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 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.

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The chronic angiotensin‐II (AngII) infusion model is commonly used to study kidney and cardiovascular injury, however the methodologies used to generate the model varies significantly in the literature. Published doses range from 100 to 3000 ng/kg*min with a wide range of blood pressure and other outcomes measured. Interestingly, there is a variety of anesthetic agents used to implant the miniosmotic pumps that deliver the AngII. While the anesthetic exposure is brief, and the anticipated physiological effects of are expected to have worn off within hours to days after administration, data in other animal models of kidney disease suggest that anesthetic influences the inflammatory response and subsequent physiological outcomes. Therefore, we set out to determine if blood pressure, renal function, or markers of kidney inflammation are affected by the anesthetic used to implant the miniosmotic pumps. All experiments were performed in accordance with protocols approved by the Liberty University IACUC and conform to the FASEB standards for the use of animals in research and education. Male C57Bl/6 mice (~20 grams) were purchased from The Jackson Laboratory and acclimated to the Liberty University vivarium for 7 days. Mice were then randomly assigned to receive miniosmotic pumps (Alzet, model 2004) delivering 500 ng/kg*min AngII (subcutaneous) using either 1) 3% isoflurane inhaled (in oxygen, ISO), 2) a ketamine/xylazine mixture) (KX) 90 and 9mg/kg intraperitoneal injection, respectively), or 3) a 2.5% tribromoethanol solution (TBE, 240mg/kg intraperitoneal injection). The tribromoethanol solution was prepared, stored and administered based on policies published by several different academic institutions. Blood pressure measurements began 3 days after pump implantation and continued for 4 weeks using the non‐invasive tail‐cuff method. At weeks 2 and 4, mice were placed in metabolic cages to assess water balance and collect urine to quantify albumin excretion. After 4 weeks, renal resistive index (renal segmental arteries) was determined for each mouse by ultrasound. Mice were then euthanized and the heart was weighed to assess for cardiac hypertrophy. Kidney tissues were also collected for quantification of inflammatory gene expression [interleukin‐6 (IL6), IL1β, CCL5, and tumor growth factor β (TGFβ)] using real‐time PCR. All data were analyzed using general linear models in SPSS. Neither AngII nor anesthetic caused a significant increase in systolic blood pressure (P=0.8) or heart weight (P=0.4). However, there was a significant effect of anesthetic on water intake and urinary output at weeks 2 and 4, with mice of the KX group consuming less water and producing less urine (P<0.05) than mice of the ISO or TBE groups. Interestingly, the TBE had 2–3 times greater (‐P=0.03) urinary albumin excretion and renal mRNA expression of CCL5 and TGFβ (P<0.04). Taken together, our data suggests that the choice of anesthetic used to implant the miniosmotic pump significantly influences outcomes of kidney health in the chronic AngII‐infusion model. More studies are needed to better characterize this valuable animal model and increase the reproducibility across different laboratories and institutions.Support or Funding InformationProject was funded by LUCOM Intramural Grant #2018‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Poor nutrition and alcohol intake are leading environmental factors associated with morbidity and mortality worldwide. However, the physiological interactions between poor diet and excessive alcohol intake remain unclear. Prior research from our lab showed diet significantly influenced measures of kidney and liver function in mice exposed to alcohol for 3 weeks. However, it was unclear whether the effects were due to diet, chronic alcohol consumption, or an additive effect of both. All experiments were performed in accordance with protocols approved by the Liberty University IACUC and conform to the FASEB standards for the use of animals in research and education. Weanling (3‐week old) male mice from the C57Bl/6 strain were purchased from The Jackson Laboratory and acclimated to a standard chow diet and housing for 7 days. Mice were then given ad libitum access to 1 of 3 diets: 1) standard laboratory chow, 2) a commercially available Western Diet (WD), or 3) a novel Americanized diet (AD). The AD was formulated to match the 50th percentile of intake for sodium, potassium, simple sugars, phosphorus, and fiber as reported in the recent What we eat in America report, based on 2011–2012 NHANES data. After 6 weeks on their assigned diet, each dietary group received distilled water or 10% (volume) ethanol solution as their only source of drinking water for an additional 4 weeks. Body weights and beverage consumption were recorded each week throughout the study. Systolic blood pressure was recorded at baseline and after 4 weeks of ethanol exposure. Following 4 weeks of ethanol intake, renal blood flow was estimated using contrast‐enhanced ultrasonography, and the mice were euthanized and kidney and liver tissues were processed for histology and quantitative real‐time RT‐PCR analysis of gene expression. All data were analyzed using general linear models in SPSS. Diet significantly influenced body weight, with both the WD and AD fed mice having a similarly (p=0.1) greater body weight as compared to mice fed chow (p<0.01). However, alcohol had no additional effect on weight gain (p=0.1). Consistent with previous findings, mice fed chow had the highest daily consumption of alcohol that was greater (p=0.05) than mice fed the WD, but not mice fed the AD (p=0.8). Histological analysis revealed hepatic steatosis in mice fed the WD and AD, and this did not appear to be worsened by alcohol intake. Although there was no difference in systolic blood pressure between the groups, mice fed the AD had a significant reduction in estimated renal blood flow as compared to mice fed WD (p=0.01) and chow (p<0.001). Interestingly, ethanol consumption caused a 25% increase in estimated renal medullary flow. Preliminary data indicate that genes (Hspa5 and Hsp90b1) associated with the cellular response to stress in the endoplasmic reticulum (ER) were expressed at higher levels in livers of mice fed the WD and AD, and this apparent ER stress was not exacerbated by alcohol intake. Taken together, the results from this study suggest that diet and alcohol significantly influence liver and renal health in mice, with a majority of the pathophysiology caused by diet.Support or Funding InformationProject was funded by LUCOM Intramural Grant #2018‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diet and alcohol consumption are significant contributors to health and disease worldwide. Liver disease is a consequence of poor diet and alcohol consumption; however preclinical animal studies have not adequately considered the combined effect of poor diet and chronic alcohol intake into study design. Given the physiological cross talk between the liver and kidney and the occurrence of hepatorenal syndrome in alcoholics, we set out to determine how poor diet and chronic alcohol intake influence liver and kidney health in mice. All experiments were performed in accordance with protocols approved by the Liberty University IACUC and conform to the FASEB standards for the use of animals in research and education. 3‐week old male C57Bl/6 mice were purchased from The Jackson Laboratory and acclimated to the new environment and chow diet for 1‐week. Mice were then given ad libitum access to chow (18% protein), our novel Americanized diet (AD), or a commercially available “Western” diet (WD) (n=6). After 6 weeks, all mice began a chronic alcohol exposure of 10% ethanol solution given as the only source of water for 3 weeks. Body weight, food, and beverage intake were recorded weekly. Systolic blood pressure was determined using a non‐invasive tail‐cuff method and renal blood flow estimated using contrast‐enhanced ultrasonography. Mice were then euthanized and blood and liver tissues collected. Blood was analyzed for circulating ALT activity and HDL, triglycerides, and glucose concentrations. All statistical analyses were performed using general linear models in SPSS with “Diet” as the independent variable. Diet had a significant effect on ethanol consumption (P<0.001), with mice fed chow having the highest ethanol intake (6 mg ethanol/g body weight) as compared to mice fed the WD (3.4 mg/g). Mice fed the WD had the greatest body weight, dietary intake, adiposity, and circulating HDL concentration as compared to mice fed chow (P<0.001). Mice fed chow had the highest circulating glucose levels (P<0.001). Histological analyses revealed excessive hepatic steatosis as the primary finding and was most severe in mice fed WD. No overt changes were noted in mice fed chow. Circulating ALT activity was elevated in mice fed the WD as compared to mice fed AD or chow, further suggesting increased liver injury in these mice. Mice fed the AD had intermediate values for all measurements with the exception of systolic blood pressure. Mice fed the AD had a 10 mmHg increase in systolic pressure as compared to mice fed chow or the WD (P=0.03). This occurred despite a lack of difference in renal hemodynamics between the different dietary groups (P≥0.3). Our data suggest that diet and alcohol have an additive effect in regards to liver damage and the development of metabolic dysfunction. The physiological mechanism by which consumption of our novel AD caused moderate hypertension in mice when combined with chronic alcohol intake will be pursued in future studies. Taken together, our findings highlight the need to consider the role of diet in preclinical animal models studying the physiological effects of chronic alcohol intake.Support or Funding InformationLUCOM Intramural Grant #2018‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.