Loss of kAE1 expression in collecting ducts of end-stage kidneys from a family with SLC4A1 G609R-associated distal renal tubular acidosis

Abstract

Distal renal tubular acidosis caused by missense mutations in kidney isoform of anion exchanger 1 (kAE1/SLC4A1), the basolateral membrane Cl^-/HCO₃^- exchanger of renal alpha-intercalated cells, has been extensively investigated in heterologous expression systems but rarely in human kidneys. The preferential apical localization of distal renal tubular acidosis (dRTA)-associated kAE1 mutants R901X, G609R and M909T in cultured epithelial monolayers has not been examined in human kidney. Here, we present kidney tissues from dRTA-affected siblings heterozygous for kAE1 G609R, characterized by predominant absence rather than mistargeting of kAE1 in intercalated cells. Thus, studies of heterologous recombinant expression of mutant proteins should be, whenever possible, interpreted in comparison to affected patient tissues.

Keywords: distal renal tubular acidosis; genetics; kAE1; kidney isoform of anion exchanger 1; slc4a1.

Fig. 1.

Pedigree of dRTA family. (A) The heterozygous kAE1/SLC4A1 mutation G609R was detected in affected family members (filled symbols), but not in unaffecteds (open symbols). SLC4A1 genotype status is listed under symbols. (B) The heterozygous SLC4A1 mutation c.1825G>A encoding the SLC4A1/AE1 missense substitution G609R was detected in the proband (III:1) by DNA sequencing of both strands of PCR-amplified cDNA prepared from total RNA isolated from whole blood, as previously described [23]. The cosegregating mutation was validated in genomic DNA of other family members by sequencing across SLC4A1 exon 15, as previously described [24]. Written consent was obtained under protocols approved by the Clinical Investigation Committees of Yale University School of Medicine and Beth Israel Deaconess Medical Center.

Fig. 2.

kAE1 immunoperoxidase localization in kidney sections from ‘normal’ patients and dRTA patients carrying the heterozygous kAE1 missense mutation G609R. (A) Basolateral kAE1 staining of intercalated cells (asterisks) shown in longitudinal and oblique section of collecting ducts from a non-cancerous, ‘normal’ kidney. Arrows indicate eAE1-stained erythrocytes in capillaries. (Inset) Basolateral kAE1 staining of intercalated cells (asterisks) in a transverse section of collecting duct from a kidney resected for renal cancer. (B) Kidney section from patient III:3, processed in the absence of primary anti-AE1 antibody, serving as control for diffuse background staining in other panels. (C and D) Kidney sections from patient III:3 (C) and from patient III:4 (D) stained with AE1 antibody, but not detecting immunostaining pattern characteristic of α-IC cells. Arrows indicate eAE1-positive erythrocytes in capillaries. Formalin-fixed, paraffin-embedded kidney tissue from prior nephrectomies were studied with written consent from proband's half-sister and her mother. Frozen sections were unavailable. Two-micrometer sections on polysine-coated slides (Fisher, Atlanta, GA, USA) were deparaffinized and rehydrated. Mounted sections were subjected to antigen retrieval (10 mM citric acid, pH 6.0, for 30 min at 90°C). Endogenous peroxidase was quenched with 3% H₂O₂ for 15 min. Sections were blocked with 2.5% normal horse serum at room temperature for 40 min, then incubated 40 min with 1:200 dilution of peptide affinity-purified antibody SA6 to mouse AE2 C-terminal aa 1224–1237 [26, 27] that cross-reacts with human kAE1 in conditions in which immunostaining of human AE2 is minimal [–30]. Specific immunolabeling was detected with the ImmPRESS HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA). Sections were developed with diaminobenzidine, counterstained with hematoxylin, dehydrated and mounted in Permount. Negative control sections were incubated without primary antibody. Scale bars, 50 µm.

Fig. 3.

Immunoperoxidase localization of kAE1 and vH-ATPase in kidney sections from dRTA patient III:3. (A and C) Non-representatative regions of tissue in which AE1 staining (diaminobenzidine substrate, Vector Laboratories) is evident in diffuse distribution in occasionally encountered intercalated cells (asterisks). Less frequently encountered are candidate intercalated cells with basal-apical or apical enhancement of kAE1 staining (arrows in A). Red blood cells are evident in interstitial spaces. (B and D) Near-consecutive sections showing intercalated cells with diffuse vH⁺-ATPase staining (NovaRed substrate, Vector Laboratories) with occasional apical enhancement. Asterisks indicate kAE1-positive cells in (A) and (B). Apical enhancement of vH⁺-ATPase staining is also evident in other collecting duct cells. (E) Unaffected ‘control’ kidney stained for vH⁺-ATPase. Scale bars: 20 µm (A–D) and 50 µm (E).