Abstract
Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN.
Highlights
Where podocytes re-enter the cell cycle and start to proliferate, inhibition of CDK2 has been shown to improve renal function by inhibiting proliferation of podocytes[20]
Immunoblotting of lysates prepared from cultured human podocytes revealed that CDK2 is expressed in both proliferating and differentiated podocytes (Fig. 1B), and immunofluorescence microscopy indicated that CDK2 concentrates in the nuclei in differentiated podocytes (Fig. 1C)
Since downregulation of CDK2 induced apoptosis and reduced phosphorylation of Akt, we analyzed whether 3-phosphoinositide dependent protein kinase-1 (PDK1), which we previously showed to function as an activator of the Akt cell survival pathway in podocytes[43], is linked to podocyte apoptosis induced by CDK2 depletion
Summary
Where podocytes re-enter the cell cycle and start to proliferate, inhibition of CDK2 has been shown to improve renal function by inhibiting proliferation of podocytes[20]. Inhibition of CDK2 has been shown to decrease mesangial and glomerular endothelial cell proliferation in passive Heymann nephritis without aggravating podocyte damage[21]. Akt is known to phosphorylate CDK2 in vitro in 293T cells, and transient activation of the pathway is necessary for cell cycle progression[25,26]. CDK2 promotes activation of Akt by phosphorylating residues Ser[477] and Thr[479] under cell cycle progression in HeLa cells[27]. Constitutively activated Akt/CDK2 pathway promotes cell death[26]. We hypothesized that CDK2 could protect podocytes from apoptosis by promoting the activation of the Akt signaling pathway, and that podocyte injury induced by high LPS activity could downregulate CDK2 and subsequently enhance podocyte apoptosis
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