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Gut microbiota, key to unlocking the door of diabetic kidney disease

Abstract This review discusses the influence of gut microbiota dysbiosis on diabetic kidney disease through metabolite profile changes and immune and inflammatory mechanisms. We also elaborate on the mechanism of dysbiosis in the onset and development of other kidney diseases.


| INTRODUCTION
Gut microbiota, comprised of trillions of microbes, are of great significance to the human body. They coevolve with humans for mutual and beneficial coexistence and play vital roles in maintaining overall health and in preventing the development of diseases. 1 Gut microbiota are relatively stable and participate in various physiological processes in the human body. 2 They maintain host homeostasis in multiple ways, with their roles including modulating metabolism, maintaining the integrity of the intestinal barrier, inhibiting pathogenic bacterial invasion and regulating physiological and immune responses. 3 In contrast, dysbiosis, or a state of imbalance in gut microbial composition and function, is closely linked to the occurrence and development of many chronic diseases, both within and outside the gastrointestinal tract. 4 We have briefly summarized the known extraintestinal diseases related to gut microbiota ( Figure 1).
Diabetic kidney disease (DKD), or diabetic nephropathy (DN)-a deleterious complication of diabetes mellitus (DM)-is an important cause of chronic kidney disease, and its prevalence in China has risen rapidly in recent years. 5 Patients often experience complications related to cardiovascular and neurological diseases and are characterized by having an increased risk for the progression of end-stage renal disease (ESRD). This increases both patient mortality and the social burden associated with DKD. Despite the impact of this, the condition's pathogenic mechanism remains unclear. However, recent studies have indicated that gut microbiota dysbiosis is one of the most characteristic changes in DKD. 6 To provide a theoretical reference for DKD pathogenesis from the perspective of gut microbiota, we reviewed the role of gut microbiota in DKD and related mechanisms. Gut microbiota surface antigens stimulate dendritic cells to produce B-cell activating factor (BAFF), whose function is to promote the class switching of B cells to IgA+ B cells, whether the process is mediated by T cells. 7 Abnormally produced gut-derived IgA can leak into the bloodstream and lead to immunocomplex deposition in the kidney, thereby promoting the occurrence and development of IgAN.
Studies have also shown that five metabolites of gut microbiota are related to increased serum BAFF levels in patients with IgAN. 8 LN is an autoimmune disease characterized by the production of pathogenic autoantibodies. Studies have found that supplementing Lactobacillus spp. in LN mice can alleviate kidney disorders. Possible mechanisms behind this effect include supplementation improving intestinal barrier integrity, increasing IL-10, and reducing IL-6 and IgG2, thereby decreasing the incidence of kidney diseases. 9 Patients with MN had higher Fusobacteria and Proteobacteria but lower Firmicutes populations than those of individuals in the healthy control (HC) group. 10 Our research revealed that lipopolysaccharide (LPS)-producing bacteria were enriched in MN and DKD. Increased LPS leaks into the bloodstream through the damaged intestinal barrier, activates the NF-κB pathway and triggers the release of inflammatory factors, resulting in the aggravation of kidney damage. 11 In recent years, emerging studies have shown that gut microbiota affect CKD progression via uremic toxin production, interactions with chronic inflammation and immune response regulation. Gut microbiota dysbiosis and intestinal barrier destruction in CKD lead to bacteria, or their metabolites, entering the bloodstream. 12 Many previous studies have shown that metabolites produced by dysbiotic gut bacterial communities can destroy the intestinal mucosal barrier and cause kidney damage. 13,14 Current studies have revealed that traditional Chinese medicine aids in the management of kidney diseases. Traditional Chinese medicine can improve the intestinal barrier, regulate intestinal flora and inhibit microinflammation induced by gut-derived metabolites in order to exert its renoprotective effects. 15,16 Zheng et al identified the keystone microbiome markers in CKD. 17 As a result of in-depth study of the gut-kidney axis, diet therapy has become a hot topic in CKD treatment. 18 After evaluating the gut microbiota of kidney transplant donors and recipients, it was found that microflora similarity may be related to the prognosis of recipients after transplantation. 19 However, relevant studies have shown that gut microbiota dysbiosis and impaired intestinal barrier function exist in oxalosis-related CKD, but microbiota-targeted therapy cannot prevent its progression. 20 Therefore, the precise molecular mechanism of the gut microbiota in kidney diseases remains unclear.
Numerous studies have also revealed that enterotypical differences in gut microbiota exist between various kidney diseases, and the dominant bacteria in different kidney diseases overlap. Therefore, further study is needed regarding whether the virulence factors of these dominant bacteria differ. 21 3 | GUT MICROBIOTA, THE CENTRAL REGULATORS OF DKD 3.1 | Gut microbiota are altered in DM As early as 2010, it was found that type 2 diabetes mellitus (T2DM) was linked to gut microbiota alterations. 22 In recent years, emerging

SUMMARY AT A GLANCE
This review presents scientific evidence on the pathophysiologic links between gut microbiota and diabetic kidney disease (DKD), highlighting the influence of gut microbiota dysbiosis on DKD through metabolite profile changes and immunologic mechanisms.
evidence from experiments in both humans and mice suggest that the gut microbiota of patients with diabetes are altered significantly as compared to that of healthy individuals, 23 even in the pre-diabetes state. 24 As for the composition of gut microbiota, a higher Firmicutes/ Bacteroidetes ratio was observed in patients with T2DM, 23 which was reported to be positively correlated with increases in plasma glucose levels and the occurrence of metabolic disorders. 25 Patients with T2DM had higher abundances of Enterobacteriaceae, Collinsella, Streptococcus, Lactobacillus and Lachnospiraceae/Ruminococcus than those of healthy individuals. These bacteria may be involved in low-grade inflammation and can aggravate insulin resistance. 23,26 However, short-chain fatty acid (SCFA)-producing probiotics, including Bacteroides, Prevotella, Lachnospira, Roseburia and Faecalibacterium, were markedly depleted in DM as compared to those in healthy individuals. 26,27 Butyrate is an SCFA that acts as an energy resource for colonic epithelial cells. However, the examination of altered T2DM gut microbiota revealed the depletion of several butyrate-producing bacteria such as Akkermansia, Bacteroides and Faecalibacterium. These bacteria, and their metabolite, butyrate, help improve glucose metabolism. 28 Thus, depletion of the butyrate-producing bacteria mentioned above, and the resulting decreased levels of butyrate, may participate in DM pathogenesis. Members of the genus Blautia decreased significantly in Japanese patients with T2DM as compared to those in control subjects. Blautia was reported to be an SCFA producer, whose abundance was negatively correlated with haemoglobin A1c and fasting plasma glucose levels. 29 In summary, the alteration of DM gut microbiota usually manifests as a decreased beneficial bacteria and an increased harmful bacteria.
In the DM state, the functions of intestinal bacteria also change to some extent. It has been reported that the profiles of gut microbial functions, such as tyrosine and butyrate production, are altered in T2DM as compared to those in controls. 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including the insulin signalling pathway and glycolysis/gluconeogenesis, were upregulated in patients with DM. 29 Many antidiabetic drugs can prevent DM progression, or alleviate symptoms by regulating the gut microbiota and reducing endotoxin-and microbe-mediated inflammation. Metformin can treat DM by regulating the structure and function of gut microbiota. 31 After metformin use, positive connections among microbial genera are strengthened, and abundances of Escherichia, Bifidobacterium and A. muciniphila increased, whereas that of Intestinibacter decreased. Bifidobacteriaceae can act as a probiotic to improve endotoxemia and glucose tolerance, 32 and A. muciniphila can relieve T2DM by reducing oxidative stress and inflammation. 33 Dapagliflozin can also improve DM by adjusting gut microflora. Using an Sprague-Dawley(SD) rat diabetic model, Mei Yang found that, while similar blood glucose-lowering effects were observed after treatments with dapagliflozin and metformin, the dominant enterotypes in the dapagliflozin group were Ruminococcaceae, whereas the dominant types in the metformin group were both Ruminococcaceae and Muribaculaceae. Dapagliflozin increased Desulfovibrionaceae (Proteobacteria), reduced Bacteroidetes/Proteobacteria and decreased the abundance of Actinobacteria and Spirochaetes, compared with those of metformin treatment. 32 Interestingly, a study performed on Zucker diabetic fatty rats found that Roux-en-Y gastric bypass surgery (RYGB) and sleeve gastrectomy (SG) induced similar glucose profile improvement, and increased microbiota diversity, compared with those of sham-operated groups. However, RYGB resulted in a higher abundance of Proteobacteria, Gammaproteobacteria, Betaproteobacteria, Fusobacteria and Clostridium than that of control groups. SG, on the other hand, resulted in more abundant Actinobacteria than that of the control groups. 34 Clostridium and Fusobacterium were also negatively correlated with serum insulin levels. 35 Overall, gut microbiota affect host metabolism, the state of which is also determined by many other intrinsic and extrinsic factors.  (Table 1).

| Changes of gut microbiota composition in patients with DKD
Like DM, DKD is associated with abnormal gut microbiota. A study from China recruited DNs diagnosed using renal biopsy, T2DMs without renal diseases (T2DM) and HCs. The research revealed that faecal samples from patients with DM contained higher abundances of Firmicutes and Proteobacteria than did those of HCs and T2DMs.
According to the authors, Proteobacteria can increase the level of circulating LPS, resulting in persistent inflammation. The richness of gut microbiota, based on operational taxonomic unit (OTUs), was significantly lower in DNs than in T2DMs, and there were obvious disparities between the microbiota compositions of patients with HC, DM and DN. Escherichia-Shigella was significantly increased in the DN group, whereas Prevotella_9 decreased, and the richness of the two genera can allow for effective distinguishment between DN and DM subjects. 36 A study on CKD suggested that the abundances of Escherichia-Shigella spp. were positively related to disease progression. The genera Escherichia-Shigella were enriched in the faecal samples of patients with advanced CKD and played a role in renal impairment by generating additional indoxyl sulphate (IS). 37 Escherichia-Shigella are also conditional pathogenic bacteria that can penetrate the intestinal epithelial barrier and aggravate gut leakiness. 36 In a C57BL/6 mouse model, with DN induced by streptozotocin (STZ) and a high-fat diet, some differences in gut microbiota composition existed between mice with severe and mild proteinuria (SP and MP, respectively). For example, Allobaculum was positively related to body weight and blood sugar, and the genus Anaerosporobacter was positively related to 24 h urine protein. Conversely, in mice with MP, Blautia was found to be negatively correlated with 24 h urine protein. Intriguingly, after the mice received faecal microbiota transplants (FMT) from DKD subjects with SP and MP, FMT-SP mice experienced more severe proteinuria after STZ injection than did FMT-MP mice. 38 Collectively, these findings indicate that gut microorganisms are linked to DKD development.

| Gut microbiota aid in DKD diagnosis
Our previous study found that patients with DKD presented with higher richness of Bacteroidetes and lower Firmicutes/Bacteroidetes ratios as compared with those of patients with MN.
Peptostreptococcus, a potential pathogen, is one of the genera enriched in patients with DKD. There were significant differences in gut microbiota composition between patients with DKD and those with MN, and microbiota profiles based on four OTUs could distinguish MN from DKD with high accuracy. As for microbial functions, modules related to the metabolic pathways of some amino acids, carbohydrates, energy and lipids were increased in the gut microbiota of DKDs. 11 Therefore, gut microbiota may also aid in the diagnosis, and differential diagnosis, of DKD.

| Gut microbiota provide potential targets for DKD treatment
To date, studies have focused on the effects of traditional Chinese In SD rats with STZ-induced DM, plasma acetate concentration and abnormal gut flora increased, accompanied by RAS system activation and renal function impairment. Nonetheless, these phenomenon can be reversed using broad-spectrum antibiotics, indicating that the presence of abnormal intestinal flora in DM can lead to renal injury by activating the RAS system through metabolites. 44 Tryptophan can be metabolized into kynurenine (Kyn) by tryptophan 2,3-dioxygenase (TDO) and indoleamine (2,3)-dioxygenase (IDO). 45 The kynurenine pathway is influenced by gut microbiota. 46 Kyn, and its derivatives, can promote vascular endothelial dysfunction and atherosclerosis in patients with CKD. 47 Kyn was found to be positively correlated with the urinary albumin-to-creatinine ratio (UACR) and negatively correlated with estimated glomerular filtration rates (eGFR) in DKD. However, the molecular mechanisms behind these relationships remain unclear. 48

| Gut microbiota increase intestinal permeability and promote inflammation in DKD
Additionally, gut microbiota can promote the occurrence of DKD by inducing or aggravating inflammation. Growing evidence has shown that, during DM and DKD, the intestinal mucosal barrier becomes more permeable owing to structural and functional abnormalities. Overactivation of C5 can induce gut microbiota dysbiosis and reduce SCFA production by intestinal microbiota. C5 can promote DKD by activating the STAT3 pathway and inducing inflammation in the kidneys, but SCFAs can partially offset its injurious effects. 53 A gut microbiome-immune axis exists in the human body. Gut microbiota-derived metabolites can pass through the intestinal epithelium and accumulate in the circulatory system. They can also be recognized by the immune system and cause a wide variety of consequences in the body through various pathways. 54 Intestinal flora, such as Firmicutes, Proteobacteria, Clostridium asparagiforme, Escherichia fergusonii and Desulfovibrio desulfuricans, can produce trimethylamine N-oxide (TMAO). 55 Escherichia coli can metabolize tryptophan into indole, which can, in turn, be converted into IS and Kyn in the liver. 56 Bacteroides, Bifidobacterium, Lactobacillus and Clostridium are all related to p-cresyl sulphate (PCS) generation. 57 PS, TMAO, Kyn, IS and PCS are uremic toxins that increase in the DKD state. 43,48,58 It has been reported that their accumulation continuously stimulates the immune system, which could result in increased inflammatory factor production and renal damage. 52,[59][60][61] In conclusion, there are various mechanisms by which bacteria affect the progression of DKD. F I G U R E 2 The mechanism of gut microbiota on the kidney. The gut microbiota itself and its metabolites can enter the Interstitial fluid through the increased permeability of the intestinal mucosa. Macrophages can phagocytose bacteria and release inflammatory factors. Metabolites and inflammatory factors enter the blood, reach the kidneys and cause damage to endothelial cells and podocytes through the activation of the RAS system, inflammation and oxidative stress. phenyl sulphate (PS); Trimethylamine-N-oxide (TMAO); Kynurenine (Kyn); Indoxyl Sulphate (IS); pcresol sulphate (PCS); Interlrukin 1 (IL-1); Interlrukin 6 (IL-6); Tumour Necrosis Factor (TNFα); Renin-angiotensin System (RAS)