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V. Medizinische Universitätsklinik (H.K., B.A.Y., F.J.v.d.W.) and Institute of Clinical Pharmacology (M.C., M.W.), Faculty of Clinical Medicine Mannheim, University of Heidelberg, 68167 Mannheim, Germany; and Department of Internal Medicine IV, J. W. Goethe University (P.C.B.), 60054 Frankfurt am Main, Germany
Address all correspondence and requests for reprints to: Prof. F. J. van der Woude, V. Medical Clinic, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany. E-mail: fokko.van-der-woude{at}med5.ma.uni-heidelberg.de.
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Abstract |
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In MC, Ptec, and Dtec, aldosterone increased [Ca2+]i within 1 min, whereas in GVEC, only a minor effect was found. Preincubation of cells with spironolactone did not blunt this effect. Hydrocortisone, used at a concentration 100-fold higher than that of aldosterone, did not affect [Ca2+]i. In MC, Ptec, and Dtec, a dose-dependent increase (
1.3- to 1.5-fold) in intracellular cAMP levels was found.
These data demonstrate a nongenomic action of aldosterone in human MC, Ptec, and Dtec. As these effects occur at concentrations close to free plasma aldosterone levels in man, they may be of physiological relevance and may contribute to renal injury.
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Introduction |
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Nongenomic steroid actions are mainly characterized by their rapid onset and insensitivity toward inhibitors of transcription and translation. Nongenomic mineralocorticoid effects can be observed at physiological concentrations of aldosterone, whereas glucocorticoids are only active at supramicromolar concentrations (15, 16, 17, 18). These actions are not inhibited by the classical mineralocorticoid receptor antagonists, canrenone and spironolactone. A specific membrane receptor for aldosterone in the plasma membrane has been suggested by indirect evidence; putative binding sites could be demonstrated in human mononuclear leukocytes, porcine kidney, and liver (19, 20, 21). Alzamora et al. (21) showed that the open ring analog RU28318 is able to block nongenomic aldosterone action and that 11-hydroxysteroid dehydrogenase may contribute to aldosterone specificity of nongenomic effects. The nongenomic action of aldosterone is mediated through the phospholipase C pathway, as aldosterone stimulation of vascular smooth muscle cells increased inositol 1,4,5-triphosphate and diacylglycerol, resulting in release of intracellular Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) (13, 22). In addition aldosterone rapidly increased cAMP and phosphorylation of the cAMP response element-binding protein (17).
Although nongenomic actions of aldosterone have been demonstrated in human collecting duct and Madin Darby canine renal cells (23, 24, 25) detailed data on human mesangial cells (MC), glomerular (visceral) epithelial cells (GVEC), and proximal and distal tubular epithelial cells (Ptec and Dtec) are lacking. The present study was conducted to investigate whether aldosterone, used at physiological concentrations, could evoke a nongenomic action in primary cultures of these cells. To this end, changes in [Ca2+]i and cAMP were evaluated after aldosterone stimulation in vitro.
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Materials and Methods |
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Aldosterone, spironolactone, HEPES, trypsin-EDTA, thrombin, fibronectin, penicillin-streptomycin solution (P/S; 104 U/ml penicillin plus 10 mg/ml streptomycin), fluorescein isothiocyanate-phalloidin, ionomycin, and collagen (calf skin) were purchased from Sigma-Aldrich (St. Louis, MO). Medium 199 and PBS were obtained from Life Technologies, Inc. (Paisley, Scotland). Fetal calf serum (FCS) and 24- and 6-well plates were obtained from Greiner Labortechnik (Frickenhausen, Germany). Renal epithelial cell growth medium was purchased from Promocell (Heidelberg, Germany). DMEM was obtained from Life Technologies, Inc. (Karlsruhe, Germany). Fura-2/acetoxymethylester (fura-2/AM) was obtained from Molecular Probes, Inc. (Eugene, OR). The cAMP RIA was manufactured by Amersham Pharmacia Biotech. Cell culture flasks (25- and 75-cm2) were obtained from Falcon (Falcon, BD Biosciences, Heidelberg, Germany). IBMX was purchased from BIOMOL (Hamburg, Germany). Isoproterenol was obtained from Fluka (Taufkirchen, Germany). Anticytokeratin antibodies were purchased from Eurodiagnostics (Apeldoorn, The Netherlands), and anti-CALLA antibodies were obtained from Dakopatts (Copenhagen, Denmark).
Cell culture
All renal cells used were isolated from normal kidney tissue from multiple sources, including allografts unsuitable for transplantation for surgical reasons and grossly normal nephrectomy specimens. Informed consent in written form and approval by the local ethics committee was obtained. The preparation of MC and GVEC has been described in detail elsewhere (26, 27, 28). Briefly, MC were subcultured from hillocks, usually appearing 3 wk after outgrowth of the glomeruli, in 24-well plates in DMEM supplemented with 10% heat-inactivated FCS and P/S. Single wells were trypsinized and seeded into a T-25 culture flask on the basis of cell morphology (multilayer, spindle-shaped) and the absence of epithelial cells with cobblestone morphology. The identity of MC was confirmed by uniform staining with fluorescein isothiocyanate-phalloidin for actin, positive staining for vimentin, and negative staining for von Willebrand factor, cytokeratin, and desmin. GVEC were passaged immediately after outgrowing from the glomeruli (1 wk) with PBS-20 mmol/liter EDTA and were grown in DMEM with 5% FCS. Characterization was performed on the basis of cell morphology (confluent monolayer of polygonal cells); positive staining with anticytokeratin (RGE3), anti-common acute lymphocytic leukemia antigen, and the monoclonal antibody TN10 [which particularly recognizes GVEC (29)]; and the absence of staining with monoclonal antibodies TN9 [specific recognition of Ptec (29)], anti-von Willebrand factor, and antidesmin. Monoclonal antibodies TN9 and TN10 were gifts from Drs. G. Müller and M. Nesper (Medizinische Klinik, Tübingen, Germany). Ptec were isolated by the method of Detrisac et al. (30) and cultured in serum-free renal epithelial cell growth medium in T-25 culture flasks coated with collagen and FCS proteins. Ptec were characterized by monoclonal antibodies directed against cytokeratin, epithelial membrane antigen, and four monoclonal antibodies against the adenosine-deaminase binding protein, designated 1071, 1072, 1079, and 1080 (gifts from Dr. Dinjens, University Hospital, Maastricht, The Netherlands). The isolation of Dtec, a gift from Dr. P. Bär (University Hospital, Frankfurt, Germany), has been described previously (31). The cells were seeded in collagen/FCS-coated, 6-well plates and cultured in medium 199 supplemented with 10% FCS and P/S.
The range of passages was the same for all cell types, cells within passages 3–8 were used. Cells exhibiting a microscopic abnormal phenotype were excluded. For each experiment a suitable positive control [100 nmol/liter angiotensin II for AT1-receptor-positive Cells (MC and Ptec) and/or 1 U/ml thrombin] was performed to ensure the examination of intact cells, which were required to exhibit an at least a 1.5-fold increase in [Ca2+]i.
Measurement of [Ca2+]i
[Ca2+]i was determined with the Ca2+-sensitive dye fura-2/AM as described previously (18). For fluorometric measurements cells were grown on glass coverslips, coated with collagen/FCS (Dtec and Ptec) or fibronectin (MC) for 2–4 d until 80% confluence was reached. Before each experiment all cells were cultured for 24 h in basal medium without serum or supplements. Thereafter, cells were washed twice with physiological saline solution [PSS; 135 mmol/liter NaCl, 5 mmol/liter KCl, 1.8 mmol/liter CaCl2, 0.8 mmol/liter MgCl2, 10 mmol/liter HEPES, and 5.5 mmol/liter glucose (pH 7.4)] and subsequently loaded with 5 µmol/liter fura-2/AM in PSS containing 0.5% dimethylsulfoxide and 0.5% Pluronic for 30–45 min at 37 C. At the end of the loading period, cells were washed twice with PSS and placed immediately in a thermostatically controlled ring chamber (37 C) in a volume of 450 µl PSS. Drugs were added in different concentrations in a volume of 50 µl. To detect fluorescence changes, a dual wavelength imaging system (Till Photonics GmbH, Gräfelfing, Germany) was attached to an Axiovert 35 (Carl Zeiss, Hanau, Germany) inverted fluorescence microscope equipped with a fluor 40/1.30 oil immersion objective and a charge-coupled device imaging camera (General Scanning, Planegg, Germany). Excitation wavelengths were separated by a dichoric mirror at 340 and 380 nm, and emitted light was collected at 510 nm. Time increments were 3.5–4 sec at an integration time of 200 msec for 340 nm and 150 msec for 380 nm. Autofluorescence was measured in each experiment by the addition of 5 µmol/liter ionomycin and 5 mmol/liter MnCl2 to quench intracellularly located dye.
Typically, a region of 5–15 cells was monitored. All administered drugs were tested for autofluorescence, which was insignificant for these conditions of excitation and emission. The stability of baseline was checked at least for 1.5 min in all readings. The criteria for a positive response to aldosterone was a rise in the 
340/380 nm ratio of more than 5 centiunits from a stable baseline (e.g. an increase of the ratio from 1.00 to 1.05) with a subsequent decline to baseline levels. In addition, cells in the same experiments must react to the positive control stimulus to qualify as responders.
At the times indicated aldosterone was added from a 10 mmol/liter stock solution in ethanol stored in glass vessels. The final ethanol concentration at lowest steroid concentrations was 0.00000001% and did not exceed 0.01% at 1 µmol/liter (except for experiments with hydrocortisone or spironolactone, which were, however, inactive) and had no effect on [Ca2+]i. A concentration of 0.01% ethanol was used in all control experiments. The 340/380 nm ratios were analyzed on serial images in two regions of interest: one in the perinuclear region of the cell, and one near the plasmalemma.
Stimulus-induced changes in intracellular cAMP levels
For the measurement of cAMP, cells were treated in a similar manner as described above. PSS buffer, used for all preincubation and washing steps, was supplemented with 0.5 mmol/liter IBMX to prevent cAMP degradation. After 30 min of preincubation in PSS/IBMX, aldosterone was added to the cells at different concentrations. Stimulation was stopped at various time points by placing the cells on ice, followed by immediate aspiration of the supernatant. Isoproterenol stimulation was used as a positive control for the receptor-mediated increase in cAMP. In addition (0.01%) ethanol was used as a negative control to exclude that an increase in cAMP was mediated by the vehicle of aldosterone or isoproterenol. Intracellular cAMP was extracted with 65% ethanol, and RIA was performed according to the manufacturer’s instructions.
Statistical analysis
A two-sided unpaired t test was used to calculate the significance of cAMP and 340/380 nm ratio increases; P less than 0.05 was considered statistically significant.
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Results |
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The effect of aldosterone on [Ca2+]i in human primary cultures of MC, Ptec, Dtec, and GVEC is shown in Fig. 1
. In MC, Ptec, and Dtec, the addition of aldosterone rapidly increased [Ca2+]i, which was maximal 1 min after stimulation and than gradually declined to the baseline level. In GVEC, however, aldosterone clearly did not induce this type of Ca response, but resulted in a gradual increase and decline in [Ca2+]i that lasted approximately 4 min. In addition, the magnitude of the increase in [Ca2+]i was different in the various cell types. Whereas in MC and Ptec the increases were 27.7 ± 15.6 and 35.3 ± 14.0 340/380 nm ratio (centiunits) fura-2 fluorescence, respectively, in Dtec this change was only 7.0 ± 1.7 340/380 nm ratio, and the change was clearly smallest in GVEC with 6.5 ± 1.3 340/380 nm ratio (P < 0.01 and P < 0.005, respectively, vs. effects in PTEC), but all effects were significant against baseline (P < 0.01). The addition of 0.01% of ethanol in PSS buffer did not affect [Ca2+]i. A second stimulation of cells within 1–2 min after the decline to baseline with aldosterone did not induce an additional increase in [Ca2+]i. The increase in [Ca2+]i after stimulation with aldosterone was not blunted by a 100-fold excess (10-5 M) of the classical mineralocorticoid receptor antagonist spironolactone (Fig. 1). Moreover, in all cells hydrocortisone did not affect [Ca2+]i, even when concentrations 100- to 1000-fold higher than that of aldosterone were used (Fig. 2).
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), dose-response experiments could not readily be performed for [Ca2+]i. With 10-6–10-7 mol/liter aldosterone, approximately 30% of MC, 65% of Ptec, 7% of Dtec, and 20% of GVEC were responsive. Due to their unique morphological shape, it is unlikely that the low percentage of DTEC responding to aldosterone merely represents a contamination by other cells.
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As it has been demonstrated that aldosterone rapidly increased cAMP in vascular smooth muscle cells (17), this second messenger was also investigated in primary cultures of renal cells. Basal cAMP concentrations in MC and Ptec were 21.7 ± 4.8 and 24.4 ± 6.8 pmol/mg protein, respectively. In Dtec the basal cAMP concentration varied, ranging widely from 2.6–47.8 pmol/mg protein, in the different cell preparations used. The influence of aldosterone on cAMP was not studied in GVEC, as, in general, cAMP increases were even smaller than calcium effects; therefore, given the minor calcium effect of aldosterone in GVEC, no visible effect on cAMP was expected. Stimulation of MC, Ptec, and Dtec with 10-8 mol/liter aldosterone caused a significant increase in intracellular cAMP concentrations, reaching maximum values after 5 min (P < 0.0001–0.05). cAMP remained elevated for up to 10 min after stimulation (Fig. 4). Aldosterone clearly induced a dose-dependent increase in cAMP, that was maximal at 10-9, 10-9, and 10-8 mol/liter aldosterone in MC, Ptec, and Dtec, respectively. The slight decrease in cAMP at higher concentrations compared with intermediate concentrations was insignificant (Fig. 5).
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Discussion |
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It is generally accepted that steroids bind to intracellular receptors and subsequently modulate nuclear transcription and, hence, protein synthesis. In addition to this genomic action, rapid steroid actions have been more widely recognized and characterized during the last decade. These rapid effects involve activation of phospholipase C, phosphoinositide turnover, free intracellular [Ca2+]i, and cAMP (15, 16, 17, 18, 23, 24, 25).
Aldosterone plays an important role in body fluid, electrolyte, and pH homeostasis (7). In the cortical collecting duct, aldosterone increases the reabsorption of sodium and water, thereby increasing blood pressure and volume (25). It is believed that this hemodynamic action of aldosterone may in part contribute to the deleterious effect of RAAS activation. Nonhemodynamic actions of aldosterone, however, may also contribute to renal injury. In vitro studies in MC have demonstrated that aldosterone directly stimulates the production of collagen type IV (12). Recently, Brown et al. (10) demonstrated that inhibition of aldosterone significantly decreased the development of glomerulosclerosis after radiation injury in the rat, accompanied by a reduction in the expression of plasminogen activator inhibitor-1.
However, there are conflicting data concerning to what extent nongenomic actions of aldosterone contribute to renal damage. Whereas Brown et al. (10) and Rocha et al. (14) demonstrated that glomerulosclerosis was significantly decreased by spironolactone, Greene et al. (11) showed that treatment of remnant rats with spironolactone did not improve glomerular injury. It should be mentioned, however, that in these studies different models were used to induce renal damage, thus possibly explaining these different findings. As nongenomic actions of aldosterone seem to be transient, it is yet unknown whether and to what extent they contribute to deleterious effects of aldosterone. The protective actions of mineralocorticoid antagonists are unquestionable, and no systematic research into the relative contributions of nongenomic mineralocorticoid actions has been performed. To date, the evidence for nongenomic effects in human renal cells is restricted to collecting duct cells. This study was therefore conducted to investigate whether nongenomic actions of aldosterone also can be induced in MC, GVEC, Ptec, and Dtec. The main findings of the present study are as follows.
The addition of aldosterone to MC, Ptec, and Dtec caused an immediate transient increase in [Ca2+]i, whereas in GVEC a small, longer lasting increase of questionable relevance was found. Tachyphylaxis was observed, in that a second stimulus after baseline had been reached again was ineffective. This phenomenon could be explained by the feedback inhibition through PKC observed earlier. Inhibition of PKC by long-term exposure to phorbol esters augmented the rapid [Ca2+]i response to aldosterone in vascular smooth muscle cells, whereas stimulation by short-term exposure decreased the response (18).
Aldosterone responses in all cell types were insensitive to blockade by the classical mineralocorticoid receptor antagonist spironolactone, even when used in up to 100-fold higher concentrations than aldosterone. Spironolactone is clearly an effective, clinically relevant antagonist to genomic responses of aldosterone, as has been shown in numerous preclinical and clinical studies.
Hydrocortisone was ineffective even at concentrations 100-fold higher (10-5 mol/liter) than aldosterone, an important property of nongenomic aldosterone effects described earlier.
Aldosterone increased the intracellular cAMP level in MC, Ptec, and Dtec, reaching a plateau after 3–5 min, with an apparent EC50 of about 10-11 mol/liter. In previous studies in vascular smooth muscle cells, a perfect congruency of pharmacological parameters for the calcium and cAMP responses to aldosterone had been firmly established (17). As the latter effects were measurable at a greater accuracy than those for calcium, dose-response curves were obtained for cAMP effects rather than calcium transients. These results thus demonstrate that nongenomic actions of aldosterone are not restricted to the collecting duct cells, but can also be induced in primary human cultures of MC, Dtec, Ptec, and, possibly, GVEC.
The rapid effects of aldosterone are thought to be mediated by membrane receptors, with high affinity for aldosterone and low affinity for glucocorticoids (19, 33, 34). Both kinetic and pharmacological properties of these receptors are distinct from those of the cytosolic type 1 receptors (35). Nonspecific membrane actions of steroids have been reported to occur only at supramicromolar concentrations (22). It is unlikely that these effects contribute to the nongenomic action of aldosterone in this paper for two reasons. First, consistent with previous observations (reviewed in Ref.34), rapid aldosterone actions occurred with an EC50 of approximately 10-11–10-10 mol/liter, a concentration range corresponding to physiological concentrations of free aldosterone in humans (35). A physiological role for these effects may therefore be postulated, as they do not require concentrations above those that occur under physiological conditions. Secondly, nongenomic effects were only observed with aldosterone, not hydrocortisone, and therefore suggest a specific aldosterone effect rather than a nonspecific steroid action. As the cultured cells used here were expensive and hard to obtain, the role of a possible involvement of 11ß-hydroxysteroid dehydrogenase by the use of inhibitors or stable glucocorticoids (e.g. dexamethasone) could not be studied, but has been demonstrated by Alzamora et al. (21). It remains speculative through which mediators aldosterone may influence glomerular injury, although it has been shown in MC that the expression of heparan sulfate is decreased by cAMP. Heparan sulfate is discussed as an endogenous protective principle stabilizing the basal membrane (36). As aldosterone has been shown to nongenomically influence cAMP levels, which subsequently induces secondary genomic effects (through cAMP response element-binding protein phosphorylation), it may be speculated that aldosterone may influence heparan sulfate expression, a hypothesis currently under investigation.
In conclusion, we have demonstrated that aldosterone induces nongenomic effects in MC, GVEC, Ptec, and, possibly, Dtec at physiological concentrations, increasing [Ca2+]i and intracellular cAMP levels. These effects are likely to be of clinical relevance, because these effectors may be important for contraction, a changed pattern of heparan sulfate production, and overproduction of extracellular matrix components of renal cells.
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Footnotes |
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Abbreviations: ACE, Angiotensin-converting enzyme; ATR, angiotensin II receptor; [Ca2+]i, intracellular Ca2+; Dtec, distal tubular epithelial cells; FCS, fetal calf serum; fura-2/AM, fura-2/acetoxymethylester; GVEC, glomerular visceral epithelial cells; MC, mesangial cells; PKC, protein kinase C; P/S, penicillin-streptomycin solution; PSS, physiological saline solution; Ptec, proximal tubular epithelial cells; RAAS, renin-angiotensin-aldosterone system.
Received February 15, 2002.
Accepted November 20, 2002.
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; n = 4–8), Ptec (•; n = 4–8), and Dtec (
; n = 2) are shown as the percent cAMP in controls (HEPES-buffered PSS, 0.5 mmol/liter IBMX plus 0.001% ethanol). Values are given as the mean ± SEM. *, p<0.05; +, p<0.0001 (vs. baseline).
