Abstract: SA-PO571
Acute Regulation of the Proximal Tubule Endocytic Pathway in Cell Culture and Ex-Vivo Kidney Slices
Session Information
- Noncystic Mendelian Diseases
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Genetic Diseases of the Kidney
- 802 Non-Cystic Mendelian Diseases
Authors
- Baty, Catherine J, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Long, Kimberly R., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
- Weisz, Ora A., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background
The proximal tubule (PT) of the kidney is highly specialized for apical endocytosis of megalin/cubilin ligands that pass through the glomerular filtration barrier into the tubule lumen. Impaired uptake of these ligands results in tubular proteinuria, which is observed in genetic diseases such as Lowe syndrome, Dent disease, and sickle cell disease. Despite the critical role of endocytosis for PT function, we know little about how the apical endocytic pathway is regulated in these cells. Studies in PT cells demonstrate that endocytosis of megalin/cubilin ligands is rapidly responsive to changes in fluid shear stress (FSS). However, currently available PT cell culture models lack many key features needed for PT function. Additionally, it is not known whether cells lining the PT in vivo also respond endocytically to changes in FSS.
Methods
We have established an OK cell culture system that dramatically enhances cell differentiation and more closely recapitulates the morphology and robust apical endocytic capacity of PT cells in vivo. We used fluorescence-based assays and confocal imaging to visualize endocytic uptake in PT cells exposed to variable levels of FSS. As a complementary approach, we optimized conditions to study endocytic uptake in ex vivo ultra-thin (<150 mm) murine kidney slices. C57Bl/6J mice (Jackson Laboratory) were anesthetized with isoflurane and kidneys were perfused, surgically harvested and immediately sectioned for culture and functional assays to visualize endocytosis.
Results
Our cell culture model demonstrates dramatically increased apical endocytic capacity compared with OK cells cultured under standard conditions. Additionally, endocytosis of megalin/cubilin ligands is rapidly and reversibly modulated by changes in FSS. Consistent with our studies in cell culture, endocytic uptake was enhanced in slices exposed to shear stress. Studies are in progress to assess the mechanism of FSS-mediated uptake in PT cells and whether this is impaired in diseases that result in tubular proteinuria.
Conclusion
Together, these complementary models offer a useful approach to dissect the effect of physiologically-relevant stimuli on the PT apical endocytic pathway and its disruption in genetic and other diseases that result in tubular proteinuria,
Funding
- NIDDK Support