Attenuation of osteoarthritis progression via locoregional delivery of Klotho-expressing plasmid DNA and Tanshinon IIA through a stem cell-homing hydrogel.

BACKGROUND: Osteoarthritis (OA) is an aging-related degenerative joint disorder marked by joint discomfort and rigidity. Senescent chondrocytes release pro-inflammatory cytokines and extracellular matrix-degrading proteins, creating an inflammatory microenvironment that hinders chondrogenesis and accelerates matrix degradation. Targeting of senescent chondrocytes may be a promising approach for the treatment of OA. Herein, we describe the engineering of an injectable peptide-hydrogel conjugating a stem cell-homing peptide PFSSTKT for carrying plasmid DNA-laden nanoparticles and Tanshinon IIA (pPNP + TIIA@PFS) that was designed to attenuate OA progression by improving the senescent microenvironment and fostering cartilage regeneration. RESULTS: Specifically, pPNP + TIIA@PFS elevates the concentration of the anti-aging protein Klotho and blocks the transmission of senescence signals to adjacent healthy chondrocytes, significantly mitigating chondrocyte senescence and enhancing cartilage integrity. Additionally, pPNP + TIIA@PFS recruit bone mesenchymal stem cells and directs their subsequent differentiation into chondrocytes, achieving satisfactory chondrogenesis. In surgically induced OA model rats, the application of pPNP + TIIA@PFS results in reduced osteophyte formation and attenuation of articular cartilage degeneration. CONCLUSIONS: Overall, this study introduces a novel approach for the alleviation of OA progression, offering a foundation for potential clinical translation in OA therapy.

Mechanism of the Fibroblast Growth Factor 23/α-Klotho Axis in Peripheral Blood Mononuclear Cell Inflammation in Alzheimer's Disease.

Alzheimer's disease (AD) is a prevalent type of dementia and threatens the health of most elderly people and poses a huge burden to families and society. The fibroblast growth factor 23 (FGF23)/α-Klotho axis is associated with multiple aging-related diseases. Hence, this study explored the mechanism of the FGF23/α-Klotho axis in AD. FGF23/α-Klotho protein contents and levels of inflammatory cytokines in AD patients were measured, and the correlation between FGF23/α-Klotho protein contents and inflammatory cytokines was analyzed. FGF23 and α-Klotho expressions were blocked in peripheral blood mononuclear cells (PBMCs) in AD patients (AD-PBMCs) to assess the effects on cell inflammation and the Wnt/ß-catenin pathway activation. The Wnt/ß-catenin pathway was inhibited to evaluate cell inflammation. Combined treatments of the cells were conducted to verify the role of the FGF23/α-Klotho axis and the Wnt/ß-catenin pathway in inflammation in AD-PBMCs. Increased FGF23 protein concentration and reduced α-Klotho protein concentration were observed in AD patients and correlated with inflammatory cytokine levels. FGF23 inhibition or α-Klotho overexpression reduced the production of inflammatory cytokines and activated the Wnt/ß-catenin pathway in AD-PBMCs. Blocking the Wnt/ß-catenin pathway increased inflammatory cytokine production in AD-PBMCs and annulled the effects of the FGF23/α-Klotho axis on AD-induced cell inflammation. We concluded that the FGF23/α-Klotho axis can regulate the AD-induced cell inflammation through the Wnt/ß-catenin pathway.

Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts.

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study, we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electric cell-substrate impedance sensing (ECIS), increased significantly during and after syncytialization, whereas the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared with nonsyncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with matrix metalloproteinase-9 (MMP-9), but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP-9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

Antiinflammatory Actions of Klotho: Implications for Therapy of Diabetic Nephropathy.

Klotho was initially introduced as an antiaging molecule. Klotho deficiency significantly reduces lifespan, and its overexpression extends it and protects against various pathological phenotypes, especially renal disease. It was shown to regulate phosphate and calcium metabolism, protect against oxidative stress, downregulate apoptosis, and have antiinflammatory and antifibrotic properties. The course of diabetes mellitus and diabetic nephropathy resembles premature cellular senescence and causes the activation of various proinflammatory and profibrotic processes. Klotho was shown to exert many beneficial effects in these disorders. The expression of Klotho protein is downregulated in early stages of inflammation and diabetic nephropathy by proinflammatory factors. Therefore, its therapeutic effects are diminished in this disorder. Significantly lower urine levels of Klotho may serve as an early biomarker of renal involvement in diabetes mellitus. Recombinant Klotho administration and Klotho overexpression may have immunotherapeutic potential for the treatment of both diabetes and diabetic nephropathy. Therefore, the current manuscript aims to characterize immunopathologies occurring in diabetes and diabetic nephropathy, and tries to match them with antiinflammatory actions of Klotho. It also gives reasons for Klotho to be used in diagnostics and immunotherapy of these disorders.

Recombinant Klotho protein enhances cholesterol efflux of THP-1 macrophage-derived foam cells via suppressing Wnt/ß-catenin signaling pathway.

BACKGROUND: Atherosclerosis (AS) is the basis of cardiovascular diseases, characterized by chronic inflammatory and lipid metabolism disorders. Although the anti-inflammatory effect of Klotho in AS has been clearly shown, its lipid-lowering effect is unclear. In this study, we examined the effects of recombinant Klotho (Re-KL) protein on lipid accumulation in foam cells. METHODS: THP-1 cells were exposed to 100 nM phorbol myristate acetate for 24 h and then to oxidized low-density lipoprotein (ox-LDL; 80 mg/mL) to induce foam cell formation. Subsequently, the foam cells were incubated with Re-KL and/or DKK1, an inhibitor of the Wnt/ß-catenin pathway. RESULTS: Oil red O staining and cholesterol intake assay revealed that the foam cell model was constructed successfully. Pre-treatment of the foam cells with Re-KL decreased total cholesterol level, up-regulated the expression of ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1), and down-regulated the expression of acyl coenzyme a-cholesterol acyltransferase 1 (ACAT1) and members of the scavenger family (SR-A1 and CD36). In addition, the expression of Wnt/ß-catenin pathway-related proteins in foam cells was significantly decreased by the stimulus of Re-KL. Interestingly, the effect of Re-KL was similar to that of DKK1 on foam cells. CONCLUSIONS: The Re-KL-induced up-regulation of reverse cholesterol transport capacity promotes cholesterol efflux and reduces lipid accumulation by suppressing the Wnt/ß-catenin pathway in foam cells.

Role of fibroblast growth factor 23 and klotho cross talk in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia that mainly affects the elderly. Several reports have demonstrated that aging is involved in the underlying pathogenic mechanisms of IPF. α-Klotho (KL) has been well characterized as an "age-suppressing" hormone and can provide protection against cellular senescence and oxidative stress. In this study, KL levels were assessed in human plasma and primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF-FB) and in lung tissue from mice exposed to bleomycin, which showed significant downregulation when compared with controls. Conversely, transgenic mice overexpressing KL were protected against bleomycin-induced lung fibrosis. Treatment of human lung fibroblasts with recombinant KL alone was not sufficient to inhibit transforming growth factor-ß (TGF-ß)-induced collagen deposition and inflammatory marker expression. Interestingly, fibroblast growth factor 23 (FGF23), a proinflammatory circulating protein for which KL is a coreceptor, was upregulated in IPF and bleomycin lungs. To our surprise, FGF23 and KL coadministration led to a significant reduction in fibrosis and inflammation in IPF-FB; FGF23 administration alone or in combination with KL stimulated KL upregulation. We conclude that in IPF downregulation of KL may contribute to fibrosis and inflammation and FGF23 may act as a compensatory antifibrotic and anti-inflammatory mediator via inhibition of TGF-ß signaling. Upon restoration of KL levels, the combination of FGF23 and KL leads to resolution of inflammation and fibrosis. Altogether, these data provide novel insight into the FGF23/KL axis and its antifibrotic/anti-inflammatory properties, which opens new avenues for potential therapies in aging-related diseases like IPF.

Endocrine regulation of ageing.

Over the past 15 years it has become clear that mutations in genes that regulate endocrine signalling pathways can prolong lifespan. Lifespan can be increased by altered endocrine signalling in a group of cells or a single tissue, which indicates that crosstalk between tissues functions to coordinate ageing of the organism. These endocrine pathways might serve as targets for the manipulation of the ageing process and prevention of age-related diseases.

Upregulation of Klotho potentially inhibits pulmonary vascular remodeling by blocking the activation of the Wnt signaling pathway in rats with PM2.5-induced pulmonary arterial hypertension.

We evaluated the effects of Klotho on pulmonary vascular remodeling and cell proliferation and apoptosis in rat models with PM2.5-induced pulmonary arterial hypertension (PAH) via the Wnt signaling pathway. After establishing rat models of PM2.5-induced PAH, these Sprague-Dawley male rats were randomized into control and model groups. Cells extracted from the model rats were sub-categorized into different groups. Activation of Wnt/ß-catenin signaling transcription factor was detected by a TOPFlash/FOPFlash assay. A serial of experiment was conducted to identify the mechanism of Klotho on PHA via the Wnt signaling pathway. VEGF levels and PaCO2 content were higher in the model group, while PaO2, NO2- /NO3- content and Klotho level was lower compared to the control group. In comparison to the control group, the model group had decreased Klotho and Bax levels, and elevated Wnt-1, ß-catenin, bcl-2, survivin, and PCNA expression, VEGF, IL-6, TNF-α, TNF-ß1, and bFGF levels, as well as the percentage of pulmonary artery ring contraction. The Klotho vector, DKK-1 and DKK-1 + Klotho vector groups exhibited reduced cell proliferation, luciferase activity, and the expression of Wnt-1, ß-catenin, bcl-2, survivin, and PCNA, as well as shortened S phase compared with the blank and NC groups. Compared with the Klotho vector and DKK-1 groups, the DKK-1 + Klotho vector groups had reduced cell proliferation, luciferase activity, and the expression of Wnt-1, ß-catenin, bcl-2, survivin, and PCNA, as well as a shortened S phase. Conclusively, Klotho inhibits pulmonary vascular remodeling by inactivation of Wnt signaling pathway.

Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo.

Cardiac inflammation and oxidative stress play a key role in the pathogenesis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho has been found to protect cells from inflammation and oxidative stress. The current study aimed to explore the cardioprotective effects of Klotho on DCM and the underlying mechanisms. H9c2 cells and neonatal cardiomyocytes were incubated with 33mM glucose in the presence or absence of Klotho. Klotho pretreatment effectively inhibited high glucose-induced inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis and hypertrophy in both H9c2 cells and neonatal cardiomyocytes. In STZ-induced type 1 diabetic mice, intraperitoneal injection of Klotho at 0.01mg/kg per 48h for 3months completely suppressed cardiac inflammatory cytokines and oxidative stress and prevented cardiac cell death and remodeling, which subsequently improved cardiac dysfunction without affecting hyperglycemia. This study revealed that Klotho may exert its protective effects by augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression and inactivating nuclear factor κB (NF-κB) activation both in vitro and in vivo. Thus, this work demonstrated for the first time that the anti-aging protein Klotho may be a potential therapeutic agent to treat DCM by inhibiting oxidative stress and inflammation. We also demonstrated the critical roles of the Nrf2 and NF-κB pathways in diabetes-stimulated cardiac injuries and indicated that they may be key therapeutic targets for diabetic complications.

Renal Klotho is Reduced in Septic Patients and Pretreatment With Recombinant Klotho Attenuates Organ Injury in Lipopolysaccharide-Challenged Mice.

OBJECTIVES: To determine the applicability of recombinant Klotho to prevent inflammation and organ injury in sepsis in man and mice. DESIGN: Prospective, clinical laboratory study using "warm" human postmortem sepsis-acute kidney injury biopsies. Laboratory study using a mouse model of endotoxemia. SETTING: Research laboratory at a university teaching hospital. SUBJECTS: Adult patients who died of sepsis in the ICU and control patients undergoing total nephrectomy secondary to renal cancer; male C57BL/6 and Klotho haploinsufficient mice. INTERVENTIONS: Lipopolysaccharide (0.05 mg/kg) injection and kill after 4, 8, and 24 hours. Mice received recombinant Klotho (0.05 mg/kg) 30 minutes prior to lipopolysaccharide (1 mg/kg) injection. Mice treated with saline were included as controls. MEASUREMENTS AND MAIN RESULTS: Quantitative reverse transcription polymerase chain reaction and immunohistochemical staining were used to quantify Klotho messenger RNA and protein expression in the kidney of sepsis-acute kidney injury patients and the kidney and brain of mice. The messenger RNA and protein expression of damage markers, inflammatory cytokine, chemokines, and endothelial adhesion molecules were also determined in mice. Renal neutrophil influx was quantified. We found significantly lower renal Klotho messenger RNA and protein levels in sepsis-acute kidney injury biopsies than in control subjects. These findings were recapitulated in the kidney and brain of lipopolysaccharide-challenged mice. Decreased Klotho expression paralleled an increase in kidney damage markers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Administration of recombinant Klotho prior to lipopolysaccharide injection attenuated organ damage, inflammation and endothelial activation in the kidney and brain of mice. Furthermore, less neutrophils infiltrated into the kidneys of recombinant Klotho mice compared with lipopolysaccharide only treated mice. CONCLUSIONS: Renal Klotho expression in human sepsis-acute kidney injury and in mouse models of sepsis was significantly decreased and correlated with renal damage. Recombinant Klotho intervention diminished organ damage, inflammation, and endothelial activation in the kidney and brain of lipopolysaccharide-challenged mice. Systemic Klotho replacement may potentially be an organ-protective therapy for septic patients to halt acute, inflammatory organ injury.

The suppressive effect of soluble Klotho on fibroblastic growth factor 23 synthesis in UMR-106 osteoblast-like cells.

Fibroblastic growth factor 23 (FGF23) is a hormone secreted primarily by bone. FGF23 is elevated in the serum of chronic kidney disease (CKD) patients, but the exact mechanism is not well known. Klotho is identified as an aging suppressor, which is mainly expressed in the kidney, and the level of soluble Klotho is negatively associated with FGF23 in CKD. The aim of this study was to investigate the effect and possible mechanism of Klotho on FGF23 synthesis in osteoblast-like UMR-106 cells. UMR-106 cells were divided into five groups: (i) control group; (ii) ß-glycerophosphate (ß-GP) group; (iii) ß-GP + Klotho group; (iv) ß-GP+ lithium chloride (LiCl, a Wnt/ß-catenin pathway agonist) group; and (v) ß-GP + Klotho + LiCl group. Subsequently, UMR-106 cells were cultured for 72 h, and the expression of FGF23, P-glycogen synthase kinase-3ß (P-GSK-3ß), and glycogen synthase kinase-3ß(GSK-3ß) were measured with Western blot analysis. The mRNA levels of FGF23 and the Wnt/ß-catenin pathway target gene c-myc were determined with RT-qPCR. The results showed that ß-GP induced increased expression of FGF23 mRNA and protein. Compared with the ß-GP group, expression of FGF23 mRNA and protein expression were downregulated in the ß-GP + Klotho group. In addition, ß-GP induced increased expression of P-GSK-3ß/GSK-3ß and c-myc, which were all downregulated in the ß-GP + Klotho group. Moreover, the expression of FGF23, P-GSK-3ß/GSK-3ß, and c-myc mRNA were upregulated when treated with LiCl. These results demonstrate that soluble Klotho suppresses FGF23 synthesis in osteoblast-like UMR-106 cells. The mechanism of this suppression may be partially through the inhibition of the Wnt/ß-catenin pathway.

Klotho Inhibits Proliferation and Migration of Angiotensin II-Induced Vascular Smooth Muscle Cells (VSMCs) by Modulating NF-κB p65, Akt, and Extracellular Signal Regulated Kinase (ERK) Signaling Activities.

BACKGROUND It has been proven that phenotype shifting, from the contractile phenotype to the synthetic phenotype, of vascular smooth muscle cells (VSMCs), plays an important role in vascular diseases such as atherosclerosis, restenosis, and hypertension. Recently, accumulating evidence suggests that Klotho is associated with many cardiovascular diseases or damage. Through the estimation of the proliferation and migration of Ang II-induced VSMCs and the related intracellular signal transduction pathways, we researched the effects of Klotho on phenotype modulation in this study. MATERIAL AND METHODS A rat vascular smooth muscle cell line was grown in vitro with or without Ang II or Klotho, and cell proliferation and migration were evaluated. RESULTS The dose-dependent inhibition of Ang II-induced proliferation and migration by Klotho was shown in VSMCs. The phenotype modulation was inhibited by Klotho co-treatment; this co-treatment promoted the expression of contractile phenotype marker proteins, including SM22α, and also the proliferation phenotype marker protein PCNA compared with Ang II alone, which was suppressed, and activated VSMCs. Furthermore, by reducing the expression of G0/G1-specific regulatory proteins such as cyclin D1, cyclin-dependent kinase (CDK) 4, cyclin E, and CDK2, cell cycle arrest was induced by Klotho at G0/G1 phase. Although Ang II strongly stimulated NF-κB, p65, Akt, and ERK phosphorylation, these activation events were diminished by co-treatment with Ang II and Klotho. CONCLUSIONS Phenotype modulation of Ang II-induced VSMCs and stimulation of the NF-κB, p65, Akt, and ERK signaling pathways were inhibited by Klotho, which suggests that Klotho may play an important role in the phenotype modulation of VSMCs.

Inhibition of TRPC6 channels ameliorates renal fibrosis and contributes to renal protection by soluble klotho.

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.

Understanding and modulating mammalian-microbial communication for improved human health.

The fact that the bacteria in the human gastrointestinal (GI) tract play a symbiotic role was noted as early as 1885, well before we began to manage microbial infections using antibiotics. However, even with the first antimicrobial compounds used in humans, the sulfa drugs, microbes were recognized to be critically involved in the biotransformation of these therapeutics. Thus, the roles played by the microbiota in physiology and in the management of human health have long been appreciated. Detailed examinations of GI symbiotic bacteria that started in the early 2000s and the first phases of the Human Microbiome Project that were completed in 2012 have ushered in an exciting period of granularity with respect to the ecology, genetics, and chemistry of the mammalian-microbial axes of communication. Here we review aspects of the biochemical pathways at play between commensal GI bacteria and several mammalian systems, including both local-epithelia and nonlocal responses impacting inflammation, immunology, metabolism, and neurobiology. Finally, we discuss how the microbial biotransformation of therapeutic compounds, such as anticancer or nonsteroidal anti-inflammatory drugs, can be modulated to reduce toxicity and potentially improve therapeutic efficacy.

Klotho ameliorates cyclosporine A-induced nephropathy via PDLIM2/NF-kB p65 signaling pathway.

Klotho, an antiaging protein, can extend the lifespan and modulate cellular responses to inflammation and oxidative stress which can ameliorate chronic kidney diseases (CKD). To investigate the molecular mechanism of Klotho on inflammation in cyclosporine A (CsA) induced nephropathy, the mice were transfected with adenovirus mediated Klotho gene and treated with cyclosporine A (CsA; 30 mg/kg/day) for 4 weeks. Also, primary human renal proximal tubule epithelial cells (RPTECs) were treated with soluble Klotho protein and LPS. The results showed that Ad-klotho significantly reduced serum creatinine (Scr) and blood urea nitrogen (BUN) caused by CsA, and significantly increased creatinine clearance. Tubule interstitial fibrosis score (TIF), renal 8-OHdG excretion, macrophage infiltration and MCP-1 were decreased after Ad-klotho gene transfer. In addition, the overexpression of Klotho led to increase in the expression of PDLIM2, decreased in the amount of NF-kB p65, and inhibited the production of inflammatory cytokines (TNFα, IL-6, IL-12) and iNOS. Accordingly, in vitro results showed, Klotho enhanced PDLIM2 expression and reduced NF-kB p65 expression, while PDLIM2 siRNA could block the inhibitory effects of Klotho on expression of NF-kB p65. Secretion of inflammatory cytokines was also inhibited by Klotho treatment, and PDLIM2 siRNA hindered regulatory effects of Klotho on the cytokines. Real-time PCR and Luciferase assay showed that Klotho markedly increased expression of PDLIM2 mRNA and PDLIM2 reporter activity in a dose-dependent manner. These findings suggest that Klotho can modulate inflammation via PDLIM2/NF-kB p65 pathway in CsA-induced nephropathy.

HSV-1 interaction to 3-O-sulfated heparan sulfate in mouse-derived DRG explant and profiles of inflammatory markers during virus infection.

The molecular mechanism of herpes simplex virus (HSV) entry and the associated inflammatory response in the nervous system remain poorly understood. Using mouse-derived ex vivo dorsal root ganglia (DRG) explant model and single cell neurons (SCNs), in this study, we provided a visual evidence for the expression of heparan sulfate (HS) and 3-O-sulfated heparan sulfate (3-OS HS) followed by their interactions with HSV-1 glycoprotein B (gB) and glycoprotein D (gD) during cell entry. Upon heparanase treatment of DRG-derived SCN, a significant inhibition of HSV-1 entry was observed suggesting the involvement of HS role during viral entry. Finally, a cytokine array profile generated during HSV-1 infection in DRG explant indicated an enhanced expression of chemokines (LIX, TIMP-2, and M-CSF)-known regulators of HS. Taken together, these results highlight the significance of HS during HSV-1 entry in DRG explant. Further investigation is needed to understand which isoforms of 3-O-sulfotransferase (3-OST)-generated HS contributed during HSV-1 infection and associated cell damage.

Klotho Improves Cardiac Function by Suppressing Reactive Oxygen Species (ROS) Mediated Apoptosis by Modulating Mapks/Nrf2 Signaling in Doxorubicin-Induced Cardiotoxicity.

BACKGROUND Anthracyclines-induced cardiotoxicity has become one of the major restrictions of their clinical applications. Klotho showed cardioprotective effects. This study aimed to investigate the effects and possible mechanisms of klotho on doxorubicin (DOX)-induced cardiotoxicity. MATERIAL AND METHODS Rats and isolated myocytes were exposed to DOX and treated with exogenous klotho. Specific inhibitors and siRNAs silencing MAPKs were also used to treat the animals and/or myocytes. An invasive hemodynamic method was used to determine cardiac functions. Intracellular ROS generation was evaluated by DHE staining. Western blotting was used to determine the phosphorylation levels of JNK, ERK, and p38 MAPKs in plasma extracts and Nrf2 in nuclear extracts. Nuclear translocation of Nrf2 in myocytes was evaluated by immunohistochemistry. Cell apoptosis was evaluated by TUNEL assay and flow cytometry. RESULTS Klotho treatment improved DOX-induced cardiac dysfunction in rats. The DOX-induced ROS accumulation and cardiac apoptosis were attenuated by klotho. Impaired phosphorylations of MAPKs, Nrf2 translocation and expression levels of HO1 and Prx1 were also attenuated by klotho treatment. However, the anti-oxidant and anti-apoptotic effects of klotho on DOX-exposed myocardium and myocytes were impaired by both specific inhibitors and siRNAs against MAPKs. Moreover, the recovery effects of klotho on phosphorylations of MAPKs, Nrf2 translocation and expression levels of HO1 and Prx1 were also impaired by specific inhibitors and siRNAs against MAPKs. CONCLUSIONS By recovering the activation of MAPKs signaling, klotho improved cardiac function loss which was triggered by DOX-induced ROS mediated cardiac apoptosis.

Direct inhibition of osteoblastic Wnt pathway by fibroblast growth factor 23 contributes to bone loss in chronic kidney disease.

Bone loss and increased fractures are common complications in chronic kidney disease. Because Wnt pathway activation is essential for normal bone mineralization, we assessed whether Wnt inhibition contributes to high-phosphorus-induced mineralization defects in uremic rats. By week 20 after 7/8 nephrectomy, rats fed a high-phosphorus diet had the expected high serum creatinine, phosphorus, parathyroid hormone, and fibroblast growth factor 23 (FGF23) levels and low serum calcium. There was a 15% reduction in tibial mineral density and a doubling of bone cortical porosity compared to uremic rats fed a normal-phosphorus diet. The decreases in tibial mineral density were preceded by time-dependent increments in gene expression of bone formation (Osteocalcin and Runx2) and resorption (Cathepsin K) markers, which paralleled elevations in gene expression of the Wnt inhibitors Sfrp1 and Dkk1 in bone. Similar elevations of Wnt inhibitors plus an increased phospho-ß-catenin/ß-catenin ratio occurred upon exposure of the osteoblast cell line UMR106-01 either to uremic serum or to the combination of parathyroid hormone, FGF23, and soluble Klotho, at levels present in uremic serum. Strikingly, while osteoblast exposure to parathyroid hormone suppressed the expression of Wnt inhibitors, FGF23 directly inhibited the osteoblastic Wnt pathway through a soluble Klotho/MAPK-mediated process that required Dkk1 induction. Thus, the induction of Dkk1 by FGF23/soluble Klotho in osteoblasts inactivates Wnt/ß-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

Klotho Ameliorates Kidney Injury and Fibrosis and Normalizes Blood Pressure by Targeting the Renin-Angiotensin System.

Loss of Klotho and activation of the renin-angiotensin system (RAS) are common pathological findings in chronic kidney diseases. However, whether these two events are intricately connected is poorly understood. We hypothesized that Klotho might protect kidneys by targeted inhibition of RAS activation in diseased kidneys. To test this hypothesis, mouse models of remnant kidney, as well as adriamycin nephropathy and unilateral ureteral obstruction, were utilized. At 6 weeks after 5/6 nephrectomy, kidney injury was evident, characterized by elevated albuminuria and serum creatinine levels, and excessive deposition of interstitial matrix proteins. These lesions were accompanied by loss of renal Klotho expression, up-regulation of RAS components, and development of hypertension. In vivo expression of exogenous Klotho through hydrodynamic-based gene delivery abolished the induction of multiple RAS proteins, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II type 1 receptor, and normalized blood pressure. Klotho also inhibited ß-catenin activation and ameliorated renal fibrotic lesions. Similar results were obtained in mouse models of adriamycin and obstructive nephropathy. In cultured kidney tubular epithelial cells, Klotho dose-dependently blocked Wnt1-triggered RAS activation. Taken together, these results demonstrate that Klotho exerts its renal protection by targeted inhibition of RAS, a pathogenic pathway known to play a key role in the evolution and progression of hypertension and chronic kidney disorders.

Heparanase stimulates chondrogenesis and is up-regulated in human ectopic cartilage: a mechanism possibly involved in hereditary multiple exostoses.

Hereditary multiple exostoses is a pediatric skeletal disorder characterized by benign cartilaginous tumors called exostoses that form next to growing skeletal elements. Hereditary multiple exostoses patients carry heterozygous mutations in the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2, but studies suggest that EXT haploinsufficiency and ensuing partial HS deficiency are insufficient for exostosis formation. Searching for additional pathways, we analyzed presence and distribution of heparanase in human exostoses. Heparanase was readily detectable in most chondrocytes, particularly in cell clusters. In control growth plates from unaffected persons, however, heparanase was detectable only in hypertrophic zone. Treatment of mouse embryo limb mesenchymal micromass cultures with exogenous heparanase greatly stimulated chondrogenesis and bone morphogenetic protein signaling as revealed by Smad1/5/8 phosphorylation. It also stimulated cell migration and proliferation. Interfering with HS function both with the chemical antagonist Surfen or treatment with bacterial heparitinase up-regulated endogenous heparanase gene expression, suggesting a counterintuitive feedback mechanism that would result in further HS reduction and increased signaling. Thus, we tested a potent heparanase inhibitor (SST0001), which strongly inhibited chondrogenesis. Our data clearly indicate that heparanase is able to stimulate chondrogenesis, bone morphogenetic protein signaling, cell migration, and cell proliferation in chondrogenic cells. These properties may allow heparanase to play a role in exostosis genesis and pathogenesis, thus making it a conceivable therapeutic target in hereditary multiple exostoses.