Regulation by Glucocorticoids of Expression and Activity of rBSC1, the Na+-K+(NH4+)-2Cl−Cotransporter of Medullary Thick Ascending Limb

Abstract

To assess whether glucocorticoids regulate rBSC1, the apical Na ؉ -K ؉ (NH 4 ϩ )-2Cl ؊ cotransporter of kidney medullary thick ascending limb (MTAL), studies were performed in normal rats, adrenalectomized (ADX) rats, and ADX rats infused with dexamethasone for 6 days. The effects of dexamethasone on rBSC1 were also studied in vitro using isolated rat MTAL segments. Cotransport activity was estimated by intracellular pH measurements; rBSC1 protein was quantified in MTAL crude membranes by immunoblotting analysis, and mRNA was quantified by quantitative reverse transcription-polymerase chain reaction. The abundance of rBSC1 protein and mRNA increased in ADX rats infused with dexamethasone compared with ADX rats (p < 0.04). In addition, application of dexamethasone for 1-3 h to MTALs caused rBSC1 protein and mRNA abundance and cotransport activity to significantly increase in a hyperosmotic medium (450 mosmol/kg of H 2 O) containing 0.7 nM arginine vasopressin, which is an in vitro experimental condition that resembles the in vivo MTAL environment. Results obtained in various media and with 8-bromocAMP indicated that stimulation of rBSC1 expression by glucocorticoids required interactions between glucocorticoid receptor-and cAMP-dependent factors. Up to 100 nM d-aldosterone had no effect on cotransport activity in vitro. Thus glucocorticoids directly stimulate MTAL rBSC1 expression and activity, which contributes to glucocorticoid-dependent effects on the renal regulation of acid-base balance and urinary concentrating ability.ϩ )-2Cl Ϫ cotransport is responsible for the apical step of NaCl and ammonia transport by the thick ascending limb (TAL) 1 of the nephron. NaCl and ammonia absorption without water by the medullary TAL (MTAL) causes transepithelial concentration differences of these solutes, which constitutes the "single effects" responsible for NaCl and ammonia accumulation in the renal medulla. This is critical both to the level of renal medullary hyperosmolality and thus to the urinary concentrating ability of the kidney and to urinary ammonia excretion and thus to the renal regulation of acid-base balance (1, 2). The MTAL apical Na ϩ -K ϩ (NH 4 ϩ )-2Cl Ϫ cotransporter (BSC1 (bumetanide-sensitive cotransporter) or NKCC2 (Na ϩ -K ϩ -Cl Ϫ cotransporter)) was recently cloned from rat (3), mouse (4, 5), rabbit (6), and human (7) kidneys. The transporter protein has been localized at the apical membrane of the TAL as well as at the macula densa (8 -10). BSC1 was recently shown to be up-regulated by chronic saline loading (9), restriction of water intake and arginine vasopressin (AVP) administration (11), and metabolic acidosis (12) and down-regulated by potassium depletion (13). However, the stimuli and cellular mechanisms of these adaptations of rBSC1 expression were not specified in the latter in vivo studies.The presence of specific glucocorticoid receptors (GR) in the MTAL has been demonstrated by binding, immunological, and mRNA detection methods (14 -17). A number of studies have suggested that in vivo gl...

Acid pH Increases the Stability of BSC1/NKCC2 mRNA in the Medullary Thick Ascending Limb

“…Indeed, BSC1 mRNA abundance significantly increased 3 h after the creation of metabolic acidosis by peritoneal dialysis, which was followed within 24 h by an augmentation of the BSC1 protein abundance (16). The augmentation of both mRNA and protein abundance persisted after 6 d of CMA induced by administration of NH 4 Cl in drinking water (16). Most important, we showed that incubation of freshly harvested MTAL fragments in an acid medium enhanced BSC1 mRNA and protein abundance (16).…”

“…Most important, we showed that incubation of freshly harvested MTAL fragments in an acid medium enhanced BSC1 mRNA and protein abundance (16). Furthermore, acid incubation stimulated BSC1 transport activity assessed in intact cells as well as in apical membrane vesicles, which was dependent on gene transcription and protein synthesis (16). These observations established acid pH as a potent regulator of BSC1 expression and explain, at least in part, the stimulation of BSC1 expression during CMA.…”

“…The augmentation of both mRNA and protein abundance persisted after 6 d of CMA induced by administration of NH 4 Cl in drinking water (16). Most important, we showed that incubation of freshly harvested MTAL fragments in an acid medium enhanced BSC1 mRNA and protein abundance (16). Furthermore, acid incubation stimulated BSC1 transport activity assessed in intact cells as well as in apical membrane vesicles, which was dependent on gene transcription and protein synthesis (16).…”

“…The method used to isolate MTAL fragments has been previously described in detail (17,18). Because we have previously shown that glucocorticoids regulate BSC1 expression (19), MTAL fragments were harvested from rats that were adrenalectomized (ADX) and given isotonic saline as drink-ing water 6 d before the experiments to avoid any interference caused by endogenous glucocorticoids. All of the following steps of the preparation of the suspensions were performed under sterile conditions with use of filter-sterilized media, as described previously in detail (17).…”

“…We recently demonstrated that CMA upregulate BSC1 expression. Indeed, BSC1 mRNA abundance significantly increased 3 h after the creation of metabolic acidosis by peritoneal dialysis, which was followed within 24 h by an augmentation of the BSC1 protein abundance (16). The augmentation of both mRNA and protein abundance persisted after 6 d of CMA induced by administration of NH 4 Cl in drinking water (16).…”