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The physiological role of microvesicles derived from macrophages M2 on chronic and acute kidney disease

Grant number: 16/25613-3
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): April 01, 2017
Effective date (End): April 30, 2019
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Alvaro Pacheco e Silva Filho
Grantee:Juan Sebastian Henao Agudelo
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Introduction: Renoprotection due to the action of Mxs M2 is primarily mediated by paracrine factors. Additionally, recently it has been shown that Mxs similarly to other eukaryotic cells may release microvesicles (MVs). MVs are defined as vesicles containing membrane fragments with determinate sizes between 100-1000 nm and capacity to carry bioactive molecules. In the case of Mxs, it has been shown that Mxs-M2 may secrete MVs with genetic material associated with M2 polarization. Actually, the physiological effect of MVs-M2 has not been verified on the kidney disease as well as macrophage polarization. In this context, our aim was to evaluate the immunoregulatory role of MVs-M2 on pro-inflammatory Mxs and verify their role in the acute renal disease. Methods: Conditioned medium of Mxs-M2 free serum and recombinant was ultracentrifugation on 100,000g for MVs extraction. Then, Bone marrow-derived Mxs were treated with doses of 9.45*109 MVs-M2 (8h in 48h) and subsequently Mxs were activated by LPS / IFN-g in 24 hours. Complementary,the effect of MVs-M2 was evaluated by focal segmental glomerulosclerosis (FSGS) induced by Adriamycin in male C57BL/6 mice. Results: Initially we verify and determine the ability of Mxs-M2 to produce microvesicles. NanoSight analysis showed that vesicles Mxs-M2 has an average size of 144.8 nm and concentration of 9.45 *1010particles/ml. Furthermore, MVs exhibited high expression of Annexin and low CD9 , together these data confirm that these vesicles would be mainly microvesicles kinds. On the other hand, we verify that MVs-M2, presented on its membrane, constituting antigens of Mxs such as CD11b, LyC6 and F4/80. In addition, when compared with MVs-M2 with MVs-M0, we find that MVs-M2 had high expression of CD206. These data would show that MVs-M2 expressed antigens associated with M2 polarization. Latter, Mxs were treated with MVs-M2 and subsequently activated com LPS/IFN-g. We found that Mxs-M1 exposed to MVs-M2 reduced costimulatory antigen CD86 and increased mannose receptor. Complementarily, Intracell revealed that MVs-M2 inhibits secretion of IL-6 and kept stable production of IL-10 in Mxs-M1. Finally, we investigated the anti-inflammatory character of the MVs-M2 in a pre-clinical model of Focal segmental glomerulosclerosis. Mice treated with MVs-M2 were protected from weight loss and proteinuria; also podocyte genes commonly affected by FSGS were up-regulation with the treatment. Conclusions: These initial results show that MVs-M2 has immunoregulatory properties on Mxs-M1 and promote renoprotection in FSGS.