Novel mechanisms of diabetic nephropathy

描述(由申请人提供)：细胞的能量状态因糖尿病而改变；其在糖尿病引起的肾脏损伤中的作用尚未得到很好的研究。糖尿病肾病以肾脏肥大和细胞外基质积聚为特征。MRNA翻译是一个耗能的过程，是蛋白质合成的限速步骤，即使基因表达受转录控制，它也是一个调控部位。然而，能量感受器在调节糖尿病引起的肾脏肥大和基质合成相关的mRNA翻译中的作用尚未得到解决。我们希望研究一种重要的能量感受器--AMP激活的蛋白激酶(AMPK)在肾脏肥大和基质积累中的作用。这些研究的理论基础是基于以下初步数据。在1型或2型糖尿病大鼠肾脏肥大阶段，AMPK的磷酸化及其活性降低，与高糖诱导的肾脏细胞蛋白质合成增加和肥大、基质蛋白质合成增加以及肾皮质和肾小球基质蛋白质合成增加有关。应用二甲双胍和AICAR可恢复1型糖尿病大鼠肾AMPK磷酸化水平的降低，并抑制肾脏肥大。然而，AMPK在1型或2型糖尿病进行性肾损害中的作用尚不清楚。我们希望验证AMPK活性调节1型和2型糖尿病患者肾脏肥大和基质堆积的假设。我们的具体目标是：具体目标1。体外研究。(A)探索参与高糖调节AMPK磷酸化的介质和信号通路。培养的肾小球上皮细胞、系膜上皮细胞和近端肾小管上皮细胞将在高血糖条件下进行研究，高血糖诱导细胞肥大，并刺激mRNA翻译和基质蛋白的合成。我们将研究AMP-ATP含量的改变、AMPK上游激酶LKB-1的活性、PI3-Kinase-Akt轴和PKC-TGF-3轴是否作为调节AMPK磷酸化和活性变化的上游信号。(B)研究AMPK对mRNA翻译起始和延伸阶段的调控。将研究AMPK在调节关键事件、mRNA翻译的起始和延伸阶段中的下游作用，并阐明介导AMPK效应的信号通路。(C)探讨AMPK在高糖诱导的肾细胞基质合成中的作用。多聚核糖体分析和体外翻译分析将被用来确定AMPK的作用。具体目的2.体内研究。探讨AMPK在1型和2型糖尿病肾脏病理中的作用。上述上游因子对AMPK磷酸化的调节及其下游效应分子的活性和调节，将在肾脏肥大的早期(4-14天)和基质沉积的建立阶段(3个月)进行研究，并与蛋白尿的功能变化和GFR的变化相关联。在1型和2型糖尿病的啮齿动物模型中，将探索AICAR和二甲双胍抑制糖尿病引起的解剖和功能变化的能力。AMPK基因敲除小鼠将被研究1型和2型糖尿病肾脏异常的演变。这些研究可能确定AMPK是一种新的损伤介质和糖尿病肾病的治疗靶点。

DESCRIPTION (provided by applicant): Energy status of cells is altered by diabetes; its role in diabetes-induced kidney injury is not well studied. Diabetic nephropathy is characterized by renal hypertrophy and extracellular matrix accumulation. mRNA translation, an energy consuming process, is the rate-limiting step in protein synthesis and is a site of regulation even when gene expression is controlled by transcription. However, the role of energy sensors in regulation of mRNA translation underlying diabetes-induced renal hypertrophy and matrix synthesis has not been addressed. We wish to examine the role of an important energy sensor, AMP-activated protein kinase (AMPK), in renal hypertrophy and matrix accumulation. The rationale for these studies is based on the following preliminary data. Phosphorylation of AMPK and its activity are reduced in association with high glucose-induced increase in protein synthesis and hypertrophy, and increase in matrix protein synthesis in renal cells, as well as, in renal cortex and glomeruli of rodents with type 1 or type 2 diabetes at the stage of renal hypertrophy. Administration of metformin and AICAR restores reduced AMPK phosphorylation and inhibits renal hypertrophy seen in the type 1 diabetic rat. However, the role of AMPK in progressive renal injury in type 1 or type 2 diabetes is not known. We wish to test the hypothesis that AMPK activity regulates renal hypertrophy and matrix accumulation in type 1 and type 2 diabetes. Our Specific Aims are: Specific aim 1. In vitro studies. (A) To explore mediators and signaling pathways involved in high glucose regulation of AMPK phosphorylation. Glomerular epithelial, mesangial and proximal tubular epithelial cells in culture will be studied under conditions of hyperglycemia that induce cell hypertrophy, and stimulate mRNA translation and synthesis of matrix proteins. We will investigate if altered AMP-ATP content, activity of LKB-1, an upstream kinase for AMPK, PI 3-kinase-Akt axis, and PKC-TGF-3 axis serve as upstream cues to regulate changes in AMPK phosphorylation and activity. (B) To investigate AMPK regulation of initiation and elongation phases of mRNA translation. The downstream effects of AMPK in regulation of critical events initiation and elongation phases of mRNA translation will be studied; signaling pathways mediating AMPK effect will be elucidated. (C) To explore the role of AMPK in high glucose induced matrix synthesis by renal cells in the context of augmented mRNA translation of matrix proteins. Polyribosomal assays and in vitro translation assays will be employed to define the role of AMPK. Specific aim 2. In vivo studies. To explore role of AMPK in renal pathology in type 1 or type 2 diabetes. Regulation of AMPK phosphorylation by aforementioned upstream factors and its activity and regulation of downstream effectors will be studied in the early stage (4 -14 days) of renal hypertrophy and established stage (3 months) of matrix accumulation and correlated with functional changes in albuminuria and changes in GFR. The ability of AICAR and metformin to inhibit both the anatomical and functional changes induced by diabetes in rodent models of type 1 and type 2 diabetes will be explored. AMPK alphal knock out mice will be studied for evolution of renal abnormalities in type 1 and type 2 diabetes. These studies may identify AMPK as a novel mediator of injury and a treatment target in diabetic nephropathy.