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Kidney Week

Abstract: FR-PO950

Reconstitution of ADPKD Cyst Formation in 3D Culture Reveals a Role for YAP 1 in Cyst Lumen Size

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic

Authors

  • Bacallao, Robert L., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Clendenon, Sherry G., Indiana University, Bloomington, Indiana, United States
  • Wells, Clark D., Indiana University School Medicine, Indianapolis, Indiana, United States
Background

Proximal tubule cells grown in 3D culture, initiate cyst emergence with uniformly small lumens upon expression of CDH8. Prior studies indicate that cyst expansion in ADPKD results from increased proliferation from increased YAP1 signaling. We tested the hypothesis that YAP1 action is sufficient to cause enlarged cyst lumens in 3D culture and YAP1 interference by AMOTL1 decreases lumen size.

Methods

Immune-blots were carried out on lysates of confluent PKD Q4004X and HmPKT cells using antibodies for SAV1, MST1/2, MOB1, p-MOB1 (thr 35), LATS1, YAP/TAZ, p-YAP (ser 127) and p-YAP (ser 397). HK2 cells were transduced with lentivirus bearing constitutively active YAP1-5SA and grown in 3D culture. Cyst emergence was activated by cell transduction with adenovirus bearing CDH8. Separately, PKD Q4004X cells were transduced with lentivirus expressing YAP1-5SA or AMOTL1 (S262E) and grown in Matrigel for 14 days. Cysts were imaged and analyzed with Image J.

Results

Cyst epithelia had lower expression of SAV1, MST2, LATS1, YAP1 and TAZ and reduced ratio of p-YAP (ser 127) over YAP1. These cells also showed increased levels of p-MOB1 and p-YAP (Ser 397). HK-2 cells transduced with lentivirus expressing YAP1-5SA formed normal tubule arrays in 3D culture. However, when cyst emergence is activated by microinjected CDH8 expressing adenovirus, large lumen cysts are formed. In separate experiments, PKD Q4004X cells were grown in Matrigel for 14 days to allow for cyst formation. PKD Q4004X cells transduced with YAP1-5AS or AMOTL1 (S262E) were compared to control PKD Q4004X cells. After 14 days, samples were fixed, stained and imaged to determine cyst size using ImageJ. PKD Q4004X cells had an average maximum area of 177.89 ± 7.16 μm2 . PKDQ 4004X cells transduced with AMOTL1 (S262E) had an average area of 122.77± 6.03 μm2 (p<0.01). PKDQ4004X cells transduced with YAP1-5SA had an average area of 6619.66 ± 773.78 μm2 (p<0.01).

Conclusion

1) Cystogenesis can be reproduced by the combined activation of CDH8 and YAP1.
2) CDH8 expression initiates decreased cell-cell adhesion that is sufficient for cyst emergence.
3) YAP1 expression in the absence of CDH8 activation does not promote cyst formation.
3) Cyst size in ADPKD cells can be inhibited by AMOTL1 and increased by YAP1.
4) Overall, YAP1 activation following cyst emergence is necessary for increased lumen size in ADPKD.

Funding

  • Private Foundation Support