Kidney Week

Abstract: PO1236

Mechanistic Interaction Between Cystin and Fibrocystin/Polyductin in Model Cell Lines and cpk/cpk Kidneys

Session Information

• Cystic Kidney Disease - I
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Zhang, Yiming Jason, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Yiming Jason Zhang

• Yang, Chaozhe, Children's National Research Institute, Washington, District of Columbia, United States

Chaozhe Yang,

• Harafuji, Naoe, Children's National Research Institute, Washington, District of Columbia, United States

Naoe Harafuji,

• Wang, Wei, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Wei Wang,

• Bell, Phillip Darwin, The University of Alabama at Birmingham, Birmingham, Alabama, United States

Phillip Darwin Bell,

• Guay-Woodford, Lisa M., Children's National Research Institute, Washington, District of Columbia, United States

Lisa M. Guay-Woodford,

• Bebok, Zsuzsanna M., The University of Alabama at Birmingham, Birmingham, Alabama, United States

Zsuzsanna M. Bebok,

Background

Cys1^cpk/cpk (cpk) mice exhibit ARPKD-like renal phenotype due to a mutation in the Cys1 gene and loss of cystin. ARPKD (MIM 263200) is caused by mutations in PKHD1, encoding FPC. Both cystin and FPC are present in the primary cilium, but no physical interaction has been reported. Using mouse CCD cell lines, we have shown that FPC levels were reduced in cpk cells by 75% relative to wt. Cystin deficiency is specifically linked to FPC reduction and did not affect cilia development, but altered ciliary architecture (ASN 2020). The current study focuses on cellular mechanisms driving FPC reduction in cystin-deficient cells, and the consequences of FPC loss. We note that FPC is necessary for proper E3 ubiquitin ligase function and consequently cellular proteome management (Kaimori 2017).

Methods

Immortalized wt and cpk mouse CCD cells. Wt and cpk mouse kidneys. siRNA silencing of Cys1; qRT-PCR, western blot, confocal microscopy, morphometry, patch clamp.

Results

Silencing Cys1 in wt cells results in a siRNA dose-dependent reduction of both cystin and FPC. Correlative studies showed marked reduction of FPC in cpk kidneys. Similar Pkhd1 mRNA levels in wt and cpk cells, and kidneys implicate FPC regulation at the protein level. Proteasome or lysosome inhibition did not recover FPC, but activation of autophagy further reduced FPC levels, suggesting a role for selective autophagy in FPC removal. Diminished FPC levels lead to E3 ubiquitin ligase defects and reduced polyubiquitination of proteins, necessary for proteome management. In cpk cells, we observed membrane retention of the epithelial sodium channel and increased sodium transport.

Conclusion

Our studies provide the first functional link between cystin and FPC in renal epithelial cells. We propose cystin as a gatekeeper for FPC at the base of the cilium and in the E3 ligase complex. In cystin-deficient cells, FPC is continuously degraded leading to dysregulated ubiquitination and altered proteome homeostasis. These data show a mechanistic connection between the renal phenotypes observed in human ARPKD and cpk mice. The recent identification of human CYS1-related ARPKD (Sci Report, in press) highlights the potential significance of the cystin-FPC mechanistic interaction in the complex pathobiology of ARPKD.

Funding

• NIDDK Support