Exosomes (EXOs) Derived from Progenitors Renal CELL (RPCs) Minimized the ACUTE Kidney Injury (AKI) By LPS MODEL

Introduction and Aim: AKI is followed by regeneration of damaged renal tubular epithelial cells. This damage is caused by toxic and ischemic insults to the kidney; it is characterized by acute tubular necrosis (ATN). There are possible sources of regenerative tubular cells as bone marrow that home to the injured kidney or resident renal stem cells as RPCs. The aim of this study was characterize RPCs and the effect of EXOs derived these cells in AKI induced by LPS. Method: The cells were obtained from neonate kidney (1^st born day) and characterized by fluorescence and confocal microscopy (CD24, PAX2 or CD133); the EXOs by transmission electronic microscopy (TEM) and fluorescence (CD63). The RPCs were preconditioned (PC) with LPS (10µg/ml) or hypoxia (12 hours) and then, the EXOs were quantified using Nanosight and Western blot. Then, the LLCP-K1 cells were treated (72hours) with LPS, PBS (CTL) or LPS+EXOs-PC and cellular viability (%), NO production (nmoles/mg protein) were evaluated by Trypan blue and Griess method, respectively. The mices (C57BL6J) received PBS (vehicle of LPS) or LPS 10mg/BW i.p. (one administration), after 24 hours from the insult, the animals received EXOs (100µg/ml) into caudal vein. After 72 hours of PBS or LPS administration, the animals were killed and the kidneys were submitted to HE (µm²), NGAL, IL18 or KIM 1 (%) quantifications and creatinine (Cr) and urea (U) evaluations (mg/dl). Results: The cells were positive for PAX2, CD24 and CD133 the EXOs were positive for CD63 and measured less than 100µm diameter. LLCP-K1 treated with LPS showed lower cellular viability when compared to CTL (79±12.6 vs.99±14.9 %; p<0.05) and higher in LPS+EXOs-PC with LPS or hypoxia (92±14.3 and 93.4±7.8, respectively, p<0.05). NO production were higher in LPS vs. CTL (189±5.6 vs.76±8.2; nmoles/mg protein, p<0.05) and lower in LPS+EXOs PC with LPS or hypoxia groups (113±11.4 and 128±18.2, p<0.05). The animals treated with LPS showed increase in Cr and U when compared to CTL group (0.34±0.02 vs.0.8±0.03; 69±3.4 vs.12±1.9 mg/dl; p<0.05; respectively). When the animals were treated with EXOs (LPS+EXOs group), the Cr and U were lower compared with LPS group (0.54±0.03 vs.0.8±0.03; 24.8±x vs. 69±3.4 mg/dl, p<0.05, respectively). We observed higher ATN (%) and labeling intensity of renal biomarkers as NGAL, KIM1 and IL18 (%) in LPS when compared to CTL, respectively (14±2.1 vs. 6.4±0.8; 26.7±5.7 vs.1±0.07; 37.3±6.2 vs.0.5; 9.9±2.3 vs.0.8±0.03; p<0.05). These parameters evaluated were minimized when the animals received EXOs (8.0±0.9; 11±2.1; 11.3±0.9; 6.9±0.07; p<o.o5; respectively). Conclusion: The treatment of AKI with EXOs derived by RPCs significantly ameliorated the ATN, functional kidney damage and renal biomarkers. When these cells were PC with LPS or hypoxia, the cellular viability were higher and NO production lower. The ability of these cells to repair renal damage are present in kidney and are activated when the kidney damage is occurring, suggesting their potential in the treatment of AKI.

Funds from: FAPESP, CNPq, CAPES and FOR