An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule

Abstract

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.

Impaired Endosome Maturation Mediates Tubular Proteinuria in Dent Disease Cell Culture and Mouse Models

“…Adapted from Shipman et al , 2022, Function . 29 (B–F) Rescue and ClC-5 KO cells on permeable supports were fixed and processed to detect megalin colocalization with the markers shown in panel A. Representative sum projection images of six planes were cropped to show a region of high colocalization of megalin with (B) EEA1, (C) Rab7, and (D) Rab11a within a single cell.…”

“…This steady state distribution was used to estimate intracellular kinetic rates with the assumption that 51% of recycling megalin traffics through apical vacuoles (AVs) (α=0.51) for all cell lines. 29 The predicted fractional rates for all three cell lines are shown in Supplemental Table S8. Supplemental Figure S7, C–E shows how the values of km,1, kDAT,f, kDAT,s change with the value of α for rescue, ClC-5 KO, and uncoupled cells.…”

“…Megalin within lysosomes is degraded at rate k d , and newly synthesized megalin (S) enters the system through AVs. Adapted from Shipman et al, 2022, Function 29. (B) Megalin steady state distribution among the endocytic compartments, surface, AEEs, AVs, DATs, and lysosomes (Lys), in rescue and ClC-5 KO cells (mean6SEM), which was calculated based on the average fractional colocalization of megalin with each marker in Figure3and the overlaps between the markers in Supplemental FigureS3.…”

“…The table (right) shows the steady state distribution of megalin in WT and ClC-5 KO mice in megalin molecules for every 10,000 molecules in WT and 1512 molecules in ClC-5 KO, based on the calculated differences in total megalin expression in panel A. Adapted from Shipman et al, 2022, Function 29.…”

It is Good to Recycle: Bringing Megalin Back to the Membrane to Stop Proteinuria

“…In a new study recently published in Function , this group used membrane surface biotinylation assays and antibody staining in OK cells to investigate rates of internalization and steady state distribution of megalin, respectively. 6 From this, they were subsequently able to generate a new mathematical model of megalin trafficking, allowing them to explore the effects of perturbing individual steps in the pathway. Moreover, by performing antibody staining for established endolysosomal markers in mouse kidney PTs, they have attempted to extrapolate key aspects of their model to the in vivo situation.…”