Membrane-bound Klotho Research Articles

Cognitive Impairment Related to Chronic Kidney Disease Is Associated with a Decreased Abundance of Membrane-Bound Klotho in the Cerebral Cortex.

Cognitive impairment (CI) is a complication of chronic kidney disease (CKD) that is frequently observed among patients. The aim of this study was to evaluate the potential crosstalk between changes in cognitive function and the levels of Klotho in the brain cortex in an experimental model of CKD. To induce renal damage, Wistar rats received a diet containing 0.25% adenine for six weeks, while the control group was fed a standard diet. The animals underwent different tests for the assessment of cognitive function. At sacrifice, changes in the parameters of mineral metabolism and the expression of Klotho in the kidney and frontal cortex were evaluated. The animals with CKD exhibited impaired behavior in the cognitive tests in comparison with the rats with normal renal function. At sacrifice, CKD-associated mineral disorder was confirmed by the presence of the expected disturbances in the plasma phosphorus, PTH, and both intact and c-terminal FGF23, along with a reduced abundance of renal Klotho. Interestingly, a marked and significant decrease in Klotho was observed in the cerebral cortex of the animals with renal dysfunction. In sum, the loss in cerebral Klotho observed in experimental CKD may contribute to the cognitive dysfunction frequently observed among patients. Although further studies are required, Klotho might have a relevant role in the development of CKD-associated CI and represent a potential target in the management of this complication.

Role of Klotho in Hyperglycemia: Its Levels and Effects on Fibroblast Growth Factor Receptors, Glycolysis, and Glomerular Filtration.

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.

Post-kidney transplant soluble Klotho levels are determined by pretransplant soluble Klotho levels in both living donors and recipients.

Soluble Klotho (sKl), the free form of membrane-bound Klotho predominantly expressed in the kidney, is detectable in serum and may have multiple pleiotropic effects. Patients with end-stage kidney disease are possibly sKl deficient, and kidney transplantation is the treatment of choice in these patients; however, little is known about changes in posttransplant sKl level and the factors influencing these changes. We conducted a prospective longitudinal study to examine changes in posttransplant sKl level in recipients for 12months after living-donor kidney transplantation and analyzed correlations between posttransplant changes in sKl levels and various influencing factors in both recipients and donors. 29 kidney transplant recipients and their living donors were included for analysis. The results showed that sKl levels transiently decreased at 1week posttransplant but progressively increased thereafter for 12months. Multivariable linear regression analysis showed that body surface area-adjusted donor sKl levels were associated with posttransplant increases in recipient sKl levels at 12months. In addition, pretransplant recipient sKl levels and body surface area-adjusted donor sKl levels were identified as an independent predictor of 12-month posttransplant sKl levels. Pretransplant sKl levels in both kidney recipients and living donors are a strong determinant of sKl levels after kidney transplantation.

MO263KLOTHO IS PROTECTIVE IN THE CONTEXT OF ACUTE GLOMERULAR INJURY BUT IS NOT EXPRESSED IN PODOCYTES

Abstract Background and Aims Podocyte integrity is crucial for the maintenance of glomerular function in health and disease. Numerous studies have reported that Klotho overexpression, or treatment with recombinant Klotho, reduces glomerular and tubular damage in mouse models of renal disease. However, the mechanism(s) of action are not fully understood. Several recent studies have also reported that Klotho is expressed in podocytes, where it protects against various types of injury. These findings conflict with previous studies, which have shown that renal Klotho expression is exclusively confined to proximal and distal tubular cells. Method To address this discrepancy and enhance our understanding of the putative glomeruloprotective effects mediated by Klotho, we examined the expression pattern of Klotho in human and mouse kidney by several different methods, and explored its protective effects by overexpressing full-length human Klotho directly in podocytes or in a distant organ (i.e. liver). Results Data at the mRNA and protein levels all converged towards an absence or very low expression of Klotho in podocytes. The generation of a podocyte-specific Klotho knockout mouse further demonstrated that its deletion did not affect glomerular structure or function. Moreover, Klotho deficiency did not worsen glomerular injury in an experimental model of glomerulonephritis (anti-GBM). However, when Klotho was overexpressed in hepatocytes (Alb-cre;hKlothofl/+ - Alb-hKL), serum Klotho increased drastically with no changes in Fgf23 or phosphate metabolism. In mice challenged with anti-GBM, renal histology and ultrastructure of the filtration barrier was less severely affected in Alb-hKL compared to WT mice. There were also significantly less albuminuria, podocyte loss and interstitial fibrosis in Alb-hKL mice compared to their WT littermates. In contrast, mice which overexpressed Klotho in podocytes (Pod-hKL) were not protected from renal injury. Conclusion Taken together, these results strongly suggest that Klotho is not expressed in any substantial amounts in human or mouse podocytes, and that membrane-bound Klotho does not play a role in podocyte biology. Importantly, our results confirm a beneficial role for soluble Klotho in protecting podocytes against injury, and in maintaining glomerular integrity and function.

Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease.

Klotho is a renal protein involved in phosphate homeostasis, which is downregulated in renal disease. It has long been considered an antiaging factor. Two Klotho gene transcripts are thought to encode membrane-bound and secreted Klotho. Indeed, soluble Klotho is detectable in bodily fluids, but the relative contributions of Klotho secretion and of membrane-bound Klotho shedding are unknown. Recent advances in RNA surveillance reveal that premature termination codons, as present in alternative Klotho mRNA (for secreted Klotho), prime mRNAs for degradation by nonsense-mediated mRNA decay (NMD). Disruption of NMD led to accumulation of alternative Klotho mRNA, indicative of normally continuous degradation. RNA IP for NMD core factor UPF1 resulted in enrichment for alternative Klotho mRNA, which was also not associated with polysomes, indicating no active protein translation. Alternative Klotho mRNA transcripts colocalized with some P bodies, where NMD transcripts are degraded. Moreover, we could not detect secreted Klotho in vitro. These results suggest that soluble Klotho is likely cleaved membrane-bound Klotho only. Furthermore, we found that, especially in acute kidney injury, splicing of the 2 mRNA transcripts is dysregulated, which was recapitulated by various noxious stimuli in vitro. This likely constitutes a novel mechanism resulting in the downregulation of membrane-bound Klotho.

Mineral adaptations following kidney transplantation.

Klotho is predominantly expressed in the kidney and reported to have antioxidant and antifibrotic properties. Soluble Klotho (sKl), the circulating protein cleaved from membrane-bound Klotho, is reduced significantly with kidney disease and inversely associated with mortality. sKl has not been thoroughly evaluated prospectively after kidney transplantation. Incident kidney transplant recipients (KTRs) were prospectively evaluated pretransplantation, 1, 12 and 52 weeks post-transplantation. Basic biochemistry, sKl and intact FGF23 were measured. Within-subject comparisons were evaluated using repeat-measure anova or Friedman's analysis. Effects of immunosuppression and biochemical parameters on sKl and FGF-23 over time were analysed using mixed-effects modelling. Median serum creatinine (sCr) at 1 week was 116 (92-142) μmol/l, and at 52 weeks, all 29 KTRs had a functioning graft with median sCr of 111 (97-131) μmol/l. Compared with baseline, sKl was increased at 52 weeks following an initial decline at 1 week (P < 0.005 and P < 0.01, respectively), while FGF23 was considerably reduced at 52 weeks (P < 0.001). In a mixed-effects model, an increased sKl was not associated with reduction in immunosuppression or evaluated biochemical parameters. Modest increase in sKl is observed one-year postkidney transplantation with excellent early graft function suggesting factors beyond renal capacity may influence circulating sKl. FGF23 normalization was observed. Longer term evaluation in transplantation, specifically addressing the effects of immunosuppression, is required to understand the pathophysiology of the sKl/FGF23 axis and potential for modification.

Klotho expression in long bones regulates FGF23 production during renal failure.

Circulating levels of bone-derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane-bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild-type (KLfl/fl ) and knockout (Prx1-Cre;KLfl/fl ) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1-Cre;KLfl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KLfl/fl mice. Consequently, Prx1-Cre;KLfl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1-α-hydroxylase expression, serum 1,25-dihydroxyvitamin D, and calcium levels than KLfl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23-treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.-Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure.

Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells.

Chronic kidney disease (CKD) is a state of Klotho deficiency and excess of the phosphaturic hormone fibroblast growth factor 23 (FGF23). Both dysregulations were shown to be associated with endothelial dysfunction in humans, but direct vascular effects of FGF23 remain largely elusive. In vitro experiments were performed to assess the effects of FGF23 (10ng/mL) in relation to its co-receptor Klotho on nitric oxide (NO) synthesis and reactive oxygen species (ROS) formation and detoxification in human coronary artery endothelial cells (HCAEC). Membrane-bound Klotho is expressed in HCAEC, and FGF23 increases the expression of the Klotho shedding protease ADAM17, and consequently the secretion of soluble Klotho. FGF23 activates FGF receptor 1 and stimulates NO release via Akt-dependent activation of endothelial NO synthase (eNOS). Both FGF receptor (FGFR)-dependent ROS formation via activation of NADPH oxidase 2 (Nox2) as well as ROS degradation via superoxide dismutase 2 (SOD2) and catalase (CAT) is stimulated by FGF23. Pre-incubation with a Klotho inhibitor blunts the FGF23-stimulated Akt-eNOS activation and NO synthesis, and decreases ROS degradation by blocking SOD2 and CAT enzymes, whereas FGF23-stimulated ROS synthesis via Nox2 is unaffected, resulting in low NO bioavailability and increased oxidative stress. Our data indicate that in the presence of Klotho, FGF23 induces NO release in HCAEC and its stimulating effects on ROS production are counterbalanced by increased ROS degradation. In states of Klotho deficiency, e.g., CKD, FGF23-mediated NO synthesis is blunted and ROS formation overrules ROS degradation. Thus, FGF23 excess may primarily promote oxidative stress and thus endothelial dysfunction.

Shedding of klotho by ADAMs in the kidney.

The anti-aging gene klotho plays an important role in Ca(2+) and phosphate homeostasis. Membrane-bound klotho is an essential coreceptor for fibroblast growth factor-23 and can be cleaved by proteases, including a disintegrin and metalloproteinase (ADAM)10 and ADAM17. Cleavage of klotho occurs at a site directly above the plasma membrane (α-cut) or between the KL1 and KL2 domain (β-cut), resulting in soluble full-length klotho or KL1 and KL2 fragments, respectively. The aim of the present study was to gain insights into the mechanisms behind klotho cleavage processes in the kidney. Klotho shedding was demonstrated using a Madin-Darby canine kidney cell line stably expressing klotho and human embryonic kidney-293 cells transiently transfected with klotho. Here, we report klotho expression on both the basolateral and apical membrane, with a higher abundance of klotho at the apical membrane and in the apical media. mRNA expression of ADAM17 and klotho were enriched in mouse distal convoluted and connecting tubules. In vitro ADAM/matrix metalloproteinase inhibition by TNF484 resulted in a concentration-dependent inhibition of the α-cut, with a less specific effect on β-cut shedding. In vivo TNF484 treatment in wild-type mice did not change urinary klotho levels. However, ADAM/matrix metalloproteinase inhibition did increase renal and duodenal mRNA expression of phosphate transporters, whereas serum phosphate levels were significantly decreased. In conclusion, our data show that renal cells preferentially secrete klotho to the apical side and suggest that ADAMs are responsible for α-cut cleavage.

Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue.

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD), a disease state that is strongly associated with loss of renal and systemic (alpha-)Klotho. Reversely, murine Klotho deficiency causes marked medial calcification. It is therefore thought that Klotho conveys a vasculoprotective effect. Klotho expression in the vessel wall, however, is disputed. We assessed Klotho expression in healthy human renal donor arteries (n = 9), CKD (renal graft recipient) arteries (n = 10), carotid endarterectomy specimens (n = 8), other elastic arteries (three groups of n = 3), and cultured human aortic smooth muscle cells (HASMCs) (three primary cell lines), using immunohistochemistry (IHC), immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, and western blotting (WB). We have extensively validated anti-Klotho antibody KM2076 by comparing staining patterns with other anti-Klotho antibodies (SC-22220, SC-22218, and AF1819), competition assays with recombinant Klotho, IHC on Klotho-deficient kl/kl mouse kidney, and WB with recombinant Klotho. Using KM2076, we could not detect full-length Klotho in vascular tissues or HASMCs. On the mRNA level, using primers against all four exon junctions, klotho expression could not be detected either. Fibroblast growth factor 23 (FGF23) injections in mice induced FGF23 signalling in kidneys but not in the aorta, indicating the absence of Klotho-dependent FGF23 signalling in the aorta. Using several independent and validated methods, we conclude that full-length, membrane-bound Klotho is not expressed in healthy or uraemic human vascular tissue.

The kidney is the principal organ mediating klotho effects.

Klotho was discovered as an antiaging gene, and α-Klotho (Klotho) is expressed in multiple tissues with a broad set of biologic functions. Membrane-bound Klotho binds fibroblast growth factor 23 (FGF23), but a soluble form of Klotho is also produced by alternative splicing or cleavage of the extracellular domain of the membrane-bound protein. The relative organ-specific contributions to the levels and effects of circulating Klotho remain unknown. We explored these issues by generating a novel mouse strain with Klotho deleted throughout the nephron (Six2-KL(-/-)). Klotho shedding from Six2-KL(-/-) kidney explants was undetectable and the serum Klotho level was reduced by approximately 80% in Six2-KL(-/-) mice compared with wild-type littermates. Six2-KL(-/-) mice exhibited severe growth retardation, kyphosis, and premature death, closely resembling the phenotype of systemic Klotho knockout mice. Notable biochemical changes included hyperphosphatemia, hypercalcemia, hyperaldosteronism, and elevated levels of 1,25-dihydroxyvitamin D and Fgf23, consistent with disrupted renal Fgf23 signaling. Kidney histology demonstrated interstitial fibrosis and nephrocalcinosis in addition to absent dimorphic tubules. A direct comparative analysis between Six2-KL(-/-) and systemic Klotho knockout mice supports extensive, yet indistinguishable, extrarenal organ manifestations. Thus, our data reveal the kidney as the principal contributor of circulating Klotho and Klotho-induced antiaging traits.

Post-Translational Loss of Renal TRPV5 Calcium Channel Expression, Ca2+ Wasting, and Bone Loss in Experimental Colitis

Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1β reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation andcytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

Partial Answers from Partial Klotho Deficiency

Membrane-bound Klotho is the coreceptor for fibroblast growth factor 23 (FGF23) that increases its affinity for FGF receptors (FGFRs).[1][1] Binding of FGF23 to FGFR-Klotho complexes in the kidney stimulates phosphaturia and inhibits production of 1,25-dihydroxyvitamin D.[2][2] In the parathyroid

Klotho as a potential biomarker and therapy for acute kidney injury

Klotho is a single-pass transmembrane protein that is highly expressed in the kidney and is known to act as a coreceptor for fibroblast growth factor 23. The extracellular domain can be produced independently or shed from membrane-bound Klotho and functions as an endocrine substance with multiple functions including antioxidation, modulation of ion transport, suppression of fibrosis, and preservation of stem cells. Emerging evidence has revealed that Klotho deficiency is an early event in acute kidney injury (AKI), and a pathogenic factor that exacerbates acute kidney damage and contributes to long-term consequences. Restoration by exogenous supplementation or stimulation of endogenous Klotho might prevent and ameliorate injury, promote recovery, and suppress fibrosis to mitigate development of chronic kidney disease. Although data are still emerging, in this Perspectives article we discuss why this renal-derived protein is a highly promising candidate as both an early biomarker and therapeutic agent for AKI.

Bone loss and renal Ca2+ wasting in experimental colitis is accompanied by downregulation of TRPV5 in renal distal convoluted tubules

Dysregulated Ca2+homeostasis likely contributes to the etiology of IBD‐associated bone loss. Experimental colitis leads to decreased expression of Klotho, a protein which supports renal Ca2+ reabsorption by stabilizing TRPV5 channel on the apical membrane of distal tubule epithelial cells. We investigated whether colitis leads to changes in renal Ca2+ handling and expression of TRPV5 utilizing TNBS, and two adoptive T‐cell transfer models (CD4+CD45RBHi or CD4+IL10−/−). Colitic mice had normal serum levels of Ca2+ and PTH. μCT analysis demonstrated changes in bone structure along with changes in serum sRANKL, IL‐6, osteocalcin, TNF, and total serum tDPD crosslinks. Increased fractional Ca2+excretion was accompanied by reduction of TRPV5 protein expression, with concomitant increase in its sialylation status. In mIMCD3 cells transduced with Ad‐TRPV5, proinflammatory cytokines reduced TRPV5 surface expression followed by its degradation. TNF induced interaction of TRPV5 with UBR4 ubiquitin ligase and siRNA‐mediated UBR4 knockdown prevented TNF induced TRPV5 degradation. The cytokine effect on TRPV5 was not observed in cells stably transfected with membrane‐bound Klotho. We propose that TNF and IFNγ act to decrease Klotho expression and activity, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin‐1, and ultimately to UBR4‐dependent endocytosis and degradation of TRPV5.

Klotho interferes with a novel FGF-signalling pathway and insulin/Igf-like signalling to improve longevity and stress resistance in Caenorhabditis elegans

Klotho exerts anti-aging properties in mammals in two different ways. While membrane-bound Klotho, which is primarily expressed in the kidney, acts as an obligate co-receptor of FGF23 to regulate phosphate homeostasis, secreted Klotho, resulting from the shedding of the KL1-KL2 ectodomain into the bloodstream, inhibits Insulin/IGF1 signalling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the biological role of Klotho in Caenorhabditis elegans. Two redundant homologues of the klotho gene exist in C. elegans and encode predicted proteins homologous to the  glucosidase-like KL1 domain of mammalian Klotho. We have used a genetic approach to investigate the functional activity of Klotho in C. elegans. Here, we report that whereas Klotho requires EGL-15 (FGFR) and EGL-17 to promote longevity and oxidative stress resistance, it is not involved in the regulation of fluid homeostasis, controlled by LET-756. Besides revealing a new post-developmental role for EGL-17, our data suggest that the KL1 form of Klotho is involved in FGF23-independent FGF signalling. We also report a genetic interaction between Klotho and the DAF-2 (Ins/IGF1R)/DAF-16 (FOXO) pathway. While the regulation of longevity requires functional DAF-2/DAF-16 signalling, the control of oxidative stress resistance involves a DAF-2- independent, DAF-16-dependent pathway, suggesting that Klotho may target either DAF-2 or DAF-16, depending of environmental conditions. Thus, the predictive KL1 form of Klotho appears to crosstalk with both FGF and Insulin/IGF1/FOXO pathways to exert anti-aging properties in C. elegans.

Klotho gene variation and expression in 20 inbred mouse strains

A defect in klotho gene expression in the mouse results in a syndrome that resembles human aging, with greatly shortened lifespan, arteriosclerosis, and defective hearing. In an effort to find functional murine variants of klotho, we sequenced the gene and examined renal expression of the secreted and membrane-bound Klotho isoforms from 16 laboratory-derived and 4 wild-derived inbred strains. Among the laboratory-derived strains, no sequence variation was found in any of the exons or intron-exon boundaries. Among the wild-derived strains, we found 45 sequence variants with six resulting in amino acid substitutions. One wild-derived strain, SPRET/Ei, had four amino acid substitutions and higher levels of the membrane form and total klotho mRNA. In addition, the membrane to secreted klotho expression ratio was elevated in three wild-derived strains with amino acid substitutions. Interestingly, SPRET/Ei mice have longer lifespans, decreased atherosclerosis risk factors, and better hearing than almost all other mouse strains.

Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane

Klotho mutant mice exhibit a set of phenotypes resembling human ageing. Although the function of Klotho remains unclear, mediation of its pleiotropic functions by putative humoral factor(s) has been presumed. Newly established antibodies against Klotho allowed the detection of secreted Klotho, a candidate for the putative humoral factor, in sera and cerebrospinal fluid. Surprisingly the secreted Klotho was 130 kDa, in contrast to the 70 kDa predicted form from klotho gene transcripts. The secreted as well as the membrane-bound Klotho proteins were suggested to form oligomerized complex. These results delineate post-translation processing of Klotho and possible regulatory mechanisms for secretion of Klotho in vivo.