**3. Results**

#### **3.1 Cilastatin as a broad nephroprotective drug: reduction of toxin-induced proximal tubular cell death**

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

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

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

Novel Strategies in Drug-Induced Acute Kidney Injury 387

Fig. 3. Effects of cilastatin on the nuclear morphology of renal proximal tubular epithelial cells (RPTECs) during treatment with toxins. RPTECs were cultured in the presence of paracetamol (30, 300 and 600 µg/ml), cyclosporine (CsA, 10, 100, and 1000 ng/ml) and tacrolimus (5, 50, and 500 ng/ml) with or without cilastatin (200 µg/ml) for 24 hours. A, Example of nuclear staining with DAPI to determine whether an apoptotic-like nuclear morphology was present. Arrows point to fragmented, apoptotic nuclei. B, C and D, Quantitative approach to staining for paracetamol, CsA and tacrolimus, respectively. Data are represented as the mean ± SEM of at least 3 separate experiments. ANOVA models

We quantified the functional impact of CsA, tacrolimus and paracetamol treatments on cell survival by measuring the percentage of adherent cells still able to reduce MTT to formazan after exposure to increasing doses of toxins. After 24 hours of incubation with toxins, the amount of surviving cells able to reduce MTT decreases progressively as the concentrations of CsA, tacrolimus, and paracetamol increase. However, in the presence of cilastatin, all surviving cells keep their capacity to reduce MTT (Fig. 4). Cilastatin is able to counteract

p<0.0001. \* cilastatin effect, p<0.05; dose effect, p<0.05.

both the structural and functional damage induced by toxins.

Fig. 1. Effect of cilastatin on nephrotoxin-induced apoptosis. Renal proximal tubular epithelial cells were exposed to CsA (1 µg/ml), gentamicin (20 mg/ml), tacrolimus (50 ng/ml), vancomycin (600 µg/ml), cisplatin (10 µM), iodinated contrast (1 mg/ml), foscarnet (1 mM), mannitol (100 mosm/l), amphotericin B (10 µg/ml), chloroform (100 µg/ml), and paracetamol (300 µg/ml) with and without cilastatin (200 µg/ml) for 48 hours. Oligonucleosomal DNA fragmentation was detected by ELISA. Data are represented as the mean ± SEM of at least 3 separate experiments. ANOVA model: p<0.0001. \*p<0.05 vs. same data with cilastatin.

Fig. 2. Reduction in nucleosomal enrichment induced by cilastatin over basal nucleosomal enrichment induced by each toxin. Individual experimental data are provided. There is a common trend for all the data, suggesting a common behavior, with cilastatin protection being proportional to basal damage. Linear regression of "cilastatin-induced reduction" vs. "basal nucleosomal enrichment", slope=0.82, r=0.839, adjusted r2=0.695, p<0.0005.

Fig. 1. Effect of cilastatin on nephrotoxin-induced apoptosis. Renal proximal tubular epithelial cells were exposed to CsA (1 µg/ml), gentamicin (20 mg/ml), tacrolimus (50 ng/ml), vancomycin (600 µg/ml), cisplatin (10 µM), iodinated contrast (1 mg/ml), foscarnet (1 mM), mannitol (100 mosm/l), amphotericin B (10 µg/ml), chloroform (100 µg/ml), and

Oligonucleosomal DNA fragmentation was detected by ELISA. Data are represented as the mean ± SEM of at least 3 separate experiments. ANOVA model: p<0.0001. \*p<0.05 vs. same

Fig. 2. Reduction in nucleosomal enrichment induced by cilastatin over basal nucleosomal enrichment induced by each toxin. Individual experimental data are provided. There is a common trend for all the data, suggesting a common behavior, with cilastatin protection being proportional to basal damage. Linear regression of "cilastatin-induced reduction" vs.

"basal nucleosomal enrichment", slope=0.82, r=0.839, adjusted r2=0.695, p<0.0005.

paracetamol (300 µg/ml) with and without cilastatin (200 µg/ml) for 48 hours.

data with cilastatin.

Fig. 3. Effects of cilastatin on the nuclear morphology of renal proximal tubular epithelial cells (RPTECs) during treatment with toxins. RPTECs were cultured in the presence of paracetamol (30, 300 and 600 µg/ml), cyclosporine (CsA, 10, 100, and 1000 ng/ml) and tacrolimus (5, 50, and 500 ng/ml) with or without cilastatin (200 µg/ml) for 24 hours. A, Example of nuclear staining with DAPI to determine whether an apoptotic-like nuclear morphology was present. Arrows point to fragmented, apoptotic nuclei. B, C and D, Quantitative approach to staining for paracetamol, CsA and tacrolimus, respectively. Data are represented as the mean ± SEM of at least 3 separate experiments. ANOVA models p<0.0001. \* cilastatin effect, p<0.05; dose effect, p<0.05.

We quantified the functional impact of CsA, tacrolimus and paracetamol treatments on cell survival by measuring the percentage of adherent cells still able to reduce MTT to formazan after exposure to increasing doses of toxins. After 24 hours of incubation with toxins, the amount of surviving cells able to reduce MTT decreases progressively as the concentrations of CsA, tacrolimus, and paracetamol increase. However, in the presence of cilastatin, all surviving cells keep their capacity to reduce MTT (Fig. 4). Cilastatin is able to counteract both the structural and functional damage induced by toxins.

Novel Strategies in Drug-Induced Acute Kidney Injury 389

Fig. 5. Effect of cilastatin on toxin-induced mitochondrial damage. Changes in the mitochondrial oxidative capacity of RPTECs were assessed by MTT reduction at 570 nm. The graphs show formation of formazan as detected in isolated cells in real time with no treatment (control) and CsA (cyclosporin, 1000 ng/ml), tacro (tacrolimus, 500 ng/ml) and para (paracetamol, 600 µg/ml) with or without 200 µg/ml cilastatin, after the incubation

**3.3 Cilastatin improves long-term recovery and viability of RPTECs after exposure to** 

To know the long-term viability of surviving RPTECs after 24 hours of exposure to CsA, tacrolimus, or paracetamol, we tested the ability of those cells to proliferate into new cell colonies. Colony-forming units (CFUs) were quantified as specified in Methods. The CFUs count decreased after 24 hours of treatment with CsA, tacrolimus, and paracetamol, and this decrease was clearly dose-dependent (Fig. 6). If the cells were exposed to toxins in the presence of cilastatin, the number of CFUs was significantly greater after 7 days of recovery for every CsA, tacrolimus, and paracetamol concentration studied. The intracellular dye was

**3.4 Cilastatin reduces intracellular accumulation of CsA, tacrolimus, and paracetamol**  In many cases, nephrotoxicity is largely dependent on the intracellular concentration of drug reached. As cilastatin is a ligand of the brush border membrane, we investigated whether it affected toxin uptake by RPTECs. To test this hypothesis, we measured the intracellular content of CsA, tacrolimus, and paracetamol by TDX analysis, as described in Methods. Cellular CsA, tacrolimus and paracetamol content increased progressively in a dose-dependent manner when RPTECs were incubated for 24 hours in the presence of different concentrations of toxins (Fig. 7). Coincubation with cilastatin consistently reduced accumulation of CsA, tacrolimus and paracetamol in the cells for every concentration studied (Fig. 7). These results confirm that adding cilastatin to primary cultures of proximal cells decreases cellular toxin accumulation. This effect may be involved in the reduced impact of CsA, tacrolimus, and paracetamol on damage to and survival and death of

extracted, and absorbance was quantified al 595 nm (Fig. 6B, C and D).

times on the X-axis.

RPTECs.

**CsA, tacrolimus, and paracetamol** 

Fig. 4. Effect of cilastatin on toxin-induced loss of cell viability determined by the ability to reduce MTT (see Methods). Renal proximal tubular epithelial cells were exposed to toxins and toxins + cilastatin (200 µg/ml) for 24 hours. Results are expressed as the percentage of the value obtained relative to control (without toxins and cilastatin) of at least 3 separate experiments. ANOVA: for CsA, \*dose effect, p≤0.05; cilastatin effect, p<0.05; for tacrolimus, \*dose effect, p<0.05; cilastatin effect, p≤0.04; for paracetamol, \*dose effect, p≤0.05; cilastatin effect, p≤0.05.

#### **3.2 Cilastatin prevents toxin-induced mitochondrial damage**

The effect of cilastatin on mitochondria may be observed very early after CsA, tacrolimus, or paracetamol is added to cell culture plates. In Fig. 5, an inverted IX-80 microscope was fitted with a black chamber, a photomultiplier, and a spectrofluorimeter (SML Aminco) to obtain absorbance readings at specific wavelengths on single (or small groups of) cells in culture. This set-up allows real time follow-up of colorimetric in vivo reactions.

Recording the first seconds after MTT addition shows the initial kinetics of MTT reduction and formazan precipitation, thus offering a first approach to the activity of the mitochondrial chain in intact cells. Although not suitable for detailed kinetic studies, this method allows a quick check of mitochondrial oxidative activity.

RPTECs exposed to toxins showed a quick and deep depression in the reduction of MTT activity compared with controls (Fig. 5). Coincubation with cilastatin partially recovers this effect, although the effect was less visible for paracetamol. Differences are observed even during the first 5 minutes of drug additions.

Fig. 4. Effect of cilastatin on toxin-induced loss of cell viability determined by the ability to reduce MTT (see Methods). Renal proximal tubular epithelial cells were exposed to toxins and toxins + cilastatin (200 µg/ml) for 24 hours. Results are expressed as the percentage of the value obtained relative to control (without toxins and cilastatin) of at least 3 separate experiments. ANOVA: for CsA, \*dose effect, p≤0.05; cilastatin effect, p<0.05; for tacrolimus, \*dose effect, p<0.05; cilastatin effect, p≤0.04; for paracetamol, \*dose effect,

The effect of cilastatin on mitochondria may be observed very early after CsA, tacrolimus, or paracetamol is added to cell culture plates. In Fig. 5, an inverted IX-80 microscope was fitted with a black chamber, a photomultiplier, and a spectrofluorimeter (SML Aminco) to obtain absorbance readings at specific wavelengths on single (or small groups of) cells in culture.

Recording the first seconds after MTT addition shows the initial kinetics of MTT reduction and formazan precipitation, thus offering a first approach to the activity of the mitochondrial chain in intact cells. Although not suitable for detailed kinetic studies, this

RPTECs exposed to toxins showed a quick and deep depression in the reduction of MTT activity compared with controls (Fig. 5). Coincubation with cilastatin partially recovers this effect, although the effect was less visible for paracetamol. Differences are observed even

p≤0.05; cilastatin effect, p≤0.05.

**3.2 Cilastatin prevents toxin-induced mitochondrial damage** 

This set-up allows real time follow-up of colorimetric in vivo reactions.

method allows a quick check of mitochondrial oxidative activity.

during the first 5 minutes of drug additions.

Fig. 5. Effect of cilastatin on toxin-induced mitochondrial damage. Changes in the mitochondrial oxidative capacity of RPTECs were assessed by MTT reduction at 570 nm. The graphs show formation of formazan as detected in isolated cells in real time with no treatment (control) and CsA (cyclosporin, 1000 ng/ml), tacro (tacrolimus, 500 ng/ml) and para (paracetamol, 600 µg/ml) with or without 200 µg/ml cilastatin, after the incubation times on the X-axis.

#### **3.3 Cilastatin improves long-term recovery and viability of RPTECs after exposure to CsA, tacrolimus, and paracetamol**

To know the long-term viability of surviving RPTECs after 24 hours of exposure to CsA, tacrolimus, or paracetamol, we tested the ability of those cells to proliferate into new cell colonies. Colony-forming units (CFUs) were quantified as specified in Methods. The CFUs count decreased after 24 hours of treatment with CsA, tacrolimus, and paracetamol, and this decrease was clearly dose-dependent (Fig. 6). If the cells were exposed to toxins in the presence of cilastatin, the number of CFUs was significantly greater after 7 days of recovery for every CsA, tacrolimus, and paracetamol concentration studied. The intracellular dye was extracted, and absorbance was quantified al 595 nm (Fig. 6B, C and D).

#### **3.4 Cilastatin reduces intracellular accumulation of CsA, tacrolimus, and paracetamol**

In many cases, nephrotoxicity is largely dependent on the intracellular concentration of drug reached. As cilastatin is a ligand of the brush border membrane, we investigated whether it affected toxin uptake by RPTECs. To test this hypothesis, we measured the intracellular content of CsA, tacrolimus, and paracetamol by TDX analysis, as described in Methods. Cellular CsA, tacrolimus and paracetamol content increased progressively in a dose-dependent manner when RPTECs were incubated for 24 hours in the presence of different concentrations of toxins (Fig. 7). Coincubation with cilastatin consistently reduced accumulation of CsA, tacrolimus and paracetamol in the cells for every concentration studied (Fig. 7). These results confirm that adding cilastatin to primary cultures of proximal cells decreases cellular toxin accumulation. This effect may be involved in the reduced impact of CsA, tacrolimus, and paracetamol on damage to and survival and death of RPTECs.

Novel Strategies in Drug-Induced Acute Kidney Injury 391

According to these results, which suggest that cilastatin interferes with intracellular administration of the nephrotoxins tested, cilastatin appears to be able to inhibit an intracellular nephrotoxin accumulation pathway as a result of its binding to renal DHP-I. We explored the possibility that cilastatin, through its interaction with DHP-I and when anchored to cholesterol lipid rafts by a glycosyl-phosphate-inositol (GPI) group (Adachi et al., 1990; Parkin et al., 2001), could block transport through lipid rafts or interfere with the cholesterol lipid raft–dependent endocytic pathway. The expression and cell membrane localization of cholera toxin, which specifically binds to its ganglioside GM1 receptor present in cholesterol lipid rafts, were assessed using confocal microscopy in RPTECs treated for very short periods. In Fig. 8, cholera toxin is identified on the cell surface after 15 minutes incubation, but it disappeared from the membrane after 1 hour (top) and accumulated in a perinuclear position. In the presence of 200 µg/ml cilastatin and after 1 hour of treatment, cholera toxin was still attached to the membrane, suggesting interference with the cholera toxin internalization site. No significant changes in FITC-cholera toxin

Fig. 8. Blockade of circulation of cholesterol rafts by cilastatin. This picture shows the change in cholera toxin fluorescence internalization over time in control cells and cells incubated in

We report that cilastatin, a powerful and specific inhibitor of DPH-I, is able to reduce both intracellular accumulation and induction of apoptosis by antibiotic, cytotoxic, anti-inflammatory, antiretroviral, anesthetic, and immunosuppressive drugs. These findings expand our previous results with cisplatin (Camano et al., 2010) and CsA (Perez et al., 2004;

staining patterns were observed at 2.5 hours in the presence of cilastatin.

**3.5 Effect of cilastatin on lipid rafts distribution** 

the presence of cilastatin (200 µg/ml). Bar, 20 µm.

**4. Conclusion** 

Tejedor et al., 2007).

Fig. 6. Cilastatin preserves long-term recovery of toxin-treated RPTECs. A, RPTECs were incubated with paracetamol, CsA (cyclosporin), or tacrolimus in the presence or absence of 200 µg/ml cilastatin for 24 hours. The number of colony-forming units was determined by staining with crystal violet after 7 days (the figure shows the experiment with paracetamol). B, C, and D, Quantification of crystal violet staining for paracetamol, CsA and tacrolimus, respectively. Data are expressed as mean ± SEM; of 3 separate experiments. ANOVA model, p<0.0001. p<0.05 vs. control; \*p≤0.05 vs. same data with cilastatin.

Fig. 7. Effects of cilastatin on accumulation of toxins by RPTECs. Intracellular accumulation was measured in the lysates of RPTECs treated with nephrotoxins for 24 hours, in the presence or absence of cilastatin (200 µg/ml), using a specific fluorescence polarization immunoassay (TDX). Cilastatin was shown to prevent entry of all nephrotoxins into RPTECs. Values were expressed as means ± SEM of drug concentrations (n=4 different experiments). ANOVA model, p<0.0001; \*, cilastatin effect p<0.05; , dose effect p<0.05.

Fig. 6. Cilastatin preserves long-term recovery of toxin-treated RPTECs. A, RPTECs were incubated with paracetamol, CsA (cyclosporin), or tacrolimus in the presence or absence of 200 µg/ml cilastatin for 24 hours. The number of colony-forming units was determined by staining with crystal violet after 7 days (the figure shows the experiment with paracetamol). B, C, and D, Quantification of crystal violet staining for paracetamol, CsA and tacrolimus, respectively. Data are expressed as mean ± SEM; of 3 separate experiments. ANOVA model,

Fig. 7. Effects of cilastatin on accumulation of toxins by RPTECs. Intracellular accumulation was measured in the lysates of RPTECs treated with nephrotoxins for 24 hours, in the presence or absence of cilastatin (200 µg/ml), using a specific fluorescence polarization immunoassay (TDX). Cilastatin was shown to prevent entry of all nephrotoxins into RPTECs. Values were expressed as means ± SEM of drug concentrations (n=4 different experiments). ANOVA model, p<0.0001; \*, cilastatin effect p<0.05; , dose effect p<0.05.

p<0.0001. p<0.05 vs. control; \*p≤0.05 vs. same data with cilastatin.

#### **3.5 Effect of cilastatin on lipid rafts distribution**

According to these results, which suggest that cilastatin interferes with intracellular administration of the nephrotoxins tested, cilastatin appears to be able to inhibit an intracellular nephrotoxin accumulation pathway as a result of its binding to renal DHP-I. We explored the possibility that cilastatin, through its interaction with DHP-I and when anchored to cholesterol lipid rafts by a glycosyl-phosphate-inositol (GPI) group (Adachi et al., 1990; Parkin et al., 2001), could block transport through lipid rafts or interfere with the cholesterol lipid raft–dependent endocytic pathway. The expression and cell membrane localization of cholera toxin, which specifically binds to its ganglioside GM1 receptor present in cholesterol lipid rafts, were assessed using confocal microscopy in RPTECs treated for very short periods. In Fig. 8, cholera toxin is identified on the cell surface after 15 minutes incubation, but it disappeared from the membrane after 1 hour (top) and accumulated in a perinuclear position. In the presence of 200 µg/ml cilastatin and after 1 hour of treatment, cholera toxin was still attached to the membrane, suggesting interference with the cholera toxin internalization site. No significant changes in FITC-cholera toxin staining patterns were observed at 2.5 hours in the presence of cilastatin.

Fig. 8. Blockade of circulation of cholesterol rafts by cilastatin. This picture shows the change in cholera toxin fluorescence internalization over time in control cells and cells incubated in the presence of cilastatin (200 µg/ml). Bar, 20 µm.

#### **4. Conclusion**

We report that cilastatin, a powerful and specific inhibitor of DPH-I, is able to reduce both intracellular accumulation and induction of apoptosis by antibiotic, cytotoxic, anti-inflammatory, antiretroviral, anesthetic, and immunosuppressive drugs. These findings expand our previous results with cisplatin (Camano et al., 2010) and CsA (Perez et al., 2004; Tejedor et al., 2007).

Novel Strategies in Drug-Induced Acute Kidney Injury 393

expression of Fas and Fas L. Fas targets brush border lipid rafts (Dimanche-Boitrel et al., 2005), binds its ligand, and triggers the extrinsic pathway of apoptosis. Internalization of

Cilastatin reduced cisplatin-induced cell apoptosis but not cell necrosis (Camano et al., 2010). When the extrinsic apoptosis pathway was checked, the initial step blocked by

Cilastatin reduces apoptosis (nuclear damage, nucleosome formation, MTT reduction capacity) and ameliorates surviving cell recovery. Both reductions in drug intake by proximal cells and blockade of lipid raft internalization are probably involved in these

Fig. 10. Diagram of the possible protective mechanism of cilastatin. Cilastatin is a dehydropeptidase-I inhibitor used in human clinical practice combined with imipenem. Dehydropeptidase inhibition affects the structure of lipid rafts by preventing hydrolysis of the lactam ring and inhibits the absorption of imipenem and other nephrotoxic drugs, thus

specific for tissue and cell type, but not for the drug tested.

Protection by cilastatin depends on its interaction with DPH-I, an enzyme that is found almost exclusively in proximal tubules. Therefore, cilastatin-induced nephroprotection is

More research is necessary to confirm the mechanism of protection, the ability to protect in animal models of acute renal failure, and the absence of an effect on the pharmacological targets of tested drugs. Nevertheless, cilastatin offers a new protective strategy, as it is a

tissue-specific designed drug, with unexpected tissue-specific antiapoptotic actions.

Fas/Fas L seems a necessary step (Camano et al., 2010).

protective actions (Fig. 10).

reducing their renal toxicity.

cilastatin was Fas L/Fas internalization (Camano et al., 2010).

Cilastatin inhibits the activity of DPH-I, but not of DPH-4, in the brush border of renal RPTECs (Fig. 9).

Fig. 9. Effect of cilastatin on the activity of dehydropeptidase I and IV. Activities were determined by the hydrolysis of specific substrates. Results are expressed as a percentage of enzyme activity compared to untreated controls (100% activity) and as the mean ± SEM of 3 experiments. ANOVA model, p<0.0001. \* p<0.01 vs. the same data without cilastatin.

Although this inhibition is probably irrelevant in the degree of nephroprotection observed none of the nephrotoxins studied have a chemical structure that could potentially be affected by dipeptidase activity—binding to DPH-I may partially explain this protection.

DPH-I is anchored to brush border lipid rafts (Pang et al., 2004; Parkin et al., 2001). Binding of FITC-labelled B-cholera toxin to lipid rafts leads to their rapid internalization. However, internalization does not occur in the presence of cilastatin.

This mechanism is probably behind the reduction observed in the intracellular concentration of the different drugs analyzed.

We previously showed that cilastatin modifies brush border membrane fluidity by interfering with membrane-bound cholesterol (Perez et al., 2004).

The drugs tested in Fig. 1 have many different chemical structures, and their mechanisms of cell permeation are not well established in some cases. However, for all those drugs, intracellular concentrations were measured and cilastatin always reduced intracellular accumulation. By inhibiting lipid raft–dependent vesicle circulation, cilastatin seems able to reduce luminal entry of drugs, even if they are not substrates for DPH-I activity.

This interference with drug entry may explain the almost instantaneous protection observed in the real-time experiments of MTT reduction. MTT reduction relies on mitochondrial oxidative chain integrity. When single cell oxidative capacity is recorded in real time, addition of the toxin inhibits MTT reduction activity relative to the single control cell, and this is evident from the first seconds. Cilastatin partially protects against this effect. The quick time course of the effect strongly suggests that a mechanism of cilastatin inhibits drug intake by the cell.

However, other mechanisms may be implicated in the broad renal protection observed. We recently published that, when exposed to toxic concentrations of cisplatin, RPTECs increase

Cilastatin inhibits the activity of DPH-I, but not of DPH-4, in the brush border of renal

Fig. 9. Effect of cilastatin on the activity of dehydropeptidase I and IV. Activities were determined by the hydrolysis of specific substrates. Results are expressed as a percentage of enzyme activity compared to untreated controls (100% activity) and as the mean ± SEM of 3 experiments. ANOVA model, p<0.0001. \* p<0.01 vs. the same data without cilastatin.

Although this inhibition is probably irrelevant in the degree of nephroprotection observed none of the nephrotoxins studied have a chemical structure that could potentially be affected by dipeptidase activity—binding to DPH-I may partially explain this protection.

DPH-I is anchored to brush border lipid rafts (Pang et al., 2004; Parkin et al., 2001). Binding of FITC-labelled B-cholera toxin to lipid rafts leads to their rapid internalization. However,

This mechanism is probably behind the reduction observed in the intracellular concentration

We previously showed that cilastatin modifies brush border membrane fluidity by

The drugs tested in Fig. 1 have many different chemical structures, and their mechanisms of cell permeation are not well established in some cases. However, for all those drugs, intracellular concentrations were measured and cilastatin always reduced intracellular accumulation. By inhibiting lipid raft–dependent vesicle circulation, cilastatin seems able to

This interference with drug entry may explain the almost instantaneous protection observed in the real-time experiments of MTT reduction. MTT reduction relies on mitochondrial oxidative chain integrity. When single cell oxidative capacity is recorded in real time, addition of the toxin inhibits MTT reduction activity relative to the single control cell, and this is evident from the first seconds. Cilastatin partially protects against this effect. The quick time course of the effect strongly suggests that a mechanism of cilastatin inhibits drug

However, other mechanisms may be implicated in the broad renal protection observed. We recently published that, when exposed to toxic concentrations of cisplatin, RPTECs increase

reduce luminal entry of drugs, even if they are not substrates for DPH-I activity.

internalization does not occur in the presence of cilastatin.

interfering with membrane-bound cholesterol (Perez et al., 2004).

of the different drugs analyzed.

intake by the cell.

RPTECs (Fig. 9).

expression of Fas and Fas L. Fas targets brush border lipid rafts (Dimanche-Boitrel et al., 2005), binds its ligand, and triggers the extrinsic pathway of apoptosis. Internalization of Fas/Fas L seems a necessary step (Camano et al., 2010).

Cilastatin reduced cisplatin-induced cell apoptosis but not cell necrosis (Camano et al., 2010). When the extrinsic apoptosis pathway was checked, the initial step blocked by cilastatin was Fas L/Fas internalization (Camano et al., 2010).

Cilastatin reduces apoptosis (nuclear damage, nucleosome formation, MTT reduction capacity) and ameliorates surviving cell recovery. Both reductions in drug intake by proximal cells and blockade of lipid raft internalization are probably involved in these protective actions (Fig. 10).

Fig. 10. Diagram of the possible protective mechanism of cilastatin. Cilastatin is a dehydropeptidase-I inhibitor used in human clinical practice combined with imipenem. Dehydropeptidase inhibition affects the structure of lipid rafts by preventing hydrolysis of the lactam ring and inhibits the absorption of imipenem and other nephrotoxic drugs, thus reducing their renal toxicity.

Protection by cilastatin depends on its interaction with DPH-I, an enzyme that is found almost exclusively in proximal tubules. Therefore, cilastatin-induced nephroprotection is specific for tissue and cell type, but not for the drug tested.

More research is necessary to confirm the mechanism of protection, the ability to protect in animal models of acute renal failure, and the absence of an effect on the pharmacological targets of tested drugs. Nevertheless, cilastatin offers a new protective strategy, as it is a tissue-specific designed drug, with unexpected tissue-specific antiapoptotic actions.

Novel Strategies in Drug-Induced Acute Kidney Injury 395

Markewitz, A.; Hammer, C.; Pfeiffer, M.; Zahn, S.; Drechsel, J.; Reichenspurner, H. &

Mraz, W.; Modic, P.K. & Hammer, C. (1992). Impact of imipenem/cilastatin on cyclosporine

Mraz. W.; Sido, B.; Knedel, M. & Hammer, C. (1987). Concomitant immunosuppressive and

Neria, F.; Castilla, M.A.; Sanchez, R.F.; Gonzalez-Pacheco, F.R.; Deudero, J.J.; Calabia, O.;

Norrby, S.R.; Alestig, K.; Björnegård, B.; Burman, L.A.; Ferber, F.; Huber, J.L.; Jones, K.H.;

*and Chemotherapy*, Vol.23, No.2, (February 1983), pp. 300-7, ISSN 0066-4804 Oh, M.S. (2010). Unconventional views on certain aspects of toxin-induced metabolic

Pabla, N. & Dong, Z. (2008) Cisplatin nephrotoxicity: mechanisms and renoprotective

Pang, S.; Urquhart, P. & Hooper, N.M. (2004). N-glycans, not the GPI anchor, mediate the

Parkin, E.T.; Turner, A.J. & Hooper, N.M. (2001). Differential effects of glycosphingolipids

Perazella, M.A. (2009). Renal vulnerability to drug toxicity. *Clinical Journal of the American Society of Nephrology*, Vol.4, No.7, (July 2009), pp. 1275-83, ISSN 1555-9041 Perez, M.; Castilla, M.; Torres, A.M.; Lázaro, J.A.; Sarmiento, E. & Tejedor, A. (2004)

*transplantation*, Vol.19, No.10, (July 2004), pp. 2445-55, ISSN 0931-0509 Servais, H.; Ortiz, A.; Devuyst, O.; Denamur, S.; Tulkens, P.M. & Mingeot-Leclercq, M.P.

pp. 865-70, ISSN 0041-1337

pp. 1704-8, ISSN 0041-1345.

4017-20, ISSN 0041-1345

0085-2538

17385997

ISSN 0021-9533

209-16, ISSN 0264-6021

(January 2008), pp. 11-32, ISSN 1360-8185

2538

Reichart, B. (1994). Reduction of cyclosporine-induced nephrotoxicity by cilastatin following clinical heart transplantation. *Transplantation*, Vol.57, No.6, (March 1994),

metabolism and excretion. *Transplantation Proceedings*, Vol.24, No.5, (October 1992),

antibiotic therapy--reduction of cyclosporine A blood levels due to treatment with imipenem/cilastatin. *Transplantation Proceedings*, Vol.19, No.5, (October 1987), pp.

Tejedor, A.; Manzarbeitia, F.; Ortiz, A. & Caramelo, C. (2009). Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity. *Kidney International*, Vol.75, No.2, (September 2008), pp. 227-34, ISSN

Kahan, F.M.; Kahan, .J.; Kropp, H.; Meisinger, M.A. & Sundelof, J.G. (1983). Urinary recovery of N-formimidoyl thienamycin (MK0787) as affected by coadministration of N-formimidoyl thienamycin dehydropeptidase inhibitors. *Antimicrobial Agents* 

acidosis. *Electrolyte & Blood Pressure*, Vol.8, No.1, (June 2010), pp.32-7, ISSN

strategies. *Kidney International*, Vol.73, No.9, (May 2008), pp. 994-1007, ISSN 0085-

apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells. *Journal of Cell Science*, Vol.117, No.Pt21, (October 2004), pp. 5079-86,

on the detergent-insolubility of the glycosylphosphatidylinositol-anchored membrane dipeptidase. *The Biochemical Journal*, Vol.358, No.Pt1, (August 2005), pp.

Inhibition of brush border dipeptidase with cilastatin reduces toxic accumulation of cyclosporin A in kidney proximal tubule epithelial cells. *Nephrology, dialysis,* 

(2008). Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms and potential approaches to modulation. *Apoptosis*, Vol. 13, No.1,

#### **5. Acknowledgments**

This work was partially supported by the Fondo de Investigaciones Sanitarias [Grants FIS-PI05/2259, FIS-PI 08/1481], Comunidad de Madrid [Grant BIO-S-0283/2006], and Fundacion Mutua Madrileña. AL holds a "Sara Borrell" post-doctoral research contract from the ISCIII.

The authors are grateful to Dr. Rafael Samaniego for help with confocal microscopy, Dr. Miguel L.F. Ruano for technical assistance with the TDX assays, and Merck Sharp & Dohme for providing cilastatin.

#### **6. References**


This work was partially supported by the Fondo de Investigaciones Sanitarias [Grants FIS-PI05/2259, FIS-PI 08/1481], Comunidad de Madrid [Grant BIO-S-0283/2006], and Fundacion Mutua Madrileña. AL holds a "Sara Borrell" post-doctoral research contract from

The authors are grateful to Dr. Rafael Samaniego for help with confocal microscopy, Dr. Miguel L.F. Ruano for technical assistance with the TDX assays, and Merck Sharp & Dohme

Adachi, H.; Tawaragi, Y.; Inuzuka, C.; Kubota, I.; Tsujimoto, M.; Nishihara, T. & Nakazato,

Birnbaum, J.; Kahan, F.M.; Kropp, H. & MacDonald, .JS. (1985). Carbapenems, a new class of

Camaño-Páez, S.; Lázaro-Fernández, A.; Callejas-Martínez, R.; Lázaro-Manero, J.A.; Castilla-

Carmellini, M.; Matteucci, E.; Boggi, U.; Cecconi, S.; Giampietro, O. & Mosca, F. (1998).

Dimanche-Boitrel, M.T.; Meurette, O.; Rebillard, A. & Lacour, S. (2005) Role of early plasma

Gruss. E.; Tomas, J.F.; Bernis, C.; Rodriguez, F.; Traver, J.A. & Fernandez-Ranada, J.M.

Lorz, C.; Benito-Martin, A.; Justo, P.; Sanz, A.B.; Sanchez-Niño, M.D.; Santamaria, B.; Egido,

*Transplantation*, Vol.64, No.1, (July 1997), pp. 164-6, ISSN 0041-1337

H. (1990). Primary structure of human microsomal dipeptidase deduced from molecular cloning. *The Journal of Biological Chemistry*, Vol.265, No.7, (March 1990),

beta-lactam antibiotics. Discovery and development of imipenem/cilastatin. *The American Journal of Medicine*, Vol.78, No.6A, (June 1985), pp. 3-21, ISSN 0002-9343 Camano, S.; Lazaro, A.; Moreno-Gordaliza, E.; Torres, A.M.; de Lucas, C.; Humanes, B.;

Lazaro, J.A.; Gomez-Gomez, M.; Bosca, L. & Tejedor, A. (2010). Cilastatin attenuates cisplatin-induced proximal tubular cell damage. *The Journal of Pharmacology and Experimental Therapeutics*, Vol.334, No.2, (April 2010), pp. 419-29,

Barba, M.; Martín-Vasallo, P.; Martínez-Escandel, A: & Tejedor-Jorge A. (2008). Study on the role of the tubule in renal vasoconstriction induced by cyclosporine. *Actas Urológicas Españolas*, Vol.32, No.1, (January 2008), pp. 128-39, ISSN 0210-4806 Carmellini. M.; Frosini, F.; Filipponi, F.; Boggi, U. & Mosca, F. (1997). Effect of cilastatin on

cyclosporine-induced acute nephrotoxicity in kidney transplant recipients.

Imipenem/cilastatin reduces cyclosporin-induced tubular damage in kidney transplant recipients. *Transplantation Proceedings*, Vol.30, No.5, (August 1998), pp.

membrane events in chemotherapy-induced cell death. *Drug Resistance Update: reviews and commentaries in antimicrobial and anticancer chemotherapy*, Vol.8, No.1-2,

(1996). Nephroprotective effect of cilastatin in allogeneic bone marrow transplantation. Results from a retrospective analysis. *Bone Marrow Transplantation*,

J. & Ortiz, A. (2006). Modulation of renal tubular cell survival: where is the evidence?. *Current Medicinal Chemistry*, Vol.13, No.4, pp. 449-54, ISSN 0929-8673

**5. Acknowledgments** 

for providing cilastatin.

pp. 3392-5, ISSN 0021-9258

ISSN 0022-3565

2034-5, ISSN 0041-1345

(April 2005), pp. 5-14, ISSN 1368-7646

Vol.18, No.4, (October 1996), pp. 761-5, ISSN 0268-3369

**6. References** 

the ISCIII.


**19** 

*México* 

**Chemical and Physical Enhancers** 

José Juan Escobar-Chávez1,\*, Isabel Marlen Rodríguez-Cruz2

*Laboratorio 12: Materiales Nanoestructurados y Sistemas Transdérmicos,* 

The application of preparations to the skin for medical purposes is as old as the history of medicine itself, with references to the use of ointments and salves found in the records of Babylonian and Egyptian medicine.(López-Castellano & Merino, 2010) The historical development of permeation research is well described by Hadgraft & Lane, 2005. Over time, the skin has become an important route for drug delivery in which topical, regional or systemic effects are desired (Domínguez-Delgado, et al., 2010). Nevertheless, skin constitutes an excellent barrier and presents difficulties for the transdermal delivery of therapeutic agents, since few drugs possess the characteristics required to permeate across the stratum corneum in sufficient quantities to reach a therapeutic concentration in the blood. In order to enhance drug transdermal absorption different methodologies have been investigated developed and patented (Rizwan et al., 2009). To date many chemical and physical approaches have been applied to increase the efficacy of the material transfer across the intact skin. These are termed 'Novel' due to recent development with satisfactory results in the field of drug delivery (Patel et al., 2010). Improvement in physical permeationenhancement technologies has led to renewed interest in transdermal drug delivery. Some of these novel advanced transdermal permeation enhancement technologies include: iontophoresis, electroporation, ultrasound, microneedles to open up the skin and the use of

transdermal nanocarriers (Díaz-Torres, 2010; Escobar-Chávez & Merino, 2010a).

**1. Introduction** 

 \*

Corresponding Author

*Nacional Autónoma de México, Carretera Cuautitlán–Teoloyucan,* 

**for Transdermal Drug Delivery** 

*Facultad de Estudios Superiores Cuautitlán-Universidad* 

*San Sebastián Xhala, Cuautitlán Izcalli, Estado de México,* 

*Facultad de Estudios Superiores Cuautitlán-Universidad* 

and Clara Luisa Domínguez-Delgado2 *1Unidad de Investigación Multidisciplinaria,* 

*2Departamento de Ingeniería y Tecnología, Sección de Tecnología Farmacéutica,* 

> *Nacional Autónoma de México, Cuautitlán Izcalli, Estado de México,*

