Severely reduced production of klotho in human chronic renal failure kidney

We recently identified a novel gene, termed klotho (kl) that is involved in the development of a syndrome in mice resembling human aging. A defect of the kl gene expression in mice leads to multiple disorders including arteriosclerosis, osteoporosis, ectopic calcification, and skin atrophy together with short life-span and infertility. Patients with chronic renal failure (CRF), develop multiple complications that are reminiscent of phenotypes observed in kl mutant mice. Furthermore, the kl gene is mainly expressed in kidney and brain. These evidences above suggest the possible involvement of Klotho function in the complications arising in CRF patients. To investigate the above possibility, we examined the kidneys of 10 clinically or histologically diagnosed CRF cases. The level of kl gene expression was measured by utilizing RNase protection assay. The expression of Klotho protein was assayed by utilizing Western blot analysis and by immunohistochemistry. The levels of kl mRNA expression were greatly reduced in all CRF kidneys. Moreover, the production of Klotho protein was also severely reduced in all CRF kidneys. These results suggest that the decrease in kl gene expression in CRF patients may underlie the deteriorating process of multiple complications in the CRF patients.     

Klotho protein deficiency leads to overactivation of mu-calpain

The klotho mouse is an animal model that prematurely shows phenotypes resembling human aging. Here we report that in homozygotes for the klotho mutation (kl(-/-)), alpha(II)-spectrin is highly cleaved, even before the occurrence of aging symptoms such as calcification and arteriosclerosis. Because alpha(II)-spectrin is susceptible to proteolysis by calpain, we examined the activation of calpain in kl(-/-) mice. m-Calpain was not activated, but mu-calpain was activated at an abnormally high level, and an endogenous inhibitor of calpain, calpastatin, was significantly decreased. Proteolysis of alpha(II)-spectrin increased with decreasing level of Klotho protein. Similar phenomena were observed in normal aged mice. Our results indicate that the abnormal activation of calpain due to the decrease of Klotho protein leads to degradation of cytoskeletal elements such as alpha(II)-spectrin. Such deterioration may trigger renal abnormalities in kl(-/-) mice and aged mice, but Klotho protein may suppress these processes.     

Reduced Klotho expression level in kidney aggravates renal interstitial fibrosis

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-β(1) (TGF-β(1)) 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-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) 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.     

Klotho蛋白在糖尿病肾病中的研究进展

Klotho蛋白是近期发现的和衰老密切相关的蛋白,主要表达于肾小管上皮细胞和脑脉络膜。Klotho蛋白的高表达可以增加机体对氧化应激的抵抗。许多研究证实,在糖尿病肾病中,肾脏klotho的表达降低,并且通过调节磷酸盐代谢、抗氧化应激、抗肾脏纤维化、抗肾小球肥大、抗凋亡、抗炎症等途径保护肾功能。本文就klotho蛋白与糖尿病肾病的关系进行综述,探寻其在糖尿病肾病中的分子生物学机制。     

Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) beta-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl beta-D-glucuronide in contrast to bovine liver beta-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of beta-glucuronidase. A number of natural beta-glucuronides were screened by competitive inhibition for KLFc beta-glucuronidase. We found that steroid beta-glucuronides such as beta-estradiol 3-beta-D-glucuronide, estrone 3-beta-D-glucuronide, and estriol 3beta-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel beta-glucuronidase and that steroid beta-glucuronides are potential candidates for the natural substrate(s) of Klotho.     

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.     

Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice

Klotho mutant mouse (kl-/-), a mouse model for human aging, exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration, skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA, however, is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variety of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain, kidney, and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were dominantly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/-mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions, were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation, which may explain why mutation of klotho gene results in a variety of phenotypes.     

Klotho as a regulator of oxidative stress and senescence

The klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. The klotho gene encodes a single-pass transmembrane protein that binds to multiple fibroblast growth factor (FGF) receptors and functions as a co-receptor for FGF23, a bone-derived hormone that suppresses phosphate reabsorption and vitamin D biosynthesis in the kidney. In addition, the extracellular domain of Klotho protein is shed and secreted, potentially functioning as a humoral factor. The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology.     

Regulation of the Na+/K+ ATPase by Klotho

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.     

Klotho attenuates diabetic nephropathy in db/db mice and ameliorates high glucose-induced injury of human renal glomerular endothelial cells

Glomerular endothelial cell injury plays an important role in the development and progression of diabetic nephropathy (DN). The expression and function of klotho in glomerular endothelial cells remain unclear. Thus, this study aimed to investigate the expression and the functional role of klotho in DN progression in mice and in high glucose (HG)-induced cell injury of human renal glomerular endothelial cells (HRGECs) and the underlying mechanism. In this study, HRGECs were cultured with media containing HG to induce endothelial cell injury and db/db mice were used as DN model mice. Klotho was overexpressed or knocked down in HRECs to evaluate its role in HG-induced HRGECs injury. klotho-overexpressing adenovirus (rAAV-klotho) was injected into db/db mice via the tail vein to further validate the protective effect of klotho in DN. Decreased klotho expression was observed in DN patients, DN mice, and HG-exposed HRGECs. Furthermore, klotho overexpression significantly abolished the HG-induced HRGECs injury and activation of Wnt/β-catenin pathway and RAAS. In contrast, klotho knockdown exerted the opposite effects. Moreover, klotho attenuated diabetic nephropathy in db/db mice, which was also associated with inhibition of the Wnt/β-catenin pathway and RAAS. In conclusion, klotho attenuates DN in db/db mice and ameliorates HG-induced injury of HRGECs.     

Association between Decreased Klotho Blood Levels and Organic Growth Hormone Deficiency in Children with Growth Impairment

Objective

Klotho is an aging-modulating protein expressed mainly in the kidneys and choroid plexus, which can also be shed, released into the circulation and act as a hormone. Klotho deficient mice are smaller compared to their wild-type counterparts and their somatotropes show marked atrophy and reduced number of secretory granules. Recent data also indicated an association between klotho levels and growth hormone (GH) levels in acromegaly. We aimed to study the association between klotho levels and GH deficiency (GHD) in children with growth impairment.

Design

Prospective study comprising 99 children and adolescents (aged 9.0±3.7 years, 49 male) undergoing GH stimulation tests for short stature (height-SDS = −2.1±0.6). Klotho serum levels were measured using an α-klotho ELISA kit.

Results

Klotho levels were significantly lower (p<0.001) among children with organic GHD (n = 11, 727±273 pg/ml) compared to both GH sufficient participants (n = 59, 1497±754 pg/ml) and those with idiopathic GHD (n = 29, 1645±778 pg/ml). The difference between GHS children and children with idiopathic GHD was not significant. Klotho levels positively correlated with IGF-1- standard deviation scores (SDS) (R = 0.45, p<0.001), but were not associated with gender, pubertal status, age or anthropometric measurements.

Conclusions

We have shown, for the first time, an association between low serum klotho levels and organic GHD. If validated by additional studies, serum klotho may serve as novel biomarker of organic GHD.     

Autophagic-lysosomal pathway functions in the masseter and tongue muscles in the klotho mouse, a mouse model for aging

Klotho mutant (kl/kl) mice, a type of short-lived mouse models, display several aging-related phenotypes. To investigate whether the atrophy of skeletal muscles is induced in these mice via activation of the ubiquitin-proteasomal pathway and/or the autophagic-lysosomal pathway through an alteration of insulin/IGF-I signaling, we analyzed the activity of the two pathways for protein degradation and components of the insulin/IGF signaling pathway in their skeletal muscles. The masseter, tongue, and gastrocnemius muscles in kl/kl showed marked reductions in muscle weight and in myofiber diameter compared with +/+. The autophagic-lysosomal pathway in kl/kl was activated in the masseter and tongue, but not in the gastrocnemius, compared with that in +/+, whereas the ubiquitin-proteasomal pathway in these three muscles of kl/kl was not altered. No marked difference in the phosphorylation levels of insulin/IGF-I signaling components, such as insulin/IGF-I receptor, Akt, and FoxO in three muscles studied were found between kl/kl and +/+, but the phosphorylation levels of signaling component at the downstream of mTOR such as 4E-BP1 and p70 S6K were suppressed in the masseter and tongue of kl/kl compared with +/+. Deficiency of essential amino acids is reported to activate the autophagy-lysosomal pathway through the down-regulation of mTOR, not through IGF-Akt-FoxO. The masseter and tongue seem to be more actively moved than limb muscles in kl/kl, because they are essential for survival activities such as mastication, swallowing, and respiration. Thus, the deficiency of amino acid by the active movement of the masseter and tongue seems to stimulate the autophagic-lysosomal pathway via the down-regulation of mTOR signalling pathway.     

Disruption of klotho gene causes an abnormal energy homeostasis in mice

klotho mice, which genetically lack klotho gene expression, are characterized with various systemic phenotypes resembling human aging, and also with growth retardation. Here we show that klotho mice have a barely detectable amount of the white adipose tissue but their brown adipose tissue (BAT) is comparably preserved. Glucose tolerance and insulin sensitivity in klotho mice are increased compared to those in wild-type mice as revealed by intraperitoneal glucose and insulin tolerance tests. Uncoupling protein-1 gene expression of BAT and body temperature in klotho mice are lower than those in wild-type mice, suggesting that klotho mice have less energy expenditure than wild-type mice. Histological examination suggests that klotho mice possess less energy storage than wild-type mice with respect to glycogen in the liver and lipid in BAT. All these changes of parameters for energy homeostasis in klotho mice are very similar to those reported under food-restricted conditions. However, the amount of food intake is not different between klotho and wild-type mice when normalized for body weight. The present study elucidates the importance of klotho gene expression for the maintenance of normal energy homeostasis.     

Noncoding variations in Cyp24a1 gene are associated with Klotho‐mediated aging phenotypes in different strains of mice

In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging‐related phenotypes caused by Klotho down‐regulation. The hypomorphic Klotho [kl/kl] allele that decreases Klotho expression in C3H, BALB/c, 129, and C57BL/6 genetic backgrounds substantially increases 1,25(OH)2D3 levels in the sera of susceptible C3H, BALB/c, and 129, but not C57BL/6 mice. This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super‐enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6‐kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.     

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.     

Effects of intra- and extracellular factors on anti-aging klotho gene expression

Inactivation of the klotho gene in mice causes serious systemic disorders, resembling human aging. However, at the molecular level, its action mechanisms are not well understood. The stimulatory or inhibitory effects of cis- and trans-regulatory factors on the klotho gene expression are also still unclear. We studied the effects of intra- and extracellular factors on human klotho gene expression. For this purpose, pHKP-Luc and pHKP-GFP reporter vectors were constructed with the 2.1-kbp upstream region of human klotho, covering its promoter region, using luciferase and GFP genes as the reporter. A series of vectors that have deletions in the upstream region of the klotho gene were constructed to assay cis-acting factors. Deletion of some parts of the klotho gene upstream region significantly affected reporter gene expression in HEK293 cells. p16 and p53 proteins inhibited reporter luciferase expression under the control of human klotho promoter in a dose-dependent manner. Calcium and phosphate ions stimulated klotho expression. p21, PTH, IGF-1, and angiotensin-II had no significant effect on klotho expression in HEK293 cells.     

Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice

C57BL/6 mice immunized with the extracellular Ig-like domain of rat myelin oligodendrocyte glycoprotein (MOG) developed experimental autoimmune encephalomyelitis (EAE) resembling that induced by rodent MOG 35-55 in its B cell independence and predominantly mononuclear CNS infiltrate. In contrast, human MOG protein-induced EAE was B cell dependent with polymorphonuclear leukocytes. Human MOG differs from rat MOG at several residues, including a proline for serine substitution at position 42. Human MOG 35-55 was only weakly encephalitogenic, and a proline substitution in rat MOG at position 42 severely attenuated its encephalitogenicity. However, human MOG 35-55 was immunogenic, inducing proliferation and IFN-gamma and IL-13 to human, but not rodent MOG 35-55 [corrected]. The B cell dependence of EAE induced by human MOG protein was not due to a requirement for Ag presentation by B cells, because spleen cells from B cell-deficient mice processed and presented human and rat MOG proteins to T cells. The different pathogenic mechanisms of human and rat MOG proteins might result from different Abs induced by these proteins. However, rat and human MOG proteins induced Abs to mouse MOG that were equivalent in titer and IgG subclass. These data demonstrate that EAE can be induced in C57BL/6 mice by two mechanisms, depending on the nature of the immunogen: an encephalitogenic T cell response to rat MOG or rodent MOG 35-55, or an encephalitogenic B cell response to epitopes on human MOG protein that most likely cross-react with mouse determinants.