Papel do sistema calicreína-cininas na recidiva de glomeruloesclerose segmentar e focal
Data
2023-10-25
Autores
Fonseca, Fernanda Maria Casimiro da 
Orientadores
Pesquero, João Bosco
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A GESF é a principal causa de doença renal no mundo, sendo o podócito a principal célula afetada. A maioria dos casos de GESF é de causa idiopática (80%). Embora o transplante seja um potencial tratamento, até 50% dos pacientes têm recidiva de GESF após o transplante. Em 1972 foi sugerido a presença de fatores circulantes, responsáveis pela recidiva de GESF primária após o transplante.
Objetivo: Estando a bradicinina relacionada com processos inflamatórios, remodelação celular e aumento da permeabilidade o objetivo deste trabalho é analisar o papel do sistema calicreína-cininas na recidiva de GESF pós-transplante; assim como mapear potenciais vias ativadas nessa patologia, a fim de melhor compreender os mecanismos envolvidos na recidiva de GESF e poder, assim, contribuir para o desenvolvimento de novas abordagens terapêuticas.
Métodos: Podócitos imortalizados foram incubados com plasma de indivíduos com ou sem recidiva de GESF pós-transplante e com bradicinina para análise morfológica. Técnica de espectrometria de massas foi utilizada para analisar a taxa de formação de bradicinina e seus metabólitos nos plasmas de indivíduos com e sem recidiva de GESF. Gel de acrilamida foi utilizado para analisar perfil proteico dos plasmas dos indivíduos com e sem recidiva de GESF pós-transplante, assim como foi utilizada análise de proteína específica por Western Blotting e análise de proteínas dos diferentes plasmas por cromatografia líquida de alta densidade.
Resultados: 15 pacientes foram selecionados para o estudo, seis com recidiva (GESF) e nove sem recidiva (nGESF) de GESF pós-transplante. Quando incubados com os plasmas nGESF, a morfologia e estrutura dos podócitos são preservadas e quando incubados com plasmas recidivados, a sua morfologia aparente é alterada. A bradicinina mostrou efeito semelhante ao plasma com GESF na morfologia dos podócitos, o qual foi inibido na presença de HOE-140. Ao incubar os podócitos com plasmas GESF e nGESF juntos na mesma proporção, os plasmas nGESF tiveram um efeito de preservar a estrutura dos podócitos na presença de plasma GESF. A produção de bradicinina no grupo com GESF ocorreu de forma mais rápida comparado ao grupo nGESF, estando o sistema de produção de bradicinina no grupo GESF 2,5 vezes mais ativado comparado ao grupo nGESF. No grupo GESF, a degradação de bradicinina também ocorre mais rapidamente, assim como a produção dos seus metabólitos, com destaque para o metabólito BK2-9, com síntese 7,5 vezes maior. No gel de acrilamida, foi possível identificar que o grupo nGESF possui uma banda entre 140 – 260 kDa 2 vezes mais presente comparado ao grupo GESF, resultado também visto por meio da cromatografia líquida, sendo esta banda identificada como dipeptidil-peptidase IV por experimento de Western Blotting. Experimentos de atividade enzimática de dipeptidil-peptidase IV mostraram que esta enzima está 2,6 vezes mais ativa no grupo nGESF, sendo as frações 15 e 16 da cromatografia líquida com maior atividade. As frações 15 e 16 de plasma nGESF foram incubadas com podócitos na presença de plasma com GESF, na qual a fração 16 foi capaz de reduzir significativamente a produção de colágeno. Tendo a DPPIV papel importante na degradação de fator 1 derivado de células estromais (SDF-1), o qual tem importante função na produção de colágeno, o inibidor do receptor de SDF-1, AMD3100, foi incubado com plasmas GESF em podócitos e foi observado que AMD3100 inibiu o efeito causado pelo plasma GESF na morfologia dos podócitos.
Conclusão: O plasma de indivíduos com recidiva de GESF é capaz de alterar a estrutura e morfologia dos podócitos in vitro, possui menor quantidade e atividade de DPP-IV, estimula síntese de colágeno pelos podócitos, assim como, sintetizam bradicinina e a degradam de forma mais rápida comparado ao plasma de indivíduos sem recidiva de GESF. O colágeno produzido pelo podócito, estimulado pelos fatores circulantes, pode interferir na composição da barreira de filtração glomerular, prejudicando o processo de filtração. A menor atividade de DPP-IV pode levar ao acúmulo de SDF-1a na circulação, e, em caso de dano aos podócitos pelos fatores circulantes, não há bloqueio do SDF-1a para que haja ativação de células-tronco progenitoras e reconstituição da barreira de filtração glomerular. Estudos para confirmação dessas hipóteses e para melhor compreensão do papel do colágeno produzido e do SDF-1a na recidiva de GESF, a fim de estabelecer a inibição de CXCL12 como potencial estratégia terapêutica em distúrbios glomerulares, se fazem necessários.
Introduction: FSGS is the main cause of kidney disease in the world, and the podocyte is the main affected cell. Most cases of FSGS are idiopathic (80%). Although transplantation is a potential treatment, up to 50% of patients have FSGS relapse after transplantation. In 1972, the presence of circulating factors was suggested, responsible for the recurrence of primary FSGS after transplantation. Objective: Since bradykinin is related to inflammatory processes, cellular remodeling and increased permeability, the objective of this work is to analyze the role of the kallikreinkinin system in posttransplant FSGS recurrence; as well as mapping potential pathways activated in this pathology, in order to better understand the mechanisms involved in FSGS recurrence and thus be able to contribute to the development of new therapeutic approaches. Methods: Immortalized podocytes were incubated with plasma from individuals with or without posttransplant FSGS recurrence and with bradykinin for morphological analysis. Mass spectrometry technique was used to analyze the formation rate of bradykinin and its metabolites in the plasma of individuals with and without FSGS recurrence. Acrylamide gel was used to analyze the protein profile of the plasmas of individuals with and without recurrence of posttransplant FSGS, as well as specific protein analysis by Western Blotting and protein analysis of different plasmas by highdensity liquid chromatography. Results: 15 patients were selected for the study, six with recurrence (FSGS) and nine without recurrence (nFSGS) of posttransplant FSGS. When incubated with nGESF plasmas, podocyte morphology and structure are preserved and when incubated with relapsed plasmas, their apparent morphology is altered. Bradykinin showed a similar effect to FSGS plasma on podocyte morphology, which was inhibited in the presence of HOE140. By incubating the podocytes with GESF and nGESF plasmas together in the same ratio, the nGESF plasmas had an effect of preserving podocyte structure in the presence of GESF plasma. Bradykinin production in the FSGS group occurred more quickly compared to the nGESF group, with the bradykinin production system in the FSGS group being 2.5 times more activated compared to the nGESF group. In the FSGS group, bradykinin degradation also occurs more quickly, as well as the production of its metabolites, especially the metabolite BK29, with 7.5 times greater synthesis. In the acrylamide gel, it was possible to identify that the nGESF group has a band between 140 260 kDa 2 times more present compared to the GESF group, a result also seen through liquid chromatography, this band being identified as dipeptidylpeptidase IV by experiment of Western Blotting. Dipeptidylpeptidase IV enzymatic activity experiments showed that this enzyme is 2.6 times more active in the nGESF group, with fractions 15 and 16 of the liquid chromatography showing the highest activity. nFSGS plasma fractions 15 and 16 were incubated with podocytes in the presence of FSGS plasma, where fraction 16 was able to significantly reduce collagen production. Since DPPIV plays an important role in the degradation of stromal cellderived factor 1 (SDF1), which plays an important role in collagen production, the SDF1 receptor inhibitor, AMD3100, was incubated with GESF plasmas in podocytes and was observed that AMD3100 inhibited the effect caused by plasma GESF on podocyte morphology. Conclusion: The plasma of individuals with FSGS recurrence is capable of altering the structure and morphology of podocytes in vitro, has a lower amount and activity of DPPIV, stimulates collagen synthesis by podocytes, as well as synthesizes bradykinin and degrades it faster than to plasma from individuals without FSGS recurrence. The collagen produced by the podocyte, stimulated by circulating factors, can interfere with the composition of the glomerular filtration barrier and impair filtration process. Lower DPPIV activity can lead to accumulation of SDF1a in the circulation, and, in case of damage to podocytes by circulating factors, there is no blockade of SDF1a for activation of progenitor stem cells and reconstitution of the barrier of glomerular filtration. Studies to confirm these hypotheses and to better understand the role of produced collagen and SDF1a in FSGS recurrence, in order to establish CXCL12 inhibition as a potential therapeutic strategy in glomerular disorders, are necessary.
Introduction: FSGS is the main cause of kidney disease in the world, and the podocyte is the main affected cell. Most cases of FSGS are idiopathic (80%). Although transplantation is a potential treatment, up to 50% of patients have FSGS relapse after transplantation. In 1972, the presence of circulating factors was suggested, responsible for the recurrence of primary FSGS after transplantation. Objective: Since bradykinin is related to inflammatory processes, cellular remodeling and increased permeability, the objective of this work is to analyze the role of the kallikreinkinin system in posttransplant FSGS recurrence; as well as mapping potential pathways activated in this pathology, in order to better understand the mechanisms involved in FSGS recurrence and thus be able to contribute to the development of new therapeutic approaches. Methods: Immortalized podocytes were incubated with plasma from individuals with or without posttransplant FSGS recurrence and with bradykinin for morphological analysis. Mass spectrometry technique was used to analyze the formation rate of bradykinin and its metabolites in the plasma of individuals with and without FSGS recurrence. Acrylamide gel was used to analyze the protein profile of the plasmas of individuals with and without recurrence of posttransplant FSGS, as well as specific protein analysis by Western Blotting and protein analysis of different plasmas by highdensity liquid chromatography. Results: 15 patients were selected for the study, six with recurrence (FSGS) and nine without recurrence (nFSGS) of posttransplant FSGS. When incubated with nGESF plasmas, podocyte morphology and structure are preserved and when incubated with relapsed plasmas, their apparent morphology is altered. Bradykinin showed a similar effect to FSGS plasma on podocyte morphology, which was inhibited in the presence of HOE140. By incubating the podocytes with GESF and nGESF plasmas together in the same ratio, the nGESF plasmas had an effect of preserving podocyte structure in the presence of GESF plasma. Bradykinin production in the FSGS group occurred more quickly compared to the nGESF group, with the bradykinin production system in the FSGS group being 2.5 times more activated compared to the nGESF group. In the FSGS group, bradykinin degradation also occurs more quickly, as well as the production of its metabolites, especially the metabolite BK29, with 7.5 times greater synthesis. In the acrylamide gel, it was possible to identify that the nGESF group has a band between 140 260 kDa 2 times more present compared to the GESF group, a result also seen through liquid chromatography, this band being identified as dipeptidylpeptidase IV by experiment of Western Blotting. Dipeptidylpeptidase IV enzymatic activity experiments showed that this enzyme is 2.6 times more active in the nGESF group, with fractions 15 and 16 of the liquid chromatography showing the highest activity. nFSGS plasma fractions 15 and 16 were incubated with podocytes in the presence of FSGS plasma, where fraction 16 was able to significantly reduce collagen production. Since DPPIV plays an important role in the degradation of stromal cellderived factor 1 (SDF1), which plays an important role in collagen production, the SDF1 receptor inhibitor, AMD3100, was incubated with GESF plasmas in podocytes and was observed that AMD3100 inhibited the effect caused by plasma GESF on podocyte morphology. Conclusion: The plasma of individuals with FSGS recurrence is capable of altering the structure and morphology of podocytes in vitro, has a lower amount and activity of DPPIV, stimulates collagen synthesis by podocytes, as well as synthesizes bradykinin and degrades it faster than to plasma from individuals without FSGS recurrence. The collagen produced by the podocyte, stimulated by circulating factors, can interfere with the composition of the glomerular filtration barrier and impair filtration process. Lower DPPIV activity can lead to accumulation of SDF1a in the circulation, and, in case of damage to podocytes by circulating factors, there is no blockade of SDF1a for activation of progenitor stem cells and reconstitution of the barrier of glomerular filtration. Studies to confirm these hypotheses and to better understand the role of produced collagen and SDF1a in FSGS recurrence, in order to establish CXCL12 inhibition as a potential therapeutic strategy in glomerular disorders, are necessary.
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Citação
FONSECA, Fernanda Maria Casimiro da. Papel do sistema calicreína-cininas na recidiva de glomeruloesclerose segmentar e focal. 2023. 116 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2023.