Urinary matrix metalloproteinase activity in diabetic kidney disease: a potential marker of disease progression

Abstract

Background: Progressive kidney fibrosis, associated with chronic kidney disease (CKD), results from an imbalance in extracellular matrix (ECM) homeostasis. Reduced matrix metalloproteinases (MMP) activity causing lower clearance of ECM proteins has been implicated mainly through an overproduction of tissue inhibitors of metalloproteinases (TIMP), but also by reduced MMP synthesis. We tested the hypothesis that MMP activity can be measured in human urine and can be used as a potential biomarker of the progression of diabetic kidney disease (DKD).

Methods: An observational prospective study was performed on 102 DKD patients using 21 diabetic patients without kidney disease and 21 healthy volunteers as controls. The Molecular Probes EnzChek Gelatinase/Collagenase Assay Kit were used to determine urinary MMP activity using DQ™ Gelatin (total MMPs), DQ™ Collagen I (interstitial collagenases) and DQ™ Collagen IV (gelatinises) substrates. A broad-spectrum synthetic inhibitor of all MMP, 1,10-phenanthroline, was used to confirm that the proteolytic activity is due to MMP activity. All MMP values were expressed per unit of urine creatinine.

Results: Overall urinary MMP activity (DQ Gelatin substrate) was significantly elevated in DKD patients (14.76 ± 3.65 Δ fl/h/mmol creatinine) compared to diabetes mellitus controls (7.09 ± 2.12 Δ fl/h/mmol creatinine) and healthy volunteers (1.87 ± 0.74 Δ fl/h/mmol creatinine) (ANOVA p = 0.01). Within the DKD cohort, there was an approximate threefold higher urinary MMP activity in nonprogressive DKD patients compared to those with progressive disease (p = 0.002). The urinary MMP activity:creatinine ratio was significantly higher in normoalbuminuric and microalbuminuric DKD compared to macroalbuminuric DKD. Positive correlations were observed between the rate of total MMP activity and interstitial collagenases (r = 0.75, p < 0.0001) and gelatinases (r = 0.59, p = 0.0001). The accuracy of MMP activity to predict the rate of annual eGFR decline (ROC analysis) was 77% compared to 64% for albuminuria.

Conclusions: Total MMP activity can be easily measured in human urine. Surprisingly and in contrast to MMP activity in the kidney, urine MMP activity is elevated in DKD. However, there is a significantly lower MMP activity in patients with progressive DKD. ROC analysis demonstrates that single urine MMP activity estimation is superior to albuminuria in predicting DKD patients with progressive disease.

Keywords: Albuminuria; Diabetic kidney disease progression; End-stage renal disease; Gelatinases; Interstitial collagenases; Matrix metalloproteinases activity; Type 2 diabetes mellitus.

Fig. 1

MMP activity:creatinine ratio in diabetic kidney disease. MMP activity was measured in urine using the cleavage of DQ Gelatin and measuring the increase in fluorescence with time. a Mean total MMP activity:creatinine ratio in healthy volunteers, DM controls and DKD patients (p < 0.0001). b MMP activity:creatinine ratio by albuminuria in DKD patients. c Total MMP activity:creatinine ratio among DKD progressors and nonprogressors (p = 0.002). The data represents mean MMP activity:creatinine ratio ± standard error of the mean (SEM). ** p < 0.001; *** p < 0.0001; ns = nonsignificant.

Fig. 2

Total MMP activities by clinical parameters. MMP activity was measured in urine using the cleavage of DQ Gelatin. Total MMP:creatinine ratio was indicated by gender (a), age category (b), cardiovascular complications (c) and diabetic retinopathy (d). Assay reactions for all samples were carried out at room temperature. Data represents mean MMP activity:creatinine ratio ± SEM. ns = Nonsignificant.

Fig. 3

ROC curve analysis of predictors of progressive DKD. A ROC analysis was performed comparing the selectivity and sensitivity of total MMP activity:creatinine ratio vs. albumin: creatinine ratio in predicting the annual decline of kidney functions. The area under the curve represents the accuracy in prediction.