EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Acute and chronic allograft rejection have been continuously an important obstacle in the follow-up of renal transplant recipients. During clinical management, several factors acting simultaneously result in acute rejection and chronic allograft nephropathy. Matrix metalloproteinases and tissue inhibitors of metalloproteinases are responsible for the organization of the extracellular matrix and play roles in cell proliferation and cellular invasion. Changes in matrix metalloproteinase expression levels have been reported to be associated with renal allograft rejection and interstitial fibrosis. In this study, we aimed to investigate functional polymorphisms of MMP2, MMP9, and TIMP2 genes in pediatric renal transplant recipients.

Materials and Methods: Our study included 68 kidney transplant recipients and 58 control patients. The kidney transplant recipient group was further divided into 2 subgroups: no graft rejection (n = 47) and graft rejection (n = 21). MMP2 -735C>T (rs2285053), MMP2 -1306C>T (rs243865), MMP2 -1575G>A (rs243866), MMP9 c.-1562C>T (rs3918242), TIMP2 -418G>C (rs8179090), and TIMP2 303C>T (rs2277698) polymorphisms were analyzed with the use of polymerase chain reaction and restriction fragment-length polymorphism methods. Allele prevalence was compared with reference values of the control group, and Hardy-Weinberg equilibrium was tested.

Results: Mean ages were 16.7 ± 3.9 years for the study group and 14.8 ± 5.6 years for the control group. The mean follow-up time after transplant was 37.7 ± 7.9 months. We compared allele frequencies in the 2 groups and calculated a statistically significant difference in rs2285053, rs243865, rs243866, rs3918242, rs8179090, and rs2277698 polymorphism frequencies between the transplant recipients and control patients. When the transplant recipient group was compared in itself with regard to allograft rejection, all investigated polymorphisms except TIMP2 -418G>C (rs8179090) revealed a statistically significant difference between those with and without rejection (P < .05).

Conclusions: Matrix metalloproteinases and their tissue inhibitors could be important predictive biological markers for the follow-up of kidney transplant recipients.

Key words : MMP2, MMP9, Renal transplantation, TIMP2

Renal transplantation is the most appropriate treatment for pediatric patients with end-stage renal disease. In pediatric patients with renal insufficiency, problems such as hospitalizations, psychosocial effects of dialysis, immature immune system, and difficult donor matching according to body size are experienced.¹ However, many studies have shown that renal transplantation in children is advantageous over dialysis treatment in factors such as overall survival, psychomotor development, social adjustment, and overall quality of life.^2,3

In the first year after transplantation, renal allograft survival depends on several clinical variables (donor and recipient characteristics, acute rejection, and blood pressure) and histologic features (interstitial fibrosis and tuberous atrophy, persistent inflammation, and transplant glomerulopathy).⁴ Acute and chronic allograft rejection episodes remain as critical impediments to successful renal transplantation.⁵ The most common cause of allograft loss in the kidney is chronic allograft injury, which is usually associated with proteinuria as a clinical feature.⁶ The emergence of proteinuria after kidney transplantation is associated with worse graft function and patient survival.⁷ Chronic allograft nephropathy remains one of the main causes of late graft loss due to multifactorial development in renal transplantation.⁸

Chronic allograft nephropathy is characterized by excessive extracellular matrix accumulation.⁸ Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are involved in the regulation of extracellular matrix homeostasis.⁹ Changes in urinary concentrations of MMP9, MMP2, and TIMP2 in patients with chronic renal failure and renal transplant have previously been associated with clinical findings of patients.¹⁰

Matrix metalloproteinases are a broad family of zinc-dependent proteolytic enzymes that participate in a variety of biological or pathological processes, such as invasion through cell proliferation, migration, and remodeling of macromolecules of extracellular matrix.¹¹ The altered expression levels in MMPs are related to renal allograft rejection pathogenesis and interstitial fibrosis process.¹² It is known that gelatinase MMP2 and MMP9 are the most important members of the MMP family, responsible for the development and progression of various renal diseases, together with TIMPs.¹³ Matrix metalloproteinases 2 and 9 are among these substrates’ gelatinases with collagen IV, fibronectin, and laminin.⁸ Matrix metalloproteinases 2 and 9 in the gelatinase subfamily of MMPs have been extensively studied in renal transplant models for acute and chronic allograft rejection.⁵ Although MMP2 (gelatinase A) predominantly degrades fibronectin and laminin, MMP9 (gelatinase B) degrades collagen types IV and V.¹⁴

Recent evidence has indicated that natural sequence variations in promoters and coding regions, particularly those involving single nucleotide polymorphisms (SNPs) in nonsynonymous SNPs of MMP genes, may cause differences in expression of MMPs between individuals.¹⁵ Also, increased expression of MMP levels was reported to be associated with heart, lung, and kidney allograft rejection.⁵ An SNP in the MMP2 gene may affect its expression or activity as well as 2 SNPs in the promoter region of the gene that affect its expression (-1306C>T, rs243865; and -735C>T, rs2285053).¹⁶ Renal MMP9 has been shown to increase during experimental renal ischemia.¹⁷ Increased MMP9 expression affects the extracellular matrix and enhances neutrophilia during renal reperfusion injury.¹⁸ Increased expression of MMP9 in acute rejection in renal transplantation has been reported, but there are still conflicting results in chronic allograft nephropathy.¹⁹

Changes in urinary concentrations of MMP9, MMP2, and TIMP2 in patients with chronic renal failure and renal transplantation have previously been associated with clinical findings of patients.

In the current study, we aimed to investigate functional polymorphisms of MMP2, MMP9, and TIMP2 genes in pediatric renal transplant recipients.

Materials and Methods

Study population
This study was evaluated by the local ethics committee, and all experimental procedures were performed after approval.

The study was performed as a retrospective, single-center, cross-sectional study on pediatric renal transplant patients. Our study included 68 kidney transplant recipients and 58 controls. Patients who had sufficient DNA samples and signed informed consent were enrolled in this study.

Genotyping
The MMP2 c.-735C>T (rs2285053), MMP2 c.-1306C>T (rs243865), MMP2 c.-1575G>A (rs243866), MMP9 c.-1562C>T (rs3918242), TIMP2 c.-418G>C (rs8179090), and TIMP2 c.303C>T (rs2277698) polymorphisms were analyzed by using polymerase chain reaction and restriction fragment-length polymorphism methods. The primer sequences and product lengths of the polymorphisms are shown in Table 1. Polymerase chain reaction conditions were 15 minutes at 95 °C for initial denaturation and also for activation of Hot-start Taq DNA polymerase, followed by 35 cycles of 1 minute at 94 °C, 1 minute at the annealing temperatures, and 1 minute at 72 °C. Annealing temperatures for the MMP2 c.-735C>T (rs2285053), MMP2 c.-1306C>T (rs243865), MMP2 c.-1575G>A (rs243866), MMP9 c.-1562C>T (rs3918242), TIMP2 c.-418G>C (rs8179090), and TIMP2 c.303C>T (rs2277698) were 65 °C, 53 °C, 66 °C, 62 °C, 60 °C, and 58 °C, respectively. The polymerase chain reaction products were digested with appropriate restriction endonuclease enzymes, which are listed in Table 1. Following restriction enzyme digestion, samples were run on 3% agarose gel electrophoresis, and genotypes were determined. Demonstrative agarose gel electrophoresis images are given in Figures 1 and 2.

Statistical analyses
Allele frequencies were compared with the frequencies in the control group, and Hardy-Weinberg equilibrium was tested. We compared the allele frequencies in the 2 groups by chi-square analysis. All statistical analyses and tests were performed with the SPSS statistical package (SPSS, version 17.0; Chicago, IL, USA). P < .05 was regarded as statistically significant.

Our study included 68 kidney transplant recipients and 58 control patients. The kidney transplant recipient group was also divided into 2 subgroups: those without rejection (n = 47) and those with rejection (n = 21). The mean ages were 16.7 ± 3.9 years in the transplant recipient group and 14.8 ± 5.6 years in the control group. The mean follow-up time after transplant was 37.7 ± 7.9 months. We compared the allele frequencies in the 2 groups and calculated a statistically significant difference in rs2285053, rs243865, rs243866, rs3918242, rs8179090, and rs2277698 polymorphism frequencies between the transplant recipient and control groups (Table 2). When the transplant recipient group was compared in itself with regard to allograft rejection, all investigated polymorphisms except TIMP2 -418G>C (rs8179090) revealed a statistically significant difference between those with and without rejection (P < .05) (Table 3).²

Matrix metalloproteinases and TIMPs are involved in the regulation of extracellular matrix homeostasis.⁹ Matrix metalloproteinases, a broad family of zinc-dependent proteolytic enzymes, are involved in various biological or pathological processes such as cell proliferation, migration, and remodeling of extracellular matrix macromolecules in the invasion.⁸ It is reported that gelatinases MMP2 and MMP9, the most important members of the MMP family, are responsible for the development and progression of various renal diseases, together with TIMPs.^8,9 Matrix metalloproteinases 2 and 9 have been extensively studied in renal transplant models for acute and chronic allograft rejection.^5,19

In this study, we analyzed the functional polymorphisms that are important for the regulation of MMP2, MMP9, and TIMP2 gene expression. In our cohort, functional polymorphisms of MMP2, MMP9, and TIMP2 genes revealed different allele frequencies in pediatric renal transplant patients and the control group as well as in allograft rejection and nonrejection patients in the study group. Previously, expression profiles of these genes and proteins have been reported in transplant recipients. To our knowledge, this is the first study investigating functional MMP2, MMP9, and TIMP2 polymorphisms in pediatric kidney transplant recipients. Matrix metalloproteinases and their tissue inhibitors could be important predictive biological markers for the follow-up of kidney transplant recipients.

In this study, we aimed to determine the possible involvement of the impact of MMP2, MMP9, and TIMP2 gene polymorphisms on allograft rejection in pediatric renal transplant recipients and its relation to clinical outcomes. To our knowledge, this is the first study investigating functional MMP2, MMP9, and TIMP2 polymorphisms in pediatric kidney transplant recipients. In our study, we found that MMP2 c.-735C>T (rs2285053), MMP2 c.-1306C>T (rs243865), MMP2 c.-1575G>A (rs243866), MMP9 c.-1562C>T (rs3918242), and TIMP2 c.303C>T (rs2277698) polymorphisms and genotype-phenotype relation were statistically significant in pediatric kidney transplant recipients. Matrix metalloproteinases and their tissue inhibitors could be important predictive biological markers for follow-up in kidney transplant recipients.

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