ABSTRACT
Chronic kidney disease (CKD) is characterized by the loss of kidney function. The molecular mechanisms underlying the development and progression of CKD are still not fully understood. Among others, the urinary peptidome has been extensively studied, with several urinary peptides effectively detecting disease progression. However, their link to proteolytic events has not been made yet. This study aimed to predict the proteases involved in the generation of CKD-associated urinary excreted peptides in a well-matched (for age, sex, lack of heart disease) case-control study. The urinary peptide profiles from CKD (n = 241) and controls (n = 240) were compared and statistically analyzed. The in-silico analysis of the involved proteases was performed using Proteasix and proteases activity was predicted based on the abundance changes of the associated peptides. Predictions were cross-correlated to transcriptomics datasets by using the Nephroseq database. Information on the respective protease inhibitors was also retrieved from the MEROPS database. Totally, 303 urinary peptides were significantly associated with CKD. Among the most frequently observed were fragments of collagen types I, II and III, uromodulin, albumin and beta-2-microglobulin. Proteasix predicted 16 proteases involved in their generation. Through investigating CKD-associated transcriptomics datasets, several proteases are highlighted including members of matrix metalloproteinases (MMP7, MMP14) and serine proteases (PCSK5); laying the foundation for further studies towards elucidating their role in CKD pathophysiology.
Subject(s)
Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 7/metabolism , Proprotein Convertase 5/metabolism , Aged , Biomarkers , Body Fluids/metabolism , Case-Control Studies , Databases, Factual , Female , Gene Expression/genetics , Gene Expression Profiling , Humans , Male , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/urine , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 7/urine , Peptide Hydrolases/metabolism , Peptides/analysis , Peptides/urine , Proprotein Convertase 5/genetics , Proprotein Convertase 5/urine , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/urine , Transcriptome/genetics , Urine/chemistryABSTRACT
Matrix metalloproteinase-9 (MMP-9) has been shown to be involved in the development of diabetic nephropathy (DNP). We studied the levels, molecular forms, and degree of activation of urinary MMP-8, -9, -14, trypsin-1 and -2, as well as tumor-associated trypsin inhibitor (TATI) of DNP patients and healthy controls. Urinary samples were analyzed for MMPs by Western blotting and gelatin zymography and for trypsin-1, -2, and TATI by time-resolved immunofluorometric assays. Total MMP-8 immunoreactivity, the proportion of active MMP-9, and gelatinolytic activity in urine were significantly higher in DNP patients than in controls. In urine of DNP patients the proportion of active polymorphonuclear neutrophil (PMN)-type (but not fibroblast-type) MMP-8 was increased. MMP-8 and MMP-9 were found to form high molecular weight complexes in DNP urine. Total immunoreactivity of soluble urinary MMP-14 and the levels of trypsin (TRY)-1 and TRY-2, but not of TATI, were also significantly increased in DNP. Zymography, Western blotting, and immunofluorometric analysis of DNP urine showed a significant association especially between activation of MMP-9 as well as PMN-type MMP-8 and TRY-2. Our findings suggest that a trypsin-MMP cascade is involved in the pathogenesis of DNP, which may offer new possibilities for diagnosis and treatment of DNP with MMP inhibitors.
