Ab0095 Study of the Role of the Urate Transporter Abcg2 in the Regulation of the Immune Response in Knockout Models of Human Colon and Kidney Cells

Background: Gout is a multifactorial metabolic disease, characterized by intra articular pain attacks due to inflammatory activation induced by monosodium urate (MSU) crystals. These crystals accumulate due to the patient's frequent hyperuricemia. ABCG2 is a membrane transporter protein that clears uric acid mainly in the intestine and kidney. ABCG2 gene has been related to hyperuricemia and gout due to polymorphism studies in different populations, however its role in the development and progression of the disease is not clear, a recent study where ABCG2 gene was silenced in THP1 cells stimulated with MSU, it suggested a possible role of ABCG2 in the regulation of inflammation in gout. Objectives: To study the role of ABCG2 as a possible regulator of inflammation induced by the presence of MSU crystals and soluble uric acid (sUA) crystals in two in vitro models of gout. Methods: Until now cultures of the human colon HCT15 and the kidney cell line HEK293T was been grown in specific media until the day of the tests with or without activation with MSU for 48 hours. Genome editing of cultures of these same cells was performed using the CRISPR CAS9 recombinant protein to silence the ABCG2 gene with Edit R- human ABCG2 crRNA and track RNA. The identification of transfected cells with non-functional ABCG2 will be through growth in culture plates to evaluate the gene and protein expression of ABCG2, later sequencing of the target area will be carried out. Activation assays with MSU of the cells silenced for 48 h will be carried out, later the expression of inflammation and ABCG2, IL-1beta, IL-6, S-100, p53 and TL3 genes will be analyzed by immunofluorescence, westen blot and real-time PCR. Results: Until now, transfected cells and not transfected cells have been stimulated with MSU and SUA, we observed that vesicles with internal crystals are formed in the colon HCT15 cells after 24 hours of exposure with MSU. ABCG2,S-100 and p53 gene expression was measured without finding differences in expression at 24 and 48 hours by immunofluorescence. Transfected HCT15 cells had higher expression of phosphorylated p53 than non-transfected cells without stimulation. So far, in our second transfection, the transfected cells express the same or less amount of abcg2 than the non-transfected cells. Conclusion: Human colon and kidney non transfected cells respond to the presence of MSU by forming vesicles with MSU crystals without finding differences in the expression of ABCG2 at the exposure times used up to now. The transfected HCT15 cells maintain their normal growth with active p53 expression. REFERENCES: [1] Dixon, S. (2023). Questions and Answers about CRISPR. Broad Institute. Https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr. [2] García Nieto, V. M., Félix Claverie, M., Moraleda Mesa, T., Ramírez, A. P., Tejera Carreño, P., Córdoba Lanus, E., Luis Yanes, M. I., & Ramos Trujillo, E. (2021). La gota asociada a reducción de la excreción renal de ácido úrico. Esa tubulopatía que no tratamos los nefrólogos. Nefrología. https://www.revistanefrologia.com/es-la-gota-asociada-reduccion-excrecion-articulo-S0211699521001429. [3] Secretaria de Salud. (2017, January 3). Gota, enfermedad crónica que afecta a tres por ciento de los mexicanos. Gobierno de México. https://www.gob.mx/salud/prensa/005-gota-enfermedad-cronica-que-afecta-a-tres-por-ciento-de-los- mexicanos. [4] Sicras-Mainar, A., Viana Álvarez, R., Galera Llorca, J., Granell Villalón, M., & Navarro Artieda, R. (2012, July 26). Estudio de pacientes con gota: características clínicas en los últimos 2 años de seguimiento. https://www.anmm.org.mx/GMM/2012/n5/GMM_148_2012_5_448-456.pdf. Acknowledgements: NIL. Disclosure of Interests: None declared.