Vitamin D independent functions of Fgf23 and Klotho

Fibroblast growth factor-23 (FGF23) is a hormone secreted from osteoblasts and osteocytes in response to elevated extracellular phosphate and vitamin D. Binding of FGF23 to FGF receptors on target cells requires the co-receptor Klotho. FGF23 downregulates renal proximal tubular reabsorption of phosphate, and inhibits renal synthesis of the vitamin D hormone through suppression of the renal 1a-hydroxlase. The exact mechanism by which FGF23 suppresses proximal tubular membrane expression of sodium-phosphate cotransporters is not known. In addition, the physiological role of the putative suppressive effect of FGF23 on parathyroid hormone secretion is currently unclear, and it has always been an enigma why FGF23 does not lower serum calcium despite suppression of vitamin D hormone synthesis. Klotho and Fgf23 knockout mice are characterized by severe hypervitaminosis D due to loss of the suppressive effect on renal 1a-hydroxlase. Therefore, the intricate association between Fgf23/Klotho signaling and vitamin D metabolism has made it difficult to clearly dissect the vitamin D independent functions of Fgf23 and Klotho in vivo. Preliminary data from 9-month-old Fgf23/vitamin D receptor (VDR) compound mutants on a rescue diet enriched with calcium, phosphorus, and lactose revealed renal calcium and sodium wasting, hyperphosphatemia, and severe secondary hyperparathyroidism in Fgf23/VDR compound mutants. Further experiments suggested that lack of Fgf23 signaling through Fgf23 or Klotho deficiency reduced distal renal tubular transport of the fully glycosylated transient receptor potential vanilloid-5 (TRPV5) channel to the plasma membrane by a mechanism involving with- no-lysine kinase-4 (WNK4) and serum- and glucocorticoid-inducible kinase-1 (SGK1) in a vitamin D independent fashion. These data suggest that Fgf23 is not only a phosphaturic but also a calcium-conserving hormone, and suggest crosstalk of Fgf23 and aldosterone signaling at the level of SGK1, establishing a novel molecular link between phosphate, calcium, and sodium homeostasis. The central aim of the current proposal is to elucidate further the vitamin D independent molecular functions of Fgf23 and Klotho in the regulation of renal calcium, phosphate, and sodium reabsorption, in the regulation of PTH secretion, and in disease progression of experimental chronic kidney disease (CKD). Our hypothesis is that Fgf23 signaling directly regulates distal tubular calcium and sodium reabsorption as well as proximal tubular phosphate reabsorption through the ERK1/2-SGK1 signaling pathway, and that Fgf23 deficiency partially protects against progression of CKD. To test this hypothesis, we propose in vivo gain-of-function experiments with recombinant FGF23 and acute loss-of-function experiments with anti-FGF23 antibodies in wild-type, VDR, Fgf23/VDR, and Klotho/VDR mutant mice, as well as in vitro experiments using isolated proximal and distal tubular segments, cultured primary proximal and distal tubular cells, and cultured primary parathyroid cells from wild-type, Fgf23, Klotho, VDR, Fgf23/VDR, and Klotho/VDR mutants. The proposed experiments will significantly advance our knowledge about the molecular role of Fgf23 and Klotho in calcium, phosphate, and sodium homeostasis, as well as in the progression of CKD. Thus, the proposed work may have important implications for human and veterinary clinical medicine.

In the current project, we have identified fibroblast growth factor-23 (FGF23) as a major dis- ease-modulating factor in chronic kidney disease (CKD). FGF23 is not a growth factor as suggested by its name, but rather a hormone secreted from bone cells in response to elevated extracellular phosphate and vitamin D. Binding of FGF23 to fibroblast growth factor receptors on target cells requires the concomitant expression of the co-receptor protein Klotho, which was named after the Greek goddess spinning the thread of life. It is well known that FGF23 downregulates reabsorption of phosphate, and inhibits synthesis of the vitamin D hormone in the kidney. However, the molecular mechanism underlying the FGF23-driven increase in renal phosphate excretion has remained unknown for a long time. In addition, clinical studies have shown that the blood levels of FGF23 are a strong predictor of survival, disease progression, and untoward cardiovascular events in CKD patients. However, the cause of this association has been an enigma. The central aim of the current project was to elucidate further the vitamin D independent molecular functions of Fgf23 and Klotho in the regulation of renal calcium, phosphate, and sodium reabsorption, and in disease progression of experimental CKD. In the current project, we have uncovered the molecular mechanism underlying the stimulation of phosphate excretion by FGF23 in the kidney. Furthermore, we found that FGF23 is not only phosphaturic, but also a calcium- and sodium-conserving hormone involved in blood pressure regulation. Our experiments in CKD mice revealed that the latter finding has major implications for the pathophysiology of CKD, because we identified excessive Fgf23 signaling as a major disease-modulating factor in CKD progression. Mechanistically, we found that high circulating concentrations of intact Fgf23 in CKD mice activate Klotho-dependent and -independent signaling pathways in the kidney, leading to renal calcium and sodium retention. Mice with genetic ablation of Fgf23 or those treated with an anti- FGF23 antibody were largely protected against the CKD-induced impairment in renal function, volume overload, hypertension, heart hypertrophy, cardiac dysfunction, hypercalcemia, and vascular calcification. This striking finding may have major implications for clinical medicine, because it suggests that the elevated circulating FGF23 levels in CKD patients may drive maladaptive processes, and that CKD patients may benefit from pharmacological inhibition of excessive FGF23 signaling.