The molecular mechanism of increase of TGF-β expression in diabetic glomeruli

本研究旨在阐明高糖条件下糖尿病肾小球或系膜细胞中TGF-β表达升高的分子机制。首先，我们使用糖尿病动物模型研究了曲格列酮（一种PPARγ激动剂）或PKCβ抑制剂（LY333531）对糖尿病肾病发展的影响。stz诱导的糖尿病大鼠肾小球DAG含量、PKC和ERK活性均较对照大鼠升高。TGF-β纤维连接表达升高，经曲格列酮治疗后正常。用2型糖尿病模型dBdB小鼠评价LY333531的作用。在dBdB小鼠肾小球中，观察到TGF-β、4型胶原和纤维连接蛋白的增加，LY333531处理可阻止这些变化。LY333531还能抑制dB/dB小鼠肾系膜扩张或尿白蛋白排泄增加。接下来，我们检测了TGF-β在培养大鼠系膜细胞中的表达调节。在高糖状态或机械拉伸刺激下的细胞，糖尿病肾小球高血压体外模型中，ERK活性升高。高糖诱导的ERK活化被PKC抑制剂抑制，而拉伸诱导的ERK活化不被PKC抑制剂抑制，而是被酪氨酸激酶抑制剂抑制。机械拉伸也增加TGF-β和纤维连接蛋白的表达，而MEK抑制剂抑制了这种增加。上述结果表明，TGF-β在糖尿病肾小球中通过pkc - erk依赖机制表达升高。糖尿病状态下ERK活性的升高可能是高糖自身与肾小球高血压产生的机械拉伸协同作用的结果，导致TGF-β和细胞外基质蛋白过量产生，从而参与糖尿病肾小球硬化的发生发展。

The aim of this study is to clarify the molecular mechanism of the increased TGF-β expression in diabetic glomeruli or mesangial cells under high glucose conditions. First, we examined the effects to troglitazone, a PPARγ agonist, or PKCβ inhibitor (LY333531) on the development of diabetic nephropathy using animal models of diabetics. In STZ-induced diabetic rat glomeruli, the content of DAG, the activities of PKC and ERK were increased compared to control rats. The expression of TGF-β fibronection was also increased, and these abnormalities were normalized by troglitazone treatment. The effect of LY333531 was evaluated using dBdB mice, a model for type2 diabetes. In the glomeruli of dBdB mice, the increased amounts of TGF-β, type4 collagen and fibronectin were observed, which were prevented by LY333531 treatment. LY333531 also prevented the mesangial expansion or the increase of urinary albumin excretion in dB/dB mice.We next examined the regulation of TGF-β expression in cultured rat mesangial cells. In the cells under high glucose condition or stimulated by mechanical stretching, in vitro model for glomerular hypertension seen in diabetes mellitus, ERK activity was increased. High glucose induced activation of ERK was inhibited by PKC inhibitors, whereas stretch-induced ERK activation of ERK was not inhibited by PKC inhibitors, but by tyrosine kinase inhibitor. Mechanical stretching also increased the expression of TGF-β and fibronectin, and these increase were inhibited by MEK inhibitor.These results indicated that the expression of TGF-β was increased via PKC-ERK-dependent mechanism in diabetic glomeruli. The increase of ERK activity in diabetic state might be the consequence of synergistic effect of high glucose itself and mechanical stretching produced by glomerular hypertension, lead to the overproduction of TGF-β and extracellular matrix proteins, and thus involve the development and progression of diabetic glomerulosclerosis.