Chronic kidney disease alters intestinal microbial flora

The population of microbes (microbiome) in the intestine is a symbiotic ecosystem conferring trophic and protective functions. Since the biochemical environment shapes the structure and function of the microbiome, we tested whether uremia and/or dietary and pharmacologic interventions in chronic kidney disease alters the microbiome. To identify different microbial populations, microbial DNA was isolated from the stools of 24 patients with end-stage renal disease (ESRD) and 12 healthy persons, and analyzed by phylogenetic microarray. There were marked differences in the abundance of 190 bacterial operational taxonomic units (OTUs) between the ESRD and control groups. OTUs from Brachybacterium, Catenibacterium, Enterobacteriaceae, Halomonadaceae, Moraxellaceae, Nesterenkonia, Polyangiaceae, Pseudomonadaceae, and Thiothrix families were markedly increased in patients with ESRD. To isolate the effect of uremia from inter-individual variations, comorbid conditions, and dietary and medicinal interventions, rats were studied 8 weeks post 5/6 nephrectomy or sham operation. This showed a significant difference in the abundance of 175 bacterial OTUs between the uremic and control animals, most notably as decreases in the Lactobacillaceae and Prevotellaceae families. Thus, uremia profoundly alters the composition of the gut microbiome. The biological impact of this phenomenon is unknown and awaits further investigation. The population of microbes (microbiome) in the intestine is a symbiotic ecosystem conferring trophic and protective functions. Since the biochemical environment shapes the structure and function of the microbiome, we tested whether uremia and/or dietary and pharmacologic interventions in chronic kidney disease alters the microbiome. To identify different microbial populations, microbial DNA was isolated from the stools of 24 patients with end-stage renal disease (ESRD) and 12 healthy persons, and analyzed by phylogenetic microarray. There were marked differences in the abundance of 190 bacterial operational taxonomic units (OTUs) between the ESRD and control groups. OTUs from Brachybacterium, Catenibacterium, Enterobacteriaceae, Halomonadaceae, Moraxellaceae, Nesterenkonia, Polyangiaceae, Pseudomonadaceae, and Thiothrix families were markedly increased in patients with ESRD. To isolate the effect of uremia from inter-individual variations, comorbid conditions, and dietary and medicinal interventions, rats were studied 8 weeks post 5/6 nephrectomy or sham operation. This showed a significant difference in the abundance of 175 bacterial OTUs between the uremic and control animals, most notably as decreases in the Lactobacillaceae and Prevotellaceae families. Thus, uremia profoundly alters the composition of the gut microbiome. The biological impact of this phenomenon is unknown and awaits further investigation. The large community of microbes residing in the intestinal tract (microbiome) constitutes a dynamic and symbiotic ecosystem that is in constant interaction with the host metabolism.1Dunne C. Adaptation of bacteria to the intestinal niche: probiotics and gut disorder.Inflamm Bowel Dis. 2001; 7: 136-145Crossref PubMed Scopus (148) Google Scholar, 2Hooper L.V. Gordon J.I. Commensal host–bacterial relationships in the gut.Science. 2001; 292: 1115-1118Crossref PubMed Scopus (1790) Google Scholar, 3Bourlioux P. Koletzko B. Guarner F. et al.The intestine and its microflora are partners for the protection of the host: report on the Danone Symposium ‘The Intelligent Intestine’, held in Paris.Am J Clin Nutr. 2003; 78: 675-683PubMed Google Scholar Under normal conditions, the gut microbiome provides trophic2Hooper L.V. Gordon J.I. 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Commensal bacteria, redox stress, and colorectal cancer: mechanisms and models.Exp Biol Med (Maywood). 2004; 229: 586-597Crossref PubMed Scopus (194) Google Scholar and obesity.13Bäckhed F. Manchester J.K. Semenkovich C.F. et al.Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.Proc Natl Acad Sci USA. 2007; 104: 979-984Crossref PubMed Scopus (1916) Google Scholar The biochemical milieu has a decisive part in shaping the structure, composition, and function of the microbial flora. Uremia can profoundly modify the biochemical milieu of the gut via heavy influx of urea into the gastrointestinal tract and secretion of uric acid and oxalate by the colonic epithelium.14Vaziri N.D. Freel R.W. Hatch M. Effect of chronic experimental renal insufficiency on urate metabolism.J Am Soc Nephrol. 1995; 6: 1313-1317PubMed Google Scholar, 15Hatch M. Vaziri N.D. Enhanced enteric excretion of urate in rats with chronic renal failure.Clin Sci. 1994; 86: 511-516Crossref PubMed Scopus (57) Google Scholar, 16Hatch M. Freel R.W. Vaziri N.D. Intestinal excretion of oxalate in chronic renal failure.J Am Soc Nephrology. 1994; 5: 1339-1343PubMed Google Scholar In addition, therapeutic interventions, including dietary restriction of fruits, vegetables, and high-fiber products to prevent hyperkalemia and oxalate overload, use of phosphate-binding agents to manage hyperphosphatemia, and administration of antibiotics to treat vascular access and other infections can modify the luminal milieu of the gut and impact its microbial flora. Alteration of microbial flora in inflammatory bowel diseases contributes to and may be exacerbated by the disruption of the gut epithelial barrier function and structure. This enables leakage of the luminal antigens and other noxious contents into the intestinal wall and the systemic circulation.17Nagalingam N.A. Lynch S.V. Role of the microbiota in inflammatory bowel diseases.Inflamm Bowel Dis. 2012; 18: 968-984Crossref PubMed Scopus (209) Google Scholar Several observations suggest that uremia impairs intestinal barrier function and promotes inflammation throughout the gastrointestinal tract. This is based on the reported increase in intestinal permeability to high-molecular-weight polyethylene glycols in uremic humans and animals,18Magnusson M. Magnusson K.E. Sundqvist T. et al.Increased intestinal permeability to differently sized polyethylene glycols in uremic rats: effects of low- and high protein diets.Nephron. 1990; 56: 306-311Crossref PubMed Scopus (68) Google Scholar,19Magnusson M. Magnusson K.E. 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The gastrointestinal tract in uremia.Dig Dis Sci. 1993; 38: 257-268Crossref PubMed Scopus (132) Google Scholar These events can clearly contribute to systemic inflammation and oxidative stress, which are constant features of advanced chronic kidney disease (CKD) and the major mediators of cardiovascular disease, cachexia, anemia, and numerous other morbidities in this population.26Carrero J.J. Stenvinkel P. Inflammation in end-stage renal disease-what have we learned in 10 years?.Semin Dial. 2010; 23: 498-509Crossref PubMed Scopus (248) Google Scholar, 27Himmelfarb J. Stenvinkel P. Ikizler T.A. et al.The elephant in uremia: oxidant stress as a unifying concept of cardiovascular disease in uremia.Kidney Int. 2002; 62: 1524-1538Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 28Vaziri N.D. Oxidative stress in uremia: nature, mechanisms, and potential consequences.Semin Nephrol. 2004; 24: 469-473Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 29Yoon J.W. Pahl M.V. Vaziri N.D. Spontaneous leukocyte activation and oxygen-free radical generation in end stage renal disease.Kidney Int. 2007; 71: 167-172Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 30Gollapudi P. Yoon J-W. Gollapudi S. et al.Effect of End Stage Renal Disease and hemodialysis on expression and activities of leukocyte toll-like receptors (TLR).Am J Nephrol. 2010; 31: 247-254Crossref PubMed Scopus (68) Google Scholar As noted above, uremia and its treatment can significantly alter the biochemical milieu of the intestinal tract and, as such, may alter the structure, composition, and function of microbial flora. This may disturb the symbiotic relationship that prevails under normal conditions and lead to the production and absorption of proinflammatory and otherwise harmful byproducts, and simultaneously limit the beneficial functions and products conferred by the normal flora. Such events can contribute to uremic toxicity, inflammation, and cardiovascular, nutritional, and other complications of CKD. The present study was designed to test the hypothesis that the biochemical modification of the gut milieu in advanced CKD can lead to significant changes in composition of the gut microbial flora. As expected, compared with the healthy control group, the ESRD patients had a significant increase in plasma concentrations of creatinine (8.6±2.9 vs. 0.8±0.1mg/dl, P<0.0001) and urea nitrogen (70±18.0 vs. 24.0±9.9mg/dl, P<0.0001) concentrations. All patients were treated with phosphate binders, erythropoiesis-stimulating agents, intravenous iron compounds, and multivitamin preparations. Strict dietary fluid and sodium, phosphorus, and potassium restrictions were prescribed to minimize fluid overload, hyperphosphatemia, and hyperkalemia. Patients received hemodialysis therapy for 3h three times weekly using cellulose triacetate dialyzers. Systemic heparinization was used for anticoagulation during hemodialysis. The Kt/V in the ESRD group was 1.5±0.3, reflecting adequacy of the dialysis regimen. The ethnic background of the ESRD group (9 Caucasians, 13 Hispanics, and 2 Asians) was similar to that of the control group (4 Caucasians, 7 Hispanics, and 1 Asian) Similarly, the body mass index in the ERSD group (30.4±8.3) was comparable to that of the control (29.2±6.1kg/m2, P=0.65). Data are summarized in Table 1. Compared with the sham-operated control group, the CKD group exhibited significant elevation of arterial pressure, increased urinary protein excretion, elevated plasma urea and creatinine concentrations, reduced hematocrit, and lower body weight.Table 1BW, blood pressure, Hct, serum creatinine and urea concentration, Ccr, and urinary protein excretion in normal control rats and rats with CRFBW (g)BP (mmHg)Hematocrit (%)Creatinine (mg/dl)Urea (mg/dl)Ccr (ml/min/kg)U Protein (mg/mg Cr)CTL407±5.9120±2.148±1.20.61±0.248±3.38.8±0.057.4±0.5CRF374±4.4155±2.5*P<0.05 compared with CTL.35±0.7*P<0.05 compared with CTL.1.14±0.2*P<0.05 compared with CTL.93±7.4*P<0.05 compared with CTL.3.4±0.03*P<0.05 compared with CTL.81.5±5.6*P<0.05 compared with CTL.Abbreviations: BP, tail arterial pressure; BW, body weight; Ccr, creatinine clearance; Cr, plasma creatinine; CRF, chronic renal failure; CTL, control; Hct, hematocrit; U protein, urine protein excretion in the CRF and control rats.Values are mean±s.d.* P<0.05 compared with CTL. Open table in a new tab Abbreviations: BP, tail arterial pressure; BW, body weight; Ccr, creatinine clearance; Cr, plasma creatinine; CRF, chronic renal failure; CTL, control; Hct, hematocrit; U protein, urine protein excretion in the CRF and control rats. Values are mean±s.d. Relative richness (the number of bacterial taxa in a sample) was assessed for subfamilies found in samples in each group. Although the mean relative richness (summarized at subphylum) for ESRD and control groups was similar (Figure 1a), the relative abundances (i.e., probe intensities) of bacterial groups within the subfamilies differed significantly. Significant increases (adjusted P<0.02) in relative abundance were found for 190 bacterial operational taxonomic units (OTUs) in the ESRD group compared with the control group. Many (159) of the OTUs that were significantly different between the study groups belonged to the Pseudomonadaceae family. Although one Pseudomonas sequence can trigger probes in several neighboring OTUs, the patterns of OTU intensity observed across the test samples here indicate that there were probably several related yet distinguishable members of the Pseudomonadaceae family with elevated abundances in the ESRD patients. Microbial families showing the largest increase in ESRD patients were from the Actinobacteria, Firmicutes (especially subphylum Clostridia), and Proteobacteria (primarily Gammaproteobacteria) phyla (Table 2). Four OTUs had two- to three-fold higher average abundances in the control samples than in the ESRD samples, although the differences were not statistically significant. Those OTUs belonged to the Sutterellaceae, Bacteroidaceae, and Lactobacillaceae families. To investigate diversity within and between groups, all significantly different OTUs were used to generate a non-metric multidimensional scaling plot (Figure 2). Despite overlapping distributions, the control group samples showed tighter clustering than did the ESRD samples. Significant inter-subject variability in human fecal microbiota has been noted in both healthy and diseased individuals in previous studies.31Martinez C. Antolin M. Santos J. et al.Unstable composition of the fecal microbiota in ulcerative colitis during clinical remission.Am J Gastroenterol. 2008; 103: 643-648Crossref PubMed Scopus (149) Google Scholar, 32Whelan K. Judd P.A. Tuohy K.M. et al.Fecal microbiota in patients receiving enteral feeding are highly variable and may be altered in those who develop diarrhea.Am J ClinNutr. 2009; 89: 240-247Crossref PubMed Scopus (52) Google Scholar, 33Eckburg P.B. Bik E.M. Bernstein C.N. et al.Diversity of the human intestinal microbial flora.Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5532) Google Scholar We assumed that compared with the recruited patients and controls, variability in the microbiome would be lower among genetically identical rats raised and maintained under similar conditions, in which we could precisely control the age, gender, and diet, as well as the onset, severity, and the underlying cause of CKD.Table 2Families of bacteria with the adjusted P-values <0.02 in the abundance of their OTUs when comparing control individuals to the ESRD patientsFamily: average intensityPhylumSubphylumFamilyExample strainOTU IDControlESRDActinobacteriaActinobacteridaeBrachybacteriumBrachybacterium nesterenkovii str. DSM 957353774828412NesterenkoniaLake Kauhako water isolate str. K2-6670634924839FirmicutesClostridiaCatabacterSwine intestine clone p-5389-2Wb5441916362617PeptostreptococcaceaeClostridium elmenteitii E2SE1-B376559736677MollicutesCatenibacteriumMidgut homogenate Pachnoda ephippiata larva clone PeM34657911,58511,678ProteobacteriaDeltaproteobacteriaPolyangiaceaeBovine fetal thymus clone EBA461110,43811,192GammaproteobacteriaAlteromonasAlteromonas macleodii201966027674Enterobacteriales_EnterobacteriaceaeEnterobacter sp. str. 16-31606365818325HalomonadaceaeHalomonas sp. str. NT N2116352446203MethylococcaceaeDeep-sea hydrothermal vent clone TAG-1153766977892MoraxellaceaeAcinetobacter sp. str. YY-5167573977878PseudomonadaceaePseudomonas sp. str. ST41179389309631ThiothrixThiothrix sp. str. KRN-B2151147185671Abbreviations: ESRD, end-stage renal disease; OUT, operational taxonomic unit.All significantly different OTUs in these families had higher average relative abundance in ESRD patients. A representative OTU and example sequence are shown. Average intensities for the significantly differing OTUs in the families are shown for each group. Open table in a new tab Figure 2Two-dimensional representation (plane of view) of non-metric multidimensional scaling analysis of standardized operational taxonomic unit (OTU) intensities (hybridized probe sets) for the dynamic subset of the microbial community consisting of 190 PhyloChip OTUs from controls or end-stage renal disease (ESRD) patients. Stress=0.10. Each point is a human fecal community from control (C) or ESRD individuals. The model is based on a Bray–Curtis similarity matrix. Connecting lines were used to aid in the visualization of the distribution of all members of a group.View Large Image Figure ViewerDownload (PPT) Abbreviations: ESRD, end-stage renal disease; OUT, operational taxonomic unit. All significantly different OTUs in these families had higher average relative abundance in ESRD patients. A representative OTU and example sequence are shown. Average intensities for the significantly differing OTUs in the families are shown for each group. In confirmation of other studies,34Islam K.B. Fukiya S. Hagio M. et al.Bile acid is a host factor that regulates the composition of the cecal microbiota in rats.Gastroenterology. 2011; 141: 1773-1781Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar,35Kataoka K. Kibe R. Kuwahara T. et al.Modifying effects of fermented brown rice on fecal microbiota in rats.Anaerobe. 2007; 13: 220-227Crossref PubMed Scopus (25) Google Scholar Firmicutes (e.g., class Clostridia, Bacilli, Mollicutes), Bacteroidetes (e.g., class Bacteroidia), Actinobacteria, and Proteobacteria were among the taxa with the greatest number of species in the fecal samples of the rats used in the present study. With the exception of the Betaproteobacteria, which had a greater number of OTUs in the chronic renal failure (CRF) rats (36 OTUs) than control rats (33 OTUs), total richness was significantly greater (Wilcoxon test, P=0.0086) in the control group compared with the CRF group, and an ANOSIM (analysis of similarity) test of the number of species per class included in Figure 1b indicated weak differences between groups (R=0.356), with class Bacilli contributing the most (19%) to the difference between groups (SIMPER data not shown, though see Supplementary Figure S1). Download .ppt (.21 MB) Help with ppt files Supplementary Figure S1 Bacterial community structure was distinctive between normal and CRF rats as shown by non-metric multidimensional scaling analysis based on standardized intensities of the hybridized probe sets of the bacterial communities (Figure 3) and supported by an Adonis test (P=0.002). There were 175 OTUs that were significantly different (adjusted P<0.05) between groups, of which certain families in the Bacteroidetes and Firmicutes were less prevalent in the CRF rats, especially Lactobacillaceae and Prevotellaceae. Of the significantly different OTUs, 81 had at least a two-fold change in average probe intensity. A heat map shows that a majority of these OTUs decreased in relative abundance in the CRF compared with the control rats (Figure 4). Previous experiments used to validate G3 PhyloChips correlating defined concentrations of target OTUs with hybridization intensity found that, to a rough approximation, as the intensity doubled, the relative abundance increased over four-fold (unpublished data). A table summarizing families with the greatest differences between CKD and control rat samples is included in the Supplementary Table S1 online.Figure 4Hierarchical clustering of probe sets representing significantly different operational taxonomic units (OTUs) between groups with a minimum two-fold change in untransformed intensities between samples to show relative abundance. Yellow indicates increased abundance and red indicates decreased abundance relative to the mean for each OTU. Columns represent rat fecal microbiota composition for control (CTL) and chronic renal failure (CRF) individual rats. Rows are OTUs with mean fold change among samples and cluster based on the similarity of their abundance profiles across the data set, with similar OTUs connected at the hierarchical tree on the left. Bars on the right represent (1) OTUs from the Firmicutes (especially Lactobacillaceae and a few Coprococcus within the Lachnospiraceae), Bacteroidetes (Prevotellaceae, two Rikenellaceae OTUs) that had higher abundances in CTL samples, or (2) OTUs from the Firmicutes (unclassified Lachnospiraceae) and other Rikenellaceae (in Bacteroides) that have higher abundances in CRF samples.View Large Image Figure ViewerDownload (PPT) Download .doc (.06 MB) Help with doc files Supplementary Table 1 As expected, there was much less variability among samples within each group for rats compared with humans (Supplementary Table S2 online). The percent coefficient of variation for OTU intensities was 14.2% for control individuals and 18.6% for ESRD patients compared with 7% for control rats and 14.4% for CKD rats. Download .doc (.03 MB) Help with doc files Supplementary Table 2 The selection pressures on the part of the host and the microbes shapes the structure, composition, and function of the microbial flora. In this context, advanced renal failure can profoundly alter the biochemical milieu of the gastrointestinal tract by several mechanisms: (1) elevation of urea concentration in the intracellular and extracellular fluids leads to its massive influx into the gastrointestinal tract via passive diffusion and incorporation in glandular secretions. Hydrolysis of urea by urease, which is expressed in some microbial species in the gut flora, results in the formation of large quantities of ammonia [CO(NH2)2 +H2O→CO2+2NH3]. This phenomenon leads to modification of the luminal pH and causes uremic enterocolitis.24Vaziri N.D. Dure-Smith B. Miller R. et al.Pathology of gastrointestinal tract in chronic hemodialysis patients: an autopsy study of 78 cases.Am J Gastroenterol. 1985; 80: 608-611PubMed Google Scholar,25Kang J.Y. The gastrointestinal tract in uremia.Dig Dis Sci. 1993; 38: 257-268Crossref PubMed Scopus (132) Google Scholar (2) Uric acid, the end product of purine metabolism in humans, is normally excreted in the urine via a complex interplay of glomerular filtration and tubular reabsorption and secretion processes. However, in advanced renal failure, the colon replaces the kidney as the primary site of uric acid excretion. This process is mediated by an adaptive rise in the secretory flux of uric acid14Vaziri N.D. Freel R.W. Hatch M. Effect of chronic experimental renal insufficiency on urate metabolism.J Am Soc Nephrol. 1995; 6: 1313-1317PubMed Google Scholar,15Hatch M. Vaziri N.D. Enhanced enteric excretion of urate in rats with chronic renal failure.Clin Sci. 1994; 86: 511-516Crossref PubMed Scopus (57) Google Scholar and accounts for the relatively minor increase in plasma uric acid despite total or near-total loss of renal function. (3) As with uric acid, the colon has a major role in excretion of oxalate in renal failure.16Hatch M. Freel R.W. Vaziri N.D. Intestinal excretion of oxalate in chronic renal failure.J Am Soc Nephrology. 1994; 5: 1339-1343PubMed Google Scholar (4) Diet has a major role in shaping the gut microbial flora. Strict dietary restrictions intended to prevent severe hyperkalemia and oxalate overload in patients with advanced CKD severely limit consumption of fruits, vegetables, and high-fiber products, which are rich in potassium and oxalate. These products normally contain most of the indigestible dietary complex carbohydrates that serve as the primary source of nutrients for the gut microbiota. Therefore, these dietary restrictions could affect the makeup and/or metabolism of the gut flora. (5) Patients with advanced CKD are invariably instructed to take large quantities of phosphate-binding agents (calcium acetate, calcium carbonate, aluminum hydroxide, and anion-exchange resins) with each meal, to control hyperphosphatemia by limiting phosphate absorption. Long-term consumption of these agents can modify the luminal milieu of the gut and affect the resident microbial flora. (6) Common use of antibiotics to treat vascular access and other infections is yet another factor that is well known to modify the composition of the enteric commensal organisms in such patients. Together, these events may alter the composition and, most likely, the function of the gut microbial flora in patients with advanced renal disease. This supposition was confirmed by the results of the present study, which showed significant differences in the abundance of 190 microbial OTUs between the ESRD and the normal control individuals. These OTUs were classified mostly in the families containing aerobic and facultative anaerobic bacteria. These findings are consistent with the results of two previous studies using the microbial culture technique. In a placebo-controlled clinical trial of a probiotic product, Rangnathan et al.36Rangnathan N. Friedman E.A. Tam P. et al.Probiotic dietary supplementation in patients with stage 3 and 4 chronic kidney disease: a 6-month pilot scale trial in Canada.Curr Med Res Opin. 2009; 25: 1919-1930Crossref PubMed Scopus (105) Google Scholar found a trend toward reduction of the numbers of culturable anaerobic bacteria in patients with CKD stages 3–4. In another study, Fukuuchi et al37Fukuuchi F. Hida M. Aiba Y. et al.Intestinal bacteria-derived putrefactants in chronic renal failure.Clin Exp Nephrol. 2002; 6: 99-104Crossref Scopus (28) Google Scholar showed a significant increase in the percentage of culturable aerobic bacteria from 0.12% in controls to