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Kidney Week

Abstract: FR-PO291

Exploring the Biology of Non-Cleavable Klotho

Session Information

Category: Bone and Mineral Metabolism

  • 501 Bone and Mineral Metabolism: Basic

Authors

  • Pastor, Johanne Virginia, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Moe, Orson W., The University of Texas Southwestern Medical Center, Dallas, Texas, United States
Background

Klotho is a type-I transmembrane protein that is a cell maintenance and cytoprotective factor. Klotho protein is primarily expressed in the kidney, but it is also found in the brain, parathyroid gland, and reproductive organs. Klotho protein exists in membrane-bound and soluble forms. Membrane- Klotho acts as a co-receptor for fibroblast growth factor (FGF)23, while the soluble form of Klotho circulates and acts systemically on distant organs. Known secretases cleave the membrane klotho to produce the soluble forms of circulating Klotho. The relative contribution of the two forms of Klotho to biology and pathobiology is not known as experimental genetic manipulation of Klotho affects both forms. Low circulating levels of Klotho is associated with numerous disorders, including chronic kidney disease, cardiovascular disease, and cognitive impairment. To dissociate the function of two forms of Klotho, we aim to generate a non-cleavable form of the Klotho protein (NCK), which will provide us with the means the creation to create of a murine model to study physiology and pathophysiology.

Methods

Using previously published data and our own mutational analysis of the putative cleavage sites, we finalized on one mutant called non-cleavable Klotho (NCK) mKLdL9-956-S958L, with the replacement of 9 aa at 956LGSGTLGFR-to GGLGGGSGG and expressed it in HEK293 cells.

Results

We proceeded to test that NCK is: 1. Expressed normally and trafficked to cell surface, 2. Functionally intact. 3. Indeed, NCK expression in cells and cell surface is comparable to WT Klotho (immunoblot, immunocytochemistry, and surface biotinylation), and. NCK is functionally intact as evident by its ability to sustain FGF23 signaling (FGF23-induced phospho-ERK/total ERK. There is no detectable NCK in the supernatant despite abundant cell surface expression. The data supports a NCK that is trafficked to the cell membrane, functionally intact, but not released from the cell surface.

Conclusion

A mouse model of NCK replacing WT Klotho is being generated which will provide a valuable tool for studying the physiological and pathological roles of membrane vs. soluble Klotho protein.

Funding

  • Other NIH Support