KL Expression Research Articles

α-Klotho prevents diabetic retinopathy by reversing the senescence of macrophages

BackgroundDiabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus (DM) and a significant cause of acquired blindness in the working-age population worldwide. Aging is considered as an important risk factor for DR development. Macrophages in aged mice bear typical M2 marker proteins but simultaneously express a pro-inflammatory factor profile. This may explain why the level of intraocular inflammation does not decrease during proliferative diabetic retinopathy (PDR) despite the occurrence of neovascularization and fibrosis (M2 activation). α-Klotho (KL) was originally discovered as a soluble anti-aging factor, which is mainly expressed in kidney tubular epithelium, choroid plexus in the brain and secreted in the blood. However, the role of KL in DR pathophysiology has not been previously reported.MethodsType 1 (streptozotocin [STZ]-induced) and type 2 (a high-fat diet along with a low dose of STZ) diabetic mouse models were established and injected with or without KL adenovirus via the tail vein for 12 weeks. Vldlr−/− mice were injected intravitreally with or without soluble KL protein from P8 to P15. The retinal structure and function were analyzed by electroretinogram and optical coherence tomography. The neovascular lesions were analyzed by retinal flat mount and RPE flat mount. The senescence markers, macrophage morphology, and KL expression levels were detected by immunofluorescence staining. A cell model was constructed using RAW264.7 cells stimulated by 4-hydroxynonenal (4HNE) and transfected with or without KL adenovirus. The senescence-associated secretory phenotypes were detected by qRT-PCR. Senescence was detected by SA-β-Gal staining. Serum, aqueous humor, and vitreous humor KL levels of proliferative diabetic retinopathy (PDR) patients were measured by enzyme‐linked immunosorbent assay. Quantitative proteomics and bioinformatics were applied to predict the change of proteins and biological function after overexpression of KL in macrophages. The effects of KL on the HECTD1 binding to IRS1 were analyzed by bioinformatics, molecular docking, and Western Blot.ResultsSerum, aqueous humor, and vitreous humor KL levels were lower in patients with PDR than in those with cataracts. KL relieved the retinal structure damage, improved retina function, and inhibited retinal senescence in diabetic mice. KL administration attenuated the neovascular lesions in VLDLR−/− mice by decreasing the secretion of VEGFA and FGF2 from macrophages. KL also protected RAW264.7 cells from 4HNE-induced senescence. Additionally, it inhibited E3 ubiquitin ligase HECTD1 expression in both diabetic mouse peripheral blood mononuclear cells and 4HNE-treated RAW264.7 cells. KL inhibited HECTD1 binding to IRS1 and reduced the ubiquitination of IRS1.ConclusionsMacrophage aging is involved in DM-induced retinopathy. KL alleviates DM-induced retinal macrophage senescence by downregulating HECTD1 and decreasing IRS1 ubiquitination and degradation. Meanwhile, KL administration attenuated the neovascular lesions by altering the activation state of macrophages and decreasing the expression of VEGFA and FGF2.

184 The metabolism of vitamin D is modulated by vitamin C supplementation during porcine reproductive and respiratory syndrome challenge in post-weaning piglets

Abstract This study evaluated the impact of different dietary vitamin D sources combined or not with vitamin C on serum 25(OH)D3 concentrations and the expression of genes related to antimicrobial peptides and to the metabolism of vitamin D, vitamin C, calcium (Ca), and phosphorus (P). From weaning (d 21) until d 68, 27 piglets were fed a post-weaning diet supplemented with: 1,500 IU of vitamin D3 (CTR; n = 9); 200 IU of vitamin D3 + 2,000 IU of 25(OH)D3 (HD; n = 8); or HD + 500 mg/kg of vitamin C (HD+C; n = 10). From d 40 to 68 piglets were exposed to porcine reproductive and respiratory syndrome (PRRS) using a natural disease challenge model. At d 68, after blood collection, all piglets were euthanized for the collection of lung and kidney samples. The concentrations of serum 25(OH)D3 were determined by HPLC and the mRNA expression of genes of interest was assessed by qPCR. Data were analyzed in a one-way analysis of variance with three treatments. At d 40 (after 19 d of supplementation), serum 25(OH)D3 concentrations were greatest for HD+C (22.48 ± 1.25 ng/mL), intermediate for HD (17.43 ± 0.84 ng/mL), and least for CTR (8.54 ± 0.65 ng/mL) treatments (P < 0.01). At d 68 (after 28 d of PRRS challenge) these values were greatest for HD (14.64 ± 2.77 ng/mL), intermediate for CTR (6.22 ± 0.59 ng/mL), and least for HD+C (2.18 ± 0.13 ng/mL; P < 0.01). No treatments effect was detected on pulmonary CYP27B1, RXRα, SLC23A1, RIGI, PR-39, and pBD1. In lungs, VDR expression was least for HD+C (P ≤ 0.06) whereas MDA5 expression was greatest for HD+C (P ≤ 0.07), and SLC23A2 expression was greatest for HD (P ≤ 0.09; Table 1). No treatments effect was detected on expression of renal FGFR1, KL, RXRα, SLC23A1, SLC23A2, and TPRV5. In kidney, CYP27B1 expression was greatest for HD+C groups (P = 0.03) and CYP24A expression was greatest for HD (P = 0.01). Renal EGR1 and NPT2c expressions were greatest for CTR groups and least for HD+C (P = 0.09) whereas WNK4 expression was greatest for HD+C and least for CTR (P = 0.10). Renal VDR expression was greatest for HD groups and least for HD+C (P ≤ 0.05). In conclusion, vitamin C supplementation increased serum 25(OH)D3 concentrations in pre-challenged piglets. These concentrations were reduced by vitamin C supplementation after PRRS challenge, which may be related to a greater synthesis of the active form of vitamin D in the kidney. Little effects were detected on pulmonary antimicrobial peptides, but the gene expression related to renal P reabsorption was influenced by HD+C whereas Ca reabsorption was apparently not impacted by treatments.

KL is a favorable prognostic factor related immune for clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a prevalent cancer in adult urology, often leading to metastasis and poor prognosis. Recently, advances in tumor immunology and aging research have opened up new possibilities for the treatment of kidney cancer. Therefore, the identification of potential targets and prognostic biomarkers for immunotherapy has become increasingly urgent.MethodsUsing GSE168845 data, we identified immune-aging-associated differentially expressed genes (IAR-DEGs) by intersecting differentially expressed immune-related genes and aging-related genes. The prognostic value of IAR-DEGs was determined via univariate and multivariate Cox regression analysis, revealing KL as an independent prognostic factor for ccRCC. We also investigated the correlation between KL and various immune-related factors, including immune cell infiltration, immune score, immune checkpoint, MSI, and TIED score. To confirm the expression of KL in ccRCC, we conducted qRT-PCR assays on both ccRCC cell lines and clinical tissue samples, and compared KL expression levels between normal kidney cell line (HK-2) and ACHN, a ccRCC cell line. Finally, we assessed KL protein expression levels in tissues using immunohistochemistry (IHC).ResultsIn this study, we utilized Venn diagram analysis to identify 17 co-expressed immune-aging related DEGs from GSE168845, import database, and MSigDB database. GO and KEGG analysis revealed that the functions of the 17 IAR-DEGs were primarily related to “aging”. Univariate and multivariate Cox analysis validated these 17 genes, and KL was determined to be an independent prognostic factor for ccRCC. The downregulation of KL was observed in ccRCC tissues and was negatively associated with T stage, M stage, pathological stage, and histologic grade (p < 0.05). This downregulation indicated disease deterioration and a shortened overall survival period. Our calibration curves and nomogram demonstrated the excellent predictive potential of KL. GSEA analysis showed that KL gene mediated immune and aging-related pathways, and was significantly correlated with immune infiltration and MS and TIED score. More research has revealed a significant reduction in KL mRNA expression levels in human renal cancer cells, particularly in ccRCC tissues compared to adjacent normal kidney tissues. Moreover, immunohistochemistry data have demonstrated a marked decrease in KL protein expression levels in ccRCC cells when compared to adjacent normal tissues.ConclusionsKL was a potential aging-related target for immunotherapy and valid prognostic biomarker for ccRCC patients.

Degradation of histone deacetylase 6 alleviates ROS-mediated apoptosis in renal ischemia-reperfusion injury

Renal ischemia reperfusion injury (RIRI) is an inevitable complication during renal surgery. Histone deacetylase 6 (HDAC6), a key member of the histone deacetylase family, is associated with multiple pathologies, including renal diseases. However, whether HDAC6 could become a potential therapeutic target for clinical application of RIRI remained to be proven. Here, we found that HDAC6 expression was abnormally enhanced by the transcription factor OSR2 in RIRI. Moreover, we were the first to validate that a selective HDAC6 degrader, proteolysis-targeting chimeras (PROTAC) NP8, could significantly improve RIRI. Further in vivo and in vitro mechanism studies have found that the reduction of HDAC6 alleviated RIRI by inhibiting ROS mediated apoptosis. Remarkably, a renal protective protein, Klotho, has been proven to be a target of HDAC6, and the degradation of HDAC6 restored KL expression, thereby ameliorating ROS mediated apoptosis. Overall, our results illustrated that the degradation of HDAC6 restrained ROS mediated apoptosis by restoring Klotho expression during RIRI. PROTAC-NP8 might be a potential therapeutic strategy for clinical prevention of RIRI.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein–umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

The Language of Returning Emigrant Children: Preliminary Insights

The article overviews the Lithuanian language of returning emigrant children. It is based on the project “Linguistic Integration of Returning Emigrant Children: Proficiency in Lithuanian and its Development” (RCL Agreement No. S-REP-21-1), implemented in 2021–2022. The data were selected from two Lithuanian language children’s corpora (returning emigrant children (KG) and permanently residing in Lithuania children (KL)) collected during the project at eight Lithuanian schools. The corpora consist of the Lithuanian language production by children aged 8–12 who returned to Lithuania from English-speaking countries 1–2 years ago and by their peers in Lithuania. The article aims to identify differences in the linguistic expression of KG and KL by comparing the word frequency lists and overviewing verb, noun, and adjective. The quantitative findings show no significant differences in the use of the selected parts of speech. The results suggest that according to the available language skills and competences, the returning emigrant children can communicate on everyday topics quite freely, and their linguistic expression is similar to the language of their peers in Lithuania.

Klotho expression in peripheral blood circulating cells is associated with vascular and systemic inflammation in atherosclerotic vascular disease

Cardiovascular disease is the leading cause of death worldwide. New therapeutic strategies are aimed to modulate the athero-inflammatory process that partially orchestrates underlying vascular damage. Peripheral blood circulating cells include different immune cells with a central role in the development of the atherogenic inflammatory response. The anti-aging protein α-Klotho has been related to protective effects against CVD. KL is expressed in monocytes, macrophages, and lymphocytes where it exerts anti-inflammatory effects. In this work, we analyse the relationships of the levels of inflammatory markers with the expression of the KL gene in PBCCs and with the serum levels of soluble KL in atherosclerotic vascular disease. For this, we conducted a cross-sectional single-center case–control study including a study group of 76 CVD patients and a control group of 16 cadaveric organ donors without medical antecedent or study indicating CVD. Vascular artery fragments and whole blood and serum samples were obtained during elective or organ retrieval surgery. Serum levels of sKL, TNFα and IL10, and gene expression levels of KL, TNF, IL10, NFKB1, DNMT1, and DNMT3A in PBCCs were measured. In these cells, we also determined KL promoter methylation percentage. Histological and immunohistochemical analyses were employed to visualize atherosclerotic lesions and to measure IL10 and TNFα levels in vascular fragments. Patients with CVD presented higher values of proinflammatory markers both at systemic and in the vasculature and in the PBCCs, compared to the control group. In PBCCs, CVD patients also presented lower gene expression levels of KL gene (56.4% difference, P < 0.001), higher gene expression levels of DNMT1 and DNMT3A (P < 0.0001, for both) and a higher methylation status of in the promoter region of KL (34.1 ± 4.1% vs. 14.6 ± 3.4%, P < 0.01). In PBCCs and vasculature, KL gene expression correlated inversely with pro-inflammatory markers and directly with anti-inflammatory markers. sKL serum levels presented similar associations with the expression levels of pro- and anti-inflammatory markers in PBCCs. The differences in KL expression levels in PBCCs and in serum sKL levels with respect to control group was even greater in those CVD patients with macroscopically observable atheromatous plaques. We conclude that promoter methylation-mediated downregulation of KL gene expression in PBCCs is associated with the pro-inflammatory status in atherosclerotic vascular disease.

The alkaloid centcyamine increases expression of klotho and lamin B1, slowing the onset of skin ageing in vitro and in vivo.

Klotho is a protein known for its beneficial effects on longevity. Centcyamine is an alkaloid present in certain plants whose extracts have an anti-inflammatory effect. Skin fibroblasts are essential to the formation and structure of the dermis. Centcyamine is an indole-based alkaloid composed of coumaric acid, a resveratrol precursor and methoxytryptamine, which can be both a precursor, or a derivative, of melatonin. Given these building blocks and their well-known bioactivities, it was of interest to explore the potential benefits of using this aryl-alkaloid, in cosmetic skincare applications. We tested cultured normal human dermal fibroblasts (NHDF) in vitro to observe how supplementation with centcyamine improves properties of the cells to counteract the effect of ageing. The expression of genes and proteins of interest was quantified. The effect on doubling time and cell function was evaluated following treatment of the cells over several replication cycles. Skin firmness, red spot index and skin isotropy were measured with Dynaskin® , Visia® and Primos® equipment, respectively, and compared over two months in a vehicle-controlled clinical trial on 60 persons. Centcyamine activates the expression of the gene KL and the related protein Klotho in dermal fibroblasts. Moreover, centcyamine slows the replicative ageing process of fibroblasts in culture. These cells retain cellular functions identical to those of young cells: the synthesis of lamin B1, a crucial regulatory protein of proliferation, as well as of collagen I and elastin is retained in aged cells. The clinical data are shown to improve skin isotropy in a majority of subjects, to reduce the red spot intensity and to maintain skin firmness in the treated group vs. the vehicle. The alkaloid centcyamine induces changes in the metabolism of the ageing process of human dermal fibroblasts. The up-to-now unobserved implication of both Klotho and lamin B1 to maintain homeostasis of the skin opens new venues for the prevention of age-related changes in skin structure. The in vitro and clinical data, while not demonstrated to be causally related, converge towards a common goal of skin repair and slower ageing processes.

Potential Antiaging Effects of DLBS1649, a Centella asiatica Bioactive Extract.

PurposeCentella asiatica is a traditional medicinal plant, especially for wound healing and as a neuroprotective agent. DLBS1649 is a bioactive extract from C. asiatica, and was studied to investigate its benefits as an antiaging agent.MethodsDLBS1649 was administered to HEK293 and 3T3L1 mammalian cells cultured in a time- or dose-dependent manner. Telomere length analysis was performed. TERT, CMYC, SIRT1, SIRT2, and KL expression were observed using reverse-transcription qPCR. Telomerase protein was studied with ELISA, while calorie restriction was observed using Oil Red O. In vivo study was conducted using Drosophila melanogaster with restricted mean survival time as the statistical method of analysis.ResultsDLBS1649 50 µg/mL showed an effect in the prevention of telomere shortening by 50% and decrease in telomerase activity by 28% compared to the controls (70% and 40%, respectively) in the HEK293 cell cultures. TERT-, CMYC-, SIRT1-, SIRT2-, and KL-expression degression was also reduced (29%, 9%, 18%, 25%, 9%, and 30%, respectively) compared to the controls (46%, 40%, 56%, 44%, and 46%, respectively) after ten serial passages. Calorie-restriction activity from DLBS1649 50 µg/mL was seen, with lower fat droplet counts being detected in the treated samples (37%) than the controls (28%) in 3T3L1 cells. DLBS1649 2 mg/mL increased restricted mean survival time in male and female D. melanogaster (23.87% [p<0.05] and 12.58%, respectively).ConclusionThe results revealed DLBS1649’s potential as an antiaging agent based on telomere-length preservation, decreased expression of aging-related genes, increased calorie restriction in vitro, and mortality reduction in D. melanogaster in vivo.

MO037EPIGENETIC REGULATION OF KLOTHO IN PERIPHERAL BLOOD CIRCULATING CELLS IS ASSOCIATED CELLS IS ASSOCIATED WITH SOLUBLE PROTEIN SERUM IN CARDIOVASCULAR DISEASE

Abstract Background and Aims The antiaging factor α-Klotho (KL) has been related to cardiovascular diseases (CVD), including coronary artery disease, heart failure and peripheral arterial disease. The soluble form of this protein (sKL), which is mainly produced by the kidneys, have demonstrated to display different protective effects on the cardiovascular system, e.g., prevention of vascular calcification, oxidative stress, or cardiac fibrosis. Likewise, different clinical studies have pointed out that CVD is a state of sKL deficiency, even when the renal function is still preserved. It has been demonstrated that peripheral blood circulating cells (PBCCs) also express KL, but little is known about its expression profile during CVD. The aim of this work is to determinate if the expression of KL gene and its promoter methylation in PBCCs are correlated with serum levels of the soluble protein, and if there exist any association with the underlying inflammatory process that occurs during CVD. Method Forty-four patients diagnosed with clinical atherosclerotic vascular disease (case group) and 15 subjects without CVD background (control group) were included in the study. Whole blood and serum samples were obtained from all participants. We performed gene expression analysis for KL expression in PBCCs by qPCR, as well as for DNMT1 and DNMT3A (members of the DNA-methyltransferases family), TNF, IL10 and NFKB1 (inflammatory parameters). We assessed the degree of methylation of the KL gene promoter in these cells by bisulfite sequencing. Furthermore, we determined circulating levels of sKL, TNFα and IL10 by ELISA immunoassay in serum samples of both groups. Results Results showed a lower expression of KL gene in PBCCs of patients with CVD compared to controls (45% reduction, P&amp;lt;0.001), which was accompanied by a higher degree of methylation of its promoter (36.7±6.3% vs. 15.9±4.8%, P&amp;lt;0.05). The case group also presented significantly higher levels of DNMT1 and DNMT3A transcripts in these cells (P&amp;lt;0.0001 for both), as well as higher values of proinflammatory parameters (gene expression of TNF and NFKB1, and TNF/IL10 ratio; P&amp;lt;0.001 for all of them). Serum levels of sKL were decreased in the CVD group compared to controls (a reduction of 57.5%, P&amp;lt;0.0001). KL expression in PBCCs correlated directly with systemic levels of sKL (r=0.20, P&amp;lt;0.05), but inversely with expression of DNMT1 (r=-0.33, P&amp;lt;0.01) and inflammatory parameters (r=-0.37, P&amp;lt;0.01 for NFKB1 expression; r=-0.38, P&amp;lt;0.01 for TNF/IL10 expression ratio; and r=-0.39, P&amp;lt;0.01 for serum TNFα/IL10 ratio). No significant associations were found between inflammatory markers and methylation degree of KL gene promoter. Conclusion These results indicate an association between epigenetic regulation of KL gene in PBCC and systemic levels of the soluble protein in CVD, which could be influenced by the proinflammatory state present in these disorders.

MO031KLOTHO GENE PROMOTER METHYLATION IN THE VASCULATURE IS RELATED TO INFLAMMATION IN HUMAN ATHEROSCLEROSIS

Abstract Background and Aims Atherosclerosis, a vascular pathological process with an important inflammatory component, underlies most of the cardiovascular diseases (CVD). Deficiencies in the antiaging factor α-Klotho (KL) have been related to the appearance and progression of atherosclerotic damage associated with CVD. In addition, some studies have demonstrated that KL gene is expressed in human arteries and that low vascular KL expression levels are related with clinical atherosclerotic disease. Among the epigenetic mechanisms that regulate the expression of the KL gene, methylation of the CpG islands in the promoter region is one of the most studied. In this pilot study, we propose that this mechanism participates in the regulation of vascular KL expression and contributes to the deficiency in vascular KL levels observed in CVD. Moreover, we determined the influence of different inflammatory components in the methylation status of KL promoter. Method We developed a case-control study that included 25 patients with diagnosis of clinical atherosclerotic vascular disease undergoing elective vascular surgery and 15 cadaveric organ donors with no medical history of CVD, respectively. Vascular fragments were retrieved from all subjects, and the degree of methylation of the KL gene promoter was assessed by bisulfite sequencing. Additionally, vascular gene expression in both groups was assessed by qPCR for KL, two DNA methyltransferases (DNMT1 and DNMT3A), and three inflammatory-related loci (TNF, IL10 and NFKB1). Serum concentrations of TNFα and IL10 were measured by ELISA immunoassay, and complementary inflammatory parameters (CRP and neutrophil/lymphocyte ratio) by standardized routine methods. Results The vascular tissue of patients in the case group presented a higher percent level of methylated positions in the KL promoter region (71.4±8.3% vs. 39.4±12.3%, P&amp;lt;0.05), as well as a significant decrease in the expression of KL gene (a 40% reduction as compared to controls, P&amp;lt;0.05). Considering the whole study population, the methylation degree was inversely related to the endogenous transcript levels of KL gene (r=-0.66, P&amp;lt;0.0001). Moreover, the expression of DNMT1 was significantly increased in the case group (P&amp;lt;0.05), as well as those of the proinflammatory genes TNF and NFKB1 (P&amp;lt;0.0001 and P&amp;lt;0.05, respectively), being all of them directly associated with the vascular methylation levels of KL gene (r=0.51, P&amp;lt;0.0001 for DNMT1; r=0.45, P&amp;lt;0.001 for TNF; r= 0.37, P&amp;lt;0.05 for NFKB1). Furthermore, vascular expression of DNMT1 and DNMT3A directly correlated with both inflammatory markers in this tissue (P&amp;lt;0.0001, for all). Systemic levels of IL10 presented a direct association with vascular KL gene expression (r=0.28, P&amp;lt;0.05), while inverse associations were observed with the methylation degree of KL gene (r=-0.38, P&amp;lt;0.05) and DNMT1 expression (r=-0,29, P&amp;lt;0.05). Conclusion These results suggest that the methylation of KL gene promoter in the vasculature is a mechanism associated with the inflammatory response that would partly explain its reduction during atherosclerotic injury.

MO440KLOTHO AS A BIOMARKER OF SUBCLINICAL CARDIOVASCULAR DISEASE IN CHRONIC KIDNEY DISEASE: A PROOF-OF-CONCEPT STUDY*

Abstract Background and Aims Cardiovascular disease (CVD) is the major cause of mortality among chronic kidney disease (CKD) patients. Reductions in serum Klotho levels are related to the prevalence of CVD in CKD patients. However, it is unclear whether circulating Klotho, and its expression in peripheral blood cells (PBCs), are associated with subclinical atherosclerotic cardiovascular disease (sCVD) in these subjects. In this proof-of-concept study, we analyzed in a group of CKD patients the relationship between Klotho and two markers of sCVD: ankle-brachial index (ABI) and carotid intima-media thickness (CIMT). Method Gene expression in PBCs and serum levels of Klotho and inflammatory cytokines (TNF, IL6 and IL10) were measured in 103 CKD patients (stages 3-4), older than 18 years of age, and without known atherosclerotic cardiovascular disease. Biochemical data were obtained following standardized clinical methods. The presence of sCVD was defined as ABI &amp;lt; 0.9 and/or CIMT ≥ 0.9 mm. Patients with ABI values ≥ 1.3 were excluded. Results Patients with sCVD presented lower serum and PBCs expression levels of Klotho (P&amp;lt;0.001 for both). Stratified analysis showed that upper tertiles of both serum and PBCs expression levels of Klotho presented significantly higher ABI (P&amp;lt;0.001 for both Klotho determinations) and lower CIMT (P&amp;lt;0.001 for serum levels and P&amp;lt;0.01 for KL expression in PBCs), which resulted in a lower prevalence of sCVD (P&amp;lt;0.001 for both determinations of Klotho). Correlation analysis showed that both serum and PBCs mRNA Klotho levels were positively correlated with ABI (r=0.556, P&amp;lt;0.0001; and r=0.373, P&amp;lt;0.0001, respectively) and inversely correlated with CIMT (r=-0.541, P&amp;lt;0.0001; and r=-0.437, P&amp;lt;0.0001, respectively). Among inflammatory markers, only serum IL6 levels presented significant associations with sCVD, being inversely related with ABI (r=-0.568, P&amp;lt;0.0001) and positively associated with CIMT (r=0.558, P&amp;lt;0.0001). Multiple regression analysis with ABI and CIMT as dependent variables demonstrated that both Klotho variables, together with serum IL6, were positively and significantly associated with ABI (adjusted R2=0.511, P&amp;lt;0,0001) and CIMT (adjusted R2=0.445, P&amp;lt;0,0001) values, independently of traditional and emergent cardiovascular risk factors. Multivariate logistic regression, using the presence/absence of sCVD as the dependent variable, showed that circulating Klotho, and its expression in PBCs constituted independent protective factors for sCVD [OR (95% CI): 0.993 (P=0.002) and 0.231 (P=0.025), respectively]. Receiver operating curve (ROC) analysis pointed to the prognostic ability for sCVD of serum Klotho (area under the curve [AUC]: 0.817, 95% CI: 0.736–0.898, P&amp;lt;0.001) and its gene expression in PBCs (AUC: 0.742, 95% CI: 0.647–0.836, P&amp;lt;0.001). Conclusion The reductions in serum soluble and PBCs expression levels of Klotho in CKD patients are independently associated with the presence of sCVD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could reduce the impact of CVD in CKD patients is warranted.

Equivalent SDOF Load Transformation Factors for One-Way Members Subjected to Air Blast Loading

The equivalent single-degree-of-freedom (SDOF) load transformation factor expressions of the simply supported, fixed end and simply-fixed one-way members subjected to air blast loading are derived by finite series-expansion method or finite point-fitting method. Moreover, figures are used to show the relative relationship between the load transformation factors KL and the ratio of stand-off distance to structural span (γ). The typical calculation conditions show that the load transformation factors are sensitive to the piecewise function of peak overpressure when γ is less than 0.5. The load transformation factor will be a smooth curve for peak overpressure adopting a single smooth continuous function. For the blast-resistant structural design, if the error of 3% in the elastic stage and 5% in the plastic stage can be allowed, γ = 1.0 is the demarcation point between near field blast and far field blast. In addition, if the error of 0.5% can be allowed, γ ≥ 2.0 can be treated as the far field blast. The calculation results of far field blast show that the values of KL of simply supported, simply-fixed, and fixed-end members are 0.64, 0.58, and 0.54, respectively, which means that the load transformation factor is insensitive to the selection of shape function in vibration mode function or static deflection curve function. The calculation accuracy of KL expression for simply supported beam structure in this paper has been verified through near field blast experiments. This paper provides a clear numerical definition for the distinction between near field and far field explosions. It provides qualification criteria for analyzing explosion categories in blast-resistant structural design and a reference for the selection of theoretical calculation formulas.

P1008THERAPY WITH SODIUM-GLUCOSE COTRANSPORTER TYPE 2 INHIBITORS INCREASES KLOTHO IN TYPE 2 DIABETIC PATIENTS

Abstract Background and Aims Klotho (KL) is a transmembrane protein that is expressed the highest in the tubular cells of the kidneys. In addition to membrane KL, a secreted form of this protein that is present in blood and urine is generated by ectodomain shedding of membrane KL from the cell surface or from the KL gene through alternative splicing. Preserved KL expression has been related to physiological protection in the human kidneys. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a new class of antidiabetic drugs with important renal benefits. The aim of the present proof-of-concept study was to analyze if treatment with SGLT2i is associated with changes in soluble KL concentrations as well as with modifications in mRNA expression levels of the KL gene in renal tubular cells. Method Thirty-four patients (18 males and 16 males; mean age 61±5 years) with type 2 diabetes (mean diabetes time higher than 10 years) and CKD G2-A2, all treated with metformin and blockers of the renin-angiotensin system during more than one year, were included in the study. Twenty-four of them received SGLT2i during 6 months (8 empagliflozin, 8 canagliflozin, 8 dapagliflozin), and their data and evolution were compared with a group of 10 patients matched by age, sex and stage of CKD who received sulfonylureas or DPP4 inhibitors. Serum and urine levels of soluble KL and tumor necrosis factor-alpha (TNFa) were determined by ELISA. In addition, the effect of SGLT2i on mRNA expression levels of KL was assessed in vitro in renal tubular cells cultures. Results Baseline median values of serum and urinary KL and TNFa were similar in both groups. Urinary KL was inversely correlated with albuminuria and TNFa excretion (r = -0.39 and r = -0.37, respectively, P&amp;lt;0.05). There were no relationship between the serum and urinary levels of these molecules, suggesting their renal origin. At the end of the study, there was a similar improvement in metabolic control, with a reduction in blood pressure significantly higher in patients treated with SGLT2i. Estimated glomerular filtration rate and albuminuria decreased by 5.8% and 17%, respectively, in subjects receiving SGLT2i (P&amp;lt;0.0001), without changes in the control group. In patients under SGLT2i therapy there was a 21% significant reduction in urinary TNFa with a concomitant 41% increase in urinary KL (P&amp;lt;0.001); there was also a small but significant rise in serum KL (P&amp;lt;0.01 vs baseline). Partial correlation analysis showed that the changes in the urinary excretion of albumin and TNFa were associated with the variation in urinary KL after controlling for other variables (r = -0.50, P&amp;lt;0.01, and r = -0.41, P&amp;lt;0.05, respectively). Finally, renal tubular cells cultured with dapagliflozin showed a significant and dose-dependent increase of mRNA expression levels of KL. Conclusion Treatment with SGLT2i induces a reduction in albuminuria and modulates inflammation (as reflected by a decrease in the urinary excretion of TNFa), which is associated with a significant increase in soluble KL concentrations as well as mRNA expression levels of the KL gene. The preservation of KL by SGLT2i may be an important mechanism of renal protection in type 2 diabetes.

Klotho Expression Predicts Poor Prognosis in Myelodysplastic Syndrome

Introduction: An important explanation for the occurrence of Myelodysplastic syndrome (MDS) in the elderly is related to hematopoietic stem-cell (HSC) senescence. Recent studies have identified aging-related alterations in mesenchymal stromal cells in MDS. During the aging process, the different HSC subtypes lose the ability to differentiate into lymphoid and myeloid precursor cells, causing a breakdown in the immune system balance. This process exposes HSC to important genotoxic factors and may induce genetic or epigenetic alterations because exposure to these factors increases the risk of hematological malignancies. A new biomarker for cancer is Klotho (α and β subtypes), which has been identified as an anti-aging gene that extended the lifespan in murine models when it was overexpressed. Several studies have demonstrated that αKlotho (KL) deficiency causes development of aging features, including increased proliferation, reduction in tumor cell apoptosis, and disruption of HSC development. Some studies have established an association between low expression of KL and risks of cancer (i.e. lymphoma) by inducing cell apoptosis, inhibiting tumor growth, and causing a decrease in insulin-like growth factor 1 receptor (IGF-1R) activation. In parallel to these studies, a recent study reported that KL downregulation was associated with the occurrence of anemia, the main clinical feature of MDS, in cancer patients. However, the expression profile of KL and βKlotho (KLB) genes in the bone marrow (BM) of healthy patients with different ages and the way in which the genes modulate the pathogenesis of MDS in the elderly are unknown. Methods: Total BM cell samples from 62 de novo MDS patients with a mean age of 66 ± 16 years old (10 MDS with single lineage dysplasia, 14 MDS with ring sideroblasts, 27 MDS with multilineage dysplasia, and 11 MDS with excess blasts based on World Health Organization's (WHO) 2016 classification) and from eleven healthy volunteer individuals were collected with informed consents. Healthy patients were stratified into young (n=6; mean 25 ± 6 years old) and elderly (n=5; mean 64 ± 9 years old) individuals. Pre-developed TaqMan gene expression assays for KL (Hs00934627_m1) and KLB (Hs00545621_m1) were used to quantify mRNA expression. Each sample was performed in duplicate, and the expression ratios were calculated using the 2−ΔCq method. Results: Our results demonstrate that the KL gene was expressed in the BM samples from all experimental groups. High KL expression was identified in MDS patients when compared to young (p &lt;0.001) and elderly (p &lt;0.001) controls (Figure 1A). In MDS, KL was downregulated in male patients (p=0.042) (Figure 1B), neutropenic MDS cases (p=0.003) (Figure 1C), MDS patients with transfusion dependence (p=0.001) (Figure 1D), and MDS with an excess of blasts (p=0.038) (Figure 1D), representing more aggressive diseases. A multiple regression was performed in order to predict KL expression from gender, absolute neutrophil count (ANC), transfusion dependence, and WHO 2016 classification. These variables significantly predicted KL expression (F [4, 27] = 3.396; p=0.023; r2=0.335). Transfusion dependence (p=0.027) and WHO 2016 classification (p=0.049) variables significantly added to the prediction. The KLB gene was only expressed in a total of 8/62 MDS patients. The feature that stood out in these patients was the presence of dyserythropoiesis (62.5%). No control sample expressed the KLB gene. Conclusions: Our results demonstrated an important expression profile of KL and KLB in BM of healthy young and old individuals and/or in MDS patients for the first time in contrast to a previous study in which it was demonstrated that human BM induces a negligible expression of these genes. Based on absence of differences between controls, our data showed that KL expression not was associated with the MDS pathogenesis-related senescence process, but its lower expression contributed to the poor prognostic MDS features, probably due to its high potential for affecting HSC number, function, and erythropoiesis. Additionally, low KLB expression in MDS patients with dyserythropoiesis suggests a compensatory response of the bone marrow environment to hematopoietic development block via clonal expansion, especially in the erythroid lineage. These approaches may define KL and KLB as new pathological markers of poor prognosis in MDS and improve future potential therapies. Disclosures No relevant conflicts of interest to declare.

Modification to L-H Kinetics Model and Its Application in the Investigation on Photodegradation of Gaseous Benzene by Nitrogen-Doped TiO2

In this paper, the Langmuir-Hinshelwood (L-H) model has been used to investigate the kinetics of photodegradation of gaseous benzene by nitrogen-doped TiO2 (N-TiO2) at 25 °C under visible light irradiation. Experimental results show that the photoreaction coefficient kpm increased from 3.992 × 10−6 mol·kg−1·s−1 to 11.55 × 10−6 mol·kg−1·s−1 along with increasing illumination intensity. However, the adsorption equilibrium constant KL decreased from 1139 to 597 m3·mol−1 when the illumination intensity increased from 36.7 × 104 lx to 75.1 × 104 lx, whereas it was 2761 m3·mol−1 in the absence of light. This is contrary to the fact that KL should be a constant if the temperature was fixed. This phenomenon can be attributed to the breaking of the adsorption-desorption equilibrium by photocatalytically decomposition. To compensate for the disequilibrium of the adsorption-desorption process, photoreaction coefficient kpm was introduced to the expression of KL and the compensation form was denoted as Km. KL is an indicator of the adsorption capacity of TiO2 while Km is only an indicator of the coverage ratio of TiO2 surface. The modified L-H model has been experimentally verified so it is expected to be used to predict the kinetics of the photocatalytic degradation of gaseous benzene.

Klotho negatively regulated aerobic glycolysis in colorectal cancer via ERK/HIF1\u03b1 axis

BackgroundKlotho (KL) was originally characterized as an aging suppressor gene, and has been identified as a tumor suppressor gene in a variety of cancers, including colorectal cancer. Recent years have witnessed the importance of metabolism transformation in cancer cell malignancies maintenance. Aberrant cancer cell metabolism is considered to be the hallmark of cancer. Our previous studies demonstrated that KL played negative roles in colon cancer cell proliferation and metastasis. However, its role in the cancer cell reprogramming has seldom been reported. The aim of this study was to examine the role of KL in aerobic glycolysis in colorectal cancer.MethodsCombining maximum standardized uptake value (SUVmax), which was obtained preoperatively via a PET/CT scan, with immunohistochemistry staining, we analyzed the correlation between SUVmax and KL expression in colorectal cancer tissues. The impact of KL on glucose metabolism and its mechanisms were further validated in vitro and in vivo.ResultsPatients with lower KL expression exhibited higher 18F-FDG uptake (P < 0.05), indicating that KL might participate in aerobic glycolysis regulation. In vitro assay by using colon cancer cell lines further supported this observation. By overexpressing KL in HTC116 and SW480 cells, we observed that the glycolysis was inhibited and the mitochondrial respiration increased, indicating that KL was a negative regulator of aerobic glycolysis. To seek for the underlying mechanisms, we tried to dig out the relation between KL and HIF1α signaling pathway, and found that KL negatively regulated HIF1α protein level and transcriptional activity. Western blot analysis showed that KL overexpression negatively regulated ERK pathway, and KL regulated aerobic glycolysis in part through its regulation of ERK/ HIF1α axis.ConclusionsTaken together, KL is a negative regulator of aerobic glycolysis and KL inhibited glucose metabolism transformation via the ERK/ HIF1α axis.

Kit Ligand Expression in granulosa cells during follicular culture and associated maturation rate of oocytes in buffalo (Bubalus bubalis)

The objectives of the study to associate the expression of Kit Ligand gene under different gonadotropins and growth factors supplementation with the maturation rate of oocytes in buffalo ovarian follicular cells during whole follicular culture at subsequent intervals of 24 hours. Intact ovarian follicles were dissected out of buffalo ovaries and cultured in Tissue Culture Media-199 supplemented with gonadotropins and growth factors for 24 hours. A semi-quantitative RT-PCR amplification was obtained in granulose cells of buffalo ovarian follicles and its expression was found to be restricted during initial stage of follicle culture. KL expression persistency in granulosa cells beyond 4 hours during follicular culture was found to be negatively correlated with maturation rate of oocytes. It was concluded that expression of Kit Ligand gene in granulose cells could be predictive role for assessing oocytes maturation rate and possibly the oocytes competence in buffalo.

Klotho Regulates Retinal Pigment Epithelial Functions and Protects Against Oxidative Stress

The retinal pigment epithelium (RPE) is a highly specialized CNS tissue that plays crucial roles in retinal homeostasis. Age-related morphological changes in the RPE have been associated with retinal degenerative disorders; our understanding of the underlying molecular mechanisms, however, remains incomplete. Here we report on a key role of Klotho (Kl), an aging-suppressor gene, in retinal health and RPE physiology. Kl(-/-) mice show RPE and photoreceptor degeneration, reduced pigment synthesis in the RPE, and impaired phagocytosis of the outer segment of the photoreceptors. Klotho protein (KL) is expressed in primary cultured human RPE, and regulates pigment synthesis by increasing the expression of MITF (microphthalmia transcription factor) and TYR (tyrosinase), two pivotal genes in melanogenesis. Importantly, KL increases phagocytosis in cultured RPE by inducing gene expression of MERTK/AXL/TYRO3. These effects of KL are mediated through cAMP-PKA-dependent phosphorylation of transcription factor CREB. In cultured human RPE, KL increases the l-3,4-dihydroxyphenylalanine synthesis and inhibits vascular endothelial growth factor (VEGF) secretion from basal membrane by inhibiting IGF-1 signaling and VEGF receptor 2 phosphorylation. KL also regulates the expression of stress-related genes in RPE, lowers the production of reactive oxygen species, and thereby, protects RPE from oxidative stress. Together, our results demonstrate a critical function for KL in mouse retinal health in vivo, and a protective role toward human RPE cells in vitro. We conclude that KL is an important regulator of RPE homeostasis, and propose that an age-dependent decline of KL expression may contribute to RPE degeneration and retinal pathology.

Oocyte-thecal Cell Regulatory Loop in the Control of Preantral Follicle Development

Oocyte-somatic cell interaction plays a crucial role in preantral folliculogenesis. Although topical research has focused on oocyte-granulosa cell interaction over the past decade, an oocyte-thecal cell regulatory loop may also play an important role during the preantral stage. Formation of the thecal cell layer is a key event that occurs during preantral folliculogenesis. Granulosal factors (e.g. IGF-I and KL) appear to stimulate the recruitment of thecal cells from stromal cells. Oocyte-derived GDF9 appears to indirectly modulate thecal cell differentiation, perhaps through regulating the granulosa/IGF-I and KL expression. Theca-produced androgens stimulate granulosa cell proliferation and preantral follicle growth. GDF9 enhances preantral follicle growth by up-regulating thecal androgen production, suggesting that a threshold level of androgens derived from thecal cells is necessary for preantral follicle growth, and that the androgen production may be controlled by the oocyte-derived GDF9. The challenge ahead is not to only understand the precise nature of these interactions, and to elucidate how dysregulation in these interactions may lead to ovarian pathologies such as polycystic ovary syndrome and gonadotropin poor-responsiveness.