RESUMO
OBJECTIVE: To investigate the effect of methylation modification on the mRNA expression of Cosmc gene in peripheral B lymphocytes from IgA nephropathy (IgAN). METHODS: Biopsy identified 22 cases of IgAN patients and 20 cases of normal control were included. Peripheral B lymphocytes were isolated and were cultured with RPMI1640 medium, with LPS (12.5 microg/mL) as well as demethylation agent (5-AZA 0.1 micromol/L, 0.5 micromol/ L and 1.0 micromol/L) for 72 hours, respectively. Expression level of Cosmc gene was measured using real-time PCR. RESULTS: The mRNA expression level of Cosmc gene in IgAN patients was significantly lower than that of control (P<0.05). The Cosmc gene expression level increased dramatically after RPMI1640 treatment (P<0.05), however, LPS could apparently reverse this effect (P<0.05). De-methylation modification up regulated the Cosmc gene expression significantly (P<0.05). CONCLUSION: De-methylation modification could effectively reverse the repressed Comsc gene mRNA expression caused by external suppressors.
Assuntos
Linfócitos B/metabolismo , Metilação de DNA/genética , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/imunologia , Chaperonas Moleculares/metabolismo , Adolescente , Adulto , Azacitidina/farmacologia , Linfócitos B/imunologia , Metilação de DNA/efeitos dos fármacos , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Chaperonas Moleculares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
OBJECTIVE: To investigate the effects of Artesunate on expression of MCP-1 and MCP-1 mRNA in renal tissue of the rat experimental IgA nephropathy model. METHODS: 40 rats were divided randomly into 4 groups with 10 rats in normal control group while the other 30 in model control group, low dose Artesunate group and high dose Artesunate group after the establishment of IgA nephropathy model. MCP-1 and MCP-1 mRNA in renal tissue were tested by immunohistochemical and RT-PCR methods. RESULTS: The expression of MCP-1 mRNA in model control group was significantly increased (0.4726+/-0.086 vs 0.1445+/-0.095, P<0.05, compared with normal control group), which was suppressed in low dose Artesunate group (0.2844+/-0.065) and high dose Artesunate group (0.2184+/-0.058) (both P<0.05, compared with model control group). In addition, hemouria, proteinuria and pathological changes in renal tissue were improved in the Artesunate groups. CONCLUSION: Artesunate shows the ability of downregulating the expression of MCP-1 in renal tissue, which may explain one of the mechanisms of Artesunate effectiveness in clinical treatment of IgA nephropathy.
Assuntos
Artemisininas/farmacologia , Quimiocina CCL2/metabolismo , Glomerulonefrite por IGA/metabolismo , Rim/metabolismo , Animais , Artesunato , Quimiocina CCL2/genética , Regulação para Baixo/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: IgA(1) aberrant O-glycosylation is one of the main pathogeneses of IgA nephropathy (IgAN), and the core I beta3-Gal-T-specific molecular chaperone (Cosmc) mRNA expression of IgAN patients was significantly decreased. This study tried to clarify whether the down-regulation was a result of genetic disorders or external suppressions. METHOD: Sixty-five IgAN patients, 23 non-IgAN glomerulonephritis patients and 21 normal controls were recruited. Genomic DNA was extracted and the Cosmc gene was PCR amplified and directly sequenced. Peripheral B lymphocytes of IgAN patients and normal controls were isolated, and cultured with RPMI-1640 alone or with lipopolysaccharide (LPS) for 72 h. The Cosmc mRNA expression levels at baseline, after RPMI culture or RPMI + LPS treatment were measured by real-time RT-PCR. RESULTS: (1) The whole coding frame region of the Cosmc gene was successfully amplified and directly sequenced. Four single nucleotide polymorphisms were detected in two IgAN patients. Two were missense mutations and the others were silent mutations. However, they are different from each other, and unrelated to expression levels; (2) the baseline Cosmc mRNA expression in IgAN patients was significantly lower than normal controls (Ct(COSMC/GAPDH) 1.29 +/- 0.08 versus 1.20 +/- 0.01, 31% of normal controls); (3) the Cosmc mRNA expression level of IgAN patients was remarkably increased after the RPMI culture (1.22 +/- 0.12 versus 1.29 +/- 0.08, 219% of the baseline level), while not in normal controls and (4) treatment with LPS (culture with RPMI + LPS) could strongly inhibit the expression of Cosmc mRNA (1.25 +/- 0.01 versus 1.22 +/- 0.12, 61% of the RPMI treatment group). CONCLUSION: No common Cosmc gene mutation was detected. Significantly increased Cosmc expression was observed in plasma-free culture, while LPS could significantly inhibit it, which suggested that it might not be genetic disorders but external suppression that causes the low Cosmc mRNA expression in IgAN.
Assuntos
Glomerulonefrite por IGA/genética , Chaperonas Moleculares/genética , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Glomerulonefrite por IGA/metabolismo , Glicosilação , Humanos , Imunoglobulina A/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To observe the effects of hepatocyte growth factor (HGF) on TGF-beta1 triggered tubular epithelial-myofibroblast transdifferentiation (TEMT) and on the expression of connective tissue growth factor (CTGF). METHODS: The morphology of transdifferentiate tubular cells was observed using phase-contrast microscopy and scanning electron microscopy. alpha-SMA was assessed by immunohistochemistry and semiquantified by mean intergrated opitical density (IOD). The level of fibronectin (FN) in the culture supernatant was measured by ELISA. CTGF mRNA expression was examined by RT-PCR. RESULTS: The TGF-beta1-induced TEMT characterized by expression of alpha-SMA was shown by immunohistochemistry. TGF-beta1 was also shown to stimulate the secretion of FN in cultured supernatant and the CTGF mRNA expression of NRK52E cells. There was no statistically significant difference between HGF-treated groups and control group in the result of alpha-SMA immunostaining and the level of FN, except that CTGF mRNA expression was slightly increased in the HGF-treated groups. The addition of HGF inhibited the TGF-beta1-induced TEMT, the secretion of FN, and the CTGF expression of NRK52E cells, there was a significant correlation between the expression of CTGF and the expression of alpha-SMA. CONCLUSION: HGF could block TEMT and FN secretion triggered by TGF-beta1, which implies that HGF could participate in renal interstitial fibrosis as a negative regulator. The negative regulation of transdifferentiation of HGF may be partially achieved by attenuation of CTGF expression.