Diabetic kidney disease (DKD) is a severe complication of diabetes, leading to significant morbidity and mortality. The gut microbiota plays a crucial role in various metabolic processes and is increasingly recognized as a key player in the pathogenesis of DKD. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been linked to cardiovascular disease and chronic kidney disease. Vitamin D has shown protective effects against DKD in previous studies. However, the interaction between vitamin D, gut microbiota, TMAO, and perirenal adipose tissue (PRAT) in the context of DKD remains unclear. This study aimed to investigate the role of vitamin D in modulating the gut microbiota, TMAO levels, and PRAT in a mouse model of DKD, shedding light on the potential mechanisms underlying its protective effects. The gut microbiota, a complex ecosystem of microorganisms residing in the intestines, is considered an essential metabolic organ. It participates in various functions including enhancing host immunity, aiding in digestion, regulating intestinal endocrine function, modulating nerve signals, and influencing drug metabolism. The symbiotic relationship between the gut microbiota and host is critical for the normal functioning of the host’s metabolic system. The metabolites produced by these gut microbes are absorbed by the host and act on receptors in the liver, intestines, adipose tissues (both brown and white), and the central nervous system (CNS), where they play a role in nutrient synthesis, intestinal motility, and mineral and electrolyte absorption. Choline, a dietary nutrient, is metabolized by the gut flora to trimethylamine (TMA), which is further oxidized in the liver to TMAO. Elevated TMAO levels have been associated with increased cholesterol accumulation in atherosclerotic plaques and impaired liver function, increasing hepatic triglyceride accumulation and lipogenesis. In humans, high TMAO levels are linked to hepatic steatosis, regulated through bile acid metabolism. A strong association exists between high TMAO levels, vitamin D deficiency, and nonalcoholic fatty liver disease (NAFLD). Furthermore, TMAO has been implicated in various diseases, including cardiovascular disease, nonalcoholic fatty liver disease, diabetes, chronic kidney disease, and colorectal cancer. Studies have shown a clear link between TMAO and the development of renal fibrosis, renal impairment, and reduced long-term survival in chronic kidney disease (CKD). The precise mechanism connecting TMAO and DKD, however, remains to be fully elucidated. The vitamin D/vitamin D receptor (VD/VDR) system is well-established for its role in kidney protection. Research indicates a strong correlation between VD, gut microbes, and inflammation. Vitamin D promotes the growth of beneficial bacteria while inhibiting pathogenic microbes, particularly under inflammatory conditions. VDR activation reduces inflammation by inhibiting NF-κB activation. Several studies have demonstrated a correlation between reduced serum 25(OH)D levels in DKD animal models and specific characteristics of the gut flora. However, the precise role of VD in influencing gut microbiota changes in DKD mice, as well as its interaction with TMAO, needs further investigation. Perirenal adipose tissue (PRAT), located in the retroperitoneal space, acts as a protective layer for the kidneys and renal vessels. Expansion of PRAT, often observed in obesity and diabetes, has been associated with various pathological abnormalities and contributes to the progression of kidney damage. PRAT, containing both white and brown adipocytes, is considered an endocrine organ that secretes adipokines, and its metabolic activity impacts the surrounding tissues, including the kidneys. Recent findings suggest that TMAO production inhibits the activity of both beige and white adipose tissue. Therefore, this study explores the relationship between vitamin D, gut microbiota, TMAO, and PRAT in the context of DKD to provide insights into the prevention and treatment of this disease.

The existing literature supports the involvement of gut microbiota in the pathogenesis of diabetic kidney disease (DKD). Studies have shown alterations in gut microbial composition in DKD patients, with a decrease in beneficial bacteria and an increase in potentially harmful bacteria. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, is linked to the progression of renal injury. Vitamin D has demonstrated renoprotective effects in various studies, likely through multiple mechanisms, including immunomodulation and reduction of inflammation. The interplay between vitamin D, gut microbiota, TMAO, and perirenal adipose tissue (PRAT) in DKD requires further investigation. Previous research has shown a correlation between low vitamin D levels and high TMAO levels, suggesting a potential interaction. However, the exact mechanisms through which vitamin D exerts its effects on the gut-kidney axis remain elusive. This study aims to further elucidate these interactions, focusing on the impact of vitamin D on the gut microbiome, TMAO production, and PRAT in a mouse model of DKD. The existing literature establishes the importance of each component individually, but the combined effects within the context of DKD are not fully understood. This integrated approach is crucial to understanding the complex pathogenesis of DKD and potential therapeutic interventions.

This study utilized a mouse model of diabetic kidney disease (DKD). Sixty 8-week-old male KKay mice (prone to type 2 diabetes) and 10 C57BL/6 mice (control group) were used. KKay mice were divided into DM (14 weeks old) and DKD (20 weeks old) groups. The DKD mice were further divided into placebo, low-dose (0.3 µg/kg/d), medium-dose (1.4 µg/kg/d), and high-dose (2.5 µg/kg/d) 1,25-(OH)2D3 groups. A separate group received medium-dose 1,25-(OH)2D3 and 0.12% TMAO. Mice were treated daily with 1,25-(OH)2D3 for 14 days, starting at 18 weeks of age. Body weight, blood glucose, and urine protein were measured. Serum TNF-α and TMAO levels were determined using ELISA and LC-MS, respectively. Gut microbiota composition was analyzed using 16S rRNA sequencing. qPCR was used to assess gene expression of TLR4, NF-κB, PGC1α, and UCP-1 in kidney and adipose tissues. Histological changes were examined using HE, PAS, Masson, and oil red staining. Immunofluorescence and immunohistochemistry detected VDR, PGC1α, podocin, and UCP-1 expression. Electron microscopy observed kidney pathological changes. A VDR knockout mouse model was created using lentiviral vectors. Statistical analyses, including t-tests, ANOVA, and Pearson correlation, were employed. Data was expressed as mean ± standard deviation (SD). P<0.05 indicated statistical significance. Specifically, the mice were fed high-sugar, high-fat, and high-cholesterol feed (KK mice feed 1042) to induce DKD. The study involved multiple analytical techniques to comprehensively assess the effects of vitamin D on renal function, gut microbiota composition, TMAO metabolism, inflammatory pathways, and PRAT characteristics. The experimental setup involved careful control groups and a range of 1,25-(OH)2D3 concentrations to determine dose-response relationships. The use of both in vivo and in vitro techniques allowed for a more robust and comprehensive examination of the underlying mechanisms. The 16S rRNA sequencing analysis provided a detailed picture of the gut microbial community changes following vitamin D treatment. Gene expression analysis using qPCR helped to elucidate the molecular mechanisms of action of vitamin D on inflammatory pathways in both kidney and adipose tissues. Histological and electron microscopy analyses were crucial in visualizing the pathological changes occurring in the kidneys and perirenal adipose tissues. Immunofluorescence and immunohistochemistry analyses contributed to the understanding of protein expression patterns in the target tissues, highlighting the role of various proteins in the observed effects. The creation of the VDR knockout mouse model was instrumental in establishing the specific role of VDR in the observed phenomena. The comprehensive statistical analyses employed ensured the reliability and validity of the research findings.

This study revealed several key findings regarding the interplay between vitamin D, gut microbiota, TMAO, and perirenal adipose tissue (PRAT) in mice with diabetic kidney disease (DKD). Firstly, administration of 1,25-(OH)2D3 significantly improved the dysbiosis of the intestinal flora in DKD mice, increasing the abundance of beneficial bacteria while decreasing the abundance of harmful bacteria. This modulation of the gut microbiota was concentration-dependent, with higher doses of 1,25-(OH)2D3 showing more pronounced effects. Secondly, serum TMAO concentrations were significantly elevated in DKD mice compared to the control group and showed a strong positive correlation with urinary albumin-creatinine ratio (UACR), a marker of kidney damage. This finding highlights the potential role of TMAO in exacerbating renal dysfunction in DKD. Thirdly, 1,25-(OH)2D3 treatment significantly reduced kidney damage in DKD mice, as evidenced by reduced UACR, improved histological findings (less glomerular hypertrophy, tubular atrophy, and interstitial fibrosis), and increased expression of podocin, a protein essential for glomerular filtration. The reduction in kidney damage was also concentration-dependent. Fourthly, 1,25-(OH)2D3 treatment stimulated the expression of PGC1α and UCP-1 in PRAT, suggesting a beneficial effect on adipose tissue metabolism. Conversely, TMAO treatment downregulated the expression of VDR in both PRAT and kidneys, indicating a potential antagonistic effect of TMAO on the renoprotective effects of vitamin D. Finally, the study demonstrated that the VDR knockout mouse model exhibited increased inflammation, reduced podocin expression, and increased collagen IV expression (a marker of fibrosis) in the kidneys, supporting the crucial role of VDR in maintaining kidney health and confirming the adverse effects of inhibiting VDR expression. These key findings provide a strong evidence base for the protective role of vitamin D in DKD and highlight the intricate interactions among gut microbiota, TMAO, PRAT, and kidney function.

The findings of this study strongly support the hypothesis that vitamin D exerts its renoprotective effects in DKD through a multifaceted mechanism that includes modulation of the gut microbiota, regulation of TMAO levels, and influence on perirenal adipose tissue (PRAT) metabolism. The observed improvement in gut dysbiosis with 1,25-(OH)2D3 administration suggests that vitamin D may directly influence the composition of the gut microbiome, promoting the growth of beneficial bacteria and suppressing potentially harmful bacteria. This could, in turn, lead to a reduction in the production of TMAO, thus mitigating its detrimental effects on the kidneys. The positive correlation between serum TMAO and UACR further underscores the contribution of TMAO to DKD pathogenesis. The stimulation of PGC1α and UCP-1 expression in PRAT by vitamin D suggests that vitamin D may improve adipose tissue metabolism, reducing fat infiltration and potentially decreasing the production of inflammatory adipokines that could contribute to kidney damage. The downregulation of VDR expression by TMAO suggests a potential mechanism through which TMAO may antagonize the renoprotective effects of vitamin D. The results of the VDR knockout study further confirm the critical role of VDR in maintaining kidney health, highlighting the importance of maintaining adequate vitamin D levels in DKD management. These findings have significant implications for the management of DKD, suggesting that strategies aimed at increasing vitamin D levels and modulating the gut microbiota might be beneficial in reducing the progression of this disease. Further research is needed to investigate the potential of combining vitamin D supplementation with microbiome-targeted therapies as a novel strategy to combat DKD.

This study demonstrates a significant interplay between vitamin D, gut microbiota, TMAO, and PRAT in the context of DKD. Vitamin D supplementation improves gut dysbiosis, reduces TMAO levels, mitigates kidney damage, and improves PRAT metabolism. These effects are likely mediated, at least in part, through the VDR pathway. Future research should focus on translating these findings into clinical practice, exploring the potential of vitamin D supplementation combined with gut microbiota modulation to improve DKD outcomes. Further investigations are needed to fully elucidate the complex interactions among these factors and to determine the optimal therapeutic strategies.

This study utilized a mouse model of DKD, which may not fully replicate the complex pathophysiology of human DKD. The relatively short treatment duration (14 days) may not be sufficient to observe long-term effects. The study focused primarily on male mice, limiting the generalizability of the findings to female mice and humans. Further investigation is needed to determine the optimal dose and duration of vitamin D supplementation for DKD patients and explore the potential interactions with other medications and dietary factors.