Evidence of glomerular epithelial cell endocytosis in vitro

The disappearance rate of protamine-heparin aggregates (PHA) from the lamina rara externa of the glomerular basement membrane was determined in vitro to better characterize the clearance mechanism. Sprague-Dawley rats were injected intravenously with protamine, followed by a neutralizing dose of heparin, and the glomeruli were isolated and incubated in Dulbecco's tissue culture medium. After obtaining a baseline sample, aliquots were taken every 10 minutes for an hour. The samples were processed for electron microscopy, and the numerical density of PHA in the lamina rara externa was determined using a computerized morphometric technique. Disappearance curves for PHA were constructed by expressing the numerical density at each point as a percentage of the baseline value and plotting the logarithm against time. At 37 degree C., the disappearance of PHA was rapid (t 1/2 = 24 minutes), followed first order kinetics (R2 = 0.9948), and was almost complete by 1 hour. The epithelial cells took up PHA by an endocytic process. Significant inhibition of PHA disappearance was achieved when the glomeruli were incubated at 4 degree C. and with 10(-3) M NaF. These data indicate that PHA disappearance is an energy-dependent cell function, and it was concluded that glomerular epithelial cell endocytosis is the principal mechanism of PHA clearance.