Cisplatin decreases HOXA13 and alphaVBeta3 integrin levels in the uterus.

OBJECTIVE: To examine the effects of cisplatin on uterine histology and implantation molecules and the possible protective role of recombinant Klotho administration on uterine histology and uterine receptivity in mice exposed to cisplatin. MATERIALS AND METHODS: This study was conducted using thirty-two adult female mice assigned to four groups with 8 mice in each group. Saline was given to the 1st group, cisplatin to the 2nd group, recombinant mouse Klotho to the 3rd group and recombinant mouse Klotho plus cisplatin to the 4th group. Uterine tissues were examined for damage histologically and immunobiologically for the uterine receptivity markers HOXA13 and alphaVBeta3 integrin. RESULTS: Apoptosis, degeneration, decrease in uterine thickness and uterine absence of gland scores were higher in the cisplatin group (3rd group) compared to the saline group (1st group) (cisplatin vs. saline p < 0.0001 for all parameters). In the recombinant Klotho plus cisplatin group (4th group), scores of apoptosis, degeneration, reduction in uterine thickness and uterine absence of gland were lower than the group receiving only cisplatin (cisplatin plus recombinant Klotho vs cisplatin, p = 0.006 for apoptosis; p = 0.017 for degeneration; p = 0.011 for the reduction in uterine thickness; p = 0.002 for the absence of gland). However, HOXA13 and alphaVBeta3 integrin staining levels were not different between the cisplatin group (group 3) and the cisplatin plus recombinant Klotho group (group 4) (p = 0.980 and p = 0.762, respectively.) CONCLUSION: Cisplatin has adverse effects on the uterus. Administration of recombinant Klotho was found to attenuate the cisplatin-induced damage but failed to preserve levels of the implantation molecules HOXA13 and alphaVbeta3. Further studies examining the effect of cisplatin toxicity using other implantation markers along with functional studies are needed.

Klotho downregulation contributes to myocardial damage of cardiorenal syndrome in sepsis.

Klotho is a type of single­pass transmembrane protein that is important for the proper function of numerous organs. The aim of the present study was to investigate the role of Klotho in sepsis­associated myocardial damage. In the present study, reverse transcription­quantitative PCR, western blotting and ELISA were conducted to examine the expression levels of function genes, and flow cytometry was performed to detect cell apoptosis and reactive oxygen species. The present study demonstrated that Klotho expression was significantly downregulated in septic mice and that the myocardial function of septic mice improved after treatment with exogenous Klotho protein. It further demonstrated that indoxyl sulfate inhibited the expression of Klotho protein. In addition, decreased Klotho protein further led to activation of the reactive oxygen species­p38 mitogen­activated protein kinase signaling pathway, finally resulting in myocardial damage. In conclusion, Klotho protein may be a key regulator in the myocardial damage of cardiorenal syndrome in sepsis. It also has a potential to be a therapeutic target for sepsis­associated myocardial damage in the future.

Central α-Klotho Suppresses NPY/AgRP Neuron Activity and Regulates Metabolism in Mice.

α-Klotho is a circulating factor with well-documented antiaging properties. However, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing neurons, energy balance, and glucose homeostasis. Intracerebroventricular administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of type 1 and 2 diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing miniature inhibitory postsynaptic currents. Experiments in hypothalamic GT1-7 cells observed that α-klotho induces phosphorylation of AKTser473, ERKthr202/tyr204, and FOXO1ser256 as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor receptor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. Phosphatidylinositol 3 kinase (PI3K) inhibition also abolished α-klotho's ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease.

Vestronidase alfa: Recombinant human ß-glucuronidase as an enzyme replacement therapy for MPS VII.

Mucopolysaccharidosis VII (MPS VII) is a rare lysosomal storage disease characterized by a deficiency in the enzyme ß-glucuronidase that has previously been successfully treated in a mouse model with enzyme replacement therapy. Here, we present the generation of a novel, highly sialylated version of recombinant human ß-glucuronidase (rhGUS), vestronidase alfa, that has high uptake, resulting in an improved enzyme replacement therapy for the treatment of patients with MPS VII. In vitro, vestronidase alfa has 10-fold more sialic acid per mole of rhGUS monomer than a prior rhGUS version (referred to as GUS 43/44) and demonstrated very high affinity at ~1 nM half maximal uptake in human MPS VII fibroblasts. Vestronidase alfa has a longer enzymatic half-life after uptake into fibroblasts compared with other enzymes used as replacement therapy for MPS (40 days vs 3 to 4 days, respectively). In pharmacokinetic and tissue distribution experiments in Sprague-Dawley rats, intravenous administration of vestronidase alfa resulted in higher serum rhGUS levels and enhanced ß-glucuronidase activity distributed to target tissues. Weekly intravenous injections of vestronidase alfa (0.1 mg/kg to 20 mg/kg) in a murine model of MPS VII demonstrated efficient enzyme delivery to all tissues, including bone and brain, as well as reduced lysosomal storage of glycosaminoglycans (GAGs) in a dose-dependent manner, resulting in increased survival after 8 weeks of treatment. Vestronidase alfa was well-tolerated and demonstrated no toxicity at concentrations that reached 5-times the proposed clinical dose. In a first-in-human phase 1/2 clinical trial, a dose-dependent reduction in urine GAG levels was sustained over 38 weeks of treatment with vestronidase alfa. Together, these results support the therapeutic potential of vestronidase alfa as an enzyme replacement therapy for patients with MPS VII.

The long-term safety and efficacy of vestronidase alfa, rhGUS enzyme replacement therapy, in subjects with mucopolysaccharidosis VII.

Vestronidase alfa (recombinant human beta-glucuronidase) is an enzyme replacement therapy (ERT) for Mucopolysaccharidosis (MPS) VII, a highly heterogeneous, ultra-rare disease. Twelve subjects, ages 8-25 years, completed a Phase 3, randomized, placebo-controlled, blind-start, single crossover study (UX003-CL301; NCT02377921), receiving 24-48 weeks of vestronidase alfa 4 mg/kg IV. All 12 subjects completed the blind-start study, which showed significantly reduced urinary glycosaminoglycans (GAG) and clinical improvement in a multi-domain responder index, and enrolled in a long-term, open-label, extension study (UX003-CL202; NCT02432144). Here, we report the final results of the extension study, up to an additional 144 weeks after completion of the blind-start study. Three subjects (25%) completed all 144 weeks of study, eight subjects (67%) ended study participation before Week 144 to switch to commercially available vestronidase alfa, and one subject discontinued due to non-compliance after receiving one infusion of vestronidase alfa in the extension study. The safety profile of vestronidase alfa in the extension study was consistent with observations in the preceding blind-start study, with most adverse events mild to moderate in severity. There were no treatment or study discontinuations due to AEs and no noteworthy changes in a standard safety chemistry panel. Out of the eleven subjects who tested positive for anti-drug antibodies at any time during the blind-start or extension study, including the baseline assessment in the blind-start study, seven subjects tested positive for neutralizing antibodies and all seven continued to demonstrate a reduction in urinary GAG levels. There was no association between antibody formation and infusion associated reactions. Subjects receiving continuous vestronidase alfa treatment showed a sustained urinary GAG reduction and clinical response evaluated using a multi-domain responder index that includes assessments in pulmonary function, motor function, range of motion, mobility, and visual acuity. Reduction in fatigue was also maintained in the overall population. As ERT is not expected to cross the blood brain barrier, limiting the impact on neurological signs of disease, and not all subjects presented with neurological symptoms, outcomes related to central nervous system pathology are not focused on in this report. Results from this study show the long-term safety and durability of clinical efficacy in subjects with MPS VII with long-term vestronidase alfa treatment.

Klotho supplementation ameliorates blood pressure and renal function in DBA/2-pcy mice, a model of polycystic kidney disease.

Klotho interacts with various membrane proteins such as receptors for transforming growth factor-ß (TGF-ß) and insulin-like growth factor (IGF). Renal expression of klotho is diminished in polycystic kidney disease (PKD). In the present study, the effects of klotho supplementation on PKD were assessed. Recombinant human klotho protein (10 µg·kg-1·day-1) or a vehicle was administered daily by subcutaneous injection to 6-wk-old mice with PKD (DBA/2-pcy). Blood pressure was measured using tail-cuff methods. After 2 mo, mice were killed, and the kidneys were harvested for analysis. Exogenous klotho protein supplementation reduced kidney weight, cystic area, systolic blood pressure, renal angiotensin II levels, and 8-epi-PGF2α excretion (P < 0.05). Klotho protein supplementation enhanced glomerular filtration rate, renal expression of superoxide dismutase, and klotho itself (P < 0.05). Klotho supplementation attenuated renal expressions of TGF-ß and collagen type I and diminished renal abundance of Twist, phosphorylated Akt, and mammalian target of rapamycin (P < 0.05). Pathological examination revealed that klotho decreased the fibrosis index and nuclear staining of Smad in PKD kidneys (P < 0.05). Our data indicate that klotho protein supplementation ameliorates the renin-angiotensin system, reducing blood pressure in PKD mice. Furthermore, the present results implicate klotho supplementation in the suppression of Akt/mammalian target of rapamycin signaling, slowing cystic expansion. Finally, our findings suggest that klotho protein supplementation attenuated fibrosis at least partly by inhibiting epithelial mesenchymal transition in PKD.

Intrathecal AAVrh10 corrects biochemical and histological hallmarks of mucopolysaccharidosis VII mice and improves behavior and survival.

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by ß-glucuronidase deficiency, prompting glycosaminoglycan accumulation in enlarged vesicles, leading to peripheral and neuronal dysfunction. Here, we present a gene therapy strategy using lumbar puncture of AAVrh10 encoding human ß-glucuronidase (AAVrh10-GUSB) to adult MPS VII mice. This minimally invasive technique efficiently delivers the recombinant vector to the cerebrospinal fluid (CSF) with a single intrathecal injection. We show that AAVrh10 delivery to the CSF allows global, stable transduction of CNS structures. In addition, drainage of AAVrh10-GUSB from the CSF to the bloodstream resulted in the transduction of somatic organs such as liver, which provided a systemic ß-glucuronidase source sufficient to achieve serum enzyme activity comparable to wild type mice. ß-glucuronidase levels were enough to correct biochemical and histopathological hallmarks of the disease in the CNS and somatic organs at short and long term. Moreover, the progression of the bone pathology was also reduced. Importantly, the biochemical correction led to a significant improvement in the physical, cognitive and emotional characteristics of MPS VII mice, and doubling their life span. Our strategy may have implications for gene therapy in patients with lysosomal storage diseases.

Therapeutic Challenge of Minicircle Vector Encoding Klotho in Animal Model.

BACKGROUND: Klotho treatment is a promising approach against kidney injury, but its clinical application is still undetermined. We developed a novel strategy to allow self-production of Klotho protein, using minicircle (MC) technology, and evaluated its feasibility in therapeutic Klotho delivery. METHODS: We engineered MC vectors to carry cassette sequences of Klotho and verified the self-production of Klotho protein from in HEK293T cells. We evaluated the location and persistence of delivered MC in vivo, and the duration of Klotho protein production from MCs by serial measurement of Klotho protein in blood. We subsequently evaluated the therapeutic potential of Klotho-encoding MCs in experimental model of renal injury. RESULTS: We confirmed the production of Klotho from MC by its significant availability in cells transfected with the MC, as well as in its conditioned medium, compared to that in cells transfected with parent vector. MCs were delivered in vivo by hydrodynamic injection via tail vein. After a single injection of MCs, red fluorescence protein was detected until 30 days in liver, and Klotho protein was maintained until 10 days in the blood, suggesting the production of Klotho protein from MCs via protein synthesis machinery in liver. Therapeutic effect of MC was confirmed by functional and histological improvement seen in mouse model of acute ischemia-reperfusion injury and unilateral ureteral obstruction. CONCLUSION: Together, these findings implied that self-generated Klotho protein, using MC technology, is functionally active and relevant as a therapeutic approach in renal injury.

Lentiviral vector-mediated overexpression of Klotho in the brain improves Alzheimer's disease-like pathology and cognitive deficits in mice.

Alzheimer's disease (AD) is the most common type of senile dementia. The antiaging gene Klotho is reported to decline in the brain of patients and animals with AD. However, the role of Klotho in the progression of AD remains elusive. The present study explored the effects and underlying mechanism of Klotho in a mouse model of AD. The upregulation of cerebral Klotho expression was mediated by an intracerebroventricular injection of a lentiviral vector that encoded Klotho (LV-KL) in 7-month-old amyloid precursor protein/presenilin 1 transgenic mice. Three months later, LV-KL significantly induced Klotho overexpression in the brain and effectively ameliorated cognitive deficit and AD-like pathology in amyloid precursor protein/presenilin 1 mice. LV-KL induced autophagy activation and protein kinase B/mammalian target of rapamycin inhibition both in AD mice and BV2 murine microglia. These results suggest that the upregulation of Klotho expression in the brain may promote the autophagic clearance of amyloid beta and protect against cognitive deficits in AD mice. These findings highlight the preventive and therapeutic potential of Klotho for the treatment of AD.

Vestronidase Alfa: A Review in Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII is an extremely rare, autosomal recessive lysosomal storage disorder characterized by a deficiency of ß-glucuronidase activity, resulting in partial degradation and accumulation of GAGs in numerous tissues throughout the body, with consequent cellular damage and organ dysfunction. Enzyme replacement therapy (ERT) with intravenous vestronidase alfa (Mepsevii™), a recombinant form of human ß-glucuronidase, is the first disease-specific therapy approved for the treatment of mucopolysaccharidosis VII in pediatric and adult patients. In the pivotal, blind start, phase 3 trial, 24 weeks of vestronidase alfa therapy significantly reduced urinary GAG (uGAG) excretion in patients with mucopolysaccharidosis VII. Based on a Multi-Domain Responder Index (MDRI; comprises six clinically important morbidity domains, with prespecified minimally important differences for each domain), most evaluable patients experienced an improvement in ≥ 1 domain during the 24-week primary assessment period (overall positive mean change of 0.5 domains). The clinical benefits of vestronidase alfa were sustained during longer-term treatment, as was the reduction in uGAG excretion. Vestronidase alfa has a manageable tolerability profile, with most adverse reactions of mild to moderate severity. Given the lack of treatment options and the clinical benefits it provides, intravenous vestronidase alfa is an important emerging ERT for patients with mucopolysaccharidosis VII.

Pharmacokinetic and Pharmacodynamic Modeling to Optimize the Dose of Vestronidase Alfa, an Enzyme Replacement Therapy for Treatment of Patients with Mucopolysaccharidosis Type VII: Results from Three Trials.

INTRODUCTION: Mucopolysaccharidosis type VII (MPS VII, Sly Syndrome) is a progressive, debilitating, ultra-rare lysosomal storage disorder caused by the deficiency of ß-glucuronidase (GUS), an enzyme required for breakdown of glycosaminoglycans (GAGs). Vestronidase alfa, a recombinant human GUS, is an enzyme replacement therapy approved in the US and EU for the treatment of MPS VII. METHODS: The pharmacokinetics (PK) and pharmacodynamics (PD) of vestronidase alfa were evaluated in 23 adult and pediatric subjects with MPS VII enrolled in phase I-III clinical trials to optimize the clinical dosing regimen of vestronidase alfa. The serum concentration-time profiles were adequately described by a two-compartment population PK model incorporating subjects' body weight as the only significant covariate. RESULTS: Model-based simulations predicted a substantially decreased time duration of serum exposures exceeding the level of Kuptake (the in vitro determined vestronidase alfa concentration corresponding to 50% maximum rate of cellular uptake) for 4 or 8 mg/kg once every 4 weeks dosing, compared with 4 mg/kg once every other week (QOW) dosing by intravenous infusion, suggesting that given the same total monthly dose, the QOW dosing frequency should result in more efficient delivery to the GUS-deficient tissue cells, and therefore superior treatment efficacy. A standard inhibitory maximal effect model reasonably explained the observed pharmacological PD responses of reduction in urinary GAGs from pretreatment baseline, which appeared to have reached the plateau of maximal effect at the 4 mg/kg QOW dose. CONCLUSION: The modeling results, together with the clinical evidence of safety and efficacy, supported the recommended 4 mg/kg QOW dosing regimen of vestronidase alfa for pediatric and adult patients with MPS VII. CLINICAL TRIAL REGISTRATION: NCT01856218, NCT02418455, NCT02230566.

Renal Klotho is Reduced in Septic Patients and Pretreatment With Recombinant Klotho Attenuates Organ Injury in Lipopolysaccharide-Challenged Mice.

OBJECTIVES: To determine the applicability of recombinant Klotho to prevent inflammation and organ injury in sepsis in man and mice. DESIGN: Prospective, clinical laboratory study using "warm" human postmortem sepsis-acute kidney injury biopsies. Laboratory study using a mouse model of endotoxemia. SETTING: Research laboratory at a university teaching hospital. SUBJECTS: Adult patients who died of sepsis in the ICU and control patients undergoing total nephrectomy secondary to renal cancer; male C57BL/6 and Klotho haploinsufficient mice. INTERVENTIONS: Lipopolysaccharide (0.05 mg/kg) injection and kill after 4, 8, and 24 hours. Mice received recombinant Klotho (0.05 mg/kg) 30 minutes prior to lipopolysaccharide (1 mg/kg) injection. Mice treated with saline were included as controls. MEASUREMENTS AND MAIN RESULTS: Quantitative reverse transcription polymerase chain reaction and immunohistochemical staining were used to quantify Klotho messenger RNA and protein expression in the kidney of sepsis-acute kidney injury patients and the kidney and brain of mice. The messenger RNA and protein expression of damage markers, inflammatory cytokine, chemokines, and endothelial adhesion molecules were also determined in mice. Renal neutrophil influx was quantified. We found significantly lower renal Klotho messenger RNA and protein levels in sepsis-acute kidney injury biopsies than in control subjects. These findings were recapitulated in the kidney and brain of lipopolysaccharide-challenged mice. Decreased Klotho expression paralleled an increase in kidney damage markers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Administration of recombinant Klotho prior to lipopolysaccharide injection attenuated organ damage, inflammation and endothelial activation in the kidney and brain of mice. Furthermore, less neutrophils infiltrated into the kidneys of recombinant Klotho mice compared with lipopolysaccharide only treated mice. CONCLUSIONS: Renal Klotho expression in human sepsis-acute kidney injury and in mouse models of sepsis was significantly decreased and correlated with renal damage. Recombinant Klotho intervention diminished organ damage, inflammation, and endothelial activation in the kidney and brain of lipopolysaccharide-challenged mice. Systemic Klotho replacement may potentially be an organ-protective therapy for septic patients to halt acute, inflammatory organ injury.

A novel Blind Start study design to investigate vestronidase alfa for mucopolysaccharidosis VII, an ultra-rare genetic disease.

BACKGROUND: Drug development for ultra-rare diseases is challenging because small sample sizes and heterogeneous study populations hamper the ability of randomized, placebo-controlled trials with a single primary endpoint to demonstrate valid treatment effects. METHODS: To overcome these challenges, a novel Blind Start design was utilized in a study of vestronidase alfa in mucopolysaccharidosis VII (Sly syndrome), an ultra-rare lysosomal disease, that demonstrates the strengths of this approach in a challenging drug-development setting. Twelve subjects were randomized to 1 of 4 blinded groups, each crossing over to active treatment in a blinded fashion at different timepoints with efficacy analysis comparing the last assessment before cross over to after 24 weeks of treatment. Study assessments included: Percentage change from baseline in urinary GAG (uGAG); a Multi-Domain Responder Index (MDRI) using prespecified minimal important differences (6-Minute Walk Test, Forced Vital Capacity, shoulder flexion, visual acuity, and Bruininks-Oseretsky Test of Motor Proficiency); fatigue as assessed by the Pediatric Quality of Life Inventory™ Multidimensional Fatigue Scale; and safety. RESULTS: Vestronidase alfa treatment for 24 weeks significantly reduced uGAG excretion (dermatan sulfate: 64.8%, p < 0.0001). Most subjects (10/12) had a clinically meaningful improvement in at least one MDRI domain with an overall mean change (±SD) of +0.5 (±0.8) at Treatment Week 24 (p = 0.0527). Exposure-adjusted incidence rates of adverse events were similar between groups. CONCLUSIONS: The Blind Start study and MDRI design improve statistical power that enhances detection of a positive treatment effect in this rare heterogeneous disease and could be utilized for other ultra-rare diseases.

Peripheral Elevation of a Klotho Fragment Enhances Brain Function and Resilience in Young, Aging, and α-Synuclein Transgenic Mice.

Cognitive dysfunction and decreased mobility from aging and neurodegenerative conditions, such as Parkinson and Alzheimer diseases, are major biomedical challenges in need of more effective therapies. Increasing brain resilience may represent a new treatment strategy. Klotho, a longevity factor, enhances cognition when genetically and broadly overexpressed in its full, wild-type form over the mouse lifespan. Whether acute klotho treatment can rapidly enhance cognitive and motor functions or induce resilience is a gap in our knowledge of its therapeutic potential. Here, we show that an α-klotho protein fragment (αKL-F), administered peripherally, surprisingly induced cognitive enhancement and neural resilience despite impermeability to the blood-brain barrier in young, aging, and transgenic α-synuclein mice. αKL-F treatment induced cleavage of the NMDAR subunit GluN2B and also enhanced NMDAR-dependent synaptic plasticity. GluN2B blockade abolished αKL-F-mediated effects. Peripheral αKL-F treatment is sufficient to induce neural enhancement and resilience in mice and may prove therapeutic in humans.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Indoxyl sulfate induces platelet hyperactivity and contributes to chronic kidney disease-associated thrombosis in mice.

Thrombosis is a common complication of chronic kidney disease (CKD), but the causes and mechanisms of CKD-associated thrombosis are not well clarified. Here, we show that platelet activity is remarkably enhanced in CKD mice, with increase of serum indoxyl sulfate (IS), a typical uremic toxin, which cannot be effectively cleared by routine dialysis. Ex vivo and in vitro experiments reveal that IS displays a distinct ability to enhance platelet activities, including elevated response to collagen and thrombin, increases in platelet-derived microparticles, and platelet-monocyte aggregates. The flow chamber assay and carotid artery thrombosis model demonstrate that IS-induced platelet hyperactivity contributes to thrombus formation. Further investigations disclose that reactive oxygen species (ROS)-mediated p38MAPK signaling plays a key role in IS-induced platelet hyperactivity. Moreover, we show that Klotho, which is expressed dominantly in the kidneys, has the capacity to counteract IS-induced platelet hyperactivity by inhibiting ROS/p38MAPK signaling, whereas Klotho reduction may aggravate the effect of IS on platelet activation in CKD and klotho+/- mice. Finally, we demonstrate that Klotho protein treatment can protect against IS-induced thrombosis and atherosclerosis in apoE-/- mice. Our findings uncover the mechanism of platelet hyperactivity induced by IS and provide new insights into the pathogenesis and treatment of CKD-associated thrombosis.

Recombinant α-Klotho may be prophylactic and therapeutic for acute to chronic kidney disease progression and uremic cardiomyopathy.

α-Klotho is highly expressed in the kidney, and its extracellular domain is cleaved and released into the circulation. Chronic kidney disease (CKD) is a state of α-Klotho deficiency, which exerts multiple negative systemic effects on numerous organs including the cardiovascular system. Since acute kidney injury (AKI) greatly escalates the risk of CKD development, we explored the effect of α-Klotho on prevention and treatment on post-AKI to CKD progression and cardiovascular disease. Therein, ischemia reperfusion injury-induced AKI was followed by early administration of recombinant α-Klotho or vehicle starting one day and continued for four days after kidney injury (CKD prevention protocol). A CKD model was generated by unilateral nephrectomy plus contralateral ischemia reperfusion injury. Late administration of α-Klotho in this model was started four weeks after injury and sustained for 12 weeks (CKD treatment protocol). The prevention protocol precluded AKI to CKD progression and protected the heart from cardiac remodeling in the post-AKI model. One important effect of exogenous α-Klotho therapy was the restoration of endogenous α-Klotho levels long after the cessation of exogenous α-Klotho therapy. The treatment protocol still effectively improved renal function and attenuated cardiac remodeling in CKD, although these parameters did not completely return to normal. In addition, α-Klotho administration also attenuated high phosphate diet-induced renal and cardiac fibrosis, and improved renal and cardiac function in the absence of pre-existing renal disease. Thus, recombinant α-Klotho protein is safe and efficacious, and might be a promising prophylactic or therapeutic option for prevention or retardation of AKI-to-CKD progression and uremic cardiomyopathy.

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.

Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy.

Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine ß-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.

Pharmacologic manipulation of lysosomal enzyme transport across the blood-brain barrier.

The adult blood-brain barrier, unlike the neonatal blood-brain barrier, does not transport lysosomal enzymes into brain, making enzyme replacement therapy ineffective in treating the central nervous system symptoms of lysosomal storage diseases. However, enzyme transport can be re-induced with alpha-adrenergics. Here, we examined agents that are known to alter the blood-brain barrier transport of large molecules or to induce lysosomal enzyme transport across the blood-brain barrier ((±)epinephrine, insulin, retinoic acid, and lipopolysaccharide) in 2-week-old and adult mice. In 2-week-old adolescent mice, all these pharmacologic agents increased brain and heart uptake of phosphorylated human ß-glucuronidase. In 8-week-old adult mice, manipulations with (±)epinephrine, insulin, and retinoic acid were significantly effective on uptake by brain and heart. The increased uptake of phosphorylated human ß-glucuronidase was inhibited by mannose 6-phosphate for the agents (±)epinephrine and retinoic acid and by L-NG-nitroarginine methyl ester for the agent lipopolysaccharide in neonatal and adult mice. An in situ brain perfusion study revealed that retinoic acid directly modulated the transport of phosphorylated human ß-glucuronidase across the blood-brain barrier. The present study indicates that there are multiple opportunities to at least transiently induce phosphorylated human ß-glucuronidase transport at the adult blood-brain barrier.