Role of Ceramide-induced Kidney Cell Death in Acute Kidney Injury

描述（由申请人提供）：横纹肌溶解是急性肾损伤（AKI）的重要原因，已知细胞凋亡和坏死在横纹肌溶解引起的AKI中起主要作用，但导致其在肾脏诱导的因素存在基本的知识空白。这种差距的持续存在是一个重大问题，因为AKI具有高死亡率，而且很少有治疗干预措施来改变这种疾病的临床病程。长期目标是揭示横纹肌溶解引起的AKI的机制，以开发新的治疗方法来保护肾脏。神经酰胺调节细胞凋亡和坏死，并在AKI期间在肾脏中升高。在肾细胞凋亡和坏死过程中调节神经酰胺产生的因素以及神经酰胺是否导致肾细胞凋亡和坏死细胞死亡是完全未知的。同样，有许多不同的神经酰胺种类和特定神经酰胺种类在AKI中的作用尚未确定。该建议将回答这些问题，以实现开发神经酰胺作为治疗横纹肌溶解后AKI的新治疗方法的目标。初步数据表明：(i) c16 -神经酰胺在肾细胞凋亡过程中通过从头合成产生，阻断其生成可抑制细胞凋亡；（ii）促凋亡BCL-2蛋白BAK是肾细胞凋亡过程中神经酰胺合成酶（CerS）和长链神经酰胺生成的关键调控因子；（iii）肾细胞坏死发生酸性鞘磷脂酶（SMase）生成c26 -神经酰胺；（iv）在横纹肌溶解诱导的AKI大鼠模型中，大鼠肾皮质cer和酸性SMase被激活，特异性神经酰胺升高超过5倍。这项不断扩大和发展的工作使我们提出以下假设：肾毒性刺激通过cer和SMase介导的途径提高特定种类的肾神经酰胺，诱导肾细胞死亡并最终导致肾衰竭。这一假设将通过三个特定目的进行验证：(1)确定BAK在肾细胞凋亡过程中调控CerS活性和特异性长链神经酰胺生成的机制；(2)确定smase产生的神经酰胺对肾细胞坏死的贡献；(3)确定特定神经酰胺在大鼠横纹肌溶解诱导的AKI和肾衰竭中的体内作用。该方法是创新的，因为它利用新颖的方法来确定单个神经酰胺物种在AKI中的特定作用，即肾皮质中单个神经酰胺物种的量化和体内特异性敲除肾脏内特定CerS亚型的表达。提出的研究是重要的，因为它通过确定在肾脏凋亡和坏死过程中调节特定神经酰胺种类产生的因素，推进了我们目前对AKI机制的认识。最终，这些知识有可能极大地改善AKI的治疗。

DESCRIPTION (provided by applicant): Rhabdomyolysis is a significant cause of acute kidney injury (AKI), and apoptosis and necrosis are known to play major roles in rhabdomyolysis-induced AKI, but there is a fundamental knowledge gap of the factors that lead to their induction in the kidney. Continued existence of this gap represents a significant problem as AKI has a high mortality rate and there are very few therapeutic interventions to alter the clinical course of this disease. The long-term goal is to uncover the mechanisms involved in rhabdomyolysis-induced AKI for the development of novel therapeutics to protect the kidney. Ceramides regulate apoptosis and necrosis and are elevated in the kidney during AKI. The factors that regulate production of ceramides during kidney apoptosis and necrosis and whether ceramides lead to apoptotic versus necrotic kidney cell death are completely unknown. Likewise, there are many different ceramide species and the roles for particular ceramide species in AKI have not been determined. This proposal will answer these questions to achieve the objective of developing ceramides as novel therapeutic approaches for the treatment of AKI following rhabdomyolysis. Preliminary data demonstrate that: (i) C16-ceramide is generated via de novo synthesis during kidney cell apoptosis and blocking its generation inhibits apoptosis; (ii) the pro-apoptotic BCL-2 protein BAK is a key regulator of ceramide synthases (CerS) and long-chain ceramide generation during kidney cell apoptosis; (iii) acid sphingomyelinase (SMase) generated C26-ceramide occurs in kidney cell necrosis; and (iv) rat kidney cortical CerS and acid SMase are activated and specific ceramides elevated more than 5-fold in a rat model of rhabdomyolysis-induced AKI. This expanding and developing body of work has led us to propose the following hypothesis: nephrotoxic stimuli elevate specific species of kidney ceramides through CerS and SMase- mediated pathways, inducing kidney cell death and ultimately kidney failure. This hypothesis will be tested with three specific aims: (1) determine the mechanism by which BAK regulates CerS activity and generation of specific long-chain ceramides during kidney cell apoptosis; (2) determine the contribution of SMase-generated ceramide to kidney cell necrosis; and (3) determine the in vivo contribution of specific ceramides to rhabdomyolysis-induced AKI and kidney failure in rats. The approach is innovative because it utilizes novel methodologies to identify the specific role of individual ceramide species in AKI, namely the quantification of the individual ceramide species in the kidney cortex and in vivo knockdown of the expression of particular CerS isoforms specifically within the kidney. The proposed research is significant as it advances our current knowledge of mechanisms of AKI by identifying factors that regulate generation of specific ceramide species during kidney apoptosis and necrosis. Ultimately such knowledge has the potential to greatly improve the treatment of AKI.