Original Investigation The Gut and Blood Microbiome in IgA Nephropathy and Healthy Controls Neal B. Shah ,1 Sagar U. Nigwekar ,2 Sahir Kalim,2 Benjamin Lelouvier,3 Florence Servant ,3 Monika Dalal,1 Scott Krinsky,2 Alessio Fasano ,4 Nina Tolkoff-Rubin,2 and Andrew S. Allegretti 2 Key Points A higher microbiome load, possibly originating from different body sites, may be playing a pathogenic role in IgA nephropathy. Several microbiome taxonomic differences between patients with IgA nephropathy and healthy controls are observed in blood and stool. Striking differences between the blood and gut microbiome confirm that the blood microbiome does not directly reflect the gut microbiome. Abstract Background IgA nephropathy (IgAN) has been associated with gut dysbiosis, intestinal membrane disruption, and translocation of bacteria into blood. Our study aimed to understand the association of gut and blood microbiomes in patients with IgAN in relation to healthy controls. Methods We conducted a case-control study with 20 patients with progressive IgAN, matched with 20 healthy controls, and analyzed bacterial DNA quantitatively in blood using 16S PCR and qualitatively in blood and stool using 16S metagenomic sequencing. We conducted between-group comparisons as well as comparisons between the blood and gut microbiomes. Results Higher median 16S bacterial DNA in blood was found in the IgAN group compared with the healthy controls group (7410 versus 6030 16S rDNA copies/ml blood, P50.04). a-andb-Diversity in both blood and stool was largely similar between the IgAN and healthy groups. In patients with IgAN, in comparison with healthy controls, we observed higher proportions of the class Coriobacteriia and species of the genera Legionella, Enhydrobacter,andParabacteroides in blood, and species of the genera Bacteroides, Escherichia-Shigella,andsome Ruminococcus in stool. Taxa distribution were markedly different between the blood and stool samples of each subject in both IgAN and healthy groups, without any significant correlation between corresponding gut and blood phyla. Conclusions Important bacterial taxonomic differences, quantitatively in blood and qualitatively in both blood and stool samples, that were detected between IgAN and healthy groups warrant further investigation into their roles in the pathogenesis of IgAN. Although gut bacterial translocation into blood may be one of the potential sources of the blood microbiome, marked taxonomic differences between gut and blood samples in each subject in both groups confirms that the blood microbiome does not directly reflect the gut microbiome. Further research is needed into other possible sites of origin and internal regulation of the blood microbiome. KIDNEY360 2: 1261–1274, 2021. doi: https://doi.org/10.34067/KID.0000132021 Introduction poorly galactosylated IgA1 in susceptible hosts, result- IgA nephropathy (IgAN) is the most common primary ing in glomerular mesangium immune complex depo- glomerulopathy and is characterized by deposition of sition, thus eliciting inflammation and tissue damage IgA antibodies, usually in the kidney mesangium (1). (2). A genome-wide association study showed that Although the exact pathogenesis remains unclear, genes involved in IgAN were associated with the ability antigens are believed to stimulate the production of of the gut-associated lymphoid tissue to regulate 1Division of Hospital Medicine, Department of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 2Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 3Vaiomer SAS, Labege, France 4Division of Pediatric Gastroenterology and Nutrition, Center for Celiac Research, Massachusetts General Hospital for Children, Boston, Massachusetts Correspondence: Dr. Neal B. Shah, Department of Medicine, Division of Hospital Medicine, Johns Hopkins Bayview Medical Center, Room 260, MFL East Tower, 5200 Eastern Avenue, Baltimore, MD 21224. E-mail: [email protected]. www.kidney360.org Vol 2 August, 2021 Copyright # 2021 by the American Society of Nephrology 1261 1262 KIDNEY360 intestinal pathogens and maintain integrity of the intestinal Materials and Methods barrier (3). These results have generated interest in the Study Design association and role of gut microbes in IgAN. We conducted a case-control study involving testing of the Previous gut microbiome studies have shown that the gut blood and stool microbiome of 20 patients with IgAN and 20 microbiome plays a vital role in host nutrition and develop- healthy control subjects. The study was approved by our ment of the immune system (4,5). This gut microbiome tends Partners Institutional Review Board and adhered to the to become imbalanced (dysbiotic) in various disease states, Declaration of Helsinki. including CKD (6,7). Gut dysbiosis associated with disrup- tion of the intestinal membrane barrier, resulting in translo- Enrollment of Study Participants cation of gut bacteria and toxins into blood, has been We recruited 20 adult patients in each group, aged 18–65 observed in CKD (8,9). Strong evidence of the gut-renal axis years, who were enrolled in our hospital electronic medical has been recently reported to be associated with the record system (Figure 1). Patients with IgAN were identified pathogenesis of IgAN (10). As a major Ig of the gut mucosal by reviewing kidney biopsy specimen reports and patient immune system, IgA in its secretory form plays a crucial role charts of individuals followed at Massachusetts General fl fi in controlling mucosal in ammation by linking to speci c Hospital. Participants with IgAN had biopsy sample–proven gut microbiota (11). A recent study by De Angelis et al. (12) IgAN with progressive disease at various stages, had an fi has shown signi cant differences in gut microbiota between eGFR of $15 ml/min using the Chronic Kidney Disease patients with IgAN and healthy subjects, with a higher Epidemiology Collaboration formula (15), were not on any proportion of species and genera of the families Rumino- oral or systemic immunosuppressants, and had never coccaceae, Lachnospiraceae, Streptococcaceae, and others received dialysis or a transplant. Healthy controls were identified in patients with IgAN. Subsequent gut micro- frequency matched by age and sex. They were recruited biome studies in a Chinese population with IgAN have primarily via advertisement of the study using an online additionally noted a higher prevalence of the genera platform named “Rally,” which is approved by the institu- Escherichia-Shigella and Bacteroides in stool when compared tional review board to foster collaboration between public with healthy controls (13,14). We were interested in under- and the research community. We excluded subjects with standing if such microbiota may be mediating their patho- diagnosed diabetes, any malignancy, inflammatory bowel genic effects by translocating into blood via a disrupted disease, history of colon surgery, or intake of antibiotics or intestinal barrier. A study simultaneously analyzing both probiotics within 30 days of the study visit. Dietary assess- blood and gut microbiome in IgAN has not been conducted ment was not performed due to unclear effects of different previously. We hypothesized that the blood microbiome in IgAN will reflect dysbiosis analogous to the gut, and differ foods on the microbiome. Notably, previous studies have from healthy controls. Our study aimed at comparing the demonstrated that the overall composition of the gut blood bacterial quantity of 16S ribosomal DNA (16S rDNA) microbiome at phylum level remains relatively stable despite and blood and stool metagenomic qualitative profiles some diurnal variations (16). between patients with IgAN and healthy controls. By analyzing human stool and blood microbiomes simulta- Study Visit and Sample Collection neously for the first time in IgAN, we also compared The study visit involved obtaining written informed concurrent stool and blood microbiome samples to better consent (per Recommendations for the Conduct, Reporting, understand the relationship of gut microbiota translocating Editing, and Publication of Scholarly Work in Medical into blood. Journals) and blood and urine samples. Blood was tested 136 total subjects screened 62 IgA nephropathy patients 74 Healthy subjects 42 excluded -Refused participation (n=16) -ESRD or transplant (n=5) 54 excluded -Age<18 or >65yrs (n=5) -Refused participation (n=31) -On immunosuppression (n=5) -Did not meet inclusion criteria -Diabetes (n=3) (antibiotic use, abnormal -Proteinuria <0.3g (n=3) urinalysis, pregnant) or not -Others (n=5) matching by age (n=23) 20 IgA nephropathy included 20 healthy included Figure 1. | Enrollment of study subjects. Out of 62 patients with IgA Nephropathy, 20 patients were recruited based on eligibility criteria detailed below. 20 out of 74 healthy subjects met the eligibility criteria for the study after matching for age and sex. KIDNEY360 2: 1261–1274, August, 2021 Blood and Gut Microbiome in IgA Nephropathy, Shah et al. 1263 for routine chemistries and the microbiome. A preprepared removed, and (4) OTUs with abundance ,0.005% of the stool kit was given to subjects and all samples were either whole dataset abundance were removed. To increase the dropped off personally or mailed to us via overnight shipping specificity of bacterial taxa truly different between the IgAN within 1–2 days of sample collection. Samples were collected and healthy groups, we lowered the sensitivity by eliminat- within 2 weeks of signing