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.

NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

“…Furosemide has been shown to increase NKCC2 protein expression, and our recent study showed an increase in NKCC2 protein expression and activity in the TAL of TNF-deficient mice (2,8). Collectively, these findings support the notion that TNF acts as a negative feedback regulator of NKCC2.…”

Endogenous vasopressin regulates Na-K-ATPase and Na+-K+-Cl−cotransporterrbsc-1in rat outer medulla

“…A ϳ160-kDa band was detected predominantly in the outer medulla. Conversely, no signal was found in the inner medulla and weak expression was observed in the renal cortex, findings consistent with previous results (7,13).…”

Hematopoietic Stem Cells Contribute to the Regeneration of Renal Tubules after Renal Ischemia-Reperfusion Injury in Mice

“…Kidney sections were serially stained with an antibody to ␤-galactosidase followed by antibodies or lectins to various renal cell markers (23)(24)(25)(26)(27). Kidney frozen sections (10-m-thick) were fixed with ice-cold 1:1 methanol:acetone for 10 min.…”

“…Thick ascending limbs of loops of Henle (TALH) and distal tubules were stained with an antibody to Tamm-Horsfall Protein (THP: Dr. John Hoyer, Children's Hospital of Philadelphia, Philadelphia, PA) at 1:100 dilution (26). TALH were also stained with an antibody to the sodium/potassium/chloride co-transporter (NKCC2; Dr. Mark Knepper, NIH, Bethesda, MD) at 1:100 dilution (27). Collecting ducts were stained with TRITC-coupled Dolichos biflorus agglutinin (DBA; Vector Laboratories) at 1:40 dilution and an antibody to the aquaporin-2 water channel (AQP2; Dr. Mark Knepper, NIH, Bethesda, MD) at 1:2000 dilution (25).…”