Kidney Week

Abstract: SA-PO451

Defects in the Exocyst-Cilia Machinery Results in Disease Development in the Kidney and Heart

Session Information

• Development and Regenerative Medicine
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 500 Development, Stem Cells, and Regenerative Medicine

Authors

• Lipschutz, Joshua H., Medical University of South Carolina, Charleston, South Carolina, United States

Joshua H. Lipschutz,

• Fulmer, Diana B., Medical University of South Carolina, Charleston, South Carolina, United States

Diana B. Fulmer,

• Norris, Russell A., Medical University of South Carolina, Charleston, South Carolina, United States

Russell A. Norris,

Background

Patients with polycystic kidney disease such as ADPKD and Joubert syndrome have elevated risk for bicuspid aortic valve disease (BAV). Non-syndromic BAV affects 1% of the population and often leads to aortic stenosis and the need for surgery. We have previously shown a link between these disorders and defects of organelles called primary cilia. Primary cilia are immotile projections of microtubules that act as signaling hubs for numerous pathways. We previously demonstrated in the kidneys that primary cilia are built by a highly-conserved octameric protein-trafficking complex, known as the exocyst. These studies leveraged the expression of the central exocyst protein, EXOC5. Knockdown of EXOC5 in MDCK cells inhibited ciliogenesis and, conversely, EXOC5 overexpression resulted in longer cilia.

Methods

We performed two human GWAS studies, generated two exoc5^-/- zebrafish knockout lines and two conditional Exoc5 mouse knockout lines. Phenotypes were characterized through immunohistochemistry, 3D reconstructions, confocal microscopy, and echocardiography.

Results

GWAS identified the exocyst as being near SNPs most associated with BAV. Next we noted that at three days post fertilization, exoc5^-/- zebrafish embryos had cardiac edema and outflow tract stenosis. This phenotype was rescued in a dose dependent manner by injection of human EXOC5 mRNA at the one cell stage; however, the rescue was significantly reduced with the introduction of a targeted mutation in the highly-conserved EXOC5 VxPx ciliary targeting sequence. These findings prompted us to examine the cardiac phenotype following Exoc5 deletion in mice. We previously reported altered ciliogenesis and nephrogenesis in Exoc5^f/f mice when bred with a kidney-specific Cre. To examine the heart, we bred these mice with endocardial-specific NfatC1-Cre, and later endothelial-specific Tie2-Cre, lines This resulted in mice with highly-penetrant BAV and aortic valve calcification. Echocardiography also revealed aortic valve stenosis and increased aortic root diameter similar to what is seen in BAV patients. There was also evidence of significant cilia disruption, and conditional homozygous mutants were embryonic lethal by E15.5, likely due to cardiac defects.

Conclusion

These data for the first time link cilia, BAV, and the exocyst, and explain the clinical association of cardiac valve abnormalities with PKD.

Funding

• NIDDK Support