Experimental Cell Line Models for Nephrotoxicity Screening

The aim of the study was to review literature data on cell models for experimental assessment of drug nephrotoxicity in vitro. Because of nephrotoxicity, 2% of new investigational medicinal products are discarded at the stage of preclinical in vivo studies in laboratory animals, and 19%—after phase 3 clinical trials. Prediction of toxicity in cell models could make drug development more cost-effective and help to reduce/avoid animal testing. At present, there are no official international guidelines for assessment of nephrotoxicity in vitro, but there is a lot of research underway. The main toxicity target in kidneys is renal proximal tubule epithelial cells, therefore the main research is focused on the development of renal proximal tubule epithelial cell lines with stable functional characteristics. Another important aspect in nephrotoxicity modeling is the choice of relevant test methods and end points which would reflect potential toxicity mechanisms. The paper reviews existing human renal proximal tubule epithelial cell lines and current test methods for assessing cytotoxicity. Promising areas for future development of cell models for nephrotoxicity assessment— are optimisation and standardisation of in vitro systems that would help to make preclinical predictions of drug nephrotoxicity in vivo.

ACTIVITIES OF PROXIMAL TUBULE ENZYMES AND ALBUMIN CONCENTRATION IN URINE OF CHILDREN TREATED WITH METHOTREXATE

In order to study methotrexate nephrotoxicity, the activities of proximal tubule epithelial cell membrane enzymes: alanine aminopeptidase (AAP) and gamma-glutamyltransferase (GGT), as well as of lysosomal N-acetyl-beta-D-glucosaminidase (NAG) and urinary albumin concentrations were determined in 12-h-urine samples of 30 patients with lymphoblastomous leukemia. The patients were i.v. receiving 4 individual methotrexate doses of 2000 mg/m2 every 15 days followed by leucovorin as a protector. Control and methotrexate-treated group, each consisting of 30 examinees, included 4–10 years old children of both sexes. Statistically significant increase of AAP and GGT activities, expressed as U/mmol creatinine was observed after the first two (p 0.05), as well as after the remaining two therapies (p 0.01) in relation to the control. Enzymatic activities of these two enzymes decreased to control value before the second and the third methotrexate application, but they increased again after the third application and remained elevated up to the end of experiments. Significant increase of NAG activity expressed as U/mmol creatinine (p 0.01), as well as urinary albumin levels (mg/mmol creatinine; p 0.01) were registered after the third methotrexate therapy and this elevation of the same statistical significance of the differences remained stable till the end of the therapy. Based on these results it can be concluded that during the time period of two first applications nephrotoxic methotrexate action is reversible and at the level of proximal tubule epithelial cell membranes. During the last two applications impairment is irreversible and at the level of cell organelles and glomerular filtration.

ALKALINE PHOSPHATASE ENZYME AND LACTATE DEHYDROGENASE ACTIVITY IN URINE OF PATIENTS TREATED WITH METHOTREXATE

In order study methotrexate nephrotoxicity, the activities of proximal tubule epithelial cell membrane enzymes: alkaline phosphatase (AP) and lactate dehydrogenase (LDH) were determined in 12-h-urine samples of 30 patients with lymphoblastomous leukemia. The patients were i.v. receiving 4 individual methotrexate doses of 2000 mg/m2 every 15 days followed by leucovorin as a protector. Control and methotrexate-treated group, each consisting of 30 examinees, included 4-10 years old children of both sexes. Statistically significant increase of AP and LDH activities, expressed as unuts/mmol creatinine was observed after the second therapy (p0.05) in relation to the control. Based on these results it can concluded that nephrotoxic methotrexate action is ireversible during the time period after the second applications at the level of proximal tubule epithelal cell.