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

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-5 E211G 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.

Fluid Shear Stress-Induced Changes in Megalin Trafficking Enhance Endocytic Capacity in Proximal Tubule Cells.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multiligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is >5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

Fluid shear stress-induced changes in megalin trafficking enhance endocytic capacity in proximal tubule cells.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

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

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.