VEGF-A: A Novel Mechanistic Link Between CYP2C-Derived EETs and Nox4 in Diabetic Kidney Disease.

Diabetes is associated with decreased epoxyeicosatrienoic acid (EET) bioavailability and increased levels of glomerular vascular endothelial growth factor A (VEGF-A) expression. We examined whether a soluble epoxide hydrolase inhibitor protects against pathologic changes in diabetic kidney disease and whether the inhibition of the VEGF-A signaling pathway attenuates diabetes-induced glomerular injury. We also aimed to delineate the cross talk between cytochrome P450 2C (CYP2C)-derived EETs and VEGF-A. Streptozotocin-induced type 1 diabetic (T1D) rats were treated with 25 mg/L of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) in drinking water for 6 weeks. In parallel experiments, T1D rats were treated with either SU5416 or humanized monoclonal anti-VEGF-A neutralizing antibody for 8 weeks. Following treatment, the rats were euthanized, and kidney cortices were isolated for further analysis. Treatment with AUDA attenuated the diabetes-induced decline in kidney function. Furthermore, treatment with AUDA decreased diabetes-associated oxidative stress and NADPH oxidase activity. Interestingly, the downregulation of CYP2C11-derived EET formation is found to be correlated with the activation of the VEGF-A signaling pathway. In fact, inhibiting VEGF-A using anti-VEGF or SU5416 markedly attenuated diabetes-induced glomerular injury through the inhibition of Nox4-induced reactive oxygen species production. These findings were replicated in vitro in rat and human podocytes cultured in a diabetic milieu. Taken together, our results indicate that hyperglycemia-induced glomerular injury is mediated by the downregulation of CYP2C11-derived EET formation, followed by the activation of VEGF-A signaling and upregulation of Nox4. To our knowledge, this is the first study to highlight VEGF-A as a mechanistic link between CYP2C11-derived EET production and Nox4. ARTICLE HIGHLIGHTS: Diabetes is associated with an alteration in cytochrome P450 2C11 (CYP2C11)-derived epoxyeicosatrienoic acid (EET) bioavailability. Decreased CYP2C11-derived EET bioavailability mediates hyperglycemia-induced glomerular injury. Decreased CYP2C11-derived EET bioavailability is associated with increased reactive oxygen species production, NADPH oxidase activity, and Nox4 expression in type 1 diabetes. Decreased CYP2C11-derived EET formation mediates hyperglycemia-induced glomerular injury through the activation of the vascular endothelial growth factor A (VEGF-A) signaling pathway. Inhibiting VEGF signaling using anti-VEGF or SU5416 attenuates type 1 diabetes-induced glomerular injury by decreasing NADPH oxidase activity and NOX4 expression.

mTORC2 Signaling Regulates Nox4-Induced Podocyte Depletion in Diabetes.

AIM: Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. We explored the hypothesis that mammalian target of rapamycin complex 2 (mTORC2) mediates podocyte injury in diabetes. RESULTS: High glucose (HG)-induced podocyte injury reflected by alterations in the slit diaphragm protein podocin and podocyte depletion/apoptosis. This was paralleled by activation of the Rictor/mTORC2/Akt pathway. HG also increased the levels of Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using small interfering RNA (siRNA)-targeting Rictor in vitro decreased HG-induced Nox1 and Nox4, NADPH oxidase activity, restored podocin levels, and reduced podocyte depletion/apoptosis. Inhibition of mTORC2 had no effect on mammalian target of rapamycin complex 1 (mTORC1) activation, described by our group to be increased in diabetes, suggesting that the mTORC2 activation by HG could mediate podocyte injury independently of mTORC1. In isolated glomeruli of OVE26 mice, there was a similar activation of the Rictor/mTORC2/Akt signaling pathway with increase in Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using antisense oligonucleotides targeting Rictor restored podocin levels, reduced podocyte depletion/apoptosis, and attenuated glomerular injury and albuminuria. INNOVATION: Our data provide evidence for a novel function of mTORC2 in NADPH oxidase-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. CONCLUSION: mTORC2 and/or NADPH oxidase inhibition may represent a therapeutic modality for diabetic kidney disease. Antioxid. Redox Signal. 25, 703-719.