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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

SDS-PAGE protein pattern, TSG101, and ubiquitin detection of pooled samples. Two μg of protein (Bradford assay) was loaded per sample in each lane. Molecular weights are expressed in kilo Dalton. H: healthy control, N: normoalbuminuric, Mi: microalbuminuric, and Ma: macroalbuminuric.
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fig2: SDS-PAGE protein pattern, TSG101, and ubiquitin detection of pooled samples. Two μg of protein (Bradford assay) was loaded per sample in each lane. Molecular weights are expressed in kilo Dalton. H: healthy control, N: normoalbuminuric, Mi: microalbuminuric, and Ma: macroalbuminuric.

Mentions: Silver stained gels showed a good overlapping pattern with a moderate interindividual variability, most likely due to variable amounts of Tamm-Horsfall Protein (THP). Interestingly, TSG101 assay also showed a progressive signal decrease in the micro- and macroalbuminuric groups, respectively. This trend was even more evident when urine pools were created for the protease and protease inhibitor arrays (Figure 2). Moreover, a detectable shift of the apparent TSG101 molecular weight (MW) was observed when pools were run in adjacent lanes in the same gel. This most likely reflects changes in posttranslational modifications of exosome components during disease course. These results were confirmed in two independent Western blots, the second of which was carried out with an optimised gradient gel to have a better separation in the TSG101 molecular weight (MW) region. In order to investigate this alteration in detail we assayed the ubiquitination state of exosomes. Although the precise molecular mechanism of the vesicle formation and protein recruitment needs to be fully elucidated, it seems apparent that this posttranslation modification (PTM) faithfully reflects the disease pathogenesis. Interestingly, ubiquitination is involved in a variety of cellular processes, including protein sorting and translocation inside the vesicle lumen during vesicle biogenesis as well as in protein degradation [37]. In support of this, Western blotting revealed a specific ubiquitination pattern in the DN groups. Anti-ubiquitin antibody used in this screening recognised free ubiquitin and monoubiquitinated protein. In all the DN groups it is possible to observe a strong signal at 8.5 kDa corresponding to the monomeric ubiquitin and 17 kDa (white rectangle) which is absent in the healthy control. Moreover a specific pattern in the normo-, micro-, and macroalbuminuric groups with an apparently relative changing for some bands (asterisk) is visible between 50 and 75 kDa.


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)

SDS-PAGE protein pattern, TSG101, and ubiquitin detection of pooled samples. Two μg of protein (Bradford assay) was loaded per sample in each lane. Molecular weights are expressed in kilo Dalton. H: healthy control, N: normoalbuminuric, Mi: microalbuminuric, and Ma: macroalbuminuric.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: SDS-PAGE protein pattern, TSG101, and ubiquitin detection of pooled samples. Two μg of protein (Bradford assay) was loaded per sample in each lane. Molecular weights are expressed in kilo Dalton. H: healthy control, N: normoalbuminuric, Mi: microalbuminuric, and Ma: macroalbuminuric.
Mentions: Silver stained gels showed a good overlapping pattern with a moderate interindividual variability, most likely due to variable amounts of Tamm-Horsfall Protein (THP). Interestingly, TSG101 assay also showed a progressive signal decrease in the micro- and macroalbuminuric groups, respectively. This trend was even more evident when urine pools were created for the protease and protease inhibitor arrays (Figure 2). Moreover, a detectable shift of the apparent TSG101 molecular weight (MW) was observed when pools were run in adjacent lanes in the same gel. This most likely reflects changes in posttranslational modifications of exosome components during disease course. These results were confirmed in two independent Western blots, the second of which was carried out with an optimised gradient gel to have a better separation in the TSG101 molecular weight (MW) region. In order to investigate this alteration in detail we assayed the ubiquitination state of exosomes. Although the precise molecular mechanism of the vesicle formation and protein recruitment needs to be fully elucidated, it seems apparent that this posttranslation modification (PTM) faithfully reflects the disease pathogenesis. Interestingly, ubiquitination is involved in a variety of cellular processes, including protein sorting and translocation inside the vesicle lumen during vesicle biogenesis as well as in protein degradation [37]. In support of this, Western blotting revealed a specific ubiquitination pattern in the DN groups. Anti-ubiquitin antibody used in this screening recognised free ubiquitin and monoubiquitinated protein. In all the DN groups it is possible to observe a strong signal at 8.5 kDa corresponding to the monomeric ubiquitin and 17 kDa (white rectangle) which is absent in the healthy control. Moreover a specific pattern in the normo-, micro-, and macroalbuminuric groups with an apparently relative changing for some bands (asterisk) is visible between 50 and 75 kDa.

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