Chronic kidney disease (CKD) is characterized by an early and progressive increase in osteocyte production of fibroblast growth factor (FGF)-23. Elevated FGF23 levels independently predict cardiovascular events, CKD progression, and mortality, suggesting a need to develop novel therapeutic approaches to reduce FGF23 levels in CKD. Our preliminary data indicate that both increased transcription and reduced cleavage of FGF23 contribute to increased circulating levels of biologically active hormone in CKD, but the mechanisms of these alterations are unknown. To date, most knowledge of FGF23 regulation comes from study of hereditary hypophosphatemic rickets caused by inactivating mutations of the osteocyte proteins, dentin matrix protein (DMP)-1 and phosphate-regulating gene with homology to endopeptidase on the X chromosome (PHEX), that cause elevated FGF23 levels. Like CKD, increased transcription and reduced cleavage of FGF23 drive increased circulating levels in states of DMP1 and PHEX inactivation. Our preliminary data suggest that direct binding of DMP1 to PHEX occurs through the ASARM motif present in the C-terminal fragment of DMP1 (cDMP1) peptide, and that this binding is an essential regulatory mechanism of FGF23 transcription and FGF23 cleavage. Although cDMP1 and PHEX are proven local regulators of FGF23 in bone, no studies have tested the hypothesis that altered PHEX and cDMP1 interactions mediate dysregulated FGF23 transcription and cleavage in CKD. In this proposal, we will apply our expertise in FGF23 regulation in hereditary hypophosphatemia to understand local bone mechanisms regulating FGF23 in CKD.
In Aim 1, we will perform interferometry and co-immunoprecipitation experiments to determine if cDMP1 is the active fragment of DMP1 that binds PHEX and inhibits FGF23 transcription.
In Aim 2, we will test whether cDMP1 inhibits GALNT3, the enzyme responsible for protecting FGF23 from degradation by glycosylating its cleavage site. In both aims, we will use calcitriol, calcium or phosphate loading as different approaches to induce FGF23 transcription in wild-type (WT) and cDMP1 transgenic (cDMP1Tg) mice and their bone marrow stromal cells (BMSC). We will also use MC3T3-E1 osteoblasts transfected with a luciferase reporter FGF23 promoter or an inducible FGF23 expression vector.
In Aim 3, we will use the validated Col4a3ko mouse model of progressive CKD to test our hypothesis that decreased cDMP1 concentrations contribute to increased FGF23 levels in CKD. We will investigate osteocyte organization and morphology using acid etched scanning electron microscopy and FITC-Imaris quantitative modelisation, we will quantify cDMP1 peptides and test whether overexpression of cDMP1 blunts the expected FGF23 increase in Col4a3ko mice, during the course of CKD. These innovative Aims form the basis of the career development plan of a new investigator working in a highly productive FGF23 research team. The results will define an essential role of the PHEX/cDMP1 axis in regulating FGF23, and identify novel therapeutic targets to treat disorders of FGF23 excess, including CKD.

Public Health Relevance

Chronic Kidney Disease (CKD) is characterized by increased production of fibroblast growth factor (FGF)- 23 in osteocytes and elevated FGF23 is associated with CKD progression, cardiovascular disease, and mortality. PHEX and DMP1 are two osteocyte proteins that regulate FGF23 locally. This project proposes to test whether the interaction between PHEX and DMP1 mediates the transcriptional and post-translational modifications of FGF23 in CKD, thus providing a potential therapeutic target for individuals with CKD.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Skeletal Biology Development and Disease Study Section (SBDD)
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Chan, Kevin E
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Northwestern University at Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
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Neuburg, Samantha; Dussold, Corey; Gerber, Claire et al. (2018) Genetic background influences cardiac phenotype in murine chronic kidney disease. Nephrol Dial Transplant 33:1129-1137