Disclosures Speakers Fees; Sandoz, Astellas, Chiesi. Advisory Board Fees; Astellas, Chiesi,Nordic Pharma Grants; Dutch Kidney Foundation, Astellas, Eu FP-7

“There’s an awful lot of money in shit.” (Roy, House of God, Chapter3, Page 35)

Feces transplantation in nephrology No guts no glory Frederike Bemelman Internist-nephrologist, Clinical Immunologist

The gut microbiota: extremely popular and associated to a wide range of diseases A role in obesity Backhed PNAS 2007 Insulin resistance ChristophePlosOne 2011

Outline presentation gut and its microbiota Terminology and normal function Changes in renal failure Solid organ transplantation Evidence and therapeutic potential Conclusions

Terminology (1) Microbiota: collective microbial community inhabiting a special environment Microbiome -Genome of the microbiota -Synonym of microbiota Metagenome: total of DNA extracted from an environment Metagenomics: study of the metagenome -Targeted: 16S ribosomal RNA en DNA -Untargeted: shotgun sequencing -Deep sequencing

Terminology (2) Probiotics live micro organisms Prebiotics non digestible food ingredients

Taxonomic ranking: Different techniques yield different resolutions Kingdom Phylum> 75% Class> 80% Order> 85% Family> 90% Genus> 95% Species> 95%

Lozupone, Nature 2012 Tremendous phyla diversity but functional homogeneity 16S rRNA analysis of the mouth and gut: phylum level Shotgun Metagenomics: Functional homogeneity

The gut microbiota Remarkable variety within and between individuals Intra individual variations might be more important than inter individual Number of unique configurations is large No easy classification in a manageable number of distinct phenotypes

The human gut microbiota a mutually beneficial co-existence Digestion capabilities Production of and increasing access to essential nutrients Shapes the immune system

Neonatal maturation of Peyers Patches mesenterial lymphnodes, IgA producing B cells Differentiation of effector T and regulatory T cells Enhancement of bactericidal function of neutrophils Reduction of threshold for Dendritic Cell activation The microbiota shapes the immune system of our gut Alegre AJT 2014

> /ml bacteria separated by 10 μm over a surface area of 200 m 2

Components of the functional Barrier; commensal bacteria have an essential role Stratification -Mucus production by goblet cells -Anti Microbial Peptides -IgA molecules formed by plasma cells in the lamina propria Maintenance of tight junctions Competition with pathogenic strains Suppression of innate responses

Bacterial products can induce regulatory T cells

Outline presentation gut and its microbiota Terminology and normal function Changes in renal failure Solid organ transplantation Evidence and therapeutic potential Conclusions

Renal failure Increased oxalate and uric acid secretion by colonic epithelium Increased uremic toxins resulting in dysbiosis Increased NH 4 OH production Enhanced proteolytic fermentation pathway Th1 type response by direct binding of LPS to macrophages driving a TH1 and a TH17 response Decreased barrier function -Frequent endotoxemia -Translocation of micro organism

Uremia: intestinal barrier dysfunction Frequent endotoxemia in the absence of overt infection Detection of bacteria in mesenteric lymph nodes of uremic rats Increased intestinal permeability to uremic solutes Curr Opin Nephrol Hypertens 2012, 21:587–592

Uremic solutes are associated with Increased mortality in CKD Cardiovascular disease Progression of CKD Endothelial dysfunction Oxidative stress Microbiota in CKD Decreased diversity

Associations IgA and IBD Coeliakie and dermatitis herpetiformis both associates with HLA-DQ2 and or DQ8 Glutenpeptides (gliadine) HLA-DQ2/8 complexes evoke specific CD4 T cells resulting in inflammation and increased permeabilitiy for all other antigens -Tissue transglutaminase 2 beaks giladin down inimmunodominant peptides IgAN patients have increased intestinal permeability, increased IgA reactive with gliadine and other alimentary antigens (bovine, lactoglobulin) 20-30% IGAN in mice can be elicited by ovalbumin, bovine gamma globulin and horse spleen ferritin

Outline presentation gut and its microbiota Terminology and normal function Changes in renal failure Solid organ transplantation Therapeutic potential Conclusions

The microbiota and solid organ transplantation: an unexplored area Search for microbiota Search for organ transplantation AND microbiota

The microbiota and solid organ transplantation: only few facts Substantial changes prior and 6 months after renal and liver transplantation but wide inter-individual variation Decreased diversity in renal, lung, liver transplantation In gut transplantation Firmicutes abundance was correlated to rejection Fricke AJT 2013 Borewicz Microbiol Lett 2013 Wu Microb Ecol 2013 Oh AJT 2012

Less diversity as compared to references Fewer bacteroidetes in diarrhea Analysis of the microbiota in renal allograft recipients Lee et al Transplantation 2014 More lactobacillus in rejection

Enterococci in gut and urinary tract Lee Transplantation 2014

Important fields to explore in nephrology and solid organ transplantation Is there a link between the local gut alloresponse and the systemic alloresponse -Do intestinal T regs recognizing microbial peptides give rise to systemic effects? -Is there cross reactivity ? Ischemia reperfusion damage might be attenuated by short chain fatty acids Andrade–Oliveira J Am Soc Nephrol 2015 Microbiota might affect (immunosuppressive) drug metabolism

Outline presentation gut and its microbiota Terminology and normal function Changes in renal failure Solid organ transplantation Therapeutic potential Conclusions

Therapeutic arsenal Probiotics Postbiotics Fecal Microbiota Transplantation Prebiotics

Fecal Microbiota Transplantation First report by Ge Hong 4 th century in Zhou Hou Bei Ji Fang (Handy medicines in emergencies) Ralph Lewin: “fresh warm camel feces as Bedouin remedy against dysentery” Eiseman et al 1958: pseudomembranous colitis

Efficacy > 90 % Minor side effects Two renal transplant recipients

Can FMT eradicate the gut of ESBL E.Coli? Urinary tract infection the most frequent infection after renal transplantation Associated with loss of transplant function The incidence of ESBL E.Coli is rising, resulting in longterm admission for intravenous administration of antibiotics Feasibility Analysis of Nasoduodenal Feces Administration to eradicate Resistant Enterobacteriaeceae (FANFARE) Aim: Eradication of the gut of renal tranplant recipients from pathogenic E.coli

Urine Feces ♀ 59y 1982 postmortal renal transplantation Recurrent urinary tract infections (≥4x/y) Despite antibiotic treatment / prophylaxis Tx E.coli K.pneumoniae E.faecalis ESBL Non-ESBL Negative culture

KEY POINTS Renal failure and solid organ transplantation are associated with profound changes in the gut microbiota -Decreased diversity -Enhanced permeability -Possibly enhanced systemic toxins Fecal Microbiota Transplantation -effective eradication of Clostridium difficile (and possibly of other pathogenic strains) -Increases insulin sensitivity Microbiota and the alloresponse?

Work in progress

Safety Retrospective multicenter study 80 subjects -HIV/AIDS (3) -IBD (36) -SOT (19) -Oncologic condition (7) -Other (15) Efficacy after a single (78%) or double FMT (89%) Side effects : 1 death after sedation for colonoscopy, one unrelated Kelly American Journal of Gastroenterology 2014

SCFA functions Improvement of renal function after injury (IR) Less systemic inflammation, oxidative stress an apoptosis More autophagy In vivo: -Less HDAC activity -Less maturation of DC -Less CD4 and CD8 cell proliferation -Better mitochondrial biogenesis of kidney epithelium

Disrupted microbiota and disease Inflammatory Bowel Disease Less diversity Manichanh Gut 2006 Less SCFA producers Tedelind World Journal of gastroenterology 2007 No randomized trials but several underway Obesity and type 2 diabetes In humans more complicated than in mice Fecal Microbiotal Transplantation from lean donors enhance insulin sensitivity Vrieze Gastroenterology 2012

Therapeutic Potential of manipulation of the microbiota Eradication of dominant harmfull strains -Clostridium difficile -Pathogenic E. coli strains Restoring resilience against pathogenic strains -Less endotoxemia -Less uremic toxins -Less insulin resistance Manipulation of the alloimmune response Decreasing ischemia reperfusion damage -SCFA reduced inflammation Vinicius JASN 2015

Resilience better overcome by foreign microbiota than pathogenic or probiotic strains Lewis rats were forced fed 14 days with pooled microbiota of Sprague-Dawley and Wistar rats Microbiome increased in diversity and resembled that of latter two strains Stable for more than 3 months Pre antibiotic courses reduced the establishment of the foreign flora Manichanh Genome Res 2010