Phosphorylation of PACSIN2 at S313 Regulates Podocyte Architecture in Coordination with N-WASP.

Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice.

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.

Adiponectin receptor agonist AdipoRon ameliorates renal inflammation in diet-induced obese mice and endotoxin-treated human glomeruli ex vivo.

AIMS/HYPOTHESIS: Chronic low-grade inflammation with local upregulation of proinflammatory molecules plays a role in the progression of obesity-related renal injury. Reduced serum concentration of anti-inflammatory adiponectin may promote chronic inflammation. Here, we investigated the potential anti-inflammatory and renoprotective effects and mechanisms of action of AdipoRon, an adiponectin receptor agonist. METHODS: Wild-type DBA/2J mice were fed with high-fat diet (HFD) supplemented or not with AdipoRon to model obesity-induced metabolic endotoxaemia and chronic low-grade inflammation and we assessed changes in the glomerular morphology and expression of proinflammatory markers. We also treated human glomeruli ex vivo and human podocytes in vitro with AdipoRon and bacterial lipopolysaccharide (LPS), an endotoxin upregulated in obesity and diabetes, and analysed the secretion of inflammatory cytokines, activation of inflammatory signal transduction pathways, apoptosis and migration. RESULTS: In HFD-fed mice, AdipoRon attenuated renal inflammation, as demonstrated by reduced expression of glomerular activated NF-κB p65 subunit (NF-κB-p65) (70%, p < 0.001), TNFα (48%, p < 0.01), IL-1ß (51%, p < 0.001) and TGFß (46%, p < 0.001), renal IL-6 and IL-4 (21% and 20%, p < 0.05), and lowered glomerular F4/80-positive macrophage infiltration (31%, p < 0.001). In addition, AdipoRon ameliorated HFD-induced glomerular hypertrophy (12%, p < 0.001), fibronectin accumulation (50%, p < 0.01) and podocyte loss (12%, p < 0.001), and reduced podocyte foot process effacement (15%, p < 0.001) and thickening of the glomerular basement membrane (18%, p < 0.001). In cultured podocytes, AdipoRon attenuated the LPS-induced activation of the central inflammatory signalling pathways NF-κB-p65, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38-MAPK) (30%, 36% and 22%, respectively, p < 0.001), reduced the secretion of TNFα (32%, p < 0.01), and protected against podocyte apoptosis and migration. In human glomeruli ex vivo, AdipoRon reduced the LPS-induced secretion of inflammatory cytokines IL-1ß, IL-18, IL-6 and IL-10. CONCLUSIONS/INTERPRETATION: AdipoRon attenuated the renal expression of proinflammatory cytokines in HFD-fed mice and LPS-stimulated human glomeruli, which apparently contributed to the amelioration of glomerular inflammation and injury. Mechanistically, based on assays on cultured podocytes, AdipoRon reduced LPS-induced activation of the NF-κB-p65, JNK and p38-MAPK pathways, thereby impelling the decrease in apoptosis, migration and secretion of TNFα. We conclude that the activation of the adiponectin receptor by AdipoRon is a potent strategy to attenuate endotoxaemia-associated renal inflammation.

Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.

Metformin, the first-line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2-overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin-treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.-Polianskyte-Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.-B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.-H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.

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.

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

Data supporting the regulation of FOXC2 in podocyte dysfunction.

This data article shows the expression levels of specific podocyte injury markers and podocyte slit diaphragm protein nephrin in obese and lean Zucker rat glomeruli. It also contains information on the effect of the overexpression of transcription factor FOXC2 on the ratio of F- and G-actin and the expression level of ZO-1 in differentiated human podocytes. The article also shows data on the effect of treatments of differentiated podocytes with various factors associated with obesity and diabetes on the expression level of FOXC2. The detailed interpretation of these data and other aspects of podocyte injury mediated by upregulation of FOXC2 can be found in "Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility [1].

Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility.

Obesity and diabetes-related kidney diseases associate with renal failure and cardiovascular morbidity, and represent a major health issue worldwide. However, the molecular mechanisms leading to their development remain poorly understood. We observed increased expression of transcription factor FoxC2 in the podocytes of obese Zucker rats that are insulin resistant and albuminuric. We also found that depletion of adiponectin, an adipocyte-derived hormone whose secretion is decreased in obesity, upregulated FOXC2 in differentiated human podocytes in vitro. Overexpression of FOXC2 in cultured human podocytes led to increased nuclear expression of FOXC2 associated with a change of cellular morphology. This was accompanied by upregulation of vimentin, a key mesenchymal marker, and active beta-catenin, associated with podocyte injury. We also observed re-organization of the actin cytoskeleton, disrupted localization of the tight junction protein ZO-1, and increased motility of podocytes overexpressing FOXC2. These data indicate that the expression of FOXC2 in podocytes needs to be tightly regulated, and that its overexpression induces a chain of cellular events leading to podocyte dysfunction. These changes may lead to podocyte detachment and depletion ultimately contributing to albuminuria. We also suggest a novel molecular mechanism linking obesity-induced decrease in adiponectin to podocyte dysfunction via upregulation of FOXC2.