Abstract
Recent evidences suggest that endoplasmic reticulum (ER) stress was involved in multi pathological conditions, including diabetic nephropathy (DN). X-box binding protein 1(XBP1), as a key mediator of ER stress, has been proved having the capability of preventing oxidative stress. In this study, we investigated the effects of spliced XBP1 (XBP1S), the dominant active form of XBP1, on high glucose (HG)-induced reactive oxygen species (ROS) production and extracellular matrix (ECM) synthesis in cultured renal mesangial cells (MCs) and renal cortex of STZ-induced diabetic rats. Real time PCR and Western blot were used to evaluate the mRNA and protein levels respectively. Transfection of recombinant adenovirus vector carrying XBP1S gene (Ad-XBP1S) was used to upregulate XBP1S expression. XBP1S siRNA was used to knockdown XBP1S expression. ROS level was detected by dihydroethidium (DHE) fluorescent probe assay. The results showed that HG treatment significantly reduced XBP1S protein and mRNA level in the cultured MCs while no obvious change was observed in unspliced XBP1 (XBP1U). In the mean time, the ROS production, collagen IV and fibronectin expressions were increased. Diphenylene-chloride iodonium (DPI), a NADPH oxidase inhibtor, prevented HG-induced increases in ROS as well as collagen IV and fibronectin expressions. Transfection of Ad-XBP1S reversed HG-induced ROS production and ECM expressions. Knockdown intrinsic XBP1S expression induced increases in ROS production and ECM expressions. Supplementation of supreoxide reversed the inhibitory effect of Ad-XBP1S transfection on ECM synthesis. P47phox was increased in HG-treated MCs. Ad-XBP1S transfection reversed HG-induced p47phox increase while XBP1S knockdown upregulated p47phox expression. In the renal cortex of diabetic rats, the expression of XBP1S was reduced while p47phox, collagen IV and fibronectin expression were elevated. These results suggested that XBP1S pathway of ER stress was involved in HG-induced oxidative stress and ECM synthesis. A downstream target of XBP1S in regulating ROS formation might be NADPH oxidase.
Highlights
Diabetic nephropathy (DN) is the leading cause of end-stage renal diseases, which confers high morbidity and mortality rates of diabetic patients [1]
Western blot result showed that the XBP1S protein level in mesangial cells (MCs) was decreased significantly after culturing the cells in high glucose (HG) media for 24 h
In agreement with the observation on XBP1S protein levels, real time PCR result showed that XBP1S mRNA levels were significantly decreased after HG treatment (Fig. 1D)
Summary
Diabetic nephropathy (DN) is the leading cause of end-stage renal diseases, which confers high morbidity and mortality rates of diabetic patients [1]. The early stage of DN is characterized by the thickness at glomerular basement membrane and glomerular hypertrophy [4]. Renal glomerular ROS generation was increased dramatically in STZ-induced diabetic animal model [7]. The MCs provide structural support for capillary loops and modulate glomerular filtration [10]. The glomerular hypertrophy, a typical event in early stage of DN, has been identified to be closely related to the excessive proliferation of glomerular MCs and extracellular matrix protein (ECM) secretion [11]. Previous evidences suggested that the increased ROS under hyperglycemic condition mediates high glucose (HG) induced MCs proliferation and ECM overproduction. As an important source of ROS generation, NADPH oxidase overactivation provided the major contribution to HG-induced oxidative stress in MCs [12,13]. The mechanism that mediates HG-induced activation of NADPH oxidase is not completely understood
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