Insulin absorption in renal proximal tubules: A quantitative immunocytochemical study

The purposes of the present study are mainly biological concerning proximal tubular handling of insulin: we will study the intracellular transport to subcellular compartments involved in insulin degradation, the specificity and saturability of the luminal endocytic absorption of insulin, the visualization of transtubular transport, and finally, if possible, the evaluation of the relative distribution (accumulation) of insulin in endocytic vacuoles and lysosomes. The second part is methodological: application of quantitative immunocytochemistry to endocytosis, quantitation of the effect of particle size and antigen density on labeling density on tissue sections, labeling at very low antigen densities, and effect of fish gelatin on background. Isolated renal proximal tubules were perfused with native insulin, ¹²⁵I-insulin, or [leucine^B-25]-insulin (2% receptor-binding ability and full immunoreactivity) or exposed to native insulin from the basolateral membranes. In conclusion, the luminal uptake of insulin is of low specificity, as native and [leucine^B-25]-insulin were accumulated to the same extent. Endocytic uptake is of high capacity and the mechanism is saturable. Insulin accumulated in endocytic vacuoles and lysosomes, thus following the classical degradation pathway. No other subcellular compartment is associated with insulin degradation. It was not possible to detect the basolateral uptake, indicating loss of immunoreactivity after binding to its receptor. Absolute quantitative immunocytochemistry is applicable in studying endocytosis. The labeling density increases nonproportionally with antigen density probably caused by steric hindrances. Reduction of the particle size (16 to 6 nm) increased the labeling density 17.6 times.