Research ArticleAldosterone induces myofibroblastic transdifferentiation and collagen gene expression through the Rho-kinase dependent signaling pathway in rat mesangial cells
Introduction
Evidence has accumulated supporting the potential role of aldosterone in the pathogenesis of cardiovascular disease and renal injury [1], [2], [3]. Treatment with mineralocorticoid receptor (MR) antagonists ameliorates renal injury in stroke-prone spontaneously hypertensive rats [4] and Dahl salt-sensitive hypertensive rats [5], [6]. Further studies show that treatment with aldosterone induces severe glomerular injury characterized by mesangial matrix expansion in rats [7], [8], [9]. Clinical studies have also shown that treatment with MR antagonists reduces albuminuria in patients with diabetic nephropathy [10], [11] or chronic kidney disease [12]. However, the precise mechanisms responsible for aldosterone-induced renal injury remain unexplored.
Rho-kinase is an important molecule that mediates various cellular functions such as contraction, adhesion, proliferation, motility or migration, cellular morphology, growth control and cytokinesis [13], [14]. Several studies have demonstrated the potential involvement of Rho-kinase in the pathogenesis of renal injury [5], [15]. The selective Rho-kinase inhibitor, Y27632, has been shown to attenuate tubulointerstitial fibrosis in mouse kidneys with unilateral urethral obstruction [16]. Fasudil, another Rho-kinase inhibitor, ameliorates glomerulosclerosis in Dahl salt-sensitive rats [17]. We recently demonstrated that treatment with fasudil markedly ameliorated aldosterone-induced glomerular mesangial injury, independent of blood pressure changes [18]. Taken together, these findings suggest that aldosterone-induced glomerular mesangial injury is involved in Rho-kinase dependent signaling pathways. However, there is no convincing evidence that supports the role of Rho-kinase in aldosterone-induced mesangial cell hypertrophy.
Recently, it has been proposed that renal fibrosis is associated with phenotypic transitions: myofibroblast transdifferentiation (MFT; for non-epithelial cells) and epithelial–mesenchymal transition (EMT; for epithelial cells) [19]. Profibrotic mediators, such as transforming growth factor-β, connective tissue growth factor, angiotensin II, endothelin-1, and fibroblast growth factor have been shown to induce MFT or EMT [20], [21], [22]. Aldosterone-mediated induction of EMT in renal epithelial (proximal tubular) cells has been recently reported [23]. However, whether aldosterone induces mesangial cell hypertrophy through MFT and its underlying mechanisms remained unclear.
In this study, we investigated the potential roles of Rho-kinase in aldosterone-induced mesangial cell hypertophy. In particular, we investigated whether Rho-kinase is involved in aldosterone-induced MFT and collagen expression in rat mesangial cells (RMCs).
Section snippets
Cell culture
RMCs were obtained from Sprague–Dawley rats and maintained as previously reported [24], [25]. Control solutions contained the appropriate amount of vehicle: ethanol for aldosterone (Wako, Co., Osaka, Japan), dimethyl sulfoxide for eplerenone (Pfizer, Inc. New York, NY), dimethyl sulfoxide for latrunculin B (Calbiochem, La Jolla, CA), and distilled water for Y27632 (Calbiochem, La Jolla, CA). In this experiment, cells were used between passage 5 and 12. After stimulation with the above-mentioned
The effect of aldosterone on MR expression in RMCs
Treatment with aldosterone (1 nmol/L) for various time points (3, 6, 12, 24, 48 and 72 h) did not affect the level of MR mRNA expression in RMCs (n = 6 for each time point, data not shown), suggesting that the effects of aldosterone were not due to an increase in MR expression.
The effect of aldosterone on Rho-kinase activity
RMCs were incubated with 1 nmol/L aldosterone for a range of incubation times from 5 to 180 min. Rho-kinase activity was measured by ELISA (n = 3 for each time point). Aldosterone increased Rho-kinase activation, reaching a
Discussion
In the present study, we have demonstrated that aldosterone stimulates MYPT-1 phosphorylation, induces MFT and increases the expression of collagen (type I, III and IV) mRNA in RMCs. In addition, these effects of aldosterone were prevented by pre-treatment with a MR antagonist and Rho-kinase inhibitor. To the best of our knowledge, this is the first report of the potential roles of Rho-kinase in aldosterone-induced mesangial cell MFT and collagen mRNA expression.
MFT (for non-epithelial cells)
Acknowledgments
This study was supported by grants from Kagawa University Project Research, Japan Research Foundation for clinical Pharmacology and the Pharmacological Research Foundation (Tokyo) to Akira Nishiyama.
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