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Background and objective Whether treatment with vitamin D receptor activators contributes to cardiovascular disease in patients with chronic kidney disease is a matter of debate. We studied mechanisms involved in vitamin D-related vascular calcifications in vivo and in vitro.
Methods Aortic calcifications were induced in subtotally nephrectomized (SNX) rats by treatment with a high dose (0.25 mu g/kg per day) of 1,25-dihydroxyvitamin D-3 (calcitriol) given for 6 weeks. Likewise, primary rat vascular smooth muscle cells (VSMCs) were incubated with calcitriol at concentrations ranging from 10(-11) to 10(-7) mol/l. Immunohistochemistry revealed that the aortic expression of osteopontin, osteocalcin and bone sialoprotein was significantly increased in calcitriol-treated SNX rats compared to untreated SNX controls. In addition, aortic expression of the transient receptor potential vanilloid calcium channel 6 (TRPV6) and calbindin D9k was significantly up-regulated by treatment with calcitriol. Furthermore, calcitriol significantly increased expression of the osteogenic transcription factor osterix. In-vitro studies showed similar results, confirming that these effects could be attributed to treatment with calcitriol.
Conclusions High-dose calcitriol treatment induces an osteoblastic phenotype in VSMC both in SNX rats and in vitro, associated with up-regulation of proteins regulating mineralization and calcium transport, and of the osteogenic transcription factor osterix.
Background/Aims: Cortisol plays an important role during pregnancy. It controls maternal glucose metabolism and fetal development. Cortisol metabolism is partially controlled by the 11b-HSD2. This enzyme is expressed in the kidney and human placenta. The activity of the enzyme is partially controlled by functional polymorphisms: the HSD11B2[CA]n microsatellite polymorphism. The impact of this functional gene polymorphism on cortisol metabolism and potential effects on the newborn's is unknown so far. Methods: In the current prospective birth cohort study in southern Asia, we analyzed the association of the HSD11B2[CA]n microsatellite polymorphisms in 187 mothers and their newborn's on maternal and newborn's serum cortisol concentrations. Results: Using multivariable regression analyses considering known confounding ( gestational age, newborn's gender, the labor uterine contraction states and the timing during the day of blood taking), we showed that the fetal HSD11B2[CA]n microsatellite polymorphisms in the first intron was related to maternal cortisol concentration ( R2=0.26, B=96.27, p=0.007), whereas as the newborn's cortisol concentrations were independent of fetal and maternal HSD11B2[CA] n microsatellite polymorphism. Conclusions: Our study showed for the first time that the fetal HSD11B2[CA]n microsatellite polymorphism of the HSD11B2 gene in healthy uncomplicated human pregnancy is associated with maternal cortisol concentration. This indicates that fetal genes controlling cortisol metabolism may affect maternal cortisol concentration and hence physiology in healthy pregnant women.
A novel common variant in DCST2 is associated with length in early life and height in adulthood
(2015)
Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 x 10(-6)) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; beta = 0.046, SE = 0.008, P = 2.46 x 10(-8), explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 x 10(-4)) and adult height (N = 127 513; P = 1.45 x 10(-5)). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
Background/Aims: Impaired pregnancy outcomes, such as low birth weight are associated with increased disease risk in later life, however little is known about the impact of common infectious diseases during pregnancy on birth weight. The study had two aims: a) to investigate risk factors of influenza virus infection during pregnancy, and b) to analyze the impact of influenza virus infection on pregnancy outcome, especially birth weight.
Methods: Prospective and retrospective observational studies found in PubMed, MEDLINE, Embase, Google Scholar, and WangFang database were included in this meta analysis. Data of included studies was extracted and analyzed by the RevMan software.
Results: Pregnant women with anemia (P=0.004, RR=1.46, 95% CI: 1.13-1.88), obesity (P<0.00001, RR=1.35, 95% CI: 1.25-1.46) and asthma (P<0.00001, RR=1.99, 95% CI: 1.67-2.37) had higher rates of influenza virus infection. Regarding birth outcomes, influenza A virus infection did not affect the likelihood for cesarean section. Mothers with influenza had a higher rate of stillbirth (P=0.04, RR=2.36, 95% CI: 1.05-5.31), and their offspring had low 5-minute APGR Scores (P=0.009, RR=1.39, 95% CI: 1.08-1.79). Furthermore, the rate for birth weight < 2500g (P=0.04, RR=1.71, 95% CI: 1.03-2.84) was increased.
Conclusion: Results of this study showed that anemia, asthma and obesity during pregnancy are risk factors influenza A virus infection during pregnancy. Moreover, gestational influenza A infection impairs pregnancy outcomes and increases the risk for low birth weight, a known risk factor for later life disease susceptibility.
Background
Riociguat is the first of a new class of drugs, the soluble guanylate cyclase (sGC) stimulators. Riociguat has a dual mode of action: it sensitizes sGC to the body’s own NO and can also increase sGC activity in the absence of NO. The NO-sGC-pathway is impaired in many cardiovascular diseases such as heart failure, pulmonary hypertension and diabetic nephropathy (DN). DN leads to high cardiovascular morbidity and mortality. There is still a high unmet medical need. The urinary albumin excretion rate is a predictive biomarker for these clinical events. Therefore, we investigated the effect of riociguat, alone and in combination with the angiotensin II receptor antagonist (ARB) telmisartan on the progression of DN in diabetic eNOS knock out mice, a new model closely resembling human pathology.
Methods
Seventy-six male eNOS knockout C57BL/6J mice were divided into 4 groups after receiving intraperitoneal high-dose streptozotocin: telmisartan (1 mg/kg), riociguat (3 mg/kg), riociguat+telmisartan (3 and 1 mg/kg), and vehicle. Fourteen mice were used as non-diabetic controls. After 12 weeks, urine and blood were obtained and blood pressure measured. Glucose concentrations were highly increased and similar in all diabetic groups.
Results
Riociguat, alone (105.2 ± 2.5 mmHg; mean±SEM; n = 14) and in combination with telmisartan (105.0 ± 3.2 mmHg; n = 12), significantly reduced blood pressure versus diabetic controls (117.1 ± 2.2 mmHg; n = 14; p = 0.002 and p = 0.004, respectively), whereas telmisartan alone (111.2 ± 2.6 mmHg) showed a modest blood pressure lowering trend (p = 0.071; n = 14). The effects of single treatment with either riociguat (97.1 ± 15.7 µg/d; n = 13) or telmisartan (97.8 ± 26.4 µg/d; n = 14) did not significantly lower albumin excretion on its own (p = 0.067 and p = 0.101, respectively). However, the combined treatment led to significantly lower urinary albumin excretion (47.3 ± 9.6 µg/d; n = 12) compared to diabetic controls (170.8 ± 34.2 µg/d; n = 13; p = 0.004), and reached levels similar to non-diabetic controls (31.4 ± 10.1 µg/d, n = 12).
Conclusion
Riociguat significantly reduced urinary albumin excretion in diabetic eNOS knock out mice that were refractory to treatment with ARB’s alone. Patients with diabetic nephropathy refractory to treatment with ARB’s have the worst prognosis among all patients with diabetic nephropathy. Our data indicate that additional stimulation of sGC on top of standard treatment with ARB`s may offer a new therapeutic approach for patients with diabetic nephropathy resistant to ARB treatment.
Selective adenosine A1 receptor antagonists targeting renal microcirculation are novel pharmacologic agents that are currently under development for the treatment of acute heart failure as well as for chronic heart failure. Despite several studies showing improvement of renal function and/or increased diuresis with adenosine A1 antagonists, particularly in chronic heart failure, these findings were not confirmed in a large phase III trial in acute heart failure patients. However, lessons can be learned from these and other studies, and there might still be a potential role for the clinical use of adenosine A1 antagonists. We review the role of adenosine A1 receptors in the regulation of renal function, and emerging data regarding the safety and efficacy of A1 adenosine receptor antagonists based on all available completed and reported clinical trials using A1 adenosine receptor antagonists. The majority of trials were done in heart failure patients. However, there is clear clinical evidence for a role of this new class in hepatorenal syndrome, hypotension on dialysis, and radiocontrast media-induced nephropathy.
Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of the nitric oxide (NO)-synthase and a biomarker of endothelial dysfunction (ED). ED plays an important role in the pathogenesis of contrast-induced nephropathy (CIN). The aim of our study was to evaluate serum ADMA concentration as a biomarker of an acute renal damage during the follow-up of 90 days after contrast medium (CM) application. Blood samples were obtained from 330 consecutive patients with diabetes mellitus or mild renal impairment immediately before, 24 and 48 hours after the CM application for coronary angiography. The patients were followed for 90 days. The composite endpoints were major adverse renal events (MARE) defined as occurrence of death, initiation of dialysis, or a doubling of serum creatinine concentration. Overall, ADMA concentration in plasma increased after CM application, although, there was no differences between ADMA levels in patients with and without CIN. ADMA concentration 24 hours after the CM application was predictive for dialysis with a specificity of 0.889 and sensitivity of 0.653 at values higher than 0.71 mu mol/L (area under the curve: 0.854, 95% confidential interval: 0.767-0.941, P<0.001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. 24 hours after the CM application, ADMA concentration in plasma was predictive for MARE with a specificity of 0.833 and sensitivity of 0.636 at a value of more than 0.70 mu mol/L (area under the curve: 0.750, 95% confidence interval: 0.602-0.897, P=0.004). Multivariate logistic regression analysis confirmed that ADMA and anemia were significant predictors of MARE. Further analysis revealed that increased ADMA concentration in plasma was highly significant predictor of MARE in patients with CIN. Moreover, patients with CIN and MARE had the highest plasma ADMA levels 24 hours after CM exposure in our study cohort. The impact of ADMA on MARE was independent of such known CIN risk factors as anemia, pre-existing renal failure, pre-existing heart failure, and diabetes. ADMA concentration in plasma is a promising novel biomarker of major contrast-induced nephropathy-associated events 90 days after contrast media exposure.
Fibroblast growth factor 23 (FGF23) is a proteohormone regulating renal phosphate transport and vitamin D metabolism as well as inducing left heart hypertrophy. FGF23-deficient mice suffer from severe tissue calcification, accelerated aging and a myriad of aging-associated diseases. Bone cells produce FGF23 upon store-operated calcium ion entry (SOCE) through the calcium selective ion channel Orai1. AMP-activated kinase (AMPK) is a powerful energy sensor helping cells survive states of energy deficiency, and AMPK down-regulates Orai1. Here we investigated the role of AMPK in FGF23 production. Fgf23 gene transcription was analyzed by qRT-PCR and SOCE by fluorescence optics in UMR106 osteoblast-like cells while the serum FGF23 concentration and phosphate metabolism were assessed in AMPKa1-knockout and wild-type mice. The AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) down-regulated, whereas the AMPK inhibitor, dorsomorphin dihydrochloride (compound C) and AMPK gene silencing induced Fgf23 transcription. AICAR decreased membrane abundance of Orai1 and SOCE. SOCE inhibitors lowered Fgf23 gene expression induced by AMPK inhibition. AMPKa1-knockout mice had a higher serum FGF23 concentration compared to wild-type mice. Thus, AMPK participates in the regulation of FGF23 production in vitro and in vivo. The inhibitory effect of AMPK on FGF23 production is at least in part mediated by Orai1-involving SOCE.
Background: Endothelin-1 (ET-1) is a multifunctional peptide, which is implicated in the renal and cardiac physicology as well as in many pathologies of these systems. ET-1's actions take place after the activation of two receptors: ETA and ETB. The expression of these receptors may be modulated during the pathologic process. The analysis of the distribution and level of expression of the receptors in animal models is therefore crucial.
Methods: We developed a protocol for non-radioactive in situ hybridization for the mRNA of the two endothelin receptors on paraffin-embedded tissue using digoxigenin-labeled RNA probes.
Results: In heart and kidney, the staining was reliable and specific. In a mouse model for endothelin/nitric oxide imbalance, cardiac ETB expression was reduced. The distribution of the receptors was in accordance with the actual knowledge. Differences in cell specific expression are discussed.
Conclusions: We developed a protocol for the in situ hybridization of the endothelin receptors in mice. Given that the endothelin system is implicated in the development of many diseases, we believe that this protocol may be useful for a number of future preclinical studies.
Background: DNA-methylation is a common epigenetic tool which plays a crucial role in gene regulation and is essential for cell differentiation and embryonic development. The placenta is an important organ where gene activity can be regulated by epigenetic DNA modifications, including DNA methylation. This is of interest as, the placenta is the interface between the fetus and its environment, the mother. Exposure to environmental toxins and nutrition during pregnancy may alter DNA methylation of the placenta and subsequently placental function and as a result the phenotype of the offspring. The aim of this study was to develop a reliable method to quantify DNA methylation in large clinical studies. This will be a tool to analyze the degree of DNA methylation in the human placenta in relationship to clinical readouts. Methods: Liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) technique was used for the quantification of the 5dmC/dG ratio in placentas from 248 healthy pregnancies. We were able to demonstrate that this method is a reliable and stable way to determine global placental DNA methylation in large clinical trials. Results/Conclusion: The degree of placental DNA methylation seen in our pilot study varies substantially from 2% to 5%. The clinical implications of this variation need to be demonstrated in adequately powered large studies.
Aims:This study aims to investigate the relationship between plasma endothelin-1 (ET-1) concentrations, ET-1 gene polymorphisms in loci rs5370, rs1630736, 3A/4A and clinical features of primary nephrotic syndrome (NS) in children.
Materials and methods: Thirty-six children with primary NS were selected as case group, and 94 healthy children were selected as control group. All subjects were genotyped for three single nucleotide polymorphisms (SNPs) (rs5370, rs10478694 [3A4A) and rs 1630736) in the ET-1 gene by gene sequencing. The plasma ET-1 concentrations were measured using a radio-immunoassay.
Key findings: Plasma ET-1 concentrations were higher in NS patients (P = 0.007) as compared to healthy children. The allele frequencies between control and NS patients were significantly different only with respect to the rs10478694 SNP of the ET-1 gene. The allele frequencies between control and NS patients for the rs5370 SNP showed a trend towards difference (P = 0.057). Plasma cholesterol in NS patients is associated with both: the Cl genotype in locus rs5370 and the 3A4A genotype in locus rs10478694 (P < 0.05 in both cases).
Significance: The ET systems might play a disease modifying role in pediatric NS. Plasma cholesterol, a hallmark of NS. seems to be associated with genetic variations within the human ET-1 gene. (C) 2014 Elsevier Inc. All rights reserved.
Background: The multidrug transporter P-glycoprotein (PGP) is expressed in the human placenta. In particular the C3435T ABCB1 polymorphism was associated with altered tissue expression of PGP in the human placenta. However, the potential functional impact of this polymorphism on the offspring is unknown so far.
Methods: We analyzed the impact of the ABCB1/C3435T polymorphism on fetal growth in 262 mother/child pairs. Fetal growth was assessed by differential ultrasound examination of the fetal body prior to birth and by measuring birth weight.
Results: The maternal ABCB1/C3435T polymorphism showed no trend for an association with birth weight or any ultrasound parameter describing late gestational fetal body shape. Genotyping the newborns, however, demonstrated that newborns carrying two copies of the T allele had a birth weight of 3176.39 g, whereas CT and CC newborns had a birth weight of 3345.04 g (p = 0.022). Adjusting for gestational age at delivery, child's gender, maternal BM1, maternal age and body weight at delivery confirmed this finding (p = 0.009). Considering gestational day of late ultrasound examination, gestational age at delivery, child's gender, maternal BMI, maternal age and maternal body weight at delivery, the fetal ABCB1/C3435T genotype revealed likewise a significant negative correlation with abdominal diameter and abdominal circumference (R-2 = 0.538, p = 0.010 and R-2 = 0.534, p = 0.005, respectively).
Conclusions: Low birth weight may be a risk factor for cardiovascular diseases in later life. The fetal ABCB1/C3435T gene polymorphism may contribute to this risk. Since PGP controls transport of various biological agents, we suggest that PGP is involved in the transport of biological agents to the fetus that are important for normal fetal growth.
Being born large for gestational age is associated with increased global placental DNA methylation
(2020)
Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p<0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p<0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p=0.001).
Being born large for gestational age is associated with increased global placental DNA methylation
(2020)
Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p<0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p<0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p=0.001).
The present study aimed to identify new key genes as potential biomarkers for the diagnosis, prognosis or targeted therapy of clear cell renal cell carcinoma (ccRCC). Three expression profiles (GSE36895, GSE46699 and GSE71963) were collected from Gene Expression Omnibus. GEO2R was used to identify differentially expressed genes (DEGs) in ccRCC tissues and normal samples. The Database for Annotation, Visualization and Integrated Discovery was utilized for functional and pathway enrichment analysis. STRING v10.5 and Molecular Complex Detection were used for protein-protein interaction (PPI) network construction and module analysis, respectively. Regulation network analyses were performed with the WebGestal tool. UALCAN web-portal was used for expression validation and survival analysis of hub genes in ccRCC patients from The Cancer Genome Atlas (TCGA). A total of 65 up- and 164 downregulated genes were identified as DEGs. DEGs were enriched with functional terms and pathways compactly related to ccRCC pathogenesis. Seventeen hub genes and one significant module were filtered out and selected from the PPI network. The differential expression of hub genes was verified in TCGA patients. Kaplan-Meier plot showed that high mRNA expression of enolase 2 (ENO2) was associated with short overall survival in ccRCC patients (P=0.023). High mRNA expression of cyclin D1 (CCND1) (P<0.001), fms related tyrosine kinase 1 (FLT1) (P=0.004), plasminogen (PLG) (P<0.001) and von Willebrand factor (VWF) (P=0.008) appeared to serve as favorable factors in survival. These findings indicate that the DEGs may be key genes in ccRCC pathogenesis and five genes, including ENO2, CCND1, PLT1, PLG and VWF, may serve as potential prognostic biomarkers in ccRCC.
Biomarkers for the prediction of mortality and morbidity in patients with renal replacement therapy
(2011)
The mortality of end-stage renal disease (ESRD) patients on dialysis remains high despite great improvement of dialysis technologies in the past decades.
These patients die due to infectious diseases (mainly sepsis), cardiovascular diseases such as myocardial infarction, heart failure, stroke, and, in particular, sudden cardiac death. End stage renal disease is a complex condition, where the failure of kidney function is accompanied by numerous metabolic changes affecting almost all organ systems of the human body. Many of the biomarker characteristics of the individually affected organ systems have been associated with adverse outcomes. These biomarkers are different in patients with ESRD compared to the general population in the prediction of morbidity and mortality. Biomarker research in this field should aim to identify patients at risk for the different disease entities.
Traditional biomarkers such as CRP, BNP, and troponins as well as new biomarkers such as fetuin, CD 154, and relaxin were analyzed in patients on dialysis. We will include observational as well as prospective clinical trials in this review. Furthermore, we will also discuss proteomics biomarker studies. The article assess the potential diagnostic value of different biomarkers in daily clinical practice as well as their usefulness for clinical drug development in end stage renal disease patients.
Background: Low birthweight is an independent risk factor of glucose intolerance and type 2 diabetes in later life. Genetically determined insulin resistance and subsequently impaired glucose uptake might explain both reduced fetal growth and elevated blood glucose. The glucose transporter 1 (GLUT!) plays an important role for fetal glucose uptake as well as for maternal-fetal glucose transfer, and it has been associated with insulin resistance in adults. The present study hypothesized that the common fetal GLUT1 XbaI polymorphism might reduce fetal insulin sensitivity and/or glucose supply in utero, thus affecting fetal blood glucose and fetal growth.
Methods: A genetic association study was conducted at the obstetrics department of the Charite University Hospital, Berlin, Germany. 119.1 white women were included after delivery, and all newborns were genotyped for the GLUT1 XbaI polymorphism. Total glycosylated hemoglobin was quantified, serving as a surrogate of glycemia during the last weeks of pregnancy.
Results: The analysis of this large population showed no significant differences in fetal glycosylated hemoglobin or birthweight for the different fetal GLUT1 XbaI genotypes. Only newborns carrying the mutated allele show the previously published inverse association between birthweight and glycosylated hemoglobin.
Conclusions: The results suggest that there is no prenatal effect of the fetal GLUT1 XbaI polymorphism on fetal insulin sensitivity, intrauterine fetal glucose supply or fetal growth. However, the polymorphism seems to modulate the inverse interaction between birthweight and fetal glycemia.
Background: Despite the beneficial effects of type 4 dipeptidyl peptidase (DPP-4) inhibitors on glucose levels, its effects on diabetic nephropathy remain unclear.
Method: This study examined the long-term renoprotective effects of DPP-4 inhibitor linagliptin in db/db mice, a model of type 2 diabetes.
Results were compared with the known beneficial effects of renin-angiotensin system blockade by enalapril. Ten-week-old male diabetic db/db mice were treated for 3 months with either vehicle (n = 10), 3 mg linagliptin/kg per day (n = 8), or 20 mg enalapril/kg per day (n = 10). Heterozygous db/m mice treated with vehicle served as healthy controls (n = 8). Results: Neither linagliptin nor enalapril had significant effects on the parameters of glucose metabolism or blood pressure in diabetic db/db mice. However, linagliptin treatment reduced albuminuria and attenuated kidney injury. In addition, expression of podocyte marker podocalyxin was normalized. We also analysed DPP-4 expression by immunofluorescence in human kidney biopsies and detected upregulation of DPP-4 in the glomeruli of patients with diabetic nephropathy, suggesting that our findings might be of relevance for human kidney disease as well.
Conclusion: Treatment with DPP-4 inhibitor linagliptin delays the progression of diabetic nephropathy damage in a glucose-independent and blood-pressure-independent manner. The observed effects may be because of the attenuation of podocyte injury and inhibition of myofibroblast transformation.
Background: Previous studies have shown that BMP4 may play an important part in the development of auditory neurons (ANs), which are degenerated in sensorineural hearing loss. However, whether BMP4 can promote sensory fate specification from mesenchymal stromal cells (MSCs) is unknown so far.
Methods: MSCs isolated from Sprague-Dawley (SD) rats were confirmed by expression of MSC markers using flow cytometry and adipogenesis/osteogenesis using differentiation assays. MSCs treated with a complex of neurotrophic factors (BMP4 group and non-BMP4 group) were induced into auditory neuron-like cells, then the differences between the two groups were analyzed in morphological observation, cell growth curve, qRT-PCR, and immunofluorescence.
Results: Flow cytometric analysis showed that the isolated cells expressed typical MSC surface markers. After adipogenic and osteogenic induction, the cells were stained by oil red O and Alizarin Red. The neuronal induced cells were in the growth plateau and had special forms of neurons. In the presence of BMP4, the inner ear genes NF-M, Neurog1, GluR4, NeuroD, Calretinin, NeuN, Tau, and GATA3 were up-regulated in MSCs.
Conclusions: MSCs have the capacity to differentiate into auditory neuron-like cells in vitro. As an effective inducer, BMP4 may play a key role in transdifferentiation.
The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and antiinflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.
To identify genetic variants associated with head circumference in infancy, we performed a meta-analysis of seven genome-wide association studies (GWAS) (N = 10,768 individuals of European ancestry enrolled in pregnancy and/or birth cohorts) and followed up three lead signals in six replication studies (combined N = 19,089). rs7980687 on chromosome 12q24 (P = 8.1 x 10(-9)) and rs1042725 on chromosome 12q15 (P = 2.8 x 10(-10)) were robustly associated with head circumference in infancy. Although these loci have previously been associated with adult height(1), their effects on infant head circumference were largely independent of height (P = 3.8 x 10(-7) for rs7980687 and P = 1.3 x 10(-7) for rs1042725 after adjustment for infant height). A third signal, rs11655470 on chromosome 17q21, showed suggestive evidence of association with head circumference (P = 3.9 x 10(-6)). SNPs correlated to the 17q21 signal have shown genome-wide association with adult intracranial volume(2), Parkinson's disease and other neurodegenerative diseases(3-5), indicating that a common genetic variant in this region might link early brain growth with neurological disease in later life.
During aging, intracranial volume remains unchanged and represents maximally attained brain size, while various interacting biological phenomena lead to brain volume loss. Consequently, intracranial volume and brain volume in late life reflect different genetic influences. Our genome-wide association study (GWAS) in 8,175 community-dwelling elderly persons did not reveal any associations at genome-wide significance (P < 5 x 10(-8)) for brain volume. In contrast, intracranial volume was significantly associated with two loci: rs4273712 (P = 3.4 x 10(-11)), a known height-associated locus on chromosome 6q22, and rs9915547 (P = 1.5 x 10(-12)), localized to the inversion on chromosome 17q21. We replicated the associations of these loci with intracranial volume in a separate sample of 1,752 elderly persons (P = 1.1 x 10(-3) for 6q22 and 1.2 x 10(-3) for 17q21). Furthermore, we also found suggestive associations of the 17q21 locus with head circumference in 10,768 children (mean age of 14.5 months). Our data identify two loci associated with head size, with the inversion at 17q21 also likely to be involved in attaining maximal brain size.
Background/Aims: Impaired birth outcomes, like low birth weight, have consistently been associated with increased disease susceptibility to hypertension in later life. Alterations in the maternal or fetal metabolism might impact on fetal growth and influence birth outcomes. Discerning associations between the maternal and fetal metabolome and surrogate parameters of fetal growth could give new insight into the complex relationship between intrauterine conditions, birth outcomes, and later life disease susceptibility. Methods: Using flow injection tandem mass spectrometry, targeted metabolomics was performed in serum samples obtained from 226 mother/child pairs at delivery. Associations between neonatal birth weight and concentrations of 163 maternal and fetal metabolites were analyzed. Results: After FDR adjustment using the Benjamini-Hochberg procedure lysophosphatidylcholines (LPC) 14:0, 16:1, and 18:1 were strongly positively correlated with birth weight. In a stepwise linear regression model corrected for established confounding factors of birth weight, LPC 16: 1 showed the strongest independent association with birth weight (CI: 93.63 - 168.94; P = 6.94x10(-11)). The association with birth weight was stronger than classical confounding factors such as offspring sex (CI: - 258.81- -61.32; P = 0.002) and maternal smoking during pregnancy (CI: -298.74 - -29.51; P = 0.017). Conclusions: After correction for multiple testing and adjustment for potential confounders, LPC 16:1 showed a very strong and independent association with birth weight. The underlying molecular mechanisms linking fetal LPCs with birth weight need to be addressed in future studies. (c) 2018 The Author(s) Published by S. Karger AG, Basel
Background/Aims: Impaired birth outcomes, like low birth weight, have consistently been associated with increased disease susceptibility to hypertension in later life. Alterations in the maternal or fetal metabolism might impact on fetal growth and influence birth outcomes. Discerning associations between the maternal and fetal metabolome and surrogate parameters of fetal growth could give new insight into the complex relationship between intrauterine conditions, birth outcomes, and later life disease susceptibility. Methods: Using flow injection tandem mass spectrometry, targeted metabolomics was performed in serum samples obtained from 226 mother/child pairs at delivery. Associations between neonatal birth weight and concentrations of 163 maternal and fetal metabolites were analyzed. Results: After FDR adjustment using the Benjamini-Hochberg procedure lysophosphatidylcholines (LPC) 14:0, 16:1, and 18:1 were strongly positively correlated with birth weight. In a stepwise linear regression model corrected for established confounding factors of birth weight, LPC 16: 1 showed the strongest independent association with birth weight (CI: 93.63 - 168.94; P = 6.94x10(-11)). The association with birth weight was stronger than classical confounding factors such as offspring sex (CI: - 258.81- -61.32; P = 0.002) and maternal smoking during pregnancy (CI: -298.74 - -29.51; P = 0.017). Conclusions: After correction for multiple testing and adjustment for potential confounders, LPC 16:1 showed a very strong and independent association with birth weight. The underlying molecular mechanisms linking fetal LPCs with birth weight need to be addressed in future studies. (c) 2018 The Author(s) Published by S. Karger AG, Basel
Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot
(2014)
Background: Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein.
Methods: In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP).
Results: Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04 - 5.71%) as assessed by coefficient of variation.
Conclusions: ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.
Background: Cardiovascular diseases are the leading cause of death in developed countries. The underlying mechanism is often atherosclerotic remodeling of blood vessels in organs such as heart, kidney, brain, and large arteries in case of peripheral arterial disease. Beside environmental and behavioral factors such as smoking or lack of physical activity, genetic variants in genes involved in lipid metabolism, blood pressure regulation, oxidative stress, and coagulation play a prominent role in the pathogenesis of atherosclerosis.
Methods: Thus, we developed and validated for clinical use and research a macroarray system for the simultaneous detection of key genetic variants in genes involved in lipid metabolism, blood pressure regulation, oxidative stress, and coagulation.
Results: When compared with standard PCR technologies to determine all these genetic variants in parallel, the macroarray system (MutaCHIP (R) ARTERO) was as accurate but faster, cheaper, and easier to handle compared to classical real time PCR based technologies.
Conclusions: MutaCHIP (R) ARTERO is a gene chip for diagnostics of a complex genetic panel involved in the pathogenesis of atherosclerosis. This method is as sensitive and precise as real time PCR and is able to replicate real time PCR data previously validated in evaluation studies.
The concept of developmental origins of diseases has gained a huge interest in recent years and is a constantly emerging scientific field. First observations hereof originated from epidemiological studies, linking impaired birth outcomes to adult chronic, noncommunicable disease. By now there is a considerable amount of both epidemiological and experimental evidence highlighting the impact of early life events on later life disease susceptibility. Albeit far from being completely understood, more recent studies managed to elucidate underlying mechanisms, with epigenetics having become almost synonymous with developmental programming. The aim of this review was to give a comprehensive overview of various aspects and mechanisms of developmental origins of diseases. Starting from initial research foci mainly centered on a nutritionally impaired intrauterine environment, more recent findings such as postnatal nutrition, preterm birth, paternal programming and putative interventional approaches are summarized. The review outlines general underlying mechanisms and particularly discusses mechanistic explanations for sexual dimorphism in developmental programming. Furthermore, novel hypotheses are presented emphasizing a non-mendelian impact of parental genes on the offspring's phenotype.
Efficacy, Safety & Modification of Albuminuria in Type 2 Diabetes Subjects with Renal Disease with LINAgliptin (MARLINA-T2D), a multicentre, multinational, randomized, double-blind, placebo-controlled, parallel-group, phase 3b clinical trial, aims to further define the potential renal effects of dipeptidyl peptidase-4 inhibition beyond glycaemic control. A total of 350 eligible individuals with inadequately controlled type 2 diabetes and evidence of renal disease are planned to be randomized in a 1:1 ratio to receive either linagliptin 5mg or placebo in addition to their stable glucose-lowering background therapy for 24weeks. Two predefined main endpoints will be tested in a hierarchical manner: (1) change from baseline in glycated haemoglobin and (2) time-weighted average of percentage change from baseline in urinary albumin-to-creatinine ratio. Both endpoints are sufficiently powered to test for superiority versus placebo after 24weeks with =0.05. MARLINA-T2D is the first of its class to prospectively explore both the glucose- and albuminuria-lowering potential of a dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes and evidence of renal disease.
Efficacy, Safety & Modification of Albuminuria in Type 2 Diabetes Subjects with Renal Disease with LINAgliptin (MARLINA-T2D), a multicentre, multinational, randomized, double-blind, placebo-controlled, parallel-group, phase 3b clinical trial, aims to further define the potential renal effects of dipeptidyl peptidase-4 inhibition beyond glycaemic control. A total of 350 eligible individuals with inadequately controlled type 2 diabetes and evidence of renal disease are planned to be randomized in a 1:1 ratio to receive either linagliptin 5mg or placebo in addition to their stable glucose-lowering background therapy for 24weeks. Two predefined main endpoints will be tested in a hierarchical manner: (1) change from baseline in glycated haemoglobin and (2) time-weighted average of percentage change from baseline in urinary albumin-to-creatinine ratio. Both endpoints are sufficiently powered to test for superiority versus placebo after 24weeks with =0.05. MARLINA-T2D is the first of its class to prospectively explore both the glucose- and albuminuria-lowering potential of a dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes and evidence of renal disease.
CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.
Background: Blood pressure (BP) regulation during pregnancy is influenced by hormones of placental origin. It was shown that the glucocorticoid system is altered in hypertensive pregnancy disorders such as preeclampsia. Epigenetic mechanism might influence the activity of genes involved in placental hormone/hormone receptor synthesis/action during pregnancy. Method: In the current study, we analyzed the association of 50-C-phosphate-G-30 (CpG) site methylation of different glucocorticoid receptor gene (NR3C1) promoter regions with BP during pregnancy. The study was performed as a nested case-control study (n = 80) out of 1045 mother/ child pairs from the Berlin Birth Cohort. Placental DNA was extracted and bisulfite converted. Nested PCR products from six NR3C1 proximal promoter regions [glucocorticoid receptor gene promotor region B (GR-1B), C (GR-1C), D (GR-1D), E (GR-1E), F (GR-1F), and H (GR-1H)] were analyzed by next generation sequencing. Results: NR3C1 promoter regions GR-1D and GR-1E had a much higher degree of DNA methylation as compared to GR-1B, GR-1F or GR-1H when analyzing the entire study population. Comparison of placental NR3C1 CpG site methylation among hypotensive, normotensive and hypertensive mothers revealed several differently methylated CpG sites in the GR-1F promoter region only. Both hypertension and hypotension were associated with increased DNA methylation of GR-1F CpG sites. These associations were independent of confounding factors, such as family history of hypertension, smoking status before pregnancy and prepregnancy BMI. Assessment of placental glucocorticoid receptor expression by western blot showed that observed DNA methylation differences were not associated with altered levels of placental glucocorticoid receptor expression. However, correlation matrices of all NR3C1 proximal promoter regions demonstrated different correlation patterns of intraregional and interregional DNA methylation in the three BP groups, putatively indicating altered transcriptional control of glucocorticoid receptor isoforms. Conclusion: Our study provides evidence of an independent association between placental NR3C1 proximal promoter methylation and maternal BP. Furthermore, we observed different patterns of NR3C1 promoter methylation in normotensive, hypertensive and hypotensive pregnancy.
Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.
Background: The need for an improved treatment for diabetic nephropathy is greatest in patients who do not adequately respond to angiotensin II receptor blockers (ARBs). This study investigated the effect of the novel dipeptidyl peptidase-4 inhibitor linagliptin alone and in combination with the ARB telmisartan on the progression of diabetic nephropathy in diabetic endothelial nitric oxide synthase (eNOS) knockout mice. Methods: Sixty male eNOS knockout C57BL/6J mice were divided into four groups after receiving intraperitoneal high-dose streptozotocin: telmisartan (1 mg/kg), linagliptin (3 mg/kg), linagliptin + telmisartan (3 mg/kg + 1 mg/kg) and vehicle. Fourteen mice were used as non-diabetic controls. Results: After 12 weeks, urine and blood were obtained and blood pressure measured. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan alone reduced systolic blood pressure by 5.9 mmHg versus diabetic controls (111.2 +/- 2.3 mmHg vs 117.1 +/- 2.2 mmHg; mean +/- SEM; P = 0.071). Combined treatment significantly reduced albuminuria compared with diabetic controls (71.7 +/- 15.3 mu g/24 h vs 170.8 +/- 34.2 mu g/24 h; P = 0.017), whereas the effects of single treatment with either telmisartan (97.8 +/- 26.4 mu g/24 h) or linagliptin (120.8 +/- 37.7 mu g/24 h) were not statistically significant. DPP-4 inhibition, alone and in combination, led to significantly lower plasma osteopontin levels compared with telmisartan alone. Histological analysis revealed reduced glomerulosclerosis after Linagliptin alone and in combination with telmisartan in comparison to non treated diabetic animals (p < 0.01 and p < 0.05). Kidney malonaldehyde immune-reactivity, a marker of oxidative stress, was significantly lower in animals treated with linagliptin. Conclusions: DPP-4 inhibition on top of ARB treatment significantly reduced urinary albumin excretion and oxidative stress in diabetic eNOS knockout mice. Linagliptin on top of an angiotensin II receptor blocker may offer a new therapeutic approach for patients with diabetic nephropathy.
DPP-4 inhibition with linagliptin delays the progression of diabetic nephropathy in db/db mice
(2012)
DPP4 inhibition prevents AKI
(2017)
Secondary activation of the endothelin system is thought to be involved in toxic liver injury. This study tested the hypothesis that dual endothelin-converting enzyme / neutral endopeptidase blockade might: be able to attenuate acute toxic liver injury.
Male Sprague-Dawley rats were implanted with subcutaneous minipumps to deliver the novel compound SLV338 (10 mg/kg*d) or vehicle. Four days later they received two intraperitoneal injections of D-galactosamine (1.3 g/kg each) or vehicle at an interval of 12 hours. The animals were sacrificed 48 hours after the first injection.
Injection of D-galactosamine resulted in very severe liver injury, reflected by strongly elevated plasma liver enzymes, hepatic necrosis and inflammation, and a mortality rate of 42.9 %. SLV338 treatment did not show any significant effect on the extent of acute liver injury as judged from plasma parameters, hepatic histology and mortality. Plasma measurements of SLV338 confirmed adequate drug delivery. Plasma concentrations of big endothelin-1 and endothelin-1 were significantly elevated in animals with liver injury (5-fold and 62-fold, respectively). Plasma endothelin-1 was significantly correlated with several markers of liver injury. SLV338 completely prevented the rise of plasma big endothelin-1 (p<0.05) and markedly attenuated the rise of endothelin-1 (p = 0.055).
In conclusion, dual endothelin-converting enzyme / neutral endopeptidase blockade by SLV338 did not significantly attenuate D-galactosamine-induced acute liver injury, although it largely prevented the activation of the endothelin system. An evaluation of SLV338 in a less severe model of liver injury would be of interest, since very severe intoxication might not be relevantly amenable to pharmacological interventions.
Background: As the prevalence of diabetes rises, its complications such as diabetic nephropathy affect an increaseing number of patients. Consequently, the need for biomarkers in rodent models which reflect the stage and course of diabetic nephropathy is high. This article focuses on Heart-type fatty acid binding protein (H-FABP), osteopontin (OPN), nephrin, and Neutrophil gelatinase-associated lipocalin (NGAL) in urine, and kidney injury molecule (KIM)-1, clusterin, and tissue inhibitior of metalloproteinases (TIMP) 1 in plasma in uni-nephrectomized rats with streptocotozin-induced type 1 diabetes mellitus, a common animal model to explore renal impairment in the setting of diabetes mellitus.
Methods: 23 male Wistar rats were uni-nephrectomized and subsequently divided into two study groups. The diabetic group received streptozotocin (STZ) via tail-vein injection, the non-diabetic group received citrate buffer without STZ. Subsequently, blood glucose, body weight, and blood pressure were checked regularly. After 18 weeks, animals were placed in metabolic cages, blood and urine obtained and subsequently organs were harvested after sacrifice.
Results: Blood glucose levels were highly increased in diabetic animals throughout the experiment, whereas systolic blood pressure did not differ between the study groups. At study end, classical biomarkers such as urinary albumin and protein and plasma cystatin c were only slightly but not significantly different between groups indicating a very early disease state. In contrast, urinary excretion of H-FABP, OPN, nephrin, and NGAL were highly increased in diabetic animals with a highly significant p-value (p<0.01 each) compared to non-diabetic animals. In plasma, differences were found for calbindin, KIM-1, clusterin, TIMP-1, and OPN. These findings were confirmed by means of the area under the receiver operating characteristic curve (ROC-AUC) analysis.
Conclusions: In summary, our study revealed elevated levels of new plasma and urinary biomarkers (urinary osteopontin, urinary nephrin, urinary NGAL, urinary H-FABP, plasma KIM-1, plasma TIMP-1) in uni-nephrectomized diabetic rats, an established rat model of diabetic nephropathy. These biomarkers appeared even before the classical biomarkers of diabetic nephropathy such as albuminuria and urinary protein excretion. The new biomarkers might offer advantage to urinary albumin and plasma cystatin c with respect to early detection.
Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) by renal 25-hydroxyvitamin D-3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)(2)D-3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)(2)D-3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)(2)D-3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 mu M CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)(2)D-3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)(2)D-3 synthesis.
Background: Uremic cardiomyopathy contributes substantially to mortality in chronic kidney disease (CKD) patients. Glucagon-like peptide-1 (GLP-1) may improve cardiac function, but is mainly degraded by dipeptidyl peptidase-4 (DPP-4).
Methodology/Principal Findings: In a rat model of chronic renal failure, 5/6-nephrectomized [5/6N] rats were treated orally with DPP-4 inhibitors (linagliptin, sitagliptin, alogliptin) or placebo once daily for 4 days from 8 weeks after surgery, to identify the most appropriate treatment for cardiac dysfunction associated with CKD. Linagliptin showed no significant change in blood level AUC(0-infinity) in 5/6N rats, but sitagliptin and alogliptin had significantly higher AUC(0-infinity) values; 41% and 28% (p=0.0001 and p=0.0324), respectively. No correlation of markers of renal tubular and glomerular function with AUC was observed for linagliptin, which required no dose adjustment in uremic rats. Linagliptin 7 mu mol/kg caused a 2-fold increase in GLP-1 (AUC 201.0 ng/l*h) in 5/6N rats compared with sham-treated rats (AUC 108.6 ng/l*h) (p=0.01). The mRNA levels of heart tissue fibrosis markers were all significantly increased in 5/6N vs control rats and reduced/normalized by linagliptin.
Conclusions/Significance: DPP-4 inhibition increases plasma GLP-1 levels, particularly in uremia, and reduces expression of cardiac mRNA levels of matrix proteins and B-type natriuretic peptides (BNP). Linagliptin may offer a unique approach for treating uremic cardiomyopathy in CKD patients, with no need for dose-adjustment.
Background/Aims: Excess maternal salt intake during pregnancy may alter fetal development. However; our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. Methods: 5.5 day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high salt treatment. RT-PCR analyses were conducted from chick retina tissues. Results: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high salt treatment. High salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt treatment was detrimental to the migration of neural crest cells. Conclusion: Taken together; our study demonstrated that high salt exposure of 5.5 day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression.
The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2 +/- 2.3 mmHg vs. 117.1 +/- 2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2 +/- 2.5 mmHg and 105.0 +/- 3.2 mmHg, respectively, vs. 117.1 +/- 2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3 +/- 9.6 mu g/24 h vs. 170.8 +/- 34.2 mu g/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4 +/- 10.6 mu g/24 h), whereas the reduction by single treatment with either telmisartan (97.8 +/- 26.4 mu g/24 h) or riociguat (97.1 +/- 15.7 mu g/24 h) was not statistically significant. The combination treatment led to a significant (p < 0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.
Objective:To investigate the effects of linagliptin alone and in combination with the angiotensin II receptor blocker (ARB), telmisartan on blood pressure (BP), kidney function, heart morphology and oxidative stress in rats with renovascular hypertension.Methods:Fifty-seven male Wistar rats underwent unilateral surgical stenosis of the renal artery [2-kidney-1-clip (2k1c) method]. Animals were randomly divided into four treatment groups (n=14-18 per group) receiving: telmisartan (10mg/kg per day in drinking water), linagliptin (89ppm in chow), combination (linagliptin 89ppm+telmisartan 10mg/kg per day) or placebo. An additional group of 12 rats underwent sham surgery. BP was measured one week after surgery. Hypertensive animals entered a 16-week dosing period. BP was measured 2, 4, 8, 12 and 16 weeks after the initiation of treatment. Blood and urine were tested for assessment of kidney function and oxidative stress 6, 10, 14 and 18 weeks after surgery. Blood and urine sampling and organ harvesting were finally performed.Results:Renal stenosis caused an increase in meanSD systolic BP as compared with the sham group (157.7 +/- 29.3 vs. 106.2 +/- 20.5mmHg, respectively; P<0.001). Telmisartan alone and in combination with linagliptin, normalized SBP (111.1 +/- 24.3mmHg and 100.4 +/- 13.9mmHg, respectively; P<0.001 vs. placebo). Telmisartan alone and in combination with linagliptin significantly prevented cardiac hypertrophy, measured by heart weight and myocyte diameter. Renal function measured by cystatin C was not affected by 2k1c surgery. Telmisartan significantly increased plasma concentration of cystatin C. 2k1c surgery initiated fibrosis in both kidneys. Telmisartan promoted further fibrotic changes in the clipped kidney, as measured by protein expression of Col1a1 and histology for interstitial fibrosis and glomerulosclerosis. In non-clipped kidneys, telmisartan demonstrated antifibrotic properties, reducing Col1a1 protein expression. Plasma levels of oxidized low-density lipoprotein were higher in the placebo-treated 2k1c rats as compared to sham-operated animals. The increase was abolished by linagliptin alone (P=0.03 vs. placebo) and in combination with telmisartan (P=0.02 vs. placebo). Combination therapy also significantly reduced plasma concentration of carbonyl proteins (P=0.04 vs. placebo).Conclusion:Inhibition of type 4 dipeptidyl peptidase with linagliptin did not counter BP-lowering effects of ARB in 2k1c rats. Linagliptin reduced lipid and protein oxidation in 2k1c rats, and this effect was BP-independent.
Background: To assess the chronic effect of the DPP-4 inhibitor, linagliptin, alone, in combination with exenatide, and during exenatide withdrawal, in diet-induced obese (DIO) rats.
Methods: Female Wistar rats were exposed to a cafeteria diet to induce obesity. Animals were then dosed with vehicle or linagliptin (3 mg/kg PO) orally once-daily for a 28 day period. In a subsequent study, rats received exenatide (either 3 or 30 mu g/kg/day) or vehicle by osmotic mini-pump for 28 days. In addition, groups of animals were dosed orally with linagliptin either alone or in combination with a 3 mu g/kg/day exenatide dose for the study duration. In a final study, rats were administered exenatide (30 mu g/kg/day) or vehicle by osmotic mini-pump for eleven days. Subsequently, exenatide-treated animals were transferred to vehicle or continued exenatide infusion for a further ten days. Animals transferred from exenatide to vehicle were also dosed orally with either vehicle or linagliptin. In all studies, body weight, food and water intake were recorded daily and relevant plasma parameters and carcass composition were determined.
Results: In contrast to exenatide, linagliptin did not significantly reduce body weight or carcass fat in DIO rats versus controls. Linagliptin augmented the effect of exenatide to reduce body fat when given in combination but did not affect the body weight response. In rats withdrawn from exenatide, weight regain was observed such that body weight was not significantly different to controls. Linagliptin reduced weight regain after withdrawal of exenatide such that a significant difference from controls was evident.
Conclusions: These data demonstrate that linagliptin does not significantly alter body weight in either untreated or exenatide-treated DIO rats, although it delays weight gain after exenatide withdrawal. This finding may suggest the utility of DPP-4 inhibitors in reducing body weight during periods of weight gain.
All components of the endothelin (ET) system are present in renal tubular cells. In this review, we summarize current knowledge about ET and the most common tubular diseases: acute kidney injury (AKI) and polycystic kidney disease. AKI originally was called acute tubular necrosis, pointing to the most prominent morphologic findings. Similarly, cysts in polycystic kidney disease, and especially in autosomal-dominant polycystic kidney disease, are of tubular origin. Preclinical studies have indicated that the ET system and particularly ETA receptors are involved in the pathogenesis of ischemia-reperfusion injury, although these findings have not been translated to clinical studies. The ET system also has been implicated in radiocontrast-dye-induced AKI, however, ET-receptor blockade in a large human study was not successful. The ET system is activated in sepsis models of AKI; the effectiveness of ET blocking agents in preclinical studies is variable depending on the model and the ET-receptor antagonist used. Numerous studies have shown that the ET system plays an important role in the complex pathophysiology associated with cyst formation and disease progression in polycystic kidney disease. However, results from selective targeting of ET-receptor subtypes in animal models of polycystic kidney disease have proved disappointing and do not support clinical trials. These studies have shown that a critical balance between ETA and ETB receptor action is necessary to maintain structure and function in the cystic kidney. In summary, ETs have been implicated in the pathogenesis of several renal tubulointerstitial diseases, however, experimental animal findings have not yet led to use of ET blockers in human beings. (C) 2015 Elsevier Inc. All rights reserved.