The Study Of The Effect And Mechanism Of Flow Shear Stress In Continuous Machine Perfusion

Objective:To investigate the mechanism of renal injury induced by changes in flow shear stress(FSS)during renal ischemia/reperfusion(I/R),and to define the mechanism of protection of donor kidney by continuous machine perfusion.Methods:In vitro,the control group was cultured in static state.The experimental group cells were loaded with 12dyn/cm~2 force for 30min,45min,and 90min in a plate fluid chamber system and modified by the laboratory.On the other hand,cells were loaded force for a certain period of time,and then cells were cultured for 1hour,3hours,8hours and 12hours respectively in the noraml incubator.In addition,different concentrations of metformin 0mml,1 mmol,2mmol,4mmol,8mmol respectively were added to HUVEC cells for 24 hours.Next,the expression of KLF-2,Bcl-2,Bax and p53 mRNA in cells was detected by RT-PCR assay,and the relatively expression of p-AMPK/AMPK protein was detected by Western blot.In vivo,20SD male rats were randomly divided into four groups using the random number table method:The experimental group was performed hypothermia machine perfusion 4hours(HMP)in self-made rat isolated kidney continuous perfusion model,5rats.The control group was treated with static cold store4hours(CS).And metformin pretreatment group was pretreated with metformin before cold store4hours(Met-CS).And the normal control group(Nromal)was just performed warm ischemia for30min in kindeys,each group 5.Finally,renal tissue apoptosis was detected by Tunel assay,and the renal injury was observed by HE staining,the protein expression of p-AMPK in renal tissue was detected by immunohistochemistry and observed the distribution of p-AMPK protein.To further explore the mechanism of protection of donor kidney by fluid shear stress provided by continuous perfusion.Result:(1)Western blot showed that FSS could up-regulate the relative expression of p-AMPK protein in HUVEC cells and increased with time(P<0.05).Notably,the relative expression of p-AMPK protein was rapidly lost after flow cessation and prolonged with time(P<0.05).Metformin can significantly activate AMPK activity with the increase of concentration(p<0.05).(2)The results of renal tissue immunohistochemistry showed that the content of p-AMPK in kidney of HMP group was significantly higher than that in CS group(p<0.05),and there was no significant difference between HMP and Normal group(p>0.05).The expression of p-AMPK protein in the metformin group was also significantly higher than that in the CS group(p<0.05).Immunohistochemical staining showed that the expression of p-AMPK in renal tissue was mainly distributed in the renal tubular epithelial cells,and few in the glomerular endothelial cells and blood vessels.(3)RT-PCR results showed that the expression KLF-2 mRNA in HUVEC cells was significantly higher than that in static cultured after loading 12dyn/cm~2 for 1hour(p<0.05),and the Bcl-2/Bax ratio was lower in FSS 1 hour group than in CTL group(p<0.05),but there was no significant difference in the relative expression of p53 mRNA between two groups(p>0.05).The apoptosis rate of HMP group and Met-CS group was significantly lower than that of CS group in renal tissue(p<0.05).Similarly,HE staining revealed that kidney tissue of CS group was severely damaged and renal tubules were swelled obviously,and tubular swelled of Met-CS group was alleviated,While the damage of renal tissue of HMP group was mild and no swelled was observed.Conclusion:The change of fluid shear stress may affect the renal injury through the AMPK pathway during renal ischemia-reperfusion in rats,while continuous perfusion preservation can provide the fluid shear stress that protects organ by activating AMPK/KLF-2 signaling,inhibiting tissue apoptosis and reducing renal IRI.Metformin can effectively activate AMPK activity,and it can also reduce renal injury caused by cold ischemia during cold storage.AMPK may be a potential drug target for the treatment of IRI after FSS cessation.