Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases.

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.