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see original article on page 732

PPAR-γ and aging: one link through klotho?Ruihua Zhang1 and Feng Zheng1

PPAR-γ, a transcription factor involved in adipogenesis, glucose homeostasis, bone turnover, and inflammation, has now been shown to increase klotho expression. Klotho is predominantly expressed by the kidney and functions at the tubule and in the circulation as an anti-aging factor and a hormone participating in mineral metabolism. The finding that klotho is upregulated by PPAR-γ may prompt further exploration of the role and mechanism of action of PPAR-γ in aging and bone diseases.Kidney International (2008) 74, 702–704. doi:10.1038/ki.2008.382

1Division of Experimental Diabetes and Aging, Department of Geriatrics, Mount Sinai School of Medicine, New York, New York, USACorrespondence: Feng Zheng, Division of Experimental Diabetes and Aging, Department of Geriatrics, Mount Sinai School of Medicine, 1468 Madison Avenue, Annenberg Building, Room 20-58, New York, New York 10029, USA. E-mail: feng.zheng@mssm.edu

1997; 273: C531–C540.25. Devor DC, Singh AK, Lambert LC et al. Bicarbonate

and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 1999; 113: 743–760.

26. Chou CC, Lunn CA, Murgolo NJ. KCa3.1: target and marker for cancer, autoimmune disorder and vascular inflammation? Expert Rev Mol Diagn 2008; 8: 179–187.

27. Tharp DL, Wamhoff BR, Wulff H et al. Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis. Arterioscler Thromb Vasc Biol 2008; 28: 1084–1089.

Aging is an inevitable but an extendable process. Both exogenous and endogenous factors may change the course of biological aging. For instance, calorie restriction has been consistently shown to prolong life span, from lower organ-isms to primates. Additionally, mutation, deletion, or overexpression of evolution-arily conserved molecules involved in the growth hormone/insulin/insulin-like growth factor-1 (IGF-1) signaling path-way alters the aging process.1

Oxidative stress, caused by an imbalance between oxidant production and antioxi-

dants, is widely believed to be a central player in aging. While the production of reactive free radicals is absolutely required for host defenses and normal cell functions, the presence of detoxificants, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, is critical in maintaining a normal redox state and in cell life-and-death decisions during stress. It is known, for example, that the prosurvival action of nuclear factor-κB is partly mediated by its increasing of the transcription of super oxide dismutase and catalase. The upregulation of antioxidants also contributes to the anti-aging function of the forkhead box O (FOXO) family of transcription factors. As mice transgenic for antioxidant molecules such as catalase and thioredoxin have an increased life span, and mice deficient in methionine sul-foxide reductase, an antioxidation enzyme, have a decreased life span, these data sup-port the important role of antioxidants in

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aging.2 Aging is commonly associated with excessive oxidant production due partly to mitochondrial dysfunction. Mitochondria as the power plants for cells are a major source of reactive oxygen species (ROS). During the process of ATP generation, ROS are a by-product generated by com-plex I and III in the electron transport chain. The function of mitochondria is progressively decreased in aging because of the accumulation of mitochondrial DNA mutation and decreased activity of enzymes and components of the electron transport chain. It is unknown, however, how ROS generation can be dispropor-tionately increased when energy produc-tion is decreased in aging. One model may be that electron stalling, due partly to the rate of electron entry into complex I and III, exceeds the rate of transition in the transport chain, the end result being ROS accumulation.3 The findings that calorie restriction reduces ROS generation while increasing mitochondrial number and respiration rate indirectly support this model. Interestingly, early aging occurs in mitochondrial DNA proofread-ing polymerase-γ knockout mice in the absence of obvious oxidative stress. As patients with mitochondrial genetic dis-eases also display early-aging phenotypes, the integrity of mitochondria may be an independent risk factor for aging. The findings that knockout of two of the DNA damage repair genes causes premature aging in the presence of increased anti-oxidants and decreased ROS production in mice4 further suggest that molecular pathways other than free radical damage are involved in aging.

Klotho is a newly identified anti-aging factor that is not directly involved in regulation of redox state and is not evo-lutionarily conserved.5 The klotho gene encodes a single-pass transmembrane protein with a long extracellular domain (two internal repeats, ~900 amino acids) and a short cytoplasmic tail (10 amino acids). The extracellular domain of klotho shares homology to mammalian lactose-phlorizin hydrolase and β-glucosidase in bacteria and plants. Klotho exists in both secreted and membranous forms. Secreted forms are derived both from the shedding of extracellular domain and as a product of alternative splicing. Klotho

is expressed predominantly in renal distal convoluted tubules and in choroid plexus in the brain. The anti-aging function of klotho has been studied in transgenic mice. Klotho-deficient mice develop a premature-aging phenotype that includes a drastically shortened life span, skin and muscle atrophy, osteoporosis, pulmonary emphysema, and extensive ectopic calci-fication.5 In contrast, mice transgenic for klotho exhibit an extended life span.6 More interestingly, Arking et al. reported that a functional variant of klotho in humans is associated with increased lon-gevity.7 One variant with a substitution of position 352 phenylalanine with valine resulted in a sixfold decrease in secreted klotho levels. Furthermore, this substi-tution may eliminate the β-glucosidase activity of klotho. Surprisingly, the 352 phenylalanine homozygote did not show increased longevity and instead had lower survival advantage over the 352 phenyla-lanine-to-valine variant heterozygote.

Inhibition of the insulin/IGF-1 signaling pathway and ROS may contribute to the

anti-aging function of klotho.5 Whereas klotho-deficient mice are hypoglycemic and demonstrate hypersensitivity to insu-lin, overexpressing mice have elevated fasting insulin levels with glucose intol-erance. The addition of klotho to L6 cells in vitro decreases both insulin and IGF-1–stimulated insulin receptor and insulin receptor substrate 1 and 2 phosphoryla-tion and phosphatidylinositol-3′-kinase and Akt/protein kinase B activation. Moreover, the administration of klotho to normal mice results in decreased glucose and insulin tolerance. These data support the involvement of klotho in the insulin/IGF-1 pathway. As klotho transgenic mice are resistant to paraquat, a robust ROS generator that induces animal death, klotho may prolong life span through the inhibition of oxidative stress. The activa-tion of FOXOs and superoxide dismutase 2 has been implicated in the anti-ROS effect of klotho.

The data are not completely clear, how-ever, regarding the anti-aging function of klotho, since it is critically involved

ROS

FOXOs

Akt/PKB

Insulin action

PPAR-γ

Klotho

FOXOs

L o n g e v i t y

InflammationInsulin resistance

PPAR-γ

Cardiovascular disease Bone remodeling

?

?

LongevityDNA repair enzymes Mitochondria integrity

figure 1 | A putative role of PPAR-γ in aging. PPAR-γ increases klotho and decreases inflammation, which may increase life span. It is unknown whether the improvement of insulin sensitivity by PPAR-γ is beneficial or harmful. As PPAR-γ activation may increase the risk of cardiovascular disease and decrease the capability of bone remodeling, a series of well-planned, careful studies in aging models is required to determine the effect of PPAR-γ activation and inhibition on the aging process. FOXOs, forkhead box O transcription factors; ROS, reactive oxygen species; PKB, protein kinase B.

704 Kidney International (2008) 74

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in phosphate homeostasis and vitamin D metabolism. A patient with transloca-tion in a breakpoint adjacent to klotho developed hypophosphatemic rickets and hyperparathyroidism in the presence of elevated klotho.8 Mice deficient in klotho have elevated plasma calcium, phosphate, and 1,25(OH)2D3 levels. Restriction of dietary phosphorus or vitamin D corrects the early-aging phenotype and prolongs the life span of klotho-deficient mice. The regulation of phosphate by klotho is probably mediated by its interaction with fibroblast growth factor-23 (FGF-23), a 30-kilodalton secreted protein that acts as a key regulator in phosphate homeostasis. FGF-23 decreases intestinal and kidney phosphate absorption. In the absence of klotho, however, FGF-23 does not bind efficiently to its receptor to induce intra-cellular signaling. This is more obvious in klotho-deficient mice, as these mice have hyperphosphatemia in the pres-ence of a nearly 1,200-fold increase in serum FGF-23. Interestingly, FGF-23 knockout mice exhibit a nearly identical early-aging phenotype.9 The defect could largely be corrected after crossing of FGF-23-null mice with 1α(OH)ase-null mice to diminish the activity of 1,25(OH)2D3. The status of phosphate homeostasis in klotho-overexpressing mice is not clear.

The presence of hypercalcemia in klot-ho-deficient mice suggests that klotho may also be involved in calcium home-ostasis. In the kidney, klotho is coex-pressed with TRPV5, a calcium channel transient receptor that is responsible for calcium reabsorption in distal convo-luted tubular cells. Klotho increases the number of TRPV5 on cell membranes and thus the influx of calcium through its glucu ronidase-like activity that hydrolyzes N-linked oligo saccharides in TRPV5. In addition to its function as a calcium influx channel in the kidney, TRPV5 also plays a role in osteoclastic activity. TRPV5 is expressed by osteoclasts. Mice lacking TRPV5 have decreased bone resorp-tion due partly to the dysfunction of osteoclasts. As mice lacking klotho have a more severe bone phenotype, klotho may also affect normal bone turnover via the regulation of phosphate and calcium homeostasis, vitamin D metabolism, and osteoclast function.

The expression of klotho is reduced in aging, chronic renal failure, type 2 diabe-tes, and acute ischemic injury. Further-more, both angiotensin II and oxidative stress decrease klotho expression. The underlying molecular mechanisms for the regulation of klotho expression are not clear. Zhang et al.10 (this issue) report that peroxisome proliferator-activated receptor-γ (PPAR-γ) increases renal tubu-lar klotho mRNA and protein expression in vitro and in vivo. More importantly, they were able to locate two non-ca-nonical PPAR-γ binding sites upstream of the klotho gene and clearly demon-strated that these sites are important for the regulation of klotho expression by PPAR-γ. Given that PPAR-γ itself func-tions as a critical regulator of glucose and lipid metabolism and may play an important role in anti-inflammation,11 this finding of an increase in klotho by PPAR-γ may help to generate interest in further examining the role of PPAR-γ in aging.

Aging is commonly associated with the metabolic syndrome, characterized by insulin resistance, obesity, dyslipi-demia, abnormal glucose metabolism, and hypertension. Forty per cent of the United States population over 40 years old has the metabolic syndrome, which carries an increased risk for cardiovas-cular diseases. Because the activation of PPAR-γ by agonists such as thiazoli-dinediones improves insulin resistance, hyperlipidemia, and glycemic levels, it constitutes an important part of therapy for the metabolic syndrome. This theo-retically would help to increase longevity, even though it may not directly affect the process of biological aging.

Additionally, aging is commonly asso-ciated with low-grade chronic inflamma-tion. As PPAR-γ has long been known as an anti-inflammatory molecule that suppresses nuclear factor-κB, AP-1, and STAT and decreases production of cytokines and chemokines, this effect of PPAR-γ may be an added benefit in the modulation of aging (Figure 1).

However, the activation of PPAR-γ is associated with increased adipocyte differentiation that promotes obesity and weight gain. Specifically in bone marrow, PPAR-γ suppresses the differentiation of

mesenchymal progenitors to osteob-lasts while steering progenitors in the direction of more adipocyte generation, which results in decreased osteoblas-togenesis and bone remodeling. This raises concern about whether the use of thiazolidinediones as PPAR-γ agonists may increase the risk of bone fracture in aging. Interestingly, the increase of klotho by PPAR-γ may partly offset this adverse effect, because klotho facili-tates bone mineralization and increases TRPV5 in osteoclasts. The major con-cern, however, is about the latest report of increasing mortality in type 2 diabetic patients treated by the PPAR-γ agonists rosiglitazone and pioglitazone. Because of this safety-versus-efficacy issue, one should be cautious regarding the ben-efit of PPAR-γ activation for aging, even though it increases klotho.

ACKNOWLEDGMENTSWe thank Gary E. Striker for critical reading of the manuscript. This work is supported by United States National Institutes of Health grant 5R01 AG027628-03 to Feng Zheng.

DISCLOSURE The authors declared no competing interests.

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