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.

Epithelial sodium channel (ENaC) subunit (alpha, beta, and gamma) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. alpha-ENaC was localized mainly in a zone in the apical domains, whereas beta- and gamma-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, alpha-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, alpha-ENaC was present in a narrow zone near the apical plasma membrane, whereas beta- and gamma-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na(+) reabsorption.

Abstract-We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT 1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the ␣ subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased ␣ENaC protein abundance in rat kidney cortex. The decrease in ␣ENaC protein abundance in response to AT 1 receptor blockade was associated with a fall in ␣ENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT 1 receptor blockade on ␣ENaC expression was not blocked by spironolactone, suggesting a direct role of the AT 1 receptor in regulation of ␣ENaC gene expression. Candesartan administration was also found to increase the abundances of the ␤ and ␥ subunits. The increase in ␤ and ␥ENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in ␤ and ␥ENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function. Key Words: receptors, angiotensin II Ⅲ angiotensin antagonist Ⅲ sodium channels Ⅲ aldosterone L ong-term control of blood pressure is closely tied to sodium balance and extracellular fluid volume regulation, both of which are controlled in part by the renin-angiotensin-aldosterone system (RAAS). 1 Angiotensin II has important nonrenal effects that are instrumental in the control of blood pressure as both a vasoconstrictor and a regulator of aldosterone secretion. In addition, angiotensin II has direct effects on the renal tubule in regulating NaCl reabsorption. 2 The direct antinatriuretic effects of angiotensin II appear to be particularly important in conditions of dietary sodium restriction or contraction of extracellular fluid volume. 1 Regulation of renal tubule sodium transport by angiotensin II has been investigated chiefly in relatively short-term experiments with observations within a few minutes of angiotensin II addition. 3-7 However, there is growing evidence that a variety of mediators of transport regulation in the kidney, such as vasopressin 8 and aldosterone, 9 work by both short-term and long-term actions. The long-term actions are associated with adaptive increases in abundance of transporter proteins, whereas short-term actions are generally associated with regulated trafficking or posttranslational modifications of the transporter proteins.The antinatriuretic effects of angiotensin II on sodium transport are...

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq · 200 g body wt−1 · day−1), rats were switched to a NaCl-deficient diet (0.02 meq · 200 g body wt−1 · day−1). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease <4% of baseline levels on day 3, of NaCl restriction. The decreased Na excretion at day 1 occurred despite the absence of a significant increase in plasma aldosterone level or in the abundance of any of the major renal Na transporters. However, after a 1-day lag, plasma aldosterone levels increased in association with increases in abundances of three aldosterone-regulated Na transporter proteins: the thiazide-sensitive Na-Cl cotransporter (NCC), the α-subunit of the amiloride-sensitive epithelial Na channel (α-ENaC), and the 70-kDa form of γ-ENaC. RNase protection assays of transporter mRNA levels revealed an increase in renal α-ENaC mRNA coincident with the increase in α-ENaC protein abundance. However, there was no change in NCC mRNA abundance, suggesting that the increase in NCC protein in response to dietary NaCl restriction was not a result of altered gene transcription. These results point to early regulatory processes that decrease renal Na excretion without an increase in the abundance of any Na transporter, followed by a late aldosterone-dependent response associated with upregulation of NCC and ENaC.

No abstract

The potential of getting a significant number of false positives (FPs) in peptide-spectrum matches (PSMs) obtained by proteomic database search has been well-recognized. Among the attempts to assess FPs, the concomitant use of target and decoy databases is widely practiced. By adjusting filtering criteria, FPs and false discovery rate (FDR) can be controlled at a desired level. Although the target-decoy approach is gaining in popularity, subtle differences in decoy construction (e.g., reversing vs. stochastic methods), rate calculation (e.g., total vs. unique PSMs), or searching (separate vs. composite) do exist among various implementations. In the present study, we evaluated the effects of these differences on FP and FDR estimations using a rat kidney protein sample and the SEQUEST search engine as an example. On the effects of decoy construction, we found that, when a single scoring filter (XCorr) was used, stochastic methods generated a higher estimation of FPs and FDR than sequence reversing methods, likely due to an increase in unique peptides. This higher estimation could largely be attenuated by creating decoy databases similar in effective size, but not by a simple normalization with a unique-peptide coefficient. When multiple filters were applied, the differences seen between reversing and stochastic methods significantly diminished, suggesting multiple filterings reduce the dependency on how a decoy is constructed. For a fixed set of filtering criteria, FDR and FPs estimated by using unique PSMs were almost twice those using total PSMs. The higher estimation seemed to be dependent on data acquisition setup. As to the differences between performing separate or composite searches, in general, FDR estimated from separate search was about three times that from composite search. The degree of difference gradually decreased as the filtering criteria became more stringent. Paradoxically, the estimated true positives in separate search were higher when multiple filters were used. By analyzing a standard protein mixture, we demonstrated that the higher estimation of FDR and FPs in separate search likely reflected an overestimation, which could be corrected with a simple merging procedure. Our study illustrates the relative merits of different implementations of the target-decoy strategy, which should be worth contemplating when large-scale proteomic biomarker discovery is to be attempted.