Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor.
Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.
Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave. ND 20, Cleveland, Ohio 44195, USA.
Nicholas
N
Ferrell
Kevin B
KB
Ricci
Joseph
J
Groszek
Joseph T
JT
Marmerstein
William H
WH
Fissell
D000418:Albumins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D019149:Bioreactors; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000502:physiology; D044085:Microfluidics / Q000379:methods*; D008970:Molecular Weight; D009893:Opossums; D021381:Protein Transport; D059748:Proteolysis
D000418:Albumins