Table3_TFP5-Mediated CDK5 Activity Inhibition Improves Diabetic Nephropathy via NGF/Sirt1 Regulating Axis.XLS

Diabetic nephropathy (DN) is one of the leading causes of chronic kidney disease (CKD), during which hyperglycemia is composed of the major force for the deterioration to end-stage renal disease (ESRD). However, the underlying mechanism triggering the effect of hyperglycemia on DN is not very clear and the clinically available drug for hyperglycemia-induced DN is in need of urgent development. Here, we found that high glucose (HG) increased the activity of cyclin-dependent kinase 5 (CDK5) dependent on P35/25 and which upregulated the oxidative stress and apoptosis of mouse podocytes (MPC-5). TFP5, a 25-amino acid peptide inhibiting CDK5 activity, decreased the secretion of inflammation cytokines in serum and kidney, and effectively protected the kidney function in db/db mouse from hyperglycemia-induced kidney injuries. In addition, TFP5 treatment decreased HG-induced oxidative stress and cell apoptosis in MPC-5 cells and kidney tissue of db/db mouse. The principal component analysis (PCA) of RNA-seq data showed that MPC-5 cell cultured under HG, was well discriminated from that under low glucose (LG) conditions, indicating the profound influence of HG on the properties of podocytes. Furthermore, we found that HG significantly decreased the level of NGF and Sirt1, both of which correlated with CDK5 activity. Furthermore, knockdown of NGF was correlated with the decreased expression of Sirt1 while NGF overexpression leads to upregulated Sirt1 and decreased oxidative stress and apoptosis in MPC-5 cells, indicating the positive regulation between NGF and Sirt1 in podocytes. Finally, we found that K252a, an inhibitor of NGF treatment could undermine the protective role of TFP5 on hyperglycemia-induced DN in db/db mouse model. In conclusion, the CDK5-NGF/Sirt1 regulating axis may be the novel pathway to prevent DN progression and TFP5 may be a promising compound to improved hyperglycemia induced DN.

糖尿病肾病（Diabetic nephropathy, DN）是慢性肾脏病（chronic kidney disease, CKD）的主要致病诱因之一，其中高血糖是推动病情进展至终末期肾病（end-stage renal disease, ESRD）的核心驱动因素。然而，高血糖诱发DN的具体分子机制尚未完全阐明，临床上针对高血糖相关DN的治疗药物仍亟待研发。本研究发现，高糖（high glucose, HG）可通过P35/25依赖性方式激活细胞周期蛋白依赖性激酶5（cyclin-dependent kinase 5, CDK5），进而上调小鼠足细胞（mouse podocytes, MPC-5）的氧化应激水平与细胞凋亡率。TFP5作为一种可抑制CDK5活性的25肽，能够降低db/db小鼠血清与肾脏组织中炎症细胞因子的分泌量，并有效保护该模型小鼠的肾功能，缓解高血糖诱导的肾损伤。此外，TFP5干预可减轻高糖诱导的MPC-5细胞及db/db小鼠肾脏组织的氧化应激与细胞凋亡。对RNA测序（RNA-seq）数据的主成分分析（principal component analysis, PCA）显示，高糖培养的MPC-5细胞与低糖（low glucose, LG）培养的细胞呈现显著的聚类分离，表明高糖对足细胞的生物学特性具有显著影响。进一步研究发现，高糖可显著降低神经生长因子（nerve growth factor, NGF）与沉默信息调节因子1（Sirtuin 1, Sirt1）的表达水平，且二者均与CDK5活性密切相关。敲低NGF可导致Sirt1表达下调，而过表达NGF则可上调Sirt1表达并减轻MPC-5细胞的氧化应激与细胞凋亡，提示足细胞中NGF与Sirt1之间存在正向调控关系。最后，本研究证实，NGF抑制剂K252a可削弱TFP5对db/db小鼠高血糖诱导肾损伤的保护作用。综上，CDK5-NGF/Sirt1调控轴有望成为阻断DN进展的全新靶点，而TFP5则可能成为治疗高血糖相关DN的潜在候选化合物。