Localization of Epidermal Growth Factor (EGF) Binding Sites on Antiluminal Plasma Membrane of Rat Kidney: Autoradiographic Study Using Nonfiltering Perfused Rat Kidney

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of ¹²⁵I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of ¹²⁵I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of ¹²⁵I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of ¹²⁵I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the ¹²⁵I-EGF binding sites. Binding of ¹²⁵I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of ¹²⁵I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of ¹²⁵I-EGF perfusion, suggesting the internalization of ¹²⁵I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of ¹²⁵ I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.