Inhibition of Src Kinase Blocks High Glucose–Induced EGFR Transactivation and Collagen Synthesis in Mesangial Cells and Prevents Diabetic Nephropathy in Mice

Abstract

Chronic exposure to high glucose leads to diabetic nephropathy characterized by increased mesangial matrix protein (e.g., collagen) accumulation. Altered cell signaling and gene expression accompanied by oxidative stress have been documented. The contribution of the tyrosine kinase, c-Src (Src), which is sensitive to oxidative stress, was examined. Cultured rat mesangial cells were exposed to high glucose (25 mmol/L) in the presence and absence of Src inhibitors (PP2, SU6656), Src small interfering RNA (siRNA), and the tumor necrosis factor-α–converting enzyme (TACE) inhibitor, TAPI-2. Src was investigated in vivo by administration of PP2 to streptozotocin (STZ)-induced diabetic DBA2/J mice. High glucose stimulated Src, TACE, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), extracellular signal–regulated kinase (ERK1/2, p38), and collagen IV accumulation in mesangial cells. PP2 and SU6656 blocked high glucose–stimulated phosphorylation of Src Tyr-416, EGFR, and MAPKs. These inhibitors and Src knockdown by siRNA, as well as TAPI-2, also abrogated high glucose–induced phosphorylation of these targets and collagen IV accumulation. In STZ-diabetic mice, albuminuria, increased Src pTyr-416, TACE activation, ERK and EGFR phosphorylation, glomerular collagen accumulation, and podocyte loss were inhibited by PP2. These data indicate a role for Src in a high glucose-Src-TACE-heparin-binding epidermal growth factor-EGFR-MAPK–signaling pathway to collagen accumulation. Thus, Src may provide a novel therapeutic target for diabetic nephropathy.

Blocking VEGF/Caveolin-1 signaling contributes to renal protection of fasudil in streptozotocin-induced diabetic rats

“…Src activation also occurs in the kidneys of mice with STZ-induced type 1 diabetes. The Src inhibitor PP2 prevents albuminuria, glomerular matrix protein accumulation, GBM thickening, and podocyte depletion [43] . Therefore, this study investigated the effects of fasudil on Src/caveolin-1 signaling.…”

EGF Receptor Deletion in Podocytes Attenuates Diabetic Nephropathy

“…15 A recent study also indicated that inhibition of c-Src kinase prevented progression of diabetic nephropathy. 27 To examine whether NADPH oxidase-dependent ROS production induced c-Src kinase-dependent activation of the EGFR-ERK signaling pathway and TGF-b-Smad2/3 signaling pathway in podocytes, WT diabetic mice were administered a mitochondria-targeted ROS scavenger, mito-tempol (10 mg/kg per day intraperitoneally), or a cell-permeable NADPHoxidase inhibitor, apocynin (5mg/kg per day intraperitoneally), for 3 weeks with the first dose being given 2 days after initiation of diabetes. Both mito-tempol and apocynin treatments inhibited phosphorylation of c-Src, EGFR (Y845), ERK1/2, and Smad2/3; prevented upregulation of TGF-b expression; and prevented the alterations of synaptopodin, Bcl2, and cleaved caspase3 expression in diabetic mouse glomeruli.…”

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

“…Nine fields per experimental condition were examined for GBM thickness and podocyte foot process width as previously described. 49,51 Quantitative Real-Time PCR Fifty milligrams of frozen renal cortex was sheared to powder using liquid nitrogen and then homogenized in 1 ml TRIzol reagent (Invitrogen) for RNA extraction. Purity and concentration of RNA were checked with a Nanodrop spectrophotometer (Thermo Fisher Scientific).…”

“…Staining of sections using the antibodies directed against collagen IV (1:2500; Rockland), TGF-b (1:2500, Acris-antibodies), nephrin (1:1000; Abnova), and Nox4 (1:2500; Santa Cruz Biotechnology) was performed by following the procedures previously described. 49 Quantitative analysis was performed on 50 glomeruli per kidney section with the aid of Visiomorph software (PLM Department, MSH, Toronto, ON).…”