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

Abstract

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.

Keywords: K+-Cl− cotransporter; Na+-Cl− cotransporter; chloride; chloride channel; potassium channel.

Fig. 1.

Kidney-specific Cl-Sensor mouse model. A: transgenic strategy used to obtain Cl-Sensor expression in renal tubule epithelial cells (see text for description). B: example of a microdissected distal convoluted tubule (DCT; left). Yellow fluorescent protein (YFP) emission signal (right) obtained from the DCT on the left. Two to five regions of interest were defined in each DCT, and a dim region of the field of view (box) was selected as background.

Fig. 2.

Cl-Sensor calibration in the microdissected distal convoluted tubule (DCT). A: example calibration experiment. Cl-Sensor responded to changes in Cl⁻ concentration near baseline levels in DCT cells. B: calibration curve fitted from the average of 7 calibration experiments. C: estimated intracellular Cl⁻ concentration ([Cl⁻]_i) was 7 mM (SD = 9.4) in DCT cells bathed in 5 mM K⁺ solution (n = 18).

Fig. 3.

ClC-Kb channel and K⁺-Cl⁻ cotransporter (KCC) mediate Cl⁻ extrusion in the basolateral membrane of distal convoluted tubule (DCT) cells. A: typical trace showing that low-Cl⁻ hypotonic stress (LCHS; 80% of baseline recording solution diluted with water) decreases the fluorescence intensity ratio of Cl⁻ (R_Cl). B: summary data of LCHS. Average R_Cl decreased by 2.1% after isolated tubules were bathed in low-Cl⁻ hypotonic solution [1.03 (SD: 0.12) vs. 1.01 (SD: 0.12) at baseline, n = 31, P < 0.001, paired t test]. C: typical trace showing when KCCs were blocked with 100 µM DIOA, R_Cl increased in DCT cells. D: summary data of KCC blockage. Average normalized R_Cl increased by 170% upon KCC blockade [2.81 (SD: 1.38) vs. 1.04 (SD: 0.21) at baseline, n = 26, P < 0.001, paired t test]. E: typical trace showing that when ClC-K2 channels were blocked with 10 µM NPPB, R_cl increased in DCT cells. F: summary data of ClC-K2 channel blockage. Average R_Cl increased by 142% upon ClC-K2 channel blockade [2.42 (SD: 0.73) vs. 1.00 (SD: 0.14) at baseline, n = 25, P < 0.001, paired t test]. Values are means ± SD.

Fig. 4.

Blockade of Kir4.1/5.1 and alterations of extracellular K⁺ concentration change intracellular Cl⁻ concentration. A: example experiment of the fluorescence intensity ratio of Cl⁻ (R_Cl) response to 4 mM BaCl₂ in 5 mM K⁺ baseline solution. B: summary data of the barium response. Average normalized R_Cl increased by 6.4% after application of 4 mM BaCl₂ [1.13 (SD: 0.13) vs. 1.06 (SD: 0.11) at baseline, n = 30, P < 0.001, paired t test]. C: example experiment of the R_Cl response to K⁺ manipulation in bath solution. D: summary data of peak R_Cl after extracellular K⁺ manipulation compared with baseline [0.96 (SD: 0.11) at 5 mM, 0.94 (SD: 0.10) at 2 mM, 0.99 (SD: 0.10) at 10 mM, n = 21, P < 0.001, paired t test]. Values are means ± SD.