Mitochondrial dynamics and cell metabolism in the kidney subjected to ischemia and reperfusion injury: study of the cytoprotective effect of dexamethasone and indomethacin

• Grant number: 20/12879-0
• Support Opportunities: Regular Research Grants
• Start date: March 01, 2022
• End date: May 31, 2024
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Alvaro Pacheco e Silva Filho
• Grantee: Alvaro Pacheco e Silva Filho
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Fernanda Paula Roncon Santana Novelli

Abstract

Kidney transplantation is one of the most performed transplants worldwide, with Brazil figuring, in 2019, as the second main center concerning the highest procedures numbers. Nevertheless, despite the restricted organ availability, one of the currently challenges across transplantation encompasses the proper graft function. In this sense, ischemia reperfusion injury (IRI) takes a relevant position among elements involved in organ viability and clinical outcomes, once it is intrinsically attached the surgical approach, and is responsible for trigger the immune and metabolic response that latter leads to tissue injury. Recently, these damage pathways have been also associated to mitochondrial structure and dynamics, either in renal tubules or leukocyte cells. In this context, organelle fission in IRI favors a pro-inflammatory pattern and a decreased kidney function, in contrast to a protective role found within fused organelles. Administration of specific drugs as heme-oxygenase-1 inductors and anti-inflammatory has been described to modulate ischemia-induced kidney disfunction on IRI and pre-conditioning models. Our laboratory already reported both medicines' roles, focusing on indomethacin IRI protection in mice, accompanied by attenuated inflammatory response. Nonetheless, although immune modulation is established in transplant procedures, its crosstalk with mitochondrial morphology in this field is still incipient. Thus, the present study intends to investigate a possible rebound between damage protection and mitochondrial patterns promoted by beneficial drugs in inflammation and transplantation, particularly, anti-inflammatory. For this aim, we will use different mice strains CX3CR1-GFP, CCR2-RFP, KAP2-Cre+PHAMflox/flox, Lyz-Cre+PHAMflox/flox, KAP2-Cre+Mitofusinaflox/flox e Lyz-Cre+Mitoflox/flox to perform in vivo and in vitro assays. In some animal group, we will use dexamethasone or indomethacin as pre-treatments. After 15 minutes or 24 hours injury, we will evaluate mitochondrial structure by electron microscopy, and PHAM fluorescence by confocal microscopy, measuring organelle mass by Mitotracker and oxidative state with Mitosox probes. The data will be supplemented through qPCR and western blot to quantify compounds involved in fusion and fission processes. Concerning inflammatory response, we will characterize infiltrated leucocytes, and its inflammatory markers on systemic and local compartments. The results will be correlated to kidney protection, evaluating organ function and tissue integrity. Finally, we will study metabolic patterns on primary tubular cells and bone-marrow derived macrophages, in vitro, in the presence and absence of proposed drugs. We believe the treatments will support a suitable metabolism followed by decreased inflammation and higher mitochondrial fusion in both renal tissue and immune cells. These results will potentially cooperate on long-term enlightenment of protective mechanisms given by anti-inflammatory drugs, as well as contribute to delimit novel mitochondrial therapeutic targets in IRI and transplant sphere. (AU)