Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium, and the glomerular afferent arteriole.

Abstract

Na-K-Cl cotransport plays an important role in the kidney in NaCl reabsorption in the thick ascending limb of Henle and a less well defined role in the inner medullary collecting duct (IMCD). Two Na-K-Cl cotransporters encoded by different genes have been identified in the mammalian kidney: BSC1/NKCC2 which localizes to the apical thick ascending limb of Henle and BSC2/NKCC1 which was isolated from a mouse IMCD cell line (mIMCD-3) but its localization has not been determined. In this study we generated a polyclonal antibody (anti-mBSC2) against the mouse BSC2/ NKCC1 protein in order to characterize and localize this protein in mouse kidney. Western blot analysis with affinity-purified anti-mBSC2 showed a protein doublet of 140 and 150 kD which was most abundant in the renal papilla but also seen in cortex and outer medulla.

Expression of the bumetanide-sensitive Na-K-Cl cotransporter BSC2 is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium.

“…This selective pattern of induction of BSC2 mRNA suggests that expression of the BSC2 gene can be differentially regulated at the tissue level, perhaps reflecting tissue-or cellspecific factors yet to be defined. In a recent report (15), positive immunofluorescence staining of constitutively expressed BSC2 protein was confined to collecting duct epithelium, glomerulus, and afferent arteriole in the unstimulated murine kidney, while most vascular structures did not display significant amounts of BSC2. Current efforts are directed at defining the cellular distribution of inducible BSC2 protein in organs such as the lung and kidney after systemic inflammatory activation.…”

“…These were fixed and permeabilized with cold methanol ( Ϫ 20 Њ C) for 5 min and incubated with the primary antibody consisting of an affinity purified polyclonal rabbit antiserum directed against the carboxy (intracellular) terminus of the murine BSC2 molecule expressed as a fusion protein (kindly provided by E. Delpire, Children's Hospital, Boston, MA). This antisera is specific for the BSC2 isoform and does not cross-react with the BSC1 molecule (15). The monolayers were then washed and incubated with goat anti-rabbit IgG conjugated to FITC.…”

“…A second gene, BSC2 (or NKCC1), encodes a more widely expressed cotransporter. This molecule (also known as the secretory form) is thought to function in intracellular volume regulation in a variety of cell types, as well as more specialized functions such as salt and water secretion across respiratory, sweat gland, and salivary epithelia (1,8,15). Both of these molecules are members of the large superfamily of 12 membrane-spanning transporters whose members subserve diverse functions ranging from the transport of inorganic and organic ions to the regulation of macrophage activation and antimicrobial activity (1,16).…”

Active sodium-urea counter-transport is inducible in the basolateral membrane of rat renal initial inner medullary collecting ducts.

“…Ϫ cotransporter: (i) NKCC2 (BSC1) in the apical membrane of the thick ascending limb (30,31); and (ii) NKCC1 (BSC2) in the basolateral membrane of the IMCD (23,32,33). In isolated IMCDs (34) and cultured IMCD cells (35), NKCC1 (BSC2) is thought to participate in NaCl and/or fluid secretion, as it does in the gastric mucosa (36).…”

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

“…Regulation of cell volume is a major function of NKCC1 (3), and in vascular endothelial cells, it may play an important role in controlling cytosolic concentrations of Cl Ϫ and K ϩ during agonist stimulation (10). In epithelial cells, it is restricted to basolateral membranes (11,12), and there is strong pharmacological evidence that it provides an entry pathway for ions that are secreted across the apical membrane.…”