**2.8 Statistical analysis**

384 Pharmacology

The cell survival assay relies on the capacity of cells to reduce 3-(4, 5- dimethylthiazol-2 yl)-2, 5-diphenyltetrazolium bromide (MTT) (Calbiochem, California, USA) to colored formazan in metabolically active cells. RPTECs were seeded onto 96-well plates and incubated with toxins alone or in combination with cilastatin. Twenty-four hours later, 0.5 mg/ml of MTT was added, plates were incubated for 3 hours in the dark at 37ºC, and 100 µL of 50% dimethylformamide in 20% SDS (pH 4.7) was added. Plates were incubated at 37ºC overnight, and absorbance was measured at 595 nm. All assays were performed in

Alternatively, MTT assays were performed in real time, following MTT reduction on single cells, with an Olympus IX70 inverted microscope fitted to a spectrofluorometer SLM

RPTECs were treated for 24 hours with CsA, tacrolimus or paracetamol in the presence or absence of cilastatin (200 µg/ml). Adherent cells were washed in saline serum, harvested with trypsin-EDTA, seeded in Petri dishes (100 mm), and cultured for 7 days in drug-free complete medium. Surviving adherent cells were fixed for 5 minutes with 5% paraformaldehyde/PBS and stained with 0.5% crystal violet/20% methanol for 2 minutes. Excess dye was rinsed with PBS. Finally, the intracellular dye was eluted with 50% ethanol/50% sodium citrate 0.1 M (pH 4.2) and quantified by spectrometry

RPTECs incubated for 24 hours with increasing concentrations of CsA, tacrolimus or paracetamol in the presence or absence of cilastatin (200 µg/ml), were scraped and lysed in 400 µL of lysis buffer at 70ºC (2.22% [w/v] SDS; 19.33 % [v/v] glycerol [87% v/v]; 790 mM Tris HCl pH 6.8 in dH2O, phenylmethylsulfonyl fluoride, and protease inhibitors). Cell lysates were heated at 100ºC for 5 minutes, homogenized in ice, and centrifuged at 12,000*g* for 5 minutes at 4ºC. The supernatant was analyzed for total protein content and the presence of nephrotoxins. The concentrations of CsA, tacrolimus and paracetamol were measured using fluorescence polarization immunoassay technology on a TDX Chemistry Analyzer (Abbot Laboratories, USA) in accordance with the instructions provided by the manufacturer. The calibrators and controls supplied with each kit were applied, and the

To study the interaction of cilastatin with cholesterol lipid rafts, we used FITC-conjugated cholera toxin B (Molecular Probes, Oregon, USA), as its internalization is mediated by

RPTECs cultured on glass coverslips were preincubated with culture medium alone or cilastatin 200 µg/ml for 15 minutes. The cells were then incubated with 10 µg/ml FITClabelled cholera toxin B for 1 and 2.5 hours. Cells were washed with PBS and fixed in 4%

AMINCO 2000. MTT was measured by reading cell absorbance at 570 nm.

**2.4 Cell viability: Quantification of colony-forming units** 

**2.5 Cellular drug transport and accumulation** 

results were expressed as ng drug/µg protein.

**2.6 Localization of lipids rafts by immunofluorescence** 

**2.3.3 Cell viability assay** 

triplicate.

at 595 nm.

lipid rafts.

Quantitative variables were expressed as the mean ± standard error of the mean (SEM). Differences were considered statistically significant for bilateral alpha values less than 0.05. Factorial ANOVA was used when more than 1 factor was considered. When a single factor presented more than 2 levels and the model showed significant differences between factors, a post-hoc analysis (least significant difference) was performed. When results are shown, they represent a minimum of at least 3 repeats. When possible, a quantification technique (e.g. dye recovery) was used to illustrate reproducibility. When figures illustrated an effect, paracetamol was chosen as the example.
