ACTION OF DICLOFENAC AND MELOXICAM ON NEPHROTOXIC CELL DEATH NG LIN ENG (BSc. Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to thank my supervisor, Professor Sit Kim Ping and Professor Barry Halliwell for providing me with the opportunity to undertake this interesting project. Prof. Sit has provided excellent guidance and deep insights, which were of great value to me throughout my project. Colleagues in the lab – Annette and Hwee Ying are greatly thanked for the technical assistance and excellent advices rendered along the way. I would also like to thank Yie Hou for providing me guidance in performing western blot and other advices in helping me to complete my project. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT vi LIST OF TABLES a LIST OF FIGURES b 1. INTRODUCTION 1.1 Overview of nonsteroidal anti-inflammatory drugs (NSAIDs) – COX-2selective and nonselective NSAIDs 1.2 Adverse effects of nonselective and COX-2-selective NSAIDs 1.3 Nephrotoxicity of diclofenac and meloxicam 1.4 Susceptibility of the kidney to toxic injury 1.5 Mechanisms of NSAID-induced nephrotoxicity 1.6 Role of mitochondria in drug-induced cell death 10 1.7 Aims of the project 11 2. MATERIALS AND METHODS 13 2.1 Chemicals 13 2.2 Isolation of rat kidney mitochondria 14 2.3 Measurement of mitochondrial respiration by oxygen consumption 14 2.4 Monitoring of mitochondrial membrane potential in isolated mitochondria by JC-1 15 2.5 Biosynthesis of ATP in isolated mitochondria 16 2.6 Measurement of NADH dehydrogenase (Complex I) activity 17 2.7 Measurement of glutamate dehydrogenase (GDH) and malate dehydrogenase (MDH) activities using mitochondrial extracts 17 2.8 Measurement of malate-aspartate shuttle activity 18 ii 2.9 Measurement of intra-mitochondrial NAD(P)H generated from glutamate/malate 19 2.10 Mammalian cell culture 19 2.11 Cell treatment with drugs 20 2.12 Phase-contrast microscopy 20 2.13 Assessment of cell viability 20 2.14 Measurement of intracellular ATP content 21 2.15 Measurement of caspase-3, -8 and -9 activities 21 2.16 Annexin V-FITC/propidium iodide (PI) double staining 22 2.17 Preparation of cytosolic fractions for western blot analysis 23 2.18 Western blot analysis 24 2.19 Statistical analysis 25 3. RESULTS 3.1 3.2 26 Action of diclofenac and meloxicam on kidney mitochondrial function 26 3.1.1 Uncoupling of oxidative phosphorylation 26 3.1.2 Loss of mitochondrial membrane potential (MMP) 29 3.1.3 Inhibition of ATP biosynthesis 32 3.1.4 Effect of diclofenac on NADH dehydrogenase (Complex I) activity 34 3.1.5 Effect of diclofenac on GDH and MDH activities 36 3.1.6 Inhibition of malate-aspartate shuttle by diclofenac 38 3.1.7 Inhibition of the intra-mitochondrial production of NAD(P)H by diclofenac 40 Studies with cultured kidney cell lines – MDCK II and LLC-PK1 42 3.2.1 Drug effects on cell viability 42 3.2.2 Effects of etoposide, meloxicam and diclofenac on the cellular morphological changes 44 iii 3.2.3 Activities of caspase-3, -8 and -9 48 3.2.4 Release of cytochrome c 50 3.2.5 Effects of drugs on intracellular ATP level 54 3.2.6 Externalization of phosphatidylserine (PS) and loss of plasma membrane integrity 56 4. DISCUSSION 4.1 4.2 61 Studies in isolated kidney mitochondria 61 4.1.1 Uncoupling of oxidative phosphorylation, decrease in mitochondrial membrane potential and inhibition of ATP biosynthesis 61 4.1.2 Inhibition of malate-aspartate shuttle by diclofenac 64 Studies in cultured kidney cells – MDCK II and LLC-PK1 67 4.2.1 Diclofenac was more toxic than meloxicam and LLC-PK1 cells were more sensitive to drug toxicity than MDCK II cells 69 4.2.2 Different mode of cell death induced by meloxicam and diclofenac in MDCK II cells 70 4.2.2.1 Cellular morphological changes of MDCK II cells 70 4.2.2.2 Caspase activation and the release of cytochrome c 71 4.2.2.3 Elevation of intracellular ATP supporting apoptosis 74 4.2.2.4 Externalization of PS and loss of membrane integrity 75 4.2.3 Meloxicam and diclofenac caused a caspase-independent necrosis in LLC-PK1 cells 77 4.2.3.1 Cellular morphological changes in LLC-PK1 cells 77 4.2.3.2 Lack of caspase activation and elevation of cytosolic ATP in spite of presence of cytosolic cytochrome c 78 4.2.3.3 Detection of early apoptosis by annexin V which eventually switched to necrosis 80 Conclusion 83 5. REFERENCES 85 4.3 iv ABSTRACT Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which inhibits both isoforms of cyclo-oxygenase: COX-1 and COX-2. Its nephrotoxicity has been reported to be fatal to vultures but this was not so with meloxicam, a COX-2 selective NSAID. The present study aimed to investigate the difference in toxicity of meloxicam and diclofenac using isolated kidney mitochondria and cultured MDCK II and LLC-PK1 renal cells, which represent renal distal and proximal tubular cells, respectively. Both drugs were shown to cause mitochondrial dysfunction by uncoupling oxidative phosphorylation resulting in a compromise in mitochondrial membrane potential and a decrease in the rate of ATP biosynthesis. However, ATP biosynthesis from the oxidation of glutamate/malate was significantly more compromised compared to that of succinate when the mitochondria were incubated with diclofenac; this phenomenon was absent under meloxicam treatment. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. It was therefore concluded that the decreased NAD(P)H production due to the inhibition of the entry of malate and glutamate via the malateaspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate/malate by diclofenac. Experiments using cultured kidney cells showed that both meloxicam and diclofenac decreased the viability of MDCK II and LLC-PK1 cells. Diclofenac was more toxic than meloxicam to both cell lines. LLC-PK1 cells were more sensitive to both drugs compared to MDCK II cells. In an attempt to elucidate the mechanism of cell death induced by diclofenac and meloxicam, it was found that exposure of MDCK II cells to meloxicam caused the appearance of v apoptotic bodies. This was accompanied by positive annexin V-FITC staining, elevation of intracellular ATP, activation of caspase-9 and caspase-3 and release of cytochrome c, implicating an intrinsic mitochondrial cell death pathway by apoptosis. Diclofenac-treated MDCK II cells on the other hand showed apoptotic features during early cell death but switched to necrosis after extended period of drug exposure as evidenced by the increased propidium iodide staining with cell remnants remained attached to the culture flasks. The mode of cell death in LLC-PK1 cells was however less well-defined with positive annexin V-FITC staining and release of cytochrome c into cytosol but minimal increase in caspase-3 activity with no elevation of intracellular ATP level, suggestive of a caspase-independent pathway. Propidium iodide staining revealed that a huge population of drug-treated LLC-PK1 cells was undergoing necrosis. In short, both diclofenac and meloxicam uncoupled oxidative phosphorylation but different modes of nephrotoxic cell death had been identified: MDCK II cells treated with meloxicam seemed to die by apoptosis, but diclofenac seemed to favor necrosis. A significant fraction of cell death induced by meloxicam and diclofenac in LLC-PK1 cells was caspase-independent and most likely to involve necrosis. vi LIST OF TABLES Table # Title Page Respiratory substrates and inhibitors used in the measurement of the rate of ATP biosynthesis in isolated kidney mitochondria. 16 Fluorogenic substrates used in the determination of caspase activity 22 Comparison of the effects of meloxicam and diclofenac on MDCK II and LLC-PK1 cells. 82 a LIST OF FIGURES Figure # Title Page Mechanism of action of NSAIDs Effect of diclofenac (A, B) and meloxicam (C, D) on mitochondrial respiration using succinate (A, C) or glutamate/malate (B, D) as substrates 27 Action of diclofenac (A, B) and meloxicam (C, D) on mitochondrial membrane potential measured with JC-1 in rat kidney mitochondria 30 Effect of diclofenac (A) and meloxicam (B) on the rate of ATP biosynthesis in rat kidney mitochondria 33 Representative recordings of NADH dehydrogenase activity using rat kidney mitochondrial extracts 35 Effect of diclofenac on (A) GDH and (B) MDH activities measured in both forward and reverse reactions 37 Inhibition of the malate-aspartate shuttle by diclofenac 39 Measurement of the production of intra-mitochondrial NAD(P)H in rat kidney mitochondria 41 Effects of meloxicam and diclofenac on cell viability 43 10 Morphological changes of MDCK II cells following drug exposure 46 11 Morphology of LLC-PK1 cells following drug exposure 47 12 Caspase activation by etoposide (Eto), meloxicam (Mel) and diclofenac (Dcf) in MDCK II cells 49 13 Caspase-3 activity measured in LLC-PK1 cells 50 14 Drug-induced release of cytochrome c in MDCK II cells 52 15 Drug-induced release of cytochrome c in LLC-PK1 cells 53 16 Intracellular ATP level following drug exposure 55 17 Mode of cell death as revealed by annexin V-FITC and propidium iodide (PI) double staining in MDCK II cells 58 b 18 Mode of cell death as revealed by annexin V-FITC and propidium iodide (PI) double staining in LLC-PK1 cells 59 19 Presentation of the flow cytometry data in the form of bar graph 60 20 Uncoupling of oxidative phosphorylation by 2,4-dinitrophenol DNP 63 21 Schematic of malate-aspartate shuttle 65 22 Two different modes of cell death 68 23 Diagram showing the effects of meloxicam and diclofenac on MDCK II cells in terms of the release of cytochrome c and caspase activation 73 24 Use of annexin V-FITC and PI for the identification of live, apoptotic and late apoptotic/necrotic cells 76 25 Diagram showing the effects of meloxicam and diclofenac on LLC-PK1 cells in terms of the release of cytochrome c and caspase activation 80 c death. Due to the existence of the possibility that the double-stained cells might be represented by the late apoptotic cells, the prevalence of necrosis in diclofenac-treated cells has to be confirmed with the results obtained from other assays. From the changes of cellular morphology of the diclofenac-treated cells, it was observed that the formation of apoptotic bodies was always less than the cells treated with either etoposide or meloxicam. The caspase assay results were then found to correlate with this observation such that the caspase activity was not much elevated by diclofenac treatment. Taken together, these results suggested that while meloxicam elicited the classical apoptotic cell death pathway similar to that of etoposide treatment in MDCK II cells, diclofenac treatment caused a switch from apoptosis to necrosis during the extended period of exposure to a relatively high dose of drug. 4.2.3 Meloxicam and diclofenac caused a caspase-independent necrosis in LLC-PK1 cells 4.2.3.1 Cellular morphological changes of LLC-PK1 cells Morphological changes in LLC-PK1 cells were observed after h of incubation with etoposide, meloxicam and diclofenac. The changes were totally different from those seen in MDCK II cells, which showed black particulates between cells and on cell surfaces and the cell boundaries became less distinct. These black particles appeared to represent discharged cellular contents giving the cells a “contaminated” appearance. Later most of the dead cells were seen to be clumped together and they floated in the media. From the morphological changes observed, it seemed to be unlikely that the cells were dying via apoptosis. Features of apoptotic cells such as cell rounding and detachment from the flask and the formation of apoptotic bodies 77 were absent. Hence, the caspase-3 activity of the drug-treated cells was examined to investigate the involvement of apoptosis in these cells. 4.2.3.2 Lack of caspase activation and elevation of cytosolic ATP in spite of presence of cytosolic cytochrome c Cellular ATP in LLC-PK1 cells remained unchanged with minimal activation of caspase-3 activity in drug-treated LLC-PK1 cells. This lack of caspase activation concurred with the morphological alterations being observed, thus suggesting that apoptosis might not be the mechanism of cell death for these drug-treated LLC-PK1 cells, at least not via the classical apoptotic pathway. Despite the low caspase-3 activity, cytochrome c was found to be released from to h post-drug exposure in meloxicam-treated cells. This indicates that apoptosis might be initiated but it involves a caspase-independent pathway (Shih et al., 2003). Such a caspaseindependent mode of cell death has been reported to occur in many cell types including primary renal proximal tubular cells (Cummings et al., 2004; Ishido et al., 1999). In the absence of caspase activity, a number of cell events associated with apoptosis can still occur including PS externalization (Leist and Jaattela, 2001); this was also observed in the current study which will be discussed in a later section. Fig. 25 summarizes the effects of meloxicam and diclofenac on cytochrome c release and caspase activation in LLC-PK1 cells. Despite the fact that most events in apoptosis appear to require a caspasemediated proteolytic step, there is evidence that apoptosis-like cell death can occur without the activation of effector caspases (Leist and Jäättelä, 2001). According to Leist and Jäättelä (2001), there are four patterns of death: apoptosis, apoptosis-like programmed cell death, necrosis-like programmed cell death and accidental necrosis. 78 Most published forms of caspase-independent apoptosis belongs to the apoptosis-like programmed cell death. In this kind of cell death, many non-caspase proteases can cleave at least some of the classic caspase substrates, but further study is needed to define the role of the individual proteases in the complex process of programmed cell death. Besides cytochrome c (Liu et al., 1996), another mitochondrial protein, the apoptosis-inducing factor (AIF) also plays a key role in apoptosis, particularly in caspase-independent apoptosis (Susin et al., 1999; Zamzami and Kroemer, 1999; Shih et al., 2003). In the study on cadmium-induced apoptosis by Shih et al. (2003), it was shown that cadmium conducts a caspase-independent pathway through the collapse of the membrane potential of mitochondria and subsequent translocation of AIF into nucleus. AIF causes partial chromatin condensation with high molecular weight DNA fragmentation that differs from those by caspase-activated-DNase (CAD) (Zamzami and Kroemer, 1999). Whether this AIF contributes to the caspase-independent cell death in drug-treated LLC-PK1 cells merits further investigation. 79 Mitochondria Meloxicam cyt.c Diclofenac cyt.c Caspase-3 AIF ?? Caspaseindependent cell death Necrosis-like cell death Fig. 25. Diagram showing the effects of meloxicam and diclofenac on LLC-PK1 cells in terms of the release of cytochrome c and caspase activation. Dotted arrows represent unidentified mechanisms involved in the meloxicam- and diclofenacinduced cell death. 4.2.3.3 Detection of early apoptosis by annexin V which eventually switched to necrosis Using the annexin V-FITC and PI double staining, it was shown that there was early apoptosis in diclofenac- and meloxicam-exposed LLC-PK1 cells, which was followed by necrosis as revealed by the strong PI staining. The early binding (3 h time point) of the drug-treated cells with annexin V without the loss of plasma membrane integrity suggested that apoptosis was initiated as an early response towards the chemical insult, which was in agreement with the results obtained from the detection 80 of the release of cytochrome c. Therefore, the early detection of apoptosis with the lack of caspase activation implied that the diclofenac- and meloxicam-induced cell death in LLC-PK1 cells was due to a caspase-independent pathway, rather than the classical apoptosis which was observed in the meloxicam-treated MDCK II cells. It is worth noting that in both cell lines, diclofenac treatment always resulted in more necrotic cells compared to meloxicam treatment. The flow-cytometric data thus corroborated the cell viability test that diclofenac is more toxic than meloxicam. The effects of diclofenac and meloxicam on MDCK II and LLC-PK1 cells are summarized and shown in Table 3. 81 Table 3. Comparison of the effects of meloxicam and diclofenac on MDCK II and LLC-PK1 cells. MDCK II LLC-PK1 Meloxicam Diclofenac Meloxicam Diclofenac Cell rounding +++ + - - Cell detachment +++ + + + Formation of apoptotic bodies +++ + - - Necrotic cell remnants remain attached to the culture flask - +++ - - Cellular discharge into media - - ++ +++ Increase in intracellular ATP ++ ++ - - Caspase-9/caspase-3 activity +++ ++ + + Cytochrome c release +++ ++ +++ + Externalization of PS +++ ++ ++ +++ Loss of membrane integrity ++ +++ ++ +++ Morphological changes Biochemical changes A scale of + to +++ is used with the latter showing highest increase compared to control; - : absent or no significant increase compared to control. 82 4.3 Conclusion From the findings obtained from the experiments using isolated kidney mitochondria and whole cells, it can be concluded that both diclofenac and meloxicam are toxic to renal cells possibly by disrupting the mitochondrial function. Both drugs were shown to act as uncouplers by dissipating the mitochondrial membrane potential and hence resulted in the compromise in ATP biosynthesis. Diclofenac was found to be more toxic due to its capability in inhibiting the malateaspartate shuttle and therefore limits the bioavailability of the two main substrates – glutamate and malate. By compromising the mitochondrial function, it is not surprising that diclofenac and meloxicam could induce nephrotoxic cell death. However, the interesting finding is that diclofenac is indeed more toxic than meloxicam and the mode of cell death varied between different drugs and cell lines. Meloxicam caused the formation of apoptotic bodies, elevation of intracellular ATP supporting caspase-9/caspase-3 activation, release of cytochrome c and PS externalization in MDCK II cells which are characteristic features of the classical intrinsic mitochondrial apoptotic pathway (Padanilam, 2003). The apparent lack of caspase-8 activity precluded the involvement of the extrinsic death-receptor mediated pathway (Padanilam, 2003). Diclofenac-induced cell death in MDCK II cells however showed a switch from apoptosis to necrosis after a prolonged period of drug exposure. The caspase-independent apoptosis in LLC-PK1 cells also resulted in eventual necrosis-like cell death with diclofenac-treated cells being more severely damaged than meloxicam-treated cells. These findings supported the preference of meloxicam over diclofenac for veterinary use in life stock as proposed by Swan et al. (2006). The more damaging cell death pathway by diclofenac in both proximal and distal kidney tubular cells could be the cause of acute renal failure reported in man and other 83 animals (Kulling et al., 1999; Rubio-Garcia and Tellez-Molina, 1992; Cicuttini et al., 1989; Schwartz et al., 1988; Schwaiger et al., 2004; Taib et al., 2004). 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Chem. 279, 43035-43045. 92 [...]... focus on their mechanism(s) of cell death, to investigate if the meloxicam- induced cell death was different from that of diclofenac Assessment of the effects of diclofenac and meloxicam on mitochondrial function was also carried out to investigate the effects of the drugs at subcellular level The role of mitochondria in cell death was examined as well in terms of the release of cytochrome c in cells... mitochondrial toxicity are inhibition of electron flow across electron transport chain, uncoupling of oxidative phosphorylation, opening of mitochondrial permeability transition pore (MPTP), inhibition of mitochondrial fatty acid metabolism, oxidation of mitochondrial DNA and inhibition of mitochondrial DNA synthesis Given that mitochondrial oxidative processes are vital in the maintenance of cellular... significant, and represented by asterisks: *, P < 0.05; **, P < 0.005 25 3 RESULTS 3.1 Action of diclofenac and meloxicam on kidney mitochondrial function 3.1.1 Uncoupling of oxidative phosphorylation The in vitro interference of diclofenac and meloxicam with the respiration of rat kidney mitochondria was evaluated for glutamate plus malate (complex I substrates) and succinate (complex II substrate) oxidation... substrate) oxidation (Fig 2) Mitochondria were preincubated with various concentrations of the drugs and their effects on state-4 and state-3 respiration were studied For succinate oxidation, both diclofenac and meloxicam at 50 µM stimulated state-4 respiration (after the addition of oligomycin) but depressed state-3 respiration (after the addition of ADP) of mitochondria (Fig 2A and C) This resulted in a decrease... induction of MPTP with subsequent rapid depletion of energy-rich phosphates and the disruption of plasma membrane integrity causes necrosis while a regulated induction of MPTP allows for the activation and action of proteases and thus giving rise to the apoptosis phenotype On the other hand, Lemasters et al (1999) suggested that the progression to necrotic and apoptotic cell killing depends in part on. .. other nonselective NSAIDs 1.3 Nephrotoxicity of diclofenac and meloxicam The adverse drug effects associated with NSAIDs are of particular concern when continuous NSAID therapy is needed, such as in treatment of various rheumatological disorders Diclofenac and meloxicam are widely used for the reduction of inflammation and pain associated with arthritis and other conditions such as rheumatoid arthritis,... containing 0.2 and 0.02 mg protein for the GDH and MDH assays, respectively (Williamson and Corkey, 1969; Heyde and Ainsworth, 1968) Mitochondrial extracts were pre-incubated in the absence or presence of 10 100 µM diclofenac prior to the initiation of the reaction The concentrations of the substrates for the GDH assay are 10 mM α-ketoglutarate and 0.2 mM NADH for the forward reaction and 20 mM glutamate... fragmentation, which ultimately translate into apoptotic cell death of kidney cells Compared to diclofenac, study on meloxicam is limited with only one report suggesting that meloxicam behaves as an uncoupler by stimulating basal respiration, inhibiting ATP biosynthesis and depressing mitochondrial membrane potential (Moreno-Sanchez et al., 1999) 1.6 Role of mitochondria in drug-induced cell death The mitochondrion... incubation medium used to measure mitochondrial respiration consisted of 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM Hepes, 110 mM sucrose and 1 g/L BSA (pH 7.1) The assay was initiated by adding a mitochondrial preparation containing 0.3 mg protein to the oxygraph cell containing the incubation medium For the examination of the effect of 14 diclofenac/ meloxicam on. .. production in untreated control cells and the cell viability was expressed as percentage of control (Mosmann, 1983) 2.14 Measurement of intracellular ATP content Cells growing on 24-well plates were lysed with 50 µl of ice-cold lysis buffer (1 M Tris, pH 7.4, 5 M NaCl, 0.5 M EDTA, 0.1 M sodium vanadate, 1% Triton X-100 and 1% NP-40) for 5 min on ice The cells were then scraped off the plate and transferred . Mechanism of action of NSAIDs 2 2 Effect of diclofenac (A, B) and meloxicam (C, D) on mitochondrial respiration using succinate (A, C) or glutamate/malate (B, D) as substrates 27 3 Action of diclofenac. ACTION OF DICLOFENAC AND MELOXICAM ON NEPHROTOXIC CELL DEATH NG LIN ENG (BSc. Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF. the effects of meloxicam and diclofenac on MDCK II cells in terms of the release of cytochrome c and caspase activation 73 24 Use of annexin V-FITC and PI for the identification of live, apoptotic