View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector original article http://www.kidney-international.org & 2006 International Society of Nephrology Differential regulation of metzincins in experimental chronic renal allograft rejection: Potential markers and novel therapeutic targets CC Berthier1,2,8, N Lods1,8, SA Joosten3, C van Kooten3, D Leppert4, RLP Lindberg4, A Kappeler5, F Raulf6, EE Sterchi7, D Lottaz7 and H-P Marti1 1Division of Nephrology/Hypertension, Inselspital, University of Bern, Bern, Switzerland; 2Division of Nephrology, University Hospital of Zu¨rich, Zu¨rich, Switzerland; 3Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; 4Department of Neurology, University of Basel, Basel, Switzerland; 5Institute of Pathology, University of Bern, Bern, Switzerland; 6Department of Transplantation, Novartis Institutes of BioMedical Research, Basel, Switzerland and 7Institute of Biochemistry and Molecular Biology, University of Bern, Bern, Switzerland Chronic renal allograft rejection is characterized by chronic renal allograft rejection. Thus, an altered pattern of alterations in the extracellular matrix compartment and in metzincins may represent novel diagnostic markers and the proliferation of various cell types. These features are possibly may provide novel targets for future therapeutic controlled, in part by the metzincin superfamily of interventions. metallo-endopeptidases, including matrix metalloproteinases Kidney International (2006) 69, 358–368. doi:10.1038/sj.ki.5000049 (MMPs), a disintegrin and metalloproteinase (ADAM) and KEYWORDS: chronic rejection; matrix metalloproteinases; meprin; meprin. Therefore, we investigated the regulation of metzincins; renal allograft; tissue inhibitors of metalloproteinases metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen The two main causes responsible for late renal allograft homogenates and paraffin sections of rat kidneys at day 0 failure are chronic rejection, a major contributor to chronic (healthy controls) and during periods of chronic rejection at allograft nephropathy, and patient death with a functioning day þ 60 and day þ 100 following transplantation. The transplant.1 Therapy and precise diagnosis of chronic messenger RNA (mRNA) expression was examined by rejection remain difficult. Therefore, the definition of new Affymetrix Rat Expression Array 230A GeneChip and by diagnostic markers and of novel therapeutic targets continue real-time Taqman polymerase chain reaction analyses. to be of great clinical relevance. Protein expression was studied by zymography, Western blot Alterations in the extracellular matrix (ECM) compartment analyses, and immunohistology. mRNA levels of MMPs and in the proliferation rates of various cell types are key (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of features in chronic allograft nephropathy, including chronic metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming rejection processes.2 The metabolism of ECM proteins in the growth factor (TGF)-b1 significantly increased during kidney is influenced substantially by the metzincin superfamily chronic renal allograft rejection. MMP-2 activity and of metalloendopeptidases, notably by matrix metalloprotein- immunohistological staining were augmented accordingly. ases (MMPs)3,4 and to some extent also by meprin5–7 and by a The most important mRNA elevation was observed in the disintegrin and metalloproteinases (ADAMs).8,9 case of MMP-12. As expected, Western blot analyses also MMPs are traditionally classified into four categories, demonstrated increased production of MMP-12, MMP-14, namely interstitial collagenases (MMP-1/-8/-13), gelatinases and TIMP-2 (in the latter two cases as individual proteins and (MMP-2/-9), stromelysins (MMP-3/-7/-10/-11/-12) and as complexes). In contrast, mRNA levels of MMP-9/-24 and membrane-type -MMP (MMP-14/-15/-16/-17).10,11 meprin a/b had decreased. Accordingly, MMP-9 protein levels The main function of MMPs is the degradation of ECM and meprin a/b synthesis and activity were downregulated proteins and to a lesser extent the regulation of cell significantly. Members of metzincin families (MMP, ADAM, proliferation.4,12–14 Reversible MMP inhibition occurs by and meprin) and of TIMPs are differentially regulated in their natural inhibitors, tissue inhibitors of metalloprotein- ases (TIMPs).15 MMPs represent important mediators in Correspondence: H-P Marti, Division of Nephrology/Hypertension, Inselspi- inflammatory diseases and also in tumor invasion, kidney tal Bern, 3010 Bern, Switzerland. E-mail: [email protected] morphogenesis and bone turnover.3,11,16–20 8Both these authors contributed equally to this investigation. MMPs may act as proinflammatory mediators in allograft 10,11 Received 25 May 2004; revised 24 June 2005; accepted 11 August 2005 rejection in several ways: direct tissue injury, augmenta- 358 Kidney International (2006) 69, 358–368 CC Berthier et al.: Metzincins in renal allograft rejection original article tion of cell proliferation and/or migration4,14,21 and facilita- for several days, followed by chronic rejection beyond day tion of tissue invasion by extrinsic cells (e.g. leukocytes).20 þ 50 after transplantation. Chronic rejection is characterized Meprin represents either a cell-surface bound or a secreted by severe histological alterations as well as by increases in zinc endopeptidase of the astacin family of the metzincins.5–7,22 microalbuminuria and serum creatinine levels, as shown in This protease is composed of a and b subunits of Figure 1 and Table 1, and as previously reported.29,30 approximately 85–110 kDa, respectively, that form homo- and hetero-oligomeric complexes.7,23 Rat meprin A exists either as Affymetrix GeneChip analysis homo-oligomers of a subunits or as heterodimers or tetramers Results of Affymetrix Rat Expression Array 230A GeneChip of a and b subunits.7,23 On the contrary, rat meprin B consists analysis are summarized in Table 2. Significant upregulation essentially of homo-oligomeric b subunits.7,23 Only the homo- was observed for MMP-11/-12/-14, ADAM-17, TIMP-1/-2 oligomers of meprin A are secreted proteins.7 Meprin B and and transforming growth factor (TGF)-b1 during chronic hetero-oligomers of meprin A remain membrane bound.7 rejection at day þ 60 and day þ 100 (with the exception Meprin is highly regulated and is strongly expressed at the of ADAM-17 and MMP-11). Levels of these two proteases brush border membranes of renal proximal tubular cells and failed to reach statistical significance at a later time point intestinal epithelial cells.5,24 Meprin cleaves a wide range of (day þ 100). peptides and proteins, including collagen type IV, laminin, In contrast, MMP-9 and meprin a messenger RNA fibronectin, and nidogen.22–25 (mRNA) substantially decreased as a result of the chronic With respect to the kidney, meprin has been investigated rejection process at both time points and MMP-24 was in experimental ischemia and reperfusion injury.5,26 Mice reduced significantly at day þ 60. expressing lower meprin A levels in the kidney showed a MMP-2/-3/-7/-8/-10/-16/-23, ADAM-10 and TIMP-3 gene decreased degree of renal damage as a result of ischemia and expression remained unchanged during the rejection process. reperfusion.27 Meprin was found to be cytotoxic to renal To complete our studies, we investigated selected metzin- tubular epithelial cells and its inhibition by actinonin cin substrates, as shown in Table 2. As expected from the ameliorated ischemia–reperfusion injury in rats.5 histological alterations commonly seen in chronic allograft The ADAMs represent a family of membrane proteins rejection and from previous descriptions of our model,29,30 containing a disintegrin and metalloprotease domain.8,9 The main substrates of ADAM are integral membrane or ECM proteins. Altered ADAM expression has been found in several disorders including asthma, arthritis, Alzheimer’s disease, Day 0 Day + 60 Day +100 atherosclerosis, and malignancy.8,9 We focused on ADAM-17 (tumour necrosis factor-a converting enzyme) due to its prominent role in inflammation.28 In our present studies, we analyzed the regulation of a wide spectrum of metzincins in the Fisher (F344) to Lewis (LEW) kidney transplant model.29,30 Two time points of chronic rejection were chosen, days þ 60 and þ 100 after transplantation.29,30 RESULTS F344 to LEW renal transplant model Figure 1 | Renal histology. Left panel: healthy control kidney (day 0). For our studies, we used the well-established F344 to LEW Middle panel: chronic rejection of an F344 renal allograft at day þ 60 kidney transplant model. This experimental model has been demonstrating glomerular and tubulo-interstitial alterations, as 29,30 scored in Table 1 (Periodic acid-Schiff staining). Right panel: chronic extensively described in the past. Importantly, LEW rejection at day þ 100 depicting similar histological alterations, as recipients develop acute rejection at approximately day þ 25 compared to day þ 60. Table 1 | Histological and functional characteristics of chronic allograft rejection Interstitial infiltrate, tubular atrophy, fibrin in glomeruli, Arterial Creatinine mesangiolysis, Interstitial fibrointimal Microalbuminurea7s.d. (serum)7s.d. glomerulitis fibrosis hyperplasia (mg/24 h) (lmol/l) Day 0a 0b 0b 0b 0.570.13 48.6773.06 Days +60 to +100c 2b 3b 1b 10.5179.74 79.43710.47 an=8. bBanff classification.29,31 cn=12 (day +60) and n=6 (day +100). Kidney International (2006) 69, 358–368 359 original article CC Berthier et al.: Metzincins in renal allograft rejection Table 2 | Affymetrix GeneChip analysis of 25 genes (12 MMP, three TIMP, two ADAM, two meprins, TGF-b1, four collagens, and a proteoglycan core protein) expressed in allografts with chronic rejection (samples at day +60 and day +100) and in healthy kidney controls (samples at day 0) RefSeq/GenBank Affymetrix Gene accession no. probe set Day 0 Day +60 Day +100 MMP-2 NM_031054 1369825_at 23.877.6 18.676.3 15.574.2 MMP-3 NM_133523 1368657_at 15.775.6 18.979.6 14.577.2 MMP-7 a NM_012864 1368766_at 2.371.0a 18.579.3a 8.