| Backgroud and objectivesA lot of study results showed that hyperlipidaemia and lipid accumulation in kidneys were important risk factors caused glomerulosclerosis since Moorhead put forward the theory "lipid nephrotoxicity" for the first time in1982, and these mechanisms of pathophysiology and histological changes of glomerulosclerosis such as dyslipidemia, endothelial damage in the early stage, mesangial cells proliferation and extracellular matrix accumulation in the late stage were similar to the pathogenesis of arteriosclerosis, Therefore, the concept of "Glomerulus Arteriosclerosis" were put forward. In recent years, the effect of oxidized low-density lipoprotein (oxLDL) on glomerulosclerosis and tubulointerstitial fibrosis were more concerned and became research focus. Clinical data and experimental studies have demonstrated that hyperlipidemia resulted in accumulation of oxLDL in kidney, inflammatory cell infiltration, proliferation and damage of renal cells, accumulation of extracellular matrix and formation of foam cell, which directly or indirectly caused glomerular sclerosis. Previous studies about kidneys damage resulted from lipid accumulation in kidneys were more focus on renal mesangial cells and endothelial cells. Joles et al reported that oxLDL caused damage in renal mesangial cells, endothelial cells and podocytes, and podocytes might be the major victim of oxLDL insult..Podocytes are a kind of highly differentiated renal cells. The foot processes of podocytes wrap the glomerular basement membrane (GBM) to form slit diaphragm (SD) complex, which plays a crucial role in the glomerular filtration barrier (GFB). Damage of podocytes is one of important causes of proteinuria and also plays a key role in the pathogenesis and development of glomerular sclerosis. Bussolati et al found that oxLDL mediated expression deficiency of SD nephrin in podocytes and which resulted in cells damage. However, the regulatory mechanism of how oxLDL induces the damage of podocytes remains to be elucidated. Therefore, understanding the mechanisms of oxLDL-induced lipid accumulation and the influence factors of oxLDL intake in podocytes may provide a novel therapeutic strategy to alleviate podocytes damage and delay the development of glomerular sclerosis.In vitro and in vivo studies of Gutwein et al have reported the presence of CXC chemokine ligand16(CXCL16) in human renal podocytes. CXCL16exists as membrane-bound CXCL16and soluble CXCL16. One of the important roles of transmembrane CXCL16is to act as a scavenger receptor for oxLDL. OxLDL enters cells by mediating of scavenger receptor on cytomembrane, leading to intracellular lipid accumulation and formation of foam cells. A disintegrin and metalloprotease10(ADAM-10) is the key factor of transformation between the two forms, which is involved in the induced shedding of CXCL16from cell membrane. The recent studies confirm that proinflammatory cytokines like interferon-γ (INF-γ) and tumor necrosis factor-a (TNF-a) can increase the expression of cellular CXCL16and the release of its soluble form from human podocytes. To our knowledge, studies on the role of transmembrane CXCL16in oxLDL intake of podocytes in vitro and the possible regulatory effect of IFN-y and ADAM10inhibitor in this process have never been reported.Statins are effective lipid drugs found in recent years. A lot experiments and clinical studies show this kind of drugs has significant lipid-lowering function. The main function of Statins in vivo is to affect cholesterol biosynthesis by blocking binding site of rate-limiting enzyme of cholesterol synthesis selectively and vicariously facilitate receptor synthesis of low density lipoprotein(LDL)at the same time, accelerate degradation of LDL, thus reduce blood fat. Studies at present find that Statins have not only the function of protecting kidneys rely on reducing blood fat, but also the function of protecting kidneys not rely on reducing blood fat such as resistant to cells proliferation、anti-inflammatory, immunoregulation and inhibit expression of scavenger receptor, but the exact mechanism is not clear. Simvastatin is the most effective and widely used statins. The effect of statins on lipid accumulation in mouse glomerulus podocytes has not been reported at present.This study observed the situation that conditional immortal mouse glomerulus podocytes ingested oxLDL by using oil red staining for the first time, and examed the expression of CXCL16, aiming to establish the model of oxLDL-induced lipid accumulation in podocytes. Therefore it may provide credible experiment basis for studying the mechanism of lipid uptake in mouse podocytes. In vitro the effects of INF-γ、CXCL16specific antibody and AD AM10inhibitor on oxLDL’s uptake as well as the variation of CXCL16expression were also explored in order to discuss initially the factors that maybe regulated lipid accumulation in podocytes. Furthermore, the effects of simvastatin on lipid accumulation and on the expression of CXCL16and nephrin were observed aiming to discuss the protective effect of simvastatin in mouse podocytesPart1Establishment the model of lipid accumulation in mouse podocytes induced by oxLDLObjectives:Observed lipid accumulation in conditional immortal mouse podocytes induced by oxLDL in order to establish the model of lipid accumulation in podocytes, which may provide credible experiment basis for studying the mechanism of oxLDL uptake and the possible factors of regulation.Methods:Conditional immortal podocytes of mouse glomerulus (MPC5) was cultured in RPIM1640complete medium without interferon-y at37℃for10-14days until differentiated and matured, added oxLDL of concentration gradient was20,40,80,160μg/ml respectively, incubated for24h and48h. Lipid accumulation in podocytes were assessed by oil red O staining, and measured quantitatively by Colorimetric cholesterol detection kit. Results:Oil red O staining showed:No obvious lipid droplets were found in cells treated with oxLDL at20,40μg/ml for24h and48h compared with control group; A few lipid droplets were found in cells treated with oxLDL at80,160μg/ml for24h, and markedly more lipid droplets were found in cells treated with oxLDL at80,160μg/ml for48h.The quantitative analysis of total cholesterol concentration in cells showed, no obvious accumulation of lipid in podocytes incubated with different concentrations of oxLDL for24h, intracellular total cholesterol concentration was respectively97.5±29.6μg/ml,101.3±36.4μg/ml,110.6±35.μg/ml,121.5±26.4μg/ml, no significant difference was found between intracellular total cholesterol concentration of each group(P>0.05)compared with control group(90.3±30.1μg/ml). Podocytes incubated with different concentrations of oxLDL for48h, no obvious changes were found in groups with low concentration oxLDL (20,40μg/ml) compared with control group (102.6±33.5μg/ml), intracellular total cholesterol concentration was respectively98.4±31.2μg/ml,103.6±31.9μg/ml (P>0.05); whereas significant difference was found in cells treated with oxLDL at80and160μg/ml for48hours and respectively was201.0±20.3μg/ml,278.0±35.1μg/ml (P<0.05, P<0.01). In addition, cells incubated with oxLDL at80and160μg/ml for48hours had significantly higher level of total cholesterol level compared with cells treated with oxLDL at corresponding concentration for24hours, respectively (P<0.05vs24hours for80μg/ml, P<0.01vs24hours for160μg/ml).Conclusions:Lipid accumulation had dose-dependent and time-dependent in certain range in mouse glomerulus podocytes induced by oxLDL. Oil red staining showed significant lipid accumulation meanwhile intracellular cholesterol content was increased significantly in vitro cultured conditional immortal mouse podocytes induced by80μg/ml oxLDL for48h,so successfully established the model of lipid accumulation. Part2The effect of different stimulating factors on lipid accumulation and CXCL16expression in mouse podocytes induced by oxLDLObjectives:Observed the effect of cytokine recombinant mouse IFN-y, CXCL16monoclonal antibody and ADAM10inhibitor on oxLDL uptake and CXCL16expression level in vitro cultured mouse glomerulus podocytes, aiming to determine the influencing factors and regulatory mechanism of lipid accumulation in mouse podocytes,Methods:Cultured conditional immortal podocytes of mouse glomerulus (MPC5) in1640complete medium without interferon-y at37℃for10-14d until differentiated and matured, respectively added different treatment factors:1. Podocytes were incubated with oxLDL (80ug/ml) and interferon-y at different concentration (5.0,10.0,20.0U/ml) for48h, and cells were incubated with oxLDL (80ug/ml) and interferon-y (20.0U/ml) for24h and48h;2. Podocytes were incubated with oxLDL (80ug/ml)and CXCL16monoclonal antibody at concentration gradient(0.625,1.25,2.5,5.0,10.0ug/ml) for48h, and cells were treated with oxLDL (80ug/ml) and CXCL16antibody (5.0ug/ml) for24h and48h;3. Podocytes were incubated with oxLDL (80ug/ml) and ADMA10inhibitor at concentration gradient (0.125,0.25,0.5,1.0,2.0ug/ml) for48h. At the same time set group Ctrl (No drugs), group oxLDL (only added80ug/ml oxLDL). Lipid accumulation in podocytes were assessed by oil red O staining, and measured quantitatively by Colorimetric cholesterol detection kit. CXCL16expression were detected by Western blot.Results:1. The effect of interferon-y on lipid accumulation and CXCL16expression in podocytes induced by oxLDL.1.1The effect of interferon-y on lipid accumulation in oxLDL-induced podocytes: The result of oil red staining showed that no visible lipid drop was observed in control cells, whereas large amount of lipid drops were found in cells treated with only oxLDL (80μg/ml) and both oxLDL and IFN-y at different concentrations. Total cholesterol assay showed significantly increased level of total cholesterol when cells were treated with both oxLDL and IFN-y at different concentrations and respectively was209.2±44.7ug/ml,211.9±22.6ug/ml,273.8±27.1ug/ml (P<0.05;P<0.01); Moreover, cells treated with oxLDL and IFN-y(20U/ml) presented with a significantly elevated total cholesterol level compared to cells treated with only oxLDL (P<0.05). Podocytes incubated with oxLDL (80μg/ml) and IFN-y(20U/ml) for24h, intracellular total cholesterol level was174.9±22.1ug/ml; After incubated for48h, intracellular total cholesterol level was275.3±12.4ug/ml, the result showed intracellular total cholesterol level was increased significantly (P<0.01) as time extended. Compared with cells treated with oxLDL only, cells treated with both oxLDL and IFN-y for24and48hours showed significantly higher level of total cholesterol (P<0.05).1.2The effect of interferon-γ on CXCL16expression in podocytes:the effect of IFN-y on oxLDL-induced CXCL16expression was determined by western blot. Compared with control group, CXCL16expression in oxLDL treated podocytes was significantly increased (P<0.05). When cells incubated with oxLDL and IFN-y, CXCL16expression was increased in an IFN-y concentration-dependent manner. Compared with group oxLDL, CXCL16expression was slightly increased in group oxLDL+IFN-y(5U/ml) and oxLDL+IFN-y(10U/ml), whereas CXCL16expression was significantly increased (P<0.05) in group oxLDL+IFN-y(20U/ml). In addition, the effect of incubation time of IFN-y on CXCL16expression was also investigated. Compared with24-hour incubation, CXCL16expression was increased in all cells. However, significant difference between24-hour incubation and48-hour incubation was only found in IFN-y and oxLDL+IFN-y treated cells (P<0.05), indicating that CXCL16expression was increased with incubation time when treated with IFN-y or oxLDL+IFN-γ.2. The effect of CXCL16monoclonal antibody on lipid accumulation and CXCL16expression in oxLDL-induced podocytes.2.1The effect of CXCL16antibody on lipid accumulation:The result of oil red staining showed no marked change was found in cells treated with oxLDL+anti-CXCL16monoclonal antibodies (0.625, and1.25p,g/ml) compared with cells treated with only oxLDL. However, lipid accumulation was remarkably decreased in cells treated with oxLDL+anti-CXCL16monoclonal antibodies at2.5,5.0and10μg/ml. Meanwhile, compared with cells treated with only oxLDL, cells treated with oxLDL+anti-CXCL16monoclonal antibodies at2.5,5.0and10μg/ml showed significantly reduced level of total cholesterol. Total cholesterol content was respectively203.8±24.8μg/ml and174.1±15.1μg/ml in cells incubated with oxLDL and anti-CXCL16at low concentration (0.625μg/ml,1.25μg/ml); Intracellular total cholesterol content was reduced as dose-dependent in groups incubated with both oxLDL and CXCL16antibody at high concentration(2.5,5.0and10.0μg/ml)for48h, and total cholesterol level was147.2±15.lug/ml,100.7±11.4ug/ml and84.1±9.1ug/ml (P<0.05; P<0.01) respectively.2.2The effect of CXCL16antibody on CXCL16expression in podocytes:The result of quantitative test from Western blot showed CXCL16expression in podocytes was reduced as dose-dependent as concentration of CXCL16antibody increased. No significant changes were found in CXCL16expression in cells incubated with both oxLDL and CXCL16antibody at low concentration (0.625μg/ml,1.25μg/ml) for48h compared with cells treated with only oxLDL; But CXCL16expression in podocytes was significantly reduced (P<0.05; P<0.01)in groups that incubated with both oxLDL and CXCL16antibody at high concentration (2.5,5.0and10.0μg/ml) for48h. Podocytes were treated with oxLDL (80μg/ml) and CXCL16antibody (5.0μg/ml) for24h and48h, compared with cells treated with only oxLDL, CXCL16expression was significantly decreased (P<0.05).3. The effect of ADAM10inhibitor on lipid accumulation and CXCL16expression in podocytes induced by oxLDL.3.1The effect of ADAM10inhibitor on intracellular lipid accumulation:Results of oil red O staining showed that no visible lipid drop was observed in control cells and cells treated with DMSO, whereas large amount of lipid drops were found in cells treated with only oxLDL (80μg/ml). Compared with cells treated with only oxLDL, no marked change was found in cells treated with oxLDL+ADAM10inhibitor (0.125and0.25μg/ml). However, lipid accumulation was remarkably increased in cells treated with oxLDL+ADAM10inhibitor at0.5,1.0and2μg/ml. The result of colorimetry showed total cholesterol level was slightly increased in groups incubated with oxLDL and ADAM10inhibitor at0.125μg/ml or0.25μg/ml compared with group oxLDL and total cholesterol level was respective211.6±12.6μg/ml and227.8±18.3μg/ml, No statistically significant difference was found (P>0.05); whereas significant difference in total cholesterol level was only found when cells incubated with oxLDL+ADAM10inhibitor at0.5,1.0and2.0μg/ml (P<0.05; P<0.01), total cholesterol level was258.5±26.8μg/ml,284.0±23.2μg/ml,338.3±26.6μg/ml respectively.3.2The effect of ADAM10inhibitor on CXCL16expression in podocytes:Western blot analysis showed CXCL16expression was slight increased in cells incubated with oxLDL+ADAM10inhibitor at0.125and0.25μg/ml, compared with cells treated with only oxLDL (P>0.05), while CXCL16expression was significant increased in cell incubated with oxLDL+ADAM10inhibitor at0.5,1.0and2.0μg/ml (P<0.05; P<0.01).Conclusions:1CXCL16expression was increased in vitro cultured mouse glomerulus podocytes induced by oxLDL, meanwhile intracellular cholesterol level was increased. Which hinted oxLDL could up-regulate the quantities of CXCL16, then was excessively ingested, therefore resulted in cholesterol accumulation in mouse podocytes2In vitro podocytes incubated with both oxLDL and IFN-y of certain concentration showed enhanced in intracellular lipid droplets and totle cholesterol content, meanwhile CXCL16expression was up-regulated significantly. Which indicated that interferon-γ could raise CXCL16expression and then facilitate oxLDL uptake in mouse glomerulus podocytes.3After treated cells with oxLDL and CXCL16antibody of certain concentration in vitro, intracellular lipid droplets showed obviously reduced accompanied with cholesterol content was significantly decreased, meanwhile CXCL16expression was also significantly dropped. That indicated blocking expression of CXCL16by CXCL16antibody could significantly suppress oxLDL uptake and lipid accumulation in mouse podocytes.4Podocytes incubated with oxLDL and ADAM10inhibitor of certain concentration showed both intracellular lipid droplets and cholesterol content were enhanced significantly, accompanied with CXCL16expression was markedly increased. That indicated ADAM10inhibitor could significantly increased CXCL16expression and then facilitated oxLDL uptake in mouse podocytes. Part3The interventional mechanism of simvastatin for lipid accumulation in mouse podocytes induced by oxLDLObjectives:Investigate the effect of simvastatin on lipid accumulation and the expression of CXCL16and nephrin in murine podocytes induced by oxLDL in order to explore its protection mechanism.Methods:Murine podocytes (MPC5) were incubated with oxLDL (80μg/ml) with/without different concentrations of simvastatin (1.0or2.0μg/ml) for48hours. Lipid accumulation in podocytes were assessed by oil red O staining, and measured quantitatively by Colorimetric cholesterol detection kit. CXCL16and nephrin expression were detected by Western blot.Results:OxLDL treated MPC5cells exhibited obvious higher intracellular lipid accumulations compared with untreated group. Colorimetric detection found that total cholesterol was90.3±30.1μg/ml in untreated cells and226.5±21.6μg/ml in oxLDL treated cells. The difference was statistically significant (P<0.01). While cells were treated with both oxLDL and simvastatin, we observed much less lipid accumulation. Total cholesterol in oxLDL+simvastatin cells were151.8±6.8μg/ml and135.5±26.9μg/ml under1.0μg/ml or2.0μg/ml of simvastatin treatment respectively. Both were statistically significantly lower than the oxLDL treated cells (P<0.05). Western blot analysis showed that CXCL16expression was significantly increased (P<0.05) in oxLDL treated cells comparing with the untreated cells, and was significantly inhibited by application of simvastatin (P<0.05). The analysis of nephrin expression showed that there were no changes in group simvastatin compared with that of control group(P>0.05). Nephrin expression was significantly reduced by treatment with oxLDL(P<0.01); and was significantly increased by application of simvastatin (P<0.05).Conclusions:1.The lipid droplets and total cholesterol concentration were obviously reduced in mouse podocytes since incubated with simvastatin and oxLDL, and the level of CXCL16expression was also markedly lower. All these indicated simvastatin treatment could significantly decrease lipid accumulation in mouse podocytes and this protective effect was realized through inhibition of the expression of CXCL16.2. The expression of nephrin was significantly decreased in mouse podocytes induced by oxLDL. After treatment with simvastatin its expression was obviously increased. That hinted nephrin loss might contribute to podocytes lipids damage at the early stage of oxLDL exposure. Simvastatin could prevent podocytes from lipids oxidative damage, which might be due to increasement the expression of nephrin. Innovations and meanings:1.This study intuitively observed lipid accumulation in mouse glomerular podocytes using oil red O staining for the first time.The method is simple, practical, economic and better repeatability. Establishment of the model of lipid accumulation in mouse podocytes induced by oxLDL may provide credible experiment basis for studying the mechanism and the factors of regulating of oxLDL uptake2. This study in vitro cell culture confirmed that CXCL16is the key molecules to mediate the lipid injury in podocytes exposed to oxLDL. Its expression could be regulated by IFN-γ and ADAM10inhibitors, thus affected podocytes to ingest lipids.All those may provide important clues for the early prevention and treatment from lipids oxidative damage in podocytes.3. This study firstly in vitro comfirmed that simvastatin could down-regulate CXCL16expression and decrease lipid accumulation in oxLDL-induced mouse podocytes, meanwhile it could enhance the expression of nephrin. All those indicated that simvastatin could alleviate lipids oxidative damage in podocytes, which may also provide some references for the clinical application of statins to protect the kidney. |
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