For Fibroblast Growth Factor Research Articles

Transfer Learning Predicts Network Biology for Fibroblast Growth Factor 23 (FGF-23) and Cardiac Disease

Transfer Learning Predicts Network Biology for Fibroblast Growth Factor 23 (FGF-23) and Cardiac Disease

Anti-Inflammatory Role of the Klotho Protein and Relevance to Aging.

The α-Klotho protein (hereafter Klotho) is an obligate coreceptor for fibroblast growth factor 23 (FGF23). It is produced in the kidneys, brain and other sites. Klotho insufficiency causes hyperphosphatemia and other anomalies. Importantly, it is associated with chronic pathologies (often age-related) that have an inflammatory component. This includes atherosclerosis, diabetes and Alzheimer's disease. Its mode of action in these diseases is not well understood, but it inhibits or regulates multiple major pathways. Klotho has a membrane form and a soluble form (s-Klotho). Cytosolic Klotho is postulated but not well characterized. s-Klotho has endocrine properties that are incompletely elucidated. It binds to the FGF receptor 1c (FGFR1c) that is widely expressed (including endothelial cells). It also attaches to soluble FGF23, and FGF23/Klotho binds to FGFRs. Thus, s-Klotho might be a roaming FGF23 coreceptor, but it has other functions. Notably, Klotho (cell-bound or soluble) counteracts inflammation and appears to mitigate related aging (inflammaging). It inhibits NF-κB and the NLRP3 inflammasome. This inflammasome requires priming by NF-κB and produces active IL-1β, membrane pores and cell death (pyroptosis). In accord, Klotho countered inflammation and cell injury induced by toxins, damage-associated molecular patterns (DAMPs), cytokines, and reactive oxygen species (ROS). s-Klotho also blocks the TGF-β receptor and Wnt ligands, which lessens fibrotic disease. Low Klotho is associated with loss of muscle mass (sarcopenia), as occurs in aging and chronic diseases. s-Klotho counters the inhibitory effects of myostatin and TGF-β on muscle, reduces inflammation, and improves muscle repair following injury. The inhibition of TGF-β and other factors may also be protective in diabetic retinopathy and age-related macular degeneration (AMD). This review examines Klotho functions especially as related to inflammation and potential applications.

The therapeutic potential of fibroblast growth factors in spinal cord injury: Roles and applications for neuroprotection

Spinal cord injury (SCI) is a severe and debilitating condition that often leads to significant and permanent neurological deficits, affecting motor, sensory, and autonomic functions. The high prevalence of SCI and the lack of effective treatments make it a critical area of medical research. Recent studies have shown promising prospects for fibroblast growth factors (FGF) in the treatment of SCI. FGF, particularly FGF-1 and FGF-2, are known to promote cell growth, differentiation, and survival, and have been demonstrated to enhance axonal regeneration and functional recovery in SCI models. This review highlights the roles of various FGF in neuroprotection, axonal regeneration, and remyelination. It also discusses the potential therapeutic applications of FGF in SCI treatment, emphasizing their ability to reduce neuroinflammation, oxidative stress, and promote motor function recovery. The comprehensive overview of existing research underscores the importance of FGF in SCI repair and suggests further exploration into their therapeutic potential.

Establishing a Reference Interval for Fibroblast Growth Factor (FGF)-23 in Cats.

Fibroblast growth factor (FGF)-23 is a phosphaturic hormone. An association between increasing FGF-23 levels and progression of chronic kidney disease (CKD) was documented in cats, dogs, and humans. The information regarding reference intervals (RIs) of FGF-23 in cats is limited. We aimed to establish RIs in a large cohort of clinically healthy cats and to investigate correlations with sex and age. A total of 118 cats with unremarkable complete blood count and serum chemistry profile were included. Clinically sick cats, cats with concurrent diseases, suspicion of CKD, or receiving renal diets were excluded. FGF-23 concentrations were measured with the FGF-23 ELISA Kit. RIs were calculated using the reference interval advisor software 2.1 (Microsoft Excel). FGF-23 concentrations were correlated with sex and age. The RI for FGF-23 concentrations spanned 85.8 to 387.0 pg/mL (90% confidence interval: lower limit 40.5 to 103.9 pg/mL, upper limit: 354.6 to 425.0 pg/mL). No significant relationships (r2 = 0.044) were detected with age (p = 0.081) or sex (p = 0.191). Other studies of the same diagnostic assay calculated RIs of 56 to 700 pg/mL in 79 cats and <336 pg/mL in 108 cats, and in concordance with the present study, did not detect any correlation with sex or age.

Evaluation of klotho expression on peripheral blood lymphocytes among hemodialysis patients and its possible contribution to their immuno-compromised status

Infection is the second most common cause of mortality among end-stage kidney disease (ESKD) patients. Uremic toxins are the main cause of impaired immune response among ESKD patients. Klotho gene, the anti-aging gene, encodes the transmembrane alpha klotho (αKL) protein which acts as an obligate coreceptor for fibroblast growth factor 23 (FGF23). Klotho protein may play a role in immune cell functions, particularly in anti-inflammatory response; however, its role is still incompletely understood. In the present study, we aimed to measure αKL protein expression on peripheral blood lymphocytes (PBLs) among hemodialysis (HD) patients, and we assumed that decreased αKL expression on PBLs may contribute to the impaired immunity among HD patients. This case-control study included 20 ESKD patients on regular hemodialysis for more than 3 months. Their ages ranged from 24 to 69 years. Patients with primary immunodeficiencies, those on systemic immunosuppressive drugs, those with ongoing infections or who had recently recovered from infections, and those with malignancies on active treatment were excluded. A control group of 20 normal subjects of comparable age and gender were also included. We compared αKL protein expression on PBLs by flow cytometry between both groups. Significant reductions in percentages of αKL protein expression on B lymphocytes (CD19), T lymphocytes (CD3), and natural killer cells (CD56) were observed among HD patients compared to controls. We also noticed a significant reduction in the percentages of natural killer cells among HD patients. The present study suggests that decreased αKL expression on PBLs may contribute to the immunocompromised status among HD patients, highlighting the importance of understanding the exact function of αKL protein on immune cells. This may offer a future diagnostic and therapeutic tool to improve the immune response among HD patients.

Mechanistic impact of fibrosis by parathyroid hormone excess

Klotho is a protein that has been found to play a role in regulating parathyroid hormone (PTH) levels. The literature suggests an intricate relationship between Klotho and PTH. Klotho acts as a co-receptor for fibroblast growth factor 23 (FGF23), a hormone that helps regulate phosphate and vitamin D metabolism. Klotho deficiency has been associated with increased PTH levels and secondary hyperparathyroidism. Several studies have investigated the relationship between Klotho and PTH levels. Some studies have shown that Klotho levels are inversely correlated with PTH levels, suggesting that higher Klotho levels may lead to lower PTH levels. Other studies have found that Klotho deficiency can contribute to PTH resistance, leading to persistent hyperparathyroidism. Additionally, the Klotho-PTH axis holds promise as a potential target for therapeutic interventions in calcium and phosphate disorders.

Comparison of Three Different Diagnostic Assays for Fibroblast Growth Factor-23 (FGF-23) Measurements in Cats: A Pilot Study.

Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aim of this pilot study was to compare three diagnostic assays and to assess how the results correlate with parameters of renal dysfunction in cats. Four groups of 10 cats each were formed retrospectively according to creatinine, based on IRIS staging. FGF-23 was measured using two different ELISAs (MyBioSource and Kainos ELISA FGF-23 Kit) and an automated assay on the DiaSorin Liaison platform. Measurements were performed in 40 cats. Spearman's rank correlation coefficient showed a strong correlation between the Kainos and DiaSorin assays (ρ = 0.742/p < 0.001) and a low correlation (ρ = 0.443/p = 0.005) between the Kainos and MyBioSource assays. The measurements with the Kainos assay strongly correlated with urea (ρ = 0.835/p < 0.001) and creatinine (ρ = 0.764/p < 0.001), and moderately correlated with SDMA (ρ = 0.580/p < 0.001) and phosphorus (ρ = 0.532/p < 0.001). The results of the MyBioSource and DiaSorin assays only showed a moderate correlation with urea (ρ = 0.624/0.572) and creatinine (ρ = 0.622/0.510) concentrations (p = 0.001 each). The Kainos assay showed the strongest correlation (ρ = 0.806) with the various creatinine concentrations according to the IRIS, followed by the MyBioSource (ρ = 0.663/p < 0.001) and DiaSorin assays (ρ = 0.580/p < 0.001). Overall, the Kainos assay demonstrated the best correlations with both biomarkers and various creatinine concentrations according to the IRIS. Individual assay-based reference values should be established to make a reliable interpretation of FGF-23 levels possible to diagnose or monitor feline CKD.

Klotho and FGF23 proteins levels and occurrence of myocardial infarction in patients with acute coronary syndrome

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Wroclaw Medical University, Poland. Background Klotho is a transmembrane and secretory protein, and acts as a co-receptor for fibroblast growth factor 23 (FGF23). Recent studies have underlined the importance of Klotho in renal disorders, where Klotho deficiency served as an early biomarker and therapeutic target [1]. Disrupted levels of Klotho and FGF23 were associated with cardiovascular disease [2]. Purpose This study aimed to analyze the level and role of Klotho and FGF23 proteins in patients during an episode of myocardial infarction (MI). Methods This study consisted of 159 patients admitted by the Department of Cardiology between 2013 and 2021. The study group comprised 129 patients diagnosed with acute coronary syndrome (ACS), who were referred for further invasive diagnosis (MI group). Blood samples were collected immediately after invasive diagnostic (t0) and between 24-48 h post-procedure (t1). The criteria for the control subjects (n=30) were no declaration of past MI and no ACS based on the examination conducted on admission (no MI group). Results The level of Klotho protein in the MI group was increased at t0 (median=622.3 pg/ml; p=0.029), and then normalized at t1 (median=510.3 pg/ml; p=0.893), as compared to no MI patients (median=502.3 pg/ml). The level of FGF23 protein in the MI group was higher at t0 (median=384.6 pg/ml; p=0.03), and continued to increase at t1 (median=825.8 pg/ml; p&amp;lt;0.0001), as compared to no MI patients (median=158.3 pg/ml). ROC analysis showed moderate accuracy for Klotho at t0 (AUC=0.633; p=0.043) and FGF23 at t0 (AUC=0.651; p=0.03), and high accuracy for FGF23 at t1 (AUC=0.866; p&amp;lt;0.0001). Conclusions The level of Klotho and FGF23 proteins are higher in patients with MI compared to the healthy population. Thus, Klotho and FGF23 can be recognized as potential biomarkers for MI in patients with ACS. The high diagnostic power of FGF23 level 24-48 h post-MI creates a great diagnostic and clinical usefulness of this parameter.

Pathobiology of the Klotho Antiaging Protein and Therapeutic Considerations.

The α-Klotho protein (henceforth denoted Klotho) has antiaging properties, as first observed in mice homozygous for a hypomorphic Klotho gene (kl/kl). These mice have a shortened lifespan, stunted growth, renal disease, hyperphosphatemia, hypercalcemia, vascular calcification, cardiac hypertrophy, hypertension, pulmonary disease, cognitive impairment, multi-organ atrophy and fibrosis. Overexpression of Klotho has opposite effects, extending lifespan. In humans, Klotho levels decline with age, chronic kidney disease, diabetes, Alzheimer’s disease and other conditions. Low Klotho levels correlate with an increase in the death rate from all causes. Klotho acts either as an obligate coreceptor for fibroblast growth factor 23 (FGF23), or as a soluble pleiotropic endocrine hormone (s-Klotho). It is mainly produced in the kidneys, but also in the brain, pancreas and other tissues. On renal tubular-cell membranes, it associates with FGF receptors to bind FGF23. Produced in bones, FGF23 regulates renal excretion of phosphate (phosphaturic effect) and vitamin D metabolism. Lack of Klotho or FGF23 results in hyperphosphatemia and hypervitaminosis D. With age, human renal function often deteriorates, lowering Klotho levels. This appears to promote age-related pathology. Remarkably, Klotho inhibits four pathways that have been linked to aging in various ways: Transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), Wnt and NF-κB. These can induce cellular senescence, apoptosis, inflammation, immune dysfunction, fibrosis and neoplasia. Furthermore, Klotho increases cell-protective antioxidant enzymes through Nrf2 and FoxO. In accord, preclinical Klotho therapy ameliorated renal, cardiovascular, diabetes-related and neurodegenerative diseases, as well as cancer. s-Klotho protein injection was effective, but requires further investigation. Several drugs enhance circulating Klotho levels, and some cross the blood-brain barrier to potentially act in the brain. In clinical trials, increased Klotho was noted with renin-angiotensin system inhibitors (losartan, valsartan), a statin (fluvastatin), mTOR inhibitors (rapamycin, everolimus), vitamin D and pentoxifylline. In preclinical work, antidiabetic drugs (metformin, GLP-1-based, GABA, PPAR-γ agonists) also enhanced Klotho. Several traditional medicines and/or nutraceuticals increased Klotho in rodents, including astaxanthin, curcumin, ginseng, ligustilide and resveratrol. Notably, exercise and sport activity increased Klotho. This review addresses molecular, physiological and therapeutic aspects of Klotho.

Growth Factors and Their Roles in Multiple Sclerosis Risk.

BackgroundPrevious studies have suggested essential roles of growth factors on the risk of Multiple Sclerosis (MS), but it remains undefined whether the effects are causal.ObjectiveWe applied Mendelian randomization (MR) approaches to disentangle the causal relationship between genetically predicted circulating levels of growth factors and the risk of MS.MethodsGenetic instrumental variables for fibroblast growth factor (FGF) 23, growth differentiation factor 15 (GDF15), insulin growth factor 1 (IGF1), insulin-like growth factor binding proteins 3 (IGFBP3) and vascular endothelial growth factor (VEGF) were obtained from up-to-date genome-wide association studies (GWAS). Summary-level statistics of MS were obtained from the International Multiple Sclerosis Genetics Consortium, incorporating 14,802 subjects with MS and 26,703 healthy controls of European ancestry. Inverse-variance weighted (IVW) MR was used as the primary method and multiple sensitivity analyses were employed in this study.ResultsGenetically predicted circulating levels of FGF23 were associated with risk of MS. The odds ratio (OR) of IVW was 0.63 (95% confidence interval [CI], 0.49–0.82; p < 0.001) per one standard deviation increase in circulating FGF23 levels. Weighted median estimators also suggested FGF23 associated with lower MS risk (OR = 0.67; 95% CI, 0.51-0.87; p = 0.003). While MR-Egger approach provided no evidence of horizontal pleiotropy (intercept = -0.003, p = 0.95). Results of IVW methods provided no evidence for causal roles of GDF1, IGF1, IGFBP3 and VEGF on MS risks, and additional sensitivity analyses confirmed the robustness of these null findings.ConclusionOur results implied a causal relationship between FGF23 and the risk of MS. Further studies are warranted to confirm FGF23 as a genetically valid target for MS.

GWAS meta-analysis followed by Mendelian randomization revealed potential control mechanisms for circulating α-Klotho levels

The protein α-Klotho acts as transmembrane co-receptor for fibroblast growth factor 23 (FGF23) and is a key regulator of phosphate homeostasis. However, α-Klotho also exists in a circulating form, with pleiotropic, but incompletely understood functions and regulation. Therefore, we undertook a genome-wide association study (GWAS) meta-analysis followed by Mendelian randomization (MR) of circulating α-Klotho levels.Plasma α-Klotho levels were measured by enzyme-linked immunosorbent assay (ELISA) in the Ludwigshafen Risk and Cardiovascular Health and Avon Longitudinal Study of Parents and Children (mothers) cohorts, followed by a GWAS meta-analysis in 4376 individuals across the two cohorts.Six signals at five loci were associated with circulating α-Klotho levels at genome-wide significance (P < 5 × 10−8), namely ABO, KL, FGFR1, and two post-translational modification genes, B4GALNT3 and CHST9. Together, these loci explained >9% of the variation in circulating α-Klotho levels. MR analyses revealed no causal relationships between α-Klotho and renal function, FGF23-dependent factors such as vitamin D and phosphate levels, or bone mineral density. The screening for genetic correlations with other phenotypes followed by targeted MR suggested causal effects of liability of Crohn’s disease risk [Inverse variance weighted (IVW) beta = 0.059 (95% confidence interval 0.026, 0.093)] and low-density lipoprotein cholesterol levels [−0.198 (−0.332, −0.063)] on α-Klotho.Our GWAS findings suggest that two enzymes involved in post-translational modification, B4GALNT3 and CHST9, contribute to genetic influences on α-Klotho levels, presumably by affecting protein turnover and stability. Subsequent evidence from MR analyses on α-Klotho levels suggest regulation by mechanisms besides phosphate-homeostasis and raise the possibility of cross-talk with FGF19- and FGF21-dependent pathways, respectively. Significance statement: α-Klotho as a transmembrane protein is well investigated along the endocrine FGF23-α-Klotho pathway. However, the role of the circulating form of α-Klotho, which is generated by cleavage of transmembrane α-Klotho, remains incompletely understood. Genetic analyses might help to elucidate novel regulatory and functional mechanisms. The identification of genetic factors related to circulating α-Klotho further enables MR to examine causal relationships with other factors. The findings from the first GWAS meta-analysis of circulating α-Klotho levels identified six genome-wide significant signals across five genes. Given the function of two of the genes identified, B4GALNT3 and CHST9, it is tempting to speculate that post-translational modification significantly contributes to genetic influences on α-Klotho levels, presumably by affecting protein turnover and stability.

Implications of Klotho Protein for Managing Kidney Disease - an Emerging Role in Therapeutics and Molecular Medicine.

Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are a growing public health problem. There is a paucity of sensitive biomarkers to detect AKI, early CKD, and ameliorate extra-renal complications. Klotho protein, detected mainly in the kidneys, regulates renal health and functions as a co-receptor for fibroblast growth factor 23 (FGF-23) signaling. It is now coming to be known for its extreme pleiotropic actions. These include cytoprotection via anti-oxidation, anti-senescence, anti-apoptosis, renoprotective effects, promotion of angiogenesis and vascularisation, inhibition of fibrogenesis, and stem cell preservation. Emerging clinical studies suggest kidney damage to be a perpetual state of renal Klotho deficiency. In AKI, Klotho levels in plasma and/or urine possibly will serve as an initial biomarker for kidney parenchymal injury. In CKD, Klotho levels may also be an indicator of early disease as well as predict the rate of progression. Earlier studies using ELISA as a technique reveal a correlation between plasma Klotho, eGFR, serum creatine, and Blood Urea Nitrogen (BUN) levels. Thereby preventing the decline of Klotho levels by various mechanisms can retard CKD advancement and improve renal function. Substantial data indicate Klotho can be therapeutically included as an individualized regimen for managing CKD patients. Considerable research is required in investigating the role of soluble Klotho as a biomarker in patients with different types and severity of kidney diseases, which will be highlighted in our review.

Klotho and calciprotein particles as therapeutic targets against accelerated ageing.

The klotho gene, named after a Greek goddess who spins the thread of life, was identified as a putative ‘ageing-suppressor’ gene. Klotho-deficient mice exhibit complex ageing-like phenotypes including hypogonadism, arteriosclerosis (vascular calcification), cardiac hypertrophy, osteopenia, sarcopenia, frailty, and premature death. Klotho protein functions as the obligate co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived hormone that promotes urinary phosphate excretion in response to phosphate intake. Thus, Klotho-deficient mice suffer not only from accelerated ageing but also from phosphate retention due to impaired phosphate excretion. Importantly, restoration of the phosphate balance by placing Klotho-deficient mice on low phosphate diet rescued them from premature ageing, leading us to the notion that phosphate accelerates ageing. Because the extracellular fluid is super-saturated in terms of phosphate and calcium ions, an increase in the phosphate concentration can trigger precipitation of calcium-phosphate. In the blood, calcium-phosphate precipitated upon increase in the blood phosphate concentration is adsorbed by serum protein fetuin-A to form colloidal nanoparticles called calciprotein particles (CPPs). In the urine, CPPs appear in the renal tubular fluid when FGF23 increases phosphate load excreted per nephron. CPPs can induce cell damage, ectopic calcification, and inflammatory responses. CPPs in the blood can induce arteriosclerosis and non-infectious chronic inflammation, whereas CPPs in the urine can induce renal tubular damage and interstitial inflammation/fibrosis. Thus, we propose that CPPs behave like a pathogen that accelerates ageing and should be regarded as a novel therapeutic target against age-related disorders including chronic kidney disease.

Performance evaluation of the new chemiluminescent intact FGF23 assay relative to the existing assay system

This study assessed the performance of a new fully automated immunoassay for fibroblast growth factor (FGF) 23 (Determinar CL FGF23 CL) among healthy individuals and those with chronic hypophosphatemia compared with the previous assay (Kainos FGF23 KI). A total of 380 serum samples from healthy participants were collected to determine the reference range of FGF23 levels with CL. A total of 200 serum samples from 22 hypophosphatemic patients were collected simultaneously to compare the difference in FGF23 levels between CL and KI. The Mann-Whitney U test and linear regression analysis were adopted to assess the differences and linearity between the two assays. The median FGF23 levels among healthy individuals was 31.7 (interquartile: 26.4-37.5) pg/mL. When the reference range was calculated as the mean ± 2 standard deviation (2SD), it was 16.1-49.3pg/mL. A total of 363 individuals (96%) among normal cases fell in this range. Among 200 samples from patients with chronic hypophosphatemic disorder, the median FGF23 levels analyzed by CL and KI were 123.0 (90.2-237.7) and 172.5 (115.8-290.7) pg/mL. KI yielded significantly higher FGF23 values than CL (p < 0.001). A linear regression model revealed the correlation between KI (x) and CL (y), which had a slope of 0.76 with a y-intercept of -0.32 and high linearity (R2 = 0.99). The new measurement kit yielded lower FGF23 values when compared with the previous assay. Clinicians should consider this discrepancy when they assay intact FGF23 values with CL.

Urinary Klotho Abnormalities in Children with Sickle Cell Disease

Background: Klotho is a trans membrane protein expressed in the renal tubules and serves as an obligatory co-receptor for fibroblast growth factor 23(FGF23) to aid in phosphorus excretion. Prior studies have shown FGF23 resistance in sickle cell disease (SCD). The purpose of the study is to investigate urinary klotho/creatinine (Ur Kl/Cr) in pediatric SCD with normal traditional markers of renal function (eGFR &gt; 90 ml/min and no micro albuminuria) and to compare it with the healthy control population. Methods: Cross-sectional observational study to compare Ur Kl/Cr in pediatric SCD and controls. To do a subgroup analysis among the study population to assess the effect of hydroxyl urea (HU) on Ur Kl/Cr. Results: 20 controls and 22 pediatric SCD were enrolled. In the SCD group, 13 were on treatment with HU. The baseline characteristics of the study and control group were the same. Wilcoxon rank-sum test was used to compare the levels of Ur Kl/Cr ratio between SCD and control. For P value of 0.05, the levels of Ur Kl/Cr were statistically significantly higher in the sickle cell group (752.7 ± 1101.0) over the control group (216.8 ± 225.3). Subgroup analysis in the SCD group showed high Urinary Kl excretion in the non-HU group (1346.7 ± 1523.4) vs. non HU group (341.4 ± 355.3) but not statistically significant. Conclusion: Children with SCD tend to have increased secretion of Ur Kl/Cr compared to control likely due to tubular receptor resistance. HU may reverse this phenomenon by its effect on preventing tubular damage.

Receptor pathways of glycated basic fibroblast growth factor affecting the proliferation and vascularization of human dermal microvascular endothelial cells

Objective: To investigate the receptor pathways of glycated basic fibroblast growth factor (bFGF) on proliferation and vascularization of human dermal microvascular endothelial cells (HDMECs). Methods: The experimental research method was used. Glycated bFGF stimulating solution was prepared with glucose and bFGF. HDMECs of the third to sixth passages were used in the experiment. Cells were divided into small interfering RNA (siRNA)-positive control group, siRNA-negative control group, siRNA-receptor for advanced glycation end product (RAGE) group, and siRNA-receptor for fibroblast growth factor (FGFR) group and transfected with siRNA-positive control glyceraldehyde-3-phosphate dehydrogenase, siRNA-negative control, siRNA-RAGE, and siRNA-FGFR for 4 to 6 hours, and then were added into HDMEC culture medium for routine culture. The transfection effect of siRNA was identified by reverse transcription polymerase chain reaction. The cells were divided into normal control group, glycated bFGF alone group, siRNA-RAGE alone group, and siRNA-RAGE+ glycated bFGF group, and seeded into 96-well plate and 6-well plate. Cells in siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group were transfected with siRNA-RAGE and then were added into HDMEC culture medium for routine culture. After two days, the original HDMEC culture medium was discarded, and cells in siRNA-RAGE alone group were routinely cultured in HDMEC culture medium, cells in siRNA-RAGE+ glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells in normal control group were routinely cultured in HDMEC culture medium, and cells in glycated bFGF alone group were routinely cultured in glycated bFGF stimulating solution. After transfection with siRNA-RAGE, cells were seeded into 48-well plate and divided into siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group. Cells in the 4 groups were conducted with the corresponding treatment as above. Cells were divided into normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group and seeded into 96-, 6-, and 48-well plates, respectively, with the corresponding treatment the same as above. Only siRNA-RAGE was replaced by siRNA-FGFR. Cell counting kit 8 method was used to determine the proliferation of cells after 2 days of culture (sample number was 6), flow cytometry was used to detect the apoptosis of cells after 2 days of culture (sample number was 3), tube forming test was used to detect the angiogenesis of cells after 6 hours of culture (sample number was 4). Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: At the 200 bp band, there were no target genes in siRNA-positive control group, siRNA-RAGE group, or siRNA-FGFR group, but target genes were detected in siRNA-negative control group, indicating the success of siRNA transfection. After 2 days of culture, the absorbance value of cells in glycated bFGF alone group was significantly lower than that of normal control group (t=2.359, P<0.05); absorbance value of cells in siRNA-RAGE+ glycated bFGF group was significantly higher than that of glycated bFGF alone group (t=3.858, P<0.01), which was similar to that of siRNA-RAGE alone group (t=2.148, P>0.05). The absorbance value of cells in siRNA-FGFR+ glycated bFGF group was similar to that of glycated bFGF alone group (t=0.805, P>0.05), but significantly lower than that of siRNA-FGFR alone group (t=4.201, P<0.01). After 2 days of culture, the apoptotic rate of cells in glycated bFGF alone group was significantly higher than that of normal control group (t=2.416, P<0.05). The apoptotic rate of cells in siRNA-RAGE+ glycated bFGF group was significantly lower than the rates in glycated bFGF alone group and siRNA-RAGE alone group (t=3.861, 2.724, P<0.05 or P<0.01). There were no statistically significant differences in apoptosis rate of cells among normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group (F=2.218, P>0.05). After 6 hours of culture, the number of tubules of cells in normal control group (636±5) was significantly more than that of glycated bFGF alone group (580±8, t=10.825, P<0.01), and the number of tubules of cells in siRNA-RAGE+ glycated bFGF group (647±10) was significantly more than those of glycated bFGF alone group and siRNA-RAGE alone group (628±4, t=13.040, 3.641, P<0.01). After 6 hours of culture, the number of tubules of cells in siRNA-FGFR+ glycated bFGF group (619±5) was more than that of glycated bFGF alone group (t=9.000, P<0.01), but less than that of siRNA-FGFR alone group (632±3, t=2.814, P<0.05). Conclusions: Glycated bFGF affects the proliferation and angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for impaired wound healing of diabetic skin.

Klotho, Aging, and the Failing Kidney.

Klotho has been recognized as a gene involved in the aging process in mammals for over 30 years, where it regulates phosphate homeostasis and the activity of members of the fibroblast growth factor (FGF) family. The α-Klotho protein is the receptor for Fibroblast Growth Factor-23 (FGF23), regulating phosphate homeostasis and vitamin D metabolism. Phosphate toxicity is a hallmark of mammalian aging and correlates with diminution of Klotho levels with increasing age. As such, modulation of Klotho activity is an attractive target for therapeutic intervention in the diseasome of aging; in particular for chronic kidney disease (CKD), where Klotho has been implicated directly in the pathophysiology. A range of senotherapeutic strategies have been developed to directly or indirectly influence Klotho expression, with varying degrees of success. These include administration of exogenous Klotho, synthetic and natural Klotho agonists and indirect approaches, via modulation of the foodome and the gut microbiota. All these approaches have significant potential to mitigate loss of physiological function and resilience accompanying old age and to improve outcomes within the diseasome of aging.

Plasma Soluble αKlotho, Serum Fibroblast Growth Factor 23, and Mobility Disability in Community-Dwelling Older Adults.

ContextαKlotho is a hormone and co-receptor for fibroblast growth factor 23 (FGF23), a hormone that downregulates active vitamin D synthesis and promotes phosphate excretion. Low αKlotho and high FGF23 occur in chronic kidney disease (CKD).ObjectiveWe aimed to assess the relationships of αKlotho and FGF23 with mobility disability in community-dwelling older adults.Design and SettingWe estimated associations of plasma-soluble αKlotho and serum FGF23 concentrations with mobility disability over 6 years. Additional analyses was stratified by CKD.ParticipantsParticipants included 2751 adults (25.0% with CKD), aged 71 to 80 years, from the 1998 to 1999 Health, Aging, and Body Composition Study visit.Main Outcome MeasuresWalking disability and stair climb disability were defined as self-reported “a lot of difficulty” or an inability to walk a quarter mile and climb 10 stairs, respectively.ResultsMedian (interquartile range [IQR]) serum FGF23 and plasma soluble αKlotho concentrations were 46.6 (36.7, 60.2) pg/mL and 630.4 (478.4, 816.0) pg/mL, respectively. After adjustment, higher αKlotho concentrations were associated with lower walking disability rates (Rate Ratio [RR] highest vs. lowest tertile = 0.74; 95% confidence interval l [CI] = 0.62, 0.89; P = 0.003). Higher FGF23 concentrations were associated with higher walking disability rates (RR highest vs. lowest tertile = 1.24; 95%CI = 1.03, 1.50; P = 0.005). Overall, higher αKlotho combined with lower FGF23 was associated with the lowest walking disability rates (P for interaction = 0.023). Stair climb disability findings were inconsistent. No interactions with CKD were statistically significant (P for interaction > 0.10).ConclusionsHigher plasma soluble αKlotho and lower serum FGF23 concentrations were associated with lower walking disability rates in community-dwelling older adults, particularly those without CKD.

Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

Parathyroid Hormone and Plasma Phosphate Are Predictors of Soluble α-Klotho Levels in Adults of European Descent.

α-klotho is an integral membrane protein that serves as a coreceptor for fibroblast growth factor 23 (FGF23) in conjunction with cognate fibroblast growth factor receptors. Proteolytic cleavage sheds the ectodomain of α-klotho (soluble α-klotho) as an endocrine substance into blood, urine, and cerebrospinal fluid. To study the relationship of soluble α-klotho to mineral metabolism in the general population with mainly preserved kidney function. Cross-sectional analysis of the associations between soluble α-klotho with laboratory markers of markers of mineral metabolism in a population-based cohort. Three centers in Switzerland including 1128 participants. Soluble full-length α-klotho levels by a specific immunoassay and markers of mineral metabolism. The median serum level of soluble α-klotho was 15.0 pmol/L. Multivariable analyses using α-klotho as the outcome variable revealed a sex-by-PTH interaction: In men, PTH was positively associated with α-klotho levels, whereas this association was negative in women. Plasma phosphate associated with soluble α-klotho levels in an age-dependent manner, changing from a positive association in young adults gradually to a negative association in the elderly. The decline of 1,25 (OH)2 vitamin D3 levels in parallel to the gradual impairment of kidney function was greatly attenuated in the setting of high circulating soluble α-klotho levels. Soluble α-klotho level is associated with plasma phosphate in an age-dependent manner and with PTH in a sex-dependent manner. Furthermore, our data reveal soluble α-klotho as a modulator of 1,25 (OH)2 vitamin D3 levels in individuals with preserved renal function.