Mechenism Of Apoptosis Induced By Knockdown Of Fibronectin In Rat Mesangial Cells

Objective: Most renal diseases progress to end-stage renal failure with glomerulosclerosis and/or tubulointerstitial fibrosis. Extracellular matrix (ECM) overaccumulation is often observed in renal fibrosis. Fibronectin (FN) is one of the major ECM components and plays an important role in cell migration, adherence and proliferation. Fibronectin accumulation can be seen in most types of glomerulosclerosis. In the present study, we used the RNA interference (RNAi) method to directly inhibit the fibronectin synthesis in rat mesangial cells. We observed the effect of the inhibition on cell proliferation and apoptosis, then explored the apoptosis pathway. Finally, we investigated the role of calcium in the apoptosis.Method: Separated and cultured primary rat mesangial cells. Transcribed and synthesized siRNA in vitro then selected the most potent target segment of FN for inhibition. Constructing the small hairpin RNA (shRNA) vector based on retroviral vector to stably inhibit the fibronectin synthesis. Fibronectin expression level was detected with real-time PCR, western-blot, and immunofluorescence. Mitochondrial function was measured with JC-1 dye. FACS was applied to calculate the apoptotic cells number. Cyclosporin A, exogenous fibronectin and caspase 3, 8 specific inhibitor were added to identify the apoptosis pathway. Calcium release from InsP3 ( 1,4,5-triphosphate receptor ) RyR ( Ryanodine receptor) under stress was detect by confocal laser scanning microscope. During the apoptosis induction, antagonists for L-type calcium channel, InsP3 receptor and RyR on endoplasmic reticulum were applied for investigating the role of...