1 Department of Nephrology and 2 Department of Laboratory Chemistry, J.W. Goethe-University Frankfurt, Germany
Correspondence and offprint request to: Binytha Wegner, Department of Nephrology, J.W. Goethe-University, Theodor, Stern-Kai 7, 60590 Frankfurt, Germany. Email: b.wegner{at}em.uni-frankfurt.de
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Abstract |
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Methods. Cells were isolated from human kidney tissue, and exposed to clinically relevant concentrations of CAS and AmB for 24 h. Total DNA content and cell viability were determined by DAPI staining and a modified MTT assay. For testing of cytotoxicity, LDH activity was measured in cell culture supernatants. To assess apoptotic effects, AnnexinV-binding assay and DAPI staining for detection of fragmented DNA were performed.
Results. DTC were more vulnerable towards the antifungal agents than PTC. In contrast to AmB, cell-damaging effects of CAS were less severe. DAPI staining revealed slight and dose-dependent antiproliferative effects of CAS at concentrations reflecting relevant plasma levels. At these concentrations, cell viability, determined by MTT assay, was not decreased in PTC and DTC. LDH release was marginally increased in a dose-dependent manner; apoptosis was not detected. Nevertheless, at CAS concentrations reflecting potential tissue concentrations, cell damaging effects were considerably more pronounced.
Conclusion. Our results suggest that CAS is less nephrotoxic than AmB in vitro. The antiproliferative and cytotoxic effects of CAS predominantly affect DTC, which seem to be more susceptible to CAS-induced damage.
Keywords: amphotericin B; caspofungin; in vitro; nephrotoxicity
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Introduction |
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Side effects of CAS assessed in clinical trials, particularly nephrotoxicity, were less severe compared to AmB. In these studies, which were carried out over an average of 14 days, nephrotoxicity was usually defined as an increase in serum creatinine, and ranged, in the majority of trials, from 0 to 1.4% [36].
Nevertheless, in one recent clinical trial, which compared CAS and AmB for treatment of invasive candidiasis, the rate of nephrotoxicity in the CAS-treated group was higher than in previous studies, and comprised 8.4% [7]. These results might indicate that the nephrotoxic potential of CAS is higher than assumed. In the quoted studies nephrotoxicity was defined as an increase in serum creatinine, but it should be borne in mind that renal damage may occur before it is reflected in an elevation of serum creatinine. Phase I studies comparing CAS to AmB only looked at changes in serum creatinine and potassium levels. To our knowledge no renal histologies were obtained in these studies (MSD, personal communication). Additionally, at the time of our study, data assessing extent and nature of CAS-associated renal cell damage in vitro were not available. Considering these facts, the aim of our study was to characterize extent and nature of cell damaging effects of CAS in vitro, and to compare them to those of AmB. Due to the fact that AmB exerts direct toxic effects on renal tubular cells [8], we selected proximal tubular cells (PTC) and distal tubular cells (DTC) for our experiments.
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Subjects and methods |
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Afterwards, PTC and DTC were seeded in 6-well plates precoated with human collagen IV (20 µg/ml; Boehringer Mannheim). Cells were grown in medium 199 (Gibco) with 10% FCS (PAA, Cölbe, Germany) at 37°C and 5% CO2 in a humidified atmosphere. Preservation of differentiation and function of cultured tubular cells were tested as previously described [9,10].
Passages 35 of PTC and DTC were used for our experiments.
Exposure of PTC and DTC to antifungal agents
PTC and DTC were grown in 8-, 12- and 96-well plates, and exposed to AmB (0.5, 2, 10, 30 µg/ml in glucose 5%; Bristol-Myers Squibb, München, Germany), and to CAS (1, 2.5, 5, 10, 50 µg/ml in sodium chloride 0.9%; MSD Haar, Germany) for 24 h in medium 199 with 10% FCS. For testing of apoptosis, exposure to 0.25 and 0.5 µg/ml CAS, and exposure times of 1, 2 and 4 h were additionally assessed.
Testing for cell proliferation and viability
Determination of total DNA content with DAPI staining (Sigma, Deisenhofen, Germany) allows for an estimation of cell proliferation [11]. PTC and DTC were grown to 50% confluence. After 24 h exposure to CAS and AmB as described above, cells were washed three times with PBS. Lysis buffer [sodium dodecyl sulfate 0.02% (v/v); Biosource, Solingen, Germany] in 15 mM sodium citrate, and 0.15 M sodium chloride (ICN Biomedicals, Eschwege, Germany) and, after 15 min, DAPI (2 µg/ml) were added. Fluorescence was measured in a microplate reader (FLUOstar, BMG) at 460 nm after excitation at 355 nm.
Cell viability and proliferation were determined with a modified MTT assay (EZ4U; Biomedica, Vienna, Austria). The test is based on reduction of tetrazolium salts into red formazan derivatives in intact mitochondria of viable cells. After 24 h exposure of PTC and DTC to CAS and AmB, cells were washed three times with PBS. Tetrazolium salt solution was pipetted into each well as recommended by the manufacturer. After 3 h incubation, plates were measured in a microplate reader (Dynatech) at 450 nm.
Testing for cell necrosis
Necrotic damage leads to loss of cell integrity with consecutive release of intracellular lactate dehydrogenase (LDH) into the environment. LDH activity was measured in cell culture supernatants of PTC and DTC that were exposed to the antifungal agents as described above. LDH activity was determined by measuring the decrease of absorbance at 340 nm, resulting from conversion of NADH to NAD in the presence of pyruvate.
Testing for apoptosis
Translocation of the phospholipid phosphatidylserine from the inner to the outer leaflet of the cell membrane occurs during early apoptosis, and was detected by annexin V-binding (Annexin V-FITC Apoptosis Detection Kit I; BD Biosciences, Pharmingen, Germany). To assess membrane integrity, cells were additionally stained with propidium iodide (PI). PTC and DTC were grown to confluence in 12-well plates, and exposed to AmB and CAS for 1, 2, 4 and 24 h at concentrations stated above. For the positive control, cells were treated with 10 µM camptothecin for 24 h or with 250 ng/ml rapamycin for 1, 2 and 4 h. After exposure to the antifungal and proapoptotic substances, cells were washed twice with ice-cold PBS, and gently detached from culture wells with accutase (PAA, Cölbe, Germany). Cells and cell culture supernatants were centrifuged for 5 min at 300 g. The cell pellet was resuspended in annexin V-binding buffer (0.01 M HEPESNaOH, pH 7.4, 0.14 M NaCl, 2.5 mM CaCl2). We stained 100 µl of the cell suspension (106 cells/ml) with 5 µl annexin VFITC and 5 µl PI. After incubation at room temperature for 15 min, 400 µl of annexin V-binding buffer were added, and a total of 10 000 cells per sample were analysed in a flow cytometer (Becton Dickinson). Data were evaluated with WinMDI software, version 2.8.
DNA fragmentation, which occurs in late apoptosis, was assessed after DAPI staining by fluorescence microscopy. Cells were grown to confluence on 8-well chamber slides, and treated with CAS and AmB for 1, 2, 4 and 24 h at concentrations stated above. Positive control cells were exposed to camptothecin and rapamycin as described above. After centrifugation at 300 g for 10 min, cells were washed three times with PBS, and fixed in acetone:methanol 1:1 (v/v) for 10 min. After washing with PBS, cells were stained with 2 µg/ml DAPI for 5 min. The number of apoptotic nuclei was determined by fluorescence microscopy, and related to the total number of cell nuclei. At least 300 nuclei were counted.
Statistical analysis
Results of DAPI, MTT and LDH assays represent mean values of at least four experiments performed in octuplets, except where indicated. Results of the apoptosis assays represent mean values of at least four experiments. Values are expressed as percent reduction or increase relative to corresponding controls ± SEM unless stated otherwise. Cells from four different preparations were used. Statistical analysis was performed using a KruskalWallis Test, followed by Dunn's Test. Each group of data was compared to the control group. Differences of results between PTC and DTC were analysed with a MannWhitney Rank Sum Test. P-values <0.05 were considered statistically significant.
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Results |
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Since measuring DNA content in lysed cells does not allow discrimination between living and dead cells, cell viability was determined with an MTT assay (Figures 2a and b). In CAS-treated cells, statistically significant decreases in viability compared to control cells were only observed at concentrations of 50 µg/ml in PTC, and at concentrations 10 µg/ml in DTC, which might be associated with a lower test sensitivity or higher spreading of data. In AmB-treated cells, at concentrations
10 µg/ml in PTC, and
2 µg/ml in DTC, we observed statistically significant dose-dependent decreases of viability. Exposure to 2 µg/ml AmB resulted in a decrease of viability of 4±3% of the control in PTC, and of 8±4% of the control (P<0.05) in DTC.
For further characterization of the nature of CAS-induced damage, LDH activity was measured in cell culture supernatants. Exposure of PTC to CAS caused a moderate, dose-dependent increase of LDH release compared to the control group: Increases of LDH release of 7±7% of the control at 1 µg/ml, and of 17±8% of the control at 2.5 µg/ml were observed (Figure 3a). In DTC, cytotoxic effects of CAS were more pronounced compared to PTC [CAS 1 µg/ml, 25±10% of the control; CAS 2.5 µg/ml, 29±11% of the control (Figure 3b)]. Statistical significance of the LDH release data was achieved at CAS concentrations 10 µg/ml in both PTC and DTC. The dose-dependent increase in LDH release of AmB-treated cells was markedly more distinct than in CAS-10 treated cells (AmB 2 µg/ml; PTC, 157±9% of control; DTC, 203±11% of control; P<0.05], and reached, at supratherapeutic doses, values of 295±9% of the control (P<0.05) in PTC, and 527±12% of the control (P<0.05) in DTC. At CAS concentrations exceeding plasma concentrations, which reflect possible concentrations in renal tissue (>10 µg/ml), decreases of proliferation and viability, and increases of LDH release, were more pronounced (Figures 13
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Differences between PTC and DTC were statistically significant for results of DAPI staining and the LDH release assay (P<0.05). Differences of the MTT data between PTC and DTC were not statistically significant.
Testing for apoptosis with annexin V- binding revealed no significant apoptotic effects of AmB and CAS after exposure times of 1, 2, 4 and 24 h (Table 1 and Figure 4). Likewise, after DAPI staining, no significant increase of DNA fragmentation was detected in CAS- and AmB-treated cells. With both assays we observed statistically significant apoptotic effects in positive control PTC and DTC treated with the proapoptotic substances camptothecin and rapamycin with values around 350% of control cells (Table 2).
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Discussion |
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We analyzed CAS-induced renal damage in vitro in comparison with toxic effects of AmB, in order to assess an underestimation of the nephrotoxic potential of CAS. At concentrations reflecting therapeutic plasma concentrations, we detected moderate and dose-dependent antiproliferative and cytotoxic effects on PTC and DTC. At higher concentrations, which reflect potential tissue concentrations these effects were marked.
We tested CAS at concentrations between 1.0 and 50 µg/ml. In vivo, plasma peak levels are not likely to exceed 10 µg/ml [1]. Mean trough concentrations range between 1 and 2.5 µg/ml. Accumulation of the drug occurs after repeated applications due to its long half-life and non-linear pharmakokinetics. Two days after administration of CAS, >90% of the dose is found in tissues [12]. Hajdu et al. [13] have shown in a mouse model that the substance rapidly accumulates in tissues with 7-fold higher drug concentrations in the kidney compared to plasma concentrations. Thus, in vivo, PTC and DTC are likely to be exposed to CAS concentrations higher than plasma concentrations. Due to the fact that tissue concentrations have not been measured in humans, we can only assume that tubular cells are exposed to concentrations around 12 µg/ml. AmB was tested in our study at concentrations between 0.5 and 30 µg/ml. In vivo, mean therapeutic plasma levels range between 0.5 and 2 µg/ml. Immediately after administration of a single therapeutic dose of AmB, plasma levels peak to concentrations around 1.4 µg/ml, and drop very slowly to trough concentrations [14]. The substance, which is highly bound to proteins, has a long half-life of up to 48 h. For this reason, accumulation occurs after repeated applications. In summary, concentrations of 12.5 µg/ml CAS, and of 0.52 µg/ml AmB reflect relevant plasma concentrations.
After 24 h exposure to clinically relevant concentrations of AmB based on therapeutic plasma levels we detected pronounced and dose-dependent cell damaging effects on tubular cells, which were explicitly reflected in the LDH release assay. At supratherapeutic doses, impressive LDH release increases with values of up to 500% of the control were observed. In rabbit PTC, Joly et al. [15] demonstrated increases in LDH release after exposure to supratherapeutic AmB concentrations. Further studies, in which AmB toxicity was evaluated in vitro, showed results that were comparable to our findings [16,17]. We did not detect significant apoptotic effects of AmB by means of annexin V and DAPI staining, even at low concentrations and short exposure times. These findings were not in concordance with in vitro results of Varlam et al. [16], who have shown that AmB induces apoptosis in renal tubular cells at low concentrations of 1 and 2.5 µg/ml, and an exposure time of 1 h. However, due to the fact that in this study canine distal and porcine proximal tubular cells were used, we cannot compare our findings with these results offhand. Furthermore, it is known that substances that cause necrosis in renal tubular cells may exhibit apoptotic effects at low concentrations, and short exposure times [18]. The discrepancy of our findings might be caused by a higher sensitivity of human tubular cells towards AmB so that in our cells necrotic damage is observed at concentrations at which apoptotic effects are induced in tubular cells derived from other species.
CAS-induced antiproliferative and cytotoxic effects at concentrations reflecting plasma concentrations were, in our study, less pronounced than in AmB-treated cells. Nevertheless, at concentrations exceeding plasma concentrations and representing potential tissue concentrations, these effects were considerably more pronounced. Further differentiation of the nature of damage pointed towards predominantly necrotic effects of CAS on PTC and DTC, reflected by data of the LDH release assay. No CAS-associated apoptotic effects were detected in PTC and DTC. Apoptosis and necrosis can represent two different mechanisms. On the other hand, as mentioned before, they might embody different grades of damage. In this case, apoptosis is characterized as an early and low-dose response, while necrosis appears as a late and high-dose response. For these reasons we additionally tested for apoptosis at lower CAS concentrations (0.25 and 0.5 µg/ml), and at shorter exposure times (10 and 30 min) (data not shown). No apoptotic effects were detectable at these test conditions, hence we can assume that CAS does not induce apoptosis, even at short exposure times and low concentrations.
We can only speculate about mechanisms by which CAS damages tubular cells. The renal clearance of CAS is very low [1]; nonetheless, Hajdu et al. [13] have demonstrated that CAS rapidly accumulates in animal kidney tissues. This observation allows the assumption that PTC and DTC, which have been shown to be vulnerable towards CAS in our in vitro studies, are indeed exposed to the substance at in vivo conditions. Considering the facts that CAS has a high molecular weight and is highly bound to serum albumin, it is not likely that the substance passes through the glomerular filter. We can assume that CAS initially reaches tubular cells via their basolateral membranes. Subsequently, in case of uptake into tubular cells and secretion of the substance into the tubular lumen, toxic effects might also be exerted via the luminal membrane. Regarding existing transport mechanisms in tubular cells, the extent of CAS-induced toxicity is likely to depend on distribution and amount of transporters that forward the echinocandin into or out of these cells, and thus have an impact on intracellular accumulation. CAS-associated tubular transport mechanisms have not been characterized yet, but considering the chemical structure of the lipopeptide CAS, organic anion transporters located at basolateral and luminal membranes of tubular cells might play an essential role. Due to the fact that CAS is not a substrate of glycoprotein P, trafficking of the substance via this transporter can be excluded [19].
The nature of AmB-associated renal cell damage is well described. The substance induces direct alterations in renal tubular cell membranes leading to the formation of transmembrane pores [8]. Tubular dysfunction with leakage of electrolytes is associated with these changes. Similarly to AmB, CAS might directly disrupt membrane integrity, initiating cascades involved in necrosis.
The proposed pathophysiological mechanisms are solely speculative. Further investigations are required in order to specify the mode of CAS-induced tubular cell damage.
We observed a different extent of CAS-associated damage in PTC compared to DTC. Except for the MTT data, our results suggest that DTC are significantly more vulnerable towards CAS than PTC. The pathomechanisms that stand behind these findings are unclear. However, it is known that different cell types display individual patterns of response to toxic agents due to differing cytokine and growth factor profiles. In a considerable number of studies, a higher sensitivity of DTC towards different toxic agents compared to PTC have been proven. Lash et al. [20] have shown that rat DTC in vitro were more vulnerable than rat PTC towards toxic damage induced by oxidants and alkylating agents targeting soft nucleophiles. As studies have shown, the increased vulnerability of DTC towards CAS gets aggravated in vivo by characteristic circumstances of these cells, namely exposition to higher sodium chloride concentrations and a lower pH environment compared to PTC [17]. In summary, our results suggest that CAS is less nephrotoxic than AmB in vitro. At concentrations reflecting plasma concentrations necrotic effects of CAS were moderate and predominantly affect DTC. Nevertheless, at concentrations that possibly represent tissue concentrations, antiproliferative and necrotic effects were markedly more pronounced so that the nephrotoxic potential of CAS should not be underestimated. Apoptotic effects do not seem to play a role in CAS-induced damage.
Subsequent studies, which specify pathomechanisms, protective conditions, and potential reversibility of CAS-induced tubular injury, are required to help develop strategies to prevent renal damage. Furthermore, longer clinical trials are needed for assessment of long term, CAS-associated renal damage. Nephrotoxitixity of the new antifungal agent CAS should be thoroughly assessed, in order to define its role as a superior alternative to AmB.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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