SUN-032 Investigation of endotoxin cumulation in kidney tissue of rats with experimental sepsis model and the protective effect of ascorbic acid in the same model

Abstract

The aim of this study was to evaluate the renal histopathology and to investigate the protective effect of ascorbic acid in rats with sepsis model. 24 young male Albino Wistar rats were divided into 4 groups (n = 6): Control (C), Ascorbic acid (AA), Lipopolysaccharide (LPS), Ascorbic acid-treated LPS group (AALPS). All groups were fed ad libitum. The AA and AALPS group were given 150 mg/kg ascorbic acid (Abdi İbrahim® Pharmaceutical Ind., Istanbul, Turkey) by gavage for 3 days. On the third day, 4 mg/kg intraperitoneal LPS (Escherichia Coli [E.coli] O 111: B4 from Sigma Aldrich) was administered to the LPS and AALPS groups 2 hours after the last dose of ascorbic acid; C and ASA groups received intraperitoneal 4 ml of physiological saline. After 6 hours, the animals' abdomens were opened under abdominal anaesthesia (Xylazine HCl 5 mg/kg and Ketamine HCl 35mg / kg) and blood samples were taken from their hearts for biochemical analysis. The kidneys were then removed and placed in 4% formaldehyde solution for light microscope (LM) and 3% glutaraldehyde solution for electron microscope (EM). Biochemistry assays: CRP, NGAL, TNFα, Presepsin (SinoGeneClon Biotech, Hangzhou, China) were examined in serum and tissue, Creatinine, Phosphorus, Ca, Cl, K, Na, Uric acid, Urea in serum. Histopathological evaluation was performed by Hematoxylin-eosin staining on an LM, uranyl acetate and pure lead citrate staining on an EM and Anti-E. coli LPS antibody (1∶100, Abcam, Cambridge, UK) on immunohistochemistry (IHC). Statistical analysis was performed by One Way Anova test and Kruskal-Wallis method using SPSS v22.0. Biochemical results are summarized in Table 1 and 2. CRP, Presepsin, NGAL, TNFα, creatinine, urea, phosphorus, calcium, chloride, potassium values showed a significant difference between the groups. LM: Histopathological examination revealed normal renal tissue in LM in the C and AA groups (Fig. 1 A). In the LPS group in LM increased blood flow in the cortical layer, loss of normal histological appearance and excessive dilation of the proximal and distal tubules are observed (Fig. 1 B; C). LPS IHC staining showed a no staining in C and AA group (Fig 2 A; C), but in LPS group increase of LPS expression in the tubular basement membrane (Fig 2 B). However, the AALPS group were showed minimal LPS expression and LM changes than LPS group (Fig. 2 D and Fig 1 D ). EM: Normal EM is observed in the C and AA group (Fig. 3A; B; Fig 4 A; B). Significant changes were made in capillary loops, endothelial basement membranes, and podocytes in ultrastructural images from the LPS group. All three layers of the basement membranes have lost their boundaries (Fig. 5 A, B and C white arrow). Mitochondrial cristae smoothing and growth in the volume of lysosomes are repeated in many endothelial cells (Fig.5 D red arrow). The most striking feature is the presence of lymphocytes (Fig.5 E white arrow). AA LPS group have weak swelling, weak hydrophilic degeneration was observed in the tissues compared with the LPS group (Figure 6A is marked with a red arrow 6B normal IV type collagen). In our study, a sepsis model was formed in accordance with the literature and the protective effect of ascorbic acid was observed in the AALPS group. Endotoxemia causes serious damage to kidney tissue. These changes may be due to renal hypoperfusion and the direct toxic effect of endotoxin.