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

糖尿病肾病（DN）是导致肾功能衰竭的主要原因。Hypertension（HG） 被确定为DN的主要风险因素，但针对已知的HG诱导的肾毒性途径的治疗， 损害没有显示出显著的益处。我们提出存在内源性保护机制， 减轻HG对DN发展的不利影响。1型糖尿病的一个独特队列的检查 糖尿病（T1 D）患者（Joslin Medalist研究; n=1007），患病50-87年，其中 只有13.2%的人患有与HbA 1c无关的严重肾脏疾病，支持这一结论。的蛋白质组学分析 奖牌获得者的死后肾小球，比较DN 0-I和IIB-III，发现了多种酶， 糖酵解、醛糖还原酶、甲基乙二醛（MG）和线粒体途径在那些受保护的 DN.此外，比较DN（eGFR <45 ml/min/1.73 m2）患者和 没有（eGFR >90 ml/min/1.73 m2）显示许多糖酵解中间体，山梨醇，MG和DAG， 降低这表明通过糖酵解（醛糖还原酶）代谢细胞内葡萄糖的能力 途径，MG降解和线粒体氧化磷酸化。代谢产物的子集比较 从没有DN的奖牌获得者到迅速进展为DN的年轻人，都支持PKM 2的保护作用。 自然血浆PKM 2水平与eGFR、MG和DN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，可能增加 通过糖酵解和线粒体氧化磷酸化途径，阻止DN的发生或停止 进展我们已经证明PKM 2激活逆转了HG诱导的甘油二酯、蛋白质和蛋白质水平的升高。 激酶C、山梨醇、MG和线粒体功能障碍，以及足细胞中的凋亡。此外，Knockdown PKM 2的表达增强了HG的毒性作用。使用PKM 2的小分子激活剂和 PKM 2足细胞特异性敲除支持PKM 2对正常化线粒体生物发生的重要性 和功能，并防止糖尿病肾病的病理在HG的存在。这些数据表明糖酵解增加 线粒体功能可能通过PKM 2的激活而发挥作用，从而预防HG所致的肾损害。具体目标 1：阐明并再现肾小球PKM 2蛋白水平与肾皮质和血清的相关性 Medalists尸检肾小球标本中的代谢物水平，以建立与PKM 2的关系 血浆水平和肾功能的临床标志物。具体目的2：比较血浆PKM 2水平、血清 Medalist人群样本中的代谢产物谱和临床肾脏标志物，持续时间较短 人群，并建立PKM 2水平与肾脏疾病进展的纵向关系。 具体目标3：通过以下方式表征导致PKM 2表达或活性降低的机制： HG及其激活对恢复HG中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.