Renal hydrolysis of absorbed protein: influence of load and lysosomal pH

Abstract

The kinetics of intracellular hydrolysis of administered protein and the effect of alkalinization of lysosomal pH on this process were studied in the isolated perfused rat kidney (IPK). Cytochrome c (CYT c) was used as a probe protein, and its hydrolysis was determined by measuring the efflux of radioactivity from IPK preloaded in vivo with [14CH3]CYT c and various doses of unlabeled CYT c. The nature of radioactivity absorbed by the kidney and released to the perfusate was analyzed by Sephadex chromatography. Administered CYT c is absorbed and hydrolyzed by the kidney, and the resulting amino acids are returned to the perfusate. At low uptake rates, the half time of hydrolysis of absorbed CYT c is about 20 min. The disposal of absorbed CYT c is a saturable function of its concentration in kidney with a Vmax = 0.60 mg CYT c X h-1 X g kidney-1 and an apparent Km = 0.55 mg CYT c/g kidney. To alkalinize the lysosomal pH, IPK were perfused in the presence of NH4Cl (10 mM) or chloroquine (0.1 mM). These lysosomotropic weak bases almost completely inhibit in a reversible manner the hydrolysis of absorbed CYT c. The results demonstrate that renal catabolism of absorbed protein is a saturable process of high capacity compared with the normal filtered loads of protein. The data are consistent with the view that normal lysosomal function is required for an adequate disposal of absorbed proteins in the kidney. It is postulated that abnormal deposition of protein absorption droplets within renal tubular cells may result from high absorbed loads and/or a deficient acidification of lysosomes.