Comparação dos efeitos de célulastronco e de suas vesículas extracelulares no tratamento de lesão renal induzida por estenose crônica da artéria renal

• Ano de defesa: 2018
• Autor(a) principal: Silva, Crysthiane Saveriano Rubiao [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Renovascular Hypertension; Hipertensão Renovascular; Chronic Kidney Disease; Mesenchymal Stem Cells; Adipose Derived Stem; Adipose Derived Stem Cells; Microvesicles; Exosome; Doença Renal Crônica; Células-Tronco Mesenquimais; Células-Tronco De Tecido Adiposo; Microvesículas; Exossomos
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=6635040; https://repositorio.unifesp.br/handle/11600/53176

Chronic renal artery stenosis is considered one of the most frequent causes of renovascular hypertension (RH), which can lead to irreversible renal tissue damage and deterioration of renal function. In this project we used the experimental model of partial stenosis of the renal artery known as 2 kidneys 1 clip model (2K1C) to induce RH and chronic renal hypoxia. Faced with limited treatment alternatives and reduced angiogenic and regenerative ability of the kidneys, stem cells appear as a promising alternative therapy for chronic renal ischemia. Previous studies have shown that mesenchymal stem cells (MSCs) obtained from bone marrow have produced significant beneficial effects on the 2K1C model through paracrine effects, stimulating angiogenesis and immunomodulation. Recent evidence indicates that MSC conditioned medium containing extracellular vesicles (EV), including microvesicles (MV) and exosomes (EX) released by MSC have therapeutic effects similar to MSCs. Therefore, the aim of this study was to investigate and compare the effects of MSC obtained from adipose tissue and MV and EX on tissue regeneration in the 2K1C model. Methods: MSCs were obtained from white adipose tissue (ASC) of Wistar rats and were characterized according to their immunophenotype and multipotentiality. Extracellular vesicles (EV) were isolated from the ASC culture medium and sizeseparated in MV and EX by ultracentrifugation. Vesicles were identified by nanoparticles tracking using Nanosight and analyzed by the presence of specific markers for each type of EV by western blotting. Male Wistar rats were divided into five groups: 1Sham Group; 2Stenotic Group; 3Stenotic Group + ASCs; 4Stenotic Group + MV; 5Stenotic Group + EX. The ASC, MV and EX were infused through the tail vein at the 3rd and 5th weeks after clamping. Systolic blood pressure (SBP) was monitored weekly. After six weeks, 24hour urine and blood were collected for biochemical analyzes. Kidneys were removed for molecular biology analysis. Gene expression was verified by quantitative realtime PCR and analyzed using the following markers: Collagen type I (Col I), TGFβ, HIF1α SDF1α, IL1β and IL10 in the renal cortex and medulla. Results: Flow cytometry analysis showed that after 48 hours the ASCs were retained mainly in the stenotic and contralateral kidneys followed by lungs and heart. After 15 days the presence of ASC decreased significantly in the lungs but was still present in both kidneys. The stenotic animals showed a progressive increase in SBP while the groups treated with ASC, MV and EX showed a stabilization of PAS in similar degrees between the different treatments. Plasma creatinine was similar between groups, however, the stenotic animals developed proteinuria which was reduced by treatment with ASC and MV an effect not observed in the EX treated group. There was an increase in the expression of Col I and TGFβ in the stenotic and contralateral kidney (cortex and medulla) which was reduced by the treatment with ASC, MV and EX. ASCtreated animals showed a significant increase in expression of the stem cells homing marker (SDF1α) in the cortex and medulla in the stenotic and contralateral kidney, the MV and EX treated groups also showed an smaller increase in the expression of SDF1α in both kidneys. The hypoxia marker (HIF1α) presented an increase in the stenotic kidney and the treatments with ASC, MV and EX were efficient in reducing this marker. There was an increase of the proinflammatory cytokine IL1β in both kidneys of the hypertensive animals being efficiently reduced in both kidneys mainly by the ASC treated group and the treatments with ASCs, MV and EX was able to induce an increase the antiinflammatory cytokine IL10. In conclusion, the results suggest that the EV released by ASC produced good results, but with lower efficiency in some parameters when compared to ASC. The use of ASCs has produced more effective effects in this model of chronic hypoxia and the use of EV in replacement to cells should be evaluated depending on the parameter to be corrected.