Select de novo gene and protein expression during renal epithelial cell culture in rotating wall vessels is shear stress dependent.

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.

Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.

Gentamicin inhibits rat renal cortical homotypic endosomal fusion: role of megalin.

Megalin, a giant glycoprotein receptor heavily concentrated in the early endosomal pathway of renal proximal tubular cells, binds gentamicin with high affinity and delivers the drug to lysosomes. Utilizing an in vitro reconstitution assay we tested whether gentamicin-induced vacuolation is associated with inhibition of early endosomal fusion, as well as whether megalin plays a role in mediating these effects. Pretreatment of rats with gentamicin inhibited rat renal proximal tubular homotypic endosomal fusion. Administered simultaneously, gentamicin and polymers of polyaspartic acid, which protect against the hemodynamic effects of gentamicin nephrotoxicity, had no net effect on fusion. Polyaspartic acid alone had no effect on fusion. Antisera to the tail of the megalin/gentamicin receptor inhibited fusion, whereas non-specific controls had no effect. Peptides matching homologous NPXY repeat sequence motifs in the cytosolic tail stimulated endosomal fusion, whereas reverse sequence control peptides had no effect. These data suggest that gentamicin inhibition of endosomal fusion in the renal proximal tubule is a damage mechanism mediated by specific peptide sequences in the cytosolic tail of the giant gentamicin-binding receptor megalin and that receptors can effect the fusion properties of membranes in which they reside.

Rat kidney papilla contains abundant synaptobrevin protein that participates in the fusion of antidiuretic hormone-regulated water channel-containing endosomes in vitro.

Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP-2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti-synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones.

Energy transfer assays of rat renal cortical endosomal fusion: evidence for superfusion.

The complex of components necessary to allow endosomal fusion includes both membrane-bound receptors and several soluble proteins. Although these factors have been isolated from cultured cell lines, and endosomal fusion has been reconstituted in vitro for vesicular systems from yeast to synaptosomes, there is a paucity of data from mammalian systems. To investigate fusion in rat renal cortical endosomes, we began by developing a fusion assay. As the immunoglobulin and avidin-based probes almost universally employed in fusion assays are excluded by the glomerular ultrafiltration barrier, it was necessary to begin by finding ultrafilterable probes which could serve as a fusion assay. We labeled the apical endosomal pathway of the renal proximal tubule by intravenous infusion of ultrafilterable fluorescent dextrans. Energy transfer from entrapped fluorescein-dextran to rhodamine-dextran had a narrow concentration dependence but allowed fluorometric assay of endosomal fusion. The "spectroscopic ruler" property of energy transfer, whereby it will only occur at < 60 A, makes fusion measurements unequivocal. The energy transfer efficiency of fluorometric (48 +/- 1%) and flow cytometry (57 +/- 1%) assays were close to the theoretical optimum (57%). Energy transfer is detected as a decrease in fluorescence of the fluorescein donor and an increase in fluorescence of the rhodamine acceptor. Our endosomal fusion assay was utilized to determine the optimal conditions for fusion of rat renal cortical light endosomes and heavy endosomes. Independent measurements of fluorescein-dextran and rhodamine-dextran on an endosome-by-endosome basis using dual-beam two-color flow cytometry demonstrated that each fusion event involves multiple endosomes rather than a single pair of endosomes. Electron microscopy analysis demonstrated that the average vesicle diameter was five times larger in the fused heavy endosomal fractions compared with control fractions without fusion. Hence, fusion of mammalian renal cortical endosomes reconstituted in vitro is consistent with multiple fusion events dubbed superfusion.

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

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.

Isolation and characterization of renal cortical membranes using an aqueous two-phase partition technique.

The aqueous two-phase partition technique is a simple, rapid and inexpensive method for the fractionation of membrane preparations. Aqueous two-phase partitioning separates according to surface properties such as charge and hydrophobicity, making it complementary to established centrifugation techniques, which separate on the basis of density. Although aqueous two-phase partitioning has been successfully applied to animal tissues, there are limited data on the functional properties of the isolated membranes. We have applied the aqueous two-phase partition technique to rat renal brush-border membrane vesicles and sheets. Our aim was to remove organelle contamination while maintaining the functional properties of the membranes. Evidence from marker enzyme analysis and electron microscopy supports the conclusion that renal brush-border membranes are fractionated separate from the mitochondria and endoplasmic reticulum. This separation procedure did not alter the Na(+)-dependent transport of brush-border membrane vesicles. Na(+)-D-glucose symporter and Na(+)-H+ antiporter activity in the fractionated preparation increased to the same extent as did the enrichment of enzyme markers for brush-border membranes.

Forward scatter pulse width signals resolve multiple populations of endosomes.

The technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification. To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H(+)-ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction. Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations.