ClC‐K2 Cl ⁻ channel allows identification of A‐ and B‐type of intercalated cells in split‐opened collecting ducts

Abstract

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid‐secreting A‐ and base secreting B‐type of intercalated cells cannot be easily separated in functional studies. We used BCECF‐sensitive intracellular pH (pHi) measurements in split‐opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell‐specific ClC‐K2−/− mice to demonstrate that ClC‐K2 inhibition enables to distinguish signals from A‐ and B‐intercalated cells. We show that ClC‐K2 Cl− channel is expressed on the basolateral side of intercalated cells, where it governs Cl−‐dependent H+/HCO3− transport. ClC‐K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC‐K2−/− mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1‐positive A‐type and decreased pHi in pendrin‐positive B‐type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A‐type and decreased proton transport in B‐type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance. Overall, we show that inhibition of ClC‐K2 can be employed to discriminate between A‐ and B‐type of intercalated cells in split‐opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A‐ and B‐type in response to systemic acid/base stimuli.

Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct

“…Thus, we next tested whether systemic acid or alkali loads can affect [Ca 2+ ] i signaling in response to Piezo1 stimulation in ICs. Mice were kept on NH 4 Cl (280 mM) or NaHCO 3 (280 mM) water for three days to induce metabolic acidosis or alkalosis, respectively, as described previously ( 39 , 40 ). Neither treatment had measurable effect on the Yoda-1-induced [Ca 2+ ] i responses in PCs, when compared to the control conditions ( Fig.…”

“…# F01825G) was injected intraperitoneally 18 h and 2 h prior to experimentation. To induce metabolic acidosis/alkalosis, animals were given water supplemented with 280 mM NH 4 Cl + 0.5% sucrose (acid load) or 280 mM NaHCO 3 + 0.5% sucrose (base load) for three days, respectively, as was described previously ( 39 , 40 ).…”

“…The procedure for isolation of the cortical collecting ducts from mouse kidneys suitable for fluorescence [Ca 2+ ] i measurements and patch clamp electrophysiology closely followed the protocols previously published by our group ( 39 , 51 ). Kidneys were cut into thin slices (<1 mm) with slices placed into an ice-cold bath solution containing (in mM): 150 NaCl, 5 KCl, 1 CaCl 2 , 2 MgCl 2 , 5 glucose, and 10 Hepes (pH 7.35).…”

“…#P10144). The labeled kidney sections were imaged with a Nikon A1R confocal microscope, as we did similarly before ( 39 ). In brief, samples were excited with 405, 640, and/or 561 nm laser diodes and emission captured with a 16 bit Cool SNAP HQ2 camera (Photometrics) interfaced to a PC running NIS elements software.…”