PTEN、SHIP和CTMP对糖尿病肾病的作用及机制研究

The glomerulosclerosis and tubular interstitial fibrosis are the main features of diabetic nephropathy, leading to decreased renal function. Many studies revealed that multiple cell signal transduction pathway involved in extracellular matrix accumulation and sclerosis of renal mesangial cells and tubular cells. At present, it was confirmed that PI3K/Akt pathway mediated the hypertrophy and secretion of extracellular matrix in renal mesangial cells and renal tubular cells in diabetes. Therefore, the relative studies aimed at PI3K/Akt pathway became gradually the hot points to prevent the injury of renal glomerulus and tulule in diabetes. Now, it is not well known whether PTEN, SHIP and CTMP, known as negative regulators of PI3K/Akt pathway, involved in the glomerulosclerosis and tubular interstitial fibrosis in diabetic nephropathy. Therefore, we plan to, in the present study, detect the expression and effect of PTEN, SHIP and CTMP on extracellular matrix secretion and fibrosis of diabetic nephropathy through diabetic mice model, in vitro cultured renal mesangial cells and renal tubular cells. The results of the present research will supply the novel content and targets for the treatment of diabetic renal glomerulosclerosis and tubular interstitial fibrosis.

肾小球硬化和肾小管间质纤维化是严重影响糖尿病肾脏功能的病理学改变。多种信号传导通路参与了肾小球系膜细胞和肾小管上皮细胞的细胞外基质分泌及硬化。近年来，PI3K/Akt通路被揭示与糖尿病肾系膜细胞及肾小管上皮细胞肥大和细胞外基质分泌相关，因此，针对PI3K/Akt通路进行调控干预，成为防治糖尿病肾小球和肾小管病变的研究方向。作为PI3K/Akt通路的负向调控因子，人第10号染色体缺失的磷酸酶及张力蛋白同源基因PTEN、SH2结构含磷酸肌醇SHIP和C末端调节蛋白CTMP是否在糖尿病肾小球硬化及肾小管间质纤维化中发挥作用及机制如何还未见系统报道。故本项目拟在糖尿病小鼠模型和体外培养的肾系膜细胞和肾小管上皮细胞，检测PTEN、SHIP和CTMP表达及对肾脏细胞外基质分泌和硬化的影响及机制，为糖尿病肾小球硬化和肾小管间质纤维化的延缓和防治提供新的研究思路，探索新的干预靶点。

本项目在前期的研究中发现糖尿病肾脏细胞外基质分泌增多和PI3K/Akt的通路过度激活有关，拟针对PI3K/Akt通路的内源性负向调控因子CTMP、PTEN和SHIP，明确其在糖尿病肾病发生发展中的作用，并调控其表达，探究是否能通过下调PI3K/Akt通路来减轻或逆转糖尿病肾病的发生发展。研究首先检测了糖尿病小鼠模型肾脏CTMP、PTEN、SHIP和磷酸化Akt的表达，又检测了细胞外基质沉积情况及相关调控因子表达，如CTGF、TGF-β1、α-SMA、E-cadherin、Col 1和Col 3。结果揭示糖尿病肾小管上皮细胞内CTMP、PTEN和SHIP较正常对照组均出现表达下调，相应的磷酸化Akt上调，并伴有促纤维化因子CTGF和TGF-β1表达增加，肾小管间质内Col 1和Col 3的沉积。本研究进一步在体外培养的肾小管上皮细胞和系膜细胞中，检测了高糖对CTMP、PTEN、SHIP、磷酸化Akt、Col 1和Col 3的影响，发现高糖可下调CTMP、PTEN或者SHIP，激活Akt，引起胶原合成分泌增多，而高表达CTMP、PTEN或者SHIP有效逆转了高糖引起的相关改变。最后，构建了CTMP质粒和SHIP的腺病毒载体，在糖尿病小鼠肾脏内上调了CTMP和SHIP的表达，检测到磷酸化Akt的表达下降，Col 1和Col 3合成减少，肾小管间质内细胞外基质沉积减少。本研究通过一系列体内外实验，证实在糖尿病肾病的发生发展过程中，PI3K/Akt通路的过度激活是一种有害因素，而针对其进行抑制是一种有效的干预手段，PI3K/Akt通路的负向调控因子CTMP、PTEN和SHIP均可作为干预靶点，通过表达上调抑制PI3K/Akt通路，以达到减轻糖尿病肾病的作用。