二次打击大鼠部分体液因子变化与肠系膜淋巴管结扎的影响

目的:观察结扎肠系膜淋巴管对二次打击大鼠部分体液因子的影响,探讨肠淋巴途径在二次打击发病学中的意义。方法:实验于2002-03/10在河北北方学院病理生理学教研室及附属第一医学检验科完成。①实验分组:选择雄性Wistar大鼠45只,按随机数字表法分为结扎组、未结扎组、假手术组,每组15只。②实验方法:所有大鼠行腹部手术,结扎组行肠系膜淋巴管结扎术;未结扎组与假手术组仅在肠系膜淋巴管下穿线,不结扎。结扎组、未结扎组大鼠手术创伤后,以失血-脂多糖方法复制二次打击模型。③实验评估:手术创伤后24h进行。采用酶联免疫吸附法测定实验前后血清肿瘤坏死因子α、白细胞介素6含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶活性、改良TBA微量法测定丙二醛水平;以硝酸还原酶法、化学显色法分别检测NO2-/NO3-浓度及一氧化氮合酶、诱导型一氧化氮合酶活性;以免疫比浊法经全自动生化分析仪检测血清免疫球蛋白IgG、IgM、IgA及补体C3、C4含量。结果:①3组大鼠存活率:实验24h期间,假手术组全部存活(15/15),未结扎组53.3%(8/15)存活,结扎组73.3%(11/15)存活,3组动物24h存活率差异有显著性意义(χ2=8.904,P<0.05)。②3组血清肿瘤坏死因子α、白细胞介素6水平比较:创伤含量高于实验前、假手术组、结扎组,差异有显著性意义(t=3.08,3.12,2.73,P<0.01;t=8.35,6.76,3.84,P<0.01)。③3组血清超氧化物歧化酶活性、丙二醛水平比较:创伤后24h,未结扎组超氧化物歧化酶活性显著低于实验前及假手术组、结扎组,差异有显著性意义(t=1.92,2.01,2.22,P<0.05);未结扎组与结扎组大鼠血清丙二醛含量均显著高于实验前及假手术组(t=3.64,2.67,1.42,P<0.05)。④3组血清一氧化氮及其合酶比较:创伤后24h,未结扎组大鼠血清NO2-/NO3-水平、一氧化氮合酶、诱导型一氧化氮合酶活性均显著高于假手术组,差异有显著性意义(t=3.54,P<0.05;t=2.05,P<0.05;t=2.57,P<0.01);结扎组大鼠血清NO2-/NO3-水平和一氧化氮合酶活性高于假手术组(t=2.10,P<0.05),NO2-/NO3-水平、诱导型一氧化氮合酶活性显著低于未结扎组(t=1.74,P<0.05;t=1.92,P<0.05)。⑤3组血清免疫球蛋白及补体水平比较:创伤后24h,未结扎组大鼠血清IgG、IgA、IgM、C3、C4均高于假手术组及结扎组,差异有显著性意义(t=2.98,P<0.01;t=2.64,2.11,2.54,2.23,P<0.05;t=2.92,2.28,P<0.01;t=1.73,2.02,1.72,P<0.05)。结论:肠系膜淋巴管结扎抑制了二次打击大鼠体液因子变化表现出的免疫反应以及过度炎症反应,肠系膜淋巴途径是二次打击过程中重要的发病环节。     

肠系膜淋巴管结扎对失血性休克大鼠肺损伤的影响

目的观察结扎肠系膜淋巴管对不同时期重症失血性休克大鼠肺组织自由基、炎症介质的影响，探讨肠淋巴途径在休克大鼠急性肺损伤（ALI）中的作用。方法78只雄性Wistar大鼠被随机分为假手术组、休克组和结扎组。休克组与结扎组复制重症失血性休克模型。结扎组于休克复苏后行肠系膜淋巴管结扎术，于休克90min、液体复苏后0、1、3、6、12和24h各处死6只大鼠，制备肺组织匀浆，检测丙二醛（MDA）、超氧化物歧化酶（SOD）、肿瘤坏死因子-α（TNF—α）、白细胞介素-6（IL-6）以及髓过氧化物酶（MPO）活性。结果休克组大鼠输液复苏后各时间点肺组织匀浆MDA、TNF—α、IL-6以及MPO活性均有不同程度的升高，3～12h持续在较高水平，均显著高于假手术组，肺组织匀浆SOD活性显著低于假手术组（P〈0．05或P〈0．01）；结扎组输液复苏后3，6、12和24h肺组织匀浆MDA、TNF-α、IL-6以及MPO活性均显著低于休克组，SOD活性高于休克组（P〈0．05或P〈0．01）。结论肠系膜淋巴管结扎可干预重症失血性休克大鼠ALI，其机制与减少肺中性粒细胞扣押，降低TNF—α、IL-6、自由基释放与SOD消耗等因素有关。     

心肺复苏后大鼠肾小管上皮细胞凋亡的实验研究

目的研究心肺复苏后大鼠肾小管上皮细胞的凋亡情况及其可能机制。方法Sprague Dawley雄性大鼠48只,随机分为6组:对照组(假手术组)以及复苏后3、6、12、24、48h组,每组各8只。采用窒息合并冰氯化钾(0·5mol/L)停跳液致大鼠心搏骤停-心肺复苏模型,心搏骤停5min后开始心肺复苏。采用TUNEL法观察复苏后肾小管上皮细胞的凋亡情况,采用免疫组化的方法观察大鼠肾小管上皮细胞Bcl-2、Fas蛋白的表达情况。结果复苏后3h大鼠肾小管上皮细胞就有明显凋亡,之后表达持续增加,至24h达高峰,平均灰度值为(19·75±4·04)%。复苏后3h肾小管上皮细胞Bcl-2有少量表达,其后表达呈上升趋势,48h达到高峰。而Fas复苏后3h呈高表达,与对照组比较(P<0·01),平均灰度值为(47·78±2·18)%,随后持续高表达,至24h达高峰。结论心肺复苏后大鼠存在肾小管上皮细胞凋亡,其中Fas的高表达介导了肾小管上皮细胞凋亡。     

肠系膜淋巴管结扎对失血性休克大鼠肺组织一氧化氮及其表达的影响

目的 观察结扎肠系膜淋巴管对不同时期重症失血性休克大鼠肺组织一氧化氮（NO）及其表达的影响，探讨肠淋巴途径在休克大鼠急性肺损伤（ALI）中的作用。方法 雄性Wistar大鼠78只，按随机数字表法分为假手术组（n=6）、休克组（n=42）和结扎组（n=30）。休克组与结扎组复制重症失血性休克模型，结扎组于休克复苏后行肠系膜淋巴管结扎术；休克组于休克后90min、输液复苏后0h，休克组及结扎组于输液复苏后1、3、6、12和24h各时间点处死大鼠，制备肺组织匀浆，检测NO及其合酶的变化；用逆转录-聚合酶链反应（RT—PCR）测定各组大鼠肺组织诱生型一氧化氮合酶（iNOS）．mRNA表达。结果 休克组大鼠复苏后3h肺组织NO含量、NOS活性及iNOSmRNA表达开始升高，复苏后6～12h持续在较高水平，均显著高于假手术组、休克后90min及复苏后0h（P〈0．05或P〈0．01）；结扎组仅于3h和6h增高，且结扎组复苏后6、12和24h肺组织NO含量、NOS活性以及iNOSmRNA表达均显著低于休克组相同时间点（P〈0．05或P〈0．01）。结论 肠系膜淋巴管结扎可降低重症失血性休克大鼠肺组织NO生成及iNOSmRNA表达，从而减轻肺损伤。     

肠淋巴途径在失血-脂多糖二次打击大鼠心肌损伤中的作用

目的 探讨肠系膜淋巴管结扎干预失血-脂多糖(LPS)致大鼠心肌损伤的作用机制.方法 将雄性Wistar大鼠按随机数字表法分为假手术组、未结扎组、结扎组;以失血-LPS复制二次打击动物模型,结扎组于失血后行肠系膜淋巴管结扎术以阻断肠淋巴液回流.创伤后24 h处死各组大鼠制备心肌组织匀浆,检测髓过氧化物酶(MPO)、ATP酶活性以及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量;制备心肌病理切片,用原位末端缺刻标记法(TUNEL)检测细胞凋亡率,免疫组化法检测bcl-2和bax蛋白表达.结果 未结扎组大鼠心肌MPO[(0.23±0.08)U/g]、TNF-α[(9.99士2.74)pg/g]、IL-6((31.57士12.71)pg/g;～均显著高于假手术组[MPO:(0.12士0.03)U/g、TNF-α:(4.17±1.35)μ/g、IL-6:(17.86±5.17)μg/g,均P＜0.01],ATP酶活性显著低于假手术组;结扎组大鼠心肌MPO[(0.13±0.03)U/g]、TNF-α[(5.57±1.65)μg/g]、IL-6[(23.24±5.95)μg/g]均显著低于未结扎组(P＜0.05或P＜0.01),ATP酶活性显著高于未结扎组.未结扎组心肌细胞凋亡率[(22.7±6.9)%)、心肌细胞bax蛋白表达(104.5±11.4)显著高于假手术组[凋亡率:(3.8±1.2)%,bax蛋白:142.1±10.9]和结扎组[调亡率:(8.4±2.8)%,bax蛋白;128.4±9.6],bcl-2蛋白表达(196.4±19.3)显著低于假手术组(132.2±12.3)和结扎组(165.1±11.6,均P＜0.01).结论 肠系膜淋巴管结扎通过降低炎症介质TNF-α、IL-6水平,提高bcl-2蛋白表达和心肌细胞膜ATP酶活性,从而干预失血-LPS致大鼠心肌损伤.     

彩超照射对中孕胎鼠肾小管上皮细胞凋亡及超微结构的影响

目的　应用彩色多普勒超声照射中孕期大鼠 ,观察胎鼠肾小管上皮细胞凋亡及超微结构的变化。方法　 32只孕 14 d SD大鼠按彩超照射时间不同随机等分为 4组 , 组 (对照组 )、 组 (照射 10 min组 )、 组 (照射 2 0 min组 )、 组 (照射 30 min组 ) ,仪器采用 Acuson公司 Sequoia- 5 12型彩色电脑声像仪 ,照射条件 :4 V1探头 ,二维频率 3.0 MHz,彩色频率 CD=3.0 MHz,Tis=1.8,MIcd=1.6。照射后 2 4 h取胎鼠肾脏标本 ,HE染色光镜观察组织结构 ;TUNEL法检测凋亡细胞 ,采用 L eica Q5 0 0 MC型图像分析仪进行图像分析 ;透射电镜观察超微结构变化。结果　各组光镜下观察肾脏组织结构均未见异常 ;Tunel法检测 ,各组胎鼠肾小管上皮内均可见散在的棕黄色淡染的 Tunel阳性表达 ,图像分析统计结果显示 : 组、 组胎鼠肾小管上皮 Tunel阳性表达强度与 组无显著差异 (P>0 .0 5 ) ; 组表达强度与 组有显著差异 (P<0 .0 5 )。照射 30 min组胎鼠肾脏电镜检查发现染色质浓缩边集、线粒体肿胀等超微结构变化。结论　彩色多普勒超声照射中孕期大鼠超过 30 min可引起胎鼠肾小管上皮细胞凋亡增多 ,超微结构发生轻微改变。     

肠系膜淋巴管结扎对急性失血大鼠血黏度的影响

目的以肠系膜淋巴管结扎为手段，观察急性失血后，阻断肠淋巴液回流对急性失血大鼠血黏度的影响。方法20只Wistar雄性大鼠均分为失血组与结扎组。所有大鼠令身肌肉麻醉，经右侧颈总动脉匀速放血（失血量为全血量的1／4，≥5．8mL，全血量以体重1/13计），结扎组失血后行肠系膜淋巴管结扎，失血组仅在肠系膜淋巴管下穿线。记录24h存活情况。24h后，将存活大鼠再次全身麻醉，经左侧颈总动脉迅速放血6mL．将实验前后放出的全血进行全血黏度、血浆黏度、红细胞压积（Hct）等指标检测。结果急性失血后24h，失血组存活6只．结扎组存活9只，结扎组存活情况略好于失血组。与实验前相比，急性失血后24h，失血组与结扎组在各切变率下的全血黏度、全血相对黏度以及Hct均显著降低，失血组全血还原黏度降低、血浆黏度升高（P〈0．01～0．05）；结扎组在1s^-1、10．3s^-1、30．7s^-1、115s^-1、300s^-1等切变率下的全血黏度、全血相对黏度以及Hct均显著高于失血组，血浆黏度降低（P〈0．05）。结论结果提示大鼠急性失血后，出现全血黏度降低，但血浆黏度升高；肠系膜淋巴管结扎可改善急性失血导致的全血黏度和血浆黏度的变化。     

草酸钙晶体对大鼠肾小管上皮细胞骨桥蛋白表达的影响

目的探讨一水草酸钙(calcium oxalate monohydrate,COM)晶体对大鼠肾小管上皮细胞中骨桥蛋白(osteopontin,OPN)表达的影响。方法选用SD大鼠的肾皮质原代培养出肾小管上皮细胞并制成爬片,将爬片的肾小管上皮细胞随机分为A、B、C、D、E5组。A组不加COM(11例);B组1 mmol/L浓度COM(17例);C组3 mmol/L浓度COM(18例);D组5 mmol/L浓度COM(18例);E组10 mmol/L浓度COM(17例)。采用免疫组织化学法(SABC)检测各组肾小管上皮细胞中OPN的表达,并进行阳性细胞计数和各组间比较。结果 OPN的表达主要定位于细胞质,呈棕褐色。各组OPN阳性表达率:A组54.5%;B组64.7%;C组77.8%;D组88.9%;E组76.5%。随着COM浓度增加,OPN表达逐渐增强,到5 mmol/L浓度COM时OPN表达最强(P0.01),而10 mmol/L浓度COM时OPN表达反而下降,接近3 mmol/L浓度COM水平(P0.05)。结论大鼠肾小管上皮细胞受COM刺激后OPN表达增强,但高浓度COM使OPN表达减弱,提示高浓度COM通过损伤肾小管上皮细胞使OPN表达下降,进而形成结石。     

百草枯致人肾小管上皮细胞凋亡及抗凋亡的PCR基因芯片研究

目的:从转录组学角度对人肾小管上皮细胞的凋亡及抗凋亡基因表达变化机制作初步探讨。方法:人肾小管上皮细胞体外培养,用CCK8试剂盒检测百草枯中毒对人肾小管上皮细胞的毒性作用,测定半数有效剂量的浓度。实验组使用一定浓度百草枯处理人肾小管上皮细胞,对照组使用无血清培养基,均培养24小时。使用光镜观察,并用流式细胞仪分析确定凋亡存在,收集两组细胞提取总RNA。采用人凋亡信号通路PCR芯片检测两组细胞的相关基因表达情况,并使用qPCR验证差异基因CASP5、CD154、Bcl-2A1的表达情况。结果:百草枯可致人肾小管上皮细胞的CASP5凋亡基因及CD154和Bcl-2A1抗凋亡基因表达升高(P0.05);用qPCR验证的结果与上述结果趋势一致。结论:CASP5可能在百草枯致人肾小管上皮细胞凋亡的过程中发挥其促凋亡的作用,而CD154及Bcl-2A1可能在百草枯致人肾小管上皮细胞抗凋亡相关的机制发挥作用。     

肠系膜淋巴管结扎对急性失血大鼠血液凝固性的影响

目的 观察结扎肠系膜淋巴管对急性失血大鼠血小板功能、体外血栓形成以及凝血功能的影响,探讨肠淋巴途径在急性失血时血液凝固性变化中的作用.方法 按随机数字表法将20只雄性Wistar大鼠分为失血组与结扎组,每组10只.采用经右颈总动脉匀速放血(失血量为全血量的1/4)的方法制备急性失血大鼠模型.结扎组失血后行肠系膜淋巴管结扎,失血组仅在肠系膜淋巴管下穿线但不结扎;记录24 h大鼠存活情况.失血后24 h将存活大鼠再次全麻,经左颈总动脉迅速放血6 ml.检测实验前后血小板黏附率、血小板聚集率、体外血栓形成率及凝血功能指标的变化,计算脑血流量.结果 急性失血后24 h,失血组存活6只(60%),结扎组存活9只(90%),结扎组存活率高于失血组,但差异无统计学意义(P>0.05).与实验前相比,两组血小板黏附率、血小板聚集率、纤维蛋白原(Fib)均显著升高,凝血酶时间(TT)显著延长,脑血流量显著降低;失血组活化部分凝血活酶时间(APTT)显著延长,血栓湿长、干长、湿重、干重和血栓形成率均显著增加(P<0.05或P<0.01).实验后结扎组血小板黏附率[(15.02±1.24)%],血小板聚集率(1 min、3 min及最大聚集率),血栓湿长、干长、湿重、干重,血栓形成率[(46.2±6.9)%],APTT[(21.04±5.53)s],Fib含量[(433.67±13.97)g/L]均显著低于失血组[分别为(18.54±1.18)%、(69.8±6.9)%、(26.35±6.26)s、(510.96±35.59) g/L],脑血流量[(485.1±41.4)ml·kg~(-1)·min-1]显著高于失血组[(417.8±42.2)ml·kg~(-1)·min-1,P<0.05或P<0.01].结论 急性失血可导致大鼠血液高凝、体外血栓形成增多,肠系膜淋巴管结扎可改善急性失血大鼠的血液高凝状态.     

Effects of mesenteric lymph duct ligation on apoptosis of renal tubule epithelial cells in rats after two-hits]

OBJECTIVE: To explore the effect of mesenteric lymph duct ligation in preventing apoptosis of renal tubule epithelial cells in rats by two-hits including hemorrhage and lipopolysaccharide (LPS) challenge. METHODS: Forty-five Wistar rats were randomly divided into three groups: the ligation group, the non-ligation group and sham operation group. The two-hits model was established by withdrawing the blood via the right side carotid artery of rats and intragastric administration 4 mg/kg LPS 6 hours after hemorrhage, and mesenteric lymph flow was blocked by ligating mesenteric lymph duct in ligation group. Twenty-four hours after operation, kidney was harvested for pathological examination, and the apoptosis cells rate of renal tubule epithelial cells were determined by method of terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), the expression of apoptosis-correlating gene bcl-2 and bax protein were determined by streptavidin-biotin complex (SABC) immunohistochemical method. RESULTS: After two-hits, the apoptosis rate and expression of bax protein of renal tubule epithelial cells in non-ligation group were significantly increased, and expression of bcl-2 protein was significantly lower as compared with those of sham operation group (all P<0.01). But the apoptosis rate and expression of bax protein of renal tubule epithelial cells in ligation group were significantly lower, and expression of bcl-2 protein was significantly increased as compared with non-ligation group (all P<0.01). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct could ameliorate the apoptosis of renal tubule epithelial cells in rats as produced by hemorrhage and LPS, and its mechanism might relate to the reduction of the down regulation of gene expression of bax protein and enhancement of the expression of bcl-2 protein after ligation of mesenteric lymph duct.     

肠淋巴途径在大鼠休克致肝脏炎症反应中的作用

目的 观察结扎肠系膜淋巴管对重症失血性休克不同时期大鼠肝脏炎症介质、自由基的变化,探讨阻断肠淋巴途径对休克大鼠肝脏炎症反应的影响.方法 78只雄性Wistar大鼠被随机分为假手术组(n=6)、休克组(n=42)和结扎组(n=30).休克组与结扎组复制重症失血性休克模型,结扎组于休克复苏后行肠系膜淋巴管结扎术.于休克90 min、输液复苏后0、1、3、6、12和24 h各处死6只大鼠,制备肝组织匀浆,检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、一氧化氮(NO)、一氧化氮合酶(NOS)、丙二醛(MDA)、超氧化物歧化酶(SOD)以及髓过氧化物酶(MPO)水平;采用逆转录-聚合酶链反应(RT-PCR)测定肝组织诱生型一氧化氮合酶(iNOS)mRNA表达.结果 休克组大鼠输液复苏后不同时间点肝组织TNF-α、IL-6、NO、NOS、MDA、MPO以及iNOS mRNA均有不同程度的升高,6～12 h持续在较高水平,均显著高于假手术组,肝组织SOD活性显著低于假手术组(P＜0.05或P＜0.01);结扎组输液复苏后3、6、12和24 h肝组织TNF-α、IL-6、NO、NOS、MDA、MPO以及iNOS mRNA均显著低于休克组相应时间点.SOD活性高于休克组相应时间点(P＜0.05或P＜0.01).结论 肠系膜淋巴管结扎可减少肝脏中性粒细胞扣押,降低TNF-α、IL-6释放,抑制iNOS mRNA表达及NO生成,减少自由基损伤与SOD消耗,从而减轻肝脏的炎症反应.     

Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation

Our objective in this study was to test the hypothesis that priming of neutrophils (PMN) in vivo by trauma-hemorrhagic shock (T/HS) is mediated by factors carried in intestinal lymph that prime PMNs by enhancing their responses to inflammatory mediators. Previous studies have shown that T/HS-induced lung injury is mediated by factors contained in mesenteric lymph and that ligation of the main mesenteric lymph duct (LDL) can prevent T/HS-induced lung injury. Since T/HS-induced lung injury is associated with PMN infiltration, one mechanism underlying this protective effect may be the prevention of PMN priming and activation. Therefore, we assessed the ability of T/HS to prime PMN responses to inflammatory agonists, and the ability of mesenteric lymph duct division to protect against such T/HS-induced PMN priming in an all-rat system. PMN were collected from male rats 6 h after laparotomy (trauma) plus hemorrhagic shock (30 mmHg for 90 min; T/HS) or trauma plus sham shock (T/SS). Uninstrumented rats were used as controls (UC). In a second set of experiments, rats were subjected to T/HS with or without mesenteric lymph duct division. PMN were then stimulated with chemokine (GRO, MIP-2) and lipid (PAF) chemoattractants, and cell calcium flux was used to quantify responses to those agonists. T/SS primed PMN responses to GRO, MIP-2. and PAF in comparison to UC rats, but the addition of shock (T/HS) amplified PMN priming in a significant manner, especially in response to GRO. Mesenteric lymph duct division prior to T/HS diminished PMN priming to the levels seen in T/SS. This reversal of priming was significant for GRO and GRO/MIP-2 given sequentially, with the other agonist regimens showing similar trends. The results support the concept that trauma and hemorrhagic shock play important additive roles in inflammatory PMN priming. Entry of gut-derived inflammatory products into the circulation via mesenteric lymph seems to play a dominant role in mediating the conversion of physiologic shock insults into immunoinflammatory PMN priming. Shock-induced gut lymph priming enhances PMN responses to many important chemoattractants, most notably the chemokines, and mesenteric lymph duct division effectively reverses such priming to priming levels seen in trauma without shock.     

Circulatory homeostasis in rats after bile duct ligation

The bile duct was ligated in rats, and their tolerance against a small blood loss was evaluated 7 days later. A 10% blood loss precipitated a large and sometimes fatal reduction in arterial blood pressure, while no reduction was seen in shamoperated rats. The plasma and erythrocytes were labelled by isotopes and the animals were rapidly frozen in liquid nitrogen. The splanchnic and pulmonary blood volumes were estimated from the isotope content in blood and tissue. These vascular beds will normally reduce their blood volumes during a blood loss and thus serve as vascular depots. In the bile duct occluded animals, the partition of blood is changed. More blood is to be found in the splanchnic vessels, and the depot function of the lung vessels is partly used for compensation. When these rats were bled, their liver blood volumes were not reduced, and only a small further reduction took place in the lung vessels. It is concluded that rats with bile duct occlusion will suffer considerably from small blood losses. This may be due to a lacking depot function of the splanchnic vessels.     

Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells

Objectives

Exosomes are 50–90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells.

Design and methods

For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines.

Results

Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells.

Conclusion

Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.