Tubular toxicity of cyclosporine A and the influence of endothelin-1 in renal cell culture models (LLC-PK1 and MDCK).
To evaluate the effect of cyclosporine A (CyA) at high concentrations (10(-4) and 10(-5) M) and the influence of endothelin-1 (ET-1) at physiological and pharmacological concentrations (10(-14) to 10(-6) M) on epithelial cell function, LLC-PK1 cells were studied as a model of the proximal tubule and MDCK cells as a model of the distal tubule/collecting duct. CyA caused time- and concentration-dependent acute toxicity. In LLC-PK1 cells, CyA caused a decrease in transepithelial resistance, indicating a loss of cell contacts, a release of lactate dehydrogenase (LDH) and villin into the supernatant, suggesting destruction of the apical membrane with loss of brush border, and finally release of uvomorulin, suggesting a disruption of the cell-cell adhesion, the zonula adherens. DNA synthesis, as evaluated by bromodeoxyuridine (BrdU) incorporation, was significantly affected at > or = 10(-5) M CyA. The toxicity of CyA was higher when given from the apical rather than the basolateral compartment. ET-1 alone was without effect, but in combination with CyA, ET-1 significantly enhanced toxicity. The ET-1 effect was partially inhibitable by an ET(B), but not an ET(A), antagonist. Immunofluorescence for alpha-catenin, another protein of the zonula adherens, demonstrated no change in polarity for this protein, and immunoprecipitation of the complex indicated relative stability of the zonula adherens despite loss of cadherin into the supernatant. In MDCK cells the effects were different. CyA was not associated with LDH release, but with an increase in transepithelial resistance, indicating increased paracellular resistance. Morphological alterations were significantly less, but BrdU incorporation was decreased. This pattern of toxicity is compatible with a direct toxic effect of CyA on cells of the proximal tubule, with predominant morphological destruction of the cells, with concomitant proximal tubular dysfunction, and a functional alteration in cells of the distal tubule associated with increased paracellular resistance, which may lead to solute and water loss.
Zimmerhackl
L B
LB
Universitäts-Kinderklinik, Albert-Ludwigs-Universität, Freiburg, Germany.
Mesa
H
H
Krämer
F
F
Kölmel
C
C
Wiegele
G
G
Brandis
M
M
D000818:Animals; D001973:Bromodeoxyuridine; D015820:Cadherins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016572:Cyclosporine / Q000633:toxicity*; D004247:DNA / Q000096:biosynthesis; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000502:physiology*; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D007166:Immunosuppressive Agents / Q000633:toxicity*; D007684:Kidney Tubules / Q000166:cytology* / Q000187:drug effects*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018374:LLC-PK1 Cells; D008840:Microfilament Proteins / Q000378:metabolism; D011233:Precipitin Tests; D013552:Swine
D015820:Cadherins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D007166:Immunosuppressive Agents; D008840:Microfilament Proteins; C026615:villin; D016572:Cyclosporine; D004247:DNA; D007770:L-Lactate Dehydrogenase; D001973:Bromodeoxyuridine