The Role of Fgf23/klotho in Mineral Metabolism and Chronic Kidney Disease

Chronic kidney disease (CKD) is a global health burden of growing incidence and prevalence. As renal function declines disturbances in mineral metabolism, such as hyperphosphatemia and secondary hyperparathyroidism, inevitably develop. These metabolic changes are closely associated with poor prognosis and survival. The bonederived hormone fibroblast growth factor-23 (FGF23) and its co-receptor Klotho represent a novel endocrine axis regulating mineral metabolism in health and disease. FGF23-Klotho signalling inhibits renal phosphate reabsorption and activation of vitamin D, and reduces secretion of parathyroid hormone (PTH). Serum levels of FGF23 rise at early stages of CKD, presumably due to increased phosphate load, and numerous studies identify elevated FGF23 as a predictor of adverse clinical outcome. In contrast, tissue expression of Klotho decreases in parallel with CKD progression and reaches low or undetectable levels in end-stage renal disease. Importantly, mice lacking Klotho develop numerous complications associated with accelerated ageing, and many patients with advanced CKD, a state of Klotho deficiency, display a similar senescencelike phenotype. Altogether, FGF23 excess and lack of Klotho may be key pathogenic factors in CKD. In the present thesis we sought to elucidate the role of renal and parathyroid FGF23-Klotho signalling in physiology and in CKD. In Study I we investigate Klotho levels in surgically resected parathyroid tissue specimen from CKD patients with secondary hyperparathyroidism, and find diminished Klotho expression paralleling the decline in renal function. Further, we demonstrate that FGF23 dose-dependently suppresses Klotho in bovine parathyroid cell culture, indicating a ligand-receptor regulatory process. In Study II we generate parathyroid-specific Klotho knockout mice (PTH-KL) using Cre-Lox recombination. PTH-KL mice display a normal gross phenotype with a preserved calcium-PTH axis. Their PTH response is similar to wildtype mice when treated with FGF23 or challenged with renal failure. Yet, FGF23 treatment activates the MAPK pathway in wild-type mice but not in PTH-KL mice. Importantly, blocking of calcineurin with cyclosporine A abolishes the FGF23mediated PTH suppression in PTH-KL mice, whereas wild-type mice remain responsive. Thus, we identify a novel calcineurin-dependent pathway in the parathyroid glands that, in the absence of Klotho, mediates acute suppression of PTH secretion by FGF23. In Study III we develop a novel, non-surgical, mouse model of tubulointerstitial nephropathy. By adding various concentrations of adenine to the diet we define an adjustable protocol for inducing and maintaining uremia in mice. In Study IV we generate distal tubule-specific Klotho knockout mice (Ksp-KL). In contrast to systemic Klotho knockout mice, Ksp-KL mice are fertile with a normal gross phenotype. Adult Ksp-KL mice are hyperphosphatemic, indicating attenuated effects of FGF23 on proximal tubular phosphate handling. Further, FGF23 is higher in Ksp-KL mice than in wild-type mice with matched serum phosphate, suggesting phosphate-independent regulation of FGF23 in Ksp-KL mice. Collectively, the studies presented in this thesis identify several novel and critical aspects of FGF23-Klotho signalling and function in health and disease, and provide important tools allowing for continuous investigation. LIST OF PUBLICATIONS I. Krajisnik T, Olauson H, Mirza MA, Hellman P, Akerström G, Westin G, Larsson TE*, Björklund P*. Parathyroid Klotho and FGF-receptor 1 expression decline with renal function in hyperparathyroid patients with chronic kidney disease and kidney transplant recipients. Kidney Int. 2010 Nov;78(10):1024-32. *Shared last authors II. Olauson H, Lindberg K, Amin R, Sato T, Ting J, Goetz R, Mohammadi M, Andersson G, Lanske B, Larsson TE. Parathyroid-specific deletion of the Klotho gene unravels a novel calcineurindependent FGF23 signalling pathway that mediates suppression of PTH secretion. Submitted manuscript III. Jia T*, Olauson H*, Lindberg K, Amin R, Edvardsson K, Lindholm B, Andersson G, Wernerson A, Sabbagh Y, Schiavi S, Larsson TE. A novel model of adenine-induced tubulointerstitial nephropathy in mice. BMC Nephrol. 2013 May 30;14(1):116. *Shared first authors IV. Olauson H, Lindberg K, Amin R, Jia T, Wernerson A, Andersson G, Larsson TE. Targeted deletion of Klotho in kidney distal tubule disrupts mineral metabolism. J Am Soc Nephrol. 2012 Oct;23(10):1641-51. RELATED PUBLICATIONS NOT INCLUDED IN THIS THESIS I. Olauson H, Larsson TE. FGF23 and Klotho in chronic kidney disease. Curr Opin Nephrol Hypertens. 2013 Jul;22(4):397-404. II. Lindberg K, Olauson H, Amin R, Ponnusamy A, Goetz R, Taylor RF, Mohammadi M, Canfield A, Kublickiene K, Larsson TE. Arterial Klotho expression and FGF23 effects on vascular calcification and function. PLoS One. 2013;8(4):e60658. III. Olauson H, Qureshi AR, Miyamoto T, Barany P, Heimburger O, Lindholm B, Stenvinkel P, Larsson TE. Relation between serum fibroblast growth factor-23 level and mortality in incident dialysis patients: are gender and cardiovascular disease confounding the relationship? Nephrol Dial Transplant. 2010 Sep;25(9):3033-8. IV. Olauson H, Brandenburg V, Larsson TE. Mutation analysis and serum FGF23 level in a patient with pulmonary alveolar microlithiasis. Endocrine. 2010 Apr;37(2):244-8. V. Larsson TE, Olauson H, Hagström E, Ingelsson E, Arnlöv J, Lind L, Sundström J. Conjoint effects of serum calcium and phosphate on risk of total, cardiovascular, and noncardiovascular mortality in the community. Arterioscler Thromb Vasc Biol. 2010 Feb;30(2):333-9. VI. Olauson H, Krajisnik T, Larsson C, Lindberg B, Larsson TE. A novel missense mutation in GALNT3 causing hyperostosishyperphosphataemia syndrome. Eur J Endocrinol. 2008 Jun;158(6):929-34. VII. Westerberg PA*, Olauson H*, Toss G, Wikström B, Morales O, Linde T, Jonsson K, Ljunggren O, Larsson TE. Preoperative tumor localization by means of venous sampling for fibroblast growth factor-23 in a patient with tumor-induced osteomalacia. Endocr Pract. 2008 Apr;14(3):362-7. *Shared first authors