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Kidney Week

Abstract: SA-PO768

Determine the Pathogenic Mechanism Underlying Infantile ADPKD Caused by a Novel Monoallelic NEK8 Mutation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Chen, Chuan, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Harris, Peter C., Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Ling, Kun, Mayo Clinic Minnesota, Rochester, Minnesota, United States
Background

ADPKD is mainly caused by mutations in PKD1 and PKD2 that encode polycystins, PC1 and PC2, respectively. Current evidence suggests that the dosage of PCs in primary cilia controls the severity and progression of ADPKD. But the molecular mechanisms mediating the trafficking of PCs into primary cilia remain unclear. NEK8 is the only known protein kinase residing in primary cilia, in the Inversin compartment, and is associated with a syndromic ciliopathy when both alleles are mutated. Interestingly, a novel monoallelic NEK8 mutation (p.Arg45Trp) was recently identified in 3 Mayo families with early onset PKD without extrarenal manifestations. This finding suggests that the mutation selectively causes PKD, and thus represents a unique tool for investigating the molecular mechanisms specifically regulating renal cystogenesis.

Methods

Mutagenesis
Transfection
CRISPR/Cas9
Immunostaining
Kinase assay
Fluorescence

Results

To understand how p.Arg45Trp (R45W) affects NEK8, we inactivated Nek8 in IMCD3 cells, and then re-expressed wild type (WT) or R45W variant NEK8. Primary cilia in Nek8-/- cells were truncated but were rescued equally well by NEK8-WT and NEK8-R45W, which show comparable expression and stability. The level of GPR161 in cilia remained the same with or without Nek8. Yet, ANKS6 as a binding partner of NEK8, lost its localization in Nek8-/- cilia, which was fully recovered by reexpressing NEK8-WT or R45W. These data suggest that the overall structure of primary cilia is not affected by the R45W mutation. However, we observed that PC2 levels significantly decreased in Nek8-null cilia; a defect rescued by NEK8-WT but not R45W, although the total protein level of PC2 appeared comparable. Our results indicate that the R45W mutation specifically impairs PC2 in cilia. Moreover, we showed that the autophosphorylation of NEK8-R45W was significantly decreased compared to NEK8-WT, implying a partial loss of kinase activity. This suggests that adequate NEK8 kinase activity is necessary for supporting the normal level of PC2 in cilia.

Conclusion

We found that a novel ADPKD variant of NEK8 (R45W) selectively repressed the ciliary level of PC2. In future, we will seek NEK8 partners in cilia that are specifically affected by this single-point mutation in NEK8. Our work will provide critical information to the understanding of PC trafficking in cilia and ADPKD etiology.

Funding

  • Other U.S. Government Support