MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidneyfibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFb exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulatingmiR-21levelsareassociatedwith renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/ TA). CirculatingmiR-21levelsare significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.061.0 vs lower grade of fibrosis: 1.561.2; p = 0.001). By contrast, circulatingmiR-21levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulatingmiR-21levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21levels and aMDRD (ß = 20.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidneyfibrosis and may represent a novel, predictive and reliable blood marker of kidneyfibrosis.
It is important to realize that endothelial dysregulation starts in the early stage of CKD (18) and is a common event identified in both chronic and acute renal failure and in ESRD (9). In addition, studies have suggested that inflammation exists in patients with ESRD and might be aggravated by hemodialysis (19). Similarly, recent stud- ies have demonstrated that miRNA can regulate vascular inflammation, endothelial homeostasis, and angiogenesis. In particular, miR-126 and miR-155 have been shown to be involved in vascular dysfunction and inflammation. MiR-126 is highly enriched in ECs and endothelial apoptotic bodies and governs the maintenance of vascular integrity and angiogenesis (20,21). Moreover, miR-126 can facilitate vascular endothelial growth factor signaling pathway (11) and modify vascular inflammation by suppressing leuko- cyte adhesion to ECs. MiR-155 regulates the expression of adhesion molecules in inflammatory ECs as well as inflammation response in ECs mediated by and angio- tensin α (22). Another study revealed that miR-155 and angiotensin α type 1 receptor (AT1R) are co-expressed in ECs and vascular smooth-muscle cells (23) and their expression is negatively correlated with the expression of the endogenous AT1R (24). A silent polymorphism (+1166 A/C) in the human AT1R has been shown to be associatedwith cardiovascular disease (CVD), which might be medi- ated by enhanced AT1R activity (23). Therefore, miR-126 and miR-155 might be responsible for endothelial activation and increased CVD risk in ESRD patients (25).
Metabolic syndrome along with the individual cardiometabolic conditions associated to it, represent the most prevalent diseases in adults , however growing evidence shows that the presence of MS traits in children and adolescents associates to increased risk of developing car- diometabolic diseases in adult life . There is no consensus regarding the diagnose of MS in pediatric population, mainly due to the absence of clear cut-off values for the classical parame- ters used for its diagnosis in adults [1, 3]. In this study, using Cook´s et al. criteria, we evaluated in children the association of different MS traits with plasma levels of miRNAs that have been reported altered in adults with cardiometabolic dysfunction. In order to provide insight on new markers associated to the risk of developing MS, we aimed to determine the plasma levels of specific miRNA in children with altered cardiometabolic traits. The strength of plasma miR- NAs as molecular markers relies on several characteristics of these molecules. First, the stability of miRNAs in plasma is comparable to proteins, and higher than mRNAs, which in combina- tion with the use of quantitative PCR on their detection rises the possibility to measure small changes in their levelswith higher precision than proteins and other biomarkers [5, 6]. At cel- lular level it is known that a single miRNA can control the expression of several mRNA and the transcriptional activity of genes [19, 27], which in most of the cases affects the gene expression of specific molecular pathways . Additionally part of plasma miRNAs circulate associated to vesicles with potential endocrine effects [29, 30] which is nowadays under study . In spite of the fact that the precise role of circulating miRNAs is unknown, evidence shows that their levelsare altered in several pathological conditions, and they change in response to pharmaco- logical treatments earlier than routine clinical markers [31, 32]. These characteristics have led to propose the use of circulating miRNA as molecular markers in cancer, metabolic disorders and cardiovascular diseases [5, 6, 33].
Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. CirculatingmiR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associatedwith breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levelsare even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR- 127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).
Tissue miRNA levels were associatedwith various clinico-pathological features (Fig 4). Tis- sue miR-378, miR-3200, miR-422a, miR-181, miR-1274a, miR-378c, and miR-18a were signifi- cantly associatedwith ER expression (P0.05). Specifically, we observed a 6-fold increase in the differential expression of miR-378 and miR-181. Previous studies have shown that miR- 378 promotes cell survival by decreasing caspase-3 activity and stimulates tumor progression and ER positivity, which are consistent with our findings . Previous studies have also found a tumor-suppressive role of miR-422a and its up-regulation in ER-positive inflammatory breast cancer, which is also consistent with our findings (Fig 4)[30, 31]. Tissue miR-21 was previously shown to be significantly associatedwith tumor local and distant recurrence and exhibits an oncogenic function in which its suppression results in enhanced sensitivity to anticancer agents. Similarly, It was shown that Serum miR-21 may be an independent poor prognostic factor for recurrence , and high stromal miR-21 expression is associatedwith significantly shorter recurrence-free survival in patients with colorectal cancer . We found that tissue miR-451 was up-regulated more than 5× and significantly associatedwith pathological stage (Fig 3). Previous studies have demonstrated that miR-451 regulates the expression of multidrug resistance 1 gene, and transfection of MCF-7/DOX-resistant cells withmiR-451 results in increased sensitivity to chemotherapy .
and lipid metabolism leading to the increase in free cholesterol associatedwith cirrhosis. A recent paper by Chatterjee et al. examined the effect of inhibiting glycosphingolipid synthesis on atherosclerosis and arterial stiffness . They observed that inhibition of glycosphingoli- pids synthesis resulted in a decrease in monohexosyl- and dihexosylceramide in the liver and this was associatedwith a decrease in serum cholesterol and triglycerides via recruitment of multiple genes/pathways of lipid metabolism. Specifically, inhibition of glycosphingolipids syn- thesis increased LDL receptor and SREBP2 gene expression and decreased HMGCoA reductase gene expression, suggesting inhibition of cholesterol biosynthesis and increased LDL uptake. The decreased monohexosyl- and dihexosylceramide were also associatedwithincreased expression of genes responsible for cholesterol efflux including ABCA1, ABCG5 and ABCG8. This inhibitory effect of lactosylceramide (a dihexosylceramide) in cholesterol efflux via the ABCA1/Apolipiprotein A-1 pathway has also been demonstrated in cultured fibroblasts . The decreased levels of lactosylceramide were also associatedwith the up regulation of the Cyp7A1 gene which encodes a 7-hydroxylase enzyme that converts cholesterol to bile acid for excretion . Thus, lactosylceramide appears to influence multiple lipid metabolic pathways leading to elevated cholesterol within the liver. Elevated cholesterol within hepatic stellate cells has been shown to increase fibrosis in ACAT-1 deficient mice  further supporting a causal role for lipid metabolism in ALC. Dihexosylceramide then, is emerging as a key mediator of fibrosis and may play an important role in the regulation of cholesterol metabolism leading to cirrhosis. This raises the dual possibility of utilising these lipids as early indicators of cirrhosis and of targeting this metabolic pathway to prevent progression to cirrhosis. It should be noted that while the underlying dysregulation of lipid metabolism likely plays an important role in ALC, whether these metabolic changes are also present in other forms of cirrhosis remains to be determined.
The current study is the first examination of rural-urban differences in circulating irisin lev- els. We have shown that rural inhabitants display higher circulating irisin levels than urban cit- izens, both in men and women. For this reason, the influence of rural/urban location prompted us to explore the irisin levels according to the physical activity in each area. Our results show that the increase in circulating irisin levels related to an active lifestyle was only observed in rural citizens and no differences were found in urban or medium inhabitants. However, it has been reported, in diverging populations, the increased cardiometabolic risk in urban popula- tions, suggesting that a good health profile appears significantly more widespread among those living in rural areas [21, 22]. Therefore, besides the augmented cardiovascular risk factors in urban areas, we cannot rule out environmental differences such as food habits or pollution lev- els between rural and urban settings that might be influencing circulating irisin concentrations. In this regard, persistent organic pollutants have been reported to impair the adipokine axis or fat metabolism, resulting in inappropiated low adiponectin levels [23, 24]. Apart from the
Serum FGF-21levels were found to closely related to lipid metabolism. A recent study has already demonstrated that serum triglycerides and total cholesterol were independently associatedwith FGF-21 . In this study, we found that apolipoprotein A1 was negatively correlated with FGF-21 concentration. Apolipo- protein A1 is the major protein component of the high-density lipoprotein (HDL) particles, which promotes cholesterol efflux from tissues to the liver for excretion. Studies have shown inverse associations between apoA1 levels and CHD [26,27], and enhancing ApoA1 is preventive against CHD. FGF21 treatment provides beneficial changes in some lipoproteins and cardiovas- cular risk factors in rodents and nonhuman primates. However, it does not change circulating ApoA1 levels . The correlation between FGF21 and ApoA1 in CHD deserves further studies.
Potential limitations still remain in this study. First, our study was a cross sectional but not a longitudinal study. Second, 81 men in our study voluntarily underwent measurements: they were therefore more likely to be health-conscious as compared with the average person. Third, we could not evaluate in women and also could not show a clear mechanism between serum IL-18 levels and physical activity. However, it seems reasonable to suggest that promoting physical activity might result in reducing serum IL-18 levels in some healthy Japanese men. To show this, further prospective and larger sample size studies are urgently required in the Japanese population.
To corroborate the CEC findings, we analyzed VEGF gene expression. This growth factor is an important mediator of angiogenesis and is involved in the patho- genesis of myeloid malignancies, including CML (19). However, there was an increase of VEGF in all phases of CML. Our results are consistent with previous studies demonstrating that the malignant cells of CML over- express VEGF. Thus, this marker has not proven useful to discriminate the different stages of CML. However, we observed a significant increase in VEGF in myeloid BC compared to lymphoid BC, corroborating the results reported by Krauth et al. (19). Those authors suggest Table 1. Minimum and maximum percentages of CECs and
Comparison of HBP levels between the different predefined diagnosis-groups (sepsis, trauma, intoxication, cardiac arrest, gastrointestinal bleeding, and other surgical or other medi- cal) was made by one-way ANOVA. A post hoc analysis with corrections for multiple compari- sons according to Bonferroni was made to find out if any groups were significantly different from any other group. For this analysis, we used the log-transformed value of admission-HBP but the actual (non-normally distributed) measured values are shown in.
Animals with reduced GFR were observed at all time points. UO was higher during fluid therapy, and polyuria or oliguria were identified at all time points (Table 3). HEIENE et al. (2001) evaluated 55 female dogs with pyometra and reported lower average GFR’s in late assessments as compared to values obtained 24h after ovariohysterectomy. This is not in agreement with the present study. The predominance of animals with reduced GFR at T4 and T5 was also observed by VERSTEGEN et al. (2008), who stated that even among non-azotemic and rehydrated female dogs with pyometra, GFR is often decreased. This suggested that other factors, such as glomerulonephritis, can alter renal perfusion. An improvement in GFR was observed at T6 and T7, suggesting restoration of kidney function in most animals although, the presence of any animals with changes in this parameter indicated the need for long term monitoring and treatment after surgery. Among the animals with reduced GFR at T6 and/or T7, only 50% showed increased phosphorus levels and only 25% showed increased creatinine levels, demonstrating greater sensitivity of GFR in detecting changes in kidney excretory function (CHEW et al., 2011).
Uromodulin (Tamm-Horsfall protein; Uniprot P07911), is an 80–90 kDa glycoprotein, exclusively synthesized by the thick ascending limb (TAL) and early distal convoluted tubule (DCT) in the kidney [1,2]. Uromodulin gene (UMOD) mutations have been confirmed in patients with familial juvenile hyperuricemia nephropathy (FJHN, OMIM 162000), medullary cystic kidney disease 2 (MCKD2, OMIM 603860) and glomerulocystic kidney disease (GCKD, OMIM 609886). Hyperuricemia, hypertension, decreased urinary uromodulin levels, tubulointerstitial nephropa- thy and progressive kidney disease are characteristics of these diseases [3,4]. Genome-wide association studies have also revealed that common variants in the promoter region of the UMOD gene areassociatedwith chronic kidney disease (CKD), glomerular filtration rate (GFR), kidney stone formation and hypertension [5,6,7,8]. In a previous study, UMOD ablation mice presented more inflammation and tubular necrosis in the face of ischemia- reperfusion injury, suggesting that uromodulin stabilizes the outer medulla of the kidney . However it is still unclear whether uromodulin influences the progression of chronic kidney disease.
Platelet characteristics and haematological parameters. Blood samples for haematological analyses were collected in 3.0 mL tubes containing EDTA (Terumo, Leuven, Belgium). In order to minimize and standardize time-dependent swelling of platelets, haematological analyses were performed within 60 minutes of blood sampling. Haematology parameters were measured using the Sysmex XE-2100 hematology analyser (Sysmex, Kobe, Japan) with upgraded software (XE IPF Master, Sysmex) allowing flow cytometric detection of immature platelets as previously described [2,20]. In brief, platelet RNA was stained with flourescent dyes (polymethine and oxazine) before stained cells were passed through a semiconductor diode laser beam. The resulting flourescence intensity (RNA content) and forward light scatter (cell volume) were measured and a preset gate (XE IPF Master software, Sysmex) discriminated between mature and immature platelets. Absolute immature platelet count (IPC) was obtained, and immature platelet fraction (IPF) was calculated as the ratio of immature platelets to the total platelet count and reported in percent. Platelet volume parameters were derived from the platelet volume distribution. MPV was calculated by dividing the platelet crit by platelet impedance count. Platelet distribution width (PDW), a measure of platelet anisocytosis, was the width of the size distribution curve in femtoliters (fL) at the 20% level of the peak. The platelet large cell ratio (P-LCR) was defined as the number of cells falling above the 12-fL threshold divided by platelet count.
A total of 44 patients (63.6% males) with a mean age of 36.1 ± 10.6 years old were selected. Additionally, 15 healthy patients were selected as controls - mean age 35.4 ± 9.2. Time from symptom onset to leprosy diagno- sis ranged from one month to 8 years, with a median time of 17 months. Twenty-six patients had positive skin-smear test (multibacillary – MB) and 18 were pau- cibacillary - PB. Clinically, there were 14 TT/BT, 19 BB and 11 LL/BL leprosy patients. Regarding renal function, no patient had chronic kidney disease according to Kidney Disease Outcomes Quality Initiative definition - glomerular filtration rate higher than 60 mL/min/1.73 m  and albu- min excretion rate less than 30 mg/g-Cr.
HbA1c levels were measured using a high performance liquid chromatography system (HLC-723G7; Tosoh Co., Tokyo, Japan). In the present study, the participants were divided into three groups according to their HbA1c levels: a Low group (<5.7% or <39 mmol/mol), a Middle group (5.7–6.0% or 39–42 mmol/mol), and a High group (>6.0% or >42 mmol/mol). Serum creatinine levels were measured using a Hitachi Automatic Analyzer (alkaline picrate, Jaffé kinetic). The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation . CKD was defined as an eGFR <60 mL/min/1.73 m 2 . Urine albumin level was measured from random samples using a turbidimetric immunoassay (Hitachi Automatic Analyzer 7600, Hitachi). Urine creatinine level was measured using a colorimetric method (Hitachi Automatic Analyzer 7600, Hitachi). Urine albumin and creatinine concentrations were measured in the same laboratory for all sur- veys. The inter-assay coefficient of variation for all laboratory work was consistenly low (<3.1%). The urine albumin-creatinine ratio (UACR) was calculated in mg per g of creatinine (mg/g). Albuminuria was defined as UACR 30 mg/g.
The interconnection between immune and neuroendocrine systems influences regulation of inflamma- tory responses. The possible relevance that this integrative response may have during the course of Cha- gas disease remains poorly characterized. In this context, our study was designed to determine the expression of vasoactive intestinal peptide (VIP), a neuropeptide with anti-inflammatory properties, in blood from the indeterminate and cardiac polarized forms of Chagas disease. Moreover, we determined whether the differential expression of VIP is associatedwith the development of cardiomyopathy in indi- viduals infected with Trypanosoma cruzi. Finally, we analyzed gene polymorphisms of VIP receptors, VPAC1 and VPAC2, and performed correlation analysis of these polymorphisms with the different clinical forms of Chagas disease. Our results demonstrated that low plasma levels of VIP were associatedwith the cardiac morbidity in Chagas disease. Accordingly, correlation analysis showed that low plasma levels of VIP were associatedwith worse cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Polymorphism analysis showed a significant association between VPAC1 and the indeterminate form of Chagas disease development. Our data indicate that VIP expression and its receptors’ polymorphism may be important in determining susceptibility to pro- gression from mild to severe forms of Chagas disease.
High fructose-induced insulin resistance is associatedwith an overproduction of superoxide anion in aorta and heart, and promotes cardiovascular alterations such as hypertension, vascular disorder and cardiac hypertrophy . Vascular and cardiac hypertrophy was associatedwith ROS generation at different times after the initiation of fructose-enriched diet . We observed significant increase in heart weight and body weight ratio, and higher expression of ANP and b-MHC in fructose fed diabetic rat heart. However, administration of raw garlic (rich with allicin) in fructose-fed rats normalised the cardiac hypertrophy along with hypertrophic gene expression. To confirm the presence of allicin we have done LC-MS study. Our data indicated that the raw garlic homogenate that used for the present study was rich with allicin and other compounds like c-glutamyl-S-allyl-L- cysteine, Alliin, S-Allyl-l-cysteine, deoxyalliin and Vinyldithiin. Previously, allicin, c-glutamyl-S-allyl-L-cysteine and S-allyl cyste- ine were reported to have antioxidant property by direct or indirect effect . However, the molecular mechanism behind the beneficial/antioxidant effect of raw garlic was not investigated in heart.
There are striking differences in chronic kidney disease between Caucasians and African descendants. It was widely accepted that this occurred due to socioeconomic factors, but recent studies show that apolipoprotein L-1 (APOL1) gene variants are strongly associatedwith focal segmental glomerulosclerosis, HIV- associated nephropathy, hypertensive nephrosclerosis, and lupus nephritis in the African American population. These variants made their way to South America trough intercontinental slave traffic and conferred an evolutionary advantage to the carries by protecting against forms of trypanosomiasis, but at the expense of an increased risk of kidney disease. The effect of the variants does not seem to be related to their serum concentration, but rather to local action on the podocytes. Risk variants are also important in renal transplantation, since grafts from donors with risk variants present worse survival.
logical actions . In fasting, FGF21 expression is induced by peroxisome proliferator-activated receptor α (PPARα) in the liver [15,16] and acts through endocrine mechanisms for adaptive starvation responses including gluconeogenesis, ke- togenesis, torpor, and inhibition of somatic growth [12,14,17]. In the fed state, FGF21 expression is induced by PPARγ in white adipose tissue and acts in an autocrine or paracrine fash- ion to increase PPARγ activity [14,18-20]. As a consequence, during feeding, the induction of FGF21 in white adipose tissue fails to increase circulatinglevels of FGF21 . Pharmaco- logical administration of FGF21 affects multiple organs and tissues including the pancreas, adipose tissue, liver, and the central nervous system . Systemic administration of FGF21 increases insulin sensitivity and energy expenditure, causing a loss of body weight and improvements in glucose and lipid metabolism in obese rodents and primates [10,11,22]. Furthermore, several reports have also shown that administra- tion of FGF21 results in a significant decrease in lipid accu- mulation in the liver of diet-induced obese mice , suggest- ing that FGF21 could be a promising drug candidate for treat- ment of metabolic syndrome, with benefits for many of the symptoms of this disease.