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Abstract: FR-PO293

Molecular Characterisation of Novel Klotho Variants Identified in Patients with Ca2+ Disturbances

Session Information

Category: Bone and Mineral Metabolism

  • 501 Bone and Mineral Metabolism: Basic

Authors

  • Grigore, Teodora, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Bayliss, Asha, University of Oxford, Oxford, United Kingdom
  • Stevenson, Mark, University of Oxford, Oxford, United Kingdom
  • Zuidscherwoude, Malou, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Carey, David J., Geisinger Health, Danville, Pennsylvania, United States
  • Haley, Jeremy S., Geisinger Health, Danville, Pennsylvania, United States
  • Smelser, Diane T., Geisinger Health, Danville, Pennsylvania, United States
  • Olauson, Hannes, Karolinska Institutet, Stockholm, Stockholm, Sweden
  • Hoenderop, Joost, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Thakker, Rajesh V., University of Oxford, Oxford, United Kingdom
Background

CKD is the long-term loss of kidney function, which exerts multiple repercussions on other numerous organs, including the cardiovascular system. The progression of CKD is significantly associated with decreased serum levels of Klotho and elevated serum levels of FGF23, however the precise underlying molecular mechanisms are yet unsolved. Although CKD has a high heterogeneity in molecular changes, the disruption of the Klotho-FGF23 axis is a common feature, considering that the decline in serum levels of Klotho as the disease progresses is constant in all stages of CKD. Klotho is a protein with a large ectodomain that can get cleaved and released into circulation. Expressed in the proximal and distal tubules, Klotho is demonstrated to affect renal PO43- excretion by interacting with NPT2a and reabsorption of Ca2+ by anchoring TRPV5.

Methods

The Klotho variants (c.1151T>C, c.1489T>C, c.1819A>G, c.2069C>T, c.2590G>A, c.2620G>A, c.3008A>G) were identified in a cohort by whole-exome sequencing. The variants were transiently transfected in HEK293 cells. The variants were characterized by investigating the shedding pattern, conditional cleavage, presence on the cell surface, cellular localization and co-localisation with TRPV5.

Results

Full-length shed Klotho (±130 kDa) and Klotho domains (±60 kDa) can be cleaved and identified by Western blotting. All results are compared to the wild-type Klotho condition. c.1151T>C, c.2590G>A and c.2620G>A do not show full-length shed Klotho nor shed Klotho domains, whereas c.1819A>G seems to have a lower molecular weight of the shed Klotho domain. In accordance with previous results, c.1151T>C, c.2590G>A and c.2620G>A show no surface expression on the cellular membrane. The variants c.1151T>C and c.2620G>A appear to have a diffused cytoplasmic localization, in contrast to wild-type Klotho, which has a membranous localization.

Conclusion

Our study indicates that the novel identified Klotho variants have an effect on the expression and shedding of Klotho, which directly affect the serum levels of circulating Klotho, leading to a potential decrease in circulating Klotho levels. These recent findings should allow for novel investigations on the effect of Klotho shedding in CKD.