The present study aimed to find the best route of adenine administration to induce CKD in female mice: oral gavage or via their standard pelleted diet. The results of this work demonstrated that: (1) Adenine treatment increased levels of urea and creatinine in plasma in both routes of administration, however, we observed an opposite response in these levels between the administration routes. In the pellet group, we found that creatinine was higher, while urea was lower. (2) The pellet and oral gavage groups showed decreased creatinine and urea urine levels with no differences among groups. (3) Both adenine-treated groups exhibited an increase in the relative weight of the kidneys, along with a rise in the necrotic debris and the tubular dilation in the kidneys. (4) Tubular dilation was greater in the oral gavage group. (5) Polyuria and polydipsia were observed with the adenine treatment, regardless of the route of administration, and (6) There was a more significant body weight loss detected in the pellet group when compared to the oral gavage group.
Regarding the route of administration, the adenine-induced CKD model was an effective approach for inducing renal injury, as indicated by the significant increase in plasma creatinine and urea levels and decreased urine levels of both creatinine and urea. Other authors have taken similar approaches, studying animals fed with adenine or administered adenine by oral gavage. They found that both adenine administration routes resulted in increased plasma levels of creatinine and urea (~ 5 to 6-fold) and decreased urine levels (~ 3-fold)22,28,33,34. Interestingly, differences in creatinine and urea levels due to the adenine administration route are consistent with the fact that the animal’s diet can influence these levels35,36,37. Adding adenine to the pellets can modify the smell and taste of the food, and animals can show aversion or difficulty eating them19,34. In the present study, peanut butter was added to the food of the pellet adenine-induced CKD group to mask the aversive taste of adenine. Different diets could result in differences in creatinine and urea plasma levels between adenine administration routes. Peanut butter can increase the protein intake in the diet (Singh, 1991), potentially leading to a substantial rise in serum creatinine levels, as we observed in the creatinine serum levels of the pellet adenine-induced CKD group. In our study, only the group receiving adenine through their pelleted diet consumed peanut butter and the pellet control group. This may limit comparisons, as the gavage group did not receive this supplement. This decision was made based on initial observations of significant reductions in food intake among the animals, leading us, in consultation with our Animal Core Facility Veterinarian, to enhance the pellets with peanut butter to ensure their welfare and maintain the experiment’s continuity. Additionally, the food intake of the pellet-adenine animals is generally lower than the food intake of the pellet control group. This could suggest that if the food had not been supplemented with peanut butter, the animals might have continued with a low feed intake. This reinforces the concern about using this route of administration, where aversions to consuming feed containing adenine have already been reported. Generally, the diet for CKD patients consists of low protein to reduce the workload on the kidneys and prevent the buildup of nitrogenous waste. However, even though the animals that received adenine via pellets consumed more protein (peanut butter), they did not show greater kidney damage (tubular dilation) compared to those that received adenine by oral gavage and did not receive peanut butter. Nevertheless, we cannot rule out any other possible effects promoted by peanut butter exclusively in the pelleted-group. Regarding kidney morphology, changes in relative kidney weight indicate renal disease, often reflecting pathological processes such as hypotrophy or hypertrophy22,38. In this study, we observed an increase in kidney weight in both the pellet and oral gavage adenine-induced CKD groups, compared to their respective controls, suggesting that kidney weight abnormalities can occur as an adaptation to manage the increasing metabolic load caused by adenine. Specifically, the increase in relative kidney weight indicates potential compensatory hypertrophy to offset the loss of function. Enlarged kidneys typically indicate an acute parenchymal process, which can also occur in chronic infiltrative kidney disease, like diabetic nephropathy39.
Additionally, the kidneys of adenine-treated animals showed significant tubular dilation and necrotic debris, which denotes pathological changes occurring within the renal structures. The marked increase in tubular dilation area, particularly in the gavage adenine-induced CKD group, highlights the severity of renal injury. This dilation likely results from tubular obstruction due to the accumulation of 2,8-DHA crystals, which impedes normal urine flow and disrupts the renal epithelium11,22. The fact that the gavage route elicited greater tubular dilatation area than the pellet route underscores the significance of administration routes with regard to disease severity. In contrast, oral gavage administration is more invasive than a pelleted route; it may yield a more pronounced pathological response, since this route ensures that the full dose of adenine is delivered. Furthermore, necrotic debris in both the pellet and oral gavage adenine-induced CKD groups support the idea that depletion in renal function accompanies the changes in renal structure. The absence of significant differences in the percentage of necrotic debris among the two administration routes suggest that, despite differences in the degree of tubular dilation area, the tissue damage induced by adenine treatment is comparable between groups.
The improvement in the ability of the kidneys to regulate water balance reflects the systemic consequences of CKD. The significant increase in water intake and urine output shown in both adenine administration routes indicates a compensatory mechanism known as hyperfiltration. This phenomenon occurs in the initial phase of kidney injury. Kidneys can compensate for the loss of nephrons by increasing their filtration rate to maintain a homeostatic balance of fluids and electrolytes40. Additionally, structural changes in the glomerular filtration barrier and increased intracapsular pressure may facilitate the filtering of more fluids and solutes, thereby contributing to hyperfiltration. Hyperfiltration results in significant fluid loss in animals, which induces a sensation of thirst, subsequently prompting an increase in water intake as a compensatory mechanism for this loss. This cycle can perpetuate hyperfiltration, as increasing water intake can further increase the filtration rate and urine output. Vázquez-Méndez et al. (2020) describe the same effects of adenine administration in hyperfiltration phenomena in rats41.
Regarding body weight and food intake results, the greater decrease in the pellet adenine-induced CKD group suggests that this administration route might impose a higher metabolic load on the animals, possibly due to their continuous adenine consumption and the taste of the food. On the other hand, while oral gavage administration may be more invasive, it provides more precise control over the dosing regimen. It generates comparable outcomes in renal injury without the metabolic stress associated with continuous adenine feeding. Not to mention that taking into consideration mice’s natural mechanism of chewing, it is known that even if one weighs the food in and food out in an attempt to dosify administration through this route, there is indeed loss of pellet content onto the cage floor and lost/mixed in the bedding that is never accounted for, thus making it an even more inaccurate method for dosing the adenine properly. In addition, animals that were administered adenine via oral gavage deteriorated more gradually, facilitating better monitoring of the disease development processes. Studies have shown that administration of adenine, either in food or by oral gavage, results in body weight loss and decreased food intake19,22,29. However, the present study found no significant differences in food intake. In the pellet adenine-induced CKD group, we observed a slight but not significant decrease in food intake. In the oral gavage adenine-induced CKD group, the response is similar to the control group. This could be due to using the lowest reported doses administered by oral gavage with renal effects19,33,42. It is important to note the exponential trend in the pellet adenine-induced CKD group’s water intake and urine production. In contrast, those parameters stabilized in the oral gavage administration group, demonstrating again that the oral gavage model is more controllable.
Kidney damage can affect digestion and nutrient absorption in the intestine. Impaired intestinal transit or problems with intestinal motility could lead to decreased stool formation, as nutrients are absorbed more efficiently43. In the pellet adenine-induced CKD group, we observed a significant decrease in stool output at the end of the experiment. In contrast, we only observed a significant decrease in the oral gavage adenine-induced CKD group 2 weeks after adenine administration. However, the oral gavage group did not completely restore stool output at 4 weeks. As mentioned above, the differences between the adenine administration routes might be due to the diet. We have observed throughout the results how the oral gavage route model is less aggressive with a more linear progression in kidney lesioning. Additionally, studies have reported alterations in the intestinal microbiota and uremia generated during adenine-induced CKD44.
Research has found that administering adenine by oral gavage is a more controlled method, yet the most commonly used route of administration is through pellets enriched with adenine. Administration through pellets has disadvantages, such as little control over the daily intake of adenine and the animal’s reluctance to consume the pellets due to their aversive taste. Furthermore, the lack of detailed reports makes it difficult to accurately prepare and replicate the dosage of adenine administration via the pellets. Animals receiving adenine via their pelleted diet experience faster deterioration than the oral gavage group, as evidenced by a sudden and drastic loss of body weight and a more markedly altered state (66.6% dehydration vs. 33.3% with the gavage tube; data not shown). Sudden weight loss and dehydration decreased the animals’ survival rate (83% survival vs. 100% with the gavage tube; data not shown). Given the present results, adenine oral gavage administration appears to be a more efficient administration route to induce CKD.
In the present study, females were used due to the higher incidence of CKD in women than in men3,6,45 and the lack of gender-based studies. Different reports suggest that female rodents are resistant to kidney damage and have demonstrated that male mice fed with adenine for 12 weeks survive less (50%) than female mice (95%)19,29,46. Evidence shows that the incidence of human CKD is higher in women than in men; however, women live longer with complications of the disease6,47. Compared to men, women experience a lower incidence of events associated with atherosclerosis, heart failure, and cardiovascular disease mortality, and studies show that kidney function in CKD patients declines faster in men29,47. Sexual differences affect physiological responses in CKD, which influence the progression and severity of the disease. However, efforts to study the progression of the disease in the female sex are still sparse48. In this regard, the present study represents a good effort to highlight the relevance of better standardizing CKD models in the context of female mice. Studies on CKD progression should consider both male and female subjects to explore gender differences.
The present results demonstrated that adenine successfully induces renal disease like CKD, as determined by several markers. The adenine-induced CKD model is the least invasive, low-cost, and highly reproducible. This model also resembles the mechanisms present in human CKD. One of the study’s limitations is the lack of evaluation of these parameters over a longer period of CKD progression. Many previous studies by others use four weeks as a timeframe to assess CKD. However, extending this timeframe could match more advanced phases of CKD in humans11. In summary, given the strong evidence that oral gavage adenine-induced CKD is a more stable, reproducible, and reliable model in female mice, we consider this method an effective experimental approach to assessing behavioral, hormonal, and metabolic alterations in CKD studies.
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