| Background In recent years, accumulating evidences indicate that hyperlipidemia accelerates the progression of glomerulosclerosis. Lipid accumulation and foam cell formation are recognized features of glomerular and tubulointerstitial injury. Evidences show that VLDL, one of triglyceride-rich lipoproteins, may be involved in the pathogenesis of renal disease. It was shown in vitro that VLDL also induces foam cell formation in human mesangial cells. However mechanisms by which VLDL mediate renal injures are unclear. Lipoprotein lipase (LPL) expressed in the arterial wall plays a key role in atherogenesis by actions of enzymolysis and molecular bridge and, thereby, leading to the formation of lipid-loaded foam cells. In kidney, LPL is expressed by glomerular mesangial cells. The precise role of LPL in mesangial cells is currently unclear. Recent observations strongly suggest that pathogenesis of glomerulosclerosis is similar to that of atherosclerosis. Then whether LPL play an important role in the promotion of VLDL-induced lipid deposition in mesangial cell is a question worthy of discussion. In this study, the role of LPL in enhancing VLDL uptake and modulating VLDL-mesangial interaction was assessed.Methods An established stable human mesangial cell line (HMCL) was used in all experiments. Human wild type LPL (hLPLwt), catalytically inactive LPL (hLPL194) or control alkaline phosphatase (hAP) were expressed in human mesangial cell line (HMCL) via adenoviral vectors. Orlistat (Tetrahydrolipstatin, THL), a specific inhibitor of the lipoidolytic activity of endogenous LPL, and heparinase, which degrades cell surface heparanase proteoglycan(HSPG) were also used to estimate the role of either enzymolysis action or "molecular bridge" action of LPL in the uptake of VLDL. Anti- low density lipoprotein receptor (LDLr) antibody and anti-LDL receptor-related protein (LRP) antibody were used to evaluate the effect of lipoprotein receptors on VLDL-induced lipid accumlation in HMCLs. Cellular lipid deposition was visualized by Oil Red O staining and analyzed quantitatively by standard enzymatic procedures. LPL protein expression and activity were measured by Western blot and a chemical analysis, respectively. Proliferation of HMCLs was evaluated by colorimetric assay using MTT. MCP-1 mRNA and secreted protein levels in treated HMCLs were determined by real-time quantitative RT-PCR, and enzyme-linked immunosorbent assay, respectively. For adhesion study, HMCLs were treated with VLDL, following by a one-hour incubation with Tamm-Horsfall protein-1 (THP-1) cells.Results1 the role of LPL in VLDL-induced lipid accumulation in HMCLs:(1) VLDL induced cellular lipid accumulation in HMCLs in time- and dose-dependent manner, and the principal component of accumulated lipid is triglyceride.(2) Human mesangial cells can express active LPL.(3) Compared with HMCLs transfected by Ad-hAP, cellular triglyceride content sharply increased in Ad-hLPLwt transfected HMCLs (109.11±5.01μg/mg protein versus 23.98±3.23μg/mg protein, P<0.01) and slightly in Ad-hLPL194 transfected HMCLs (36.33±2.64μg/mg protein versus 23.98±3.23μg/mg protein, P<0.05).(4) Orlistat (final concentration: 0.1μM, 1μM, and 10μM) inhibited LPL activity by 26%( P<0.05), 68%( P<0.01) and 80%( P<0.01), respectively. Orlistat also reduced cellular lipid deposition in a dose-dependent manner. Intracellular lipid assay showed that cellular triglyceride content was reduced from 84.4054±1.7319μg/mg protein(DMSO group, DMSO was solvent of orlistat) to 62.6526±2.0883μg/mg protein(0.1μM orlistat, P < 0.05), 46.4751±1.3543μg/mg protein(1μM orlistat, P<0.01) and 34.67122±0.8934μg/mg protein (10μM orlistat, P <0.01) in a dose-dependent manner. No significant modifications were observed in cellular total cholesterol levels in HMCLs incubated with VLDL in the presence of orlistat.(5) Pretreatment of the cells with heparinase slightly reduced cellular triglyceride accumulation in the present of high concentration of VLDL. Cellular TC has no significant different between two groups.(6) The blockade of some lipoprotein receptors, such as LDLr, didn't significantly reduce cellular triglyceride accumulation. 2 the role of LPL in VLDL-mediated mesangial injuries:(1) VLDL stimulated the proliferation of mesangial cells and up-regulated MCP-1 expression in time- and dose-dependent manner. Such an effect was accompanied by increase adhesion of monocytes to HMCLs.(2) LPLwt amplified VLDL-driven mesangial cells proliferation. Compared to the HMCL-AdhAP, MCP-1 mRNA and protein expression increasd by 39% (P<0.05) and 118% (P<0.01) in HMCL-AdhLPLwt, and number of THP-1 cells adhering to HMCL-AdhLPLwt increased 1.69-fold (P<0.05), While there were no significant differences between HMCL-AdhLPL194 and HMCL-AdhAP.(3) Orlistat inhibited the proliferation of mesangial cells and MCP-1 expression in a dose-dependent manner.(4) VLDL slightly up-regulated LPL expression in human mesangial cells in time- and dose-dependent manner.Conclusion(1) LPL can be expressed by glomerular mesangial cells.(2) VLDL-induced triglyceride accumulation in human mesangial cells is largely mediated by LPL, and enzymolysis action of LPL could be major factor in this process.(4) VLDL up-regulated LPL expression in human mesangial cells in time- and dose-dependent manner.(4) LPL enhanced VLDL-induced cellular lipid accumulation and proliferative effect on human glomerular mesangial cells and enhanced monocyte adhesion to mesangial cells through upregulation of MCP-1. Hence, LPL may be an important factor participating in initiation and progression of triglyceride-rich lipoproteins-mediated lipid renal injury.
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