胰激肽原酶通过调节自噬减轻免疫制剂诱导肾损伤的机制

Pancreatic kininogenase alleviates the mechanism of kidney injury induced by immune agents byregulating autophagy

目的按探讨他克莫司(TAC)诱导大鼠使用胰激肽原酶(PK)之后的肾脏损伤治疗效果,分析胰激肽原酶的药用机理。方法选取48只雄性Sprague-Dawledy大鼠作为样本,随机将大鼠分为VH组、VH+PK组、TAC组、TAC+PK组,每组各12只。VH组大鼠进行皮下橄榄油1mL·(kg·d)^(-1)、腹腔生理盐水2mL·(kg·d)^(-1)注射;VH+PK组大鼠进行皮下橄榄油1 mL·(kg·d)^(-1)和腹腔PK为7.2U·(kg·d)^(-1)"注射;TAC组进行皮下TAC1.5mg·(kg·d)^(-1)和腹腔生理盐水2mL·(kg·d)^(-1)注射;TAC+PK组进行皮下TAC1.5mg·(kg·d)^(-1)和腹腔PK7.2U·(kg·d)^(-1)注射,每组大鼠的给药周期为4周,在给药结束后采集大鼠尿液样本,检测尿蛋白水平(UPRO);在大鼠颈部右侧静脉部位采集血液样本,检测血肌酐(Scr)、血尿素氮(BUN)、TAC水平;使用电子显微镜观察肾组织的超微结构改变,运用蛋白免疫印迹法对组织细胞内的自噬相关因子轻链蛋白3B(LC3B)、P62、Beclin的变化进行分析。结果与VH组比较,TAC组大鼠的饮水量增加,尿量提高,而体重明显下降,差异均有统计学意义(均P<0.05)。TAC+PK组大鼠的体重大于TAC组(P<0.05)。TAC组大鼠的Scr、BUN、UPRO大于VH组,差异均有统计学意义(均P<0.05)。TAC+PK治疗组的Scr、BUN和UPRO均较TAC组明显下降,差异均有统计学意义(均P<0.05)。VH+PK组大鼠在电子显微镜下的各项变化和VH组无差异,而TAC组不仅出现了肾小管基膜增厚,还伴有肾小管萎缩,肾小管间质炎性细胞增多以及肾间质胶原纤维明显增生,同时出现了上皮细胞空泡变形,大量自噬体形成。相较于VH组,TAC组大鼠组织细胞中的LC3B-Ⅱ表达下降,而P62和Beclin表达显著增强(均P<0.05);TAC+PK组大鼠的LC3B-Ⅱ表达大于TAC组,而P62、Beclin表达低于TAC组,差异均有统计学意义(均P<0.05)。结论自噬可参与免疫抑制剂诱导肾损伤的发生发展,TAC诱导肾脏损伤大鼠在采用PK给药治疗后的肾脏损伤程度有所下降,其原因可能源于PK对肾脏细胞自噬功能的调节作用。

Objective To investigate the therapeutic effect of tacrolimus(TAC)induced renal injury after the use of pancreatic kininogenase(PK)in rats,and analyze the medicinal mechanism of pancreatic kininogenase.Methods Forty-eight male Sprague-Dawledy rats were selected as samples and randomly divided into VH group,VH+PK group,TAC group and TAC+PK group,with 12 rats in each group.The rats in VH group were injected with 1 mL·(kg·d)^(-1)subcutaneous olive oil and 2 mL·(kg·d)^(-1)intraperitoneal saline.The rats in VH+PK group were given 1 mL·(kg·d)^(-1)subcutaneous olive oil and 7.2 U.(kg·d)^(-1)PK intraperitoneally.In TAC group,subcutaneous TAC 1.5 mg·(kg·d)^(-1)and abdominal normal saline 2 mL·(kg·d)^(-1)were injected.TAC+PK group was injected with subcutaneous TAC 1.5 mg·(kg·d)^(-1)and peritoneal PK 7.2 U:(kg·d)^(-1).The administration cycle of each group was 4 weeks.Urine samples were collected after administration to detect urinary protein level(UPRO).Serum creatinine(Scr),blood urea nitrogen(BUN)and TAC levels were detected by blood samples collected from the right neck vein of rats.The ultrastructural changes of renal tissue were observed by electron microscope,and the changes of autophagy related factors light chain protein 3B(LC3B),P62 and Beclin were analyzed by Western blot.Results Compared with VH group,the drinking water volume and urine volume of rats in TAC group increased,while the body weight decreased significantly,with statistical significance(all P<0.05).The body weight of rats in TAC+PK group was higher than that in TAC group(P<0.05).Scr,BUN and UPRO in TAC group were higher than those in VH group,and the differences were statistically significant(all P<0.05).Scr,BUN and UPRO in TAC+PK treatment group were significantly decreased compared with TAC group,and the differences were statistically significant(all P<0.05).Under electron microscope,the changes of VH+PK group were no different from those of VH group,while in TAC group,not only the basal membrane of renal tubule was thickened,but also the renal tubule atrophy,the increase of renal tubule interstitial inflammatory cells,and the renal interstitial collagen fiber hyperplasia were observed.At the same time,vacuolar deformation of epithelial cells and a large number of autophagosomes were formed.Compared with VH group,LC3B-Ⅱ expression in TAC group decreased,while P62 and Beclin expression increased significantly.The expression of LC3B-Ⅱ in TAC+PK group was higher than that in TAC group,while the expression of P62 and Beclin was lower than that in TAC group,with statistical significance(all P<0.05).Conclusions Autophagy is involved in the development of renal injury induced by immunosuppressants.The degree of renal injury in rats with renal injury induced by tacrolimus decreased after administration of pancreatic kallikrein.The reason for this phenomenon is that pancreatic kallikrein can actively regulate the autophagy of renal cells.