Aldosterone induces CTGF in mesangial cells by activation of the glucocorticoid receptor.

BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.     

Early nongenomic events in aldosterone action in renal collecting duct cells: PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response

Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation. E-mail: marcel.blot-chabaud@pharmacie.univ-mrs.fr     

Impairment of sodium balance in mice deficient in renal principal cell mineralocorticoid receptor

Germline inactivation of the mineralocorticoid receptor (MR) gene in mice results in postnatal lethality as a result of massive loss of sodium and water. The knockout mice show impaired epithelial sodium channel (ENaC) activity in kidney and colon. For determination of the role of renal MR in aldosterone-driven ENaC-mediated sodium reabsorption, mice with principal cell MR deficiency were generated using the Cre-loxP system. For driving Cre recombinase expression in principal cells, the regulatory elements of the mouse aquaporin 2 (AQP2) gene were used. Mutant mice (MR(AQP2Cre)) were obtained by crossing AQP2Cre mice with mice that carried a conditional MR allele. Under standard diet, MR(AQP2Cre) mice develop normally and exhibit unaltered renal sodium excretion but show strongly elevated aldosterone levels. Increased renal sodium and water excretion, resulting in continuous loss of body weight, occur under low-sodium diet. Immunofluorescence revealed that the loss of MR and apical ENaC staining is restricted to principal cells of the collecting duct (CD) and late connecting tubule (CNT) and that MR is crucial for ENaC trafficking to the apical membrane. These results demonstrate that inactivation of MR in CD and late CNT can be compensated under standard diet but no longer when sodium supply is limited. Because the mutant mice show preserved renal ENaC activity, this study provides evidence that the late distal convoluted tubule and early CNT can compensate to a large extent deficient ENaC-mediated sodium reabsorption in late CNT and CD.     

Mineralocorticoid receptor knockout mice: lessons on Na+ metabolism

The mineralocorticoid receptor (MR) binds aldosterone and glucocorticoids with equal affinity. In aldosterone target tissues, like the epithelial cells of the distal colon and the principal cells of the collecting ducts in the kidney, the MR is protected from glucocorticoids by the action of the enzyme 11beta-hydroxysteroid-dehydrogenase type 2 (11betaOHSD2), allowing aldosterone to specifically activate the receptor. However, in MR-expressing cells, which lack 11betaOHSD2, like the neurons of the limbic system in the brain, MR is mainly activated by glucocorticoids. MR knockout mice die in the second week after birth, showing at day 8 symptoms of pseudohypoaldosteronism with hyponatremia, hyperkalemia, high renal salt wasting, and a strongly activated renin-angiotensin-aldosterone system (RAAS). The activity of the amiloride-sensitive epithelial Na+ channel (ENaC) is strongly reduced in colon and kidney, but there is no down-regulation of the mRNA abundance of the three ENaC subunits. Daily subcutaneous injections of isotonic NaCl solution until weaning and continued oral NaCl supply lead to survival of the MR knockout mice. The NaCl-rescued MR knockout mice display a strongly enhanced fractional renal excretion of Na+, hyperkalemia, and a persistently strongly activated RAAS. There is almost no renal ENaC activity. The renal mRNA abundance of alphaENaC is reduced by 30%, whereas betaENaC and gammaENaC are not altered.     

Mineralocorticoid versus glucocorticoid receptor occupancy mediating aldosterone-stimulated sodium transport in a novel renal cell line

Aldosterone controls sodium balance by regulating an epithelial sodium channel (ENaC)-mediated sodium transport along the aldosterone-sensitive distal nephron, which expresses both mineralocorticoid (MR) and glucocorticoid receptors (GR). Mineralocorticoid specificity is ensured by 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes cortisol or corticosterone into inactive metabolites that are unable to bind MR and/or GR. The fractional occupancy of MR and GR by aldosterone mediating the sodium transport response in the aldosterone-sensitive distal nephron cannot be studied in vivo. For answering this question, a novel mouse cortical collecting duct cell line (mCCD(cl1)), which expresses significant levels of MR and GR and a robust aldosterone sodium transport response, was used. Aldosterone elicited a biphasic response: Low doses (K(1/2) = approximately 0.5 nM) induced a transient and early increase of sodium transport (peaking at 3 h), whereas high doses (K(1/2) = approximately 90 nM) entailed an approximately threefold larger, long-lasting response. At 3 h, the corticosterone dose-response curve was shifted to the right compared with that of aldosterone by more than two log concentrations, an effect that was fully reverted in the presence of the 11beta-hydroxysteroid dehydrogenase type 2 inhibitor carbenoxolone. Low doses of dexamethasone (0.1 to 1 nM) failed to induce an early response, but high doses elicited a long-lasting response (K(1/2) = approximately 8 nM), similar to that observed for high aldosterone concentrations. Equilibrium binding assays showed that both aldosterone and corticosterone bind to a high-affinity, low-capacity site, whereas dexamethasone binds to one site. Within the physiologic range of aldosterone concentrations, sodium transport is predicted to be controlled by MR occupancy during circadian cycles and by MR and GR occupancy during salt restriction or acute stress.     

Enhancement of epithelial sodium channel expression in renal cortical collecting ducts cells by advanced glycation end products.

BACKGROUND: The epithelial sodium channel (ENaC) is a complex, and the alphaENaC subunit has a crucial role in sodium uptake induced by aldosterone in the distal nephron. Although experimental animal models of diabetes have demonstrated up-regulation of alphaENaC expression in renal cortical collecting duct (CCD) cells, the molecular mechanism remains unclear. Advanced glycation end products (AGEs) are by-products of long-term hyperglycaemia and comprise a significant pathogenic factor in diabetic nephropathy. We hypothesize that AGEs play a role in regulating alphaENaC gene expression. METHODS: Mouse CCD cells (mpkCCDcl(4)) were cultured with AGE to determine the effects of AGE on alphaENaC expression and sodium uptake. Gene expressions of ENaC were measured by real-time PCR and sodium uptake was measured with fluorescent dye as a sodium indicator (SBFI-AM). This study analysed mitogen-activated protein kinases signalling pathways by western blotting. Cells co-transfected with plasmids of the alphaENaC promoter carrying a luciferase reporter and plasmids expressing wild-type or mutant serum- and glucocorticoid-induced kinase 1 (Sgk1) mRNA were stimulated with AGE to identify the signalling pathway. RESULTS: The AGEs, stimulated in a time- and dose-dependent manner, enhanced alphaENaC mRNA expression and sodium uptake in mpkCCDcl(4) cells. The AGEs also significantly stimulated Sgk1 mRNA and Sgk1 activity in a time- and dose-dependent manner. Co-transfected with plasmid expressing mutant Sgk1 significantly limited stimulated alphaENaC promoter-driven luciferase activity by AGEs in mpkCCDcl(4) cells. CONCLUSION: Experimental results indicate that AGEs induced alphaENaC expression and increased sodium uptake in renal CCD cells. The mechanism through which AGEs activate alphaENaC expression may be via activation of Sgk1 in mpkCCDcl(4) cells.     

Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells

BACKGROUND: Aldosterone at physiological levels induces rapid (<5 min) increases in intracellular protein kinase C (PKC) activity and a rise in calcium and pH in mineralocorticoid hormone target epithelia, such as distal colon and sweat gland. The end targets of these rapid responses in epithelia are Na+/H+ exchange and K+ channels. METHODS: The mouse cortical collecting duct (CCD) M-1 cell line was grown to confluency and loaded with Fura-2 for spectrofluorescence measurements of intracellular free calcium at 37 degrees C bathed in Krebs solution. RESULTS: Aldosterone (1 nmol/L) produced a rapid, transient peak increase in [Ca2+]i in M-1 cells. This effect was abolished upon removal of extracellular Ca2+, but was unaffected by pretreatment with spironolactone (10 micromol/L) or actinomycin D (10 micromol/L). However, pretreatment with the specific PKC inhibitor chelerythrine chloride (1 micromol/L) prevented the aldosterone-induced rise in [Ca2+]i. Dexamethasone, at a concentration 10,000-fold higher than aldosterone (10 micromol/L), also produced a transient increase in [Ca2+]i, but this response was significantly smaller than that of aldosterone. In contrast, hydrocortisone had no effect on [Ca2+]i at either nmol/L or micromol/L concentrations. Both of the sex steroids, 17beta-estradiol (10 nmol/L) and progesterone (10 nmol/L), induced protein kinase C-dependent increases in [Ca2+]i. CONCLUSIONS: Aldosterone and sex steroid hormones activate intracellular calcium signaling in CCD cells via a nongenomic PKC-dependent pathway, which may have important implications for renal transport.     

Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis

Edema and ascites in nephrotic syndrome mainly result from increased Na+ reabsorption along connecting tubules and cortical collecting ducts (CCD). In puromycin aminonucleoside (PAN)-induced nephrosis, increased Na+ reabsorption is associated with increased activity of the epithelial sodium channel (ENaC) and Na+,K+-ATPase, two targets of aldosterone. Because plasma aldosterone increases in PAN-nephrotic rats, the aldosterone dependence of ENaC activation in PAN nephrosis was investigated. For this purpose, (1) the mechanism of ENaC activation was compared in nephrotic and sodium-depleted rats, and (2) ENaC activity in PAN-nephrotic rats was evaluated in the absence of hyperaldosteronemia. The mechanism of ENaC activation was similar in CCD from nephrotic and sodium-depleted rats, as demonstrated by (1) increased number of active ENaC evaluated by patch clamp, (2) recruitment of ENaC to the apical membrane determined by immunohistochemistry, (3) shift in the electrophoretic profile of gamma-ENaC, and (4) increased abundance of beta-ENaC mRNA. Corticosteroid clamp fully prevented all PAN-induced changes in ENaC but did not alter the development of a full-blown nephrotic syndrome with massive albuminuria, amiloride-sensitive sodium retention, induction of CCD Na+,K+-ATPase, and ascites. It is concluded that in PAN-nephrosis, (1) ENaC activation in CCD is secondary to hyperaldosteronemia, (2) sodium retention and induction of Na+,K+-ATPase in CCD are independent of hyperaldosteronemia, and (3) ENaC is not necessarily limiting for sodium reabsorption in the distal nephron.     

Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-beta1 and aldosterone

Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta1 (TGF-beta1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I(Na)) were measured. The effect of TGF-beta1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I(Na) by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-beta1 significantly increased protein expression of PN-1 by 3.8+/-0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7+/-6.7%. Basolateral, but not apical, application of TGF-beta1 significantly reduced I(eq). To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I(eq) by 1.6+/-0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.     

Inhibition of prostasin secretion by serine protease inhibitors in the kidney

A serine protease, prostasin, has been shown to stimulate the activity of amiloride-sensitive sodium channels (ENaC). Prostasin is a glycosylphosphatidylinositol-anchored protein that is found free in physiologic fluids and tissue culture medium, but the mechanism by which prostasin is secreted from the cells has not been elucidated. The current studies found that serine protease inhibitor aprotinin blocked the secretion of prostasin in a mouse cortical collecting duct (CCD) cell line (M-1 cells). A synthetic serine protease inhibitor, nafamostat mesilate (NM), which is commonly used for the treatment of pancreatitis and disseminated intravascular coagulation in Japan, also inhibited the secretion of prostasin in M-1 cells. Continuous infusion of NM into rats resulted in a substantial decrease in urinary prostasin and urinary sodium excretion. p-guanidinobenzoic acid and 6-amidino-2-naphtol, catalytically inactive metabolites of NM, had no effect on prostasin secretion both in M-1 cells and in rats. These findings suggest that a serine protease-sensitive mechanism is involved in the secretion of prostasin in vitro as well as in vivo. Potassium secretion in the CCD is tightly linked to sodium reabsorption through EnaC; therefore, NM-induced decrease in prostasin secretion and subsequent inhibition of ENaC activity could account for the side effects of hyponatremia and/or hyperkalemia that are found sometimes in patients treated with NM. The results indicate an important role for prostasin in sodium reabsorption in the kidney under pathophysiologic conditions.     

Basolateral translocation by vasopressin of the aldosterone-induced pool of latent Na-K-ATPases is accompanied by alpha1 subunit dephosphorylation: study in a new aldosterone-sensitive rat cortical collecting duct cell line

The regulation of plasma membrane Na(+)-K(+)-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD(2). This cell line has maintained many characteristics of the CCD-in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds (3)H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10(-9) M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the alpha subunit of the epithelial sodium channel (sixfold) and the alpha1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10(-9) M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD(2) cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the alpha(1) catalytic subunit of NKA.