Abstract
Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.
Highlights
Exosomes are small-sized nanovesicles originated inside the cell traffic network and secreted through the fusion of multivesicular bodies with the cell membrane [1,2]
Emphasizing the potential importance of these cytokines for kidney pathophysiology, a human kidney transcriptomics approach disclosed that TRAIL and its decoy receptor osteoprotegerin (OPG/OCIF/ TNFRSF11B) [19] were the apoptosis-related genes most highly expressed in diabetic nephropathy (DN), the most frequent form of chronic kidney disease (CKD) [16]
We have shown for the first time that cultured human proximal tubular epithelial cells constitutively secrete exosome-like vesicles that contain the decoy receptor OPG as well as several other proteins, some not previously known to be exosomal proteins
Summary
Exosomes are small-sized nanovesicles originated inside the cell traffic network and secreted through the fusion of multivesicular bodies with the cell membrane [1,2]. T cell exosomes contain members of the TNF superfamily of proapoptotic cytokines such as TRAIL, TNF and FasL and their presence in exosomes is key to death of T cell target cells [10,11]. Tubular cells express functional TNF superfamily proapoptotic cytokines such as TNF, Fas ligand, TRAIL and TWEAK [14,15,16,17,18]. Emphasizing the potential importance of these cytokines for kidney pathophysiology, a human kidney transcriptomics approach disclosed that TRAIL and its decoy receptor osteoprotegerin (OPG/OCIF/ TNFRSF11B) [19] were the apoptosis-related genes most highly expressed in diabetic nephropathy (DN), the most frequent form of chronic kidney disease (CKD) [16]. Serum levels of OPG are increased in CKD patients and have been associated with vascular calcification [22]
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