Separation of human urinary proximal tubular cells from familial hypercholesterolemic homozygotes by Ficoll gradient centrifugation

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Abstract
The isolation of renal proximal tubular (PT) cells from the fresh urinary sediment (UrS) of familial hypercholesterolemic (FH) homozygotes using discontinuous Ficoll gradient centrifugation was studied. For comparative purposes, cultured cells derived from normal human UrS or kidney were also studied. Unfractionated PT cells were nucleated and 90–99% of the cells excluded trypan blue. Both the unfractionated and fractionated cultured normal PT cells contained numerous empty cytoplasmic vesicles. In contrast, similar preparations from the UrS of FH homozygotes contained membrane-enclosed cytoplasmic vesicles that stained with the Papanicolaou (Pap) reagent and were strongly positive with a fluorescein-labeled antibody against lactosylceramide. The PT cells of FH homozygotes contained 2.0 to 2.5 fold higher activity of γ-glutamyltransferase and alkaline phosphatase, respectively, than the unfractionated UrS cells. We conclude that human PT cells can be separated from other UrS cells by Ficoll gradient centrifugation, and that most, if not all of the LacCer present in the UrS of FH homozygotes is associated with the PT cells. Purified PT cells should provide a useful model to test the biochemical mechanisms of LacCer accumulation in FH.