The Experimental Study Of The Protection Of Ulinastatin To Acute Kidney Injury After Cardiopulmonary Resuscitation In Rats

Objective To explore the mechanism and evaluate the protective effect of ulinastatinto acute kidney injury in rats after cardiopulmonary resuscitation induced by suffocation byinjecting of different doses of ulinastatin by the femoral vein of rats,studying the changes ofthe serum renal function and inflammatory factors in different time points of each group,andobserving the changes of kidney tissue pathology and expression of inflammatory cytokinesin renal tissue.Methods80healthy SD rats were randomly divided into five groups:the sham group（Cgroup）,the saline group（NS group）,Ulinastatin group (according to the doses divided to be thelow-dose group （50,000u/kg, U1group）, middle-dose group （100000u/kg, U2group）, thehigh-dose group （200000u/kg, U3group） and there were16rats in each group.In the shamgroup（C group）,the rat was only intubated and puncture the femoral artery and femoralvein,then indwelling the puncture tube,without of establishing a model of cardiopulmonaryresuscitation; In the saline group（NS group）, the model of cardiopulmonary resuscitation inrat was established by suffocation.After intubating and puncturing the femoral artery andfemoral vein, the intubation of trachea was clippinged in end-expiratory into cardiacarrest.Cardiopulmonary resuscitation was started7minutes later, then injected saline with2ml/kg into the femoral vein of rat immediately at restoration of spontaneous circulation（ROSC）;In the Ulinastatin group, the model of cardiopulmonary resuscitation in rat wasestablished by suffocation.After intubating and puncturing the femoral artery and femoralvein,the intubation of trachea was clippinged in end-expiratory into cardiacarrest.Cardiopulmonary resuscitation was started7minutes later, according to the ulinastatin group inject ulinastatin to the femoral vein with50,000u/kg in the group U1,100,000u/kgin the U2group,200,000u/kg in the U3group immediately at restoration of spontaneouscirculation （ROSC）.Collected rat venous blood samples at ROSC immediately,6hours and24hours after ROSC to test the serum BUN and Cr,also test the serum TNF-α and NGAL level atthe aforementioned three time points by ELISA;the kidney tissue was collected at6h,24h afterthe ROSC,then embedded and do hematoxy-lin-eosin（HE）staining to observe pathologicalchanges,and assess the degree of tubular injury with Mc Whinnie semi-quantitative method.Immunohistochemical staining was completed to detect the expression and localization ofTNF-α.Results1.The pathological changes in kidney in different groups.Kidney of C group inrats with HE staining observed by light microscopy:renal organizational structure was clearat T6,and there was no obvious degeneration and necrosis in tubular epithelial cells, therewere no significant difference between T₂₄and T6. kidney of NS group in rats with HEstaining observed by light microscopy:the renal tissue is abnormality a little,renal tubularepithelial cells was swelling,and the brush border of epithelial cells disappeared at T6,the renaltissue is abnormality severely,the structure of the kidney tissue was disordered,renalinterstitial was edemaed, renal tubular epithelial cells was swelling and nuclei disappeared,thebrush border of epithelial cells disappeared,and the basement membrane was bared atT₂₄,kidney of U1,U2and U3group in rats with HE staining observed by light microscopy:therenal tissue is abnormality a little,renal tubular epithelial cells was swelling,and the brushborder of epithelial cells disappeared at T6,compared with the NS group,the damage of kidneytissue significantly reduced at T₂₄,there was only a small amount of vacuolar degeneration oftubular epithelial cells, brush border loss,and the structure of tubular maintain good.Compared with C group,tubular injury score of NS group6h and24h after CPR wassignificantly higher,and it was significantly lower than the NS group’s,and renal tubular injuryscore decreased with increaseing of Ulinastatin dose.2.Renal function in different groups:the serum BUN and Cr in different time was no significantly difference in C group（P＞0.05）.Compare with the C group the serum BUN and Cr was higher in U1,U2,U3groups and theNS group at T6and T₂₄（P＜0.05）;the BUN and Cr levels was significantly higher at T6andT₂₄than at T0（P＜0.05）.The serum BUN and Cr levels in U1,U2,U3groups was significantlylower than the NS Group（P＜0.05）;Compared with U1group, U2group and U3group wassignificantly lower at T6and T₂₄（P＜0.05）;and compared with U2group, U3group wassignificantly lower at T6and T₂₄（P＜0.05）.3. Comparison of the serum NGAL in differenttimes between different experimental groups：there was no significant differences in everygroups at T0（P＞0.05）,the serum NGAL levels was higher at T6and T₂₄than T0in NSgroup（P＜0.05）;and at T6,the levels the C group was lower than the NS group,U1group,U2group and U3group（P＜0.05）,and the serum NGAL was lower in U1group,U2group and U3group than the NS group（P＜0.05）,the serum NGAL was lower in U2group than the U1group,and the serum NGAL was lower in U3group than the U2group（P＜0.05）;In the NSgroup，the level of the serum NGAL at T₂₄than at T6，but it is still was significant higher thanT0（P＜0.05）,and at T₂₄,it was also higher in the NS group than U1,U2,U3groups（P＜0.05）;however at T₂₄,there was no significant differences between the U1,U2,U3groups（P＞0.05）.4.Comparison of the serum TNF-α in different experimental group.There was nosignificantly differences in C group between the three time points（P＞0.05）.Compared with Cgroup,the serum TNF-αwas significant higher in NS,U1,U2,U3groups at T6and T₂₄（P＜0.05）；and compared with T0，the level of the serum TNF-α in NS group was significantlyhigher at T6and T₂₄（P＜0.05）,compared with the NS group,it is significantly lower inU1,U2,U3groups at T6and T₂₄（P＜0.05）;and it is significantly lower in U2,U3groups thanU1group（P＜0.05）.And also compared with U2group, U3group was significantly lower atT6and T₂₄（P＜0.05）.5.The Expression of the TNF-α was little or rare in nomal renal tissue inrats.At T6,the IOD of TNF-α in C group was lower than the NS,U1,U2and U3groups（P＜0.05）;compared with the NS group,the IOD of TNF-α was lower than the U1,U2,U3groups at T6（P＜0.05）,and the IOD of U2group was lower than U1group（P＜0.05）,and the U3groupwas also lower than U2group（P＜0.05）;At T₂₄, the IOD of TNF-α in NS,U1,U2and U3groupwas lower than at T6（P＜0.05）,but it is significant higher than C group（P＜0.05）; At T₂₄,theIOD of TNF-α in the U1,U2and U3groups was lower than the NS group（P＜0.05）,and theU2group was lower than U1group（P＜0.05）,the U3group was also lower than U2group （P＜0.05）.Conclusion There was acute kidney injury in the early after CPR in rats; serumNGAL can be a biomarker of acute kidney injury in rats after CPR; The inflammatoryresponse is one of the mechanisms of the development in early kidney damage after CPR; Ulinastatinhas a protective effect to acute kidney injury in rats after CPR by inhibiting the inflammatoryresponse,and its protective effect was related with the dose of ulinastatin.