KL Gene Research Articles

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.

An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus.

Several novel non-antibiotic therapeutics for the critical priority bacterial pathogen, Acinetobacter baumannii, rely on specificity to the cell-surface capsular polysaccharide (CPS). Hence, prediction of CPS type deduced from genes in whole genome sequence data underpins the development and application of these therapies. In this study, we provide a comprehensive update to the A. baumannii K locus reference sequence database for CPS typing (available in Kaptive v. 2.0.1) to include 145 new KL, providing a total of 237 KL reference sequences. The database was also reconfigured for compatibility with the updated Kaptive v. 2.0.0 code that enables prediction of 'K type' from special logic parameters defined by detected combinations of KL and additional genes outside the K locus. Validation of the database against 8994 publicly available A. baumannii genome assemblies from NCBI databases identified the specific KL in 73.45 % of genomes with perfect, very high or high confidence. Poor sequence quality or the presence of insertion sequences were the main reasons for lower confidence levels. Overall, 17 KL were overrepresented in available genomes, with KL2 the most common followed by the related KL3 and KL22. Substantial variation in gene content of the central portion of the K locus, that usually includes genes specific to the CPS type, included 34 distinct groups of genes for synthesis of various complex sugars and >400 genes for forming linkages between sugars or adding non-sugar substituents. A repertoire of 681 gene types were found across the 237 KL, with 88.4 % found in <5 % of KL.

KL-FGF23-VD Axis in Improving Late-Onset Alzheimer's Disease by Modulating IKK/NF-κB Signal Pathway.

Materials and Methods LOAD rats and Aβ microglia were constructed by using Aβ1-40 and IBO mixture. The effect of KL-FGF23-VD axis on LOAD was investigated by transfecting overexpressing and interfering with KL gene adenovirus, and IKK-16 was added to Aβ microglia to explore the effect of KL-FGF23-VD axis on regulation of IKK/NF-κB signaling pathway. Results The results showed that, in KL-OE group, FGF23 was decreased in the hippocampus of LOAD rats compared with control and KL-si, and the trend was opposite in the KL-si group. The KL-FGF23-VD axis can alleviate inflammatory response, reduce the deposition of Aβ, and inhibit activation of the NF-κB pathway and neuron apoptosis in brain tissue of LOAD rats. In Aβ microglia, the expression of KL-FGF23-VD axis was consistent with animal experiments. The KL-FGF23-VD axis can inhibit the expression of Aβ microglia inflammatory factors and the activation of microglia and NF-κB pathway. Meanwhile, IKK expression was decreased in KL-OE group compared with KL-si and Control. In the IKK-16 addition group, the ability of KL-FGF23-VD axis to inhibit the activation of microglia and NF-κB pathway was enhanced. Conclusions These findings suggest a potential role of the KL-FGF23-VD axis in AD treatment by regulating the IKK/NF-κB pathway.

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.

MO640: Treatment with Sodium–Glucose Cotransporter 2 Inhibitors Increases Klotho in Type 2 Diabetic Patients

Abstract BACKGROUND AND AIMS Klotho (KL) is a transmembrane protein mainly expressed by the tubular cells of the kidneys, with an additional soluble form that is present in blood and urine. Preserved KL has been related to physiological protection of the kidneys. Sodium–glucose cotransporter type-2 inhibitors (SGLT2i) are a new class of anti-diabetic drugs with important renal benefits. The aim of the present study was to analyze the effect of treatment with SGLT2i on soluble KL concentrations as well as on mRNA expression levels of the KL gene in renal tubular cells. METHOD Fifty-seven patients (30 males and 27 females; mean age 61 ± 5 years) with type 2 diabetes and chronic kidney disease stage G2-A2, all treated with metformin and blockers of the renin–angiotensin system, were included in the study. All patients were in need of intensified anti-diabetic therapy at the discretion of their physicians in accordance with the routine clinical practice. Forty-five received SGLT2i during 3 months (15 empagliflozin, 15 canagliflozin and 15 dapagliflozin), and the evolution was compared with a group of 12 patients matched by age, sex and stage of CKD who received DPP4 inhibitors (control group). Serum and urine levels of soluble KL and tumor necrosis factor-alpha (TNFa) were determined by ELISA. In addition, the effect on mRNA expression levels of KL was assessed in vitro in renal tubular cells cultures. RESULTS Baseline values of serum and urinary KL and TNFa were similar in both groups. Urinary KL was inversely correlated with albuminuria (r = −0.45, P &amp;lt; 0.001) and urine TNFa excretion (r = −0.40, P &amp;lt; 0.01). At the end of the study, there was a similar improvement in metabolic control in both groups; however, only patients treated with SGLT2i showed a significant reduction in albuminuria and urinary TNFa (P &amp;lt; 0.01). Serum and urinary KL increased by 5.2% and 38.9%, respectively, only in patients receiving SGLT2i (P &amp;lt; 0.001 versus baseline), without changes in controls (P &amp;lt; 0.01 between groups). There were no differences among the three drugs in the SGLT2i group. The reduction in albuminuria (β = −0.35, P &amp;lt; 0.01) and urinary TNFa (β = −0.57, P &amp;lt; 0.001) were independently associated with the increase in urine KL, while the change in albuminuria was related to the increase in serum KL (β = −0.39, P &amp;lt; 0.05). Cultured renal tubular cells under high glucose, inflammatory stimulus with TWEAK or serum bovine albumin decreased Klotho mRNA and protein expression within 24 h, an effect that was prevented by all the SGLT2i when added to the cultures. CONCLUSION In type 2 diabetic patients, 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. In addition, SGLT2i prevent the reduction in protein and mRNA expression levels of the KL gene in renal tubular cells. The preservation of KL by SGLT2i may be an important mechanism of renal protection.

α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt-β-catenin pathway.

Randall's plaques (RP) are well established as precursor lesions of idiopathic calcium oxalate (CaOx) stones, and the process of biomineralization driven by osteogenic-like cells has been highlighted in RP formation, but the mechanism is poorly understood. Given the inhibitory role of α-Klotho (KL), an aging suppressor protein with high expression in kidneys, in ectopic calcification and the close association between KL gene polymorphisms and urolithiasis susceptibility, we determined the potential role of KL in RP formation. This study found that both soluble KL (s-KL) and transmembrane KL (m-KL) were downregulated, and that s-KL but not m-KL was inversely correlated with upregulation of osteogenic markers in RP tissues. Additionally, s-KL expression was markedly suppressed in human renal interstitial fibroblasts (hRIFs) and slightly suppressed in HK-2 cells after osteogenic induction, intriguingly, which was echoed to the greater osteogenic capability of hRIFs than HK-2 cells. Further investigations showed the inhibitory effect of s-KL on hRIF osteogenic differentiation in vitro and in vivo. Moreover, coculture with recombinant human KL (r-KL) or HK-2 cells suppressed osteogenic differentiation of hRIFs, and this effect was abolished by coculture with KL-silenced HK-2 cells or the β-catenin agonist SKL2001. Mechanistically, s-KL inactivated the Wnt-β-catenin pathway by directly binding to Wnt2 and upregulating SFRP1. Further investigations identified activation of the Wnt-β-catenin pathway and downregulation of SFRP1 and DKK1 in RP tissues. In summary, this study identified s-KL deficiency as a pathological feature of RP and revealed that s-KL released from HK-2 cells inhibited osteogenic differentiation of hRIFs by inactivating the Wnt-β-catenin pathway, not only providing in-depth insight into the role of s-KL in renal interstitial biomineralization but also shedding new light on the interaction of renal tubular epithelial cells with interstitial cells to clarify RP formation.

Soluble Klotho-integrin β1/ERK1/2 pathway ameliorates myocardial fibrosis in diabetic cardiomyopathy.

Soluble Klotho (sKL) is closely related to insulin resistance, which is a major factor in the progression of diabetic cardiomyopathy (DCM). The purpose of this study was to investigate the role of sKL in the regulation of DCM and the mechanism involved. A mouse model of type 2 diabetes was induced by high-fat diet and streptozotocin injection. An insulin-resistant cardiac fibroblast model was established by high glucose and high insulin. KL gene overexpression was achieved in vivo and vitro through transfection with an adenovirus-harboring KL-cDNA. Gene overexpression was used to evaluate the role of sKL in the pathophysiologic characteristics of DCM. Insulin-resistant cardiac fibroblasts reduced sKL expression and collagen deposition. Diabetic mice constructed by streptozotocin exhibited severe insulin resistance, inflammation, fibrosis, left ventricular dysfunction, and sKL downregulation. The overexpression of sKL mitigated insulin resistance and metabolic disturbance; inflammation, fibrosis, and upregulated collagen I/III content ratio in diabetic state were significantly reduced. Our findings were accompanied by notable moderation of cardiac function. Further, blunted phosphorylation of Akt was restored with sKL gene overexpression, and activated phosphorylation of extracellular signal-regulated kinase 1/2 in DCM was reduced. Our results suggest that sKL protein overexpression exerts a defensive measure by ameliorating selective insulin resistance in mouse DCM, thus revealing its underlying mechanism for potential human DCM treatment.

Epigenetic regulation of KLOTHO in peripheral blood circulating cells is associated with soluble protein serum levels in cardiovascular disease

Background and Aims: The soluble form of Klotho protein (sKL), which is mainly produced by the kidneys, have demonstrated to display different protective effects on cardiovascular diseases (CVD). It has been demonstrated that peripheral blood circulating cells (PBCCs) also express KL, but little is known about its expression profile during CVD. We 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.

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.

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.

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.

TO007PLASMA EXTRACELLULAR VESICLES MEDIATE ENDOTHELIAL TO MESENCHYMAL TRANSITION AND TUBULAR SENESCENCE IN RENAL ANTIBODY MEDIATED REJECTION BY COMPLEMENT ACTIVATION

Abstract Background and Aims EVs (Extracellular vesicles) are circulating microparticles able to mediate cell-to cell communication by carrying proteins, DNA, RNA or antibodies on their surface. EV are emerging as pivotal in renal Antibody-mediated rejection (AMR), one of the major causes of transplant failure associated to a massive complement activation. AMR is characterized by endothelial to mesenchymal transition (EndMT) and the occurrence of tubular accelerated senescence process, known as graft “Inflammaging”. However, the potential role of EVs in accelerating the renal aging and graft fibrosis after AMR is not well understood. Method Plasma EVs were isolated from 10 Acute AMR (AAMR), 10 Chronic AMR (CAMR) patients, 5 stable graft transplanted patients and 5 healthy volunteers. EVs were isolated by ultracentrifugation at 100.000 g for 1h at 4°C, quantified by Nanoparticle Tracking Analysis (Nanosight, NTA EV/ml) and characterized by FACS (Attune Nxt). RPTEC and endothelial cell culture were incubated with EVs (5e+4 EVs/cells target for 24h. MTT test was performed to assess cell viability. Cellular senescence was investigated by qPCR for p21, p53, Klotho and CYP1B1 and SA-β-gal staining were performed. To assess EndMT analysis for CD31, VE-cadherin, Vimentin, Collagen I was performed by FACS. mRNA level of C3 and CFH were also measured by qPCR and C4d deposits were evaluated in endothelial cell colture by IF. Renal biopsies were then analyzed for inflammaging (p16INK4a) and EndMT markers (CD31/αSMA) by IHC and IF. Results The Nanosight analysis showed significant differences in the amount of-EVs count per ml of plasma in AMR patients compared to healthy subjects. EVs appeared to be significantly augmented in acute AMR, in a higher manner than chronic AMR (NTA, p=0.0154). By cytofluorimetric analysis, the endothelial markers CD31 and VE- cadherin appeared to be significantly increased compared of healthy control, indicating a predominant endothelial EV origin (p&amp;lt;0.05). Furthermore, an increase for CD3, CD4, CD8, CD40 and CD40L lymphocyte/monocytes markers was found. In vitro, the exposure of RPTEC and endothelial cells to AMR-derived EVs did not induce the loss of cell proliferation. However, AMR-derived EVs induced senescence in RPTEC, as observed by increase in SA-β-gal positive cells, upregulation of p21, p53, CYP1B1 and downregulated KL gene expression (p&amp;lt;0.05). EVs induced the EndMT as observed by FACS with the downregulation of CD31, VE-cadherin (CD144) and increase of Vimentin and Collagen I (p=0.025). To evaluate the contribution of EVs to local complement activation, C3 and CFH qPCR analysis were performed. EVs from AMR patients induced a significant increase in C3 gene expression with concomitant downregulation in CFH in RPTEC. EV exposition induced the classical and lectin pathway activation in endothelial culture medium, and C4d deposition. These data support the hypothesis that circulating EVs can amplify local complement activation in systemic endothelial and tubular cells during AMR, therefore leading to accelerated senescence and fibrosis as later effects. Finally, renal AMR biopsies showed significant tubular senescence as indicated by p16 expression; p16 was significantly upregulated in Chronic compared with Acute AMR biopsies (p&amp;lt;0.05). The AMR biopsies showed positivity for EndMT, as indicated by CD31 decrease and interstitial αSMA upregulation (p&amp;lt;0.05). Conclusion These results suggest a putative role for circulating AMR derived-EVs in inducing the tubular inflammaging by local complement activation and early fibrosis by EndMT. The EVs cargo characterization that is ongoing might highlight novel targets for therapeutic intervention.

MO036SINGLE NUCLEOTIDE POLYMORPHISMS OF THE CYP24A1 AND KL GENES ARE ASSOCIATED WITH THE EXTENT OF ARTERIAL CALCIFICATION IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Individuals with chronic kidney disease (CKD) constitute a population with an exceptionally high cardiovascular risk. Vascular calcification, a common finding in these patients, is a known contributor to cardiovascular disease and several studies suggest it has a certain genetic component. Single nucleotide polymorphisms (SNPs) associated with the extent of calcification in CKD patients could therefore be used to predict individual susceptibility to calcification and subsequent increased cardiovascular risk. We consequently searched for associations of SNPs from candidate genes of known implication in the pathogenesis of vascular calcification (encoding cytokines, extracellular matrix proteins, members of the RANK/RANKL/OPG axis and vitamin D metabolism proteins) with the extent of arterial calcium deposits in order to improve the risk prediction of cardiovascular events in CKD patients. Method The study was performed in 1439 individuals from the NEFRONA population, which include CKD patients (stages 2-3, 4-5, and dialysis) as well as healthy controls recruited from 81 Spanish hospitals. Individuals were genotyped with the iPLEXGOLD MassARRAY technology and Assay Design v4 software for 61 SNPs from 22 genes. A continuous vascular calcification score was calculated from the echogenicity of atherosclerotic plaques detected by ultrasonography in the carotid and femoral arteries. Among several other clinical variables, the presence of cardiovascular events during a 4-year follow up was collected. Association of SNPs with calcification extent was identified by univariate linear regression models. Multiple linear regression with backward elimination was used for the selection of an appropriate SNP-based model adjusted by age, sex and CKD stages. Finally, a Cox proportional hazard regression model was applied for the prediction of cardiovascular risk. Results Arterial calcification scores were higher with increasing age, male sex, and advanced CKD stages (all p&amp;lt;0.001), as expected. Univariate linear regression analyses of all SNPs with the arterial calcification score as dependent variable retrieved p-values &amp;lt;0.05 for 6 six of them (rs11568820, rs2248359, rs2296241, rs3102735, rs385564 and rs495392), which were selected for subsequent analyses. These polymorphisms were next included in a multivariate linear regression model with CKD stage, age and sex as additional independent variables. Only rs2296241 of CYP24A1 (estimate 0.36, 95% CI 0.14 to 0.58, p=0.001 for homocygous GG) and rs495392 of KL (estimate -0.39, 95% CI -0.69 to -0.09, p=0.011 for homocygous TT) remained independently associated with the extent of calcium deposits. Finally, using Cox regression models, it was determined that both the CKD stage (HR [for dialysis stage]=8.34, 95% CI 4.59 to 15.15, p&amp;lt;0.001) and the calcification score (HR=2.05, 95% CI 1.67 to 2.54, p&amp;lt;0.001) predicted the development of cardiovascular events. Considering all possible risk factors, no differences were found in the rate of development of cardiovascular events according to the genotype for the two associated polymorphisms: rs2296241 HR=1.13, 95% CI 0.87 to 1.48, p=0.36; rs495392 HR=0.80 95% CI 0.63 to 1.03, p=0.08. Conclusions Polymorphisms of KL and CYP24A1 genes are associated with the extent of calcification in CKD individuals although they lack the capacity to predict cardiovascular events. However, the echogenic determination of the extent of arterial calcium deposits in CKD patients seems a promising non-invasive, non-irradiating method for the scoring of calcification and even the prediction of cardiovascular events. Further genetic association studies using this technique could therefore yield valuable results.

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

ANALYSIS OF ASSOCIATION OF rs9536314 OF THE KL GENE WITH ANTHROPOMETRIC AND BIOCHEMICAL INDICATORS IN MEN OF NOVOSIBIRSK (WEST SIBERIA)

Purpose of the study. To determine the frequency of the rs9536314 (F325V) polymorphism of the KL gene and the association of this variant with a number of biochemical and anthropometric parameters in men of the study group and in the Caucasian population of Western Siberia. Materials and methods. The study group (69 men, average age 61.2 ±11.5 years) was randomly formed from a sample of persons who applied to the clinic and polyclinic of NIITPM - a branch of the ICG SB RAS and GBUZ NSO Hospital of war veterans No. 3 (178 men, aged 50-65 years old and over 80 years old). The population group was randomly selected (219 people) from the sample surveyed within the framework of the International Multicenter Project Risk Factors for Cardiovascular Diseases in Eastern Europe HAPIEE (9360 participants, 45–69 years old, mean age 53.8±7 years old, Caucasians > 90 %). Biochemical parameters were determined by standard enzymatic methods. Serum concentration of Klotho protein was measured by ELISA. Genomic DNA was amplified by polymerase chain reaction in a standard reaction mixture and further digested with TaqI B restriction enzyme. Results. The frequency of genotypes (TT, TG, and GG) and alleles (T and G) rs9536314 of the KL gene in the study group corresponds to the data in the population of Western Siberia, as well as the population of Western and Eastern Europe. There were no statistically significant differences in the mean values of the studied clinical and biochemical parameters depending on the rs9536314 genotypes of the KL gene in the study group and in the population. In the study group, the level of Klotho protein in the blood and the glomerular filtration rate in men with coronary heart disease and arterial hypertension did not differ in the autosomal dominant and autosomal recessive models for the rs9536314 KL gene. Conclusion . Thus, the frequency of the rs9536314 polymorphism of the KL gene in the study group corresponds to the frequency of the rs9536314 polymorphism of the KL gene in the Caucasian population of Western Siberia. Biochemical and anthropometric parameters, as well as the Klotho protein, do not have statistically significant differences depending on the genotypes of rs9536314 of the KL gene in the examined men.

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.

Klotho Protein Reduced the Expression of Matrix Metalloproteinase-1 (MMP-1) and Matrix Metalloproteinase-3 (MMP-3) in Fibroblasts from Patients with Pelvic Organ Prolapse (POP) by Down-Regulating the Phosphorylation of ERK1/2.

BackgroundPelvic organ prolapse (POP) is due to age-related atrophy and the weakening of the tissues of the pelvic floor, with degradation of collagen and extracellular matrix (ECM) by metalloproteinases (MMPs). This study aimed to investigates the role of the age-related enzyme klotho, encoded by the KL gene, in cultured fibroblasts obtained from patients with POP and the levels of reactive oxygen species (ROS), interleukin-6 (IL-6), and MMPs.Material/MethodsPelvic floor fibroblasts were obtained from connective tissue from three patients with POP and three normal subjects. Cell proliferation and ROS production were measured using a cell counting kit-8 (CCK-8) assay and flow cytometry. Levels of interleukin-6 (IL-6), klotho, metalloproteinase-1 (MMP-1), MMP-3, extracellular signal-regulated kinases 1/2 (ERK1/2), and p-ERK1/2 were measured by enzyme-linked immunoassay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot.ResultsIn cultured pelvic floor fibroblasts from patients with POP, the expression of klotho protein and klotho mRNA were significantly down-regulated in fibroblasts from patients with POP compared with normal fibroblasts. Klotho supplementation in cultured fibroblasts for patients with POP included increased cell growth, reduced expression of ROS reduction, and reduced the secretion of IL-6. Using qRT-PCR and Western blot, klotho supplementation of fibroblasts from patients with POP increased cell growth and reduced the levels of IL-6 and ROS in a dose-dependent way.ConclusionsKlotho protein reduced the expression of MMP-1 and MMP-3 in fibroblasts from patients with POP by down-regulating the phosphorylation of ERK1/2.

The K90 capsular polysaccharide produced by Acinetobacter baumannii LUH5553 contains di-N-acetylpseudaminic acid and is structurally related to the K7 polysaccharide from A. baumannii LUH5533

Acinetobacter baumannii isolate LUH5553 carries the KL90 capsule gene cluster, which includes genes for three glycosyltransferases (Gtrs) and the ItrA3 initiating transferase, as well as a set of genes for synthesis of a higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac). The K90 capsular polysaccharide (CPS) has a tetrasaccharide repeat (K90 unit), which begins with d-GlcpNAc and contains Pse5Ac7Ac. The higher sugar was cleaved by mild acid hydrolysis of the CPS, and structures of the initial and modified polysaccharides were established by 1D and 2D 1H and 13C NMR spectroscopy. K90 contains α-d-Galp-(1 → 6)-d-GlcpNAc and α-d-GlcpNAc-(1 → 3)-d-GlcpNAc fragments, and formation of these glycosidic linkages is catalysed respectively by Gtr14 and Gtr15. The gtr14 and gtr15 genes occur in several A. baumannii KL gene clusters, including KL5 and KL7 that carry itrA2 rather than itrA3. As ItrA2 introduces d-GalpNAc rather than d-GlcpNAc as the first monosaccharide, Gtr15 can transfer d-GlcpNAc to either of these amino sugars, suggesting that this enzyme has relaxed specificity. Consequently, the third, novel glycosyltransferase, Gtr163, forms the β-(2 → 3) linkage between Pse5Ac7Ac and d-Galp. Wzy polymerases encoded by KL90 and KL7 are 54% identical and form the same linkage between the K units to give branched polysaccharides with the same main chain but different disaccharide side chains, β-Pse5Ac7Ac-(2 → 3)-d-Galp in K90 and α-Leg5Ac7Ac-(2 → 6)-d-Galp in K7.

Low bone mineral density and renal malformation in Mexican patients with Turner syndrome are associated with single nucleotide variants in vitamin D-metabolism genes

Turner syndrome (TS) is a common genetic disorder. TS-phenotype includes short stature, gonadal dysgenesis, cardiac and kidney malformations, low bone mineral density (low-BMD) and thyroiditis. TS-phenotype varies from patient to patient and the cause is not clear, the genomic background may be an important contributor for this variability. Our aim was to identify the association of specific single nucleotide variants in the PTPN22, VDR, KL, and CYP27B1 genes and vitamin D-metabolism, heart malformation, renal malformation, thyroiditis, and low-BMD in 61 Mexican TS-patients. DNA samples were genotyped for SNVs: rs7975232 (VDR), rs9536282 (KL), rs4646536 (CYP27B1), and rs1599971 (PTPN22) using the KASP assay. Chi-square test under a recessive model and multifactorial dimensionality reduction method were used for analysis. We found a significant association between renal malformation and the rs9536282 (KL) variant and between rs4646536 (CYP27B1) and low-BMD, these variants may have modest effects on these characteristics but contribute to the variability of the TS phenotype. In addition, we identified gene–gene interactions between variants in genes KL, CYP27B1 and VDR related to vitamin D-metabolism and low-BMD in TS-patients. Our results support the idea that the genetic background of TS-patients contributes to the clinical variability seen in them.

Fibroblast Growth Factor 23 Genotype and Cardiovascular Disease in Patients Undergoing Hemodialysis

Background: Elevated serum concentrations of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality in patients with chronic kidney disease and those undergoing dialysis. Objectives: We tested the hypotheses that polymorphisms in FGF23, its co-receptor alpha-klotho (KL), and/or FGF23 receptors (FGFR) are associated with cardiovascular events and/or mortality. Methods: We used 1,494 DNA samples collected at baseline from the Evaluation of Cinacalcet HCl Therapy to Lower Cardiovascular Events Trial, in which patients were randomized to the calcimimetic cinacalcet or placebo for the treatment of secondary hyperparathyroidism. We analyzed European and African Ancestry samples separately and then combined summary statistics to perform a meta-analysis. We evaluated single-nucleotide polymorphisms (SNPs) in FGF23, KL, and FGFR4 as the key exposures of interest in proportional hazards (Cox) regression models using adjudicated endpoints (all-cause and cardiovascular mortality, sudden cardiac death, and heart failure [HF]) as the outcomes of interest. Results: rs11063112 in FGF23 was associated with cardiovascular mortality (risk allele = A, hazard ratio [HR] 1.32, meta-p value = 0.004) and HF (HR 1.40, meta-p value = 0.007). No statistically significant associations were observed between FGF23 rs13312789 and SNPs in FGFR4 or KL genes and the outcomes of interest. Conclusions: rs11063112 was associated with HF and cardiovascular mortality in patients receiving dialysis with moderate to severe secondary hyperparathyroidism.