Traditional Chinese Medicine: An Exogenous Regulator of Crosstalk between the Gut Microbial Ecosystem and CKD

Chronic kidney disease (CKD) is often accompanied by an imbalance in the gut microbial ecosystem. Notably, the imbalanced gut microbiota and impaired intestinal barrier are the keys to the crosstalk between the gut microbial ecosystem and CKD, which was the central point of previous studies. Traditional Chinese medicine (TCM) has shown considerable efficacy in the treatment of CKD. However, the therapeutic mechanisms have not been fully elucidated. In this review, we explored therapeutic mechanisms by which TCM improved CKD via the gut microbial ecosystem. In particular, we focused on the restored gut microbiota (i.e., short-chain fatty acid- and uremic toxin-producing bacteria), improved gut-derived metabolites (i.e., short-chain fatty acid, indoxyl sulfate, p-Cresyl sulfate, and trimethylamine-N-oxide), and intestinal barrier (i.e., permeability and microbial translocation) as therapeutic mechanisms. The results found that the metabolic pattern of gut microbiota and the intestinal barrier were improved through TCM treatment. Moreover, the microbiota-transfer study confirmed that part of the protective effect of TCM was dependent on gut microbiota, especially SCFA-producing bacteria. In conclusion, TCM may be an important exogenous regulator of crosstalk between the gut microbial ecosystem and CKD, which was partly attributable to the mediation of microbiota-targeted intervention.


Introduction
Chronic kidney disease (CKD) is defned as the presence of progressive and irreversible destruction of renal structure or function. It is an important public health concern, afecting 10.6%-13.4% of the general population worldwide [1]. However, risk factors for abnormal renal structure or function are diverse, and the pathogenesis has not been fully elucidated [2]. Terefore, currently available strategies for slowing the progression of CKD are few and incomplete. Clearly, additional therapeutic avenues to accessing efective treatment of CKD must be recognized, and public health strategies must be developed to overcome current barriers, including the management, control, and delay of CKD [3].
Notably, researches in recent years have linked alterations in the gut microbiota (a condition known as "dysbiosis") and its mediation on the intestinal barrier with chronic diseases outside the digestive system (e.g., CKD) [4,5]. Te imbalanced gut microbiota and impaired intestinal barrier are key to the crosstalk between the gut microbial ecosystem and CKD, which was the central point of previous studies ( Figure 1). Briefy, CKD-related changes in gut microbiota lead to abrupt shifts in the production of gut-derived metabolites, accompanied by an impaired intestinal barrier. Te alteration of the intestinal barrier allows the translocation of bacterial components from the gut into the bloodstream, ultimately contributing to renal infammation [6,7]. Terefore, restoring the gut microbial ecosystem (i.e., microbiota, gut-derived metabolites, and intestinal barrier) or engaging in microbiota-targeted interventions may be potential strategies for the prevention and management of CKD.
Accumulating evidence suggests that traditional Chinese medicine (TCM) has perfect therapeutic efects for alleviating diseases (e.g., diabetes, obesity, ulcerative colitis) based on gut microbiota and its metabolites [8][9][10]. Te gut microbiota can alter the chemical composition of individual herbs or herbal extracts to have diferent bioavailability, bioactivity, or toxicity than their precursors. Bidirectionally, TCM herbs or herbal extracts can also remodel the diversity of gut microbiota to alleviate related diseases [11]. Several recent studies have determined that TCM can signifcantly infuence the progression of CKD [12,13]. However, there is still a lack of a comprehensive summary of the efects and mechanisms of TCM on CKD from the perspective of microecology. Terefore, this review focused on the core crosstalk between the gut microbial ecosystem and CKD, that is, gut microbiota and intestinal barrier, to explore the therapeutic mechanisms of TCM on CKD progression.

Te Imbalanced Gut Microbiota and CKD.
Symbiosis is considered a close and long-term biological interaction between two symbionts (e.g., gut microbiota and the human body). Healthy gut microbiota can produce corresponding dynamic changes with the body's biological rhythms to maintain host homeostasis. On the contrary, gut dysbiosis (e.g., altered microbiota composition and its metabolic capacity) may contribute to the development and progression of chronic diseases, including CKD. For example, changes in microbiota composition can transform normally symbiotic gut microbiota into a pathogenic factor that adversely afects renal function. Encouragingly, in recent years, large-scale clinical studies on the gut microbiota (e.g., composition, abundance, symbiotic relationship, functional prediction) of CKD patients have gradually increased [14,15]. At the same time, breakthroughs have also been made in exploring the potential pathogenesis of CKD through animal models based on gut microbiota [16,17]. Te goal of these studies is to seek therapeutic targets that may be used to improve morbidity and survival in patients with CKD.
Tere is increasing evidence of gut microbiota dysbiosis in CKD. Vaziri et al. found that patients with stage V of CKD had 190 signifcantly diferent microbial taxonomic units (OTUs) compared to healthy controls. Similar results were obtained in animal experiments, that is, the model group of 5/ 6 nephrectomy-induced CKD rats had signifcant diferences in bacterial OTUs compared with the sham-operated group [18].
Moreover, research on the marker microbiota and the metabolic pattern of the gut microbiota in CKD are also increasing. As reported in previous studies on animals and patients with CKD, the relative abundance of Lactobacillus was signifcantly reduced. In contrast, Enterobacteriaceae is overgrown with a marked increase in relative abundance [19,20]. Jiang et al. found that the relative abundance of short-chain fatty acid (SCFA)-producing bacteria in CKD patients was signifcantly reduced, which promoted the metabolic pattern of the gut microbiota from saccharolytic fermentation to protein fermentation. Ultimately, these changes may cause a shift in the enterotype of CKD patients [21]. On the other hand, the bidirectionality of imbalanced gut microbiota and CKD has also been experimentally confrmed. In a study of 30 patients without receiving dialysis, bacterial DNA was detected in the blood of 6 of them (20%), and its bacterial genera were found to overgrow in the guts of these patients. In addition, these 6 patients had signifcantly elevated C-reactive protein and IL-6, a marker of low-grade infammation, compared with the 24 patients in which bacterial DNA was not detected. Tese fndings confrmed gut microbiota dysbiosis in CKD patients. Furthermore, overgrown bacteria could translocate through the gut into the bloodstream to induce low-grade infammation and ultimately promote CKD progression [22].
Gut microbiota dysbiosis in CKD patients is closely associated with diet restrictions, medications, slow colonic transit, and changes in the gut environment (Figure 1(a)). Te above four points are not only attached to the background of CKD but also the trigger factors of imbalanced gut microbiota in CKD patients. Specifcally: (1) Dietary restriction: dietary fber generally refers to the nondigestible carbohydrates present in food. Foods rich in dietary fber include fruits, vegetables, beans, whole grains, etc. High dietary fber intake can reduce the substrate required for protein fermentation, and reduce colonic transit time by stimulating intestinal mucosa to increase secretion and promote intestinal motility [23]. For the general population, the current recommended dietary fber intake is 20-30 g/ d [24]. For CKD patients, there are no specifc recommended doses in related guidelines. High dietary fber intake will increase potassium and phosphorus levels, leading to imbalanced electrolytes in CKD patients. Terefore, these patients are generally characterized by reduced dietary fber intake. However, insufcient intake can induce the imbalance of saccharolytic and proteolytic microbiota, leading to a shift in the metabolic pattern from saccharolytic fermentation to protein fermentation [25]. Ultimately, two major gut-derived metabolites, SCFAs and gut-derived uremic toxins (GDUT) are deregulated [26]. (2) Medications: CKD patients are often exposed to antibiotics to treat vascular access infections or other infectious diseases. However, antibiotics can deplete key bacterial taxa that maintain gut homeostasis, while reducing bacterial diversity and metabolic capacity [27]. On the other hand, for CKD patients with anemia or calcium-phosphorus metabolism disorders, the long-term administration of iron supplementation or phosphate binders may induce changes in the gut environment and afect the colonization of microbiota, leading to imbalanced gut microbiota [28,29]. (3) Slow colonic transit: prolonged colonic transit time can reduce the availability of carbohydrates in the colon, thereby inducing an increase in proteolytic microbiota, and ultimately leading to the imbalance of saccharolytic and proteolytic microbiota in CKD patients [30]. (4) Changes in the gut environment: urea concentrations are signifcantly elevated in CKD patients [31]. It has been confrmed that the increased infux of urea into the intestinal lumen contributes to the proliferation of urease-producing bacteria [32,33]. Lau et al. confrmed that the relative abundance of urease-producing bacteria was signifcantly increased in CKD patients (stage V) compared with healthy controls [34]. Urea is decomposed by urease to produce ammonia. Ammonia raises the pH of the intestinal lumen and alters the composition of the gut microbiota, leading to gut dysbiosis [35].
IS and pCS: specifcally, dietary tryptophan is catabolized into indole by gut Escherichia coli under the action of tryptophanase. After indole is absorbed from the gut into the portal circulation, it is converted to hydroxyindole and IS by two hepatic cytochrome oxidases, CYP 2E1 and SULT1A1, respectively. As for pCS, dietary tyrosine and phenylalanine are catabolized by gut anaerobic bacteria to 4hydroxyphenylacetic acid, and then decarboxylated to pcresol, which is converted to pCS by SULT1A1 in the liver [39]. For details, see Figure 3(a). Serum IS and pCS concentrations were observed to be extremely low in healthy populations, around 10 μmol and 60 μmol, respectively. Both are mainly excreted by renal tubular secretion ( Figure 3(b)) under normal renal function [40]. However, IS and pCS cannot be efectively eliminated in the state of renal dysfunction, resulting in a large accumulation. In ESRD patients, the concentrations of both could be 10-50 times higher than those in healthy controls [41]. Te key toxic efects of IS and pCS on renal cells mainly include induction of oxidative stress [42], increased infammatory response [43], enhanced profbrotic expression [44], and downregulated expression of nephroprotective proteins (e.g., Klotho protein) [45]. IS and pCS are protein-bound uremic toxins that bind tightly through albumin-binding site II with up to 90% binding. Te current clinical dialysis strategies are extremely limited in the clearance of these two uremic toxins [46,47].
TMAO: Te main sources of TMAO are L-carnitine, choline, and betaine. Tese precursors are metabolized by gut microbiota to trimethylamine (TMA). Te absorbed TMA enters the liver through the portal venous circulation and is rapidly oxidized to TMAO by favin monooxidase (FMO3) [48]. See Figure 3(a) for details. TMAO is associated with an increased risk of cardiovascular disease and the progression of CKD. Notably, cardiovascular disease is the leading cause of death in CKD patients [49]. TMAO is Evidence-Based Complementary and Alternative Medicine normally excreted by glomerular fltration and tubular secretion (the main pathway) (Figure 3(b)), and then excreted in the urine [50]. Circulating TMAO concentrations gradually increased with the progression of CKD. A previous study found that patients with CKD (stages III-V) had higher plasma TMAO concentrations than non-CKD subjects [15]. Compared with patients with CKD (stage IIIb), patients with CKD (stage IV) had higher plasma TMAO concentrations [51]. Te serum TMAO concentration of ESRD patients was 20 times higher than that of healthy  controls [52]. Te serum TMAO concentration of patients who successfully received renal transplantation could quickly return to the normal range [53]. Notably, unlike IS and pCS, TMAO can be efectively removed by conventional dialysis [54].
Notably, although the small intestine provides the main site for the host's digestive activities, the production site of SCFAs is mainly concentrated in the colon, especially the ascending colon [55]. SCFA-producing bacteria, such as Lactobacillaceae, Ruminococcaceae, and Lachnospiraceae, can efectively degrade nondigestible carbohydrates to produce SCFAs [56]. Most of them can be rapidly absorbed by the intestinal epithelium through specifc transporters or by difusion, and are the energy source suppliers of colon tissue [57]. Among them, acetate is an important cofactor for bacterial growth [58]. Propionate and butyrate are key metabolites that provide the primary energy source for the colonocytes [59]. Most of the absorbed SCFAs are used as energy sources [60], while a small part is consumed by the liver [61]. Ultimately, the remaining SCFAs can pass through the circulatory system to target organs and tissues, where they can perform certain functions [62]. Mechanistic studies continue to provide evidence for the importance of SCFAs in diseases (e.g., hypertension, infammatory bowel disease, and CKD) [63][64][65]. Terefore, the homeostasis of SCFAs may provide clues and evidence for the balance between the gut microbiota and the host. At present, an increasing number of studies have focused on the interplay among SCFAs, intestinal barrier, and CKD [66,67]. Signifcantly decreased SCFA concentrations were observed in CKD patients compared to healthy controls [68]. Recent evidence suggested that concentrations of SCFAs, especially acetate and butyrate, are almost completely suppressed in patients and animal models with CKD [69,70]. In addition, there is increasing evidence that reduced concentrations of SCFAs contribute to renal dysfunction [71]. Conversely, supplementation with SCFAs, especially butyrate, can improve the intestinal barrier and control microbial translocation, and ultimately achieve nephroprotective efects [72]. Terefore, targeting the gut microbiota, especially SCFA-producing bacteria, may provide a promising therapeutic approach for CKD. Te mechanism by which SCFAs improve the intestinal barrier will be elaborated in section 3.1 of this review.

Te Impaired Intestinal Barrier and CKD.
Te intestinal epithelium is a single layer of columnar epithelium that separates the intestinal lumen from the lamina propria. It plays an important role in nutrient absorption while acting as a natural barrier to prevent and inhibit microbial translocation. Tese columnar epithelial cells are adjacent to each other by tight junctions, forming the "seal" of the intestinal barrier [73]. In a healthy population, the characteristics of gut ecosystem homeostasis include the following: (1) Te gut microbiota structure is characterized by the predominance of commensal bacteria (e.g., SCFAproducing bacteria, etc.), accompanied by few pathogenic bacteria (e.g., p-Cresol-and indole-producing bacteria). (2) Te intestinal barrier structure and function are intact (Figure 4(a)).
Te intestinal barrier of CKD was shown in Figure 4(b). Due to factors such as diet restrictions, medications, slow colonic transit, and changes in the gut environment, drastic changes in the gut microbiota of CKD patients are caused. Imbalanced gut microbiota can further lead to an impaired intestinal barrier (characterized by increased intestinal permeability) and microbial translocation. Ultimately, the translocated bacterial components can fow into the kidney through systemic circulation, exacerbating renal infammation. Te specifc mechanisms of impaired intestinal barrier caused by the imbalanced gut microbiota in CKD are as follows: (1) CKD patients have signifcantly elevated urea, which difuses into the intestinal lumen and further contributes to the expansion of urease-producing bacteria. Urea is hydrolyzed by urease to produce ammonia, which results in increased ammonia production in the intestinal lumen due to unregulated urease. Tis results in increased intestinal PH and a damaged intestinal wall, ultimately leading to increased intestinal permeability [33]. (2) Imbalanced SCFA-producing bacteria and reduced concentration of SCFAs resulted in a dramatic reduction in the nutrient and energy sources of colon tissue. Teoretically, these changes could lead to an impaired intestinal barrier [74]. (3) Impaired intestinal barrier stimulates leukocyte infltration. Local infammation and associated proinfammatory cytokines induced the endocytosis of intestinal epithelial tight junction proteins, which further contributes to increased intestinal barrier permeability [75].

Mechanisms of TCM in the Treatment of CKD via the Gut Microbial Ecosystem
3.1. Te Potential Terapeutic Mechanisms. TCM treatment could improve the clinical symptoms and renal function indexes of CKD patients. Previous animal experiments also found that CKD progression could be delayed by TCM treatment, which was characterized by improved renal function (pathological) indicators and systemic infammation. In addition, the regulatory efects of TCM on the gut microbial ecosystem had also been confrmed (Table 1). Notably, the above studies provided evidence that the protective efect of TCM was partially attributable to the mediation of the gut microbial ecosystem. Terefore, TCM may be an important exogenous regulator of crosstalk between the gut microbial ecosystems and CKD. Te mechanisms of TCM in the treatment of CKD via the gut microbial ecosystem were reviewed as follows: A recent study showed that JPYS had signifcant efects on improving renal function and modulating gut microbiota in CKD rats. Specifcally, JPYS increased the relative abundance of SCFA-producing bacteria (Coprococcus, Phascolarctobacterium, and Parasutterella), whereas the relative abundance of GDUT-producing bacteria (Clostridium XIVb) was decreased. Te metabolic pattern of gut microbiota shifted from saccharolytic fermentation to protein fermentation, which contributed to the imbalanced SCFA and GDUT-producing bacteria in CKD [76]. Terefore, improving the imbalance between SCFAand GDUT-producing bacteria may play a role in the treatment of CKD. (2) Regulation of Imbalanced Gut-Derived Metabolites. Ji et al. preliminarily confrmed that signifcantly elevated TMAO levels were observed in 5/6 nephrectomized rats, and rhubarb enema could efectively reduce circulating TMAO and alleviate renal function in CKD rats, which may be related to the regulation of TMAO-producing bacteria (Intestinimonas, Methanobrevibacter, Parasutterella, Anaerostipes, Catabacter, Ruminiclostridium, Desulfovibrio, and Clostridia) [17]. TMAO, IS, and pCS are the most representative gut-derived uremic toxins in CKD. GDUTs could directly act on renal cells by inducing oxidative stress, increasing infammatory response, enhancing profbrotic expression, and downregulating the expression of nephroprotective protein levels. Notably, the current clinical dialysis strategies are extremely limited in the clearance of IS and pCS. However, Based on the gut microbial ecosystem, previous studies have continuously provided clues and scientifc evidence that improved gut microbiota and intestinal barrier may be important entry points for CKD treatment. Preliminary studies had found that SCFAs, as important metabolites of gut microbiota, participate in the aforementioned processes, and the specifc manifestations were as follows: (1) Imbalanced SCFA-producing bacteria in CKD: the metabolic pattern of gut microbiota shifted from saccharolytic fermentation to protein  Sun et al. [12] Evidence-Based Complementary and Alternative Medicine  (2) improved kidney appearance (color, capsule, border); (3) improved renal function (Scr↓, BUN↓, 24 h urinary protein↓); (4) histopathologic evaluation of renal tissue; 1) H&E (infammation infltration↓, mesangial expansion↓, tubular atrophy and dilation↓, glomerular sclerosis↓, and interstitial fbrosis↓); 2) histopathological indicators (glomerular fbrosis area↓, tubulointerstitial fbrosis area↓); (5) regulates markers of infammation in renal tissue (IL-6↓); (6) microbiota-transfer study showed that the protective efect of Yishen Qingli Heluo granule was partly attributed to the mediation of the gut microbiota, especially the SCFA-producing bacteria Sun et al. [79] 8 Evidence-Based Complementary and Alternative Medicine fermentation, which contributed to the inhibition of SCFAs.
(3) Regulate intestinal pH value: provide a suitable environment for the production of acetate, propionate, and butyrate, which is conducive to shaping a perfect gut microbial ecosystem. For example, butyrate is the main energy substrate of colonocytes, providing about 70% of the important energy required for cell growth and diferentiation. Propionate is also an energy source for colonocytes, which has the efects of regulating cholesterol levels and antilipogenesis. Furthermore, acetate acts as the predominant SCFA, which is an important cofactor for bacterial growth [80]. Tis in turn reduces intestinal permeability. A recent study found that 12 weeks of Lycium ruthenicum anthocyanins supplementation in high-fat dietinduced mice could induce the production of SCFAs by regulating the gut microbiota, thereby attenuating intestinal barrier dysfunction [82]. Te relationship between SCFAs and the gut is not limited to this, and related clinical and animal studies have been advancing in recent years. (5) Concentration of SCFAs afects CKD progression. Reduced concentration of SCFAs led to renal dysfunction. Conversely, supplementation with SCFAs, especially butyrate, could improve the intestinal barrier and control microbial translocation, and ultimately achieve nephroprotective efects [83]. Hence, targeting the gut microbiota, especially SCFAproducing bacteria, may provide a new strategy for the treatment of CKD.   decreased SCFA concentrations, and local infammation of the intestinal wall. Ultimately, the translocated bacterial components can fow into the kidney through systemic circulation, exacerbating renal infammation. A recent study confrmed that rhubarb enema could reduce renal interstitial fbrosis and delay the progression of CKD. Specifcally, rhubarb increased the SCFA-producing bacteria (Akkermansia muciniphila, Lactobacillus acidophilus, Bacteroides caccae, and Faecalibaculum rodentium) in CKD rats, thereby increasing SCFA (propionic acid, butyric acid) concentrations and ultimately contributing to an improved intestinal barrier and controlled gut microbiota [67].

Evidence-Based Complementary and Alternative Medicine
Animal experiment found [78] that FZHY treatment hindered disease progression in CKD rats, manifested as improvements in renal function and fbrosis, decreased expression of renal fbrosis-related indicators (LN, FN, Col-I, Col-III), and systemic infammation markers (CRP, TNFα, IL-6, IL-1). In addition, FZHY signifcantly reduced the pathogenic bacteria (Monoglobus, Papillibacter, Eubacterium nodatum, Family XIII AD3011) and the precursor of gut-derived uremic toxins, and upregulated the expression of intestinal tight junction proteins (ZO-1, Occludin, Claudin-1). Elevated ammonia levels had been shown to promote disruption of the intestinal barrier. A previous study found that Monoglobus was positively correlated with blood ammonia levels. Te inhibition of Monoglobus by FZHY may have a protective efect on the intestinal barrier, which was consistent with the increased expression of intestinal tight junction proteins in this study. In addition, increased GDUT-related bacteria (Family XIII AD3011) or metabolites (indoles, phenols, etc.) act on renal cells and contribute to renal fbrosis and infammation, ultimately promoting CKD progression. In short, the underlying mechanism of FZHY alleviating CKD is mainly through the interrelationship between gut microbiota and gut-derived metabolites (Figure 6 Clinical studies had shown that clinical symptoms and Scr levels in CKD patients could be improved by YQHG. In addition, YQHG also delayed progression from stage III to stage IV in CKD patients [84].Sun et al. showed [79] that YQHG treatment signifcantly halted the progression of CKD, characterized by increased body weight, improved renal appearance and function, and reduced tissue damage in 5/6 nephrectomized rats. Importantly, the study demonstrated that 5/6 nephrectomized rats treated with YQHG showed signifcant improvement in renal fbrosis, such as reduced glomerular and tubulointerstitial fbrosis areas. Notably, they found that YQHG modulated bacterial communities, particularly increasing the relative abundance of SCFA-producing bacteria (i.e., Lactobacillaceae, Lactobacillus, and Lactobacillus gasseri), which in turn improved SCFA (i.e., total SCFA, acetic acid, butyric acid) concentrations and intestinal barrier (decreased FITC-dextran concentration). Ultimately, controlled microbial translocation (reduced bacterial signals) contributes to alleviating renal infammation (reduction of IL-6 expression) (Figure 6(b)). Interestingly, to further confrm the importance of the gut microbiota for YQHG in CKD treatment, they reshaped the bacterial community by conducting a microbiota-transfer study (cohousing and fecal microbiota transplantation). Impressively, the kidneys of CKD rats were profoundly protected after the microbiota-transfer study, characterized by the remission of renal infammation, fbrosis, and dysfunction. Te results suggested that the protective efect of YQHG was partly attributable to the mediation of gut microbiota, especially SCFA-producing bacteria.

Conclusions
In this review, we explored the therapeutic mechanisms of TCM to improve CKD via the gut microbial ecosystem. We summarized from the following three aspects: (1) TCM could regulate the metabolic pattern of gut microbiota: the metabolic pattern of gut microbiota shifted from saccharolytic fermentation to protein fermentation through TCM treatment. Specifcally, TCM treatment contributed to elevated SCFA and reduced GDUT. (2) TCM could improve the intestinal barrier: TCM increased SCFA concentrations (i.e., total SCFA, acetic acid, butyric acid), which in turn improved the intestinal barrier. Ultimately, controlled microbial translocation contributed to alleviating renal infammation. (3) Terapeutic efect mediated by the gut microbiota: the microbiota-transfer study confrmed that the protective efects of TCM were partly attributable to the mediation of gut microbiota, especially SCFA-producing bacteria (i.e., Lactobacillaceae, Lactobacillus, and Lactobacillus gasseri). Tese fndings propose a microbiota-targeted intervention and suggest that TCM may be a promising therapeutic avenue for overcoming current CKD-related barriers.

Conflicts of Interest
Te authors declare that they have no conficts of interest.