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Summary The kidneys balance many byproducts of the metabolism of dietary components. Previous studies examining dietary effects on kidney health are generally of short duration and manipulate a single macronutrient. Here, kidney function and structure were examined in C57BL/6J mice randomized to consume one of a spectrum of macronutrient combinations (protein [5%–60%], carbohydrate [20%–75%], and fat [20%–75%]) from weaning to late-middle age (15 months). Individual and interactive impacts of macronutrients on kidney health were modeled. Dietary protein had the greatest influence on kidney function, where chronic low protein intake decreased glomerular filtration rates and kidney mass, whereas it increased kidney immune infiltration and structural injury. Kidney outcomes did not align with cardiometabolic risk factors including glucose intolerance, overweight/obesity, dyslipidemia, and hypertension in mice with chronic low protein consumption. This study highlights that protein intake over a lifespan is an important determinant of kidney function independent of cardiometabolic changes., Graphical abstract, Highlights • Chronic high macronutrient intake from any source increases kidney function (GFR) • Low protein intake led to greater kidney tubular structural injury and inflammation • Lower protein intake decreased kidney mass and glomerular filtration capacity • Kidney outcomes did not align with longevity or cardiometabolic outcomes, Physiology; Animal physiology; Systems biology; Metabolomics

Macronutrients and dietary energy influence aging, age-related health, and life span. Reduction in telomere length has been proposed as one mechanism for aging. Therefore, this study investigated the effects of varying ratios of dietary macronutrients and energy on telomere length in older adult mice. C57Bl/6 mice were fed ad libitum their entire life on one of 25 diets varying in protein, carbohydrates, fat, and energy. Average telomere length ratio (ATLR) was measured by polymerase chain reaction in livers of a subset of 161 mice aged 15 months. There was a significant positive relationship between ATLR and carbohydrate intake and a negative relationship with protein intake, but no relationships with fat or energy intake. Analysis using the Geometric Framework and Generalized Additive Models confirmed that carbohydrate intake was positively associated with ATLR, while the longest ATLR was achieved by mice restricted to low protein, high carbohydrate diets. ATLR distribution across the diets was parallel to median life-span results previously published. ATLR was associated with blood levels of some amino acids (asparagine, glutamate, taurine) but not with blood levels of fatty acids, hepatic mitochondrial function, or nutrient sensing pathways. In conclusion, mice on low protein, high carbohydrate diets have the longest hepatic telomeres and longest life span.

Fibroblast growth factor 21 (FGF21) is the first known endocrine signal activated by protein restriction. Although FGF21 is robustly elevated in low-protein environments, increased FGF21 is also seen in various other contexts such as fasting, overfeeding, ketogenic diets, and high-carbohydrate diets, leaving its nutritional context and physiological role unresolved and controversial. Here, we use the Geometric Framework, a nutritional modeling platform, to help reconcile these apparently conflicting findings in mice confined to one of 25 diets that varied in protein, carbohydrate, and fat content. We show that FGF21 was elevated under low protein intakes and maximally when low protein was coupled with high carbohydrate intakes. Our results explain how elevation of FGF21 occurs both under starvation and hyperphagia, and show that the metabolic outcomes associated with elevated FGF21 depend on the nutritional context, differing according to whether the animal is in a state of under- or overfeeding.

Nutrition influences both hepatic function and aging, but mechanisms are poorly understood. Here, the effects of lifelong, ad libitum-fed diets varying in macronutrients and energy on hepatic gene expression were studied. Gene expression was measured using Affymetrix mouse arrays in livers of 46 mice aged 15 months fed one of 25 diets varying in protein, carbohydrates, fat, and energy density from 3 weeks of age. Gene expression was almost entirely influenced by protein intake. Carbohydrate and fat intake had few effects on gene expression compared with protein. Pathways and processes associated with protein intake included those involved with mitochondrial function, metabolic signaling (PI3K-Akt, AMPK, mTOR) and metabolism of protein and amino acids. Protein intake had variable effects on genes associated with regulation of longevity and influenced by caloric restriction. Among the genes of interest with expression that were significantly associated with protein intake are Cth, Gls2, Igf1, and Nnmt, which were increased with higher protein intake, and Igf2bp2, Fgf21, Prkab2, and Mtor, which were increased with lower protein intake. Dietary protein has a powerful impact on hepatic gene expression in older mice, with some overlap with genes previously reported to be involved with regulation of longevity or caloric restriction.

SummaryBoth caloric restriction (CR) and low-protein, high-carbohydrate (LPHC) ad-libitum-fed diets increase lifespan and improve metabolic parameters such as insulin, glucose, and blood lipids. Severe CR, however, is unsustainable for most people; therefore, it is important to determine whether manipulating macronutrient ratios in ad-libitum-fed conditions can generate similar health outcomes. We present the results of a short-term (8 week) dietary manipulation on metabolic outcomes in mice. We compared three diets varying in protein to carbohydrate ratio under both CR and ad libitum conditions. Ad libitum LPHC diets delivered similar benefits to CR in terms of levels of insulin, glucose, lipids, and HOMA, despite increased energy intake. CR on LPHC diets did not provide additional benefits relative to ad libitum LPHC. We show that LPHC diets under ad-libitum-fed conditions generate the metabolic benefits of CR without a 40% reduction in total caloric intake.

In invertebrates, reproductive output and lifespan are profoundly impacted by dietary macronutrient balance, with these traits achieving their maxima on different diet compositions, giving the appearance of a resource-based tradeoff between reproduction and longevity. For the first time in a mammal, to our knowledge, we evaluate the effects of dietary protein (P), carbohydrate (C), fat (F), and energy (E) on lifespan and reproductive function in aging male and female mice. We show that, as in invertebrates, the balance of macronutrients has marked and largely opposing effects on reproductive and longevity outcomes. Mice were provided ad libitum access to one of 25 diets differing in P, C, F, and E content, with reproductive outcomes assessed at 15 months. An optimal balance of macronutrients exists for reproductive function, which, for most measures, differs from the diets that optimize lifespan, and this response differs with sex. Maximal longevity was achieved on diets containing a P:C ratio of 1:13 in males and 1:11 for females. Diets that optimized testes mass and epididymal sperm counts (indicators of gamete production) contained a higher P:C ratio (1:1) than those that maximized lifespan. In females, uterine mass (an indicator of estrogenic activity) was also greatest on high P:C diets (1:1) whereas ovarian follicle number was greatest on P:C 3:1 associated with high-F intakes. By contrast, estrous cycling was more likely in mice on lower P:C (1:8), and the number of corpora lutea, indicative of recent ovulations, was greatest on P:C similar to those supporting greatest longevity (1:11).

Werner syndrome is a progeric syndrome characterized by premature atherosclerosis, diabetes, cancer, and death in humans. The knockout mouse model created by deletion of the RecQ helicase domain of the mouse Wrn homologue gene (Wrn(∆hel/∆hel)) is of great interest because it develops atherosclerosis and hypertriglyceridemia, conditions associated with aging liver and sinusoidal changes. Here, we show that Wrn(∆hel/∆hel) mice exhibit increased extracellular matrix, defenestration, decreased fenestration diameter, and changes in markers of liver sinusoidal endothelial cell inflammation, consistent with age-related pseudocapilliarization. In addition, hepatocytes are larger, have increased lipofuscin deposition, more frequent nuclear morphological anomalies, decreased mitochondria number, and increased mitochondrial diameter compared to wild-type mice. The Wrn(∆hel/∆hel) mice also have altered mitochondrial function and altered nuclei. Microarray data revealed that the Wrn(∆hel/∆hel) genotype does not affect the expression of many genes within the isolated hepatocytes or liver sinusoidal endothelial cells. This study reveals that Wrn(∆hel/∆hel) mice have accelerated typical age-related liver changes including pseudocapillarization. This confirms that pseudocapillarization of the liver sinusoid is a consistent feature of various aging models. Moreover, it implies that DNA repair may be implicated in normal aging changes in the liver.

The nutrient sensing protein, SIRT1 influences aging and nutritional interventions such as caloric restriction in animals, however, the role of SIRT1 in human aging remains unclear. Here, the role of SIRT1 single-nucleotide polymorphisms (SNPs) and serum-induced SIRT1 protein expression (a novel assay that detects circulating factors that influence SIRT1 expression in vitro) were studied in the Concord Health and Ageing in Men Project (CHAMP), a prospective cohort of community dwelling men aged 70 years and older. Serum-induced SIRT1 expression was not associated with age or mortality, however participants within the lowest quintile were less likely to be frail (odds ratio (OR) 0.34, 95% confidence interval (CI) 0.17–0.69, N = 1,309). Serum-induced SIRT1 expression was associated with some markers of body composition and nutrition (height, weight, body fat and lean % mass, albumin, and cholesterol) but not disease. SIRT1 SNPs rs2273773, rs3740051, and rs3758391 showed no association with age, frailty, or mortality but were associated with weight, height, body fat and lean, and albumin levels. There were some weak associations between SIRT1 SNPs and arthritis, heart attack, deafness, and cognitive impairment. There was no association between SIRT1 SNPs and the serum-induced SIRT1 assay. SIRT1 SNPs and serum-induced SIRT1 expression in older men may be more closely associated with nutrition and body composition than aging and age-related conditions. NHMRC 512364, Sydney Medical School Foundation, Ageing and Alzheimers Research

Myocardial inflammation following acute myocardial infarct (AMI) is associated with risk of congestive heart failure. Pro-inflammatory neutrophils were recruited to the damaged myocardium 24 h after permanent coronary ligation in rats to induce AMI as judged by the presence of immune-positive myeloperoxidase (MPO) in the tissues; MPO generates the oxidant hypochlorous acid (HOCl). Neutrophils were absent in hearts from Control (untreated) and surgical Sham. Similarly, rats exposed to 1 h coronary ligation (Ischemia) showed no neutrophil infiltrate. Concomitantly, MPO activity increased in left ventricular (LV) homogenates prepared from the AMI group and this was inhibited by paracetamol and the nitroxide TEMPO. The same LV-homogenates showed increased 3-chlorotyrosine/tyrosine ratios (biomarker for MPO-activity). Combined 2D gel/Western blot indicated cardiac myoglobin (Mb) was modified after AMI. Subsequent MALDI-TOF and LC-MS/MS analysis of isolated protein spots revealed increased Mb oxidation in hearts from the AMI group relative to Control, Sham and Ischemia groups. Peptide mass mapping revealed oxidation of Met9 and Met132 to the corresponding sulfoxides yet Cys67 remained unmodified. Therefore, neutrophil-generated HOCl can oxidize cardiac Mb after AMI and this may impact on its function within the affected myocardium: oxidized Mb maybe a useful marker of myocardial inflammation.

Diet influences health and patterns of disease in populations. How different diets do this and why outcomes of diets vary between individuals are complex and involve interaction with the gut microbiome. A major challenge for predicting health outcomes of the host-microbiome dynamic is reconciling the effects of different aspects of diet (food composition or intake rate) on the system. Here we show that microbial community assembly is fundamentally shaped by a dichotomy in bacterial strategies to access nitrogen in the gut environment. Consequently, the pattern of dietary protein intake constrains the host-microbiome dynamic in ways that are common to a very broad range of diet manipulation strategies. These insights offer a mechanism for the impact of high protein intake on metabolic health and form the basis for a general theory of the impact of different diet strategies on host-microbiome outcomes.

We investigated whether cosupplementation with synthetic tetra-tert-butyl bisphenol (BP) and vitamin C (Vit C) ameliorated oxidative stress and acute kidney injury (AKI) in an animal model of acute rhabdomyolysis (RM). Rats were divided into groups: Sham and Control (normal chow), and BP (receiving 0.12% w/w BP in the diet; 4 weeks) with or without Vit C (100mg/kg ascorbate in PBS ip at 72, 48, and 24h before RM induction). All animals (except the Sham) were treated with 50% v/v glycerol/PBS (6 mL/kg injected into the hind leg) to induce RM. After 24h, urine, plasma, kidneys, and aortae were harvested. Lipid oxidation (assessed as cholesteryl ester hydroperoxides and hydroxides and F(2)-isoprostanes accumulation) increased in the kidney and plasma and this was coupled with decreased aortic levels of cyclic guanylylmonophosphate (cGMP). In renal tissues, RM stimulated glutathione peroxidase (GPx)-4, superoxide dismutase (SOD)-1/2 and nuclear factor kappa-beta (NFκβ) gene expression and promoted AKI as judged by formation of tubular casts, damaged epithelia, and increased urinary levels of total protein, kidney-injury molecule-1 (KIM-1), and clusterin. Supplementation with BP±Vit C inhibited the two indices of lipid oxidation, down-regulated GPx-4, SOD1/2, and NF-κβ gene responses and restored aortic cGMP, yet renal dysfunction and altered kidney morphology persisted. By contrast, supplementation with Vit C alone inhibited oxidative stress and diminished cast formation and proteinuria, while other plasma and urinary markers of AKI remained elevated. These data indicate that lipid- and water-soluble antioxidants may differ in terms of their therapeutic impact on RM-induced renal dysfunction.

Despite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity and mortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpins myocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediate gene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. This study sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules (DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR. It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulating its downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1β-activated endothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcine model of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei of cardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarct size and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this large animal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, and the reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zone of the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route after IR injury in pigs have potential therapeutic implications in human MI.

To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hep1 cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep1 cells had pores; some of which appeared to be fenestrations (diameter 55 ± 28 nm, porosity 2.0 ± 1.4%), rudimentary sieve plates, bristle-coated micropinocytotic vesicles and expressed caveolin-1, von Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 ± 2.6% ( P < 0.01) and pore diameter to 104 ± 59 nm ( P < 0.001). GFP-actin was expressed throughout the cells, whereas GFP-caveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hep1 cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus.

Background Mice lacking plasminogen ( PG −/− ) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG −/− mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type ( PG +/+ ) mice. Methods Phagocytic activity of PMNs from PG −/− and PG +/+ mice was compared following exposure to Staphylococcus aureus ( S. aureus ) particles and the expression of Mac-1 was assessed in parallel by flow cytometric analysis. Resistance to phorbol-12-myristate-13-acetate (PMA)-induced cell death was compared between PMNs from the different genotypes. Results Stimulation of PG −/− PMNs by opsonized S. aureus diluted in PG −/− plasma significantly increased phagocytosis (15-fold) compared with stimulation of PG +/+ PMNs in PG +/+ plasma. Incubation of PG −/− PMNs with PG +/+ plasma (control) or PG −/− plasma supplemented with human PG inhibited this increased phagocytic activity. Mac-1 cell surface density increased 6.2±1.0-fold in PG −/− PMNs versus 2.9±0.6-fold in PG +/+ PMNs ( P PG −/− PMNs with an anti-Mac-1 antibody in PG −/− plasma inhibited phagocytic activity. In addition, physiologic PG blocked Mac-1 accessibility at the surface of PMNs. Addition of PMA resulted in 33% death of PMNs from PG −/− mice versus 68% in PG +/+ controls ( P Conclusions PMNs from PG −/− mice exhibit a Mac-1 dependent increase in phagocytic activity that is suppressed with human PG, an anti-Mac-1 antibody or the plasma from PG +/+ mice. The propensity for PMNs from PG −/− mice to be activated in response to PMA together with their relative resistance to PMA-toxicity may contribute to increased PMN half-life and enhanced fibrin(ogen) clearance in the setting of PG deficiency.

Polycystic ovary syndrome (PCOS) is associated with reproductive, endocrine, and metabolic abnormalities. Because hyperandrogenism is the most consistent PCOS feature, we used wild-type (WT) and androgen receptor (AR) knockout (ARKO) mice, together with a mouse model of PCOS, to investigate the contribution of genomic AR-mediated actions in the development of PCOS traits. PCOS features were induced by prenatal exposure to dihydrotestosterone (250 μg) or oil vehicle (control) on days 16-18 of gestation in WT, heterozygote, and homozygote ARKO mice. DHT treatment of WT mice induced ovarian cysts (100% vs 0%), disrupted estrous cycles (42% vs 100% cycling), and led to fewer corpora lutea (5.0±0.4 vs 9.8±1.8). However, diestrus serum LH and FSH, and estradiol-induced-negative feedback as well as hypothalamic expression of kisspeptin, neurokinin B, and dynorphin, were unaffected by DHT treatment in WT mice. DHT-treated WT mice exhibited a more than 48% increase in adipocyte area but without changes in body fat. In contrast, heterozygous and homozygous ARKO mice exposed to DHT maintained comparable ovarian (histo)morphology, estrous cycling, and corpora lutea numbers, without any increase in adipocyte size. These findings provide strong evidence that genomic AR signaling is an important mediator in the development of these PCOS traits with a dose dependency that allows even AR haplosufficiency to prevent induction by prenatal androgenization of PCOS features in adult life.

Iatrogenic and spontaneous downstream microembolization of atheromatous material is increasingly recognized as a source of cardiovascular morbidity and mortality. Devising ways of reducing this distal embolization using a variety of mechanical means--distal protection--is currently under intense and diverse investigation. This review therefore summarizes the present status of distal protection. It examines the problem of distal embolization, describes the available distal protection devices, reviews those areas of cardiovascular medicine where distal protection devices are being investigated, and discusses potential future developments.

SummaryAnimal models of cardiovascular pathology contribute towards understanding and treatment of a broad range of conditions. Specifically in the context of acute myocardial infarction (AMI), rat models have been commonly used in studies of pathogenesis, investigation and novel therapies, although there has often been difficulty in translating experimental findings to clinical benefit. However, recent years have seen two important changes to our clinical approaches to AMI. First, there is increasing recognition that the pathophysiology of human AMI is a process occurring at many levels, not just within the epicardial coronary artery, but also within the microvasculature and the myocardium. Second, contemporary treatments are shifting away from thrombolytic dissolution of epicardial coronary thrombus to direct mechanical approaches using angioplasty and stents. These changes in our understanding of AMI have implications for the relevance of these animal models. The following discussion therefore reviews and examines the current rat models of AMI, places them in a clinical context, discusses their advantages and limitations, and outlines likely future developments, providing an overview of the place of these important models of AMI.

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naive CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype.

Polycystic ovary syndrome (PCOS) affects 5–10% of women of reproductive age, causing a range of reproductive, metabolic and endocrine defects including anovulation, infertility, hyperandrogenism, obesity, hyperinsulinism, and an increased risk of type 2 diabetes and cardiovascular disease. Hyperandrogenism is the most consistent feature of PCOS, but its etiology remains unknown, and ethical and logistic constraints limit definitive experimentation in humans to determine mechanisms involved. In this study, we provide the first comprehensive characterization of reproductive, endocrine, and metabolic PCOS traits in 4 distinct murine models of hyperandrogenism, comprising prenatal dihydrotestosterone (DHT, potent nonaromatizable androgen) treatment during days 16–18 of gestation, or long-term treatment (90 days from 21 days of age) with DHT, dehydroepiandrosterone (DHEA), or letrozole (aromatase inhibitor). Prenatal DHT-treated mature mice exhibited irregular estrous cycles, oligo-ovulation, reduced preantral follicle health, hepatic steatosis, and adipocyte hypertrophy, but lacked overall changes in body-fat composition. Long-term DHT treatment induced polycystic ovaries displaying unhealthy antral follicles (degenerate oocyte and/or > 10% pyknotic granulosa cells), as well as anovulation and acyclicity in mature (16-week-old) females. Long-term DHT also increased body and fat pad weights and induced adipocyte hypertrophy and hypercholesterolemia. Long-term letrozole-treated mice exhibited absent or irregular cycles, oligo-ovulation, polycystic ovaries containing hemorrhagic cysts atypical of PCOS, and displayed no metabolic features of PCOS. Long-term dehydroepiandrosterone treatment produced no PCOS features in mature mice. Our findings reveal that long-term DHT treatment replicated a breadth of ovarian, endocrine, and metabolic features of human PCOS and provides the best mouse model for experimental studies of PCOS pathogenesis.

Nutrition influences skin structure; however, a systematic investigation into how energy and macronutrients (protein, carbohydrate and fat) affects the skin has yet to be conducted. We evaluated the associations between macronutrients, energy intake and skin structure in mice fed 25 experimental diets and a control diet for 15 months using the Geometric Framework, a novel method of nutritional analysis. Skin structure was associated with the ratio of dietary macronutrients eaten, not energy intake, and the nature of the effect differed between the sexes. In males, skin structure was primarily associated with protein intake, whereas in females carbohydrate intake was the primary correlate. In both sexes, the dermis and subcutaneous fat thicknesses were inversely proportional. Subcutaneous fat thickness varied positively with fat intake, due to enlarged adipocytes rather than increased adipocyte number. We therefore demonstrated clear interactions between skin structure and macronutrient intakes, with the associations being sex-specific and dependent on dietary macronutrient balance.

SummaryThe fundamental questions of what represents a macronutritionally balanced diet and how this maintains health and longevity remain unanswered. Here, the Geometric Framework, a state-space nutritional modeling method, was used to measure interactive effects of dietary energy, protein, fat, and carbohydrate on food intake, cardiometabolic phenotype, and longevity in mice fed one of 25 diets ad libitum. Food intake was regulated primarily by protein and carbohydrate content. Longevity and health were optimized when protein was replaced with carbohydrate to limit compensatory feeding for protein and suppress protein intake. These consequences are associated with hepatic mammalian target of rapamycin (mTOR) activation and mitochondrial function and, in turn, related to circulating branched-chain amino acids and glucose. Calorie restriction achieved by high-protein diets or dietary dilution had no beneficial effects on lifespan. The results suggest that longevity can be extended in ad libitum-fed animals by manipulating the ratio of macronutrients to inhibit mTOR activation.

Background. End-stage renal failure is associated with a low-output cardiomyopathy, left ventricular hyper- rectomy model; uraemic cardiomyopathy; whole-cell patch-clamp trophy and increased QTc dispersion. Cardiac dysfunction is prevalent in patients at the beginning of dialysis and is an important predictor of mortality. Ca2+ influx through voltage-gated L-type Ca2+ channels plays a Introduction key role in the excitation–contraction coupling of cardiac myocytes. The purpose of this study was to End-stage renal failure is associated with a 10-fold examine the effect of subtotal nephrectomy (SNx) in increase in age-matched cardiovascular mortality [1] the rat on both cardiac L-type Ca2+ currents and and a cardiomyopathy associated with low output left action potential duration. ventricular failure [2], left ventricular hypertrophy [3], Methods. Wistar rats underwent two-stage SNx; con- myocardial fibrosis [4] as well as increased intra-ECG trol rats (C ) underwent bilateral renal decapsulation. variation in QT interval (‘QT dispersion’), which has Animals were sacrificed after 8 weeks, and ventricular been associated with an increased risk of sudden death myocytes were isolated. SNx rats showed a 2-fold [5]. The aetiology of this cardiomyopathy is complex, increase in plasma urea and creatinine compared with but is likely to include hypertension, anaemia, secondC rats. Whole-cell patch clamp techniques were used ary hyperparathyroidism, acidosis and uraemic toxins. to examine L-type Ca2+ channel currents in isolated The cardiomyopathy is prevalent in patients just begincardiac myocytes at 37°C. In separate experiments, the ning regular dialysis therapy and is an important epicardial monophasic action potentials of isolated indicator of subsequent mortality [6 ]. perfused whole hearts from C and SNx rats were It is clear from animal work that renal failure is

Some aspects of vascular reactivity are altered in mild experimental uraemia, as shown by increased myogenic tone and a reduced lumen diameter in the femoral artery. This study was conducted to investigate the prevention of these uraemia-induced vascular abnormalities by the angiotensin-converting enzyme inhibitor (ACE-I) Ramipril. Ten male Wistar rats were rendered uraemic (U) by 5/6th nephrectomy, and 10 control (C) rats were concurrently sham-operated. After 4 weeks, both groups were given daily subcutaneous injections of 3 μg of Ramipril for a further 4 weeks. Tail-cuff systolic blood pressure was then recorded and the rat was killed. Isolated femoral arteries were mounted on a pressure myograph and pressurized at 40 mmHg for baseline measurements of the lumen internal diameter. Myogenic tone was then assessed over a range of intravascular pressures from 40 to 160 mmHg. Biochemically, serum urea and creatinine were significantly higher in the uraemic (U) group [urea: U, 23±3 mmol/l; C, 6±1 mmol/l (P < 0.001); creatinine: U, 147±17 mmol/l, C, 72±11 mmol/l (P < 0.01)]. Systolic blood pressure was the same in both groups (U, 127±7 mmHg; C, 127±3 mmHg). The mean baseline internal diameter was the same in both groups (U, 756±22 μm; C, 721±34 μm, not significant), as was mean myogenic tone (U, 4.7±1%; C, 3.4±1%). In conclusion, there were no differences in baseline lumen diameter or myogenic tone in uraemic compared with control femoral arteries of rats treated with Ramipril, which suggests that Ramipril may prevent the development of elevated myogenic tone and decreased lumen diameter previously observed in this model of uraemia. These results suggest that these specific vascular abnormalities in uraemia may be mediated by renin or bradykinin, or by the direct action of angiotensin II on vascular smooth muscle.

Particulate and histopathologic examination of atherosclerotic material collected during carotid artery stenting is presented, illustrating the limitations of current knowledge regarding the use of distal protection devices (DPD) during this novel vascular intervention.

Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di-tert-butyl-bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical-scavenging activity than vitamin E, crosses the blood-brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre-treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down-regulation of hypoxia inducible factor-1α and glucose transporter-1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro-apoptotic factors that together enhanced cerebral tissue viability after injury. That pre-treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection. We demonstrate that pre-treatment with ditert-butyl bisphenol(Di-t-Bu-BP) inhibits lipid, protein, and total thiol oxidation and decreases caspase activation and infarct size in rats subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. These data suggest that inhibition of oxidative stress contributes significantly to neuroprotection.

Dmitri Svistounov1, Svetlana N. Zykova1,2, Victoria C. Cogger1, Alessandra Warren1, Aisling C. McMahon1, Robin Fraser 3 and David G. Le Couteur1 1Centre for Education and Research on Ageing and ANZAC Research Institute, University of Sydney and Concord RG Hospital, Sydney, 2Department of Nephrology, University Hospital of Northern Norway, Tromso, 3University of Otago, Christchurch, 1Australia 2Norway 3New Zealand

Erythropoietin therapy for uraemic anaemia is associated with a high rate of hypertensive and thrombotic complications. The mechanism is unknown, but a change in cellular calcium control may be relevant to changes in blood pressure and thrombosis. Platelets were utilized as a model of vascular smooth muscle cells. The effects of erythropoietin therapy on platelet cellular calcium, assessed by fura-2, were measured in 25 patients receiving renal replacement therapy during a 6-month treatment period. Three patients failed to reach a target haemoglobin and were excluded from the analysis. Blood pressure increased in 11 of the remaining 22 subjects, eight requiring an increase in antihypertensive medication. There were no differences in cellular calcium control between the group in whom blood pressure rose and patients with stable blood pressure. Overall there was a fall of 24% in resting cytosolic calcium (baseline 69.2 +/- 5.1 to 52.5 +/- 3.0 nmol/l, P < 0.05) after 3 months of erythropoietin therapy. There was no change in the thrombin-stimulated peak response in the presence of extracellular calcium during therapy, although thrombin-stimulated intracellular release also fell at 3 months (baseline 769 +/- 61 versus 3 months 559 +/- 49 nmol/l, P < 0.01). This study suggests that intracellular free calcium control within platelets improves in response to erythropoietin therapy. However these changes appear not to be related to the development of hypertension.

Diabetes mellitus is associated with oxidative injury to the vasculature. Here, the link between oxidative stress and ultrastructural changes in the hepatic microcirculation was investigated as well as the effects of a synthetic antioxidant, tert-butyl bisphenol (tBP). The study focused on the impact of experimental diabetes on liver sinusoidal endothelial cell (LSEC) fenestrations, which are pores in the liver endothelium that facilitate substrate transfer between blood and hepatocytes. Adult male rats were rendered diabetic using streptozotocin (60 mg/kg) and administered 1–2 IU insulin daily. After 8 weeks, animals received either 100 mg/kg tBP or vehicle alone, on 2 consecutive days. Livers were harvested 24 h later under isofluorane anaesthesia (5% v/v in O2(g) by inhalation) and fixed for scanning electron microscopy to evaluate fenestrations or for immuno-histochemical assessment of nitrotyrosine, a marker of nitrosative stress. Median fenestration diameter increased significantly following 8 weeks of diabetes (80 nm vs. 70 nm controls; P

Rhabdomyolysis (RM) caused by severe burn releases extracellular myoglobin (Mb) that accumulates in the kidney. Extracellular Mb is a pro-oxidant. This study tested whether supplementation with tert-butyl-bisphenol (BP) or vitamin E (Vit E, as α-tocopherol) at 0.12% w/w in the diet inhibits acute renal failure (ARF) in an animal model of RM. After RM-induction in rats, creatinine clearance decreased (p0.01), proteinuria increased (p0.001) and renal-tubule damage was detected. Accompanying ARF, biomarkers of oxidative stress (lipid oxidation and hemeoxygenase-1 (HO-1) gene and protein activity) increased in the kidney (p0.05). Supplemented BP or Vit E decreased lipid oxidation (p0.05) and HO-1 gene/activity and restored aortic cyclic guanylyl monophosphate in control animals (p0.001), yet ARF was unaffected. Antioxidant supplementation inhibited oxidative stress, yet was unable to ameliorate ARF in this animal model indicating that oxidative stress in kidney and vascular cells may not be causally related to renal dysfunction elicited by RM.

Circulating factors that have an effect on SIRT1 expression are influenced by caloric restriction. To determine the association between frailty and such circulating factors, we measured serum-induced SIRT1 expression from a nested cohort of frail (n = 77) and robust (n = 82) participants from Concord Health and Ageing in Men Project, a population-based study of community-dwelling men older than 70 years. Serum-induced SIRT1 expression was not different between frail and robust men (103.1 ± 17.0 versus 100.4 ± 19.3 μg/L). However, subsequent analyses showed that men with the lowest values (first quartile) were less likely to be frail (odds ratio = 0.5, 95% confidence interval = 0.2-1.0, p = .04) and had higher total body lean mass (p = .001) than the other participants. Serum-induced SIRT1 expression did not correlate with age, diseases, medications, albumin, fasting glucose, or lipids. Overall, there was no association between frailty and serum-induced SIRT1 expression; however, post hoc analysis suggested that there might be a paradoxical association between low serum-induced SIRT1 expression and robustness.

The liver undergoes changes in structure and function in old age. There are age-related changes in liver mass, blood flow and hepatocyte and sinusoidal cell morphology. These changes are associated with significant impairment of many hepatic metabolic and detoxification activities. This has implications for systemic aging and age-related disease. For example the age-related impairment of the hepatic metabolism of lipoproteins predisposes to cardiovascular disease. The effects of caloric restriction in the liver are beneficial in terms of ameliorating many of deleterious phenotypic effects of ageing, but from the mechanistic point of view, caloric restriction does not just simply reverse or delay age-related cellular changes. Instead caloric restriction appears to act via discrete cellular mechanisms such as sirtuin pathways that impact cellular bioenergetics, apoptosis and other cellular functions. The liver has as a pivotal coordinating role when dietary intake is reduced, therefore many of the effects of caloric restriction are mediated by the liver.

The medical and socio–economic burden of ischemic stroke is vast. Current thrombolytic therapies have a time-limited therapeutic window and do not provide significant benefits beyond tissue reperfusion. The detrimental effect of oxidative stress caused by excessive oxidant production due to cerebral reperfusion injury is a neglected consequence of ischemic stroke and warrants special consideration. Strategies directed at preventing or reducing oxidative damage in the brain post-ischemic stroke have the potential to improve neurological outcome and reduce morbidity and mortality from this common disease. Significantly, the prospect of increasing the size of the treatment window for thrombolytic therapies, perhaps by synergistic effects with other medications given in parallel, is also an avenue worthy of further investigation. This perspective outlines the current status of thrombolytic therapy for the treatment of ischemic stroke and explores the possibility of improving and expanding this potential therapy. Furthermore, the implications of directly treating damage caused by oxidative stress with novel antioxidant therapy are discussed.

Female androgen receptor (AR) knockout mice (AR−/−) generated by an in-frame Ar exon 3 deletion are subfertile, but the mechanism is not clearly defined. To distinguish between extra- and intraovarian defects, reciprocal ovarian transplants were undertaken. Ovariectomized AR−/− hosts with wild-type (AR+/+) ovary transplants displayed abnormal estrus cycles, with longer cycles (50%, P < 0.05), and 66% were infertile (P < 0.05), whereas AR+/+ hosts with either AR−/− or surgical control AR+/+ ovary transplants displayed normal estrus cycles and fertility. These data imply a neuroendocrine defect, which is further supported by increased FSH (P

Both morbidity and mortality from coronary artery disease are higher in patients with diabetes. Moreover, percutaneous treatment of coronary artery disease using coronary stents – with either bare metal or the newer drug-eluting stents – is associated with poorer outcomes in patients with diabetes compared to those without, adding to their burden of disease. Surprisingly, given these broadly poor outcomes in patients with diabetes, there is a paucity of data using animal models examining the responses to stenting in a diabetic context. Such an animal model is proposed below. Five adult lean Zucker rats underwent stenting of the left common carotid artery. This was performed following an open cutdown under general anaesthesia (using 2% isofluorane) and an arteriotomy. Balloon-mounted human coronary stents (2.0 x 7mm, BiodivYsio, Abbott Vascular, Illinois, US) were placed in the common carotid artery under direct vision (OPMI Pico-microscope, Zeiss, Germany), advanced to a mid-arterial position and deployed at 12 atmospheres. The balloon was then removed, the arteriotomy ligated, and the animals allowed to recover. There was one peri-operative death, thought to be secondary to anaesthesia. After two weeks, the remaining four animals were sacrificed and the carotids dissected free en bloc. The tissue block was placed in 10% buffered formalin, then plasticised in methylmethacrylate. Sections (100 μm thick) were cut using a band cutting system (Exakt GmbH, Norderstedt, Germany), polished, stained with haematoxylin and eosin and imaged using light microscopy. A representative section is shown (figure 1). This processing technique leaves individual stent struts in situ. These were observed stretching and compressing, but not lacerating the media, suggesting a mild degree of vessel injury. The medial stretch and compression was associated with variable, but concentric, neointima (NI) formation (figure 1). This technique therefore demonstrates the potential to provide a specific model of in-stent restenosis in the setting of diabetes. Clearly, these are preliminary observations. The degree of NI formation has not yet been quantified in relation to the degree of vessel stretch; the molecular and cellular characterisation of the NI is yet to be elucidated; and the response to drug-eluting stent deployment has not yet been established. However, the technique is straightforward, low cost, readily available and importantly, to our knowledge, the first such description of carotid stenting in the Zucker rat, a readily available animal model of diabetes. In an environment where the clinical need for such models is clear, we trust that these preliminary observations will assist researchers in this important area of investigation. RESEARCH LETTER

sICAM-1 measurements are here shown to be independent of processing method (serum, platelet rich and platelet poor plasma) and sampling size (venous or arterial blood) in patients with coronary disease.

The last decade has seen a number of important advances in the use of animal models of atherosclerosis progression. Small animal models, particularly mouse knockouts and rabbit models, are finding increasing use. This review discusses those models of particular research utility, highlights their advantages and limitations, and specifically addresses methodologies and current developments, in what is a rapidly changing field.

Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C–deficient (Lmna–/–) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna–/– myocytes show reduced shortening with normal baseline and peak amplitude of Ca2+ transients. Lmna–/– LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna–/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARγ expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna–/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna–/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.

Background. Cardiovascular disease is the most common cause of premature death in patients with end-stage renal disease, possibly due to a specific ‘uraemic cardiomyopathy’. This study was designed to investigate the cardiac changes induced by a moderate impairment of renal function in a model of uraemia. Methods. Male Wistar rats (ns11) were rendered uraemic by 5u6 nephrectomy or sham operated (ns11). After 4 weeks, cardiac dimensions were measured from fixed tissue sections using a digital image analysis technique. In parallel groups of animals, cardiac myocytes were isolated and studied for evidence of functional changes attributable to uraemia. After steady-state field stimulation at 0.5 Hz, intracellular Ca 2q handling (using Fura-2) was investigated. Up to 20 consecutive transients were averaged as the extracellular Ca 2q was increased. Results. The 5u6 nephrectomy group had a 75% reduction in glomerular filtration rate, and a 2- to 3-fold increase in serum urea and creatinine compared with sham-operated control animals (P-0.0001). However, the blood pressure was found to be similar in each group. Histology of the intact hearts (five pairs) showed a significant increase in tissue cross-sectional area (14%; P-0.04), cross-sectional area of the left ventricle (22%; P-0.04), and a significant increase in left ventricular wall thickness (15%; P-0.03). In the single cardiac cell study, under basal conditions (1–2 mM extracellular Ca 2q ) no significant differences in intracellular Ca 2q were observed, but in high extracellular Ca 2q the uraemic cells were slower to return to diastolic intracellular Ca 2q levels (P-0.05). Conclusions. The data provide evidence of altered myocardial structure and function in early experimental uraemia. The changes described are consistent with concentric hypertrophy of the left ventricle, which occurs in the absence of hypertension.

Little is known about the renal handling of endogenous ouabain-like compound (OLC). The aim of this study was to determine the normal renal clearance of OLC and the effect of mild experimental uremia on plasma OLC and its clearance.Male Wistar rats were studied 8 weeks after subtotal (5/6th) nephrectomy (n = 8) and compared with a control sham-operated group (n = 8).Plasma creatinine and OLC were higher in uremic animals compared with controls (creatinine 76+/-5.6 micromol/L v 45+/-9.6 micromol/L, respectively, P.00005; OLC 195+/-62 pmol/L v 121+/-62 pmol/L, P.02). Creatinine clearance and OLC clearance were lower in uremic animals compared with controls (creatinine 1.06+/-0.12 mL/min v 1.58+/-0.32 mL/min, respectively, P.002; OLC 23.6+/-10.4 microL/min v 33.2+/-11.4 microL/min, P.05). There were no significant differences (all P.05) between the uremic and control groups in the fractional clearance of OLC (uremic 2.3%+/-1.0% v control 2.2%+/-1.0%), OLC excretion rate (uremic 6.2+/-2.4 pmol/24 h v control 5.0+/-1.1 pmol/24 h) or in the mean systolic blood pressure (BP) (uremic 132+/-13 mm Hg v control 126+/-3 mm Hg). The amount of OLC excreted per unit of functioning nephron mass was 78% higher in uremic animals than in controls. The rate of tubular absorption varied linearly with filtered load, did not differ between groups, and showed no evidence of saturation.The kidneys are an important excretion route for plasma OLC and moderate but significant increases may occur without inducing hypertension in the short term. The low fractional clearance of OLC is most likely due to tubular absorption and/or catabolism.

1. Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodelling and the localization of atherosclerotic plaque. Uraemia is a proatherogenic process and is expected to be associated with impaired vascular reactivity. 2. To study this, 12 male Wistar rats were rendered uraemic by five-sixths nephrectomy and 12 control rats were sham operated simultaneously. After 8 weeks a tail-cuff systolic blood pressure was recorded, blood samples were taken and the animals killed. Isolated femoral arteries were dissected and mounted on a pressure myograph and myogenic tone was assessed over a range of intravascular pressures from 40 to 160 mmHg. Histologically the arteries were comparatively examined for gross morphology, calcification and deposition of collagen. 3. Biochemically the serum urea and creatinine were greater in the uraemic compared with the control rats (urea: 23.5±6 mmol/l and 6.8±01 mmol/l respectively, P not significant; creatinine: 130.7±13 mmol/l and 70.3±5 mmol/l respectively, P < 0.01) but systolic blood pressure was the same in both groups (control, 97±1 mmHg; uraemic, 98±2 mmHg), compatible with mild uraemia. 4. Myogenic tone was significantly greater in uraemic vessels (7.3±1.8% versus 2.3±0.4% in control, P = 0.01). The actual vessel lumen diameter was also smaller in pressurized uraemic vessels compared with control vessels (471±30 μm versus 604±33 μm, P< 0.01) after equilibration in physiological salt solution. However, when incubated in calcium-free physiological salt solution, the passive internal diameter was similar in uraemic vessels (538±25 μm compared with 595±31 μm in control). Histologically, there were no differences between the two groups. 5. We conclude that some aspects of vascular reactivity are altered in mild experimental uraemia as shown by a reduced internal lumen diameter and increased myogenic tone. Furthermore, these changes are apparent in the absence of hypertension and precede structural changes.