No abstract

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na-Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptidedirected antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunof luorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ؎ 21% of control diet). Immunof luorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ؎ 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, f ludrocortisone, increased TSC expression (656 ؎ 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.The mineralocorticoid hormone aldosterone regulates urinary sodium excretion by increasing the rate of renal epithelial sodium absorption (1). It is widely held that the major site of aldosterone action in the mammalian kidney is the cortical collecting duct, where aldosterone regulates apical sodium entry via the amiloride-sensitive epithelial sodium channel (1-3). However, the results of recent renal micropuncture studies (4) and studies of [ 3 H]metolazone binding in renal cortical membranes (4, 5) suggest that the distal convoluted tubule is an additional renal tubule site of mineralocorticoid action. Apical sodium entry into the distal convoluted tubule is mediated by a thiazide-sensitive Na-Cl cotransporter (TSC) (6). Thus, it is likely that putative actions of aldosterone to regulate sodium transport in the distal convoluted tubule would result from regulation of the thiazide-sensitive Na-Cl entry pathway.Investigation of the function and regulation of the TSC has been facilitated by the recent cloning of cDNAs for the cotransporter from flounder bladder (7) and rat kidney (8), which has led to the development of molecular tools for localization of TSC expression. Based on experiments using in situ hybridization (9) and reverse transcription-PCR (10), it was concluded that TSC mRNA is found virtually exclusively in the distal convoluted tubule in the rat kidney. Immunohistochemical studies using fusion protein-derived antibodies to TSC also have demonstrated that expression of the TSC protein in the rat kidney is limited to the distal convoluted tubule cells (11)....

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (alpha, beta, and gamma) in rat kidney. Seven-day infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of beta- and gamma-ENaC (to 238% and 288% of vehicle, respectively), whereas alpha-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of beta- and gamma- but no significant change in alpha-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.