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Objective • To extract serum IgA1 from patients with IgA nephropathy (IgAN) (end stage renal disease vs long-term stable renal function) to explore the effect on proliferation rate of human mesangial cells (HMCs) and the level of transforming growth factor-β1 (TGF-β1). Methods • Nine patients with primary IgAN were divided into rapidly progressive group (n=6) and long-term stable group (n=3). Jacalin affinity chromatography and sephacryl S-200HR molecular sieves were used to distract serum IgA1. HMCs were cultured and co-cultivated with different IgA1 concentration (10, 50, 250 and 1 000 μg/mL)at point of 12 h and 24 h respectively. The proliferation rate was measured by cell counting kit-8 (CCK8). The expression of TGF-β1 mRNA was measured via quantitative real-time PCR (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was used to detect TGF-β1 protein. Results • Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) activated proliferation of HMC significantly, presenting with time-dependence and concentration-dependence. The highest value showed at 250 μg/mL and 1 000 μg/mL respectively. Serum IgA1 in two groups of patients statistically increased the expression of TGF-β1 mRNA and protein. In group with end stage renal disease, the summit stood at 10 μg/mL and started to decrease by degrees afterwards; while in group with long-term stable renal function, the level of TGF-β1 increased in a parallel manner with the serum IgA1. Conclusion • Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) can both promote the proliferation of HMC. There is no dramatical difference observed with in10-50 μg/mL, but the IgA1 from group with end stage renal disease reveals a stronger effect on TGF-β1, in accordance with the pathological type of the patients (IgA sclerosis), suggesting the prognostic value of serum IgA.
Résumé
Objective·To extract serum IgA1 from patients with IgA nephropathy (IgAN) (end stage renal disease vs long-term stable renal function) to explore the effect on proliferation rate of human mesangial cells (HMCs) and the level of transforming growth factor-β1 (TGF-β1). Methods?·?Nine patients with primary IgAN were divided into rapidly progressive group (n=6) and long-term stable group (n=3). Jacalin affinity chromatography and sephacryl S-200HR molecular sieves were used to distract serum IgA1. HMCs were cultured and co-cultivated with different IgA1 concentration (10, 50, 250 and 1?000?μg/mL)at point of 12?h and 24?h respectively. The proliferation rate was measured by cell counting kit-8 (CCK8). The expression of TGF-β1 mRNA was measured via quantitative real-time PCR (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was used to detect TGF-β1 protein. Results?·?Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) activated proliferation of HMC significantly, presenting with time-dependence and concentration-dependence. The highest value showed at 250?μg/mL and 1?000?μg/mL respectively. Serum IgA1 in two groups of patients statistically increased the expression of TGF-β1 mRNA and protein. In group with end stage renal disease, the summit stood at 10?μg/mL and started to decrease by degrees afterwards; while in group with long-term stable renal function, the level of TGF-β1 increased in a parallel manner with the serum IgA1. Conclusion?·?Serum IgA1 from IgAN patients with different renal functions (end stage renal disease vs long-term stable renal function) can both promote the proliferation of HMC. There is no dramatical difference observed with in10-50?μg/mL, but the IgA1 from group with end stage renal disease reveals a stronger effect on TGF-β1, in accordance with the pathological type of the patients (IgA sclerosis), suggesting the prognostic value of serum IgA.
Résumé
Objective:To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs).Methods:The HMCs were cultured.The subcellular location of IgA was detected by immunofluorescence staining;the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing.The expressions of Igαt and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction.The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose.The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) data-base.Results:By immunofluorescence staining,we detected the presence of IgA heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λ mRNA in the NCBI database exhibited that the similarities were 99％,97％,98％ and 97％,respectively.Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant.To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry.The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No.52 and No.104,amino acids between No.154 and No.221,amino acids between No.276 and No.327 from Ig Cα1 and amino acids between No.52 and No.113,amino acids between No.151 and No.204,amino acids between No.251 and No.314 from Ig Cα2 were the same with those derived from B cells.Conclusion:Our findings suggested that HMCs could synthesize and secret IgA.
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Objective To investigate the effect of high glucose and losartan on cell proliferation and cyelooxygenase-2 (COX2) expression in normal human mesangial cells (NHMCs), and to examine the effect of losartan on COX2 and transforming growth factor-betal (TGF-β1) expression in a model of diabetic nephropathy (DN). Methods NHMCs were cultured in vitro in high glucose media with or without losartan. NHMCs proliferation and COX2 expression were determined by WST-1, Western blot,and RT-PCR. The rat model of DN was produced by injections of streptozocin (STZ). After the treatment with losartan for 4 weeks, glomerular hypertrophy, urinary thromboxane B2(TXB2) and 24 h urinary pro-tein counts were measured,and COX2 and TGF-β1 expressions were investigated using immunohistochem-ical techniques and RT-PCR. Results Losartan dose-dependently inhibited the proliferation of NHMCs in response to high glucose. Losartan also decreased COX2 expression in NHMCs at high or low glucose concentrations. In vivo experiments found kidney weight/body weight (KW/BW), urinary TXB2 and 24 hurinary protein counts increased significantly in the DN group. Losartan reduced KW/BW, urinary TXB2,and 24 h urinary protein counts and significantly suppressed the over-expression of COX2 and TGF-β1.Conclusion Losartan reduces COX2 expression in NHMCs, especially at high glucose concentrations.Losartan could suppress the expression of COX2 and TGF-β1 in the kidney of DN rats and attenuate the renal lesions caused by DN.
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Objective:To investigate the effects of troglitazone on cell proliferation and expression of intercellular adhesion molecule-1(ICAM-1) induced by tumor necrosis factor-?(TNF-?) in human mesangial cells.Methods:Human mesangial cells were cultured in RPMI-1640 media containing TNF-? with or without troglitazone.Cell proliferation was assessed by MTT;the mRNA and protein expression of ICAM-1 were measured by RT-PCR and ELISA;the protein expression of NF-?B was measured by immunohistochemistry.Results:Troglitazone inhibited the proliferation of mesangial cell.The mRNA and protein expression of ICAM-1 of the cell induced by TNF-? increased significantly(P