Heavy endosomes isolated from the rat renal cortex show attributes of intermicrovillar clefts

Abstract

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. A fraction containing entrapped fluorescein was isolated from a cortical homogenate after differential centrifugation and Percoll density gradient centrifugation. This fraction has been dubbed heavy endosomes. To our surprise, small-particle flow cytometry techniques demonstrated that heavy endosomes are homogeneous for entrapped fluorescein dextran and the presence of H(+)-adenosinetriphosphatase activity. The abundance of heavy endosomes, combined with the findings that true endosomal populations are identifiable in other renal cortical fractions, led us to test whether heavy endosomes had the attributes of intermicrovillar clefts. First, we tested whether heavy endosomes vesiculate in vivo or in vitro. Vesicle-by-vesicle flow cytometry analysis of uptake of fluorescein dextran added to the homogenate demonstrated that virtually all the vesicles form in vitro (99 +/- 2%, n = 4). Second, the fraction contains markers associated with intermicrovillar clefts: clathrin light chains, actin, glycoprotein gp280, and gp330, the "Heymann antigen." The presence of the brush border enzyme markers gamma-glutamyl transpeptidase and leucine aminopeptidase in> 99% of the heavy endosomes confirms that the vesicles are of apical origin. The activity of the enzymes colocalized with entrapped markers but was tenfold less than in brush-border membrane vesicles. Heavy endosomes isolated from the rat renal cortex vesiculate in vitro and contain several intermicrovillar markers.

Megalin binds and internalizes angiotensin-(1–7)

“…Purified human megalin and polyclonal antibodies against megalin were provided by Dr. Pierre J. Verroust (Institut National de la Santé et de la Recherche Medicale, Paris, France). These antibodies were raised against proteins purified by immunoaffinity chromatography using previously reported monoclonal antibodies coupled to Sepharose 4B (13,20,22,23). Antibodies were determined to be monospecific by immunoblotting on whole brush-border preparations and by immunoprecipitation of biosynthetically labeled yolk sac epithelial cells in culture.…”

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

“…A549-GFPa1 cells were incubated for 10 minutes with 50 M FSK at 37°C, washed twice with ice-cold phosphate-buffered saline (PBS), and basolateral membranes were purified according to Hammond and Verroust by using a 16% Percoll gradient (Hammond et al, 1994).…”

Megalin mediates renal uptake of heavy metal metallothionein complexes

“…Rat renal cortical brush-border membrane vesicles were isolated by magnesium precipitation techniques as described previously (3,19,39). The binding of MT was investigated in the presence of 100-to 3,300-fold dilutions of anticubilin or anti-megalin polyclonal antibodies that recognize the holoprotein (3,19,39,40).…”

“…The binding of MT was investigated in the presence of 100-to 3,300-fold dilutions of anticubilin or anti-megalin polyclonal antibodies that recognize the holoprotein (3,19,39,40). Antibodies to the AT1 receptor and anti-NK1 peptide antibodies were chosen as negative controls for nonspecific interference by binding because they bind brush-border membrane vesicles at the same titer as the anti-megalin antisera.…”