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• W1497182509 abstract "Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohisto- chemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR. Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohisto- chemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR. Short-term kidney graft survival has improved considerably over the past two decades, whereas long-term survival rate has not changed significantly and remains unsatisfactory (1Meier-Kriesche HU Schold JD Srinivas TR Kaplan B Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era..Am J Transplant. 2004; 4: 378-383Crossref PubMed Scopus (986) Google Scholar). Chronic humoral rejection (CHR) is a distinctive form of rejection mediated by antibodies specific to the allograft. CHR carries a poor prognosis, as there is no known effective treatment. A set of specific criteria recently proposed to further define CHR (2Sis B Mengel M Hass M et al.Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups..Am J Transplant. 2010; 10: 464-474Crossref PubMed Scopus (633) Google Scholar). These criteria include serologic evidence of donor-specific antibodies (DSA), deposition of the complement cleavage product C4d in the graft tissue, especially in the peritubular capillaries, as well as specific histological features (3Colvin RB Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis..J Am Soc Nephrol. 2007; 18: 1046-1056Crossref PubMed Scopus (440) Google Scholar). Based on these characteristics, it is now estimated that a significant number of late graft losses may result from CHR (4Hourmant M Cesbron-Gautier A Terasaki PI et al.Frequency and clinical implications of development of donor-specific and nondonor-specific HLA antibodies after kidney transplantation..J Am Soc Nephrol. 2005; 16: 2804-2812Crossref PubMed Scopus (257) Google Scholar, 5Terasaki PI Ozawa M Predictive value of HLA antibodies and serum creatinine in chronic rejection: Results of a 2-year prospective trial..Transplantation. 2005; 80: 1194-1197Crossref PubMed Scopus (132) Google Scholar, 6Terasaki PI Ozawa M Castro R Four-year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival..Am J Transplant. 2007; 7: 408-415Crossref PubMed Scopus (284) Google Scholar). The biological mechanisms of renal tissue injury associated with CHR are still poorly defined. A better understanding of the physiological processes leading to graft dysfunction in CHR would help design novel treatments for this complication. Of the known pathological features of CHR, histological hallmarks include fibrosis and accumulation of extracellular matrix (ECM) (7Racusen LC Solez K Colvin RB et al.The Banff 97 working classification of renal allograft pathology..Kidney Int. 1999; 55: 713-723Abstract Full Text Full Text PDF PubMed Scopus (2773) Google Scholar). Matrix metalloproteinases (MMPs) are thought to contribute to ECM modification (8Baricos WH Cortez SL el-Dahr SS Schnaper HW ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade..Kidney Int. 1995; 47: 1039-1047Abstract Full Text PDF PubMed Scopus (212) Google Scholar,9Lovett DH Johnson RJ Marti HP Marti J 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: 85-98PubMed Google Scholar). The implication of individual MMPs in allograft rejection has previously been demonstrated in animal and human studies. Specifically, elevated serum levels of MMP-2 were found in patients with ‘chronic transplant nephropathy’ without further classification. Such increases correlated with proteinuria and elevated serum creatinine, but specific linkage to CHR was not sought (10Rodrigo 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: 2041-2045Crossref PubMed Scopus (40) Google Scholar). In a rat model of antibody- mediated glomerulonephritis, sharing a number of features with CHR, active MMP-2 was found in glomeruli and correlated with proteinuria (11Hayashi K Horikoshi S Osada S Shofuda K Shirato I Tomino Y Macrophage-derived MT1-MMPand increased MMP-2 activity are associated with glomerular damage in crescentic glomerulonephritis..J Pathol. 2000; 191: 299-305Crossref PubMed Scopus (29) Google Scholar). In this model, anti-glomerular basement membrane (GBM) polyclonal antibodies injected to rats trigger crescentic glomerulonephritis through a mechanism dependent on the activation of MMP-2. Collectively, these findings suggest that MMP-2 may have a role in glomerular injury and proteinuria. As a type IV collagenase, MMP-2 efficiently digests collagen structures in various basement membranes. We hypothesized that MMP-2 contributes to GBM injury in CHR, resulting in proteinuria associated with this complication. The goal of our study was to examine the presence of active MMP-2 in the glomeruli and urine of kidney transplant recipients experiencing CHR to assess its potential contribution to graft dysfunction. All patients included in this study were single organ, primary kidney transplant recipients followed at the Massachusetts General Hospital (MGH) Transplant Center between 1999 and 2007. Cases of CHR (N = 27) were diagnosed according to the consensus criteria established previously (3Colvin RB Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis..J Am Soc Nephrol. 2007; 18: 1046-1056Crossref PubMed Scopus (440) Google Scholar,12Racusen LC Colvin RB Solez K et al.Antibody-mediated rejection criteria -an addition to the Banff 97 classification of renal allograft rejection..Am J Transplant. 2003; 3: 708-714Crossref PubMed Scopus (949) Google Scholar). These criteria include: (1Meier-Kriesche HU Schold JD Srinivas TR Kaplan B Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era..Am J Transplant. 2004; 4: 378-383Crossref PubMed Scopus (986) Google Scholar) Serological evidence of DSA. (2Sis B Mengel M Hass M et al.Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups..Am J Transplant. 2010; 10: 464-474Crossref PubMed Scopus (633) Google Scholar) Evidence for the deposition of the complement byproduct C4d in graft tissue around peritubular capillaries. (3Colvin RB Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis..J Am Soc Nephrol. 2007; 18: 1046-1056Crossref PubMed Scopus (440) Google Scholar) Histological evidence of at least two of the following characteristics: arterial intimal fibrosis; duplication of the GBM; multilaminaion of the peritubular capillary basement membrane and interstitial fibrosis (IF). Kidney transplant recipients without CHR (non-CHR; N = 65) included those with stable allograft function, calcineurin inhibitor nephrotoxicity (CNI), BK virus nephropathy (BKVN) and chronic kidney diseases other than CHR with proteinuria greater than 150 µg/mL, including diabetes mellitus, membranous glomerulonephritis, systemic lupus erythematosus, IgA nephropathy and focal segmental glomerulosclerosis. We also examined biopsies from patients with native kidney diseases (N = 13), specifically those with nephrotic range proteinuria. Use of patient urine samples and biopsy specimens in our study was approved by the MGH institutional review board. Biopsies were performed as clinically indicated. Urine samples were collected in sterile containers, kept at 4°C and processed within 24 h. The urine was spun at 3000 rpm and the supernatant was stored at –80°C. Staining was performed on paraffin tissue sections using a rabbit polyclonal antibody specific for MMP-2 (Abcam; Cambridge, MA, USA). This antibody recognizes both pro and truncated, that is inactive and forms of MMP- 2. Sections were deparaffinized with xylene, rehydrated with ethanol and treated with 4.5% hydrogen peroxide in methanol for 5 min to block endogenous peroxidase. Sections were then incubated with 2% normal goat serum diluted in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) and Avidin (Vector Laboratories; Burlingame, CA, USA) for 20 min at room temperature to minimize nonspecific binding and block endogenous biotin. Rabbit anti-MMP-2 primary antibody was diluted (1:500) in 1% BSA/PBS and used to stain sections over night at 4°C. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories). Color development was carried out using Streptavidin horseradish peroxidase (Vector Laboratories) followed by treatment with 3- amino-9-ethylcarbazole (Biocare Medical; Concord, CA, USA). All sections were counterstained with Harris hematoxylin (Richard Allen Scientific; Kalamazoo, MI, USA), dehydrated with graded concentrations of ethanol and then mounted. The 'IF and tubular atrophy' (IFTA) scores were determined for all biopsy samples by visual inspection by a pathologist. After thawing, urine samples were first centrifuged at 2000 rpm for 30 min in 4°C to remove cellular debris. To readjust pH and salt concentration, samples were then dialyzed overnight against 10 mM Tris-HCl (pH 7.4) using a 5 kDa cutoff dialysis plate (Harvard Apparatus; Holliston, MA, USA).Following dialysis, the assessment of MMP-2 concentration was carried out using beads coated with antihuman MMP-2 monoclonal antibody (Mab) (R&D Systems; Minneapolis, MN, USA) and a multiplex strategy. Dialyzed urine samples were diluted 1:2 in PBS, incubated with MMP-2-specific beads and revealed with a second MMP-2-specific Mab conjugated to horseradish peroxidase. Detection and quantification of MMP-2 was carried out with the Luminex 200 analyzer (Luminex; Austin, TX, USA). Concentration of urinary MMP-2 was determined by comparison to a standard curve obtained using recombinant MMP-2 (Calbiochem; Gibbstom, NJ, USA). Urine protein content was measured using the micro pyrogallol red method. Briefly, 4 µL of dialyzed urine was incubated at 30°C with 200 iLof pyrogallol red-molybdate solution (Sigma-Aldrich; St. Louis, MO, USA) for 3 min to allow complex formation. Absorbance was then read at 600 nm using a SpectroMax Plus384 spectrophotometer (Molecular Devices; Sunnyvale, CA, USA). The concentration of total proteinuria was calculated based on the following formula: (absorbance of sample/absorbance of standard) × (concentration of provided standard). The enzymatic activity of MMP-2 in urine samples was assessed using a fluorometric assay adapted from the Enzolyte MMP assay (Anaspec Inc.; Fremont CA, USA). This assay measured the digestion, in real-time, of a synthetic peptide containing the specific location of the catalytic cysteine residue for MMP-2 activation. The peptide substrate QXLTM520-Pro-Leu- Gly-Met-TrpSer-Arg-Lys(5-FAM)-NH2, which included a FAM fluorescent dye at its C-terminus and a quencher at its N-terminus was purchased from Anaspec (Fremont, CA, USA). The substrate is incubated with biological samples for which the level of active MMP-2 is to be assessed. Upon cleavage, the fluorescent dye is released from its quencher and can be detected. To detect the dye we used a Stratagene 3500 quantitative PCR machine (Stratagene; Cedar Creek, TX, USA). The enzymatic reaction was carried out at 37°C using 40 µL of dialyzed urine diluted 1:40 in 50 mM Hepes buffer (Sigma-Aldrich; St. Louis, MO, USA), pH 7.4, with 10 mM CaCl2, 0.2 M NaCl and 1 iM of the MMP-2-specific substrate mentioned above. A 1 µM reference standard simµLar in composition to the proteolytic cleavage product of the substrate was used to ensure the fluorochrome reaction worked properly. Deionized water was used as a negative control to account for background fluorescence emitted by the substrate alone. Measurements were taken at excitation/emission of 490 nm/520 nm every 5 min. Categorical data were summarized as percentages and were compared between patients with CHR and non-CHR control groups using the Fisher's exact test. Continuous variables were presented as medians and tested for possible differences between groups using the Wilcoxon rank-sum tests. The association between urine levels of MMP-2 and proteinuria within CHR group and within non-CHR group was determined using Pearson correlation coefficients. A p-value of <0.05 was considered significant. All analyses were performed using STATA-version 10.0 (StataCorp; College Station, TX, USA). Glomerular staining paraffin embedded sections of allograft biopsies from kidney transplant recipients with CHR (N = 7) or without CHR (N = 13) were stained for the presence of MMP-2. All biopsies had some level of staining in the tubular epithelium, without any significant difference between CHR and non-CHR patients (Figure 1, Table 1). In contrast, distinctive staining patterns were observed in glomeruli, indicating a different MMP-2 distribution. As µLlustrated inFigure 1, no glomerular MMP-2 staining was detected in tissue from healthy kidneys or control allografts without diagnostic abnormalities (Figure 1A and B, respectively). Some biopsies revealed intense staining of periglomerular fibrosis (Figure 1C). No podocyte staining was observed in any of the 13 biopsies collected from non-CHR patients, even though some of these pa-tients had nephrotic range proteinuria at time of sampling (Figure 1D; Table 1). Some non-CHR biopsies showed intense staining of the tubular epithelium without any staining of the glomeruli (Figure 1 B and D). In contrast, all seven biopsies obtained from CHR patients showed unequivocal podocyte staining for MMP-2 (Figure 1E and F, Table 1). The differential staining was highly significant (p = 0.004). Parietal glomerular epithelial cells also appeared to express MMP-2 in CHR and some non-CHR patients. Additionally, we carried out immunostaining on biopsies collected from native kidney diseases associated with nephrotic range proteinuria. A minority of these biopsies showed MMP- 2 expression in the podocytes (Table S1), although staining in surrounding glomeruli structures was clearly visible (Figure S1).Table 1MMP-2 staining and IFTA scores in kidney allograft biopsiesDiagnosisPodocyte1MMP-2 staining intensity: 0, no staining.Tubular epithelium1MMP-2 staining intensity: 0, no staining.IFTA2Interstitial fibrosis tubular atrophy. score (%)Chronic humoral rejection case #1+Low to moderate stainingintense staining0Chronic humoral rejection case #2+Low to moderate stainingintense staining40Chronic humoral rejection case #3+Low to moderate stainingintense staining15Chronic humoral rejection case #4intense stainingintense staining30Chronic humoral rejection case #5intense stainingintense staining15Chronic humoral rejection case #6intense stainingintense staining20Chronic humoral rejection case #7intense stainingintense staining40Total7/77/7Acute tubular necrosis0+Low to moderate staining5BK virus nephropathy0+Low to moderate staining70Diabetic glomerulosclerosis0+Low to moderate staining10Diabetic glomerulosclerosis0intense staining50Focal segmental glomerulosclerosis0+Low to moderate staining40Focal segmental glomerulosclerosis0+Low to moderate staining25Glomerulonephritis not otherwise specified0+Low to moderate staining10IgA nephropathy0+Low to moderate staining5Interstitial nephritis0+Low to moderate staining10Interstitial Nephritis0+Low to moderate staining50Membranoproliferative glomerulonephritis0intense staining5No diagnostic abnormalities recognized0intense staining5No diagnostic abnormalities recognized0intense staining5Total0/1313/13+ Low to moderate staining++ intense staining1 MMP-2 staining intensity: 0, no staining.2 Interstitial fibrosis tubular atrophy. Open table in a new tab We next examined whether MMP-2 could be detected in the urine of transplant recipients with CHR as a reflection of its accumulation in the glomeruli. Urine samples from 27 CHR and 65 non-CHR kidney transplant recipients were collected and assessed for the presence of MMP-2. Urine samples were collected at the time of biopsy, which documented CHR or other kidney allograft pathology. Table 2 summarizes patient characteristics. Patients with CHR had higher levels of serum creatinine and proteinuria compared with patients without CHR (p < 0.001).Table 2Patient characteristicsNon-CHRCHRp-valuesN65 (8 BKVN,1BK virus nephropathy. 9 CNI,2Calcineurin inhibitor toxicity. 23 other kidney disease,3Includes: Diabetic nephropathy (N = 8), focal segmental glomeru-losclerosis (N = 5), membranoproliferative glomerulonephritis (N = 2), membranous glomerulonephritis (N = 2), interstitial nephritis (N = 2), acute tubular necrosis (N = 1), IgA nephropathy (N = 1), lambda light chain deposition disease (N = 1), systemic lupus erythematosus (N = 1). 25 stable)27Median age (years)49470.45Female27 (42%)7 (26%)0.23Years posttransplant4Median.2.16.8<0.001Creatinine (mg/dL)4Median.1.52.5<0.001Proteinuria (mg/dL)4Median.2931829<0.001DonationDeceased16 (59%)35 (54%)0.95Living related6 (22%)15 (23%)Living unrelated5 (19%)15 (23%)ImmunosuppressionThree drug regimen48 (74%)17 (63%)0.21Two or less drug regimen17 (26%)10 (37%)1 BK virus nephropathy.2 Calcineurin inhibitor toxicity.3 Includes: Diabetic nephropathy (N = 8), focal segmental glomeru-losclerosis (N = 5), membranoproliferative glomerulonephritis (N = 2), membranous glomerulonephritis (N = 2), interstitial nephritis (N = 2), acute tubular necrosis (N = 1), IgA nephropathy (N = 1), lambda light chain deposition disease (N = 1), systemic lupus erythematosus (N = 1).4 Median. Open table in a new tab Concentration of MMP-2 in the urine was measured using a Luminex-based technique. As shown in Figure 2, patients with CHR had higher levels of urinary MMP-2 than transplant recipients without this complication (p < 0.001). Even though patients with CHR were biopsied at later times posttransplantation compared with non-CHR patients (Table 2; p < 0.001), we could not find any correlation between time posttransplant and urine concentration of MMP-2 (Figure S2). The difference in urine MMP-2 levels was significant for all patient groups when compared with the CHR group (Table 3). Two transplant recipients with kidney diseases other than CHR also displayed abnormally elevated urine concentrations of MMP-2. The values for these two patients (17 952 and 17 049 pg/mL) are responsible for the extended range shown in Figure 2. The urine creatinine concentration was also assessed in all available samples and used to normalize urinary MMP-2 levels. As reported in Figure S3, normalized urinary MMP-2 measurements were significantly higher in CHR patients than in non-CHR controls. Finally, we assessed urinary MMP-2 levels in nontransplant patients with advanced kidney diseases. Elevated urinary MMP-2 levels comparable with that of CHR patients were also found for this group (data not shown; Wilcoxon rank sum test; p = 0.84).Table 3Comparison of urinary MMP-2 levelsPatient groupsMMP-2 (pg/mL)1Median value.p-Value2Wilcoxon rank-sum test.CHR (N = 27) versus4942BKVN (N = 8)8910.02CNI (N = 9)7160.003Other kidney disease3See Table 2 (N = 23)11330.04Stable (N = 25)298<0.0011 Median value.2 Wilcoxon rank-sum test.3 See Table 2 Open table in a new tab The molecular weight of the active form of MMP-2 is approximately 72 kDa, which largely excludes it from the glomerular filtrate under normal physiological conditions. We therefore assessed whether MMP-2 in the urine was linked to proteinuria. Overall, there was a positive correlation between urinary MMP-2 levels and proteinuria in both CHR and non-CHR patients (p < 0.001 for both groups). In ourstudies, we arbitrarily considered urine MMP-2 concentration above 8000 pg/mL as 'high' and below 2000 pg/mL as 'low' (dotted lines in Figure 3). All patients with high urinary levels of MMP-2 also had proteinuria (>500 µg/mL; Figure 3). However, several CHR and non-CHR patients with proteinuria above 500 µg/mL did not show any significant levels of MMP-2 (arrows in Figure 3). These findings indicate that the presence of MMP-2 in the urine was not merely due to glomerular damage leading to proteinuria. As indicated with stars in Figure 3, the two non-CHR patients mentioned above with high MMP-2 concentrations in the urine also had proteinuria greater than 2000 µg/mL. Biopsy evaluation indicated acute tubular injury with podocyte effacement in one (Figure 4A) and de novo membranous glomerulonephritis in the other (Figure 4B). We investigated whether the glomerular MMP-2 staining pattern for these two patients was comparable with that of patients with CHR. As depicted in Figure 4, immunohistochemistry revealed a comparable MMP-2 staining of the podocytes for these two patients as was observed for the CHR patients.Figure 4Immunohistochemical MMP-2 staining for two non-CHR patients with high urinary MMP-2 concentration. Staining for MMP-2 was carried out in biopsies of two kidney transplant recipients with high urinary MMP-2 levels. (A) Acute tubular injury. Urinary MMP-2 and total protein concentrations were 17 952 pg/mL and 2317 µg/mL, respectively. (B) De novo membranous glomerulonephritis. MMP-2 and proteinuria concentrations were 17 049 pg/mL and 3744 ug/mL, respectively. Podocyte staining is indicated with arrowheads.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether detection of MMP-2 in the urine coincided with CHR, we assessed serial samples avaµLable from four patients with this complication. For all four patients, elevation in MMP-2 levels was found in urine samples taken between 0 and 58 days prior to the biopsy documenting CHR (Figure 5). The MMP-2-specific antibodies used in our Luminex-based assays and in immunohistochemistry experiments did not distinguish between the active and inactive forms of the enzyme. We therefore utilized a quantitative enzymatic assay to determine if MMP-2 released in the patient urine had been activated in vivo. This functional assay made use of a quantitative PCR machine to measure the cleavage over time of a fluorescent peptide including a binding motif specific for MMP-2. The sensitivity of this assay allows the reproducible detection of MMP-2 concentrations as low as 50 pg/mL (Figure S4). Using this assay, we also verified that the enzymatic activity of MMP-2 was maintained even after three freeze/thaw cycles (Figure S5). The presence of the active form of MMP-2 was assessed with this method in dialyzed urine samples from three CHR patients with high concentrations of MMP-2 (>15 000 pg/mL), two CHR patients with low MMP-2 (<2 000 pg/mL) and two non-CHR patients with MMP-2 below 1000 pg/mL. As µLlustrated in Figure 6, MMP-2 activity was detected in the urine of the three CHR patients for whom we had previously shown elevated urinary levels of this enzyme. In contrast, samples with low MMP-2 levels from the two CHR and the two non-CHR patients did not show any proteolytic activity. CHR is a serious complications following kidney transplantation (4Hourmant M Cesbron-Gautier A Terasaki PI et al.Frequency and clinical implications of development of donor-specific and nondonor-specific HLA antibodies after kidney transplantation..J Am Soc Nephrol. 2005; 16: 2804-2812Crossref PubMed Scopus (257) Google Scholar, 5Terasaki PI Ozawa M Predictive value of HLA antibodies and serum creatinine in chronic rejection: Results of a 2-year prospective trial..Transplantation. 2005; 80: 1194-1197Crossref PubMed Scopus (132) Google Scholar, 6Terasaki PI Ozawa M Castro R Four-year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival..Am J Transplant. 2007; 7: 408-415Crossref PubMed Scopus (284) Google Scholar). At this time, conventional therapies for rejection have not proven effective for the treatment of CHR and better strategies are needed. Most current therapies target the recipient alloimmunity, which is thought to be responsible for CHR. However, complementary treatments to reverse tissue damage associated with this complication have not been considered. This is partly due to the fact that mechanisms of tissue destruction selectively associated with CHR are largely unknown. Our studies examined the presence of the extracellular zinc dependent protease MMP-2 in kidney transplant recipients with CHR or other complications. A novel finding is the MMP-2 staining of podocytes in patients with CHR compared with the non-CHR kidney transplant recipients, including patients with high proteinuria. Previous studies carried out in rats, have already detected the expression of the MMP-2 gene in situ by PCR during chronic allograft rejection (13Cheng S Pollock AS Mahimkar R Olson JL Lovett DH Matrix metalloproteinase 2 and basement membrane integrity: A unifying mechanism for progressive renal injury..FASEB J. 2006; 20: 1898-1900Crossref PubMed Scopus (196) Google Scholar). Zymogram experiments further confirmed the presence of active MMP-2 in the graft tissue. In humans, MMP-2 was detected in several glomerular diseases (14Endo T Nakabayashi K Sekiuchi M Kuroda T Soejima A Ya-mada A Matrix metalloproteinase-2, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 in the peripheral blood of patients with various glomerular diseases and their implication in pathogenetic lesions: Study based on an enzyme-linked assay and immunohistochemical staining..Clin Exp Nephrol. 2006; 10: 253-261Crossref PubMed Scopus (24) Google Scholar,15Sanders JS Huitema MG Hanemaaijer R van Goor H Kallenberg CG Stegeman CA Urinary matrix metalloproteinases reflect renal damage in anti-neutrophµL cytoplasm autoantibody-associated vasculitis..Am J Physiol Renal Physiol. 2007; 293: F1927-F1934Crossref PubMed Scopus (31) Google Scholar). These studies, however, did not reveal the localization of MMP-2 in the glomeruli. The source of MMP-2 is also stµLl uncertain. MMP-2 may be produced locally either directly by podocytes, which can secrete other types of MMPs (16Bai Y Wang L Li Y et al.High ambient glucose levels modulates the production of MMP-9 and alpha5(IV) collagen by cultured podocytes..Cell Physiol Biochem. 2006; 17: 57-58Crossref PubMed Scopus (53) Google Scholar), endothelial cells (17Serluca FC Drummond IA Fishman MC Endothelial signaling in kidney morphogenesis: A role for hemodynamic forces..Curr Biol. 2002; 12: 492-497Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) or by intracapµLlary monocytes (18Fahim T Bohming GA Exner M et al.The cellular lesion of humoral rejection: Predominant recruitment of monocytes to peritubular and glomerular capillaries..Am J Transplant. 2007; 7: 385-393Crossref PubMed Scopus (84) Google Scholar). Alternatively, MMP-2 could originate from the plasma where the inactive form of this enzyme can be found in both healthy subjects and kidney transplant recipients with rejection (10Rodrigo 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: 2041-2045Crossref PubMed Scopus (40) Google Scholar). Its accumulation in the glomeruli of CHR would then result from the ongoing immune response and lead to its cleavage and activation in the graft. Glomerular staining MMP-2 has been observed in cases of antineutrophµL cytoplasmic antibodies (ANCA) glomerulonephritis (19Sanders JS van Goor H Hanemaaijer R Kallenberg CG Stegeman CA Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis..Nephrol Dial Transplant. 2004; 19: 1412-1419Crossref PubMed Scopus (50) Google Scholar). We have recently shown that CHR is accompanied by the development of antibodies to a broad range of self-antigens (20Porcheray F Devito J Yeap BY et al.Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses..Transplantation. 2010; 89: 1239-1246Crossref PubMed Scopus (71) Google Scholar). These findings draw a parallel between the presence of MMP-2 in glomeruli and serum autoantibodies. As mentioned above, we also observed elevated urinary MMP-2 levels in native kidney diseases unrelated to humoral immunity. These findings indicate that MMP-2 involvement in kidney diseases is not specific to CHR. More comprehensive studies are now required to examine MMP-2 distribution in native kidney diseases, especially those with glomerular dysfunction. Whereas high urinary MMP-2 levels were always accompanied by proteinuria, the reverse was not true. Several patients had proteinuria, but undetectable or low levels of MMP-2 in the urine, thereby indicating that increased urinary MMP-2 concentration was not merely due to proteinuria. Moreover, results from our enzymatic assays showed that MMP-2 in the urine of these CHR patients was in its active form. Others have reported urinary MMP-2 as a reflection of kidney damage in patients with autoantibody-associated vasculitis ANCA (15Sanders JS Huitema MG Hanemaaijer R van Goor H Kallenberg CG Stegeman CA Urinary matrix metalloproteinases reflect renal damage in anti-neutrophµL cytoplasm autoantibody-associated vasculitis..Am J Physiol Renal Physiol. 2007; 293: F1927-F1934Crossref PubMed Scopus (31) Google Scholar). As mentioned previously, we have recently shown that the humoral response in CHR includes an autoimmune component (20Porcheray F Devito J Yeap BY et al.Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses..Transplantation. 2010; 89: 1239-1246Crossref PubMed Scopus (71) Google Scholar). Remarkably, tubular epithelial cells also express significant levels of MMP-2 and could therefore add to the total urinary MMP-2 level. This contribution is uncertain, however, as several biopsies showed intense tubular epithelial staining without evidence of MMP-2 in the urine. Overall, our retrospective studies provide evidence for the presence of active MMP-2 in glomeruli, especially in podocytes, of CHR patients. Based on our findings obtained from a limited series of transplant recipients, we cannot establish a causal link between this association and podocyte injury observed in CHR. Subsequent mechanistic studies are now warranted to determine if this enzyme is directly involved in tissue destruction and can represent a potentially interesting therapeutic target for the treatment of CHR. We are indebted to Drs Yongguang Yang and Robert Hawley for their thoughtful review of our manuscript. This work was supported by the Fahd and Nadia Alireza's Research Fund as well as the MµLton Fund, Boston, MA. The authors of this manuscript have no conflicts of interest to disclose as described in the American Journal ofTransplantation. Additional Supporting Information may be found in the online version of this article: Download .doc (.23 MB) Help with doc files Supplementary Material Figure S1. Immunohistochemical staining for MMP-2 in native kidney biopsies. (A) Focal segmental glomerulosclerosis, (B) Focal segmental glomerulosclerosis, (C) Focal segmental glomerulosclerosis, (D) Focal segmental glomerulosclerosis. Figure S2. Urinary levels of MMP-2 and sampling time posttransplant. Urinary MMP-2 measurements are depicted as a function of sampling time posttransplant for CHR and non-CHR patients. Figure S3. Normalized urinary MMP-2 levels. Creatinine content was assessed by ELISA in available dialyzed urine samples collected from CHR (N = 19) and non- CHR patients (N = 36) and used to normalized urinary MMP-2 levels as follows: Norm. MMP-2x = MMP-2x × (creatx/creatmax). Normalized MMP-2 values are depicted using a box plot representation, showing values for the median, range, 25th and 75th percentiles. Figure S4. Sensitivity of the assay for the detection of urinary MMP-2 enzymatic activity. The enzymatic activity of MMP-2 was measured in serial dilutions of two representative urine samples known to have MMP-2 (Sample 1 and 2) as well as a patient urine sample that did not contain MMP-2 (Sample 3). The final MMP-2 concentration is reported in the figure. Figure S5. Functional assessment of urinary MMP-2 activity after freezing/thawing. The enzymatic activity of MMP-2 was measured in a representative urine sample after 1, 2 or 3 freezing/thawing cycles. Table S1: MMP-2 Staining in native kidney biopsies Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article." @default.
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