Mechanical Role Of Glycogen Synthase Kinase-3β In Severe Acute Pancreatitis Associated Kidney Injury In Rats

Part1. The study of expression and function of glycogen synthase kinase-3p in kidney damage of severe acute pancreatitis in ratsObjective:To explore the change of pathology features, ultrastructure and renal function in the rat model of severe acute pancreatitis (SAP). To investigate the change and roles of glycogen synthase kinase-3β (GSK-3β) in kidney injury progress after severe acute pancreatitis.Methods:Sixty SPF male Wistar rats were randomly divided into5groups (N=12). Sham operation group (SO group) and severe acute pancreatitis model groups (SAP3h,6h,12h and24h four subgroups). Pancreatitis model was established by retrograde infusion of5%sodium taurocholate into the bilio-pancreatic duct. In the sham operation group, pancreas and duodenum was reversed after a midline incision. Serum amylase (AMY), lipase (LIPA), creatinine (Cr) and urea nitrogen (BUN) levels were detected. Histology in pancreas and kidney stained with HE (hematoxylin and eosin stain) for light microscopy were analyzed. Kidney cell ultrastructure changes were observed by projection electron microscope. GSK-3β protein expression and their phosphorylated form p-GSK-3p ser9in kidney tissue were quantitated by western blot.Results:With the progress of pancreatitis, pancreatic enzyme indicators AMY, LIPA, and pancreatic histology score were elevated significantly. Cr, BUN and renal pathological grading in SAP3h,6h,12h group showed increased gradually, while SAP12h, SAP24h group presented statically, there was no statistically difference (P>0.05). GSK-3β protein expression was higher in SAP group than in SO group, but didn’t appear increased gradually with the pancreatitis progress. While p-GSK-3β ser9protein expression was higher in SAP3h group than SO group, the difference was statistically significant (P＜0.05), and with the progress of pancreatitis, the expression of p-GSK-3β ser9protein presents a weakening trend, SAP6h,12h,24h group, all expression levels belower than SO group (P＜0.05).Conclusion:To reveal the function and morphology changes of pancreatitis-associated kidney injury in severe acute pancreatitis model rats. p-GSK-3β ser9activation rise shows in time-dependent in kidney tissue in rats with severe acute pancreatitis, has effects on renal cells apoptosis and necrosis, suggesting that p-GSK-3β ser9may plays a crucial role in pancreatitis-associated kidney injury in severe acute pancreatitis. Part2. To explore the effect of different types of GSK-3β inhibitor and dose-response relationship in SAP associated kidney injury in ratsObjective:To observe the dose-response relationship of the GSK-3β inhibitor TDZD-8in severe acute pancreatitis (SAP) associated kidney injury in rats. In order to identify the most effective class of GSK-3β inhibitor and its effective and reasonable safe dose in SAP associated kidney injury model in rats by comparing three kinds of frequently-used GSK-3β inhibitor TDZD-8, lithium chloride (LiCL), SB216763in this model.Method:96SPF male Wistar rats were randomly divided into8groups (N=12): sham operation group (SO group), severe acute pancreatitis group (SAP group), TDZD-8pretreatment groups (TD group, marked TD、TD2、TD3、TD4group, respectively) at different dosage (0.25,0.5,1and2mg/kg), LiCL pretreatment groups (L group,40mg/kg) and SB216763pretreatment group (SB group,1mg/kg). SAP model was induced by retrograde infusion of5%sodium taurocholate into the biliopancreatic duct. The rats of SO group and SAP group were injected with10%DMSO (0.1ml/100g) by femoral vein30min before the operation. TD groups were injected different dosage of TDZD-8dissolved in10%DMSO before the operation, L group and SB group were injected LiCL and SB216763dissolved in10%DMSO before the operation. Rats in each group were sacrificed at12hr after operation to measure the mortality, quantity of ascites, serum AMY, Cr, BUN and ALT and to observe the pathological changes of pancreatic tissues and kidney tissues.Results:SO, SAP, TD3, L and SB group rats mortality rates were0%,33.3%,0%,8.3%and16.6%; Compared with SO group, the levels of ascites, serum AMY, Cr, BUN, ALT and pancreatic and renal pathologic score in SAP group were all significantly increased (P<0.05); TD1group almost no relief from SAP; TD2, TD3, TD4groups can reduce quantity of ascites, serum AMY, Cr, BUN,ALT and pancreatic tissue pathological grading in different degrees, the difference were statistically significant(P<0.05); TD2, TD3can reduce ALT values in different degrees contrast with SAP group (P＜0.05), while TD4group ALT value is higher, similar to the SAP group; TD2compared with TD3group, has effect on various indexes to SAP, but TD3group was better than TD2, which shows all the indexes have significant difference (P＜0.05); Compared with TD3group(the best group in TD group), the levels of ascites and serum ALT in L group and SB group were no significant difference(P>0.05), but the levels of AMY, Cr, BUN, ALT, pancreatic and renal pathologic score were significantly reduced in TD3group than L and SB groups (P<0.05); L group compared with SB group, the values of Cr, BUN, pancreatic and renal pathologic score were lower (P＜0.05).Conclusion:Through comparing three different GSK-3β inhibitor roles on SAP associated with kidney injury in rats, we can concluded that TDZD-8is the most effective GSK-3β inhibitor for this model. The GSK-3β inhibitor TDZD-8lmg/kg administered intravenously was safe, effective and optimal dosage for attenuating the severity of severe acute pancreatitis associated with kidney injury.Part3. Protection mechanism of GSK-3β inhibitor TDZD-8on acute kidney injury of severe acute pancreatitis in ratsObjective:The aim of this study is to explore the effects of GSK-3β activity changes on activation of nuclear factor-KB and NF-KB-dependent gene expression, and to investigate the protection mechanism of GSK-3β inhibitor TDZD-8on severe acute pancreatitis-associated kidney injury in rats.Method:80SPF male Wistar rats were randomly divided into four groups (N=20). Sham operation group (SO group, sham+vehicle group), severe acute pancreatitis group (SAP group, SAP+vehicle group), TDZD-8treatment group (SAP+TDZD-8group) and TDZD-8drug-control group (sham+TDZD-8group). The rats of SO group and SAP group were injected with10%DMSO (O.lml/100g) by femoral vein30min before the operation. TDZD-8treatment group were injected TDZD-8(lmg/kg) dissolved in10%DMSO. TDZD-8drug control group was chosen optimal dosage according to the effect of TDZD-8. Rats in each group were sacrificed at12hr after operation to measure the serum AMY, LIPA Cr, and BUN and to observe the pathological changes of pancreatic tissues and kidney tissues. Ultrastructural change of renal cells was analyzed by transmission electron microscopy. Serum interleukin-1β (IL-lβ) and interleukin-6(IL-6) levels were evaluated using ELISA assay, kidney tissue myeloperoxidase (MPO) activity was detected by chromatometry (reflects neutrophil infiltration). The activation of nuclear factor-KB (NF-kB) was evaluated using an immunohistochemistry assay. The expression of GSK-3β, phospho-GSK-3β (Ser9), NF-kB p65, tumour necrosis factor a (TNF-a), intercellular adhesion molecule-1(ICAM-1), inducible nitric oxide synthase (iNOS) and interleukin-10(IL-10) protein in the kidney was characterised using western blot analysis.Results:Compared with SAP model group, TDZD-8reduced serum pancreatic enzyme indicators and histology score, alleviated histopathological and ultrastructural changes in kidney, downregulated serum IL-1β, IL-6and myeloperoxidase activity (P<0.05), NF-kB p65expression in TDZD-8treatment group is mainly in the nucleus, but decreased significantly than SAP group. GSK-3β protein expression were detected in SAP group and TDZD-8treatment group higher than in SO and TDZD-8drug-control group, but GSK-3β protein expression no difference between SAP group and TDZD-8treatment group. p-GSK-3β ser9protein expression in SAP group was lower than SO group, while p-GSK-3β ser9protein expression in TDZD-8treatment group higher than SAP group. NF-kB p65, TNF-a, ICAM-1and iNOS protein expressions increased in kidney tissue after induction of pancreatitis, and IL-10protein expression decreased, but inhibition of GSK-3P activity reduced the activation of NF-kB, TNF-a, ICAM-1and iNOS, meanwhile, increased activation of IL-10.Conclusion:Blockade of GSK-3β activity by TDZD-8exerts the protective effect against kidney injury by inhibition the inflammation signaling pathway in severe acute pancreatitis. It could alleviate histopathological and ultrastructural changes in kidney injury, which protection mechanism was mediated by NF-kB and its related inflammatory mediators(TNF-α IL-β、IL-6、ICAM-1、iNOS), neutrophil infiltration.