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Kidney Week

Abstract: SA-PO084

Identification of Enhancers and Their Impact on the Kidney Injury Regulator Klotho

Session Information

  • AKI: Mechanisms - III
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Jankowski, Jakub, Veterinarmedizinische Universitat Wien, Wien, Wien, Austria
  • Lee, Hye kyung, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
  • Wilflingseder, Julia, Veterinarmedizinische Universitat Wien, Wien, Wien, Austria
  • Hennighausen, Lothar, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
Background

Enhancer activity provides new insights into the gene expression machinery and is emerging as a valuable target of research and therapy. Enhancer disruption is involved in development of human disease, but can also be used as a tool to investigate genes key for disease progression and recovery.
There is a well-known connection between kidney function and Klotho. While decrease in Klotho is a known sign of kidney disease, deletion of the gene in mice results in severe phenotype and greatly shortened lifespan, making investigating Klotho knockouts challenging.
Previously, we identified two putative enhancers of Klotho, which lost activity in a kidney injury setting. In this study, we used a novel way of gaining insight into the role of Klotho. We alteried gene regulatory elements, rather than the gene itself, to investigate the impact of the two putative enhancers on Klotho expression, and physiological effects of their deletion.

Methods

We used Crispr/Cas9 to generate six viable mouse lines with deletions in the distal, proximal or both Klotho enhancers, including selective deletion of HNF1b transcription factor binding site. We used qPCR and RNA-seq to investigate gene expression in the kidney, and a blood biochemistry panel and ELISA to investigate phenotype. We also used Chip-seq to analyze changes in histone modifications with focus on the Klotho promoter and enhancer regions to assess their activity and verify deletions.

Results

Deletion of distal, but not proximal enhancer resulted in 50% lower Klotho expression, but only when HNF1b binding site was impacted. Inactivity of the proximal enhancer was confirmed by lack of further expression decrease in double knockout mice. Despite significant Klotho depletion, mice did not display Klotho knockout phenotype, as lifespan, weight, serum phosphate, calcium and FGF23 levels were not different compared to controls. RNA-seq analysis revealed altered expression of several genes associated with kidney disease.

Conclusion

Our study provides new insight into Klotho regulation. We identified a new regulatory element of the Klotho gene as well as the transcription factor responsible for its activity. Despite significantly lower Klotho expression levels, normal phenotype is preserved. Susceptibility of the mice to injury remains to be investigated and will further elucidate Klotho’s impact on renal health.

Funding

  • NIDDK Support