Altered bicarbonate‐dependent intracellular pH (pHi) regulation in a cortical collecting duct (CCD) principal cell line from a mouse model of autosomal recessive polycystic kidney disease (ARPKD)

Abstract

ARPKD in the kidney primarily affects the collecting ducts and leads to tubule dilation, altered cell polarity and morphology, as well as dysfunctional ion transport that promotes Na+ hyperabsorption. Little is known about dysfunctional ion transport associated with pHi regulation. We used fluorescence imaging with the pH‐sensitive dye BCECF to examine the regulation of pHi in a cilium‐deficient (“mutant”) CCD principle cell line derived from a mouse model of ARPKD that lacks the Tg737 gene encoding the ciliary protein Polaris, as well as a cilium‐competent (“rescued”) cell line transfected with Tg737 cDNA. We characterized HCO3−‐dependent pHi regulation following NH4+ prepulse‐induced acid loads by exposing both the apical and basolateral membranes of polarized monolayers simultaneously to 5% CO2/22 mM HCO3– in the presence or absence of 200 μM DIDS, and in the presence of 50 μM HOE‐642 to eliminate Na‐H exchanger activity. As previously reported, HOE‐642 nearly blocked the pHi recovery from an acid load in both cell monolayers bathed in HEPES‐buffered solutions. However, subsequently switching to the HCO3− solution stimulated a pHi recovery that was slower in the mutant vs. rescued cells. During pHi recoveries, total acid extrusion in the pHi range 6.33–7.13 was 29–45% less in the mutant vs. rescued cells; DIDS‐insensitive acid extrusion was similar. DIDS‐sensitive acid extrusion in the pHi range 6.40–6.90 was reduced 37–49% in the mutant vs. rescued cells. Applying DIDS selectively to the basolateral but not apical membrane of rescued cells inhibited the HCO3−‐induced pHi recovery. Reduced HCO3−‐dependent acid extrusion may contribute to Na+ hyperabsorption in this principal cell model of ARPKD. NIH DK067343, NIH T35 HL007473