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Perturbations of the Gut Microbiome and Metabolome in Children with Calcium Oxalate Kidney Stone Disease

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Perturbations of the Gut Microbiome and Metabolome in Children with Calcium Oxalate Kidney Stone Disease CLINICAL RESEARCH www.jasn.org Perturbations of the Gut Microbiome and Metabolome in Children with Calcium Oxalate Kidney Stone Disease Michelle R. Denburg,1,2,3 Kristen Koepsell,4 Jung-Jin Lee ,5 Jeffrey Gerber,3,6 Kyle Bittinger,5 and Gregory E. Tasian2,3,4 1Division of Nephrology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 2Department of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 3Center for Pediatric Clinical Effectiveness, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 4Division of Pediatric Urology, Department of Surgery, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 5Division of Gastroenterology, Department of Pediatrics, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 6Division of Infectious Diseases, Department of Pediatrics, The Children’s Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania ABSTRACT Background The relationship between the composition and function of gut microbial communities and early-onset calcium oxalate kidney stone disease is unknown. Methods We conducted a case-control study of 88 individuals aged 4–18 years, which included 44 indi- viduals with kidney stones containing $50% calcium oxalate and 44 controls matched for age, sex, and race. Shotgun metagenomic sequencing and untargeted metabolomics were performed on stool samples. Results Participants who were kidney stone formers had a significantly less diverse gut microbiome com- pared with controls. Among bacterial taxa with a prevalence .0.1%, 31 taxa were less abundant among individuals with nephrolithiasis. These included seven taxa that produce butyrate and three taxa that degrade oxalate. The lower abundance of these bacteria was reflected in decreased abundance of the gene encoding butyryl-coA dehydrogenase (P50.02). The relative abundance of these bacteria was cor- related with the levels of 18 fecal metabolites, and levels of these metabolites differed in individuals with kidney stones compared with controls. The oxalate-degrading bacterial taxa identified as decreased in those who were kidney stone formers were components of a larger abundance correlation network that included Eggerthella lenta and several Lactobacillus species. The microbial (a) diversity was associated with age of stone onset, first decreasing and then increasing with age. For the individuals who were stone formers, we found the lowest a diversity among individuals who first formed stones at age 9–14 years, whereas controls displayed no age-related differences in diversity. Conclusions Loss of gut bacteria, particularly loss of those that produce butyrate and degrade oxalate, associates with perturbations of the metabolome that may be upstream determinants of early-onset calcium oxalate kidney stone disease. JASN 31: 1358–1369, 2020. doi: https://doi.org/10.1681/ASN.2019101131 Correspondence: Dr. Gregory E. Tasian, Division of Urology, Received October 31, 2019. Accepted March 22, 2020. Children’s Hospital of Philadelphia, Wood Center, 3rd Floor, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104. Published online ahead of print. Publication date available at Email: [email protected] www.jasn.org. Copyright © 2020 by the American Society of Nephrology 1358 ISSN : 1046-6673/3106-1358 JASN 31: 1358–1369, 2020 www.jasn.org CLINICAL RESEARCH Kidney stone disease (nephrolithiasis) is highly prevalent, in- Significance Statement creasingly common, and is characterized by painful stone events that cause considerable morbidity. In addition, as a Although antibiotics have been associated with an increased risk of disorder of mineral metabolism, nephrolithiasis is associated kidney stones, particularly early in life, perturbations of the gut with increased risks of kidney function loss,1,2 decreased microbiome and metabolome in early-onset nephrolithiasis have not been investigated. Using shotgun metagenomic sequencing 325 bone2pt mineral density and fracture, and cardiovascular and untargeted metabolomics of stool samples in a study of 44 2 disease.6 8 Kidney stones affect one in 11 people in the United children with kidney stones and 44 controls matched for age, sex, States9 and result in annual healthcare costs .$10 billion.10 and race, the authors found that 31 bacterial taxa—including seven The prevalence of nephrolithiasis has increased by 70% over butyrate-producing taxa and three that degrade oxalate—were less 20 years.9,11 Our group and others have discovered dispropor- abundant among children with calcium oxalate stones. Levels of 18 metabolites differed between cases and controls and correlated tionate increases in the incidence of nephrolithiasis among with the fecal bacteria that were less abundant among children with 2 children, adolescents, and women.12 14 The shift in neph- nephrolithiasis. Such disruptions in the gut microbiome and me- rolithiasis to a younger age of onset has caused increasing tabolome may thus be determinants of early-onset disease and may hospitalizations, surgeries, and healthcare expenditures.15 explain the association between antibiotics and nephrolithiasis. In addition, the morbidity associated with nephrolithiasis appears to be more pronounced in younger individuals, and 50% was calcium oxalate) that spontaneously passed or were stone recurrence rates may be higher in children than removed surgically within the prior 3 years (stone analysis 4,16 adults. The reasons for this shift in the epidemiology of done by mass spectroscopy at various clinical laboratories). nephrolithiasis are unclear. However, the rapidity of the change We used a 3-year time window since the last stone event to suggests that the driving forces are external exposures such as capture individuals most likely to have active stone disease.16 diet and antibiotics. Many of these exposures may disrupt the Cases were recruited during outpatient visits to the CHOP gut-kidney axis, which is the complex interplay between the Kidney Stone Center. 17 intestinal and urinary tracts in human health and disease. Controls were healthy volunteers matched to cases on age Prior investigations have demonstrated perturbations of (62 years), sex, race, and ethnicity. The study was nested the gut microbiome among adults with nephrolithiasis. These within the CHOP healthcare system so that cases and controls studies found that the gut microbiome of those who formed arose from the same source population, and equal application 18,19 kidney stones is less diverse than controls and that bacteria of eligibility criteria for both cases and controls ensured that that degrade oxalate were less abundant in the stool of adults both groups were healthy, with the exception that cases were 20 with kidney stones. Currently, the composition and function individuals with kidney stones. To increase study efficiency, of the gut microbiome and metabolome among individuals participants with kidney stones were first matched to healthy with early-onset calcium oxalate kidney stone disease, the participants (n517) who had enrolled as controls in an in- 21 most common form of nephrolithiasis, is unknown. Discov- dependent study that used the same stool collection and ex- ery of the identity and function of microbial communities posure questionnaire as our study. These control participants and downstream metabolites perturbed in early-onset calcium were recruited from the CHOP emergency and dermatology oxalate kidney stone disease could reveal targets for novel ther- departments, oral surgery clinic, and urgent care centers. The apeutics for kidney stone prevention across the life span and remaining 27 control participants were recruited from seven help determine causes of the rapid shift in the epidemiology of suburban and urban practices in the Pediatric Research Con- nephrolithiasis. sortium, which includes the network of CHOP primary care practices in Pennsylvania and New Jersey. METHODS Study Procedures Participants completed a baseline questionnaire, including Study Design and Population past medical history, early lifetime exposures (e.g., vaccina- We conducted a matched case-control study of 88 individuals tions), tobacco use, recent hospitalizations, and probiotic aged 4–18 years who received care in The Children’s Hospital use. In addition, research nutritionists administered 24-hour of Philadelphia (CHOP) healthcare system. We excluded in- dietary recalls over the telephone on 3 days (2 weekdays, dividuals who took antibiotics in the last 3 months and those 1 weekend day) to estimate participants’ daily nutrient and with inflammatory bowel disease, prior bariatric surgery, mineral intake. The 24-hour dietary recalls were collected using monogenic causes of kidney stones, cancer, immobility, cystic the Nutrition Data System for Research developed at the University fibrosis, celiac disease, diabetes, congenital anomalies of the of Minnesota.22 Dietary intake data were gathered by a multiple- kidney and urinary tract, urinary tract obstruction, and renal pass interview approach,23 and values for 165 nutrients, nutrient tubular acidosis. The Institutional Review Board at CHOP ratios, and other food components were generated from a database approved this study. that includes .18,000 foods.24,25 Antibiotics, including route Cases included individuals
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