| Background:The diabetes mellitus and chronic kidney disease are world-wide health problems. Patients with diabetes mellitus or end-stage kidney disease are often accompanied with disturbances of lipid metabolism and abnormally high levels of adanced glycation end products (AGEs). Present epidemiological survey found that lipid metabolism disorders (including lower high density lipoprotein HDL, and higher low density lipoprotein, and higer cholesterol TC and triglyceride TG) and AGEs (including adanced glycation end products of albumin, lipoprotein Andβ-microglobulin protein) increased, often herald an increased risk of cardiovascular events and rate of mortalityAdvanced end protein products (AGEs) are metabolites proteins which generated by Mailard and have strongly pathophysiological role. The normal reaction in the body is very slowly. But when once protein is modified by AGE, they lost its original physiological function and obtain a new pathophysiological function. AGE-modified proteins are uptaked and degradation,mainly through AGE receptor (RAGE) or the CD36 receptor on the monocyte/macrophage cell surface. With diabetes, end-stage kidney disease and other pathological conditions, due to glycosylated response acceleration or AGE-related remove obstacles,there are over-modified proteins in the body, AGE modified proteins, through receptors of RAGE, or CD36 on vascular endothelial cells surface, mediated stimulation of pro-inflammatory mediator release, which resulting in blood vessels and other tissue injury. The experiments confirmed that AGEs can stimulate human vein endothelial cells and human peripheral blood monocyte express monocyte chemoattractant protein (MCP-1); In the pathogenesis of diabetic nephropathy, AGEs can promote glomerular mesangial cells and renal tubular epithelial produce TNF-a and IL-1β; AGE-RAGE interaction can lead to oxidation of emergency response, through activation of NF-κB and AP-1 and other nuclear transcription factor and mediate VCAM-1, MCP-1 and other genes.Lipid metabolism disorders, particularly the increase in plasma LDL, often form some oxidation or glycated LDL, such as oxLDL (oxidized low density lipoprotein), AGE-LDL (advanced glycation end product of low density lipoprotein), mmLDL (minimally modified low density lipoprotein), et al. These modified LDL, often has more strongly pathophysiological role than the lipid itself, In renal disease, these modified LDL can damage endothelial cells and glomerular basement membrane, induce platelet aggregation, macrophages infiltration and foam cells formation, lead to mesangial cell proliferation and extracellular matrix accumulation, influence produced and release of peanut acid analogues, aggravate and accelerate the development of kidney disease.Advanced glycation end products of low-density lipoprotein (AGE-LDL) is lipoprotein modified by glycosylation, which can trigger apoptosis and reduce nitric oxide synthesis, mediate express VCAM-1 and macrophage scavenger receptor. Epidemiology found in plasma of patients with diabetes, AGE-LDL higer levels have a positive correlation with atherosclerosis and coronary heart disease.Recently AGE-LDL was found that can bind to RAGE and by CD36, activate cytokines inflammation pathway of p38MAPK and NF-κB, inducing superoxide and activating NADPH, upregulating inflammatory cytokines and monocyte-macrophage Cells chemotaxis. Recently Hodgkinson found that a AGE-LDL can through TLR4 (Toll-like receptor 4) receptor on human coronary artery endothelial cells and human and mouse macrophages, activate p38a, JNK, ERK1 kinase and AP1, Elk1, NF-κB and other transcription factors to produce cytokines, which causing the diabetic patients easily concurrent atherosclerosis.Toll-like receptor 4 is a subtype of toll-like receptor which belong to IL-1 family receptor and are main receptor of pathogenic microorganisms, widely expressed in various tissues and play an important role in the innate immune response. TLR4 is the main transmembrane signal transduction receptor mediated by bacteria lipopolysaccharidel and is essential for is gram-negative bacteria infectious inflammation.Recent studies showed that, TLR4 recognizes not only LPS and FimH, other endogenous ligands such as HSP60, HSP70 and the extracellular matrix degradation products such as hyaluronan, Tamm-Hosfall glycoprotein, pulmonary surfactant A, modified LDL, also can interact directly with TLR4 leads to an intracellular signal transduction, resulting in biological effects. Present known caused TLR4 intracellular signal transduction including MyD88 dependence ways and the dependence way. MyD88-dependent pathway mainly induced NF-κB activation, cytokine production, while the MyD88-independent pathway is responsible for LPS-induced INF expression and dendritic cell maturation.The renal tubular epithelial cells and mesangial cells express TLR1,2,3,4,6. clinical and experimental studies have proved that Toll-like receptors are involved in kidney disease such as acute kidney injury (AKI), kidneys immune rejection and other autoimmune glomerulonephritis,. The cause of the disease and the pathophysiology are different, but in the beginning are accompanied with activation of Toll-like receptors, and caused intracellular signal transduction pathways to produce inflammatory cytokines and chemical factors, leading to inflammation and fibrosis at last.In obesity, diabetes and chronic renal failure patients, there are systemic micro-inflammation and oxidative stress, meanwhile plasma AGE-LDL also significantly increased. Renal tubular epithelial cells in patients gradually atrophic necrosis and tubulointerstitium also gradually fibrosis.Therefore, we Hypothesis AGE-LDL may be as immune media, through the innate immune receptor TLR4, caused renal tubular injury. The main purpose of this experiment is to explore whether AGE-LDL tubular epithelial cells can through the TLR4 receptor and its signaling pathway, cause renal tubular and interstitial inflammation and fibrosis; Using culture human renal tubular epithelial cells (HK-2) cells in vitro, search for AGE-LDL induced tubulointerstitial inflammation and fibrosis is through Toll-4 receptor and the Toll signaling pathway; and clear the pathophysiological role of AGE-LDL in tubulointerstitial inflammation and fibrosis process. establish new crosstalk between natural immunity and oxidatie stress.Methods1. Preparation of AGEs1.1 Preparation of AGE-BSAAGE-BSA was prepared in vitro according to the method described by our laboratory. AGE-BSA was generated by incubating D-100 mM of glucose in 400 mM phosphate buffer solution for three months at 37℃. Nonglycated BSA was used as negative control, without the presence of reducing sugars. Glycation was confirmed by analyzing the fluorescence (ex.335/em.460) emitted by AGE-BSA. he endotoxin levels in the preparations were tested using the Amebocyte Lysate Kit (Sigma Chemical) and were found to below0.025 EU/ml.1.2 Preparation of AGE-LDLAGE-LDL was prepared by incubation of nLDL (2 mg protein/ml) with D(+)glucose (0.2M final concentration), for 4 weeks, at 37℃, under sterile conditions with antioxidants (lmg/ml EDTA and 10μM BHT). Prior to the experiments, AGE-LDL was extensively dialyzed against PBS, pH 7.4,4℃. he endotoxin levels in the preparations were tested using the Amebocyte Lysate Kit (Sigma Chemical) and were found to below 0.025 EU/ml.2. Cell cultureA human renal tubular epithelial cell line (HK-2) was obtained from ATCC(Manassas, VA, USA). Cells were cultured in DMEM/F-12 supplemented with 10% fetal bovin serum (PAA, German), 100U/ml penicillin and 100μg/ml streptomycin at 37℃,5%CO2. Passages 6-10 cells were used for the experiments.For all experiments, the cells were grown to 70%-90% confluence on 6-well plates or 100×20mm plastic petri dishes (Numc, Roskilde, Denmark) and maken quiescent by incubation in serum-free DMEM for 24 hours before stimulation with LDL AGE-LDL HSA AGE-HSA LPS. All reagents used were certified to be endotoxin free.3. Determination and location of Toll-like receptors 4 in HK-2 cell3.1 Determination TLR4 in HK-2 cell by flow cytometryHK-2 cells were cultured in T75 culture flask. After digested cells, FITC fluorescently-labeled TLR4 protein on HK-2, and detected TLR4 in HK-2 cell by flow cytometry.3.2 Observation of the location of TLR4 protein on HK-2 cellsHK-2 cells were cultured in 6-wells plates. The location of FITC fluorescently-labeled TLR4 protein on HK-2 was observed under the confocal microscope by immunofluorescent staining.4. AGE-LDL upreglated IL-6 and IFN-βin HK-2 cell4.1 Determination of IL-6 mRNA induced by AGEs by qRT-PCRHK-2 cells were treated with 100μg/ml LDL HSA AGE-LDL or 200μg/ml unmodified LDL and HSA for 6hours, Total RNA of HK-2 was extracted with TRIZOL reagent. IL-6 mRNA and IFN-βwas detected by qRT-PCR according to the manufacturer’s protocol.4.2 Determination of IL-6 mRNA by qRT-PCRHK-2 cells were treated with 25,50, 100μg/ml AGE-LDL or 100μg/ml unmodified LDL for 0,3,6,12,24 hours, Total RNA of HK-2 was extracted with TRIZOL reagent. IL-6 mRNA was detected by QRT-PCR according to the manufacturer’s protocol.4.3 Determination of IL-6 protein expressionHK-2 cells were treated with25,50, 100μg/ml AGE-LDL or 100μg/ml unmodified LDL for 0,3,6,12,24 hours. Supernatants were collected at the end of the experimental period and IL-6 in the media were determined by Enzyme-Linked immunosorbent Assays Kits.5. The role of TLR4 in AGE-LDL upreglated IL-6 in HK-2 cell5.1 AGE-LDL induced IL-6 expression though TLR4 in HK-2 cellHK-2 cells transfected with TLR4 siRNA or scramble siRNA, then were exposed to 100μg/ml LDL HSA AGE-LDL or 200μg/ml unmodified LDL and HSA for 6 hours, and IL-6 mRNA detected by qRT-PCR.5.2 Anti-TLR4 antibody diminished IL-6 protein induced by AGE-LDLHK-2 cells preincabuted with anti-TLR4 antibody or IgG antibody, then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 mRNA detected by qRT-PCR.5.3 Blocked TLR4 diminished IL-6 protein excrete induced by AGE-LDLHK-2 cells preincabuted with anti-TLR4 antibody or IgG antibody or transfected with TLR4 siRNA or scramble siRNA, then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 protein detected by ELISA.5.4 AGE-LDL not induced expression charged of TLR4In brief, HK-2 cells incabuted with 100μg/ml AGE-LDL for various time periods up to 24 hours, TLR4 protein detected by western-blot.6. The role of Myd88 and TRIF in AGE-LDL upreglated IL-6 in HK-2 cell6.1 AGE-LDL not induced expression charged of Myd88 and TRIF.In brief, HK-2 cells incabuted with 100μg/ml AGE-LDL for various time periods up to 24 hours, Myd88 and TRIF protein detected by western-blot.6.2 Blocked Myd88/TRIF diminished IL-6 induced by AGE-LDLHK-2 cells transfected with Myd88 siRNA or TRIF siRNA or scramble siRNA, then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 mRNA detected by qRT-PCR.6.3 After blocked TLR4, expression of Myd88 and TRIF not chargedHK-2 cells preincabuted with anti-TLR4 antibody or IgG antibody or transfected with TLR4 siRNA or scramble siRNA, then were exposed to 100μg/ml AGE-LDL for 6 hours, Myd88 and TRIF protein detected by western-blot.7. The role of MAPK in AGE-LDL upreglated IL-6 in HK-2 cell7.1 AGE-LDL activated MAPK in HK-2 cell In brief, HK-2 cells incabuted with 100μg/ml AGE-LDL for various time periods up to 1 hours, then phosphorylation of p38 JNK and ERK and total of non-phosphorylation p38 JNK and ERK protein detected by western-blot.7.2 After blocked TLR4, AGE-LDL activated MAPK in HK-2 cellHK-2 cells preincabuted with anti-TLR4 antibody or IgG antibody or transfected with TLR4 siRNA or scramble siRNA, then were exposed to 100μg/ml AGE-LDL for various time periods up to 1 hours, then phosphorylation of p38 JNK and ERK and total of non- phosphorylation p38 JNK and ERK protein detected by western-blot.7.3 Inhibition of MAPK diminished IL-6 induced by AGE-LDLHK-2 cells preincabuted with various MAPK inhibitors then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 mRNA detected by qRT-PCR8. The role of NF-κB in AGE-LDL upreglated IL-6 in HK-2 cell8.1 AGE-LDL activated NF-κB in HK-2 cellIn brief, HK-2 cells incabuted with 100μg/ml AGE-LDL for various time periods up to 1 hours, then phosphorylation of p65 and total of non-phosphorylation p65 protein detected by western-blot.8.2 Observation of the nuclear translation of p65 protein in HK-2 cellsHK-2 cells were cultured in 6-wells plates. The location of FITC fluorescently-labeled TLR4 protein on HK-2 was observed under the confocal microscope by immunofluorescent staining.8.3 After blocked TLR4, AGE-LDL activated NF-κB in HK-2 cellHK-2 cells preincabuted with anti-TLR4 antibody or transfected with TLR4 siRNA, then were exposed to 100μ,g/ml AGE-LDL for various time periods up to 1 hours, then phosphorylation of p65 and total of non-phosphorylation p65 protein detected by western-blot.8.4 After blocked Myd88/TRIF, AGE-LDL activated NF-κB in HK-2 cellHK-2 cells transfected with Myd88 siRNA or TRIF siRNA or scramble siRNA, then were exposed to 100μg/ml AGE-LDL for various time periods up to 1 hours, then phosphorylation of p65 and total of non-phosphorylation p65 protein detected by western-blot.8.5 Inhibition of NF-κB diminished IL-6 induced by AGE-LDLHK-2 cells transfected with p65 siRNA or preincabuted with NF-κB inhibitors Bay11-7082, then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 mRNA detected by qRT-PCR9 Blocked CD36 diminished IL-6 induced by AGE-LDLHK-2 cells preincabuted with anti-CD36 antibody or transfected with CD36 siRNA, then were exposed to 100μg/ml AGE-LDL for 6 hours, and IL-6 mRNA detected by qRT-PCR.10. StatisticsAll data were expressed as mean±SEM. The significance of differences among mean values was determined by One-way ANOVA, followed by LSD method When P≤0.05. Differences of the variables between two time points were determined by independent samples t test. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was <0.05.Results1. AGEs induced expression of IL-6 mRNA in HK-2 cell100μg/ml LDLã€100μg/ml AGE-LDLã€200μg/ml AGE-BSA upregulated expression of IL-6 mRNA (P<0.05), but 100μg/ml HSA did not change expression of IL-6 mRNA (P>0.05). 100μg/ml LDLã€100μg/ml AGE-LDLã€200μg/ml AGE-BSA did not change expression IFN-βmRNA (P>0.05), but 10μg/ml LPS upregulated expression of IFN-(3 mRNA (P<0.05).2. TLR4 was expressed in HK-2 cell and located on HK-2 cell mamberance2.1 TLR4 was expressed in HK-2 cell by FCMHK-2 cell tagged with anti-TLR4 antibody was 13.36±1.80% more than cells tagged with IgG antibody was 0.92±0.075%(P<0.05).2.2 TLR4 was located on HK-2 cell mamberance by IHCHK-2 cell tagged with anti-TLR4 displayed flavo-green on cell mamberance. 3. AGE-LDL induced expression of IL-6 mRNA through TLR4 in HK-2 cell3.1 AGE-LDL induced expression of IL-6 mRNA through TLR4 in HK-2 cellInhibition of TLR4 with TLR4 siRNA can significantly decrease expression of IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05), but not charge expression of IL-6 mRNA in HK-2 cell by LDL and AGE-BSA (P>0.05).3.2 Decrease expression of IL-6 mRNA in HK-2 cell by TLR4 anti-bodyInhibition of TLR4 with TLR4 anti-body can significantly decrease expression of IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05).3.3 Blocked TLR4 decreased secresion of IL-6 in HK-2 cellInhibition of TLR4 with TLR4 anti-body or TLR4 siRNA can significantly decrease secresion of IL-6 in HK-2 cell by AGE-LDL (P<0.05).4. AGE-LDL induced the expression IL-6 in HK-2 cell4.1 Influence of AGE-LDL on IL-6 gene expressionAfter the stimulation of HK-2 cell with 100μg/ml AGE-LDL for 0,3,6,12,24 hours, the mRNA expression of IL-6 increase gradually with the prolonging of time (P<0.05).After the stimulation of HK-2 cell with 100μg/ml AGE-LDL for 0,3,6,12,24 hours, the excretion. of IL-6 increase gradually with the prolonging of time (P<0.05).4.2 Influence of AGE-LDL on IL-6 gene expressionAfter the stimulation of HK-2 cell with 25,50,100μg/ml AGE-LDL for 12 hours, the mRNA expression of IL-6 increase gradually with the prolonging of dose (P<0.05).After the stimulation of HK-2 cell with 25,50,100μg/ml AGE-LDL for 12 hours, the excretion of IL-6 increase gradually with the prolonging of dose (P<0.05).4.3 AGE-LDL uncharged the expression of TLR4 in HK-2 cellAGE-LDL did not charge the expression of TLR4 in HK-2 cell in various time (P>0.05).5. The role of Myd88 and TRIF in the AGE-LDL signal way5.1 AGE-LDL uncharged the expression of Myd88/TRIF in HK-2 cellAGE-LDL did not charge the expression of Myd88/TRIF in HK-2 cell in various time (P>0.05).5.2 AGE-LDL induced expression of IL-6 mRNA through Myd88Inhibition of TLR4 with Myd88 siRNA can significantly decrease expression of IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05), but not charge expression of IL-6 mRNA in HK-2 cell by inhibition of TRIFwith TRIF siRNA (P>0.05).5.3 Blocked TLR4 not charge expression of Myd88/TRIF in HK-2 cellInhibition of TLR4 with TLR4 anti-body or TLR4 siRNA can not charge expression of Myd88 and TRIF in HK-2 cell by AGE-LDL (P>0.05).6. The role of MAPK in the AGE-LDL signal way6.1 AGE-LDL induced activation of MAPK in HK-2 cellAGE-LDL induced the phosphorylation of p38 and JNK in a time dependent way (ratio to total P<0.05), while phosphorylation of ERK was uncharged after AGE-LDL stimulation (P>0.05). And LPS induced the phosphorylation of p38 and JNK and ERK in a time dependent way (P<0.05).6.2 Blocked TLR4 decrease phosphorylation of MAPK in HK-2 cellInhibition of TLR4 with TLR4 anti-body or TLR4 siRNA can significantly decreas phosphorylation of MAPK(ratio to tota) in HK-2 cell by AGE-LDL (P<0.05).6.3 AGE-LDL induced expression of IL-6 mRNA through MAPK Inhibition of MAPK with MAPK inhibitor (P98059 SB203580 SP420119) can significantly decrease expression of IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05), but not charge expression of IL-6 mRNA in HK-2 cell by inhibition of ERK inhibitor with UO126(P>0.05).7. The role of NF-κB in the AGE-LDL signal way7.1 AGE-LDL induced activation of NF-κB in HK-2 cellAGE-LDL induced the phosphorylation of p65 in a time dependent way (ratio to total P<0.05).7.2 AGE-LDL induced nuclear translation of p65 in HK-2 cellsAGE-LDL induced p65 tagged with anti-TLR4 displayed flavo-green translation transfer from cytoplasm into nuclear in HK-2 cell.7.3 Blocked TLR4 decrease phosphorylation of p65 in HK-2 cell Inhibition of TLR4 with TLR4 anti-body or TLR4 siRNA can significantly decreas phosphorylation of p65 (ratio to tota) in HK-2 cell by AGE-LDL (P<0.05).7.4 Blocked Myd88/TRIF decrease phosphorylation of p65 in HK-2 cellInhibition of Myd88 with Myd88 siRNA can significantly decreas phosphorylation of p65 (ratio to total) in HK-2 cell by AGE-LDL (P<0.05).7.5 AGE-LDL induced expression of IL-6 mRNA through NF-κBInhibition of NF-κB with IκB phosphorylation inhibitor Bay 11-7082 or p65 siRNA can significantly decreas IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05).8. Blocked CD36 decrease expression of IL-6 mRNA in HK-2 cellInhibition of TLR4 with CD36 anti-body or CD36 siRNA can significantly decrease expression of IL-6 mRNA in HK-2 cell by AGE-LDL (P<0.05).ConclusionOur results revealed that AGE-LDL induced IL-6 upregulation through a TLR4-mediated signals way involving Myd88, activator mitogen-activated protein(MAP) kinase (p38 and JNK) and activator nuclear transcription factorκB (NF-κB) in cultured human renal tubular epithelial cells(HK-2 cell). That AGE-LDL induced inflammation in HK-2 cell combined with our previous data may show a new way to intervention for the disorder of lipid metabolism and AGEs. |
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