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

2. 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

3. Welcome to your microbial life!
Presentation Title Here |

4. 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
trillion organisms
>1000 different species
Bacteria, fungi, viruses

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

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

7. 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, (18 October 2007)
Bacteroidetes
Firmicutes
Nature 449,

8. Gut Microbiota have a Role in Health and Disease

10. 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

11. 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

12. 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 December 24; 330(6012): 1768–1773.
doi: /science
PMCID: PMC
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

13. Gut barrier function and microbiota

14. A barrier exists between microbes and the immune system
TOLERANCE

15. 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)

16. Tight junctions maintain barrier between epithelial cells

17. 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

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

19. 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.

20. 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.

21. 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:

22. 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.

23. 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

24. 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:

25. 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)
Plasma indoxyl sulphate (mg/dL)
T W Meyer et al. JASN 2011; 22:
Kibow Biotech, Inc.

26. 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

27. 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

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

29. 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

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

31. 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

32. 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

33. 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
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

34. Effects of probiotics are strain-specific
Benefit
Product
Bifidobacterium animalis DN (marketed as Bifidis Regularis)
Decreased transit time – help with constipation
Dannon Activia yogurt
Lactobacillus casei DN (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

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

36. 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

37. 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

38. Probiotics also modulate immune function

39. Probiotic Effects on Immune Cells

40. 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

41. Probiotics can alter both existing microbe and host metabolism

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

43. 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

44. 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

45. Clinical applications
What is the evidence?

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

47. 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

48. 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

49. THE END