Therapeutic targeting of GSK3beta: A novel approach for podocyte protection

描述(申请人提供)：蛋白尿是大多数类型慢性肾脏疾病患者的一种不变的发现，它是少数几个可改变的长期预后和进展为终末期肾功能衰竭的危险因素之一。不管最初的病因是什么，肾小球蛋白尿的病理基础都是足细胞功能障碍或损伤。有证据表明，糖原合成酶3(GSK)是一种多任务的激酶，通过调节多种致病细胞事件，如线粒体通透性转换(MPT)、促炎性NFB激活和细胞骨架紊乱，在包括肾脏在内的多个实体器官的急慢性损伤中发挥重要作用。抑制GSK3可以预防肾脏损伤，是一种新的肾脏保护策略。本文提出的研究将破译GSK3在足细胞功能障碍中的假定作用，并测试阻断GSK3保护足细胞免受损伤、改善蛋白尿和减缓肾小球硬化进展的新假设。目的1研究GSK3在阿霉素诱导的足细胞损伤中的作用。足细胞中的GSK3活性将通过RNA干扰或非活性或不可抑制的突变体GSK3的异位表达来特异性地操纵。将评估GSK3对阿霉素诱导的足细胞损伤的调节作用，包括MPT和随之而来的足细胞死亡，促炎的NFB激活和共刺激分子B7-1的从头表达，NFB的靶基因，以及足细胞的形状变化和潜在的细胞骨架破坏；目的2将确定多西环素诱导的足细胞特异性GSK3敲除对阿霉素诱导的小鼠足细胞病变和蛋白尿的影响。这些研究对于最终阐明GSK3在体内足细胞损伤中的作用至关重要，因为选择性GSK3抑制剂可能具有非特异性作用，而足细胞特异性阻断GSK3在药理学上是不可能的。在Aim1中确定的GSK3促进足细胞损伤的致病机制将在体内在基因敲除小鼠中得到验证。目的研究新型GSK3非三磷酸腺苷竞争性小分子抑制剂TDZD-8对阿霉素肾病的防治作用。TDZD-8的疗效将与低剂量锂进行比较，低剂量锂是FDA批准的一种安全有效的药物，具有强大的GSK3抑制作用，已存在数十年，并可在肾脏特异性GSK3阻断之前数年用于临床试验。TDZD-8或锂对阿霉素诱导的足细胞损伤的影响及其相关机制将被描述。总而言之，这些研究将使具有GSK3抑制活性的现有药物迅速进入临床试验，以改善足细胞损伤，诱导蛋白尿缓解，并减缓人类肾小球硬化的进展。 公共卫生相关性：蛋白尿在大多数类型的慢性肾脏疾病患者中是不变的发现，也是导致肾小球硬化和终末期肾功能衰竭的最终共同途径。肾小球蛋白尿的病理基础是足细胞功能障碍和损伤。现有的改善蛋白尿的策略，包括糖皮质激素和细胞毒性药物，都有严重的副作用和有限的效用。本文提出的研究将提供一种新的细胞信号分子在足细胞损伤调节中的重要作用的全面图景，并探索一种全新的基于阻断该分子来减轻蛋白尿的治疗方法。这些研究应该允许现有药物的临床试验快速进展，这些药物具有阻断活性，以改善足细胞损伤，诱导蛋白尿缓解，并减缓人类肾小球硬化的进展。

DESCRIPTION (provided by applicant): Proteinuria is an invariable finding in patients with most types of chronic kidney disease and it is one of the few modifiable risk factors for long-term prognosis and progression to end stage renal failure. Regardless of the original etiology, the pathologic basis for glomerular proteinuria is podocyte dysfunction or injury. Evidence suggests that glycogen synthase kinase (GSK) 3, a multi-tasking kinase, plays an important role in mediating acute and chronic injuries in multiple solid organs including the kidney through regulating multiple pathogenic cellular events, such as mitochondria permeability transition (MPT), proinflammatory NFB activation, and cytoskeleton disorganization. Inhibition of GSK3 prevents kidney injury and represents a novel renoprotective strategy. The studies proposed here will decipher the putative role of GSK3 in podocyte dysfunction and test the novel hypothesis that blockade of GSK3 protects podocyte from injury, improves proteinuria and slow progression of glomerulosclerosis. Aim 1 will examine the role of GSK3 in adriamycin induced podocyte injury in cultured podocytes. GSK3 activity in podocytes will be specifically manipulated by RNA interference or ectopic expression of either inactive or non-inhibitable mutant GSK3. The regulatory effect of GSK3 on adriamycin induced podocyte injuries will be assessed, including MPT and the ensuing podocyte death, proinflammatory NFB activation and de novo expression of the costimulatory molecule B7-1, an NFB target gene, as well as podocyte shape changes and the underlying cytockeleton disorganization; Aim 2 will determine the effect of doxycycline inducible podocyte specific GSK3 knockout on adriamycin induced podocytopathy and proteinuria in adult mice. These studies are essential to conclusively elucidate the role of GSK3 in podocyte injury in vivo because selective GSK3 inhibitors may have nonspecific effects and podocyte specific blockade of GSK3 is impossible pharmacologically. Pathogenic mechanisms identified in Aim1 by which GSK3 promotes podocyte injury will be validated in vivo in the knockout mice. Aim 3 will test the preventive and rescue effects of TDZD-8, a novel non-ATP competitive small molecule inhibitor of GSK3 on adriamycin induced nephropathy. The efficacy of TDZD-8 will be compared with low dose lithium, a safe and effective FDA approved drug that possesses potent GSK3 inhibitory actions, already exists for decades and could be used for clinical trials years before kidney specific GSK3 blockade is possible. The effects of TDZD-8 or lithium on adriamycin induced podocyte injury and related mechanisms will be delineated. Collectively, these studies should allow rapid progress to clinical trials of existing drugs with GSK3 inhibitory activities to improve podocyte injury, induce proteinuria remission, and slow progression of glomerulosclerosis in man. PUBLIC HEALTH RELEVANCE: Proteinuria is an invariable finding in patients with most types of chronic kidney disease and is the final common pathway driving progression to glomerulosclerosis and end stage renal failure. The pathologic basis for glomerular proteinuria is podocyte dysfunction and injury. Existing strategies to improve proteinuria, including glucocorticoids and cytotoxic drugs, have severe side effects and limited utility. The studies proposed here will provide a comprehensive picture of a new cellular signaling molecule important in the regulation of podocyte injury and investigate an entirely novel therapeutic approach to attenuate proteinuria based on blocking this molecule. These studies should allow rapid progress to clinical trials of existing drugs with blocking activities to improve podocyte injury, induce proteinuria remission, and slow progression of glomerulosclerosis in man.