Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Histochem J. 2002 Jan-Feb;34(1-2):27-33. doi: 10.1023/a:1021391724643.

Abstract

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin-Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24 h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction. The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24 h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Phosphatase / analysis
  • Acid Phosphatase / metabolism
  • Alkaline Phosphatase / analysis
  • Alkaline Phosphatase / metabolism
  • Animals
  • Cell Line
  • Cell Survival / drug effects
  • Cyclosporine / toxicity*
  • Dogs
  • Immunohistochemistry
  • Immunosuppressive Agents / toxicity*
  • Kidney Tubules / cytology
  • Kidney Tubules / drug effects*
  • Kidney Tubules / enzymology
  • L-Lactate Dehydrogenase / analysis
  • L-Lactate Dehydrogenase / metabolism
  • LLC-PK1 Cells
  • Succinate Dehydrogenase / analysis
  • Succinate Dehydrogenase / metabolism
  • Swine
  • Trypan Blue / chemistry

Substances

  • Immunosuppressive Agents
  • Cyclosporine
  • L-Lactate Dehydrogenase
  • Succinate Dehydrogenase
  • Alkaline Phosphatase
  • Acid Phosphatase
  • Trypan Blue