Article Blood Microbiome Profile in CKD A Pilot Study Neal B. Shah ,1 Andrew S. Allegretti,1 Sagar U. Nigwekar,1 Sahir Kalim,1 Sophia Zhao,1 Benjamin Lelouvier,2 Florence Servant,2 Gloria Serena,3 Ravi Ishwar Thadhani,1,4 Dominic S. Raj,5 and Alessio Fasano3 Abstract Background and objectives The association between gut dysbiosis, high intestinal permeability, and endotox- 1 fl Division of emia-mediated in ammation is well established in CKD. However, changes in the circulating microbiome in Nephrology, patients with CKD have not been studied. In this pilot study, we compare the blood microbiome profile between Department of patients with CKD and healthy controls using 16S ribosomal DNA sequencing. Medicine, Massachusetts General Hospital, Design, setting, participants, & measurements Blood bacterial DNA was studied in buffy coat samples Boston, quantitatively by 16S PCR and qualitatively by 16S targeted metagenomic sequencing using a molecular pipeline Massachusetts; specifically optimized for blood samples in a cross-sectional study comparing 20 nondiabetic patients with CKD 2Vaiomer, Department and 20 healthy controls. of Research and Development, fi Bioinformatics Results There were 22 operational taxonomic units signi cantly different between the two groups. 16S metagenomic Division, Labe`ge, sequencing revealed a significant reduction in a diversity (Chao1 index) in the CKD group compared with healthy France; 3Division of controls (127618 versus 145631; P=0.04). Proteobacteria phylum, Gammaproteobacteria class, and Enterobacter- Pediatric iaceae and Pseudomonadaceae families were more abundant in the CKD group compared with healthy controls. Gastroenterology and fi Nutrition, Center for Median 16S ribosomal DNA levels did not signi cantly differ between CKD and healthy groups (117 versus 122 Celiac Research, copies/ng DNA; P=0.38). GFR correlated inversely with the proportion of Proteobacteria (r=20.54; P#0.01). Massachusetts General Hospital for Conclusions Our pilot study demonstrates qualitative differences in the circulating microbiome profile with lower Children, Boston, a fi Massachusetts; diversity and signi cant taxonomic variations in the blood microbiome in patients with CKD compared with 4Department of healthy controls. Biomedical Sciences, Clin J Am Soc Nephrol 14: 692–701, 2019. doi: https://doi.org/10.2215/CJN.12161018 Cedars-Sinai Medical Center, Los Angeles, California; and 5Division of Renal Introduction human donors, has been found using 16S ribosomal Diseases and Complex assemblages of microorganisms unique to DNA (rDNA) measurement (11). We hypothesized Hypertension, The different human body sites are emerging as important George Washington that patients with CKD would have a different quan- University, modulators of human health and diseases (1). High- titative and qualitative microbial profile in blood com- Washington, DC throughput sequencing methods have demonstrated paredwithcontrols.Thisdeviationinthemicrobiome that the human gut is symbiotically inhabited by has the potential to provide an insight into the etiology Correspondence: thousands of bacterial species in diverse communities and mechanisms of inflammation and associated out- Dr. Neal B. Shah, helping the host in digestion of complex carbohy- comes in the CKD population. The aim of our pilot study Division of drates, short-chain fatty acid generation, vitamin and fi Nephrology, was to measure the blood microbiome pro le in Massachusetts General amino acid synthesis, and shaping of the immune nondialysis patients with CKD and compare them Hospital, 165 system (2,3). An imbalanced intestinal microbial with healthy controls by comparing their quantitative Cambridge Street, Suite community has been associated with various diseased 16S rDNA levels and qualitative metagenomic profiles. 302, Boston, MA states and is referred to as gut dysbiosis (4). Several 02114. Email: [email protected]. studies have demonstrated an association between edu. gut dysbiosis, inflammation, and CKD (5,6). Gut Methods dysbiosis leading to disruption in intestinal barrier Study Design function in CKD permits translocation of gut-derived We conducted a cross-sectional pilot study includ- toxins, bacterial products, and intact bacteria into the ing 20 patients with CKD and 20 healthy controls who circulation, resulting in inflammation (7,8). This cir- had blood buffy coat samples in our hospital biobank. culating microbiome in CKD is indirectly evidenced by higher levels of endotoxins, LPS levels, and gut Study Participants uremic toxins measured in blood (9,10). Recently, the Buffy coat samples of eligible patients were obtained existence of a highly diversified blood microbiome, from Partners Healthcare biobank. The Partners including metagenomic profiles even in healthy biobank comprises samples voluntarily obtained 692 Copyright © 2019 by the American Society of Nephrology www.cjasn.org Vol 14 May, 2019 Clin J Am Soc Nephrol 14: 692–701, May, 2019 Blood Microbiome in CKD, Shah et al. 693 from individuals at any Partners-affiliated hospitals to foster sterile precautions. DNA extraction, 16S rDNA quantitative research. Patients and healthy volunteers affiliated with the PCR, and qualitative 16S targeted metagenomic sequenc- Partners Healthcare system can contribute their blood sam- ingwasperformedbyVaiomer(Vaiomer,Toulouse, ples to the biobank after consenting and electronically filling France). The testing laboratory was blinded to the two out a health information survey. Their blood samples are groups. The primary aim was to compare quantitative 16S drawn during a scheduled visit with a biobank staff or at the rDNA levels and qualitative bacterial profiles between the time of their clinic visit. These samples are made available to two groups. The secondary aim was to look for any Partners Healthcare investigators with appropriate approval correlation between 16S rDNA levels and GFR. from the Partners Institutional Review Board (IRB protocol No. 2009P002312). They are linked to clinical data from the 16S DNA Extraction and Quantification electronic medical record and categorized into healthy and DNA was extracted from these samples using an optimized various disease states as well as by other clinical character- tissue-specific technique. Total genomic DNA was collected istics enabling researchers to identify patients relevant to in a final 50 ml extraction volume. Total DNA concentrations their study. The Partners ethics committee approved this were determined by ultraviolet spectroscopy. Total 16S rDNA study. The study adhered to the Declaration of Helsinki. present in the samples was measured by quantitative PCR in Patients with CKD of various nondiabetic etiologies, triplicates using 16S universal primers targeting the V3–V4 who were aged 30–65 years, with GFR 10–59 ml/min, and region of the bacterial 16S ribosomal gene and normalized never on dialysis were eligible for inclusion. Patients with using a plasmid-based standard scale, details of which have diabetes were excluded because of its ability to influence been described previously (11,13). In these experiments, the 16S rDNA levels, demonstrated by prior studies (12). efficiency calculated from the standard curve was required to Patients with clearly documented etiology were preferred. be between 80% and 120%, and the R2 of the standard curve Healthy controls matched by age group 30–65 years and was .0.980. After successful extraction and amplification, 16S available buffy coat samples were randomly chosen using rDNA was measured as 16S copy number per nanogram of estimated 10-year survival .95%, determined by Charlson DNA in triplicates and fell within the standard curve range. comorbidity index. Any patient with a white blood cell . 9 (WBC) count 10 /L, evidence of active infection or on 16S Metagenomic Sequencing fl antibiotics during sample collection, or history of in am- Library preparation was performed by two-step PCR matory bowel disease or prior intestinal surgery was amplification using 16S universal primers targeting the excluded. Charts of eligible patients were reviewed pre- V3–V4 region of the bacterial 16S ribosomal gene (Vaiomer ferring the latest available blood samples until 20 patients universal 16S primers) as described previously (14). in each group were included (Figure 1). Demographics and The resulting amplicon of approximately 467 base pairs covariates were obtained from electronic medical records. (as in Escherichia coli 16S) was sequenced using 23300 paired-end MiSeq kit V3. For each sample, a sequencing Sample Collection library was generated by addition of sequencing adapters. Microbiome in blood predominantly exists in the buffy The detection of the sequencing fragments was performed coat (11). For analysis, samples were first thawed under using MiSeq Illumina technology. 237 non-diabetic CKD and 5163 healthy subjects aged 30-65yrs with biobank samples* (2010-2017) 95 CKD and 1440 Healthy samples with missing buffy coat samples 142 CKD samples 3723 Healthy samples 64 older samples 3643 older samples excluded excluded 78 Charts reviewed 80 Charts reviewed 58 excluded subjects 60 excluded subjects -GFR>59 (n=35) -Missing WBC or GFR (n=34) -Unclear CKD cause (n=11) -Genetic or autoimmune diseases (n=10) -Elevated or missing WBC (n=9) -GI pathology or taking antibiotic (n=10) -On antibiotic (n=3) -H/o AKI or CKD (n=6) 20 CKD included 20 Healthy included *Healthy with estimated 10 year survival based on Charlson Comorbidity Index >95% Figure 1. | Enrollment of study participants. GI, gastrointestinal.