No 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...

Abstract. Chronic metabolic acidosis enhances the ability of the medullary thick ascending limb (MTAL) to absorb NH 4 ϩ at least in part by stimulating the mRNA and protein expression of BSC1/NKCC2, the MTAL apical Na ϩ -K ϩ (NH 4 ϩ )-2Cl Ϫ co-transporter. For assessing the mechanism by which an acid pH enhances the BSC1 mRNA abundance, MTAL were harvested from adrenalectomized rats and incubated in control (pH 7.35) and acid (pH 7.10) 1:1 mixtures of Ham's nutrient mixture F-12 and DME. rBSC1 mRNA abundance and gene transcription rate were quantified by quantitative reverse transcription-PCR and run-off assay, respectively. Acid incubation enhanced mRNA abundance within 4 h in whole cell (P Ͻ 0.02) but not in nucleus. BSC1 gene transcription rate was not affected by acid incubation. In contrast, under conditions in which gene transcription was blocked, rBSC1 mRNA decreased within 6 h by 38 Ϯ 11% in control but only by 15 Ϯ 15% in acid medium (P Ͻ 0.02), which represented an increase in the BSC1 mRNA half-life from approximately 7 to approximately 17 h. Furthermore, in a mouse TAL cell line, acid incubation for 16 h significantly increased (P Ͻ 0.02) the amount of BSC1 mRNA in cells transfected with the fulllength mBSC1 cDNA but not in cells transfected with a mBSC1 cDNA lacking the 3'-UTR. These results demonstrate that acid pH enhances the stability of BSC1 mRNA probably by activating pathways that act on the AU-rich 3'-UTR of BSC1 mRNA, which contributes to the renal response to metabolic acidosis. Increased urinary NH 4ϩ excretion, which augments acid excretion, has long been recognized to be quantitatively the major compensatory response of the kidney against chronic metabolic acidosis (CMA) (1). It is established that most of ammonia that leaves the proximal tubule is absorbed by the medullary thick ascending limb (MTAL), which causes ammonia accumulation in the medullary interstitium followed by its secretion in adjacent medullary collecting tubule (2). The role of MTAL ammonia absorption may be particularly important in states of CMA because micropuncture experiments have shown that the NH 4 ϩ amount absorbed by the loop of Henle is increased under the latter condition (3,4). In this regard, Good (5) has shown that rat MTAL isolated and perfused in vitro have an increased ability to absorb NH 4 ϩ in response to CMA. This adaptive response in MTAL would substantially augment NH 4 ϩ excretion during metabolic acidosis.It is established that the luminal step of MTAL NH 4 ϩ absorption largely involves the MTAL bumetanide-sensitive Na ϩ -K ϩ (NH 4 ϩ )-2Cl Ϫ apical co-transporter (6 -10) that was recently cloned in various species and named BSC1 or NKCC2 (11)(12)(13)(14)(15). 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...