**2.3 Cell death studies**

## **2.3.1 Nuclear morphology**

Cell nuclei were visualized following DNA staining with the fluorescent dye DAPI (Sigma-Aldrich, Missouri, USA). Briefly, cells were seeded on cover slips in a 24-well plate, fixed in 4% formaldehyde for 10 minutes, and permeabilized with 0.5% Triton X-100. They were then rinsed with PBS and incubated with DAPI (12.5 µg/ml) for 15 minutes. Excess dye was removed. Cells imaging was performed with the 40X PL-APO 1.25 NA oil objective of a Leica-SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). DAPI was excited with a 405 nm laser-diode. Emission between 420 nm and 490 nm was collected following the manufacturer's recommendations. Six fields with ~200 cells per field were examined in each condition to estimate the percentage of nuclei with an apoptosis-like appearance.

#### **2.3.2 Nucleosomal quantification**

To evaluate DNA fragmentation in the context of apoptosis, RPTECs were incubated for 48 hours under specific conditions with the nephrotoxic compounds selected. At the end of this period RPTECs were lysed and centrifuged at 200*g* for 10 minutes to remove cell debris. DNA and histones present in the soluble fraction were quantified using an enzyme-linked immunosorbent assay (*Cell Death Detection ELISAPLUS* kit, Boehringer Mannheim, Germany), as previously described (Camano et al., 2010; Perez et al., 2004).

Novel Strategies in Drug-Induced Acute Kidney Injury 385

formaldehyde in PBS for 10 minutes before being rinsed with PBS. The nuclei were counterstained with DAPI. After washing, cells were mounted in fluorescent mounting medium (Dako North America, Inc., Carpinteria, California). Images of the distribution of cholera toxin immunolocalization across membranes were obtained with the 20X PL-APO 0.7-numerical aperture objective of a Leica-SP2 confocal microscope (Leica Microsystems).

RPTECs were incubated overnight with Gly-Phe-*p*-nitroanilide (DHP-I substrate; Sigma-Aldrich) 1mM in PBS for DHP-I activity determination or with Gly-Pro-*p*-nitroanilide (DHP-IV substrate; Sigma-Aldrich) 1 mM for DHP-IV activity determination. Both activities were measured in the presence or absence of cilastatin (200 µg/ml). *P*-Nitroanilide was quantified

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,

**3.1 Cilastatin as a broad nephroprotective drug: reduction of toxin-induced proximal** 

After 48 hours of exposure to the drugs tested, apoptosis of RPTECs measured as nucleosomal DNA fragmentation and migration from nuclei to cytosol was quantified and compared with apoptosis under the same conditions, although in the presence of cilastatin (Fig. 1). RPTECs exposed to toxins present different increases in the number of nucleosomes recovered from cytosol. Cilastatin significantly partially or totally prevented these changes

When the magnitude of cilastatin protection was plotted against the magnitude of basal cell death under every treatment tested, a clear linear trend was observed (r=0.839, p<0.0005).

We made a detailed study of the effect of 3 of these drugs: CsA, tacrolimus and paracetamol. A more selective qualitative estimation of apoptotic cell death was also obtained in adherent cells treated with CsA, tacrolimus, and paracetamol and stained with DAPI (Fig. 3). Incubation with toxins led to cell shrinkage with significant nuclear condensation, fragmentation, and formation of apoptosis-like bodies (see arrows). Cilastatin was able to reduce nuclear damage in all cases. Apoptosis-like nuclei are

None of the drugs tested differed significantly from this trend (Fig. 2).

**2.7 Dehydropeptidase I and IV activity assays** 

**2.8 Statistical analysis** 

**3. Results** 

**tubular cell death** 

paracetamol was chosen as the example.

in most of the selected drugs (Fig. 1).

quantified in Fig. 3B, C, and D.

in aliquots from supernatants by measuring at 410 nm absorbance.

#### **2.3.3 Cell viability assay**

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 triplicate.

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

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

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 at 595 nm.

#### **2.5 Cellular drug transport and accumulation**

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 results were expressed as ng drug/µg protein.

#### **2.6 Localization of lipids rafts by immunofluorescence**

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 lipid rafts.

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% formaldehyde in PBS for 10 minutes before being rinsed with PBS. The nuclei were counterstained with DAPI. After washing, cells were mounted in fluorescent mounting medium (Dako North America, Inc., Carpinteria, California). Images of the distribution of cholera toxin immunolocalization across membranes were obtained with the 20X PL-APO 0.7-numerical aperture objective of a Leica-SP2 confocal microscope (Leica Microsystems).
