oxLDL诱导小鼠足细胞脂质蓄积和CXCL16表达以及辛伐他汀干预的研究
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摘要
研究背景和目的：
自1982年,Moorhead等首次提出“脂质肾毒性”学说以来,大量的研究结果表明,高脂血症和脂质在肾脏沉积是发生肾小球硬化的重要危险因素,而肾小球硬化的病理生理和组织学变化,如血脂异常、病变早期表现内皮损害、后期出现系膜细胞增殖及细胞外基质积聚,这些机制与动脉粥样硬化的发病机制相似,因此提出了“肾小球动脉硬化”这一概念。近年来,氧化低密度脂蛋白(oxLDL)在肾小球硬化及间质纤维化中的作用越来越受到关注,成为研究热点。临床和实验研究表明,高脂血症可以使oxLDL在肾脏沉积、炎症细胞浸润、肾脏固有细胞增生和损伤、细胞外基质积聚及泡沫细胞形成,直接或间接地导致肾小球硬化。既往对脂质在肾脏沉积导致肾损害的研究,多集中在肾脏系膜细胞及内皮细胞,而Joles的实验证明oxLDL可以同时对肾脏系膜细胞、内皮细胞和足细胞造成损伤,在诸多损伤中足细胞可能是最主要的受害者。
足细胞是一种高度分化的肾脏固有细胞,在体内足细胞伸出足突包绕在肾小球基底膜(GBM)上,形成裂孔隔膜(slit diaphragm,SD)复合体,这一结构在维持肾小球滤过屏障中起重要作用。足细胞损伤是肾病蛋白尿形成的重要原因之一,在肾小球硬化的发生、发展中起重要作用。Bussolati等研究发现oxLDL能够介导足细胞SD蛋白nephrin表达缺失而致细胞损伤,但有关oxLDL介导足细胞损伤的具体调控机制,尚未得到深入的研究,因此建立可靠的oxLDL诱导肾小球足细胞脂质蓄积模型,探索影响足细胞摄取oxLDL的机制,对于减轻足细胞损伤以及延缓肾小球硬化而言,可能是一种极具前景的治疗策略。
Gutwein等的体内和体外实验证明在人肾组织足细胞上存在CXC趋化因子配体16(CXC chemokine ligand16,CXCL16)的表达。CXCL16在体内有两种存在形式：膜结合型CXCL16和可溶型CXCL16。跨膜CXCL16的一个重要作用是oxLDL的清道夫受体。oxLDL通过细胞膜上的清道夫受体介导进入细胞,造成细胞内脂质大量积聚,导致泡沫细胞形成。金属蛋白水解酶10(a disintegrin and metalloprotease10, ADAM-10)是两种形式转化的关键因素,参与诱导CXCL16从细胞膜的脱落。新近的研究认为,炎性细胞因子如干扰素-γ (INF-γ)和肿瘤坏死因子-a (TNF-a)可以增加CXCL16在人足细胞内以及可溶形式的表达。有关跨模型CXCL16是否参与体外培养鼠源性肾小球足细胞oxLDL摄取,以及干扰素-γ和ADAM10抑制剂可能的调控作用,国内外研究甚少。
他汀类药物是近年来发现的有效的调脂药。大量实验和临床研究资料显示,该类药具有显著的降脂作用。他汀类药物在体内主要的作用是通过选择性阻断胆固醇合成的限速酶结合位点,影响胆固醇生物合成,同时代偿性促进低密度脂蛋白(LDL)受体合成,加速LDL降解,从而降低血脂。目前研究发现,他汀类药物不仅有依赖降血脂的肾脏保护作用,而且还有抗细胞增殖、抗炎症、免疫调节及抑制清道夫受体表达等非依赖降血脂的肾脏保护作用,但确切机制尚不明确。辛伐他汀是最有效、应用最为广泛的一种他汀类药物,目前关于辛伐他汀对清道夫受体介导的鼠源肾小球足细胞脂质蓄积的影响尚未见报道。
本研究首次采用油红染色观察小鼠肾小球条件性永生足细胞对oxLDL摄取情况,探讨足细胞摄取oxLDL与CXCL16蛋白表达的关系,旨在建立oxLDL诱导足细胞脂质蓄积模型,为研究足细胞摄取oxLDL的机制及其可能存在的调控因素提供可靠的实验基础。通过体外观察INF-γ、CXCL16特异性抗体和ADAM10抑制剂对足细胞摄取oxLDL的影响以及CXCL16蛋白表达的变化,初步探讨可能调控足细胞脂质氧化损伤的因素。此外,通过观察辛伐他汀对oxLDL诱导鼠源肾小球足细胞脂质蓄积及CXCL16、nephrin蛋白表达的影响,探讨辛伐他汀对足细胞的保护作用机制。
第一部分oxLDL诱导小鼠足细胞脂质蓄积模型的建立
目的：观察oxLDL诱导小鼠肾小球条件永生性足细胞脂质蓄积情况,旨在建立oxLDL诱导足细胞脂质蓄积模型,为研究足细胞摄取oxLDL的机制及其可能存在的调控因素提供可靠的实验基础。
方法：采用体外培养的小鼠肾小球条件永生性足细胞株(MPC5)作为研究对象,在37℃无干扰素-γ的1640完全培养基培养10-14d至其分化成熟后,分别加入梯度浓度为20,40,80,160μg/ml的oxLDL,孵育24h及48h后,油红0染色观察细胞内脂质蓄积情况；比色法检测细胞内总胆固醇浓度。结果：油红0染色可见：与对照组相比,20、40μg/ml oxLDL组作用24h及48h,细胞中均未发现明显脂滴；80、160μg/ml oxLDL组作用24h有少许脂滴出现,作用48h均发现明显脂滴。
细胞内总胆固醇浓度定量分析结果显示,不同浓度oxLDL孵育足细胞24h脂质蓄积不明显,细胞内总胆固醇浓度分别为97.5±29.6μ g/ml、101.3±36.4μ g/ml、110.6±35.1μ g/m1、121.5±26.4u g/ml,与对照组组(90.3±30.1u g/m1)相比,各组细胞内总胆固醇浓度均无显著性差异(P均>0.05)。不同浓度oxLDL孵育足细胞48h,与对照组(102.6±33.5μ g/m1)相比,低浓度oxLDL(20、40μ g/m1)组无明显变化,细胞内总胆固醇浓度分别为98.4±31.2μg/ml、103.6±31.9μg/m1(P>0.05)；高浓度oxLDL(80及160μg/m1)组,细胞内总胆固醇浓度显著增高,分别为201.0±20.3u g/ml、278.0±35.1μg/m1(P<0.05,P<0.01)；且高浓度oxLDL组孵育48h与24h比较,随着孵育时间延长,细胞内总胆固醇浓度显著增高(P<0.05,P<0.01)。
结论：首次用油红染色观察足细胞脂质蓄积,方法简便实用。oxLDL诱导鼠源肾小球足细胞脂质蓄积在一定范围内呈剂量依赖性和时间依赖性。80μg/ml oxLDL诱导体外培养小鼠肾小球条件永生足细胞株48h,油红染色可见显著脂质沉积,细胞内胆固醇含量显著增高,成功构建脂质蓄积模型。
第二部分不同刺激因素对oxLDL诱导足细胞脂质蓄积和CXCL16表达的影响
目的：观察重组小鼠细胞因子IFN-γ、 CXCL16单克隆抗体和ADAM10抑制剂对体外培养的小鼠肾小球足细胞摄取oxLDL以及足细胞CXCL16蛋白表达的影响,初步探讨足细胞脂质蓄积的影响因素和调控机制。
方法：体外培养小鼠肾小球条件永生性足细胞(MPC5),在37℃无干扰素-γ的1640完全培养基培养10～14d至其分化成熟后,分别加不同处理因素：1.oxLDL(80ug/ml)与不同浓度干扰素-γ(5.0,10.0,20.OU/ml)共同孵育细胞48h和oxLDL(80ug/ml)与干扰素-γ(20.0U/ml)共同孵育细胞24h及48h;2.oxLDL(80ug/ml)与梯度浓度CXCL16单克隆抗体(0.625,1.25,2.5,5.0,10.0ug/ml)共同孵育细胞48h和oxLDL(80ug/ml)与CXCL16抗体(5.0ug/ml)共同孵育细胞24h及48h；3.oxLDL(80ug/ml)与梯度浓度ADMA10抑制剂(0.125,0.25,0.5,1.0,2.0ug/ml)共同孵育细胞48h。另设Ctrl组(不给药),oxLDL组(只加80ug/ml oxLDL)。油红染色观察足细胞脂质蓄积情况,比色法检测足细胞内总胆固醇浓度,Western blot检测足细胞CXCL16的蛋白表达。
结果：1.干扰素-γ对oxLDL诱导足细胞脂质蓄积及CXCL16蛋白表达的影响1.1干扰素-γ对oxLDL诱导足细胞脂质蓄积的影响：油红染色结果显示,oxLDL组及oxLDL与各浓度IFN-γ共同孵育组均出现大量脂滴。与Ctrl组相比,oxLDL+各浓度IFN-γ共同孵育组,细胞内总胆固醇含量显著增加,分别为209.2±44.7ug/ml、211.9±22.6ug/ml、273.8±27.1ug/ml(P<0.05；P<0.01)；与oxLDL组比较,oxLDL+IFN-γ(20U/ml)组细胞内总胆固醇含量显著增加(P<0.05)。用oxLDL(80μg/ml)与IFN-γ(20U/ml)共同孵育足细胞24h及48h,细胞内总胆固醇含量分别为174.9±22.1ug/ml及275.3±12.4ug/ml,结果显示随着作用时间延长,细胞内总胆固醇含量显著增加(P<0.01)。此外,oxLDL与IFN-γ共同孵育组,细胞内总胆固醇含量均高于同期oxLDL组(P<0.05)。1.2干扰素-γ对足细胞内CXCL16蛋白表达的影响：Western blot定量分析结果显示,oxLDL组及oxLDL+各浓度IFN-γ孵育组足细胞内CXCL16蛋白表达显著高于Ctrl组(P<0.05)；与oxLDL组比较,oxLDL+IFN-γ(20U/ml)组CXCL16蛋白表达显著增加(P<0.05)。时效性研究发现,oxLDL组CXCL16的蛋白表达在孵育48h与24h比较无明显变化,不随时间延长而增加；而oxLDL与IFN-γ(20U/ml)共同孵育组,细胞内CXCL16的蛋白水平在48h与24h比较,随作用时间延长而显著增加(P<0.05)。
2.CXCL16单克隆抗体对oxLDL诱导足细胞脂质蓄积及CXCL16蛋白表达的影响
2.1CXCL16抗体对足细胞内脂质蓄积的影响：油红染色结果显示,与oxLDL组相比,oxLDL与低浓度(0.625μg/ml、1.25μg/ml) CXCL16抗体共同孵育48h油红染色无明显变化；而oxLDL与高浓度(2.5、5.0和10.0μg/ml) CXCL16抗体共同孵育48h,细胞内脂滴均明显减少。细胞内胆固醇检测结果显示,与oxLDL组比较,oxLDL与低浓度(0.625μ g/m1、1.25μ g/m1)CXCL16抗体共同孵育48h,细胞内总胆固醇含量分别为203.8±24.8u g/ml及174.1±15.1μ g/ml,差异无统计学意义(P>0.05)；oxLDL与高浓度(2.5、5.0和10.0μ g/m1)CXCL16抗体共同孵育48h组,细胞内总胆固醇含量呈剂量依赖性降低,分别为147.2±15.1ug/ml、100.7±11.4ug/ml及84.1±9.1ug/ml(P<0.05；P<0.01)。
2.2CXCL16抗体对足细胞内CXCL16蛋白表达的影响：Western blot定量检测结果显示,足细胞内CXCL16蛋白表达水平随CXCL16抗体浓度增加呈剂量依赖性降低。与oxLDL组比较,oxLDL与低浓度(0.625μg/ml、1.25μg/ml) CXCL16抗体共同孵育48h组,CXCL16蛋白的表达无显著变化；而oxLDL与高浓度(2.5、5.0和10.0μg/ml) CXCL16抗体共同孵育48h组,细胞内CXCL16蛋白的表达显著降低(P<0.05；P<0.01)。以oxLDL(80μg/ml)与同一浓度(5.0μ g/m1)CXCL16抗体共同孵育足细胞24h及48h后,与oxLDL组相比,oxLDL与CXCL16抗体共同孵育组足细胞内CXCL16蛋白表达均显著降低(P<0.05)。
3. ADAM10抑制剂对oxLDL诱导足细胞脂质蓄积和CXCL16表达的影响
3.1ADAM10抑制剂对细胞内脂质蓄积的影响：油红染色结果显示,与oxLDL组相比,oxLDL分别与0.125μg/m1及0.25μg/mlADAM10抑制剂共同孵育48h,细胞内脂滴无显著变化；oxLDL分别与0.5μ g/ml,1.0μ g/m1和2.0ug/mlADAM10抑制剂共同孵育48h,细胞内脂滴显著增加。比色法结果显示,与oxLDL组比较,oxLDL与0.125μ g/ml及0.25μg/mlADAM10抑制剂共同孵育48h组,细胞内总胆固醇的含量略增高,分别为211.6±12.6μ g/m1及227.8±18.3μ g/ml,但差异无统计学意义(P>0.05)；oxLDL与0.5μ g/ml,1.0μ g/ml和2.0u g/mlADAM10抑制剂共同孵育48h组,细胞内总胆固醇的含量显著增加(P<0.05；P<0.01),分别为258.5±26.8u g/ml、284.0±23.2μg/ml、338.3±26.6μ g/ml。
3.2ADAM10抑制剂对细胞内CXCL16蛋白表达的影响：Western blot定量分析结果显示,与oxLDL组比较,oxLDL与0.125μg/ml及0.25μg/mlADAM10抑制剂共同孵育组,足细胞内CXCL16蛋白表达略增加,但差异无统计学意义(P>0.05)；oxLDL与0.5μg/ml、1.0μg/ml和2.0μg/mlADAM10抑制剂共同孵育组,足细胞内CXCL16蛋白表达显著增加(P<0.05；P<0.01)。结论：1oxLDL诱导体外培养鼠源肾小球足细胞内CXCL16蛋白表达显著增加,提示oxLDL可能上调足细胞内清道夫受体CXCL16的数量并被过度摄取,导致细胞内胆固醇聚集。
2在体外培养条件下,oxLDL与一定浓度的IFN-γ共同孵育足细胞,可使细胞内脂滴增多,总胆固醇含量增高,同时发现足细胞内CXCL16蛋白表达显著增加。提示干扰素-Y通过诱导鼠源肾小球足细胞CXCL16表达上调,促进足细胞对oxLDL的摄取。
3在体外培养条件下,oxLDL与一定浓度的CXCL16抗体共同孵育足细胞,可见细胞内脂滴明显减少,总胆固醇含量显著降低,同时发现足细胞内CXCL16蛋白水平显著降低。提示应用CXCL16抗体阻断足细胞CXCL16蛋白表达,可显著降低足细胞对oxLDL的摄取及脂质蓄积。
4在体外培养条件下,oxLDL与一定浓度的ADAM10抑制剂共同孵育足细胞,可见细胞内脂滴显著增多,总胆固醇含量显著增加,同时发现足细胞内CXCL16蛋白水平显著增加。提示ADAM10抑制剂可显著增加足细胞CXCL16表达,从而促进足细胞对oxLDL的摄取。
第三部分辛伐他汀对oxLDL诱导鼠源肾小球足细胞脂质蓄积干预机制的探讨
目的：观察辛伐他汀干预前后oxLDL诱导鼠源肾小球足细胞脂质蓄积及CXCL16、 nephrin蛋白表达的变化,探讨其对小鼠肾小球足细胞的保护机制。
方法：用80μg/ml oxLDL与不同浓度的辛伐他汀(1.0μg/ml、2.0μg/ml)共同孵育小鼠肾小球条件永生性足细胞48h,另设空白对照组(不给药),oxLDL组(只加80μg/ml oxLDL),辛伐他汀组(只加1.0μg/ml辛伐他汀)。油红0染色观察细胞内脂质蓄积情况,比色法检测细胞总胆固醇浓度,Western blot检测足细胞CXCL16/nephrin蛋白的表达。
结果：油红0染色可见,与oxLDL组比较,oxLDL+辛伐他汀各组细胞内脂滴明显减少。比色法检测发现,对照组细胞内总胆固醇含量为90.3±30.1μ g/ml,辛伐他汀组为93.3±28.6μg/ml,oxLDL组为226.5±21.6u g/ml,oxLDL+辛伐他汀组分别为151.8±6.8μ g/ml和135.5±26.9μ g/ml。与对照组比较,辛伐他汀组细胞总胆固醇含量无显著变化(P>0.05)；与oxLDL组比较,oxLDL+辛伐他汀各组细胞总胆固醇含量显著降低(P<0.05)。Western blot检测足细胞CXCL16蛋白的表达水平,与对照组比较,辛伐他汀组CXCL16蛋白表达无改变(P>0.05)；与oxLDL组比较,oxLDL+辛伐他汀各组CXCL16表达显著降低(P<0.05)。nephrin蛋白的表达检测结果显示,与对照组比较,辛伐他汀组nephrin蛋白表达无改变(P>0.05),oxLD组nephrin表达显著降低(P<0.01)；与oxLDL组比较,oxLDL+辛伐他汀各组nephrin表达水平显著增加(P<0.05)。结论：1.经辛伐他汀和oxLDL共同孵育的鼠源足细胞内脂滴明显减少,总胆固醇浓度显著降低,足细胞CXCL16蛋白表达水平亦显著降低。提示辛伐他汀下调oxLDL诱导的鼠源肾小球足细胞CXCL16蛋白表达,减少足细胞吞噬脂质,从而减轻足细胞内脂质蓄积。
2.oxLDL诱导鼠源足细胞nephrin蛋白表达显著下降,经辛伐他汀和oxLDL共同孵育的足细胞内nephrin蛋白表达显著增加,提示oxLDL诱导鼠源足细胞nephrin蛋白表达缺失而致足细胞损伤,辛伐他汀能够上调oxLDL诱导的鼠源肾小球足细胞nephrin蛋白表达,对于稳定足细胞功能意义重大。
创新和意义：
1.首次用油红0染色直观地观察oxLDL诱导鼠源肾小球足细胞内脂质蓄积情况,方法简便实用、经济,可重复性强。oxLDL诱导足细胞脂质蓄积模型的建立,为研究足细胞摄取oxLDL的机制及其调控因素提供可靠的实验基础。
2.通过体外细胞培养,证实CXCL16是介导足细胞脂质损伤的关键分子,其表达可被IFN-γ、ADAM10抑制剂等调控,从而影响足细胞摄取脂质,为临床早期防治足细胞脂质氧化损伤提供重要线索。
3.首次通过体外实验证实,辛伐他汀干预能够下调oxLDL诱导的鼠源肾小球足细胞CXCL16蛋白表达,减轻足细胞内脂质蓄积；同时上调足细胞nephrin蛋白表达,从而减轻足细胞损伤,初步探讨辛伐他汀对足细胞的保护作用,为临床应用他汀类药物保护肾脏提供参考依据。
Backgroud and objectives
A 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 podocytes
Part1Establishment the model of lipid accumulation in mouse podocytes induced by oxLDL
Objectives: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 oxLDL
Objectives: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 podocytes
2In 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 oxLDL
Objectives: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 uptake
2. 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.
自1982年,Moorhead等首次提出“脂质肾毒性”学说以来,大量的研究结果表明,高脂血症和脂质在肾脏沉积是发生肾小球硬化的重要危险因素,而肾小球硬化的病理生理和组织学变化,如血脂异常、病变早期表现内皮损害、后期出现系膜细胞增殖及细胞外基质积聚,这些机制与动脉粥样硬化的发病机制相似,因此提出了“肾小球动脉硬化”这一概念。近年来,氧化低密度脂蛋白(oxLDL)在肾小球硬化及间质纤维化中的作用越来越受到关注,成为研究热点。临床和实验研究表明,高脂血症可以使oxLDL在肾脏沉积、炎症细胞浸润、肾脏固有细胞增生和损伤、细胞外基质积聚及泡沫细胞形成,直接或间接地导致肾小球硬化。既往对脂质在肾脏沉积导致肾损害的研究,多集中在肾脏系膜细胞及内皮细胞,而Joles的实验证明oxLDL可以同时对肾脏系膜细胞、内皮细胞和足细胞造成损伤,在诸多损伤中足细胞可能是最主要的受害者。
足细胞是一种高度分化的肾脏固有细胞,在体内足细胞伸出足突包绕在肾小球基底膜(GBM)上,形成裂孔隔膜(slit diaphragm,SD)复合体,这一结构在维持肾小球滤过屏障中起重要作用。足细胞损伤是肾病蛋白尿形成的重要原因之一,在肾小球硬化的发生、发展中起重要作用。Bussolati等研究发现oxLDL能够介导足细胞SD蛋白nephrin表达缺失而致细胞损伤,但有关oxLDL介导足细胞损伤的具体调控机制,尚未得到深入的研究,因此建立可靠的oxLDL诱导肾小球足细胞脂质蓄积模型,探索影响足细胞摄取oxLDL的机制,对于减轻足细胞损伤以及延缓肾小球硬化而言,可能是一种极具前景的治疗策略。
Gutwein等的体内和体外实验证明在人肾组织足细胞上存在CXC趋化因子配体16(CXC chemokine ligand16,CXCL16)的表达。CXCL16在体内有两种存在形式：膜结合型CXCL16和可溶型CXCL16。跨膜CXCL16的一个重要作用是oxLDL的清道夫受体。oxLDL通过细胞膜上的清道夫受体介导进入细胞,造成细胞内脂质大量积聚,导致泡沫细胞形成。金属蛋白水解酶10(a disintegrin and metalloprotease10, ADAM-10)是两种形式转化的关键因素,参与诱导CXCL16从细胞膜的脱落。新近的研究认为,炎性细胞因子如干扰素-γ (INF-γ)和肿瘤坏死因子-a (TNF-a)可以增加CXCL16在人足细胞内以及可溶形式的表达。有关跨模型CXCL16是否参与体外培养鼠源性肾小球足细胞oxLDL摄取,以及干扰素-γ和ADAM10抑制剂可能的调控作用,国内外研究甚少。
他汀类药物是近年来发现的有效的调脂药。大量实验和临床研究资料显示,该类药具有显著的降脂作用。他汀类药物在体内主要的作用是通过选择性阻断胆固醇合成的限速酶结合位点,影响胆固醇生物合成,同时代偿性促进低密度脂蛋白(LDL)受体合成,加速LDL降解,从而降低血脂。目前研究发现,他汀类药物不仅有依赖降血脂的肾脏保护作用,而且还有抗细胞增殖、抗炎症、免疫调节及抑制清道夫受体表达等非依赖降血脂的肾脏保护作用,但确切机制尚不明确。辛伐他汀是最有效、应用最为广泛的一种他汀类药物,目前关于辛伐他汀对清道夫受体介导的鼠源肾小球足细胞脂质蓄积的影响尚未见报道。
本研究首次采用油红染色观察小鼠肾小球条件性永生足细胞对oxLDL摄取情况,探讨足细胞摄取oxLDL与CXCL16蛋白表达的关系,旨在建立oxLDL诱导足细胞脂质蓄积模型,为研究足细胞摄取oxLDL的机制及其可能存在的调控因素提供可靠的实验基础。通过体外观察INF-γ、CXCL16特异性抗体和ADAM10抑制剂对足细胞摄取oxLDL的影响以及CXCL16蛋白表达的变化,初步探讨可能调控足细胞脂质氧化损伤的因素。此外,通过观察辛伐他汀对oxLDL诱导鼠源肾小球足细胞脂质蓄积及CXCL16、nephrin蛋白表达的影响,探讨辛伐他汀对足细胞的保护作用机制。
第一部分oxLDL诱导小鼠足细胞脂质蓄积模型的建立
目的：观察oxLDL诱导小鼠肾小球条件永生性足细胞脂质蓄积情况,旨在建立oxLDL诱导足细胞脂质蓄积模型,为研究足细胞摄取oxLDL的机制及其可能存在的调控因素提供可靠的实验基础。
方法：采用体外培养的小鼠肾小球条件永生性足细胞株(MPC5)作为研究对象,在37℃无干扰素-γ的1640完全培养基培养10-14d至其分化成熟后,分别加入梯度浓度为20,40,80,160μg/ml的oxLDL,孵育24h及48h后,油红0染色观察细胞内脂质蓄积情况；比色法检测细胞内总胆固醇浓度。结果：油红0染色可见：与对照组相比,20、40μg/ml oxLDL组作用24h及48h,细胞中均未发现明显脂滴；80、160μg/ml oxLDL组作用24h有少许脂滴出现,作用48h均发现明显脂滴。
细胞内总胆固醇浓度定量分析结果显示,不同浓度oxLDL孵育足细胞24h脂质蓄积不明显,细胞内总胆固醇浓度分别为97.5±29.6μ g/ml、101.3±36.4μ g/ml、110.6±35.1μ g/m1、121.5±26.4u g/ml,与对照组组(90.3±30.1u g/m1)相比,各组细胞内总胆固醇浓度均无显著性差异(P均>0.05)。不同浓度oxLDL孵育足细胞48h,与对照组(102.6±33.5μ g/m1)相比,低浓度oxLDL(20、40μ g/m1)组无明显变化,细胞内总胆固醇浓度分别为98.4±31.2μg/ml、103.6±31.9μg/m1(P>0.05)；高浓度oxLDL(80及160μg/m1)组,细胞内总胆固醇浓度显著增高,分别为201.0±20.3u g/ml、278.0±35.1μg/m1(P<0.05,P<0.01)；且高浓度oxLDL组孵育48h与24h比较,随着孵育时间延长,细胞内总胆固醇浓度显著增高(P<0.05,P<0.01)。
结论：首次用油红染色观察足细胞脂质蓄积,方法简便实用。oxLDL诱导鼠源肾小球足细胞脂质蓄积在一定范围内呈剂量依赖性和时间依赖性。80μg/ml oxLDL诱导体外培养小鼠肾小球条件永生足细胞株48h,油红染色可见显著脂质沉积,细胞内胆固醇含量显著增高,成功构建脂质蓄积模型。
第二部分不同刺激因素对oxLDL诱导足细胞脂质蓄积和CXCL16表达的影响
目的：观察重组小鼠细胞因子IFN-γ、 CXCL16单克隆抗体和ADAM10抑制剂对体外培养的小鼠肾小球足细胞摄取oxLDL以及足细胞CXCL16蛋白表达的影响,初步探讨足细胞脂质蓄积的影响因素和调控机制。
方法：体外培养小鼠肾小球条件永生性足细胞(MPC5),在37℃无干扰素-γ的1640完全培养基培养10～14d至其分化成熟后,分别加不同处理因素：1.oxLDL(80ug/ml)与不同浓度干扰素-γ(5.0,10.0,20.OU/ml)共同孵育细胞48h和oxLDL(80ug/ml)与干扰素-γ(20.0U/ml)共同孵育细胞24h及48h;2.oxLDL(80ug/ml)与梯度浓度CXCL16单克隆抗体(0.625,1.25,2.5,5.0,10.0ug/ml)共同孵育细胞48h和oxLDL(80ug/ml)与CXCL16抗体(5.0ug/ml)共同孵育细胞24h及48h；3.oxLDL(80ug/ml)与梯度浓度ADMA10抑制剂(0.125,0.25,0.5,1.0,2.0ug/ml)共同孵育细胞48h。另设Ctrl组(不给药),oxLDL组(只加80ug/ml oxLDL)。油红染色观察足细胞脂质蓄积情况,比色法检测足细胞内总胆固醇浓度,Western blot检测足细胞CXCL16的蛋白表达。
结果：1.干扰素-γ对oxLDL诱导足细胞脂质蓄积及CXCL16蛋白表达的影响1.1干扰素-γ对oxLDL诱导足细胞脂质蓄积的影响：油红染色结果显示,oxLDL组及oxLDL与各浓度IFN-γ共同孵育组均出现大量脂滴。与Ctrl组相比,oxLDL+各浓度IFN-γ共同孵育组,细胞内总胆固醇含量显著增加,分别为209.2±44.7ug/ml、211.9±22.6ug/ml、273.8±27.1ug/ml(P<0.05；P<0.01)；与oxLDL组比较,oxLDL+IFN-γ(20U/ml)组细胞内总胆固醇含量显著增加(P<0.05)。用oxLDL(80μg/ml)与IFN-γ(20U/ml)共同孵育足细胞24h及48h,细胞内总胆固醇含量分别为174.9±22.1ug/ml及275.3±12.4ug/ml,结果显示随着作用时间延长,细胞内总胆固醇含量显著增加(P<0.01)。此外,oxLDL与IFN-γ共同孵育组,细胞内总胆固醇含量均高于同期oxLDL组(P<0.05)。1.2干扰素-γ对足细胞内CXCL16蛋白表达的影响：Western blot定量分析结果显示,oxLDL组及oxLDL+各浓度IFN-γ孵育组足细胞内CXCL16蛋白表达显著高于Ctrl组(P<0.05)；与oxLDL组比较,oxLDL+IFN-γ(20U/ml)组CXCL16蛋白表达显著增加(P<0.05)。时效性研究发现,oxLDL组CXCL16的蛋白表达在孵育48h与24h比较无明显变化,不随时间延长而增加；而oxLDL与IFN-γ(20U/ml)共同孵育组,细胞内CXCL16的蛋白水平在48h与24h比较,随作用时间延长而显著增加(P<0.05)。
2.CXCL16单克隆抗体对oxLDL诱导足细胞脂质蓄积及CXCL16蛋白表达的影响
2.1CXCL16抗体对足细胞内脂质蓄积的影响：油红染色结果显示,与oxLDL组相比,oxLDL与低浓度(0.625μg/ml、1.25μg/ml) CXCL16抗体共同孵育48h油红染色无明显变化；而oxLDL与高浓度(2.5、5.0和10.0μg/ml) CXCL16抗体共同孵育48h,细胞内脂滴均明显减少。细胞内胆固醇检测结果显示,与oxLDL组比较,oxLDL与低浓度(0.625μ g/m1、1.25μ g/m1)CXCL16抗体共同孵育48h,细胞内总胆固醇含量分别为203.8±24.8u g/ml及174.1±15.1μ g/ml,差异无统计学意义(P>0.05)；oxLDL与高浓度(2.5、5.0和10.0μ g/m1)CXCL16抗体共同孵育48h组,细胞内总胆固醇含量呈剂量依赖性降低,分别为147.2±15.1ug/ml、100.7±11.4ug/ml及84.1±9.1ug/ml(P<0.05；P<0.01)。
2.2CXCL16抗体对足细胞内CXCL16蛋白表达的影响：Western blot定量检测结果显示,足细胞内CXCL16蛋白表达水平随CXCL16抗体浓度增加呈剂量依赖性降低。与oxLDL组比较,oxLDL与低浓度(0.625μg/ml、1.25μg/ml) CXCL16抗体共同孵育48h组,CXCL16蛋白的表达无显著变化；而oxLDL与高浓度(2.5、5.0和10.0μg/ml) CXCL16抗体共同孵育48h组,细胞内CXCL16蛋白的表达显著降低(P<0.05；P<0.01)。以oxLDL(80μg/ml)与同一浓度(5.0μ g/m1)CXCL16抗体共同孵育足细胞24h及48h后,与oxLDL组相比,oxLDL与CXCL16抗体共同孵育组足细胞内CXCL16蛋白表达均显著降低(P<0.05)。
3. ADAM10抑制剂对oxLDL诱导足细胞脂质蓄积和CXCL16表达的影响
3.1ADAM10抑制剂对细胞内脂质蓄积的影响：油红染色结果显示,与oxLDL组相比,oxLDL分别与0.125μg/m1及0.25μg/mlADAM10抑制剂共同孵育48h,细胞内脂滴无显著变化；oxLDL分别与0.5μ g/ml,1.0μ g/m1和2.0ug/mlADAM10抑制剂共同孵育48h,细胞内脂滴显著增加。比色法结果显示,与oxLDL组比较,oxLDL与0.125μ g/ml及0.25μg/mlADAM10抑制剂共同孵育48h组,细胞内总胆固醇的含量略增高,分别为211.6±12.6μ g/m1及227.8±18.3μ g/ml,但差异无统计学意义(P>0.05)；oxLDL与0.5μ g/ml,1.0μ g/ml和2.0u g/mlADAM10抑制剂共同孵育48h组,细胞内总胆固醇的含量显著增加(P<0.05；P<0.01),分别为258.5±26.8u g/ml、284.0±23.2μg/ml、338.3±26.6μ g/ml。
3.2ADAM10抑制剂对细胞内CXCL16蛋白表达的影响：Western blot定量分析结果显示,与oxLDL组比较,oxLDL与0.125μg/ml及0.25μg/mlADAM10抑制剂共同孵育组,足细胞内CXCL16蛋白表达略增加,但差异无统计学意义(P>0.05)；oxLDL与0.5μg/ml、1.0μg/ml和2.0μg/mlADAM10抑制剂共同孵育组,足细胞内CXCL16蛋白表达显著增加(P<0.05；P<0.01)。结论：1oxLDL诱导体外培养鼠源肾小球足细胞内CXCL16蛋白表达显著增加,提示oxLDL可能上调足细胞内清道夫受体CXCL16的数量并被过度摄取,导致细胞内胆固醇聚集。
2在体外培养条件下,oxLDL与一定浓度的IFN-γ共同孵育足细胞,可使细胞内脂滴增多,总胆固醇含量增高,同时发现足细胞内CXCL16蛋白表达显著增加。提示干扰素-Y通过诱导鼠源肾小球足细胞CXCL16表达上调,促进足细胞对oxLDL的摄取。
3在体外培养条件下,oxLDL与一定浓度的CXCL16抗体共同孵育足细胞,可见细胞内脂滴明显减少,总胆固醇含量显著降低,同时发现足细胞内CXCL16蛋白水平显著降低。提示应用CXCL16抗体阻断足细胞CXCL16蛋白表达,可显著降低足细胞对oxLDL的摄取及脂质蓄积。
4在体外培养条件下,oxLDL与一定浓度的ADAM10抑制剂共同孵育足细胞,可见细胞内脂滴显著增多,总胆固醇含量显著增加,同时发现足细胞内CXCL16蛋白水平显著增加。提示ADAM10抑制剂可显著增加足细胞CXCL16表达,从而促进足细胞对oxLDL的摄取。
第三部分辛伐他汀对oxLDL诱导鼠源肾小球足细胞脂质蓄积干预机制的探讨
目的：观察辛伐他汀干预前后oxLDL诱导鼠源肾小球足细胞脂质蓄积及CXCL16、 nephrin蛋白表达的变化,探讨其对小鼠肾小球足细胞的保护机制。
方法：用80μg/ml oxLDL与不同浓度的辛伐他汀(1.0μg/ml、2.0μg/ml)共同孵育小鼠肾小球条件永生性足细胞48h,另设空白对照组(不给药),oxLDL组(只加80μg/ml oxLDL),辛伐他汀组(只加1.0μg/ml辛伐他汀)。油红0染色观察细胞内脂质蓄积情况,比色法检测细胞总胆固醇浓度,Western blot检测足细胞CXCL16/nephrin蛋白的表达。
结果：油红0染色可见,与oxLDL组比较,oxLDL+辛伐他汀各组细胞内脂滴明显减少。比色法检测发现,对照组细胞内总胆固醇含量为90.3±30.1μ g/ml,辛伐他汀组为93.3±28.6μg/ml,oxLDL组为226.5±21.6u g/ml,oxLDL+辛伐他汀组分别为151.8±6.8μ g/ml和135.5±26.9μ g/ml。与对照组比较,辛伐他汀组细胞总胆固醇含量无显著变化(P>0.05)；与oxLDL组比较,oxLDL+辛伐他汀各组细胞总胆固醇含量显著降低(P<0.05)。Western blot检测足细胞CXCL16蛋白的表达水平,与对照组比较,辛伐他汀组CXCL16蛋白表达无改变(P>0.05)；与oxLDL组比较,oxLDL+辛伐他汀各组CXCL16表达显著降低(P<0.05)。nephrin蛋白的表达检测结果显示,与对照组比较,辛伐他汀组nephrin蛋白表达无改变(P>0.05),oxLD组nephrin表达显著降低(P<0.01)；与oxLDL组比较,oxLDL+辛伐他汀各组nephrin表达水平显著增加(P<0.05)。结论：1.经辛伐他汀和oxLDL共同孵育的鼠源足细胞内脂滴明显减少,总胆固醇浓度显著降低,足细胞CXCL16蛋白表达水平亦显著降低。提示辛伐他汀下调oxLDL诱导的鼠源肾小球足细胞CXCL16蛋白表达,减少足细胞吞噬脂质,从而减轻足细胞内脂质蓄积。
2.oxLDL诱导鼠源足细胞nephrin蛋白表达显著下降,经辛伐他汀和oxLDL共同孵育的足细胞内nephrin蛋白表达显著增加,提示oxLDL诱导鼠源足细胞nephrin蛋白表达缺失而致足细胞损伤,辛伐他汀能够上调oxLDL诱导的鼠源肾小球足细胞nephrin蛋白表达,对于稳定足细胞功能意义重大。
创新和意义：
1.首次用油红0染色直观地观察oxLDL诱导鼠源肾小球足细胞内脂质蓄积情况,方法简便实用、经济,可重复性强。oxLDL诱导足细胞脂质蓄积模型的建立,为研究足细胞摄取oxLDL的机制及其调控因素提供可靠的实验基础。
2.通过体外细胞培养,证实CXCL16是介导足细胞脂质损伤的关键分子,其表达可被IFN-γ、ADAM10抑制剂等调控,从而影响足细胞摄取脂质,为临床早期防治足细胞脂质氧化损伤提供重要线索。
3.首次通过体外实验证实,辛伐他汀干预能够下调oxLDL诱导的鼠源肾小球足细胞CXCL16蛋白表达,减轻足细胞内脂质蓄积；同时上调足细胞nephrin蛋白表达,从而减轻足细胞损伤,初步探讨辛伐他汀对足细胞的保护作用,为临床应用他汀类药物保护肾脏提供参考依据。
Backgroud and objectives
A 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 podocytes
Part1Establishment the model of lipid accumulation in mouse podocytes induced by oxLDL
Objectives: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 oxLDL
Objectives: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 podocytes
2In 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 oxLDL
Objectives: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 uptake
2. 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.
引文
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