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Kidney Week

Abstract: SA-PO468

Polycystin 1 Regulates Cilia Length and Cyst Formation by Controlling Centrosomal ARHGAP35-Dependent RhoA Activation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Streets, Andrew J., University of Sheffield, Sheffield, United Kingdom
  • Prosseda, Philipp Paolo, University of Sheffield, Sheffield, United Kingdom
  • Boudiffa, Maya, University of Sheffield, Sheffield, United Kingdom
  • Ong, Albert C., University of Sheffield, Sheffield, United Kingdom

Group or Team Name

  • Kidney Genetics Group
Background

Mutations in PKD1 (encoding for PC1) account for most patients with ADPKD but it is still unclear how these result in the highly complex cellular phenotype of ADPKD. It had been reported that primary cilia length is normal in ADPKD cells implying an abnormality in function (flow-dependent signalling); however this hypothesis has been recently challenged. Unexpectedly, we observed consistently shorter cilia in patient-derived PKD1 cystic cells compared to normal tubular cells and its correction by cytochalasin-D, an inhibitor of actin polymerisation.

Methods

RhoA activity (GST pulldown) and its localisation (RhoA biosensor) were determined in ciliated cells; siRNA knockdown of reported centrosomal ARHGAPs identified from ciliome and centrosome databases and their effect on cilia length; PKD1 null human tubular cells were generated by CrispR/Cas9 mutagenesis; BioID proximity assay using a centrosome targeting sequence (PACT) to confirm centrosomal localisation of ARHGAPs; effect of a selective ROCK inhibitor (hydroxyfasudil) in a Pkd1 mouse model.

Results

We confirmed that primary cilia are shorter in vivo (human ADPKD and mouse Pkd1 kidneys) and in PKD1 null CrispR cells. In ciliated cells, increased RhoA (but not Cdc42 or Rac1) activation was observed in PKD1 cystic cells with a localised increase at the cilia base. Cilia length could be normalised by Rho kinase (ROCK) inhibitors or expression of dominant negative RhoA (but not Cdc42 or Rac1). Knockdown of several centrosomal ARHGAPs (5, 29, 35) resulted in shorter cilia but only ARHGAP35 centrosomal localisation was reduced in the absence of PKD1. Specific binding of ARHGAP35 to the PC1 C-terminal domain was shown by co-IP. Finally, we demonstrate that selective ROCK inhibition reduced cyst growth in vitro (PKD1 human cystic cells) and in vivo (Pkd1 mouse).

Conclusion

PC1 appears to regulate centrosomal RhoA activity through the recruitment or stabilisation of ARHGAP35. Mutations in PKD1 lead to shorter cilia due to increased RhoA and ROCK activity. Interestingly a recently reported ARHGAP35 hypomorphic mouse develops shorter cilia and glomerular cysts. Inhibition of ROCK normalised cilia length and reduced cyst expansion suggesting that the RhoA/ROCK pathway is a potential new axis to develop therapies to inhibit cyst initiation in ADPKD.

Funding

  • Government Support - Non-U.S.