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MMP-9 and potential benefit of an MMP inhibitor in experimental acute kidney allograft rejection
Transplant Immunology 11 (2003) 137–145
Differential expression of MMP-2yMMP-9 and potential benefit of an MMP inhibitor in experimental acute kidney allograft rejection ` Lods, Mahmoud Hammoud, Hans-Peter Marti* Monica Ermolli, Martin Schumacher, Nadege Division of Nephrology and Hypertension, Inselspital Bern, CH-3010 Bern, Switzerland Received 15 April 2002; accepted 7 August 2002
Abstract Acute cellular allograft rejection is characterized by leukocyte invasion and tissue destruction, associated with qualitative and quantitative alterations in the extracellular matrix (ECM) compartment. Metabolism of ECM proteins is mainly regulated by matrix metalloproteinases (MMP), that are zinc depended endoproteinases. MMP, especially basement membrane degrading MMP2 and MMP-9, also facilitate tissue invasion of leukocytes. In addition, MMP-2 exerts a direct pro-inflammatory effect upon glomerular mesangial cells. Therefore, the investigation of the role of MMP in transplant rejection may lead to novel approaches in the therapy of rejection processes. To our knowledge, this is the first study of acute allograft rejection, formally addressing expression and activity of MMP, including the effect of a MMP inhibiting agent. For our studies, we used the orthotopic kidney allograft model in the stringent Dark Agouti-to-Lewis rat strain combination. Animals were divided into four groups: group A, healthy untreated Lewis rats (ns3); group B, sham operated Lewis rats (ns3); group C, transplanted Lewis rats treated with vehicle solution only (ns12); group D, transplanted Lewis rats treated with MMP inhibitor BB-94 (ns12). Respective animals were treated once daily intraperitonealy with BB-94 (30 mgykg) or vehicle solution only. Treatment lasted from the third preoperative day until the end of the experiment, the time of severe rejection at day q7. Acute kidney allograft rejection led to alterations in the expression and activity of MMP. Overall MMP activity slightly increased despite severe destruction of kidney histology. The MMP inhibitor BB-94 successfully inhibited MMP activity to a high extent. MMP expression did not show uniform findings, since acute rejection led to differential expression of MMP-2 and MMP-9. During the rejection process, MMP-9 showed a small but significant increase, whereas MMP-2 production decreased substantially. Interestingly, BB-94 was able to keep proteinuria at a low level in transplanted animals. In conclusion, MMP—especially MMP-9—appear to represent new mediators involved in acute kidney transplant rejection. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Matrix metalloproteinases; MMP-2; MMP-9; BB-94; Batimastat; Acute allograft rejection
1. Introduction Chronic and relentless loss of kidney graft function remains a crucial problem in kidney transplantation. Acute rejection episodes and chronic allograft nephropathy are principal causes of tissue destruction leading to graft loss. Importantly, severe acute allograft rejection may not only lead to immediate graft failure but may also initiate chronic allograft nephropathy with fibrosis and sclerosis. Therefore, the search for new mediators contributing to the development of rejection remains pivotal. *Corresponding author. Tel.: q41-31-632-31-44; fax: q41-31632-94-44. E-mail address: [email protected] (H.-P. Marti). 0966-3274/03/$ 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 2 . 0 0 1 5 0 - 8
Acute cellular rejection episodes of solid organs are characterized by invasion of leukocytes and tissue destruction, features associated with qualitative and quantitative alterations in the extracellular matrix (ECM) compartment. Turnover of ECM proteins is mainly regulated by extracellularly active matrix metalloproteinases (MMP), which represent the major group of zinc depended matrix degrading proteases w1,2x. Furthermore, MMP are involved in the regulation of tissue invasion by lymphocytes w3,4x. Therefore, the investigation of the role of MMP in transplant rejection may lead to new approaches to the therapy of rejection processes. MMP are proteolytic enzymes involved in ECM degradation and secreted by various cell lines in normal
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and pathological conditions. They are involved in a variety of biological processes, such as proliferation, migration and invasion of cells, organ development, tumor metastasis and angiogenesis w5–7x. In our studies, we focused on MMP-2 and MMP-9, which in particular degrade collagen IV, the main structural component of basement membranes. Most MMP are secreted in a soluble pro-enzyme form and their activity is regulated through multiple events including pro-enzyme activation and interaction with the tissues inhibitors of metalloproteinases, TIMP1-4 w7–9x. It has been shown that MMP activity is up-regulated in different types of acute inflammatory events w3,10,11x. Acute cell-mediated kidney transplant rejection represents a severe form of inflammation, characterized by tissue invasion of mononuclear leukocytes resulting in cellular and ECM destruction. Therefore, it is conceivable that MMP expression may be up-regulated in this disorder. Furthermore, MMP inhibition may favorably influence features involved in allograft rejection, such as proteolytic tissue destruction and possibly invasion of lymphocytes. Therefore, we analyzed the production of MMP, especially MMP-2 and MMP-9 in an experimental rat model of acute kidney allograft rejection. In parallel, we assessed the effect of Batimastat, BB-94 (British Biotech Pharmaceuticals Ltd, Oxford, UK). This agent represents a hydoxamic acid-based synthetic MMP inhibitor, designed as an anti-invasive and anti-metastatic drug particularly well inhibiting MMP-2, MMP-9 and MMP3 w12–14x. To our knowledge, this is the first study formally addressing expression and activity of MMP, including the effect of an MMP inhibiting agent, in the setting of acute kidney allograft rejection. 2. Aims and objectives The aim of this study was to demonstrate if MMP play a role in acute experimental kidney allograft rejection. Specifically, we wanted to demonstrate: (i) how MMP, especially MMP-2 and MMP-9, are regulated during kidney transplant rejection; and (ii) whether MMP inhibition by the use of a synthetic MMP inhibitor shows a benefit with regard to kidney histology and proteinuria. 3. Materials and methods 3.1. Animals Male Dark Agouti (DA; RT1a haplotype) rats and male Lewis (RT11 haplotype) rats were purchased from Charles River (Sulzfeld, Germany). Their body weight at the outset of the studies was 200–220 g. The rats were housed in individual metabolic cages at constant temperature with a 12-h light and 12-h dark cycle and
with free access to water and food tanks. Approval for animal studies was obtained from the commission for animal studies, a local government agency, in accordance to the Guide of Care and Use of Laboratory Animals of the Veterinary Institute of the University of Bern. 3.2. Matrix metalloproteinases inhibitor The synthetic MMP inhibitor BB-94 (Batimastat), w4-(N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)-succinylx-L-phenylalanine-N-methylamide, MW 478, was provided by British Biotech Pharmaceuticals Ltd w14x. BB-94 concentrations demonstrating 50% MMP inhibition were as follows: MMP-1, 5 nmolyl; MMP-2, 4 nmolyl, MMP-3, 20 nmolyl; and MMP-9, 1–10 nmolyl w14x. BB-94 solution (3 mgyml) was prepared in PBSyTween-80 (0.01%) as vehicle by sonication until a uniform suspension was achieved. Animals were treated with a one daily intraperitoneal (ip) injection of 30 mg BB-94 per kg body weight w14x. As a control, an identical volume of vehicle solution only was given in the same fashion. 3.3. Kidney transplantation Orthotopic kidney allotransplantation in male Lewis rats, using donor organs from male DA rats, was performed as published previously w15,16x. 3.3.1. Donor procedure A midline laparotomy incision was made from the xyphoid to the pubic area. A self-retaining retractor was inserted after bowel evisceration. The urethra was sectioned at 10 mm distal of the renal hilus; mobilization of the left kidney from the perinephric fat was performed without encapsulating the kidney from the fat. Without disturbance of the renal hilus, renal artery and renal vein were dissected. A total of 1.5 ml of 200 IU heparin was injected into the inferior vena cava of donors to avoid thrombosis. Euthanasia of the donors was performed by exsanguination. 3.3.2. Recipient procedure Opening and evisceration of the peritoneal cavity was done as described above. The ureter was divided at the level of the lower pole of the kidney. The renal artery was divided proximal to its bifurcation and renal vein was divided at the junction of its middle and lateral third. End-to-end anastomosis of the renal artery was performed first, followed by the anastomosis of the renal vein using 10-0 Ethilon䉸 (Ethicon, Johnson & Johnson). The ureteric end-to-end anastomosis was carried out with 11-0 Ethilon䉸. Thereafter, 4 ml of normal saline were instilled into the abdominal cavity to compensate for the blood loss. Abdominal wall and skin were closed using 4-0 Suturamid䉸. Duration of surgery did not
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exceed 80 min and times of warm ischemia were always below 40 min. Postoperatively, analgesia with paracetamol (100–300 mgykg body weighty4 h) was given directly into the drinking water. 3.4. Experimental design The animals were divided into four groups: group A, healthy untreated Lewis rats (ns3); group B, sham operated Lewis rats (ns3); group C, transplanted Lewis rats treated with vehicle solution only (ns12); group D, transplanted Lewis rats treated with BB-94 (ns12). Respective animals were treated daily with MMP inhibitor or vehicle solution only, from the third preoperative day until the end of the experiment. In previous studies, no effect of administration of vehicle solution only on kidney histology or MMP was described and MMP inhibitor did not affect renal histology in healthy animals w17,18x. At the 7th postoperative day, all animals were killed and kidneys were harvested. Kidneys were cut in halves and were either used for histology or were deep frozen for subsequent zymography and fluorometry, as described below. Blood and 24-h urine samples were collected at days y3 (urine only), 0 and q7 with respect to transplantation. Urinary ratios of proteinycreatinine (mgymmol) as well as plasma sodium (mmolyl) and potassium (mmolyl) concentrations and plasma creatinine (mmoly l) levels were determined by the central laboratory of our institution. 3.5. Zymography Zymography for the analyses of MMP-2 and MMP-9 activities was performed using kidney homogenates. Briefly, frozen half-kidneys were minced on a cold petri dish and transferred into separate tubes. Thereafter, 1 ml of lysis buffer (0.1 M Tris–HCl containing 0.1% Tween-80, pH 7.5) per 60 mg of wet tissue was added. The homogenates were centrifuged twice at 10 000=g for 10 and 5 min, respectively. Extracted supernatants were stored at y20 8C. For subsequent analyses of MMP activity, protein contents in supernatants were determined using the BCA protein assay reagent kit (Pierce, Lausanne, Switzerland). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on precast 10-well, 10% polyacrylamide minigels of 0.75-mm thickness containing 0.1% gelatin (Novex, San Diego, CA), as described previously w17–19x. Briefly, samples adjusted to an uniform protein content of 15 mg in a volume of 10 ml were diluted with the same volume of double-strength, non-reducing sample buffer (0.5 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue). Subsequently, 18 ml of this mixture
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was loaded into each well and electrophoresed at constant voltage (15 Vycm) for approximately 2 h. After incubation in renaturating buffer (2.5% Triton X-100 in 50 mM Tris–HCl, pH 8.0) for 30 min, gels were exposed at 37 8C to proteolysis buffer (50 mM Tris– HCl, pH 8.0, 5 mM CaCl2, 1 mM ZnCl2) for 24 h with gentle agitation. Subsequently, zymograms were fixed and zones of lyses were visualized by staining with 0.2% Coomassie Blue R250. Molecular weight standard (噛161-0372, Bio-Rad, Glattbrugg, Switzerland) was used for assignment of molecular weight. 3.6. Fluorometric analyses Samples were prepared as described above. Thereafter, MMP activity was investigated as described w18,19x. In brief, 5 ml samples were diluted in Hanks buffer to a final volume of 100 ml, containing the quenched fluorescent substrate (7-methoxycoumarin-4-yl)AcetylPro-Leu-Gly-Leu-(3-w2,4-dinitrophenylx-L2,3-diaminopropionyl)-Ala-Arg-NH2 (Bachem, Bubendorf, Switzerland), at a final concentration of 5 mM. This agent represents a very sensitive substrate for activity assays for a broad-spectrum of MMP; examples are MMP-2 (kcatyKms629 000ys M) and MMP-7 (kcaty Kms169 000ys M) w20x. These reaction mixtures were supplemented with or without 2 mM phenylmethylsulfonyl (PMSF; Sigma-Aldrich, Buchs, Switzerland) or 5 mM phenantroline (Sigma–Aldrich). Fluorogenic activity was calculated by subtracting the increase in emission of the blank samples consisting of Hanks buffer only, from the samples containing kidney homogenates. The kinetic of fluorescence activity demonstrating linearity of cleavage rates over time was measured (excitation wavelength 328 nm, emission wavelength 393 nm) each 2 min for 1 h at 37 8C by Spectra Max Gemini XS (Paul Bucher Company, Basel, Switzerland). Results are expressed as Vmax (Uys) per mg of total protein, determined as described above. 3.7. Real-time TaqMan PCR for MMP-2 and for MMP9 3.7.1. RNA extraction Five paraffin-embedded tissue sections of 20 mm were collected from each sample and used for total RNA extraction by Ambion’s ‘Paraffin Block RNA Isolation Kit’, including DNase treatment (噛1902, Ambion, Europe Ltd, Huntingdon, Cambridgeshire, UK). First strand cDNA was synthesized with oligo(dT)12-18 primer (Life Technologies, Basel) as follows. A total of 5 mg extracted RNA was incubated at 65 8C for 5 min with 1 ml of oligo(dT)12-18 primer (0.5 mgyml); volume was adjusted to 12 ml with RNase free water. Thereafter, a solution containing 4 ml of 5= first strand buffer (250 mM Tris–HCl, pH 8.3, 375 mM
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KCl, 15 mM MgCl2), 2 ml of 0.1 M DTT, 1 ml of dNTPs (10 mM of each dNTP) and 1 ml of M-MLV reverse transcriptase (200 Uyml, Gibco Life Techonologies AG, Switzerland) was added. This entire mixture was incubated at 37 8C for 1 h. Subsequently, reverse transcriptase was denatured by heating the reaction solution to 90 8C for 5 min. 3.7.2. PCR for MMP-2 and MMP-9 The polymerase chain reaction (PCR) conditions were established according to the instructions of PE Biosystems (Rotkreuz, Switzerland). Two microliters of RT reaction solution containing 5 mg of total RNA were added to the PCR reaction mixtures of 23 ml. Reaction mixtures were composed as follows: Taqman Universal PCR Master Mix containing AmpliTaq Gold DNA Polymerase (PE Biosystems) at final concentration 1=, 300 nM forward primer and 900 nM reverse primer for GAPDH and MMP-2 assays, respectively, as well as 900 nM forward primer and 300 nM reverse primer for MMP-9 assays. The primer concentrations for each gene were determined previously following the PE Biosystems Manual. The sequences of primers and probes used were as follows: Rat GAPDH (accession no. AF106860; National Center of Biotechnology Information, NCBI, USA): primer forward 59 ccg agg gcc cac taa agg 39, reverse primer 59 tgc tgt tga agt cac agg aga ca 39, probe 59 cat cct ggg cta cac tga gga cca gg 39. Rat MMP-2 (accession no. U65656; NCBI): primer forward 59 gcc tga gct ccc gga aaa 39, reverse primer 59 cct gcg aag aac aca gcc ttc t 39, probe 59 att gat gcc gtg tac gag gcc cc 39. Rat MMP-9 (accession no. U24441; NCBI): primer forward 59 tgc aat gtg gat gtt ttt gat 39, reverse primer 59 acc gac cgt cct tga aga aa 39, probe 59 cat tgc tga tat cca ggg cgc tct g 39. Thermal cycle profile was: 94 8C for 15 s, 60 8C for 1 min, cycles: 40. All the samples were amplified by real-time PCR (ABI Prism 7700 Sequence Detector, PE Biosystems) in triplicate; to exclude contaminations, all analyses included negative control samples without cDNA. The results were visualized by Sequence Detector program V. 1.7 (PE Biosystems). Results were expressed as mean and standard deviation (S.D.) of the differences between the Ct (threshold cycle) of GAPDH and MMP assays following instructions of the PE Biosystems manual. 3.8. Renal histology Kidney tissues were fixed for 48 h in 5% neutralbuffered formalin, dehydrated and embedded in paraffin. Subsequently, kidneys were cut longitudinally into 2 mm sections and stained with periodic acid Schiff (PAS) reagent.
3.9. Statistical analyses Results of MMP activity and MMP expression as well as proteinuria were analyzed by the Mann–Whitney test for comparisons of two groups (InSTAT 2.05 Software; Macintosh, USA). Results were expressed as mean"S.D. For all experiments, probability of error (P values) were included; values for P-0.05 were regarded as significant. 4. Results 4.1. Reduced MMP activity in allograft rejection Total MMP activity in kidneys was determined by a continuously recording fluorescent assay, as shown in Fig. 1a. In comparison to sham operated animals, acute allograft rejection showed a trend towards an increase in MMP activity. Importantly, compared to vehicle, MMP inhibitor BB-94 successfully inhibited MMP activity close to two thirds in allografts during acute rejection. To confirm the specific cleavage of the fluorogenic substrate by MMP, activity of each sample was measured in presence of 2 mM PMSF (serine proteinase inhibitor) or 5 mM phenantroline (zinc chelating agent). As expected, PMSF had no effect, but phenantroline almost totally inhibited MMP activity (data not shown). Furthermore, in these and other assays (see below), there was no difference between sham operated Lewis rats and healthy Lewis rats or healthy DA rats (data not shown). Therefore, only results obtained by the sham operated Lewis rats were reported. 4.2. Zymography of MMP-2 and of MMP-9 activity Results of MMP activity in allograft rejection were extended and further specified by substrate zymography for the analyses of MMP-2 and of MMP-9. In comparison to the sham operated group, gelatin zymography demonstrated a clear reduction of MMP-2 and MMP-9 activities in both groups of transplanted animals, such as in vehicle only exposed animals and in BB-94 treated rats (Fig. 1b). 4.3. Differential expression of MMP-2 and MMP-9 The gelatinases MMP-2 and MMP-9 are crucial for the metabolism of collagen type IV, the major basement membrane constituent. Therefore, we selectively assessed the expression of these two enzymes by the use of real-time TaqMan PCR. Kidney allograft rejection led to a substantial, approximately 60%, decrease in the mRNA level of MMP-2, as demonstrated in Fig. 2a. Interestingly, MMP-9 mRNA level demonstrated a small but noticeable increase of
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as a result of BB-94 treatment. Renal histology both in vehicle and BB-94 treated groups displayed a high grade of rejection, which was identical to the severity, reported by others w21x. 4.5. Effect of MMP inhibitor on proteinuria As a result of acute allograft rejection, transplanted animals showed a clear trend towards an increase in proteinuria at the time of nephrectomy (P)0.05). This trend was almost completely abrogated by the therapy with an MMP inhibitor. Results are summarized in Fig. 4. Compared to vehicle exposed transplanted animals, MMP inhibitor treatment demonstrated an approximate 4-times lesser amount of proteinuria at day q7, the time of sacrifice of the animals. Interestingly, 3 animals in the vehicle treated group but none of the inhibitor treated rats displayed comparatively high proteinuria
Fig. 1. (a) Fluorometric analyses of MMP activity. MMP activity remained constant in vehicle solution (VT) treated allografts. To the contrary, MMP inhibitor BB-94 (BB-94) greatly reduced MMP activity in kidney transplants (P-0.05), compared to VT and sham operated animals (Sh); * denotes statistical significance between BB-94 and either VT (P-0.0001) or Sh (P-0.0001). (b) Zymography of MMP-2 and of MMP-9 activities. Gelatin zymography depicts decreased activities of MMP-2 and of MMP-9 in VT and in BB-94 treated kidney allografts, compared to the Sh group.
7% (BB-94 treated group) to 17% (vehicle only exposed group) during rejection, that just reached statistical significance. These results are depicted in Fig. 2b. 4.4. Kidney histology in allograft rejection As expected, a severe acute allograft rejection was observed at day q7. Acute rejection displayed severe tissue destruction, characterized by marked cellular infiltration leading to tubulitis with tubular damage and by signs of vascular necrosis, as shown in Fig. 3a–d. There was no obvious amelioration of renal tissue destruction
Fig. 2. (a) Expression of MMP-2 by real-time PCR. Expression of MMP-2 decreased to approximately 40% both in vehicle solution (VT) and in BB-94 (BB-94) treated kidney allografts, as compared to the sham operated (Sh) group. MMP-2 expression in Sh is depicted as 100%; * denotes statistical significance between VT and Sh (Ps 0.0357), and between BB-94 and Sh (Ps0.021). (b) Expression of MMP-9 by real-time PCR. Expression of MMP-9 increased to 117% in VT and to 107% in BB-94 treated kidney allografts, as compared to the Sh group. MMP-9 expression in Sh is depicted as 100%; * denotes statistical significance beween VT and Sh (Ps0.03), and between BB-94 and Sh (Ps0.0485).
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Fig. 3. Renal histology 7 days after transplantation. (a) Kidney from sham operated Lewis rat; (b) transplanted kidney from vehicle only exposed rat; (c and d) kidney from BB-94 treated transplanted animals. Severe acute celluar rejection with vascular involvement (d) is depicted in (b)– (d). PAS stain, magnification =400.
with protein (mg)ycreatinine (mmol) ratios of 00.3 during allograft rejection. Proteinuria in the groups of healthy rats and sham operated rats remained minimal and did not differ from the values obtained at day 0 from the two other groups (data not shown). In addition, proteinuria at day y3 was identical to day 0 (data not shown). Plasma levels of sodium, potassium and creatinine obtained at day q7 were almost identical between the three experimental groups (for all comparisons, P) 0.05), as shown in Table 1. Furthermore, there were no noticeable differences between values obtained at day 0 and day q7 (data not shown).
Table 1 Concentrations of sodium, porassium and creatinine in plasma, obtained at day q7
Fig. 4. Effect of allograft rejection on proteinuria. Proteinuria is expressed as ratios of protein (mg)ycreatinine (mmol), obtained from 24-h urine specimen at days 0 and q7, after kidney transplantation. Vehicle only treated (VT) animals demonstrated a distinct trend towards an elevated proteinuria at the time of rejection (P)0.05). Treatment with the MMP inhibitor (BB-94) was able to keep proteinuria at a low level during allograft rejection.
SH (ns3) VT (ns12) BB-94 (ns12)
Naq (mmolyl)
Kq (mmolyl)
Creatinine (mmolyl)
145"3.21 144"0.87 144"1.75
4.6"0.87 3.8"0.35 3.7"0.71
58"52.9 50"2.73 51"5.46
Sh, sham operated group; VT, vehicle solution only treated transplanted group; and BB-94, MMP inhibitor treated transplanted group.
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5. Discussion The present study focused on the expression and activity of MMP in experimental kidney allograft rejection. We have used an established animal model, the orthotopic kidney allografting in the DA-to-Lewis rat strain combination w15,16,21x. This model is reported to be very stringent because of the major histocompatibility mismatch between donor and recipient animals w21x. In the transplanted kidney, severe acute cellular rejection with some degree of vascular involvement occurs within 7 days post transplantation w16,21x. MMP are zinc-dependent metalloendopeptidases. They metabolize and remodel ECM in conjunction with serine proteinases and membrane-anchored a disintegrin and metalloproteinase w7,22,23x. Most prominent examples of MMP are interstitial collagenases (MMP-1y-8y -13), gelatinases (MMP-2y-9), stromelysins (MMP-3y7y-10y-11) and membrane-type(MT)-MMP (MT1y2y 3y4-MMP). MMP activity is regulated and contained by the tissue inhibitors of metalloproteinases (TIMP) w7,23,24x. Meanwhile, there exist several synthetic low molecular weight MMP inhibitors, such as hydroxamic acid derivatives like BB-94 w14,24,25x. BB-94 represents a particularly well studied broad-spectrum MMP inhibitor w14,26x. Importantly, some of these MMP inhibitors may also inhibit the activity of metalloproteinases closely related to MMP that cleave transmembrane cytokine precursors w23x. Augmented expression or activity of renal MMP was found by us and others in several forms of acute and chronic inflammatory kidney disorders w7,27–30x. In the past, we have successfully demonstrated a distinct antiinflammatory effect of MMP inhibitors on glomerular mesangial cells in vitro and in vivo, using cell cultures and an experimental model of mesangial proliferative glomerulonephritis w17,19x. The anti-proliferative effect of MMP inhibitors appeared to be a result of the induction of mesangial cell cycle arrest followed by apoptosis w31x. Recently, the pro-inflammatory role of MMP in the kidney was reviewed by us in this journal in more detail w32x. Importantly, MMP-2 as shown to exert a direct pro-inflammatory effect upon the glomerular mesangial cells w1x. The biologic function of MMP in kidney transplantation remains to be defined. A potential role of MMP in renal allograft fibrosis has been advocated by some authors w33x. There are no formal studies analyzing the biological role of MMP in kidney transplantation, apart from a few descriptive investigations. For instance, inpatients with chronic transplant nephropathy, serum levels of MMP-2 and MMP-3 were found to be increased w34x. Interestingly, the former correlated with proteinuria and the latter with serum creatinine. In the same study, serum MMP-1 was increased in cases with acute rejec-
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tion w34x. In contrast, another study failed to demonstrate alterations in the expression of fibrosis associated genes, including MMP-2, in isolated renal glomeruli of patients with acute kidney allograft rejection w35x. With regard to the effect of immunosuppressive therapy, cyclosporine and tacrolimus were found to lead to almost identical levels of MMP-2 in isolated glomeruli obtained from protocol biopsies of transplanted patients w36x. Interestingly, with respect to transplantation medicine, BB-94 was recently shown to prevent influx of leukocytes into the liver in a mouse model of lethal hepatitis induced by tumor necrosis factor w26x. Furthermore, MMP have been shown previously to play a role in mesangial proliferative glomeruloneophritis w10,17,32x. Since this disease often reappears in allografts, MMP may also play a role in the recurrence of primary renal diseases. In our study, we found a small increase in overall MMP activity as a result of severe kidney transplant rejection. Although this difference appeared to be insignificant, it occurred despite severe tissue destruction with reduced synthetic capacity of cellular elements. Better preservation of kidney histology in a less severe form of acute rejection may well demonstrate a steep increase in MMP activity reflecting acute inflammation. Interestingly, expression of MMP-2 and of MMP-9 was differentially regulated. As a result of acute rejection, MMP-2 was down-regulated, but MMP-9 increased. The differential regulation of these two MMP may argue against a non-specific effect of kidney destruction on MMP synthesis. The divergent results with regard to mRNA levels and activity of MMP-9 may be explained by its extracellular degradation or inhibition w24x. In accordance with our results, the inflammatory cytokine interleukin-1b was previously shown to increase production of MMP-9 but not of MMP-2 w37x. Despite our measurements, we could not exclude the presence of additional and relevant MMP that were not fully assessed in our activity assay systems. Therefore, we included an MMP inhibitor in our studies, regardless of the overall only marginally increased MMP activity in allograft rejection. Importantly, only inhibition studies may ultimately elucidate the functional role of the MMP. As expected, renal damage was very severe in our rejection model. As a consequence of the only minimally elevated MMP activity, there was no discernable benefit with respect to preservation of kidney structure as a result of the MMP inhibitor treatment. Nevertheless, BB-94 successfully inhibited MMP activity and it was able to keep proteinuria at a low level during the rejection process. Therefore, it may be still possible that besides MMP-9, some not yet defined and noxious MMP are also up-regulated during the rejection process. Furthermore, other MMP may only be increased during an earlier phase of the acute rejection process. Once the kidney is seriously destroyed, activity
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of many MMP, possibly with the exclusion of MMP-9, may decrease by itself. According to our results MMP may offer opportunities for complementary therapy of acute allograft rejection. Nevertheless, since these proteinases do not appear to represent prime targets in such severe forms of acute rejection, future studies are required to evaluate the full impact of MMP in this context. Importantly, the role of MMP may be better definable in experimental models with attenuated forms of acute rejection, achieved by the concomitant use of low dose cyclosporine A w38x. Furthermore, such studies may more mimic clinical practice and should include time-course analyses of MMP activity and expression. MMP may play an even more prominent role in chronic allograft rejection promoting fibrosis and sclerosis. Established models of chronic kidney transplant rejection, such as in Lewis rat recipients of Fisher kidney transplants, offer unique opportunities for the analyses of MMP w39,40x. In conclusion, severe kidney allograft rejection affected the expression and activity of MMP. Overall MMP activity only slightly increased, possibly mitigated by the overwhelming renal tissue destruction. In addition, the effect of the rejection process on MMP was not uniform, since MMP-2 and MMP-9 were differentially regulated. MMP activity was successfully reduced by the MMP inhibitor BB-94. In this respect, BB-94 prevented the rise in proteinuria in transplanted animals. Therefore, MMP appear to represent new mediators involved in acute kidney transplant rejection. This may be especially relevant for MMP-9.
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We thank Professor Brigitte Frey from our institution for corrections of the manuscript. We are also thankful to A. Galloway (British Biotech Pharmaceuticals Ltd) for providing BB-94 as a gift. This work was supported by the Swiss National Foundation for Scientific Research by the grant 噛31-55779.98 of Hans-Peter Marti. References w1x Turk J, Pollock AS, Lee LK, Marti HP, Lovett DH. Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation. J Biol Chem 1996;271:15074 –15083. w2x Woessner JF. Matrix metalloproteinase inhibition. From the Jurassic to the third millennium. Ann NY Acad Sci 1999;878:388 –403. w3x Leppert D, Lindberg RL, Kappos L, Leib SL. Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis. Brain Res Brain Res Rev 2001;36(2–3):249 –257. w4x Tarlton JF, Whiting CV, Tunmore D, et al. The role of upregulated serine proteases and matrix metalloproteinases in the
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pathogenesis of a murine model of colitis. Am J Pathol 2000;157(6):1927 –1935. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991;64:327 –336. Brown PD, Giavazzi R. Matrix metalloproteinases inhibition: a review of anti-tumour activity. Ann Oncol 1995;6:967 –974. Lenz O, Elliot SJ, Stetler-Stevenson WG. Matrix metalloproteinases in renal development and disease. J Am Soc Nephrol 2000;11:574 –581. Birkedal-Hansen . Proteolytic remodelling of extracellular matrix. Curr Opin Cell Biol 1995;7:728 –735. Emmert-Buck MR, Emonard HP, Corcoran ML, Krutzsch HC, Foidart JM, Stetler-Stevenson WG. Cell surface binding of Timp-2 and pro-MMP-2yTIMP-2 complex. FEBS Lett 1995;364:28 –32. Lovett DH, Johnson RJ, Marti HP, Davies M, Couser WG. Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis. Am J Pathol 1992;141:85 –98. Belleguic C, Corbel M, Germain N, et al. Increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients. Clin Exp Allergy 2002;32(2):217 –223. Prontera C, Mariani B, Rossi C, Poggi A, Rotilio D. Inhibition of gelatinase a (MMP-2) by Batimastat and Captopril reduces tumour growth and lung metastases in mice bearing Lewis lung carcinoma. Int J Cancer 1999;81:761 –766. Yamamoto M, Tsujishita H, Hori N, et al. Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket. J Med Chem 1998;41:1209 –1217. Zempo N, Koyama N, Kenagy RD, Lea HJ, Clowes AW. Regulation of vascular smooth muscle cell migration and proliferation in vitro and in injured rat arteries by a synthetic matrix metalloproteinase inhibitor. Arterioscler Thromb Vasc Biol 1996;16(1):28 –33. Schuler W, Sedrani R, Cottens S, et al. SDZ RAD a new rapamycin derivative. Transplantation 1997;64:36 –42. Schuurman HJ, Cottens S, Fuchs S, et al. Transplantation 1997;64:32 –35. ¨ M, Ziswiler R, Marti HP. Inhibition Steinmann-Niggli K, Kung of matrix metalloproteinases attenuates anti-Thy1.1 nephritis. J Am Soc Nephrol 1998;9:397 –407. Ziswiler R, Daniel C, Franz E, Marti HP. Renal matrix metalloproteinase activity is unaffected by experimental ischemia-reperfusion injury and matrix metalloproteinase inhibition does not alter outcome of renal function. Exp Nephrol 2001;9(2):118 –124. Steinmann-Niggli K, Lukes M, Marti HP. Rat mesangial cells and matrix metalloproteinase inhibitor: inhibition of 72-kDa type IV collagenase (MMP-2) and of cell proliferation. J Am Soc Nephrol 1997;8:395 –405. Knight CG, Willenbrock F, Murphy G. A novel coumarinlabelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett 1992;296(3):263 –266. Beckmann N, Joergensen J, Bruttel K, Rudin M, Schuurman HJ. Magnetic resonance imaging for the evaluation of rejection of a kidney allograft in the rat. Transplant Int 1996;9:175 – 183. Mahimkar RM, Baricos WH, Visaya O, Pollock AS, Lovett DH. Identification, cellular distribution and potential function of the metalloproteinase-disintegrin MDC9 in the kidney. J Am Soc Nephrol 2000;11:595 –603.
M. Ermolli et al. / Transplant Immunology 11 (2003) 137–145 w23x Woessner JF, Nagase H. Matrix metalloproteinases and TIMPs. New York, USA: Oxford University Press, Inc, 2000. p. 1–10 and 109–35 w24x Lelongt B, Legallicier B, Piedagnel R, Ronco PM. Do matrix metalloproteinases MMP-2 and MMP-9 (gelatinases) play a role in renal development, physiology and glomerular diseases? Curr Opin Nephrol Hypertens 2001;10(1):7 –12. w25x Skotnicki JS, Zask A, Nelson FC, Albright JD, Levin JI. Design and synthetic considerations of matrix metalloproteinase inhibitors. Ann NY Acad Sci 1999;878:61 –72. w26x Wielockx B, Lannoy K, Shapiro SD, et al. Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy. Nat Med 2001;7(11):1202 –1208. w27x Koide H, Nakamura T, Ebihara I, Tomino Y. Increased mRNA expression of metalloproteinase-9 in peripheral blood monocytes from patients with immunoglobulin A nephropathy. Am J Kidney Dis 1996;28:32 –39. w28x Marti HP, McNeil L, Thomas G, Davies M, Lovett DH. Molecular characterization of a low molecular weight matrix metalloproteinase secreted by glomerular mesangial cells as Pump-1. Biochem J 1992;285:899 –905. w29x Ebihara I, Nakamura T, Noriaki S, Koide H. Increased plasma metalloproteinase-9 concentrations precede development of microalbuminuria in non-insulin dependent diabetes mellitus. Am J Kidney Dis 1998;32:544 –550. w30x McMillan JI, Riordan JW, Couser WG, Pollock AS, Lovett DH. Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy. J Clin Invest 1996;97(4):1094 –1101. w31x Daniel C, Duffield J, Brunner T, Steinmann-Niggli K, Lods N, Marti HP. Matrix metalloproteinase inhibitors cause cell cycle arrest and apoptosis in glomerular mesangial cells. J Pharmacol Exp Ther 2001;297(1):57 –68.
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w32x Marti HP. The role of matrix metalloproteinases in the activation of mesangial cells. Transplant Immunol 2002;9(2–4):97 – 100. w33x Waller JR, Nicholson ML. Molecular mechanisms of renal allograft fibrosis. Br J Surg 2001;88(11):1429 –1441. w34x Rodrigo E, Lopez-Hoyos M, Escallada R, et al. Circulating levels of matrix metalloproteinases MMP-3 and MMP-2 in renal transplant recipients with chronic transplant nephropathy. Nephrol Dial Transplant 2000;15(12):2041 –2045. w35x White SA, Bicknell GR, Jain S, et al. Effect of acute rejection on expression of fibrosis associated genes in renal transplant recipients. Transplant Proc 2000;32(1):19 –20. w36x Bicknell GR, Williams ST, Shaw JA, Pringle JH, Furness PN, Nicholson ML. Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants. Br J Surg 2000;87(11):1569 – 1575. w37x Martin J, Steadman R, Knowlden J, Williams J, Davies M. Differential regulation of matrix metalloproteinases and their inhibitors in human glomerular epithelial cells in vitro. J Am Soc Nephrol 1998;9(9):1629 –1637. w38x Schuurman HJ, Pally Ch, Fringeli-Tanner M, Papageorgiou Ch. Comparative efficacy of mycophenolate sodium (MPS) and mycophenolate mofetil (MMF) with and without cyclosporine in rat transplantation models. Transplantation 2001;72:1776 – 1783. w39x Yin JL, Pilmore HL, Yan YQ, et al. Expression of growth arrest-specific gene 6 and its receptors in a rat model of chronic renal transplant rejection. Transplantation 2002;73(4):657 – 660. w40x Laskowski IA, Pratschke J, Wilhelm MJ, et al. Anti-CD28 monoclonal antibody therapy prevents chronic rejection of renal allografts in rats. J Am Soc Nephrol 2002;13(2):519 – 527.
Differential expression of MMP-2yMMP-9 and potential benefit of an MMP inhibitor in experimental acute kidney allograft rejection ` Lods, Mahmoud Hammoud, Hans-Peter Marti* Monica Ermolli, Martin Schumacher, Nadege Division of Nephrology and Hypertension, Inselspital Bern, CH-3010 Bern, Switzerland Received 15 April 2002; accepted 7 August 2002
Abstract Acute cellular allograft rejection is characterized by leukocyte invasion and tissue destruction, associated with qualitative and quantitative alterations in the extracellular matrix (ECM) compartment. Metabolism of ECM proteins is mainly regulated by matrix metalloproteinases (MMP), that are zinc depended endoproteinases. MMP, especially basement membrane degrading MMP2 and MMP-9, also facilitate tissue invasion of leukocytes. In addition, MMP-2 exerts a direct pro-inflammatory effect upon glomerular mesangial cells. Therefore, the investigation of the role of MMP in transplant rejection may lead to novel approaches in the therapy of rejection processes. To our knowledge, this is the first study of acute allograft rejection, formally addressing expression and activity of MMP, including the effect of a MMP inhibiting agent. For our studies, we used the orthotopic kidney allograft model in the stringent Dark Agouti-to-Lewis rat strain combination. Animals were divided into four groups: group A, healthy untreated Lewis rats (ns3); group B, sham operated Lewis rats (ns3); group C, transplanted Lewis rats treated with vehicle solution only (ns12); group D, transplanted Lewis rats treated with MMP inhibitor BB-94 (ns12). Respective animals were treated once daily intraperitonealy with BB-94 (30 mgykg) or vehicle solution only. Treatment lasted from the third preoperative day until the end of the experiment, the time of severe rejection at day q7. Acute kidney allograft rejection led to alterations in the expression and activity of MMP. Overall MMP activity slightly increased despite severe destruction of kidney histology. The MMP inhibitor BB-94 successfully inhibited MMP activity to a high extent. MMP expression did not show uniform findings, since acute rejection led to differential expression of MMP-2 and MMP-9. During the rejection process, MMP-9 showed a small but significant increase, whereas MMP-2 production decreased substantially. Interestingly, BB-94 was able to keep proteinuria at a low level in transplanted animals. In conclusion, MMP—especially MMP-9—appear to represent new mediators involved in acute kidney transplant rejection. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Matrix metalloproteinases; MMP-2; MMP-9; BB-94; Batimastat; Acute allograft rejection
1. Introduction Chronic and relentless loss of kidney graft function remains a crucial problem in kidney transplantation. Acute rejection episodes and chronic allograft nephropathy are principal causes of tissue destruction leading to graft loss. Importantly, severe acute allograft rejection may not only lead to immediate graft failure but may also initiate chronic allograft nephropathy with fibrosis and sclerosis. Therefore, the search for new mediators contributing to the development of rejection remains pivotal. *Corresponding author. Tel.: q41-31-632-31-44; fax: q41-31632-94-44. E-mail address: [email protected] (H.-P. Marti). 0966-3274/03/$ 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 2 . 0 0 1 5 0 - 8
Acute cellular rejection episodes of solid organs are characterized by invasion of leukocytes and tissue destruction, features associated with qualitative and quantitative alterations in the extracellular matrix (ECM) compartment. Turnover of ECM proteins is mainly regulated by extracellularly active matrix metalloproteinases (MMP), which represent the major group of zinc depended matrix degrading proteases w1,2x. Furthermore, MMP are involved in the regulation of tissue invasion by lymphocytes w3,4x. Therefore, the investigation of the role of MMP in transplant rejection may lead to new approaches to the therapy of rejection processes. MMP are proteolytic enzymes involved in ECM degradation and secreted by various cell lines in normal
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and pathological conditions. They are involved in a variety of biological processes, such as proliferation, migration and invasion of cells, organ development, tumor metastasis and angiogenesis w5–7x. In our studies, we focused on MMP-2 and MMP-9, which in particular degrade collagen IV, the main structural component of basement membranes. Most MMP are secreted in a soluble pro-enzyme form and their activity is regulated through multiple events including pro-enzyme activation and interaction with the tissues inhibitors of metalloproteinases, TIMP1-4 w7–9x. It has been shown that MMP activity is up-regulated in different types of acute inflammatory events w3,10,11x. Acute cell-mediated kidney transplant rejection represents a severe form of inflammation, characterized by tissue invasion of mononuclear leukocytes resulting in cellular and ECM destruction. Therefore, it is conceivable that MMP expression may be up-regulated in this disorder. Furthermore, MMP inhibition may favorably influence features involved in allograft rejection, such as proteolytic tissue destruction and possibly invasion of lymphocytes. Therefore, we analyzed the production of MMP, especially MMP-2 and MMP-9 in an experimental rat model of acute kidney allograft rejection. In parallel, we assessed the effect of Batimastat, BB-94 (British Biotech Pharmaceuticals Ltd, Oxford, UK). This agent represents a hydoxamic acid-based synthetic MMP inhibitor, designed as an anti-invasive and anti-metastatic drug particularly well inhibiting MMP-2, MMP-9 and MMP3 w12–14x. To our knowledge, this is the first study formally addressing expression and activity of MMP, including the effect of an MMP inhibiting agent, in the setting of acute kidney allograft rejection. 2. Aims and objectives The aim of this study was to demonstrate if MMP play a role in acute experimental kidney allograft rejection. Specifically, we wanted to demonstrate: (i) how MMP, especially MMP-2 and MMP-9, are regulated during kidney transplant rejection; and (ii) whether MMP inhibition by the use of a synthetic MMP inhibitor shows a benefit with regard to kidney histology and proteinuria. 3. Materials and methods 3.1. Animals Male Dark Agouti (DA; RT1a haplotype) rats and male Lewis (RT11 haplotype) rats were purchased from Charles River (Sulzfeld, Germany). Their body weight at the outset of the studies was 200–220 g. The rats were housed in individual metabolic cages at constant temperature with a 12-h light and 12-h dark cycle and
with free access to water and food tanks. Approval for animal studies was obtained from the commission for animal studies, a local government agency, in accordance to the Guide of Care and Use of Laboratory Animals of the Veterinary Institute of the University of Bern. 3.2. Matrix metalloproteinases inhibitor The synthetic MMP inhibitor BB-94 (Batimastat), w4-(N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)-succinylx-L-phenylalanine-N-methylamide, MW 478, was provided by British Biotech Pharmaceuticals Ltd w14x. BB-94 concentrations demonstrating 50% MMP inhibition were as follows: MMP-1, 5 nmolyl; MMP-2, 4 nmolyl, MMP-3, 20 nmolyl; and MMP-9, 1–10 nmolyl w14x. BB-94 solution (3 mgyml) was prepared in PBSyTween-80 (0.01%) as vehicle by sonication until a uniform suspension was achieved. Animals were treated with a one daily intraperitoneal (ip) injection of 30 mg BB-94 per kg body weight w14x. As a control, an identical volume of vehicle solution only was given in the same fashion. 3.3. Kidney transplantation Orthotopic kidney allotransplantation in male Lewis rats, using donor organs from male DA rats, was performed as published previously w15,16x. 3.3.1. Donor procedure A midline laparotomy incision was made from the xyphoid to the pubic area. A self-retaining retractor was inserted after bowel evisceration. The urethra was sectioned at 10 mm distal of the renal hilus; mobilization of the left kidney from the perinephric fat was performed without encapsulating the kidney from the fat. Without disturbance of the renal hilus, renal artery and renal vein were dissected. A total of 1.5 ml of 200 IU heparin was injected into the inferior vena cava of donors to avoid thrombosis. Euthanasia of the donors was performed by exsanguination. 3.3.2. Recipient procedure Opening and evisceration of the peritoneal cavity was done as described above. The ureter was divided at the level of the lower pole of the kidney. The renal artery was divided proximal to its bifurcation and renal vein was divided at the junction of its middle and lateral third. End-to-end anastomosis of the renal artery was performed first, followed by the anastomosis of the renal vein using 10-0 Ethilon䉸 (Ethicon, Johnson & Johnson). The ureteric end-to-end anastomosis was carried out with 11-0 Ethilon䉸. Thereafter, 4 ml of normal saline were instilled into the abdominal cavity to compensate for the blood loss. Abdominal wall and skin were closed using 4-0 Suturamid䉸. Duration of surgery did not
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exceed 80 min and times of warm ischemia were always below 40 min. Postoperatively, analgesia with paracetamol (100–300 mgykg body weighty4 h) was given directly into the drinking water. 3.4. Experimental design The animals were divided into four groups: group A, healthy untreated Lewis rats (ns3); group B, sham operated Lewis rats (ns3); group C, transplanted Lewis rats treated with vehicle solution only (ns12); group D, transplanted Lewis rats treated with BB-94 (ns12). Respective animals were treated daily with MMP inhibitor or vehicle solution only, from the third preoperative day until the end of the experiment. In previous studies, no effect of administration of vehicle solution only on kidney histology or MMP was described and MMP inhibitor did not affect renal histology in healthy animals w17,18x. At the 7th postoperative day, all animals were killed and kidneys were harvested. Kidneys were cut in halves and were either used for histology or were deep frozen for subsequent zymography and fluorometry, as described below. Blood and 24-h urine samples were collected at days y3 (urine only), 0 and q7 with respect to transplantation. Urinary ratios of proteinycreatinine (mgymmol) as well as plasma sodium (mmolyl) and potassium (mmolyl) concentrations and plasma creatinine (mmoly l) levels were determined by the central laboratory of our institution. 3.5. Zymography Zymography for the analyses of MMP-2 and MMP-9 activities was performed using kidney homogenates. Briefly, frozen half-kidneys were minced on a cold petri dish and transferred into separate tubes. Thereafter, 1 ml of lysis buffer (0.1 M Tris–HCl containing 0.1% Tween-80, pH 7.5) per 60 mg of wet tissue was added. The homogenates were centrifuged twice at 10 000=g for 10 and 5 min, respectively. Extracted supernatants were stored at y20 8C. For subsequent analyses of MMP activity, protein contents in supernatants were determined using the BCA protein assay reagent kit (Pierce, Lausanne, Switzerland). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on precast 10-well, 10% polyacrylamide minigels of 0.75-mm thickness containing 0.1% gelatin (Novex, San Diego, CA), as described previously w17–19x. Briefly, samples adjusted to an uniform protein content of 15 mg in a volume of 10 ml were diluted with the same volume of double-strength, non-reducing sample buffer (0.5 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue). Subsequently, 18 ml of this mixture
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was loaded into each well and electrophoresed at constant voltage (15 Vycm) for approximately 2 h. After incubation in renaturating buffer (2.5% Triton X-100 in 50 mM Tris–HCl, pH 8.0) for 30 min, gels were exposed at 37 8C to proteolysis buffer (50 mM Tris– HCl, pH 8.0, 5 mM CaCl2, 1 mM ZnCl2) for 24 h with gentle agitation. Subsequently, zymograms were fixed and zones of lyses were visualized by staining with 0.2% Coomassie Blue R250. Molecular weight standard (噛161-0372, Bio-Rad, Glattbrugg, Switzerland) was used for assignment of molecular weight. 3.6. Fluorometric analyses Samples were prepared as described above. Thereafter, MMP activity was investigated as described w18,19x. In brief, 5 ml samples were diluted in Hanks buffer to a final volume of 100 ml, containing the quenched fluorescent substrate (7-methoxycoumarin-4-yl)AcetylPro-Leu-Gly-Leu-(3-w2,4-dinitrophenylx-L2,3-diaminopropionyl)-Ala-Arg-NH2 (Bachem, Bubendorf, Switzerland), at a final concentration of 5 mM. This agent represents a very sensitive substrate for activity assays for a broad-spectrum of MMP; examples are MMP-2 (kcatyKms629 000ys M) and MMP-7 (kcaty Kms169 000ys M) w20x. These reaction mixtures were supplemented with or without 2 mM phenylmethylsulfonyl (PMSF; Sigma-Aldrich, Buchs, Switzerland) or 5 mM phenantroline (Sigma–Aldrich). Fluorogenic activity was calculated by subtracting the increase in emission of the blank samples consisting of Hanks buffer only, from the samples containing kidney homogenates. The kinetic of fluorescence activity demonstrating linearity of cleavage rates over time was measured (excitation wavelength 328 nm, emission wavelength 393 nm) each 2 min for 1 h at 37 8C by Spectra Max Gemini XS (Paul Bucher Company, Basel, Switzerland). Results are expressed as Vmax (Uys) per mg of total protein, determined as described above. 3.7. Real-time TaqMan PCR for MMP-2 and for MMP9 3.7.1. RNA extraction Five paraffin-embedded tissue sections of 20 mm were collected from each sample and used for total RNA extraction by Ambion’s ‘Paraffin Block RNA Isolation Kit’, including DNase treatment (噛1902, Ambion, Europe Ltd, Huntingdon, Cambridgeshire, UK). First strand cDNA was synthesized with oligo(dT)12-18 primer (Life Technologies, Basel) as follows. A total of 5 mg extracted RNA was incubated at 65 8C for 5 min with 1 ml of oligo(dT)12-18 primer (0.5 mgyml); volume was adjusted to 12 ml with RNase free water. Thereafter, a solution containing 4 ml of 5= first strand buffer (250 mM Tris–HCl, pH 8.3, 375 mM
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KCl, 15 mM MgCl2), 2 ml of 0.1 M DTT, 1 ml of dNTPs (10 mM of each dNTP) and 1 ml of M-MLV reverse transcriptase (200 Uyml, Gibco Life Techonologies AG, Switzerland) was added. This entire mixture was incubated at 37 8C for 1 h. Subsequently, reverse transcriptase was denatured by heating the reaction solution to 90 8C for 5 min. 3.7.2. PCR for MMP-2 and MMP-9 The polymerase chain reaction (PCR) conditions were established according to the instructions of PE Biosystems (Rotkreuz, Switzerland). Two microliters of RT reaction solution containing 5 mg of total RNA were added to the PCR reaction mixtures of 23 ml. Reaction mixtures were composed as follows: Taqman Universal PCR Master Mix containing AmpliTaq Gold DNA Polymerase (PE Biosystems) at final concentration 1=, 300 nM forward primer and 900 nM reverse primer for GAPDH and MMP-2 assays, respectively, as well as 900 nM forward primer and 300 nM reverse primer for MMP-9 assays. The primer concentrations for each gene were determined previously following the PE Biosystems Manual. The sequences of primers and probes used were as follows: Rat GAPDH (accession no. AF106860; National Center of Biotechnology Information, NCBI, USA): primer forward 59 ccg agg gcc cac taa agg 39, reverse primer 59 tgc tgt tga agt cac agg aga ca 39, probe 59 cat cct ggg cta cac tga gga cca gg 39. Rat MMP-2 (accession no. U65656; NCBI): primer forward 59 gcc tga gct ccc gga aaa 39, reverse primer 59 cct gcg aag aac aca gcc ttc t 39, probe 59 att gat gcc gtg tac gag gcc cc 39. Rat MMP-9 (accession no. U24441; NCBI): primer forward 59 tgc aat gtg gat gtt ttt gat 39, reverse primer 59 acc gac cgt cct tga aga aa 39, probe 59 cat tgc tga tat cca ggg cgc tct g 39. Thermal cycle profile was: 94 8C for 15 s, 60 8C for 1 min, cycles: 40. All the samples were amplified by real-time PCR (ABI Prism 7700 Sequence Detector, PE Biosystems) in triplicate; to exclude contaminations, all analyses included negative control samples without cDNA. The results were visualized by Sequence Detector program V. 1.7 (PE Biosystems). Results were expressed as mean and standard deviation (S.D.) of the differences between the Ct (threshold cycle) of GAPDH and MMP assays following instructions of the PE Biosystems manual. 3.8. Renal histology Kidney tissues were fixed for 48 h in 5% neutralbuffered formalin, dehydrated and embedded in paraffin. Subsequently, kidneys were cut longitudinally into 2 mm sections and stained with periodic acid Schiff (PAS) reagent.
3.9. Statistical analyses Results of MMP activity and MMP expression as well as proteinuria were analyzed by the Mann–Whitney test for comparisons of two groups (InSTAT 2.05 Software; Macintosh, USA). Results were expressed as mean"S.D. For all experiments, probability of error (P values) were included; values for P-0.05 were regarded as significant. 4. Results 4.1. Reduced MMP activity in allograft rejection Total MMP activity in kidneys was determined by a continuously recording fluorescent assay, as shown in Fig. 1a. In comparison to sham operated animals, acute allograft rejection showed a trend towards an increase in MMP activity. Importantly, compared to vehicle, MMP inhibitor BB-94 successfully inhibited MMP activity close to two thirds in allografts during acute rejection. To confirm the specific cleavage of the fluorogenic substrate by MMP, activity of each sample was measured in presence of 2 mM PMSF (serine proteinase inhibitor) or 5 mM phenantroline (zinc chelating agent). As expected, PMSF had no effect, but phenantroline almost totally inhibited MMP activity (data not shown). Furthermore, in these and other assays (see below), there was no difference between sham operated Lewis rats and healthy Lewis rats or healthy DA rats (data not shown). Therefore, only results obtained by the sham operated Lewis rats were reported. 4.2. Zymography of MMP-2 and of MMP-9 activity Results of MMP activity in allograft rejection were extended and further specified by substrate zymography for the analyses of MMP-2 and of MMP-9. In comparison to the sham operated group, gelatin zymography demonstrated a clear reduction of MMP-2 and MMP-9 activities in both groups of transplanted animals, such as in vehicle only exposed animals and in BB-94 treated rats (Fig. 1b). 4.3. Differential expression of MMP-2 and MMP-9 The gelatinases MMP-2 and MMP-9 are crucial for the metabolism of collagen type IV, the major basement membrane constituent. Therefore, we selectively assessed the expression of these two enzymes by the use of real-time TaqMan PCR. Kidney allograft rejection led to a substantial, approximately 60%, decrease in the mRNA level of MMP-2, as demonstrated in Fig. 2a. Interestingly, MMP-9 mRNA level demonstrated a small but noticeable increase of
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as a result of BB-94 treatment. Renal histology both in vehicle and BB-94 treated groups displayed a high grade of rejection, which was identical to the severity, reported by others w21x. 4.5. Effect of MMP inhibitor on proteinuria As a result of acute allograft rejection, transplanted animals showed a clear trend towards an increase in proteinuria at the time of nephrectomy (P)0.05). This trend was almost completely abrogated by the therapy with an MMP inhibitor. Results are summarized in Fig. 4. Compared to vehicle exposed transplanted animals, MMP inhibitor treatment demonstrated an approximate 4-times lesser amount of proteinuria at day q7, the time of sacrifice of the animals. Interestingly, 3 animals in the vehicle treated group but none of the inhibitor treated rats displayed comparatively high proteinuria
Fig. 1. (a) Fluorometric analyses of MMP activity. MMP activity remained constant in vehicle solution (VT) treated allografts. To the contrary, MMP inhibitor BB-94 (BB-94) greatly reduced MMP activity in kidney transplants (P-0.05), compared to VT and sham operated animals (Sh); * denotes statistical significance between BB-94 and either VT (P-0.0001) or Sh (P-0.0001). (b) Zymography of MMP-2 and of MMP-9 activities. Gelatin zymography depicts decreased activities of MMP-2 and of MMP-9 in VT and in BB-94 treated kidney allografts, compared to the Sh group.
7% (BB-94 treated group) to 17% (vehicle only exposed group) during rejection, that just reached statistical significance. These results are depicted in Fig. 2b. 4.4. Kidney histology in allograft rejection As expected, a severe acute allograft rejection was observed at day q7. Acute rejection displayed severe tissue destruction, characterized by marked cellular infiltration leading to tubulitis with tubular damage and by signs of vascular necrosis, as shown in Fig. 3a–d. There was no obvious amelioration of renal tissue destruction
Fig. 2. (a) Expression of MMP-2 by real-time PCR. Expression of MMP-2 decreased to approximately 40% both in vehicle solution (VT) and in BB-94 (BB-94) treated kidney allografts, as compared to the sham operated (Sh) group. MMP-2 expression in Sh is depicted as 100%; * denotes statistical significance between VT and Sh (Ps 0.0357), and between BB-94 and Sh (Ps0.021). (b) Expression of MMP-9 by real-time PCR. Expression of MMP-9 increased to 117% in VT and to 107% in BB-94 treated kidney allografts, as compared to the Sh group. MMP-9 expression in Sh is depicted as 100%; * denotes statistical significance beween VT and Sh (Ps0.03), and between BB-94 and Sh (Ps0.0485).
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Fig. 3. Renal histology 7 days after transplantation. (a) Kidney from sham operated Lewis rat; (b) transplanted kidney from vehicle only exposed rat; (c and d) kidney from BB-94 treated transplanted animals. Severe acute celluar rejection with vascular involvement (d) is depicted in (b)– (d). PAS stain, magnification =400.
with protein (mg)ycreatinine (mmol) ratios of 00.3 during allograft rejection. Proteinuria in the groups of healthy rats and sham operated rats remained minimal and did not differ from the values obtained at day 0 from the two other groups (data not shown). In addition, proteinuria at day y3 was identical to day 0 (data not shown). Plasma levels of sodium, potassium and creatinine obtained at day q7 were almost identical between the three experimental groups (for all comparisons, P) 0.05), as shown in Table 1. Furthermore, there were no noticeable differences between values obtained at day 0 and day q7 (data not shown).
Table 1 Concentrations of sodium, porassium and creatinine in plasma, obtained at day q7
Fig. 4. Effect of allograft rejection on proteinuria. Proteinuria is expressed as ratios of protein (mg)ycreatinine (mmol), obtained from 24-h urine specimen at days 0 and q7, after kidney transplantation. Vehicle only treated (VT) animals demonstrated a distinct trend towards an elevated proteinuria at the time of rejection (P)0.05). Treatment with the MMP inhibitor (BB-94) was able to keep proteinuria at a low level during allograft rejection.
SH (ns3) VT (ns12) BB-94 (ns12)
Naq (mmolyl)
Kq (mmolyl)
Creatinine (mmolyl)
145"3.21 144"0.87 144"1.75
4.6"0.87 3.8"0.35 3.7"0.71
58"52.9 50"2.73 51"5.46
Sh, sham operated group; VT, vehicle solution only treated transplanted group; and BB-94, MMP inhibitor treated transplanted group.
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5. Discussion The present study focused on the expression and activity of MMP in experimental kidney allograft rejection. We have used an established animal model, the orthotopic kidney allografting in the DA-to-Lewis rat strain combination w15,16,21x. This model is reported to be very stringent because of the major histocompatibility mismatch between donor and recipient animals w21x. In the transplanted kidney, severe acute cellular rejection with some degree of vascular involvement occurs within 7 days post transplantation w16,21x. MMP are zinc-dependent metalloendopeptidases. They metabolize and remodel ECM in conjunction with serine proteinases and membrane-anchored a disintegrin and metalloproteinase w7,22,23x. Most prominent examples of MMP are interstitial collagenases (MMP-1y-8y -13), gelatinases (MMP-2y-9), stromelysins (MMP-3y7y-10y-11) and membrane-type(MT)-MMP (MT1y2y 3y4-MMP). MMP activity is regulated and contained by the tissue inhibitors of metalloproteinases (TIMP) w7,23,24x. Meanwhile, there exist several synthetic low molecular weight MMP inhibitors, such as hydroxamic acid derivatives like BB-94 w14,24,25x. BB-94 represents a particularly well studied broad-spectrum MMP inhibitor w14,26x. Importantly, some of these MMP inhibitors may also inhibit the activity of metalloproteinases closely related to MMP that cleave transmembrane cytokine precursors w23x. Augmented expression or activity of renal MMP was found by us and others in several forms of acute and chronic inflammatory kidney disorders w7,27–30x. In the past, we have successfully demonstrated a distinct antiinflammatory effect of MMP inhibitors on glomerular mesangial cells in vitro and in vivo, using cell cultures and an experimental model of mesangial proliferative glomerulonephritis w17,19x. The anti-proliferative effect of MMP inhibitors appeared to be a result of the induction of mesangial cell cycle arrest followed by apoptosis w31x. Recently, the pro-inflammatory role of MMP in the kidney was reviewed by us in this journal in more detail w32x. Importantly, MMP-2 as shown to exert a direct pro-inflammatory effect upon the glomerular mesangial cells w1x. The biologic function of MMP in kidney transplantation remains to be defined. A potential role of MMP in renal allograft fibrosis has been advocated by some authors w33x. There are no formal studies analyzing the biological role of MMP in kidney transplantation, apart from a few descriptive investigations. For instance, inpatients with chronic transplant nephropathy, serum levels of MMP-2 and MMP-3 were found to be increased w34x. Interestingly, the former correlated with proteinuria and the latter with serum creatinine. In the same study, serum MMP-1 was increased in cases with acute rejec-
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tion w34x. In contrast, another study failed to demonstrate alterations in the expression of fibrosis associated genes, including MMP-2, in isolated renal glomeruli of patients with acute kidney allograft rejection w35x. With regard to the effect of immunosuppressive therapy, cyclosporine and tacrolimus were found to lead to almost identical levels of MMP-2 in isolated glomeruli obtained from protocol biopsies of transplanted patients w36x. Interestingly, with respect to transplantation medicine, BB-94 was recently shown to prevent influx of leukocytes into the liver in a mouse model of lethal hepatitis induced by tumor necrosis factor w26x. Furthermore, MMP have been shown previously to play a role in mesangial proliferative glomeruloneophritis w10,17,32x. Since this disease often reappears in allografts, MMP may also play a role in the recurrence of primary renal diseases. In our study, we found a small increase in overall MMP activity as a result of severe kidney transplant rejection. Although this difference appeared to be insignificant, it occurred despite severe tissue destruction with reduced synthetic capacity of cellular elements. Better preservation of kidney histology in a less severe form of acute rejection may well demonstrate a steep increase in MMP activity reflecting acute inflammation. Interestingly, expression of MMP-2 and of MMP-9 was differentially regulated. As a result of acute rejection, MMP-2 was down-regulated, but MMP-9 increased. The differential regulation of these two MMP may argue against a non-specific effect of kidney destruction on MMP synthesis. The divergent results with regard to mRNA levels and activity of MMP-9 may be explained by its extracellular degradation or inhibition w24x. In accordance with our results, the inflammatory cytokine interleukin-1b was previously shown to increase production of MMP-9 but not of MMP-2 w37x. Despite our measurements, we could not exclude the presence of additional and relevant MMP that were not fully assessed in our activity assay systems. Therefore, we included an MMP inhibitor in our studies, regardless of the overall only marginally increased MMP activity in allograft rejection. Importantly, only inhibition studies may ultimately elucidate the functional role of the MMP. As expected, renal damage was very severe in our rejection model. As a consequence of the only minimally elevated MMP activity, there was no discernable benefit with respect to preservation of kidney structure as a result of the MMP inhibitor treatment. Nevertheless, BB-94 successfully inhibited MMP activity and it was able to keep proteinuria at a low level during the rejection process. Therefore, it may be still possible that besides MMP-9, some not yet defined and noxious MMP are also up-regulated during the rejection process. Furthermore, other MMP may only be increased during an earlier phase of the acute rejection process. Once the kidney is seriously destroyed, activity
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of many MMP, possibly with the exclusion of MMP-9, may decrease by itself. According to our results MMP may offer opportunities for complementary therapy of acute allograft rejection. Nevertheless, since these proteinases do not appear to represent prime targets in such severe forms of acute rejection, future studies are required to evaluate the full impact of MMP in this context. Importantly, the role of MMP may be better definable in experimental models with attenuated forms of acute rejection, achieved by the concomitant use of low dose cyclosporine A w38x. Furthermore, such studies may more mimic clinical practice and should include time-course analyses of MMP activity and expression. MMP may play an even more prominent role in chronic allograft rejection promoting fibrosis and sclerosis. Established models of chronic kidney transplant rejection, such as in Lewis rat recipients of Fisher kidney transplants, offer unique opportunities for the analyses of MMP w39,40x. In conclusion, severe kidney allograft rejection affected the expression and activity of MMP. Overall MMP activity only slightly increased, possibly mitigated by the overwhelming renal tissue destruction. In addition, the effect of the rejection process on MMP was not uniform, since MMP-2 and MMP-9 were differentially regulated. MMP activity was successfully reduced by the MMP inhibitor BB-94. In this respect, BB-94 prevented the rise in proteinuria in transplanted animals. Therefore, MMP appear to represent new mediators involved in acute kidney transplant rejection. This may be especially relevant for MMP-9.
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