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.

Diet and diet‐induced morbidities (such as obesity) are leading risk factors for the development of various forms of cancer worldwide. However, the physiological mechanisms responsible for the interaction of diet and cancer growth are still unclear. Mechanistic studies in animals support the interaction of diet with cancer development and progression, however the animal diets used do not adequately model the true nutritional quality of many Americans. We have developed a novel disease inducing rodent diet referred to as the Americanized diet (AD) that embodies the complex nutritional profile of Americans while also accounting for the differences in human and rodent nutritional requirements. We set out to determine how consumption of the AD would influence the growth of the MC‐38 murine adenocarcinoma cells in vivo. 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 C57Bl/6 male mice were given ad libitum access to the AD (n=5) or standard rodent chow (n=5) and ddH2O. Food intake and body weights were calculated weekly and circulating total cholesterol (CHOL), HDL‐cholesterol (HDL), triglycerides, and glucose were quantified at baseline and again at the end of the study. After 6 weeks, mice were injected with 0.5×106 MC‐38 adenocarcinoma cells in the rear flank and injection site was monitored for the presence of a palpable tumor. Tumor volume was calculated using calipers and, after 3 weeks, tumor blood flow was estimated using contrast‐enhanced ultrasonography. Animals were then euthanized and tumor tissues collected for assessment of leukocyte content by flow cytometry and quantification of interleukin‐10 (IL‐10), major histocompatibility complex H2K, and vascular endothelial growth factor (VEGF) gene expression by RT‐PCR. Body weights and tumor volumes were analyzed using generalized linear models and the remaining parameters were analyzed using GLM multivariate analyses in SPSS. Mice assigned to ad libitum feeding of the AD consumed more calories and gained more weight (P=0.01) as compared to mice consuming standard chow. After 6 weeks of consuming the diet, mice fed the AD had greater HDL cholesterol (P=0.03); however this difference was lost after MC‐38 innoculation (P=0.2). MC‐38 growth was significantly reduced (P=0.004 and 0.07 at weeks 1 and 2, respectively) in mice consuming the AD as compared to mice consuming chow. Interestingly, no palpable tumor was observed in 2 of the 5 mice consuming the AD while all 5 mice consuming chow developed tumors. There was a greater relative tissue perfusion (P=0.02) in the tumor‐bearing mice fed the AD as compared to the mice fed chow. No difference was observed in the proportion of tumor leukocytes amongst the diets. Similarly, RT‐PCR revealed no difference in IL‐10, H2K or VEGF gene expression between treatment groups (P= 0.25, 0.98, 0.62 respectively). Based on this study, mice fed the AD had reduced growth of the MC‐38 murine adenocarcinoma cell line in vivo despite having greater body weight and caloric intake. While we were unable to detect a significant difference in tumor leukocyte content and/or the expression of IL10, H2K, and VEGF at the end of the study, it is possible these outcomes differed during earlier time points (weeks 1 and 2) and contributed to the reduced tumor growth observed in mice fed the AD.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diet has an established relationship with morbidity and mortality worldwide. While poor diet is primarily associated with cardiovascular disease, there is evidence that poor diet has a direct negative influence on renal health that can also contribute to deteriorating cardiovascular health. However, due to the lack of a well‐designed disease inducing rodent diet, the physiological link between diet and the cardiovascular and renal systems remain unclear. We have developed a novel Americanized diet (AD) that is based on the nutritional intake of Americans and includes modifications to several nutrients while also accounting for the difference in human and mouse nutritional requirements. We hypothesized that mice consuming the AD will have greater weight gain and exacerbated renal injury, as compared to mice fed standard chow, in response to low‐dose angiotensin II (AngII) administration. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conforms to the FASEB Statement of Principles for the use of animals in research and education. Weanling (3‐week old) male C57Bl/6J mice were given ad libitum access to the AD (n=5) or standard laboratory chow (n=5) and ddH2O. 24‐hour dietary intake and body weights were recorded weekly. After 5 weeks, a baseline urine sample was collected and a subcutaneous pump delivering AngII (250 ng/kg*min) was implanted in the rear flank. Systolic blood pressure was recorded daily during AngII infusion using a non‐invasive tail‐cuff volume pressure recording method. After 4 weeks of AngII infusion, another urine sample was collected and urinary albumin concentrations were quantified by ELISA. Prior to euthanasia, renal blood flow was estimated using contrast‐enhanced ultrasonography. Body weight and blood pressure data were analyzed using generalized linear models (Two‐Way Repeated Measures), while the remaining parameters were analyzed using a multivariate GLM procedure. All statistical analyses were performed using SPSS. Mice consuming the AD had greater weight gain (P<0.01) and urinary concentration of albumin (P=0.01) prior to AngII administration. Interestingly, the difference in weight was abolished upon initiation of AngII infusion despite a continued difference in estimated daily caloric intake. Albuminuria persisted in the mice consuming the AD after 4 weeks of AngII (38±10 vs 20±5 ug/24 hr, P=0.02), despite a lack of difference in blood pressure between the AD and chow fed mice (P=0.8). Estimated renal blood flow was also greater in mice fed the AD (P=0.02), and likely explains the tendency for higher 24 hour urine output (P=0.2) as compared to mice consuming chow and receiving AngII. In conclusion, mice consuming an AD had greater weight gain and renal damage (albuminuria) despite the absence of a change in systolic blood pressure. These data show that the nutritional quality of a “typical” American diet has direct negative impact on renal health in rodents and further studies are needed to further elucidate the consequences of these findings.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sepsis affects more than a million Americans each year and is responsible for more deaths in the United States than prostate cancer, breast cancer, and AIDS combined. Obesity has been positively associated with the severity of sepsis amongst patients in the ICU, suggesting diet significantly influences the pathophysiology of disease. Animal studies support the interaction of diet with sepsis pathogenesis and severity; however the diets used have a drastically different nutrient profile as compared to the reported nutritional intake of Americans. Therefore, the goal of the current application was to determine how a novel rodent diet that adequately models the complex nutrient composition of Americans (Americanized diet ‐ AD) influences the severity of sepsis in mice. 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 male C57Bl/6 mice were fed either chow (n=5) or the AD (n=5) ad libitum for 8 weeks with body weights and 24‐hour dietary intake collected weekly. After 8‐weeks, mice underwent cecal‐ligation and puncture to induce polymicrobial peritonitis. 24 hours later blood was collected, animals were euthanized, and kidney and spleen tissues were collected. Circulating interleukin‐6 (IL6) and blood urea nitrogen (BUN) concentrations were determined using commercially available assays. Kidney and splenic IL6, TNFα, and C3 gene expression were measured using RT‐PCR and expression normalized to GAPDH using the delta‐Ct method. All data were analyzed using general linear model analyses in SPSS. Mice consuming the AD tended (P=0.05) to have significantly greater body weights starting after 4 weeks of feeding as compared to mice consuming chow. Following CLP, systemic IL6 values were significantly greater (P=0.04) in mice fed the AD (80±22 μg/mL plasma) as compared to chow fed mice (18±10). There was also a numerical increase in BUN in the AD fed mice following CLP (120±22 mg/dL) as compared to mice fed chow (70±35); however this did not reach statistical significance (P=0.16). No difference was observed in TNFα or C3 mRNA expression in the kidney or spleen; however, there was a 4‐fold increase in splenic (but not renal) IL6 mRNA expression in mice fed the AD as compared to mice fed chow (n=3). Taken together, these data highlight the significance of diet to the immunopathology of polymicrobial sepsis in mice and suggest that diet has a direct impact on IL‐6 production in the spleen.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.