Clinical immunology
Serum levels of MMP-9 in children and young adults with chronic kidney disease treated conservatively and undergoing hemodialysis

Introduction

End-stage renal disease (ESRD) is a major health problem worldwide. In this disorder depressed immune responses have been observed frequently but some immunological factors are also activated and overproduced. ESRD is associated with chronic inflammatory reaction. However, the mechanisms involved in causing this injury still remain unknown.
Chronic inflammation and complications occurring in the end-stage renal disease patients may lead to significant morbidity and mortality. Many studies documented various complications observed in uremic and hemodialyzed adults. But in the past years much more studies involved also pediatric patients. The important complications are e.g. infections, osteodystrophy, malnutrition, vascular and heart failure, hemorrhagic stroke, cerebrovascular accidents [1-4]. These disorders have a special significance when are concerned in the period of development child organism. Chronic inflammation in ESRD patients may be partly due to the uremic intoxication and also is increased by hemodialysis treatment with frequent contact between patients’ blood and dialyzer membranes. The types of artificial membranes play a significant role as well. Moreover, the types of fistulae and the catheter-related bloodstream infection are very important for the health status of hemodialyzed patients.
Many cells and mediators are involved in the acute and chronic inflammatory process. It may be associated with the overproduction of proinflammatory cytokines, oxygen radicals and enzymes [5, 6]. One of the most important proteolytic enzymes is zinc-dependent endopeptidase - matrix metalloproteinase-9 (MMP-9) (gelatinaseB/92kDa type IV collagenase). It is secreted as inactive proenzyme mainly from neutrophils, macrophages, eosinophils, T lymphocytes, osteoclast and endothelial cells in response to various stimuli, and can be activated extracellulary by several factors.
MMP-9 plays an important role in both normal and pathological tissue remodeling, angiogenesis, tissue repair, apoptosis [7]. It is a factor of cells migration across basement membrane by the ability to degrade type IV and V collagen, type I gelatin, elastin fibres [8]. This enzyme may also influence on cytokine release and is implicated in chronic inflammatory conditions that occurred in some pathological disorders in experimental and clinical studies, e.g. arthritis, cardiovascular disease, nephritis, chronic lung diseases, systemic sclerosis, cancer and other [9-13]. Various studies have shown that elevated levels and mRNA expression of MMP-9 in chronic kidney disease (CKD) and in dialysis patients may contribute to the pathogenesis of some complications by enhancing inflammation [14].
In the available literature we have not found the study on the role of MMP-9 in children with chronic kidney disease. The aim of this study was to assess whether serum concentrations of MMP-9 differed between patients suffering from chronic kidney disease treated conservatively and undergoing hemodialysis therapy. In order to evaluate the influence of hemodialysis on levels of MMP-9, we studied serum before and after the single hemodialysis session.

Material and methods


Subjects

The study was carried out on the group of 34 patients with chronic kidney disease (table 1). Among them 21 were on regular hemodialysis treatment (group A), aged 12 to
48 years, from both sexes and 13 treated conservatively, aged 4 to 17.5 years, from both sexes (group B). None of these patients had acute diseases in the course of the investigation. Patients of both groups were admitted (and treated) to the Department of Pediatric Nephrology in Wroclaw, Poland.
The patients were hemodialyzed from 0.5-11 years, 2 or 3 times weekly, with cuprophan (n=16) or polysulfone (n=5) dialyzers membrane and a routine used heparin as anticoagulants and dialysate with bicarbonate and low calcium content. The time of a single session of hemodialysis (HD) was 4-6 hours. The causes of CKD in group A were as follows: chronic glomerular nephritis (n= 9), hydronephrosis (n=4), neurogenic bladder (n=1), polycystic kidney disease (n=1), hypoplasia renum (n=1), systemic lupus erythomatosus (n=3), rheumatoid arthritis (n=2). The causes of CKD
in group B were as follows: chronic glomerular nephritis
(n=4), hydronephrosis (n=1), polycystic kidney disease (n=4), hypoplasia renum (n=1), chronic interstitial nephritis (n=1), urethrae valves (n=2).
Control group (C) consisted of 20 healthy subjects, both sexes, without chronic diseases and without treatment in anamnesis.
Blood samples were taken from the peripheral vein in all subjects. Venous blood was allowed to clot at room temperature for 60 min before centrifugation to obtain serum. Serum samples were stored in aliquotes at –80°C until assayed. In group A the analysis was performed before (A1) and after (A2) a single of the HD session.
MMP-9 assay
The total serum concentrations (free proMMP-9 and free MMP-9) of MMP-9 were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc, Minneapolis, MN, USA). The detection limits of the MMP-9 concentration was 0.156 ng/mL. The analyses were performed according to the manufacturers’ recommendations.

Statistical analysis

Statistical analysis was preceded by using the Walda-Wolfowitza and Manna-Whitneya test for independent and Wilcoxon test for dependent variables. Correlations between parameters were tested with the Spearman test. The level of statistical significance was assumed to be p<0.05.
Statistical analysis was performed using the Statistica PL computer program.

Results


Circulating MMP-9 values

Serum MMP-9 levels are shown in table 2. In the group of patients before HD (A1) serum levels of MMP-9 ranged from 31.13 to 673.3 ng/mL with a median of 189.69 ng/mL and a mean of 31.13±673.3 ng/mL, after HD (A2) ranged from 35.42 to 313 ng/mL with a median of 142.63 ng/mL and a mean of 35.42±313.49 ng/mL, and in not undergoing dialysis group (B) ranged from 30.4 to 325.8 ng/mL with
a median of 173.6 ng/mL and a mean of 239.65±186.39 ng/mL.
There was no difference between serum MMP-9 concentration in the patients treated conservatively and undergoing dialysis and those found in the controls which ranged from 73 to 322 ng/mL with a median of 194.6 ng/mL and a mean of 188.63±73.28 ng/mL.
Although MMP-9 levels in patients at the end of HD were not significantly lower than those at the start of HD, nevertheless in 11/21 (52%) individual cases concentrations of MMP-9 were lower after HD than before HD (figure 1). We found that 3/21(14%) sera from A1, 3/21 (14%) from A2 group and 6/13 (46%) sera of patients from group B had increased levels above normal range. No significant differences were identified in serum MMP-9 concentrations between group A1, A2 and B.

Discussion

It is difficult to understand the inflammatory process occurring in the end-stage renal disease because of variety cells and metabolites playing a pivotal role in the connective tissue destruction.
MMP-9 is one of the major proteolytic enzymes capable of degrading the extracellular matrix (ECM) and basement membrane compounds increasing the migration of pro- and anti-inflammatory cells and has immunomodulatory properties. In this reason MMP-9 is implicated in tissue remodeling processes and takes part in the development of the inflammatory state. To the tissue damage may contribute: the imbalance between the MMP-9 and endogenous tissue inhibitors of metalloproteinase (TIMP-1), regulation its activity by transcription of the gene some cytokines (e.g.
IL-8) or several serine proteases (trypsin, cathepsin G, elastase), and also other MMPs [10, 15].
A recent study showed that MMP-9 plays multifunctional significant roles in pathogenesis of some disorders occurring in uremic and hemodialyzed patients.
In the present paper, we demonstrated that the serum concentration of MMP-9 did not statistically differ in children and young adults suffering from uremia undergoing dialysis and treated conservatively compared to healthy donors. But we also have shown that in individual cases the level of MMP-9 was much more above normal range in both groups especially in group treated conservatively. In our opinion the rise in MMP-9 production could be due to injury processes and may contribute to the development of some complications. Concentration changes occurred in the course of HD treatment may be of prognostic importance. Thus, in these cases children should be covered by a special prevention and even more intense treatment. Our data also showed that in 52% of sera samples the decreased concentrations of MMP-9 were the result of HD treatment. Chou et al. in similar studies demonstrated that hemodialysis process tends to decrease plasma concentration of MMP-9 (from 187±148 ng/mL before to 124±72 ng/mL after HD) but TIMP-1 was not affected by HD [16].
The lower levels of MMP-9 may contribute to an increased extracellular matrix deposition and to irreversible fibrosis. Tissue remodeling by initiating the degradation of the ECM by MMP-9 can make the migration of host defense cells to inflammatory foci easier. These activated neutrophils, macrophages, lymphocytes, fibroblast as well as their products can also cause and support an inflammatory reaction, and additionally may be a source of the MMP-9 secreted into the circulation, fueling the vicious circle of inflammation.
All forms of ESRD are associated with chronic inflammatory reaction resulting in interstitial fibrosis. Various immune effectors cells and mediators take part in these processes. The inflammatory cascade is initiated in several situations – infection, trauma, surgery. The frequent contact of blood and dialytic membrane in patients requiring HD additionally results in a wide range of abnormalities of the immune system.
Neutrophils and other activated inflammatory cells produce a wide array of proteases including matrix metalloproteinase, elastase, cathepsin G, reactive oxygen species, cytokines (e.g. IL-8, IL-1, TNF-αlpha), adhesion molecules. These products are not only involved in defense against a broad spectrum of microorganisms but upon release may also cause tissue injury and may also be involved in the subsequent repair processes.
Cells activation occurs during hemodialysis, and its intensity depends on the type of dialyzer and whether it is new or reused. Ebihara et al. reported that in HD patients MMP-9 mRNA levels did not differ among the types of membranes [14].
Patients with chronic renal failure have a disturbed defense, which causes infections (bacteremia, metastatic infection, sepsis) often leading to death of the patients. The role of MMP-9 in the innate immune response to serious infectious is also discussed in the literature [17]. Data in animals’ models suggest that MMP-9 may be an essential component of an effective host response to E. coli peritonitis. Mice with MMP-9 gene deficient neutrophils showed
a reduced phagocytosis of E. coli [18]. On the other hand, MMP-9 may serve as sensitive and early markers for cell activation in acute inflammatory responses. The significantly elevated levels of MMP-9 in severe sepsis were found [19].
Matrix metalloproteinases play an important role in the degradation of extracellular matrix and basement membranes by destroying the elastic lamina. It is also well known that oxidative stress has been identified as one of the most important causes of vascular injury also in renal failure. On the other hand, Tepel et al. described that regular hemodialysis sessions using biocompatible membranes have no effect on the elevated intracellular reactive oxygen species in patients with end-stage renal failure [20].
It is well known that patients with CKD are at increased risk for cardiovascular morbidity and mortality. The data indicate that in HD patients cardiovascular diseases are associated with oxidative stress and with an imbalance in antioxidant status. Moreover, reactive oxygen species production was significantly positive correlated with
MMP-9/TIMP-1 system. In contrast, the levels of MMP-9 in HD patients (investigated in pre-dialysis period) with and without cardiovascular disease were similar to the controls [21]. The results published by Nakamura et al. indicate that when low density lipoprotein (LDH) was removed from blood of the hemodialysis patients with arteriosclerosis obliterans, in the process of apheresis, plasma levels of MMP-9 and serum levels of inhibitor TIMP-1 decreased significantly compared to those before LDH apheresis [22]. The data demonstrated that inflammation and increased oxidant stress together with early cardiovascular damage were found in non dialysis, peritoneal dialysis and on HD children with chronic renal disease [23, 24]. Also in children with ESRD treated conservatively lipid metabolism disturbances were found [25].
Vascular inflammation of arteriovenous fistulas is very important for the later complication (e.g. the thrombotic process). Chang et al. basing on the obtained results concluded that elevated expression of MMP-9 by macrophages at luminal edge may cause disruption of the anticoagulant endothelial barrier and may contribute to the luminal thrombosis of arteriovenous fistulas [26]. Elliot at al. demonstrated that treatment with pentosan polysulfate in hemodialysis patients inhibited smooth muscle cells proliferation and reduced the accumulation of type I and IV collagens in stenosis vascular, and they suggested that this effect was associated with increase of MMP-9 in the tissue [27].
Malnutrition, systemic inflammation, and atherosclerosis, known as MIA syndrome [28, 29], is associated with high cardiovascular mortality rate in dialysis patients [30].
It is very essential to prevent of renal osteodystrophy for the development of the young organism in children [31]. Also MMP-9 may play a pathogenic role in cartilage degradation and in the regulation of angiogenesis near the epiphyseal growth plate. The data indicate that growth plates from MMP-9-null mice in culture show a delayed release of an angiogenic activator [32].
Early diagnosis is very important for health condition of children and young adults with ESRD and long-term dialysis in order to prevent from the occurrence of any complications. For this reason the use of new biochemical indices of inflammatory stage should be crucial.
Better understanding of the biology of MMP-9 in modulation of inflammatory processes in children with ESRD is needed for more effective therapies. Based on knowledge of the MMP-9 actions the objective of many investigations is the search of the MMP-9 antagonists as new therapeutic targets for many disorders [33]. The use of the new treatment preventing from early and future complications will improve the quality of life and will lower the cost of treatment.

Conclusions

Obtained results indicate that elevated levels of MMP-9 only in small part of patients from both investigated group were found. We conclude that concentration changes occurring in the course of HD treatment may be of prognostic importance. In these cases increased levels of MMP-9 may be serving as marker of risk for complication. A better understanding of the inflammatory reactions involved in end-stage renal disease will enable the development of new treatment strategies. Usefulness of determination of serum MMP9 in pediatric patients with end-stage renal disease on hemodialysis or treated conservatively should be verified by further study.


Acknowledgements

Our study was reviewed and approved by the Research Ethics Committee at the Medical University of Wroclaw. The study was carried out and financed within the framework of the University Research Project no. 498.
The authors thank MSc Danuta Karabiniewicz from Laboratory of Department of Pediatric Nephrology for help with technical preparation of blood samples for this study.

References

1. Groothoff JW, Gruppen MP, Offringa M, et al. (2002): Mortality and causes of death of end-stage renal disease in children: a Dutch cohort study. Kidney Int 61: 621-629.
2. Pecoits-Filho R, Sylvestre LC, Stenvinkel P (2005): Chronic kidney disease and inflammation in pediatric patients: from bench to playground. Pediatr Nephrol 20: 714-720.
3. Onder MA, Chandar J, Coakley S, et al. (2006): Predictors and outcome of catheter-related bacteremia in children on chronic hemodialysis. Pediatr Nephrol 21: 1452-1458.
4. Zwolińska D, Grzeszczak W, Szczepańska M, et al. (2006): Lipid peroxidation and antioxidant enzymes in children on maintenance dialysis. Pediatr Nephrol 21: 705-710.
5. Kimmel PL, Phillips TM, Simmens SJ, et al. (1998): Immunologic function and survival in hemodialysis patients. Kidney Int 54: 236-244.
6. Bayes B, Pastor MC, Bonal J, et al. (2006): Oxidative stress, inflammation and cardiovascular mortality in haemodialysis - role of seniority and intravenous ferrotherapy: analysis at 4 years of follow-up. Nephrol Dial Transplant 21: 984-990.
7. Massova I, Kotra LP, Fridman R, Mobashery S (1998): Matrix metalloproteinases: structures, evolution, and diversification. FASEB J 12: 1075-1095.
8. Murphy G, Cockett MI, Ward RV, Docherty AJ (1991): Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP). Biochem J 277 (Pt 1): 277-279.
9. Mautino G, Oliver N, Chanez P, et al. (1997): Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics. Am J Respir Cell Mol Biol 17: 583-591.
10. Nagase H, Woessner JF Jr (1999): Matrix metalloproteinases. J Biol Chem 274: 21491-21494.
11. Kim WU, Min SY, Cho ML, et al. (2005): Elevated matrix metalloproteinase-9 in patients with systemic sclerosis. Arthritis Res Ther 7: R71-R79.
12. Polańska B, Niemczuk M, Jankowski A (2003): Neutrophil elastase complexed with inhibitor alfa1-proteinase and matrix metalloproteinase-9 in children with recurrent respiratory tract diseases. Przeg Ped 33: 237-241.
13. Benjamin IJ (2001): Matrix metalloproteinases: from biology to therapeutic strategies in cardiovascular disease. J Investig Med 49: 381-397.
14. Ebihara I, Nakamura T, Tomino Y, et al. (1998): Metalloproteinase-9 mRNA expression in monocytes from with chronic renal failure. Am J Nephrol 18: 305-310.
15. Goetzl EJ, Banda MJ, Leppert D (1996): Matrix metalloproteinases in immunity. J Immunol 1: 1-4.
16. Chou FP, Chu SC, Cheng MC, et al. (2002): Effect of hemodialysis on the plasma level of type IV collagenases and their inhibitors. Clin Biochem 35: 383-388.
17. Albert J, Radomski A, Soop A, et al. (2003): Differential release of matrix metalloproteinase-9 and nitric oxide following infusion of endotoxin to human volunteers. Acta Anaesthesiol Scand 47: 407-410.
18. Renckens R, Roelofs JJ, Florquin S, et al. (2006): Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis. J Immunol 176: 3735-3741.
19. Hoffmann U, Bertsch T, Dvortsak E, et al. (2006): Matrix-metalloproteinases and their inhibitors are elevated in severe sepsis: prognostic value of TIMP-1 in severe sepsis. Scand
J Infect Dis 38: 867-872.
20. Tepel M, Echelmeyer M, Orie NN, Zidek W (2000): Increased intracellular reactive oxygen species in patients with end-stage chronic renal failure: effect of hemodialysis. Kidney Int 58: 867-872.
21. Pawlak K, Pawlak D, Myśliwiec M (2005): Circulating beta-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients – role of oxidative stress. Cytokine 31: 18-24.
22. Nakamura T, Matsuda T, Suzuki Y, et al. (2003): Effects of low-density lipoprotein apheresis on plasma matrix metalloproteinase-9 and serum tissue inhibitor of metalloproteinase-1 levels in diabetic hemodialysis patients with arteriosclerosis obliterans. ASAIO J 49: 430-434.
23. Ece A, Gurkan F, Kervancioglu M, et al. (2006): Oxidative stress, inflammation and early cardiovascular damage in children with chronic renal failure. Pediatr Nephrol 21: 545-552.
24. Zwolińska D, Makulska I, Berny U, et al. (2000): The condition of circulatory system in children and adolescents with end-stage renal failure treated continuous ambulatory peritoneal dialysis and hemodialysis. Pol Merk Lekarski 8: 450-453.
25. Puziewicz-Zmonarska A, Zwolińska D, Zmonarski SC, Makulska I (2004): Lipid metabolism disturbances in children with chronic renal failure treated conservatively. Przeg Ped
34: 46-51.
26. Chang CJ, Ko YS, Ko PJ, et al. (2005): Thrombosed arteriovenous fistula for hemodialysis access is characterized by a marked inflammatory activity. Kidney Int 68: 1312-1319.
27. Elliot SJ, Striker LJ, Connor E, et al. (2000): Pentosan polysulfate decreases proliferation and extracellular matrix deposition by vascular smooth muscle cells isolated from failed hemodialysis access grafts. Clin Nephrol 54: 121-127.
28. Pawlaczyk K, Oko A, Lindholm B, Czekalski S (2003): Malnutrition – inflammation – atherosclerosis (MIA syndrome) in patients with renal failure. Pol Merk Lekarski 15: 334-341.
29. Yao Q, Axelsson J, Heimburger O, et al. (2004): Systemic inflammation in dialysis patients with end-stage renal disease: causes and consequences. Minerva Urol Nefrol 56: 237-248.
30. Yao Q, Pecoits-Filho R, Lindholm B, Stenvinkel P (2004): Traditional and non-traditional risk factors as contributors to atherosclerotic cardiovascular disease in end-stage renal disease. Scand J Urol Nephrol 38: 405-416.
31. Klaus G, Watson A, Edefonti A, et al. (2006): Prevention and treatment of renal osteodystrophy in children on chronic renal failure: European guidelines. Pediatr Nephrol 21: 151-159.
32. Vu TH, Shipley JM, Bergers G, et al. (1998): MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93: 411-422.
33. Rudek MA, Venitz J, Figg WD (2002): Matrix metalloproteinase inhibitors: do they have a place in anticancer therapy? Pharmacotherapy 22: 705-720.