Sustained Klotho delivery reduces serum phosphate in a model of diabetic nephropathy.

Diabetic nephropathy (DN) is a primary cause of end-stage renal disease and is becoming more prevalent because of the global rise in type 2 diabetes. A model of DN, the db/db uninephrectomized ( db/db-uni) mouse, is characterized by obesity, as well as compromised renal function. This model also manifests defects in mineral metabolism common in DN, including hyperphosphatemia, which leads to severe endocrine disease. The FGF23 coreceptor, α-Klotho, circulates as a soluble, cleaved form (cKL) and may directly influence phosphate handling. Our study sought to test the effects of cKL on mineral metabolism in db/db-uni mice. Mice were placed into either mild or moderate disease groups on the basis of the albumin-to-creatinine ratio (ACR). Body weights of db/db-uni mice were significantly greater across the study compared with lean controls regardless of disease severity. Adeno-associated cKL administration was associated with increased serum Klotho, intact, bioactive FGF23 (iFGF23), and COOH-terminal fragments of FGF23 ( P < 0.05). Blood urea nitrogen was improved after cKL administration, and cKL corrected hyperphosphatemia in the high- and low-ACR db/db-uni groups. Interestingly, 2 wk after cKL delivery, blood glucose levels were significantly reduced in db/db-uni mice with high ACR ( P < 0.05). Interestingly, several genes associated with stabilizing active iFGF23 were also increased in the osteoblastic UMR-106 cell line with cKL treatment. In summary, delivery of cKL to a model of DN normalized blood phosphate levels regardless of disease severity, supporting the concept that targeting cKL-affected pathways could provide future therapeutic avenues in DN. NEW & NOTEWORTHY In this work, systemic and continuous delivery of the "soluble" or "cleaved" form of the FGF23 coreceptor α-Klotho (cKL) via adeno-associated virus to a rodent model of diabetic nephropathy (DN), the db/db uninephrectomized mouse, normalized blood phosphate levels regardless of disease severity. This work supports the concept that targeting cKL-affected pathways could provide future therapeutic avenues for the severe mineral metabolism defects associated with DN.

Chronic Hyperphosphatemia and Vascular Calcification Are Reduced by Stable Delivery of Soluble Klotho.

αKlotho (αKL) regulates mineral metabolism, and diseases associated with αKL deficiency are characterized by hyperphosphatemia and vascular calcification (VC). αKL is expressed as a membrane-bound protein (mKL) and recognized as the coreceptor for fibroblast growth factor-23 (FGF23) and a circulating soluble form (cKL) created by endoproteolytic cleavage of mKL. The functions of cKL with regard to phosphate metabolism are unclear. We tested the ability of cKL to regulate pathways and phenotypes associated with hyperphosphatemia in a mouse model of CKD-mineral bone disorder and αKL-null mice. Stable delivery of adeno-associated virus (AAV) expressing cKL to diabetic endothelial nitric oxide synthase-deficient mice or αKL-null mice reduced serum phosphate levels. Acute injection of recombinant cKL downregulated the renal sodium-phosphate cotransporter Npt2a in αKL-null mice supporting direct actions of cKL in the absence of mKL. αKL-null mice with sustained AAV-cKL expression had a 74%-78% reduction in aorta mineral content and a 72%-77% reduction in mineral volume compared with control-treated counterparts (P<0.01). Treatment of UMR-106 osteoblastic cells with cKL + FGF23 increased the phosphorylation of extracellular signal-regulated kinase 1/2 and induced Fgf23 expression. CRISPR/Cas9-mediated deletion of fibroblast growth factor receptor 1 (FGFR1) or pretreatment with inhibitors of mitogen-activated kinase kinase 1 or FGFR ablated these responses. In summary, sustained cKL treatment reduced hyperphosphatemia in a mouse model of CKD-mineral bone disorder, and it reduced hyperphosphatemia and prevented VC in mice without endogenous αKL. Furthermore, cKL stimulated Fgf23 in an FGFR1-dependent manner in bone cells. Collectively, these findings indicate that cKL has mKL-independent activity and suggest the potential for enhancing cKL activity in diseases of hyperphosphatemia with associated VC.

Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.

Circulating αKlotho influences phosphate handling by controlling FGF23 production.

The FGF23 coreceptor αKlotho (αKL) is expressed as a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as an endoproteolytic cleavage product of mKL (cKL). Previously, a patient with increased plasma cKL as the result of a translocation [t(9;13)] in the αKLOTHO (KL) gene presented with rickets and a complex endocrine profile, including paradoxically elevated plasma FGF23, despite hypophosphatemia. The goal of this study was to test whether cKL regulates phosphate handling through control of FGF23 expression. To increase cKL levels, mice were treated with an adeno-associated virus producing cKL. The treated groups exhibited dose-dependent hypophosphatemia and hypocalcemia, with markedly elevated FGF23 (38 to 456 fold). The animals also manifested fractures, reduced bone mineral content, expanded growth plates, and severe osteomalacia, with highly increased bone Fgf23 mRNA (>150 fold). cKL activity in vitro was specific for interactions with FGF23 and was FGFR dependent. These results demonstrate that cKL potently stimulates FGF23 production in vivo, which phenocopies the KL translocation patient and metabolic bone syndromes associated with elevated FGF23. These findings have important implications for the regulation of αKL and FGF23 in disorders of phosphate handling and biomineralization.