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.

Free vitamin D is independently associated with systolic blood pressure in diabetic patients with impaired kidney function.

Vitamin D contributes to blood pressure (BP) regulation. We compared the association of BP in diabetic patients with either total vitamin D - the standard way of analyzing the vitamin D status - or free vitamin D, because only free vitamin D passes the cell membrane and interacts with the nuclear vitamin D receptor (VDR). An analytical cross-sectional study was conducted with 178 diabetic patients with impaired kidney function. Free and total vitamin D concentrations were measured in all patients. Multiple linear regression analysis considering patient age, sex, body mass index, height, smoking and drinking situation, the use of antihypertensive drugs, cholecalciferol treatment, C-reactive protein and estimated glomerular filtration rate as confounding factors were conducted to compare the association of free and total vitamin D with systolic and diastolic blood pressure (SBP and DBP). Multiple linear regression analysis revealed that neither SBP nor DBP was correlated with total vitamin D (SBP, 95% CI -0.405 ~ 0.159, p = 0.390; DBP, 95% CI -0.131 ~ 0.142, p = 0.933) (Table 2). However, the concentration of free vitamin D was independently associated with SBP (95% CI -2.691 ~ -0.210; p = 0.022) (Table 3), but not with DBP (95% CI -0.934 ~ 0.285; p = 0.293). In conclusion, free - but not total - vitamin D serum concentrations in patients with diabetes and impaired kidney function are inversely correlated with SBP. This study suggests that free vitamin D measurements might be more clinically relevant - as compared to measurements of total vitamin D - to adjust vitamin D therapy in diabetic patients with impaired kidney function.

Relationship Between Vitamin D and Hormones Important for Human Fertility in Reproductive-Aged Women.

Vitamin D deficiency is very common in women of reproductive age. Studies in animals suggests a link between vitamin D and reproductive hormone biosynthesis. A systematic analysis of the correlation of reproductive hormones in reproductive-aged women with both total and free vitamin D was, however, not done so far. This cross-sectional study was performed in 351 healthy reproductive age Caucasian women (median age, 28.0 years; interquartile ranges, 24.7-31.0 years). We measured serum levels of both total and free 25(OH)D, endocrinological, hematological and biochemical parameters. Spearman's rank correlations were performed to assess the correlation between 25(OH)D metabolites and selected parameters. Total vitamin D and free vitamin D measurements correlated well (rho=0.912, p < 0.0001). Both total 25(OH)D and free 25(OH)D showed significant negative correlation with FAI (rho=-0.229, p<0.0001 and rho=-0.195, p<0.0001 for total and free 25(OH)D, respectively); LH (rho=-0.177, p=0.001 and rho=-0.114, p=0.04 for total and free 25(OH)D, respectively), testosterone (rho=-0.174, p=0.001 and rho=-0.190, p<0.0001 for total and free 25(OH)D, respectively) and AMH (rho=-0.130, p=0.015 and rho=-0.107, p=0.047 for total and free 25(OH)D, respectively). Our study showed comparable correlations of both total and free 25(OH)D with endocrinological parameters, i.e. inverse correlations with free androgen index, luteinizing hormone, testosterone, LH/FSH ratio, androstenedione and anti-Müllerian hormone, and also with hematological and biochemical parameters, i.e. inverse correlations with erythrocytes, hsCRP and leukocytes count, and positive correlation with transferrin saturation, mean corpuscular hemoglobin and mean corpuscular volume in healthy reproductive age women.

Opposite correlation of 25-hydroxy-vitamin D- and 1,25-dihydroxy-vitamin D-metabolites with gestational age, bone- and lipid-biomarkers in pregnant women.

25-Hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)2D) need to be bound to carrier proteins to be transported to their target cells. The majority of either 25OHD or 1,25(OH)2D is bound to vitamin D-binding protein (DBP), a smaller fraction is bound to albumin and only very small amounts of 25OHD or 1,25(OH)2D are free. Albumin-bound 25OHD or 1,25(OH)2D is relatively easily available after dissociation from albumin. Thus, the sum of free and albumin-bound forms is called bioavailable 25OHD and bioavailable 1,25(OH)2D. Total 25OHD and 1,25(OH)2D are defined as the sum of free, albumin-bound and DBP-bound 25OHD and 1,25(OH)2D, respectively. This cross-sectional study in 427 pregnant women compared the correlation of the six vitamin D compounds with biomarkers of bone health, lipid metabolism, kidney function, endocrine parameters, and group B water-soluble vitamins. Among the 25OHD metabolites analysed, total 1,25(OH)2D showed clearly the best correlation with calcium, bone-specific alkaline phosphatase, adiponectin, LDL, HDL, urea, thyroxine, and group B water-soluble vitamins. When comparing the three 25OHD metabolites, both free 25OHD and bioavailable 25OHD showed overall good correlations with calcium, bone-specific alkaline phosphatase, adiponectin, LDL, HDL, urea, thyroxine, triiodothyronine, and group B water-soluble vitamins, The correlations of 1,25(OH)2D and 25OHD metabolites went always in opposite directions. Only PTH correlates always inversely with all six vitamin D compounds. In conclusion, free 25(OH)D and bioavailable 25(OH)D are more precise determinants of the vitamin D status than total 25(OH)D in normal pregnancy, whereas total 1,25(OH)2D is superior to free and bioavailable 1,25(OH)2D. Except for PTH, correlations of 25(OH)D and 1,25(OH)2D metabolites with typical clinical chemistry readouts go in opposite directions.

Linagliptin and telmisartan induced effects on renal and urinary exosomal miRNA expression in rats with 5/6 nephrectomy.

Dipeptidyl peptidase 4 inhibitors and angiotensin II receptor blockers attenuate chronic kidney disease progression in experimental diabetic and non-diabetic nephropathy in a blood pressure and glucose independent manner, but the exact molecular mechanisms remain unclear. MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of nephropathy. miRNAs are present in urine in a remarkably stable form, packaged in extracellular vesicles. Here, we investigated linagliptin and telmisartan induced effects on renal and urinary exosomal miRNA expression in 5/6 nephrectomized rats. In the present study, renal miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from the following groups of rats: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus telmisartan; and 5/6 nephrectomy plus linagliptin. TaqMan Array miRNA Cards were used to evaluate which of the deregulated miRNAs in the kidney are present in urinary exosomes. In kidneys from 5/6 nephrectomized rats, the expression of 13 miRNAs was significantly increased (>1.5-fold, P < 0.05), whereas the expression of 7 miRNAs was significantly decreased (>1.5-fold, P < 0.05). Most of the deregulated miRNA species are implicated in endothelial-to-mesenchymal transition and inflammatory processes. Both telmisartan and linagliptin suppressed the induction of pro-fibrotic miRNAs, such as miR-199a-3p, and restored levels of anti-fibrotic miR-29c. In conclusion, the linagliptin and telmisartan-induced restorative effects on miR-29c expression were reflected in urinary exosomes, suggesting that miRNA profiling of urinary exosomes might be used as a biomarker for CKD progression and monitoring of treatment effects.

Mechanisms of GLP-1 receptor-independent renoprotective effects of the dipeptidyl peptidase type 4 inhibitor linagliptin in GLP-1 receptor knockout mice with 5/6 nephrectomy.

Dipeptidyl peptidase type 4 (DPP-4) inhibitors were reported to have beneficial effects in experimental models of chronic kidney disease. The underlying mechanisms are not completely understood. However, these effects could be mediated via the glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP1R) pathway. Here we investigated the renal effects of the DPP-4 inhibitor linagliptin in Glp1r-/- knock out and wild-type mice with 5/6 nephrectomy (5/6Nx). Mice were allocated to groups: sham+wild type+placebo; 5/6Nx+ wild type+placebo; 5/6Nx+wild type+linagliptin; sham+knock out+placebo; 5/6Nx+knock out+ placebo; 5/6Nx+knock out+linagliptin. 5/6Nx caused the development of renal interstitial fibrosis, significantly increased plasma cystatin C and creatinine levels and suppressed renal gelatinase/collagenase, matrix metalloproteinase-1 and -13 activities; effects counteracted by linagliptin treatment in wildtype and Glp1r-/- mice. Two hundred ninety-eight proteomics signals were differentially regulated in kidneys among the groups, with 150 signals specific to linagliptin treatment as shown by mass spectrometry. Treatment significantly upregulated three peptides derived from collagen alpha-1(I), thymosin ß4 and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and significantly downregulated one peptide derived from Y box binding protein-1 (YB-1). The proteomics results were further confirmed using western blot and immunofluorescence microscopy. Also, 5/6Nx led to significant up-regulation of renal transforming growth factor-ß1 and pSMAD3 expression in wild type mice and linagliptin significantly counteracted this up-regulation in wild type and Glp1r-/- mice. Thus, the renoprotective effects of linagliptin cannot solely be attributed to the GLP-1/GLP1R pathway, highlighting the importance of other signaling pathways (collagen I homeostasis, HNRNPA1, YB-1, thymosin ß4 and TGF-ß1) influenced by DPP-4 inhibition.

Comparison of free and total 25-hydroxyvitamin D in normal human pregnancy.

Vitamin D status correct monitoring during pregnancy is critically important for both maternal and fetal health. 25-Hydroxyvitamin D (25(OH)D) - a prohormone of a biologically active 1,25-dihydroxyvitamin D (1,25(OH)2D), despite the lack of biological activity, during the past decades has been routinely used as a main biomarker characterizing vitamin D status. About 85% of 25(OH)D in the bloodstream is bound to its specific carrier - vitamin D-binding protein (DBP), the remaining 15% are loosely bound to albumin, and only less than 0.1% are free in the circulation ("free 25(OH)D"). Total 25(OH)D is the sum of DBP-bound, albumin-bound and free 25(OH)D. According to a "free hormone hypothesis", only free 25(OH)D is able to induce a biological effect. Normal pregnancy is characterized by elevated serum DBP levels, and due to this fact the diagnostic strength of serum total 25(OH)D has been questioned. Free 25(OH)D might be a better characteristic of vitamin D status in this settings. We aimed to compare the diagnostic strength of a routine total 25(OH)D with directly measured free 25(OH)D in normal pregnancy by comparing the association strength between free and total 25(OH)D with biomarkers of bone health (PTH, calcium, bone-specific alkaline phosphatase (BSAP)), lipid metabolism (adiponectin, LDL, HDL), kidney function (urea), endocrine parameters (T4, T3, TSH), and group B water-soluble vitamins. The study was conducted in 368 healthy white pregnant women - residents of north-east Germany. Free 25(OH)D showed an overall better associations with gestational age, markers of bone metabolism (calcium (rho = 0.141, p = 0.007 with free 25(OH)D; rho = 0.060, p = 0.251 with total 25(OH)D) and BSAP (rho = -0.203, p < 0.001 with free 25(OH)D; rho = -0.108, p = 0.038 with total 25(OH)D), lipid metabolism parameters (adiponectin (rho = 0.142, p = 0.008 with free 25(OH)D; rho = 0.054, p = 0.307 with total 25(OH)D), LDL cholesterol (rho = -0.191, p < 0.001 with free 25(OH)D; rho = 0.033, p = 0.539 with total 25(OH)D)) and a kidney function marker (urea (rho = 0.114, p = 0.032 with free 25(OH)D; rho = 0.008, p = 0.887 with total 25(OH)D)) than total 25(OH)D. In conclusion, the current study revealed that free 25(OH)D is a more precise determinant of the vitamin D status during normal human pregnancy than total 25(OH)D. In the settings of normal pregnancy, free 25(OH)D revealed better associations with markers of bone metabolism (calcium, BSAP), lipid metabolism (adiponectin, LDL cholesterol, LDL/HDL ratio) and kidney function (urea) than total 25(OH)D.

Folate treatment of pregnant rat dams abolishes metabolic effects in female offspring induced by a paternal pre-conception unhealthy diet.

AIMS/HYPOTHESIS: Paternal high-fat diet prior to mating programmes impaired glucose tolerance in female offspring. We examined whether the metabolic consequences in offspring could be abolished by folate treatment of either the male rats before mating or the corresponding female rats during pregnancy. METHODS: Male F0 rats were fed either control diet or high-fat, high-sucrose and high-salt diet (HFSSD), with or without folate, before mating. Male rats were mated with control-diet-fed dams. After mating, the F0 dams were fed control diet with or without folate during pregnancy. RESULTS: Male, but not female offspring of HFSSD-fed founders were heavier than those of control-diet-fed counterparts (p < 0.05 and p = 0.066 in males and females, respectively). Both male and female offspring of HFSSD-fed founders were longer compared with control (p < 0.01 for both sexes). Folate treatment of the pregnant dams abolished the effect of the paternal diet on the offspring's body length (p Ë‚ 0.05). Female offspring of HFSSD-fed founders developed impaired glucose tolerance, which was restored by folate treatment of the dams during pregnancy. The beta cell density per pancreatic islet was decreased in offspring of HFSSD-fed rats (-20% in male and -15% in female F1 offspring, p Ë‚ 0.001 vs controls). Folate treatment significantly increased the beta cell density (4.3% and 3.3% after folate supplementation given to dams and founders, respectively, p Ë‚ 0.05 vs the offspring of HFSSD-fed male rats). Changes in liver connective tissue of female offspring of HFSSD-fed founders were ameliorated by treatment of dams with folate (p Ë‚ 0.01). Hepatic Ppara gene expression was upregulated in female offspring only (1.51-fold, p Ë‚ 0.05) and was restored in the female offspring by folate treatment (p Ë‚ 0.05). We observed an increase in hepatic Lcn2 and Tmcc2 expression in female offspring born to male rats exposed to an unhealthy diet during spermatogenesis before mating (p Ë‚ 0.05 vs controls). Folate treatment of the corresponding dams during pregnancy abolished this effect (p Ë‚ 0.05). Analysis of DNA methylation levels of CpG islands in the Ppara, Lcn2 and Tmcc2 promoter regions revealed that the paternal unhealthy diet induced alterations in the methylation pattern. These patterns were also affected by folate treatment. Total liver DNA methylation was increased by 1.52-fold in female offspring born to male rats on an unhealthy diet prior to mating (p Ë‚ 0.05). This effect was abolished by folate treatment during pregnancy (p Ë‚ 0.05 vs the offspring of HFSSD-fed male rats). CONCLUSIONS/INTERPRETATION: Folate treatment of pregnant dams restores effects on female offspring's glucose metabolism induced by pre-conception male founder HFSSD.

Urinary cGMP predicts major adverse renal events in patients with mild renal impairment and/or diabetes mellitus before exposure to contrast medium.

BACKGROUND: The use of iodine-based contrast agents entails the risk of contrast induced nephropathy (CIN). Radiocontrast agents elicit the third most common cause of nephropathy among hospitalized patients, accounting for 11-12% of cases. CIN is connected with clinically significant consequences, including increased morbidity, prolonged hospitalization, increased risk of complications, potential need for dialysis, and increased mortality rate. The number of in-hospital examinations using iodine-based contrast media has been significantly increasing over the last decade. In order to protect patients from possible complications of such examinations, new biomarkers are needed that are able to predict a risk of contrast-induced nephropathy. Urinary and plasma cyclic guanosine monophosphate (cGMP) concentrations are influenced by renal function. Urinary cGMP is primarily of renal cellular origin. Therefore, we assessed if urinary cGMP concentration may predict major adverse renal events (MARE) after contrast media exposure during coronary angiography. METHODS: Urine samples were prospectively collected from non-randomized consecutive patients with either diabetes or preexisting impaired kidney function receiving intra-arterial contrast medium (CM) for emergent or elective coronary angiography at the Charité Campus Mitte, University Hospital Berlin. Urinary cGMP concentration in spot urine was analyzed 24 hours after CM exposure. Patients were followed up over 90 days for occurrence of death, initiation of dialysis, doubling of plasma creatinine concentration or MARE. RESULTS: In total, 289 consecutive patients were included into the study. Urine cGMP/creatinine ratio 24 hours before CM exposure expressed as mean±SD was predictive for the need of dialysis (no dialysis: 89.77±92.85 µM/mM, n = 277; need for dialysis: 140.3±82.90 µM/mM, n = 12, p = 0.008), death (no death during follow-up: 90.60±92.50 µM/mM, n = 280; death during follow-up: 169.88±81.52 µM/mM, n = 9; p = 0.002), and the composite endpoint MARE (no MARE: 86.02±93.17 µM/mM, n = 271; MARE: 146.64±74.68 µM/mM, n = 18, p<0.001) during the follow-up of 90 days after contrast media application. cGMP/creatinine ratio stayed significantly increased at values exceeding 120 µM/mM in patients who developed MARE, required dialysis or died. CONCLUSIONS: Urinary cGMP/creatinine ratio ≥ 120 µM/mM before CM exposure is a promising biomarker for the need of dialysis and all-cause mortality 90 days after CM exposure in patients with preexisting renal impairment or diabetes.

Reference intervals for measured and calculated free 25-hydroxyvitamin D in normal pregnancy.

The determination of free 25-hydroxyvitamin D (25(OH)D) as compared to the analysis of total 25-hydroxyvitamin D might reflect better the vitamin D status during pregnancy, since vitamin D-binding protein (DBP) concentrations increase throughout pregnancy and the vast majority of 25(OH)D is tightly bound to DBP thus strongly influencing total 25(OH)D. The concentration of the biologically active free 25(OH)D - on the other hand - is much less dependent on the DBP concentrations. The study was conducted in May-June 2016 in 368 Caucasian pregnant healthy women - residents of Northeastern Germany. Free 25(OH)D was either measured directly by commercial ELISA kit or assessed by calculation via total 25(OH)D, DBP, and albumin serum concentrations. Regardless of the detection method, free 25(OH)D lowers in the 3rd trimester comparing to the 1st trimester (by 12% and 21%, p < 0.05 and p < 0.001, for measured and calculated free 25(OH)D, respectively), whereas total 25(OH)D was not decreased in late pregnancy. DBP rises with gestational age. Total 25(OH)D was not correlated with serum calcium (p = 0.251), whereas free 25(OH)D was significantly (p = 0.007 for measured free 25(OH)D and p < 0.001 for calculated free 25(OH)D) positively correlated with calcium. All 25(OH)D isoforms were significantly negatively correlated with bone-specific alkaline phosphatase (BSAP), however the correlation strength was the lowest with total 25(OH)D (rho = -0.108, p = 0.038), whereas both measured and calculated free 25(OH)D revealed better associations with BSAP (rho = -0.203 and rho = -0.211 for measured and calculated free 25(OH)D, respectively, p < 0.001 for both). We established pregnancy trimester-specific reference intervals for free measured and calculated 25(OH)D and DBP. Both measured and calculated free 25(OH)D showed better correlations with parameters of the endocrine vitamin D system (calcium and BSAP). Both ways of measuring free 25(OH)D in pregnant women are suitable as novel laboratory parameter for vitamin D status monitoring during human pregnancy and might replace in the future the routine total 25(OH)D assessment.

Why should we measure free 25(OH) vitamin D?

Vitamin D, either in its D2 or D3 form, is essential for normal human development during intrauterine life, kidney function and bone health. Vitamin D deficiency has also been linked to cancer development and some autoimmune diseases. Given this huge impact of vitamin D on human health, it is important for daily clinical practice and clinical research to have reliable tools to judge on the vitamin D status. The major circulating form of vitamin D is 25-hydroxyvitamin D (25(OH)D), although it is not the most active metabolite, the concentrations of total 25-hydroxyvitamin D in the serum are currently routinely used in clinical practice to assess vitamin D status. In the circulation, vitamin D - like other steroid hormones - is bound tightly to a special carrier - vitamin D-binding protein (DBP). Smaller amounts are bound to blood proteins - albumin and lipoproteins. Only very tiny amounts of the total vitamin D are free and potentially biologically active. Currently used vitamin D assays do not distinguish between the three forms of vitamin D - DBP-bound vitamin D, albumin-bound vitamin D and free, biologically active vitamin D. Diseases or conditions that affect the synthesis of DBP or albumin thus have a huge impact on the amount of circulating total vitamin D. DBP and albumin are synthesized in the liver, hence all patients with an impairment of liver function have alterations in their total vitamin D blood concentrations, while free vitamin D levels remain mostly constant. Sex steroids, in particular estrogens, stimulate the synthesis of DBP. This explains why total vitamin D concentrations are higher during pregnancy as compared to non-pregnant women, while the concentrations of free vitamin D remain similar in both groups of women. The vitamin D-DBP as well as vitamin D-albumin complexes are filtered through the glomeruli and re-uptaken by megalin in the proximal tubule. Therefore, all acute and chronic kidney diseases that are characterized by a tubular damage, are associated with a loss of vitamin D-DBP complexes in the urine. Finally, the gene encoding DBP protein is highly polymorphic in different human racial groups. In the current review, we will discuss how liver function, estrogens, kidney function and the genetic background might influence total circulating vitamin D levels and will discuss what vitamin D metabolite is more appropriate to measure under these conditions: free vitamin D or total vitamin D.

Impact of vitamin D on pregnancy-related disorders and on offspring outcome.

Observational studies from all over the world continue to find high prevalence rates of vitamin D insufficiency and deficiency in many populations, including pregnant women. Beyond its classical function as a regulator of calcium and phosphate metabolism, vitamin D elicits numerous effects in the human body. Current evidence highlights a vital role of vitamin D in mammalian gestation. During pregnancy, adaptations in maternal vitamin D metabolism lead to a physiologic increase of vitamin D levels, mainly because of an increased renal production, although other potential sources like the placenta are being discussed. A sufficient supply of mother and child with calcium and vitamin D during pregnancy ensures a healthy bone development of the fetus, whereas lack of either of these nutrients can lead to the development of rickets in the child. Moreover, vitamin D insufficiency during pregnancy has consistently been associated with adverse maternal and neonatal pregnancy outcomes. In multitudinous studies, low maternal vitamin D status was associated with a higher risk for preeclampsia, gestational diabetes mellitus and other gestational diseases. Likewise, several negative consequences for the fetus have been reported, including fetal growth restriction, increased risk of preterm birth and a changed susceptibility for later-life diseases. However, study results are diverging and causality has not been proven so far. Meta-analyses on the relationship between maternal vitamin D status and pregnancy outcomes revealed a wide heterogeneity of studied populations and the applied methodology in vitamin D assessment. Until today, clinical guidelines for supplementation cannot be based on high-quality evidence and it is not clear if the required intake for pregnant women differs from non-pregnant women. Long-term safety data of vitamin D supplementation in pregnant women has not been established and overdosing of vitamin D might have unfavorable effects, especially in mothers and newborns with mutations of genes involved in vitamin D metabolism. Reliable data from large observational and interventional randomized control trials are urgently needed as a basis for any detailed and safe recommendations for supplementation in the general population and, most importantly, in pregnant women. This is of utmost importance, as ensuring a sufficient vitamin D-supply of mother and child implies a great potential for the prevention of birth complications and development of diseases.

Pre-Interventional Kynurenine Predicts Medium-Term Outcome after Contrast Media Exposure Due to Coronary Angiography.

BACKGROUND/AIMS: Contrast induced acute kidney injury (CI-AKI) remains a serious complication of contrast media enhanced procedures like coronary angiography. There is still a lack of established biomarkers that help to identify patients at high risk for short and long-term complications. The aim of the current study was to evaluate plasma kynurenine as a predictive biomarker for CI-AKI and long-term complications, measured by the combined endpoint "major adverse kidney events" (MAKE) up to 120 days after CM application. METHODS: In this prospective cohort study 245 patients undergoing coronary angiography were analyzed. Blood samples were obtained at baseline, 24h and 48h after contrast media (CM) application to diagnose CI-AKI. Patients were followed for 120 days for adverse clinical events including death, the need for dialysis, and a doubling of plasma creatinine. Occurrence of any of these events was summarized in the combined endpoint MAKE. RESULTS: Preinterventional plasma kynurenine was not associated with CI-AKI. Patients who later developed MAKE displayed significantly increased preinterventional plasma kynurenine levels (p<0.0001). ROC analysis revealed that preinterventional kynurenine is highly predictive for MAKE (AUC=0.838; p<0.0001). The optimal cutoff was found at ≥3.5 µmol/L Using this cutoff, the Kaplan-Meier estimator demonstrated that concentrations of plasma kynurenine ≥3.5 µmol/L were significantly associated with a higher prevalence of MAKE until follow up (p<0.0001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. CONCLUSION: Preinterventional plasma kynurenine might serve as a highly predictive biomarker for MAKE up to 120 days after coronary angiography.

Head-to-head comparison of structurally unrelated dipeptidyl peptidase 4 inhibitors in the setting of renal ischemia reperfusion injury.

BACKGROUND AND PURPOSE: Results regarding protective effects of dipeptidyl peptidase 4 (DPP4) inhibitors in renal ischaemia-reperfusion injury (IRI) are conflicting. Here we have compared structurally unrelated DPP4 inhibitors in a model of renal IRI. EXPERIMENTAL APPROACH: IRI was induced in uninephrectomized male rats by renal artery clamping for 30 min. The sham group was uninephrectomized but not subjected to IRI. DPP4 inhibitors or vehicle were given p.o. once daily on three consecutive days prior to IRI: linagliptin (1.5 mg·kg-1 ·day-1 ), vildagliptin (8 mg·kg-1 ·day-1 ) and sitagliptin (30 mg·kg-1 ·day-1 ). An additional group received sitagliptin until study end (before IRI: 30 mg·kg-1 ·day-1 ; after IRI: 15 mg·kg-1 ·day-1 ). KEY RESULTS: Plasma-active glucagon-like peptide type 1 (GLP-1) increased threefold to fourfold in all DPP4 inhibitor groups 24 h after IRI. Plasma cystatin C, a marker of GFR, peaked 48 h after IRI. Compared with the placebo group, DPP4 inhibition did not reduce increased plasma cystatin C levels. DPP4 inhibitors ameliorated histopathologically assessed tubular damage with varying degrees of drug-specific efficacies. Renal osteopontin expression was uniformly reduced by all DPP4 inhibitors. IRI-related increased renal cytokine expression was not decreased by DPP4 inhibition. Renal DPP4 activity at study end was significantly inhibited in the linagliptin group, but only numerically reduced in the prolonged/dose-adjusted sitagliptin group. Active GLP-1 plasma levels at study end were increased only in the prolonged/dose-adjusted sitagliptin treatment group. CONCLUSIONS AND IMPLICATIONS: In rats with renal IRI, DPP4 inhibition did not alter plasma cystatin C, a marker of glomerular function, but may protect against tubular damage.

Global Overexpression of ET-1 Decreases Blood Pressure - A Systematic Review and Meta-Analysis of ET-1 Transgenic Mice.

BACKGROUND/AIMS: ET-1 has independent effects on blood pressure regulation in vivo, it is involved in tubular water and salt excretion, promotes constriction of smooth muscle cells, modulates sympathetic nerve activity, and activates the liberation of nitric oxide. To determine the net effect of these partially counteracting mechanisms on blood pressure, a systematic meta-analysis was performed. METHODS: Based on the principles of Cochrane systematic reviews, we searched in major literature databases - MEDLINE (PubMed), Embase, Google Scholar, and the China Biological Medicine Database (CBM-disc) - for articles relevant to the topic of the blood pressure phenotype of endothelin-1 transgenic (ET-1+/+) mice from January 1, 1988 to March 31, 2016. Review Manager Version 5.0 (Rev-Man 5.0) software was applied for statistical analysis. In total thirteen studies reported blood pressure data. RESULTS: The meta-analysis of blood pressure data showed that homozygous ET-1 transgenic mice (ET-1+/+ mice) had a significantly lower blood pressure as compared to WT mice (mean difference: -2.57 mmHg, 95% CI: -4.98∼ -0.16, P = 0.04), with minimal heterogeneity (P = 0.86). A subgroup analysis of mice older than 6 months revealed that the blood pressure difference between ET-1+/+ mice and WT mice was even more pronounced (mean difference: -6.19 mmHg, 95% CI: -10.76∼ -1.62, P = 0.008), with minimal heterogeneity (P = 0.91). CONCLUSION: This meta-analysis provides robust evidence that global ET-1 overexpression in mice lowers blood pressure in an age-dependent manner. Older ET-1+/+ mice have a somewhat more pronounced reduction of blood pressure.

Paternal programming of offspring cardiometabolic diseases in later life.

Early - intrauterine - environmental factors are linked to the development of cardiovascular disease in later life. Traditionally, these factors are considered to be maternal factors such as maternal under and overnutrition, exposure to toxins, lack of micronutrients, and stress during pregnancy. However, in the recent years, it became obvious that also paternal environmental factors before conception and during sperm development determine the health of the offspring in later life. We will first describe clinical observational studies providing evidence for paternal programming of adulthood diseases in progeny. Next, we describe key animal studies proving this relationship, followed by a detailed analysis of our current understanding of the underlying molecular mechanisms of paternal programming. Alterations of noncoding sperm micro-RNAs, histone acetylation, and targeted as well as global DNA methylation seem to be in particular involved in paternal programming of offspring's diseases in later life.

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner.

Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood.

The dipeptidyl peptidase inhibitor linagliptin and the angiotensin II receptor blocker telmisartan show renal benefit by different pathways in rats with 5/6 nephrectomy.

Dipeptidyl peptidase (DPP)-4 inhibitors delay chronic kidney disease (CKD) progression in experimental diabetic nephropathy in a glucose-independent manner. Here we compared the effects of the DPP-4 inhibitor linagliptin versus telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. Animals were allocated to 1 of 4 groups: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus linagliptin; and 5/6 nephrectomy plus telmisartan. Interstitial fibrosis was significantly decreased by 48% with linagliptin but a non-significant 24% with telmisartan versus placebo. The urine albumin-to-creatinine ratio was significantly decreased by 66% with linagliptin and 92% with telmisartan versus placebo. Blood pressure was significantly lowered by telmisartan, but it was not affected by linagliptin. As shown by mass spectrometry, the number of altered peptide signals for linagliptin in plasma was 552 and 320 in the kidney. For telmisartan, there were 108 peptide changes in plasma and 363 in the kidney versus placebo. Linagliptin up-regulated peptides derived from collagen type I, apolipoprotein C1, and heterogeneous nuclear ribonucleoproteins A2/B1, a potential downstream target of atrial natriuretic peptide, whereas telmisartan up-regulated angiotensin II. A second study was conducted to confirm these findings in 5/6 nephrectomy wild-type and genetically deficient DPP-4 rats treated with linagliptin or placebo. Linagliptin therapy in wild-type rats was as effective as DPP-4 genetic deficiency in terms of albuminuria reduction. Thus, linagliptin showed comparable efficacy to telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. However, the underlying pathways seem to be different.

Urinary Vitamin D Binding Protein and KIM-1 Are Potent New Biomarkers of Major Adverse Renal Events in Patients Undergoing Coronary Angiography.

BACKGROUND: Vitamin-D-binding protein (VDBP) is a low molecular weight protein that is filtered through the glomerulus as a 25-(OH) vitamin D 3/VDBP complex. In the normal kidney VDBP is reabsorbed and catabolized by proximal tubule epithelial cells reducing the urinary excretion to trace amounts. Acute tubular injury is expected to result in urinary VDBP loss. The purpose of our study was to explore the potential role of urinary VDBP as a biomarker of an acute renal damage. METHOD: We included 314 patients with diabetes mellitus or mild renal impairment undergoing coronary angiography and collected blood and urine before and 24 hours after the CM application. Patients were followed for 90 days for the composite endpoint major adverse renal events (MARE: need for dialysis, doubling of serum creatinine after 90 days, unplanned emergency rehospitalization or death). RESULTS: Increased urine VDBP concentration 24 hours after contrast media exposure was predictive for dialysis need (no dialysis: 113.06 ± 299.61 ng/ml, n = 303; need for dialysis: 613.07 ± 700.45 ng/ml, n = 11, Mean ± SD, p<0.001), death (no death during follow-up: 121.41 ± 324.45 ng/ml, n = 306; death during follow-up: 522.01 ± 521.86 ng/ml, n = 8; Mean ± SD, p<0.003) and MARE (no MARE: 112.08 ± 302.00 ng/ml, n = 298; MARE: 506.16 ± 624.61 ng/ml, n = 16, Mean ± SD, p<0.001) during the follow-up of 90 days after contrast media exposure. Correction of urine VDBP concentrations for creatinine excretion confirmed its predictive value and was consistent with increased levels of urinary Kidney Injury Molecule-1 (KIM-1) and baseline plasma creatinine in patients with above mentioned complications. The impact of urinary VDBP and KIM-1 on MARE was independent of known CIN risk factors such as anemia, preexisting renal failure, preexisting heart failure, and diabetes. CONCLUSIONS: Urinary VDBP is a promising novel biomarker of major contrast induced nephropathy-associated events 90 days after contrast media exposure.