Neuropeptides 45 (2011) 113–121

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Neuropeptides

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Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP)show increased susceptibility to in vivo renal ischemia/reperfusion injury

Peter Szakaly a, Eszter Laszlo b, Krisztina Kovacs c, Boglarka Racz c, Gabriella Horvath b, Andrea Ferencz d,Andrea Lubics b, Peter Kiss b, Andrea Tamas b, Reka Brubel b, Balazs Opper b, Akemichi Baba e,Hitoshi Hashimoto e, Jozsef Farkas b, Attila Matkovits b, Tamas Magyarlaki f, Zsuzsanna Helyes g,Dora Reglodi b,⇑a Department of Surgery, University of Pecs, Hungaryb Department of Anatomy, University of Pecs, Hungaryc Department of Biochemistry and Medical Chemistry, University of Pecs, Hungaryd Department of Surgical Research and Techniques, University of Pecs, Hungarye Graduate School of Pharmaceutical Sciences, Osaka University, Japanf Department of Laboratory Medicine, University of Pecs, Hungaryg Department of Pharmacology and Pharmacotherapeutics, University of Pecs, Hungary

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 August 2010Accepted 7 December 2010Available online 5 January 2011

Keywords:HistologyNephroprotectionPACAP knockoutKidneyMouseCytokineSuperoxide dismutase

0143-4179/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.npep.2010.12.003

⇑ Corresponding author. Address: Department of A7624 Pecs, Szigeti u 12, Hungary. Tel.: +36 72 536001

E-mail address: dora.reglodi@aok.pte.hu (D. Reglod

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with well-known cytopro-tective effects. We have reported earlier that PACAP decreases mortality and the degree of tubular atro-phy in a rat model of renal ischemia/reperfusion injury. Recently, we have shown that kidney culturesisolated from PACAP deficient mice show increased susceptibility to renal oxidative stress. Based on theseprevious studies, we raised the question whether PACAP deficient mice display increased sensitivity toin vivo kidney ischemia/reperfusion. PACAP�/� mice underwent 45 or 60 min of renal ischemia followedby 2 weeks reperfusion. Kidneys were processed for histological analysis. Sections stained with PAS–hae-matoxylin were graded for the following parameters: degree of tubular dilation, Bowmann’s capsule dila-tion, lymphocyte and macrophage infiltration, thyroidization and the disappearance of the PAS-positiveglycocalyx from under the brush border. In other sets of experiments, tissue cytokine expression and thelevel of the endogenous antioxidant superoxide dismutase (SOD) were also determined after 60 minischemia/reperfusion. Our results show that while intact kidneys were not different between wild-typeand PACAP deficient mice, marked differences were observed in the histological structures in groups thatunderwent ischemia/reperfusion. PACAP deficient mice had a worse histological outcome, with signifi-cantly higher histological scores for all tested parameters. Cytokine expression was also markedly differ-ent between wild-type and PACAP deficient mice. In addition, the level of SOD was significantly lower inPACAP�/� animals after ischemia/reperfusion. In conclusion, the lack of endogenous PACAP leads tohigher susceptibility to in vivo renal ischemia/reperfusion, suggesting that PACAP has an endogenousrenoprotective effect.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Pituitary adenylate cyclase activating polypeptide (PACAP) wasfirst isolated as a hypothalamic neuropeptide (Vaudry et al., 2009).Since its discovery in 1989, PACAP and its receptors (PAC1R, VPAC1and VPAC2) have been described in the nervous system and inperipheral organs (Vaudry et al., 2009). PACAP has, among otherfunctions, neuroprotective and general cytoprotective effects

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natomy, University of Pecs,x5398; fax: +36 72 536393.i).

against different types of injuries. In the nervous system, PACAPexerts anti-apoptotic and anti-inflammatory actions, providingprotection of neurons against various types of harmful stimuli(Somogyvari-Vigh and Reglodi, 2004). PACAP has been proven tobe protective in animal models of neurodegenerative diseases,traumatic and ischemic conditions (Kovesdi et al., 2008; Ohtakiet al., 2008; Reglodi et al., 2006). Similar effects have been de-scribed in non-neural tissues. PACAP promotes survival of endo-thelial cells, cardiomyocytes, lymphocytes, Schwann cells, smallintestine and ovarian follicular cells against various noxious stim-uli (Delgado and Ganea, 2000; Ferencz et al., 2009; Giunta et al.,2010; Guillot et al., 2008; Lee et al., 1999; Racz et al., 2007, 2010).

114 P. Szakaly et al. / Neuropeptides 45 (2011) 113–121

PACAP also occurs in the urinary system and exerts several dis-tinct effects. In the kidney, PACAP binding has been shown and di-rect evidence for the receptors by RT-PCR has been provided (Liet al., 2010). We have recently proven the occurrence of the pep-tide itself in the kidney (Horvath et al., 2010a). The peptide has ef-fect on the renal blood flow and renin synthesis (Gardiner et al.,1994; Hautmann et al., 2007). Increasing body of evidence suggeststhat PACAP is a promising protective agent in renal damage. PACAPdecreases the effects of renal injury induced by ischemia/reperfu-sion, gentamicin, cisplatin, oxidative stress, experimental diabeticand myeloma nephropathy (Arimura et al., 2006a,b; Horvathet al., 2010b, 2011; Li et al., 2008, 2010; Szakaly et al., 2008).

PACAP deficient (PACAP�/�) mice have been generated to studythe effects of endogenous PACAP (Hashimoto et al., 2001). Thesemice display several abnormalities. For example, abnormal axonalarborization, altered cerebellar development and abnormal behav-ior have been described (Allais et al., 2007; Hashimoto et al., 2001;Yamada et al., 2010). Several peripheral abnormalities have alsobeen reported, such as formation of colorectal tumors (Nemetzet al., 2008), altered pain responses (Sandor et al., 2010) and re-duced white adipose tissue along with increased insulin sensitivity(Tomimoto et al., 2008).

Regarding the urinary system, it has been recently reported thatPACAP deficient mice have bladder dysfunction and altered so-matic sensitivity in the urinary system (May and Vizzard, 2010).It has also been reported that PACAP deficient mice display a lackof light-induced elevation of renal sympathetic nerve activity(Hatanaka et al., 2008). We have recently described that cell cul-tures from kidneys of mice lacking endogenous PACAP display in-creased sensitivity to in vitro oxidative stress and hypoxia(Horvath et al., 2010a,c). It is not known, however, whether thesemice show increased sensitivity to renal injury in vivo. Therefore,the primary aim of the present study was to compare the degreeof renal damage ’’in wild-type and PACAP deficient (PACAP�/�)mice subjected to renal ischemia/reperfusion. Furthermore, weinvestigated the cytokine and antioxidant profile of wild-typeand PACAP deficient mice following kidney ischemia/reperfusion.

2. Materials and methods

2.1. Animals

Generation by a gene targeting technique, maintenance andbackcrossing of CD1 PACAP�/� mice has been reported previously(Hashimoto et al., 2001). Mice were kept under 12-h light/dark cy-cle with free access to food and water. Animal breeding, housing,care and application of experimental procedures were in accor-dance with institutional guidelines under approved protocols(No. BA02/2000-20/2006, University of Pecs).

2.2. Renal ischemia/reperfusion

Animals were anesthetized with pentobarbital (50 mg/kg body-weight). Temperature of animals was measured with a rectal ther-mometer and was kept constant during the operation with awarming blanket. Low molecular weight heparin (dalteparin-so-dium, 1.25 U/animal) was injected into the jugular vein before lap-arotomy. Total median laparotomy was performed, renal vesselswere freed and warm ischemic damage was induced by cross-clamping renal pedicles for 45 or 60 min in PACAP deficient andwild-type mice. Clamping was performed only on one side (left),while the other side was left intact. Sham-operated mice wereanesthesized and underwent laparotomy, but renal pedicles wereleft intact (n = 5). These mice served as sham-operated controlmice.

2.3. Histology

Animals (n = 10 wild-type and n = 10 PACAP deficient mice)were sacrificed 2 weeks after 45 and 60 min ischemia/reperfusion.Mice were perfused by 4% paraformaldehyde containing 15% picricacid under pentobarbital anesthesia and kidneys were removed.The parenchyme was freed from the capsule, kidneys were cut lon-gitudinally into two halves and then were placed in 4% paraformal-dehyde for fixation. Serial, 10 lm thick sections from the kidneywere made and stained with routine haematoxylin–eosin and peri-odic acid Schiff (PAS)–haematoxylin staining. Digital photomicro-graphs were captured with a Nikon FXA photomicroscopeattached to a digital camera (Spot RT Color camera). Histologicalevaluation of the kidneys was made in a blinded fashion. The de-gree of renal tubular atrophy was determined in the cortex andmedulla of the kidney according to slight modifications of standardgradings described previously (Gulmen et al., 2009; Kiris et al.,2008; Miller et al., 1992; Senturk et al., 2008). The followingparameters were graded on a scale 0–2 (0 – absent, 1 – mild-mod-erate, 2 – severe): lymphocyte infiltration, macrophage accumula-tion, tubular dilation, dilation of the Bowmann’s capsule,disappearance of the PAS-positive glycocalyx from under the brushborder and the degree of thyroidization. For lymphocyte and mac-rophage infiltration, as well as to assess the degree of tubular dila-tion, 10 fields in each kidney were examined under 40�magnification, and cellular infiltration was scored as absent, mildor severe (0, 1, 2, respectively). Thyroidization of the tubules wasexamined also in 10 fields in each kidney, by assessing the percent-age of the tubules presenting thyroidization: under 20% grade 0,between 20% and 60%: grade 1, and above 60% grade 2. Bowmann’scapsule dilation was examined in the whole kidney: the entire cor-tex was evaluated, and all renal corpuscles were scored as no dila-tion, mild or severe, bubble-like appearance (grade 0, 1, 2,respectively). The PAS-positive glycocalyx is normally well-pro-nounced under the brush border of the proximal tubuli (grade 0).In mild ischemic damage, a thinning of this layer was observedwith occasional missing staining (grade 1). In severe cases, noPAS-positive layer could be observed (grade 2). For this parameter,also 10 fields in each kidney were scored. Finally, adding up allscores, a total grading score was obtained which included all exam-ined parameters. Data are given as mean ± SEM. Statistical analysiswas performed with Mann–Whitney non-parametric test, andP < 0.05 was considered as statistically significant difference.

2.4. Cytokine array after in vivo ischemia/reperfusion

Kidneys from wild-type and PACAP deficient mice (n = 5 in bothgroups) were removed 24 h after 60-min ischemia/reperfusion andwere processed for cytokine array studies. Control kidneys were re-moved from sham-operated mice (n = 3). Cytokine array from tis-sue homogenates was performed using Mouse Cytokine ArrayPanel A Array kit from R&D Systems (Biomedica Hung., Budapest,Hungary). Kidneys were excised then homogenized in PBS withprotease inhibitors. Triton X-100 was added to a final concentra-tion of 1%. The samples were stored at �80 �C prior to use. The ar-ray was performed as described by the manufacturer. Afterblocking the array membranes for 1 h and adding the reconstitutedDetection antibody coctail for another 1 h at room temperature,the membranes were incubated with 1 ml of tissue homogenatesat 2–8 �C overnight on a rocking platform. After washing with buf-fer three times and addition of horseradish peroxidase-conjugatedstreptavidin to each membrane, we exposed them to a chemilumi-nescent detection reagent (Amersham Biosciences, Hungary), thenside up to an X-ray film cassette (Jin et al., 2008). The developedfilms were scanned and the pixel volumes of the spot were deter-mined by using GenePix Pro 3.0 software. Pixel volumes of the spot

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of interest were normalized to the control. The array was repeatedthree times. Statistical analysis was performed by analysis of vari-ance and Student’s t test.

2.5. Superoxide dismutase (SOD) assay

In order to elucidate whether the oxidative stress markers differbetween PACAP deficient and wild-type mice after in vivo renal

Fig. 1. Representative photomicrographs of PAS-stained kidney sections from wild type (after 45 min (C and D) or 60 min (E and H) ischemia. Scale bar: 50 lm. (A and B) Normalmouse) and cortex (B: PACAP deficient mouse). Note the PAS-positive glycocalyx onBowmann’s capsules show normal width (asterisks). (C) Cortex of a wild-type mouse aftecapsules (asterisks) are slightly widened. In the connective tissue around the tubuli there(double arrows). (D) Cortex of a PACAP deficient mouse after 45 min ischemia and 2(thyroidization, arrows). The Bowmann’s capsules are extremely enlarged (asterisks), theCortex of a wild-type mouse after 60 min ischemia and 2 weeks reperfusion. Altough theis still preserved (arrows). Some of the tubuli show thyroidization (arrowheads). (F and HThe tubuli are enlarged and the lining epithelial cells are flattened. Almost all of the tubutubuli (asterisks). Complete disappearance of glycocalyx is visible.

ischemia/reperfusion, kidneys from a further group of mice (n = 5wild-type and n = 5 PACAP deficient mice) were processed for bio-chemical analysis of superoxide dismutase (SOD), the main endog-enous antioxidant marker. SOD was measured in homogenatesusing a superoxide dismutase assay kit (Calbiochem-NovabiochemCorp, Darmstadt, Germany.), as described previously (Ferenczet al., 2009). One reagent of the kit underwent alkaline autooxida-tion, which was accelerated by SOD. Autooxidation of this reagent

left column) and PACAP deficient mice (right column), without ischemia (A and B) orly structured kidney of control, intact animals, corticomedullar border (A: wild-typethe luminal surface of the tubuli lined by cuboidal epithelial cells (arrows). Ther 45 min ischemia and 2 weeks reperfusion. The tubuli (arrows) and the Bowmann’s

are some PAS-stained macrophages (arrowheads). The glycocalyx is still preservedweeks reperfusion. Numerous wide tubuli are filled with PAS-positive substanceconnective tissue contains a lot of PAS-stained macrophages (arrowheads). (E and G)tubuli are widened, the PAS-positive glycocalyx on the luminal surface of the tubuli) Cortex of a PACAP deficient mouse after 60 min ischemia and 2 weeks reperfusion.li display thyroidization (arrows). A severe lymphatic infiltration appears around the

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yielded a chromophore, which absorbed maximally at 525 nm. Re-sults are given in mean values of activity (units per gram) ± stan-dard error of the mean (SEM). Data were analyzed with one-wayANOVA. The level of significance was set at P < 0.05.

Fig. 2. Mean score obtained from the all the examined parameters in wild-type andPACAP deficient mice. Data are expressed as mean ± SEM. ⁄⁄⁄P < 0.001 vs wild-typemice.

Fig. 3. Scores of individual histopathological parameters in wild-type and PACAP deficieBowmann’s capsule; (D) tubular dilation; (E) disappearance of PAS-positive glycocalyx; (Fvs wild-type mice.

3. Results

3.1. Histology

Sham-operated mice showed normal histological structure ofthe kidneys. Histological scores were 0 in both wild-type and PA-CAP deficient mice in all examined parameters in these groups(Fig. 1A and B). Representative photomicrographs from kidneysafter ischemia/reperfusion are shown in Fig. 1C–H. The total scorecalculated from all scores showed a significant difference on boththe non-clamped and clamped sides, after both 45 and 60 minischemia, with PACAP deficient mice having significantly worsescores (Fig. 2).

Analyzing the individual scores it was found that all scores weresignificantly higher on the ischemic side compared to the contra-lateral sides (Fig. 3). Lymphocyte infiltration was higher on thenon-clamped sides of the PACAP deficient animals. In the 45 mingroup, no difference was found between the two groups on theischemic side, but in wild-type mice the degree of lymphocyteinfiltration was not increased further with 60 min ischemia, whilein PACAP deficient mice it was significantly higher (Fig. 3A). Mac-rophage infiltration showed a significant difference in the 45 min

nt mice. (A) lymphocyte infiltration; (B) macrophage infiltration; (C) dilation of the) thyroidization. Data are expressed as mean ± SEM. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001

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group (Fig. 3B). Dilation of the Bowmann’s capsule was very prom-inent in PACAP deficient mice, while it was only slightly increasedin wild-type animals (Fig. 3C). The degree of tubular dilation wassignificantly higher in PACAP deficient mice in the 45 min group,but differences could no longer be detected with 60 min ischemia

Fig. 4. Representative cytokine arrays of kidneys from control wild-type (panel a), cont(panel c) and PACAP deficient (panel d) mice. Table shows the cytokines of the array irepresent loading controls (ctr).

(Fig. 3D). The disappearance of the PAS-positive glycocalyx wasapparent after 45 min ischemia in both groups, but scores furtherincreased in PACAP deficient mice after 60 min ischemia (Fig. 3E).Thyroidization was not visible on the contralateral sides, while itwas very well expressed in PACAP deficient mice in the ischemic

rol PACAP deficient (panel b), 24 h after 60 min ischemia/reperfusion of wild-typen the representative double spots. Left and right upper as well as left lower spots

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kidneys after both 45 and 60 min ischemia compared to wild-typeanimals, where only a small degree of tubular thyroidization wasobserved (Fig. 3F).

3.2. Cytokine array

There was no marked difference in the several cytokines/che-mokines examined between wild-type and PACAP deficient controlmice (Fig. 4, panel a and b). Twenty-four hours after 60 min ische-mia/reperfusion, marked changes were observed in both animalgroups. Several cytokines/chemokines were upregulated afterischemia/reperfusion, with manifest differences between ligatedsides of wild-type and PACAP deficient animals (Fig. 4, panel cand d).

BLC (B lymphocyte chemoattractant) was slightly expressedafter ischemia/reperfusion in wild-type, while it was very stronglyexpressed in PACAP�/� mice (Fig. 4, spot A1). Similar patterns wereobserved in G-CSF (granulocyte colony stimulating factor) and IL-1ra (interleukin-1ra) (Fig. 4, spots A2,3 and 11, respectively), inIL-6 (Fig. 4, spot B4), in KC (keratinocyte-derived chemokine),MCP-1 (monocyte chemoattractant protein-1) and MIP-2 (macro-phage inflammatory protein-2) (Fig. 4, spots C3, C5, C10, respec-tively), in TIMP-1 (tissue inhibitor of metalloproteinases-1) andTREM-1 (triggering receptor expressed on myeloid cells-1)(Fig. 4, spots D2 and D4). In contrast, some cytokines were lessstrongly expressed in PACAP�/� mice than in wild-type animalsafter ischemia/reperfusion: sICAM-1 (soluble intercellular adhe-sion molecule-1), interferon-c, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL7, IL-10, IL-13, IL-16, IL-17, IL-23, IL-27, IP-10, M-CSF (macrophage col-

Fig. 5. Graphs showing quantification of the cytokine array. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0intensity of the spots were above 50,000.

ony stimulating factor), MCP-5 (monocyte chemotactic protein-5),MIG (monokine induced by gamma interferon) and SDF-1 (stromalcell-derived factor-1) (Fig. 4, spots A7,8,9,10,12,B1,2,5,6,7,9,10,11,12, C1,4,6,7 and C12, respectively). The resultsof the quantitative analysis are found in Fig. 5, with only those ar-rays represented where the mean pixel intensity of the spots wereabove 50,000.

3.3. SOD activity

The activity of the endogenous scavenger SOD showed a slightlyreduced level in PACAP deficient mice compared to their wild-typemates under control conditions. In wild-type mice, levels of SODdecreased after 60 min ischemia/reperfusion, however, it was notsignificantly different from the control levels. A significant de-crease in SOD level was observed in PACAP deficient mice afterischemia/reperfusion (Fig. 6).

4. Discussion

The present study showed that PACAP deficient mice have re-duced resistance to renal ischemia/reperfusion injury in vivo.While intact kidneys were not different between wild-type and PA-CAP deficient mice, marked differences were observed both after45 and 60 min ischemia/reperfusion. PACAP deficient mice showedmore severe histological outcome than wild-type mice in all histo-pathological parameters examined, implying that endogenous PA-CAP has renoprotective effects. Furthermore, cytokine profile of thekidney was markedly altered in PACAP deficient mice and the level

.001 vs wild-type animals. Only those arrays are represented where the mean pixel

Fig. 6. Changes in kidney tissue superoxide dismutase (SOD) activity in controlwild-type and PACAP deficient mice as well as following 60 min ischemia/reperfusion. Data are given as mean ± SEM. ⁄⁄P < 0.01 vs control kidney.

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of SOD was significantly reduced in these mice after ischemia/reperfusion. These observations suggest that PACAP, either derivedfrom the kidney itself or reaching the kidney via blood vessels, ispart of the endogenous protective machinery. The endogenous pro-tection by PACAP is most probably receptor-mediated, since in ourprevious study adding exogenous PACAP to a kidney cell culturederived from PACAP deficient mice could reverse the increasedin vitro susceptibility of these cells to oxidative stress (Horvathet al., 2010a).

Several lines of evidence suggest that the observed cytoprotec-tive action of PACAP is a general phenomenon in the nervous sys-tem and also in peripheral organs. It has been suggested thatPACAP acts as an endogenous protective agent against differentstimuli (Vaudry et al., 2009). Our present observations are in goodaccordance with several other reports, which have shown that thenormal structure of several organs of PACAP deficient mice displayno major alteration, but marked differences exist when a harmfulstimulus acts in the system. For example, in the nervous systemit has been described that cultured cerebellar granule cells ob-tained from PACAP deficient mice react to oxidative stress with de-creased survival (Vaudry et al., 2005). In a model of cerebralischemia, PACAP deficient mice have an increased infarct volume,along with increased edema formation (Nakamachi et al., 2010;Ohtaki et al., 2008). In a model of axonal regeneration, motor neu-ron survival after axotomy was not significantly different in PACAPdeficient versus wild-type mice, but recovery was significantly de-layed (Armstrong et al., 2008). Similar observations have beenmade in peripheral tissues in a few studies. It has been reportedthat PACAP deficient mice do not have altered intestinal structurein intact animals, but in a model of colitis, more severe histologicaloutcome can be observed (Azuma et al., 2008; Nemetz et al., 2008).Recent studies have revealed that PACAP deficient mice are moresusceptible also in small intestinal ischemia/reperfusion, with sim-ilar intestinal structure in intact animals (Ferencz et al., 2010a,b).This is in accordance with our present findings in the kidney: theintact kidneys showed no apparent histological difference betweenwild-type and PACAP deficient mice. However, when they weresubjected to ischemia/reperfusion injury, marked differences wereobserved. Our present study proves that the higher susceptibilityof PACAP deficient mice to renal damage observed in vitro (Horv-ath et al., 2010a), is also present in vivo. Histopathological scoresincreased on both non-ligated and on ligated sides, with ligatedsides displaying obviously much more severe injuries. It is knownthat the non-ligated kidney also undergoes compensatory andpathological changes (Durrani et al., 2006; Ouellette et al., 1990;

Tokuyama et al., 2007). These changes were also more severe inPACAP deficient mice, the reason for which is not known at themoment.

Two main mechanisms have been proposed for the cytoprotec-tive effects of PACAP: anti-apoptotic and anti-inflammatory effects,both of which processes have major roles in the ischemia/reperfu-sion-induced damage of the kidney. In the present study we exam-ined the differences in cytokine expression and the level of theendogenous scavenger SOD in wild-type and PACAP deficient mice.Cytokines and chemokines are inflammatory mediators, involvedin the pathogenesis of renal ischemia/reperfusion injury (Basileet al., 1998; Jain et al., 2000; Vannay et al., 2009). Several cyto-kines/chemokines were strongly upregulated after ischemia/reper-fusion, with a markedly different expression in PACAP deficientmice. The exact role of some of these cytokines in renal injuriesis not elucidated yet. Several members of the cytokines and che-mokines have a role not only in the initiation and progression of re-nal inflammation, but also during the restorative phase afterinjuries (Stroo et al., 2010). Cytokine expression shows a temporalexpression pattern after kidney ischemia/reperfusion (Stroo et al.,2010), some having pro- while other anti-inflammatory actions.Our present results show that several cytokines were morestrongly, while others less strongly expressed in PACAP deficientmice. For example, B lymphocyte chemoattractant was morestrongly expressed in PACAP�/� mice. The increase of this chemo-attractant factor is known to precede B cell trafficking and it hasbeen shown that the infiltration of B cells limit the repair processafter kidney ischemia/reperfusion (Jang et al., 2010). Similarly, re-duced expression of MCP-1 and TIMP-1 has been associated withreduced ischemic damage (Prakash et al., 2008). IL-16, on the otherhand, was less strongly expressed in PACAP deficient mice. Inacti-vation of IL-16 has been shown to provide nephroprotection inischemia/reperfusion injury (Wang et al., 2008). Although the exactfunctional significance of endogenous PACAP is not known basedon the present data, but the changed cytokine profile and the morepronounced infiltration of lymphocytes and macrophages in PACAPdeficient mice observed in the present study support the role ofPACAP as an endogenous anti-inflammatory peptide, similar toits effect as an exogenously administered therapeutic agent. PACAPhas been shown to reduce the expression of several pro-inflamma-tory cytokines in several models of kidney injury. PACAP reducedthe expression of TNF-a in cisplatin-induced renal damage and ina rat model of diabetic nephropathy (Li et al., 2008). PACAP alsoinhibited the myeloma light chain-induced elevation of TNF-aand interleukin-6 (Li et al., 2008). The anti-inflammatory effectsof PACAP have also been demonstrated in vivo, in renal ischemia(Riera et al., 2001). We have recently shown that several cytokineswere up-regulated following 45 and 60 min ischemia in rats, whichwas partially counteracted by exogenous PACAP administration(Horvath et al., 2010b). In the present study, we also found that PA-CAP deficient mice had significantly decreased level of SOD, a ma-jor endogenous antioxidant scavenger enzyme. It is not known atthe moment, whether this is a direct influence on the SOD expres-sion, or an indirect effect by preserving more cells producing SOD.Similar findings have been previously described, in intestinalischemia and cold preservation injury in PACAP�/� mice (Ferenczet al., 2010a,b). These results support the hypothesis that PACAPinfluences the endogenous antioxidant/scavenger system.

The anti-apoptotic effect of PACAP has been shown previously,in cisplatin-induced tubular apoptosis and in addition, PACAP alsoattenuated necrosis in light chain-induced tubular cell damage(Arimura et al., 2006b). In a recent study we have shown that PA-CAP attenuated the ischemia/reperfusion-induced decrease in theanti-apoptotic molecule bcl-2, which is in accordance with anotherstudy showing similar effects after cisplatin treatment (Horvathet al., 2010b; Li et al., 2010).

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In summary, our present results show that the previously ob-served renoprotective effects of exogenously administered PACAPexist also endogenously, and PACAP is part of the endogenous pro-tective system of the kidney, the lack of which results in increasedsusceptibility to renal ischemia/reperfusion injury.

Acknowledgements

Support by Grants OTKA F67830, K72592, CNK78480, T73044,PD77474, Richter Foundation, Bolyai Scholarship, PTE AOK Re-search Grant 2009, Grants-in-Aid for Scientific Research from JapanSociety for the Promotion of Science.

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