Determination of uromodulin in human urine: influence of storage and processing.

BACKGROUND: Uromodulin (Tamm-Horsfall protein) is the most abundant protein excreted in the urine under physiological conditions. It is exclusively produced in the kidney and secreted into the urine via proteolytic cleavage. The involvement of UMOD, the gene that encodes uromodulin, in rare autosomal dominant diseases, and its robust genome-wide association with the risk of chronic kidney disease suggest that the level of uromodulin in urine could represent a critical biomarker for kidney function. The structure of uromodulin is complex, with multiple disulfide bonds and typical domains of extracellular proteins. METHODS: Thus far, the conditions influencing stability and measurement of uromodulin in human urine have not been systematically investigated, giving inconsistent results. In this study, we used a robust, in-house ELISA to characterize the conditions of sampling and storage necessary to provide a faithful dosage of uromodulin in the urine. RESULTS: The levels of uromodulin in human urine were significantly affected by centrifugation and vortexing, as well as by the conditions and duration of storage. CONCLUSIONS: These results validate a simple, low-cost ELISA and document the optimal conditions of processing and storage for measuring uromodulin in human urine.

Endocytosis of the somatostatin analogue, octreotide, by the proximal tubule-derived opossum kidney (OK) cell line.

BACKGROUND: Nephrotoxicity of cancer therapy using radiolabeled somatostatin analogues such as octreotide is due to ultrafiltration and reuptake by proximal tubular cells (PTCs). The mechanism of uptake is unknown. It could occur either by receptor-mediated endocytosis via a somatostatin receptor or, alternatively, the multiligand megalin/cubilin tandem receptor, or by fluid-phase endocytosis. To define the mechanisms of internalization and to identify potential receptors, we have studied the uptake and processing of octreotide by the PTC-derived opossum kidney (OK) cell line. METHODS: We compared the kinetics of uptake and fate of (111)In-diethylenetriamine pentaacetic acid (DTPA)-D-Phe(1)-octreotide and (125)I-human serum albumin ((125)I-HSA). To determine the contribution of receptor-mediated endocytosis, we tested competition for uptake by octreotide and somatostatin and by various megalin/cubilin ligands [receptor-associated protein (RAP), albumin, transferrin, insulin, polymixin B] or basic amino acids. The subcellular localization of fluorescein isothiocyanate (FITC)-D-Phe(1)-octreotide was studied by confocal microscopy. RESULTS: Kinetics of uptake of (111)In-DTPA-D-Phe(1)-octreotide and (125)I-HSA by OK cells were comparable, but only the somatostatin analogue was significantly retained intact. All megalin/cubilin ligands and basic amino acids strongly inhibited (125)I-HSA uptake, but these could not compete for >50% of (111)In-DTPA-D-Phe(1)-octreotide uptake. The same was found for somatostatin and octreotide. The noncompetable uptake of (111)In-DTPA-D-Phe(1)-octreotide was comparable to the clearance of Lucifer Yellow, a marker of fluid-phase endocytosis. By confocal microscopy, FITC-D-Phe(1)-octreotide colocalized with transferrin in endosomes, then accumulated in lysosomes. CONCLUSION: Receptor-mediated endocytosis via megalin/cubilin and fluid-phase endocytosis contribute about equally to the uptake of radiolabeled somatostatin analogues by OK cells.