Tissue Inhibitors of Metalloproteinase System in Renal Transplant Recipients With Chronic Allograft Injury

Imbalance of Metallaproteinase/Tissue Inhibitors of Metalloproteinase System in Renal Transplant Recipients With Chronic Allograft Injury O. Mazanowska, D. Kamin´ska, M. Krajewska, M. Z˙abin´ska, W. Kopec´, M. Boratyn´ska, P. Chudoba, D. Patrzalek, and M. Klinger ABSTRACT Introduction. Nowadays, renal allografts continue to be lost at the rate of 2% to 4% per year beyond the first year after transplantation due to chronic allograft injury. Excessive accumulation of extracellular matrix results from overproduction and/or defective degradation by proteolytic enzymes, among which metalloproteinases (MMPs) play a major role. The aim of this study was to assess the role of MMPs in renal transplant recipients (RTR) in the context of allograft injury or proteinuria. Materials and methods. Plasma and urine MMP-2 and MMP-9 and tissue inhibitors of metalloproteinases (TIMPs) were assessed by enzyme-linked immunoassay in 150 RTR including 66% males with an overall mean age of 49.2 ⫾ 11.5 years. The subjects were examined at a mean of 73.4 ⫾ 41.2 months (range ⫽ 12–240) after kidney transplantation. Thirty-seven healthy volunteers including 54% male with an overall mean age of 48.4 ⫾ 14.1 years served as a control group. Results. Renal transplant recipients displayed significantly decreased plasma MMP-2 activity compared with healthy controls (P ⬍ .000) probably due to increased inhibitory plasma (p) TIMP-2 activity (P ⫽ .0029), and lower plasma MMP-2:TIMP-2 index (P ⬍ .0001). Plasma MMP-9 and pTIMP-1 activities were twofold increased in RTR compared with controls (P ⫽ .0015 and P ⬍ .000) but with a nearly stable plasma MMP-9:TIMP-1 index (P ⫽ NS). There was no difference between RTR and controls according to urine (u) MMP-2 activity, but uMMP-9 was increased in RTR compared with healthy controls (P ⫽ .0032). Urine MMP-9 potential was probably diminished by increased uTIMPs (uTIMP-2, P ⫽ .017; uTIMP-1, P ⫽ .000), which contributed to graft impairment or proteinuria. Conclusion. Our study revealed profibrotic MMP/TIMP constellations in RTR that show an imbalance in plasma MMP-2 and MMP-9 with increased plasma and urinary TIMPs. The net proteolytic potential of increased plasma and urinary MMP-9 may be diminished significantly by enhanced plasma and urine TIMP activities.

R

ENAL ALLOGRAFT SURVIVAL has not improved much over the past 30 years.1 Renal allografts continue to be lost at the rate of 2% to 4% per year beyond the first year after transplantation.2 Progressive interstitial fi-

brosis and tubular atrophy is a leading cause of chronic allograft injury (CAI).3 Both immune and nonimmune factors have been implicated in the pathogenesis of CAI, which results in excessive accumulation of extracellular

From the Department of Nephrology and Transplantation Medicine (O.M., D.K., M.K., M.Z., W.K., M.B., M.K.) and Department of Vascular, General and Transplant Surgery (P.C., D.P.), Wroclaw Medical University, Wroclaw, Poland. This study was supported by research grant (GR 1645) of Wroclaw Medical University.

Address reprint requests to Oktawia Mazanowska, Department of Nephrology and Transplantation Medicine, ul. Borowska 213, 50-556 Wroclaw, Poland. E-mail: o.mazanowska@gmail. com

0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.08.012

© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

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Transplantation Proceedings, 43, 3000 –3003 (2011)

IMBALANCE OF MMP/TIMP SYSTEM

matrix (ECM) components due to overproduction and/or defective ECM degradation by proteolytic enzymes, among which metalloproteinases (MMPs) play the major role.4 The activity of MMPs is controlled through the activation of proenzymes and inhibited by tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 inhibits the pro- and active forms of MMP-9; whereas, TIMP-2 is the major inhibitor of proMMP-2.5 TIMPs form stoichiometric 1:1, noncovalent complexes with MMPs, which can dissociate to yield enzyme.6 MMP-2 and MMP-9 can degrade components of basement membrane types IV and V collagens, aggrecan, elastin and gelatins, fibronectin, as well as laminin. However, the ECM substrate specificity and renal expression of MMP-2 and MMP-9 are not totally identical. Unlike MMP-2, which is mostly constitutively expressed, MMP-9 shows a restricted pattern of expression in developmental and adult tissues.7 MMP-9 has been identified in a variety of tissues and body fluids under pathophysiological conditions including smoking, cancers, and polycystic kidney disease. Improving the long-term survival of renal allografts is nowadays an important challenge. Noninvasive laboratory tests to guide therapeutic decisions based upon relevant early indicators of chronic graft injury may help to design better management strategies for renal transplant recipients.1 The aim of this study was to assess the potential role of MMP/TIMP content in renal transplant recipients with long-functioning allografts experiencing CAI or proteinuria.

3001 Table 1. Patient and Control Characteristic and Mean Plasma and Urine MMP and TIMP Concentrations Feature

Gender, male (%) sCr ⱕ 1.5 vs ⬎ 1.5 mg/dL Proteinuria, yes (%) pMMP-2 (ng/mL) ⫾ SD pTIMP-2 (ng/mL) ⫾ SD pMMP2:pTIMP-2 index (for medians) pMMP-9 (ng/mL) ⫾ SD pTIMP-1 (ng/mL) ⫾ SD pMMP-9:pTIMP-1 index (for medians) uMMP-2 (ng/mg) ⫾ SD* uTIMP-2 (ng/mg) ⫾ SD* uMMP-9 (ng/mg) ⫾ SD* uTIMP-1 (ng/mg) ⫾ SD*

Patients

Controls

20/37 (54%) 79/71 37/0 44/150 (29%) 0 (0%) 217 ⫾ 51 248 ⫾ 41 92 ⫾ 24 74 ⫾ 29 2.3 3.1

P

99/150 (66%)

7.53e-06 .0029 ⬍.0001

147 ⫾ 148 159 ⫾ 59 0.67

70 ⫾ 44 87 ⫾ 16 0.71

.0015 2.2e-16 .67

0.7 ⫾ 2.7 7.4 ⫾ 20.4 2.1 ⫾ 11.4 2.5 ⫾ 4.3

0.1 ⫾ 0.4 2.1 ⫾ 2.6 0.1 ⫾ 0.04 1.3 ⫾ 3.9

.39 .017 .0032 3.4e-05

MMP, metalloproteinase; TIMP, tissue inhibitors of metalloproteinase; sCr, serum creatinine; p, plasma; u, urine; SD, standard deviation. *Urine activities standardized to urine creatinine concentrations.

METHODS

volunteers (control group) gave informed consent to participate in the study. Statistical analysis was performed using SPSS, Statistica 9.0, R and Medcalc. The data are presented as mean values, standard deviations and medians. Wilcoxon sum rank test, Mann-Whitney U, Welch t test, Spearman and Kruskal-Wallis test (for nonparametric data) were applied because the data were not normally distributed. Correlations between parameters were assessed by logistic regression. Differences at P ⬍ .05 were considered statistically significant.

The study was approved by our University Ethics Committee and was performed in accordance with the Helsinki declaration.

RESULTS

Labs In the morning under fasting conditions urine samples and venous blood was collected, in heparinized tubes for MMPs and EDTA tubes for TIMPs. Plasma immediately separated at 4°C, was aliquoted and frozen at ⫺70°C. For urine MMPs (uMMP) and TIMPs (uTIMP), spot samples (5 mL) were centrifuged (3000 rpm for 30 minutes at 4°C) to remove suspended particles for storage as aliquots at ⫺70°C. Plasma and urine MMP-2 and MMP-9 activities were measured by use of commercial enzyme-linked immunoassay kits (R&D), concentrations were determined by interpolation from standard curves, with results expressed in ng/mL (for plasma activities). uMMPs/urine creatinine (uCr) and uTIMPs/uCr ratios were calculated to standardize the samples; urine concentrations were expressed in ng/mg uCr.

Participants The study was conducted in 150 renal transplant recipients (RTR) and in 37 healthy volunteers (control group) with no renal pathology. The primary disease represented by these RTR was chronic glomerulonephritis (n ⫽ 122) and hypertensive nephropathy (n ⫽ 28). We excluded patients with systemic diseases causing secondary glomerulonephritis, pyelonephritis, diabetic nephropathy, polycystic kidney disease, as well as those with current general infections or urinary tract infection because of their possible additional impact on the MMP/TIMP system. All patients and healthy

At assessment patients and healthy controls had mean ages of 49.2 ⫾ 11.5 and 48.4 ⫾ 14.1 years (P ⫽ .74; NS). In RTR the mean time since renal transplantation was 73.4 ⫾ 41.2 months (range ⫽ 12–240). Mean serum creatinine concentration and estimated GFR (measured by abbreviated MDRD formula) for RTR were 1.65 ⫾ 0.67 mg/dL and 48.4 ⫾ 15.94 mL/min/1.73 m2 and for controls 1.0 ⫾ 0.1 mg/dL and 73 ⫾ 10 mL/min/1.73 m2, respectively. All subjects demonstrated CRP levels below 5 mg/L: the mean CRP in RTR was 1.6 ⫾ 1.2 mg/L and among controls, 1.4 ⫾ 0.9 mg/L. Table 1 summarizes the subjects’ characteristics, data, and results. Plasma MMP and TIMP Concentrations

Renal transplant recipients showed significantly lower plasma MMP2 (pMMP2) and simultaneously higher plasma TIMP-2 (pTIMP-2) activities than the controls. The MMP2:TIMP-2 index was lower in RTR, namely, medians values of: 2.3 versus 3.1; (P ⬍ .0001) showing a relative MMP-2 deficiency and TIMP-2 overabundance. There was a significant excess of pMMP-2 over pTIMP-2 in controls (P ⬍ .0001) and RTR (P ⬍ .0001), more pronounced in controls than in RTR (Mann-Whitney U test, P ⬍ .0001). Renal transplant recipients demonstrated twofold higher

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pMMP-9 and pTIMP-1 concentrations compared with healthy controls, but there was no difference in plasma MMP-9:TIMP-1 index; median values of 0.67 versus 0.71 (P ⫽ .67 ⫽ NS). The deficiency of pMMP-9 in relation to pTIMP-1 was significant in controls (P ⫽ .0007) and in RTR (P ⬍ .0001). There was no significant difference in pMMP-9:pTIMP-1 index between controls and RTR (Welch t test, P ⫽ .761 ⫽ NS). uMMPs and uTIMPs Concentrations

There was no difference in uMMP-2 in RTR compared with healthy controls, but RTR showed significantly higher uTIMP-2. Urine MMP-2:TIMP-2 index (⫻100%) was low in both RTR and controls (9.5% vs 4.8%). RTR displayed significantly higher uMMP-9 and uTIMP-1 than controls, with a tenfold increase (84% vs 7.7%) in urine MMP-9: TIMP-1 index among RTR (⫻100%). RTR universally showed increased plasma as well as urine TIMP concentrations and an imbalance in plasma MMP activities. DISCUSSION

MMPs are usually considered to be protective due to their antifibrotic activities, but this view is too simplistic and too optimistic. MMPs can influence the behavior of glomerular cells via the generation of ECM cleavage products and can regulate the local availability of growth factors via a direct action on their synthesis or degradation, their release from ECM, or the cleavage of binding proteins. Increased levels of MMPs are usually associated with disease activity and the influx of inflammatory cells. Plasma and urine MMPs and TIMPs have been assessed by many authors mostly in kidney diseases; there are only a few reports in kidney transplantation with inconsistent results. Bauvois showed significant differences between plasma MMP-2, MMP-9, and TIMP-1 concentrations in various glomerular diseases: decreased MMP-2, MMP-9 and increased TIMP-1 in IgA nephropathy; decreased MMP-9 and increased MMP-2 and TIMP-1 in membranous nephropathy, and increased MMP-2 and TIMP-1 in minimal change disease and focal segmental glomerulosclerosis (FSGS), thus suggesting the involvement of different mechanisms in the regulation of fibrosis in glomerular diseases.8 In contrast Endo and coworkers showed increased or no change in serum MMP-9 concentrations in immunoglobulin A nephropathy and other nephropaties.9 The contradictory observations show the inconsistencies in research depending on methodology or clinical status of the patients. In chronic kidney disease, Chang et al reported pMMP-2 activity to be significantly higher (P ⬍ .001), whereas pMMP-9 was lower (P ⬍ .001) in patients than in controls.10 Based on a linear regression analysis Chang et al found that serum creatinine concentration correlated positively with pMMP-2 activity (Y ⫽ 2.35X⫹82.925, R ⫽ .288, P ⬍ .05) and negatively with pMMP-9 level (Y ⫽ -4.873X⫹111.1034, R ⫽ .344, P ⬍ .01).10 Nagano et al showed that serum MMP-2, adjusted to

MAZANOWSKA, KAMIN´SKA, KRAJEWSKA ET AL

age and serum creatinine concentration, was significantly increased (P ⫽ .001) in proportion to the increased proteinuria among patients with various diseases leading to chronic kidney disease.11 Imbalances between MMPs and TIMPs have been observed in various acute and chronic kidney diseases.11–13 In pediatric patients with vesicoureteral reflux, Taranta-Janusz et al observed higher serum and urine TIMP-1 and TIMP-2 concentrations than among controls.14 Moreover, MMP-9 concentration was higher in urine and MMP-2 in serum. Urine TIMPs were increased proportionally to the increased uMMPs concentrations, with unchanged urine MMP-2:TIMP-2 and MMP9:TIMP-1 indices.14 In mice Eddy et al confirmed the vital role of TIMP-1 in the pathogenesis of renal fibrosis, based upon the correlations between TIMP-1 and transforming growth factor ␤1.15 Hörstrup et al16 observed in 54 patients with various primary and secondary kidney diseases and lowered glomerular filtration rate, increased values of pTIMP-1 (6- to 18-fold) in glomerulonephritis, nephrosclerosis, and diabetic nephropathy, and uTIMP-1/uCr were elevated as well. Increased pMMP-2 concentrations were observed in RTR experiencing chronic humoral rejection; it correlated with proteinuria and elevated serum creatinine concentrations.17 MMP’s which are rapidly released from cells after biosynthesis in the glomerulus by mesangial and epithelial cells, act extracellularly in the matrix, but easily diffuse into the blood or urine. In this context MMPs may refill tubular excretion; yet at the same time they are difficult to detect in situ.18 The advantage of our research was the fairly homogenous group of RTR, without systemic disease leading to renal failure in their native kidneys, without general or urinary infection and more than 1 year after kidney transplantation, a time when chronic graft injury begins to impair allograft function. Our study revealed a profibrotic constellation of MMP/ TIMP in RTR with a significant imbalance between plasma MMP-2 and MMP-9 activities with decreased pMMP-2 and increased pMMP-9 as well as increased pTIMPs activities. In total, uMMP-9 activity was increased, probably due to increased intrarenal production and excretion, but the net proteolytic potential may be diminished by enhanced uTIMP concentrations.

REFERENCES 1. Chapman JR: Clinical renal transplantation: where are we now, what are our key challenges? Transplant Proc 42(9 suppl):S3, 2010 2. Hariharan S, Johnson CP, Bresnahan BA, et al: Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 342:605, 2000 3. Chapman JR, O’Connell PJ, Nankivell BJ: Chronic renal allograft dysfunction. J Am Soc Nephrol 16:3015, 2005 4. Waller JR, Nicholson ML: Molecular mechanisms of renal allograft fibrosis. Br J Surgery 88:1429, 2001 5. Baricos WH: Chronic renal disease: do metalloproteinase inhibitors have a demonstrable role in extracellular matrix accumulation? Curr Opin Nephrol Hypertens 4:365, 1995

IMBALANCE OF MMP/TIMP SYSTEM 6. Murphy G, Willenbrock F: Tissue inhibitors of metalloendopeptidases. Methods Enzymol 248:496, 1995 7. Lelongt B, Legallicier B, Piedagnel R, et al: Do matrix metalloproteinases MMP-2 and MMP-9 (gelatinases) play a role in renal development, physiology and glomerular diseases? Curr Opin Nephrol Hypertens 10:7, 2001 8. Bauvois B: Specific changes in plasma concentrations of matrix metalloproteinase-2 and -9, TIMP-1 and TGF-␤1 in patients with distinct types of primary glomerulonephrits. Nephrol Dial Transplant 22:1115, 2007 9. Endo T, Nakabayashi K, Sekiuchi M, et al: Matrix metalloproteinase-2, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 in the peripheral blond 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 10:253, 2006 10. Chang H-R, Yang S-F, Li M-L, et al: Relationship between circulating matrix metalloproteinase-2 and -9 and renal function in patients with chronic kidney disease. Clin Chim Acta 366:243, 2006 11. Nagano M, Fukami K, Yamagishi S, et al: Circulating matrix metalloproteinase-2 is an independent correlate of proteinuria in patients with chronic kidney disease. Am J Nephrol 29:109, 2009

3003 12. Chromek M, Tullus K, Hertting O, et al: Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in acute pyelonephritis and renal scarring. Pediatr Res 53:698, 2003 13. Jiang Z, Sui T, Wang B: Relationships between MMP-2, MMP-9, TIMP-1, TIMP-2 levels and their pathogenesis in patients with lupus nephritis. Rheumatol Int 30:1219, 2010 14. Taranta-Janusz K, Zoch-Zwierz W, Wasilewska A, et al: Serum and urinary concentrations of selected metalloproteinases and their tissue inhibitors in children with vesicoureteral reflux (in polish). Pol Merk Lek 29:88, 2010 15. Eddy AA, Kim H, Lopez-Guisa J, et al: Interstitial fibrosis in mice with overload proteinuria: deficiency of TIMP-1 is not protective. Kidney Int 58:618, 2000 16. Hörstrup JH, Gehrmann M, Schneider B, et al: Elevation of serum and urine levels of TIMP-1 and tenascin in patients with renal disease. Nephrol Dial Transplant 17:1005, 2002 17. Wong W, DeVito J, Nguyen H, et al: Chronic humoral rejection of human kidney allografts is associated with MMP-2 accumulation in podocytes and its release in the urine. Am J Transplant 10:2463, 2010 18. Rodrigo E, López-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 15:2041, 2000