I published an introduction to the remarkable “anti-aging gene” Klotho in the October 2009 blog entryKlotho anti-aging gene in the news, and a September 2010 update Klotho, phosphates, cola drinks and longevity. Since then in little more than a year, something like 100 new Klotho-related research publications have appeared. There is too much to cover in these 100 publications but I report here selectively on new insights related to Klotho, picking up where the previous blog entries leave off. Also, there are important insights here relating Klotho to vitamin D receptors and the biological actions of some phyto-substances like curcumin.
Discovered in 1997, the Klotho gene has attracted increasing attention. In small animals, insufficient or knocked-down Klotho expression leads to short-lived disease-ridden animals while forced over-expression of Klotho can produce mice that live 20% to 30% longer than their wild type counterparts. And the same pathways appear to work in humans. More-recently, research has been decoding the relationship of Klotho expression to several important disease processes.
Among the statements documented in the above-cited blog entries or known for some time are:
- There are two forms of Klotho: alpha which is a circulating hormone and beta which relates to cell surface receptors.
- Mutation of the Klotho gene can produce a syndrome that resembles aging and Klotho expression generally declines with age.
- Klotho knockout mice have only half to one-third the body weight of normal mice, live only a median of 60 days, have arteriosclerosis, emphysema, osteoporosis, skin atrophy, cognitive impairment, markers of oxidative damage, ectopic calcification, hypervitaminosis D, hypercalcemia, and hyperphosphatemia.
- “Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1 (IGF-1); and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety(ref).”.
- One of the aging mechanisms that may be accelerated by insufficient expression of Klotho is the buildup of advanced glycation endproducts (AGEs).
- One viewpoint is that Klotho derives much of its anti-aging capability from the protein acting “by increasing the cell’s ability to detoxify harmful reactive oxygen species. Over-expression of Klotho can protect against oxidative stress.
- Klotho gene delivery in mice increases natural SOD antioxidant protection in aorta, liver and kidneys.
- Klotho overexpression suppresses insulin and inhibits the IGF-1 channel; its deficiency leads to expression of IGF-1.
- Inhibition of the IGF-1 channel signaling partially ameliorates aging and age-related problems in Klotho knockout mice.
- Klotho deficiency leads to overexpression of vitamin D3 which leads to premature aging. The mechanism is associated with Klotho’s function in regulating FGF23.
- Secretion of Klotho appears to be partially regulated by insulin.
- Klotho appears to regulate nitric oxide production in the endothelium. Insufficient Klotho leads to diminished angiogenesis and vasculogenesis..
- Defects in Klotho expression can lead to underexpression of FGF23 and accumulation of phosphates. Accumulated phosphates can accelerate aging. Phosphate retention can lead to an aging phenotype.
- FGF23 and its relationship to Klotho are linked to a number of bone and joint diseases.
- Klotho is a regulator of oxidative stress and cell senescence.
- Klotho inhibits growth and promotes apoptosis in some cancer lines.
- Klotho protein protects against endothelial dysfunction and hypertension.
- Control of Klotho expression comes about through epigenetic mechanisms; some cancers can silence Klotho expression.
- Consuming cola drinks rich in phosphoric acid when coupled with Klotho insufficiency may exert a pro-aging effect.
- In humans, studies suggest that Klotho KL-VS gene polymorphisms may be associated with stroke, coronary artery disease and longevity.
- Klotho pathways might be targets for anti-aging interventions.
Unless you are already thoroughly familiar with Klotho, I strongly suggest you review the above-mentioned two blog entries as background before reading this one. In generating this blog entry I found there was a very large amount of information to digest and see in context.
Additional observations gleaned from more-recent publications
Plasma Klotho level is inversely associated with cardiovascular disease.
The August 2011 publication Plasma Klotho and Cardiovascular Disease in Adultslooked at “One thousand twenty-three men and women aged 24 to 102 participating in the Invecchiare in Chianti (InCHIANTI) study.” — Anthropometric measures, plasma klotho, fasting plasma total, high-density lipoprotein cholesterol (HDL-C), triglycerides, glucose, creatinine, C-reactive protein (CRP). Clinical measures: medical assessment, diabetes mellitus, hypertension, coronary heart disease, heart failure, stroke, peripheral artery disease, cancer, chronic kidney disease. Logistic regression models were used to examine the relationship between plasma klotho and prevalent CVD. — RESULTS: Of 1,023 participants, 259 (25.3%) had CVD. Median (25th, 75th percentile) plasma klotho concentrations were 676 pg/mL (530, 819 pg/mL). Plasma klotho was correlated with age (correlation coefficient (r)=-0.14, P<.001), HDL-C (r=0.11, P<.001), and CRP (r=-0.10, P<.001) but not systolic blood pressure, fasting plasma glucose, or renal function. Plasma klotho age-adjusted geometric means were 626 pg/mL (95% confidence interval (CI)=601-658 pg/mL) in participants with CVD and 671 pg/mL (95% CI=652-692 pg/mL) in those without CVD (P=.001). Adjusting for traditional cardiovascular risk factors (age, sex, smoking, total cholesterol, HDL-C, systolic blood pressure, and diabetes mellitus), log plasma klotho was associated with prevalent CVD (odds ratio per 1 standard deviation increase=0.85, 95% CI=0.72-0.99). — CONCLUSION: In community-dwelling adults, higher plasma klotho concentrations are independently associated with a lower likelihood of having CVD.”
The InChianti study established in a substantial population that many of the Klotho effects observed in mice apply also to humans; for example, low circulating Klotho is associated with shorter lifespans, greater incidents of cardiovascular diseases.
An interesting association is reported in the July 2011 publication Relationship of low plasma klotho with poor grip strength in older community-dwelling adults: the InCHIANTI study. “Handgrip strength is a strong indicator of total body muscle strength and is a predictor of poor outcomes in older adults. The aging suppressor gene klotho encodes a single-pass transmembrane protein that is secreted as a circulating hormone. In mice, disruption of klotho expression results in a syndrome that includes sarcopenia, atherosclerosis, osteoporosis, and shortened lifespan, and conversely, overexpression of klotho leads to a greater longevity. The objective was to determine whether plasma klotho levels are related to skeletal muscle strength in humans. We measured plasma klotho in 804 adults, ≥65 years, in the InCHIANTI study, a longitudinal population-based study of aging in Tuscany, Italy. Grip strength was positively correlated with plasma klotho at threshold <681 pg/mL. After adjusting for age, sex, education, smoking, physical activity, cognition, and chronic diseases, plasma klotho (per 1 standard deviation increase) was associated with grip strength (beta = 1.20, standard error = 0.35, P = 0.0009) in adults with plasma klotho <681 pg/mL. These results suggest that older adults with lower plasma klotho have poor skeletal muscle strength.’
The August 2011 report Relationship of serum fibroblast growth factor 23 with cardiovascular disease in older community-dwelling womenlooks at the relationship of FGF23to cardiovascular disease in a cohort of 659 women, aged 70–79. “Conclusion Elevated serum FGF23 concentrations are independently associated with prevalent cardiovascular disease in older community-dwelling women.”
See also (January 2011) Klotho: An Elixir of Youth for the Vasculature?
Klotho expression is a key factor in phosphate metabolism, which in turn can be an important factor in cardiovascular, kidney and bone diseases
A number of different studies point to this conclusion.
The 2010 publication Overview of the FGF23-Klotho axis sets the scene. “Recent studies have identified a novel bone-kidney endocrine axis that maintains phosphate homeostasis. When phosphate is in excess, fibroblast growth factor-23 (FGF23) is secreted from bone and acts on the kidney to promote phosphate excretion into urine and suppress vitamin D synthesis, thereby inducing negative phosphate balance. One critical feature of FGF23 is that it requires Klotho, a single-pass transmembrane protein expressed in renal tubules, as an obligate coreceptor to bind and activate FGF receptors. Several hereditary disorders that exhibit inappropriately high serum FGF23 levels are associated with phosphate wasting and impaired bone mineralization. In contrast, defects in either FGF23 or Klotho are associated with phosphate retention and a premature-aging syndrome. The aging-like phenotypes in Klotho-deficient or FGF23-deficient mice can be rescued by resolving hyperphosphatemia with dietary or genetic manipulation, suggesting a novel concept that phosphate retention accelerates aging. Phosphate retention is universally observed in patients with chronic kidney disease (CKD) and identified as a potent risk of death in epidemiological studies. Thus, the bone-kidney endocrine axis mediated by FGF23 and Klotho has emerged as a novel target of therapeutic interventions in CKD.”
The October 2011 publication Phosphate Metabolism in Cardiorenal Metabolic Disease reports “Hyperphosphatemia is a major risk factor for cardiovascular disease, abnormalities of mineral metabolism and bone disease, and the progression of renal insufficiency in patients with chronic renal disease. In early renal disease, serum phosphate levels are maintained within the ‘normal laboratory range’ by compensatory increases in phosphaturic hormones such as fibroblast growth factor-23 (FGF-23). An important co-factor for FGF-23 is Klotho; a deficiency in Klotho plays an important role in the pathogenesis of hyperphosphatemia, renal tubulointerstitial disease, and parathyroid and bone abnormalities. Clinical hyperphosphatemia occurs when these phosphaturic mechanisms cannot counterbalance nephron loss. Hyperphosphatemia is associated with calcific uremic arteriolopathy and uremic cardiomyopathy, which may explain, in part, the epidemiologic connections between phosphate excess and cardiovascular disease. However, no clinical trials have been conducted to establish a causal relationship, and large, randomized trials with hard endpoints are urgently needed to prove or disprove the benefits and risks of therapy. In summary, hyperphosphatemia accelerates renal tubulointerstitial disease, renal osteodystrophy, as well as cardiovascular disease, and it is an important mortality risk factor in patients with chronic kidney disease.”
See also (August 2011) Downregulation of NaPi-IIa and NaPi-IIb Na-coupled phosphate transporters by coexpression of Klotho, (Mar 2011) Phosphate levels and cardiovascular disease in the general population, (March 2011) Derangements in phosphate metabolism in chronic kidney diseases/endstage renal disease: therapeutic considerations, and (July 2005) Fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease, (April 2011) Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate. “Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification.”
Klotho-deficient mice and FGF23-deficient mice exhibit identical phenotypes.
Several publications mention this observation including the June 2011 publication Klotho and the aging process. This publication treats a number of topics discussed elsewhere in this blog entry and includes a discussion of the roles of secreted Klotho in promoting longevity. “Secreted Klotho regulates the activity of multiple growth factors, including insulin/insulin-like growth factor-1 (IGF-1) , Wnt , and transforming growth factor (TGF)-β1 . Because adequate suppression of insulin/IGF-1 signaling pathway has been identified as an evolutionarily conserved mechanism for extending life span , the anti-aging properties of Klotho may stem partly from its ability to suppress insulin/IGF-1 signaling. In fact, transgenic mice that overexpress Klotho are long-lived and slightly resistant to insulin and IGF-1 without overt diabetes . The mechanism by which secreted Klotho suppresses insulin/IGF-1 signaling remains to be determined. However, secreted Klotho inhibits Wnt signaling by directly binding to Wnt ligands and preventing them from binding to their receptors. Wnt signaling is enhanced in Klotho-deficient mice, which results in exhaustion of stem cells in highly proliferative tissues such as skin and intestine and may partly contribute to atrophy of these tissues in Klotho-deficient mice . — We recently found that secreted Klotho suppresses TGF-β1 signaling; secreted Klotho directly binds to type-II TGF-β receptor (TGFβR2) on the cell surface and prevents TGF-β1 binding to TGFβR2 . TGF-β1 is the most potent inducer of the epithelial-to-mesenchymal transition (EMT) . EMT is a cellular process whereby epithelial cells lose epithelial characters and undergo a phenotypic transition to acquire mesenchymal characters, including the ability to migrate and proliferate. EMT is essential for tissue repair in response to injury but can result in fibrosis under pathological conditions in the kidney as well as in many other tissues, including the liver, lung, and heart . Furthermore, cancer cells undergo EMT and acquire the ability to migrate and proliferate, leading to metastasis . Thus, the ability of secreted Klotho to inhibit TGF-β1 activity may counteract EMT and prevent tissue fibrosis and cancer metastasis. In fact, injecting secreted Klotho prevents renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts in mice . These activities of secreted Klotho may also contribute to life span extension by Klotho overexpression in mice .”
Besides serving as cofactor for FGF23, the Klotho gene family encodes proteins that are cofactors for regulation of tissue-specific metabolic activities of the endocrine fibroblast growth factors (FGF19, FGF21, and FGF23).
See the 2008 publication The Klotho gene family and the endocrine fibroblast growth factors “In addition to the Klotho-FGF23 axis, recent studies have shown that betaKlotho, a Klotho family protein, also functions as a cofactor required for FGF19 and FGF21 signaling and determines tissue-specific metabolic activities of FGF19 and FGF21.” And see the 2010 publicationRelevant use of Klotho in FGF19 subfamily signaling system in vivo. “Alpha-Klotho (alpha-Kl) and its homolog, beta-Klotho (beta-Kl) are key regulators of mineral homeostasis and bile acid/cholesterol metabolism, respectively. FGF15/ humanFGF19, FGF21, and FGF23, members of the FGF19 subfamily, are believed to act as circulating metabolic regulators. Analyses of functional interactions between alpha- and beta-Kl and FGF19 factors in wild-type, alpha-kl(-/-), and beta-kl(-/-) mice revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of alpha-Kl, FGF23, and 1,25(OH)(2)D and an analogous regulatory network composed of beta-Kl, FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Contrary to in vitro data, beta-Kl is not essential for FGF21 signaling in adipose tissues in vivo, because (i) FGF21 signals are transduced in the absence of beta-Kl, (ii) FGF21 could not be precipitated by beta-Kl, and (iii) essential phenotypes in Fgf21(-/-) mice (decreased expressions of Hsl and Atgl in WAT) were not replicated in beta-kl(-/-) mice. These findings suggest the existence of Klotho-independent FGF21 signaling pathway(s) where undefined cofactors are involved. One-to-one functional interactions such as alpha-Klotho/FGF23, beta-Klotho/FGF15 (humanFGF19), and undefined cofactor/FGF21 would result in tissue-specific signal transduction of the FGF19 subfamily.”
Low levels of Klotho may serve as an early warning biomarker for kidney disease and cardiovascular complications
More than 26 million people in the U.S. are affected by chronic kidney disease. The November 2010 publication Klotho Deficiency Causes Vascular Calcification in Chronic Kidney Disease reports “Soft-tissue calcification is a prominent feature in both chronic kidney disease (CKD) and experimental Klotho deficiency, but whether Klotho deficiency is responsible for the calcification in CKD is unknown. Here, wild-type mice with CKD had very low renal, plasma, and urinary levels of Klotho. In humans, we observed a graded reduction in urinary Klotho starting at an early stage of CKD and progressing with loss of renal function. Despite induction of CKD, transgenic mice that overexpressed Klotho had preserved levels of Klotho, enhanced phosphaturia, better renal function, and much less calcification compared with wild-type mice with CKD. Conversely, Klotho-haploinsufficient mice with CKD had undetectable levels of Klotho, worse renal function, and severe calcification. The beneficial effect of Klotho on vascular calcification was a result of more than its effect on renal function and phosphatemia, suggesting a direct effect of Klotho on the vasculature. In vitro, Klotho suppressed Na+-dependent uptake of phosphate and mineralization induced by high phosphate and preserved differentiation in vascular smooth muscle cells. In summary, Klotho is an early biomarker for CKD, and Klotho deficiency contributes to soft-tissue calcification in CKD. Klotho ameliorates vascular calcification by enhancing phosphaturia, preserving glomerular filtration, and directly inhibiting phosphate uptake by vascular smooth muscle. Replacement of Klotho may have therapeutic potential for CKD.”
The September 2011 publication Cross talk between the renin-angiotensin-aldosterone system and vitamin D-FGF-23-klotho in chronic kidney disease provides additional insight related to Klotho and renal disease. “There is increasingly evidence that the interactions between vitamin D, fibroblast growth factor 23 (FGF-23), and klotho form an endocrine axis for calcium and phosphate metabolism, and derangement of this axis contributes to the progression of renal disease. Several recent studies also demonstrate negative regulation of the renin gene by vitamin D. In chronic kidney disease (CKD), low levels of calcitriol, due to the loss of 1-alpha hydroxylase, increase renal renin production. Activation of the renin-angiotensin-aldosterone system (RAAS), in turn, reduces renal expression of klotho, a crucial factor for proper FGF-23 signaling. The resulting high FGF-23 levels suppress 1-alpha hydroxylase, further lowering calcitriol. This feedback loop results in vitamin D deficiency, RAAS activation, high FGF-23 levels, and renal klotho deficiency, all of which associate with progression of renal damage. Here we examine current evidence for an interaction between the RAAS and the vitamin D-FGF-23-klotho axis as well as its possible implications for progression of CKD.”
The negative health consequences of hypercalcemia due to underexpression of Klotho can be very serious.
Expression of Klotho is highly relevant to maintenance of calcium homeostasis. The 2011 publication Calcium metabolism & hypercalcemia in adults reports “Calcium is essential for many metabolic process, including nerve function, muscle contraction, and blood clotting. The metabolic pathways that contribute to maintain serum calcium levels are bone remodeling processes, intestinal absorption and secretion, and renal handling, but hypercalcemia occurs when at least 2 of these 3 metabolic pathways are altered. Calcium metabolism mainly depends on the activity of parathyroid hormone (PTH). Its secretion is strictly controlled by the ionized serum calcium levels through a negative feed-back, which is achieved by the activation of calcium-sensing receptors (CaSRs) mainly expressed on the surface of the parathyroid cells. The PTH receptor in bone and kidney is now referred as PTHR1. The balance of PTH, calcitonin, and vitamin D has long been considered the main regulator of calcium metabolism, but the function of other actors, such as fibroblast growth factor-23 (FGF-23), Klotho, and TPRV5 should be considered. Primary hyperparathyroidism and malignancy are the most common causes of hypercalcemia, accounting for more than 90% of cases. Uncontrolled hypercalcemia may cause renal impairment, both temporary (alteration of renal tubular function) and progressive (relapsing nephrolithiasis), leading to a progressive loss of renal function, as well as severe bone diseases, and heart damages. Advances in the understanding of all actors of calcium homeostasis will be crucial, having several practical consequences in the treatment and prevention of hypercalcemia. This would allow to move from a support therapy, sometimes ineffective, to a specific and addressed therapy, especially in patients with chronic hypercalcemic conditions unsuitable for surgery.”
Klotho suppresses renal fibrosis.
The February 211 publication Phosphate and Klothorelates “Klotho is a putative aging suppressor gene encoding a single-pass transmembrane co-receptor that makes the fibroblast growth factor (FGF) receptor specific for FGF-23. In addition to multiple endocrine organs, Klotho is expressed in kidney distal convoluted tubules and parathyroid cells, mediating the role of FGF-23 in bone–kidney–parathyroid control of phosphate and calcium. Klotho–/– mice display premature aging and chronic kidney disease-associated mineral and bone disorder (CKD-MBD)-like phenotypes mediated by hyperphosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter). CKD can be seen as a state of hyperphosphatemia-induced accelerated aging associated with Klotho deficiency. Humans with CKD experience decreased Klotho expression as early as stage 1 CKD; Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases, and hypovitaminosis D. Secreted Klotho protein, formed by extracellular clipping, exerts FGF-23-independent phosphaturic and calcium-conserving effects through its paracrine action on the proximal and distal tubules, respectively. We contend that decreased Klotho expression is the earliest biomarker of CKD and the initiator of CKD-MBD pathophysiology. Maintaining normal phosphate levels with phosphate binders in patients with CKD with declining Klotho expression is expected to reduce mineral and vascular derangements.”
Hyperphosphatemia associated with to reduced Klotho expression may also play a key role in obesity.
The November 2011 publicationGenetic induction of phosphate toxicity significantly reduces the survival of hypercholesterolemic obese mice reports: “OBJECTIVE: The adverse effects of metabolic disorders in obesity have been extensively studied; however, the pathologic effects of hyperphosphatemia or phosphate toxicity in obesity have not been studied in similar depth and detail, chiefly because such an association is thought to be uncommon. Studies have established that the incidence of obesity-associated nephropathy is increasing. Because hyperphosphatemia is a major consequence of renal impairment, this study determines the in vivo effects of hyperphosphatemia in obesity. METHODS AND RESULTS: We genetically induced hyperphosphatemia in leptin-deficient obese (ob/ob) mice by generating ob/ob and klotho double knockout [ob/ob-klotho(-/-)] mice. As a control, we made ob/ob mice with hypophosphatemia by generating ob/ob and 1-alpha hydroxylase double knockout [ob/ob-1α(OH)ase(-/-)] mice. Compared to the wild-type mice, all three obese background mice, namely ob/ob, ob/ob-klotho(-/-), and ob/ob-1α(OH)ase(-/-) mice developed hypercholesterolemia. In addition, the hyperphosphatemic, ob/ob-klotho(-/-) genetic background induced generalized tissue atrophy and widespread soft-tissue and vascular calcifications, which led to a shorter lifespan; no such changes were observed in the hypophosphatemic, ob/ob-1α(OH)ase(-/-) mice. Significantly, in contrast to the reduced survival of the ob/ob-klotho(-/-) mice, lowering serum phosphate levels in ob/ob-1α(OH)ase(-/-) mice showed no such compromised survival, despite both mice being hypercholesterolemic. CONCLUSION: These genetic manipulation studies suggest phosphate toxicity is an important risk factor in obesity that can adversely affect survival.”
Inflammatory cytokines inhibit expression of Klotho via NF-kappaB.
The July 2011 publication The inflammatory cytokines TWEAK and TNFα reduce renal klotho expression through NFκB reports: “Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNFα, and siRNA-mediated knockdown of IκBα all activated NFκB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFκB with parthenolide prevented TWEAK- or TNFα-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAK-induced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNFα, downregulate Klotho expression through an NFκB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.”
The vitamin D receptor (VDR) is a complicit partner in the feedback loops involving FGF23 and Klotho signaling. The VDR upregulates FGF23 and Klotho.
The October 2011 publication Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells reports: “Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. — Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.”
Klotho and FGF23 expression, via vitamin D3 vitamin D responsive elements (VDREs) in target genes, can be affected by dietary supplements including vitamin D3, omega3/omega6 polyunsaturated fatty acids (PUFAs) and curcumin.
This inference can be arrived at by combining the previously mentioned results with those mentioned in the 2007 publication Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands. “The vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)(2)D(3) is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real-time PCR and reporter gene transfection assays were used to probe VDR-mediated transcriptional control by 1,25(OH)(2)D(3). Reporter gene and mammalian two-hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)(2)D(3) induces FGF23 78-fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)(2)D(3) synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)(2)D(3)-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)(2)D(3)-FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)(2)D(3)-PTH axis that regulates calcium. 1,25(OH)(2)D(3) also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)(2)D(3) supports a cloverleaf model, whereby VDR-RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co-modulators and chromatin remodeling enzymes. VDR also selectively binds certain omega3/omega6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR-RXR complexes. Moreover, the turmeric-derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)(2)D(3)-independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR-containing tissues.”
The September 2011 publication The role of vitamin D in the FGF23, klotho, and phosphate bone-kidney endocrine axis amplifies on the above. “1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.”
Also relevant here is the September 2010 publication Reciprocal control of 1,25-dihydroxyvitamin D and FGF23 formation involving the FGF23/Klotho system. “Fibroblast growth factor 23 (FGF23) is a circulating hormone that is synthesized by osteocytes and osteoblasts. This glycosylated peptide controls phosphate balance by modulating urinary phosphate excretion and indirectly intestinal phosphate absorption by reducing expression of the renal and intestinal sodium phosphate transporters. In a feedback loop, 1,25-dihydroxyvitamin D and phosphate intake control FGF23 production. FGF23 is inactivated by cleavage by a still unidentified enzyme. FGF23 cleavage occurs within cells and probably in the circulation. Klotho, a protein expressed at the cell surface of few organs, forms complexes with FGF receptors, which increases their affinity for FGF23. Klotho is also released into the plasma and urine by an enzymatic cleavage. FGF23 plays a central role in vitamin D metabolism: It inhibits calcitriol synthesis in the kidney and stimulates the catabolism of active vitamin D sterols. In turn, calcitriol stimulates FGF23 and Klotho expression. In chronic kidney diseases, FGF23 concentration increases as GFR declines, whereas Klotho tissue expression decreases. The modifications of FGF23 and Klotho expression are probably involved in the genesis of hyperparathyroidism and the resistance to vitamin D receptor (VDR) activation in chronic kidney disease. Low vitamin D, high FGF23 concentrations, and defects in VDR activation are associated with similar risks, which evoke the possibility that potential FGF23 toxicity might be partly mediated by FGF23-induced decrease in calcitriol or 25-hydroxyvitamin D. Conversely, VDR activators could be used to modulate Klotho or FGF23 expression.”
See also (2007) Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands, (2007)1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism, .and (2005) 1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts.
In fact, it well be that some of the health-producing affects of some dietary polyphenols may be mediated by its effects on VDEEs and expression of Klotho and FGF23.
The December 2010 publication Curcumin: a novel nutritionally derived ligand of the vitamin D receptor with implications for colon cancer chemoprevention states: “Numerous studies have shown chemoprotection by CM (curcumin) against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDR-expressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.” I (Vince) think we have enough evidence to go the next step and hypothesize “Curcumin facilitates expression of Klotho and its health benefits via activation of vitamin D receptor elements.”
Klotho expression can be modulated by dehydration.
See the October 2011 items Dehydration: a new modulator of klotho expression and Downregulation of Klotho expression by dehydration. “Klotho, a transmembrane protein, protease, and hormone mainly expressed in renal tissue counteracts aging. Overexpression of Klotho substantially prolongs the life span. Klotho deficiency leads to excessive formation of 1,25(OH)2D3, growth deficit, accelerated aging, and early death. Aging is frequently paralleled by dehydration, which is considered to accelerate the development of age-related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR, and Klotho protein abundance was detected by Western blotting in renal tissue from hydrated and 36-h-dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase in plasma osmolarity as well as plasma ADH, aldosterone, and 1,25(OH)2D3 levels. Antidiuretic hormone (ADH; 50 nM) and aldosterone (1 μM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.”
There are more publications on how cancers epigenetically silence the expression of Klotho.
The October 2011 publication Epigenetic silencing of the tumor suppressor klotho in human breast cancer. Reports “We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.”
The November 10, 2011 publication Klotho is silenced through promoter hypermethylation in gastric cancer reports “As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. — Demethylation with 5-aza-2′-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.”
Age-related decline in Klotho may be related to promoter methylation.
The September 2011 publication Promoter methylation and age-related downregulation of Klotho in rhesus monkeyrelates “While overall DNA methylation decreases with age, CpG-rich areas of the genome can become hypermethylated. Hypermethylation near transcription start sites typically decreases gene expression. Klotho (KL) is important in numerous age-associated pathways including insulin/IGF1 and Wnt signaling and naturally decreases with age in brain, heart, and liver across species. Brain tissues from young and old rhesus monkeys were used to determine whether epigenetic modification of the KL promoter underlies age-related decreases in mRNA and protein levels of KL. The KL promoter in genomic DNA from brain white matter did not show evidence of oxidation in vivo but did exhibit an increase in methylation with age. Further analysis identified individual CpG motifs across the region of interest with increased methylation in old animals. In vitro methyl modification of these individual cytosine residues confirmed that methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5′ regulatory region.“
Though this has been a long blog entry, the literature related to Klotho during the last 14 months has more things to say than I have been able to report here. Klotho is one of the three Greek Morie fates, moon goddesses. Klotho spins the thread of life, so she is quite important from the viewpoint of longevity. Her two sister fates are important too: Lachesis measures the thread of life. She decides just how long each person will live. “Lachesis is the measurer of the thread woven by Clotho’s spindle, and in some texts, determines Destiny, or thread of life.” Atropos cuts the thread of life. She is the killer. “It was Atropos who chose the mechanism of death and ended the life of each mortal by cutting their thread with her abhorred shears.” They are the daughters of night and Erebus. My writings in this blog are all about their work.
Based on the above, it would seem prudent to supplement with Curcumin, Omega-3 and Vit D, correct? Nice to read what’s above, however I guess very little has filtered down concerning nutritional approaches, etc, towards upregulating Klotho.
Correct. And your comment that little is known about nutritional approaches to upregulating Klotho seems to be correct too. I also wish more was known about the relationships of Klotho to other known life-extending approaches like inhibition of mTOR and dietary restriction. They all seem to have certain downstream effects in common such as inhibition of the IGF-1 pathway and fewer age-related diseases but we know little about upstream causal effects among them. In fact, given the complex interacting feedback systems in biological organisms, simple causation may not be a good way to think about matters.
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