Untreated Male Rats Research Articles

Determining optimal pretreatment in cardiac surgery: an experimental study.

Heart failure patients with reduced ejection fraction are currently treated with four drug combinations: angiotensin receptor/neprilysin inhibitors, beta-blockers, mineralocorticoid receptor antagonists, and sodium-glucose cotransporter 2 inhibitors, resulting in improved survival outcomes. Herein, we examined whether myocardial protection by esaxerenone or sacubitril/valsartan may present a counter-effect to the harm caused by cardioplegic arrest. Male Wistar rats fed a normal diet were orally administered esaxerenone (3mg/kg; Esax) or sacubitril/valsartan (68mg/kg; SaV) once a day for 2weeks from 6weeks of age. Age-matched, untreated male Wistar rats served as controls (Control). Isolated rat hearts were aerobically Langendorff-perfused and subjected to 2min of St Thomas' Hospital 2 cardioplegia (STH2) infusion and 28min of normothermic global ischemia followed by 60min of reperfusion. The recovery of function was measured during 60min of reperfusion. Additionally, troponin T levels were measured after reperfusion as myocardial injury. The final recovery of left ventricular developed pressure (presented as the percentage of preischemic value) in the Control, Esax, and SaV groups was 50.7 ± 6.2%, 68.5 ± 7.4%*, and 69.3 ± 14.3%*, respectively (*p < 0.05 vs. Control). Troponin T (ng per gram wet weight) levels in the Control, Esax, and SaV groups were 166.8 ± 78.1, 77.0 ± 14.6*, and 74.2 ± 36.6*, respectively (*p < 0.05 vs. Control). Oral administration of esaxerenone or sacubitril/valsartan to rats 2weeks prior to surgery enhanced the myocardial protection afforded by STH2 and may attenuate the myocardial injury caused by hyperkalemic cardioplegic arrest.

Therapeutic hypothermia demonstrates sex-dependent improvements in motor function in a rat model of neonatal hypoxic ischemic encephalopathy

Neonatal hypoxic ischemic encephalopathy (HIE) is a neurological disease caused by restricted oxygen and blood flow to the brain at or around the time of birth. Long term cognitive and motor sequelae are common and demonstrate sexual dimorphism in animal studies. Therapeutic hypothermia (TH) is the standard of care for HIE, but provides incomplete neuroprotection. Using the Vannucci model of neonatal HIE, term-equivalent 11-day old rat pups were subjected to mild-moderate hypoxic-ischemic injury (HII), and a subset of animals were treated with TH. Sex-dependent neuroprotection was measured with gross and fine motor control assays, and functional deficits detected with these assays were correlated to injury in specific brain structures. At the equivalent of human adolescence and adulthood (P51–89), accelerod and beam walking tests were used to assess gross motor function, and string-pulling and food handling tests were used to assess fine motor function. At necropsy (P94–97), brain lesions were primarily focused to the posterior cerebrum and characterized by variable reduction in cortical, thalamic and hippocampal regions and glial scarring. Gross motor impairment was detected in male rats with untreated and TH-treated HIE in the accelerod test, but beam walk test data was confounded by the lower body mass of untreated male rats. HIE animals of both sexes demonstrated deficit in the forelimb contralateral to ischemic surgery, observed as unilaterally impaired food handling behaviors, and in string pulling as decreased string contacts and increased in bracing behavior. However, kinematic analyses revealed sex-specific decreases in peak speeds in string reaching and pulling movements. In both sexes, treatment with TH improved body mass, some measures of contralateral forelimb impairment, and the severity of brain lesions to levels not different to Sham surgery rats. Unique differences in behavior following TH were observed in female rats, who took longer to consume food items but traversed beams and approached strings faster than untreated and Sham females. Future use of these motor assays may unravel the subtle, sex-specific differences in HIE outcomes and in developing a customized therapeutic approach to neonatal brain injury.

Behavioural multigenerational effects induced by the administration of very low doses of zinc during pregnancy, lactation, and prepuberal period in the rat

In studies from this laboratory, the chronic administration of ZnTe during pregnancy, lactation, and prepuberal stages of litter (F1 generation) modified the behavioral patterns of motivated exploration, lateralized exploration, social activity, and survival responses of maturing rats. To determine whether these affected behaviors would extend to the next generation, F1 litter rats previously exposed to tellurium (Te) up to 30-day-old were left at rest with no further treatment up to 90-day-old. Then, F1 female rats were mated with normal untreated male rats, and in the next generation (F2), the litter rats at 30-day-old preserved the modified behaviors previously observed in their parents. The study revealed that Te effects were intergenerational. Here, considering that ZnTe was used in the previous study and that Zn ion has many physiological functions in the cell, experiments were conducted to elucidate if Zn would have an intergenerational effect similar to Te. Working with the same experimental setup as in the previous study but using ZnCl2 instead of ZnTe, results revealed that none of the behavioral responses studied were affected by the F1 generation. However, in the F2 generation, lateralized exploration and survival behavior were inhibited, suggesting that Zn also has an intergenerational effect.

DNA methylation and hydroxymethylation have distinct genome-wide profiles related to axonal regeneration

ABSTRACT Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

Passiflora edulis seed oil from west Cameroon: Chemical characterization and assessment of its hypolipidemic effect in high-fat diet-induced rats.

In this study, the in vivo hypolipidemic effect of west Cameroonian Passiflora edulis variety seed oil (PE) was assessed in female and male Wistar rats. The chemical properties of the oil were evaluated through the determination of the peroxide, iodine, and thiobarbituric acid values, as well as its fatty acid composition using gas chromatography. Results showed that the oil extraction yield was 19.90% and its quality indexes were as follows: peroxide value = 2.10 ± 0.20 meq O2/kg; thiobarbituric acid value = 0.25 ± 0.00 ppm; and iodine value = 97.40 ± 0.45 g I2/100 g. Its fatty acid composition showed that it contains about 84.88% of unsaturated fatty acid, linoleic acid being the most represented (68.39%), followed by oleic acid (14.31%). The administration of this oil resulted in a significant reduction (p < .05) in the level of triglycerides, total cholesterol, and low‐density lipoprotein‐cholesterol in rats. The PE groups showed a significant increase (p < .05) in high‐density lipoprotein‐cholesterol compared with untreated male rats. A similar trend was observed with female rats for triglycerides, but lowest values were observed with olive oil at 1 ml. This study suggests that P. edulis seed oil is rich in linoleic acid, which might be responsible for its hypolipidemic effect comparable to that of olive oil.

Tellurium epigenetic transgenerational effects on behavioral expression of coping behavior in rats.

An increasing interest has been developed in the past 15 years in the relationship between trace elements and cell functioning. In the present work the possibility of transgenerational effects of Te was investigated in rats. F1 generation exposed to K2TeO3 (1.55nM) from day 1 of pregnancy until litters were 30 day old, these animals with no other treatment than tap water and food were let to reach 60-70 day old. At this age, female rats were mated with normal untreated male rats. The F2 generation also without any Te treatment was allowed to grow until 30 days of age. At this age, behavioral tests measuring exploration induced by novelty, lateralized exploration, social interaction and survival behavior were applied. Results showed that head-dipping, rearing, lateralized exploration, social interaction, and survival behaviors, affected by Te treatment in F1 generation, also were modified in the same manner in F2 generation. These data show that Te effects on coping behavior in rats are preserved epigenetically in the next generation.

Effect of intranasal insulin on peripheral glucose profile in dexamethasone-induced insulin resistance in Wistar rats

This study evaluates the therapeutic potential of intranasal insulin (INI) in dexamethasone-induced insulin resistance in Wistar rats. In the first phase of the study, thirteen, healthy, untreated male Wistar rats were divided into 2 groups and administered either vehicle (0.9% Normal saline, 20 µl) or insulin (2 IU) intranasally to assess intranasal delivery of insulin in brain. In the second phase of experiments, to evaluate the acute effects of intranasal insulin on peripheral blood glucose, intranasal or intraperitoneal insulin was co-administered with or without dexamethasone 10 mg/kg to 26 male Wistar rats and blood glucose monitored. To evaluate effect of intranasal insulin in peripheral metabolic disease model, insulin or vehicle was administered via intranasal or intraperitoneal (IP) route to control or dexamethasone (Dex)-treated (0.5 mg/kg, IP) female Wistar rats for seven consecutive days. Twenty-four hours after last dose, trunk blood was collected via cardiac puncture. Biochemical assay of glucose, lipid and insulin was performed on serum while enzyme activity – glucokinase and glucose-6-phosphatase (G6Pase) or lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) were assayed from liver and brain homogenates respectively. Acute intranasal but not intraperitoneal insulin elevated brain insulin after 30 min. In animals administered single dose of 10 mg/kg dexamethasone, intranasal and intraperitoneal insulin lowered blood glucose within one hour. However, only the former’s effect was maintained at the 3rd and fourth hour. Dex-induced hyperglycemia was associated with increased hepatic glucose-6-phosphatase activity and decreased high-density lipoprotein (HDL), these effect were attenuated by subchronic (INI) administration. Also INI did not induce oxidative stress in the brain which suggests no brain damage during the period of study. Subchronic administration of INI was able to reduce the effect of Dex-induced hyperglycemia that is associated with increased hepatic glucose-6-phosphatase activity and decreased high-density lipoprotein (HDL) without damage to the brain. We demonstrate the potential of brain targeting with intranasal insulin in a rat model of insulin resistance and peripheral metabolic disease.

Survival rates of homozygotic Tp53 knockout rats as a tool for preclinical assessment of cancer prevention and treatment

BackgroundThe gene that encodes tumor protein p53, Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention.MethodsA small colony of Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves.ResultsThe four consecutive quarterly survival curves were highly reproducible. They were combined into a single “master” curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals (when assessed using the t-test with a power of 80% and alpha set at 0.05). As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative.ConclusionFurther evaluation of the Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment.

Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro

In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.

Age and sex differences in kidney microRNA expression during the life span of F344 rats.

BackgroundGrowing evidence suggests that epigenetic mechanisms of gene regulation may play a role in susceptibilities to specific toxicities and adverse drug reactions. MiRNAs in particular have been shown to be important regulators in cancer and other diseases and show promise as predictive biomarkers for diagnosis and prognosis. In this study, we characterized the global kidney miRNA expression profile in untreated male and female F344 rats throughout the life span. These findings were correlated with sex-specific susceptibilities to adverse renal events, such as male-biased renal fibrosis and inflammation in old age.MethodsKidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. Differential expression was determined using filtering criteria of ≥1.5 fold change and ANOVA or pairwise t-test (FDR <5%) to determine significant age and sex effects, respectively. Pathway analysis software was used to investigate the possible roles of these target genes in age- and sex-specific differences.ResultsThree hundred eleven miRNAs were found to be expressed in at least one age and sex. Filtering criteria revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2-week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified.ConclusionsThe expression of 56% of detected renal miRNAs was found to vary significantly with age and/or sex during the life span of F344 rats. Pathway analysis suggested that 2-week-expressed miRNAs may be related to organ and cellular development and proliferation pathways. Male-biased miRNA expression at older ages correlated with male-biased renal fibrosis and mononuclear cell infiltration. These miRNAs showed high representation in renal inflammation and nephritis pathways, and included miR-214, miR-130b, miR-150, miR-223, miR-142-5p, miR-185, and miR-296*. Analysis of kidney miRNA expression throughout the rat life span will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-014-0019-1) contains supplementary material, which is available to authorized users.

Adipose tissue-derived mesenchymal stem cells repair germinal cells of seminiferous tubules of busulfan-induced azoospermic rats.

CONTEXT:Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are less invasive than bone marrow mesenchymal stem cells to obtain for cell therapy.AIMS:The aims of this study were to evaluate the germinal cells characteristics and repairs in seminiferous tubules of busulfan-induced azoospermic rats after AT-MSCs transplantation.SETTINGS AND DESIGN:Experimental case-control study.MATERIALS AND METHODS:In the present experimental study, donors AT-MSCs were isolated from subcutaneous adipose tissue of two Sprague-Dawley rats. The recipients (n = 5) were received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia by busulfan, rats were injected with the AT-MSCs into the efferent duct of right testes. After 60 days, the right testes were injected AT-MSCs were compared to left azoospermic testes. Five untreated male rats served as negative control.STATISTICAL ANALYSIS USED:Stereological indices were analyzed by one-way ANOVA and LSD post-hoc test. The spermatogenesis index was compared using Mann–Whitney U test.RESULTS:After stereological analyses, the seminiferous tubules treated with AT-MSCs had normal morphology. The untreated seminiferous tubules were empty. Spermatogenesis was observed in most cell-treated seminiferous tubules.CONCLUSIONS:The testis of busulfan-induced azoospermic rats accepted transplanted AT-MSCs. The transplanted AT-MSCs could induce spermatogenesis in seminiferous tubules of the rat.

Fumaric acid esters can block pro-inflammatory actions of human CRP and ameliorate metabolic disturbances in transgenic spontaneously hypertensive rats.

Inflammation and oxidative stress have been implicated in the pathogenesis of metabolic disturbances. Esters of fumaric acid, mainly dimethyl fumarate, exhibit immunomodulatory, anti-inflammatory, and anti-oxidative effects. In the current study, we tested the hypothesis that fumaric acid ester (FAE) treatment of an animal model of inflammation and metabolic syndrome, the spontaneously hypertensive rat transgenically expressing human C-reactive protein (SHR-CRP), will ameliorate inflammation, oxidative stress, and metabolic disturbances. We studied the effects of FAE treatment by administering Fumaderm, 10 mg/kg body weight for 4 weeks, to male SHR-CRP. Untreated male SHR-CRP rats were used as controls. All rats were fed a high sucrose diet. Compared to untreated controls, rats treated with FAE showed significantly lower levels of endogenous CRP but not transgenic human CRP, and amelioration of inflammation (reduced levels of serum IL6 and TNFα) and oxidative stress (reduced levels of lipoperoxidation products in liver, heart, kidney, and plasma). FAE treatment was also associated with lower visceral fat weight and less ectopic fat accumulation in liver and muscle, greater levels of lipolysis, and greater incorporation of glucose into adipose tissue lipids. Analysis of gene expression profiles in the liver with Affymetrix arrays revealed that FAE treatment was associated with differential expression of genes in pathways that involve the regulation of inflammation and oxidative stress. These findings suggest potentially important anti-inflammatory, anti-oxidative, and metabolic effects of FAE in a model of inflammation and metabolic disturbances induced by human CRP.

Sexual odor discrimination and physiological profiles in adult male rats after a neonatal, short term, reversible nasal obstruction.

The present study was designed to examine behavioral responses (interpreted as preferences) to olfactory cues (nest bedding odor and odors of estrous and anestrus females) in adult male rats after they had a short term reversible, bilateral, nasal obstruction (RbNO) as developing rat pups. These results were compared to behavior of control (untreated) and sham operated male littermates. Behavioral tests and physiological parameters were analyzed 90 days after recovery of nasal breathing. Experiments investigated the time spent in arms or the center of a maze of male rats in response to odors from the nest bedding or from adult females. There were no differences in responses between untreated, sham and RbNO adult male rats to fresh and nest bedding odors. RbNO males spent more time in the center of the maze when given a choice of estrus or anestrus female odors, or bedding odors from untreated or sham operated female rats. In contrast untreated and sham male rats preferred the odors of estrous females and of untreated or sham females. Plasma corticosterone levels in the males increased during the behavioral tests. Plasma testosterone levels were significantly lower in RbNO males compared to untreated males and did not increase during the behavioral tests compared to sham operated males. Males from all groups had similar preferences for the odor of bedding from adult RbNO females. Plasma levels of cholesterol and triglycerides were increased in RbNO adults. In conclusion, short term nasal obstruction in males while juvenile has long term consequences on hormones and behavioral preferences, thus potential partner selection when adult.

Short-term esmolol improves coronary artery remodeling in spontaneously hypertensive rats through increased nitric oxide bioavailability and superoxide dismutase activity.

The aim of this study was to assess the effects of short-term esmolol therapy on coronary artery structure and function and plasma oxidative stress in spontaneously hypertensive rats (SHR). For this purpose, 14-month-old male SHR were treated for 48 hours with esmolol (SHR-E, 300 μg/kg/min). Age-matched untreated male SHR and Wistar Kyoto rats (WKY) were used as hypertensive and normotensive controls, respectively. At the end of intervention we performed a histological study to analyze coronary artery wall width (WW), wall-to-lumen ratio (W/L), and media cross-sectional area (MCSA). Dose-response curves for acetylcholine (ACh) and sodium nitroprusside were constructed. We also assessed several plasma oxidative stress biomarkers, namely, superoxide scavenging activity (SOSA), nitrites, and total antioxidant capacity (TAC). We observed a significant reduction in WW (P < 0.001), W/L (P < 0.05), and MCSA (P < 0.01) and improved endothelium-dependent relaxation (AUCSHR-E = 201.2 ± 33 versus AUCSHR = 97.5 ± 21, P < 0.05) in SHR-E compared with untreated SHR; no differences were observed for WW, MCSA, and endothelium-dependent relaxation by ACh at higher concentrations (10−6 to 10−4 mol/l) for SHR-E with respect to WKY. SOSA (P < 0.001) and nitrite (P < 0.01) values were significantly higher in SHR-E than in untreated SHR; however, TAC did not increase after treatment with esmolol. Esmolol improves early coronary artery remodeling in SHR.

The inhibitory effect of anandamide on oxytocin and vasopressin secretion from neurohypophysis is mediated by nitric oxide

The neurohypophyseal hormones oxytocin (OT) and vasopressin (VP) are involved in behavioral, autonomic and neuroendocrine functions. Both peptides are synthesized in magnocellular neurons of paraventricular and supraoptic nuclei at hypothalamic level whose axons terminate in the neurohypophysis (NH), from where OT and VP are released into the systemic circulation. NH contains abundant nitric oxide (NO) synthase suggesting that NO plays a role in the release of these neuropeptides. The endocannabinoid system is present in magnocellular neurons of the hypothalamic neurohypophyseal system, and we have previously demonstrated that endocannabinoids modulate OT secretion at hypothalamic level.In the present work, we investigated the in vitro effect of the endocannabinoid anandamide (AEA) on OT and VP release from NH of untreated adult male rats and the involvement of NO in this action.Our results showed that AEA decreased OT and VP secretion from NH. AEA action was mediated by NO, since the inhibition of NO synthesis completely blocked this inhibitory effect. We found that cannabinoid receptor type 2 (CB2) and transient receptor potential cation channel subfamily V member 1 (TRPV1) are involved in the inhibitory effect of AEA because AM630 and capsazepine, CB2 and TRPV1 antagonists respectively, but not AM251, a CB1 antagonist, blocked AEA effect at neurohypophyseal level.These findings revealed an interaction between endocannabinoid, nitric oxide and oxytocin/vasopressin systems that could be involved in the modulation of homeostatic, behavioral and reproductive processes.

Life Cycle Analysis of Kidney Gene Expression in Male F344 Rats

Age is a predisposing condition for susceptibility to chronic kidney disease and progression as well as acute kidney injury that may arise due to the adverse effects of some drugs. Age-related differences in kidney biology, therefore, are a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of genes expressed in the kidney at various life cycle stages will impact susceptibility to adverse drug reactions. Therefore, establishing changes in baseline expression data between these life stages is the first and necessary step in evaluating this hypothesis. Untreated male F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age. Kidneys were collected for histology and gene expression analysis. Agilent whole-genome rat microarrays were used to query global expression profiles. An ANOVA (p<0.01) coupled with a fold-change>1.5 in relative mRNA expression, was used to identify 3,724 unique differentially expressed genes (DEGs). Principal component analyses of these DEGs revealed three major divisions in life-cycle renal gene expression. K-means cluster analysis identified several groups of genes that shared age-specific patterns of expression. Pathway analysis of these gene groups revealed age-specific gene networks and functions related to renal function and aging, including extracellular matrix turnover, immune cell response, and renal tubular injury. Large age-related changes in expression were also demonstrated for the genes that code for qualified renal injury biomarkers KIM-1, Clu, and Tff3. These results suggest specific groups of genes that may underlie age-specific susceptibilities to adverse drug reactions and disease. This analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Estrogen modulates the influence of cardiac inflammatory cells on function of cardiac fibroblasts

BackgroundInflammatory cells play a major role in the pathology of heart failure by stimulating cardiac fibroblasts to regulate the extracellular matrix in an adverse way. In view of the fact that inflammatory cells have estrogen receptors, we hypothesized that estrogen provides cardioprotection by decreasing the ability of cardiac inflammatory cells to influence fibroblast function.MethodsMale rats were assigned to either an untreated or estrogen-treated group. In the treated group, estrogen was delivered for 2 weeks via a subcutaneous implanted pellet containing 17β-estradiol. A mixed population of cardiac inflammatory cells, including T-lymphocytes (about 70%), macrophages (about 12%), and mast cells (about 12%), was isolated from each rat and cultured in a Boyden chamber with cardiac fibroblasts from untreated adult male rats for 24 hours. To examine if tumor necrosis factor-alpha (TNF-α) produced by inflammatory cells represents a mechanism contributing to the stimulatory effects of inflammatory cells on cardiac fibroblasts, inflammatory cells from the untreated group were incubated with cardiac fibroblasts in a Boyden chamber system for 24 hours in the presence of a TNF-α-neutralizing antibody. Cardiac fibroblasts were also incubated with 5 ng/mL of TNF-α for 24 hours. Fibro-blast proliferation, collagen synthesis, matrix metalloproteinase activity, β1 integrin protein levels, and the ability of fibroblasts to contract collagen gels were determined in all groups and statistically compared via one-way analysis of variance.ResultsInflammatory cells from the untreated group resulted in: 1) an increased fibroblast proliferation, collagen production and matrix metalloproteinase activity; and 2) a loss of β1 integrin protein and a reduced ability to contract collagen gels. In contrast, inflammatory cells from the treated group resulted in: 1) an attenuated fibroblast proliferation; 2) a nonsignificant reduction in collagen production; 3) the prevention of matrix metalloproteinase activation and the loss of β1 integrin by fibroblasts and 4) a preservation of the fibroblasts’ ability to contract collagen gels. The TNF-α neutralizing antibody attenuated or prevented the untreated inflammatory cell-induced fibroblast proliferation, collagen production, matrix metalloproteinase activation and loss of β1 integrin protein as well as preserved fibroblast contractile ability. Incubation with TNF-α yielded changes in the cardiac fibroblast parameters that were directionally similar to the results obtained with untreated inflammatory cells.ConclusionThese results and those of our previous in vivo studies suggest that a major mechanism by which estrogen provides cardioprotection is its ability to modulate synthesis of TNF-α by inflammatory cells, thereby preventing inflammatory cell induction of cardiac fibroblast events that contribute to adverse extracellular matrix remodeling.

Age and sex dependent changes in liver gene expression during the life cycle of the rat

BackgroundAge- and sex-related susceptibility to adverse drug reactions and disease is a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of hepatic genes expressed at various life cycle stages will impact susceptibility to adverse drug reactions. Understanding the basal liver gene expression patterns is a necessary first step in addressing this hypothesis and will inform our assessments of adverse drug reactions as the liver plays a central role in drug metabolism and biotransformation. Untreated male and female F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age. Liver tissues were collected for histology and gene expression analysis. Whole-genome rat microarrays were used to query global expression profiles.ResultsAn initial list of differentially expressed genes was selected using criteria based upon p-value (p < 0.05) and fold-change (+/- 1.5). Three dimensional principal component analyses revealed differences between males and females beginning at 2 weeks with more divergent profiles beginning at 5 weeks. The greatest sex-differences were observed between 8 and 52 weeks before converging again at 104 weeks. K-means clustering identified groups of genes that displayed age-related patterns of expression. Various adult aging-related clusters represented gene pathways related to xenobiotic metabolism, DNA damage repair, and oxidative stress.ConclusionsThese results suggest an underlying role for genes in specific clusters in potentiating age- and sex-related differences in susceptibility to adverse health effects. Furthermore, such a comprehensive picture of life cycle changes in gene expression deepens our understanding and informs the utility of liver gene expression biomarkers.

Renal expression of organic anion transporter Oat5 in rats and mice exhibits the female-dominant sex differences.

The organic anion transporter 5 (Oat5, Slc22a19) was previously localized to the brush-border of proximal tubule (PT) S3 segment in rat and mouse kidneys. Here we report on sex hormone-regulated expression of Oat5 in rat kidneys, after reinvestigating: a) expression of its mRNA by end-point and real time RT-PCR in the tissue, b) abundance of its protein by Western blotting (WB) in isolated membranes, and c) immunolocalization in tissue cryosections. In untreated male (M) and female (F) adult rats, the expression of Oat5 mRNA was predominant in the outer stripe (OS), exhibiting sex differences (M<F), upregulated by castration, and unaffected by ovariectomy. In castrated M, testosterone treatment strongly downregulated, whereas estradiol and progesterone treatment weakly upregulated its expression. By WB, a single protein band of ~72 kDa in variously-treated animals exhibited a density pattern comparable to that of mRNA. By immunostaining, Oat5 protein was localized to the brush-border of S1/S2 in the cortex (CO) (weakly) and in S3 of the OS and medullary rays (strongly) with the F-dominant intensity. In variously-treated rats, the immunostaining pattern matched that of mRNA and WB data. In prepubertal rats, the renal expression of Oat5 mRNA and protein was weak and sex-independent. In adult mice, the sex-dependent pattern of renal Oat5 protein expression was comparable to that in rats. Therefore, the renal expression of Oat5 in rats (and mice) exhibits zonal (CO<OS) and sex differences (M<F), which appear after puberty, largely due to androgen-driven downregulation of its mRNA and protein expression.

Hypercholesterolemia and 3-hydroxy-3-methylglutaryl coenzyme A reductase regulation in aged female rats

Coronary heart disease is less prevalent in pre-menopausal women than in men, but increases at the onset of menopause. This delay is due to estrogen protective effects. The rise of cholesterolemia is one of the main risk factors for coronary disease. Since 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the cholesterol biosynthetic pathway, it plays a pivotal role in cholesterol homeostasis maintenance. Aim of this study is to investigate whether HMGR is involved in the cholesterolemia increase that occurs during aging, and to consider its potential role as a target for estrogen protective effects. “In vivo” studies have been performed using the livers of 12-month-old female rats (whose estrogen level decrease is comparable to the one detected at the occurrence of human menopause), 12-month-old female rats treated with 17-β-estradiol, and 3-month-old untreated male and female rats. The results indicated hypercholesterolemic status and a significant increase of HMGR activity according to a reduced activation of AMPK detected in treated rats compared to controls. Furthermore, 17-β estradiol treatment reduced HMGR activity restoring AMPK activation. These findings highlight the correlation between estrogen and HMGR short-term regulation, and suggest the presence of another mechanism underlying the protective role of estrogen in age-related diseases.