Insulin-like growth factor binding protein-3 mediates high glucose-induced apoptosis by increasing oxidative stress in porcine proximal tubular epithelial cells

Abstract

[한글]

[영문]Purpose: Insulin-like growth factor binding protein-3 (IGFBP-3) is the major circulating carrier protein for insulin-like growth factors (IGFs), and also acts as a potent antiproliferative agent that blocks the cell cycle and induces apoptosis in various cell types. Recently, IGFBP-3 was reported to mediate high glucose-induced apoptosis in mesangial cells and glomerular epithelial cells (podocytes). When exposed to elevated levels of plasma glucose, renal tubular epithelial cells also develop intracellular hyperglycemia, resulting in hyperglycemic damage. This study investigated the role of IGFBP-3 in high glucose-induced apoptosis in proximal tubular epithelial cells (PTECs). Methods: Expression of IGFBP-3 protein in a porcine PTEC line (LLC-PK1 cells) was measured by Western immunoblot after exposure to either low (5.5 mM) or high glucose (30 mM) medium, and mRNA expression was assayed by reverse transcriptase-PCR and real-time quantitative PCR. Apoptosis was measured by annexin V-fluorescein isothiocyanate staining and DNA fragmentation ELISA. To investigate the specific role of IGFBP-3 in high glucose-induced apoptosis, we quantified apoptosis in LLC-PK1 cells after treatment with small interfering RNA against IGFBP-3 (siRNA:IGFBP-3) in high glucose media, or in cells that overexpressed IGFBP-3 or GGG-IGFBP-3, a mutant IGFBP-3 that cannot bind to IGFs. To determine whether IGFBP-3 influences oxidative stress, we measured reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate staining in LLC-PK1 cells in high glucose media, in the absence or presence of siRNA:IGFBP-3, or IGFBP-3 overexpression. To determine whether ROS influences IGFBP-3 expression, we measured IGFBP-3 expression in LLC-PK1 cells in high glucose medium, after treatment with an antioxidant, or with hydrogen peroxide. Results: IGFBP-3 protein and mRNA expression in LLC-PK1 cells was higher in high glucose medium than in low glucose medium. Exposure to high glucose medium increased apoptosis in LLC-PK1 cells, and high glucose-induced apoptosis was abolished by treatment with siRNA:IGFBP-3, suggesting that IGFBP-3 mediates high glucose-induced apoptosis. In addition, IGFBP-3 overexpression by transfection of IGFBP-3 cDNA induced PTEC apoptosis. GGG-IGFBP-3 overexpression also increased apoptosis, suggesting that IGFBP-3 promotes PTEC apoptosis in an IGF-independent manner. Both high glucose medium and overexpression of IGFBP-3 increased oxidative stress, and siRNA:IGFBP-3 abolished this increase, suggesting that IGFBP-3 is involved in inducing oxidative stress by high glucose. Antioxidant treatment of LLC-PK1 cells decreased IGFBP-3 expression and apoptosis, while oxidative stress from hydrogen peroxide treatment increased IGFBP-3 expression. This finding suggested that oxidative stress increases IGFBP-3 expression. Conclusion: Our results suggest that increased IGFBP-3 expression by high glucose mediate high glucose-induced apoptosis in PTECs. Elevated levels of oxidative stress from high glucose increase IGFBP-3 expression, inducing apoptosis. Increased expression of IGFBP-3 by high glucose induces additional oxidative stress, which may result in amplification of hyperglycemic damage in PTECs.