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

Abstract

aKlotho (aKL) regulates mineral metabolism, and diseases associated with aKL deficiency are characterized by hyperphosphatemia and vascular calcification (VC). aKL 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 aKL-null mice. Stable delivery of adeno-associated virus (AAV) expressing cKL to diabetic endothelial nitric oxide synthase-deficient mice or aKL-null mice reduced serum phosphate levels. Acute injection of recombinant cKL downregulated the renal sodium-phosphate cotransporter Npt2a in aKL-null mice supporting direct actions of cKL in the absence of mKL. aKL-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 aKL. 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.

Novel functions of circulating Klotho

“…Since DN is currently the leading cause of CKD-MBD 42, 43 , identifying novel pathways for pharmacological interventions are needed. cKL delivery to db/db-eNOS −/− mice resulted in a reduction in serum phosphate with increased FGF23 despite no improvements in renal function or pathology 39 . Although the molecular mechanisms remain to be determined, these results support that at pharmacologic levels cKL may reduce serum phosphate during compromised renal function.…”

“…To address the question whether providing cKL to a model of CKD-MBD would be therapeutically beneficial, AAV-cKL was administered to mouse models with phenotypes that parallel those of patients. To test cKL’s pharmacological effects the db/db-eNOS −/− mouse model of diabetic nephropathy (DN) was used 39 . The db/db-eNOS −/− mouse model is characterized by a loss of activity of the leptin receptor and disruption of eNOS causing mice to develop exceedingly high blood glucose, and progressive renal damage including glomerular and interstitial fibrosis 40, 41 .…”

“…The αKL-null mice also provide a biological platform for examining cKL effects that are independent of all other αKL isoforms. Therefore AAV-cKL was provided to αKL-null mice for four weeks 39 . Interestingly, the prevailing hyperphosphatemia in αKL-null mice was significantly reduced in parallel with further FGF23 increases above the elevated baseline FGF23 levels 39 .…”

“…Therefore AAV-cKL was provided to αKL-null mice for four weeks 39 . Interestingly, the prevailing hyperphosphatemia in αKL-null mice was significantly reduced in parallel with further FGF23 increases above the elevated baseline FGF23 levels 39 . In WT mice cKL delivery increased serum PTH and suppressed serum 1,25D, consistent with a reduction in the renal 1α-OHase ( Cyp27b1 ) and an increase in the 24-OHase ( Cyp24a1 ) mRNAs.…”

Treating Systemic Klotho Deficiency

“…Challenges in optimization of preparation, achieving adequate bioactivity, and prolongation of its short half-life notwithstanding, recombinant Klotho is still the only method that allows dosing and precise control of therapeutic levels. Klotho driven by strong universal pro- moter in conventional cDNA plasmids has also been either directly injected [6] or delivered while encapsulated by adeno-associated virus which is not pathogenic [7]. Although only selective studies are cited, these methods have been used by multiple laboratories and Klotho expression and biologic effects were all verified.…”