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

描述(申请人提供)：横纹肌溶解是急性肾损伤(AKI)的一个重要原因，细胞凋亡和坏死在横纹肌溶解诱导的AKI中起主要作用，但在肾脏中导致它们诱导的因素存在基本的知识空白。这一差距的持续存在是一个重大问题，因为AKI的死亡率很高，而且很少有治疗干预措施来改变这种疾病的临床进程。其长期目标是揭示横纹肌溶解导致AKI的机制，为开发保护肾脏的新疗法奠定基础。神经酰胺调节细胞凋亡和坏死，在急性肾损伤时肾脏中神经酰胺含量升高。在肾脏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的凋亡性和坏死性死亡是完全未知的。同样，有许多不同的神经酰胺种类，特定神经酰胺种类在AKI中的作用尚未确定。这项提案将回答这些问题，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)在肾脏细胞凋亡过程中，C16-神经酰胺是通过从头合成产生的，而阻断其产生可以抑制细胞凋亡；(Ii)在横纹肌溶解诱导的大鼠AKI模型中，促凋亡的bcl2蛋白BAK是神经酰胺合成酶(CERs)和长链神经酰胺产生的关键调节因子；(Iii)酸性鞘磷脂酶(SMase)产生的C26-神经酰胺存在于肾脏细胞坏死中；(Iv)在横纹肌溶解诱导的AKI大鼠模型中，大鼠肾脏皮质CER和酸性神经酰胺酶被激活，特异性神经酰胺增加5倍以上。这一不断扩大和发展的工作使我们提出了以下假设：肾毒性刺激通过CERs和sMase介导的途径增加特定种类的肾神经酰胺，诱导肾细胞死亡，最终导致肾功能衰竭。这一假说将以三个具体目标进行验证：(1)确定BAK调节CERs活性和肾脏细胞凋亡过程中特定长链神经酰胺生成的机制；(2)确定sMase生成的神经酰胺对肾脏细胞坏死的贡献；以及(3)确定特定神经酰胺在横纹肌溶解诱导的大鼠急性肾损伤和肾功能衰竭中的作用。这种方法是创新的，因为它利用新的方法来确定单个神经酰胺物种在AKI中的特定作用，即量化肾脏皮质中的单个神经酰胺物种，并在体内敲除特定CERs亚型在肾脏中的表达。这项拟议的研究具有重要意义，因为它通过识别在肾脏凋亡和坏死过程中调节特定神经酰胺物种生成的因素，促进了我们对AKI机制的现有认识。归根结底，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.