Circulating levels of matrix metalloproteinases MMP-3 and MMP-2 in renal transplant recipients with chronic transplant nephropathy

Emilio Rodrigo, Marcos López-Hoyos1, Rafael Escallada, Gema Fernández-Fresnedo, Juan C. Ruiz, Celestino Piñera, Julio G. Cotorruelo, Jose A. Zubimendi, Angel L. M. de Francisco and Manuel Arias

Nephrology and 1 Immunology Units, University Hospital Marqués de Valdecilla, Insalud, 39008 Santander, Spain



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Chronic transplant nephropathy remains the major cause of graft loss after the first year post transplant, with the exception of death with functioning graft. The histological hallmark of chronic kidney rejection is progressive fibrosis in which extracellular matrix turnover plays an important role. This turnover is regulated by several systems of connective tissue proteases, the matrix metalloproteinases family being one of them. Every metalloproteinase exerts a different function over extracellular matrix proteins and can contribute to the pathogenesis of several diseases, such as rheumatoid arthritis and glomerulonephritis. The role of metalloproteinases in the pathogenesis of chronic transplant nephropathy and in kidney transplantation has not yet been addressed.

Methods. We measured the serum levels of proMMP-1, proMMP-2 and proMMP-3 by ELISA in 40 patients with chronic transplant nephropathy, 30 with acute rejection, 30 with stable graft function for a time equivalent to chronic transplant nephropathy, 30 with stable graft function for a time equivalent to acute rejection, and 30 healthy age-paired blood donors.

Results. Serum proMMP-2 and proMMP-3 were significantly higher in patients with chronic transplant nephropathy than in patients with acute rejection, stable graft function and healthy donors. The most striking finding was the significant positive correlation observed between serum levels of proMMP-3 and serum creatinine, and between circulating levels of proMMP-2 and proteinuria. Serum concentration of proMMP-1 was increased in patients with acute rejection compared with those with stable graft function and healthy donors.

Conclusions. Serum proMMP-2 and proMMP-3 reflect the changes of glomerular and interstitial extracellular matrix in chronic transplant nephropathy, suggesting that they could play a role in the pathogenesis of this condition. Acute rejection is associated with increased levels of proMMP-1, which could be a reflection of the stimulation induced by a number of inflammatory cytokines produced in such a process.

Keywords: matrix metalloproteinase; chronic transplant nephropathy



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
About one-half to three-quarters of graft losses after the first post-transplant year are explained by patient death with a functioning kidney, while chronic transplant nephropathy (CTN) accounts for an important percentage of graft failures in surviving patients [1]. CTN is characterized by mild or moderate tissue infiltration by T cells and macrophages, and variable structural lesions of the vessels, the glomeruli and the tubulointerstitial compartment [1]. Progressive interstitial fibrosis is a characteristic histological feature of CTN of renal allograft [2] and of all progressive renal diseases [3]. Vascular changes of CTN include deposition of extracellular matrix material, and mesangial matrix expansion is one of the abnormalities of transplant glomerulopathy [1]. All these changes include not only the accumulation of normal matrix proteins but qualitative changes in the molecular composition of matrix proteins [3].

Matrix metalloproteinases (MMPs) comprise a family of extracellular matrix degrading enzymes that are believed to play a role in tissue remodelling and repair. Excessive or inappropriate expression of MMPs may contribute to the pathogenesis of many tissue-destructive processes, such as rheumatoid arthritis, tumour progression or cardiovascular diseases [4]. Recent studies have established that MMPs play a role in the development of glomerular sclerosis, a common final result of glomerular diseases as diabetic nephropathy [5] and a number of glomerulonephritis [6]. In addition, several experimental models of interstitial fibrosis have demonstrated changes in the production of MMPs [7,8].

To date 16 MMPs have been identified, which are classified into four groups according to protein sequence and substrate specificity. Collagenases (MMP-1) hydrolyse all three {alpha}-chains of interstitial collagens, making them susceptible to degradation by gelatinases and other proteases. Gelatinases (MMP-2 or gelatinase A) degrade collagen IV and V and denatured forms of collagen I, contributing to basement membrane turnover and interstitial matrix turnover. Stromelysin (MMP-3) degrades different types of collagen and other matrix proteins. There is a fourth type of MMP, anchored to the membrane, which is involved in the activation of inactive forms of MMPs (proMMPs). MMPs activities are regulated by tissue inhibitors of metalloproteinases (TIMPs) [7]. All of the 16 MMPs described exert different functions and play a role in renal matrix remodelling (glomerular or interstitial). While MMP-1 degrades interstitial collagens and is involved in the extracellular matrix (ECM) turnover that occurs in interstitial renal fibrosis [7], the role of MMP-2 and MMP-3 in mesangial expansion has been promoted in recent years [9,10].

Due to the key role of ECM changes in CTN histopathology, we studied whether MMPs could be involved in the pathogenesis of these changes. We determined serum levels of the three above-described MMPs in kidney graft recipients diagnosed with CTN and acute rejection (AR) and correlated them with renal function and proteinuria.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum samples were obtained from four groups. The chronic transplant nephropathy group (CTN) consisted of 40 kidney transplant recipients whose sera was taken simultaneously with the biopsy that diagnosed chronic transplant nephropathy. The acute rejection group (AR) consisted of 30 kidney transplant recipients whose sera was taken simultaneously with the biopsy that diagnosed acute rejection. The stable renal grafts group consisted of 30 patients with stable renal graft for a time equivalent to acute rejection (ASTx) and 30 patients with stable renal graft for a time equivalent to chronic transplant nephropathy (CSTx). The control group consisted of 30 healthy age-paired subjects (HS).

All sera from transplant patients were collected and stored at -70°C until analysis, which was performed at the same time for all samples. Serum levels of MMP-1, MMP-2, and MMP-3 were determined by ELISA, using standard kits provided by The Binding Site (Cambridge, UK). The antibodies used in the kits are raised to the pro-enzyme forms of MMP-1, MMP-2 and MMP-3, due to the impossibility of measuring active enzymes in serum complexed to the non-specific protease inhibitor {alpha}2-macroglobulin. Sera were diluted according to manufacturer instructions. The concentration of these molecules was calculated by reference to the standard curves performed with the corresponding provided molecules. Results were expressed in nanograms per millilitre.

All the transplant patients received the same immunosuppressive therapy with cyclosporin, prednisone and azathioprine. Clinical and laboratory data are presented in Table 1Go.


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Table 1. Clinical features of patients under study

 
The statistical analysis performed included correlation and ANOVA, with the Newman–Keuls test for multiple comparisons among groups.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to assess whether circulating levels of proMMPs could be modified during CTN, the concentration of these molecules was measured in 40 patients with diagnosis of CTN, and was compared with levels found in 30 patients with CSTx, 30 patients with AR, 30 patients with ASTx, and 30 HS.

High blood levels of proMMP-2 and proMMP-3 during chronic transplant nephropathy
Serum MMP-2 levels, measured as total amount of the precursor form of MMP-2 ng/ml, were significantly higher in patients with CTN (272.3 ng/ml±89.3) than in subjects with CSTx (203.2 ng/ml±87.9, P<0.05) (Fig. 1Go). Patients with AR showed comparable levels of proMMP-2 (201.8 ng/ml±96.6, P<0.05 with CTN) to those of CSTx and ASTx (193.8 ng/ml±88.7, P<0.05 with CTN). Likewise, serum proMMP-3 concentration was significantly higher in patients with CTN (331.2 ng/ml±203.7) than in subjects with CSTx (171.6 ng/ml±97.7, P<0.05) (Fig. 1Go). However, different from proMMP-2, patients with AR had increased serum levels of proMMP-3 (272.8 ng/ml±167.7) in comparison with ASTx (193.2 ng/ml±143.4, not significant), although those levels were significantly lower than in CTN (P<0.05).



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Fig. 1. Serum concentrations of proMMP-1, proMMP-2 and proMMP-3 in the different groups: CTN, AR, CSTx and ASTx groups, and HS. Results are expressed as mean±standard deviation. Statistical differences between groups are indicated in the figure by lines.

 

Serum levels of proMMP-1 were increased during acute graft rejection
As shown in Figure 1Go, there were significant differences in serum levels of proMMP-1 obtained during AR (105.4±104.5) as compared with the samples obtained in ASTx (51.4±48.9, P<0.05) and HS (42.1±18.9, P<0.05). However, there was no correlation between proMMP-1 levels and renal function (Table 2Go), measured by serum creatinine. Although MMPs have been demonstrated to play some role in vascular diseases [4], we could not correlate serum concentration of pro-MMPs with the vascular lesion component of the Banff classification due to the low number of cases. In CTN, serum proMMP-1 levels increased slightly (75.8±68.7) compared with CSTx (41.0±45.8) and HS, but did not reach the levels or significance of AR.


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Table 2. Relation of serum creatinine and proteinuria with serum concentration of proMMPsa

 

Deterioration of renal function increased the serum levels of proMMP-3
As we found increased serum levels of proMMP-2 and proMMP-3 in CTN, which is characterized by a deterioration of renal function, we analysed whether the serum concentration of those MMPs had any relation with renal function. Our results indicated a positive correlation of serum proMMP-3 concentration with serum creatinine expressed in mg/dl (r=0.44, P=0.005) in CTN patients (Fig. 2AGo and Table 2Go). However, there was no correlation between proMMP-2 (and proMMP-1) levels and serum creatinine in the CTN group. Serum proMMP-3 was also correlated with renal function in the other three groups of transplant patients (Table 2Go).



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Fig. 2. Correlation between serum levels of proMMP-3 and serum creatinine (A), and between proMMP-2 and proteinuria (B) in the patients with chronic transplant nephropathy. There was a positive correlation in both cases (P<0.01). Correlation coefficients are indicated in the upper right corner of each figure.

 

Correlation between serum levels of proMMP-2 and proteinuria
Proteinuria of varying degree is the outstanding clinical feature associated with transplant glomerulopathy. We analysed whether MMPs had any correlation with proteinuria, in order to attribute a pathogenic role of MMP in the development of glomerulopathy during CTN. The serum proMMP-2 levels had a direct correlation with proteinuria in patients with CTN (r=0.48, P<0.01) (Fig. 2BGo and Table 2Go), whereas no correlation was found between proteinuria and proMMP-1 or proMMP-3 levels in patients with CTN. No correlation was observed between proMMP-2 concentration and proteinuria in the other groups (Table 2Go), which is explained by the low proteinuria observed in those patients with lesser glomerular damage.



   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MMPs play a role in glomerular sclerosis [5,6] and interstitial kidney fibrosis [8]. Although ECM changes have major importance in CTN, there are very few studies that measure expression of MMPs mRNA in situ in experimental models of chronic rejection (Inkinen et al., ESOT 99, Oslo) or cyclosporin nephrotoxicity [7]. Moreover, there is no previous work evaluating serum levels of MMPs in human kidney transplant recipients.

Currently, available information regarding synthesis and release of MMPs in the kidney is scant and contradictory. Since MMPs are rapidly released from cells after biosynthesis and then act extracellularly in the matrix, they may easily diffuse into the blood and serve as indicators of collagen turnover, yet at the same time MMPs may be difficult to detect in situ. Thus, levels of several circulating MMPs can change in chronic hepatitis C [11], rheumatoid arthritis [12], hypertension [13], and renal diseases as lupus nephritis [14] or glomerulonephritis [6]. Due to the fast release to the extracellular medium of these enzymes this study analyses serum concentration of the pro-enzyme forms of several MMPs in kidney transplantation.

Our results showed increased serum proMMP-2 concentration during CTN that is characterized by a progressive glomerulosclerosis. During inflammatory states mesangial cells acquire a characteristic spectrum of features that may be defined as ‘the inflammatory phenotype’, coupled with high-level synthesis of MMP-2 that directly mediates the evolution to the sclerotic state [15]. Furthermore, in several experimental models of nephritis in rodents, the increased expression of ECM components correlates with MMP-2 production, and even mesangial proliferation could be suppressed by inhibiting MMP-2 [1618]. In an experimental model of chronic rejection in rat renal transplantation, Inkinen et al. have shown an enhanced activity and gene expression of MMP-2 only in chronic rejection grafts (ESOT 99, Oslo). The increase of circulating proMMP-2 found in CTN supports these results. Both mesangial cells and leukocytes that participate in CTN could increase MMP-2 production, contributing to ECM turnover. The good correlation of proMMP-2 with proteinuria, a basic hallmark of CTN, supports the importance of the role of MMP-2 in chronic glomerular damage.

Circulating levels of MMP-3 are increased in patients with IgA nephropathy and lupus nephritis, which are types of mesangial proliferative glomerulonephritis [6] and even they are closely associated with clinical features of lupus nephritis like circulating immune complexes, decreased C4, or decreased creatinine clearance [14]. Similarly, in transplant glomerulopathy mesangial matrix is expanded. Thus, the significantly higher concentration of proMMP-3 in CTN suggests that it can play a role in the pathogenesis of this condition. However, the correlation between serum creatinine and proMMP-3 in all transplant groups suggests that stromelysin-1 (molecular weight of 55 kDa) could be, at least in part, modified by renal filtration. As CTN is associated with the worsening of renal function, we must be very cautious when analysing these results. Though previous studies conclude that MMP-3 is capable of affecting the glomerular filtration barrier directly and that it may play a role in causing proteinuria in glomerular disease [19], we did not find correlation between proteinuria and MMP-3.

Finally, MMP-1 plays a role in diseases with extracellular matrix changes like rheumatoid arthritis, where MMP-1 is elevated in the synovial joint fluid [12]. Our results showed a significant increase in circulating levels of proMMP-1 during acute rejection in patients with stable renal grafts for the same time of evolution. The production of interstitial collagenase from fibroblasts and endothelial cells is markedly enhanced by pro-inflammatory cytokines [20], for example interleukin-1 and tumour necrosis factor {alpha} that are overexpressed during AR [21]. Thus, the inflammatory environment of graft rejection would increase MMP-1 levels and justify the ECM changes occurring in this process. However, it may be possible that the role of MMP-1 during AR was to repair ECM damage caused by inflammatory infiltrate [4] as in wound healing. Furthermore, increased proMMP-1 levels in AR may be attributed to immunosuppression or surgical injury. However, the ASTx group received similar drugs and had equivalent follow-up times.

In summary, our data suggest an important role for MMPs, mainly MMP-2, in the fibrotic changes of CTN. The accumulation of ECM proteins in CTN may be explained by a dysregulation of MMP-2 and MMP-3, but the elevation of both MMPs may be an attempt to increase the turnover of ECM components. In addition, interstitial collagenase seems to be the main MMP involved in acute graft rejection. We must be very cautious when interpreting these data since increased secretion of MMPs can be stimulated unspecifically by different cytokines. Moreover, increased concentration may be a reflection of a decrease in the elimination of these molecules. TIMPs have not been addressed and they may help elucidate the importance of our findings. Thus, further studies that assess the exact role of MMPs in renal transplantation are needed, not only in order to understand the pathogenic mechanism of renal transplant fibrosis, but also to improve current therapeutic strategies. Recent developments of synthetic inhibitors of MMPs [22] and the influence of different immunosuppressive therapies in MMP production through cytokines, like IL1-ß, TNF-{alpha} or TGF-ß, may change the progression of fibrotic events in kidney transplantation and improve graft survival.



   Notes
 
Correspondence and offprint requests to: Dr Manuel Arias, Servicio de Nefrologia, Hospital Universitario Marqués de Valdecilla, E-39008 Santander, Spain. Back



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 7.12.99
Revision received 29. 5.00.



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