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Kidney Week

Abstract: PO1221

Investigation of Lad1 as a Candidate Modifier of Polycystic Kidney Disease in Pkd1 Mouse Models

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Pearson, Elisabeth, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
  • Menezes, Luis F., National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
  • Germino, Gregory G., National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
Background

Polycystic Kidney Disease (PKD) is a monogenic disease caused by mutations in either PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. One proposed function for PC1 is to regulate cell migration and cell-cell interaction, possibly through modulation of the cytoskeleton. In prior transcriptomic studies of Pkd1 mutant kidneys, we had identified Lad1 as one of the few genes whose expression was dysregulated early in the course of the disease. The limited available literature suggests Lad1 may encode a cytoskeleton protein that acts as an anchoring filament to the basement membrane of epithelial cells. The goal of this study is to investigate the role of Lad1 in mediating/modifying PKD phenotypes.

Methods

We quantified Lad1 gene and protein expression in the tissues of Pkd1 conditional mutant mice, kidney epithelial cell lines derived from Pkd1 conditional mice and human keratinocyte HaCat cells by quantitative polymerase chain reaction (qPCR) and western blot analyses. We used CRISPR technology to generate two Lad1 mutant mouse lines that have large deletions spanning most of the coding region of Lad1 and characterized the phenotype of homozygous mutants. Lad1 mutants were crossed with Pkd1cond/cond mice with Ksp-Cre and tamoxifen-Cre to test for genetic interaction in early and late onset PKD models.

Results

We confirmed that Pkd1 mutant kidneys and epithelial cells have lower Lad1 expression levels compared to wild type samples. Mice with homozygous deletion of Lad1 exons 3 to 8 were born at normal mendelian ratios and lacked obvious abnormalities up to 1 year of age. Histopathologic analyses of the kidney and organs with the highest expression of Lad1 also were normal. Pkd1/Lad1 double mutants were born at expected rates and lacked distinctive phenotypic features. However, early data suggest that Pkd1cond/cond; Ksp-Cre+/ Lad1 homozygous mutants have increased kidney-body weight ratios with worse cystic disease. Further analysis, including Lad1 CRISPR knock-out animals crossed with late-stage conditional Pkd1 knock-out animals, is ongoing.

Conclusion

Lad1 expression is reduced in Pkd1 mutant kidneys. CRISPR knock-out of Lad1 alone shows no obvious phenotype. Loss of Lad1 may worsen disease in the early onset Pkd1-cystic model.

Funding

  • NIDDK Support