Selected Metal Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases as Potential Biomarkers for Tubulointerstitial Fibrosis in Children with Unilateral Hydronephrosis.

Renal tubulointerstitial fibrosis caused by congenital ureteropelvic junction obstruction (UPJO) may lead to the development of obstructive nephropathy (ON) and the impairment of kidney function. Hence, the identification of early biomarkers of this condition might be of assistance in therapeutic decisions. This study evaluates serum and urinary metalloproteinases MMP-1, MMP-2, and MMP-9 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 as potential biomarkers of ON in children with congenital unilateral hydronephrosis (HN) caused by UPJO. Forty-five (45) children with congenital HN of different grades of severity and twenty-one (21) healthy controls were enrolled in the study. Urinary and serum concentrations of MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured using specific ELISA kits. The urinary excretions were expressed as biomarker/creatinine (Cr) ratios. To evaluate the extracellular matrix remodelling process activity, the serum and urinary MMP-1, -2, -9/TIMP-1, -2 ratios were also calculated. In comparison with the controls, patients with HN, independent of the grade, showed significantly increased median serum MMP-9, TIMP-1, and TIMP-2, median urinary MMP-9/Cr, and TIMP-2/Cr ratios. Lower median values of serum MMP-2/TIMP-1, MMP-9/TIMP-1 in patients with HN were also revealed. Additionally, higher urinary MMP-2/Cr, lower urinary MMP-2/TIMP-2, and lower serum MMP-9/TIMP-2 ratios were observed in patients with HN grades 3 and 4. Patients with ON diagnosed by renal scintigraphy had a significantly higher median serum MMP-9 concentration and lower median serum MMP-9/TIMP-1, -2 ratios in comparison with those without this condition. Patients with nonglomerular proteinuria had a significantly higher median serum TIMP-1 concentration, a higher median urinary TIMP-2/Cr ratio, and a lower serum MMP-9/TIMP-1 ratio compared to those without this symptom. The relationship between the measured biomarkers and the relative function of the obstructed kidney showed no correlations. The ROC curve analysis showed a promising diagnostic profile for the detection of ON for serum MMP-9 and the serum MMP-9/TIMP-1 and MMP-9/TIMP-2 ratios. In conclusion, the results of this study suggest that patients with HN, particularly with grades 3 and 4, are at higher risk of renal tubulointerstitial fibrosis. The noninvasive markers of this condition considered are urinary MMP-2/Cr and MMP-9/Cr, serum MMP-9, serum and urinary MMP-2, MMP-9/TIMP-1, -2. Additionally, serum MMP-9 and MMP-9/TIMP-1, -2 may become promising markers of ON.

Performance of candidate urinary biomarkers for pancreatic cancer - Correlation with pancreatic cyst malignant progression?

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are precursors of pancreatic cancer. Potential biomarkers of IPMN progression have not been identified in urine. A few urinary biomarkers were reported to be predictive of pancreatic ductal adenocarcinoma (PDAC). Here, we seek to assess their ability to detect high-risk IPMN. METHODS: Urine was collected from patients undergoing pancreatic resection and healthy controls. TIMP-1(Tissue Inhibitor of Metalloproteinase-1), LYVE-1(Lymphatic Vessel Endothelial Receptor 1), and PGEM(Prostaglandin E Metabolite) levels were determined by ELISA and analyzed by Kruskal-Wallis. RESULTS: Median urinary TIMP-1 levels were significantly lower in healthy controls (n = 9; 0.32 ng/mg creatinine) compared to PDAC (n = 13; 1.95) but not significantly different between low/moderate-grade (n = 20; 0.71) and high-grade/invasive IPMN (n = 20; 1.12). No significant difference in urinary LYVE-1 was detected between IPMN low/moderate (n = 16; 0.37 ng/mg creatinine) and high/invasive grades (n = 21; 0.09). Urinary PGEM levels were not significantly different between groups. CONCLUSIONS: Urinary TIMP-1, LYVE-1, and PGEM do not correlate with malignant potential of pancreatic cysts.

Serum Levels and Urinary Excretion of Tenascin-C and TIMP-1 in Acute Kidney Injury.

BACKGROUND: Both the extracellular matrix molecule tenascin-C (Tn-C) and tissue inhibitors of metalloproteinases (TIMPs) have a role in tissue injury, inflammation, and remodeling. In this pilot study, we tried to evaluate the role of these markers in acute kidney injury (AKI). METHODS: A total of 52 subjects were enrolled in this study. Group 1 consisted of 27 patients with AKI (stage 1, 2, and 3), and Group 2 consisted of 25 age- and gender-matched healthy subjects. Serum and urine samples (to determine Tn-C and TIMP-1) were obtained from the participants at the beginning of the study. Second samples were obtained from Group 1 patients when renal function improved (at discharge). RESULTS: Serum TIMP-1 concentrations (admission and discharge) were higher in Group 1 than Group 2 (p = 0.0001 for both comparisons). Tn-C excretion in spot urine was significantly higher in healthy controls than at the admission levels of the patient group (p = 0.036). However, TIMP-1 excretion in spot urine was lower in healthy controls than in admission and discharge levels of the patient group (p = 0.0001 for both comparisons). CONCLUSIONS: Our results show that these biomarkers (especially TIMP-1) may have a role in the pathophysiology of AKI. Further studies are needed in this field.

Protein biomarkers in serum and urine for determining presence or absence of hip dysplasia in a canine model.

This study compares serum and urine concentrations of relevant protein biomarkers among adult dogs with or without radiographic canine hip dysplasia (CHD). Adult (≥2 years of age), client-owned dogs (n = 74) radiographically categorized as having at least "good" hips (n = 49) or having "mild," "moderate," or "severe" hip dysplasia (n = 25) by the Orthopedic Foundation for Animals (OFA). Urine and serum samples were obtained from each dog at a single time-point and processed and analyzed for relevant protein biomarkers. Urinary concentrations of CTX-II (p < 0.001) and TIMP-1 (p = 0.002) were significantly lower in dogs with CHD compared to dogs with no CHD. ROC curve analyses were successful in establishing a panel of four biomarkers (urinary CTX-I and II, serum MMP-9, and serum PIICP) with high discriminatory capability for the presence or absence of hip dysplasia in adult dogs (AUC = 0.89). Urine and serum biomarkers can distinguish adult dogs with radiographic CHD from those with no CHD with a sensitivity of 0.95 and specificity of 0.77 using ROC analysis with AUC 0.89. Clinical Significance: This finding suggests that this simple, minimally invasive diagnostic technique has potential for discriminating dysplastic dogs from dogs with normal hips, with possible translational application to humans based on similar etiopathogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-5, 2019.

Urine Levels of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases in Children with Type 1 Diabetes Mellitus

Objective: Histopathological changes in the kidney in type 1 diabetes mellitus (T1DM) begin before detection of microalbuminuria. Therefore, there is interest in finding a better biomarker for the early detection of diabetic kidney injury. The aim of this present study was to determine whether urinary indicators of fibrosis are detectable early in the development of T1DM in children and if they may predict progressive renal injury. Methods: Urinary matrix metalloproteinase 2 and 9 (MMP2 and MMP9), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2) and transforming growth factor-ß1 (TGF-ß1) were assessed in 33 patients with T1DM with normal renal functions and in 24 healthy controls. Microalbuminuria was not present in the patient group with the exception of three patients. The results were adjusted to urine creatinine (Cr) and the differences between patients and controls were evaluated. These measurements were repeated after one year and the results were compared with the first year results. Results: Urine MMP2/Cr, MMP9/Cr, TIMP1/Cr, TIMP2/Cr, TGF-ß1/Cr were not different between the patient and control groups (p>0.05). There were also no significant differences between the first and second year results for these biomarkers (p>0.05). None of these parameters were correlated with hemoglobin A1c, body mass index and duration of T1DM. Interestingly, all parameters were negatively correlated to age of onset of T1DM (p<0.05). Conclusion: Our findings suggest that urinary biomarkers of fibrosis do not show an increase in diabetic children without microalbuminuria. The results also indicate that the risk of early fibrosis may increase as age of onset of T1DM decreases.

Analysis of the differential urinary protein profile in IgA nephropathy patients of Uygur ethnicity.

BACKGROUND: IgA nephropathy (IgAN) is one of the most common forms of idiopathic glomerular diseases and might lead to end-stage kidney disease. Accurate and non-invasive biomarkers for early diagnosis are required for early intervention and consequent therapy for IgAN patients. Because variance in the disease incidence and predisposing genes of IgAN has been detected among different ethnicities, the ethnicity factor should be considered in IgAN biomarker discovery. The differences in the protein profiles and pathological mechanisms of IgAN in patients of Uygur ethnicity need to be clearly illustrated. METHODS: In this study, we used urinary proteomics to discover candidate biomarkers of IgAN in patients of Uygur ethnicity. The urinary proteins from Uygur normal control and Uygur IgAN patients were extracted and analyzed using 2D-LC-MS/MS and isobaric tags for relative and absolute quantitation (iTRAQ) analysis. RESULTS: A total of 277 proteins were found to be differentially represented in Uygur IgAN compared with the respective normal controls. The bioinformatics analysis revealed that the immune response, cell survival, and complement system were activated in Uygur IgAN. Many differentially expressed proteins were found to be related to nephropathy and kidney injuries. Four candidate biomarkers were validated by Western blot, and these results were consistent with the iTRAQ results. ICAM1, TIMP1, SERPINC1 and ADIPOQ were upregulated in Uygur IgAN. Bioinformatic analysis revealed that the increase of ICAM1 and TIMP1 might be caused by IgAN, but the increase of SERPINC1 and ADIPOQ might be caused by proteinuria. SERPINC1 and ICAM1 were identified as the candidate biomarkers with excellent area-under-the-curve (AUC) values (0.84) for distinguishing Uygur IgAN from normal controls. CONCLUSIONS: Using urinary proteomic analysis, we identified several candidate biomarkers for IgAN in patients of Uygur ethnicity. These results will prove helpful for exploring the pathological mechanism of IgAN in patients of Uygur ethnicity and for developing better treatments for these patients.

Matrix Metalloproteinase-9 and Tissue Inhibitor of Matrix Metalloproteinase-1 in Sepsis after Major Abdominal Surgery.

BACKGROUND: The role of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in sepsis after major abdominal surgery and sepsis-associated organ dysfunction is unexplored. MATERIALS AND METHODS: Fifty-three patients with sepsis after major abdominal surgery were compared to 50 operated and 50 nonoperated controls. MMP-9, TIMP-1, biomarkers of inflammation, kidney and liver injury, coagulation, and metabolic disorders were measured daily during 96 h following diagnosis of sepsis and once in controls. MMP-9/TIMP-1 ratios and disease severity scores were calculated. Use of vasopressors/inotropes, mechanical ventilation, and survival were recorded. RESULTS: Septic patients had lower MMP-9 and MMP-9/TIMP-1 ratios but higher TIMP-1 levels compared to controls. AUC-ROC for diagnosis of sepsis was 0.940 and 0.854 for TIMP-1 and 0.924 and 0.788 for MMP-9/TIMP-1 ratio (sepsis versus nonoperated and sepsis versus operated controls, resp.). Lower MMP-9 and MMP-9/TIMP-1 ratio and higher TIMP-1 levels were associated with shorter survival. MMP-9, TIMP-1, and MMP-9/TIMP-1 ratio correlated with biomarkers of inflammation, kidney and liver injury, coagulation, metabolic disorders, and disease severity scores. Use of vasopressors/inotropes was associated with higher TIMP-1 levels. CONCLUSIONS: MMP-9, TIMP-1, and MMP-9/TIMP ratio were good diagnostic or prognostic biomarkers of sepsis after major abdominal surgery and were linked to sepsis-associated organ dysfunction.

Acute- and late-phase matrix metalloproteinase (MMP)-9 activity is comparable in female and male rats after peripheral nerve injury.

BACKGROUND: In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. METHODS: Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. RESULTS: The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. CONCLUSION: The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes.

DNA methylation of extracellular matrix remodeling genes in children exposed to arsenic.

Several novel mechanistic findings regarding to arsenic's pathogenesis has been reported and some of them suggest that the etiology of some arsenic induced diseases are due in part to heritable changes to the genome via epigenetic processes such as DNA methylation, histone maintenance, and mRNA expression. Recently, we reported that arsenic exposure during in utero and early life was associated with impairment in the lung function and abnormal receptor for advanced glycation endproducts (RAGE), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) sputum levels. Based on our results and the reported arsenic impacts on DNA methylation, we designed this study in our cohort of children exposed in utero and early childhood to arsenic with the aim to associate DNA methylation of MMP9, TIMP1 and RAGE genes with its protein sputum levels and with urinary and toenail arsenic levels. The results disclosed hypermethylation in MMP9 promotor region in the most exposed children; and an increase in the RAGE sputum levels among children with the mid methylation level; there were also positive associations between MMP9 DNA methylation with arsenic toenail concentrations; RAGE DNA methylation with iAs, and %DMA; and finally between TIMP1 DNA methylation with the first arsenic methylation. A negative correlation between MMP9 sputum levels with its DNA methylation was registered. In conclusion, arsenic levels were positive associated with the DNA methylation of extracellular matrix remodeling genes;, which in turn could modifies the biological process in which they are involved causing or predisposing to lung diseases.

Diastolic Left Ventricular Function in Relation to Urinary and Serum Collagen Biomarkers in a General Population.

Current knowledge on the pathogenesis of diastolic heart failure predominantly rests on case-control studies involving symptomatic patients with preserved ejection fraction and relying on invasive diagnostic procedures including endomyocardial biopsy. Our objective was to gain insight in serum and urinary biomarkers reflecting collagen turnover and associated with asymptomatic diastolic LV dysfunction. We randomly recruited 782 Flemish (51.3% women; 50.5 years). We assessed diastolic LV function from the early and late diastolic peak velocities of the transmitral blood flow and of the mitral annulus. By sequencing urinary peptides, we identified 70 urinary collagen fragments. In serum, we measured carboxyterminal propeptide of procollagen type 1 (PICP) as marker of collagen I synthesis and tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1), an inhibitor of collagen-degrading enzymes. In multivariable-adjusted analyses with Bonferroni correction, we expressed effect sizes per 1-SD in urinary collagen I (uCI) or collagen III (uCIII) fragments. In relation to uCI fragments, e' decreased by 0.183 cm/s (95% confidence interval, 0.017 to 0.350; p = 0.025), whereas E/e' increased by 0.210 (0.067 to 0.353; p = 0.0012). E/e' decreased with uCIII by 0.168 (0.021 to 0.316; p = 0.018). Based on age-specific echocardiographic criteria, 182 participants (23.3%) had subclinical diastolic LV dysfunction. Partial least squares discriminant analysis contrasting normal vs. diastolic LV dysfunction confirmed the aforementioned associations with the uCI and uCIII fragments. PICP and TIMP-1 increased in relation to uCI (p<0.0001), whereas these serum markers decreased with uCIII (p≤0.0006). Diastolic LV dysfunction was associated with higher levels of TIMP-1 (653 vs. 696 ng/mL; p = 0.013). In a general population, the non-invasively assessed diastolic LV function correlated inversely with uCI and serum markers of collagen I deposition, but positively with uCIII. These observations generalise previous studies in patients to randomly recruited people, in whom diastolic LV function ranged from normal to subclinical impairment, but did not encompass overt diastolic heart failure.

Urinary Metalloproteinases-9 and -2 and Their Inhibitors TIMP-1 and TIMP-2 are Markers of Early and Long-Term Graft Function After Renal Transplantation.

BACKGROUND/AIMS: Renal ischemia-reperfusion (I-R) injury (IRI) is an inseparable feature of organ transplantation and may have a negative impact on the graft, its function and survival. Acute tubular necrosis, which is reversible thanks to the regenerative capacity of renal tubular epithelial cells, is the main cause of acute renal failure secondary to IRI. MMP-2 and MMP-9 are proteolytic enzymes involved in digesting proteins that are components of the extracellular matrix (ECM) and the basement membrane of the nephrons. This way post-reperfusion MMP activation allows the inflammatory process to spread. METHODS: In our studies, we focused on identifying whether the concentrations of MMP-2 and MMP-9 and their natural inhibitors TIMP-1 and TIMP-2 in urine sample at day 1 and day 30 as well as after 12 months following renal transplantation are markers of early and long-term renal function during meanly five-years observation. Moreover, in urine sampled at months 6 and 12 after renal transplantation, we determined the content of TGF-ß as a graft fibrosis indicator. RESULTS: MMP-9 concentration in the early post-transplant period is a major marker of early and long-term function of the transplanted kidney. Its increased concentration was correlated with lesions related to tubular atrophy and fibrosis in renal biopsies performed at months 3 and 12 after transplantation. Its concentration is correlated with TGF-ß content in a later period. CONCLUSIONS: TIMP-1 and-2 are primarily markers of an early function of the transplanted kidney. Early post-transplant concentration of MMP-2 is a marker of proteinuria in early and long-term post-transplant periods.

Urinary Emmprin, matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 as potential biomarkers in children with ureteropelvic junction narrowing on conservative treatment.

BACKGROUND: Rapid diagnosis and initiation of the treatment on congenital obstructive nephropathy are important for young children to slow down renal injury. The aim of our study was to investigate the role of urinary extracellular matrix metalloproteinase inducer (Emmprin), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the long-term follow-up of children with ureteropelvic junction (UPJ) narrowing on conservative treatment. METHODS: The study included 40 children with non-obstructed hydronephrosis due to unilateral UPJ narrowing who were treated conservatively and followed up for 24 months. Voided urine samples were collected at diagnosis and at 3, 9, 15 and 24 months of follow-up, respectively. Three enzymes concentrations were measured in urine. RESULTS: During the follow-up, 25 children showed renal function stabilization (non-obstructed group) and 15 children renal function deterioration (obstructed group). In the non-obstructed group, a comparison between urine three enzymes levels at the last follow-up and at baseline showed no significant differences (all P > 0.05). Glomerular filtration rate (GFR) and split renal function (SRF) showed similar trends. In the obstructed group, a comparison between the three enzymes levels at diagnosis and at basal condition showed a significant increase (all P < 0.01). But GFR and SRF showed a marked reduction at diagnosis (all P < 0.001). Receiver operator characteristic (ROC) analyses revealed a better diagnostic profile for uEmmprin, uMMP-9 and uTIMP-1 in identifying children with abnormal SRF (<40%) at 24 months of follow-up [area under the curve (AUC) 0.877, 0.727 and 0.823, respectively]. CONCLUSIONS: Urinary Emmprin, MMP-9 and TIMP-1 may be noninvasive potential biomarkers that could be used for long-term follow-up of children with UPJ narrowing on conservative treatment to determine those who might develop obstruction.

Advanced age of renal transplant recipients correlates with increased plasma concentrations of interleukin-6, chemokine ligand 2 (CCL2), and matrix metalloproteinase 2, and urine concentrations of CCL2 and tissue inhibitor of metalloproteinase 1.

BACKGROUND: Advanced age of renal transplant recipients (RTRs) has a negative impact on kidney allograft survival through impaired extracellular matrix degradation by the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) system. Moreover, older RTRs are at risk of smoldering inflammation, known as inflammaging. AIM: The aim of the study was to assess the impact of a RTR's age on plasma and urine concentrations of interleukin 6 (IL-6), chemokine ligand 2 (CCL2), and the MMPs/TIMPs system. MATERIAL AND METHODS: One hundred fifty adult RTRs (8.7% ≥ 65 years) and 37 adult healthy volunteers (10.8% ≥ 65 years) were enrolled in the study. The studied factors (IL-6, CCL2, MMP-2, MMP-9, TIMP-1 and TIMP-2) were quantified in plasma and urine with enzyme-linked immunosorbent assay. The Mann-Whitney U test and Spearman's (rs) rank correlation were applied, and differences with a P < .05 were considered statistically significant. RESULTS: There was a weak but significant positive correlation between increasing RTR's age and plasma IL-6 (rs = 0.18, P = .028), CCL2 (rs = 0.27, P = .001), and MMP-2 (rs = 0.20, P = .017), as well as urine CCL2 (rs = 0.16, P = 0.050) and TIMP-1 (rs = 0.20, P = .014) concentrations. CONCLUSIONS: Advancing age of RTRs correlates with increasing plasma IL-6 and CCL2 concentrations, reflecting smoldering inflammation (known as inflammaging) and alterations in MMPs/TIMPs profiles, especially with increased plasma MMP-2 and urine TIMP-1 concentrations.

Urinary TIMP-1 and MMP-2 levels detect the presence of pancreatic malignancies.

BACKGROUND: A majority of patients with pancreatic malignancies, including both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumours (pNETs), present with advanced disease due to a lack of specific symptoms and current diagnostic limitations, making this disease extremely difficult to detect. Our goal was to determine whether urinary matrix metalloproteases (uMMPs) and/or their endogenous inhibitors, urinary tissue inhibitor of metalloproteases (uTIMPs), could be detected in the urine of patients with pancreatic malignancies and whether they may serve as independent predictors of disease status. METHODS: Retrospective analyses of urine samples (n=139) from PDAC and pNET patients as well as age- and sex-matched controls were conducted. Urinary MMP-2 and uTIMP-1 levels were determined using ELISA and zymography. Biomarker expression in tumour and normal pancreatic tissues was analysed via immunohistochemistry (IHC). RESULTS: Multivariable logistic regression analyses indicated that, when controlling for age and sex, uMMP-2 (P<0.0001) and uTIMP-1 (P<0.0001) but not uMMP-9, were significant independent predictors for distinguishing between PDAC patients and healthy controls. Our data also indicated that uMMP-2 was an independent predictor of the presence of pNET. In addition, uTIMP-1 levels could differentiate the two cancer groups, PDAC and pNET, respectively. Immunohistochemistry analysis confirmed that MMP-2 and TIMP-1 protein expression is significantly upregulated in PDAC tissue compared with the normal pancreas. CONCLUSIONS: Taken together, our results suggest that the detection of uMMP-2 and uTIMP-1 may have diagnostic value in the detection of pancreatic malignancies and that uTIMP-1 may be useful in distinguishing between pancreatic adenocarcinoma and neuroendocrine tumours.

The role of urinary TIMP1 and MMP9 levels in predicting vesicoureteral reflux in neonates with antenatal hydronephrosis.

BACKGROUND: Antenatal hydronephrosis (AH) is commonly found on evaluation of pregnant women and 20-30 % of neonates have vesicoureteral reflux (VUR). In order to diagnose VUR, we required invasive testing and exposure of the neonate to radiation. The concentrations of a matrix metalloproteinase, MMP9, and its inhibitor TIMP1, were analyzed in hydronephrotic newborns with VUR and were compared to those without reflux. METHODS: The neonates with a history of prenatal hydronephrosis were enrolled in two groups based on imaging study results, the neonates with VUR and without VUR. Neonates with a normal prenatal history and postnatal ultrasound were placed in a third group. We measured the random urinary levels of MMP9, TIMP1, and creatinine, their cut-off values and the MMP9/Cr and MMP9/TIMP1/Cr ratio was calculated, and an ROC curve was drawn. RESULTS: Sixty-nine neonates were enrolled in three groups; 27 patients (20 male, seven female) with AH and VUR were in group 1, 23 neonates (19 male, four female) without VUR were placed in group 2, and 19 (15 male, four female) acted as controls in group 3. The differences between the three groups and the normal and total hydronephrotic groups were statistically significant for MMP9, the MMP9/Cr, MMP9/TIMP1, and MMP9/TIMP1/Cr ratios. The urinary TIMP1 and TIMP1/Cr ratios were not significantly different between the groups. A cut-off value of MMP9 was measured as 358.5 ng/ml (sensitivity [sens] 74 %, specificity [spec] 78 %) and was used to compare groups 1 and 2. For groups 2 and 3, this cut-off was 181.00 pg/ml (sens 91 %, spec 89 %). The cut-off values measured for the MMP9/TIMP1 ratio were 30.32 (sens 70 %, spec 61 %) and 9.85 (sens 96 %, spec 89 %) to compare groups 1 and 2, and 2 and 3, respectively. We found no valuable cut-offs for the TIMP1 and TIMP1/Cr values. There was no difference between neonates with mild, moderate, and severe VUR according to urinary biomarker concentrations. CONCLUSIONS: Evaluation of urinary levels of MMP9, or the MMP9/Cr, MMP9/TIMP1, or MMP9/TIMP1/Cr ratios may help us to differentiate the newborns with hydronephrosis and VUR from those without reflux.

Correlation of serum and urinary matrix metalloproteases/tissue inhibitors of metalloproteases with subclinical allograft fibrosis in renal transplantation.

Progressive interstitial fibrosis and tubular atrophy (IF/TA) is a leading cause of chronic allograft dysfunction. Increased extracellular matrix remodeling regulated by matrix metalloproteases (MMPs) and their inhibitors (TIMPs) has been implicated in the development of IF/TA. The aim of this study was to investigate whether urinary/serum MMPs/TIMPs correlate with subclinical IF/TA detected in surveillance biopsies within the first 6months post-transplant. We measured eight different MMPs/TIMPs simultaneously in urine and serum samples from patients classified as normal histology (n=15), IF/TA 1 (n=15) and IF/TA 2-3 (n=10). There was no difference in urinary MMPs/TIMPs among the three groups, and only 1/8 serum MMPs/TIMPs (i.e. MMP-1) was significantly elevated in biopsies with IF/TA 2-3 (p=0.01). In addition, urinary/serum MMPs/TIMPs were not different between surveillance biopsies demonstrating an early development of IF/TA (i.e. delta IF/TA≥1 compared to a previous biopsy obtained three months before; n=11) and stable grade of IF/TA (i.e. delta IF/TA=0; n=20). Next, we investigated whether urinary/serum MMP/TIMP levels are elevated during acute subclinical tubulitis in surveillance biopsies obtained within the first 6months post-transplant (n=25). Compared to biopsies with normal histology, serum MMPs/TIMPs were not different; however, all urinary MMP/TIMP levels were numerically higher during subclinical tubulitis (MMP-1, MMP-7, TIMP-1 with p≤0.04). We conclude that urinary/serum MMPs/TIMPs do hardly correlate with existing or early developing IF/TA in surveillance biopsies obtained within the first 6months post-transplant. This could be explained by the dynamic process of extracellular matrix remodeling, which seems to be active during acute tubulo-interstitial injury/inflammation, but not in quiescent IF/TA.

Urinary MMP-9/NGAL ratio as a potential marker of FSGS in nephrotic children.

BACKGROUND: The study was undertaken to develop a potential new markers for distinguishing minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS) in children. We hypothesized that matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin (MMP-9/NGAL) is a better marker of focal sclerosis in the glomerulus then matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1) and matrix metalloproteinase-2/tissue inhibitor of metalloproteinase-2 MMP2/TIMP-2. METHODS: The present study used a sample of 36 children and adolescents subdivided into two groups: I - 20 children with MCNS, subjected to examination twice: A - in relapse of nephrotic syndrome, before treatment and B - after regression of proteinuria; II - 16 children with FSGS. MMPs and TIMPs and NGAL levels were measured in the urine using ELISA kit. MMP-9/TIMP-1, MMP-2/TIMP-2 and MMP-9/NGAL ratios were calculated. RESULTS: Median NGAL/cr. was significantly higher in MCNS and FSGS patients when compared to healthy controls. Both, NGAL and MMP-9 urinary levels were significantly elevated in FSGS subjects, as compared with control subjects. Contrary to FSGS children, in MCNS group, before treatment only NGAL/cr., but not MMP-9/cr. was increased. Urinary concentrations of NGAL and MMP-9 were highly associated with each other (NGAL/cr. vs. MMP-9/cr., r=0.485, p<0.01). Median urine MMP-9/NGAL ratio in FSGS patients was significantly higher than in patients with MCNS. We also found that significant increase in MMP-9/NGAL was associated with FSGS [odds ratio (OR) - 9.0; confidence interval (CI) 1.97-41.07]. CONCLUSION: MMP-9/NGAL ratio may serve as differentiation marker between MCNS and FSGS in nephrotic children.

Matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 in vesicoureteral reflux.

The aim of this study was to investigate whether urine levels of matrix metalloproteinase 9 (uMMP9) and tissue inhibitor of metalloproteinase 1 (uTIMP1) are novel biomarkers of vesicoureteral reflux (VUR) and to determine the optimal cut-off levels of these enzymes to predict VUR in children. The study group consisted of 67 children with VUR and 20 healthy children. Urine MMP9 and TIMP1 levels were measured by an enzyme-linked immunosorbent assay. Children with VUR had significantly higher uMMP9 (1,539.8 vs. 256.4 pg/mL; p = 0.0001) and uTIMP1 (182 vs. 32.6 pg/mL; p = 0.0001) levels than healthy children. For the prediction of VUR, the sensitivity of uMMP9 was 67%, with a specificity of 85% [cut-off value 1,054 pg/mL; area under the curve (AUC) 0.77], and the sensitivity of uTIMP1 was 74%, with a specificity of 65% (cut-off value 18.7 pg/mL; AUC 0.73). Both uMMP9 and uTIMP1 levels were significantly higher in patients with renal scar (uMMP9: 3,117.3 vs. 1,234.15 pg/mL; p = 0.0001; uTIMP1: 551.05 vs. 128.64 pg/mL; p = 0.0001). Urine MMP9 levels had a sensitivity of 81.2%, with a specificity of 85% to predict renal scar in the VUR group (cut-off 1,054 pg/mL; AUC 0.88). The sensitivity of uTIMP1 was 75%, with a specificity of 90% to predict renal scar (cut-off 243.7 pg/mL; AUC 0.82). Based on these results, we suggest that uTIMP1 may be a useful marker to predict renal scarring with a different cut-off value from VUR and a high specificity at this cut-off point. Although uMMP9 seemingly cannot distinguish renal scar from VUR, the simultaneous increase in the level of both markers may indicate ongoing renal injury due to VUR.

Imbalance of metallaproteinase/tissue inhibitors of metalloproteinase system in renal transplant recipients with chronic allograft injury.

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.

Distinct metalloproteinase excretion patterns in focal segmental glomerulosclerosis.

Metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) degrade type IV collagen, and represent important tissue remodeling enzymes in several kidney disorders. In this study, we measured urinary levels of MMP-2, MMP-9, and the tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in patients with steroid-sensitive nephrotic syndrome (SSNS, n = 18, median age 5) and focal segmental glomerulosclerosis (FSGS, n = 16, median age 15). We found that urinary concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2 were significantly elevated in FSGS patients as compared to SSNS in both relapse and remission (p < 0.002). Furthermore, urinary levels of these enzymes are increased early on in the FSGS disease process (chronic kidney disease stages 1 and 2). The findings from this pilot study suggest that MMPs and TIMPs have the potential to represent candidate, early non-invasive biomarkers for diagnosis and/or response to therapy. In addition, they may represent therapeutic targets for preventing chronic kidney disease progression in FSGS.