Effects Of Advanced Glycation End-products On Expression Of TGF-β₁, CTGF MRNA And Oxidative Stress In NRK-49F Cells

BackgroundDiabetic nephropathy ( DN) is a mainly microvascular complication of diabetes mellitus (DM) , and accounts for disabilities and the high mortality rates in patients. Hyperglycemia can not only affect the normal metabolism of tissues and cells in DM, but also cause structural and functional abnormalities through the formation of advanced glycation end - products (AGEs) by react non - enzy-matically with the free amino groups on proteins, lipids and nucleic acids. Over-expression of transforming growth factor - β ( TGF - β ) and connective tissue growth factor (CTGF) , which are very important profibrogenic factors, is associated with AGEs in DN. Moreover, AGEs - induced reactive oxygen species ( ROS) is a mediator of AGEs - induced lesions. This study aims to determine whether AGEs can up - regulate TGF - β₁ CTGF mRNA expression and ROS generation in NRK -49F cells, and whether antioxidant can attenuate AGEs -induced lesions.Materials and MethodsMaterialsAlannarBlue; DMEM; bovine serum albumin; fetal bovine serum; N -acetylcysteine;2' 7'- dichlorofluorescein diacetate; UNIQ - 10 Column Total RNA Isolation Kit; TaKaRa RNA PCR Kit(AMV) Ver.3.0.ApparatusLabsystem Ascent plate reader; Flowcytometry; PTC - 100 Peltier Thermal Cycler; GDS8000 gel analyzer; Fluorescent reader; centrifuger. Methods1. Preparation of AGEs - BSAAGEs - BSA was prepared as previously described. AGEs - BSA fluorescence was measured with spetrofluorophotometer at excitation of 370nm and e-mission of 440 nm.2. Cell culture and treatmentNRK - 49 F cells were grown in Dulbecco 's modified Eagle 's medium ( DMEM) containing 5.5mmol/L glucose and 10% FBS under standard cell cultured conditions( humidified atmosphere, 5%CO₂, 37℃) , passaged as usual. Cells were randomly divided into four groups: (1) cells were treated with different dose of AGEs -BSA( 0,50,100,200μg/ml) for 24 hours, and 100μg/ml AGEs -BSA for 0,12,24,48 hours. (2) cells were treated with high glucose (25mmol/L) for 24 hours; (3) cells were treated with both high glucose and 100μg/ml AGEs - BSA for 24 hours; (4) Cells were incubated with control medium or medium containing NAC (10 mmol/L) for 24 hours, and then treated with 100μg/ml AGEs - BSA for another 24 hours.3. Detection of cell proliferationCells were plated at 1 x 10Vml/well in 96 - well plates. When the cells were grown to 85% confluence, incubated with 6. 25 12. 5 25 50, 100 200 400 600 800 1000 2000.4000 μg/ml AGEs - BSA for 24 hours . Add Alamar-blue to the medium, after 4 hours of incubation, analyzed by Labsystem Ascent plate reader. Alamarblue reduction rates were then calculated.4. Measurement of intracellular ROS generationThe intracellular generation of ROS was detected using the fluorescent probe CM - H2DCFDA. After treated ,cells were loaded with 20μmol/L CM -H₂DCFDA, incubated for 30 minutes at 37℃ ,and analyzed by Flowcytometry (excitation/emission 488/525nm) and Cell Quest software. A total of 10,000 events were analyzed for each sample, and in every experiment a control tube with cells alone was performed.5.RT-PCRExpression of TGF - β₁ and CTGF mRNA were performed by a semi -quantitative RT - PCR technique. Total RNA was extracted using UNIQ - 10 Column Total RNA Isolation Kit, according to manufacturer' s instructions. Ultraviolet spectrophotometer was used to estimate the purity and concentration of total RNA. Using TaKaRa RNA PCR Kit( AMV) Ver. 3.0, total RNA(0.3μg) was reverse transcribed into single - stranded DNA, GAPDH gene was used as inner control gene, then amplificated the TGF - β₁ CTGF and GAPDH gene. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Analyzed with GDS8000 gel analyzer and Gelworks ID software. TGF - β₁ CTGF mRNA levels were presented by the ratio of TGF - β ₁ CTGF band density to GAPDH band density.6. Statistical analysisAll values were presented as mean ± SD, One - way ANOVA were used for comparison between experimental groups using the SPSS10. 0 software . A level of P <0.05 was considered as statistically significant.Results1. Fluorescence intensity of AGEs - BSA and BSAFluorescence intensity of AGE - BSA was increased 10 - fold in comparison with control nonglycated BSA (AGEs - BSA 141. 1 ± 0. 61 U/mg vs. control nonglycated BSA 13.53 ±0.01 U/mg).2. Effects of AGEs - BSA on cell proliferationCompared with control conditions (nontreated with AGEs - BSA) , treatment with 6.25 to 200μg/ml AGEs - BSA decreased the AlamarBlue reduction rates, but not significantly. While treated with 400μg/ml or higher concentration of AGEs - BSA, the reduction rates of AlamarBlue were significantly decreased (P < 0.05).3. Effects of AGEs - BSA on intracellular ROS generationCompared with control conditions (normal glucose concentration and non-treated with AGEs - BSA) , both AGEs - BSA and high glucose increased ROSgeneration in NRK -49F cells, and AGEs increased ROS production in a dose- dependent and time - course manner. Compared with high glucose, AGEs (100,200μg/ml)and AGEs +high glucose significantly increased ROS generation.4. Effects of AGEs on expression of TGF - β₁ and CTGF mRNA Compared with control conditions (normal glucose concentration and non-treated with AGEs - BSA) and high glucose, AGEs - BSA significantly increased expression of TGF - β₁ and CTGF mRNA in a dose - dependent and time- course manner. Compared with control, high glucose also increased TGF - β₁ mRNA expression,but not CTGF. Incubated with both high glucose and AGEs -BSA significantly increased expression of TGF - β₁ and CTGF mRNA , compared with AGEs - BSA and high glucose respectively (P < 0.05).5. The correlation between TGF - β₁ and CTGF mRNA expressionThe correlation between TGF - β₁ and CTGF mRNA expression in high glucose group was not significant, but there was significantly positive correlation between TGF - β₁, and CTGF mRNA expression in control , AGEs and AGEs + high glucose group.6. Effects of antioxidant on AGEs - induced ROS generation and TGF - β₁ and CTGF mRNA expressionPreincubation with NAC attenuated AGEs - induced ROS production and AGEs - induced TGF - β₁, and CTGF mRNA expression (P <0.05).DiscussionDN is characterized by the hypertrophy of glomerular and tubular, basement membrane thickening of glomerular and tubular, mesangial extracelluar matrix accumulation and tubulointerstitial fibrosis. Along with the study on tubu-lointersitium in progression and prognosis in kidney diseases, scholars found that hypertrophy of tubular epithelial cells and basement membrane thickening of tubular already occurred in early DN, and the level of tubulointersitial lesions was associated directly with prognosis. In DN, AGEs mainly deposit in nodular glo-merulosclerosis area, glomerular mesangium, basement membrane, meanwhilecan be observated in other kidney tissues such as tubular, tubulointerstitium and vessel wall. We studied on the major cell type of interstitium —fibroblasts to investigate effects of AGEs - induced tubulointerstitium damages.Our data showed that high concentration of AGEs inhibited cell proliferation activities and induced overexpression of TGF - β₁ mRNA . So we deduced that AGEs up - regulated TGF - β₁ expression, induced alteration in cell cycle and inhibition of cell proliferation, resulted in cell hypertrophy and overproduction of ECM, played a critical role in progressive tubulointerstitial fiborsis in DN.There is a growing body of evidence suggesting that AGEs play a pivotal role in the pathogenesis of DN, but the effects of AGEs on TGF - β₁ and CTGF expression in renal fibroblasts remain unclear. This study suggested that AGEs directly and significantly increased expression of TGF - β₁ and CTGF mRNA in a dose - dependent and time - course manner in NRK -49F cells. TGF - β₁ is a potent profibrotic growth factor in fibrosis, has a broad spectrum of bioactivities, such as acceleration of ECM synthesis and tissue fibrosis, anti - proliferation, anti - immunization, plays an important role in glomerulosclerosis and tubulointerstitial fibrosis of DN. CTGF is a cysteine - rich polypeptide, acts as a downstream mediator of TGF - β- induced fibrosis. In both in vivo and in vitro studies CTGF is up - regulated by TGF - β, which acts through a distinct element in the promoter region of the CTGF gene. This study showed that there was a positive correlation between TGF - β₁ and CTGF expression after AGEs treatment , suggesting that AGEs up - regulated CTGF expression partly through TGF - β₁ pathway.Our study also showed that compared with normal glucose, high glucose also increased TGF - β₁ mRNA expression, but not CTGF. Incubation with both AGEs - BSA and high glucose significantly increased expression of TGF - β₁, and CTGF mRNA , compared with AGEs - BSA and high glucose respectively, which demonstrated that there was a cooperation between AGEs and high glucose, and the AGEs - induced interstitium fibrosis lesions might be enhanced when hyper-glycemia. So we inferred that both AGEs and high glucose up - regulated expression of TGF - β₁ and CTGF through some identical signal transduction pathways , and these processes of signal transduction were amplified in presence of...