Role of gut microbiota‐derived metabolites on vascular calcification in CKD

Abstract The interaction between gut microbiota and the host has gained widespread concern. Gut microbiota not only provides nutrients from the ingested food but also generates bioactive metabolites and signalling molecules to impact host physiology, especially in chronic kidney disease (CKD). The development of CKD, accompanied by changed diet and medication, alters the gut flora and causes the effect in distant organs, leading to clinical complications. Vascular calcification (VC) is an actively regulated process and a high prevalence of VC in CKD has also been linked to an imbalance in gut microbiota and altered metabolites. In this review, we focused on gut microbiota‐derived metabolites involved in VC in CKD and explained how these metabolites influence the calcification process. Correcting the imbalance of gut microbiota and regulating microbiota‐derived metabolites by dietary modification and probiotics are new targets for the improvement of the gut‐kidney axis, which indicate innovative treatment options of VC in CKD.

to the development and progression of VC. 9 Accumulating evidence demonstrated that gut microbiota might play an essential role in VC in CKD patients. 10,11 Therefore, this review intended to provide an overview of gut microbiota-derived metabolites on the facilitation of VC and to propose new thoughts based on the interference of gut microbiota-derived metabolites to retard VC in CKD patients.

| VC IN CK D
The vascular wall is composed of three differently structured layers from the periphery to the lumen of the vessel. Blood vessels contain two primary cell types, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), which exert essential functions to maintain vascular homeostasis. Being different from ECs, VSMCs are not terminally differentiated and preserve their plasticity. VC can develop in the intimal and medial layers of arteries. Alternatively, medial calcification is characterized by VSMCs' transformation into osteoblastlike cells and is more common in CKD patients. 12 Overproduction of reactive oxygen species (ROS) in VSMCs is related to vascular dysfunction, which is a risk factor for CKD patients. VC is an active biological process associated with hydroxyapatite crystallisation in the vascular wall. The decline of inhibitors such as Matrix Gla Protein (MGP), Gla-rich protein (GRP), osteoprotegerin (OPG), bone morphogenetic protein 7 (BMP-7) and the increase of calcification inducers lead to more extensive VC in the CKD population. 13,14 Furthermore, uremic toxins, calcium and phosphate metabolism dysfunction may directly influence VSMCs' physiological function, leading to irregular senescence, proliferation and migration of VSMC, ultimately leading to VC.

| THE G UT MI CROB I OTA IN CK D
The human gastrointestinal tract is inhabited with 100 trillion different microbes, including bacteria (Lactobacillus), viruses (primarily phage), fungi, archaea and so on. 15 There are two characteristics of gut microbiota in healthy adults; one is taxonomical diversity, such as Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Verrucomicrobia all be seen in the healthy gut. 16 The other is functional diversity, such as enhancing the host against enteral pathogens, modulating systemic immunity. In the body, approximately 70% of the immune cells reside in the gut, cutting down bacterial dissemination and producing vitamins and essential metabolites that are not synthesized by the host. 17 A study amongst end-stage renal disease (ESRD) patients found high phyla Firmicutes, Proteobacteria F I G U R E 1 The kidney plays an important role in nutritional homeostasis. Base on Chronic kidney disease causes kidney damage, Increased susceptibility to malnutrition injury. In CKD, a reduction in renal filtering capacity results in the deposition and accumulation of waste products in the blood, which eventually develops into uremia. In addition, complications of uremia include uremic neuropathy, which can contribute to gustatory dysfunction leads to an imbalanced diet. Dysbiosis caused by an imbalanced diet (for example, a diet high in protein and animal fat) leads to excessive production and accumulation of p-cresol and indoxyl sulfate in the gut. This accumulation destroys the intestinal barrier, thereby increasing the permeability of the intestine. Therefore, it can cause kidney damage (like inflammation of the kidneys). Metabolite causes activation of immune cells and factors, and continuous destruction of the intestinal barrier. This process into a vicious circle and Actinobacteria, and a decrease in Lactobacilli, Roseburia and Phytoalexin. 18 Currently, several studies proposed the role of the gut-kidney axis ( Figure 1). For example, excessive protein intake in the diet can cause intra-glomerular hypertension, which leads to kidney ultrafiltration and glomerular damage. Long-term high protein intake may lead to CKD. In CKD patients, a reduction in renal filtering capacity results in the deposition and accumulation of waste products in the blood, which eventually develops into uremia. Uremia can lead to malnutrition, which leads to an imbalance of intestinal flora and abnormal metabolites, increased intestinal permeability. Finally, kidney damage is aggravated again. 19 In another study with CKD patients, it was demonstrated that the reduction infiltration capacity of the kidneys led to the deposition and accumulation of toxic waste products in the blood. 7 The genome of the gut microbiome contains 3.3 million genes. It is 150 times the human genome. Recently, a study has shown that dual-omics (metagenomic and metabolomics) data can reveal the connections between gut microbes and circulating metabolites perturbed in CKD. In the early stage of CKD, microbial genes to secondary bile acid biosynthesis were differentially abundant. In the advanced stage that lipid metabolism and lipopolysaccharide biosynthesis were enriched. However, the research lacks a replication cohort. 20

| INFLUEN CE OF D IE T ON G UT MICROB IOTA
Diet strongly affects human health. Most of the beneficial effects are obtained by modulating gut microbiome composition. According to the dominant bacterial system type, the gut microbiota of adults can be divided into two main types. Both intestinal types are closely related to long-term diet. The main bacterial population of intestinal type 1 is Bacteroides, which mainly metabolizes proteins, while gut type 2 is mainly glycolytic chlorella. 21 Previously, animal studies reported that intake of a high-unsaturated fat-rich diet would increase Actinobacteria (Lactobacillus and Streptococcus) and Verrucomicrobia. Probiotics intake, such as cultured milk products and yogurt, is a source of ingestible microorganisms. 22 Diet containing high fat can increase Lipopolysaccharides (LPS) translocation. 23 Common food like fruits, vegetables and tea are all rich in polyphenols. 24 Probiotics and Polyphenols both enhance Bifidobacterium and Lactic acid-producing bacteria and reduce enteropathogenic bacteria. 25 The renal diet (low potassium, low phosphorus), which is lacking in plant fibre, can lead to the overgrowth of bacteria with harmful metabolites like uremic toxins. 26 A study reported that older adults with CKD had a higher taste sensation for phosphate-containing salts. Hyperphosphatemia can accelerate VSMCs transdifferentiating and directly participate in the deposition of the calcium-containing osteoid matrix in vascular media. Therefore, CKD with gustatory dysfunction can develop metabolism perturbation and contribute to uremic neuropathy. If the gustatory function in CKD patients can be preserved, food intake can be improved and reduce the production of uremic solutes, leading to a lower risk of VC. 27 Therefore, diet can adjust the type of gut microbiota and metabolites. In CKD patients, more attention should be paid to the impact of diet in VC.

| THE REL ATI ON S HIP B E T WEEN G UT MICROB IOTA-DERIVED ME TABOLITE S AND VC IN CK D
Altered gut microbiota and metabolites in CKD are believed to be involved in the VC process. 28 LPS and bacterial DNA could directly induce inflammation and immune response, leading to end-organ damage. 29 Gut microbiota-derived metabolites such as uremic toxins, trimethylamine N-oxide (TMAO), bile acid are all associated with VC in CKD by regulating vascular phenotype, oxidative stress and epigenetics. 30,31 The following is a detailed introduction of gut microbiota-derived metabolites on vascular calcification ( Figure 2).

| Uremic toxins
Gut microbiota-derived metabolites of amino acids are uremic toxins, including indole-3 acetic acid, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), which translocate into the bloodstream and cause extensive oxidative stress-induced damage to the kidneys. Uremic toxins can lead to endothelial dysfunction, vascular senescence, vascular inflammation in CKD patients. It is supporting the link between uremic toxins and vascular dysfunction. 32 Silvia D et al demonstrated that uremic toxins impaired the autophagic flux leading to endothelial dysfunction. 33 Generally, gut bacteria metabolize tryptophan into indole, further exchanged into indoxyl sulfate in the liver after intestinal absorption. 34 Gut bacteria also metabolize aromatic amino acids into tyrosine phenylalanine and p-cresol, which are bio-transformed by sulfotransferase into PCS in the liver. 35,36 In CKD patients, the production of uremic toxins (such as IS, PCS) by bacteria increases.
IS, a critical protein-bound uremic toxin, can be described as a significant risk factor of VC in CKD patients. 37 Research has shown that uremic toxins (mainly Pi) are responsible for the high prevalence of VC in the CKD population. 38 In a healthy human beings, IS concentration ranges from 10 to 130 mg/day. But excessive IS induces the production of free radicals in both renal cells and VSMC through oxidative stress and inflammation to cause tissue injury. 39 In CKD patients, clinical evidence has shown that IS plasma levels are as-  42 Also, IS induced methyltransferase-like (METTL14)dependent N6-methyladenosine (m6A) to regulate VC. 43 PCS is a prototype protein-bound molecule. 44 PCS's concentration ranges between 2.8 ± 1.7 mg/L and 6.6 ± 3.7 mg/L in healthy human plasma. However, PCS is significantly increased in end-stage renal disease (ESRD) patients (21.8 ± 12.4 mg/L and 106.9 ± 44.6 mg/L). 45 PCS induced inflammatory factors that triggered monocyte-endothelial cell interaction and incriminated oxidative stress in human VSMCs. 46 In addition, the effect of PCS on renal injury has been reported. Sun et al demonstrated that PCS was activated by the renal renin-angiotensin-aldosterone system and then induced epithelial-mesenchymal transition, contributing to kidney injury. 47 Moreover, a study has demonstrated that IS and PCS both could potentially induce endothelial dysfunction and distinct calcification in the arteries of CKD rats. 31 54 IS also down-regulated miR-29b and activated Wnt/β-catenin signalling to F I G U R E 2 In chronic kidney disease (CKD) patients, decreased renal function leads to decreased glomerular filtration rate (GFR), increased proteinuria and uremic toxins, and damage to glomeruli and tubulointerstitial. Also cause metabolic acidosis, accelerated protein catabolism, resulting in malnourished patients. Malnutrition also leads to an imbalance of intestinal homeostasis, which is characterized by increased mucosal inflammation, increased intestinal permeability and abnormally increased gut microbiota-derived metabolites like (p-cresol, indoxyl sulfate and Trimethylamine N-oxide (TMAO)). It can directly or indirectly affect vascular smooth muscle cells or vascular endothelial cells and induce vascular calcification. And high phosphorus caused by chronic kidney disease, parathyroid hormone (PTH), inflammatory cytokines, oxidative stress and uremic toxins can induce vascular calcification induce vascular activation. 55 In conclusion, IS and PCS are considered harmful vascular toxins and promote VC in CKD patients. MVs and miRNAs might be therapeutic targets to prevent vascular disease in CKD (Figure 3).

| Trimethylamine N-oxide (TMAO)
Trimethylamine N-oxide is a product of the gut microbiome. Choline and phosphatidylcholine are catalysed into trimethylamine (TMA) in intestinal microbiota (such as phosphatidylcholine, betaine and I-carnitine), which is further oxidized as TMAO in the human liver. 56 In vitro experiments showed that TMAO promoted VC only in a calcifying medium, explaining that high calcium and phosphate were critical for TMAO-induced VC. In vivo study demonstrated that TMAO promoted VC in CKD rats with high calcium/ phosphorus (Ca/P) diet. 57 Besides, TMAO was shown to increase the expression of pro-inflammatory genes such as interleukin-18 (IL-18), interleukin-6 (IL-6) and interleukin-1β (IL-1β) adhesion molecules and chemokines. 58  In summary, the elevated TMAO level in CKD is a considerable promoter of VC. Several approaches are being explored to reduce TMAO levels, like oral broad-spectrum antibiotics or promoting the growth of bacteria that use TMAO as matrix and baicalin. [62][63][64] Recently, the Pretest-posttest study showed that a diet supplemented with β-glucan was potentially efficient in lowering serum concentrations of TMAO in patients with CKD. 30 But whether these methods are able to prevent VC still needs further research.

| Bile acids
Bile acids are amphipathic molecules that have two types. One is primary bile acids and another is secondary bile acids. Primary bile acids produced by cholesterol in the liver are processed into secondary bile acids by gut microbiota. Bile acid metabolism affects host metabolism through the regulation of the cholesterol cholestero7-α hydroxylase (CYP7A1) and G protein-coupled receptor (TGR5, GPBAR1). 65,66 In CKD patients, circulate bile acids present a high level. When bile acids are perturbed, primary bile acid is decreased, and secondary bile acid, deoxycholic acid (DCA) are increased.
DCA is directly toxic to VSMCs. A clinical trial proved that high DCA is an independent risk factor for VC in CKD patients. 67 DCA can induce mineralisation and osteogenic differentiation of VSMCs by regulating endoplasmic reticulum (ER) stress. Emerging data implicate the role of ER stress as a new mechanism for VC. 68 Primary bile acids (BAs) increase the colon Retinoid-related orphan receptor)

ROR acids + Regulatory T (Treg) cells improve host susceptibility.
Tregs can modulate both innate and adaptive immune responses to suppress VC in CKD. 69 Researchers have discovered that the genome-wide biliary network interaction between intestinal bacteria and the host can control the host's immune homeostasis. 70 Besides,

F I G U R E 3
In patients with chronic kidney disease (CKD), the effect of gut microbiota-derived metabolites indoxyl sulfate (IS) and p-cresyl sulfate (PCS) on vascular smooth muscle cell (VSMC). IS down-regulated miR-29b and activated Wnt/β-catenin signalling to induce vascular activation. IS induced reactive oxygen species (ROS) also promote the activation of Nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) pathways, increase inflammation and damage the endothelial cell Bas and their nuclear receptor, such as farnesoid X receptor (FXR), are found in macrophages and vasculature. Importantly, activation of FXR was shown to reduce VC in the CKD model. 71

| Lipopolysaccharide (LPS)
LPS, also called endotoxin, is a specific type of gut microbiotaderived metabolite. It is a component of the outer membrane of gram-negative bacteria. If the intestinal barrier is impaired by bacterial translocation and gut microbiota disorder, LPS can enter the circulation through the gut wall and induce a systemic inflammatory response. 72,73 Evidence suggested that pro-inflammatory cytokines such as IL-6 induced VSMC mineralisation and osteogenic transition, playing a pivotal role in the progression of VC. 74 The research demonstrated that IL-18 also contributed to VC in pro-inflammatory conditions. 75

| Short-chain fatty acids (SCFAs)
Short-chain fatty acids (SCFAs) are another significant gut microbiota-derived metabolite. SCFAs are produced through the fermentation of dietary fibres by anaerobic gut bacteria in the caecum and proximal colon. 81 SCFAs are mainly composed of acetate, propionate and butyrate. Butyrate is primarily metabolized by the colonic commensal bacteria, which regulates cell growth and differentiation. 82 SCFAs can be used to maintain the gut barrier and inhibit pathogenic microbe proliferation in acidic PH conditions. 83 It is also mediated by G protein-coupled receptors GRP419 (FFAR3) and GRP43 (FFAR2), predominantly expressed in the immune cell. SCFAs also can contribute to improving vascular phenotypes. 84,85

| P OTENTIAL THER APEUTI C S TR ATEG IE S AG AIN S T VC
The intervention of gut microbiota-derived metabolites is a potential strategy to reduce calcification in CKD patients. Resveratrol is a dietary polyphenol compound. It has anti-inflammatory, antioxidative properties. Recently, studies also showed that resveratrol is a phytoalexin and scavenger for many free radicals. Resveratrol can decreased plasma TMAO and regulated sirtuin-1 (Sirt-1) and nuclear factor-E2associated factor 2 (Nrf2) signalling pathway to ameliorate VC vascular calcification. 86 Besides, Resveratrol can reverse the effect of IS. Also, F I G U R E 4 IS enhances the CpG hypermethylation of Klotho and epigenetic modification of klotho to promote the process of VC in CKD and induced methyltransferase-like (METTL14) -dependent N6-methyladenosine (m6A) regulated vascular calcification in VSMC. TMAO activates endothelial cell mitogenactivated protein kinase (MAPK) and vascular smooth muscle cell (VSMC) through nuclear factor-κB (NF-κB) pathway, leading to inflammatory gene expression and augmenting Ca2+ release from intracellular stores resveratrol was shown to inhibit IS-activated aryl hydrocarbon receptor (AHR) and regulate VE-cadherin and permeability-induced increase in Src activation. 87 Resveratrol could protect rat VMSCs against oxidative injury in VC. A range of studies has highlighted that interfering with some intestinal flora metabolites is beneficial for vascular suppression (Table 1). [88][89][90][91][92][93][94][95] S-equol is produced naturally in the gut by the bacterial biotransformation of daidzein, a soy isoflavone. A number of studies suggested that S-equol can inhibit vascular remodelling in the protective vascular system. S-equol can protect vasculature against cardiovascular diseases. Thus, S-equol should have an important role in the field of vascular research. 96 It is thus proposed that the use of oral iron supplements might improve the gut microbiome. However, iron supplements can lead to a decreased abundance of Lactobacillus and Bifidobacterium species. 97 Recently, many ongoing experiments have been focusing on the diet being the most significant modifiable factor capable of changing gut microbiota. 98 The development of diet (such as probiotics) as a primary management option to regulate the intestinal flora is pivotal to retard the development and progression of VC.

| CON CLUS ION
Gut microbiota is closely related to human health. The gut microbiota and metabolites have significant effects on CKD patients especially with the complication of VC. In this review, we focused on intestinal microbiota-derived metabolites such as uremic toxins, TMAO and SCFAs in VC. The multi-factorial mechanism of VC suggests that the intervention of gut microbiota-derived metabolites could be one of the important strategies to inhibit VC in CKD.
There are existing methods like dietary or pharmacological intervention affecting genomics, intestinal absorption, gut-kidney axis to reduce the production of harmful substances in the intestine and might further to improve VC. Thus, considering the significance of gut microbiota, future research should further explore the direct relationship between gut microbiota-derived metabolites and VC.  TA B L E 1 Change in gut microbiotaderived metabolites to treat VC