C-terminal fragment of fibroblast growth factor 23 improves heart function in murine models of high intact fibroblast growth factor 23.

Cardiovascular disease (CVD) is the major cause of death in chronic kidney disease (CKD) and is associated with high circulating fibroblast growth factor (FGF)23 levels. It is unresolved whether high circulating FGF23 is a mere biomarker or pathogenically contributes to cardiomyopathy. It is also unknown whether the C-terminal FGF23 peptide (cFGF23), a natural FGF23 antagonist proteolyzed from intact FGF23 (iFGF23), retards CKD progression and improves cardiomyopathy. We addressed these questions in three murine models with high endogenous FGF23 and cardiomyopathy. First, we examined wild-type (WT) mice with CKD induced by unilateral ischemia-reperfusion and contralateral nephrectomy followed by a high-phosphate diet. These mice were continuously treated with intraperitoneal implanted osmotic minipumps containing either iFGF23 protein to further escalate FGF23 bioactivity, cFGF23 peptide to block FGF23 signaling, vehicle, or scrambled peptide as negative controls. Exogenous iFGF23 protein given to CKD mice exacerbated pathological cardiac remodeling and CKD progression, whereas cFGF23 treatment improved heart and kidney function, attenuated fibrosis, and increased circulating soluble Klotho. WT mice without renal insult placed on a high-phosphate diet and homozygous Klotho hypomorphic mice, both of whom develop moderate CKD and clear cardiomyopathy, were treated with cFGF23 or vehicle. Mice treated with cFGF23 in both models had improved heart and kidney function and histopathology. Taken together, these data indicate high endogenous iFGF23 is not just a mere biomarker but pathogenically deleterious in CKD and cardiomyopathy. Furthermore, attenuation of FGF23 bioactivity by cFGF23 peptide is a promising therapeutic strategy to protect the kidney and heart from high FGF23 activity.NEW & NOTEWORTHY There is a strong correlation between cardiovascular morbidity and high circulating fibroblast growth factor 23 (FGF23) levels, but causality was never proven. We used a murine chronic kidney disease (CKD) model to show that intact FGF23 (iFGF23) is pathogenic and contributes to both CKD progression and cardiomyopathy. Blockade of FGF23 signaling with a natural proteolytic product of iFGF23, C-terminal FGF23, alleviated kidney and cardiac histology, and function in three separate murine models of high endogenous FGF23.

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

In vivo evidence for therapeutic applications of beclin 1 to promote recovery and inhibit fibrosis after acute kidney injury.

Autophagy regulator beclin 1 activity determines the severity of kidney damage induced by ischemia reperfusion injury, but its role in kidney recovery and fibrosis are unknown and its therapeutic potentials have not been tested. Here, we explored beclin 1 effects on kidney fibrosis in three models of acute kidney injury (AKI)-ischemia reperfusion injury, cisplatin kidney toxicity, and unilateral ureteric obstruction in mouse strains with three levels of beclin 1 function: normal (wild type), low (heterozygous global deletion of beclin 1, Becn1+/-), and high beclin 1 activity (knockin gain-of-function mutant Becn1, Becn1FA). Fourteen days after AKI induction, heterozygous mice had more, but knockin mice had less kidney fibrosis than wild-type mice did. One day after ischemia reperfusion injury, heterozygous pan-kidney tubular Becn1 null mice had more severe kidney damage than homozygous distal tubular Becn1 null mice did, which was similar to the wild-type mice, implying that proximal tubular beclin 1 protects the kidney against ischemia reperfusion injury. By 14 days, both pan-kidney heterozygous Becn1 null and distal tubular homozygous Becn1 null mice had poorer kidney recovery than wild-type mice did. Injection of beclin 1 peptides increased cell proliferation in kidney tubules in normal mice. Beclin 1 peptides injection either before or after (2-5 days) ischemia reperfusion injury protected the kidney from injury and suppressed kidney fibrosis. Thus, both endogenous beclin 1 protein expression in kidney tubules and exogenous beclin 1 peptides are kidney protective via attenuation of acute kidney damage, promotion of cell proliferation, and inhibition of kidney fibrosis, consequently improving kidney recovery post-AKI. Hence, exogenous beclin 1 peptide may be a potential new therapy for AKI.

Phosphate and Cellular Senescence.

Cellular senescence is one type of permeant arrest of cell growth and one of increasingly recognized contributor to aging and age-associated disease. High phosphate and low Klotho individually and synergistically lead to age-related degeneration in multiple organs. Substantial evidence supports the causality of high phosphate in cellular senescence, and potential contribution to human aging, cancer, cardiovascular, kidney, neurodegenerative, and musculoskeletal diseases. Phosphate can induce cellular senescence both by direct phosphotoxicity, and indirectly through downregulation of Klotho and upregulation of plasminogen activator inhibitor-1. Restriction of dietary phosphate intake and blockage of intestinal absorption of phosphate help suppress cellular senescence. Supplementation of Klotho protein, cellular senescence inhibitor, and removal of senescent cells with senolytic agents are potential novel strategies to attenuate phosphate-induced cellular senescence, retard aging, and ameliorate age-associated, and phosphate-induced disorders.

The tripartite interaction of phosphate, autophagy, and αKlotho in health maintenance.

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.

High-Phosphate Diet Induces Exercise Intolerance and Impairs Fatty Acid Metabolism in Mice.

BACKGROUND: Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in the Western diet. Physical inactivity, a common feature of Western societies, is associated with increased cardiovascular morbidity and mortality. It is unknown whether dietary Pi excess contributes to exercise intolerance and physical inactivity. METHODS: To determine an association between Pi excess and physical activity in humans, we assessed the relationship between serum Pi and actigraphy-determined physical activity level, as well as left ventricular function by cardiac magnetic resonance imaging, in DHS-2 (Dallas Heart Study phase 2) participants after adjusting for relevant variables. To determine direct effects of dietary Pi on exercise capacity, oxygen uptake, serum nonesterified fatty acid, and glucose were measured during exercise treadmill test in C57/BL6 mice fed either a high-Pi (2%) or normal-Pi (0.6%) diet for 12 weeks. To determine the direct effect of Pi on muscle metabolism and expression of genes involved in fatty acid metabolism, additional studies in differentiated C2C12 myotubes were conducted after subjecting to media containing 1 to 3 mmol/L Pi (pH 7.0) to simulate in vivo phosphate conditions. RESULTS: In participants of the DHS-2 (n=1603), higher serum Pi was independently associated with reduced time spent in moderate to vigorous physical activity ( P=0.01) and increased sedentary time ( P=0.004). There was no association between serum Pi and left ventricular ejection fraction or volumes. In animal studies, compared with the control diet, consumption of high-Pi diet for 12 weeks did not alter body weight or left ventricular function but reduced maximal oxygen uptake, treadmill duration, spontaneous locomotor activity, fat oxidation, and fatty acid levels and led to downregulation of genes involved in fatty acid synthesis, release, and oxidation, including Fabp4, Hsl, Fasn, and Pparγ, in muscle. Similar results were recapitulated in vitro by incubating C2C12 myotubes with high-Pi media. CONCLUSIONS: Our data demonstrate a detrimental effect of dietary Pi excess on skeletal muscle fatty acid metabolism and exercise capacity that is independent of obesity and cardiac contractile function. Dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.

Beclin 1/Bcl-2 complex-dependent autophagy activity modulates renal susceptibility to ischemia-reperfusion injury and mediates renoprotection by Klotho.

Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho's known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 (Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 (Becn1+/-), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 (Bcl2AAA). Klotho was increased by transgenic overexpression (Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/- and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/-;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/- or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.

Dietary vitamin D interacts with high phosphate-induced cardiac remodeling in rats with normal renal function.

BACKGROUND: Vitamin D (VD) and phosphate (Pi) load are considered as contributors to cardiovascular disease in chronic kidney disease and the general population, but interactive effects of VD and Pi intake on the heart are not clearly illustrated. METHODS: We fed normal male rats with three levels of dietary VD (100, 1100 or 5000 IU/kg chow) and Pi (0.2, 0.6 or 1.6%) (3X3 design) for 8 weeks and examined renal and cardiac function and histology. RESULTS: High dietary Pi decreased plasma and renal Klotho and plasma 25-hydroxyvitamin D, and increased plasma Pi, fibroblast growth factor 23 and parathyroid hormone without affecting renal function, while low Pi increased plasma and renal Klotho. Both low and high VD diets enhanced high Pi-reduced Klotho expression. Low dietary VD reduced-plasma Klotho was rescued by a low Pi diet. High dietary Pi reduced-cardiac ejection fraction was not modified by a low or high VD diet, but the dietary VD effects on cardiac pathologic changes were more complex. High dietary Pi-induced cardiac hypertrophy was attenuated by a low VD and exacerbated by a high VD diet. In contrast, high dietary Pi -induced cardiac fibrosis was magnified by a low VD and attenuated by a high VD diet. CONCLUSIONS: High Pi diet induces hypertrophy and fibrosis in left ventricles, a low VD diet accelerates high Pi-induced fibrosis, and a high VD diet exacerbated high Pi -induced hypertrophy. Therefore, cardiac phosphotoxicity is exacerbated by either high or low dietary VD in rats with normal kidney function.

Role of αKlotho and FGF23 in regulation of type II Na-dependent phosphate co-transporters.

Alpha-Klotho is a member of the Klotho family consisting of two other single-pass transmembrane proteins: ßKlotho and γKlotho; αKlotho has been shown to circulate in the blood. Fibroblast growth factor (FGF)23 is a member of the FGF superfamily of 22 genes/proteins. αKlotho serves as a co-receptor with FGF receptors (FGFRs) to provide a receptacle for physiological FGF23 signaling including regulation of phosphate metabolism. The extracellular domain of transmembrane αKlotho is shed by secretases and released into blood circulation (soluble αKlotho). Soluble αKlotho has both FGF23-independent and FGF23-dependent roles in phosphate homeostasis by modulating intestinal phosphate absorption, urinary phosphate excretion, and phosphate distribution into bone in concerted interaction with other calciophosphotropic hormones such as PTH and 1,25-(OH)2D. The direct role of αKlotho and FGF23 in the maintenance of phosphate homeostasis is partly mediated by modulation of type II Na+-dependent phosphate co-transporters in target organs. αKlotho and FGF23 are principal phosphotropic hormones, and the manipulation of the αKlotho-FGF23 axis is a novel therapeutic strategy for genetic and acquired phosphate disorders and for conditions with FGF23 excess and αKlotho deficiency such as chronic kidney disease.

Effects of erythropoietin receptor activity on angiogenesis, tubular injury, and fibrosis in acute kidney injury: a "U-shaped" relationship.

The erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knockin of wild-type human EpoR, which is hypofunctional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, more apoptosis at the acute stage (<7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knockin of a constitutively active human EpoR had higher autophagic flux, milder kidney damage, and better renal function at the acute stage but, surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shaped" relationship. The direct effects of EpoR on tubular cells were confirmed in vitro by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal erythropoietin (EPO)/EpoR signaling in renal tubules provides defense against renal tubular injury maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.

Cisplatin nephrotoxicity as a model of chronic kidney disease.

Cisplatin (CP)-induced nephrotoxicity is widely accepted as a model for acute kidney injury (AKI). Although cisplatin-induced chronic kidney disease (CKD) in rodent has been reported, the role of phosphate in the cisplatin-induced CKD progression is not described. In this study, we gave a single peritoneal injection of CP followed by high (2%) phosphate diet for 20 weeks. High dose CP (20 mg/Kg) led to high mortality; whereas a lower dose (10 mg/Kg) resulted in a full spectrum of AKI with tubular necrosis, azotemia, and 0% mortality 7 days after CP injection. After consuming a high phosphate diet, mice developed CKD characterized by low creatinine clearance, interstitial fibrosis, hyperphosphatemia, high plasma PTH and FGF23, low plasma 1,25(OH)2 Vitamin D3 and αKlotho, and classic uremic cardiovasculopathy. The CP model was robust in demonstrating the effect of aging, sexual dimorphism, and dietary phosphate on AKI and also AKI-to-CKD progression. Finally, we used the CP-high phosphate model to examine previously validated methods of genetically manipulated high αKlotho and therapy using exogenous soluble αKlotho protein supplementation. In this CP CKD model, αKlotho mitigated CKD progression, improved mineral homeostasis, and ameliorated cardiovascular disease. Taken together, CP and high phosphate nephrotoxicity is a reproducible and technically very simple model for the study of AKI, AKI-to-CKD progression, extrarenal complications of CKD, and for evaluation of therapeutic efficacy.

Dopamine reduces cell surface Na+/H+ exchanger-3 protein by decreasing NHE3 exocytosis and cell membrane recycling.

The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels, including translation, degradation, endocytosis, and protein phosphorylation. Because cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10-6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A and by brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the ε-subunit of coatomer protein based on yeast-two-hybrid and coimmunoprecipitation. Because NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical reinsertion assay and showed that reinsertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, among the many mechanisms by which DA reduces apical membrane NHE3 and induces proximal tubule natriuresis, one additional mechanism is inhibition of exocytotic insertion and reinsertion of NHE3 in the apical cell surface.

Recombinant α-Klotho may be prophylactic and therapeutic for acute to chronic kidney disease progression and uremic cardiomyopathy.

α-Klotho is highly expressed in the kidney, and its extracellular domain is cleaved and released into the circulation. Chronic kidney disease (CKD) is a state of α-Klotho deficiency, which exerts multiple negative systemic effects on numerous organs including the cardiovascular system. Since acute kidney injury (AKI) greatly escalates the risk of CKD development, we explored the effect of α-Klotho on prevention and treatment on post-AKI to CKD progression and cardiovascular disease. Therein, ischemia reperfusion injury-induced AKI was followed by early administration of recombinant α-Klotho or vehicle starting one day and continued for four days after kidney injury (CKD prevention protocol). A CKD model was generated by unilateral nephrectomy plus contralateral ischemia reperfusion injury. Late administration of α-Klotho in this model was started four weeks after injury and sustained for 12 weeks (CKD treatment protocol). The prevention protocol precluded AKI to CKD progression and protected the heart from cardiac remodeling in the post-AKI model. One important effect of exogenous α-Klotho therapy was the restoration of endogenous α-Klotho levels long after the cessation of exogenous α-Klotho therapy. The treatment protocol still effectively improved renal function and attenuated cardiac remodeling in CKD, although these parameters did not completely return to normal. In addition, α-Klotho administration also attenuated high phosphate diet-induced renal and cardiac fibrosis, and improved renal and cardiac function in the absence of pre-existing renal disease. Thus, recombinant α-Klotho protein is safe and efficacious, and might be a promising prophylactic or therapeutic option for prevention or retardation of AKI-to-CKD progression and uremic cardiomyopathy.

αKlotho Mitigates Progression of AKI to CKD through Activation of Autophagy.

AKI confers increased risk of progression to CKD. αKlotho is a cytoprotective protein, the expression of which is reduced in AKI, but the relationship of αKlotho expression level to AKI progression to CKD has not been studied. We altered systemic αKlotho levels by genetic manipulation, phosphate loading, or aging and examined the effect on long-term outcome after AKI in two models: bilateral ischemia-reperfusion injury and unilateral nephrectomy plus contralateral ischemia-reperfusion injury. Despite apparent initial complete recovery of renal function, both types of AKI eventually progressed to CKD, with decreased creatinine clearance, hyperphosphatemia, and renal fibrosis. Compared with wild-type mice, heterozygous αKlotho-hypomorphic mice (αKlotho haploinsufficiency) progressed to CKD much faster, whereas αKlotho-overexpressing mice had better preserved renal function after AKI. High phosphate diet exacerbated αKlotho deficiency after AKI, dramatically increased renal fibrosis, and accelerated CKD progression. Recombinant αKlotho administration after AKI accelerated renal recovery and reduced renal fibrosis. Compared with wild-type conditions, αKlotho deficiency and overexpression are associated with lower and higher autophagic flux in the kidney, respectively. Upregulation of autophagy protected kidney cells in culture from oxidative stress and reduced collagen 1 accumulation. We propose that αKlotho upregulates autophagy, attenuates ischemic injury, mitigates renal fibrosis, and retards AKI progression to CKD.

Renal Production, Uptake, and Handling of Circulating αKlotho.

αKlotho is a multifunctional protein highly expressed in the kidney. Soluble αKlotho is released through cleavage of the extracellular domain from membrane αKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating αKlotho production and handling is incompletely understood, however. Here, we found higher αKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum αKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of αKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous αKlotho in anephric rats was four- to five-fold longer than that in normal rats, and exogenously injected labeled recombinant αKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that αKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in αKlotho homeostasis, producing and releasing αKlotho into the circulation and clearing αKlotho from the blood into the urinary lumen.

Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism.

The metabolically active and perpetually remodeling calcium phosphate-based endoskeleton in terrestrial vertebrates sets the demands on whole-organism calcium and phosphate homeostasis that involves multiple organs in terms of mineral flux and endocrine cross talk. The fibroblast growth factor (FGF)-Klotho endocrine networks epitomize the complexity of systems biology, and specifically, the FGF23-αKlotho axis highlights the concept of the skeleton holding the master switch of homeostasis rather than a passive target organ as hitherto conceived. Other than serving as a coreceptor for FGF23, αKlotho circulates as an endocrine substance with a multitude of effects. This review covers recent data on the physiological regulation and function of the complex FGF23-αKlotho network. Chronic kidney disease is a common pathophysiological state in which FGF23-αKlotho, a multiorgan endocrine network, is deranged in a self-amplifying vortex resulting in organ dysfunction of the utmost severity that contributes to its morbidity and mortality.

Klotho connects intermedin1-53 to suppression of vascular calcification in chronic kidney disease.

Vascular calcification is one of the leading causes of death in chronic kidney disease (CKD) patients. Klotho protein encoded by the anti-aging gene, Klotho, and intermedin1-53 have both been shown to protect the vasculature from calcification in CKD. The study by Chang and colleagues links Klotho and intermedin1-53 in prevention of vascular calcification in CKD by showing that intermedin1-53 increased renal, vascular, and plasma Klotho, and reduced vascular calcification in CKD rats. Intermedin1-53 may be a novel therapeutic agent for CKD.