Avaliação da toxicidade urêmica de p-cresol e p-cresilsulfato na expressão de MCP-1 via nf-kB em células musculares lisas humanas (VSMC)

Abstract

Resumo: O p-cresol (PC) e seu conjugado p-cresilsulfato (PCS) sao toxinas uremicas de baixo peso molecular que quando ligadas a proteinas, sobretudo a albumina, podem desencadear via NF-ƒÈB a producao da quimiocina monocyte chemoattractant protein-1 (MCP-1). Essa quimiocina e responsavel pela quimioatracao de monocitos para a camada intima do vaso durante os eventos que precedem a aterosclerose. Desta forma, o objetivo do presente estudo foi investigar in vitro o papel do PC e PCS na expressao de MCP-1, via fator de transcricao NF-ƒÈB p65. O PCS foi sintetizado a partir da sulfatacao do PC com acido clorosulfonico e caracterizado por cromatografia de camada delgada (CCD). As concentracoes de PC e PCS utilizadas nos experimentos seguiu o preconizado pelo European Group of Uremic Toxins (EuTox). Sendo assim, utilizaram-se as concentracoes normais, PC1 (0,60 mg/L) e PCS1 (2,87 mg/L), minimas uremicas PC2 (20,10 mg/L) e PCS2 (15,60 mg/L) e maximas uremicas PC3 (40,70 mg/L) e PCS3 (47,20 mg/L). Como modelo experimental foram utilizadas celulas musculares lisas humanas (VSMC) extraidas atraves de digestao enzimatica de veia de cordao umbilical. A fim de avaliar a viabilidade das celulas frente as toxinas foi realizado o ensaio de MTT. Para verificar a expressao de MCP-1, as celulas foram tratadas com PC e PCS em cinetica de 0 e 3 horas nas concentracoes descritas acima. Para fins de comparacao, as VSMC tambem foram tratadas com 1 ƒÊg/mL de lipopolissacarideo (LPS), outra conhecida toxina uremica de baixo peso molecular. Todos os ensaios foram realizados na presenca e ausencia de inibidor da via NF-ƒÈB p65. Atraves do ensaio de MTT verificou-se que nao houve diferenca significativa na viabilidade celular entre o controle e todas as concentracoes testadas de PC e PCS. As VSMC expostas a PC2 e PC3 produziram niveis significativos de MCP-1 apos 3h de tratamento (149,8 } 16 pg/mL, p<0,001 e 143,5 } 28,7 pg/mL, p<0,05 respectivamente) quando comparados ao tempo 0h. PCS3 estimulou de forma mais intensa a producao de MCP-1 do que as VSMC tratadas com PC3 (162,7 } 7,5 pg/mL vs 143,7 } 28 pg/mL, p<0,005) em 3h. Na presenca do inibidor de NF-ƒÈB p65, houve uma diminuicao significativa na producao de MCP-1 apos o tratamento por 3h com PC2 (149,8 } 16 pg/mL vs 36,4 } 10,5 pg/mL; p<0,001) e PC3 (143,6 } 28 pg/mL vs 50 } 18 pg/mL; p<0,05). Apos tratamento com PCS1 houve diferenca significativa nos niveis de MCP-1 na presenca de inibidor (156,49 } 15,9 pg/mL vs 102,3 } 4,6 pg/mL; p<0,05). Apos 3h de tratamento com LPS, nao houve producao significativa de MCP-1 em relacao ao controle (35,5 } 8,9 pg/mL vs 34 } 8,9 pg/mL). Ainda, na presenca do inibidor, verificou-se uma leve diminuicao nos niveis de MCP-1, porem nao significativa (35,5 } 8,9 pg/mL vs 25,6 } 6,5 pg/mL). Com base em nossos resultados, concluimos que apesar de PC e PCS nao serem citotoxicas, induzem a expressao de niveis significativos de MCP-1, sendo o PCS mais efetivo na inducao da expressao de MCP-1 que seu precursor PC. Assim como PC e PCS, LPS tambem ativa a producao de MCP-1 via NF-ƒÈB. Desta forma sugerimos que a inibicao da via NF-ƒÈB p65 poderia diminuir niveis de MCP-1 e consequentemente a progressao do desenvolvimento da aterosclerose na DRC. Palavras chave: doenca renal cronica, toxicidade uremica, p-cresol, p-cresilsulfato, LPS, MCP-1 e NF-ƒÈB.

Abstract: p-cresol (PC) and its conjugate p-cresyl sulfate are low molecular weight uremic toxins that when attached to proteins, especially albumin, can trigger via NF-êB the production of chemokine monocyte chemoattractant protein-1 (MCP-1). This chemokine is responsible for monocytes chemoattraction to the vessel intimal layer during the events that precede atherosclerosis. Thus, the aim of this study was to investigate the in vitro role of PC and PCS in MCP-1 expression via transcription factor NF-êB p65. PCS was synthesized from PC sulfation with chlorosulphonic acid and characterized by thin layer chromatography (TLC). The PCS and PC concentrations were used according to the recommendations of the European Uremic Toxins Work Group (EuTox). So we used normal, PC1 (0.60 mg/L) and PCS1 (2.87 mg/L), uremic minimum PC2 (20.10 mg/L) and PCS2 (15.60 mg/L) and maximum uremic concentrations PC3 (40.70 mg/L) and PCS3 (47.20 mg/L). As experimental model we used human smooth muscle cells (VSMC) extracted by enzymatic digestion of umbilical cord vein. In order to assess the cells viability after toxins treatment, MTT assay was performed. To verify MCP-1 expression, cells were treated with PC or PCS in a kinetics of 0 and 3 h using the concentrations described above. For comparison purposes, the VSMC were also treated with lipopolysaccharide (LPS) (1 ìg/mL), other low molecular weight uremic toxin. All assays were performed in the presence and absence of NF-êB p65 inhibitor pathway. MTT assay showed that there was no significant difference in cell viability between control and all concentrations of PC and PCS tested. VSMC exposed to PC2 and PC3 produced significant levels of MCP-1 after 3h treatment (149.8 ± 16 pg/mL, p<0.001, and 143.5 ± 28.7 pg/mL, p<0.05 respectively) when compared to time 0h. PCS3 stimulated more intensively MCP-1 production in VSMC treated with the PC3 (162.7 ± 7.5 pg/mL vs 143.7 ± 28 pg/mL, p<0.005) after 3h. In the presence of NF-êB p65 inhibitor, there was a significant decrease in MCP-1 production after 3h treatment with PC2 (149.8 ± 16 pg/mL vs 36.4 ± 10.5 pg/mL, p<0.001) and PC3 (143.6 ± 28 pg/mL vs 50 ± 18 pg/mL, p<0.05). After PCS1 treatment there was a significant difference in MCP-1 levels in the presence of inhibitor (156.49 ± 15.9 pg/mL vs 102.3 ± 4.6 pg/mL, p<0.05). Finally after 3h treatment with LPS, there was no significant production of MCP-1 compared to control (35.5 ± 8.9 pg/mL vs 34 ± 8.9 pg/mL), and , in the presence of the inhibitor, there was a slightly decrease in MCP-1 levels, but not significantly (35.5 ± 8.9 pg/mL vs. 25.6 ± 6.5 pg/mL). Based on our findings, we concluded that in spite of PCS and PC are not cytotoxic, they induce significantly MCP-1expression and PCS is more effective than its precursor PC. As PC and PCS, LPS also activates MCP-1 production via NF-êB. Therefore, we suggest that the inhibition of NF-êB could decrease MCP-1 levels and hence the development of atherosclerosis progression of CKD. Keywords: chronic kidney disease, uremic toxicity, p-cresol, p-cresyl sulfate, LPS, MCP-1 and NF-êB.