Endocytic adaptation to functional demand by the kidney proximal tubule

Abstract

The kidney proximal tubule (PT) efficiently recovers the low level of albumin and other proteins that normally escape the glomerular filtration barrier. Two large receptors, megalin and cubilin/amnionless (CUBAM), bind to and efficiently retrieve these predominantly low molecular-weight proteins via clathrin-mediated endocytosis. Studies in cell culture models suggest that PT cells may sense changes in shear stress to modulate recovery of filtered proteins in response to normal variations in filtration rate. Impairments in PT endocytic function lead to the excretion of filtered proteins into the urine (tubular proteinuria). Remarkably, when the glomerular filtration barrier is breached, the PT is able to recover excess albumin with a capacity that is orders of magnitude higher than normal. What mediates this excess capacity for albumin uptake under nephrotic conditions, and why doesn't it compensate to prevent tubular proteinuria? Here we propose an integrated new working model to describe the PT recovery of filtered proteins under normal and nephrotic states. We hypothesize that uptake via the fluid phase provides excess capacity to recover high concentrations of filtered proteins under nephrotic conditions. Further, concentration of tubular fluid along the tubule axis will enhance the efficiency of uptake in more distal regions of the PT. By contrast to cells where fluid phase and receptor-mediated uptake are independent pathways, expression of megalin is required to maintain apical endocytic pathway integrity and is essential for both uptake mechanisms. This model accounts for both the high-affinity and the high-capacity responses to filtration load in physiological and pathological states.

Keywords: albumin; cubilin; endocytosis; glomerular filtration rate; megalin; proteinuria.

Figure 1.. PT organization and endocytic uptake of filtered ligands

A, the proximal tubule consists of convoluted (PCT) and straight (PST) regions, and can be further subdivided into S1, S2 and S3 segments. The S3 segment extends into the outer stripe of the medulla and may have different lengths in cortical vs. juxtamedullary nephrons. B, ligands that normally escape the filtration barrier are internalized by receptor mediated endocytosis in clathrin-coated pits (CCPs) that invaginate from the base of apical microvilli on PT cells. Ligands dissociate from their receptors as acidified apical early endosomes (AEEs) mature into apical vacuoles (AVs) and are delivered to lysosomes (Lys) for degradation, whereas receptors are recycled to the apical surface in dense apical tubules (DAT). C, megalin and cubilin receptors mediate the internalization of filtered ligands by PT cells. Megalin contains a single transmembrane domain whereas cubilin trimers traffic as a CUBAM complex together with the membrane spanning protein amnionless (AMN). Megalin and AMN cytoplasmic tails contain motifs that engage the Dab2 endocytic adaptor protein, which facilitates receptor recruitment to clathrin-coated pits. Figure created with BioRender.com.

Figure 2.. Possible mechanisms for modulation of apical endocytic capacity by flow

A, endocytosis of megalin and cubilin normally proceeds via clathrin-coated pits (CCP) that bud from the base of PT microvilli. We speculate that under conditions of increased flow, there could be an increase in the number or maturation of pits that form (B) and/or an increase in the average size of clathrin-coated structures (C). Bending of the microvilli and/or the primary cilium at the centre of the apical plasma membrane may mediate flow-dependent changes in endocytic uptake. Figure created with BioRender.com.

Figure 3.. Models for PT uptake of normal and nephrotic levels of albumin

A, under normal conditions, filtered albumin is retrieved largely in the S1 segment and is mediated by direct binding to cubilin receptors. Tubular proteinuria results from loss of either megalin or cubilin function: loss of cubilin prevents high-affinity albumin uptake, whereas loss of megalin ablates the low-affinity uptake site and also compromises the integrity of the apical endocytic pathway. Modulation of endocytic capacity may enable PT cells to accommodate normal variations in GFR as shown in Fig. 2. B, when high concentrations of albumin enter the tubule lumen, as occurs under nephrotic conditions, uptake of cubilin- and megalin-bound albumin along the tubule axis becomes saturated. Under these conditions, non-saturable fluid-phase uptake provides a reservoir for retrieval of excess albumin. Uptake via this pathway occurs via the same endocytic compartments that mediate the internalization of receptor-bound albumin. Fluid phase uptake becomes more efficient in distal regions of the PT because the concentration of ligand increases as tubular fluid is progressively removed by ion transport mechanisms along the PT axis. Although albumin uptake in the figure is depicted to increase preferentially towards the distal end of the PT to illustrate this point, the actual profile of albumin recovery in S1, S2 and S3 segments will reflect the balance between the availability of binding sites and kinetics of receptor-mediated endocytosis, the endocytic volume that accompanies receptor-mediated uptake, and the tubular concentration of albumin along the tubule axis. Figure created with BioRender.com. [Correction made on 15 July 2021, after first online publication: Figure 3 has been updated to re-instate the albumin “dots” in the cells, which were previously missing.]