Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells.

Thiazides block Na reabsorption while enhancing Ca reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl channels, however the nature of these Cl channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl current at extracellular pH7.4. This constitutive Cl current was more permeable to larger anions (Eisenman sequence I; I>Br≥Cl) and was substantially inhibited by >100mM [Ca], which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl current was blocked by NPPB, along with other Cl channel inhibitors (4,4'-diisothiocyanatostilbene-2,2'-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH<5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl current. This acid-induced Cl current was also anion permeable (I>Br>Cl), but was distinguished from the constitutive Cl current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl current would be particularly relevant in the acidic IMCD (pH<5.5). To our knowledge, the properties of these Cl currents are unique and provide the mechanisms to account for the Cl efflux previously speculated to be present in MDCT cells.