Evaluation of P-glycoprotein activity and expression in primary cultured human renal tubular cells : effect of longer-term exposure to cyclosporin

Abstract

Cyclosporin A has had the greatest effect on renal transplant survival, by reducing the incidence of acute rejection.  There is still a significant long-term attrition rate, presumed to be a result of cyclosporine toxicity.  The mechanisms of this are not fully understood, but seem to relate to intra-renal cellular accumulation of cyclosporin.

To investigate this chronic accumulation in vitro, human cells in culture need to be exposed to cyclosporine over a period of weeks.

The first part of this thesis describes a model for quiescent culture of primary human renal tubular epithelial cells, which maintain viability and phenotypes as close to normal as possible for at least 6 weeks.  Characterisation by light and electron microscopy, enzyme activity and epitope expression by immunofluorescence and flow cytometry are described.

Renal tubular cells, among others, possess a transmembrane protein called P-glycoprotein, whose physiological role is not fully understood, but which is involved in effluxing (potentially toxic) hydrophobic macromolecules from the cell cytoplasm.  One such macromolecule is cyclosporin.

The second part of the thesis describes the function of P-glycoprotein in human primary cultured renal tubular epithelial cells in normal fully defined growth medium, the retention of normal function in quiescent cells for up to 6 weeks in culture, and then investigates the effects of co-incubation with cyclosporine for at least 3 weeks.  Incubation of quiescent cells with higher pharmacological cyclosporine doses impairs the cellular character.  Incubation with lower pharmacological concentrations of cyclosporine increases the efflux activity of P-glycoprotein, as measured by a fluorescent substrate, but does not affect the membrane expression of the protein. 

This is the first time that (i) such a quiescence model has been described, (ii) P-glycoprotein has been investigated in primary cultured human renal epithelial cells, and (iii) the effect of prolonged exposure to cyclosporine in culture has been studied.

More information

Identifiers

Catalogue record

Export record

ASCII CitationAtomBibTeXData Cite XMLDublin CoreDublin CoreEP3 XMLEndNoteHTML CitationHTML CitationHTML ListJSONMETSMODSMPEG-21 DIDLOpenURL ContextObjectOpenURL ContextObject in SpanRDF+N-TriplesRDF+N3RDF+XMLRIOXX2 XMLReferReference ManagerSimple Metadata