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Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

Musante L, Tataruch D, Gu D, Liu X, Forsblom C, Groop PH, Holthofer H - J Diabetes Res (2015)

Bottom Line: Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively.Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups.Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bioanalytical Sciences (CBAS), Dublin City University, Dublin 9, Ireland.

ABSTRACT
Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

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Related in: MedlinePlus

Protease chromogenic activity. Protease activities were assessed by specific chromogenic substrates for DDPIV Gly-Pro-p-nitroanilide (GP-pNA), leukocyte proteinase 3, and elastase (N-methoxysuccinyl Ala-Ala-Pro-Val-p-nitroanilide M-MeAAPV-pNA). Two μg of protein (Bradford assay) was used per replica. The bar represents the standard deviation of the technical triplicate.
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fig4: Protease chromogenic activity. Protease activities were assessed by specific chromogenic substrates for DDPIV Gly-Pro-p-nitroanilide (GP-pNA), leukocyte proteinase 3, and elastase (N-methoxysuccinyl Ala-Ala-Pro-Val-p-nitroanilide M-MeAAPV-pNA). Two μg of protein (Bradford assay) was used per replica. The bar represents the standard deviation of the technical triplicate.

Mentions: Beside the relative quantitative amount we checked the activity of some of these proteases by gelatine zymography for metalloproteases (Figure 4) which confirmed a clear increase of gelatinase activity in the normoalbuminuric group and by chromogenic substrates for DPP IV, Kallikreins, cathepsins, and PRTN3 (Figure 5). Spectrophotometric assays were performed in native condition and after organic delipidation to release proteases which can be localized in the vesicle lumen. After delipidation only the Kallikrein family was significantly affected (full loss of activity) while all the other protease activities maintained the same profile. Although there is a substantial increase of the cathepsin expression in the protease array, the colorimetric assay showed a decrease of the activity in the DN groups. Moreover, it is interesting to notice that the activity of DPP IV was much lower in the DN groups independently of the levels detected in the array. Proteinase 3 activity was high in the normoalbuminuric group but in the other groups it remained unchanged despite the higher levels especially in the microalbuminuric group. Since proteomic profiling of UEVs reported the presence of several protease inhibitors [18, 19], such discrepancy between activity and expression levels could be caused by the presence of protease inhibitors.


Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

Musante L, Tataruch D, Gu D, Liu X, Forsblom C, Groop PH, Holthofer H - J Diabetes Res (2015)

Protease chromogenic activity. Protease activities were assessed by specific chromogenic substrates for DDPIV Gly-Pro-p-nitroanilide (GP-pNA), leukocyte proteinase 3, and elastase (N-methoxysuccinyl Ala-Ala-Pro-Val-p-nitroanilide M-MeAAPV-pNA). Two μg of protein (Bradford assay) was used per replica. The bar represents the standard deviation of the technical triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383158&req=5

fig4: Protease chromogenic activity. Protease activities were assessed by specific chromogenic substrates for DDPIV Gly-Pro-p-nitroanilide (GP-pNA), leukocyte proteinase 3, and elastase (N-methoxysuccinyl Ala-Ala-Pro-Val-p-nitroanilide M-MeAAPV-pNA). Two μg of protein (Bradford assay) was used per replica. The bar represents the standard deviation of the technical triplicate.
Mentions: Beside the relative quantitative amount we checked the activity of some of these proteases by gelatine zymography for metalloproteases (Figure 4) which confirmed a clear increase of gelatinase activity in the normoalbuminuric group and by chromogenic substrates for DPP IV, Kallikreins, cathepsins, and PRTN3 (Figure 5). Spectrophotometric assays were performed in native condition and after organic delipidation to release proteases which can be localized in the vesicle lumen. After delipidation only the Kallikrein family was significantly affected (full loss of activity) while all the other protease activities maintained the same profile. Although there is a substantial increase of the cathepsin expression in the protease array, the colorimetric assay showed a decrease of the activity in the DN groups. Moreover, it is interesting to notice that the activity of DPP IV was much lower in the DN groups independently of the levels detected in the array. Proteinase 3 activity was high in the normoalbuminuric group but in the other groups it remained unchanged despite the higher levels especially in the microalbuminuric group. Since proteomic profiling of UEVs reported the presence of several protease inhibitors [18, 19], such discrepancy between activity and expression levels could be caused by the presence of protease inhibitors.

Bottom Line: Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively.Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups.Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bioanalytical Sciences (CBAS), Dublin City University, Dublin 9, Ireland.

ABSTRACT
Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

Show MeSH
Related in: MedlinePlus