The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized and high capacity apical endocytic pathway that is necessary to recover essential nutrients and to maintain a protein-free urine. Proteins in the ultrafiltrate bind to the multiligand receptors megalin and cubilin on the apical surface of PT cells and are internalized via receptor-mediated endocytosis. Despite the critical role of this pathway in maintaining PT function, the molecular identities of the compartments involved in sorting and recycling in PT cells, and thus the mechanisms by which they are regulated, are largely unknown. Impaired PT endocytosis results in urinary excretion of filtered proteins [termed low molecular weight (LMW) proteinuria]. Dent disease is an X-linked disorder caused by mutations in the CLCN5 gene that encodes CLC-5, an electrogenic 2Cl-/H+ exchanger. Patients with Dent disease present with LMW proteinuria and typically progress to renal failure. Loss of CLC-5 has been shown to decrease the endocytic uptake of filtered ligands in mouse models of Dent disease. PTs from CLCN5 knockout mice exhibit a marked reduction in megalin protein expression (without altered RNA levels) that likely accounts for the reduced endocytic capacity of these cells. It has been suggested that loss of CLC-5 causes enhanced degradation of megalin, but this has not been directly tested and the mechanism by which this might occur is unclear. A significant barrier to understanding the regulation of receptor-mediated apical endocytosis of filtered proteins has been the lack of a highly differentiated cell culture model that retains the organization and high capacity of the PT apical endocytic pathway. The Weisz lab has optimized a cell culture model of differentiated PT cells that develops morphological specializations, metabolism, and endocytic capacity similar to the PT in vivo. Using this system, we can now determine the itinerary, kinetics, and regulation of PT apical endocytic traffic. To this end, I will develop a model to describe the route and kinetics of megalin traffic in PT cells using data acquired from imaging and biochemical approaches, and the itinerary of megalin trafficking will be verified in mouse proximal tubules. This model will be used to make testable predictions for how changes in megalin trafficking result in LMW proteinuria in Dent disease. I will test these predictions in a Dent disease cell culture model, generated using CRISPR/Cas9 technology. I hypothesize that loss of CLC-5 alters megalin trafficking, by impairing recycling and shifting the receptors toward degradative pathways, leading to reduced megalin expression and to LMW proteinuria. Completing the proposed studies will enhance our understanding of how disease can lead to LMW proteinuria and provide insight into new approaches to manipulate PT endocytic capacity to preserve kidney function in proteinuric diseases.

Public Health Relevance

Projective Narrative Recovery of filtered proteins by the proximal tubule by receptor-mediated endocytosis is necessary to maintain protein-free urine and is impaired in many types of kidney disease, including Dent disease. The proposed studies aim to enhance our understanding of the trafficking of megalin, an essential receptor for the uptake of filtered protein, including the organization of the endocytic pathway, trafficking kinetics, and how alterations to this pathway result in disease. My studies will provide insight into the development of therapeutics to manipulate endocytic capacity in proteinuric disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31DK121394-01A1
Application #
9832454
Study Section
Special Emphasis Panel (ZDK1)
Program Officer
Maric-Bilkan, Christine
Project Start
2019-07-01
Project End
2022-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15260