Ramipril inhibits AGE-RAGE-induced matrix metalloproteinase-2 activation in experimental diabetic nephropathy

Background: Advanced glycation end products (AGE)-receptor for AGE (RAGE) axis and renin-angiotensin system (RAS) play a role in diabetic nephropathy (DN). Matrix metalloproteinase-2 (MMP-2) activation also contributes to DN. However, the pathological interaction among AGE-RAGE, RAS and MMP-2 in DN remains unknown. We examined here the involvement of AGE and RAS in MMP-2 activation in streptozotocin (STZ)-induced diabetic rats and in AGE-exposed rat renal proximal tubular cells (RPTCs).Methods. Experimental diabetes was induced in 6-week-old male Sprague-Dawley (SD) rats by intravenous injection of STZ. Diabetic rats received ramipril (3 mg/kg body weight/day) or vehicle for 32 weeks. AGE-modified rat serum albumin (AGE-RSA) or RSA was intraperitoneally administrated to 6-week-old male SD rats for 16 weeks. RPTCs were stimulated with 100 μg/ml AGE-modified bovine serum albumin (AGE-BSA) or BSA in the presence or absence of 10 M ramiprilat, an inhibitor of angiotensin-converting enzyme or 100 nM BAY11-7082, an IκB- phosphorylation inhibitor.Results: AGE and RAGE expression levels and MMP-2 activity in the tubules of diabetic rats was significantly increased in association with increased albuminuria, all of which were blocked by ramipril. AGE infusion induced tubular MMP-2 activation and RAGE gene expression in SD rats. Ramiprilat or BAY11-7082 inhibited the AGE-induced MMP-2 activation or reactive oxygen species generation in RPTCs. Angiotensin II increased MMP-2 gene expression in RPTCs, which was blocked by BAY11-7082.Conclusions: Our present study suggests the involvement of AGE-RAGE-induced, RAS-mediated MMP-2 activation in experimental DN. Blockade of AGE-RAGE axis by ramipril may protect against DN partly via suppression of MMP-2

A rapid extraction method for glycogen from formalin-fixed liver

Liver glycogen, a highly branched polymer, acts as our blood-glucose buffer. While past structural studies have extracted glycogen from fresh or frozen tissue using a cold-water; sucrose-gradient centrifugation technique, a method for the extraction of glycogen from formalin-fixed liver would allow the analysis of glycogen from human tissues that are routinely collected in pathology laboratories. In this study, both sucrose-gradient and formalin-fixed extraction techniques were carried out on piglet livers, with the yields, purities and size distributions (using size exclusion chromatography) compared. The formalin extraction technique, when combined with a protease treatment, resulted in higher yields (but lower purities) of glycogen with size distributions similar to the sucrose-gradient centrifugation technique. This formalin extraction procedure was also significantly faster, allowing glycogen extraction throughput to increase by an order of magnitude. Both extraction techniques were compatible with mass spectrometry proteomics, with analysis showing the two techniques were highly complementary. (C) 2014 Elsevier Ltd. All rights reserved

Modulation of the Cellular Expression of Circulating Advanced Glycation End-Product Receptors in Type 2 Diabetic Nephropathy

Background. Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease. Methods. Flow cytometry was used to measure the predictive capacity for kidney impairment of the AGE receptors RAGE, AGE-R1, and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE containing diets and in obese type 2 diabetic and control human subjects. Results. Diets high in AGE content fed to diabetic mice decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated Albumin excretion rates (AERs), and high AGE fed dbdb mice had declining Glomerular filtration rate (GFR). Cell surface AGE-R1 expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE R3 was decreased in diabetic dbdb mice or with a low AGE diet. Conclusions. The most predictive PBMC profile for renal disease associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts

Change in adiposity is associated with change in glycoprotein acetyls but not hsCRP in adolescents with severe obesity.

BACKGROUND Obesity-associated chronic inflammation mediates the development of adverse cardiometabolic outcomes. There are sparse data on associations between severe obesity and inflammatory biomarkers in adolescence; most are cross-sectional and limited to acute phase reactants. Here, we investigate associations between adiposity measures and inflammatory biomarkers in children and adolescents with severe obesity both cross-sectionally and longitudinally. METHODS From the Childhood Overweight Biorepository of Australia (COBRA) study, a total of n = 262 participants, mean age 11.5 years (SD 3.5) with obesity had measures of adiposity (body mass index, BMI; % above the 95th BMI-centile, %>95th BMI-centile; waist circumference, WC; waist/height ratio, WtH; % total body fat, %BF; % truncal body fat, %TF) and inflammation biomarkers (glycoprotein acetyls, GlycA; high-sensitivity C-Reactive Protein, hsCRP; white blood cell count, WBC; and neutrophil/lymphocyte ratio, NLR) assessed at baseline. Ninety-eight individuals at mean age of 15.9 years (3.7) participated in a follow-up study 5.6 (2.1) years later. Sixty-two individuals had longitudinal data. Linear regression models, adjusted for age and sex for cross-sectional analyses were applied. To estimate longitudinal associations between change in adiposity measures with inflammation biomarkers, models were adjusted for baseline measures of adiposity and inflammation. RESULTS All adiposity measures were cross-sectionally associated with GlycA, hsCRP and WBC at both time points. Change in BMI, %>95th BMI-centile, WC, WtH and %TF were associated with concomitant change in GlycA and WBC, but not in hsCRP and NLR. CONCLUSION GlycA and WBC but not hsCRP and NLR may be useful in assessing adiposity-related severity of chronic inflammation over time

Sex and puberty-related differences in metabolomic profiles associated with adiposity measures in youth with obesity

BackgroundSpecific patterns of metabolomic profiles relating to cardiometabolic disease are associated with increased weight in adults. In youth with obesity, metabolomic data are sparse and associations with adiposity measures unknown.ObjectivesPrimary, to determine associations between adiposity measures and metabolomic profiles with increased cardiometabolic risks in youth with obesity. Secondary, to stratify associations by sex and puberty.MethodsParticipants were from COBRA (Childhood Overweight BioRepository of Australia; a paediatric cohort with obesity). Adiposity measures (BMI, BMI z-score, %truncal and %whole body fat, waist circumference and waist/height ratio), puberty staging and NMR metabolomic profiles from serum were assessed. Statistics included multivariate analysis (principal component analysis, PCA) and multiple linear regression models with false discovery rate adjustment.Results214 participants had metabolomic profiles analyzed, mean age 11.9years (SD3.1), mean BMI z-score 2.49 (SD +/- 0.24), 53% females. Unsupervised PCA identified no separable clusters of individuals. Positive associations included BMI z-score and phenylalanine, total body fat % and lipids in medium HDL, and waist circumference and tyrosine; negative associations included total body fat % and the ratio of docosahexaenoic acid/total fatty acids and histidine. Stratifying by sex and puberty, patterns of associations with BMI z-score in post-pubertal males included positive associations with lipid-, cholesterol- and triglyceride-content in VLDL lipoproteins; total fatty acids; total triglycerides; isoleucine, leucine and glycoprotein acetyls.Conclusion In a paediatric cohort with obesity, increased adiposity measures, especially in post-pubertal males, were associated with distinct patterns in metabolomic profiles.</p

Decreasing severity of obesity from early to late adolescence and young adulthood associates with longitudinal metabolomic changes implicated in lower cardiometabolic disease risk

Background Obesity in childhood is associated with metabolic dysfunction, adverse subclinical cardiovascular phenotypes and adult cardiovascular disease. Longitudinal studies of youth with obesity investigating changes in severity of obesity with metabolomic profiles are sparse. We investigated associations between (i) baseline body mass index (BMI) and follow-up metabolomic profiles; (ii) change in BMI with follow-up metabolomic profiles; and (iii) change in BMI with change in metabolomic profiles (mean interval 5.5 years). Methods Participants (n = 98, 52% males) were recruited from the Childhood Overweight Biorepository of Australia study. At baseline and follow-up, BMI and the % >95th BMI-centile (percentage above the age-, and sex-specific 95th BMI-centile) indicate severity of obesity, and nuclear magnetic resonance spectroscopy profiling of 72 metabolites/ratios, log-transformed and scaled to standard deviations (SD), was performed in fasting serum. Fully adjusted linear regression analyses were performed.Results Mean (SD) age and % >95th BMI-centile were 10.3 (SD 3.5) years and 134.6% (19.0) at baseline, 15.8 (3.7) years and 130.7% (26.2) at follow-up. Change in BMI over time, but not baseline BMI, was associated with metabolites at follow-up. Each unit (kg/m2) decrease in sex- and age-adjusted BMI was associated with change (SD; 95% CI; p value) in metabolites of: alanine (-0.07; -0.11 to -0.04; p p p p p = 0.003), monounsaturated fatty acids (-0.04; -0.07 to -0.01; p = 0.004), ratio of ApoB/ApoA1 (-0.05; -0.07 to -0.02; p = 0.001), VLDL-cholesterol (-0.04; -0.06 to -0.01; p = 0.01), HDL cholesterol (0.05; 0.08 to 0.1; p = 0.01), pyruvate (-0.08; -0.11 to -0.04; p p = 0.005) and 3-hydroxybuturate (0.07; 0.02 to 0.11; p = 0.01). Results using the % >95th BMI-centile were largely consistent with age- and sex-adjusted BMI measures.Conclusions In children and young adults with obesity, decreasing the severity of obesity was associated with changes in metabolomic profiles consistent with lower cardiovascular and metabolic disease risk in adults.</p

Modest decrease in severity of obesity in adolescence associates with low arterial stiffness

Background and aimsChildhood obesity is associated with cardiovascular risk factors (CVRF), subclinical cardiovascular phenotypes (carotid intima-media thickness, cIMT; pulse-wave velocity, PWV; and carotid elasticity), and adult cardiovascular disease (CVD) mortality. In youth with obesity (body mass index, BMI ≥95th centile), we investigated associations between changes in adiposity and CVRF in early adolescence and subclinical cardiovascular phenotypes in late adolescence.MethodsParticipants had adiposity measures (the severity of obesity in percentage >95th BMI-centile (%>95th BMI-centile)), waist circumference (WC), percentage total body fat (%BF) and CVRF (systolic blood pressure, SBP; glycoprotein acetyls, GlycA; and low-density lipoprotein cholesterol) assessed in early (mean age 10.2 ± 3.5y) and late (15.7 ± 3.7y) adolescence. Subclinical cardiovascular phenotypes were assessed in late adolescence. Multivariable regression analysis was performed.ResultsDecreasing the %>95th BMI-centile was associated with carotid elasticity (0.945%/10 mmHg, p = 0.002) in females, and with PWV in males (−0.75 m/s, p p p μmol-increase) were associated with elasticity (−0.162%/10 mmHg, p = 0.042), and changes in SBP (per 10 mmHg-increase) were associated with PWV (0.260 m/s, p μm, p = 0.006).ConclusionsIn youth with obesity, decreasing or maintaining the severity of obesity, and decreasing the levels of SBP and GlycA from early to late adolescence was associated with low arterial stiffness.</p

Mapping Time-course Mitochondrial Adaptations in the Kidney in Experimental Diabetes

Abstract Oxidative phosphorylation drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homeostasis. In diabetic kidney disease, mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics, have not been previously documented. Here, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks) a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells of diabetic kidneys were clearly apparent, but no change urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary Kidney injury molecule (Kim)-1 excretion, where an increase in Complex I-linked oxygen consumption rate, in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of diabetic kidney disease. Summary statement We identified that dysfunction of cellular power stations, mitochondria, may precede the development of kidney disease in diabetes. This suggests that mitochondrial dysfunction is a primary cause of diabetic nephropathy, which could be targeted to improve the burden of this disease. Short title: Mitochondrial adaptations in diabetic nephropath

Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes

Abstract Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H 2 O 2 ) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP , indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD

Evidence for Protein Leverage in Children and Adolescents with Obesity

Objective The aim of this study was to test the protein leverage hypothesis in a cohort of youth with obesity.Methods A retrospective study was conducted in a cohort of youth with obesity attending a tertiary weight management service. Validated food questionnaires revealed total energy intake (TEI) and percentage of energy intake from carbohydrates (ì), fats (ï), and proteins (%EP). Individuals with a Goldberg cutoff >= 1.2 of the ratio of reported TEI to basal metabolic rate from fat-free mass were included. A subgroup had accelerometer data. Statistics included modeling of percentage of energy from macronutrients and TEI, compositional data analysis to predict TEI from macronutrient ratios, and mixture models for sensitivity testing.Results A total of 137 of 203 participants were included (mean [SD] age 11.3 [2.7] years, 68 females, BMI z score 2.47 [0.27]). Mean TEI was 10,330 (2,728) kJ, mean ì was 50.6% (6.1%), mean ï was 31.6% (4.9%), and mean %EP was 18.4% (3.1%). The relationship between %EP and TEI followed a power function (L coefficient -0.48; P Conclusions In youth with obesity, protein dilution by either carbohydrates or fats increases TEI. Assessment of dietary protein may be useful to assist in reducing TEI and BMI in youth with obesity.</p