1. Protective effects of chitosan oligosaccharide on paraquat-induced nephrotoxicity in rats Byung Chul Shin, Wan Soo Lee, Dae Wong Kang, Jong Hoon Chung and Hyun Lee Kim Division of Nephrology, Department of Internal Medicine, College of Medicine, Chosun University Protective effects of chitosan oligosaccharide on paraquat-induced nephrotoxicity in rats Byung Chul Shin, Wan Soo Lee, Dae Wong Kang, Jong Hoon Chung and Hyun Lee Kim Division of Nephrology, Department of Internal Medicine, College of Medicine, Chosun University Introduction Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) - world wide used herbicide and has a very high mortality rate in human Chitosans - N-deacylated derivative of chitin in crab and shrimp shells - Physiological activities : antitumor activities, immuno-enhancing effect, ant-micorbial effect, cholesterol reducing effect and antioxidant effects. Apoptosis PUMA (p53 upregulated modulator of apoptosis): pro-apoptotic member of the Bcl-2 protein family and induction of programmed cell death 6). [ [ APE (Apurinic/apyrimidinic endonuclease): repair of DNA damage and inhibits pro-apoptotic functions of P53 7). Caspse-3: important role in the apoptotic process in two ways 8). First: apoptotic gene PUMA and an effector of the caspase cascade, caspase-3, were evaluated to gain insight into apoptotic signaling during PQ- induced nephrotoxicity. Second,: protective effects of COS were estimated through restoration of serological parameters and by PUMA and caspase-3 expression. Finally: roles of apurinic/apyrimidinic endonuclease (APE) were also as- 103 sessed in PQ-induced nephrotoxicity. RatsMice Male Sprague–Dawley rats (8–10 weeks old, 200–250 g) Experimental protocol Western Blot electrophoresed on 10% SDS–PAGE gels transferred to polyvinyldifluoridine membranes (GE, Healthcare Bio- Sciences Corp., Piscataway, NJ). detected using the iNtRON Biotech Enhanced Chemiluminescence Detect System (Seoul, Republic of Korea) and quantified using ImageQuant 350 (GE Healthcare Korea, Seoul, Republic of Korea) densitometric units of each primary antibody relative to b-actin and in reference to the value of the control sample for each gel. Cytoarchitecture and Immunohistochemistry 5-lm-thick serial sections were cut using a Leica RM2155 rotary microtome (Nussloch, Germany) and mounted on slides coated with 3-aminopropyl-tri- ethoxy-silane (Sigma–Aldrich, St. Louis, MO). Immunohistochemical staining was carried out by the routine method. Statistical analysis analyzed using Mann–Whitney test and Kruskal–Wallis test (SPSS version 11.0, Chicago, IL, USA). A p value of less than 0.05 was considered significant. Body weight and serological aspects Methods Results-2 Purpose Western blot analysis Western blot analysis of apurinic/apyrimidinic endonuclease (APE), p53-upregulated modulator of apoptosis (PUMA), caspase-3, and cleaved caspase- 3 expression in the kidneys of paraquat (PQ)-intoxicated rats. The levels of these proteins increased in a time-dependent manner in the control group after PQ administration. Pretreatment with chitosan oligosaccharide (COS) resulted in a higher level of these proteins before PQ injection, which decreased at the end of experiment. Densitometric results in the control and chitosan oligosaccharide (COS) groups in paraquat (PQ)-intoxicated rats. Changes are compared bewteen groups at the same time point. *p < 0.05; #p = 0.05; Cas-3, caspase-3; Cl. Cas-3, cleaved caspase-3. Histology (PAS stain) Immunohistochemistry PQ concentration, BUN/s-Cr level -Significantly increased in PQ group and decreased in COS-PQ group with time dependant manner (p<0.05). Caspase-3 and PUMA protein expression -Significantly increased in PQ group and decreased after 24hours in COS-PQ group compared with PQ group. APE protein expression -Significantly increased after 12hours in COS-PQ group compared with PQ group. PQ induced apoptosis via elevated cleaved caspase-3 and PUMA in the rat kidneys, which might contribute to the clinical manifestations of PQ intoxication. COS had renoprotective effects on PQ-induced nephrotoxicity by suppressing apoptosis and increasing anti-renin activity following an increase in APE. This study has provided supporting evidence for the renoprotective effects of COS in PQ-induced nephrotoxicity, suggesting that it may be a potential aid to manage patients suffering from PQ intoxication. Periodic acid Schiff (PAS) staining in the deep renal cortex in paraquat (PQ)-intoxicated rats. It clearly shows the proximal convoluted tubules in both groups ((A) control group; (C) chitosan oligosaccharide (COS) group) before PQ injection and also shows that the findings had disappeared 24 h after PQ administration (B). Only a few proximal tubules bear brush borders (asterisks). COS pretreatment prevented the degenerative changes in proximal tubules (D), whereas the distal tubules and collecting ducts were not affected by PQ administration. Scale bar = 50 um. Immunohistochemistry with apurinic/apyrimidinic endonuclease (APE) (upper column) and cleaved caspase-3 (lower column) in the kidneys of paraquat (PQ)-intoxicated rats. APE and cleaved caspase-3 were mainly seen in the proximal tubules of the control renal cortex. They were found in whole components of nephrons from 4 h after PQ injection, but degenerated proximal tubules (asterisks) were devoid of immunoreactivity 24 h after PQ injection. Chitosan oligosaccharide (COS)-pretreated kidneys showed ubiquitous distribution of immunolocalized APE and cleaved caspase-3. The immunolocalization did not change until 24 h after PQ injection, even in the proximal convoluted tubules. G, glomeruli. Scale bar = 100 um. Results-1 SummarySummary ConclusionConclusion