Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis.

Lack of CD2-associated protein (CD2AP) in mice increases podocyte apoptosis and leads to glomerulosclerosis and renal failure. We showed previously that SHIP2, a negative regulator of the PI3K/AKT signalling pathway, interacts with CD2AP. Here, we found that the expression level and activity of SHIP2 and production of reactive oxygen species (ROS) are increased in cultured CD2AP knockout (CD2AP-/-) mouse podocytes. Oxidative stress was also increased in CD2AP-/- mouse glomeruli in vivo. We found that puromycin aminonucleoside (PA), known to increase ROS production and apoptosis, increases SHIP2 activity and reduces CD2AP expression in cultured human podocytes. PDK1 and CDK2, central regulators of AKT, were downregulated in CD2AP-/- or PA-treated podocytes. Downregulation of PDK1 and CDK2, ROS generation and apoptosis were prevented by CD2AP overexpression in both models. Notably, inhibition of SHIP2 activity with a small molecule inhibitor AS1949490 ameliorated ROS production in CD2AP-/- podocytes, but, surprisingly, further reduced PDK1 expression and aggravated apoptosis. AKT- and ERK-mediated signalling was diminished and remained reduced after AS1949490 treatment in the absence of CD2AP. The data suggest that inhibition of the catalytic activity of SHIP2 is beneficial in reducing oxidative stress, but leads to deleterious increase in apoptosis in podocytes with reduced expression of CD2AP.

Cyclin-dependent kinase 2 protects podocytes from apoptosis.

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.

Lipid phosphatase SHIP2 downregulates insulin signalling in podocytes.

Podocyte injury plays an important role in the development of diabetic nephropathy. Podocytes are insulin-responsive and can develop insulin resistance, but the mechanisms are unknown. To study the role of CD2-associated protein (CD2AP) in podocyte injury, we performed a yeast two-hybrid screening on a glomerular library, and found that CD2AP bound to SH2-domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2), a negative regulator of insulin signalling. SHIP2 interacts with CD2AP in glomeruli and is expressed in podocytes, where it translocates to plasma membrane after insulin stimulation. Overexpression of SHIP2 in cultured podocytes reduces Akt activation in response to insulin, and promotes apoptosis. SHIP2 is upregulated in glomeruli of insulin resistant obese Zucker rats. These results indicate that SHIP2 downregulates insulin signalling in podocytes. The upregulation of SHIP2 in Zucker rat glomeruli prior to the age of onset of proteinuria suggests a possible role for SHIP2 in the development of podocyte injury.