The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

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)....

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

“…However, the employed transgenic mouse model Although we were able to detect a stimulation of NCC by dietary Na + restriction, we did not find any evidence for a regulatory role of the MR for NCC abundance and phosphorylation. This contrasts with the conclusions drawn from several previous in vivo studies in which the effect of Na + restriction [11,35], aldosterone infusion [11,35], furosemide-treatment [1], and MR inhibition [16] on NCC was studied at the level of the whole animal. However, all these experimental maneuvers do not only change plasma aldosterone levels and/or interfere with MR signaling, but may provoke numerous confounding effects on plasma hormone levels and/or tubular flow that may interfere with NCC regulation as well.…”

“…Renal MR expression is not restricted to the CS, but is also found in the distal convoluted tubule (DCT) and the thick ascending limb (TAL) [2,5]. Increased plasma levels of aldosterone induced by exogenous aldosterone infusion, dietary Na + restriction or diuretic treatment correlate with an increased abundance and phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the DCT [11,12,34,35]. The stimulatory effect of aldosterone on NCC was also reported to occur in DCT cells in vitro and was suggested to involve the activation of various different kinases including SGK1 [32], WNK4 [34], and SPAK [12] as well as the ubiquitin ligase Nedd4-2 [14,27].…”

Angiotensin II inhibits NaCl absorption in the rat medullary thick ascending limb

“…In the more distal part of the nephron, angiotensin II also directly stimulates sodium reabsorption by activating Na ϩ /H ϩ exchange and the amiloridesensitive Na ϩ channel (2,23,37). Finally, angiotensin II stimulates aldosterone synthesis and thereby indirectly enhances sodium reabsorption via the epithelial sodium channel in the cortical collecting duct (26) and likely via the thiazidesensitive sodium-chloride cotransporter in the distal convoluted tubule (21).…”

Role of the alternative splice variant of NCC in blood pressure control

“…Hence, PKA and PKC activation can stimulate WNK4 signaling, indirectly leading to NCC activation. The NCC cell surface abundance and phosphorylation are also increased by the mineralocorticoid aldosterone, likely through a mechanism involving the serum glucocorticoid regulated kinase 1 (SGK1)-Nedd4-2 pathway or WNK4 [45,61] (Figure 2). However, recent evidence suggests that modulation of NCC in response to aldosterone is mediated by secondary changes in plasma potassium concentration that result from aldosterone effects on ENaC [62,63].…”