Probiotics and Gastrointestinal Concerns of the Dialysis Patient Karen Madsen, PhD University of Alberta Karen Madsen, PhD University of Alberta Edmonton, Alberta, Canada

Objectives To gain an understanding of the human gut microbiome and how it can influence human health To learn what probiotics are and their mechanisms of action To gain knowledge of the efficacy of probiotic therapy in patients with renal disease

Welcome to your microbial life! Presentation Title Here |

What kinds of microbes are found in the gut? Bacteria >50 different phyla ~5 phyla found in gut Over 50 known bacterial phyla Generally only 6 phyla found in gut Bacteroidetes* Firmicutes* Actinobacteria Proteobacteria Verrucomicrobria Fusobacteria 10-100 trillion organisms >1000 different species Bacteria, fungi, viruses

Microbial species and abundance change over the length of the GI track

Microbial Ecology of the Gut Species have a characteristic geographic distribution along both the length and the diameter of the gut

Bacterial phyla have specific site-distribution in healthy humans MOUTH SKIN ESOPHAGUS STOMACH VAGINA COLON The area of the chart for each site represents the average number of distinct phylotypes (approximate species-level taxa, based on 16S rRNA gene-sequence analysis) per individual. (The mean number of phylotypes per individual is shown in parentheses; 3–11 individuals were studied per habitat.) The coloured wedges represent the proportion of phylotypes belonging to different phyla. More than 50 bacteria phyla exist, but human microbial communities are overwhelmingly dominated by the 4 that are shown. The relative abundance of these phyla at most sites tends to be consistent across individuals: for example, in almost all humans studied so far, Bacteroidetes and Firmicutes predominate in the colon. By contrast, the composition of the vaginal microbiota is more variable; most women have a preponderance of Firmicutes with few other representatives, whereas a minority of women have a preponderance of Actinobacteria with few other representatives. An estimated 20–80% of human-associated phylotypes (depending on habitat) are thought to have eluded cultivation so far. Data taken from refs 1An ecological and evolutionary perspective on human–microbe mutualism and disease Les Dethlefsen, Margaret McFall-Ngai & David A. Relman Nature 449, 811-818(18 October 2007) Bacteroidetes Firmicutes Nature 449, 811-818. 2007

Gut Microbiota have a Role in Health and Disease

A fine balance of gut microbes University of Alberta Faculty of Medicine & Dentistry A fine balance of gut microbes COMMENSALS PATHOGENS Inhibit pathogen growth Convert pro-drugs to active metabolites Degrade polysaccharides of plant origin Produce folate and Vitamin K Produce short-chain fatty acids Stimulate and modulate immune function Regulate body fat storage Maintain barrier function and stimulate epithelial repair Stimulate gut motility Sepsis, infection Inflammation Liver damage Production of carcinogens Diarrhea, constipation © Do not copy or distribute without permission

Low diversity and imbalances in gut microbiota are associated with human disease states Health High biodiversity and richness Stable Primarily Bacteroides and Firmicutes Disease Low biodiversity Unstable Increased abundances of Proteobacteria, Fusobacteria, Verrucomicrobia C. Difficile colitis, IBD, IBS, obesity, metabolic syndrome, peripheral vascular disease, renal disease, diabetes

A “dysbiosis” of the gut microbiota can result from different mechanisms… Anti Pro Healthy Balanced Pro-inflammatory microbes Excess “bad” bacteria How the microbiome and the human genome contribute to inflammatory disease. In a simplified model, the community composition of the human microbiome helps to shape the balance between immuneregulatory (Treg) and pro-inflammatory (Th17) T cells. The molecules produced by a given microbiome network work with the molecules produced by the human genome to determine this equilibrium. (A) In a healthy microbiome, there is an optimal proportion of both pro- and anti-inflammatory organisms (represented here by SFBs and B. fragilis), which provide signals to the developing immune system (controlled by the host genome) that leads to a balance of Treg and Th17 cell activities. In this scenario, the host genome can contain ‘autoimmune specific’ mutations (represented by the stars), but disease does not develop. (B, C) The genome of patients with multiple sclerosis, type I diabetes, rheumatoid arthritis and Crohn’s disease contain a spectrum of variants that are linked to disease by genome wide association studies (reviewed in (61)). Environmental influences, however, are risk factors in all of these diseases. Altered community composition of the microbiome due to lifestyle, known as dysbiosis, may represent this disease modifying component. An increase in pro-inflammatory microbes, for example SFBs in animal models, may promote Th17 cell activity to increase and thus predispose genetically susceptible people to Th17-mediated autoimmunity (B). Alternatively, a decrease or absence in anti-inflammatory microbes, for example B. fragilis in animal models, may lead to an under-development of Treg cell subsets (C). The imbalance between Th17 cells and Tregs ultimately leads to autoimmunity. Science. 2010 December 24; 330(6012): 1768–1773. doi: 10.1126/science.1195568 PMCID: PMC3159383 NIHMSID: NIHMS314237 Has the microbiota played a critical role in the evolution of the adaptive immune system? Yun Kyung Lee and Sarkis K. Mazmanian* Anti-inflammatory microbes Too few “good” bacteria

Gut barrier function and microbiota

A barrier exists between microbes and the immune system TOLERANCE

Components of the intestinal barrier Image adapted from: Hooper LV (2009) Nat Rev Microbiol 7(5):367-74 Physical barrier (the epithelium) Chemical barrier (mucus layer) Immunological barrier (immune cells of the lamina propria) Microbial barrier (commensal bacteria) Muscle layers (smooth muscle intestinal wall)

Tight junctions maintain barrier between epithelial cells

A breakdown in gut barrier function has been linked with numerous diseases Inflammatory bowel disease Chronic kidney disease Sepsis Necrotizing pancreatitis Celiac disease Type 1 diabetes Food allergies Alcoholic liver disease

What is the role of the microbiota and gut permeability in kidney disease?

Secretory Excretory Regulatory Kidney Function Secretory Erythropoietin Vitamin D, Renin Excretory Urea, uric acid, creatinine, nitrogenous wastes Regulatory Maintains homeostasis; Na, K, Po4,trace elements. Kibow Biotech, Inc.

Impaired Kidney Function results in Waste Accumulation Toxins Retained Urea Uric acid Creatinine Indoxyl sulphate Parathyroid hormone Para cresyl sulphate Phenol P-cresol Oxalate Blood with waste Renal artery Filtered blood Renal vein Water Toxins\ Waste in urine Kibow Biotech, Inc.

Dialysis-induced hypotension Hemodialysis Urea Accumulation Fluid retention Urea influx into gut Dialysis-induced hypotension Increase urease-expressing microbes Generation of urea-derived ammonia Bowel ischemia Disruption of epithelial tight junctions Bowel edema End-stage renal disease results in: (1) accumulation of urea in the body fluids and its diffusion into the gastro-intestinal tract, expanding urease-expressing bacterial species, forming urea-derived ammonia and ammonium hydroxide, a caustic product that damages tight junctions, (2) fluid retention causing generalized and bowel edema which can impair intestinal epithelial barrier function, (3) intermittent hemodialysis complicated by intra-dialytic and post-dialytic hypotension associated with bowel ischemia impairs intestinal epithelial barrier function and facilitates influx of endotoxin and bacterial translocation and (4) influx of endotoxin and translocation of microbial components trigger local and systemic inflammation which further amplifies epithelial barrier dysfunction Translocation of endotoxin and microbial components Local and systemic inflammation Wong et al. Am J Nephrology 39:230. 2014

Nosratola D Vaziri et al. Kidney International 19th Sept 2012, Unbalanced microbiota in CKD patients has higher number of pathogens CKD Patients Increased Actinobacteria Clostridia Proteobacteria Relative richness of the gut microbiome in the study groups. Relative richness comprised of the average number of (a) subfamilies per subphylum for control (CTL) or end-stage renal disease (ESRD) patients or (b) species per class for control (CTL) and chronic renal failure (CRF) rats. A subfamily or species had to be present in at least three replicates of a treatment group and also had to have an average of four subfamilies or species present in a subphylum or class to be included in the figure. Kibow Biotech, Inc. Nosratola D Vaziri et al. Kidney International 19th Sept 2012, Kibow Biotech, Inc. Kibow Biotech, Inc.

How does a gut dysbiosis alter metabolism in the colon? Chronic renal failure is characterized by a progressive retention of a number of microbial metabolic end products(urea, uric acid, creatinine,phenols, indoles and polyamines). Secondly small intestinal assimilation(digestion and/or absorption) of proteins is impaired in the case of renal failure, resulting in an increased availability of proteins for fermentation in the colon. Fermentation of these proteins in the distal region of the colon leads to production of toxic metabolites (Phenols, thiols, indoles, amines and ammonia) Short Chain Fatty Acids Butyrate Acetate Propionate Tyrosine Typtophan p-cresol indole

p-Cresyl sulphate and indoxyl sulphate originate from dietary amino acid bacterial fermentation the colon CKD Decreased protein absorption in small intestine Prolonged colonic transit time Increased luminal pH secondary to increased colonic urea diffusion Meijers et al Nephrol. Dial. Transplant. 2011;26:759-761

Role of the colon in systemic levels of uremic solutes Dialysis patients with intact colon and colectomized patients were studied. Metabolite Normal Control (n=7 to 10) Dialysis Colectomy (n=6) Dialysis Intact colon (n=9) Plasma p-cresyl sulphate (mg/dL) 0.19+0.13 0.06+0.09 4.1+1.6 Plasma indoxyl sulphate (mg/dL) 0.06+0.02 0.08+0.06 2.8+1.3 T W Meyer et al. JASN 2011; 22:1769-1776 Kibow Biotech, Inc.

Healthy Kidney Uremia/ CKD/ ESRD Hypothetical concept about how a failing kidney and the intestinal microbiota affect each other. (Left part) Under physiological conditions, the predominance of symbiotic bacteria, an intact intestinal barrier, defensins production, mucus integrity, and immunoglobulin A (IgA) secretion support the symbiosis between the host and its gut microbiota. An intramural innate immunity controls pathobiont overgrowth inside the lumen of the intestinal tract. (Right part) The metabolic changes that are associated with the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD) change the balance of symbionts and pathobionts in a way that favors pathobiont overgrowth, that is dysbiosis. Pathobiont overgrowth induces inflammation and loss of barrier function that in turn promotes increased translocation of bacterial components and even living bacteria into the host’s internal environment. This process will activate innate immunity characterized by production of proinflammatory cytokines that define a state of systemic inflammation. This process potentially modulates a number of clinically relevant processes in CKD such as the progression of CKD, accelerated atherogenesis, and protein wasting. Anders et al. Kidney Int Jan 16, 2013

How does CKD/ESRD induce a gut dysbiosis? Metabolic acidosis Retention of uremic toxins Volume overload with intestinal wall congestion Frequent use of antibiotics Immune dysfunction Diet restrictions Oral iron usage

Can therapies aimed at modulation of gut microbiota help patients with kidney disease?

How can you change your microbiota? Antibiotics Kill both good and bad bacteria Original microbiota usually return once drugs are removed Can allow for the growth of pathogens Probiotics Giving back live beneficial microorganisms Do not colonize Prebiotics Non-digestible food substances that provide substrate for existing beneficial microbes already present in the gut Diet Changes activity of existing microbes Fecal transplants Changing complete gut ecosystem

Lactobacillius rhamnosus GG Names of Probiotics Brand name Lactobacillus rhamnosus St11 = Lactobacillus fortis Scientific name Commercial name Lactobacillius rhamnosus GG Kibow Biotech, Inc.

Common Probiotics Lactobacillus Bifidobacteria Streptococcus Others L acidophilus L casei GG L rhamnosom L salavarius L delbruecki L reuteri L brevis L plantarum L. bulgaricus B bifidum B infantis B longum B thermophilum B adolescents B. Lactis B. breve S thermophilus S lactis S salivarius E. Coli Nissle 1917 Serotype O6:K5:H1 Saccharomyces boulardi

Some examples of food with probiotics…. 1 billion/100 gm B. lactis (B. regularis) 1 billion/100 gm B. Lactis and L. acidophilus 10 billion/100 ml L casei 1 billion/100 gm B. Lactis and L. acidophilus

Some Probiotic Supplements 15 billion CFU L. acidophilus, B. lactis L. Bulgaricus, B. longum L. rhamnosus, L. brevis, S. thermophilus, L. casei, L. salivarius L. lactis, B. breve, L. plantarum L. paracasei, B. bifidum 450 billion CFU B. breve B. longum B. infantis L. acidophilus L. plantarum L. paracasei L. bulgaricus S. thermophilus 1 billion CFU Bifidobacterium infantis 35624. 1.5 billion CFU Lactobacillus gasseri (KS-13) Bifidobacterium bifidum (G9-1) Bifidobacterium longum (MM-2) 30 billion CFU S. Thermophilus KB19 L. Acidophilus KB27 B. Longum KN31

Effects of probiotics are strain-specific Benefit Product Bifidobacterium animalis DN-173 010 (marketed as Bifidis Regularis) Decreased transit time – help with constipation Dannon Activia yogurt Lactobacillus casei DN-114 001 (marketed as L. casei immuntas) Stimulates immune system Dannon’s DanActive dairy drink Bifidobacterium infantis 35624 Alleviates symptoms of irritable bowel syndrome Procter and Gambles ALIGN supplement Bifidobacterium lactis Bb-12 Yo-Plus yogurt, Nestle Good Start Infant Formula Lactobacillus casei Shirota Yakult fermented dairy drink Lactobacillus rhamnosus GR-1 in combination with L. reuteri Helps eradicate vaginal infections RepHresh Pro-B and Fem-Dophilus dietary supplements BB-12® Bifidobacterium lactis, and LA-5® Lactobacillus acidophilus Iogo Yogurt Lactobacillus reuteri 55730 Reduce antibiotic-associated diarrhea BioGaia tablets, drops, and lozenges Saccharomyces boulardii Reduces antibiotic-associated diarrhea Florastor dietary supplement

Probiotics interact with cells along the entire intestinal tract but they do not colonize Immune cells Epithelial cells Microflora Immune Function Barrier Function Metabolism

Probiotics interact with all components of the gut barrier Effects of probiotic bacteria and yeast on intestinal epithelial barrier function. Probiotics affect the epithelial barrier in numerous, diverse ways. This multifactorial approach to enhancing intestinal barrier function aids in developing and maintaining homeostasis. Depending on the strain of bacteria or yeast and the model used, probiotics target the epithelial barrier in the following 3 areas. A: direct effects on the epithelium. Probiotics can increase mucin expression and secretion by goblet cells, thereby limiting bacterial movement across the mucous layer. Augmentation of β-defensin expression and secretion into the mucus by epithelial cells can prevent the proliferation of commensals and pathogens, thus also contributing to barrier integrity. Finally, probiotics can enhance tight junction stability, which decreases epithelial permeability to pathogens and their products. B: effects on mucosal immunity. Probiotics can increase levels of IgA-producing cells in the lamina propria and promote secretory IgA (sIgA) secretion into the luminal mucous layer. These antibodies limit epithelial colonization by binding bacteria and their antigens, thus contributing to gut homeostasis. C: effects on other surrounding or infecting bacteria. Probiotics can alter the microbiota composition and/or gene expression, leading to indirect enhancement of the barrier through the commensal bacteria. Furthermore, some probiotics can directly kill or inhibit growth of pathogenic bacteria via expression of antimicrobial factors such as bacteriocins. Probiotics can also compete with pathogens or commensals for binding sites on mucins or epithelial cells, thereby preventing detrimental colonization and contributing to barrier function. Modulate neural-muscular system Induce the expression of µ-opioid and cannabinoid receptors Modulate visceral hypersensitivity Ohland C L Am J Physiol 2010;298:G807-G819

Gut Microbes Modulate Gut Permeability The type and quantity of bacterial species present in the gut has a definitive role in modulating intestinal permeability Some microbes enhance barrier function Bifidobacterium infantis, Lactobacillus plantarum Some microbes decrease barrier function

Probiotics also modulate immune function

Probiotic Effects on Immune Cells

Effects of probiotics on immune function Depending on the strain and host environment, probiotics can: Stimulate immune function Increase phagocytosis (Lactobacillus casei, L. acidophilus, B. breve) Increase sIgA secretion Have an anti-inflammatory effect Reduce secretion of pro-inflammatory cytokines Increase secretion of anti-inflammatory cytokines Modulate NF-κ activity Have no effect at all

Probiotics can alter both existing microbe and host metabolism

Probiotics rapidly alter microbial and host metabolic activity Gut microbial activity Host Metabolism Altered Metabolites Systemic Effects

How could probiotics help patients with kidney disease? Altering bacterial composition to reduce production of metabolites Probiotics could increase SCFA and decrease colonic pH Probiotics could repress activity of bacteria that produce toxic metabolites Reducing colonic transit time Some strains have direct effect on gut motility Improving gut barrier function Through effects on tight junction proteins and mucous production Modulating immune function

Are probiotics safe? Commercially available probiotic strains are considered to be GRAS (Generally regarded as safe) due to their long term usage in fermented foods Risks appear to be minimal in most patients Few side effects – primarily gas and bloating which are usually temporary Isolated case reports of systemic infections linked to Lactobacillus rhamnosus (critically ill; severely immunosuppressed) and S. boulardii (intravenous catheters) Richard N Fedorak, MD 2007

Clinical applications What is the evidence?

Meta-analysis for pre-pro- and synbiotic therapy on serum indoxyl-sulfate in patients undergoing haemodialysis for ESKD Int J Nephrol. 2012; 2012: 673631.

Clinical Trial Results Type Strain Patient Type and Number Effect Open label pilot study Simenhoff Miner Electrolyte Metabol 1996 L. acidophilus Hemodialysis N=8 Serum dimethylamine Nitrosodimethylamine Prospective DBPRC crossover Ranganathan et al. Curr Med Res Opin 2009 S. thermophilus, L. acidophilus, B. longum 90 x 109 cfu/d CKD Stages 3 and 4 N=13 6 months BUN Creatinine Uric acid Ranganathan Adv Ther 2010 N=46 Improved QOL Open label single arm Nakabayashi Nephrol Dial Transplant 2011 L. Casei Shirota B. Breve Yakult + galactooligosaccharides 1x 108 4 weeks Serum p-cresol Improved stool consistency Randomized control trial Alatriste Nutr Hosp 2014 Lactobacillus casei shirota 8x109 16x109 N=30 8 weeks blood urea in high dose group

Conclusions The gut microbiota has an important role in human health and in pathogenesis of disease Evidence is supportive of a role for colonic metabolism contributing to uremia Manipulation of the gut microbiota is a promising new therapeutic strategy for patients with renal disease However, to date, limited clinical trials have been done Limitations due to sample size, varying concentrations and types of pro- and prebiotics used, dietary confounders

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