**Novel Strategies in Drug-Induced Acute Kidney Injury**

Alberto Lázaro, Sonia Camaño, Blanca Humanes and Alberto Tejedor *Renal Physiopathology Laboratory, Department of Nephrology, Hospital General Universitario Gregorio Marañón, Madrid, Spain* 

#### **1. Introduction**

380 Pharmacology

Wood, D.M., Davies, S., Puchnarewicz, M., Button, J., Archer, R. et al. (2009). Recreational

and confirmed by toxicological screening, *Clinical Toxicology,* Vol.47:7331. Wood, D.M., Greene, S.L. & Dargan, P.I. (2010b). Clinical pattern of toxicity associated with

*of Medical Toxicology*, Vol.6:327–330.

doi:10.1136/emj.2010.092288

methylmethcathinone, 4-MMC) with associated sympathomimetic toxicity, *Journal* 

use of 4-methylmethcathinone (4-MMC) presenting with sympathomimetic toxicity

the novel synthetic cathinone mephedrone, *Emergency Medical Journal,*

#### **1.1 Renal toxicity**

Renal toxicity associated with commonly prescribed drugs lengthens hospital stay, worsens prognosis, and limits the potential benefits obtained from therapy (Peracella, 2011; Servais et al., 2008).

Proximal tubule preservation is a clue in strategies aimed to prevent nephrotoxicity. The proximal tubule is a target for filtered drugs that are reabsorbed by solvent drag or pinocytosis, but also for drugs that are secreted into the luminal side.

Proximal tubules recover more than 60% of total filtered load, i.e., a single molecule of toxin that is filtered and reabsorbed will pass through the proximal tubule cell more than 50 times per day. Such a high degree of exposure implies a risk of cell damage causing a variety of clinical syndromes, from proximal acidosis and acquired Fanconi syndrome to tubular cell necrosis (Oh, 2010). This spectrum of diseases is known as acute kidney injury (AKI), which also includes cell death by apoptosis, anoikis, necrosis, or cell dysfunction (Lorz et al., 2006).

Nephrotoxicity can often be expected with certain drugs, such as vancomycin, gentamicin, foscarnet, cisplatin, cyclosporine A (CsA), and tacrolimus. Less often, the toxic effect is unexpected and not predictable, as is the case with iodinated contrast agents and paracetamol.

#### **1.2 Cell death mediation**

Intrinsic pathway–mediated apoptosis and extrinsic pathway–mediated apoptosis are both involved in toxic proximal tubule cell death (Pabla & Dong, 2008; Servais et al., 2008; Xiao et al., 2011). With most of toxins, cell death is followed by detachment and anoikis. Paracetamol is a notable exception to this behavior. Caspases activation, mitochondrial depolarization, release of cytochrome C from mitochondria, cell membrane modification, and nucleosome formation are all hallmarks of apoptosis that are regularly observed in toxin-damaged proximal tubules (Camano et al., 2010). Nitric oxide, soluble oxygen radicals, and proinflammatory cytokines are released by damaged proximal tubules, thus amplifying the lesion.

Novel Strategies in Drug-Induced Acute Kidney Injury 383

Novartis Farmaceutica S.A., Spain), tacrolimus (Prograf®, Fujisawa S.A., Spain), and paracetamol (Perfalgan, Bristol Myers Squibb). The concentrations used were similar to the

Crystalline cilastatin was provided by Merck Sharp & Dohme S.A. (Madrid, Spain). A dose of 200 µg/ml was chosen, because it is cytoprotective and falls within the reference range for

All drug dilutions were performed with sterile culture medium and cilastatin, and the tested

Porcine RPTECs were obtained as previously described (Camano et al., 2010; Perez et al., 2004). Briefly, the cortex was sliced and incubated for 30 minutes at 37°C with 0.6 mg/ml of collagenase A (Boehringer Mannheim, Germany) in Ham's F-12 medium. Digested tissue was then filtered through a metal mesh (250 µm), washed 3 times with Ham's F-12 medium, and centrifuged using an isotonic Percoll gradient (45% [v/v]) at 20,000*g* for 30 minutes. Proximal tubules were recovered from the deepest fraction, washed, and resuspended in supplemented DMEM/Ham's F-12 at a 1:1 ratio (with 25 mM HEPES, 3.7 mg/ml sodium bicarbonate, 2.5 mM glutamine, 1% non-essential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, 5 x 10–8 M hydrocortisone, 5 mg/ml insulin-transferrin-sodium selenite media supplement, and 2% fetal bovine serum). Proximal tubules were seeded at a density of 0.66 mg/ml and incubated at 37°C in a 95% air/5% CO2 atmosphere. Culture medium was renewed every 2

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

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),

pharmacologically active recommended plasma level.

clinical use (Camano et al., 2010; Perez et al., 2004).

**2.2 Primary cultures of renal proximal tubule epithelial cells** 

days. RPTECs were used after they had reached confluence (80%).

as previously described (Camano et al., 2010; Perez et al., 2004).

drugs were added simultaneously.

**2.3 Cell death studies** 

appearance.

**2.3.1 Nuclear morphology** 

**2.3.2 Nucleosomal quantification** 

#### **1.3 Nephrotoxicity prevention strategies**

Overhydration is the most common maneuver to prevent toxic concentrations in urine and, consequently, inside the cell. However, nephrotoxicity usually requires dose adjustment or drug withdrawal, thus limiting effectiveness.

Other strategies aimed at inhibiting cell drug transport or interfering with mediation of apoptosis also tend to interfere with the therapeutic targets and, consequently, limit the effectiveness of therapy (Pabla & Dong, 2008; Servais et al., 2008).

During the last 5 years, our work on nephrotoxicity has enabled us to better understand the role of proximal tubule behavior in the adaptation of the kidney to toxic aggressions (Camano et al., 2010; Camaño-Paez et al., 2008; Neria et al., 2009; Perez et al., 2004; Tejedor et al., 2007).

Therefore, not surprisingly, the search for alternative protective strategies against toxic damage to the proximal tubule is an important area of investigation today.

#### **1.4 Ability of cilastatin to prevent drug toxicity targeting the proximal tubule**

Cilastatin is an inhibitor of brush border dehydropeptidase I (DHP-I), which is present in renal proximal tubular epithelial cells (RPTECs). It was initially designed to inhibit hydrolysis and uptake of the carbapenem antibiotic imipenem, thus enabling it to be more easily recovered from urine (Birnbaum et al., 1985; Norbby et al., 1983). However, cilastatin is also able to inhibit uptake of CsA and cisplatin by RPTECs by decreasing in a dosedependent way the toxic effect of CsA and cisplatin on RPTECs (Camano et al., 2010; Perez et al., 2004). Clinical studies also support this protective role of cilastatin against CsAinduced nephrotoxicity (Carmellini et al., 1997, 1998; Gruss et al., 1996; Markewitz et al., 1994; Mraz et al., 1987, 1992; Tejedor et al., 2007). Experimental evidence suggests that cilastatin binding to brush border DHP-I could interact with apical cholesterol lipid rafts (Camano et al., 2010; Perez et al., 2004; Tejedor et al., 2007).

The aim of this brief report is to determine whether cilastatin is able to interfere with the direct toxic effect of several known nephrotoxic drugs on cultured RPTECs. We investigated the effect of cilastatin on the toxicity of gentamicin, vancomycin, iodinated contrast agent, amphotericin B, foscarnet, cisplatin, mannitol, chloroform, paracetamol, CsA and tacrolimus.

We describe for the first time the effects of a drug that specifically targets the renal proximal tubule brush border and seems to be able to reduce accumulation and toxicity of the main nephrotoxic drugs by inhibiting internalization of brush border–bound lipid rafts.

#### **2. Methods**

#### **2.1 Drugs**

We used commercially available parenteral formulations of gentamicin (powder, Guinama, Alboraya, Spain), vancomycin (powder, Combino Pharm, Barcelona, Spain), iodinate contrast agent (iopamidol, Laboratorios Farmacéuticos Rovi, Madrid, Spain), amphotericin B (Bristol Myers Squibb, Madrid, Spain), foscarnet (Foscavir, AstraZeneca, Madrid, Spain), cisplatin (Pharmacia, Barcelona, Spain), mannitol 20%, (Osmofundin®, Braun Medical S.A., Barcelona, Spain), chloroform (Scharlau, Barcelona, Spain), CsA (Sandimmun Neoral®,

Overhydration is the most common maneuver to prevent toxic concentrations in urine and, consequently, inside the cell. However, nephrotoxicity usually requires dose adjustment or

Other strategies aimed at inhibiting cell drug transport or interfering with mediation of apoptosis also tend to interfere with the therapeutic targets and, consequently, limit the

During the last 5 years, our work on nephrotoxicity has enabled us to better understand the role of proximal tubule behavior in the adaptation of the kidney to toxic aggressions (Camano et al., 2010; Camaño-Paez et al., 2008; Neria et al., 2009; Perez et al., 2004; Tejedor et

Therefore, not surprisingly, the search for alternative protective strategies against toxic

Cilastatin is an inhibitor of brush border dehydropeptidase I (DHP-I), which is present in renal proximal tubular epithelial cells (RPTECs). It was initially designed to inhibit hydrolysis and uptake of the carbapenem antibiotic imipenem, thus enabling it to be more easily recovered from urine (Birnbaum et al., 1985; Norbby et al., 1983). However, cilastatin is also able to inhibit uptake of CsA and cisplatin by RPTECs by decreasing in a dosedependent way the toxic effect of CsA and cisplatin on RPTECs (Camano et al., 2010; Perez et al., 2004). Clinical studies also support this protective role of cilastatin against CsAinduced nephrotoxicity (Carmellini et al., 1997, 1998; Gruss et al., 1996; Markewitz et al., 1994; Mraz et al., 1987, 1992; Tejedor et al., 2007). Experimental evidence suggests that cilastatin binding to brush border DHP-I could interact with apical cholesterol lipid rafts

The aim of this brief report is to determine whether cilastatin is able to interfere with the direct toxic effect of several known nephrotoxic drugs on cultured RPTECs. We investigated the effect of cilastatin on the toxicity of gentamicin, vancomycin, iodinated contrast agent, amphotericin B, foscarnet, cisplatin, mannitol, chloroform, paracetamol, CsA and tacrolimus. We describe for the first time the effects of a drug that specifically targets the renal proximal tubule brush border and seems to be able to reduce accumulation and toxicity of the main

We used commercially available parenteral formulations of gentamicin (powder, Guinama, Alboraya, Spain), vancomycin (powder, Combino Pharm, Barcelona, Spain), iodinate contrast agent (iopamidol, Laboratorios Farmacéuticos Rovi, Madrid, Spain), amphotericin B (Bristol Myers Squibb, Madrid, Spain), foscarnet (Foscavir, AstraZeneca, Madrid, Spain), cisplatin (Pharmacia, Barcelona, Spain), mannitol 20%, (Osmofundin®, Braun Medical S.A., Barcelona, Spain), chloroform (Scharlau, Barcelona, Spain), CsA (Sandimmun Neoral®,

nephrotoxic drugs by inhibiting internalization of brush border–bound lipid rafts.

**1.3 Nephrotoxicity prevention strategies** 

drug withdrawal, thus limiting effectiveness.

al., 2007).

**2. Methods 2.1 Drugs** 

effectiveness of therapy (Pabla & Dong, 2008; Servais et al., 2008).

(Camano et al., 2010; Perez et al., 2004; Tejedor et al., 2007).

damage to the proximal tubule is an important area of investigation today.

**1.4 Ability of cilastatin to prevent drug toxicity targeting the proximal tubule** 

Novartis Farmaceutica S.A., Spain), tacrolimus (Prograf®, Fujisawa S.A., Spain), and paracetamol (Perfalgan, Bristol Myers Squibb). The concentrations used were similar to the pharmacologically active recommended plasma level.

Crystalline cilastatin was provided by Merck Sharp & Dohme S.A. (Madrid, Spain). A dose of 200 µg/ml was chosen, because it is cytoprotective and falls within the reference range for clinical use (Camano et al., 2010; Perez et al., 2004).

All drug dilutions were performed with sterile culture medium and cilastatin, and the tested drugs were added simultaneously.

#### **2.2 Primary cultures of renal proximal tubule epithelial cells**

Porcine RPTECs were obtained as previously described (Camano et al., 2010; Perez et al., 2004). Briefly, the cortex was sliced and incubated for 30 minutes at 37°C with 0.6 mg/ml of collagenase A (Boehringer Mannheim, Germany) in Ham's F-12 medium. Digested tissue was then filtered through a metal mesh (250 µm), washed 3 times with Ham's F-12 medium, and centrifuged using an isotonic Percoll gradient (45% [v/v]) at 20,000*g* for 30 minutes. Proximal tubules were recovered from the deepest fraction, washed, and resuspended in supplemented DMEM/Ham's F-12 at a 1:1 ratio (with 25 mM HEPES, 3.7 mg/ml sodium bicarbonate, 2.5 mM glutamine, 1% non-essential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, 5 x 10–8 M hydrocortisone, 5 mg/ml insulin-transferrin-sodium selenite media supplement, and 2% fetal bovine serum). Proximal tubules were seeded at a density of 0.66 mg/ml and incubated at 37°C in a 95% air/5% CO2 atmosphere. Culture medium was renewed every 2 days. RPTECs were used after they had reached confluence (80%).
