title: Role of NKCC in BK channel-mediated net K⁺ secretion in the CCD.
abstract: Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (J(K)) and Na absorption (J(Na)) at slow (∼1) and fast (∼5 nl·min(-1)·mm(-1)) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal J(K), J(Na), or the flow-induced [Ca(2+)](i) transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce J(K). Basolateral Cl removal reversibly inhibited FIKS but not basal J(K) or J(Na). Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH(4)(+) at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH(4)(+) addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH(4)(+) entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.
authors.affiliation: Division of Pediatric Nephrology, Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Carlos
authors.initials: C
authors.lastname: Schreck
authors.forename: Richard A
authors.initials: RA
authors.lastname: Coleman
authors.forename: James B
authors.initials: JB
authors.lastname: Wade
authors.forename: Yubelka
authors.initials: Y
authors.lastname: Hernandez
authors.forename: Beth
authors.initials: B
authors.lastname: Zavilowitz
authors.forename: Richard
authors.initials: R
authors.lastname: Warth
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Satlin
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000502:physiology*; D008297:Male; D009399:Nephrons / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
chemical_list: D002412:Cations; D004232:Diuretics; D005456:Fluorescent Dyes; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; C064719:iberiotoxin; D011188:Potassium; D002118:Calcium