Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.

Effects of podocin transfection on CD2AP distribution in HEK293 cells

The aim of this paper is to construct a podocin fluorescence expression vector and observe the effects of podocin transfection on CD2AP distribution in HEK293 cells.The pGEMT-easy vector containing the full-length cDNA encoding human podocin was cloned and digested with BamHI and XhoI.The digested full-length podocin was subcloned into pEGFP-C2.The constructed plas-mids were transfected into HEK293 cells and its effects on CD2AP distribution were observed by immunofluo-rescence.The pEGFP-NPHS2 expression vector was successfully constructed and podocin exclusively located on the HEK293 cell membrane.After podocin transfec-tion,CD2AP redistributed from the perinucleus to the cytoplasm in HEK293 cells.It can be concluded that podocin can recruit CD2AP to redistribute from the perinucleus to the cytoplasm in HEK293 cells.