The Increase in Circulating Levels of Pro-Inflammatory Chemokines, Cytokines, and Complement C5 in Canines with Impaired Kidney Function.

Chronic low-grade inflammation is a key contributor to the progression of kidney disease. The release of cytokines and other pro-inflammatory proteins may further contribute to detrimental kidney health by increasing interstitial edema and renal fibrosis. The aim of the present study was to investigate the inflammatory markers in canines who developed renal disease naturally and were diagnosed with renal disease either during life or following necropsy, as assessed by a veterinarian. RNA was isolated from canine blood obtained at necropsy and stored as bioarchived samples from ten canines with renal disease (9.6−14.7 yr) and ten controls (10.1−14.8 yr). At the time of death, the mean blood creatinine concentration and BUN were elevated in dogs with renal disease compared to control (both p < 0.01). Samples were assessed for changes in gene expression using the Canine cytokine RT2 Profiler PCR Array for inflammation. There was a significant increase in C-C Motif Chemokine Ligand 16 (CCL16), C-X-C Motif Chemokine Ligand 5 (CXCL5), Interleukin 16 (IL-16), and Complement Component 5 (C5) (all p < 0.05 vs. con). In addition, there was also a statistically non-significant increase in 49 genes and a down-regulation in 35 genes from a panel of total 84 genes. Pro-inflammatory genes including CCL16, CXCL5, IL-16, and C5 can all contribute to renal inflammation and fibrosis through different signaling pathways and may lead to a progressive impairment of kidney function. Blockade of their activation may be important in ameliorating the initiation and/or the progression of renal disease.

Metabolomic changes in cats with renal disease and calcium oxalate uroliths.

INTRODUCTION: There is a significant incidence of cats with renal disease (RD) and calcium oxalate (CaOx) kidney uroliths in domesticated cats. Foods which aid in the management of these diseases may be enhanced through understanding the underlying metabolomic changes. OBJECTIVE: Assess the metabolomic profile with a view to identifying metabolomic targets which could aid in the management of renal disease and CaOx uroliths. METHOD: This is a retrospective investigation of 42 cats: 19 healthy kidney controls, 11 with RD, and 12 that formed CaOx nephroliths. Cats were evaluated as adults (2 through 7 years) and at the end of life for plasma metabolomics, body composition, and markers of renal dysfunction. Kidney sections were assessed by Pizzolato stain at the end of life for detection of CaOx crystals. CaOx stone presence was also assessed by analysis of stones removed from the kidney at the end of life. RESULTS: There were 791 metabolites identified with 91 having significant (p < 0.05, q < 0.1) changes between groups. Many changes in metabolite concentrations could be explained by the loss of renal function being most acute in the cats with RD while the cats with CaOx stones were intermediate between control and RD (e.g., urea, creatinine, pseudouridine, dimethylarginines). However, the concentrations of some metabolites differentiated RD from CaOx stone forming cats. These were either increased in the RD cats (e.g., cystathionine, dodecanedioate, 3-(3-amino-3-carboxypropyl) uridine, 5-methyl-2'-deoxycytidine) or comparatively increased in the CaOx stone forming cats (phenylpyruvate, 4-hydroxyphenylpyruvate, alpha-ketobutyrate, retinal). CONCLUSIONS: The metabolomic changes show specific metabolites which respond generally to both renal diseases while the metabolomic profile still differentiates cats with RD and cats with CaOx uroliths.

Dietary Protein and Carbohydrate Levels Affect the Gut Microbiota and Clinical Assessment in Healthy Adult Cats.

BACKGROUND: Relative levels of dietary protein and carbohydrate intake influence microbiota and their functional capabilities, but the effect has not been well documented in cats. OBJECTIVES: The impact of 3 foods with different protein:carbohydrate ratios on the gut microbiota and functional attributes in healthy adult cats was evaluated. METHODS: Male and female cats (n = 30; mean age: 5.1 y; mean body weight: 5.26 kg) were fed 1 of 3 foods [P28 (28.3% protein, dry matter basis), P35 (35.1%), and P55 (54.8%)] for 90 d in a Williams Latin Square design. Each food had a 1:1 ratio of animal (dried chicken) to plant (pea) protein; protein replaced carbohydrate as protein level increased. Fecal microbiota and their functional capability were assessed with 16S sequencing and the Kyoto Encyclopedia of Genes and Genomes database, respectively. RESULTS: Fecal pH, ammonia, and branched-chain fatty acids (BCFAs) were higher when cats consumed P55 food than when they consumed P28 and P35. Clear separation of samples between P28 and P55 based on bacterial genera was observed, with partitioning into saccharolytic and proteolytic functions, respectively. Significantly higher α diversity was seen with P55 than with P28 and P35. Amino acid metabolism, mucin foraging pathways, and urea metabolism were higher with P55 than with P28, whereas feces from cats fed P28 had higher concentrations of carbohydrate-active enzymes and enzymes involved in SCFA pathways than with P55. Bacterial genera that showed positive associations with amino acid catabolism also showed positive associations with mucin degradation. CONCLUSIONS: Despite higher protein digestibility and less protein arriving to the colon, when healthy adult cats consumed the highest level of protein (P55), their gut microbiota exhibited higher mucin glycan foraging and amino acid metabolism, leading to higher fecal pH, ammonia, and BCFAs. This is likely due to lower availability of carbohydrate substrates and dietary fiber as protein replaced carbohydrate in the food.

Experimental Biology 2020 Meeting Abstracts.

Bone morphogenetic proteins (BMPs) are growth factors that belong to the transforming growth factor-ß (TGF-ß) superfamily, and till date 15 BMPs have been described. BMPs, first described for their role in bone and cartilage formation, also play a role in renal fibrosis in chronic kidney disease (CKD). There is evidence to indicate that in rodent models of CKD, administration of recombinant BMP1-3 increases renal fibrosis whereas administration of a BMP1-3-neutralizing antibody or BMP-7 antibody reduces renal fibrosis and preserves renal function. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy, to identify BMPs associated with renal dysfunction in cats and potential fibrosis. At time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone-forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a modest, but significant, increase in the expression of BMP-1 in cats with kidney disease (2.48 fold) and stone formers (1.72 fold), compared to controls (both p<0.01). While the increase in BMP-2 in CaOx cats was significant (1.46 fold; p<0.05 vs Con), the increase in cats with kidney disease was not (1.23 fold; NS). BMP2K, a BMP-2 inducible kinase, was significantly increased in both kidney disease (1.43 fold) and CaOX (1.46 fold) (both p<0.05). In contrast, a significant decrease in BMP4 was observed in both groups (<2.2 fold and 1.68 fold in kidney disease and CaOx, respectively; both p<0.001 vs Con). A decrease was also seen in CRIM 1, a protein associated with podocyte filtration function and whose reduction is associated with fibrosis, in both groups. BMP-7, whose potential therapeutic role in treating CKD and reversing fibrosis has been documented, was modestly decreased in both groups (both less than 1.5 fold) compared to controls. Given that there was an increase in all three forms of TGFß (TGFß1, TGFß2, and TGFß3), a potent initiator of renal fibrosis, in both groups, and a decline in BMP-7, an endogenous inhibitor of TGFß signaling in fibrosis, compared to controls, our results profile the BMPs potentially associated with renal fibrosis in cats that may contribute to kidney dysfunction. In summary, a nutritional therapy to slow the progression of kidney dysfunction may benefit from the inclusion of dietary ingredients that attenuate renal fibrosis in cats. SUPPORT OR FUNDING INFORMATION: This study was funded by Hill's Pet Nutrition, Inc.

Consumption of identically formulated foods extruded under low and high shear force reveals that microbiome redox ratios accompany canine immunoglobulin A production.

Digestion-resistant starch (RS) can provide health benefits to the host via gut microbiome-mediated metabolism. This study tested the physiological effects on healthy dogs of identically formulated foods processed under high (n = 16) or low (n = 16) shear extrusion conditions resulting in respective lower and higher levels of RS. Faecal samples collected at weeks 3 and 6 were assayed for stool score, proximate analysis, short-chain fatty acids (SCFA), immunoglobulin A (IgA) and microbiome; faecal metabolome was characterized at week 6. Proximate and digestibility analyses of the foods and stool scores and stool proximate analysis showed few differences between the two shear methods except for increased apparent fibre digestibility in the low shear food. In contrast, levels of butyrate (p = .030) and total SCFA (p = .043) were significantly greater in faeces at week 6 from dogs who consumed the low versus high shear food. Faecal IgA levels were significantly higher at week 3 (p = .001) but not week 6 (p = .110) in the low shear food. Significant differences in 166 metabolites between consumption of the two foods were identified via faecal metabolomic analysis, with changes in sugars, bile acids, advanced glycation end products and few amino acids. Strikingly, consumption of the low shear food resulted in elevated levels of the reduced members of redox couples derived from metabolized sugars and branched-chain and phenyl amino acids. Alpha diversity of the microbiome showed significantly higher species richness in faeces from the low shear group at week 6, though other measures of diversity were similar for both foods. Twelve genus-level operational taxonomic units (OTU; half Firmicutes) significantly differed between the food types. Six OTU significantly correlated with RS-derived sugars and ratios of the redox couples. Taken together, these data show that RS impacts microbiome-mediated metabolism in the gut, resulting in changes in the reducing state.

When fed foods with similar palatability, healthy adult dogs and cats choose different macronutrient compositions.

Dogs and cats make short-term food choices based on palatability. We hypothesized that, if palatability were masked, long-term food choices would be based on physiological requirements, and circulating metabolite concentrations would reflect those choices. Four experimental foods with similar palatability, but varying in macronutrient composition, were prepared for healthy adult dogs (n=17) and cats (n=27). Food 1 was high protein; food 2 was high fat; food 3 was high carbohydrates; and food 4 was balanced for macronutrients. By choosing any combination of foods, dogs and cats could individually set their macronutrient intake. Plasma metabolomic profiles were determined at baseline and after animals had consumed their food intake of choice for 28 days. Based on food intake calculations over 28 days, dogs on average chose to consume most of their calories from fat (41.1±4.3%) and then carbohydrate (35.8±3.7%), whereas cats on average chose to consume most of their calories from carbohydrate (43.1±4.0%) and then protein (30.3±3.9%; all P<0.001). Age and lean or fat body mass also influenced protein intake. Younger, leaner cats consumed more protein compared with older cats, whereas younger, leaner dogs consumed less protein compared with dogs having more fat body mass. Older cats with moderate protein intake had lower circulating docosahexaenoic acid (DHA) concentrations as well as higher concentrations of sulfated microbial catabolic products compared with younger, leaner cats. In summary, when fed foods with similar palatability, dogs and cats consume different macronutrient compositions, and concentrations of circulating metabolites in cats reflect food choices.

Effect of dietary antioxidants on free radical damage in dogs and cats.

Alpha-tocopherol (vitamin E) is an antioxidant that is largely involved in immune defense and enhancing the ability of biological systems to respond to oxidative stress. During the process of free radical scavenging, vitamin C supports the regeneration of vitamin E. Although the functions of antioxidants and their importance have been widely studied, the intricate interplay between antioxidants has yet to be fully elucidated, especially in dogs and cats. As such, the objective of the present study was to determine the effect of a combination of dietary antioxidants on DNA damage and antioxidant status in dogs and cats. Forty adult mixed-breed dogs and 40 adult domestic shorthair cats were randomly assigned to one of four treatment groups per species. Dogs and cats remained in these groups for the 84-d duration of the study. The food differed in antioxidant supplementation with the control food meeting all of the Association of American Feed Control Officials requirements for complete and balanced nutrition, including sufficient vitamin E to exceed the published minimum. The treatment diets were targeted to include either 500, 1,000, or 1,500 IU vitamin E/kg as well as 100 ppm of vitamin C and 1.5 ppm of ß-carotene in the food. The effect of vitamin E supplementation level on serum vitamin E concentration, DNA damage, and total antioxidant power was evaluated. Feeding diets enriched with antioxidants resulted in an increased (P < 0.05) circulating vitamin E concentration, increased (P < 0.05) immune cell protection, reduced (P < 0.05) DNA damage in dogs, and an improved (P < 0.05) antioxidant status. Overall, these data demonstrated that feeding a dry kibble with an antioxidant blend inclusive of vitamin E, vitamin C, and ß-carotene enhanced cell protection and improved antioxidant status in dogs and cats.

Animals have an impressive array of defenses to excessive reactive oxygen species in the body. The antioxidant defense system is complex and sophisticated. vitamin E, vitamin C, and ß-carotene are known to scavenge free radicals that are created during times of oxidative stress. To evaluate the effect of the various antioxidants, dogs and cats were fed one of four diets for 84 d. Diets included a control group that had vitamin E concentrations that exceeded regulatory minimums and four treatment groups that were targeted to include 500, 1,000, or 1,500 IU vitamin E/kg as well as 100 ppm of vitamin C and 1.5 ppm of ß-carotene in the food. To assess the effectiveness of the different vitamin E concentrations provided in the foods, circulating vitamin E, DNA damage, and total antioxidant power were assessed. Results from the parameters assessed showed that dogs and cats benefit from supplementing their diet with a blend of antioxidants targeted to include 100 ppm of vitamin C, 1.5 ppm of ß-carotene, and have varying benefits to increased vitamin E/kg in the food.

Betaine and L-Carnitine Synergistically Influence the Metabolome and Immune Response in Dogs.

This study used thirty-two dogs, which were assigned to a preferred period of 14 days and then assigned to one of the four treatment foods: control (containing no added betaine, no added L-carnitine), control with 0.5% added betaine (Treatment 2), control with no added betaine and 300 ppm added L-carnitine (Treatment 3), or control with 0.5% added betaine and 300 ppm added L-carnitine (Treatment 4). All treatment foods were fed for ninety days. Untargeted blood metabolomic analysis and immune response were measured at the beginning and end of the 90-day feeding trial. Feeding betaine increased single-carbon metabolites while decreasing many carnitine-containing metabolites. Feeding L-carnitine increased many carnitine metabolites, while the combination synergistically influenced the metabolome. The combination of betaine and L-carnitine increased the cytokines released in a Tru-culture system in response to stimulation while numerically decreasing their release when unstimulated. Therefore, the combination of dietary betaine and L-carnitine could have the dual positive effects of reducing cytokine stimulation, controlling inflammation during health, and providing a robust response to bacterial infection.

Increased lipogenesis and lipidosis of gallbladder epithelium in dogs with gallbladder mucocele formation.

BACKGROUND: Gallbladder disease in people is frequently associated with disorders of lipid metabolism and metabolic syndrome. A recently emergent gallbladder disease of dogs, referred to as mucocele formation, is characterized by secretion of abnormal mucus by the gallbladder epithelium and is similarly associated with hyperlipidemia, endocrinopathy, and metabolic dysfunction. The cause of gallbladder mucocele formation in dogs is unknown. METHODS: A prospective case-controlled study was conducted to gain insight into disease pathogenesis by characterization of plasma lipid abnormalities in 18 dogs with gallbladder mucocele formation and 18 age and breed matched control dogs using direct infusion mass spectrometry for complex plasma lipid analysis. This analysis was complemented by histochemical and ultrastructural examination of gallbladder mucosa from dogs with gallbladder mucocele formation and control dogs for evidence of altered lipid homeostasis of the gallbladder epithelium. RESULTS: Gallbladder mucocele formation in dogs carried a unique lipidomic signature of increased lipogenesis impacting 50% of lipid classes, 36% of esterified fatty acid species, and 11% of complex lipid species. Broad enrichment of complex lipids with palmitoleic acid (16:1) and decreased abundance within complex lipids of presumptive omega-3 fatty acids eicosapentaenoic (20:5) and docosahexaenoic (22:6) was significant. Severe lipidosis of gallbladder epithelium pinpoints the gallbladder as involved causally or consequently in abnormal lipid metabolism. CONCLUSION: Our study supports a primary increase in lipogenesis in dogs with mucocele formation and abnormal gallbladder lipid metabolism in disease pathogenesis.

Increased dietary vitamin D was associated with increased circulating vitamin D with no observable adverse effects in adult dogs.

Introduction: There is no consensus for the optimum concentration of vitamin D, although a minimum concentration of 100 ng/mL (250 nM) of circulating vitamin D, measured as 25(OH) D, has been suggested in order to support optimal health in dogs. Few studies have examined the relationship between dietary vitamin D3 (cholecalciferol) intake and the resulting concentrations of circulating 25(OH) D in adult dogs. Recommendations for dog foods for adult maintenance report a safe upper limit of 3,200 IU vitamin D/kg on a dry matter basis. However, these recommendations were not based on studies of adult maintenance requirements. Understanding the relationship between dietary vitamin D and circulating vitamin D is necessary to utilize dietary vitamin D to influence health in dogs. Methods: Five groups of adult dogs (each n = 8) were fed food of approximately 4,000 kcal/kg containing one of the following dry matter concentrations of vitamin D for 6 months: 795.7, 3087.3, 5510.9, 7314.0, and 9992.5 IU/kg. Body weight was recorded at baseline and measured weekly, and daily food intake was recorded. Blood samples were taken at baseline and at the end of the 26-week study period. Results: There were no clinical signs of vitamin D deficiency or excess. Serum concentrations of creatinine, blood urea nitrogen, albumin, hematocrit, hemoglobin, alkaline phosphatase, phosphorus, total calcium, ionized calcium, and parathyroid hormone were maintained within reference values in all groups. Circulating 25(OH) D increased in all groups except those that consumed food with 795.7 IU/kg vitamin D, and increased in a linear and quadratic fashion in response to dietary vitamin D concentration. All of the dogs fed food with 5510.9 IU/kg vitamin D or above met or exceeded 100 ng/mL (250 nM) circulating 25(OH) D. Discussion: Dietary vitamin D was positively associated with increased circulating concentrations in concentrations up to 9992.5 IU/kg dry matter, with no observable adverse effects. Consumption of ≥5510.9 IU/kg vitamin D resulted in all dogs with at least the 100 ng/mL (250 nM) circulating concentration.

Feeding of fish oil and medium-chain triglycerides to canines impacts circulating structural and energetic lipids, endocannabinoids, and non-lipid metabolite profiles.

Introduction: The effect of medium-chain fatty acid-containing triglycerides (MCT), long-chain polyunsaturated fatty acid-containing triglycerides from fish oil (FO), and their combination (FO+MCT) on the serum metabolome of dogs (Canis familiaris) was evaluated. Methods: Dogs (N = 64) were randomized to either a control food, one with 7% MCT, one with FO (0.18% eicosapentaenoate and 1.3% docosahexaenoate), or one with FO+MCT for 28 days following a 14-day washout period on the control food. Serum metabolites were analyzed via chromatography followed by mass spectrometry. Results: Additive effects of serum metabolites were observed for a number of metabolite classes, including fatty acids, phospholipids, acylated amines including endocannabinoids, alpha-oxidized fatty acids, and methyl donors. Some effects of the addition of FO+MCT were different when the oils were combined compared with when each oil was fed separately, namely for acylcarnitines, omega-oxidized dicarboxylic acids, and amino acids. Several potentially beneficial effects on health were observed, including decreased circulating triglycerides and total cholesterol with the addition of FO (with or without MCT) and decreases in N-acyl taurines with the addition of MCT, FO, or FO+MCT. Discussion: Overall, the results of this study provide a phenotypic characterization of the serum lipidomic response to dietary supplementation of long-chain n3-polyunsaturated and medium-chain saturated fats in canines.

Betaine and Soluble Fiber Improve Body Composition and Plasma Metabolites in Cats with Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is a common condition in cats and cachexia (loss of lean body mass) is a concern. A nutrition-based intervention was investigated in cats with CKD for its effects on body composition, the plasma metabolome, and possible implications on health. METHODS: After a 4-week prefeed period with the control food, cats with CKD (N = 24) were randomized to one of six groups to consume a control food; a food supplemented with 0.5% betaine, 0.39% oat beta-glucan, and 0.27% short-chain fructooligosaccharides (scFOS, test food 1); and a food supplemented with 0.5% betaine, 0.59% oat beta-glucan, and 0.41% scFOS (test food 2) in a William's Latin Square design, each for 10 weeks. Body composition was assessed via dual-energy X-ray absorptiometry measurements, and the plasma metabolome was characterized. RESULTS: Despite no significant differences in daily intake among the three foods, significant increases in total body mass, lean body mass, and lean plus bone mineral composition were observed when cats with CKD consumed test food 1 compared with the control food; numerical increases were seen with test food 2 versus the control food. Plasma metabolomics indicated increased one-carbon metabolism following consumption of test food 1 and/or 2, with significant increases in sarcosine and numerical increases in methionine. Lower levels of plasma trans-4-hydroxyproline and N-methylproline following consumption of test foods 1 and 2 indicates reduced collagen breakdown and perhaps reduced fibrosis. Several acylcarnitines and branched-chain fatty acids associated with CKD were also reduced when cats ate test food 1 or 2 versus the control food. Higher plasma levels of sphingomyelins with consumption of test food 1 or 2 may reflect less severe CKD. CONCLUSIONS: Consumption of foods with supplemental betaine and fibers by cats with CKD led to improvements in body composition and changes in the plasma metabolome that correspond to better kidney health.

Predictive equations for dietary energy are improved when independently developed for dry and wet food which could benefit both the pet and the environment.

Introduction: Measuring energy availability through metabolizable energy feeding studies is the "gold standard" for establishing metabolizable energy concentration. However, predictive equations are often used to estimate metabolizable energy in dog and cat pet foods. The goal of this work was to evaluate the prediction of energy density and compare those predictions to each other and the energy needs of the individual pets. Methods: Feeding studies used 397 adult dogs and 527 adult cats on 1,028 canine and 847 feline foods. Individual pet results for the estimate of metabolizable energy density were used as outcome variables. Prediction equations were generated from the new data and compared to previously published equations. Results and discussion: On average the dogs consumed 747 kilocalories (kcals) per day (SD = 198.7) while cats consumed 234 kcals per day (SD = 53.6). The difference between the average prediction of energy density and the measured metabolizable energy varied from the modified Atwater prediction 4.5%, 3.4% (NRC equations), 1.2% (Hall equations) to the new equations calculated from these data at 0.5%. The average absolute values of the differences between measured and predicted estimates in pet foods (dry and canned, dog and cat) are: 6.7% (modified Atwater), 5.1% (NRC equations), 3.5% (Hall equations) and 3.2% (new equations). All of these estimates resulted in significantly less variation in the estimate of the food expected to be consumed than the observed variation associated with actual pet consumption to maintain body weight. When expressed as a ratio of energy consumed to metabolic body weight (weight in kilograms3/4) the within species variation in energy consumed to maintain weight was still high as compared to the energy density estimates variance from measured metabolizable energy. The amount of food offered as the central point in a feeding guide, based on the prediction equations, would on average result in an average variance between 8.2% error in the worst case estimate (feline dry using modified Atwater estimates) and approximately 2.7% (the new equation for dry dog food). All predictions had relatively small differences in calculating food consumed when compared to the differences associated with the variation in normal energy demand.

Relative supersaturation values distinguish between feline urinary and non-urinary foods and align with expected urine analytes contributions to uroliths.

Introduction: Uroliths are concretions formed in the urinary tract. These can be problematic in humans and companion animals such as cats. Magnesium ammonium phosphate (struvite) and calcium oxalate (CaOx) are the most common forms of uroliths. The relative supersaturation (RSS) is a relative risk index of crystal formation. Here, an updated program for calculating RSS, EQUIL-HL21, was used to detect differences in RSS values when cats were fed foods formulated for urinary and non-urinary conditions. In addition, the contributions of urinary analytes to RSS values were examined via regression analyses. Methods: Historical data from feeding trials including foods indicated for use in urinary or non-urinary conditions were analyzed for nutrient composition and urinary parameters. RSS was calculated by EQUIL-HL21. The relationship between RSS values calculated by EQUIL-HL21 and urinary analytes was examined by regression models, which were selected by R2 and stepwise methods. Results: Cats that consumed urinary foods had significantly greater levels of urinary sodium and chloride compared with those that consumed non-urinary foods, consistent with the greater amounts of sodium and chloride in the urinary foods. Those that consumed non-urinary foods had higher urine pH, ammonium, potassium, phosphorus, magnesium, oxalate, citrate, and sulfate. Struvite RSS value and number of urinary crystals were significantly lower in cats fed the urinary foods. Mean CaOx RSS values were similar in both foods, though the number of CaOx crystals were significantly higher in cats that consumed non-urinary foods. A model predicting the natural log of struvite RSS values indicated that these values would increase with increasing urine pH, ammonium, chloride, calcium, phosphorus, and magnesium, and would decrease with increasing urine citrate and sulfate. CaOx RSS was predicted to increase as urinary chloride, calcium, and oxalates increased, and would decrease as urine pH, sodium, phosphorus, citrate, and sulfate increased. Discussion: These analyses demonstrate that the EQUIL-HL21 program can accurately detect expected differences between foods formulated for urinary and non-urinary indications. Regression models showed the eight urinary analytes that, respectively, contribute to the predicted RSS values for struvite and CaOx.

Comparison of two software programs used to determine the relative supersaturation of urine ions.

Introduction: Relative supersaturation (RSS) values for urine crystals are a measure of the risk of urinary stone formation and have been shown to be lowered in foods shown to aid in the management of urolithiasis. In order to calculate RSS in pets, computer programs have been developed to calculate RSS and aid in the understanding of stone formation in veterinary medicine. However, some older programs have not been updated for use in animals, and the specific coefficients used are not publically available. One of the first RSS programs was developed in BASIC computer language and published in 1985 which was called EQUIL2. The EQUIL2 program was updated to a compiled version compatible with a PC platform. However, the formulas could not be read or altered. Methods: This study evaluates a new program with known coefficients to the original EQUIL2 program. The RSS values of the two programs were compared through a t-test, calculating the r2 from correlation analysis, Lin's concordance correlation coefficient, and by a Bland-Altman analysis of outputs from the two programs using urine samples from healthy dogs and cats. Results and Discussion: Our results show that for both magnesium ammonium phosphate (struvite) and calcium oxalate, the RSS values of the original program could be calculated from the new programs RSS values. Although the actual RSS values were different (as might be expected through the use of the updated coefficients and different thermodynamic stability constants in the calculations) the results were highly correlated, finding elevations and reductions in RSS proportionally in the same urine samples. The current work creates a foundation for using the modernized program to calculate RSS and provides a shared method for understanding the risk of struvite and calcium oxalate stone formation.

Feeding cats with chronic kidney disease food supplemented with betaine and prebiotics increases total body mass and reduces uremic toxins.

Cats with chronic kidney disease (CKD) have a decreased ability to maintain body weight. As CKD advances, loss of body weight contributes to morbidity and mortality. The goal of this study was to evaluate the combined effects of feeding betaine and prebiotics on body weight of both CKD and healthy cats. The pre-trial food (control food) was a complete and balanced dry food designed to aid in the management of CKD. Test food was the control food supplemented with betaine (0.500%) and prebiotics: long-chain oat beta-glucan (0.586%) and 0.407% short chain fructooligosaccharides (scFOS). The CKD cats (n = 7) were fed pre-trial food for 28 days and then randomly assigned to control food or test food. Each food was fed for 8 weeks in a cross-over study design. In a second study, healthy cats received control food or test food for 8 weeks (n = 8 each group). Blood, urine, and fecal samples were collected to evaluate concentrations of relevant kidney function biomarkers and metabolites at the end of each feeding period for CKD cats, and blood samples were collected monthly to evaluate concentrations of plasma metabolites for healthy cats. Body weight and composition were measured using dual-energy X-ray absorptiometry (DEXA) scan at baseline and after each feeding period. Total body mass was significantly higher in CKD cats after consuming test food compared with control food (P = 0.004), with no significant difference in food intake while consuming test or control food (P = 0.34). Test food did not affect total body mass or composition of healthy cats. Indole compounds produced by bacterial metabolism were decreased in urine and increased in feces of CKD cats fed test food, and plasma concentrations were negatively correlated with the level of kidney function, indicating a potential benefit of consuming test food. In healthy cats, consuming test food resulted in significantly decreased concentrations of plasma P-cresol sulfate (P = 0.004) and increased concentrations of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA; both P < 0.05), despite the fact that both control and test foods had similar concentrations of these long-chain fatty acids, 0.03% and 0.02%, respectively. These results suggest that the addition of betaine and prebiotics to the control food formula may have increased total body mass in CKD cats by enhancing one-carbon metabolism and by modulating the gut microbiome.

Dietary Betaine and Fatty Acids Change Circulating Single-Carbon Metabolites and Fatty Acids in the Dog.

In order to evaluate the interaction of betaine and n-3 PUFA in foods consumed by the dog, six extruded dry foods were formulated. The control food had no specific source of added betaine or n-3 fatty acids, while the test foods were supplemented with betaine, flax or fish oil in a 2 × 3 factorial design (no added n-3 source, added flax, added menhaden fish oil, and all with or without added betaine). Forty eight adult dogs were used in this study. All dogs were assigned to one of the six dietary treatments and consumed that food for the length of the 60-day study. Blood was analyzed for metabolomics (plasma), fatty acids and selected health-related analytes (serum) at the beginning and the end of the study. Added dietary betaine increased single-carbon metabolites (betaine, dimethyl glycine, methionine and N-methylalanine), decreased xenobiotics (stachydrine, N-acetyl-S-allyl-L-cysteine, 4-vinylguaiacol sulfate, pyrraline, 3-indoleglyoxylic acid, N-methylpipecolate and ectoine) and enhanced the production of eicosapentaenoic acid (EPA). Dietary betaine also decreased the concentration of circulating carnitine and a number of carnitine-containing moieties. The addition of the n-3 fatty acids alpha-linolenic, EPA and docosahexaenoic acid (DHA) increased their respective circulating concentrations as well as those of many subsequent moieties containing these fatty acids. The addition of alpha-linolenic acid increased the concentration of EPA when expressed as a ratio of EPA consumed.

A Diet Supplemented with Polyphenols, Prebiotics and Omega-3 Fatty Acids Modulates the Intestinal Microbiota and Improves the Profile of Metabolites Linked with Anxiety in Dogs.

A nutrition-based approach was utilized to examine the effects of fish oil and a polyphenol blend (with or without tomato pomace) on the fecal microbiota and plasma/fecal metabolomes. Forty dogs, aged 5-14 years, were fed a washout food, then randomized to consume a control (fish oil and polyphenol blend without tomato pomace) or test (fish oil and polyphenol blend with tomato pomace) food, then the washout food, and crossed over to consume the test or control food; each for 30 days. Several metabolites differed when comparing consumption of the washout with either the control or test foods, but few changed significantly between the test and control foods. Plasma levels of 4-ethylphenyl sulfate (4-EPS), a metabolite associated with anxiety disorders, demonstrated the largest decrease between the washout food and the control/test foods. Plasma 4-EPS levels were also significantly lower after dogs ate the test food compared with the control food. Other plasma metabolites linked with anxiety disorders were decreased following consumption of the control/test foods. Significant increases in Blautia, Parabacteroides, and Odoribacter in the fecal microbiota correlated with decreases in 4-EPS when dogs ate the control/test foods. These data indicate that foods supplemented with polyphenols and omega-3 fatty acids can modulate the gut microbiota to improve the profile of anxiety-linked metabolites.

Dietary Betaine Interacts with Very Long Chain n-3 Polyunsaturated Fatty Acids to Influence Fat Metabolism and Circulating Single Carbon Status in the Cat.

Six foods were used to evaluate the interaction of dietary betaine and n-3 PUFA in the cat. There was no ingredient added to the control food to specifically increase betaine or n-3 fatty acids. The experimental design was a 3 × 2 factorial (fatty acids were varied from the control food which had no added source of n-3 fatty acids, flax was included as a source of 18 carbon n-3, or menhaden fish oil as a source of EPA and DHA). Foods were then formulated using these three foods as a base with added betaine or without added betaine. Forty eight cats were used in this study. Equal numbers of cats were allotted by age and gender to each of the six dietary treatments. The cats were offered food amounts to maintain weight and consumed the food to which they were assigned for the length of the study (60 days). Metabolomics, selected circulating analytes and fatty acids were analyzed at the beginning and end of the feeding period. There was an increase in single carbon metabolites (betaine, dimethyl glycine, and methionine) with the consumption of dietary betaine. Betaine also increased the concentration of specific PUFA (ARA, αLA, DHA, and the sum of all circulating PUFA). The combination of dietary betaine and fish oil resulted in a reduction of circulating 3-indoxyl sulfate which suggests a renal benefit from their combined dietary presence.

Cats with Genetic Variants of AGXT2 Respond Differently to a Dietary Intervention Known to Reduce the Risk of Calcium Oxalate Stone Formation.

This study was completed to evaluate a genotype-specific nutritional intervention for reducing the risk of calcium oxalate stone formation. Serum metabolomic profiles and genotypes of 445 cats in the colony at Hill's Pet Nutrition, Inc (Topeka, KS, USA)were assessed in a genome-wide association study, and revealed an association between genetic variants of alanine-glyoxylate aminotransferase 2 (AGXT2) and 2-oxoarginine. The most significant single nucleotide polymorphisms (SNP) associated with 2-oxoarginine was at position chrA1:212069607, [G/A] (p < 3.687 × 10−17). This SNP explained approximately 15% of the variance in 2-oxoarginine concentrations. The distribution of genotype frequencies was 0.07 AA, 0.39 AG, and 0.54 GG, with a mean relative 2-oxoarginine concentration for each genotype of 0.45 AA, 0.92 AG, and 1.27 GG, indicating a subtractive effect of the minor allele (A). Serum concentrations of two AGXT2 substrates, symmetric/asymmetric dimethylarginines (SDMA/ADMA) and ß-aminoisobutyrate (BAIB) were also strongly associated with SNP chrA1:212069607 (p < 1.43 × 10−12 and p < 2.30 × 10−14, respectively). These two AGXT2 substrates were increased with the minor allele (A), indicating that the variant of the AGXT2 gene results in decreased aminotransferase activity. Additionally, the lifetime history of stone incidence showed that cats with the AA variant of AGXT2 SNP had a 2.515× increased incidence of stones compared with cats having the GG variant (p = 0.019). In a subsequent study assessing AGXT2 genotypes, cats (n = 10 GG, 4 AG, 9 AA) were fed control or test food (containing betaine at 0.500%, and the botanicals green tea, fenugreek and tulsi at 0.25, 0.025, and 0.0015%, respectively) in a cross-over study design. Stone risk analysis was conducted on urine samples after feeding control or test food for 28 days each. A calcium oxalate titration test (COT) was performed to assess the amount of added Ox−2 (per L) required to initiate calcium oxalate crystal formation. Cats with the GG variant of the AGXT2 SNP required more added oxalate to initiate urine crystal formation after consuming test food compared with control food, indicating a decreased risk of oxalate crystal formation in GG cats. In addition, urine oxalate concentrations showed an overall effect of test food independent of genotype (p = 0.0009), which resulted in lower oxalate concentrations after consuming test food compared with control food. These data indicate that cats with the GG-specific variant of AGXT2 should benefit from a reduced risk of calcium oxalate stone formation after consuming a betaine and botanical dietary enhancement.