Procoagulant activity in normal human urine associated with subcellular particles

Abstract

Procoagulant activity (PCA) in normal human urine was found to be sedimented by centrifugation at X 100,000g. Therefore, studies were done to identify the structures associated with the procoagulant activity. Transmission electron microscopy of the X 100,000g pellet revealed numerous membrane-bound vesicles as well as fibrous material. Filtration of normal urine through a 0.2-micron filter removed more than 90% of the procoagulant activity. Scanning electron microscopy of the filter surface revealed 0.1 to 1.1 micron particles and fibrous material. By centrifugation at pH 3 and 5 the fibrous material and particles were separated. The procoagulant activity remained with the particles in each case. The fibrous material was shown to be Tamm-Horsfall protein by SDS-PAGE and Western blotting using anti-Tamm-Horsfall protein serum. Purified Tamm-Horsfall protein itself was not procoagulant. Therefore, PCA in normal human urine is associated with particles 0.1 to 1.1 micron in diameter which appear to be lipid membranes in various arrangements.

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

“…The absence of THG from the CF fraction demonstrates that most of the protein in the sieved fraction is particulate. This is consistent with the observation that centrifugation of sieved urine causes a large reduction (91.2%) in the number of particles, while filtration removes the remaining 8.8% (6), and confirms early observations that THG in normal urine exists as very large polymeric structures that are easily removed by centrifugation and filtration (5,(43)(44), as well as more recent reports that levels of THG and other proteins are reduced by these procedures (6,32). Thus the high inhibitory effect of the S urine fractions is almost certainly ascribable to the fact that S urine contains the highest concentration of polymerized particulate THG.…”

Discovery of Urinary Biomarkers

“…These components can be isolated by centrifugation at moderate speed, typically 17,000 ϫ g. Additionally urine contains numerous exosomes, derived from virtually every epithelial cell type facing the urine including glomerular podocytes, renal tu- In an additional experiment, TammHorsfall protein was removed from the final pellet by reduction with DTT and reultracentrifugation. The appearance of Tamm-Horsfall protein in the soluble fraction after ultracentrifugation at 200,000 ϫ g in this experiment is consistent with reports that most TammHorsfall protein in urine has been released from the membrane-bound state (76). B, Tamm-Horsfall protein networks and constrained vesicles in urine demonstrated by immunogold electron microscopy of urinary low density membranes probed with antibody to TammHorsfall protein (a kind gift from Dr. John R. Hoyer).…”

Urine Podocyte mRNAs Mark Progression of Renal Disease

“…For the studies described, the centrifugation step would predominantly pellet whole cells; however, it is possible that podocyte mRNAs could be packaged and protected from degradation within lipid membranes as microvesicles and that their association with larger structures in urine (e.g., polymerized Tamm-Horsfall protein) might allow them to be pelletted under the conditions used for the assay. 30,32 Both RNA and micro-RNAs have been shown to travel via microvesicles. 35,36 Further studies will be required to evaluate the possibility that downstream nephron signaling can occur via this mechanism.…”

“…30 We also demonstrated the presence of microvesicles in normal human urine (termed "urosomes"). 32 Knepper and colleagues 33,34 characterized urine exosomes and defined a urine exosome proteome that includes podocin and podocalyxin. For the studies described, the centrifugation step would predominantly pellet whole cells; however, it is possible that podocyte mRNAs could be packaged and protected from degradation within lipid membranes as microvesicles and that their association with larger structures in urine (e.g., polymerized Tamm-Horsfall protein) might allow them to be pelletted under the conditions used for the assay.…”