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Abstract: PO1588

Toward a Molecular Mechanism for Low-Molecular-Weight Proteinuria in Dent Disease

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

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

Dent disease is a progressive X-linked disorder caused by loss of function of the Cl-/H+ exchanger CLC-5. Early symptoms include low molecular weight (LMW) proteinuria resulting from inefficient recovery of filtered proteins by megalin and cubilin receptors in the proximal tubule (PT). Knockout of Clc-5 in mice recapitulates the LMW proteinuria observed in human disease and decreases protein (but not mRNA) levels of megalin and cubilin. How loss of CLC-5 leads to reduced receptor expression remains unknown. Previous gene expression studies in Clcn5 KO mice suggest there are alterations in cholesterol and lipid metabolism. Elevated cholesterol levels have been demonstrated to alter the organization of the endocytic pathway and impair receptor recycling in cultured cells. We hypothesize that altered cholesterol metabolism impairs megalin traffic through the recycling pathway and promotes its degradation.

Methods

We used siRNA knockdown (KD) and CRISPR/Cas9 knockout (KO) and rescue approaches in an opossum kidney (OK) cell culture model that recapitulates morphologic and functional features of the PT in vivo to study the role of Clc-5 in the endocytic pathway. Additionally, we assessed PT function, megalin/cubilin expression, and cholesterol distribution in newly generated CRISPR/Cas9 Clc-5 KO mice.

Results

KD or KO of Clc-5 resulted in significantly decreased endocytic uptake of fluorescently labeled albumin that was fully rescued by heterologous expression of wild-type human CLC-5. Additionally, the half-life of megalin was reduced in Clc-5 depleted cells. We confirmed LMW proteinuria in the KO mice. Heterozygous females also have reduced PT albumin uptake and megalin expression. We observed an accumulation and a redistribution of cholesterol in PTs of heterozygous mice and in Clc-5 KD OK cells.

Conclusion

Our new cellular models for Dent disease should enable us to identify the molecular mechanism that results in reduced megalin/cubilin expression and determine whether altered cholesterol metabolism contributes to the LMW proteinuria observed in Dent disease.

Funding

  • NIDDK Support