Hammond T G, Verroust P J
University of Wisconsin Hospitals and Clinics, Madison.
Am J Physiol. 1994 Jun;266(6 Pt 1):C1783-94. doi: 10.1152/ajpcell.1994.266.6.C1783.
The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Using Percoll gradient centrifugation, we isolated and characterized a previously undetected renal cortical endosomal fraction slightly lighter than basolateral membranes. Assay of entrapped dextran on a vesicle-by-vesicle basis using small-particle flow cytometry techniques demonstrates homogeneity for entrapped dextran. Flow cytometry colocalization of entrapped markers with brush-border enzymes in > 99% of the vesicles and the absence of Na-K-adenosinetriphosphatase (ATPase) suggest both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Furthermore, two glycoproteins derived from intermicrovillar clefts are detectable in this fraction. Ultrastructurally the vesicles are heterogeneous, consisting of multivesicular bodies together with vesicles of diverse size and coating. Populations of vesicles can be cleanly separated from each other and basolateral membranes according to their surface charge by high-resolution free-flow electrophoresis. Multiparameter flow cytometry analysis demonstrates that a more abundant population of smaller vesicles has brisker H(+)-ATPase activity, whereas a less abundant population of larger vesicles has slower H(+)-ATPase activity. In contrast, brush-border membrane vesicles contained no entrapped markers and lacked H(+)-ATPase activity. Analysis of vesicles prepared after addition of dextran to the homogenate confirms that the vesicles form in vivo. Hence a heterogeneous renal cortical endosomal population is of apical origin.
通过静脉输注足够小的荧光葡聚糖来标记大鼠肾皮质的内体途径，该荧光葡聚糖能够穿过肾小球超滤屏障，并被近端小管的管腔胞吞作用摄取。使用Percoll梯度离心法，我们分离并鉴定了一种先前未检测到的肾皮质内体组分，其比重略低于基底外侧膜。使用小颗粒流式细胞术技术逐个囊泡地测定包裹的葡聚糖，结果表明包裹的葡聚糖具有均一性。在超过99%的囊泡中，包裹的标记物与刷状缘酶的流式细胞术共定位以及钠钾-三磷酸腺苷酶（ATP酶）的缺失表明，这些囊泡均起源于顶端，并且顶端酶会运输到内体成分中。此外，在该组分中可检测到两种源自微绒毛间裂的糖蛋白。在超微结构上，这些囊泡是异质性的，由多囊泡体以及大小和包被各异的囊泡组成。通过高分辨率自由流动电泳，可根据囊泡的表面电荷将不同群体的囊泡彼此以及与基底外侧膜清晰地分离。多参数流式细胞术分析表明，数量较多的较小囊泡群体具有更活跃的H(+) - ATP酶活性，而数量较少的较大囊泡群体具有较慢的H(+) - ATP酶活性。相比之下，刷状缘膜囊泡不包含包裹的标记物且缺乏H(+) - ATP酶活性。对向匀浆中添加葡聚糖后制备的囊泡进行分析证实，这些囊泡在体内形成。因此，异质性的肾皮质内体群体起源于顶端。
