Kidney Week

Abstract: SA-PO630

Characterization of a Novel Mutation Associated with Renal Tubular Acidosis

Session Information

• Genetic Kidney Diseases: Models, Mechanisms, and Therapies
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Bernert, Christopher M., Western University of Health Sciences College of Osteopathic Medicine of the Pacific-Northwest, Lebanon, Oregon, United States

Christopher M. Bernert

• Mendelsohn, Bryce, Kaiser Permanente, Oakland, California, United States

Bryce Mendelsohn, MD, PhD

• Salyer, Anne S., Kaiser Permanente, Oakland, California, United States

Anne S. Salyer, MD

• Breen, Anna K., Western University of Health Sciences College of Osteopathic Medicine of the Pacific-Northwest, Lebanon, Oregon, United States

Anna K. Breen

• Wasko, Brian, Western University of Health Sciences College of Osteopathic Medicine of the Pacific-Northwest, Lebanon, Oregon, United States

Brian Wasko, PhD

Background

Primary Distal Renal Tubular Acidosis (dRTA) is a condition involving an impaired ability of the distal renal tubules to remove protons from blood. ATP6V1B1 encodes a kidney specific protein subunit of the V-ATPase proton pump involved in acid secretion into urine. A patient with classic dRTA has been identified with a de novo single nucleotide polymorphism (SNP) in ATP6V1B1, resulting in a Phe365Leu mutation of uncharacterized pathogenicity. Evolutionary conservation between Homo sapiens ATP6V1B1 and Saccharomyces cerevisiae (yeast) VMA2 allows for the use of yeast to elucidate the impact of the novel ATP6V1B1 mutation on V-ATPase function.

Methods

CRISPR/Cas9 was used to generate yeast strains expressing VMA2 Tyr352Leu, which serves as an analogous mutation to ATP6V1B1 Phe365Leu. Additional yeast strains were generated containing Tyr352Phe as well as a frameshift mutation for use as controls. V-ATPase function was assessed by assaying S. cerevisiae growth under conditions previously shown to inhibit growth of V-ATPase mutants. The growth assays included control YPD media, YPD with 50 mM CaCl₂, YPD with 1 mM paraquat (forming reactive oxygen species), and glycerol containing YPG media (to induce respiration).

Results

S. cerevisiae haploid strains containing a VMA2 Tyr352Leu mutation were found to exhibit growth phenotypes consistent with a complete loss of V-ATPase activity, including an inability to grow when exposed to 50 mM CaCl_2, an inability to grow under respiratory conditions, and sensitivity to reactive oxygen species. Yeast containing the humanized VMA2 Tyr352Phe mutation were not found to display these phenotypes.

Conclusion

Our results provide supportive evidence that a Phe365Leu mutation within ATP6V1B1 results in a loss of function and thus is highly probable to contribute to dRTA.

Growth of haploid yeast VMA2 mutants under various assay conditions.