Elsevier

Experimental Cell Research

Volume 314, Issue 20, 10 December 2008, Pages 3654-3662
Experimental Cell Research

Research Article
Aldosterone induces myofibroblastic transdifferentiation and collagen gene expression through the Rho-kinase dependent signaling pathway in rat mesangial cells

https://doi.org/10.1016/j.yexcr.2008.09.018Get rights and content

Abstract

There is accumulating evidence indicating the role of aldosterone in the pathogenesis of hypertension and renal injury. In this study, we investigated the role of the Rho-kinase dependent signaling pathway in aldosterone-induced myofibroblastic transdifferentiation and collagen gene expression in rat mesangial cells (RMCs). Stimulation with aldosterone (1 nmol/L) significantly increased phosphorylation of myosin phosphatase target subunit-1 (MYPT-1), a marker of Rho-kinase activity, with a peak at 20 min in RMCs. Pre-incubation with a selective mineralocorticoid receptor antagonist, eplerenone (10 µmol/L), or a specific Rho-kinase inhibitor, Y27632 (10 µmol/L), attenuated the aldosterone-induced increase in MYPT-1 phosphorylation. Aldosterone also induced hypertrophy in RMCs, accompanied by an increase in actin polymerization and expression of α-smooth muscle actin (α-SMA), a myofibroblastic transdifferentiation marker. Collagen type I, III and IV mRNA levels were also increased with aldosterone stimulation. Pre-treatment with eplerenone or Y27632 prevented the aldosterone-induced cell hypertrophy, actin polymerization, the increase in α-SMA expression and the increases of collagen type I, III, IV mRNA levels in RMCs. These results suggest that aldosterone-induced mesangial cell hypertrophy is associated with cell transformation, leading to an increase in collagen gene expression via the Rho-kinase dependent signaling pathway.

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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