Journal of Cellular Biochemistry 103:1999–2009 (2008) Atrial Natriuretic Peptide Attenuates High Glucose-Activated Transforming Growth Factor-b, Smad and Collagen Synthesis in Renal Proximal Tubular Cells Chao-Sheng Lo, 1 Zhao-Hong Chen, 1 Tusty-Jiuan Hsieh, 2 and Shyi-Jang Shin 2,3 * 1 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Graduate Institute of Medical Genetics, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Division of Endocrinology and Metabolism, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Abstract Atrial natriuretic peptide, besides its role in the regulation of volume homeostasis, has been noted to exert cytoprotective effects in several cell types from hypoxia. The present study was performed to explore the effect of ANP on high glucose-activated transforming growth factor-beta1 (TGF-b1), Smad and collagen synthesis in renal proximal epithelial cells. Cultured NRK-52E cells were divided into five groups: (1) normal glucose (5.5 mM), (2) high glucose (35 mM), (3) D-mannitol (29.5 mM), (4) high glucose plus ANP (10 6 –10 9 M), and (5) high glucose plus ANP (10 6 M) and guanylate cyclase inhibitor LY83583 (10 7 M) groups. Messenger RNA levels of TGF-b1, Smad2, and collagens were measured by RT-PCR. ELISA, immunocytochemistry and Western blotting were used to detect protein levels of TGF-b1, Smad2, phospho-Smad 2/3 and collagen type 1. We found high glucose to significantly increase mRNA levels of TGF-b1, Smad 2, collagen types I and III and protein levels of TGF-b1, phospho-Smad 2/3 and collagen type 1, but mannitol did not affect their expression. The addition of ANP significantly attenuated high glucose-enhanced mRNA and protein levels of TGF-b1, Smad and collagens. LY83583 blocked the influence of ANP on high glucose-activated TGF-b1, Smad and collagen synthesis. This is the first study to demonstrate that activation of TGF-b1, Smad and collagen synthesis stimulated by high glucose can also be inhibited by exogenous ANP in renal tubular epithelial cells. J. Cell. Biochem. 103: 1999– 2009, 2008. ß 2007 Wiley-Liss, Inc. Key words: ANP; high glucose; TGF-beta1; Smad; collagen Atrial natriuretic peptide (ANP) has been known to play an important role in the regu- lation of extracellular fluid volume and blood pressure [de Bold et al., 1981; Brenner et al., 1990]. However, ANP also is so known to be cytoprotective in several cell types, including hepatocytes [Gerbes et al., 1998; Kiemer et al., 2000], cardiomyocytes [Kato et al., 2005; Nishikimi et al., 2006], endothelial cells [Kiemer et al., 2002, 2003; Irwin et al., 2005], vascular smooth muscular cells [Baldini et al., 2005], and macro- phage [Tsukagoshi et al., 2001]. ANP protects these cells from hypoxia, ischemia-reperfusion, inflammation, and vasoconstrictors. In cultured renal mesangial and tubular cells, ANP also inhibits hypertrophy, and proliferation caused by angiotensin II [Hannken et al., 2001], cyclo- sporin A [Polte et al., 2002], and high sodium infusion [Roso ´ n et al., 2006]. Diabetes mellitus, one of the most common chronic diseases in the developed countries, associated with a threefold risk of all cardiovascular diseases and is a major cause of renal failure and blindness [Garcia et al., 1974]. Hyperglycemia is the initial trigger ß 2007 Wiley-Liss, Inc. Chao-Sheng Lo and Zhao-Hong Chen equally contributed to this work. Grant sponsor: National Science Council of Taiwan; Grant numbers: NSC 92-2314-B-037-057, NSC 93-2314-B-037- 065. *Correspondence to: Shyi-Jang Shin, Professor, Division of Endocrinology and Metabolism, Kaohsiung Medical Uni- versity Hospital, 100 Shih-Chuan 1st Rd, Kaohsiung 80708, Taiwan. E-mail: sjshin@kmu.edu.tw Received 11 March 2007; Accepted 31 August 2007 DOI 10.1002/jcb.21590