Protective Effect Of Pancreatic Kallikrein On Tacrolimus-induced Renal Injury And Its Mechanism

ObjectiveTo investigate the protective effect and mechanism of pancreatic kallikrein(PK)on renal injury induced by tacrolimus(TAC)in rats.MethodsIn vivo study,48 male SD rats were randomly divided into 4 groups(12 rats in each group).The vehicle(VH)groupreceived subcutaneous injection of olive oil 1mL/kg/day and intraperitoneal injection of normal saline 2mL/kg/day;VH+PK group received subcutaneous injection of olive oil 1mL/kg/day and intraperitoneal injection of PK 7.2u/kg/day;TAC group received subcutaneous injection of TAC 1.5mg/kg/day and intraperitoneal injection of normal saline 2mL/kg/day;TAC+PK group received subcutaneous injection of TAC 1.5mg/kg/day and intraperitoneal injection of PK 0.72ug/kg/day.Each group was administrated for 4 weeks.4 weeks later,the 24-hour urine of each group was collected for the 24h UPRO detection by the automatic urine analyzer;Blood was collected from the right internal jugular vein for the Scr,BUN and TAC blood concentration detection by the automatic biochemical analyzer;IPGTT was used to detect the rats' blood Glucose in Omin,30min,60min,90min and 120min,which could be used to calculate the AUCg.The urinary 8-OHdG and serum 8-OHdG excretion rate were measured by the ELISAKidney tissue samples were collected,embedded and sectioned with paraffin routinely.The TIF was observed by Masson trichrome staining;the glomerular lesion was observed by the PAS staining;the microstructure of cells is observed by electron microscope.The apoptotic cells in renal tissue were observed by the TUNEL staining.The expressions of WT1,OPN,ED-1 and MnSOD in renal tissues were detected by immunohistochemistry.In vitro study,Hk-2 cell lines were cultured in vitro and divided into control(CON)group,PK(6pg/mL)group,TAC(50ug/mL)group,and TAC(50ug/mL)+PK(6pg/mL)group.The treatment time of each group was 24h.the cell viability of each group was determined by the CCK-8;The production of intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescence coupled with flow cytometry;the expressions of LC3B,Beclin,Bax and Bcl-2 was detected by the Western blotResultsIn vivo experiment1.Compared with the VH group,the body weight of the rats in the TAC group was significantly decreased.However,the 24h urine volume,24h UPRO,BUN and Scr increased(P<0.05).Compared with the TAC group,the body weight was significantly increased in the TAC+PK group(P<0.05)and the 24h UPRO,BUN and Scr decreased in the TAC+PK group(P<0.05).2.Compared with the VH group,the glucose tolerance was obviously abnormal in the TAC group(P<0.05).Compared with the TAC group,the abnormal glucose tolerance was significantly improved in the TAC+PK group(P<0.05).3.Compared with the VH group,the renal tubules in the TAC group were significantly damaged and TIF was aggravated(P<0.05).In the TAC+PK group,renal tubular injury and TIF were reduced.4.Compared with the VH group,the mesangial area of the glomerulus was enlarged in the TAC group(P<0.05).The expression of WT-1 in renal tissue of the TAC group decreased significantly(P<0.05).Compared with the TAC group,the mesangial area shrinked and the expression of WT-1 increased significantly in the TAC+PK group(P<0.05).5.Compared to the VH group,under electron microscope,in the TAC group the renal tubule and glomeruli injuries were observed,moreover,the TAC+PK group showed different degrees of improvement in the ultrastructural changes of renal tissue.6.Compared with the VH group,the expression of TGF?-1 in renal tissue of the TAC group increased significantly(P<0.05).Compared with the TAC group,the expression of TGF?-1 in the TAC+PK group decreased significantly(P<0.05)7.Compared with the VH group,urinary 8-OHdG excretion rate and serum 8-OHdG increased in TAC group(P<0.05).Compared with the TAC group,the urinary 8-OHdG excretion rate and serum 8-OHdG decreased significantly(P<0.05).Compared with VH group,the expression of MnSOD and HO-1 in TAC group decreased but the expression of NOX2 and NOX4 increased significantly(P<0.05).Compared with TAC group,the expression of MnSOD and HO-1 in the TAC+PK group increased significantly accompany with the expression of NOX2 and NOX4 decreased(P<0.05).8.Compared with the VH group,the expression of OPN,ED-1and IL-6 in renal tissue of the TAC group increased significantly but the expression of HO-1 decreased significantly(P<0.05).Compared with the TAC group,the expression of OPN,ED-1 and IL-6 in the TAC+PK group decreased significantly and the expression of HO-1 increased significantly(P<0.05).9.Compared with the VH group,the expression of LC3B-II,P62 and Beclin in TAC group increased significantly(P<0.05).Compared with the TAC group,the expression of LC3B-?,P62 and Beclin decreased significantly in the TAC+PK group P<0.05).10.Compared with the VH group,in the TAC group the expression of TUNEL positive cells increased significantly(P<0.05).However the ratio of Bcl-2/Bax decreased(P<0.05).Compared with the TAC group,the number of TUNEL positive cells reduced in the TAC+PK group accompany with the ratio of Bcl-2/Bax increased(P<0.05).In vitro experiment1.Compared with the CON group,the cell viability of HK-2 decreased in the TAC group(P<0.05).Compared with the TAC group,the cell viability of HK-2 increased in the TAC+PK group(P<0.05).2.Compared with the CON group,the generation of ROS increased in the TAC group(P<0.05).Compared with the TAC group,the generation of ROS decreased significantly in the TAC+PK group(P<0.05).3.Compared with the CON group,the expression of LC3B-? and Beclin in TAC group increased significantly in the TAC group(P<0.05).Compared with the TAC group,the expression of LC3B-? and Beclin decreased significantly in the TAC+PK group(P<0.05).4.Compared with the CON group,the expression of the ratio of Bcl-2/Bax decreased in the TAC group(P<0.05).Compared with the TAC group,the expression of the ratio of Bcl-2/Bax increased in the TAC+PK group(P<0.05).ConclusionIn vivo and vitro study PK has a protective effect on tubulointerstitial fibrosis and glomerular injury induced by TAC,via the reduction of blood glucose,the inhibition of TGF-?1 expression,anti-inflammatory,anti-oxidative stress,regulation of autophagy and anti-apoptosis.