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Abstract: TH-PO738

Model of Megalin Trafficking in Differentiated Proximal Tubule Cells and Its Application in Dent Disease

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Shipman, Katherine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Long, Kimberly R., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Rbaibi, Youssef, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Baty, Catherine J., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Weisz, Ora A., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized, high capacity apical endocytic pathway necessary to maintain a protein-free urine. The multiligand receptor megalin plays an essential role in the binding and uptake of proteins in the ultrafiltrate. The molecular identities of the compartments involved in sorting of ligands and recycling of receptors in PT cells and the kinetics of megalin trafficking through them are unknown. Dysfunctional PT endocytosis results in tubular proteinuria that can progress to renal failure, as is commonly observed in Dent disease, an X-linked disorder caused by mutations in the Cl-/H+ exchanger CLC-5. Reduced endocytic uptake in Dent disease is likely due to decreased expression of megalin receptors (without altered mRNA levels); however, the mechanism by which this occurs is unclear.

Methods

We previously discovered that OK cells cultured under continuous fluid shear stress develop morphological and functional features similar to that of the PT in vivo, including high apical endocytic capacity and increased megalin expression. Using biochemical techniques, we have estimated endocytic and recycling rates and the half-life of surface megalin. These data were used to construct a model of megalin trafficking in differentiated PT cells. To study megalin trafficking in a Dent disease cell model, we knocked-down CLC-5 expression in these cells using siRNA. Typical knockdown efficiency was ~70%.

Results

We present an ordinary differential equation model of megalin trafficking describing surface and internalized pools of megalin with estimated kinetic parameters. The model is capable of achieving a steady-state with a much larger pool of internalized megalin compared to surface within a range of realistic parameters; this is consistent with observations made by our biochemical assays and by indirect immunofluorescence of endogenous megalin. In our Dent disease cell model, we observed decreased megalin expression as observed in vivo, and we are quantifying megalin trafficking kinetics in these cells to identify the affected step(s).

Conclusion

Our new PT cell culture model provides an ideal system in which to determine the organization of the apical endocytic pathway and to delineate the mechanistic basis of genetic diseases that cause tubular proteinuria.

Funding

  • NIDDK Support