Estímulo da hidroxicloroquina e anti-hipertensivos em células mesangiais humanas imortalizadas: modulação do sistema Renina Angiotensina Aldosterona e suas vias de sinalização
Data
2023-06-29
Autores
Bello, Marina de Moura [UNIFESP]
Orientadores
Casarini, Dulce Elena [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A ocorrência do primeiro caso de COVID-19 (doença do coronavírus) foi descrita em Wuhan (China) no fim de 2019, e logo foi considerada uma pandemia, devido ao alto grau de transmissão do SARS-CoV-2 (coronavírus da síndrome respiratória aguda grave 2). Esse vírus possui uma proteína de membrana que interage com um receptor da superfície de células hospedeiras, a Enzima Conversora de Angiotensina 2 (ECA2), que é essencial para a infecção viral. Esta enzima é um componente do Sistema Renina-Angiotensina-Aldosterona (SRAA), que é responsável pela manutenção da homeostase e regulação da pressão arterial, dentre outras, pela sua importante ação nos rins. A unidade básica do rim, o néfron, possui células mesangiais, que fazem parte tanto do glomérulo quanto da matriz mesangial, e sintetiza os componentes do SRAA. O desequilíbrio nesse sistema pode levar à hipertensão arterial, uma das comorbidades associadas a um maior grau de severidade dos sintomas, como a indução de lesão renal aguda, e piora no desfecho de COVID-19. No início da pandemia, não haviam estudos clínicos que comprovassem a eficácia do uso de drogas e seus efeitos colaterais. Nesse contexto, a hidroxicloroquina (HCQ), um antimalárico, foi bastante indicada como tratamento para COVID-19. Objetivos: Avaliar os efeitos do estímulo com HCQ e anti-hipertensivos nos componentes do SRAA e suas vias de sinalização em células humanas mesangiais imortalizadas (CHMI). Métodos: As CMHI foram tratadas com HCQ, em diferentes concentrações, e anti-hipertensivos (losartana – Los 1 μM, enalapril – Em 1 μM e espironolactona – Spi 10 nMol/L) por 4 e 12 horas, para posterior avaliação da viabilidade celular e de atividade proteolítica (renina, ECA e ECA2), além da análise de expressão da ECA2 por imunofluorescência. Foi também realizada a análise de vias de sinalização de receptores acoplados à proteína G (GPCR’s) por PCR array. Resultados: A atividade da renina foi reduzida frente aos tratamentos com Los+HCQ[50 μM] e aumentada com En+ HCQ[50 μM], bem como nos tratamentos com Spi+[HCQ50 μM]. Houve um aumento de atividade de ECA quando estimulada com En+HCQ[150 μM], comparado ao tratamento único de En. Nos tratamentos concomitantes aos estímulos com HCQ por 12 horas, observou-se diminuição da atividade da ECA2. Em relação aos genes da via de sinalização dos GPCR’s, 90,47% dos genes testados apresentaram modulação de expressão gênica positiva ou negativa quando expostos a 150 μM de HCQ. Conclusão: A HCQ apresentou efeitos deletérios, em tratamento único e concomitante com anti-hipertensivos, sendo capaz de modular as principais enzimas do SRAA, de forma a suprimir efeitos protetores dos anti-hipertensivos, além de reduzir a viabilidade das CMHI. O fármaco induziu o aumento da expressão da ECA2 frente aos tratamentos concomitantes, além da regulação, de forma positiva ou negativa, dos genes envolvidos em vias de sinalização do SRAA, de processos pró-inflamatórios, fibróticos e oncogênicos. Os resultados deste trabalho sugerem que o uso de HCQ, juntamente com drogas anti-hipertensivas, durante a pandemia poderia vir a agravar as manifestações clínicas, no âmbito de pacientes hipertensos com COVID-19.
Introduction: The occurrence of the first COVID-19 (coronavirus disease) case was described in Wuhan (China) by the end of 2019, and soon it became a pandemic due to the high transmission rate of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). This virus possesses a membrane protein that binds to a host cell membrane receptor, the angiotensin-converting enzyme 2 (ECA2), which is essential for the viral infection. This enzyme is a Renin-Angiotensin-Aldosterone System (SRAA) component and is responsible for maintaining the homeostasis, blood pressure regulation, among other functions, for its important action in the kidney. The kidney’s functional unit, the nephron, possesses mesangial cells, which are part of both the glomerulus and the mesangial matrix, and synthetizes the SRAA components. The imbalance of this system may lead to arterial hypertension, one of the comorbidities associated with a higher symptom severity, such as the induction of acute kidney injury, and a worse COVID-19 outcome. In the beginning of the pandemic there were not clinical studies that proved any drug efficacy and its side effects. In this context, hydroxychloroquine (HCQ), a drug for treating malaria, was highly recommended as COVID-19 treatment. Objectives: To assess the stimuli effects of HCQ and anit-hypertensives on the SRAA components and its signaling pathways in human immortalized mesangial cells (CHMI). Methods: CHMI cells were treated with different concentrations of HCQ, and with one of the following anti-hypertensives (losartan (Los) 1 μM, enalapril (En) 1μM and spironolactone (Spi) 10 nMol/mL) for 4 and 12 hours, for later assessment of cell viability and proteolytic activities (renin, ECA and ECA2), and for the ECA2 expression by immunofluorescence. It was also conducted the analysis of the signaling pathways of G protein-coupled receptors (GPCR’s) by PCR array. Results: The renin activity was reduced after treatment with Los+HCQ [50 μM] and increased after En+HCQ [50 μM] and Spi+HCQ [50 μM]. The ECA activity increased after stimulation with En+HCQ [150 μM] when compared to En treatment alone. In the 12-hour-HCQ-concomitant treatments, a reduction in the ECA2 activity was observed. Concerning the GPCR’s signaling pathways genes, 90,47% of the tested genes showed either a positive or negative modulation when exposed to HCQ 150 μM. Conclusion: The HCQ showed deleterious effects, in both alone and concomitant treatments with anti-hypertensives, being able to modulate the main SRAA enzymes, suppressing its anti-hypertensive protective effects, and by reducing CHMI viability. The drug induced a higher expression of ECA2 when compared to the concomitant treatments, as well as the positive or negative modulation of genes involved in the SRAA signaling pathways, pro-inflammatory, fibrotic and oncogenic processes. This study’s results suggest that the use of HCQ, in combination with anti-hypertensive drugs, during the pandemic may aggravate the clinical manifestations in COVID-19 hypertensive patients
Introduction: The occurrence of the first COVID-19 (coronavirus disease) case was described in Wuhan (China) by the end of 2019, and soon it became a pandemic due to the high transmission rate of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). This virus possesses a membrane protein that binds to a host cell membrane receptor, the angiotensin-converting enzyme 2 (ECA2), which is essential for the viral infection. This enzyme is a Renin-Angiotensin-Aldosterone System (SRAA) component and is responsible for maintaining the homeostasis, blood pressure regulation, among other functions, for its important action in the kidney. The kidney’s functional unit, the nephron, possesses mesangial cells, which are part of both the glomerulus and the mesangial matrix, and synthetizes the SRAA components. The imbalance of this system may lead to arterial hypertension, one of the comorbidities associated with a higher symptom severity, such as the induction of acute kidney injury, and a worse COVID-19 outcome. In the beginning of the pandemic there were not clinical studies that proved any drug efficacy and its side effects. In this context, hydroxychloroquine (HCQ), a drug for treating malaria, was highly recommended as COVID-19 treatment. Objectives: To assess the stimuli effects of HCQ and anit-hypertensives on the SRAA components and its signaling pathways in human immortalized mesangial cells (CHMI). Methods: CHMI cells were treated with different concentrations of HCQ, and with one of the following anti-hypertensives (losartan (Los) 1 μM, enalapril (En) 1μM and spironolactone (Spi) 10 nMol/mL) for 4 and 12 hours, for later assessment of cell viability and proteolytic activities (renin, ECA and ECA2), and for the ECA2 expression by immunofluorescence. It was also conducted the analysis of the signaling pathways of G protein-coupled receptors (GPCR’s) by PCR array. Results: The renin activity was reduced after treatment with Los+HCQ [50 μM] and increased after En+HCQ [50 μM] and Spi+HCQ [50 μM]. The ECA activity increased after stimulation with En+HCQ [150 μM] when compared to En treatment alone. In the 12-hour-HCQ-concomitant treatments, a reduction in the ECA2 activity was observed. Concerning the GPCR’s signaling pathways genes, 90,47% of the tested genes showed either a positive or negative modulation when exposed to HCQ 150 μM. Conclusion: The HCQ showed deleterious effects, in both alone and concomitant treatments with anti-hypertensives, being able to modulate the main SRAA enzymes, suppressing its anti-hypertensive protective effects, and by reducing CHMI viability. The drug induced a higher expression of ECA2 when compared to the concomitant treatments, as well as the positive or negative modulation of genes involved in the SRAA signaling pathways, pro-inflammatory, fibrotic and oncogenic processes. This study’s results suggest that the use of HCQ, in combination with anti-hypertensive drugs, during the pandemic may aggravate the clinical manifestations in COVID-19 hypertensive patients