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Background PCOS appears to be associated with an increased risk of metabolic aberrations, including insulin resistance and hyperinsulinemia, type 2 diabetes mellitus, dyslipidemia, and cardiovascular disease throughout womens’ lifespan. Obesity is a state of chronic low-grade systemic inflammation. This chronic inflammation is characterized by abnormal cytokine production and activation of inflammatory signaling pathways in adipose tissues, which contributes to insulin resistance and its related diseases such as PCOS and metabolic syndrome. Objective To evaluate Lipocalin-2 level in a sample of Egyptian females with PCOS and study the effect of metformin therapy on Lipocalin-2 level in patients with PCOS. Methods This case control study was conducted on 52 females, collected from the outpatient obstetrics and gynaecology clinics of Ain Shams University Hospitals from June 2018 to March 2019, their age ranged between 17-43 years old. divided into 2 groups: Group (I) 32 women with polycystic ovary syndrome According to the Rotterdam diagnostic criteria of PCOS, and Group (II) 20 healthy women old with normal ovulatory cycle as a control group. All subjects were subjected to full medical history taking, thorough physical examination including BMI and waist circumference. Fasting plasma glucose, Fasting s.insulin, HOMA-IR, lipocalin-2 level were assessed. Results Serum lipocalin-2 levels did not differ between patients with PCOS and BMI-matched healthy controls (P-value 0.193), and there was no significant difference between the 2 studied groups as regard HOMA-IR (p = 0.375). Metformin therapy for 3 months in patients with PCOS in the present study, resulted in significant reduction in lipocalin-2 level (p-value Conclusion PCOS per se is not associated with elevated serum lipocalin-2 levels. Metformin therapy induces a significant reduction in serum lipocalin-2 levels, weight, BMI, waist circumference and HOMA-IR in patients with PCOS. Reduction of BMI was the only significant confounder affecting lipocalin level after metformin therapy. So the reduction in lipocalin level following metformin therapy in patients with PCOS in our study may be due to weight reduction perse.

Introduction Lupus nephritis (LN) affects almost two-thirds of Systemic lupus erythematosus (SLE) patients. Despite initial aggressive therapy, up to 25% of patients with LN will progress to end stage renal disease (ESRD). Conventional serum markers for LN lack the sensitivity of an ideal biomarker. Urinary Neutrophil gelatinase-associated lipocalin (UNGAL) is an excellent biomarker for early diagnosis of acute kidney injury and predicting renal outcomes. Objectives To measure UNGAL among LN patients correlating its levels with renal disease activity to investigate its predictive performance in response to induction therapy. Patients and Methods 40 SLE patients with biopsy-proven LN, class III, IV, or V were randomly selected. The study was conducted in internal medicine department and Outpatient clinic in Ain Shams University Hospitals on and completed after six months. UNGAL was measured at baseline and after complete induction therapy. Results In LN patients at baseline; mean UNGAL levels of complete response, partial response, and no response groups were 14.48 ±2.99 ng/ml, 34.49 ±4.09, and 62.07 ±14.44 ng/ml respectively. Based on ROC curve, we found better performance of baseline UNGAL to discriminate complete response group from partial and non-response groups to predict response to induction, outperforming conventional biomarkers. The area under the curve was 0.943 (92.31% sensitivity, 88.89% specificity), and the best cut-off level was 26.5 ng/ml. Conclusion UNGAL performed better than conventional biomarkers in predicting response to treatment of active LN.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Rigshospitalets Forskningsfond (Grant number: 07IO) Hjerteforeningen (Grant number A6024) Lundbeckfonden (Grant number R186-2015-2132) BACKGROUND In patients with ST-elevation myocardial infarction (STEMI) increased inflammatory response is associated with development of cardiogenic shock (CS). Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a glycoprotein released from mature neutrophils and the plasma concentration of NGAL likely increases immediately after STEMI. PURPOSE We aimed to assess whether admission NGAL plasma concentration in patients with STEMI was associated with CS development after leaving the catheterization laboratory (late CS) and 30-day all-cause mortality. MATERIALS AND METHODS From 1892 consecutive patients with STEMI 1626 (86%) had plasma NGAL concentration measured upon hospital admission before angiography throughout a 1-year period at two tertiary heart centers in Denmark. Patients were stratified according to NGAL quartiles (Q1-4). To assess late CS development, we adjusted for the Observatoire Régional Breton sur l’Infarctus (ORBI) risk score for late CS. For mortality assessment, we adjusted for gender, age, post-PCI culprit Thrombolysis in myocardial infarction (TIMI) flow, left ventricular ejection fraction (LVEF), kidney dysfunction, admission lactate concentration and being comatose after cardiac arrest. RESULTS Increasing NGAL concentration was associated with higher age, more comorbidities (hypertension, diabetes, heart failure, previous stroke, and kidney dysfunction) and more critical patient conditions at presentation including lower blood pressure and LVEF. Plasma NGAL concentration was associated with both late CS development and 30-day mortality (Figure). When adjusted for factors associated with poor outcome, NGAL remained independently associated with both late CS development (Q4 vs. Q1-3) (OR (95% CI) 2.64 (1.57-6.03)) and 30-day mortality (HR (95% CI) 3.18 (1.46-6.93)). CONCLUSION High admission plasma concentration of NGAL in patients with STEMI was independently associated late CS development and 30-day all-cause mortality. Abstract Figure. NGAL quartiles and late CS/mortality

Background Neutrophil gelatinase-associated lipocalin (NGAL) is marker of renal function and is strongly associated with presence of comorbidities. AHEAD comorbidity score is commonly used to predict survival in acute heart failure patients and could predict events even in chronic heart failure. Methods 547 stable patients with chronic heart failure patients with left ventricular ejection fraction Results Median age was 66 years, 80.3% were men. The etiology of heart failure was in 54% ischemic heart disease, in 40% dilatated cardiomyopathy, in 0.5% hypertrophic cardiomyopathy. 60% of patients were in NYHA class II. In the first two years of follow-up, 74 events (13.5%) occurred, including all-cause death, left ventricle assist device implantation or orthotopic heart transplantation. The AHEAD comorbidity score (Atrial fibrillation, low Haemoglobin level 70 years; Abnormal renal parameters with creatinine >130 μmol/L, Diabetes mellitus; 1 point for each comorbidity present) was set in this registry. Patients with AHEAD 0–1 survived without event in 89.2%, AHEAD 2–3 in 82.4% and AHEAD 4–5 only in 63.5% (p Also levels of NGAL are higher when comorbidities from AHEAD score are present: Atrial fibrillation (62 vs. 50 ng/mL; p NGAL levels are singificantly higher in patients with higher AHEAD score. Mean level of NGAL in AHEAD 0–1 (N=320) is 51 ng/mL, in AHEAD 2–3 (N=190) is 78 ng/mL and in AHEAD 4–5 (N=37) is 142 ng/mL (Kruskal-Wallis test p Conclusion In stable chronic heart failure registry FAR NHL, comorbidity score AHEAD can predict events. Serum NGAL level is significantly higher when AHEAD score comorbidities are present: Atrial fibrillation, low Haemoglobin, Eldery, Abnormal renal function and Diabetes mellitus. Funding Acknowledgement Type of funding source: None

Background and Aims Urinary microRNA (miRNA)-21 is reported to be a biomarker for detection of acute kidney injury (AKI). Analysis of urinary exsome may serve as a novel diagnostic approach in kidney disease. The aim of this study is to investigate the clinical significance of urinary exosomal miRNA-21 for AKI in patients with scrub typhus. Method In a cross-sectional study, we collected 138 urine samples at the time of admission from 145 patients with scrub typhus. For 25 patients with scrub typhus-associated AKI and 25 age, sex-matched scrub typhus patient without AKI, we measured miRNA-21 in urinary exosomal fraction and compared diagnostic value in predictiong AKI. Results Compared with patients in the non-AKI group, patients in the AKI group were more likely to have one or more comorbidity such as diabetes (50% vs. 5%, P Conclusion Urinary exosomal miRNA-21 could be a surrogate markers for the diagnosis of scrub typhus–associated AKI.

Background and Aims Transgelin (SM22) is a cytoskeletal actin-binding protein involved in differentiation of smooth muscle cells, osteoblasts and adipocytes. It is present in fibroblasts, some epithelium, immune cells and acts as a tumor suppressor. It was found as a marker of interstitial fibrosis and glomerulosclerosis. Transgelin upregulation depends on the etiology of the disease in various cells (glomerular parietal or visceral, or tubular interstitial cells). Elevated SM22 expression was detected in glomerular and in tubulointerstitial injury. Among patients with multiple myeloma (MM) up to 50% suffer from renal insufficiency (RI) in early stages. Free light chains (FLCs), potential nephrotoxic substances, where lambda light chains are more commonly associated with renal damage, appear to have a prognostic value in MM and indicate the stages of MM. The aim of the study was to analyse the utility of transgelin in case of renal impairment in MM patients and investigate its relationship with acclaimed parameters of renal failure and markers of MM stages. Method The analysis included 126 patients (73 women and 53 men) in mean age 66 ± 10 years. Symptomatic MM was diagnosed in 119 patients, including 84 (67%) with ISS stage 1, 20 (16%) with stage 2 and 15 (12%) with stage 3; 7 (6%) patients had smoldering MM. Mean baseline eGFR (CKD-EPICr) was 74 ± 24 ml/min/1.73 m2 and 31 (25%) patients had eGFR Results Median serum transgelin in the whole studied group was 84.1 (IQR: 65.4; 116.4) ng/ml. Transgelin concentrations were higher in men (median 96.2 versus 78.8 ng/ml; p=0.022) and in patients with smoldering MM (median 149.2 versus 82.4 ng/ml; p=0.003). Transgelin did not differ according to ISS stage (p=0.3) or disease state (regression, stable disease or progression) (p=0.3). Significant correlations were detected between transgelin and serum creatinine (R=0.29; p=0.001), eGFR (CKD-EPICr) (R=-0.25; p=0.007), uric acid (R=0.19; p=0.036), alanine (R=0.18; p=0.048) and aspartate (R=0.26; p=0.003) aminotransferases, ferritin (R=-0.22; p=0.049), hepcidin (R=-0.25; p=0.033), and urine cystatin C (R=0.19; p=0.042). Moreover, after exclusion of patients with smoldering MM, transgelin significantly correlated with serum FLC lambda (R=0.18; p=0.047) and serum periostin (R=-0.22; p=0.013). After median follow-up of 21 (IQR: 15; 24) months, eGFR decreased in 47 (37%) of patients and increased in 71 (56%) patients (no follow-up data were available in 8, i.e. 6% of patients). Patients in whom eGFR decreased had higher transgelin (median 106.6 versus 83.9 ng/ml), although the difference was marginally significant (p=0.05). However, baseline transgelin positively correlated with serum creatinine after follow-up (R=0.37; p Conclusion Transgelin may be useful in investigating renal damage because of its proven role in regulation of fibrosis, especially in kidney. As a biomarker it may indicate diagnosis and etiology of MM-related chronic kidney disease, which can lead to beginning of early therapeutic interventions and improved overall survival and quality of life in patients with MM. Moreover, elevated transgelin at the beginning of the disease may induce future development of chronic kidney disease.

Background and Aims Acute kidney injury (AKI) is a widespread disease affecting mostly old people. Dietary restriction (DR), based on the reduction of food intake, is believed to be one of the most efficient approaches ameliorating damage in different pathological conditions including age-associated diseases. The aim of the study was to investigate the protective mechanisms of DR in the model of AKI in young and old rats. Method All experiments were made on young (3-4 months) and old (22-24 months) male rats. DR was performed by limiting the amount of food for 35% of the ad libitum (AL) daily intake. Since earlier, we showed ineffectiveness of 4-weeks DR in old rats, in this study we applied 35% DR lasting 8 weeks for old rats and 4 weeks for young rats. During DR, we registered the weight loss and measured the level of adiponectin, as this hormone is closely associated with adipose tissue metabolism. Renal ischemia/reperfusion (I/R) was used as a model of ischemic AKI. I/R was performed by clamping the left renal pedicle for 40 minutes followed by reperfusion with simultaneous contralateral nephrectomy. The severity of AKI was evaluated by measuring blood urea nitrogen (BUN), serum creatinine (SCr) and the levels of protein biomarkers of AKI (NGAL and L-FABP) in urine. Proliferation in kidney epithelium in response to I/R was analyzed by PCNA protein level in kidney tissue. We evaluated the function of mitochondria by measuring TMRE/MitoTracker Green ratio in vital kidney slices; in kidney homogenates, we also analyzed levels of Bcl-XL and Bcl-XS proteins. The production of reactive oxygen species (ROS) was evaluated by staining vital kidney slices with DCF. The content of lipid peroxidation products was measured using Image-iT Lipid Peroxidation Kit, and the level of carbonylated proteins was determined by OxyBlot Protein Oxidation Detection Kit. The activation of autophagic-lysosomal system was estimated by western blotting to LC3 II/LC3 I ratio and LAMP1 level, as well as by staining vital kidney slices with LysoTracker Green probe. Results The body weight of rats during DR dropped as far as 20% by the end of 4 weeks in young rats and 30% by the end of 8 weeks in old rats. Nevertheless, adiponectin concentration elevated during DR only in the serum of young rats. DR strongly influenced mitochondria function, in particular, elevated mitochondrial membrane potential both in kidney cells of young and old rats. DR also resulted in increasing the Bcl-XL level. We revealed the decrease of ROS and lipid peroxidation products in vital kidney slices, but only in kidneys of young rats. However, DR reduced the content of carbonyl groups more than 2 times in animals of both ages. We showed that activation of autophagy in response to DR and I/R occurred only in the kidneys of young rats, indicating deterioration of autophagy signaling in old animals. We also found that 48 h after I/R PCNA level increased 19 times in young kidney, although old rats showed only 4-fold elevation of kidney cells proliferation. Estimation of kidney injury markers (NGAL, L-FABP) in urine revealed that 2-month DR led to some protection in old rats. Nonetheless, despite all positive alterations in kidney tissue of old rats, DR was not able to ameliorate impairment of kidney function after I/R, whereas all young rats showed significant improvement of SCr and BUN levels. Conclusion Short-term DR has a significant nephroprotective effect against renal I/R in young rats. Old animals require longer periods of food restriction, after which some protective alterations are observed. We propose, protection of kidney in old and young rats is implemented through slightly different mechanisms and some of them are missing in old animals.

Background and Aims Acute Kidney Injury (AKI) is a common complication of acute coronary syndromes (ACS), and associated with higher mortality and adverse outcomes. Despite advances in research over the past years, effective treatments for current AKI are not available. Prevention and early intervention remain the most effective strategies for AKI of any entity. This study aimed to explore a risk factors and biomarkers for predictive and early diagnostic of AKI in ACS. Method Study was prospective and cohort, patients hospitalized with ACS in Pavlov First Saint Petersburg State Medical University were included. In case of exclusion of ACS, patients were determined in the comparison group, in case of confirmation of the diagnosis of ACS - in the study group. Biomaterial (blood and urine) was taken at admission (T1), 1 day after admission (T2) and 2 days after admission (T3). For the diagnosis of AKI, KDIGO 2012 criteria were used for the diagnosis of AKI. The measured biomarkers at each point were NGAL, KIM-1, cystatin C, sST2, troponin I. Results The study included 73 patients, the diagnosis of ACS was confirmed in 40 patients and AKI development was in 15 patients, all from the ACS group. The most significant for predictive diagnosis was the assessment of the parameters of systemic hemodynamics and the severity of acute heart failure (AHF): heart rate>90 (AUC=0,798, p=0,001), GRACE Risk Score>133 (AUC=0,926, p=0,005). In evaluation the suitability of biomarkers in terms of prognostic diagnosis of AKI, urine NGAL>32 ng/ml (AUC=0,814 p=0,04) and sST2>23.4 ng/ml (AUC=0,718, p=0,02) showed the best results. Conclusion In study of biomarkers efficiency, the use of urine sST2 and NGAL is most promising. Together with hemodynamic parameters, biomarkers have high predictive ability in the diagnosis of AKI in ACS.

Background and Aims Renal inflammation is a protective response to several types of renal insults. Resolution is the ideal outcome of acute inflammation, however fail in resolution leads to chronic inflammation, progressive renal fibrosis and finally to end-stage renal failure. Recently is has been established that resolution of inflammation is mediated not only by disappearance of pro-inflammatory signals (including lipid mediators such as leukotrienes and prostaglandins) but also by activation of specialized pro-resolving mediators (SPM) including lipid mediators such as protectins (PD) and resolvins (Rvs) derived from omega-3 fatty acids precursors. Several authors have explored the role of different SPM in the treatment of experimental nephropathies. In the experimental model of bilateral ischemia/reperfusion pro-resolving mediators such as RvDs and PD1 were endogenously produced in the kidney in response to the injury. In this model, administration of RvD1 or PD1 before the ischemia or after the reperfusion showed a renoprotective effect. Moreover in the unilateral ureteral obstruction model RvE1 and RvD1administration inhibited interstitial fibrosis. One of the key mediators of renal injury is Angiotensin II (AngII) that participates in the pathogenesis of renal diseases through the regulation of two key processes, inflammation and fibrosis. Therefore, the aim of this study was to investigate the effect of the pro-resolving mediator Resolvin D2 (RvD2) in AngII-induced renal injury Method We used C57BL/6 mice that were infused with AngII (1440 µg/kg/day o 1000 ng/Kg/min). After 7 days of AngII treatment, when hypertension was already established, mice were treated with RvD2 (100ng/mouse ip) every two days for additional 7 days. At day 14 mice were sacrificed and kidneys were removed for protein and gene expression analysis and for histological examination. Results In our experimental model RvD2 ameliorated renal injury assessed by expression of NGAL both at mRNA and protein levels. In addition AngII-treated mice presented significant reduction in glomerular size that was increased by RvD2 treatment reaching values comparable to control. In addition, RvD2 inhibited the activation of inflammatory markers including COX-2, IL-6 and MCP-1 induced by AngII and reduced the number of F4/80+ infiltrated macrophages. We next evaluated the effect of RvD2 in the activation of NFκB, a key signaling pathway in inflammation. We observed that RvD2 inhibited AngII-induced NFkB activation. Moreover, RT-PCR analysis indicated that Ang II increased the expression of Tenascin C, a component of the fibrogenic niche in kidney fibrosis, as well as Type-I Collagen and Fibronectin and these levels were significantly reduced by the treatment with RvD2. These results were also confirmed by Masson-Goldner trichrome staining. Importantly, these effects were independent of changes in blood pressure. We finally studied whether the effects of RvD2 might be related to the modulation of RvDs endogenous biosynthesis pathway or RvD2 receptor GPR-18. mRNA analysis showed that AngII did not modified the expression levels of RvD2 biosynthesis enzymes including LOX-5 and LOX-15 nor GPR-18. However, RvD2 treatment increased the expression of LOX-15 and GPR-18 and downregulated LOX-5 indicating a possible auto-regulatory mechanism of RvD2 Conclusion Our results evidence a dual beneficial effect of the pro-resolvin mediator RvD2 in Ang II-induced renal injury as RvD2 treatment reversed not only renal inflammation but also tubulo-interstitial fibrosis. Hence, synthetic pro-resolving mediators would be an interesting therapeutic option for renal diseases.

Introduction Kidney injury doubles burn mortality—thus, early prediction of acute kidney injury (AKI) in the burn population could benefit from artificial intelligence (AI) and machine learning (ML). Our objective in this study was to build and assess the theoretical performances of such AI/ML algorithms and to develop generalizable models that could augment AKI recognition. Methods Two databases containing patients that received neutrophil gelatinase associated lipocalin (NGAL), creatinine, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and urine output (UOP) measurements at admission were used to train, test, and generalize the AI/ML models. Models were first optimized in Cohort A for predicting AKI in Cohort B. Cohort A (n = 50) was based on a retrospective dataset of adult (age³18 years) burn patients, while Cohort B (n = 51) consisted of prospectively enrolled adult burned or non-burned trauma patients at risk for AKI. We employed a grid search and cross validation approach in building 68,100 unique ML models from five distinct ML approaches: logistic regression (LR), k-nearest neighbor (k-NN), support vector machine (SVM), random forest (RF), and deep neural networks (DNN) which enabled us to find the most accurate ML models. Results The best generalization accuracy (86%), sensitivity (91%), and specificity (85%) with NGAL alone was noted with LR, SVM and RF models. Generalizability prediction accuracy, sensitivity and specificity were respectively highest with the optimized DNN model (92%, 100%, and 90%) and the k-NN model (92%, 91%, and 93%) when tested with Cohort B using all four biomarkers. k-NN provided best generalization accuracy (84%) without NGAL using only NT-proBNP and creatinine, followed by DNN using creatinine only with an accuracy of 82%. AI/ML algorithms using results obtained at admission accelerated the average (SD) time to AKI prediction by 61.8 (32.5) hours. Conclusions NGAL is analytically superior to traditional AKI biomarkers such as creatinine and UOP. With machine learning, the AKI predictive capability of NGAL can be further enhanced and accelerated when combined with NT-proBNP, UOP, and creatinine. Applicability of Research to Practice Without NGAL, machine learning models continue to provide robust means in accelerating the prediction of AKI using both common and biomarkers of cardiorenal dysfunction.

Background Ustekinumab (UST) is an effective therapy for moderate-to-severe ulcerative colitis (UC).1 A subset of patients responded to the induction dose of UST and achieved clinical remission at maintenance Week 44 (M-Week 44) following placebo (PBO) maintenance. The clinical and molecular characteristics of this group are unknown. Methods Three subsets of patients in the UNIFI phase 3 studies were compared: (1) UST induction Week 8 (I-Week 8) responders who achieved clinical remission at M-Week 44 following PBO maintenance (n = 32; referred to as 1UST-R); (2) UST I-Week 8 responders who did not achieve clinical remission at M-Week 44 following PBO maintenance (n = 87; 1UST-NR); (3) patients who had no clinical response following two doses of UST induction (n = 93; 2UST-NR). Clinical characteristics, histologic activity, inflammation burden, and genetic risk were compared among the three groups at induction baseline. Serum biomarkers related to the IL-12 and IL-23 pathways and/or UC in general (IFNg, IL-17A, IL-22, MMPs, SAA, and NGAL) were analysed in ~60% of patients within the three groups and compared with patients who achieved M-Week 44 clinical remission following UST maintenance and normal controls. Results 1UST-R had lower disease activity and inflammation burden (CRP, faecal calprotectin, and faecal lactoferrin) at induction baseline than 1UST-NR and 2UST-NR. They had shorter disease duration, a lower frequency of biologic therapy failure, and a trend of lower polygenic genetic risk scores. Disease (MMPs and NGAL) and pathway-related biomarkers (IL17A and IL22) for this group were comparatively less elevated at induction baseline. Histologic measures of architectural change and chronic inflammation were higher in 2UST-NR than in 1UST-R or 1UST-NR at induction baseline. IFNg, IL17A, IL22, MMPs, and SAA in UC were partially normalised by UST induction and further improved with UST maintenance therapy. In contrast, there was no further improvement in these serum markers among the 1UST-R patients during the maintenance phase. Conclusion Patients with a durable response to UST induction had lower disease activity, serum biomarkers indicative of inflammation, and IL-12 and IL-23 pathway activity. Although the molecular effects of UST induction were retained through M-Week 44 in this group, the lack of further improvement suggests a molecular benefit of UST maintenance therapy. Reference

Background Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI). However, the current program for NGAL detection is not extensively applied in clinics due to the high expense of antibodies. Nucleic acid aptamers are single-strand DNAs or RNAs which could bind to targets with high specificity and affinity, and they have been widely used in the diagnosis and therapy for multiple diseases. It is valuable for us to develop a new method for NGAL detection using aptamers instead of antibodies to achieve increased efficiency and decreased cost. Methods Nucleic acid aptamers against NGAL were obtained after SELEX process using magnetic beads, and an enzyme-linked aptamer analysis (ELAA), which can be widely used in clinical diagnosis at low cost, were successfully established. The feasibility of ELAA was further validated with urine samples harvested from 43 AKI patients and 30 healthy people. Results Three candidate aptamers, including NA36, NA42 and NA53, were obtained after 8 rounds of SELEX process with magnetic beads and verified by quantitative polymerase chain reaction (qPCR), and the Kd value of each aptamer was 43.59, 66.55 and 32.52 nM, respectively. Moreover, the linear relationship was consistent at the range of 125–4000 ng/mL, and the detection limit of ELAA assay was 30.45 ng/mL. We also found that NGAL could be exclusively detected with NA53, and no cross-reaction between NA53 and human albumin or globulin occurred, the coefficient of variation (CV) between inner-plate and inter-plate was less than 15%, and the recovery rate was between 80 and 110%. Moreover, the sensitivity and specificity of ELAA assay in this study are 100% and 90%, respectively. Consistently, these results could also diagnose whether the occurrence of AKI in lots of patients, which has been demonstrated with the ELAA method we established after using NA53. Conclusions Taken together, NA53, the best candidate aptamer targeting NGAL protein, can be applied in clinical testing. Electronic supplementary material The online version of this article (10.1186/s12967-019-1955-7) contains supplementary material, which is available to authorized users.

The tumor microenvironment plays a critical regulatory role in cancer progression, especially in central nervous system metastases. Cancer cells within the cerebrospinal fluid (CSF)-filled leptomeninges face substantial microenvironmental challenges, including inflammation and sparse micronutrients. To investigate the mechanism by which cancer cells in these leptomeningeal metastases (LM) overcome these constraints, we subjected CSF from five patients with LM to single-cell RNA sequencing. We found that cancer cells, but not macrophages, within the CSF express the iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17. These macrophages generate inflammatory cytokines that induce cancer cell LCN2 expression but do not generate LCN2 themselves. In mouse models of LM, cancer cell growth is supported by the LCN2/SLC22A17 system and is inhibited by iron chelation therapy. A Phase Ia/1b clinical trial focused on this novel treatment approach is underway.

The role of fermentable dietary fibers in patients with inflammatory bowel disease (IBD) is not understood. Herein, we elucidated the effect of dietary fiber guar gum, commonly added to a wide range of processed foods, on colonic inflammation. The use of three different IBD models allowed us to examine the effect of guar gum on various aspects of human IBD, such as immune hyperactivity [IL-10 receptor (IL-10R) neutralization], epithelial injury [dextran sulfate sodium (DSS)], and infection [Citrobacter rodentium (CR)]-mediated inflammation. Wild-type (WT, C57BL/6) mice fed either control cellulose (insoluble fiber, aka non-fermentable fiber) or guar gum (soluble fiber, aka fermentable fiber, 7.5% w/w) were administered four weekly injections of IL-10R neutralizing antibody (α-IL-10R) to induce immune-hyperactivation mediated chronic colitis. Guar gum treated mice developed robust α-IL-10R mediated colitis. Guar gum fed mice had splenomegaly, colomegaly, elevated systemic proinflammatory markers [serum amyloid A (SAA), lipocalin 2 (Lcn2) and keratinocyte-derived chemokine (KC)] and elevated colonic Lcn2 and interleukin (IL)-1β, and histopathology scores compared to control, cellulose-fed, mice. Similar results were observed in Toll-like receptor 5 deficient mice, which are prone to develop microbiota-dependent colitis. Next, to examine the effect of guar gum on the epithelial injury model, mice were treated with DSS (1.4% w/v in drinking water) for seven days. The guar gum fed group developed severe colitis, including reduced body weight, diarrhea, rectal bleeding, shortening of colon length, and elevated levels of pro-inflammatory markers (Lcn2, KC, and SAA)compared to the control group. The last model to be tested was infection-induced colitis. Since inflammation is required to clear the infection, we hypothesized that guar gum fed mice might clear CR infection better than controls. To our surprise, guar gum fed WT mice shed higher numbers of CR in the feces than the cellulose group. The guar gum fed mice had lower body weights, colomegaly, an elevated level of colonic and serum Lcn2, and higher serum SAA than the control group. Collectively, guar gum failed to protect against CR-induced colonic pathology. Altogether, the work demonstrates that guar gum feeding may exacerbate colonic inflammation following immune-hyperactivation, chemical, and infectious injury. Cautioning IBD patients to monitor their consumption of guar gum fiber might be a way to reduce the severity of intestinal inflammation.

The mechanisms by which the intestinal microbiome affects intestinal inflammation and disease activity in IBD is not clear. Most of the current data on the association between the intestinal microbiome and IBD is focusing on the intestinal microbial taxa and very little is known about the functionality of the microbiome in this condition. Aim: To construct a live bacterial consortium for reduction of intestinal inflammation in IBD based on a high-resolution functional microbial analysis, and its association with anti-inflammatory processes. Methods: Fecal metagenomics samples (n=581) and corresponding fecal calprotectin (FCP) levels from patients with Crohn’s disease and Ulcerative Colitis were downloaded from the NCBI (SRA SRP057027, http://hmpdacc.org, SRA SRP002163 and SRP056641 (BioProject PRJNA275349)). Computational analysis was performed using PRISM (Biomica Ltd, Rehovot Israel), a proprietary metagenomics analysis platform integrating assembly-based and reference-based analyses for functional genomics profiling and utilizing BioCOGS, Biomica’s internal comprehensive catalogue of microbial gene-orthology groups. We compared microbial functions between subjects based on severity of inflammation (FCP250 and FCP >250 vs FCP Validation of the anti-inflammatory activity of BMC322 was performed using a DSS-induced colitis mouse model. Results: Out of the dozens of identified functions, 32 were related to host anti-inflammatory processes, intestinal barrier integrity and colonocyte function. Our functional microbiome investigation has pointed to novel metabolic mechanisms by which specific strains may attenuate mucosal inflammation. Using functional annotations, we infer the biological processes governing microbe-host interactions contributing to reduced inflammation, and identify 16 bacteria of which 4 strains comprise the full array of the identified microbial functions associated with lower disease severity. These strains comprise BMC322- a novel live-bacterial consortium for inflammation reduction in IBD. The anti-inflammatory activity of these strains was validated in in-vitro studies and demonstrated efficacy in preliminary a mouse model of DSS-induced colitis; including shortening period of recovery, disease activity index and fecal lipocalin 2. Conclusion: High-resolution functional genomics profiling of the gut microbiome enables identification of microbial-derived functions relevant to inflammatory disease activity, and microbial strains bearing specific inflammation-reduction activity. These strains have provided the basis for the construction of a 4-strain consortium for intervention in IBD. Preliminary work has validated the anti-inflammatory activity of these strains and their potential as novel therapeutic modality for IBD.

Multiple factors contribute to inflammatory bowel disease (IBD) pathogenesis, including diet. Although correlations exist between the introduction of processed foods and a rise in IBD prevalence, it is not understood what components of processed foods drive disease pathogenesis or how. Despite a FDA classification of Generally Regarded As Safe (GRAS), food additives significantly impact microbe communities and phenotypes. We hypothesized that common food additives, such as maltodextrin (MDX) and carboxymethyl cellulose (CMC), alter intestinal microbes and increase the onset and progression of IBD. We tested this hypothesis using the interleukin-10 deficient (IL10KO) mouse model of spontaneous colitis. To standardize disease onset, IL10KO mice were pre-conditioned with fecal material from nucleotide-binding oligomerization domain 2 deficient mice (NOD2KO) to promote intestinal inflammation. Pre-conditioned IL10KO mice were randomized to chow diets containing either 1% MDX, 1% CMC, or a control diet. MDX and CMC accelerated the onset of colitis compared to the control diet. This was accompanied by elevated fecal lipocalin-2 (p

Purpose: The aim of this study was to investigate and compare the effects of udenafil and mannitol in an experimental renal ischemia-reperfusion (I/R) injury model. Materials and Methods: A total of 64 female Wister Albino rats were used. Right nephrectomy was performed in all groups. In the control group; I/R injury was not performed. In the I/R group; left renal pedicle was clamped for 45 minutes and then underwent 60 minutes and 24 hours of reperfusion. In the mannitol group; 1 mL 20% mannitol was given intravenously 15 minutes before clamping. In the udenafil group; 10-mg/kg udenafil was given orally 1 hour before clamping. Creatinine (Cr), blood urea nitrogen (BUN), Cr clearance, malondialdehyde, neutrophil gelatinase associated lipocalin (NGAL), histological examination and DNA damage (Comet Assay method) levels were compared in tissue, serum and urine samples. Results: Udenafil had a better protective effect than mannitol according to biochemical parameters (Cr, BUN, Cr clearance, and NGAL levels) and histopathological findings when compared with the I/R group. In the Comet sampling analysis no significant difference was detected. Conclusions: Udenafil has a better renoprotective effect than mannitol against I/R injury and this effect supports more functional improvements. Further clinical trials are needed to demonstrate those effects and clinical utility of udenafil for that purpose in humans.

Background— In patients with heart failure, renal dysfunction is associated with a poor outcome. We aimed to assess the prognostic value of plasma neutrophil gelatinase–associated lipocalin (NGAL), a novel marker of renal tubular damage, in patients with heart failure with or without renal dysfunction, and compare it with 2 frequently used biomarkers of chronic kidney disease. Methods and Results— Plasma NGAL, estimated glomerular filtration rate (eGFR), and cystatin C were assessed in 562 patients with heart failure. Chronic kidney disease was defined as eGFR2 . Outcome was all-cause mortality at 36 months. Mean age was 71±11 years, 61% were men, and 97% were in New York Heart Association functional class II/III. Mean baseline eGFR was 54±20 mL/min per 1.73 m 2 , mean cystatin C was 11.2 (7.7–16.2) mg/L, and median plasma NGAL was 85 (60–123) ng/mL. Higher plasma NGAL levels were independently associated with an increased risk of all-cause mortality, in patients with and without chronic kidney disease (hazard ratio [per SD increase in log NGAL]=1.45 [1.22–1.72]; P P =0.023, respectively). Similarly, both in patients with high and low cystatin C (median cut-off), higher plasma NGAL levels were independently associated with an increased risk of all-cause mortality. Moreover, when NGAL was entered in the multivariable risk prediction model, eGFR ( P =0.616) and cystatin C ( P =0.937) were no longer associated with mortality. Conclusions— Plasma NGAL predicts mortality in patients with heart failure, both in patients with and without chronic kidney disease and is a stronger predictor for mortality than the established renal function indices eGFR and cystatin C.

Purpose: The aim of this study was to investigate and compare the effects of udenafil and mannitol in an experimental renal ischemia-reperfusion (I/R) injury model., Materials and Methods: A total of 64 female Wister Albino rats were used. Right nephrectomy was performed in all groups. In the control group; I/R injury was not performed. In the I/R group; left renal pedicle was clamped for 45 minutes and then underwent 60 minutes and 24 hours of reperfusion. In the mannitol group; 1 mL 20% mannitol was given intravenously 15 minutes before clamping. In the udenafil group; 10-mg/kg udenafil was given orally 1 hour before clamping. Creatinine (Cr), blood urea nitrogen (BUN), Cr clearance, malondialdehyde, neutrophil gelatinase associated lipocalin (NGAL), histological examination and DNA damage (Comet Assay method) levels were compared in tissue, serum and urine samples., Results: Udenafil had a better protective effect than mannitol according to biochemical parameters (Cr, BUN, Cr clearance, and NGAL levels) and histopathological findings when compared with the I/R group. In the Comet sampling analysis no significant difference was detected., Conclusions: Udenafil has a better renoprotective effect than mannitol against I/R injury and this effect supports more functional improvements. Further clinical trials are needed to demonstrate those effects and clinical utility of udenafil for that purpose in humans.

Background: Acute kidney injury (AKI) is common after cardiac surgery, the incidence varying between 7.7% and 28.1%. It significantly increases morbidity and mortality. Creatinine considerably delays the diagnosis with its own attended demerits. Novel urinary biomarkers are emerging which help in rapid diagnosis thus reducing the morbidity and mortality. Biomarkers of our study were neutrophil gelatinase-associated lipocalin (NGAL) and Interleukin-18 (IL-18). Objectives: To find out the incidence of AKI in post-cardiac surgery patients in our hospital, the ability of the two biomarkers in early diagnosis in predicting the severity of AKI based on RIFLE’s criteria and their ability to discriminate pre-renal from intrinsic AKI. Patients and Methods: One-hundred patients who underwent cardiac surgery were selected. Midstream urine samples were collected at 3 time intervals (baseline before surgery, 24 hours and 7 days after surgery). Biomarkers were measured by ELISA using BIORAD processors. Fractional excretion of sodium and urea were used to discriminate pre-renal from intrinsic AKI. Results: Out of 100 patients, 31 had AKI, 11 being pre-renal and 20 intrinsic AKI. Four patients required renal replacement therapy (12.9% among AKI cases and 4% in the overall study cohort). Four among 31 expired in intensive care unit. Identifiable risk factors for AKI included insulin requiring diabetes mellitus, chronic obstructive pulmonary disease, increased cardio-pulmonary bypass time, combined valvular surgery and coronary artery bypass grafting, employment of intra-aortic balloon counter pulsation, left main coronary artery occlusion and an ejection fraction of < 40%. NGAL was extremely sensitive (area under curve-0.96) in detecting intrinsic AKI at 24 hours followed by IL-18 ratio with an area under curve of 0.89. Creatinine at 24 hours was able to detect only 31.6% of intrinsic AKI. None of the pre-renal cases showed rise in the urinary biomarker levels. Patients with higher stages of AKI had higher levels of both biomarkers than those at lower stages. Conclusions: NGAL and IL-18 obviated the disadvantages of creatinine. They were efficient in early detection of AKI, in differentiating pre-renal from intrinsic AKI and in predicting the severity of AKI reliably in post-cardiac surgery patients.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a promising biomarker for acute kidney injury (AKI). Our objectives were to evaluate the NGAL Test(TM) from Bioporto for both urine NGAL and plasma NGAL on the Cobas 6000 c501 (Roche Diagnostics, Rotkreuz, Switzerland) with matched measurements run on Hitachi 917, the method's linearity on the Cobas 6000 in urine, EDTA and Lithium-Heparin (Li-Hep), the influence of using EDTA or Li-Hep tubes and, finally, the impact of freezing and thawing on the sample.Forty matched samples of Li-Hep and EDTA plasma and 40 urine samples were analyzed for method, anticoagulant, and freeze-thaw comparisons. Linearity was assessed using high NGAL samples diluted in urine, EDTA, and Li-Hep plasma. Commercial internal controls were used for the imprecision study.The Cobas 6000 measured identically with the Hitachi 917, however, not in EDTA plasma (Median Difference = 17.50 μg/L, p0.0001). Freeze-thaw process reduced NGAL ((EDTA: Mean Difference = = 15.13 μg/L, p = 0.0014)(Li-Hep: Median Difference = = 6.5 μg/L, p = 0.0129)). NGAL results were higher in Li-Hep plasma than in EDTA plasma ((Non-thawed: Median Difference = = 14.5 μg/L, p0.0001), (Thawed: Median Difference = = 21.5 μg/L, p = 0.0003)). Linearity agreements were observed in all three specimens. Imprecision (CV%) was below 3%.The NGAL Test(TM) can be applied on the Cobas 6000 with acceptable performance, although the Cobas 6000 measured higher than the Hitachi 917 in EDTA plasma. Though clinically insignificant, we found that the freeze-thaw process had a reduced effect. NGAL results were higher in Li-Hep tubes than in EDTA tubes. Thus, for blood samples we recommend use of EDTA tubes for NGAL measurements.

Unaccustomed strenuous physical exertion in hot environments can result in heat stroke and acute kidney injury (AKI). Both exercise-induced muscle damage and AKI are associated with the release of interleukin-6, but whether muscle damage causes AKI in the heat is unknown. We hypothesized that muscle-damaging exercise, before exercise in the heat, would increase kidney stress. Ten healthy euhydrated men underwent a randomized, crossover trial involving both a 60-min downhill muscle-damaging run (exercise-induced muscle damage; EIMD), and an exercise intensity-matched non-muscle-damaging flat run (CON), in random order separated by 2 wk. Both treatments were followed by heat stress elicited by a 40-min run at 33°C. Urine and blood were sampled at baseline, after treatment, and after subjects ran in the heat. By design, EIMD induced higher plasma creatine kinase and interleukin-6 than CON. EIMD elevated kidney injury biomarkers (e.g., urinary neutrophil gelatinase-associated lipocalin (NGAL) after a run in the heat: EIMD-CON, mean difference [95% CI]: 12 [5, 19] ng/ml) and reduced kidney function (e.g., plasma creatinine after a run in the heat: EIMD-CON, mean difference [95% CI]: 0.2 [0.1, 0.3] mg/dl), where CI is the confidence interval. Plasma interleukin-6 was positively correlated with plasma NGAL ( r = 0.9, P = 0.001). Moreover, following EIMD, 5 of 10 participants met AKIN criteria for AKI. Thus for the first time we demonstrate that muscle-damaging exercise before running in the heat results in a greater inflammatory state and kidney stress compared with non-muscle-damaging exercise. Muscle damage should therefore be considered a risk factor for AKI when performing exercise in hot environments.

Aim: To assess and compare the roles of plasma and urine concentrations of neutrophil gelatinase associated lipocalin (NGAL) and Cystatin C for early diagnosis of septic acute kidney injury (AKI) in adult critically ill patients.Methods: Patients were divided into three groups as sepsis-non AKI, sepsis-AKI and non sepsis-non AKI. Plasma samples for NGAL and Cystatin C were determined on admission and on alternate days and urinary samples were collected for every day until ICU discharge.Results: One hundred fifty one patients were studied; 66 in sepsis-non AKI, 63 in sepsis-AKI, 22 in non-sepsis-non-AKI groups. Although plasma NGAL performed less well (AUC 0.44), urinary NGAL showed significant discrimination for AKI diagnosis (AUC 0.80) with a threshold value of 29.5 ng/ml (88% sensitivity, 73% specificity). Both plasma and urine Cystatin C worked well for the diagnosis of AKI (AUC 0.82 and 0.86, thresholds 1.5 and 0.106 mg/L respectively).Conclusion: Plasma and urinary Cystatin C and urinary NGAL are useful markers in predicting AKI in septic critically ill patients. Plasma NGAL raises in patients with sepsis in the absence of AKI and should be used with caution as a marker of AKI in septic ICU patients.

Udgivelsesdato: 2010-Sep Abstract Background. Neutrophil Gelatinase-Associated Lipocalin (NGAL) has been described as an excellent marker of acute kidney injury (AKI) using the enzyme-linked immunosorbent assay (ELISA) from BioPorto Diagnostics, DK. Validation of the ELISA kit and investigation of stability of the NGAL protein is a prerequisite before introducing NGAL as a marker for AKI in clinical research. Methods. Plasma and urine samples from a healthy adult and from 16 children undergoing surgery for congenital heart disease were used to validate the 036 NGAL ELISA kit from BioPorto Diagnostics and study stability of the NGAL protein. Results. Median intra-assay variation in plasma and urine from the healthy adult was

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a 20 kDa monomeric protein secreted by activated human neutrophils. NGAL is believed to bind small lipophilic substances such as bacteria-derived lipopolysaccharides, siderophores, formylpeptides, and may function as a modulator of inflammation. Clinical studies have shown NGAL can serve as an early diagnostic marker for acute kidney injury (AKI). We have developed a sensitive immunoassay to measure NGAL level in patient urine. During the course of assay development, we employed a variety of biophysical methods to characterize the physical and binding properties of several anti-NGAL antibody candidates and a recombinant NGAL protein. CD spectroscopy was used to study the structure and stability of NGAL; Forster Resonance Energy Transfer (FRET) was used to determine the binding affinity of NGAL toward anti-NGAL mAbs; Dual-Color Fluorescence Cross-Correlation Spectroscopy (DC-FCCS) was used to compare the capability of two antibodies forming a sandwich with NGAL; NMR was used to identify epitopic regions of NGAL for each antibody candidate. Two antibodies, which have the highest binding affinity toward NGAL and recognize distinct discontinuous epitope regions on NGAL, were chosen as the reagents for a sandwich type microparticle based immunoassay. This work demonstrates a modern strategy and biophysical tools, which are necessary to build a sensitive and robust diagnostic assay.