Department of Clinical Pharmacology, Faculty of Clinical Medicine Mannheim, Heidelberg University, Mannheim, Germany
Correspondence and offprint requests to: Martin Wehling, MD, Department of Clinical Pharmacology, Faculty of Clinical Medicine Mannheim, Heidelberg University, Theodor-Kutzer Ufer 1-3, D-68167 Mannheim, Germany. Email: martin.wehling{at}kpha.ma.uni-heidelberg.de
Keywords: aldosterone; early induced genes; kidney; non-genomic effects; renal disease
Introduction
Steroid-hormone action in the classical sense means binding to intracellular receptors, binding of the hormonereceptor complex to DNA and activation or repression of transcription of target genes. However, all classes of steroid hormones can additionally act through non-genomic mechanisms and thereby change physiological processes [1]. In contrast to genomic steroid actions, these non-genomic effects are characterized with rapid onset and insensitivity towards inhibitors of transcription and translation. Non-genomic effects of aldosterone on various target organs and cells have been described, i.e. vascular smooth muscle cells and endothelial cells [2], skeletal muscle cells [3], lymphocytes [4], cardiac myocytes [5], colonic epithelial cells [6] and kidney cells [7]. In the classical target organ, the kidney, aldosterone stimulates Na+ (re)absorption and K+ secretion through both mechanisms, genomic as well as non-genomic [8].
Mechanisms involved in non-genomic aldosterone action
Little is known about the primary target of non-genomic aldosterone effects, whereas genomic aldosterone action is mediated via the type 1 mineralocorticoid receptor (MR). A distinct specific membrane receptor different from the classical intracellular MR has been postulated on the basis of several lines of evidence. Specific high-affinity binding sites for aldosterone have been described in membranes of different cells or tissues. For example, in pig and rat kidney, receptors for aldosterone have been found in membranes. Their binding affinity is an order of magnitude higher than that for intracellular binding of aldosterone [9,10]. These membrane binding sites could, in contrast to MR, distinguish aldosterone from hydrocortisone [9]. Furthermore, such aldosterone membrane binding sites have antagonistic properties different from the classical MR, i.e. they are insensitive towards classical MR antagonists such as canrenone or spironolactone. We found recently that short-term aldosterone treatment of the cortical collecting duct cell line M-1 induced mitogen-activated protein kinase (MAPK) phosphorylation. This phosphorylation could not be blocked by four different MR antagonists (K. Rossol-Haseroth et al., submitted for publication). In this context it should be mentioned that in skin fibroblasts from knockout mice lacking the MR, aldosterone led to a rapid increase in intracellular levels of calcium and cAMP [11]. All these results taken together strongly imply that a membrane aldosterone receptor unrelated to MR exists.
The postulated membrane aldosterone receptor has not been isolated and cloned so far. In human mononuclear leukocytes a membrane protein with a MW of 50 kDa was identified, which binds aldosterone with an affinity constant of
0.1 nM [12]. Efficient enrichment of this protein for further sequencing and cloning has not yet been achieved. However, since the first membrane steroid receptor has been cloned recently, namely the membrane progestin receptor [13], it is expected that related proteins binding other steroid hormones, including the membrane aldosterone receptor, will be cloned very soon.
Upon binding to the membrane receptor, aldosterone can elicit a number of rapid effects, such as rise in second messenger concentration and activation of protein kinase cascades with subsequent phosphorylation and activation of target proteins. Mechanisms of action have been studied in freshly dissected kidney tissue and several cell-culture systems, mimicking different cell types of the kidney, e.g. A6 cells (Xenopus laevis renal epithelial cells), MadinDarby canine kidney cells (MDCK, sharing properties with renal cortical collecting duct epithelium) and M-1 cells (a mouse principal cortical collecting duct cell line).
Second messengers that have been demonstrated to be induced by aldosterone include cAMP [14], calcium, diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). Yet, most commonly reported is the increase in intracellular calcium concentrations shortly after aldosterone administration.
In MDCK cells there was a 3-fold increase of intracellular Ca2+ within seconds of aldosterone application. Simultaneously, rapid stimulation of a plasma membrane proton conductance and cytosolic acidification was observed. These two effects led to the activation of Na+/H+ exchange, rise in intracellular Na+ and to an increase of plasma membrane K+ conductance [15]. As a consequence, cell volume increases. Inhibition of protein kinase C and G-proteins reduced the effect, suggesting the involvement of a heterotrimeric G-protein coupled receptor [16]. Also, inhibition of MAPK ERK1/2 prevented the activation of Na+/H+ exchange [17]. Further studies demonstrated that aldosterone uses epidermal growth factor receptor (EGFR) as a heterologous signal transducer to stimulate the MAPK signalling cascade [18]. However, aldosterone does not bind directly to EGFR. It can affect EGFR activity via src kinase activation; it is so far unknown how aldosterone leads to src kinase activation [19].
In another kidney cell line, M-1 cells, aldosterone also produced a rapid calcium signal [20]. We recently demonstrated that aldosterone rapidly induced MAPK phosphorylation in M-1 cells (K. Rossol-Haseroth et al., submitted for publication). Thus, calcium and MAPK phosphorylation may, in general, be mediators of the aldosterone effects and activate renal Na+/H+ exchange.
In medullary thick ascending limb (MTAL), inhibition of HCO-3 absorption has been reported, which is induced by a non-genomic aldosterone mediated pathway [21]. The exact mechanism of this inhibition is not clear. However, absorption of HCO-3 by the MTAL is mediated virtually completely by the apical membrane Na+/H+ exchanger NHE3. Thus, aldosterone might not only rapidly activate but also inhibit Na+/H+ exchange possibly in a cell-specific manner.
Besides the Na+/H+ antiporter, the epithelial sodium channel (ENaC) controls Na+ reabsorption. Studies by Zhou and Bubien [22] on freshly isolated principal cells from rabbit displayed rapidly increased ENaC sodium current within 2 min after aldosterone administration. Interestingly, methylation appeared to play a role in the non-genomic signal transduction pathway between aldosterone and ENaC, suggesting that a methylation reaction regulates ENaC activity. In addition, ENaC is regulated by aldosterone induced phosphorylation [23].
Crosstalk between non-genomic and genomic aldosterone actions
Non-genomic modulation of intracellular signalling has, most likely, an impact on genomic steroid action. We have suggested a two-step model of steroid action integrating both genomic and non-genomic aspects and their possible interaction [1,24]. First, steroid hormones elicit rapid activation of protein kinases, e.g. protein kinase A (PKA) and MAPKs, through non-genomic mechanisms, which in turn can phosphorylate transcription factors and co-factors and thereby activate early genomic responses.
According to our own data, cAMP responsive element binding protein (CREB) might be such a transcriptional factor influenced by rapid aldosterone action. In porcine coronary vascular smooth muscle cells, both increases in intracellular cAMP levels and CREB phosphorylation were observed [25]. Phosphorylated CREB could trigger gene transcription independent of MR.
On the other hand, modulation of transcription rates has also been observed after stimulation with mineralocorticoids in cells transfected with MR during co-incubation with 8-bromo-cAMP [26]. In this case, PKA mediated phosphorylation of co-factors of the MR leading to transcriptional activation of MR have been suggested to explain the observed effect.
Thus, early aldosterone-induced genes might fall into two classes: (i) genes dependent on transcription factors other than MR, which are activated by non-genomic mechanisms and (ii) genes dependent on MR and additional non-genomically induced effects, such as phosphorylation of MR co-factors.
We have recently identified the gadd153 gene among early aldosterone upregulated genes in renal cells (M. Kellner et al., submitted for publication). The induction of this gene could not be blunted by either glucocorticoid receptor (GR) or MR antagonists. gadd153 seems to be the first example of an early aldosterone induced gene whose transcription is independent of the MR.
Aldosterone as a mediator of renal dysfunction
Aldosterone is an important pathogenic factor in progressive renal disease. Patients with renal insufficiency have increased aldosterone levels compared with healthy individuals. Furthermore, a relationship between augmented aldosterone level and disease status exists [27,28]. Highest aldosterone levels were found among patients with greatest impairment of renal function.
Also, several animal models demonstrated the involvement of aldosterone in renal failure. In the remnant kidney model (subtotal renal ablation), aldosterone contributes significantly to manifestation of proteinuria, hypertension and glomerulosclerosis [29]. Treatment with the MR antagonist spironolactone reduced only transiently proteinuria and lowered arterial pressure. However, glomerular structural injury was not notably reduced. It might be reasonable to assume that part of the deleterious aldosterone effects on the kidney are mediated by the putative membrane receptor and non-genomic signalling mechanisms.
In line with this assumption is the fact that the good prognostic effects of aldosterone antagonists in heart failure do not seem to be related to the intrarenal effects, with the exception of preventing hypokalaemia [30].
Further elucidation of non-genomic aldosterone signalling mechanisms will be necessary to better understand progression of renal disease. The identification of the putative aldosterone membrane receptor could lead to the development of new types of aldosterone antagonists and to more effective treatment of renal dysfunction.
Conflict of interest statement. None declared.
References