Kidney Week

Abstract: SA-PO1004

Characterization of the Effects of a Vacuolar H+-ATPase (V-ATPase) Mutation Linked to Distal Renal Tubular Acidosis (dRTA)

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Juarez, Jordan J., Keck School of Medicine of USC, Los Angeles, California, United States

Jordan J. Juarez,

• Pham, Jessica, Keck School of Medicine of USC, Los Angeles, California, United States

Jessica Pham,

• Ho, Pei-Yin, Keck School of Medicine of USC, Los Angeles, California, United States

Pei-Yin Ho,

• Mancino, Valeria, Keck School of Medicine of USC, Los Angeles, California, United States

Valeria Mancino,

• Bhalla, Vivek, Stanford University, Stanford, California, United States

Vivek Bhalla,

• Pastor-Soler, Nuria M., Keck School of Medicine of USC, Los Angeles, California, United States

Nuria M. Pastor-Soler,

Background

Distal renal tubular acidosis (dRTA) Type I is a disease affecting the kidney’s ability to secrete non-volatile acid. This disease can lead to severe kidney and systemic sequelae. Improper functioning of collecting duct (CD) intercalated cells (ICs) due to autoimmune or infectious conditions can lead to dRTA. More recently, single point mutations in transport proteins in ICs have been found to result Type I dRTA. One such mutation is the 1231G>T in ATP6V0A4, the a4 isoform of the V₀domain of the V-ATPase. This mutation results in a D411Y (Aspartate to Tyrosine) variant. Carriers of this mutation are seemingly healthy and therefore this mutation likely acts in an autosomal recessive fashion. We identified a 26 year-old man with refractory dRTA that was homozygous for this mutation by a NextGen dRTA sequencing panel. One of his half-siblings has presented with metabolic acidosis and hypokalemia, consistent with dRTA. Published reports from Brazil and Mexico identify this mutation as causative for dRTA. However, the exact effects of this mutation on the expression and activity of the V-ATPase has yet to be determined. We hypothesized that this mutation in a reportedly non-transmembrane region of the a4 subunit would have effects on the subcellular localization of the subunit as the underlying mechanism for decreased V-ATPase membrane function.

Methods

We performed transfections of FLAG-Tagged wild-type (WT) vs. D411Y pTracer plasmids into HEK and Clone C cells (of IC origin), followed by immunoblot and immunofluorescent labeling and confocal imaging.

Results

Our results demonstrate low expression of both WT- and D411Y ATP6V0A4 in Clone-C cells, although an anti-FLAG antibody detected a band at ~100 kDa by immunoblot in transfected Clone-C lysates. By immunolabeling and confocal microscopy we detected the WT subunit at the apical domain of transfected Clone-C polarized monolayers on transwell filters. In contrast, the D411Y mutant subunits were detected in a cytosolic distribution.

Conclusion

Our preliminary findings indicate that the D411Y mutation interferes with normal accumulation of the V-ATPase at the apical membrane of ICs, thus offering a potential mechanism for defective V-ATPase function and dRTA patients with this mutation.

Funding

• Private Foundation Support