidneys derived from hospitalized patients who have, in addition to a range of disease, usually been exposed to a wide and uncontrolled range of medications, which may alter the cell spectrum through injury or apoptosis [21]. The absence to date of a published immunohistological census of CCD cell types in human kidney only highlights its importance for the future.
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Acknowledgments
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We thank Professors Kerstin Amann and Eberhard Ritz (University of Heidelberg) for tissue blocks and discussion. This work was supported by NIH grants DK43495 (S.L.A.) and DK34854 (the Harvard Digestive Diseases Center). C.S. is currently supported by Thai Research Foundation and Siriraj Grant for Research Development.
Conflict of interest statement. None declared.
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Received for publication: 11. 6.03
Accepted in revised form: 17. 9.03