黒尾 誠

BACKGROUND: αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study. METHODS: We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form. RESULTS: Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency. CONCLUSION: Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.

Although several recent studies have investigated the major phylogenetic relationships within Hynobiidae, their evolutionary history remains partially resolved and the phylogenetic positions of some genera, particularly Pachyhynobius and Salamandrella are still disputed. Notably, previous studies relied primarily on mitochondrial DNA data and concatenated analyses; thus, a new investigation based on multiple nuclear genes and species-tree inference is needed. Here, we provide an in-depth phylogenetic analysis, based on 29 nuclear genes comprising 29,232bp of data from a comprehensive taxonomic sampling (24 hynobiids and 7 outgroups), using both concatenated and species-tree methods. Our results robustly resolved most genus-level relationships within Hynobiidae, including the placement of Salamandrella as the sister group to a clade containing Batrachuperus, Liua and Pseudohynobius, and the placement of Pachyhynobius as the sister group to a clade containing all hynobiids excluding Onychodactylus, Paradactylodon and Ranodon. Time estimates based on our data suggest that the major group of living hynobiids (excluding Onychodactylus) originated approximately 40Ma, ∼50% younger than estimates from mtDNA data (62.5Ma) but 10% older than estimates from three nuclear genes (36Ma). Our results highlight the benefits of using a large number of nuclear loci to infer both phylogeny and time for relatively old lineages.

Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1)/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1) the ratio of free ASK1 to thioredoxin (Trx)-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2) the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of-bound Trx; and 3) that 14-3-3ζ is hyper phosphorylated (Ser-58) in the transgene which correlated with increased monomer forms. In addition, we evaluated the in vivo robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.

The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

Klotho is a single-pass transmembrane protein predominantly expressed in the kidney. The extracellular domain of Klotho is subject to ectodomain shedding and is released into the circulation as a soluble form. Soluble Klotho is also generated from alternative splicing of the Klotho gene. In mice, defects in Klotho expression lead to complex phenotypes resembling those observed in dialysis patients. However, the relationship between the level of serum soluble Klotho and overall survival in hemodialysis patients, who exhibit a state of Klotho deficiency, remains to be delineated. Here we prospectively followed a cohort of 63 patients with a mean duration of chronic hemodialysis of 6.7 ± 5.4 years for a median of 65 months. Serum soluble Klotho was detectable in all patients (median 371 pg/mL, interquartile range 309-449). Patients with serum soluble Klotho levels below the lower quartile (<309 pg/mL) had significantly higher cardiovascular and all-cause mortality rates. Furthermore, the higher all-cause mortality persisted even after adjustment for confounders (hazard ratio 4.14, confidence interval 1.29-13.48). We conclude that there may be a threshold for the serum soluble Klotho level associated with a higher risk of mortality.

α-Klotho exerts pleiotropic biological actions. Heterozygous α-Klotho haplo-insufficient mice (kl/+) appear normal at baseline except for age-related changes in the lung, suggesting heightened pulmonary susceptibility to α-Klotho deficiency. We used in vivo and in vitro models to test whether α-Klotho protects lung epithelia against injury. Normally, α-Klotho is not expressed in the lung, but circulating α-Klotho levels are reduced -40% in kl/+ mice and undetectable in homozygous α-Klotho-deficient mice (kl/kl). kl/+ mice show distal air space enlargement at a given airway pressure, with elevated lung oxidative damage marker (8-hydroxydeoxyguanosine; 8-OHdG); these abnormalities are exacerbated in kl/kl mice. Studies were performed in A549 lung epithelial cells and/or primary culture of alveolar epithelial cells. Hyperoxia (95% O2) and high inorganic phosphate concentrations (Pi, 3-5 mM) additively caused cell injury (lactate dehydrogenase release), oxidative DNA damage (8-OHdG), lipid oxidation (8-isoprostane), protein oxidation (carbonyl), and apoptosis (caspase-8 activity and TUNEL stain). Transfection of transmembrane or soluble α-Klotho, or addition of soluble α-Klotho-containing conditioned media, increased cellular antioxidant capacity (Cu- and Fe-based assays) via increased nuclear factor erythroid-derived 2-related factors 1 and 2 (Nrf1/2) transcriptional activity and ameliorated hyperoxic and phosphotoxic injury. To validate the findings in vivo, we injected α-Klotho-containing conditioned media into rat peritoneum before and during hyperoxia exposure and found reduced alveolar interstitial edema and oxidative damage. We conclude that circulating α-Klotho protects the lung against oxidative damage and apoptosis partly via increasing endogenous antioxidative capacity in pulmonary epithelia. Cytoprotection by α-Klotho may play an important role in degenerative diseases of the lung.

Extracellular phosphate is toxic to the cell at high concentrations. When the phosphate level is increased in the blood by impaired urinary phosphate excretion, premature aging ensues. When the phosphate level is increased in the urine by dietary phosphate overload, this may lead to kidney damage (tubular injury and interstitial fibrosis). Extracellular phosphate exerts its cytotoxicity when it forms insoluble nanoparticles with calcium and fetuin-A, referred to as calciprotein particles (CPPs). CPPs are highly bioactive ligands that can induce various cellular responses, including osteogenic transformation of vascular smooth muscle cells and cell death in vascular endothelium and renal tubular epithelium. CPPs are detected in the blood of animal models and patients with chronic kidney disease (CKD) and associated with adaptation of the endocrine axes mediated by fibroblast growth factor-23 (FGF23) and Klotho that regulate mineral metabolism and aging. These observations have raised the possibility that CPPs may contribute to the pathophysiology of CKD. This notion, if validated, is expected to provide new diagnostic and therapeutic targets for CKD.

AIM: Suicidal erythrocyte death or eryptosis is characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes may adhere to the vascular wall by binding of phosphatidylserine to endothelial CXC chemokine ligand 16 (CXCL16). Triggers of eryptosis include osmotic shock or energy depletion. Susceptibility to eryptosis is modified by Klotho, a protein with profound effect on ageing and lifespan. Klotho deficiency leads to accelerated ageing and early death. The percentage of eryptotic erythrocytes is significantly larger in klotho-deficient mice (klotho(-/-) ) than in their wild-type littermates (klotho(+/+) ). The present study explored whether the accelerated eryptosis of klotho-deficient mice is paralleled by enhanced adhesion. METHODS: Phosphatidylserine-exposing erythrocytes were identified by measurement of annexin V binding and adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labelled erythrocytes in a flow chamber. RESULTS: Annexin V binding was higher in klotho(-/-) erythrocytes than in klotho(+/+) erythrocytes. Osmotic shock for 1 h (addition of 550 mm sucrose) and energy depletion (12-h glucose depletion) increased annexin V binding to values again significantly larger in klotho(-/-) erythrocytes than in klotho(+/+) erythrocytes. klotho(-/-) erythrocytes were particularly sensitive to osmotic shock. Both osmotic shock and energy depletion enhanced erythrocyte adhesion, an effect again more pronounced in klotho(-/-) erythrocytes than in klotho(+/+) erythrocytes. The adhesion was significantly decreased by coating of phospatidylserine with annexin V (5 μL mL(-1) ) or by coating of CXCL16 with neutralizing antibodies (4 μg mL(-1) ). CONCLUSIONS: klotho(-/-) erythrocytes are particularly sensitive to osmotic shock, and enhanced eryptosis of klotho(-/-) erythrocytes is paralleled by enhanced adhesion to endothelial CXCL16.

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to - 1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho downregulation. © FASEB.

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β1 (TGF-β1) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β1 receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β1 activity and is a cause of renal fibrosis. On the other hand, TGF-β1 reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression. © 2012 the American Physiological Society.

Previous work found that different Japanese lineages of salamanders had quite different levels of species and genetic diversity. Lineages vary from having one to several species and the extent of genetic variation among lineages differs substantially. Most speciose, genus Hynobius contains 18 species and several potential cryptic species. We explore genetic diversity in this genus by combining comprehensive sampling and mitochondrial DNA sequences. Based on this and previous analyses of salamanders, relative times of divergence are employed to evaluate the relationship between age and diversity among the four major lineages whose distributions broadly overlap on the islands. For Hynobius, our analyses are congruent with the previously reported high level of cryptic diversity in morphology and allozymes, particularly in species composed of non-sister matrilines. Both species and genetic diversity correlate with the relative ages of the lineages. This correlation indicates that the variation in levels of diversity can be explained, to a considerable extent, by the hypothesis that older insular lineages have accumulated greater diversity. In addition to the Korean Peninsula, H. leechii might have survived in another Pleistocene glacial refugium north of the peninsula and this refugium provided a source of colonization after the last glacial maximum.

In the practice of molecular dating, substitution saturation will bias the results if not properly modeled. Date estimates based on commonly used mitochondrial DNA sequences likely suffer from this problem because of their high substitution rate. Nevertheless, the patterns and extent of such expected bias remain unknown for many major evolutionary lineages, which often differ in ages, available calibrations, and substitution rates of their mitochondrial genome. In this case study of salamanders, we used estimates based on multiple nuclear exons to assess the effects of saturation on dating divergences using mitochondrial genome sequences on a timescale of ~200-300 My. The results indicated that, due to saturation for older divergences and in the absence of younger effective calibration points, dates derived from the mitochondrial data were considerably overestimated and systematically biased toward the calibration point for the ingroup root. The overestimate might be as great as 3-10 times (about 20 My) older than actual divergence dates for recent splitting events and 40 My older for events that are more ancient. For deep divergences, dates estimated were strongly compressed together. Furthermore, excluding the third codon positions of protein-coding genes or only using the RNA genes or second codon positions did not considerably improve the performance. In the order Caudata, slowly evolving markers such as nuclear exons are preferred for dating a phylogeny covering a relatively wide time span. Dates estimated from these markers can be used as secondary calibrations for dating recent events based on rapidly evolving markers for which mitochondrial DNA sequences are attractive candidates due to their short coalescent time. In other groups, similar evaluation should be performed to facilitate the choice of markers for molecular dating and making inferences from the results.