The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly-differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically-quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

Significance Statement Loss of function of the 2Cl−/H+ antiporter ClC-5 in Dent disease causes an unknown impairment in endocytic traffic, leading to tubular proteinuria. The authors integrated data from biochemical and quantitative imaging studies in proximal tubule cells into a mathematical model to determine that loss of ClC-5 impairs endosome acidification and delays early endosome maturation in proximal tubule cells, resulting in reduced megalin recycling, surface expression, and half-life. Studies in a Dent mouse model also revealed subsegment-specific differences in the effects of ClC-5 knockout on proximal tubule subsegments. The approach provides a template to dissect the effects of mutations or perturbations that alter tubular recovery of filtered proteins from the level of individual cells to the entire proximal tubule axis. Background Loss of function of the 2Cl−/H+ antiporter ClC-5 in Dent disease impairs the uptake of filtered proteins by the kidney proximal tubule, resulting in tubular proteinuria. Reduced posttranslational stability of megalin and cubilin, the receptors that bind to and recover filtered proteins, is believed to underlie the tubular defect. How loss of ClC-5 leads to reduced receptor expression remains unknown. Methods We used biochemical and quantitative imaging data to adapt a mathematical model of megalin traffic in ClC-5 knockout and control cells. Studies in ClC-5 knockout mice were performed to describe the effect of ClC-5 knockout on megalin traffic in the S1 segment and along the proximal tubule axis. Results The model predicts that ClC-5 knockout cells have reduced rates of exit from early endosomes, resulting in decreased megalin recycling, surface expression, and half-life. Early endosomes had lower [Cl−] and higher pH. We observed more profound effects in ClC-5 knockout cells expressing the pathogenic ClC-5E211G mutant. Alterations in the cellular distribution of megalin in ClC-5 knockout mice were consistent with delayed endosome maturation and reduced recycling. Greater reductions in megalin expression were observed in the proximal tubule S2 cells compared with S1, with consequences to the profile of protein retrieval along the proximal tubule axis. Conclusions Delayed early endosome maturation due to impaired acidification and reduced [Cl−] accumulation is the primary mediator of reduced proximal tubule receptor expression and tubular proteinuria in Dent disease. Rapid endosome maturation in proximal tubule cells is critical for the efficient recovery of filtered proteins.

The proximal tubule (PT) of the kidney is comprised of cells that are uniquely specialized for efficient apical uptake of albumin and other proteins that escape the glomerular filtration barrier. These proteins bind to the multiligand receptors megalin and cubilin and are internalized via clathrin-dependent endocytosis into apical early endosomes that rapidly mature into larger apical vacuoles (AVs). Dissociated ligands are delivered from AVs to lysosomes for degradation, while receptors are collected into dense apical tubules for recycling to the cell surface. When cells are grown under continuous fluid shear stress (FSS), the endocytic uptake is approximately 9-fold greater than static cells. Additionally, cells cultured in this manner rapidly and reversibly adjust their endocytic capacity in response to changes in FSS. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are combining biochemical, imaging, and mathematical modeling approaches to determine the mechanism(s) by which cells chronically and acutely modulate endocytic uptake. Increased levels of megalin transcripts (~2x) and protein (~4x) is insufficient to account for the large increase in endocytic uptake observed in cells cultured under FSS vs static conditions. We used cell surface biotinylation experiments to compare the fraction of megalin at the surface, the fraction endocytosed, and the half-life of surface-tagged megalin under FSS and static conditions. These data were used to adapt a kinetic model that describes kinetics of megalin biosynthesis, trafficking, and degradation in cells. Overall, the cells grown under FSS have a greater endocytic rate and recycling rate compared with static grown cells. By contrast, megalin degradation is faster than recycling in static-grown cells. Colocalization of megalin with known markers of apical compartments will enable us to extend our model to determine whether changes in endosomal maturation rates contribute to these differences. Additionally, cells cultured under continuous FSS rapidly and reversibly adjust their endocytic capacity in response to changes in shear stress. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are currently adapting our model to determine how acute changes in shear stress trigger alterations in endocytic uptake. National Institute of Health: 5T32GM133353-03, R01 DK118726, R01 DK125049 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.