High salt intake is a risk factor for CKD and even hypertension [16]. The non-pharmacological strategy of diet modification to achieve renal protection is the cornerstone of CKD treatment [1]. The sulfur-containing amino acids in the diet are metabolized by gut microbiota to produce sulfide, which can be modified by sulfur-sulfhydrylation of tryptophan enzyme of E. coli, thereby reducing the generation of urinary toxin and renal injury in mice [17]. Through post-translational modification, diet can bring new enlightenment to tune microbiota function to support healthy host physiology [17, 18].
In this work, the interaction between HSD and gut microbiota was used to investigate the specific mechanism of gut microbiota activating the gut-kidney axis in HSD-related CKD. This work reveals that high dietary salt promotes renal injury development dependent on gut microbiota in mice. CKD typically appears alongside other comorbidities, highlighting underlying complex pathophysiology thought to be vastly modulated by the bidirectional gut-kidney crosstalk. CKD is a metabolic disease involving multiple tissue interactions, and its complex physiopathological and pathological functional changes are regulated by gut-kidney bidirectional regulation. The transplantation of fecal microbiota from mice on HSD into ordinary mice revealed that the gut microbiota in HSD group changed and induced renal injury and dysfunction, indicating that the interaction between diet and gut microbiota can be transmitted and changed and that the intestinal ecological imbalance induced by HSD is an important cause of renal injury [19]. Meanwhile, antibiotics can reverse the pathological features of intestinal leakage, early renal injury, and blood pressure rise induced by HSD in mice [19]. We discovered that HSD reduced the α diversity of the microbial community, reduced the relative abundances of Rikenella and Christensenella, and increased the relative abundances of Atopobium at the genus level in previous study [9]. Therefore, gut microbiota play the important role in HSD-related disease.
The renal injury involves pathophysiology, inflammation, immunity, and physiological functions. Most previous studies have revealed that HSD leads to the composition and structure of gut microbiota, immune dysfunction, and renal function injury [9]. Regulating macrophage function through the gut-kidney axis helps reduce renal fibrosis, which is a pathological change in chronic kidney disease [20]. Alterations in the gut-kidney axis have been found in CP-induced renal injury after enhancing the GLP-1 signaling pathway [21]. Manipulating the microbiota by oral administration of oxalobacter formigenes or probiotics containing inulin can prevent the formation of oxalate kidney stones, suggesting that manipulating the gut microbiota is a potential strategy for treating kidney stone diseases [22]. In our study, metabolomics, transcriptomics, and other methods were used to analyze the transcriptome characteristics of conventional and GF mice induced by HSD, the transcriptome characteristics of intestinal tissues, and the metabolic profiling of intestinal contents. Further, a combined analysis of transcriptomics and metabolomics was used to reveal the mechanism of gut microbiota in renal injury induced by HSD. First, this study revealed the changes in the renal transcriptome and intestinal transcriptome of gut microbiota involved in HSD-induced renal injury. Furthermore, the changes in metabolome were used to verify the molecular mechanism of gut microbiota regulating the gut-kidney axis involved in HSD-induced renal injury. Finally, the key genes and metabolites were verified in the present work.
The levels of Scr and BUN involved in evaluating the physiological function of the kidney, TNFα, IL-1β, IL-6, and IL-10, can be used to evaluate the inflammatory state of the kidney [23]. SIgA and NHE3 are related to the immune level and Na permeability [24, 25]. Our study discovered that HSD increased renal function-related factors, inflammatory response, and pathological damage in conventional mice. Compared with conventional mice, HSD did not cause pathological damage to renal function in GF mice, reflecting no changes in immunity, inflammation, or physiological function. Among them, elevated renal function factors were found in conventional mice, and decreased renal function factors were found in GF mice. These findings suggest that gut microbiota is a necessary intermediate medium for HSD-induced renal injury, and gut microbiota plays a promoting role. HSD led to the imbalance of gut microbiota and renal injury, and HSD cannot lead to renal injury without gut microbiota. Previous studies have found that antibiotics can reverse pathological features such as HSD-induced renal injury, consistent with our findings [19]. The loss or destruction of gut microbiota then blocked or reversed the protection of renal injury [19]. Furthermore, HSD has effectively inhibited tumor growth by up-regulating the number of NK cells and activation markers [26]. When we used antibiotics to destroy the gut microbiota of mice and fed them HSD, the results demonstrated that gut microbiota consumption induced by antibiotics destroyed the tumor inhibition and anti-tumor NK cell function mediated by HSD and that the gut microbiota was involved in regulating HSD and tumor [27].
To reveal the characteristics of renal transcriptome changes caused by gut microbiota participation in HSD, we first established the renal gene expression profiling changes of renal tissue involved in gut microbiota. For the first time, we systematically compared the changes of host transcription profiles in renal tissue of mice with or without gut microbiota interacting with HSD. We found 284 differential genes, including 107 up-regulated and 177 down-regulated, from the interaction between gut microbiota and HSD in the kidney. The up-regulated genes were significantly enriched in parathyroid hormone synthesis, secret, and action, and the down-regulated genes were significantly enriched in basal cell carcinoma, the wnt signaling pathway, ECM receiver interaction, and other top 20 signaling pathways. Parathyroid hormone is a key regulator of calcium and phosphorus homeostasis, which is related to CKD [28, 29]. Developing basal cell carcinoma is associated with constitutive activation of sonic hedgehog signaling, which is related to CKD, renal cell carcinoma, and clear cell renal cell carcinoma [30]. The wnt/β-catenin signaling pathway plays an important role in renal development and is re-expressed in the injured kidney [31]. ECM-receptor interactions associated with kidney development are also leveraged for regenerative bioactivity [32]. These findings indicate the potential mechanism in the renal injury induced by high dietary salt dependent on gut microbiota in mice.
To Further reveal the gut-kidney axis-related response mechanism of the interaction between gut microbiota and HSD, we established the gene expression profiling changes of intestinal tissue involved in gut microbiota and compared the changes of intestinal tissue transcription profiling of mice with or without gut microbiota and HSD for the first time. We found 426 genes in intestinal tissue, of which 286 were up-regulated and 140 were down-regulated. The up-regulated genes were significantly enriched in the pathways of primary immunodeficiency, African trypanosomias, mineral absorption, phospholipase D signaling pathway, etc. The down-regulated genes were significantly enriched in the pathways of Herpes simplex virus 1 infection, ECM receiver interaction, glycosphingolipid biosynthesis (globo and isoglobo series), and steroid hormone biosynthesis. In the axial relationship, a metabolic signal pathway can respond to two organs and tissues simultaneously. For instance, the MPAK signaling pathway reveals the axial relationship of the reno-cardiac axis [33]. Our study revealed that during the interaction between HSD and gut microbiota, steroid hormone biosynthesis responds synchronously in the down-regulated genes of the gut and kidney, which is also the only differential metabolic pathway with a common response, indicating that the steroid hormone biosynthesis pathway may be the key signal pathway of gut-kidney axis. No reports have confirmed the relationship between the steroid hormone biosynthesis pathway and the gut-kidney axis. Specifically, the steroid hormone biosynthesis pathway, the only common metabolism-associated pathway found in our work, belongs to the lipid metabolism pathways related to various diseases [34]. The adrenal cortex can synthesize various kinds of steroid hormones. The sex hormone produced by the adrenal gland is mainly dehydroepiandrosterone, which has a weak androgen effect and acts as other hormone precursors [35]. A combined transcriptome and metabolome analysis revealed that lovastatin, red yeast rice, and monascus pigment improved lipid metabolism in a high-fat model related to steroid hormone biosynthesis [36]. A previous study revealed that steroid hormone biosynthesis was significantly different between the benign tumor group and the renal cell carcinoma (RCC) group the urine metabolomes in a cohort of 61 patients with renal tumors and 68 healthy controls [37]. To verify whether the interaction between HSD and gut microbiota completes the gut and kidney interaction through the steroid hormone biosynthesis pathway, our work revealed 196 differential metabolites produced by the interaction between gut microbiota and HSD. The 88 up-regulated metabolites were enriched in thiamine metabolism, sulfur relay system, steroid hormone biosynthesis, protein digestion and absorption, and central carbon metabolism in cancer. The significant metabolites enrichment included the steroid hormone biosynthesis pathway. Therefore, to sum up, the interaction between HSD and gut microbiota completes the signal transmission of gut microbiota-related metabolites through the steroid hormone biosynthesis pathway, which provides strong scientific evidence to understand the significance of the gut-kidney axis in HSD-related CKD.
Furthermore, to identify the more core metabolites in the steroid hormone biosynthesis pathway, we first revealed the interaction between core genes through PPI network; second, we revealed the relationship between core metabolites and host genes using correlation analysis; third, these metabolites and genes can indeed be significantly distinguished using ROC curve; finally, the relative abundance of transcriptome and metabolome also further confirmed the differences of these metabolites (3-alpha,21-dihydroxy-5beta-pregnane-11,20-dione and dehydroepiandrosterone) and genes including Cyp1a1, Cyp2b10, Hsd17b1, Srd5a2, Cyp3a44. Currently, there is no report on the correlation between 3-alpha,21-dihydroxy-5beta-pregnane-11,20-dione and CKD. Dehydroepiandrosterone significantly reduced the basal expression of CYP1A1, and dehydroepiandrosterone inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels [38]. CYP1A1 is an important xenobiotic metabolizing enzyme associated with gene polymorphism in patients with CKD with unknown etiology [39]. To avoid differences caused by subjective factors, we selected the core genes of HSD and gut microbiota involved in the gut-kidney axis for experimental verification according to the following criteria: (1) they belonged to core genes in PPI network; (2) significant differences simultaneously existed between the two different metabolites; (3) AUC area in ROC analysis was larger; (4) renal and intestinal tissues responded simultaneously; (5) FC had a large variation multiple. Therefore, we used RT-qPCR to detect Cyp1a1 gene expression in intestinal and renal tissues. A further experiment verified that HSD induced low expression of the Cyp1a1 gene that plays an important role in the gut-kidney axis. In addition, we found that the sensitivity of Cyp1a1 gene to HSD in GF mice is different from that in SPF mice, which may be related to the physiological functions of gut microbiota. Previous studies have shown that the gut microbiota influences CYP1A [40, 41]. The additional animal experiments demonstrated that dehydroepiandrosterone produced by gut microbiota and high dietary salt inhibited the Cyp1a1 mRNA expression in the gut and kidney of mice, suggesting that the core differential metabolites and genes enriched in common steroid hormone biosynthesis pathway are related to HSD-related renal injury. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in a reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice [42]. Actinomycin D chase experiments in T47D cells revealed that dehydroepiandrosterone increased the rate of CYP1A1 mRNA degradation [43]. Therefore, our present work suggests that the interaction mechanism of HSD and gut microbiota in HSD-related renal injury is closely associated with the steroid hormone biosynthesis pathway.
Our work provides more detailed evidence of the gut-kidney axis in HSD-related renal injury, the study confirms that HSD promotes renal injury by manipulating the gutkidney axis via gut microbiota and strengthening the steroid hormone biosynthesis pathway. Increasing evidence has demonstrated a bidirectional relationship between host and gut microbiota in patients with various kidney diseases41. Our findings provide new insights into therapeutic strategies to prevent or alleviate HSD-related kidney disease. Through in-depth study, we found that gut microbes play an important regulatory role in HSD-related renal injury. We found that by regulating the composition and function of the intestinal microbial community, we can effectively intervene in the development of HSD-related kidney disease. In addition, our study also revealed the interaction network in the gut-kidney axis, including the interaction between intestinal microbes and intestinal mucosal barrier and kidney. However, this study has several limitations: the use of only male mice creates a single-sex model; the HSD intervention followed a single fixed duration; and dynamic phenotype monitoring was lacking. Although elevated dehydroepiandrosterone was observed, the specific bacterial species responsible for its production and the regulatory mechanisms were not elucidated. These aspects warrant more comprehensive investigation in future research.
The findings provide important clues for us to further understand the regulatory mechanism of the gut-kidney axis. Based on these findings, we can explore new therapeutic strategies to prevent or alleviate the development of HSD-related kidney disease. It will help to reduce the pathological process of HSD-related kidney disease. Furthermore, we can also use the metabolites of intestinal microorganisms as potential therapeutic targets. By regulating the metabolic activity of the intestinal microbial community, it affects the signal transduction pathway in the gut-kidney axis, thereby improving the pathological process of HSD-related kidney disease.
Increasing evidence has demonstrated a bidirectional relationship between host and gut microbiota in patients with various kidney diseases [44]. Previous studies have demonstrated that the gut microbiota affects the expression of kidney genes in a gender-specific manner, which may affect the physiological function of the kidney, while, the related mechanism has not been confirmed. Even microbiota from patients transplanted to renal injured GF or antibiotic-treated rats induces higher production of serum uraemic toxins and aggravated renal fibrosis and oxidative stress more than microbiota from controls [45]. Therefore, the more detailed mechanisms were required further exploration.