Distal Renal Tubular Acidosis in Mice Lacking the AE1 (Band3) Cl⁻/HCO₃⁻ Exchanger (slc4a1)

Measurement of Cl⁻/HCO₃⁻ exchanger activity in single type A intercalated cells (A-IC) in isolated outer medullary collecting duct (OMCD). (A and B) Representative original tracings of intracellular pH measurements (BCECF ratio fluorimetry) in the presence of HCO₃⁻/CO₂ and the alternating presence or absence of extracellular chloride. Addition of chloride caused a rapid acidification as a result of activation of Cl⁻/HCO₃⁻ exchange activity. The intracellular acidification rate was reduced by 400 nM of the AE1 inhibitor diBA(5)C4. Intracellular acidification was partially reduced in A-IC from Ae1−/− mice, and the remaining activity was insensitive to diBA(5)C4. (C) Summarized data from all experiments using the inhibitors diBA(5)C4 (400 nM) or DIDS (50 μM; n = 4 to 5 mice with five to six OMCD for each condition and 100 to 150 A-IC each). *P < 0.001 versus wild-type.

Expression of anion exchangers in kidney. (A) reverse transcriptase–PCR with primers detecting specific isoforms of several anion exchanger mRNA as well as KCC4 and aquaporin-6 (AQP6), two transport proteins implicated in A-IC function, did not reveal consistent changes in mRNA abundance in kidneys from Ae1−/− mice. β-Actin was used as a reference transcript. (B) Immunoblot showed that abundance of AE4/Slc4a9 polypeptide was not changed in kidneys from Ae1−/− mice. The abundance of pendrin/Slc26a4 anion exchanger polypeptide was strongly reduced in kidneys from Ae1−/− mice.

Severe urinary concentrating defect in Ae1−/− mice. (A) Under control conditions, urine volume normalized to body weight was approximately three-fold elevated in AE1-deficient mice and remained two-fold elevated during the severe dehydration produced by acid loading (see serum osmolarity, Table 2). (B) Urine osmolarity was much lower in AE1-deficient mice and did not increase during acid load–induced dehydration. (C) The acid load–induced loss of body weight in Ae1−/− mice was greatly increased. *P < 0.001 versus wild-type.

Dysregulation of AQP2 water channel in kidney of AE1-deficient mice. Immunoblots reveal increased AQP2 protein abundance in kidney cortex (A) and medulla (B) of Ae1−/− mice in control conditions. (C) Confocal immunofluorescence microscopy of outer medulla (top) shows increased AQP2 immunostaining (green) with strong apical localization in AE1-deficient mice. In contrast, in the inner medulla of Ae1−/− kidneys (bottom), AQP2 staining was confined mainly to intracellular vesicular structures, whereas AQP2 in wild-type kidney was predominantly luminal. AE1 staining is in red. Magnification, ×600.