Chronic kidney disease (CKD), with a global prevalence of 10–12%, poses a significant health burden, increasing morbidity, mortality, and reducing quality of life. Advanced CKD, progressing to end-stage renal disease, is associated with high cardiovascular mortality. The disease manifests with a premature aging phenotype, and the survival rate of dialysis patients is lower than that of most solid-organ cancer patients. Established mechanisms linking CKD to cardiovascular morbidity and mortality include inflammation, oxidative stress, cellular senescence, and metabolic dysfunction. The gut microbiome plays a role in both cardiovascular disease (CVD) and CKD, directly impacting pathophysiology through food metabolism and being influenced by dietary habits and disease states. The trimethylamine N-oxide (TMAO) pathway is a promising target for microbiota-directed treatments in various lifestyle diseases. Gut microbiota metabolizes dietary substrates, including choline, to produce trimethylamine (TMA), which is then oxidized to TMAO in the liver. High choline and TMAO levels are independently associated with CVD risk, while betaine insufficiency is linked to adverse vascular risk profiles. Importantly, elevated TMAO levels are observed in CKD, correlating with mortality risk and inducing renal fibrosis. While the association between TMAO and CKD/CVD is well-established, the links between betaine and choline with CVD are mediated through TMAO. This research utilizes a comparative biomimetic approach, drawing inspiration from animal adaptations, to investigate the circulating levels of betaine, choline, and TMAO in humans with CKD, various animal species, and hibernating animals to understand metabolic regulation of the microbiota and identify potential therapeutic targets for human diseases.

Existing literature establishes a strong link between trimethylamine N-oxide (TMAO), a gut microbial metabolite derived from choline and other dietary components, and cardiovascular disease (CVD) and chronic kidney disease (CKD). Studies have shown that high TMAO levels are associated with increased risk of atherosclerosis, heart failure, and mortality in CKD patients. This elevation in TMAO is attributed to both impaired renal function, which reduces TMAO excretion, and gut dysbiosis, leading to increased microbial TMA production. Conversely, betaine, a metabolite related to choline, exhibits a protective effect, counteracting some of the adverse effects of TMAO. Low betaine levels are associated with components of the metabolic syndrome and increased cardiovascular risk. Previous research highlights the potential of modulating the gut microbiota to influence TMAO and betaine levels as a therapeutic strategy for CKD and CVD. This study leverages the unique metabolic adaptations observed in hibernating animals, particularly bears, to explore these relationships further and identify potential novel treatment strategies.

This study employed a comparative biomimetic approach to analyze circulating levels of betaine, choline, and TMAO across different populations and species. The study included three main groups: 1. **Human Subjects:** Fasting plasma samples were collected from Caucasian CKD stage 3 (n=30) and stage 5 (n=121) patients, and 80 age- and sex-matched healthy controls. Exclusion criteria included age <18 years, active hepatitis B/C, HIV, or acute infections. Ethical approval was obtained from the Karolinska Institutet Ethics Committee. 2. **Animal Species:** The study encompassed a diverse range of animals, including free-ranging brown bears (*Ursus arctos*), captive brown bears, captive garden dormice (*Eliomys quercinus*), wild boars (*Sus scrofa*), captive lions, and captive tigers. Sampling methods varied across species; free-ranging bears were sampled during hibernation and the active period under anesthesia, while captive animals were sampled during routine veterinary health checks under anesthesia. Garden dormice were sampled post-mortem during hibernation and active periods. Ethical approval and relevant permits were obtained for each animal group. 3. **Comparative Analysis:** To assess the impact of diet and hibernation, the researchers compared free-ranging and captive brown bears and analyzed the changes in metabolites in garden dormice at different stages of hibernation. A comparison group included animals with CKD-like status (hibernating bears and felids) and human patients with CKD stage 3 to contrast metabolite levels. **Assays:** Betaine, choline, and TMAO levels were quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Other parameters, such as liver enzymes, renal function markers, and electrolytes, were measured using standard laboratory methods. **Statistical Analysis:** Statistical analysis was performed using SPSS and GraphPad Prism. Non-parametric tests (Kruskal-Wallis, Wilcoxon signed-rank test, Mann-Whitney U test) were employed to compare groups and assess significant differences.

The study revealed significant differences in betaine, choline, and TMAO levels across species, dietary habits, and hibernation status. Key findings include: * **Humans with CKD:** Betaine levels decreased significantly with deteriorating renal function, while choline and TMAO levels increased. Patients with CKD stage 5 had the lowest betaine levels and the highest choline and TMAO levels compared to healthy controls and CKD stage 3 patients. * **Across Species:** Significant differences in betaine, choline, and TMAO levels were observed across various species. Free-ranging bears had notably low TMAO levels (undetectable in most), in contrast to captive bears that exhibited measurable TMAO. Carnivorous animals (lions and tigers) showed high TMAO levels. * **Hibernation vs. Active State (Brown Bears):** Free-ranging brown bears displayed a dramatic increase in betaine (422%) and a smaller increase in choline (18%) during hibernation, with undetectable TMAO. This contrasts sharply with their active state and other species. * **Hibernation vs. Active State (Garden Dormice):** In contrast to bears, hibernating dormice had significantly lower betaine and choline levels compared to their active state. TMAO levels were low in both states but slightly higher in active dormice. * **Free-ranging vs. Captive Bears:** Free-ranging bears had significantly higher betaine and lower choline levels compared to captive bears, and TMAO was almost exclusively found in captive bears, implicating captivity in altering microbiota. * **CKD-like status:** A comparison between hibernating bears and felids revealed dramatically different metabolite profiles. Hibernating bears had significantly higher betaine and lower choline and TMAO levels than felids and human CKD patients.

The study's primary finding is the dramatic increase in betaine in free-ranging hibernating bears, suggesting a metabolic switch that shunts choline toward betaine production instead of TMAO. This metabolic shift may offer several protective benefits during hibernation, including anti-inflammatory, antioxidant, and osmoprotective effects. The absence of detectable TMAO in free-ranging bears contrasts with the high levels observed in carnivorous species and humans with CKD, implicating diet and gut microbiota composition in TMAO production. The protective effect of high betaine levels during hibernation may involve various mechanisms, including anti-inflammation, methyl group donation, osmoprotection, and protection against hyperhomocysteinemia. The observed difference between free-ranging and captive bears highlights the impact of captivity on gut microbiota and subsequent TMAO production. The similar pattern seen in garden dormice, though less pronounced, suggests the metabolic switch might be linked to temperature changes and energy conservation mechanisms during hibernation.

This comparative biomimetic study suggests a metabolic switch in hibernating bears and dormice, leading to increased betaine production and reduced TMAO levels. This highlights a potential protective mechanism against CKD and other lifestyle diseases. The association between carnivorous diets and elevated TMAO levels is also evident. Future research should focus on characterizing this metabolic switch and exploring its potential for therapeutic manipulation in humans. Investigating the specific gut microbial changes and their interplay with endogenous choline metabolism during hibernation is critical to understanding these adaptive strategies.

The study has several limitations. The sample sizes for some groups (especially human controls and certain animal species) were relatively small, limiting statistical power and the ability to account for individual variation. Age and weight were not perfectly matched between free-ranging and captive bears. A lack of detailed dietary information for free-ranging bears could affect the interpretation of metabolic findings. The absence of fecal or circulatory microbiota analyses prevents a deeper understanding of the relationship between microbiota composition and metabolite levels. Using S-creatinine as a proxy for renal function is limited as it is also affected by muscle mass and meat intake.