播散性血管内凝血急性非少尿型肾损伤大鼠尿水通道蛋白-1水平与炎性因子的相关性
|
摘要
目的探讨播散性血管内凝血(disseminated intravascular coagulation, DIC)急性非少尿型肾损伤大鼠尿水通道蛋白-1(aquaporin-1, AQP-1)水平变化及与白细胞介素(interleukin, IL)-18、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)的相关性。方法 24只Wistar大鼠随机分为脂多糖(lipopolysaccharide, LPS)4、6、8 h组各5只,对照4、6、8 h组各3只。LPS 4、6、8 h组经股静脉滴注LPS溶液30 mg/kg,持续时间分别为4、6、8 h,制备DIC急性非少尿型肾损伤模型;对照4、6、8 h组分别经股静脉滴注等量生理盐水4、6、8 h。收集滴注LPS溶液或生理盐水期间尿量,测定AQP-1、IL-18、TNF-α、NGAL水平;滴注结束采集各组心脏血液,检测血小板计数、纤维蛋白原、凝血酶原时间、活化部分凝血活酶时间,及血浆D-二聚体、肌酐水平;采血后处死大鼠,取一侧肾脏及肺组织行HE或纤维素染色,光镜下观察肺、肾组织毛细血管充血、微血栓形成情况;Pearson法分析尿AQP-1与IL-18、TNF-α、NGAL水平的相关性。结果对照4、6、8 h组尿AQP-1、IL-18、TNF-α、NGAL水平,血小板计数、纤维蛋白原、凝血酶原时间、活化部分凝血活酶时间,及血浆D-二聚体、肌酐水平比较差异均无统计学意义(P>0.05);LPS 4、6、8 h组血小板计数、纤维蛋白原较对照4、6、8 h组降低,凝血酶原时间、活化部分凝血活酶时间较对照4、6、8 h组延长,血浆D-二聚体、肌酐较对照4、6、8 h组升高,尿量均较对照4、6、8 h组增多(P<0.05);LPS 4、6、8 h组血小板计数、纤维蛋白原依次降低(P<0.05),D-二聚体依次升高(P<0.05);LPS 8 h组凝血酶原时间、活化部分凝血活酶时间较LPS 4、6 h组延长(P<0.05),LPS 4 h组与LPS 6 h组比较差异无统计学意义(P>0.05);LPS 6、8 h组尿量较LPS 4 h组增加,肌酐较LPS 4 h组增高(P<0.05),且LPS 8 h组肌酐均较LPS 6 h组增高,尿量与LPS 6 h组比较差异无统计学意义(P>0.05);对照4、6、8 h组肺组织纤维素染色镜下无明显异常病理改变,LPS 4、6、8 h组毛细血管呈不同程度的充血及微血栓形成;对照4、6、8 h组和LPS 4 h组HE染色镜下肾小管上皮细胞未发生明显变化,LPS 6、8 h组可见少量炎症细胞浸润、上皮细胞肿胀;LPS 4、6、8 h组尿AQP-1[(114.83±2.35)、(124.18±0.51)、(140.37±2.09)ng/L]、NGAL[(5.69±0.02)、(6.32±0.08)、(7.03±0.02)μg/L]、TNF-α[(39.84±0.39)、(41.96±1.20)、(47.42±1.16)ng/L]、IL-18[(23.61±0.17)、(24.96±0.12)、(28.49±0.79)ng/L]依次升高,且均高于对照4、6、8 h组(P<0.05);LPS 4、6、8 h组尿AQP-1与TNF-α、IL-18、NGAL水平呈正相关(r=0.959,P<0.001;r=0.962,P<0.001;r=0.978,P<0.001)。结论 DIC急性非少尿型肾损伤大鼠尿AQP-1明显升高,且与TNF-α、IL-18、NGAL呈正相关,可作为评估肾损伤的敏感标志物。
Objective To investigate the level of urinary aquaporin-1(AQP-1) in rats with non-oliguric acute kidney injury induced by disseminated intravascular coagulation(DIC) and its correlations with inflammatory cytokines as interleukin(IL)-18, tumor necrosis factor-α(TNF-α) and neutrophil gelatinase-associated lipocalin(NGAL). Methods Twenty-four Wistar rats were randomly divided into lipopolysaccharide(LPS)4,6 and 8 hgroups,with 5 rats in each group,as well as control 4,6 and 8 h groups,with 3 rats in each group.LPS 4,6 and 8 h groups received intravenous injection of LPS(30 mg/kg)within 4,6 and 8 h via femoral vein to prepare DIC-induced non-oliguric acute kidney injury rats models,while control 4,6 and 8 h groups received intravenous injection of equivalent volume of normal saline within 4,6 and 8 h via femoral vein.The urine volume was collected during injection to detect the levels of AQP-1,IL-18,TNF-α and NGAL.After injection,the blood sample from the heart in every group was collected to detect the levels of platelet count,fibrinogen,prothrombin time(PT),activated partial thromboplastin time(APTT),D-Dimer(D-D)and serum creatinine(SCr).All rats were sacrificed to obtain the kidney and lung from one side for HE stain or cellulose dyeing.The capillary congestion and microthrombosis in kidney and lung tissue were observed under light microscope.The correlations of urinary AQP-1 with urinary IL-18,TNF-αand NGAL levels were analyzed by Pearson correlation method.Results There were no significant differences in the levels of AQP-1,IL-18,TNF-α,NGAL,platelet count,fibrinogen,PT,APTT,D-D and SCr among control 4,6 and 8 h groups(P>0.05).LPS 4,6 and 8 h groups had lower platelet count and fibrinogen levels,longer PT and APTT,higher D-D and SCr levels,and larger urine volume than control 4,6 and 8 groups(P<0.05).The levels of platelet count and fibrinogen decreased,while D-D increased gradually in LPS 4,6 and 8 h groups in turns(P<0.05).PT and APTT were longer in LPS 8 h group than those in LPS4 and 6 groups(P<0.05),and showed no significant differences between LPS 4 h group and LPS 6 h group(P>0.05).The urine volume was significantly larger,while the level of SCr was significantly higher in LPS 6 and 8 h groups than that in LPS 4 h group(P<0.05).The level of SCr was significantly higher in LPS 8 hgroup than that in LPS 6 h group(P<0.05),while there was no significant difference in urine volume between LPS 8 h group and LPS 6 hgroup(P>0.05).No obvious abnormal histopathological change was found in lung tissue under microscope after cellulose dyeing in control 4,6 and 8 h groups,while LPS 4,6 and 8 h groups were found congestion and microthrombosis in different degree.HE stain showed no obvious changes in renal tubular epithelial cell in control 4,6 and 8 groups as well as LPS 4 group,and slight inflammatory cytokines infiltration and epithelial cell swelling in LPS 6 and 8 hgroups.The levels of AQP-1((114.83±2.35),(124.18±0.51),(140.37±2.09)ng/L),NGAL((5.69±0.02),(6.32±0.08),(7.03±0.02)μg/L),TNF-α((39.84±0.39),(41.96±1.20),(47.42±1.16)ng/L)and IL-18((23.61±0.17),(24.96±0.12),(28.49±0.79)ng/L)increased gradually in LPS 4,6,8 h groups in turns,significantly higher than those in control 4,6 and 8 hgroups(P<0.05).The level of AQP-1 was positively correlated with TNF-α,IL-18 and NGAL(r=0.959,P<0.001;r=0.962,P<0.001;r=0.978,P<0.001)in LPS 4,6 and 8 groups.Conclusion In DIC-induced non-oliguric acute kidney injury rats,the level of urinary AQP-1 is obviously elevated,and is positively correlated with TNF-α,IL-18 and NGAL,therefore it can be used as a sensitive marker to evaluate kidney injury.
Objective To investigate the level of urinary aquaporin-1(AQP-1) in rats with non-oliguric acute kidney injury induced by disseminated intravascular coagulation(DIC) and its correlations with inflammatory cytokines as interleukin(IL)-18, tumor necrosis factor-α(TNF-α) and neutrophil gelatinase-associated lipocalin(NGAL). Methods Twenty-four Wistar rats were randomly divided into lipopolysaccharide(LPS)4,6 and 8 hgroups,with 5 rats in each group,as well as control 4,6 and 8 h groups,with 3 rats in each group.LPS 4,6 and 8 h groups received intravenous injection of LPS(30 mg/kg)within 4,6 and 8 h via femoral vein to prepare DIC-induced non-oliguric acute kidney injury rats models,while control 4,6 and 8 h groups received intravenous injection of equivalent volume of normal saline within 4,6 and 8 h via femoral vein.The urine volume was collected during injection to detect the levels of AQP-1,IL-18,TNF-α and NGAL.After injection,the blood sample from the heart in every group was collected to detect the levels of platelet count,fibrinogen,prothrombin time(PT),activated partial thromboplastin time(APTT),D-Dimer(D-D)and serum creatinine(SCr).All rats were sacrificed to obtain the kidney and lung from one side for HE stain or cellulose dyeing.The capillary congestion and microthrombosis in kidney and lung tissue were observed under light microscope.The correlations of urinary AQP-1 with urinary IL-18,TNF-αand NGAL levels were analyzed by Pearson correlation method.Results There were no significant differences in the levels of AQP-1,IL-18,TNF-α,NGAL,platelet count,fibrinogen,PT,APTT,D-D and SCr among control 4,6 and 8 h groups(P>0.05).LPS 4,6 and 8 h groups had lower platelet count and fibrinogen levels,longer PT and APTT,higher D-D and SCr levels,and larger urine volume than control 4,6 and 8 groups(P<0.05).The levels of platelet count and fibrinogen decreased,while D-D increased gradually in LPS 4,6 and 8 h groups in turns(P<0.05).PT and APTT were longer in LPS 8 h group than those in LPS4 and 6 groups(P<0.05),and showed no significant differences between LPS 4 h group and LPS 6 h group(P>0.05).The urine volume was significantly larger,while the level of SCr was significantly higher in LPS 6 and 8 h groups than that in LPS 4 h group(P<0.05).The level of SCr was significantly higher in LPS 8 hgroup than that in LPS 6 h group(P<0.05),while there was no significant difference in urine volume between LPS 8 h group and LPS 6 hgroup(P>0.05).No obvious abnormal histopathological change was found in lung tissue under microscope after cellulose dyeing in control 4,6 and 8 h groups,while LPS 4,6 and 8 h groups were found congestion and microthrombosis in different degree.HE stain showed no obvious changes in renal tubular epithelial cell in control 4,6 and 8 groups as well as LPS 4 group,and slight inflammatory cytokines infiltration and epithelial cell swelling in LPS 6 and 8 hgroups.The levels of AQP-1((114.83±2.35),(124.18±0.51),(140.37±2.09)ng/L),NGAL((5.69±0.02),(6.32±0.08),(7.03±0.02)μg/L),TNF-α((39.84±0.39),(41.96±1.20),(47.42±1.16)ng/L)and IL-18((23.61±0.17),(24.96±0.12),(28.49±0.79)ng/L)increased gradually in LPS 4,6,8 h groups in turns,significantly higher than those in control 4,6 and 8 hgroups(P<0.05).The level of AQP-1 was positively correlated with TNF-α,IL-18 and NGAL(r=0.959,P<0.001;r=0.962,P<0.001;r=0.978,P<0.001)in LPS 4,6 and 8 groups.Conclusion In DIC-induced non-oliguric acute kidney injury rats,the level of urinary AQP-1 is obviously elevated,and is positively correlated with TNF-α,IL-18 and NGAL,therefore it can be used as a sensitive marker to evaluate kidney injury.
引文
[1] JIN Y Y, YU G Z, PENG P, et al. Down-regulated expression of AQP5 on lung in rat DIC model induced by LPS and its effect on the development of pulmonary edema[J]. Pulm Pharmacol Ther,2013,26(6):661-665.
[2] MACLOUGHLIN R J, HIGGINS B D, DEVANEY J, et al. Aerosol-mediated delivery of AAV2/6-I kappa B alpha attenuates lipopolysaccharide-induced acute lung injury in rats[J]. Hum Gene Ther,2015,26(1):36-46.
[3] ZARBOCK A, GOMEZ H, KELLUM J A. Sepsis-induced acute kidney injury revisited: pathophysiology, prevention and future therapies[J]. Curr Opin Crit Care,2014,20(6):588-595.
[4] 李文,程帆,魏捷,等.HMGB1调控急性肾损伤失控性炎症反应的研究进展[J].临床急诊杂志,2018,19(10):713-718.
[5] PREVICH L E, MA L, WRIGHT J C, et al. Progress in AQP research and new developments in therapeutic approaches to ischemic and hemorrhagic stroke[J]. Int J Mol Sci,2016,17(7):1146.
[6] 沈明,郭志福,鲍礼智,等.肾脏水通道蛋白2的短时调控机制研究进展[J].第二军医大学学报,2017,38(3):349-354.
[7] DHANAPRIYA J, GOPALAKRISHNAN N, ARUN V, et al. Acute kidney injury and disseminated intravascular coagulation due to mercuric chloride poisoning[J]. Indian J Nephrol,2016,26(3):206-208.
[8] RUIZ-ORTEGA M, ORTIZ A, RAMOS A M. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and kidney disease[J]. Curr Opin Nephrol Hypertens,2014,23(1):93-100.
[9] 王美钧,郑钧元,柴红燕,等.尿白细胞介素-18联合血清胱抑素C早期诊断造影剂肾病价值[J].中华实用诊断与治疗杂志,2015,29(5):473-475.
[10] 吴颖,何再明.NGAL作为急性肾损伤生物标志物的研究现况[J].医学综述,2016,22(6):1058-1061.
[11] 朱红,马丽娜,杨伟,等.老年冠心病慢性心力衰竭患者血清中性粒细胞明胶酶相关脂质运载蛋白水平变化及意义[J].中华实用诊断与治疗杂志,2014,28(8):767-769.
[2] MACLOUGHLIN R J, HIGGINS B D, DEVANEY J, et al. Aerosol-mediated delivery of AAV2/6-I kappa B alpha attenuates lipopolysaccharide-induced acute lung injury in rats[J]. Hum Gene Ther,2015,26(1):36-46.
[3] ZARBOCK A, GOMEZ H, KELLUM J A. Sepsis-induced acute kidney injury revisited: pathophysiology, prevention and future therapies[J]. Curr Opin Crit Care,2014,20(6):588-595.
[4] 李文,程帆,魏捷,等.HMGB1调控急性肾损伤失控性炎症反应的研究进展[J].临床急诊杂志,2018,19(10):713-718.
[5] PREVICH L E, MA L, WRIGHT J C, et al. Progress in AQP research and new developments in therapeutic approaches to ischemic and hemorrhagic stroke[J]. Int J Mol Sci,2016,17(7):1146.
[6] 沈明,郭志福,鲍礼智,等.肾脏水通道蛋白2的短时调控机制研究进展[J].第二军医大学学报,2017,38(3):349-354.
[7] DHANAPRIYA J, GOPALAKRISHNAN N, ARUN V, et al. Acute kidney injury and disseminated intravascular coagulation due to mercuric chloride poisoning[J]. Indian J Nephrol,2016,26(3):206-208.
[8] RUIZ-ORTEGA M, ORTIZ A, RAMOS A M. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and kidney disease[J]. Curr Opin Nephrol Hypertens,2014,23(1):93-100.
[9] 王美钧,郑钧元,柴红燕,等.尿白细胞介素-18联合血清胱抑素C早期诊断造影剂肾病价值[J].中华实用诊断与治疗杂志,2015,29(5):473-475.
[10] 吴颖,何再明.NGAL作为急性肾损伤生物标志物的研究现况[J].医学综述,2016,22(6):1058-1061.
[11] 朱红,马丽娜,杨伟,等.老年冠心病慢性心力衰竭患者血清中性粒细胞明胶酶相关脂质运载蛋白水平变化及意义[J].中华实用诊断与治疗杂志,2014,28(8):767-769.