Only bioactive forms of PTH (n-oxPTH and Met18(ox)-PTH) inhibit synthesis of sclerostin - evidence from in vitro and human studies.

Sclerostin (SOST) is produced by osteocytes and is known as a negative regulator of bone homeostasis. Parathyroid hormone (PTH) regulates calcium, phosphate as well as vitamin D metabolism, and is a strong inhibitor of SOST synthesis in vitro and in vivo. PTH has two methionine amino acids (positions 8 and 18) which can be oxidized. PTH oxidized at Met18 (Met18(ox)-PTH) continues to be bioactive, whereas PTH oxidized at Met8 (Met8(ox)-PTH) or PTH oxidized at Met8 and Met18 (Met8, Met18(di-ox)-PTH) has minor bioactivity. How non-oxidized PTH (n-oxPTH) and oxidized forms of PTH act on sclerostin synthesis is unknown. The effects of n-oxPTH and oxidized forms of PTH on SOST gene expression were evaluated in UMR106 osteoblast-like cells. Moreover, we analyzed the relationship of SOST with n-oxPTH and all forms of oxPTH in 516 stable kidney transplant recipients using an assay system that can distinguish in clinical samples between n-oxPTH and the sum of all oxidized PTH forms (Met8(ox)-PTH, Met18(ox)-PTH, and Met8, Met18(di-ox)-PTH). We found that both n-oxPTH and Met18(ox)-PTH at doses of 1, 3, 20, and 30 nmol/L significantly inhibit SOST gene expression in vitro, whereas Met8(ox)-PTH and Met8, Met18(di-ox)-PTH only have a weak inhibitory effect on SOST gene expression. In the clinical cohort, multivariate linear regression showed that only n-oxPTH, but not intact PTH (iPTH) nor oxPTH, is independently associated with circulating SOST after adjusting for known confounding factors. In conclusion, only bioactive PTH forms such as n-oxPTH and Met18(ox)-PTH, inhibit SOST synthesis.

Renal and cardiac effects of the PDE9 inhibitor BAY 73-6691 in 5/6 nephrectomized rats.

It has been suggested that the novel selective phosphodiesterase 9 (PDE9) inhibitor may improve cardiac and renal function by blocking 3',5'-cyclic guanosine monophosphate (cGMP) degradation. 5/6 nephrectomized (5/6Nx) rats were used to investigate the effects of the PDE9 inhibitor (BAY 73-6691) on the heart and kidney. Two doses of BAY 73-6691 (1 mg/kg/day and 5 mg/kg/day) were given for 95 days. The 5/6Nx rats developed albuminuria, a decrease in serum creatinine clearance (Ccr), and elevated serum troponin T levels. Echocardiographic data showed that 5/6 nephrectomy resulted in increased fractional shortening (FS), stroke volume (SV), and left ventricular ejection fraction (EF). However, 95 days of PDE9 inhibitor treatment did not improve any cardiac and renal functional parameter. Histopathologically, 5/6 nephrectomy resulted in severe kidney and heart damage, such as renal interstitial fibrosis, glomerulosclerosis, and enlarged cardiomyocytes. Telmisartan attenuated renal interstitial fibrosis and glomerulosclerosis as well as improved cardiomyocyte size. However, except for cardiomyocyte size and renal perivascular fibrosis, BAY 73-6691 had no effect on other cardiac and renal histologic parameters. Pathway enrichment analysis using RNA sequencing data of kidney and heart tissue identified chronic kidney disease pathways, such as phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, complement and coagulation cascades, and nuclear factor kappa B (NF-κB) signaling pathway. PDE9i did not affect any of these disease-related pathways. Two dosages of the PDE9 inhibitor BAY 73-6691 known to be effective in other rat models have only limited cardio-renal protective effects in 5/6 nephrectomized rats.

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

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

Both partial inactivation as well as activation of NF-κB signaling lead to hypertension and chronic kidney disease.

BACKGROUND AND HYPOTHESIS: Activation of NF-κB-signalling is key in the pathogenesis of chronic kidney diseases (CKD). However, a certain level of NF-κB activity is necessary to enable tissue repair. METHODS: To investigate the relationship between activated and inactivated NF-κB signaling on the pathogenesis of CKD using mouse models of NF-κB partial inactivation (mutating cysteine at position 59 of the sixth exon on the NF-κB gene into alanine) and activation (mutating cysteine at position 59 of the sixth exon on the NF-κB gene into serine). RESULTS: The density of CD3, CD8, CD68 positive cells, as well as the expression of IL-6, TRAF-1, and NAF-1 in the kidney tissues of NF-κBC59A mice were reduced, whereas an opposing pattern was observed in the NF-κBC59S mice. Blood pressure, kidney fibrosis (analyzed by PAS-, Masson trichrome-, and Sirius-Red-staining as well as α-SMA immunofluorescence), serum creatinine and urinary albumin-to-creatinine-ratio are markedly increased in NF-κB activated and inactivated mice compared to controls. Transmission electron microscopy indicated that the glomerular basement membrane was thicker in both NF-κBC59A and NF-κBC59S mice compared to wild-type mice. CONCLUSIONS: Using mice models with partially activated and inactivated NF-κB pathways suggests that there is an apparently U-shaped relationship between blood pressure, kidney function as well as morphology and the activation of the NF-κB pathway. A certain optimal activity of the NF-κB pathway seems to be important to maintain optimal kidney function and morphology.

Reno- and cardioprotective molecular mechanisms of SGLT2 inhibitors beyond glycemic control: from bedside to bench.

Large placebo-controlled clinical trials have shown that sodium-glucose cotransporter-2 inhibitors (SGLT2i) delay the deterioration of renal function and reduce cardiovascular events in a glucose-independent manner, thereby ultimately reducing mortality in patients with chronic kidney disease (CKD) and/or heart failure. These existing clinical data stimulated preclinical studies aiming to understand the observed clinical effects. In animal models, it was shown that the beneficial effect of SGLT2i on the tubuloglomerular feedback (TGF) improves glomerular pressure and reduces tubular workload by improving renal hemodynamics, which appears to be dependent on salt intake. High salt intake might blunt the SGLT2i effects on the TGF. Beyond the salt-dependent effects of SGLT2i on renal hemodynamics, SGLT2i inhibited several key aspects of macrophage-mediated renal inflammation and fibrosis, including inhibiting the differentiation of monocytes to macrophages, promoting the polarization of macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, and suppressing the activation of inflammasomes and major proinflammatory factors. As macrophages are also important cells mediating atherosclerosis and myocardial remodeling after injury, the inhibitory effects of SGLT2i on macrophage differentiation and inflammatory responses may also play a role in stabilizing atherosclerotic plaques and ameliorating myocardial inflammation and fibrosis. Recent studies suggest that SGLT2i may also act directly on the Na+/H+ exchanger and Late-INa in cardiomyocytes thus reducing Na+ and Ca2+ overload-mediated myocardial damage. In addition, the renal-cardioprotective mechanisms of SGLT2i include systemic effects on the sympathetic nervous system, blood volume, salt excretion, and energy metabolism.

Hyperfiltration can be detected by transcutaneous assessment of glomerular filtration rate in diabetic obese mice.

We addressed if hyperfiltration can be assessed transcutaneously in male diabetic obese mice (BTBRob/ob) at 12 and 24 wk and how this relates to glomerular parameters indicative for hyperfiltration. Transcutaneous assessment of FITC-Sinistrin clearance [transcutaneous assessment of glomerular filtration rate (tGFR)] was compared against classical plasma clearance. Kidney from SV620C-01-PEI perfused mice were harvested at 24 wk and processed for tissue clearing and classical histology. Perfusion patterns of glomerular capillaries, glomerular size, and vasodilation of the afferent arterioles were assessed. Although at 12 wk FITC-Sinistrin half-life (t1/2) for both tGFR and plasma clearance suggested hyperfiltration, this was not significant anymore at 24 wk. In kidneys of diabetic mice the diameter of the afferent arteriole was significantly larger and positively correlated with glomerular size. Glomerular perfusion pattern in these mice was heterogeneous ranging from non- to well-perfused glomeruli. Nonperfused glomerular areas displayed a strong periodic acid-Schiff's (PAS) positive staining. Collectively our data demonstrate that tGFR is a valid method to detect hyperfiltration. Hyperfiltration occurs early in BTBRob/ob mice and disappears with disease progression as a consequence of a reduced filtration surface. It remains to be assessed if tGFR is also a valid method in diabetic mice with severely compromised renal function.NEW & NOTEWORTHY tGFR measurement is a relatively new method to assess kidney function in conscious rodents, which can be repeated multiple times in the same animal to track the course of the disease and/or the effect of potential treatments. Since the literature was inconclusive on the suitability of this technique in obese mice, we validated it for the first time against classical plasma clearance in the commonly used BTBRob/ob mouse model.

Renoprotective effects of empagliflozin are linked to activation of the tubuloglomerular feedback mechanism and blunting of the complement system.

The mechanisms of nephroprotection in nondiabetic chronic kidney disease (CKD) models by sodium-glucose cotransporter 2 (SGLT2) inhibitors are not well defined. Five groups were established: sham-operated rats, placebo-treated rats with 5/6 nephrectomy (5/6Nx), 5/6Nx + telmisartan (5 mg/kg/day), 5/6Nx + empagliflozin (3 mg/kg/day), and 5/6Nx + empagliflozin (15 mg/kg/day). Treatment duration was 95 days. Empagliflozin showed a dose-dependent beneficial effect on the change from baseline of creatinine clearance (Ccr). The urinary albumin-to-creatinine ratio likewise improved in a dose-dependent manner. Both dosages of empagliflozin improved morphological kidney damage parameters such as renal interstitial fibrosis and glomerulosclerosis. 5/6 nephrectomy led to a substantial reduction of urinary adenosine excretion, a surrogate parameter of the tubuloglomerular feedback (TGF) mechanism. Empagliflozin caused a dose-dependent increase in urinary adenosine excretion. The urinary adenosine excretion was negatively correlated with renal interstitial fibrosis and positively correlated with Ccr. Immunofluorescence analysis revealed that empagliflozin had no effect on CD8+ and CD4+ T cells as well as on CD68+ cells (macrophages). To further explore potential mechanisms, a nonhypothesis-driven approach was used. RNA sequencing followed by quantitative real-time polymerase chain reaction revealed that complement component 1Q subcomponent A chain (C1QA) as well as complement component 1Q subcomponent C chain (C1QC) gene expression were upregulated in the placebo-treated 5/6Nx rats and this upregulation was blunted by treatment with empagliflozin. In conclusion, empagliflozin-mediated nephroprotection in nondiabetic CKD is due to a dose-dependent activation of the TGF as well as empagliflozin-mediated effects on the complement system.

Adenosine signalling in T-cell activation favours development of IL-17 positive cells with suppressive properties.

Evidence suggests that the anti-inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (Treg )/helper (Th17) T-cell balance in favour of Treg . Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T-cells. Proliferation, phenotype and cytokine production of stimulated T-cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan-adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) skews human CD3+ T-cell responses towards non-inflammatory Th17 cells. Addition of NECA during T-cell activation increased the development of IL-17+ cells with a CD4+ RORγt+ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non-inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell-like molecular signature and induced surface expression of the adenosine-producing ectoenzymes CD39 and CD73. Thus, T-cells cultured under Th17-inducing conditions together with NECA were capable of suppressing responder T-cells. Finally, genome-wide gene expression profiling revealed metabolic quiescence previously associated with non-pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non-inflammatory Th17 cells in human T-cell differentiation, potentially through regulation of metabolic pathways.

A paired-kidney allocation study found superior survival with HLA-DR compatible kidney transplants in the Eurotransplant Senior Program.

The Eurotransplant Senior Program (ESP) has expedited the chance for elderly patients with kidney failure to receive a timely transplant. This current study evaluated survival parameters of kidneys donated after brain death with or without matching for HLA-DR antigens. This cohort study evaluated the period within ESP with paired allocation of 675 kidneys from donors 65 years and older to transplant candidates 65 years and older, the first kidney to 341 patients within the Eurotransplant Senior DR-compatible Program and 334 contralateral kidneys without (ESP) HLA-DR antigen matching. We used Kaplan-Meier estimates and competing risk analysis to assess all cause mortality and kidney graft failure, respectively. The log-rank test and Cox proportional hazards regression were used for comparisons. Within ESP, matching for HLA-DR antigens was associated with a significantly lower five-year risk of mortality (hazard ratio 0.71; 95% confidence interval 0.53-0.95) and significantly lower cause-specific hazards for kidney graft failure and return to dialysis at one year (0.55; 0.35-0.87) and five years (0.73; 0.53-0.99) post-transplant. Allocation based on HLA-DR matching resulted in longer cold ischemia (mean difference 1.00 hours; 95% confidence interval: 0.32-1.68) and kidney offers with a significantly shorter median dialysis vintage of 2.4 versus 4.1 yrs. in ESP without matching. Thus, our allocation based on HLA-DR matching improved five-year patient and kidney allograft survival. Hence, our paired allocation study suggests a superior outcome of HLA-DR matching in the context of old-for-old kidney transplantation.

Fibroblast growth factor 23 is associated with the development of gestational diabetes mellitus.

BACKGROUND: Besides its established impact on bone and mineral metabolism, it was suggested that fibroblast growth factor 23 (FGF23) might play an important role in the pathogenesis of type 2 diabetes. The impact of FGF23 on gestational diabetes mellitus (GDM), however, is not well understood. iFGF23 ELISAs measure the intact FGF23 molecule, whereas cFGF23 assays measure intact FGF23 as well as degradation products of FGF23. OBJECTIVES: The aim of this study is to compare the association of maternal and foetal cFGF23 and iFGF23 with GDM in a German birth cohort. METHODS: cFGF23 and iFGF23 were analysed in 826 random mother/child pairs from the Berlin Birth Cohort. RESULTS: Mothers who developed GDM had higher concentrations of iFGF-23 compared to mothers who did not suffer from GDM (19.73 vs. 13.23 pg/mL, p < 0.0001), but not higher concentrations of cFGF-23. Multivariant regression analyses showed that gestational diabetes is associated with iFGF23 independently of confounding factors such as age, BMI, ethnic background, family history of diabetes, smoking during pregnancy, and recurrent pregnancy loss. This, however, was only seen when using an iFGF23 ELISA measuring just the full length FGF23 and not in addition FGF23 fragments. No differences in both iFGF23 and cFGF23 concentrations between the GDM and non-GDM groups were detected in cord blood samples of the offspring. CONCLUSIONS: This study of a representative German birth cohort showed that maternal but not foetal iFGF23 is independently associated with GDM.

High-fat, sucrose and salt-rich diet during rat spermatogenesis lead to the development of chronic kidney disease in the female offspring of the F2 generation.

Effects of feeding male rats during spermatogenesis a high-fat, high-sucrose and high-salt diet (HFSSD) over two generations (F0 and F1) on renal outcomes are unknown. Male F0 and F1 rats were fed either control diet (F0CD+F1CD) or HFSSD (F0HD+F1HD). The outcomes were glomerular filtration rate and urinary albumin excretion in F1 and F2 offspring. If both outcomes were altered a morphological and molecular assessment was done. F2 offspring of both sexes had a decreased GFR. However, increased urinary albumin excretion was only observed in female F2 F0HD+F1HD offspring compared with controls. F0HD+F1HD female F2 offspring developed glomerulosclerosis (+31%; p < .01) and increased renal interstitial fibrosis (+52%; p < .05). RNA sequencing followed by qRT-PCR validation showed that four genes (Enpp6, Tmem144, Cd300lf, and Actr3b) were differentially regulated in the kidneys of female F2 offspring. lncRNA XR-146683.1 expression decreased in female F0HD+F1HD F2 offspring and its expression was (r = 0.44, p = .027) correlated with the expression of Tmem144. Methylation of CpG islands in the promoter region of the Cd300lf gene was increased (p = .001) in female F2 F0HD+F1HD offspring compared to controls. Promoter CpG island methylation rate of Cd300lf was inversely correlated with Cd300lf mRNA expression in F2 female offspring (r = -0.483, p = .012). Cd300lf mRNA expression was inversely correlated with the urinary albumin-to-creatinine ratio in female F2 offspring (r = -0.588, p = .005). Paternal pre-conceptional unhealthy diet given for two generations predispose female F2 offspring to chronic kidney disease due to epigenetic alterations of renal gene expression. Particularly, Cd300lf gene promotor methylation was inversely associated with Cd300lf mRNA expression and Cd300lf mRNA expression itself was inversely associated with urinary albumin excretion in F2 female offspring whose fathers and grandfathers got a pre-conceptional unhealthy diet.

Association of BMAL1 clock gene polymorphisms with fasting glucose in children.

BACKGROUND: The brain and muscle Arnt-like protein-1 (BMAL1) gene is an important circadian clock gene and previous studies have found that certain polymorphisms are associated with type 2 diabetes in adults. However, it remains unknown if such polymorphisms can affect fasting glucose in children and if other factors modify the associations. METHODS: A school-based cross-sectional study with 947 Chinese children was conducted. A multivariable linear regression model was used to analyze the association between BMAL1 gene polymorphisms and fasting glucose level. RESULTS: After adjusting for age, sex, body mass index (BMI), physical activity, and unhealthy diet, GG genotype carriers of BMAL1 rs3789327 had higher fasting glucose than AA/GA genotype carriers (b = 0.101, SE = 0.050, P = 0.045). Adjusting for the same confounders, rs3816358 was shown to be significantly associated with fasting glucose (b = 0.060, SE = 0.028, P = 0.032). Furthermore, a significant interaction between rs3789327 and nutritional status on fasting glucose was identified (Pinteraction = 0.009); rs3789327 was associated with fasting glucose in the overweight/obese subgroup (b = 0.353, SE = 0.126, P = 0.006), but not in non-overweight/non-obese children. CONCLUSIONS: BMAL1 polymorphisms were significantly associated with the fasting glucose level in children. Additionally, the observed interaction between nutritional status and BMAL1 supports promoting an optimal BMI in children genetically predisposed to higher glucose level. IMPACT: Polymorphisms in the essential circadian clock gene BMAL1 were associated with fasting blood glucose levels in children. Additionally, there was a significant interaction between nutritional status and BMAL1 affecting fasting glucose levels. BMAL1 rs3789327 was associated with fasting glucose only in overweight/obese children. This finding could bring novel insights into mechanisms by which nutritional status influences fasting glucose in children.

Blood Pressure and Mortality in the 4D Study.

INTRODUCTION: Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) are risk factors for cardiovascular mortality (CVM). Pulse pressure (PP) is an easily available parameter of vascular stiffness, but its impact on CVM in chronic dialysis patients with diabetes is unclear. METHODS: Therefore, we have examined the predictive value of baseline, predialytic PP, SBP, DBP, and MAP in the German Diabetes and Dialysis (4D) study, a prospective, randomized, double-blind trial enrolling 1,255 patients with type 2 diabetes on hemodialysis in 178 German dialysis centers. RESULTS: Mean age was 66.3 years, mean blood pressure 146/76 mm Hg, mean time suffering from diabetes 18.1 years, and mean time on maintenance dialysis 8.3 months. Considered as continuous variables, PP, MAP, SBP, and DBP could not provide a significant mortality prediction for either cardiovascular or all-cause mortality. After dividing the cohort into corresponding tertiles, we also did not detect any significant mortality prediction for PP, SBP, DBP, or MAP, both for all-cause mortality and CVM after adjusting for age and sex. Nevertheless, when comparing the HR plots of the corresponding blood pressure parameters, a pronounced U-curve was seen for PP for both all-cause mortality and CVM, with the trough range being 70-80 mm Hg. DISCUSSION: In patients with end-stage renal disease and long-lasting diabetes mellitus predialytic blood pressure parameters at study entry are not predictive for mortality, presumably because there is a very high rate of competing mortality risk factors, resulting in overall very high rates of all-cause and CVM that may no longer be significantly modulated by blood pressure control.

Lipopolysaccharide Tolerance in Human Primary Monocytes and Polarized Macrophages.

Innate immune memory allows macrophages to adequately respond to pathogens to which they have been pre-exposed. To what extent different pattern recognition receptors, cytokines and resolution signals influence innate immune memory needs further elucidation. The present study assessed whether lipopolysaccharide (LPS) tolerance in monocytes and macrophages is affected by these factors. Human CD14+ cells were isolated from peripheral blood, stimulated by LPS and re-stimulated after 3 days of resting. Hereafter, immune-responsive gene 1 (IRG-1), heme oxygenase 1 (HO-1), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expression were assessed. Our study revealed the following findings: (1) While pre-stimulation with the Toll-like receptor 4 ligand LPS inhibits the induction of IRG-1, TNF-α and IL-6 expression, pre-stimulation with TLR 1/2 ligands only affects cytokine production but not IRG-1 expression upon subsequent TLR4 engagement. (2) Prior TNF-α stimulation does not affect LPS tolerance but rather increases LPS-mediated cytokine expression. (3) Dimethyl itaconate (DMI) inhibits the expression of IRG-1 in a dose-dependent manner but does not affect TNF-α or IL-6 expression. (4) Docosahexaenoic acid (DHA) partly inhibits IRG-1 expression in monocytes but not in M(IFNγ) and M(IL-4) polarized macrophages. LPS tolerance is not affected in these cells by DHA. The data presented in this study partly corroborate and extend previous findings on innate immune memory and warrant further studies on LPS tolerance to gain a better understanding of innate immune memory at the molecular level.

Integrated proteome and acetylome analyses unveil protein features of gestational diabetes mellitus and preeclampsia.

Gestational diabetes mellitus (GDM) and preeclampsia (PE) are associated with maternal and infant health. Although the pathogenesis of PE and GDM remains controversial, oxidative stress is involved in the underlying pathology of GDM and PE. Protein lysine acetylation (Kac) plays an important regulatory role in biological processes. There is little data regarding the association of the maternal acetylome with GDM and PE. This study aimed to assess the potential value of the proteome and acetylome for GDM and PE. In our study, we included placental tissues from healthy individuals (n = 6), GDM patients (n = 6), and PE patients (n = 6) to perform 4D-label free quantification proteomics analysis and PRM analysis. We identified 22 significantly regulated proteins and 192 significantly regulated acetylated proteins between the GDM and PE groups. Furthermore, 192 significantly regulated acetylated proteins were mainly enriched in endoplasmic reticulum stress (ERS) and ferroptosis pathways. Seventeen acetylated sites in these two pathways were verified by PRM analysis. Our comprehensive analysis revealed key features of GDM/PE-significantly regulated acetylated proteins in the placentas from GDM and PE. The results of signaling pathway analysis focused on ERS and ferroptosis. These findings may help explore the underlying pathology, new biomarkers, and therapeutic targets of GDM and PE.

Paternal eNOS deficiency in mice affects glucose homeostasis and liver glycogen in male offspring without inheritance of eNOS deficiency itself.

AIMS/HYPOTHESIS: It was shown that maternal endothelial nitric oxide synthase (eNOS) deficiency causes fatty liver disease and numerically lower fasting glucose in female wild-type offspring, suggesting that parental genetic variants may influence the offspring's phenotype via epigenetic modifications in the offspring despite the absence of a primary genetic defect. The aim of the current study was to analyse whether paternal eNOS deficiency may cause the same phenotype as seen with maternal eNOS deficiency. METHODS: Heterozygous (+/-) male eNOS (Nos3) knockout mice or wild-type male mice were bred with female wild-type mice. The phenotype of wild-type offspring of heterozygous male eNOS knockout mice was compared with offspring from wild-type parents. RESULTS: Global sperm DNA methylation decreased and sperm microRNA pattern altered substantially. Fasting glucose and liver glycogen storage were increased when analysing wild-type male and female offspring of +/- eNOS fathers. Wild-type male but not female offspring of +/- eNOS fathers had increased fasting insulin and increased insulin after glucose load. Analysing candidate genes for liver fat and carbohydrate metabolism revealed that the expression of genes encoding glucocorticoid receptor (Gr; also known as Nr3c1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a; also known as Ppargc1a) was increased while DNA methylation of Gr exon 1A and Pgc1a promoter was decreased in the liver of male wild-type offspring of +/- eNOS fathers. The endocrine pancreas in wild-type offspring was not affected. CONCLUSIONS/INTERPRETATION: Our study suggests that paternal genetic defects such as eNOS deficiency may alter the epigenome of the sperm without transmission of the paternal genetic defect itself. In later life wild-type male offspring of +/- eNOS fathers developed increased fasting insulin and increased insulin after glucose load. These effects are associated with increased Gr and Pgc1a gene expression due to altered methylation of these genes.

Influence of carnosine and carnosinase-1 on diabetes-induced afferent arteriole vasodilation: implications for glomerular hemodynamics.

Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Two cohorts of mice including appropriate controls were studied: i.e., diabetic mice that received oral carnosine supplementation (cohort 1) and human (h)CN1 transgenic (TG) diabetic mice (cohort 2). The lumen area ratio (LAR) of the afferent arterioles and glomerular parameters were measured by conventional histology. Three-dimensional analysis using a tissue clearing strategy was also used. In both cohorts, LAR was significantly larger in diabetic BTBRob/ob versus nondiabetic BTBRwt/ob mice (0.41 ± 0.05 vs. 0.26 ± 0.07, P < 0.0001 and 0.42 ± 0.06 vs. 0.29 ± 0.04, P < 0.0001) and associated with glomerular size (cohort 1: r = 0.55, P = 0.001 and cohort 2: r = 0.89, P < 0.0001). LAR was partially normalized by oral carnosine supplementation (0.34 ± 0.05 vs. 0.41 ± 0.05, P = 0.004) but did not differ between hCN1 TG and wild-type BTBRob/ob mice. In hCN1 TG mice, serum CN1 concentrations correlated with LAR (r = 0.90, P = 0.006). Diabetic mice displayed decreased nephrin expression and increased glomerular hypertrophy. This was not significantly different in hCN1 TG BTBRob/ob mice (P = 0.06 and P = 0.08, respectively). In conclusion, carnosine and CN1 may affect intraglomerular pressure in an opposing manner through the regulation of afferent arteriolar tone. This study corroborates previous findings on the role of carnosine in the progression of DKD.NEW & NOTEWORTHY Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Our results provide evidence that carnosine feeding and CN1 overexpression likely affect intraglomerular pressure through vasoregulation of the afferent arteriole.

Even high normal blood pressure affects live birth rate in women undergoing fresh embryo transfer.

STUDY QUESTION: Do differences in blood pressure within the normal range have any impacts on the live birth rate (primary outcome) or biochemical pregnancy rate (beta-hCG positivity), clinical pregnancy rate (heart beating in ultrasound), abortion rate and ectopic pregnancy rate (secondary outcomes) of fresh embryo transfer in women undergoing their IVF/ICSI treatment? SUMMARY ANSWER: Even rather small differences in baseline blood pressure in women with normal blood pressure according to current guidelines undergoing fresh embryo transfer after IVF/ICSI affects substantially the live birth rate. WHAT IS KNOWN ALREADY: Pre-pregnancy hypertension is a well-known risk factor for adverse pregnancy events such as preeclampsia, fetal growth restriction, placental abruption and adverse neonatal events. It is likewise well known that hypertension during pregnancy in women undergoing ART is associated with adverse pregnancy outcomes. However, whether blood pressure at the high end of the normal range has an impact on ART is unknown. STUDY DESIGN, SIZE, DURATION: It is a prospective observational cohort study based on a single IVF center between January 2017 and December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two thousand four hundred and eighteen women with normal blood pressure undergoing fresh embryo transfer after IVF/ICSI at the Reproductive and Genetic Hospital of CITIC-Xiangya were enrolled in this study. MAIN RESULTS AND THE ROLE OF CHANCE: Blood pressure was measured at the first visit when women consulted the IVF center due to infertility. In women with a successful pregnancy outcome (1487 live births out of 2418 women undergoing fresh embryo transfer after IVF/ICSI), systolic blood pressure (SBP) (114.1 ± 9.48 mmHg versus 115.4 ± 9.8 mmHg, P = 0.001) and diastolic blood pressure (DBP) (74.5 ± 7.5 mmHg versus 75.3 ± 7.34 mmHg, P = 0.006) were lower than in those who did not achieve live births. Multivariate logistic regression analysis revealed that SBP (OR: 0.987, 95% CI: 0.979-0.996, P = 0.004) and DBP (OR: 0.986, 95% CI: 0.975-0.998, P = 0.016) were negatively associated with live birth. Similarly, SBP was significantly negatively related to clinical pregnancy rate (OR: 0.990, 95% CI: 0.981-0.999, P = 0.033), while for DBP the association was not statistically significant (OR: 0.994, 95% CI: 0.982-1.006, P = 0.343). However, both SBP and DBP were positively associated with miscarriage OR: 1.021 (95% CI: 1.004-1.037, P = 0.013) and OR: 1.027 (95% CI: 1.005-1.049, P = 0.014), respectively. Both SBP and DBP were unrelated to biochemical pregnancy (hCG positivity), implantation and ectopic pregnancy rate. LIMITATIONS, REASONS FOR CAUTION: Whether lowering blood pressure before initiating ART treatment in women with SBP or DBP higher than the thresholds defined in our study will confer a benefit is unknown. Also, we cannot exclude bias due to different ethnicities. Moreover, participants in our study only received fresh embryo transfer, whether the results could apply to frozen embryo transfer is unclear. WIDER IMPLICATIONS OF THE FINDINGS: Our study challenges the current blood pressure goals in women undergoing fresh embryo transfer after IVF/ICSI. Further studies are needed to figure out the mechanism and effective approach to increase IVF/ICSI pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S): Hunan Provincial Grant for Innovative Province Construction (2019SK4012). The authors declare that there were no conflicts of interest in this study. TRIAL REGISTRATION NUMBER: N/A.

Angiopoietin-2 predicts all-cause mortality in male but not female end-stage kidney disease patients on hemodialysis.

BACKGROUND: Angiopoietin-2 (Ang-2) plays a pivotal role in pathological vascular remodeling and angiogenesis. Both vascular mechanisms are active in patients with end-stage renal disease (ESRD) and may contribute to the high mortality in these patients. The aim of this multicenter prospective cohort study was to investigate baseline serum Ang-2 concentrations in ESRD patients on hemodialysis (HD) for their ability to predict all-cause mortality. METHODS: We conducted a prospective cohort study in 340 stable HD patients from different chronic dialysis centers in Berlin, Germany. The primary endpoint was all-cause mortality during a 5-year follow-up period. Blood samples and clinical data were collected at baseline. Serum Ang-2 was measured with a validated enzyme-linked immunosorbent assay (Biomedica, Vienna, Austria). RESULTS: A total of 313 HD patients (206 men and 107 women) were finally included in the study. Receiver operating characteristic (ROC) analysis of Ang-2 concentrations yielded an area under the curve (AUC) of 0.65 (P < 0.0001) for predicting all-cause mortality in the entire study population and was used to determine the optimal cut-off (111.0 pmol/L) for all-cause mortality. Kaplan-Meier survival analysis indicated that male but not female end-stage kidney disease patients on HD with higher Ang-2 concentrations had a significantly lower survival (log-rank test, P < 0.0001 and P = 0.380 for male and female patients, respectively). Multivariable Cox regression analyses adjusted for age, comorbidity, smoking, dialysis vintage, serum creatinine, hemoglobin, C-reactive protein, serum albumin, intact parathyroid hormone (iPTH), low-density lipoprotein (LDL) and Kt/V likewise indicated that elevated Ang-2 concentrations are associated with all-cause mortality in male {hazard ratio [HR] 3.294 [95% confidence interval (CI) 1.768-6.138]; P = 0.0002} but not in female end-stage kidney disease patients on HD [HR 1.084 (95% CI 0.476-2.467); P = 0.847]. CONCLUSION: Ang-2 at baseline is independently associated with all-cause mortality in male ESRD patients on HD.

Heterologous immunization with BNT162b2 followed by mRNA-1273 in dialysis patients: seroconversion and presence of neutralizing antibodies.

INTRODUCTION: The vital renal replacement therapy makes it impossible for dialysis patients to distance themselves socially. This results in a high risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and developing coronavuris disease 2019, with excess mortality due to disease burden and immunosuppression. We determined the efficacy of a 100-µg booster of mRNA-1273 (Moderna, Cambridge, MA, USA) 6 months after two doses of BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, USA) in 194 SARS-CoV-2-naïve dialysis patients. METHODS: Anti-SARS-CoV-2 spike antibodies were measured with the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, Mannheim, Germany) 4 and 10-12 weeks after two doses of BNT162b2 as well as 4 weeks after the mRNA-1273 booster. The presence of neutralizing antibodies was measured by the SARS-CoV-2 Surrogate Virus Neutralization Test (GenScript Biotech, Piscataway, NJ, USA). Two different cut-offs for positivity were used, one according to the manufacturer's specifications and one correlating with positivity in a plaque reduction neutralization test (PRNT). Receiver operating characteristics analyses were performed to match the anti-SARS-CoV-2 spike antibody cut-offs with the cut-offs in the surrogate neutralization assay accordingly. RESULTS: Any level of immunoreactivity determined by the anti-SARS-CoV-2 spike antibody assay was found in 87.3% (n = 144/165) and 90.6% (n = 164/181) of patients 4 and 10-12 weeks, respectively, after two doses of BNT162b2. This was reduced to 68.5% or 60.6% 4 weeks and 51.7% or 35.4% 10-12 weeks, respectively, when using the ROC cut-offs for neutralizing antibodies in the surrogate neutralization test (manufacturer's cut-off ≥103 U/mL and cut-off correlating with PRNT ≥196 U/mL). Four weeks after the mRNA-1273 booster, the concentration of anti-SARS-CoV-2 spike antibodies increased to 23 119.9 U/mL and to 97.3% for both cut-offs of neutralizing antibodies. CONCLUSION: Two doses of BNT162b2 followed by one dose of mRNA-1273 within 6 months in patients receiving maintenance dialysis resulted in significant titres of SARS-CoV-2 spike antibodies. While two doses of mRNA vaccine achieved adequate humoral immunity in a minority, the third vaccination boosts the development of virus-neutralizing quantities of SARS-CoV-2 spike antibodies (against wild-type SARS-CoV-2) in almost all patients.