Pyruvate kinase M2 levels and activation as protective factors for diabetic nephropathy

在美国，糖尿病肾病是导致肾衰竭的主要原因。高血糖(HG)一直是 被认为是糖尿病肾病的主要危险因素，但针对HG诱导的肾脏的已知途径的治疗 损害没有显示出显著的好处。我们提出了存在的内源性保护机制 减轻HG对糖尿病肾病发展的不利影响。对类型1的独特队列的检查 糖尿病(T1D)患者(Joslin勋章获得者研究；n=1007)，患有这种疾病50-87年，其中 只有13.2%的人有独立于HbA1c的显著肾脏疾病支持这一结论。蛋白质组学分析 奖牌获得者的尸检肾小球，比较DN0-I级和IIB-III级发现在肾脏中的多种酶 糖酵解、醛糖还原酶、甲基乙二醛(MG)和线粒体途径在受保护的人中更高 DN。此外，代谢分析比较了糖尿病肾病(EGFR和lt；45ml/min/1.73m2)和 无(EGFR&GT；90ml/min/1.73m2)显示许多糖酵解中间体，山梨醇、MG和DAG是 减少了。这表明了一种通过糖酵解代谢细胞内葡萄糖的能力，即醛糖还原酶 途径、MG降解和线粒体氧化磷酸化。代谢物的子集比较 从没有糖尿病肾病的奖牌获得者到进步迅速的年轻人到糖尿病肾病支持PKM2的S保护 大自然。奖牌获得者血浆PKM2水平与EGFR、MG、糖尿病肾病分级相关。因此，激活 关键的调节变构糖酵解酶，丙酮酸激酶(PK)，特别是M2亚型，可能会增加 糖酵解和线粒体氧化磷酸化途径的通量和预防糖尿病肾病的发生或停止 进步。我们已经记录了PKM2的激活逆转了HG诱导的二酰甘油、蛋白质的升高 激酶C、山梨醇、MG和线粒体功能障碍，以及足细胞的凋亡。更进一步，击倒 PKM2的表达夸大了HG的毒性作用。使用PKM2和PKM2小分子激活剂的体内研究 PKM2足细胞特异性敲除支持PKM2对线粒体生物发生正常化的重要性 和功能，预防HG存在时糖尿病肾病的病理改变。这些数据表明糖酵解增加。 线粒体功能可能通过激活PKM2来预防HG所致的肾脏损伤。特定目标 1：验证和复制肾小球PKM2蛋白水平与肾皮质和血清的关系 奖牌获得者死后肾小球标本中代谢产物水平与PKM2的关系 肾功能的血浆水平和临床标志物。具体目标2：比较血浆PKM2水平、血清 奖牌获得者人群样本中的代谢物分布和临床肾脏标志物 并与PKM2水平和肾脏疾病的进展建立纵向关系。 具体目标3：确定导致PKM2表达或活性降低的机制 HG及其激活对恢复线粒体和细胞功能以及足细胞存活的影响 条件。

Diabetic nephropathy (DN) is the leading cause of renal failure in the US. Hyperglycemia (HG) has been identified as the primary risk factor for DN, but therapies targeting the known pathways of HG-induced renal damage have not shown significant benefits. We proposed endogenous protective mechanisms exist which mitigate the adverse effects of HG that contribute to DN development. Examination of a unique cohort of type 1 diabetic (T1D) patients (Joslin Medalist Study; n=1007) who have had the disease for 50–87 years, of whom only 13.2% have significant renal disease independent of HbA1c support this conclusion. Proteomic analysis of Medalists' post-mortem renal glomeruli, comparing DN class 0-I to IIB –III identified multiple enzymes in the glycolytic, aldose reductase, methylglyoxal (MG) and mitochondrial pathways as higher in those protected from DN. Additionally, metabolomic analyses comparing those with DN (eGFR <45 ml/min/1.73 m2) and those without (eGFR >90 ml/min/1.73 m2) showed many glycolytic intermediates, sorbitol, MG and DAG to be decreased. This suggests an ability to metabolize intracellular glucose via glycolysis, the aldose reductase pathway, MG degradation and mitochondrial oxidative phosphorylation. A subset comparison of metabolites from Medalists' without DN to younger individuals who progressed rapidly to DN supports PKM2's protective nature. Plasma PKM2 levels from the Medalists correlated with eGFR, MG and DN class. Therefore, activating the key regulatory allosteric glycolytic enzyme, pyruvate kinase (PK), especially the M2 isoform, may increase flux via glycolysis and the mitochondrial oxidative phosphorylation pathways and prevent the onset or stop DN progression. We have documented PKM2 activation reversed HG-induced elevations of diacylglycerol, protein kinase C, sorbitol, MG, and mitochondrial dysfunction, as well as apoptosis in podocytes. Further, knockdown of PKM2 exaggerated the toxic effects of HG. In vivo studies using a small molecule activator of PKM2 and PKM2 podocyte specific knock-outs support the importance of PKM2 to normalize mitochondrial biogenesis and function, and prevent pathology of DN in the presence of HG. These data indicate increases in glycolysis and mitochondrial function, possibly by PKM2 activation, can prevent renal damage due to HG. Specific Aim 1: Validate and reproduce the association of glomeruli PKM2 protein levels with renal cortex and serum metabolite levels in post-mortem glomerular specimens from Medalists to establish a relationship with PKM2 plasma levels and clinical markers of renal function. Specific Aim 2: Compare plasma PKM2 levels, serum metabolite profiles and clinical renal markers in samples from the Medalist population and in a shorter duration population, and establish a longitudinal relationship with PKM2 levels and the progression of renal disease. Specific Aim 3: Characterize the mechanism that causes the decreased expression or activities of PKM2 by HG and the effect of its activation to restore mitochondria and cellular function, and survival of podocytes in HG conditions.