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Abstract: FR-PO510

Acute and Chronic Modulation of Kidney Proximal Tubule Endocytic Capacity by Fluid Shear Stress

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Lackner, Emily, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Shipman, Katherine E., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Cowan, Isabella, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Long, Kimberly R., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Weisz, Ora A., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
Background

The kidney proximal tubule (PT) is uniquely specialized for efficient apical uptake of proteins that escape the glomerular filtration barrier. These proteins bind to the multiligand receptors megalin and cubilin and are internalized 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. We have extensively characterized an opossum kidney (OK) PT cell model cultured under continuous fluid shear stress (FSS) that has several-fold higher levels of megalin, cubilin, and endocytic capacity compared with static-grown cells. Additionally, cells cultured in this manner rapidly and reversibly adjust their endocytic capacity in response to acute changes in FSS. We hypothesize that flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration, though these mechanisms are unknown.

Methods

We combined biochemical, imaging, and mathematical modeling approaches to compare the effects of chronic vs acute changes in FSS on megalin trafficking. Biochemical measurements of surface expression, endocytic kinetics, and half-life were combined with quantitative imaging to determine subcellular distribution of megalin with markers of endocytic compartments. Data were fit to a previously validated mathematical model.

Results

Cells cultured under continuous FSS have faster megalin endocytic and recycling rates and slower degradation rates compared with static grown cells. These cells also have reduced steady state fraction of surface megalin due to the enhanced endocytic flux. By contrast, surface expression and endocytic kinetics of megalin were not significantly altered when cells were removed from FSS for 1h, despite an overall reduction in endocytic capacity. Our model predicts that this decrease reflects slower endosomal maturation and megalin recycling.

Conclusion

Chronic vs acute changes in FSS mediate alterations in endocytic capacity via distinct mechanisms. Whereas the primary rate affected when cells are cultured under continuous FSS is megalin internalization, acute drops in FSS preferentially alter intracellular trafficking steps to reduce endocytic uptake.

Funding

  • NIDDK Support