Podocytes degrade endocytosed albumin primarily in lysosomes.
Podocytes degrade endocytosed albumin primarily in lysosomes.
复制标题
DOI：
10.1371/journal.pone.0099771
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Blaine J
中科院分区：
文献类型：
--
作者：
Carson JM;Okamura K;Wakashin H;McFann K;Dobrinskikh E;Kopp JB;Blaine J
白蛋白尿是慢性肾脏疾病进展的一个强有力的独立预测因子。我们推测，足细胞通过溶酶体处理白蛋白可能是足细胞损伤和损失的一个重要决定因素。人尿源性足细胞样上皮细胞（HUPEC）系用于体外实验。在用荧光素标记（FITC）白蛋白加载HUPEC后，通过Western印迹定量白蛋白摄取。通过共聚焦显微镜测定白蛋白与溶酶体的共定位。通过Western印迹定量HUPEC裂解物中的FITC-白蛋白丰度来测量白蛋白降解。使用氯喹（一种溶酶体抑制剂）或MG-132（一种蛋白酶体抑制剂）处理的HUPEC重复降解实验。通过光漂白后荧光恢复（FRAP）测量溶酶体活性。通过ELISA测量细胞因子产生。通过台盼蓝染色确定细胞死亡。在体内，用溶酶体相关膜蛋白-1（LAMP-1）对来自肾病综合征的Denys-Drash转基因小鼠模型的组织进行染色。HUPEC内吞白蛋白，其与溶酶体共定位。氯喹，而不是MG-132，抑制白蛋白降解，表明降解发生在溶酶体。通过FRAP测量的组织蛋白酶B活性在暴露于白蛋白（对照组活性的12.5%）和氯喹（12.8%）的HUPEC中显著降低，并且在暴露于白蛋白加氯喹（8.2%，p<0.05）时进一步降低。细胞因子的产生和细胞死亡显着增加HUPECs暴露于白蛋白和氯喹单独，这些影响被加强暴露于白蛋白加氯喹。与野生型小鼠相比，Denys-Drash小鼠中LAMP-1的肾小球染色显著增加，并且似乎在足细胞中最突出。这些数据表明，溶酶体参与了足细胞内吞白蛋白的加工，溶酶体功能障碍可能导致足细胞损伤和蛋白尿性疾病中的肾小球硬化。溶酶体活性修饰剂可能通过增强足细胞加工和降解白蛋白的能力，在减缓肾小球硬化进展方面具有治疗潜力。
Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.
登录
查看更多内容
DOI：
10.1097/01.asn.0000016684.07368.75
发表时间：
2002-06-01
影响因子：
13.6
作者：
Alroy, J;Sabnis, S;Kopp, JB
通讯作者：
Kopp, JB
DOI：
10.1152/ajprenal.00272.2006
发表时间：
2007-02-01
影响因子：
4.2
作者：
Eyre, Jeanette;Ioannou, Kyriakos;Topham, Peter S.
通讯作者：
Topham, Peter S.
影响因子：
13.6
作者：
Cina, Davide P.;Onay, Tuncer;Quaggin, Susan E.
通讯作者：
Quaggin, Susan E.
影响因子：
4.5
作者：
DAVIES, DJ;MESSINA, A;RYAN, GB
通讯作者：
RYAN, GB
影响因子：
30.8
作者：
Boute, N;Gribouval, O;Antignac, C
通讯作者：
Antignac, C