Hemoglobin binding sites on renal brush-border membranes

Prolonged exposure of renal tubules to hemoglobin markedly reduces kidney function and eventually leads to acute renal failure called pigment nephropathy. Intracellular hemoglobin toxicity is one of main pathomechanisms involved in the disease development. However, the process in which hemoglobin is taken up by renal tubular epithelium has not been characterized so far. Isolated renal brush-border membranes of the rat and radioiodinated rat and human hemoglobins were used. Binding properties were examined by the use of rapid filtration technique. Partial isolation of hemoglobin binding proteins was achieved by affinity chromatography. Our experiments showed that both human and rat hemoglobins can be specifically bound to renal brush-border membranes by one class of low affinity (Kd, 7.7 microM) and high capacity (Bmax, 0.18 nmol/mg protein) binding sites. The sites were relatively selective for hemoglobin. Albumin did not compete with hemoglobin. Cationic molecules cytochrome C and lysine exhibited some competition while strong competition of myoglobin was observed. The binding was affected by EGTA indicating a Ca2+ requirement for the interaction. There was a rise in binding in pH 5.4. Fall in binding activity after preincubation of the membranes with peptidases suggested the proteinaceous nature of the binding sites. Affinity chromatography of membrane proteins extract yielded heterogeneous preparation consisting of proteins with molecular masses of 110, 72, 38 and 27 kDa respectively. The existence of binding sites for hemoglobin in renal brush-border membranes strongly suggests that uptake of the protein by tubular epithelia occurs via adsorptive endocytosis. Increased binding of hemoglobin to the membranes under acidic conditions may explain exacerbation of hemoglobinuric acute renal failure in aciduric states.