BACKGROUND Increased DAN protein (Grem1, Grem2, Grem3, Cerberus, NBL1, SOST, and USAG1) levels are often associated with severe disease-states in adult kidneys. Grem1, SOST, and USAG1 have been demonstrated to be upregulated and play a critical role in the progression of diabetic nephropathy (DN); however, the expression and the role of other DAN family members in DN have not been reported yet. In this study, we investigated the expression and the role of Grem2 in the development of renal lesions in mice with type 2 DN. METHODS Fourteen-week-old BTBRob/ob (a mouse model of type 2 diabetes mellitus) and control (BTBR, wild type) mice were evaluated for renal functional and structural biomarkers. Urine was collected for protein content assay, and renal tissues were harvested for molecular analysis with real-time PCR, Western blotting, and immunohistochemistry. In vitro studies, human podocytes were transfected with Grem2 plasmid and were evaluated for apoptosis (morphologic assay and Western blotting). To evaluate the Grem2-mediated downstream signaling, the phosphorylation status of Smad2/3 and Smad1/5/8 was assessed. To establish a causal relationship, the effect of SIS3 (an inhibitor for Samd2/3) and BMP-7 (an agonist for Smad1/5/8) was evaluated on Germ2-induced podocyte apoptosis. RESULTS BTBRob/ob mice showed elevated urinary protein levels. Renal tissues of BTBRob/ob mice showed an increased expression of Grem2; both glomerular and tubular cells displayed enhanced Grem2 expression. In vitro studies, high glucose increased Grem2 expression in cultured human podocytes, whereas, Grem2 silencing partially protected podocyte from high glucose-induced apoptosis. Overexpression of Grem2 in podocytes not only increased Bax/Bcl2 expression ratio but also promoted podocyte apoptosis; moreover, an overexpression of Grem2 increased the phosphorylation of Smad2/3 and decreased the phosphorylation of Smad1/5/8; furthermore, SIS3 and BMP-7 attenuated Grem2-induced podocyte apoptosis. CONCLUSIONS High glucose increases Grem2 expression in kidney cells. Grem2 mediates podocyte apoptosis through Smads.

中文翻译：

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Grem2在高葡萄糖环境中介导足细胞凋亡。

背景DAN蛋白（Grem1，Grem2，Grem3，Cerberus，NBL1，SOST和USAG1）水平升高通常与成年肾脏的严重疾病状态相关。已经证明，Grem1，SOST和USAG1在糖尿病性肾病（DN）的进展中被上调并起关键作用。但是，尚无关于DN中其他DAN家族成员的表达和作用的报道。在这项研究中，我们调查了2型DN小鼠中Grem2在肾脏病变发展中的表达及其作用。方法评估了14周龄的BTBRob / ob（2型糖尿病小鼠模型）和对照组（BTBR，野生型）小鼠的肾功能和结构生物标志物。收集尿液进行蛋白含量测定，收集肾组织进行实时PCR，蛋白质印迹，和免疫组化。在体外研究中，用Grem2质粒转染了人类足细胞，并评估了其凋亡（形态分析和蛋白质印迹）。为了评估Grem2介导的下游信号传导，评估了Smad2 / 3和Smad1 / 5/8的磷酸化状态。为了建立因果关系，对Germ2诱导的足细胞凋亡评估了SIS3（Samd2 / 3抑制剂）和BMP-7（Smad1 / 5/8激动剂）的作用。结果BTBRob / ob小鼠尿蛋白水平升高。BTBRob / ob小鼠的肾脏组织显示Grem2表达增加。肾小球和肾小管细胞均显示增强的Grem2表达。在体外研究中，高葡萄糖可提高培养的人足细胞中Grem2的表达，而Grem2沉默可部分保护足细胞免受高糖诱导的细胞凋亡的影响。足细胞中Grem2的过表达不仅增加了Bax / Bcl2的表达率，而且还促进了足细胞的凋亡。此外，Grem2的过表达增加了Smad2 / 3的磷酸化，降低了Smad1 / 5/8的磷酸化。此外，SIS3和BMP-7减弱了Grem2诱导的足细胞凋亡。结论高糖可增加肾细胞中Grem2的表达。Grem2通过Smads介导足细胞凋亡。