Effect of mycophenolate mofetil on nitric oxide production and inducible nitric oxide synthase gene expression during renal ischaemia-reperfusion injury

Sing Leung Lui1,, Loretta Yuk Yee Chan1, Xiao Hui Zhang2, Wen Zhu2, Tak Mao Chan1, Peter Chin Wan Fung2 and Kar Neng Lai1

1 Division of Nephrology and 2 Division of Medical Physics, University Department of Medicine, Queen Mary Hospital, Hong Kong



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Recent animal data suggest that inducible nitric oxide synthase (iNOS) derived nitric oxide (NO) plays an important role in the pathogenesis of renal ischaemia-reperfusion injury (IRI) and that inhibition of iNOS ameliorates IRI. Mycophenolate mofetil (MMF), a lymphocyte selective anti-proliferative agent, has been shown to inhibit NO production in vitro. The aim of this study is to evaluate the effect of MMF on NO production and iNOS gene expression in vivo during renal IRI.

Methods. Renal IRI was induced by clamping the left renal pedicle of male BALB/c mice for 30 min, followed by 15 min of reperfusion. The mice received placebo or MMF at 40, 80 or 120 mg/kg/day by oral gavage for 5 days before the operation. Sham-operated mice served as the operation control. The amount of NO produced and the level of iNOS gene expression in the kidney tissue during IRI was assessed by spin trapping electron paramagnetic resonance (EPR) spectroscopy and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) respectively.

Results. In the sham-operated kidneys, only low levels of NO and iNOS mRNA were detected. In mice with renal IRI, the amount of NO detected was significantly increased, which was reduced in a dose dependent fashion by pre-treatment with MMF. Pre-treatment with MMF also substantially reduced iNOS gene expression in the kidney tissue.

Conclusions. We conclude that pre-treatment with MMF inhibits the production of NO and the induction of iNOS gene expression in the kidney during IRI. These results suggest that MMF might have the potential to ameliorate renal IRI.

Keywords: ischaemia-reperfusion injury; mycophenolate mofetil; nitric oxide; renal



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Ischaemia-reperfusion injury (IRI) is an important cause of early allograft dysfunction after renal transplantation [1] and may adversely affect the long-term survival of the allograft [2]. Therapeutic strategies capable of ameliorating IRI may, therefore, improve the outcome of renal transplantation.

Nitric oxide (NO) is a labile gaseous free radical, which plays an important role in a wide range of physiological and pathological conditions, including IRI [3]. Several recent studies have suggested that increased inducible nitric oxide synthase (iNOS) derived NO production during renal IRI is harmful to the kidney and that inhibition of iNOS activity can ameliorate IRI [47]. It is believed that under ischaemic conditions, NO will react with superoxide anion to form peroxynitrite, which induces renal injury via direct oxidant injury and protein tyrosine nitration [8].

Mycophenolate mofetil (Cellcept®, MMF), a prodrug of mycophenolic acid, is widely used for the prevention of acute rejection after renal transplantation [9]. It acts by inhibiting inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo purine synthesis pathway [10]. In addition to its anti-proliferative effect, MMF has also been shown to inhibit cytokine-induced NO biosynthesis in vitro [11]. However, it is not known whether MMF can exert a similar effect on NO production in vivo.

The aim of this study is to determine the effect of MMF on NO production and iNOS gene expression in the kidney in vivo during IRI.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Mice
Male BALB/c mice aged 8 to 10 weeks were obtained from the Laboratory Animal Unit at the University of Hong Kong. The mice were maintained in the animal colony of the Laboratory Animal Unit under standard conditions. All experiments conformed to approved animal care protocols.

Induction of renal ischaemia-reperfusion injury
The mice were anaesthetized by intraperitoneal injection of midazolam and fentanyl citrate. The left renal pedicle was identified through a midline incision and occluded with a micro-vascular clamp for 30 min. The kidney was then allowed to reperfuse for 15 min. Sham-operated mice underwent a simple laparotomy under identical conditions. The mice were scarified after 45 min of IRI. The left kidneys were then harvested and snapped frozen in liquid nitrogen. Each experimental group consisted of 8 to 10 mice.

Drug administration
MMF was obtained in powder form as a gift from Hoffmann-La Roche Inc. A suspension of MMF in carboxymethyl-cellulose (CMC) in saline vehicle was prepared fresh daily before use. The dose of MMF (40, 80, 120 mg/kg of body weight/day) in 0.1 ml of the CMC vehicle was administered to the mice by oral gavage for 5 days prior to the induction of IRI. The control mice were given the CMC vehicle alone on the same schedule. The period of 5 days' of MMF treatment was chosen arbitrarily to ensure that an adequate level of the drug is present in the mouse's body to deplete intracellular stores of guanosine and deoxyguanosine nucleotides before the IRI. The various doses of MMF were well tolerated by the mice.

NO spin trapping procedure
Spin trapping electron paramagnetic resonance (EPR) spectroscopy is a well-established technique to measure the in vivo production of NO, which has a very short half-life, in various biological models [12,13]. We utilized this technique to detect the production of NO in vivo during IRI as previously described [13]. Briefly, 2 spin trapping agents, diethyldithiocarbamate (DETC) (500 mg/kg), intra-peritoneally, and iron sulphate (50 mg/kg)/sodium citrate (250 mg/kg), subcutaneously, were administered to each mouse 20 min prior to the induction of renal ischaemia. These spin trapping agents served to trap the NO produced during IRI and form with it stable paramagnetic mononitrosyl complex, which can then be detected by an EPR spectrometer. After reperfusion, the mice were sacrificed and the kidneys harvested. The whole left kidney was then sliced into small pieces, fitted into plastic EPR tubes and snapped frozen in liquid nitrogen for EPR analysis.

EPR spectroscopy
EPR spectra were measured using a Bruker ER300E spectrometer. The EPR measuring conditions were: 10 mW microwave power, 9.34 GHz microwave frequency, 100 kHz modulation frequency, 0.52 mT modulation amplitude, 10.24 s time constant, 0.48 mT/s sweep rate and 2.0x104 receiver gain. The NO-Fe-DETC complex is characterized by a triplet EPR signal with g{bot}=2.035 and g||=2.02.

RNA isolation
Total RNA from kidney specimen was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction [14]. The total RNA was air-dried and re-suspended in diethyl pyrocarbonate-treated water. The concentration of RNA was quantified by measuring the absorbance at 260 nm. The quality of the RNA was monitored by using the OD 260/280 ratio. The RNA was electrophoresed through formaldehyde agarose gel and the RNA integrity was verified by examination of the 28S and 18S ribosomal RNA bands under ultraviolet irradiation after staining with ethidium bromide.

Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR was performed as previously described [15]. Briefly, 4 µg of total RNA was reverse transcribed in a final volume of 20 µl containing 10 mM dithiothreitol, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 25 mM dNTPs, 75 ng random hexamer, 40U RNase inhibitor (Promega, Madison, WI) and 200 U of Superscript II RNase H reverse transcriptase (Gibco BRL, Grand Island, NY). The RNA sample was heat denatured at 95°C before the addition of reverse transcriptase and RNase inhibitor. The reaction was incubated for 60 min at 42°C. PCR was performed in a 20 µl reaction volume containing 1 µl of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 1 U of AmpliTaq gold DNA polymerase (Perkin Elmer-Cetus, Foster City, CA) and 0.8 µM or 1.2 µM of sense and antisense primers for ß-actin and iNOS, respectively. Samples were overlaid with mineral oil, heated to 95°C for 10 min, and then amplified for 23 cycles (ß-actin) or 33 cycles (iNOS) consisting of 45 s denaturation at 95°C, 45 s annealing at 63°C (ß-actin) or at 58°C (iNOS) and 1 min extension at 72°C. The experimental conditions and number of cycles of PCR were predetermined to ensure that the amount of iNOS and ß-actin amplicons were within the linear range of amplification. The sequences of the PCR sense and antisense primers and the size of amplicons are shown in Table 1Go.


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Table 1. The sequences of the PCR sense and antisense primers and the size of amplicons

 

Semi-quantitative measurement of iNOS mRNA
The iNOS mRNA was semi-quantified by normalizing the differences occurring during reverse transcription and PCR using a housekeeping gene, ß-actin. After amplification, 20 µl of each PCR reaction mixture were electrophoresed through a 1.8% agarose gel with ethidium bromide (0.5 µg/ml). The PCR products of ß-actin and iNOS were electrophoresed in the same gel to eliminate gel to gel variance. The gel image was captured by video gel documentation system (Bio-Rad Laboratories, Hercules, CA) under the same exposure and integration. The computer image was analysed by IPLab gel software from Signal Analytics Corporation (Virginia, USA) for quantitation. The results were expressed as a ratio calculated from the integrated signal of iNOS amplicon over that of the ß-actin amplicon.

Statistical analysis
Data were expressed as mean±SD. Statistical differences between the control and the experimental groups were analysed using Kruskal-Wallis one-way analysis of variance (ANOVA). A P value of <0.05 was considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study, we found that there was a significant increase in NO production and iNOS gene expression in the kidney during IRI. Pre-treatment with MMF resulted in a dose dependent reduction in both NO production and iNOS gene expression.

Figure 1Go shows a typical EPR spectrum of NO-Fe-DETC complex, obtained from a preparation of iron citrate and DETC after incubation with a NO donor compound, S-nitroso-N-acetylpenicillamine. The EPR spectrum consists of a triplet waveform with a centre at g=2.035 and a hyperfine coupling constant aN of 13G. In the absence of NO, no triplet EPR spectrum is observed from the Fe-DETC complex. The size of the NO signal was obtained by measuring the height of the first peak of the standard triplet waveform from the baseline. It has been shown that the concentration of NO-Fe-(DETC) corresponds directly to the height of the first peak of the standard triplet waveform [12,13]. We have verified this method by showing that the height of the EPR signal has a linear relationship with NO concentration up to at least 20 µmol/l using saturated NO solution.



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Fig. 1. Typical EPR spectrum of NO-Fe-DETC complex: the EPR spectrum is obtained from a preparation of DETC and iron sulphate with sodium citrate after incubation with 2 nmol concentration of the NO donor compound S-nitroso-N-acetylpenicillamine. It consists of a triplet waveform with a centre at g=2.035 and a hyperfine coupling constant aN of 13G (arrowed). The size of the NO signal was obtained by measuring the height of the first peak of the standard triplet waveform from the baseline. EPR measuring conditions were: 10 mW microwave power, 9.34 GHz microwave frequency, 100 kHz modulation frequency, 0.52 mT modulation amplitude, 10.24 s time constant, 0.48 mT/s sweep rate and 2.0x104 receiver gain.

 
Effect of MMF on NO production in the kidney during IRI
Figure 2Go shows a representative EPR spectrum from the various experimental groups. In the sham-operated mice, only a weak spectrum of the triplet NO-Fe-DETC complex was observed. In mice subjected to renal IRI and treated with placebo, a prominent triplet NO adduct signal was observed. Pre-treatment with MMF resulted in a dose dependent reduction in the size of the NO adduct signals. Figure 3Go shows the relative amplitudes of the NO-Fe-DETC EPR spectra of the various experimental groups. In mice with renal IRI and treated with placebo, there was a more than 2-fold increase in the amplitude of the NO signals. The amplitude of the NO signals was significantly reduced by pre-treatment with MMF.



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Fig. 2. Typical EPR spectra from mouse kidneys: effect of MMF on NO generation during IRI. The mouse kidneys were subjected to 30 min of ischaemia followed by 15 min of reperfusion. Two spin trapping agents, DETC, intraperitoneally, and iron citrate, subcutaneously, were administered to the mice 20 min before the onset of ischaemia. The arrows indicate the position of the EPR signals of the NO-Fe-DETC complex. The EPR spectra were recorded as described in the legend of Figure 1Go.

 


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Fig. 3. Relative amplitude of the NO-Fe-DETC signal in the mouse kidney during IRI: effect of MMF administration. Data are shown as the mean±SD, n=6–8. **Significant difference between vehicle treated and sham-operated mice (P<0.05); *significant difference between MMF treated and vehicle treated mice (P<0.05).

 

Effect of MMF on iNOS mRNA expression in the kidney during IRI
In order to determine whether the decrease in NO production in the kidney tissue during IRI in mice treated with MMF is related to a reduced iNOS gene expression, we studied the expression of iNOS mRNA in the kidney tissue by semi-quantitative RT-PCR. The amount of iNOS mRNA present in the kidney tissue was expressed as a ratio calculated from the integrated signal of the iNOS amplicon over that of the ß-actin amplicon. The effect of MMF on the steady-state mRNA levels for iNOS in the mouse kidneys subjected to IRI is shown in Figure 4AGo. In mice with IRI and treated with vehicle, the iNOS mRNA expression was increased as compared with the sham-operated mice. Pre-treatment with MMF resulted in substantial reduction in the iNOS gene expression. Figure 4BGo shows the relative ratio of the integrated signal of iNOS amplicon over that of the ß-actin amplicon. There was a significant increase in iNOS expression in the vehicle treated mice as compared to sham-operated mice. The induction in iNOS gene expression in the vehicle treated mice was reduced in a dose dependent manner in mice pre-treated with MMF.



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Fig. 4. Effect of MMF on iNOS mRNA expression in the mouse kidney during IRI. Total RNA was extracted from the kidney of individual mouse and analysed by RT-PCR for iNOS and a housekeeping gene, ß-actin. The PCR products were electrophoresed in the same gel to eliminate gel to gel variance and were visualized with ethidium bromide and UV light. (A) Representative RT-PCR gel for iNOS mRNA. RT-PCR amplification of the housekeeping gene ß-actin was positive at 367 bp in all samples (data not shown). (B) Relative ratio of the integrated signal of iNOS amplicon over that of the ß-actin amplicon as shown in (A). Data are shown as the mean±SD, n=8–10. **Significant difference between vehicle treated and sham-operated mice (P<0.05); *significant difference between MMF treated and vehicle treated mice (P<0.05).

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This report documents the effect of MMF on in vivo NO production and iNOS gene expression in the kidney during IRI. We found that IRI increases the production of NO and induces the expression of iNOS mRNA in the kidney and that pre-treatment with MMF inhibits both the production of NO and the induction of iNOS gene expression in a dose dependent manner.

Nitric oxide is generated by the enzyme NOS, which exists in 3 isofoms (endothelial, inducible and neural), all of which can be expressed within the kidney [16]. The exact role of NO in the pathogenesis of IRI appears to depend on the isoform of NOS involved in the production of the NO [8]. Substantial evidence has accumulated to suggest that increased iNOS derived NO production plays an important major role in the pathogenesis renal IRI [47]. Inhibition of iNOS activity by antisense oligodeoxynucleotide to iNOS mRNA has been shown to decrease renal tubular damage after IRI [5]. In addition, it has been demonstrated that {alpha}-melanocyte-stimulating hormone, which inhibits the induction of iNOS activity, significantly reduces the tubular injury and loss of renal function associated with IRI [6]. Furthermore, it has been reported that renal tubules obtained from iNOS knockout mice are not susceptible to hypoxic injury [7]. Our findings that MMF inhibits NO production and iNOS gene expression in the kidney during IRI raise the possibility that MMF may be capable of ameliorating renal IRI.

The results of our study suggest that there is a very rapid induction of iNOS and increase in NO production during IRI. These findings might appear somewhat unexpected in view of the relatively short duration of ischaemia and reperfusion. However, several recent reports have shown similar results as those of ours. In one study using a rat model of renal IRI, significantly increased NO production was observed after 30 min of ischaemia and 20 min of reperfusion [4]. In another study, significant induction of iNOS in the myocardium was observed after 30 min of ischaemia and 60 min of reperfusion, thus, implying a super-induction of iNOS in the heart [17]. It thus seems possible that the induction of iNOS gene expression and increased NO production do occur during the early phase of IRI although the exact mechanism responsible for such rapid induction of iNOS remains to be elucidated.

In this study, we have not examined the effect of MMF on the functional changes in the kidney after IRI because the primary aim of this study was to determine the effect of MMF on in vivo NO production and iNOS gene expression. Nevertheless, there are experimental data to support a protective role of MMF in IRI. In a rat model of heart transplantation, it has been demonstrated that donor pre-treatment with MMF protects cardiac grafts exposed to prolonged tepid storage against primary non-function [18]. In another study using a rat model of renal IRI, it has been shown that treatment with MMF reduces renal injury and facilitates tissue repair [19]. In the light of our findings, it is plausible to postulate that the beneficial effect of MMF on IRI may be attributed in part to its inhibitory effect on NO production.

The exact mechanism whereby MMF suppresses NO production during IRI has not been elucidated completely. Previous studies have suggested that MMF might reduce NO production via the inhibition of the biosynthesis of tetrahydrobiopterin (BH4), an essential co-factor of NOS [11]. The results of our study indicate that MMF may also reduce the production of NO directly by suppressing the induction of iNOS at the transcription level. The induction of iNOS after IRI is dependent on the transcription factor NF{kappa}B and is inducible by various pro-inflammatory cytokines such as tumour necrosis factor, interleukin-1 and interferon-{gamma}. MMF does not have a direct effect on cytokine production on a per cell basis [10] but may limit the total amount of cytokine produced by limiting clonal expansion of the triggered lymphocytes [20]. This effect may also contribute to the overall reduction in iNOS gene expression during IRI.

In this study, we have pre-treated the mice with MMF for 5 days before the induction of IRI. Valentin et al. also used a pre-treatment regimen to study the effect of MMF on IRI in their experimental model of cardiac transplantation [18]. It is obvious that such pre-treatment regimen is not directly applicable to current clinical practice with the exception of perhaps living related transplantation. Nevertheless, as many inflammatory processes will have already been initiated in the donor organ long before it is being harvested, we believe that the approach of pre-treating the donor with immunosuppressive agents is clinically relevant and might have the potential of minimizing the injury to the donor organ.

In summary, our preliminary data show that pre-treatment with MMF inhibits NO generation and iNOS gene expression in vivo during renal IRI. Given the important role played by iNOS-derived NO in the pathogenesis of IRI, we postulate that MMF might have the potential of ameliorating renal IRI. Further studies are warranted to verify this possible application of MMF.



   Acknowledgments
 
This study was supported by a grant (#337/041/0069) from the Committee on Research and Conference, University of Hong Kong. The authors would like to thank Hoffmann-La Roche Inc. for providing the MMF powder. We are also grateful to Dr K. S. Lo and Mr J. C. K. Leung for their excellent technical assistance.



   Notes
 
Correspondence and offprint requests to: Dr Sing Leung Lui, Division of Nephrology, University Department of Medicine, Queen Mary Hospital, Pokfulam, Hong Kong. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 9.00
Revision received 6. 4.01.