title: Localization and regulation of the rat renal Na(+)-K(+)-2Cl- cotransporter, BSC-1.
abstract: To investigate the role of the thick ascending limb (TAL) Na(+)-K(+)-2Cl- cotransporter in regulation of water excretion, we have prepared a peptide-derived polyclonal antibody based on the cloned cDNA sequence of the rat type 1 bumetanide-sensitive cotransporter, BSC-1 (also termed "NKCC-2"). Immunoblots revealed a single broad 161-kDa band in membrane fractions of rat renal outer medulla and cortex but not from rat colon or parotid gland. A similar protein was labeled in mouse kidney. Immunoperoxidase immunohistochemistry in rat kidney revealed labeling restricted to the medullary and cortical TAL segments. Because long-term regulation of urinary concentrating ability may depend on regulation of Na(+)-K(+)-2Cl- cotransporter abundance, we used immunoblotting to evaluate the effects of several in vivo factors on expression levels of BSC-1 protein in rat kidney outer medulla. Chronic oral saline loading with 0.16 M NaCl markedly increased BSC-1 abundance. However, long-term vasopressin infusion or thirsting of rats did not affect BSC-1 abundance. Chronic furosemide infusion caused a 9-kDa upward shift in apparent molecular mass and an apparent increase in expression level. These results support the previous identification of BSC-1 as the TAL Na(+)-K(+)-2Cl- transporter and demonstrate that the expression of this transporter is regulated.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda 20892, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Ecelbarger
authors.forename: J
authors.initials: J
authors.lastname: Terris
authors.forename: J R
authors.initials: JR
authors.lastname: Hoyer
authors.forename: S
authors.initials: S
authors.lastname: Nielsen
authors.forename: J B
authors.initials: JB
authors.lastname: Wade
authors.forename: M A
authors.initials: MA
authors.lastname: Knepper
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001127:Arginine Vasopressin / Q000494:pharmacology; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D005665:Furosemide / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D028021:Sodium-Potassium-Chloride Symporters; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D028021:Sodium-Potassium-Chloride Symporters; D001127:Arginine Vasopressin; D005665:Furosemide