Diet Significantly Influences the Immunopathology and Severity of Kidney Injury in Male C57Bl/6J Mice in a Model Dependent Manner

Diet is a leading causative risk factor for morbidity and mortality worldwide, yet it is rarely considered in the design of preclinical animal studies. Several of the nutritional inadequacies reported in Americans have been shown to be detrimental to kidney health; however, the mechanisms responsible are unclear and have been largely attributed to the development of diabetes or hypertension. Here, we set out to determine whether diet influences the susceptibility to kidney injury in male C57Bl/6 mice. Mice were fed a standard chow diet, a commercially available “Western” diet (WD), or a novel Americanized diet (AD) for 12 weeks prior to the induction of kidney injury using the folic acid nephropathy (FAN) or unilateral renal ischemia reperfusion injury (uIRI) models. In FAN, the mice that were fed the WD and AD had worse histological evidence of tissue injury and greater renal expression of genes associated with nephrotoxicity and monocyte infiltration as compared to mice fed chow. Mice fed the AD developed more severe renal hypertrophy following FAN, and gene expression data suggest the mechanism for FAN differed among the diets. Meanwhile, mice fed the WD had the greatest circulating interleukin-6 concentrations. In uIRI, no difference was observed in renal tissue injury between the diets; however, mice fed the WD and AD displayed evidence of suppressed inflammatory response. Taken together, our data support the hypothesis that diet directly impacts the severity and pathophysiology of kidney disease and is a critical experimental variable that needs to be considered in mechanistic preclinical animal studies

Determining Microbial and Neuroimmunological Differences Associated with Adolescent Alcohol Use

Early alcohol use is an important risk factor for alcohol and substance use disorders, but the neurobiological changes that lead to increased vulnerability are not well understood. Alcohol-induced alterations in the microbiome at an early age may lead to neural changes that affect the escalation of alcohol use. Consequently, the microbiome is a novel area of research for adolescent alcohol use disorder (AUD). The present studies investigated differences in the oral microbiome of human adolescents (ages 17-19) who engage in heavy drinking (n=21, 52.4% female) compared to non-drinking control participants (n=18, 44.4% female). We explored associations between the oral microbiome, oral cytokine mRNA levels (interleukin 6 [IL-6], interleukin 1 beta [IL-1β], tumor necrosis factor alpha [TNF-α]), and proton magnetic resonance spectroscopy markers of neuroinflammation (myo-inositol, total choline, total creatine, N-Acetylaspartate). As hypothesized, adolescents who engaged in heavy drinking had significant differences in the diversity and composition of the oral microbiome. Compared to the control group, the alcohol-using group exhibited lower evenness and higher abundances of Rothia and Corynebacterium. We observed no significant differences in cytokine mRNA levels between the groups; however, levels of IL-1β correlated with the abundances of several genera in both groups, including abundances of Rothia. IL-1β was also associated with a higher number of alcohol use days in the past 90 days and phosphatidylethanol concentrations in the alcohol-using group. While there were no significant group differences in neuroinflammation markers, abundances of Rothia were associated with significantly higher concentrations of total creatine-containing metabolites across the whole sample. The associations between Rothia, mRNA cytokine levels, and neurometabolite levels are intriguing, as Rothia is well known for its high capacity to covert alcohol into acetaldehyde, suggesting a potential link between the oral microbiome and neurobiological processes related to alcohol use. Taken together, these studies indicate that the oral microbiome may be affected by alcohol use and is associated with cytokine mRNA levels and neurometabolite concentrations in individuals who use alcohol and controls. The microbiome may aid in prevention and intervention efforts for adolescent AUD by providing new targets for treatment and/or diagnostic and therapeutic response biomarkers

Neutrophil Depletion Influences Renal Outcomes in Male Mice with Chronic Angiotensin II Infusion

Hypertension is the leading cause of morbidity and mortality worldwide; yet the exact cause for many cases remain unknown. Recent research has highlighted the significance of the immune system in the development and progression of HTN in animal models. Much of this research has focused on classical immune pathways involving antigen presenting cells and subsequent activation of the adaptive immune response (primarily CD8+ T-cells). These activated lymphocytes then support the development of HTN by mediating end-organ damage in organs tasked with regulating blood pressure and extracellular volume (such as the kidneys). In the current study, we set out to determine the role of neutrophils in a murine model of HTN – the chronic angiotensin II (AngII) infusion model. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conform to the FASEB Statement of Principles for the use of animals in research and education. Baseline blood pressure was recorded in male C57Bl/6 mice (N=10) for 3 weeks using the CODA-8 tail cuff volume pressure recording system. Neutrophil depletion was accomplished via intraperitoneal injections of a monoclonal anti-Ly6G antibody (1A8, n=5), or IgG control (2A3, n=5) every 48 hours (250μg/mouse). 18 hours following the first antibody injection, a mini-osmotic pump was implanted into the rear flank delivering 500ng/kg*min AngII. 3 days after pump implantation, blood pressure readings resumed and continued for 14 days. After 14 days, mice were individually housed in metabolic cages and urine was collected for quantification of albuminuria. Renal blood flow was estimated in anesthetized mice using contrast-enhanced ultrasonography. Following renal blood flow estimation, mice were euthanized and blood, kidney, and spleens collected for determination of leukocyte content by flow cytometry. All data were analyzed using general linear models procedures in SPSS. Significant differences in blood pressure were observed periodically during the 14 days of AngII infusion (P≤0.05 for days 8, 12, and 14); however, the overall effect of treatment (1A8 vs. 2A3) did not reach statistical significance (P\u3c0.2). Mice receiving the 1A8 neutrophil depleting antibody also tended (P≤0.07) to have a higher urinary output and albuminuria as compared to mice receiving the isotype control (respectively). No difference in estimated renal blood flow was detected between the treatment groups. Tissue analysis by flow cytometry confirmed the depletion and/or significant reduction of circulating and splenic neutrophils in mice receiving 1A8, but not 2A3, injections after 2 weeks. In the kidneys, mice receiving the 1A8 neutrophil depleting antibody had reduced (P=0.03) CD45+ leukocyte content. Further analysis of renal single cell suspensions revealed a significant reduction (P=0.04) in B-cells and higher (P=0.03) CD8+ T-cells in 1A8 treated animals. Taken together, our data suggest that neutrophil depletion alters several renal outcomes in the AngII murine model of HTN, with neutropenic mice tending to have higher blood pressure, greater urinary output and albuminuria as compared to control mice. These effects could be mediated by the significant differences in renal leukocyte content between the treatment groups. Further studies are needed to confirm our results and delineate the molecular mechanisms responsible for altered renal function and inflammation due neutrophil depletion in this animal model

Determining the Influence of a Novel Rodent Diet on Body Weight Gain and Renal Health in Male Mice from 2 Different Strains

Diet has an established relationship with health in humans; yet it is poorly represented or significantly oversimplified in most preclinical animal studies. Based on recent data, the “typical” American adult consumes a diet that is inadequate in several nutrients and high in salt and energy yielding compounds (particularly simple sugars). However, rodents diets used to model the nutritional composition of developed (or “Western”) societies do not embody these same inadequacies. Interestingly, many of the nutritional inadequacies noted in developed countries have been independently shown to negatively affect renal health in animal models. However, to our knowledge no animal studies have adequately determined the physiological consequences of consuming a diet modeling the complex nutritional inadequacies of Americans. Based on this information, we hypothesized that mice consuming an “Americanized diet” (AD) would have greater weight gain and evidence of renal injury as compared to mice fed chow. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conform to the FASEB Statement of Principles for the use of animals in research and education. Weanling (3-week old) male 129 Svlm/J (129, n=10) and C57Bl/6J mice (B6, n=10) were obtained from Jackson Laboratory and given 1 week to acclimate to solid diet. Mice were then assigned (n=5 per strain) to remain on a commercially available chow diet or our novel AD formulated to have similar nutritional inadequacies as reported by recent NHANES anlyses. Mice were given ad libitum access to their respective diets for 6 weeks and 24-hour dietary intake and body weights were recorded throughout the study. At the end of the 6-week feeding study, mice were individually housed in metabolic cages to collect urine for determination of albuminuria by ELISA. Renal blood flow was estimated using contrast-enhanced ultrasonography. All statistical analyses were performed using general linear models in SPSS. The B6 mice had higher average 24-hour caloric intake and resulted in a significant increase in body weight in the B6 mice (P=0.01), with the B6 mice consuming the AD having the highest food intake and overall body weight. Strain and diet both significantly influenced urinary output regardless of water intake (covariate), with higher (P=0.03) urinary output in the 129 strain (0.9±0.1 vs 0.4± 0.1 mL urine/day, 129 and C57Bl/6 respectively) and in mice consuming the AD diet (0.8±0.1 vs 0.5±1 mL urine/day). Regardless of strain, all mice consuming the AD tended (P=0.06) to have a greater 24 hour albumin excretion (26.7±4 μg) as compared to mice consuming chow (14.7±4 μg). Strain also significantly influenced estimated renal blood flow regardless of diet, with the B6 mice having a greater (P=0.02) estimated renal blood flow as compared to the 129 strain. Taken together, our data strongly suggest that a rodent diet formulated to match the nutritional quality of a “typical” American significantly influences renal health, particularly albuminuria and daily urinary volume. These data also highlight a strain difference in regards to renal outcomes alone or in combination with diet. Further studies are needed to elucidate the physiological mechanisms dictating these differences and provide insight into the nutrients involved in initiating renal dysfunction

Brain metabolite alterations related to alcohol use: a meta-analysis of proton magnetic resonance spectroscopy studies.

Alcohol misuse and alcohol use disorder (AlUD) have neurobiological consequences. This meta-analysis of proton magnetic resonance spectroscopy (MRS) studies aimed to assess the differences in brain metabolite levels in alcohol misuse and AUD relative to controls (PROSPERO registration: CRD42020209890). Hedges g with random-effects modeling was used. Sub-group and meta-regression techniques explored potential sources of demographic and MRS parameter heterogeneity. A comprehensive literature review identified 43 studies, resulting in 69 models across gray and white matter (GM, WM). Lower N-acetylaspartate levels were found in frontal, anterior cingulate cortex (ACC), hippocampal, and cerebellar GM, and frontal and parietal WM, suggesting decreased neuronal and axonal viability. Lower choline-containing metabolite levels (all metabolites contributing to choline peak) were found in frontal, temporal, thalamic, and cerebellar GM, and frontal and parietal WM, suggesting membrane alterations related to alcohol misuse. Lower creatine-containing metabolite levels (Cr; all metabolites contributing to Cr peak) were found in temporal and occipital cortical GM, while higher levels were noted in midbrain/brainstem GM; this finding may have implications for using Cr as an internal reference. The lack of significant group differences in glutamate-related levels is possibly related to biological and methodological complexities. The few studies reporting on GABA found lower levels restricted to the ACC. Confounding variables were age, abstinence duration, treatment status, and MRS parameters (echo time, quantification type, data quality). This first meta-analysis of proton MRS studies consolidates the numerous individual studies to identify neurometabolite alterations within alcohol misuse and AUD. Future studies can leverage this new formalized information to investigate treatments that might effectively target the observed disturbances

The glucagon-like peptide-1 system is modulated by acute and chronic alcohol exposure: Findings from human laboratory experiments and a post-mortem brain study

Growing evidence suggests that the glucagon-like peptide-1 (GLP-1) system modulates alcohol seeking and consumption, and GLP-1 analogues may represent novel pharmacotherapies for alcohol use disorder (AUD). Accordingly, it is important to understand the potential effects of alcohol on the endogenous GLP-1 system. In a series of secondary analyses of previous human laboratory experiments, we first examined the effects of alcohol administration, with different doses and routes of administration, on peripheral active GLP-1 concentrations in heavy-drinking individuals with AUD enrolled in placebo-controlled pharmacological studies (only placebo conditions were analysed here). Alcohol administration resulted in a significant reduction of GLP-1 levels across the four experiments (oral alcohol, variable dose: F3,28 = 6.52, p = 0.002; oral alcohol, fixed dose: F7,75.94 = 5.08, p \u3c 0.001; intravenous alcohol, variable dose: F4,37.03 = 20.72, p \u3c 0.001; intravenous alcohol, fixed dose: F4,13.92 = 10.44, p \u3c 0.001). Next, central expression of the GLP-1 receptor (GLP-1R) in post-mortem brain tissues (amygdala, ventral tegmental area, nucleus accumbens, hippocampus and prefrontal cortex) was compared between individuals with AUD and controls. Fold change of GLP-1R mRNA in the hippocampus was significantly higher in individuals with AUD, compared to controls (F1,21 = 6.80, p = 0.01). A trend-level effect with the same direction was also found in the prefrontal cortex (F1,20 = 3.07, p = 0.09). Exploratory analyses showed that GLP-1R gene expression levels were correlated with behavioural measures of alcohol drinking (hippocampus) and cigarette smoking (hippocampus and prefrontal cortex). Collectively, these data provide novel information on the crosstalk between alcohol and GLP-1 in a clinically relevant sample. Further studies are needed to understand the underlying mechanisms of this link

Differential association between the GLP1R gene variants and brain functional connectivity according to the severity of alcohol use

Growing evidence suggests that the glucagon-like peptide-1 (GLP-1) system is involved in mechanisms underlying alcohol seeking and consumption. Accordingly, the GLP-1 receptor (GLP-1R) has begun to be studied as a potential pharmacotherapeutic target for alcohol use disorder (AUD). The aim of this study was to investigate the association between genetic variation at the GLP-1R and brain functional connectivity, according to the severity of alcohol use. Participants were 181 individuals categorized as high-risk (n = 96) and low-risk (n = 85) alcohol use, according to their AUD identification test (AUDIT) score. Two uncommon single nucleotide polymorphisms (SNPs), rs6923761 and rs1042044, were selected a priori for this study because they encode amino-acid substitutions with putative functional consequences on GLP-1R activity. Genotype groups were based on the presence of the variant allele for each of the two GLP-1R SNPs of interest [rs6923761: AA + AG (n = 65), GG (n = 116); rs1042044: AA + AC (n = 114), CC (n = 67)]. Resting-state functional MRI data were acquired for 10 min and independent component (IC) analysis was conducted. Multivariate analyses of covariance (MANCOVA) examined the interaction between GLP-1R genotype group and AUDIT group on within- and between-network connectivity. For rs6923761, three ICs showed significant genotype × AUDIT interaction effects on within-network connectivity: two were mapped onto the anterior salience network and one was mapped onto the visuospatial network. For rs1042044, four ICs showed significant interaction effects on within-network connectivity: three were mapped onto the dorsal default mode network and one was mapped onto the basal ganglia network. For both SNPs, post-hoc analyses showed that in the group carrying the variant allele, high versus low AUDIT was associated with stronger within-network connectivity. No significant effects on between-network connectivity were found. In conclusion, genetic variation at the GLP-1R was differentially associated with brain functional connectivity in individuals with low versus high severity of alcohol use. Significant findings in the salience and default mode networks are particularly relevant, given their role in the neurobiology of AUD and addictive behaviors

Effects of exogenous ghrelin administration and ghrelin receptor blockade, in combination with alcohol, on peripheral inflammatory markers in heavy-drinking individuals: Results from two human laboratory studies

The ghrelin system has been garnering interest for its role in different neuropsychiatric disorders, including alcohol use disorder (AUD). Accordingly, targeting the ghrelin system is under investigation as a potential novel therapeutic approach. While alcohol provokes the immune system and inflammatory responses, ghrelin has potent immunomodulatory and anti-inflammatory properties. The present study aimed to shed light on the “crosstalk” between ghrelin and inflammation by examining the effects of exogenous ghrelin administration and ghrelin receptor blockade on peripheral inflammatory markers in the context of two human laboratory studies with alcohol administration. Non-treatment-seeking, heavy-drinking individuals with alcohol dependence, the majority of whom were African American males, were enrolled. In the first randomized, crossover, double-blind, placebo-controlled human laboratory study, participants underwent two experimental paradigms – an intravenous alcohol self-administration (IV-ASA) and an intravenous alcohol clamp (IV-AC) – each consisting of two counterbalanced sessions (ghrelin, placebo). A loading dose of intravenous ghrelin (3 mcg/kg) or placebo, followed by a continuous ghrelin (16.9 ng/kg/min) or placebo infusion was administered. In the second dose-escalating, single-blind, placebo-controlled human laboratory phase 1b study, participants were dosed with an oral ghrelin receptor blocker (PF-5190457) and underwent an oral alcohol challenge. Repeated blood samples were collected, and plasma concentrations of the following inflammatory markers were measured: C-reactive protein (CRP), interleukin (IL)-6, IL-10, IL-18, and tumor necrosis factor alpha (TNF-α). During the IV-ASA experiment, significant drug × time interaction effects were observed for IL-6 (F3,36 = 3.345, p = 0.030) and IL-10 (F3,53.2 = 4.638, p = 0.006), indicating that ghrelin, compared to placebo, significantly reduced blood concentrations of the proinflammatory cytokine IL-6, while increasing blood concentrations of the anti-inflammatory cytokine IL-10. No significant drug × time interaction effects were observed during the IV-AC experiment, possibly because of its much shorter duration and/or smaller sample. Treatment with PF-5190457, compared to placebo, had no significant effect on the inflammatory markers investigated. In conclusion, a supraphysiologic pharmacological challenge with exogenous ghrelin in heavy-drinking individuals produced anti-inflammatory effects in the context of intravenous alcohol administration. On the contrary, ghrelin receptor blockade did not lead to any change in the inflammatory markers included in this study. Mechanistic studies are required to better understand the interaction between ghrelin, alcohol, and inflammatory processes