The Inhibition of Atrial Natriuretic Peptide on the High Glucose-Stimulated TGFβ1/Smads/Collagen Signaling Pathway in Renal Proximal Epithelial Cells

• Main Authors: Zhao-Hong Chen, 陳肇閎
• Other Authors: Shyi-Jang Shin(辛錫璋)
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/36470596200149332881

碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 93 === Atrial natriuretic peptide (ANP) has been known to be a natriuretic, diuretic and vasodilatory hormone that regulates blood pressure and volume homeostasis. But in recent researches, ANP is also discovered to have cytoprotective function. Our previous studies demonstrate that ANP gene expression increases markedly in diabetic renal epithelial tubular cells. Transforming growth factor-β1 (TGF-β1) plays a key role in tubular fibrosis and glomerular-sclerosis in diabetic nephropathy, and it promotes extracellular matrix (ECM) accumulation through Smads signal transduction. In this study we aim to investigate whether ANP regulates high glucose-induced activation of TGF-β1/Smad/ECM signaling pathway in renal tubular epithelial cells. We culture normal rat kidney tubular epithelial cell line (NRK52E) with five different conditions: (1) normal glucose concentration (5.5mM); (2) D-mannitol (35mM); (3) high glucose concentration (35mM); (4) high glucose concentration and ANP treated (10-6 – 10-9M); and (5) high glucose concentration treated ANP (10-6M) and guanylate cyclase inhibitor,LY-83583. With RT-PCR ,we measured TGF-β1, Smad 2, Collagen I and III mRNA. We applied enzyme immunoassay to determine TGF-β1 concentration, and applied immunocytochemistry, as well as Western blot, to detect phosphorylation of Smad 2. Moreover, we can also measure Collagen I protein with Western blot method. Our data shows that high glucose concentration stimulates mRNA expression of TGF-β1, Smad 2, collagenⅠand Ⅲ.TGF-β1, phosphorylation of Smad 2/3, and CollagenⅠalso increase via stimulation of high glucose concentration. Such activation can be significantly weakened by adding ANP (10-6-10-8M) and the attenuation can be reversed with guanylate cyclase inhibitor,LY-83583. Therefore we conclude that ANP can inhibit the high glucose-induced activation of TGF-β1/Smad/Collagen signaling pathway in renal epithelial tubular cells, and such inhibition of ANP can be reversed via guanylate cyclase inhibitor.