FP418
ANGIOPOIETIN2 INDUCES MESANGIAL CELLS APOPTOSIS VIA SOC5STAT3 SIGNALING IN DIABETIC NEPHROPATHY MICROENVIRONMENT

INTRODUCTION AND AIMS: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD) and accounts for 30~50% of people starting renal replacement therapy in the world. Endothelial dysfunction has been associated with renal progression. The angiopoietin (Ang)/Tie ligand-receptor system tightly controls the endothelial phenotype. Ang-2 destabilizes the quiescent endothelium and modulates the inflammatory and angiogenic cytokines. The aim of this study is to examine the effect of Ang-2 on mesangial cells apoptosis in DN microenvironment.

METHODS: In vitro study, Ang-2 and Ang-1 mRNA expression in mesangial cells and glomerular endothelial cells (GECs) treated with normal glucose (NG), high glucose (HG) were measured by RT-PCR. Mouse mesangial cells were treated with NG, HG or combining with Ang-2 (300ng/ml) to detect cell viability by using the Cell Counting Kit-8 (CCK-8) assay or apoptosis by flow cytometry with Annexin-V/PI, and caspase 3 and 9 activity. The protein expression of suppressor of cytokine signaling 5 (SOCS5), signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma 2 (Bcl-2) were determined by western blotting analysis. SOCS5 siRNA transfection by lipofectamine 2000 was done to examine consequent signaling and apoptosis. In vivo study, IHC stain of Ang-2, SOCS5 and STAT3, TUNEL and DAPI was performed in db/m and db/db mice.

RESULTS: Ang-2, not Ang-1 mRNA was elevated in GECs treated with HG for 24h. Ang-2 mRNA did not increase in mesangial cells treated with HG, but Ang-2 mRNA increased in mesangial cells with co-culture of GECs treated with HG for 24h. Ang-2 increased in glomerulus of 12wk db/db mice and the source of Ang-2 could be GECs. Ang-2 caused mesangial apoptosis and low cell viability under NG for 48h, and Ang-2 aggravates these phenomena induced by HG. Ang-2 blocker could diminish mesangial cells apoptosis caused by GECs co-culture treated with HG for 48h. Ang-2 increased caspase 9 activity and decreased Bcl-2 protein in mesangial cells treated with NG and HG for 36h. Ang-2 augmented SOCS5 expression and ameliorated STAT3 expression in mesangial cells treated with NG and HG at 24h. SOCS5 siRNA suppressed mesangial cells apoptosis induced by Ang-2 under HG.

CONCLUSIONS: Ang-2 induced mesangial cells apoptosis under HG via SOC5-STAT3 signaling. Blocking Ang-2/SOCS5 signaling could be a potential therapeutic target to prevent mesangial apoptosis in DN.