Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury

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Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
Page 1 of 32Articles in PresS. Am J Physiol Renal Physiol (May 14, 2008). doi:10.1152/ajprenal.00567.2007

                Indoleamine 2,3-dioxygenase (IDO) expression promotes renal
                                 ischemia reperfusion injury

            KANISHKA MOHIB1,2, SHUANG WANG2, QIUNONG GUAN3, ANDREW L. MELLOR,4
            HONGTAO SUN5,6, CAIGAN DU3, ANTHONY M. JEVNIKAR1,2,5,6
            1
              Departments of Medicine and Microbiology & Immunology, The University of Western
            Ontario, London, Ontario, Canada
            2
              The Robarts Research Institute, London, Ontario, Canada
            3
              Department of Urologic Sciences, The University of British Columbia, Vancouver, British
            Columbia, Canada
            4
              Department of Medicine, Medical College of Georgia, Augusta, Georgia
            5
              The Lawson Health Research Institute, London, Ontario, Canada
            6
              The Multi-Organ Transplant Program, London Health Sciences Center, London, Ontario,
            Canada

            Running Head: IDO expression enhances ischemia/reperfusion injury

            Reprint requests to Dr. Anthony M. Jevnikar, Division of Nephrology, University Hospital, 339
            Windermere Road, Room A10-113, London, ON, Canada N6A 5A5
            E-mail: jevnikar@uwo.ca

                               Copyright © 2008 by the American Physiological Society.
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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ABSTRACT

Indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan to N-formyl kunurenine and has a

pro-apoptotic role in renal tubular epithelial cells (TEC) in response to IFN- and TNF- in-

vitro. TEC produce abundant amounts of IDO in vitro in response to inflammation but a

pathological role for IDO in renal injury remains unknown. We investigated the role of IDO in a

mouse model of renal ischemia-reperfusion injury (IRI). IRI was induced by clamping the renal

pedicle of C57BL/6 mice for 45 minutes at 32°C. Here we demonstrate up-regulation of IDO in

renal tissue at 2 hrs after reperfusion which reached maximal levels at 24 hrs. Inhibition of IDO

following IRI prevented the increase in serum creatinine observed in vehicle treated mice

(86.4±25 µmol/L, n=11) when compared to mice treated with 1-methyl-d-tryptophan (1-MT), a

specific inhibitor of IDO, (33.7±8.7µmol/L, n=10, p=0.031). The role of IDO in renal IRI was

further supported by results in IDO-KO mice which maintained normal serum creatinine levels

(32.5±2.0µmol/L, n= 6) following IRI as compared to wild type (WT) mice (123±30µmol/L, n=

9, p= 0.008). Our data suggest that attenuation of IDO expression within the kidney may

represent a novel strategy to reduce renal injury as a result of ischemia/reperfusion.

Key words: tubular epithelial cell, apoptosis, renal function
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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               INTRODUCTION

                      Ischemia reperfusion injury (IRI) is invariably associated with solid organ

               transplantation. As therapeutic agents that completely or predictably prevent IRI are currently

               unavailable, IRI remains a major problem in renal transplantation. The degree of injury sustained

               under such conditions influences both the immediate and potentially the long-term outcome of a

               transplant [17, 36, 44]. One of the deleterious consequences of renal IR involves tubular

               epithelial cell (TEC) injury and death [25, 26, 42]. Kidney TEC undergo apoptosis as well as

               necrosis as a result of IRI [26, 37, 43]. As TEC are central to the function of the nephron and

               thus the kidney, strategies that limit or prevent TEC death may improve long-term renal allograft

               function.

                      Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine

               enzymatic pathway and is expressed in various tissues and cells including dendritic cells and

               macrophages in response to IFN- , TNF- , and LPS [3, 8, 21, 38, 46-50]. IDO expression has

               been shown to cause activation induced cell death (AICD) of T cells by enhancing Fas mediated

               apoptosis [24]. The activity of the enzyme has also been demonstrated to augment anti-Fas

               mediated cell death of human tumor cell lines [19]. Previous studies have associated IDO

               expression in the kidney with renal acute rejection and chronic failure [5, 39, 45]. Specifically,

               the expression of IDO in renal tubules and the presence of high concentration of kynurenine,

               found in sera and urine of recipients, during acute rejection and chronic renal failure suggest IDO

               may participate in graft injury [5, 39, 45]. Recently, we have demonstrated that IDO augments

               Fas/FasL mediated self injury and death of TEC in response to IFN- and TNF- in-vitro [32].

               However a role for IDO in promoting renal injury following IRI has not been determined in-vivo.

               The aim of this study was to determine if renal expression of IDO could promote IRI by
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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augmenting tubular cell death and worsening function. Using IDO-KO mice as well as the IDO

specific inhibitor 1-methyl-d-tryptophan (1-MT) in wild type mice, we tested for renal injury

following renal artery clamping in uni-nephrectomized mice. Our studies demonstrate that renal

IDO expression is deleterious during renal IR and elimination or inhibition of IDO prevented

injury with maintenance of normal histology and renal function. Therapeutic inhibition of IDO is

clinically feasible in the time frame of IRI, and as such these results may be considerably

significant in renal transplantation.

METHODS

Animals:

        C57BL/6 (B6) mice (male, 8–10 weeks old) were obtained from the Jackson Laboratory

(Bar Harbor, ME,). Both wild type (WT) B6 and IDO KO mice [31] (C57BL/6 background)

were maintained in the animal facility at the University of Western Ontario (London Ontario,

Canada). Animal experiments were conducted in accordance with the Canadian Council on

Animal Care guidelines under protocols approved by the Animal Use Subcommittee at our

institution.

Renal ischemia reperfusion injury induction:

        Ischemic injury was induced by clamping the renal pedicle of the left kidney for 45

minutes at 32°C. After the clamps were released, reperfusion of the kidneys was confirmed

visually, and the right kidney was removed. For the IDO inhibitor treatment, wild type B6 mice

received intra peritoneal injections of 3mg of 1-MT (Sigma-Aldrich, Mississauga, ON) dissolved
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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               in PBS or PBS alone (vehicle) twice a day for a total period of 48 hours following reperfusion.

               Some mice received 1-MT one hour prior to ischemia as well as following reperfusion. After 48

               hours of reperfusion the mice were sacrificed at which point serum and tissue samples were

               collected. Uni-nephrectomized mice that did not undergo IR were also included in the study as

               sham operated controls. Renal tubular epithelial cells (TEC) were subjected to ischemic

               conditions in vitro. NG 1.1 [32] TEC (2.0 x 106) were seeded to each well of 6-well plates

               overnight. Medium was replaced in confluent monolayer cultures with K1 medium without

               serum or growth factors to arrest cell division. To mimic ischemia injury in vitro, sterile mineral

               oil (sigma) was added to the wells to cover the medium, followed by continued culture at 37 C,

               At various time points, oil was removed and TEC RNA was collected by using TRIzol at the

               different time points (Baseline, 0h, 1h, 2h, 4h, 8h, 24h).

               RNA isolation and RT-PCR:

                      Kidney tissue pooled from 3 mice were added to 1-2 mL of Trizol (Invitrogen GIBCO,

               Burlington, ON) and ground up using a tissue sonicator. RNA was isolated according to

               manufacturer’s instructions. RT-PCR was carried out using total isolated RNA. Briefly, 5Lg of

               RNA from each sample was used to carry out reverse transcription with Superscript II

               (Invitrogen GIBCO). Subsequently PCR amplification of IDO cDNA was carried out using a

               specific pair of primers 5’-CATAAGACAGAATAGGAGGC (sense) and 5’-

               GAAGATGTGGGCTTTGCTCTA (anti-sense) for 32 cycles. Glyceraldehyde-3-phospate

               dehydrogenase (GAPDH) mRNA levels were used as an internal control for the amount of RNA

               loaded in each sample. PCR products were visualized on a 1% agrose gel with 0.5Lg/mL-
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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ethidiumbromide. Although semi-quantitative, comparisons of expression of IDO mRNA levels

between sample times were made using densitometry with GAPDH mRNA expression levels.

Western blot analysis:

       Kidney tissue samples pooled from 3 different mice were added to lysis buffer [10mM

HEPES (pH 7.9), 10m KCl, 0.10 mM EDTA, 0.10 mM EGTA, 0.10% NP 40, 1.0 mM DTT and

a protease cocktail (Roche Diagnostics, Mannheim Germany)] and ground up using a sonicator.

An equal amount of 4x SDS sample buffer [20mM Tris- HCl (pH 6.8), 5.0% (wt/vol) SDS, 10%

vol/vol beta-mercaptoethanol, 2.0 mM EDTA, and 0.020% bromophenol blue) was added to the

ground up tissue. The samples were run on a 12% SDS-polyacrylamide gel and subsequently

transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane

was blocked using 5.0% milk in TBS-T (20 mM Tris-HCl, pH 7.6, 0.14 M NaCl, 0.10% Tween

20) for one hour. The IDO protein (~45kda) was detected using a primary polyclonal rabbit anti-

IDO antibody [31] (1:3000), in TBS-T containing 2.5% milk and a goat anti-rabbit peroxidase

conjugated secondary antibody (Sigma Aldrich). The bound protein on the membrane was

visualized by an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech,

Buckinghamshire, England). Blots were re-probed using total ERK antibody (Sigma-Aldrich) as

a loading control. Quantification of IDO protein levels at various sample times was made using

densitometry and comparing IDO levels to total ERK levels.

Densitometry analysis:

       RT-PCR or western blot images were scanned using a GS700 Imaging densitometry

scanner (BioRad Inc. Mississauga, Ontario). Analysis of the images was carried out with BioRad
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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               Densitometry software (BioRad Inc.). Individual blot/gel images were examined by analyzing

               the intensity of each band within a fixed volume. The density ratio was determined by dividing

               the intensity of the target mRNA/protein by the respective GAPDH/total ERK band intensities.

               Tissue immuno-histochemical staining:

                     Paraffin embedded tissue sections were immersed for 5 minutes in 100% xylene twice, 1:1

               (xylene:ethanol), 100%, 90%, 75% ethanol and washed 3 times in Tris buffer saline (TBS) (pH

               7.4). The slides were transferred into already boiling 10mM sodium citrate buffer (pH 6.0) for 40

               minutes and then left to cool in room temperature for 20 minutes. Three 5 minutes washes in

               TBS were carried out and the sections were treated with 100µL of 2.0% H2O2 for a period of 10

               minutes followed by permeabilization with 0.20% Triton X-100 for 10 min at room temperature.

               The sections were washed 2 times with TBS and blocked for 1 hour using 4.0% rabbit and 2.0%

               goat serum. Subsequently the slides were incubated with 1:100 of primary anti-IDO antibody

               overnight at 4°C. Three 10 minutes TBS washes were carried out and the samples were

               incubated with 1:200 of secondary biotin conjugated antibody for 2 hours. Three TBS washes

               were carried out and the slides were incubated with 1:200 of avidin solution for 45 minutes at

               RT. Antibody binding was visualized using 3,3'-diaminobenzidine (DAB) as substrate (Sigma

               Aldrich) followed by counterstaining with hematoxylin for 5 minutes. All slides contained

               duplicate sections from which one served as a control for secondary-antibody binding specificity.

               Determination of renal function, injury and cellular infiltration:

                      Kidney function was determined through measurement of serum creatinine and blood

               urea nitrogen (BUN), by a Jaffe reaction method with an automated CX5 clinical analyzer
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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(Beckman Instruments, Fullerton, CA, USA). Histological evaluation of tubular injury in kidney

sections was performed in a blinded fashion. Formalin-fixed and paraffin-embedded sections

(5µm thickness) were stained by hematoxylin and eosin (H&E) and periodic acid Schiff (PAS)

method. These sections were examined in a blinded fashion by a pathologist. Kidney injury was

assessed as percentage of tissue involved with histological changes in the cortex and medulla

using a semi-quantitative scale and an evaluation of area involved with tubular injury and

neutrophil infiltration on a 5-point scale as follows: 0, no change ; 1, 10–25%; 2, 25–50%; 3, 50–

75% and 4, 75–100%. Scores were averaged for at least 10 non-overlapping fields for each

kidney. Neutrophil (PMN) infiltration was assessed by direct counting of sections at 400x.

Results are expressed as PMN ± SEM per 5 high power fields (hpf).

TUNEL assay:

       Buffered-formalin-fixed sections were processed with proteinase K (30 min at 37°C),

followed by quenching endogenous peroxidase in 3.0% H2O2 in TBS (pH 7.4) for 30 min at

room temperature, then permeabilizing with 0.20% Triton X-100 for 10 min at room

temperature. After washing with TBS, the sections were incubated with terminal transferase and

FITC-conjugated dUTP (Roche Diagnostics) for 60 min at 37°C. FITC-dUTP-labeled nuclei

were detected using anti-FITC antibody conjugated with phosphatase (Sigma-Aldrich), and

visualized with phosphatase substrate Sigma Fast Red (Sigma-Aldrich). The number of TUNEL-

positive cells representing apoptotic/necrotic cells in the section of each kidney were counted in

at least 10 non-overlapping high power fields (hpf) under 400x magnification and averaged in a

blinded fashion.
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               Statistical analysis:

                       Two-way ANOVA and t-tests (two-tailed distribution) were used for comparisons

               between groups using Statview software (SAS Institute, Cary, CA, USA). A p-value of S0.05

               was considered significant.

               RESULTS

               Upregulation of IDO following renal ischemia/reperfusion:

                       The activity of IDO has diverse physiological functions including suppression of

               activated T cells by placental tissues in fetal-maternal tolerance, tryptophan starvation of

               intracellular pathogens and UV protection in the cornea [33, 41, 46]. Recently, we have

               demonstrated the ability of IDO to enhance renal TEC fratricide mediated through TEC

               expression of Fas/FasL in response to inflammatory cytokines [32]. Therefore, we hypothesized

               that if IDO is expressed in the kidney in response to inflammation following IRI, it may promote

               this form of TEC self-injury. In order to determine whether the expression of IDO is up regulated

               in response to renal IRI, wild type mice were subjected to renal IRI and sacrificed at various

               times points. Sham operated mice, used as controls, displayed basal expression levels of IDO

               mRNA and those levels remained unchanged in mice that were subjected to 45 minutes of

               ischemia and 0.5 hour, and 1 hour of reperfusion (Fig. 1a). However, at 2 hours post reperfusion

               there was a noticeable increase in the level of IDO mRNA that became most pronounced at 4

               hours and 24 hours (Fig. 1a). Similar to IDO mRNA expression kinetics, the level of IDO protein
Indoleamine 2,3-dioxygenase (IDO) expression promotes renal ischemia reperfusion injury
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initially increased at 2 hours post reperfusion and remained up regulated at 4 and 24 hours as

compared to sham control (Fig. 1b). IDO mRNA was detected in quiescent TEC but was not

increased following ischemic conditioning in vitro (not shown), suggesting enhanced IDO

expression in TEC is related to the reperfusion associated inflammation, rather than ischemia

alone. Although RT-PCR and western blot analysis are sensitive in detecting mRNA and protein

levels of the desired target, these methods do not distinguish location of expression within the

kidney. Immuno-staining was performed on sections obtained from sham operated and IR treated

mice that were sacrificed 24 hours following reperfusion. IDO staining was detected very weakly

within TEC of control mice (Fig. 2a). In contrast, IR subjected mice had intense and diffuse IDO

staining within the TEC of the renal cortex (Fig. 2b). These results suggest that tubular cells

appear to be primarily responsible for the elevated expression of IDO noted, but do not

distinguish proximal or distal tubules. IDO expression in TEC was not clearly localized to

specific cellular compartments in these tissue sections, although expression appeared to be

primarily cytoplasmic in cultured TEC (not shown). It is unknown whether IDO can translocate

in epithelial cells to possibly facilitate immune-modulation given its reported role in promoting T

cell apoptosis.

IDO deficient kidneys have improved function following IRI:

       Previous studies of renal IDO expression have established an association with acute

rejection, decreased glomerular filtration and renal insufficiency. However these studies have not

directly linked renal IDO to acute or chronic injury [5, 39, 45]. To address this, we carried out

IRI studies using IDO-KO mice [31]. Following 48 hours of reperfusion, sera from the mice

were collected and creatinine and BUN levels were measured to assess kidney function. Clamped
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                IDO-KO mice had markedly lower level of creatinine (32.5 ± 2.0 µmole/L, n = 6) compared to

                (123 ± 30 µmole/L, n = 9, p = 0.008) in wild type mice (Fig. 3a) reflecting protection of these

                kidneys from IRI. Clamped IDO-KO mice also had improved kidney function reflected by

                preserved sera BUN concentrations. IDO-KO mice had significantly lower levels of urea (13.0 ±

                2.0 mmol/L, n = 6) versus wild type mice (49.6 ± 11 mmol/L, n = 9, p = 0.006) (Fig. 3b).

                Collectively these data demonstrate that enhanced IDO expression occurs with IRI and that renal

                expression of IDO can have a deleterious effect on kidney function.

                Administration of IDO specific inhibitor 1-MT improves renal function following IRI:

                       Down regulation of IDO expression might be useful as a strategy to prevent renal injury

                including transplantation. 1-MT is a potent inhibitor of IDO [7] and hence we used this agent to

                substantiate data obtained with IDO-KO mice. Wild type mice subjected to IR and treated with

                1-MT pre and post ischemia had creatinine levels (33.7 ± 8.7 µmole/L, n=10) substantially lower

                than vehicle treated mice (86.4 ± 25 µmole/L, n=11, p = 0.031) (Fig. 4a) and was equivalent to

                that observed in IDO-null mice. Similarly BUN concentrations in 1-MT treated mice (20.9 ± 4.6

                mmol/L, n=10) were lower than those from wild type mice (43.4 ± 8.3 mmol/L, n=11, p = 0.036)

                (Fig. 4b). Therefore, pharmacological inhibition of IDO with 1-MT is consistent with the benefit

                observed using IDO-KO mice when administered 1 hour prior to ischemia induction. However

                no benefit was observed with 1-MT treatment post ischemia as creatinine levels (111.7 ± 30

                umol/l, n=4) were not different from control mice. Higher levels and greater delivery of 1-MT

                post ischemia was limited by its low solubility and its rapid clearance, and the relatively large

                volumes required for injection.
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IDO expression contributes to tubular epithelial cell damage:

       Histological assessments were carried out to establish a correlation between the

functional protection observed and the degree of injury sustained by the kidney. Tissue sections

were paraffin embedded, and stained with hematoxylin and eosin (H&E) or PAS for scoring,

which was in a blinded fashion as previously described [11]. Wild type kidney sections were

characterized by extensive damage that included disrupted tubules, loss of normal architecture,

flattening of cells with loss of tubular brush border, non cellular cast formation and changes

consistent with acute tubular necrosis (ATN) (Fig. 5b). In contrast, sections obtained from IDO-

KO mice showed preservation of normal architecture of cells with subtle injury in comparison to

wild type mice. Accordingly, tubular necrosis and vacuolization (%) scoring confirmed a

considerable difference in tissue involved in wild type kidneys (51 ± 5.1%, n = 9) versus IDO-

KO kidneys (23 ± 2.1 %, n = 6) p = 0.001 (Fig. 5d). Histology was also improved in mice

treated with 1-MT prior to ischemia (Fig. 6c) with less tubular damage and minimal cast

formation compared to vehicle treated mice (Fig. 6b). Scoring of the sections and subsequent

comparison confirmed that WT-PBS treated mice had higher amounts of tubular necrosis and

vacuolization (45 ± 3.0%, n =11) compared to pre and post 1-MT treated mice (29 ± 3.3%, n =

10), p = 0.002 (Fig 6d). As IR injury has been associated with cellular infiltration, we also

carried out H&E staining to assess this. WT PBS treated kidney sections had greater neutrophil

infiltration (10.4±9/ 5hpf, n=8) than pre-ischemia 1-MT treated mice (2.4±5, n=7, p=0.02 vs WT

control) or IDO null kidneys (0±0.5/5hpf, n=6, p=0.007 vs WT control) (Fig 7 a, b, c). Hence,

the functional improvement in IDO-KO and 1-MT mice following IRI is consistent with a

reduction in histological kidney injury compared to their respective controls.
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                IDO-KO kidneys have reduced cellular apoptosis/necrosis following IRI:

                         TUNEL staining of the fixed sections was performed to study the effect of IDO

                elimination on kidney apoptosis and necrosis, in relation to the observed protection in IDO null

                kidneys. Wild type mice had significantly higher numbers of TUNEL positive cells (6.5 ±

                0.9/hpf, n = 9) in the tubular compartment, than IDO-KO mice (2.1 ± 0.6/hpf, n = 6, p = 0.03)

                (Fig 8). These data suggest IDO expression contributes to apoptosis/necrosis within the kidney

                following IR. As control kidneys had significantly more infiltrate than either 1-MT or IDO null

                kidneys, it is difficult to unequivocally identify which cells were undergoing apoptosis in tissue

                sections. However the absence of diffuse and prominent infiltrates in control sections and

                morphology of cells, would suggest that apoptosis was largely occurring within TEC. Apoptosis

                was also largely absent in ischemic IDO null kidneys, consistent with reduced TEC injury and

                correlation to improved function .

                DISCUSSION

                         Ischemia reperfusion injury in kidneys represents an orchestrated series of inflammatory

                events leading to organ injury in which there is rapid and significant upregulation of numerous

                pro-inflammatory factors that are relevant to renal injury including IFN- , TNF- , and MHC I,

                II, as well as Fas/FasL [9, 15, 14, 18, 30, 29]. The degree of injury sustained to the kidney as a

                result of IR may not only influence graft acceptance through links between innate and adaptive

                immunity but also the longevity of the graft as nephrons continue to be injured by inflammation

                [1-3].

                         There are several key observations that suggest IDO may be in a position to affect renal

                IRI. TEC express basal levels of both Fas and FasL and increase Fas expression in response to
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LPS, inflammatory cytokines such as IFN- and TNF- , as well oxidative stress, which may lead

to injury [1, 4, 10, 20, 22, 23, 28, 34, 35, 40]. IRI and transplant studies carried out with MRL-

lpr and MRL-gld mice have established that loss of functional Fas/FasL can limit IRI and

consequently be beneficial to the organ [34]. Recently, we have shown that IDO promotes

Fas/FasL mediated TEC death in response to IFN- /TNF- in-vitro [32]. Moreover, the

expression and activity of IDO associated with renal acute rejection and chronic failure indirectly

implicates a role for IDO in immune injury following transplantation. In the present study, we

have found that increased IDO expression is part of the acute response of IR as both mRNA and

protein levels of the enzyme noticeably increased within 2 hours post reperfusion and peaked

between 4 hours and 24 hours. The expression of IDO appears to be in response to reperfusion

associated inflammatory cells and the cytokines that they produce, rather than ischemia per se.

TEC augment IDO expression in response to pro-inflammatory cytokines [32] but IDO mRNA

did not change with ischemic in vitro conditioning in the present study (data not shown). The in

vivo results in the present study examining kidneys of IDO wild type mice therefore are

consistent with our results in cytokine exposed TEC, although IDO expression may differ in non

TEC cells.

       This study is the first to describe the induction of IDO in response to renal IR. In

addition, we elucidated that both IDO-KO and WT mice treated with the IDO inhibitor had

improved kidney function, and decreased tissue injury with reduced numbers of TEC undergoing

apoptosis/necrosis. As a result, our studies suggest that therapeutic inhibition of IDO may be

beneficial in the early stages of transplant injury that involve IR, particularly as this is when TEC

express high levels of both IDO and Fas/FasL. Although our data would suggest that inhibition

of IDO expression in kidneys is required prior to the onset of ischemia, IDO inhibition may be
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                therapeutically relevant to some forms of renal injury. With renal transplantation, the addition of

                1-MT or alternate pharmacological agents to block IDO or downstream metabolites to organ

                preservation solutions may be of greater benefit than treating recipients post transplant.

                       Traditionally IDO has been described to be beneficial to tissues expressing the enzyme as

                it is able to prevent the growth of tryptophan dependent pathogens and suppress T cells by

                inhibiting proliferation in addition to inducing Fas/FasL mediated cell death [3, 6, 8, 46]. Several

                groups have been able to prolong survival of transplanted lungs, pancreatic beta cells, and skin

                [2, 13, 27, 33]. More recently, Liu et al. have demonstrated that IDO plays a protective role in

                lung IR [27]. Although, our findings are not consistent with the above studies we believe the

                disparity may be explained by the differential expression of Fas/FasL in parenchymal cells of the

                various organs. While over expression of IDO may have potential beneficial effects to promoting

                long-term graft survival by evading the immune system, we suggest that caution should be taken

                in cells such as TEC in which Fas and FasL are co-expressed. Future studies examining this

                strategy are needed in order to determine potential immune protective effects of IDO bestowed to

                TEC following transplantation.

                       The mechanism(s) by which IDO augments injury in TEC have not yet been clearly

                delineated. Interestingly, the expression of IDO was associated with increased numbers of

                infiltrating cells as well as greater apoptosis in tissue sections. Our previous results using

                cultured TEC supports a role for IDO in promoting tubular cell self injury through Fas-FasL

                expression in response to inflammatory cytokines [32] may hold true in-vivo as our data does

                not exclude the possibility that IDO associated renal injury in IRI is related to enhanced cytokine

                release by infiltrated cells. While we cannot exclude that apoptosis was also occurring in the

                infiltrating cells, the worsened function in wild type IDO kidneys and improved function in the
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absence of IDO would suggest that the beneficial effect was on TEC and renal parenchymal

cells. As neutrophils are observed with severe IRI, the reduction of neutrophil infiltration with

attenuation of IDO was likely due to reduced tissue injury. However the reduction of

inflammatory infiltrates may have had a further benefit in limiting renal injury. Several studies

have associated an increase in IDO related, downstream kynurenine pathway metabolites with

acute rejection and chronic renal failure [5, 39, 45]. Accordingly, our previous studies

demonstrate a potentiating effect of cytokine-mediated TEC death, upon exposure to downstream

metabolites picolinic acid and quinolinic acid [32]. These findings are substantiated by previous

reports of the ability of quinolinic acid to directly activate caspase-8 and cause the release of

cytochrome c [12, 16]. Strategies that limit down stream kynurenine pathway enzymes more

specifically than 1-MT in donor kidneys prior to transplantation may suppress TEC injury while

allowing IDO expression to persist and provide immune protection.

CONCLUSIONS

       In conclusion IDO expression following IR has a deleterious effect on kidney injury and

function. Our data suggests that attenuation of IDO may represent a novel strategy to reduce

renal IRI and thereby potentially improve kidney graft function and survival.
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                ACKNOWLEDGMENTS

                We thank Ms. Pamela Gardner for secretarial assistance, Drs. Joaquim Madrenas, Ewa Cairns,

                and the late Dr. Robert Zhong for their invaluable advice.

                Grants:

                This work was supported by a grant from the Canadian Institutes of Health Research (CIHR)

                (A.M.J and C.D.) and the Multi-Organ Transplant Program of London Health Sciences Centre.

                Disclosures:

                A.L. Mellor has intellectual property interests in the therapeutic use of IDO and IDO inhibitors

                and receives consulting income from NewLink Genetics Inc.
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                                                                                               22

Figure Legends:

Figure 1) Ischemia/reperfusion induces renal IDO expression. The renal pedicle of uni-

nephrectomized C57BL/6 mice was clamped for 45 minutes at 32°C and sacrificed at 0.5h, 1h,

2h, 4h, and 24h following reperfusion. Renal tissue was collected and pooled from 3 separate

mice for each time point. a) IDO mRNA levels were detected by RT-PCR. GAPDH was used to

control for the amount of RNA used in each sample. mRNA changes by this method are semi-

quantitative and densitometry is provided as a ratio of IDO:GAPDH to demonstrate increases

following IRI b) IDO protein (~45kDa) was detected using a rabbit-anti-IDO antibody in

Western blot. Total ERK was used as a protein loading control. Densitometry of IDO:ERK levels

is shown. Data are representative of 2 separate experiments.

Figure 2) Immuno-histochemical staining displays tubular expression of IDO in response to

IR. Paraffin embedded fixed kidney section were probed with anti-IDO antibody in order

pinpoint IDO expression by a specific cell type due to IR. (a) Sham operated mice. (b) Mice

exposed to 45 minutes of ischemia at 32 °C and sacrificed 24hrs following reperfusion. Data are

representative of 3 separate experiments.

Figure 3) IDO-KO mice have improved kidney function subsequent to IR. C57BL/6 IDO-

KO and wild type age matched C57BL/6 mice were exposed to 45 minutes of ischemia at 32°C

and sacrificed 48 hours post reperfusion. a) Serum creatinine levels of IDO-KO mice compared

to WT mice, p = 0.008. b) Serum urea levels of IDO-KO mice compared to WT mice, p = 0.006.

Data are represented as mean ± SEM, n = 6 for IDO-KO, n = 9 for WT, and n = 4 for sham

operated mice.
Page 23 of 32

                                                                                                               23

                Figure 4) IDO expression contributes to renal ischemia reperfusion injury. IDO expression

                contributes to renal ischemia reperfusion injury: C57BL/6 wild type mice were pre-treated with

                3mg of 1-MT dissolved in PBS or PBS alone 1 hour prior to 45 minutes of ischemia at 32°C.

                Following reperfusion, the mice were injected IP with the same dosage, 1-MT or PBS alone,

                twice a day for 48 hours a) Serum creatinine of 1-MT treated compared to PBS treated, p =

                0.031. b) Serum urea levels of 1-MT treated compared to PBS treated, p = 0.036. Data are

                presented as mean ± SEM (n = 10 for 1-MT, n = 11 for WT, and n = 4 for sham operated mice).

                Figure 5) IDO-KO mice are resistant to renal IRI. IDO-KO mice are resistant to renal IRI.

                C57BL/6 IDO-KO and wild type mice were exposed to 45 minutes of ischemia at 32°C and

                sacrificed 48 hours post reperfusion. Kidneys were formalin fixed, paraffin embedded and

                sections (5µm) were PAS stained. a) sham-operated, b) WT, and c) IDO-KO d) Tubular necrosis

                and vacuolization score. Blinded scoring of 10 non-overlapping fields (400x) was performed and

                averaged for WT and IDO-KO sections, p = 0.001. Images are representative of each group. Data

                are presented as mean ± SEM (n = 6 for IDO-KO, n = 9 for WT, and n = 4 for sham operated

                mice).

                Figure 6) IDO expression contributes to renal ischemia reperfusion injury: C57BL/6 wild

                type mice were pre-treated with 3mg of 1-MT dissolved in PBS or PBS alone 1 hour prior to 45

                minutes of ischemia at 32 °C. Following reperfusion, the mice were injected IP with the same

                amount of 1-MT or PBS alone, twice a day for 48 hours. Kidneys were formalin fixed, paraffin

                embedded and sections (5µm) were PAS or H&E stained. a) sham-operated, b) PBS-control,
Page 24 of 32

                                                                                               24

and c) 1-MT treated mice d) Tubular necrosis and vacuolization score. Blinded scoring of 10

non-overlapping fields (400x) were performed for PBS control and 1-MT treated mice, p =

0.002. Data are presented as mean ± SEM. (n = 10 for 1-MT, n = 11 for WT, and n = 4 for sham

operated mice).

Figure 7) IDO-KO kidneys have reduced infiltrate following IRI: a, b, c) H&E staining of

PBS control, 1-MT pre-ischemia treated and IDO-KO kidney sections was carried out to detected

infiltration. Images are representative for each group. n=8 for WT, n=7 for 1-MT, n=6 for IDO-

KO).

Figure 8) IDO-KO kidneys have reduced apoptosis in response to IRI. Kidney sections from

a) sham, b) WT, and c) IDO-KO mice were fixed, and cell death was determined by TUNEL

staining. d) Blinded scoring of 10 non-overlapping fields was performed and averaged for WT

and IDO-KO (400x), p = 0.03. Images are representative for each group. Data are presented as

mean ± SEM. (n = 6 for IDO-KO, n = 9 for WT).
Page 25 of 32

          Figure 1.0

                       a

                                                        IRI mRNA

                                             0.5   1      2       4       24        sham   (h)

                             IDO

                             GAPDH

                            IDO: GAPDH
                            Density ratio

                       b
                                                       IRI Protein

                                            0.5    1          2       4        24      sham (h)

                           IDO

                           Total ERK

                           IDO: ERK
                           Density ratio
Page 26 of 32

Figure 2.0

             a   b
Page 27 of 32

          Figure 3.0

                       a                 b
                           * p = 0.008       * p = 0.006
Page 28 of 32

Figure 4.0

             a                 b

                 * p = 0.031       * p = 0.036
Page 29 of 32

          Figure 5.0

                       d

                                                  60
                           Tubular necrosis/vac

                                                            p = 0.001

                                                  40
                                   (%)

                                                  20

                                                  0
                                                       WT   IDO-KO
Page 30 of 32

Figure 6.0

             d

                                    p = 0.002
             Tubular necrosis/vac
                     (%)
Page 31 of 32

          Figure 7.0
                a                 b          c

                    PBS control       1-MT       IDO null
Page 32 of 32

Figure 8.0

             a   c

             b   d

                     p = 0.03
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