Therapeutic targeting of GSK3beta: A novel approach for podocyte protection

描述（由申请人提供）：蛋白尿是大多数慢性肾脏疾病患者的一个不变的发现，它是少数可改变的长期预后和进展到终末期肾衰竭的危险因素之一。无论最初的病因如何，肾小球蛋白尿的病理基础是足细胞功能障碍或损伤。有证据表明，糖原合成酶激酶（GSK） 3是一种多任务激酶，通过调节线粒体通透性转化（MPT）、促炎NFB激活和细胞骨架破坏等多种致病细胞事件，在介导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可预防肾损伤，是一种新的肾保护策略。本文提出的研究将揭示GSK3在足细胞功能障碍中的作用，并验证阻断GSK3可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将研究GSK3在阿霉素诱导的培养足细胞损伤中的作用。足细胞中的GSK3活性将通过RNA干扰或非活性或非抑制性突变GSK3的异位表达来特异性地操纵。GSK3对阿霉素诱导的足细胞损伤的调控作用将被评估，包括MPT和随后的足细胞死亡、促炎NFB激活和共刺激分子B7-1 （NFB靶基因）的重新表达，以及足细胞形状改变和潜在的细胞骨架紊乱；目的2将确定多西环素诱导的足细胞特异性GSK3基因敲除对阿霉素诱导的成年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，并且在药理学上不可能对足细胞进行特异性阻断。在Aim1中发现的GSK3促进足细胞损伤的致病机制将在体内敲除小鼠中得到验证。目的3将测试新型非atp竞争性GSK3小分子抑制剂TDZD-8对阿霉素肾病的预防和挽救作用。TDZD-8的疗效将与低剂量锂进行比较，低剂量锂是一种安全有效的FDA批准的药物，具有有效的GSK3抑制作用，已经存在了几十年，可以在肾脏特异性GSK3阻断可能之前用于临床试验。TDZD-8或锂对阿霉素诱导足细胞损伤的影响及相关机制将被阐明。总的来说，这些研究将使现有具有GSK3抑制活性的药物的临床试验取得快速进展，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.