Distal convoluted tubule Cl-concentration is modulated via K+ channels and transporters

Cl-sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-K2 or K+-Cl- cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]_i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl-]_i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl-]_i was ~7 mM. Stimulation of Cl- exit with low-Cl- hypotonic solutions decreased [Cl-]_i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl-]_i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl-]_i. Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl-]_i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]_i, whereas raising extracellular K+ transiently increased [Cl-]_i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]_i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl-]_i in the DCT.