Laboratory Investigation

Kidney International (1981) 20, 71–77; doi:10.1038/ki.1981.106

Analytical cell fractionation of isolated rabbit renal proximal tubules

J Thomas Hjelle1, Jean-Paul Morin1 and André Trouet1

1International Institute of Cellular and Molecular Pathology, University Catholic de Louvain, Brussels, Belgium

Correspondence: Dr J T Hjelle, Peoria School of Medicine, 123 S.W. Glendale Ave., Peoria, Illinois 61605, USA

Received 5 June 1980; Revised 14 November 1980.

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Abstract

Analytical cell fractionation of isolated rabbit renal proximal tubules. Proximal tubules were isolated in highly pure form from rabbit cortices by a mechanical procedure that is known to preserve the structural and metabolic aspects of the tubular cells. Postnuclear supernates prepared from the isolated tubules were subjected to isopycnic centrifugation in linear sucrose gradients. The enzyme activities associated with the plasma membrane (gamma-glutamyl transpeptidase, amino-peptidase M, alkaline phosphatase, Na-K-ATPase, and phosphodiesterase I) exhibited sharp unimodal frequency-density profiles with a median density near 1.16 g/ml, which shifted to a heavier density when treated with digitonin. The lysosomal enzymes, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and cathepsin B, and the peroxisomal enzyme catalase exhibited particle-associated activity near a density of 1.22 g/ml. Disruption of these particles by freezing and thawing resulted in these activities appearing in the rho = 1.10 g/ml region of the gradient where the soluble cytosolic enzyme, phosphoglucomutase, exhibited activity. Cytochrome oxidase activity typical of mitochondria gave a sharp unimodal profile at rho = 1.18 g/ml. Microsomal glucose-6-phosphatase and NADPH: cytochrome c reductase activities gave median densities near 1.16 g/ml, which did not change after incubation with digitonin. Galactosyl transferase activity gave a skewed profile at rho = 1.16 g/ml and showed a slight shift to heavier density after digitonin. This study of the enzyme activities and density gradient distribution of the components of the proximal tubule cells provides the methodology for the further study of the cellular processing of endogenous and exogenous substances by this vital cell type.

Fractionnement cellulaire analytique de tubes proximaux isolés de lapin. Des tubes proximaux ont été isolés en une préparation hautement purifiée à partir de cortex de lapin par un procédé mécanique qui préserve les caractéristiques structurales et métaboliques des cellules tubulaires. Les surnageants préparés à partir des tubes isolés ont été soumis à une centrifugation isopycnique dans des gradients linéaires de sucrose. Les activités enzymatiques associées à la membrane plasmique, gamma-glutamyl transpeptidase, aminopeptidase M, phosphatase alcaline, Na-K-ATPase et phosphodiestérase I ont mis en évidence des profils unimodaux de fréquence de densité avec une densité médiane proche de 1,16 g/ml qui s'élève après traitement par la digitonine. Les enzymes lysosomiaux, N-acétyl-beta-glucosaminidase, alpha-mannosidase et cathepsine B et l'enzyme peroxysomale, la catalase, avaient une activité associée à la fraction particulaire près d'une densité de 1,22 g/ ml. La destruction des particules par congélation et décongélation a eu pour résultat l'apparition de ces activités dans la région de rho = 1,10 g/ml du gradient où l'enzyme soluble, la phosphoglucomutase, a son activité. L'activité cytochrome oxydase typique de la mitochondrie avait un profil unimodal à rho = 1,18 g/ml. Les activités de la glucose-6-phosphatase microsomale et de la NADPH cytochrome c réductase donnaient des densités médianes proches de 1,16 g/ml, non modifiées par l'incubation avec la digitonine. L'activité de la galactosyl transférase avait un profil non aigu à rho = 1,16 g/ml avec un faible déplacement vers des densités plus élevées par la digitonine. Cette étude des activités enzymatiques et de la distribution selon le gradient de densité des composants du tube proximal fournit une méthodologie pour l'étude ultérieure de ce type cellulaire.

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