{'fraction_non_alphanumeric': 0.04030196988164982, 'fraction_numerical': 0.025360751779184523, 'mean_word_length': 5.344225492641557, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Gut Microbiome Characteristics in IgA Nephropathy: Qualitative and Quantitative Analysis from Observational Studies

Background Recent data indicate the importance of gut-kidney axis in the pathogenesis of Immunoglobulin A nephropathy (IgAN). Growing evidence suggests the alterations of diversity and composition of gut microbiome among patients with IgAN, however, the details are not yet fully understood. Methods Eligible studies comparing the gut microbiome between patients with IgAN and non-IgAN individuals were systematically searched from PubMed, Embase, Web of Science, Cochrane Library, China National Knowledge Infrastructure, and ClinicalTrials.gov. The primary outcomes were alpha- and beta-diversity, and the differences in gut microbiota composition between patients with IgAN and non-IgAN persons. Qualitative analysis and meta-analysis were performed according to available data. Results Eleven cross-sectional studies, including 409 patients with IgAN and 243 healthy controls, were enrolled. No significant differences in the diversity and enrichment of gut bacteria were found between IgAN and healthy individuals, whereas the beta-diversity consistently showed significant microbial dissimilarities among the two groups. Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia were the dominant phyla, however, no significant differences were found between IgAN patients and healthy controls at the phylum level. The genera, Streptococcus and Paraprevotella showed a higher proportion in patients with IgAN compared to healthy individuals, whereas Fusicatenibacter showed a lower abundance according to meta-analysis. Qualitative analyses suggested that Escherichia-Shigella might be increased in IgAN patients; the genera, Clostridium, Prevotella 9,and Roseburia, members of Ruminococcaceae and Lachnospiraceae families, were likely to have decreased abundances in patients with IgAN compared to healthy individuals. Conclusion Gut microbiota dysbiosis was demonstrated in IgAN, which might be involved in the pathogenesis of IgAN. Further studies are needed to confirm the findings of this study, due to the substantial heterogeneity. Systematic Review Registration https://www.crd.york.ac.uk/prospero/, identifier PROSPERO (CRD42022304034).


INTRODUCTION
Immunoglobulin A nephropathy (IgAN) is the most common immune-associated primary glomerulonephritis worldwide, characterized by the deposition of IgA, specifically, galactosedeficient IgA1 (Gd-IgA1), in the glomerular mesangium (Monteiro and Berthelot, 2021). Up to 40% of patients ultimately progress to end-stage kidney disease (Selvaskandan et al., 2022). Although the underlying pathogenesis has not been completely elucidated, the "multi-hit-hypothesis" is a widely accepted immunological interpretation for IgAN, that is, the overproduction of polymeric Gd-IgA1, anti-Gd-IgA1 and the formation of circulating immune complex containing Gd-IgA1, the mesangial deposition of Gd-IgA1 immunocomplex, and subsequent inflammation and fibrosis (Knoppova et al., 2021). Mesangial Gd-IgA1 deposits resemble mucosal IgA, mostly produced by mucosal B lymphocytes located in the Peyer's patches, have been considered as an important pathogenetic factor of IgAN (Ohyama et al., 2021). Increasing evidence suggest a pivotal role of mucosal immunity in IgAN, which can be triggered by antigenic stimulation from the commensal microflora, more specifically, the gut microbiota dysbiosis and subsequent IgA production (Ichinohe et al., 2011). Intestinal mucosal hyperresponsiveness and abnormal production of Gd-IgA1 have been found in patients with IgAN, which was associated with specific fecal microbiota (Sallustio et al., 2021). The targetedrelease of the glucocorticosteroid budesonide targeting excessive intestinal mucosal immune responses via releasing the drug to Peyer's patches, showed a significant reduction of proteinuria compared to placebo in patients with IgAN (Fellström et al., 2017). Depleting of fecal microbiota by a broad-spectrum antibiotic prevented human IgA1 mesangial deposition, glomerular inflammation, and the development of proteinuria in a humanized mice model of IgAN . Patients with IgAN achieved partial remission after intensive fecal microbiota transplantation regularly for 6 months (Zhao et al., 2021). This evidence indicates the importance of gut-kidney axis in IgAN. Since the first human study by De Angelis et al. reporting the gut dysbiosis of IgAN (De Angelis et al., 2014), a growing number of studies have focused on the diversity and composition of gut microbiome in IgAN, however, the details are not yet fully understood. In addition, the gut microbiome is dynamic and differs in different populations, ages, sexes, seasonal variations, geographies, ethnicities, diets, and lifestyles (Gupta et al., 2017;Koliada et al., 2020;Koliada et al., 2021). Hence, this systematic review was conducted to comprehensively assess the diversity and abundance of the gut microbiome in patients with IgAN compared with non-IgAN individuals.

Design and Registration
This systematic review was registered prospectively at PROSPERO (CRD42022304034) and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement (Supplementary Table S1) (Page et al., 2021).

Search Strategy
Eligible studies comparing the gut microbiome between patients with IgAN and non-IgAN individuals before March 1, 2022, were systematically searched from the following databases and registers: PubMed, Embase, Web of Science (WOS), Cochrane Library, China National Knowledge Infrastructure (CNKI), and ClinicalTrials.gov. A combination of MeSH with free text search was applied using the keywords gut microbiome, IgAN, and their associated subject words. The specific retrieval strategies are detailed in Supplementary Table S2.

Eligible Criteria and Outcome Measures
Studies eligible for inclusion were original research that compared the diversity and composition of gut microbiome between biopsy-proven IgAN patients and healthy controls or non-IgAN persons. Exclusion criteria were as follows: (i) study included secondary IgAN or end-stage renal disease; (ii) unavailable data of gut microbiome.
The primary outcomes were as follows: (i) alpha-and betadiversity; (ii) gut microbiome composition. Alpha diversity was evaluated to describe the community richness and diversity of gut microbiota. The Chao1 index, ACE index, and number of observed species/operational taxonomic units (OTUs) are estimated for microbial richness, whereas the Shannon and Simpson indices are calculated for microbial diversity. Beta diversity is a comparative analysis of microbial composition differences between patients with IgAN and controls. The secondary outcome of interest was the association of microbial signature and characteristics of IgAN.
The following data were extracted: first author, year of publication, location, design of study, baseline characteristics for all cohorts (sample size, age, sex, estimated glomerular filtration rate [eGFR] or serum creatinine level, urinary protein excretion), DNA extraction method, sequencing platform, bioinformatics pipelines, and outcomes.

Quality Assessment
The Newcastle-Ottawa Scale (NOS) for the case-control study was adopted for quality assessment (Wells et al.). This scale consists of three dimensions with eight items, including selection, comparability, and exposure. The selection module contains four questions, including adequate definition of cases, representativeness of cases, selection of controls, and definition of controls. Comparability focuses on the controls of confounding factors. As the assessment of exposure, ascertainment of exposure, same method of ascertainment, and non-response rate should be answered. A maximum of nine scores can be awarded for a study, including one score for each item of the selection and exposure categories, and a maximum of two scores for comparability. A total score of ≥ 7 was considered as high quality (Suriyong et al., 2022).

Data Synthesis and Analysis
For quantitative synthesis, the differences in bacterial diversity indices and relative abundances between patients with IgAN and non-IgAN individuals were estimated using standardized mean difference (SMD) and 95% confidence intervals (CIs). Heterogeneity was assessed utilizing Cochran I 2 test, which was considered significant when P < 0.10 or I 2 > 50% (Higgins et al., 2003). A fixed-effects model or a random-effects model meta-analysis was performed to calculate pooled SMD based on heterogeneity. Sensitivity analysis was conducted by omitting each study in turn.
For qualitative data analysis, the number of studies reporting statistically significant differences between IgAN group and control group for each prespecified outcome were recorded. We also carried out a funnel plot to compare the proportion of studies reporting significantly higher or lower relative abundances of specific intestinal bacteria, using the funnelR script and calculating a binomial Poisson distribution score2 with significant levels set at 50%, 80%, and 95% CIs (Woodall et al., 2022). Considering that several factors are associated with human gut microbiota composition, such as age, sex, seasonal variation, geography, ethnicity, diet, and lifestyle (Gupta et al., 2017;Koliada et al., 2020;Koliada et al., 2021), we further performed sensitivity analyses of matched confounding factors for the quantitative synthesis and qualitative analysis. Statistical analysis and graphical presentation were implemented by Stata (version 14.0), RStudio (Open source edition 2021.09.2), and GraphPad Prism (version 8.0).
All the studies consecutively included biopsy-proven IgAN, and at least matched age and sex between IgAN group and control group. Six studies were awarded NOS scores of nine (De Angelis et al., 2014;Dong et al., 2020;Hu et al., 2020;Chai et al., 2021;He et al., 2021;Weng et al., 2021). Five studies were given eight scores, because they did not report the details of recruitment for controls (Zhong et al., 2020;Sugurmar et al., 2021;Tian et al., 2021;Wu et al., 2021;Ya et al., 2021). Two independent cohorts were set in the study by He et al., which were the cohorts matched with age, sex, and BMI, and the other cohorts recruited without selection (He et al., 2021). NOS scores were assessed as 9 and 7, respectively.

Alpha-and Beta-Diversity
At the individual study level, 10 studies reported the observed species or OTUs. Compared with healthy controls, only one study showed an increased observed species in patients with IgAN (Tian et al., 2021), three studies reported significant decreased OTUs (De Angelis et al., 2014;Dong et al., 2020;Hu et al., 2020), and unchanged OTUs were reported in five studies (Chai et al., 2021;Sugurmar et al., 2021;Weng et al., 2021;Wu et al., 2021;Suriyong et al., 2022). The ACE index was found to be significantly higher in IgAN than in control in two studies (Dong et al., 2020;Tian et al., 2021), lower in one study (Hu et al., 2020), and not significantly changed in three studies (Zhong et al., 2020;Wu et al., 2021;Weng et al., 2021). One study indicated a significant increase of the Chao1 index in patients with IgAN compared with healthy controls (Dong et al., 2020), three studies showed a significant decrease (De Angelis et al., 2014;Hu et al., 2020;Weng et al., 2021), and five studies with six cohorts reported no significant differences (Zhong et al., 2020;Chai et al., 2021;Wu et al., 2021;He et al., 2021;Tian et al., 2021). The Shannon index, reported in nine studies, was found to be significantly higher in IgAN patients compared to healthy controls in one study (Weng et al., 2021), lower in three studies (De Angelis et al., 2014;Chai et al., 2021;Wu et al., 2021), and not significantly changed in five studies (Dong et al., 2020;Hu et al., 2020;Zhong et al., 2020;Sugurmar et al., 2021;Tian et al., 2021). As for the Simpson index, five of six studies showed no significant differences between patients with IgAN and healthy controls (Chai et al., 2021;Dong et al., 2020;Tian et al., 2021;Weng et al., 2021;Zhong et al., 2020), a significant increase in the IgAN group was observed by Wu et al. (2021) (Figure 2A).

Microbial Composition at Phylum Level
All the included studies reported the intestinal microbial composition at the phylum level ( Figure 3A). The relative abundances of six phyla accounted for more than 99% of the total community, including Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia.
For Firmicutes, only one study observed a significantly increased abundance in patients with IgAN (Weng et al., 2021), two studies reported significantly decreased proportions (Zhong et al., 2020;Ya et al., 2021), and eight studies showed no significant differences between IgAN and healthy individuals ( Figure 3A). Regarding Bacteroidetes, no significant differences in the relative abundance were found between IgAN group and control group in eight studies, two studies observed significantly higher abundances in IgAN (Zhong et al., 2020;Weng et al., 2021), whereas one study reported a significantly lower proportion of abundance in patients with IgAN (Wu et al., 2021). Actinobacteria (Chai et al., 2021;Weng et al., 2021) and Proteobacteria (Dong et al., 2020;Wu et al., 2021) were reported to have significantly higher abundances in IgAN groups compared to the control groups in two studies, whereas a significantly lower proportion of relative abundance was found in IgAN patients in one study each (Zhong et al., 2020;Weng et al., 2021), no statistically significant differences were observed in the remaining studies. The relative abundances of Fusobacteria were found to be significantly higher among IgAN patients in four studies (Hu et al., 2020;Zhong et al., 2020;Sugurmar et al., 2021;Tian et al., 2021), whereas a lower proportion of abundance in IgAN group was reported in one study (Dong et al., 2020). For Verrucomicrobia, 10 studies did not find significant differences between sufferers of IgAN and healthy controls, and one study observed a higher abundance in IgAN group (Ya et al., 2021).
We also compared the reported average abundance of each phylum at the study level, using paired t-test and Wilcoxon ranksum test. Proteobacteria was found to have a higher average proportion of abundance in patients with IgAN compared to healthy persons, and Bacteroidetes showed a lower abundance in IgAN, however, statistical differences were not reached ( Figures 3C, D).

Microbial Composition at Genus Level
All the included studies reported the data of relative abundance between IgAN and healthy persons at the genus level. A total of 76 bacteria showed significant differences between the two groups. Escherichia-Shigella showed a higher relative abundance in patients with IgAN than in healthy persons in four studies (Dong et al., 2020;Hu et al., 2020;Ya et al., 2021;Zhong et al., 2020). Five genera, including Clostridium, Prevotella 9, Roseburia, members of Ruminococcaceae and Lachnospiraceae families, were found to be significantly lower in IgAN groups in at least three studies ( Figure 4A; Supplementary Table S3).
Opposite results were reported in five genera, including Streptococcus, Bacteroides, Megamonas, Bifidobacterium, and Enterococcus. The proportion of studies showed significantly changed abundances of each bacterium and the total number of reported studies did not exceed the upper 95%CI in any genus, according to the funnel plot ( Figure 4B). Available data from four studies were used for meta-analysis, Streptococcus (SMD=0.47, 95%CI 0.001, 0.94) and Paraprevotella (SMD=0.35, 95%CI 0.08, 0.62) were found to have higher abundances in patients with IgAN than in healthy individuals; the genus Fusicatenibacter was found to have a lower proportion among IgAN sufferers than among healthy individuals (SMD=-0.43, 95%CI -0.84, -0.03) ( Figure 5). Although the direction of estimates did not change, the statistical differences disappeared in sensitivity analyses for the three genera (Supplementary Figure S2).

Secondary Outcome
Spearman correlation between fecal microbiota and clinical parameters of IgAN was reported in two studies (Dong et al., 2020;Hu et al., 2020). The genera, Blautia, Veillonella, Anaerostipes, and Bifidobacterium were found to have a positive association with eGFR, but Escherichia-Shigella, Sneathia, Plesiomonas, and Defluviitaleaceae were negatively correlated with eGFR. The enrichments of Escherichia-Shigella, Sneathia, Parabacteroide, Defluviitaleaceae, and Anaerotruncus were related to higher urinary protein excretion in IgAN, whereas Rectale was negatively correlated with urinary protein.

Sensitivity Analysis of Matched Confounding Factors
All the included studies have matched age and sex between patients with IgAN and healthy controls, and most studies (10/ 11) were conducted in Asia. Seven studies reported the timeline for recruiting subjects across summer and winter, and four studies did not describe the specific time of fecal sample collection. Therefore, we performed an analysis of intestinal flora in diet and lifestyle matched cohorts based on four studies (Hu et al., 2020;Zhong et al., 2020;Sugurmar et al., 2021;Wu et al., 2021). Non-significant differences in alphadiversity and significant dissimilarities of gut bacteria between IgAN and healthy individuals were found (Supplementary Figures S3A, B). There were no significant differences in intestinal bacteria abundances between IgAN and healthy persons at the phylum level ( Supplementary Figures S3C-F).
Escherichia-Shigella showed a significantly higher abundance in patients with IgAN than in healthy controls in two studies (Hu et al., 2020;Zhong et al., 2020). Three genera, including Prevotella 9, members of Ruminococcaceae family, and Coprococcus were found to be significantly lower in IgAN in at least two studies (Supplementary Figure S3G). These results were consistent with the findings from the qualitative and quantitative analyses of all the included 11 studies.

DISCUSSION
The gut-kidney axis comes central to the pathogenesis in IgAN, and gut dysbiosis has been proven closely associated with IgAN (Coppo, 2018). This is the first systematic review comparing the differences in gut microbiome between patients with IgAN and healthy individuals involving 11 studies and 652 participants. Although we did not find significant differences in the diversity and enrichment of intestinal bacteria according to the alpha-diversity indexes of OTUs, ACE, Chao1, Shannon, and Simpon, the betadiversity consistently showed significant microbial dissimilarities between IgAN and healthy persons, indicating gut dysbiosis of IgAN. More specifically, at the phylum level, we found an increase of Proteobacteria, but a decrease of Bacteroidetes among patients with IgAN, which is consistent with the subgingival microbiome of IgAN sufferers (Cao et al., 2018), although the difference was not statistically significant. At the genus level, Streptococcus and paraprevotella showed a higher proportion in cases of IgAN compared to healthy individuals, whereas, Fusicatenibacter showed a lower proportion according to meta-analysis. Qualitative analyses suggested that Escherichia-Shigella might be increased in IgAN patients because four studies reported higher relative abundances and three studies showed no significant differences when compared with healthy controls. The genera, Clostridium, Prevotella 9, Roseburia, members of Ruminococcaceae and Lachnospiraceae families, were found to be decreased in patients with IgAN in at least three studies, but no reports of increased abundances compared to healthy individuals. The enrichment of Proteobacteria is considered a potential microbial diagnostic signature of dysbiosis and increases the risk of host diseases (Shin et al., 2015). Proteobacteria was also found to have a higher abundance of circulating microbiome profile in patients with chronic kidney disease (CKD) than healthy controls and correlated inversely with eGFR (Shah et al., 2019). Additionally, the abundances of Proteobacteria were depleted in two cases with IgAN, which showed alleviation of proteinuria after fecal microbiota transplantation (Zhao et al., 2021). The abundances of Streptococci, Paraprevotella, and Escherichia-Shigella probably increased in gut microbiota of patients with IgAN according to our meta-analysis and qualitative analysis. Streptococcal antigens binding with IgA were found to be deposited in renal tissue of patients with IgAN (Schmitt et al., 2010); additionally, IgAN is often induced or aggravated after suffering upper respiratory tract infection or gastrointestinal tract infections. This evidence indicated the potentially harmful role of Streptococcus in the pathological mechanism of IgAN. Escherichia-Shigella is a gram-negative, oxidase-negative, rod-shaped bacterium from the Proteobacteria phylum, which can result in intestinal infection under conditions van den Beld et al., 2022). An increase of Escherichia-Shigella may cause local infection and activate gut immune responses, leading to the excessive synthesis of IgA (Tao et al., 2019). Moreover, patients with IgAN enriched with Escherichia-Shigella in the gut had higher urinary albumin excretion rate, worse hematuria, and lower eGFR (Hu et al., 2020;Zhong et al., 2020). The increase of Escherichia-Shigella also exacerbated gut leakiness by reducing butyrate biosynthesis and increasing oxidative stress to penetrate the intestinal epithelial barrier (Croxen et al., 2013). Paraprevotella was also found to be enriched in the fecal samples of patients with CKD, and to be superior in discriminating CKD from the healthy individuals . Many intestinal bacteria have been shown to be associated with the production and metabolism of various short-chain fatty acids (SCFA). SCFAs have been documented as having important roles in maintaining health, such as acting as a nutrient source of the gut epithelium, protecting the intestinal mucosal barrier, and inhibiting inflammation (Zhang et al., 2015). SCFAs, especially acetate and butyrate, were found to inhibit the proliferation of glomerular mesangial cells and oxidative stress induced by lipopolysaccharides and high glucose in vitro (Huang et al., 2017). Patients with IgAN and CKD were found to have decreased levels of SCFAs (Felizardo et al., 2019). Some genera with lower abundances in cases of IgAN compared with healthy controls, according to our results, including Clostridium, Prevotella 9, Roseburia, members of Ruminococcaceae and Lachnospiraceae families, were confirmed as important bacteria involving the production and metabolism of SCFAs (Baxter et al., 2019;Fei et al., 2020;Sun et al., 2022;Xie et al., 2021). Barnesiella is one of the most abundant genera that has anti-inflammatory protective effects, which were also decreased in patients with IgAN, according to our results (Weiss et al., 2014). A chimeric fusion of Fc segment of human IgG1 and AK183, which is an IgA protease from the genus Clostridium, was found to promote renal clearance of IgA and obliteration of blood IgA and remove C3 deposits in the glomerulus (Xie et al., 2022). Therefore, the findings of this study demonstrated the gut dysbiosis of IgAN, characterized by an increase of pathogenic bacteria and a decrease of beneficial bacteria, especially the SCFA-associated species.
The strength of this systematic review is that we conducted a comprehensive search to ensure all relevant studies reporting the gut microbiome between IgAN and non-IgAN individuals. All the included studies adopted high throughput 16S sequencing to analyze the composition of intestinal flora. Additionally, patients who took medications that might result in modifications of gut microbiome before fecal specimen collection were excluded from all the selected studies. All the included studies had a NOS score of ≥ 8, suggesting high methodological quality.
Several limitations should be considered. First, the sample sizes of included studies are relatively small, which might lead to more uncertainty and less precision in the findings. Second, meta-analyses can only be performed among five studies, because some studies did not report sufficient data on the diversity and relative abundance of the gut microbiome for quantitative synthesis, therefore, the results of this review might not be able to fully reflect the current evidence. Third, we did not perform a subgroup analysis stratified by ethnicity and pathological severity, due to the limited data, although these factors may influence the composition of intestinal flora.

CONCLUSIONS
In conclusion, this study presents a comprehensive analysis of the intestinal microbiota in patients with IgAN, and showed significant differences in gut bacterial composition between IgAN and healthy individuals. Due to the potential limitation and substantial heterogeneity, high-quality studies with large sample sizes are needed to confirm the detailed gut dysbiosis of IgAN.

AUTHOR CONTRIBUTIONS
LS and YW designed this study; SH and YL conducted the literature search, data extraction, quality assessment; SH completed data analyses and manuscript writing. All authors approved the final manuscript.