The Expression Of Lectin-like Oxidized Low-density Lipoprotein Receptor In Human Glomerular Mesangial Cells And Renal Cortex Of Diabetic Rat

Abstract BackgroundDiabetic nephropathy is one of the most prevailing chronic complication of diabetes. It is the second cause of kidney transplantation in Europe and the first in USA. It costs a large amount of expenses on therapy every year. It should be paid more attention because we know less on its pathological mechanism.Researches during the past decade showed the level of ox-LDL in plasma was related with atherosclerosis and glomerulosclerosis, suggested ox-LDL pla-ying a significant role in the progression of DN.Ox-LDL can chemoattract inflammatory cell and adhere to vascular wall, induce endothelical/glomerular mesangial cell apoptosis. It is very toxic.Ox-LDL is cleaned by its scavenger receptor. Many scavenger receptorsare recognized, such as traditional SR-A(?)/(?), CD36, SR-BI, CD38 and SR-EC,however,their functions are not fully elucidated. Lectin like oxidized lowdensity lipoprotein receptor is arousing more attention because of its specificallybinding with ox-LDL and involving in the progression of atherosclerosis.LOX-1 is a noval receptor for atherogenic ox-LDL, cloned in vascular bovine aortic endothelial cell. It can bind specifically with ox-LDL. Under physiologically condition, LOX-1 may serve to clean up damaged or apoptotic cell debris, bacteria or other related materials. In pathological states, LOX-1 may be enhanced by TNF-(?), TGF-(?)1, endothelin, PMA, Ang(?) and shear stress. LOX-1 is involved in the bingding of ox-LDL and cellular ligands to activate endothelial cells, transformation of SMCs, accumulation of lipids in mac- rophages, and other events which related with the development of atherosclero-sis.Although atherosclerosis is generally considered a macrovascular disease and glomerulosclerosis is mostly a microvascular disease, researches during the past decade have provided ample evidences shown that there are so much strik-ing similarities between these two disease processes that one researcher de-scribed "glomerulosclerosis" as "glomerular atherosclerosis". Both processes are initiated by endothelial insult, followed by trapping and oxidation of LDL, mon-ocyte recruitment and activation, smooth muscle (mesangial) cell proliferation, and fibrous tissue formation. They share many common pathogenic factors, such as blood glucose(?)AGE-BSA and ox-LDL which are all related with diabetic cardiovascular and nephropathy.Diabetes accelerates the development of atherosclerosis. Recently it was de-termined that LOX-1 expression was enhanced by diabetes-associated condi-tions both in vitro and in vivo. Jono showed AGE-BSA had high affinity to LOX-1．In cultured BAECs, LOX-1 binded with AGE-BSA specifically, it was inhibited by LOX-1 antibody, too. These findings suggested LOX-1 act as scavenger receptor for AGE,too.There still exist reversials on the effect of D-glucose on LOX-1 expres-sion. Chen showed that the serum of diabetic rat added with high concentration D-glucose did not change the expression of LOX-1．On the contrary, Li showed D-glucose upregulated the expression of LOX-1 on human aortic endothelial cell and monocyte derived macrophage and promote monocyte adhesion to macro-phage, induced macrophage to formate foam cell. These effects can be abolished by antioxidant,NF-(?)B inhibitor,protein kinase C inhibitor and MAPK inhibitor . Mobility shift analysis data showed high concentration glucose enhanced nuclear protein binding with NF-(?)B regulatory element in LOX-1 gene in HAECs. These studies suggested abnormal LOX-1 playing a key role in endothelial cell dysfunction , monocyte adhesion as well as atherosclerosis.Glomerular mesangial cell as a mainly injured target cell proliferates in the early stage of diabetic nephropathy, secretes large amount of cytokines and influ-ences the balance of synthesis and degradation of ECM protein, eventually lead- ing to glomerulosclerosis and renal failure. Transforming growth factor(?)1 ( TGF-(?)1) is one of cytokines secreted by glomerular mesangial cells. It is consid-ered to be a key mediator and plays a principal role in the progression of diabetic nephropathy. Many reports showed that high D-glucose and ox-LDL stimula-ted mesangial cells to overproduce TGF-(?)1．However ,its molecular mechanism has not been fully elucidated. In the present study, we examined the expression of LOX-1 and TGF-(?)1 in HGMCs exposed to D-glucose or ox-LDL condi-tion and in STZ induced diabetic rat of different stages.The development of 3-hydroxyl-3-methylglutaryl coenzyme A ( HMG-coA) reductase inhibitors has been a major milestone in the prevention of coro-nary heart disease and nephropathy, its molecular mechanism is also needed to be studied. In the present study, we examined the effect of atorvastatin on the expression of LOX-1 and TGF-(?)1 in HGMCs exposed to ox-LDL and STZ induced diabetic rat of different stages.Materials and methods1．HGMCs culture and Characterization6-8 cm blocks of cortex were cut from the cortex of kidney from the healthy segment of nephroectomy from a tumor patient. The kidney fragments were further minced into a paste-like preparation, which was then gently pressed with the rod of a glass syringe through a 100 mesh nylon sieve. Glomer-uli were collected from underneath the 200 mesh sieve. The glomerular suspen-sion was then spun for 10 min at 900g, resuspended in a tube containing 0．6mg/ml type (?) collagenase. RPMI1640 media and gently stirred at 37(?) in a Dubnoff bath for 15min, checking digestion of the glomeruli on the inverted mi-croscope every 5 min. When the " core" about half the sized of initial tuft, glo-meruli were spun again 5 min and resuspended in 12ml RPMI1640 media sup-plemented with 2mM glutamine, 15% fetal bovine serum and antibiotics (100U/ml penicillin and 100(?)g/ml streptomycin) and plated into two flasks. The flasks were placed in a 37(?) incubator with a controlled humidified atmos-phere of 5% CO. The media were changed every 2 to 3 days. Cell were diges- ted with trypsin solution (0．2% trypsin 0．02% EDTA in Ca(?)Mg-free PBS) for 2 to 3 min, stopped by adding 2 ml 15% FBSRPMI1640 medium and diluted 1:2 or 1:3, depending on the desired cell density and plated into appro-priate flasks. The 4-10th passage cells were used to study.After the first two subculture, the cells was pure based on morphology and characterized by DAB staining for Desmin antigen and actin antigen is posi-tive, whereas cytokeratin antigen is negative. Oil red "O" staining confirmed that ox-LDL was intaked by HGMCs.2．Diabetic rat model was established with STZ intravenous injection and confirmed when blood glucose over 16．7mmol/L. The 36 rats were divided into control group (n = 10) , diabetic group (n = 13) and Atorvastatin (Atorv ) ther-apy group (n = 13) . Atorv was given by gorge on the dosage of l5mg/Kg/d. The rats were killed on the 4th and 8 th week to determine the expression of LOX-1, TGF(?)1 and p38MAPK.3．MTTThe number of living cells in the human mesangial cell cultures was assayed by MTT method. Cells were seeded into culture dishes and grown under normal conditions. On the third day, the cells were washed with PBS and were serum starved for 24h. After starvation, the cells were preincubated with atorvastatin for 30min, then the cells were exposed to ox-LDL for 24h, controls were vehi-cle treated. After the addition of RMPI 1640 medium containing 10% MTT, the cells were incubated at 37(?) for 4 hour. After the medium was aspirated, the cells were lysed by the addition of 150(?)l dimethylsulfoxide ( DMSO). After the sample was mixed, the optical density of each sample at the wavelength of 492nm was measured with immunosorbant assay system.4．Semi-quantitative RT-PCRTo determine the mRNA expression of LOX-1 and TGF(?)1 induced by ox-LDL or D-glucose, HGMCs were incubated with ox-LDL for 24h. To de-termine the effect of atorvastatin on the expression of TGF(?)1, the cells were pre-incubated with atorvastatin for 30min before exposed to ox-LDL. Total RNA (2(?)g) extracted from cultured HGMCs was reverse-transcribed with Oligo dT and AMV reverse transcriptase at 42(?) for 1h. Then 5(?)l of the reverse tran- scribed material was amplified with Taq DNA polymerase using specific primers for human LOX-1 and TGF(?)1．The products of the PCR-amplified samples were visualized on 1．5% agarose gel by use of ethidium bromide. Each specific mRNA band was normalized with the GAPDH mRNA band.5．Western Blot AnalysisTo determine the intracellular signal mechanism, the protein expression of p38MAPK and LOX-1 was examined after HGMCs incubated with ox-LDL or D-glucose for 24h. To determine the effect of atorvastatin on signaling mecha-nism , the cells were preincubated with atorvastatin for 30min, before exposed to ox-LDL or D-glucose for 24h. HGMCs lysate from each experiment (40(?)g per lane) were separated by SDS-PAGE and transferred to nitrocellulose mem-branes. After incubation with blocking solution (4% non fat milk) , membrane was incubated overnight with 1:3000 dilution primary antibody ( LOX-1 anti-body or p38MAPK antibody) at 4(?). Membranes were washed and incubated with 1:1000 dilution of peroxidase goat anti-mouse IgG for 1 hour at room tem-perature. Relative intensity of protein band were analyzed by Scan gel it soft-ware.6．ELISAThe concentration of TGF(?)1 protein in conditioned media was measured by TGF(?)1 ELISA kit according to the manufacture protocol. The time incubating with primary antibody and DAB staining was prolonged.7．StatisticsAll Data were expressed as mean (?) standard deviation. Analysis of vari-ance and post hoc scheffe F test was used to test the statistical significance of the results. P value of less than 0．05 was considered to be statistically significant. Pearson correlation analysis was used to examine the relation of blood glucose(?) HbA1c(?)blood lipid (?)24h urine albumin of diabetic rat with the expression of LOX-1 mRNA relatively content in rat renal cortex. SPSS 13．0 edition was used. Results1．Effect of ox-LDL on expression of LOX-1 in human glomerular me-sangial cellsThe results showed ox-LDL (80(?)g/ml) increased the expression of LOX-1 in mesangial cell in time dependent manner within 24 h, and the effect was the most significant in 24h, the effect in 48h was slightly weakened due to cell-apoptosis. ox-LDL increased the expression of LOX-1 in concentration de-pendent manner, the effect was the most significant in 80(?)g/ml group, and the effect was inhibited by JTX92 (10(?)g/ml).2．Effect of ox-LDL on expression of TGF-(?)1 in human glomerular me-sangial cellsThe results showed ox-LDL (80(?)g/ml) increased the expression of TGF-(?)1 in mesangial cell in time dependent manner within 24h,and the effect was the most significant in 24h, the effect in 48h was slightly weakened due to cell apoptosis. ox-LDL increased the expression of TGF-(?)1 in concentration de-pendent manner, the effect was the most significant in 80(?)g/ml group, and the effect was inhibited by JTX92 (10(?)g/ml).3．Effect of ox-LDL on expression of p38MAPK in human glomerular me-sangial cellsThe results showed ox-LDL increased the expression of p38MAPK in me-sangial cell in concentration dependent manner,and the effect was the most sig-nificant in 80(?)g/ml group, and the effect was inhibited by JTX92 (10(?)g/ml).4 Effect of atorvastatin on mesangial cells proliferation in ox-LDL environ-mentThe results showed us that ox-LDL (80(?)g/ml) increased mesangial cell proliferation significantly compared with control group. Atorvastatin inhibited mesangial cell proliferation in dose-dependent manner.5．Effect of ox-LDL on LOX-1 expression and modulation by atorvastatinThe results showed us that incubation of HGMCs with ox-LDL ( 10 to 80(?)g/ml ) for 24 hours increased the expression of LOX-1 in concentration dependent manner. Atorvastatin (12(?)g/ml) downregulated ox-LDL-induced expression of LOX-1 significantly compared with 80(?)g/ml ox-LDL group.6．Intracellular mechanism of effect of atorvastatinThe results showed us tha ox-LDL increased the expression of p38MAPK in concentration dependent manner (10 to 80(?)g/ml) compared with control group. Atorvastatin decreased the activation of p38MAPK pathway induced by ox-LDL significantly (vs. group ox-LDL 80(?)g/ml).7．Effect of D-glucoes on expression of LOX-1 in human mesangial cells The results showed that D-glucose (600mg/dl) increased the expressionof LOX-1 in mesangial cell in time dependent manner, and the effect was the most significant in 48h. D-glucose increased the expression of LOX-1 in con-centration dependent manner, the effect was the most significant in 600mg/dl group, and the effect was inhibited by JTX92 (10(?)g/ml).8．Effect of D-glucoes on expression of TGF-(?)1 in human mesangial cellsThe results showed that D-glucose (600mg/dl) increased the expression of LOX-1 in mesangial cell in time dependent manner, and the effect was the most significant in 48h. D-glucose increased the expression of TGF-(?)1 in concentration dependent manner, the effect was the most significant in 600mg/dl group, and the effect was inhibited by JTX92 (10(?)g/ml).9．Effect of D-glucoes on expression of p38MAPK in human mesangial cellsThe results showed that D-glucose (600mg/dl) increased the expression of p38MAPK in mesangial cell in time dependent manner, D-glucose increased the expression of p38MAPK in concentration dependent manner, the effect was the most significant in 600mg/dl group, and the effect was inhibited by JTX92 (10(?)g/ml) ,too.10．Expression of LOX-1 in diabetic rat renal cortexThe results showed that the expression of LOX-1 in diabetic renal cortex was upregulated compared with control group (4week,p < 0．01;8week,p < 0．01) . The expression of LOX-1 in the 8 th week was increased compared with 4th week in diabetic group ( p < 0．01) . The expression was significantly de- creased in Atorv treatment group(4week,p <0．01 ;8week,p <0．01) .11．Expression of TGF-(?)1 in diabetic rat renal cortexThe results showed that the expression of TGF-(?)1 in diabetic renal cortex was upregulated compared with control group (4week,p <0．01 ;8week,p <0．01) . The expression of TGF-(?)1 in the 8th week was increased compared with 4th week in diabetic group ( p < 0．01) . The expression was significantly de-creased after Atorv treatment (4week, p <0．01; 8week, p <0．01) .12．Expression of p38MAPK in diabetic rat renal cortexThe results showed that the expression of p38MAPK in diabetic renal cortex was upregulated compared with control group (4week, p <0．01; 8week, p <0．01) . The expression of p38MAPK in the 8th week was increased compared with 4th week in diabetic group ( p < 0．01 ). The expression was significantly de-creased in treatment group (4week, p <0．01; 8week, p <0．01) .13．Correlation analysisAfter analysis we found blood glucose and HbA1c of diabetic rat related with the expression of LOX-1 mRNA relatively content in rat renal cortex (r= 0．619, p<0．01;r=0．828, p <0．01) ,blood lipid related with the ex-pression of LOX-1 mRNA relatively content in rat renal cortex (r =0．589,p <0．01 ,r =0．430,p <0．05 ) (?)24h urine albumin related with the expression of LOX-1 mRNA relatively content (r =0．801 ,p <0．01) ,too.Conclusion1．ox-LDL increased the expression of LOX-1 and TGF-(?)1 in mesang-ial cell in time and concentration dependent manner,the effect was inhibited by JTX92, suggested LOX-1 mediate the upregulation of TGF-(?)1．2．ox-LDL activated the p38MAPK pathway in mesangial cell in concen-tration dependent manner,the effect was inhibited by JTX92,suggested ox-LDL binding with LOX-1 stimulate TGF-(?)1 synthesis and secretion via p38MAPK pathway.3．ox-LDL induced cell proliferation and upregulated TGF(?)1 expression in human glomerular mesangial cells. Atorvastatin decreased the effect of ox-LDL on human glomerular mesangial cells was associated with p38MAPK activation. 4．The Expression of LOX-1 and TGF-(?)1 in diabetic rat renal cortex was upregulated ,p38MAPK pathway was activated . Atorvastatin exerts its renal pro-tecting action perhaps by inhibiting LOX-1(?)TGF-(?)1 expression and antagoni-zing p38MAPK pathway activation.