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ARTICLE Effects of serum lipoproteins on cyclosporine A cellular uptake and renal toxicity in vitro Dion R. Brocks, Hetal R. Chaudhary, Mohamed Ben-Eltriki, Marwa E. Elsherbiny, and Ayman O.S. El-Kadi

Abstract: In-vitro studies were performed to shed light on previous findings that showed increased uptake of cyclosporine A in the kidneys and liver of hyperlipidemic rats, and increased signs of kidney toxicity. Hepatocytes were obtained from rats, cultured, and exposed to a diluted serum from hyperlipidemic rats. Some cells were also exposed to lipid-lowering drugs. After washing out the rat serum or lipid-lowering drugs, cells were exposed to cyclosporine A embedded in serum lipoproteins. Pretreatment with hyperlipidemic serum and lipid-lowering drugs was associated with an increased uptake of cyclosporine A. As expected, atorvastatin caused an increase in low density lipoprotein receptor and a decrease in MDR1A mRNA in the hepatocytes. A decrease in NRK-52E rat renal tubular cellular viability caused by cyclosporine A was noted when cells were preincubated with diluted hyperlipidemic serum. This was matched with evidence of hyperlipidemic-serum-associated increases in the NRK-52E cellular uptake of cyclosporine A and rhodamine-123. The findings of these experiments suggested that in hyperlipidemia the expression and (or) the functional activity of P-glycoprotein was diminished, leading to greater hepatic and renal uptake of cyclosporine A, and renal cellular toxicity. Key words: hepatocytes, hyperlipidemia, lipoprotein receptors, NRK-52E cells, P-glycoprotein. Résumé : Des études in vitro ont été réalisées afin de faire la lumière sur des résultats obtenus précédemment, qui ont révélé une captation accrue de cyclosporine A dans les reins et le foie de rats hyperlipidémiques ainsi que des signes accrus de toxicité rénale. Des hépatocytes de rats ont été isolés, cultivés et exposés a` du sérum de rat hyperlipidémique dilué. Certaines cellules ont aussi été exposées a` des hypolipidémiants. Après avoir rincé les cellules du sérum de rat ou des hypolipidémiants, elles ont été exposées a` la cyclosporine A enrobée dans des lipoprotéines sériques. Le prétraitement au sérum hyperlipidémique et aux hypolipidémiants était associé a` une captation accrue de cyclosporine A. Comme attendu, l'atorvastatine induisait une augmentation de l'expression du récepteur des lipoprotéines de faible densité ainsi qu'une diminution de l'expression de l'ARNm de MDR1A dans les hépatocytes. Une diminution la viabilité des cellules de tubules rénaux de rats NRK-52E induite par la cyclosporine A a été notée lorsque les cellules étaient préincubées avec le sérum hyperlipidémique dilué. Cela coïncidait avec l'augmentation de la captation de cyclosporine A et de rhodamine-123 associée au sérum hyperlipidémique dans les cellules NRK-52E. Les résultats de ces expériences suggèrent qu'en situation d'hyperlipidémie, l'expression et (ou) l'activité fonctionnelle de la P-glycoprotéine était diminuée, conduisant a` une captation accrue de clyclosporine A par le foie et les reins, ainsi qu'a` une toxicité cellulaire rénale. [Traduit par la Rédaction] Mots-clés : hépatocytes, hyperlipidémie, récepteurs de lipoprotéines, cellules NRK-52E, P-glycoprotéine.

Introduction In rats, experimental hyperlipidemia (HL) is associated with increased concentrations of the immunosuppressive agent, cyclosporine A (CyA) in the kidneys and liver (Aliabadi et al. 2006). This finding has particular relevance for several reasons: CyA is extensively, but slowly, metabolized by cytochrome P450 (CYP) isoenzymes in the liver (most notably CYP3A), and, an increased amount of drug in liver could be an indicator of an increased transport (net influx) of drug. Although this could increase metabolism of the drug if the number of available enzymes remained the same, an increased hepatic concentration could also be caused independently by a slower rate of metabolism in HL. The kidney is the organ principally affected by CyA toxicity, and increased renal concentrations are of particular concern. Indeed, in HL rats, CyA has been associated with increased signs of kidney toxicity (Bohdanecka et al. 1999; Aliabadi et al. 2006). The cause of the increased kidney concentrations and renal toxicity remains unknown. CyA is known to be a substrate for binding or sequestration by serum lipoproteins, which are increased in HL. In rats in which HL was induced by the intraperitoneal admin-

istration of 1 g·(kg body mass)–1 poloxamer 407, a decrease in the plasma unbound fraction of almost 65% was observed (Brocks et al. 2006). Because the drug is extensively metabolized, but has a low hepatic extraction ratio, this increased binding would normally be expected to not lead to significant changes in overall tissue concentrations of the drug (Mehvar 2006). Lipoprotein binding, however, differs from binding to other serum proteins in that the particles can be selectively taken up by lipoprotein receptors present in specific organs (Wasan et al. 2008). Two important classes of lipoprotein receptors are the low (LDLr) and very low (VLDLr) lipoprotein receptor subtypes. In liver, it is the LDLr that is primarily present. The LDLr is down-regulated by exposure to high concentrations of LDL and VLDL (Takahashi et al. 2004). LDLr is controlled by negative feedback, being up- or down-regulated, respectively, by depleted or enhanced concentrations of cellular cholesterol (CHOL) (Peteherych and Wasan 2001). Some lipid-lowering drugs, such as statins, decrease the expression/ function of LDLr (Pocathikorn et al. 2010). Drug distribution or clearance in HL might also be affected by changes in the activities or

Received 9 July 2013. Accepted 21 November 2013. D.R. Brocks, H.R. Chaudhary, M. Ben-Eltriki, M.E. Elsherbiny, and A.O.S. El-Kadi. 2-142H Katz Group Centre for Pharmacy and Health Research, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada. Corresponding author: Dion R. Brocks (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 140–148 (2014) dx.doi.org/10.1139/cjpp-2013-0250

Published at www.nrcresearchpress.com/cjpp on 25 November 2013.

Brocks et al.

expressions of other transport proteins. Although LDLr and VLDLr are not likely to impart much effect on CyA uptake in a tissue such as the kidney (not replete with lipoprotein receptors), there are other transport proteins that, if affected by HL, could lead to changes in cellular uptake and (or) effect. This is a possible concern for CyA, as P-glycoprotein (P-gp) is involved in its transmembrane cellular flux (Magnarin et al. 2004). We have recently reported that the exposure of isolated rat hepatocytes to diluted HL serum was associated with a decrease in metabolism of 2 drugs with a high affinity for lipoproteins (Patel et al. 2012; Brocks et al. 2013). There were also decreased levels of mRNA coding for a variety of drug metabolizing enzymes and transport proteins, including P-gp. These findings may also explain some of the tissue uptake of the drug and the renal toxicity seen in HL rats treated with CyA; the purpose of this report is to describe the results of some experiments designed to give insight into these issues. It was hypothesized that the exposure of cells to serum lipoproteins enhanced CyA-induced reduction in kidney cell viability by reducing the activity of P-gp, and caused changes in the cellular uptake of the drug. Drugs known to modify either the expression of lipoprotein receptors or P-gp, or the uptake of lipoproteins, were used to provide a gauge of the possible importance of each of these proteins in the uptake of the CyA into the cells. Cellular toxicity was also estimated.

Materials and methods Chemicals Chemicals and media components were obtained from Fisher Scientific (Fair Lawn, New Jersey, USA), Caledon Laboratories Ltd. (Georgetown, Ontario, Canada), Sigma–Aldrich (St. Louis, Missouri, USA), and GIBCO (Invitrogen Corporation, Carlsbad, California, USA). To assay for lactate dehydrogenase (LDH), a cytotoxic-one homogeneous membrane integrity assay kit was used (Cyto-Tox-ONE kit; Promega, Madison, Wisconsin, USA). High capacity cDNA (cDNA) reverse transcription kits, and 96-well optical reaction plates with optical adhesive films were purchased from Applied Biosystems (Foster City, Calif.). SYBR-Green Super Mix was purchased from Applied Biosystems (Warrington, Cheshire, UK). Real-time PCR primers were synthesized and supplied by Integrated DNA Technologies, Inc. (Coralville, Indiana, USA). The forward primer sequences for MDR1A/B, LDLr, and GAPDH were 5=-GACAGGACATCAGGACCATCAAT-3=, 5=CAACGGTGGCTGCCAGTAC-3=, and 5=-CAAGGTCATCCATGACAACT TTG-3=, respectively. The corresponding reverse primer sequences for the three genes were 5=-GACGTTTTCTCGGCCATAGC-3=, 5=-GAA CTTGGGTGAGTGGGCAC-3=, and 5=-GGGCCATCCACAGTCTTCTG-3=, respectively. Animals The protocols involving animal use were approved by the University of Alberta Health Sciences Animal Care and Use Committee. Sprague–Dawley rats (Charles River, Quebec, Canada) with body weight ranging from 250–350 g were used as a source of serum and hepatocytes for the studies. The animals were housed in temperature-controlled rooms with 12 h light per day and were fed standard rodent chow containing 4.5% fat (Lab Diet 5001, PMI nutrition LLC, Brentwood, Mo.). The rats had free access to food and water prior to experimentation. To obtain HL serum, rats were administered a single intraperitoneal dose of 1 g·(kg body mass)–1 P407 in saline (Chaudhary and Brocks 2013). The rats were allowed free access to water and food for 36 h. Then, while the rats were anesthetised with isoflurane, blood was collected by cardiac puncture into glass test tubes. The blood was kept at room temperature for 30 min for clotting, then centrifuged for 10 min at 2500g. The serum was separated and stored at –20 °C until used. Hepatocytes were isolated from the rat livers according to previously published methods (Brocks et al. 2012; Patel et al. 2012).

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Cell lines LLC-PK1 (pig epithelial-like kidney cell line) (Perantoni and Berman 1979) and NRK-52E (rat renal proximal tubular cell line) were purchased from the American Type Culture Collection (ATCC). To keep the cells at optimal density for continued growth and to stimulate further proliferation, the cells were subcultured or passaged, and fresh medium was supplied according to the guidelines provided by the supplier. Cells were incubated in O2–CO2 at a ratio of 95%:5%, at 37 °C, in a humidified atmosphere. LLC-PK cells were grown in Medium 199 with a subculture ratio of 1:6 (i.e., cells of one 75 cm2 80% confluent flask were equally divided among six 75 cm2 flasks), whereas NRK-52E cells were grown in Dulbecco's Modified Eagle's media (DMEM) in a subculture ratio of 1:4. During the growth period all media contained 10% FBS, 100 U·mL–1 penicillin, and 100 ␮g·mL–1 streptomycin. For splitting the cells, a monolayer was washed with phosphate-buffered saline (PBS) and cells were detached with 5 mL of trypsin (in a 0.25% solution of PBS–EDTA). For the toxicity, CyA uptake, and P-gp mRNA assessments the LLC-PK1 and NRK-52E cell lines were plated onto 96-well plates (0.2 mL volume). For the rhodamine-123 uptake assessment, NRK-52E cells were plated onto 24-well plates (0.5 mL volume). The final concentration of cells in the wells was approximately 5 × 105 cells·mL–1. Stock solutions CyA stock solutions for hepatocyte experiments were prepared in DMSO. Further dilutions were made in media to obtain the required concentrations. Atorvastatin and fenofibrate were similarly diluted in DMSO. The final concentration of DMSO that the cells were exposed to was less than 0.5%. For the toxicity experiments involving renal tubular epithelial cell lines, Sandimmune formulation (Novartis) was used as a source of CyA (50 mg·mL–1, with each millilitre containing 650 mg Cremophor EL (polyethoxylated castor oil)). It was diluted in normal saline and then diluted further in media when necessary to obtain the required concentrations. The final concentration of Cremophore EL in the CyA-containing incubation wells ranged from 0.013% to 0.065%. For P-gp activity experiments involving NRK-52E cell lines, rhodamine-123 stock solution (121 ␮mol·L–1) was prepared in 0.23% methanol in autoclaved water, and dilutions of this stock (to yield 10 ␮mol·L–1; final concentration) in the cell culture medium were used for cell treatment. Uptake studies Hepatocytes Experiments were conducted to examine the role of lipoproteins and lipid-lowering drugs on CyA hepatic uptake by hepatocytes. Hepatocytes isolated from the 3 normolipidemic (NL) rats were divided into 2 main groups: (i) the pre-incubated serum treatment group, and (ii) the co-incubated serum treatment group. The pre-incubated groups were further subdivided into 4 subgroups, being pre-exposed to either: (i) 10% normolipidemic rat serum; (ii) 10% HL rat serum; (iii) 5 ␮mol·L–1 atorvastatin in 10% NL rat serum; or (iv) 5 ␮mol·L–1 fenofibrate in 10% NL rat serum. Each preincubation phase lasted 24 h at a temperature of 37 °C. Thereafter, medium containing rat serum and drugs was removed and treatment was initiated with the drug, which was incubated with the media containing rat serum. For the rat serum co-incubation groups, 2.5 mg·L–1 CyA was pre-incubated with NL and HL rat serum for 1 h at 37 °C in a shaking water bath. This was carried out to promote the association of CyA with serum lipoproteins. The pre-incubated mixture of drug and serum was then further diluted to 2% in medium, and then added to the wells containing hepatocytes. Thereafter, the media were entirely removed from the wells at 0, 5, 15, 30, and 60 min from the start of incubation to determine the amount of drug accumulated inside the cells. Drug uptake was measured at each time point, with 6 wells being allocated per time point, per rat. Because of technical feasibility issues, hepatocyte Published by NRC Research Press

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sampling had to be split differentially over 3 days using a different rat each day. Therefore a randomly assigned selection of sampling points was made using hepatocytes from each rat on each day of hepatocyte harvesting. For example, in rat 1 the selected sampling times were conducted at 5 min and 30 min for CyA in 2% NL serum incubations for each pretreatment group, and 5 min for 2% HL serum incubations. This was repeated in a different combination of sampling times for the other 2 rats, until 6 replicates of samples for each selected time and CyA+serum treatment was achieved. At each time point, cells were washed twice with 500 ␮L of ice cold PBS. Then, 20 ␮L of methanol, 40 ␮L internal standard solution, 80 ␮L of 1 N NaOH, and 300 ␮L HPLC water were added to the well contents. The well contents were then transferred into microcentrifuge tubes and frozen at –20 °C until the LC-MS analysis was performed for CyA measurement. NRK-52E cells NRK-52 E cells were grown in either rifampin medium (RF, a known P-gp inducer) (Magnarin et al. 2004) or non-RF medium and treated with 10% of NL or HL rat serum, or media only, as mentioned above. Then, after 24 h of pre-incubation, cells were washed with PBS and serum-free medium, and then CyA-containing media (10 or 30 mg·L–1) was added to each well. After treatment with CyA, the medium containing the drug was removed at selected time points (0, 5, 10, 60, and 1440 min), and then the cells were washed with PBS and serum-free medium. HPLC water (500 ␮L) was added to each well and the plates were stored at –20 °C. Internal standard (IS) was added to each well and samples were frozen at –20 °C until assayed. As a measure of P-gp function, there was a preliminary assessment of rhodamine-123 uptake using NRK-52E cells. One day after culture, the cells were exposed to either medium only or media containing 20% NL or HL rat serum. Serum exposure was continued for 24 h. Serum-containing medium was then aspirated and the cells were washed thrice with PBS (37 °C). Thereafter, serumfree medium containing 10 ␮mol·L–1 of rhodamine-123 was added to the cells. At different time points, the medium was aspirated and the cells were washed thrice with ice-cold PBS. Then the cells were lysed by adding 0.5 mL of 0.3 mol·L–1 NaOH that was neutralized with 0.3 mol·L–1 HCl. Rhodamine-123 cellular levels were measured using fluorometry at excitation and emission wavelengths of 485 and 535 nm, respectively. Determination of cell viability Hepatocytes: pretreatment conditions After isolating the hepatocytes, the initial cell viability was determined using the trypan blue exclusion method. For this purpose, 50 ␮L of cell suspension in DMEM was added to 50 ␮L of 0.2% trypan blue solution, after which the cells were counted in 16 microscopic squares and viable cells expressed in millions of cells per millilitre (excluding the dead cells). To assess the effect of time and treatments on hepatocyte viability, both MTT and LDH tests were used as described previously (Korashy and El-Kadi 2008). Briefly, freshly isolated rat hepatocytes were kept in medium containing 10% FBS for 36 h. Hepatocytes were treated for 24 h in the presence of 10% NL/HL rat serum or 5 ␮mol·L–1 of each lipid-lowering drug (atorvastatin or fenofibrate). The medium was removed and replaced with cell culture medium containing 1.2 mmol·L–1 MTT dissolved in PBS (pH 7.4). After 2 h of incubation, the purple crystals that formed were dissolved in isopropanol. The intensity of the color in each well was measured at a wavelength of 550 nm using a Bio-Tek EL 312e 96-well microplate reader (Bio-Tek Instruments, Winooski, Vermont, USA). The percentage of cell viability was calculated relative to the control wells grown in a medium without drugs or rat serum. LDH release was measured with a 10 min coupled enzymatic assay (Cyto-Tox-ONE) that results in the conversion of resazurin into florescent resorufin. The amount of fluorescence (EL 312e microplate reader with excitation/emission wavelengths of 560 and 590 nm; BIO-TEK Instruments) was proportional to the number of cells with a

Can. J. Physiol. Pharmacol. Vol. 92, 2014

damaged membrane. The extent of LDH leakage was calculated relative to controls (cell media only). For MTT and LDH, 3 independent experiments were performed (3 rats and 8 wells per group). Toxicity studies of CyA involving LLC-PK1 and NRK-52E cell lines The relative toxicity of CyA on LLC-PK1 and NRK-52E cell lines in the absence of rat serum was studied using an MTT assay (Korashy and El-Kadi 2008). Cells were divided into 2 groups and grown with or without RF media for 6 days, with CyA treatment initiated on day 7. Cells were then treated with 0, 10, 30, or 50 mg·L–1 of CyA (Sandimmune) for up to 48 h in 96-well plates (n = 6 wells per treatment, per concentration, per time point). Sandimmune was used in these experiments because CyA is given clinically in intravenous doses using this formulation, we used this formulation in our previous studies with rats, and the vehicle may contribute to its toxicity. Percentage cell viability using MTT was calculated relative to the viability of the control group wells. The effects of 10% HL or NL serum pre-incubation on LLC-PK1 and NRK 52E cell viability was also assessed. The respective cells were pre-incubated with NL/HL- or FBS-containing media for 24 h followed by replacement of media with rat-serum-free media containing CyA. Cell viability with the MTT assay was measured as described above for 0 and 24 h. Cytotoxicity was also measured using LDH release as indicated above. mRNA measures using real-time PCR An increase in MDR1 gene expression for LLC-PK1 cells treated with RF-containing medium has been previously demonstrated (Magnarin et al. 2004); however, this has not been assessed for NRK 52E cells. Therefore, NRK-52E MDR1 gene expression was assessed with RF and non-RF treatment using real time PCR. For total isolation of RNA, cells were grown in 6-well plates. Total RNA was isolated from cells using TRIzol reagent according to the manufacturer's protocol. RNA concentration measurement was conducted using a UV spectrometer at 260/280 nm wavelengths. First strand cDNA synthesis was performed according to the manufacturer's instruction, using a High-Capacity cDNA reverse transcription kit (Applied Biosystems). The mRNA of MDR1 and LDLr was determined in the hepatocytes that were pretreated with the NL or HL serum, or the lipid-lowering drugs as described above under the section on “CyA uptake studies”. The methods have been previously described (Brocks et al. 2013), and were similar to those mentioned above for the NRK-52E cells. Cyclosporine A assay A validated LC-MS method was used for the analysis of CyA with minor modification (Kanduru et al. 2010). Briefly, to 100 ␮L of cell suspension was added 40 ␮L of amiodarone (10 ␮g·mL–1) as the internal standard for each sample. Then 500 ␮L of HPLC water and 80 ␮L of 1 mol·L–1 NaOH were added. CyA and internal standard were extracted by adding 4 mL of 95:5 v/v ether–methanol followed by vortex mixing for 60 s. The samples were then centrifuged at 3000g for 10 min. The supernatant was transferred into clean glass tubes and evaporated to dryness in vacuo. The dried samples were then reconstituted with 300 ␮L methanol and 10 ␮L was injected into the LC-MS system. Data analysis For the hepatocyte uptake study, the CyA area under the concentration vs. time curve (AUC) of percent of CyA accumulated in the cells over 1 h was determined. Gene expression data was assessed without log transformation; if normally distributed, the data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test. Non-normal data were analyzed using a Kruskal–Wallis one-way ANOVA followed by Dunn's method of pairwise multiple comparison procedure. The Bailer's method of assessing for significance of comparisons between AUC values was used with Bonferroni correction; ANOVA Published by NRC Research Press

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Fig. 1. CyA uptake (mean ± SD, expressed as % added) by primary rat hepatocytes. Cells were obtained from normolipidemic rats and pre-incubated for 24 h with either 10% normolipidemic (NL) rat serum, hyperlipidemic (HL) rat serum, 5 ␮mol·L–1 atorvastatin in 10% NL serum, or 5 ␮ mol·L–1 fenofibrate in 10% NL serum. After 24 h the media was replaced with media containing CyA in 2% NL or HL rat serum. Livers from 3 rats were used as the source of hepatocytes for each group with 6 wells per time point per rat. Statistically significant differences between amounts accumulated at each time points are shown. CyA, cyclosporine A.

Table 1. Summary of the area under the curve (AUC) for CyA in primary rat hepatocytes when treated with diluted (2%) normolipidemic (NL) or hyperlipidemic (HL) rat serum. Co-incubation CyA in 2% NL serum

CyA in 2% HL serum

CyA

Preincubation conditions (24 h)

AUC0−1 h (mg·min–1·L–1)

% Difference vs. NL (preincubated)

AUC0–1 h (mg·min–1·L–1)

% Difference vs. NL (preincubated)

% Difference, HL vs. NL

A. 10% NL B. 10% HL C. 5 ␮mol·L–1 atorvastatin in 10% NL D. 5 ␮mol·L–1 fenofibrate in 10% NL Ranking

60.0±1.8 66.7±1.6 73.0±1.2

— 11.2 21.6

47.5±0.96 74.0±1.7 57.5±1.2

— 55.8 21.1

−20.8* 10.9* −21.2*

73.8±1.6

23.0

52.5±1.2

10.5

−28.9*

[C = D] > B > A

B>C>D>A

Note: The calculations are based on the data depicted in Fig. 1. *, P < 0.05 for CyA coincubated in 2% HL compared with NL under the same preincubation conditions. The Bailer method of assessing for significance of comparisons between AUC values was used with a Bonferroni correction.

followed by Dunn's post-hoc test was used to assess the significance of differences in concentration between means of the treatment groups at each time point. For comparisons between cell line incubations, t-tests and the Bonferroni correction were used. Data are the mean ± SD, unless otherwise indicated. For all comparisons, results were considered statistically significant when P < 0.05.

Table 2. The relative mRNA levels of MDR1 A and LDLr in hepatocytes. Pretreatment

LDLr

MDR1 A

10% NL Atorvastatin in 10% NL Fenofibrate in 10% NL

1.02±0.23 4.26±0.43a 0.79±0.14a

1.00±0.14 0.68±0.16b 0.90±0.25

Results

Note: GAPDH was used as the housekeeping gene. a, P < 0.05 compared with the other treatments; b, P < 0.05 compared with 10% normolipidemic (NL) only.

Hepatocyte studies Compared with the incubations without rat serum, there was no evidence of cellular toxicity, based on LDH or MTT measures, after 24 h incubations of hepatocytes with either 10% NL or HL serum, 5 ␮mol·L–1 atorvastatin in 10% rat NL serum, or 5 ␮mol·L–1 fenofibrate in 10% rat NL serum (data not shown). In the hepatocytes exposed to CyA–2% NL rat serum, there was an initial spike in the uptake of CyA by the cells regardless of pretreatment (Fig. 1). The rate of uptake thereafter progressively declined over the next 55 min in most of the treatments until an apparent plateau in uptake was reached. Some differences were noticed that depended on whether the drug was added with 2% NL

or HL serum, or between pre-incubation treatments. A plateau in the CyA concentration vs. time curve seemed to be reached earlier with the CyA-2% NL incubations than those involving CyA–2% HL. At each time point there was no clear trend as to which treatment caused higher hepatocyte concentrations of CyA, so the AUC over 1 h was used (Table 1). Pretreatment with 10% NL serum led to the lowest uptake of CyA, depending on whether the cells were coincubated with 2% NL or HL serum. Within the CyA–2% NL incubation group, pre-treatment with the lipid-lowering drugs was associated with a small (⬃20%) but significantly higher uptake of CyA than pretreatment with rat serum. When CyA was added to the cells in a lipoprotein-rich medium (2% HL serum), the highest Published by NRC Research Press

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Fig. 2. LLC-PK1 and NRK-52E cell viability (compared with the CyA-free group at same time) measured by MTT assay after exposure of cells to CyA for 0, 24, and 48 h (n = 6 wells per group). A clear dose- and time-dependent toxicity was seen for NRK-52E, but not LLC-PK1 cells. a, P < 0.05 compared with the same concentration at other time points; *, P < 0.05 compared with the same time point for different concentrations. CyA, cyclosporine A.

uptake of drug into the cells was seen after pretreatment with 10% HL rat serum, followed by atorvastatin and fenofibrate. This increase of CyA in the 10% HL pretreatment group was not only significant, but also substantial (55% greater by comparison with the 10% NL pretreated cells). In comparing CyA–2% HL with the CyA–2% NL treatments, the 2% HL incubations, with the exception of pretreatment with 10% HL, were associated with reduced uptake of CyA (Table 1). The relative mRNA levels of 2 genes possibly involved in lipoprotein-associated CyA uptake were examined in the hepatocytes. As expected, atorvastatin caused a significant and sizable increase in the mRNA of LDLr. It also caused a decrease in MDR1A levels compared with the 10% NL pretreatment. Fenofibrate was found to cause a significant decrease in LDLr mRNA (Table 2). Kidney cell toxicity studies A preliminary assessment was made to assess the relative toxicity of Sandimmune with the rat and pig tubular epithelial cell lines (Fig. 2). A noticeably higher susceptibility of the rat kidney cell line to toxicity in the presence of CyA in vehicle was noted compared with the pig kidney cell line (Fig. 2). Consequently, owing to the relative resistance of LLC-PK1 cells to the toxic effects of CyA, the NRK-52E cell line was used for all of the remaining experiments. In the absence of rat serum, a concentration of 10 mg·L–1 CyA as Sandimmune caused little change in MTT levels in the NRK-52E cells compared with the increased CyA concentrations (Fig. 2). This was consistent with LDH release from the cells exposed to 10 mg·L–1 CyA (Fig. 3, middle panel). Concentrations of 30 and 50 mg·L–1, however, were highly toxic if the cells were exposed to it for 24 h or more. Pretreatment of the NRK-52E cells with either 10% NL or HL rat serum caused no decrease in cell viability (Fig. 3, upper panel). The comparative effects of CyA on NRK-52E viability are depicted in Fig. 3. Compared with the drug-free cells at the same time point, the low dose of CyA (10 mg·L–1) caused no significant toxicity regardless of whether the cells were treated with rifampin or not (Fig. 3). Concentrations of 30 and 50 mg·L–1 of CyA as Sandimmune caused large increases in LDH release into the cell medium at 24 and 48 h after incubation. At the 24 h incubation time, there was more toxicity with the 50 than the 30 mg·L–1 concentration, although there was little difference in LDH release at 48 h for either concentration. Rifampin pretreatment was generally associated with lower LDH release at both 24 and 48 h after incubation, compared with non-rifampin treated cells.

The combined toxic effects of CyA, rifampin, and pretreatment with NL or HL rat serum are shown in Fig. 4. CyA concentrations of 10 mg·L–1 led to small increases in toxicity measures over 48 h. Preincubation with NL or HL rat serum followed by exposure to CyA at 30 and 50 mg·L–1 led to large and significant increases in LDH release by 24 after exposure to the drug. In the uninduced (rifampin-free) cells, there was little difference in LDH release between NL and HL pretreatments, with 2 exceptions: after 24 h at 30 mg·L–1, and after 48 h at 50 mg·L–1. In the rifampin-treated cells, there were generally lower levels of LDH release from the cells. There was also a general increase in LDH release in the HL-pretreated cells compared with those pretreated with NL rat serum. Preincubation with 10% HL rat serum was generally associated with a higher LDH release compared with the NL serum (Fig. 4). In the rifampin-treated cells, there was generally a lower level of LDH release compared with similarly treated uninduced cells. The differences between NL- and HL-rat serum amongst the rifampintreated groups were more numerous than in the uninduced cells, where most groups displayed differences between NL and HL pretreatment conditions. An attempt was made to determine total LDH in the wells by lysing the NRK-52E cells using the lysis solution supplied in the kit. It was found that lysis was not uniform after treatments. With the CyA-free medium and 10 mg·L–1 CyA medium, cells were found to be relatively resistant to lysis. In contrast, those cells treated with 30 and 50 mg·L–1 CyA readily lysed. For this reason it was not possible to normalize the LDH present in the cell media to overall LDH content in the wells. In the measurements of the CyA concentrations in the NRK-52E cells (Fig. 5), cellular concentrations of CyA were significantly higher in the cells treated with 30 mg·L–1 than in the cells treated with 10 mg·L–1, in 14 of the 16 comparator groups, with the differences generally being in the range of 2- to 3-fold. In general, the mean concentrations of CyA were higher in the HL than in the similarly treated NL rat serum treated cells, although significance based on the measured concentrations was only apparent in 3 of the 16 groups. In general, the mean value of CyA uptake into rifampin-treated cells seemed lower than in non-rifampin treated cells, but only in 2 of the 8 comparisons was significance established in the 8 groups treated with 10 mg·L–1, and none in the 8 groups treated with 30 mg/L. To assess the presence of differences between the effects of induction and the effects of rat serum, the data were compiled for all time points and both CyA concentrations to derive the mean Published by NRC Research Press

Brocks et al.

Fig. 3. Effect of CyA concentration, incubation time, and rifampin pretreatment on the release of lactate dehydrogenase (LDH) from NRK-52E cells that were either treated with 10% hyperlipidemic (HL) or normolipidemic (NL) rat serum (no CyA), or without rat serum (untreated) but with 10, 30, or 50 mg·L–1 of CyA (n = 6 wells per group). Each bar represents the mean ± SD of the measured LDH in the media compared with that from cells incubated without drug for the same length of time, with the same pretreatment conditions. *, P < 0.05 for cells incubated at the same concentration compared with other incubation durations; †, P < 0.05 compared with 0 h for the same concentration; ‡, P < 0.05 compared with the rifampintreated cells at the same time and concentration. RF, rifampintreated cells; C, control cells; CyA, cyclosporine A.

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and confidence intervals for the ratios of HL to NL, and for the control groups compared with the rifampin-treated cells. For the control cells, the mean intracellular CyA concentration ratio of HL:NL and the 95% confidence interval (CI) was 1.26 (1.04–1.48); for rifampin-treated cells the corresponding values were 1.53 (1.33– 1.72). The overall control to rifampin mean ratio and CI was 1.34 (1.12–1.55). Since none of the CI encompassed 1.0, this suggested that overall, HL caused a relative increase in CyA uptake, and that rifampin caused a decrease in uptake. There was a 7.2 ± 3.7 fold increase in MDR1A noted in the rifampintreated NRK-52E cells. To assess the functional activity of P-gp in the presence of rat serum, cells were spiked with rhodamine-123 and the uptake was assessed. In the cells not treated with rat serum, a preliminary experiment was undertaken to assess the initial influx of MDR1A into the cells. A linear uptake by the cells was noted over the first 6 h after exposure to rhodamine-123 (Fig. 6 left panel). The uptake was then carried out in cells that were pretreated with 20% NL or HL rat serum (Fig. 6 right panel). HL-pretreated cells were associated with a significantly higher uptake of rhodamine-123 compared with the NL rat serum, at each of the selected time points.

Discussion The cause of increased kidney and liver CyA concentrations in HL vs. NL rats observed in vivo was the main impetus for performing these experiments (Aliabadi et al. 2006). In HL rats the plasma protein binding of several drugs with a low hepatic extraction ratio (including CyA) is known to be increased. Although this is expected to have an overall neutral effect on tissue uptake, increases were actually observed in some tissues (Shayeganpour et al. 2008; Patel et al. 2009). For CyA, this notably included increases in kidney, the main site of toxicity, and liver, its primary site of elimination (mostly by metabolism). CyA is also a known substrate for P-gp, which may influence its absorption and its tissue concentrations. Inhibition of P-gp can worsen the renal toxicity caused by CyA (Anglicheau et al. 2006). There is also some information suggesting that in the intestine, lipids, and (or) lipoproteins might inhibit P-gp expression or activity (Kim et al. 2004; Custodio and Benet 2005). CyA is extensively but slowly metabolized (primarily by CYP3A), is highly plasma protein bound (>70% in rat) (Brocks et al. 2006), and is a substrate for association with lipoproteins. In contrast to expectations based on an increase in plasma protein binding, healthy subjects given a fatty meal with CyA were seen to have an increased clearance and volume of distribution (Gupta and Benet 1990). A similar trend was seen in the plasma concentrations of rats given peanut oil together with the drug, which suggested a postprandial lipoprotein-mediated increase in the cellular uptake of drug by the liver. This was likewise also seen in HL rats in not only liver, but also in kidney (Brocks et al. 2006). It was hypothesized that for CyA, there may have been an increase in clearance owing to an increased delivery of drug to the liver, secondary to the effects of LDLr, leading to increased metabolism and clearance. When an attempt was made to upregulate LDLr by administration of ethinyl estradiol to rats, however, there was no increase in uptake of CyA into the isolated perfused rat livers when coinfused with human LDL. This leaves open the possibility that another factor may be the cause, or that perhaps the findings were affected by experimental factors related to the in-vitro methodology used. In the hepatocyte uptake experiments, the influence of preincubation with HL serum and lipid-lowering drugs was explored combined with co-incubation in CyA with NL or HL rat serum. The concentration of 10% or 20% HL serum represented lipid levels about 3- to 12-fold higher than what would be present in the plasma of NL rats in vivo. This level of increase approximately matches what would be seen in plasma of HL vs. NL patients (Nikkila et al. 1976; Chaudhary and Brocks 2013). We used 2% rat serum for the coincubations to minimize any direct cellular changes (e.g., P-gp or Published by NRC Research Press

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Fig. 4. Effect of CyA concentration, incubation time, and rifampin (RF) pretreatment on the release of lactate dehydrogenase (LDH) from NRK-52E cells that were pretreated with normolipidemic (NL) or hyperlipidemic (HL) rat serum (n = 6 wells per group). Each bar represents the mean ± SD of the measured LDH in the media compared with that from cells incubated without drug for the same length of time, with the same pre-treatment conditions. CyA was added as Sandimmune. *, P < 0.05 compared with cells incubated with the same serum type and RF-treated cells (same concentration and incubation time); †, P < 0.05 compared with cells of the same induction status and HL serum (same concentration and incubation time). CyA, cyclosporine A.

Fig. 5. Measured cyclosporine concentrations within NRK-52E cells (n = 3–4 wells per group) exposed to either 10 mg·L–1 (left panel) or 30 mg·L–1 CyA (right panel) as Sandimmune. The incubation times at which the measurements were taken were 0.083, 0.167, 1, or 24 h after incubation with CyA. CyA, cyclosporine A; C, control cells (untreated); R, rifampin-treated cells; NL, cells exposed to normolipidemic rat serum; HL, cells exposed to hyperlipidemic rat serum. *, P < 0.05 compared with cells incubated in 30 mg·L–1 for the same time and preincubation conditions; †, P < 0.05 compared with cells incubated with HL or NL rat serum for the same duration, concentration, and rifampin status; ‡, P < 0.05 compared with the cells treated with rifampin for the same duration and rat serum status.

LDLr expression) while allowing as much as possible of the drug to be incorporated (especially with HL coincubations) into lipoprotein particles, and thus facilitate lipoprotein-receptor mediated uptake of the drug. Our intention was also to minimize binding of the drug to other proteins such as rat albumin. Fibrates exert their lipidlowering action through modification of the density of LDL particles, which effectively increases the proportion LDL particles with a higher density (Farnier 2008; Guerin et al. 1996). The statin drugs upregulate LDL, thus increasing the clearance of LDL particles by the liver (Dergunov et al. 2008; Pocathikorn et al. 2010). Both of these effects of fenofibrate and atorvastatin would be expected to increase the hepatocellular uptake of CyA-laden lipoprotein particles. As expected, we found that there was a significant increase in LDLr mRNA in hepatocytes that were exposed to atorvastatin (Table 1). We have recently reported that in rat hepatocytes treated

with 5% HL rat serum there were decreases in the mRNA of a number of cytochrome P450 isoforms, transport proteins, and lipoprotein receptors, including LDLr and MDR1A (Brocks et al. 2012). In general, the uptake of CyA into hepatocytes co-incubated with 2% NL serum showed relatively minor differences, with initial uptake being somewhat higher in cells pretreated with lipid-lowering drugs. When coincubated with 2% HL serum, the highest uptake was seen with the cells preincubated with 10% HL serum. Indeed, when comparing HL with NL co-incubation for each of the other pre-incubation groups, this was the only group in which the uptake was increased; a finding inconsistent with an increase in LDLr receptors. For the lipidlowering drugs and 10% NL serum treatment groups, a decrease in uptake with HL co-incubations could be attributed to increased plasma protein binding, but this would also have been the case for the combined pre- and co-treatment with HL group. Hence, plasma Published by NRC Research Press

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Fig. 6. Rhodamine-123 uptake into attached NRK-52E cells (expressed in pmol·mL–1 equivalents). Left panel: uptake into cells vs. time when not exposed (unexposed) to rat serum. Right panel: relative effects of rhodamine-123 uptake into cells after being exposed to 20% normolipidemic (NL) or hyperlipidemic (HL) rat serum for 24 h. The uptake is expressed as the relative uptake of rhodamine-123 in the absence of rat serum proteins (left panel). At each time point (n = 6 wells per group) there was a significant difference between HL and NL serum pretreatment (P < 0.05).

protein binding did not seem to be involved in the differences between the groups. The exposure of HL serum to hepatocytes decreases the mRNA levels of both LDLr and MDR1A, but especially the latter (Brocks et al. 2012). A decrease in LDLr would have been expected to decrease the uptake of CyA into the cells. On the other hand a decrease in MDR1A mRNA and its coded protein P-gp would be expected to cause an increase in CyA uptake. There was some increase in CyA uptake in the 10% HL serum pre-incubation group when CyA was co-incubated with 2% NL serum, which would be consistent with a downregulation of MDR1A. When the drug was co-incubated with 2% HL serum and the same type of pretreatment, however, the effect was magnified. As one possible explanation, this could indicate a direct interaction of lipoproteins with P-gp thereby magnifying the effect beyond that seen with 2% NL serum coincubations. Regardless, the finding is consistent with the observed increase in drug uptake in liver of HL rats in vivo (Brocks et al. 2006). With respect to renal cellular toxicity assessed using MTT measures and LDH release, we found that the NRK-52E cell line was more sensitive to CyA than was the LLC-PK1 cell line over the concentration range 10 to 50 mg·L–1 (Fig. 2). Although in the NRK-52E cell line 10 mg·L–1 CyA caused little increase in LDH release, a noticeable increase was noted in response to CyA concentrations of 30 and 50 mg·L–1. The release of LDH also progressively increased up to 48 h after incubation. For this reason further experiments were conducted using the NRK-52E cell line. This also had the advantage of being a rat cell line, the same species for which much of the in vivo comparator data was based and from which serum was used as a source of lipoproteins. This diminished any concerns regarding possibly diminished interspecies-related LDLr-apoprotein recognition, which is present if LDL from another species is used. One of the hypotheses for the increased kidney CyA concentrations in HL rats in vivo was related to a reduction in P-gp activity. It was previously shown that P-gp was induced in response to exposure of LLC-PK1 cells to rifampin (Magnarin et al. 2004). We likewise found that rifampin was capable of increasing the levels of mRNA for MDR1A in NRK–52E cells, which presumably led to higher P-gp function in the cells. The Sandimmune formulation was used for these toxicity experiments in NRK-52E cells, to match our experiments with those done in vivo where injectable Sandimmune was administered to rats (Aliabadi et al. 2006; Brocks et al. 2006). Cremophore EL, which is a constituent of the formulation, has been shown to cause a low level of damage to kidney cell lines within the range

of concentrations used in our experiments (Sokol et al. 1990; Nassberger et al. 1991; Jiang and Acosta 1993). The data indicated that rifampin pretreatment afforded a protective effect against the toxicity of CyA, which was in line with the increase in MDR1A mRNA in rifampin-treated cells. The findings also suggested that pretreatment with HL serum caused some reduction in the protective effect of rifampin in the NRK-52E cell line. An attempt was made to link the toxicity data with that of CyA cellular concentrations (Fig. 5), at the 2 lower concentrations of drug (10 and 30 mg·L–1). The higher CyA concentration was associated with higher cellular concentrations of CyA, consistent with the higher level of toxicity seen as measured by LDH release (Fig. 4). The effects of rifampin and lipoproteins were less clear, although the 95% confidence intervals of HL:NL and uninduced:induced CyA cellular concentrations did not include one (Fig. 5). In general, the CyA cellular concentrations were consistent with the toxicity data and suggested that HL diminished the function of P-gp, which was in turn consistent with the increase in cellular rhodamine-123 when the cells were preincubated with HL serum (Fig. 6). The rhodamine 123 assessment was performed prior to the CyA toxicity studies, and involved 20% rat serum. To reduce the possibility of incomplete washout of rat serum in the follow-up studies involving CyA and the associated confounding effect of lipoprotein binding, a lower concentration of 10% rat serum was used in those studies. Our results in the NRK-52E cells could be compared with those of another study (Peteherych and Wasan 2001). Using LLC-PK1 cells and human lipoproteins, it was reported that human LDL (co-incubated, with or without pre-incubation) caused a decrease in signs of toxicity (as measured by radiolabeled leucine uptake) and of intracellular levels of radiolabelled CyA. In human plasma, the unbound fraction of CyA in vivo is considerably lower than that in rat plasma, suggesting that human lipoproteins bind more readily than rat lipoproteins to CyA (Brocks et al. 2006). The protective effect of lipoproteins in LLC-PK1 cells could partly be explained by a high binding of CyA to lipoproteins containing human apoproteins, which might not be readily recognized by the pig LDLr (Peteherych and Wasan 2001). Besides different study goals and the use of a different cell line, the experiments differed in a number of methodological factors, including the presence or absence of lipoproteins during incubation with CyA, use of same species lipoproteins, use of a specific assay for CyA, and use of different measures for assessment of toxicity. Published by NRC Research Press

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Conclusions In hepatocytes, the HL-associated increase in CyA concentrations seemed to be attributable primarily to the effects of decreased P-gp activity, more so than increased LDLr. A diminishment of P-gp activity was also observed in HL pretreated cells, which appeared to contribute to an increase in the HL:NL ratio of CyA concentrations and an increase in toxicity. Although caution is needed in comparing in vitro experimental data with that in vivo, the in vitro data are nevertheless consistent with in vivo findings where increases in AUC of CyA in kidney and liver, and histological changes in kidney cells, were observed.

Acknowledgements M.B.E. was a recipient of a studentship award from the Libyan Ministry of Education and Scientific Research.

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Effects of serum lipoproteins on cyclosporine A cellular uptake and renal toxicity in vitro.

In-vitro studies were performed to shed light on previous findings that showed increased uptake of cyclosporine A in the kidneys and liver of hyperlip...
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