SCF-SKP2 E3 ubiquitin ligase links mTORC1/ER stress/ISR with YAP activation in murine renal cystogenesis.

The Hippo pathway nuclear effector Yes-associated protein (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1), driven by ERK1/2 and PI3K/AKT cascades, induced ER proteotoxic stress. To reduce this stress by reprogramming translation, the protein kinase R-like ER kinase-eukaryotic initiation factor 2α (PERK/eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α phosphorylation-deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress/ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan impeded these processes. Restoring ER homeostasis and/or interfering with the SKP2-YAP interaction represent potential therapeutic avenues for stemming the progression of renal cystogenesis.

Burosumab Treatment for Autosomal Recessive Hypophosphatemic Rickets Type 1 (ARHR1).

CONTEXT: Autosomal recessive hypophosphatemic rickets (ARHR) are rare, heritable renal phosphate-wasting disorders that arise from overexpression of the bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) leading to impaired bone mineralization (rickets and osteomalacia). Inactivating mutations of Dentin matrix protein 1 (DMP1) give rise to ARHR type 1 (ARHR1). Short stature, prominent bowing of the legs, fractures/pseudofractures, and severe enthesopathy are prominent in this patient population. Traditionally, treatment consists of oral phosphate replacement and the addition of calcitriol but this approach is limited by modest efficacy and potential renal and gastrointestinal side effects. OBJECTIVE: The advent of burosumab (Crysvita), a fully humanized monoclonal antibody to FGF23 for the treatment of X-linked hypophosphatemia and tumor-induced osteomalacia, offers a unique opportunity to evaluate its safety and efficacy in patients with ARHR1. RESULTS: Monthly administration of burosumab to 2 brothers afflicted with the disorder resulted in normalization of serum phosphate, healing of pseudofracture, diminished fatigue, less bone pain, and reduced incapacity arising from the extensive enthesopathy and soft tissue fibrosis/calcification that characterizes this disorder. No adverse effects were reported following burosumab administration. CONCLUSION: The present report highlights the beneficial biochemical and clinical outcomes associated with the use of burosumab in patients with ARHR1.

CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders.

CYP24A1 (hereafter referred to as CYP24) enzymatic activity is pivotal in the inactivation of vitamin D metabolites. Basal renal and extrarenal CYP24 is usually low but is highly induced by its substrate 1,25-dihydroxyvitamin D. Unbalanced high and/or long-lasting CYP24 expression has been proposed to underlie diseases like chronic kidney disease, cancers, and psoriasis that otherwise should favorably respond to supplemental vitamin D. Using genetically modified mice, we have shown that renal phosphate wasting hypophosphatemic states arising from high levels of fibroblast growth factor 23 (FGF23) are also associated with increased renal Cyp24 expression, suggesting that elevated CYP24 activity is pivotal to the pathophysiology of these disorders. We therefore crossed 2 mouse strains, each with distinct etiology for high levels of circulating FGF23, onto a Cyp24-null background. Specifically, we evaluated Cyp24 deficiency in Hyp mice, the murine homolog of X-linked dominant hypophosphatemic rickets, and transgenic mice that overexpress a mutant FGF23 (FGF23R176Q) that is associated with the autosomal dominant form of hypophosphatemic rickets. Loss of Cyp24 in these murine models of human disease resulted in near-complete recovery of rachitic/osteomalacic bony abnormalities in the absence of any improvement in the serum biochemical profile. Moreover, treatment of Hyp and FGF23R1760-transgenic mice with the CYP24 inhibitor CTA102 also ameliorated their rachitic bones. Our results link CYP24 activity to the pathophysiology of FGF23-dependent renal phosphate wasting states and implicate pharmacologic CYP24 inhibition as a therapeutic adjunct for their treatment.

Impairment of spatial learning and memory in transgenic mice overexpressing human fibroblast growth factor-23.

Fibroblast growth factor-23 (FGF-23) is a potent circulating phosphaturic factor associated with renal phosphate wasting. Effects of FGF-23 on skeleton, phosphate homeostasis, and cardiovascular system have been investigated; however, the effect of FGF-23 on the central nervous system (CNS) is unknown. To assess whether FGF-23 influences the function and structure of the CNS and whether the effect of FGF-23 on the CNS is mediated by FGF receptors directly or by hypophosphatemia indirectly, FGF-23 transgenic mice and their wild-type littermates were fed a normal diet or a high-phosphate diet containing a normal diet plus 1.25% phosphate in drinking water from weaning for 5weeks and the phenotypes of the CNS were compared between FGF-23 transgenic mice and their wild-type littermates on the same diet. At the end of this time period, transgenic animals on the normal diet showed impaired spatial learning and memory. Furthermore, these mice exhibited the impairment of long-term potentiation in hippocampal CA1 region, and the reduction of hippocampal adenosine-triphosphate content and of choline acetyltransferase-positive neurons in basal forebrain, possibly as pathogenetic factors contributing to the cognitive deficit. The central nervous phenotypes of transgenic mice were rescued following improved hypophosphatemia by the high-phosphate diet intake. This study demonstrates that FGF-23 overexpression can result in abnormalities in the CNS mediated by the secondary severe hypophosphatemia.

Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype.

Transgenic mice overexpressing fibroblast growth factor (FGF23) (R176Q) (F(Tg)) exhibit biochemical {hypophosphatemia, phosphaturia, abnormal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] metabolism} and skeletal (rickets and osteomalacia) abnormalities attributable to FGF23 action. In vitro studies now implicate the aging-related factor Klotho in the signaling mechanism of FGF23. In this study, we used a mouse genetic approach to validate in vivo the pivotal role of Klotho in the metabolic and skeletal derangements associated with FGF23 (R176Q) overexpression. To this end, we crossed mice heterozygous for the hypomorphic Klotho allele (Kl(+/-)) to F(Tg) mice and obtained F(Tg) transgenic mice homozygous for the Kl-hypomorphic allele (F(Tg)/Kl(-/-)). Mice were killed on postnatal day 50, and serum and tissues were procured for analysis and comparison with F(Tg), wild-type, and Kl(-/-) controls. From 4 wk onward, F(Tg)/Kl(-/-) mice were clearly distinguishable from F(Tg) mice and exhibited a striking phenotypic resemblance to the Kl(-/-) controls. Serum analysis for calcium, phosphorus, parathyroid hormone, 1,25(OH)(2)D(3), and alkaline phosphatase activity confirmed the biochemical similarity between the F(Tg)/Kl(-/-) and Kl(-/-) mice and their distinctness from the F(Tg) controls. The characteristic skeletal changes associated with FGF23 (R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence the wide, unmineralized growth plates and the osteomalacic abnormalities apparent in trabecular and cortical bone were completely reversed in the F(Tg)/Kl(-/-) mice. Nevertheless, independent actions of Klotho on bone were suggested as manifested by alterations in mineralized bone, and in cortical bone volume which were observed in both the Kl(-/-) and F(Tr)/Kl(-/-) mutants. In summary, our findings substantiate in vivo the essential role of Klotho in the mechanism of action of FGF23 in view of the fact that Klotho ablation converts the biochemical and skeletal manifestations resulting from FGF23 overexpression to a phenotype consistent with Klotho deficiency.

Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders.

Fibroblast growth factor 23 (FGF23) is a recently characterized protein likely involved in the regulation of serum phosphate homeostasis. Increased circulating levels of FGF23 have been reported in patients with renal phosphate-wasting disorders, but it is unclear whether FGF23 is the direct mediator responsible for the decreased phosphate transport at the proximal renal tubules and the altered vitamin D metabolism associated with these states. To examine this question, we generated transgenic mice expressing and secreting from the liver human FGF23 (R176Q), a mutant form that fails to be degraded by furin proteases. At 1 and 2 months of age, mice carrying the transgene recapitulated the biochemical (decreased urinary phosphate reabsorption, hypophosphatemia, low serum 1,25-dihydroxyvitamin D(3)) and skeletal (rickets and osteomalacia) alterations associated with these disorders. Unexpectantly, marked changes in parameters of calcium homeostasis were also observed, consistent with secondary hyperparathyroidism. Moreover, in the kidney the anticipated alterations in the expression of hydroxylases associated with vitamin D metabolism were not observed despite the profound hypophosphatemia and increased circulating levels of PTH, both major physiological stimuli for 1,25-dihydroxyvitamin D(3) production. Our findings strongly support the novel concept that high circulating levels of FGF23 are associated with profound disturbances in the regulation of phosphate and vitamin D metabolism as well as calcium homeostasis and that elevated PTH levels likely also contribute to the renal phosphate wasting associated with these disorders.

The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency.

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumor-bearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH)(2)D(3) levels by increasing renal expression of 25(OH)D(3)-1alpha-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D(3)-24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.

Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression.

Inactivating mutations and/or deletions of PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans and in the murine homolog Hyp. The predominant osteoblastic expression of Phex has implicated a primary metabolic osteoblast defect in the pathophysiology of this disorder. By targeting PHEX expression to osteoblasts in the Hyp genetic background, we aimed to correct the corresponding biochemical and morphological abnormalities and obtain information on their pathogenetic mechanism. When transgene Phex expression, driven by a mouse pro-alpha1(I) collagen gene promoter, was crossed into the Hyp background, it improved the defective mineralization of bone and teeth but failed to correct the hypophosphatemia and altered vitamin D metabolism associated with the disorder. Ex vivo bone marrow cultures confirmed the amelioration in the Hyp-associated matrix mineralization defect after Phex expression. These findings suggest that while the Hyp bone and teeth abnormalities partially correct after PHEX gene transfer, additional factors and/or sites of PHEX expression are likely critical for the elaboration of the appropriate molecular signals that alter renal phosphate handling and vitamin D metabolism in this disorder.