Factors involved in the progression of renal injury induced by crystal nephropathy or ischemia/reperfusion: in vivo and in vitro studies

Abstract

Crystalline nephropathy (CN) and renal ischemia followed by reperfusion (IR) induce acute kidney injury (AKI), but there are not enough data regarding treatment, tissue repair molecules or the progression of AKI to the chronic phase in both pathologies, and recurrence in the case of CN. Recent studies by our group evidenced an increase in the inflammatory and fibrotic profiles of the renal tissue, as well as a significant decrease in the expression of megalin and Tamm Horsfall (THP) proteins during the acute phase of CN and IR in experimental models. Our hypothesis is that despite the ability to repair the kidney tissue after an insult, the inflammatory and fibrotic factors present in the acute phase can become relevant molecules in the progression of kidney damage to the chronic phase, or relapse in the case of CN, interfering in renal function. The aim of the present study is to identify the factors involved with AKI, in renal tissue repair after the insult or in the progression from the acute to the chronic phase in experimental models in vivo (crystalline nephropathy, ischemia/reperfusion) and in vitro (cell culture of components of the nephron and the immune system). To this end, two subprojects are presented. In subproject 1 - crystalline nephropathy: the factors involved in tissue injury and repair in the acute, progressive and post-recurrence phases will be investigated, focusing on inflammatory and fibrotic factors and the possible renoprotective role of THP. In subproject 2 - ischemia/reperfusion: the tubular injury factors (inflammation, apoptosis and fibrosis) and the autophagy process (cellular repair) in the renal tissue of the acute and chronic groups will be investigated. To answer the objectives, male mice of the lineage C57BL/6J aged 8 weeks will be used. In subproject 1, the animals will be organized into control groups, treated with sodium oxalate (NaOx, 90 mg/kg of body weight, single ip injection to induce CN) or co-treated with NaOx + synthetic THP (170 µg/kg of body weight, single ip injection) and monitored for 24 h or 30 days, the latter group being subdivided into chronic and chronic + recurrence with NaOx (90 mg/kg of body weight) and followed for 24 h. In subproject 2, the animals will be organized into control groups (Sham) and submitted to renal ischemia (30 min) followed by reperfusion for 48 h 28 days. The in vitro studies will be performed in cell co-cultures of the thick ascending segment of the loop of Henle, dendritic cells, macrophages, proximal tubule, and glomerulus cells, which will be treated with calcium oxalate suspension (200µg/mL) and/ or THP (10 µM) or leupeptin (10 µM, hepsin inhibitor - enzyme that catalyzes the cleavage of THP) for 48 h. For ischemia/reperfusion in cell cultures of the proximal tubule and thick ascending loop of Henle, the monolayers will be treated with antimycin A for 3 h and reperfused with regular culture medium for 1, 3, 6, 12 and 24 h. The techniques used include clearance and renal function, cell culture, western blotting, quantitative PCR, and morphological analysis by microscopy. (AU)