BACKGROUND: Aldosterone plays an important role in fibrosis. Recent studies showed that Na(+)-H(+) exchanger isoform 1 (NHE1) is involved in mineralocorticoid-induced organ fibrosis. In this study, we examined the role of NHE1 in aldosterone-induced fibronectin accumulation in rat mesangial cells and the signaling pathway involved. METHODS: We detected the expression of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase 2 in rat glomeruli by immunochemistry and Western blot. Then the eukaryotic vectors of shRNA with insert targeting on NHE1 were successfully constructed and transfected into rat mesangial cells. The mRNA was quantified by real-time PCR. We measured the protein abundance of NHE1, ERK1/2 and phosphor-ERK1/2 by Western blot and the level of fibronectin by ELISA. RESULTS: First we demonstrated the local action of aldosterone on rat glomeruli in vivo. Then, after exposure to aldosterone, the NHE1 protein abundance was found increased in rat mesangial cells. Aldosterone treatment markedly increased the fibronectin level, which can be reduced by PD98059, spironolactone and shRNA-NHE1. PD98059 substantially reduced the aldosterone-induced NHE1 expression, while the knocking down of NHE1 did not alter aldosterone-stimulated phospho-ERK1/2 stimulation. CONCLUSION: The present study suggests that NHE1 may play an important role in aldosterone-mediated fibronectin accumulation in rat mesangial cells via the ERK1/2 pathway. Copyright 2009 S. Karger AG, Basel.
BACKGROUND:Aldosterone plays an important role in fibrosis. Recent studies showed that Na(+)-H(+) exchanger isoform 1 (NHE1) is involved in mineralocorticoid-induced organ fibrosis. In this study, we examined the role of NHE1 in aldosterone-induced fibronectin accumulation in rat mesangial cells and the signaling pathway involved. METHODS: We detected the expression of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase 2 in rat glomeruli by immunochemistry and Western blot. Then the eukaryotic vectors of shRNA with insert targeting on NHE1 were successfully constructed and transfected into rat mesangial cells. The mRNA was quantified by real-time PCR. We measured the protein abundance of NHE1, ERK1/2 and phosphor-ERK1/2 by Western blot and the level of fibronectin by ELISA. RESULTS: First we demonstrated the local action of aldosterone on rat glomeruli in vivo. Then, after exposure to aldosterone, the NHE1 protein abundance was found increased in rat mesangial cells. Aldosterone treatment markedly increased the fibronectin level, which can be reduced by PD98059, spironolactone and shRNA-NHE1. PD98059 substantially reduced the aldosterone-induced NHE1 expression, while the knocking down of NHE1 did not alter aldosterone-stimulated phospho-ERK1/2 stimulation. CONCLUSION: The present study suggests that NHE1 may play an important role in aldosterone-mediated fibronectin accumulation in rat mesangial cells via the ERK1/2 pathway. Copyright 2009 S. Karger AG, Basel.