protecting the peritoneal membrane: future...

Protecting the Peritoneal Membrane: Future Strategies

Michael F. Flessner, MD, PhD Bethesda, Maryland

No Disclosures

Take-Home Points • Currently used dialysis solutions, even so-called

“biocompatible” ones do not promote peritoneal health. Are there alternatives to glucose?

• The PD catheter acts as a foreign body and elicits significant inflammation within hours after insertion. Needed: better biomaterials that preclude biofilm formation.

• Patient genetics affect peritoneal function, opening the door for a “personalized medical approach” to the selection of PD as a dialysis modality and future care of a specific patient (PDOPPS). Expression profiling (miRNA) may provide new diagnostic and therapeutic targets.

• Novel pharmacologic measures to preserve the peritoneum need clinical evaluation in patients.

Topics

• Theory • Solutions • Catheter • Genetics • Pharmacology

Needed: Comprehensive Understanding of:

Overview of Wound Repair and Fibrosis Infections

Toxins Drugs

Trauma

Epithelial/ endothelial damage

Recurrent inflammation

TSLP IL-25 IL-33

IL-1 IL-6 TNF

IL-1, TNF, free radicals, coagulation pathway activation,

EMT

Adaptive immune activation

Innate immune

activation

Fibroblast activation

Treg

TH2

TH17

IFN-γ

PMN

EOS Macrophage

TH1 B

Ab

Baso Mast IL-18 TGFβ1 IL-17

↑ Smooth muscle actin ↑ Collagen synthesis ↑ Matrix deposition

Inflammation and cell recruitment

Myofibroblast activation Tissue repair

Persistent myofibroblast activation

Irritant removed?

NO

YES Healing and homeostasis

Persistent

irritant

Fibrosis

Fibrosis

Wynn TA Nature Med 18:1028, 2012

Katie

Vica

ri

IL-5 IL-4

GM-CSF

IFN-γ

LPS

IL-17

IL-13 LMWHA

AAM CAM EPO MBP EDN

Arginase Relm-α Chi3l3 MMP2 MMP9

TGF-β1

iNOS TNF-α ROS IL-1β

RNS ROS Phagocytosis

Eosinophil Macrophage Neutrophil

Macrobicidal

activity Myofibroblast

activation

Microbicidal activity

Repair? Fibrosis?

Katie

Vic

ari

Naive CD4+

T cell

Intracellular bacteria, viruses,

protozoa

IL-12, IFN-γ

Extracellular bacteria,

fungi

TGF-β, IL-6

Intrinsic or converted

TGF-β Extracellular helminths,

fungi, allergens

IL-25, IL-33, TSLP

TH1 TH17 Treg TH2

IL-2, IFN-γ,

IL-13

IL-17A, IL-17F,

IL-10, TGF-β1,

IL-4, IL-5,

TNF-α Rx

IL-22 IL-35 Rx IL-13 IL-13Rα2

Rx

Increased

Increased Decreased Increased

• Monocytes/ macrophages

• iNOS • ROS • Necrosis

• Intracellular

• PMN • ROS • Cell death

• Extracellular • Microbicidal

• Inflammation

• Profibrotic?

• Monocytes/

macrophages • Eosinophils • Mast cells • Arginase-1 • RELM-α • MUC5AC

• Microbicidal Failure to resolve insult

• Apoptosis

Fibrosis

• Extracellular

parasite killing • Wound

Adaptive Immune Pathways in Fibrosis

Innate Immune Cells in Fibrosis

Wynn TA Nature Med 18:1028, 2012

There are multiple pathways and cells involved in fibrosis.

Role of Tyrosine Kinase Receptors in Peritoneal Fibrosis Wang L, Zhuang S PDI 35:497-505; 2015

Another Part of the Puzzle

FIBROSIS IN CKD Grgic, Ped Neph 2012

Is peritoneal fibrosis like that of the kidney, liver, lung, and skin?

?

?

?

Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury

JASN 25:2847-2858, 2014 Yi-Ting Chen,*†‡ Yu-Ting Chang,* Szu-Yu Pan,† Yu-Hsiang Chou,*† Fan-Chi Chang,*† Pei-Ying Yeh,* Yuan-Hung Liu,§ Wen-Chih Chiang,† Yung-Ming Chen,† Kwan-Dun Wu,† Tun-Jun Tsai,† Jeremy S. Duffield,| and Shuei-Liong Lin*† National Taiwan University

• Used inducible genetic fate mapping to trace cells after inflammation in 3 transgenic mouse (C57BL6) animal models (hypochlorite, hyperglycemic solutions, TGF-β1)

• Demonstrated mesothelial cells repair the mesothelium and myofibroblasts arise from submesothelial fibroblasts

• Found a possible target (PDGFRβ) for blockade to attenuate fibrosis

Chen YT et al JASN 2014

Model of Peritoneal Inflammation/Fibrosis

80% labeled

65% labeled

More basic research is needed using lineage tracing and other

molecular manipulations in order to sort out mechanisms and develop new targets for

combating peritoneal fibrosis.

Topics


Control daily 4% Glucose

micrographs at 200x

Chronic Exposure to Biocompatible (pH=7.4, low GDP) Glucose Solution

Flessner AJP 2007

-10 x thicker -Angiogenesis -Extensive matrix deposition -Many activated myofibroblasts

8 weeks

Clinical Research: Search for Alternative Osmotic Agent to Glucose

• Does not produce GDP • Does not produce metabolic changes • Does not increase uremia • Does not inflame or alter the peritoneal

barrier • Exerts effective osmotic pressure over 4-6

hours • Is relatively inexpensive

HYPERBRANCHED POLYGLYCEROL IS AN EFFICACIOUS AND BIOCOMPATIBLE NOVEL OSMOTIC AGENT IN A RODENT MODEL OF

PERITONEAL DIALYSIS Mendelson AA et al PDI 33:15; 2013 Du C et al Biomaterials 35:1378; 2014

• Tested HPG, 3kDa polymer in 2.5-15% concentrations (osmolality: 279-424 mOsm/kg) in same electrolyte solution as Dextrose solution

• Compared UF, urea clearance to 2.5% Dextrose: both were superior at 7.5-15% to the 2.5% D solution

• Histology of abdominal wall and WBC flow cytometry demonstrated better biocompatibility

HPG: Questions/Concerns prior to Human Testing

• Metabolism of hyperbranched polyglycerol?

• Use in uremic animals? • Multiple exchanges: efficacy and

biocompatibility? • Toxicity? Deposition in cells/organs? • Rodents vs humans?

Topics


PD Catheter = Foreign Body

Progressive inflammatory response over 1-7 days from sterile polyethylene

catheters in rats E Gomez-Sanchez-Flessner ASN 2010

Trichrome

control 1 day 3 days 7 days

Mouse Adherent Cell Layer at 2 weeks on Polyethylene Catheter

Bacteria on Catheters in patients undergoing PD Pihl M PDI 2013;33:51

• Cultured cells from catheters of 15 patients without signs of infection

• Visualized bacteria with confocal microscopy • Detected bacteria on 12/15 catheters • Most common: Staph Epidermidis • Up to 4 different species on some catheters

• These may provide a continuous source of inflammation and subsequent peritonitis.

Staphylococcal biofilm on peritoneal catheter Dasgupta Sem Dial 15:338, 2002

Can we decrease or eliminate inflammation induced by the

catheter? Can we decrease or eliminate

bacterial biofilm?

Molecular Determinants of Biocompatibility

Tang, Hu Expert Rev. Med. Devices 2005, 2:493

• Host cells interact predominantly with adsorbed plasma proteins, which have altered conformations and present sites for cell binding.

• Exposure of the P1/P2 epitopes of fibrinogen bound to biopolymers may be critical for binding of inflammatory cells.

• Different biopolymers have different affinities for protein-cell interactions.

• Blockade of protein sites alters cell binding.

PET=dacron; PE=polyethylene; PVC=polyvinyl chloride; PEU = polyurethane; PDMS = polydimethyl siloxane

Hu, Eatorn, Tang Blood 98:1231, 2001.

Quantitative binding site measurement versus material

Phagocyte accumulation on various biomaterials.

Biocompatible?

Surface Modification of Silicone for Biomedical Applications Requiring Long-Term Antibacterial, Antifouling, and

Hemocompatible Properties Li M et al: Langmuir 28:16408; 2012

• Covalently grafted poly(poly(ethylene glycol) dimethacrylate) – P(PEGDMA)) to medical silicone

• Added polysulfobetaine polymer (P(DMAPS)) to enhance anti-fouling

4 hr Exposure of polymers to S. aureus ( ) Li et al; Langmuir 28:16408; 2012 810 /cells ml

10 μm

3T3 Fibroblast adherence to polymers (24 hr) Li et al Langmuir 28:16408; 2012

200 μm

Platelet adherence to polymers (1 hour) Li et al Langmuir 28:16408; 2012

10 μm

Antifouling and Antibacterial Multifunctional Polyzwitterion/Enzyme Coating on Silicone

Catheter Material Prepared by Electrostatic Layer-by-Layer Assembly Vaterrodt A et al Langnuir 2016

• Utilized 3-layers: – copolymer with zwitterion/quaternary ammonium side

groups – Contact biocidal derivative of that polymer with octyl

groups – Antibacterial hydrogen peroxide producing enzyme

cellobiose dehydrogenase – Assembled these layer by layer

• Decreased bacterial adherence by 60%

Catheter Inflammation and Biofilm

• Remains a major problem for IP, IV, and urethral applications

• Biomaterials needed to decrease protein adsorption and cell adhesion

• NIDDK SBIR: PA-13-050 and PA-13-051: Improved Biomaterials for Urinary and Dialysis Catheters

Topics


Uremia alone alters genetic pathways: Immunity (Table 3 from: R Scherer Alteration in the human blood cell transcriptome BMC Medical Genomics 2013 6:23.) Principal gene pathways altered in uremia p-value Ratio* Transport: Clathrin-coated vesicle cycle 8.039E-23 60 / 71 Cytoskeleton remodeling: TGF, WNT 2.990E-19 77 / 111 Cytoskeleton remodeling: Cytoskeleton remodeling 3.226E-17 70 / 102 Development: EPO-induced Jak-STAT pathway 2.658E-16 33 / 35 Translation: Regulation of EIF4F activity 2.083E-15 43 / 53 Chemotaxis: CXCR4 signaling pathway 2.445E-14 31 / 34 Development: GM-CSF signaling 4.953E-14 40 / 50 Immune response: T cell receptor signaling pathway 5.938E-14 41 / 52 Immune response: IL-2 activation and signaling path 1.410E-13 39 / 49 Oxidative phosphorylation 1.787E-13 66 / 105 Immune response : Immunological synapse formation 2.407E-13 44 / 59 Development: Flt3 signaling 2.595E-13 36 / 44 Signal transduction: Activation of PKC via G protein-coupled receptor 5.244E-13 40 / 52 Cell cycle: Influence of Ras and Rho proteins on G1/S Transition 1.552E-12 40 / 53 Immune response: Role of DAP12 receptors in NK cells 4.346E-12 40 / 54 Immune response: BCR pathway 4.346E-12 40 / 54 Transcription: NF-kB signaling pathway 4.945E-12 32 / 39 Development: PIP3 signaling in cardiac myocytes 9.777E-12 36 / 47 Development: signaling pathway 1.026E-11 44 / 63

Some patients appear more inflamed. In a study of a subset of the EAPOS cohort of 233, 13 of 22 patients displayed stable solute transport over 5 yrs, while the other 9 had progressive increase in solute transport. The latter group had earlier loss of residual function and required significantly more hypertonic solution. There were no differences in rates of peritonitis between groups. Davies SJ. JASN 12:1046, 2001.

Can we predict who will do poorly on PD, based on their

genetics?

Genetic Polymorphisms and Peritoneal Membrane Function

Siddique I; Margetts PJ PDI 35:517; 2015

• Systematic review of literature for rapid transport status, risk of peritonitis, EPS, patient/technique survival

• 18 relevant studies – Solute transport correlated with:

• IL-6 • Endothelial nitric oxide synthase and receptor for AGEs

– 2 studies demonstrated predisposition for peritonitis – 1 study found predisposition for EPS – Survival ~ VEGF polymorphism – Technique failure ~ IL-1 receptor antagonist

• Most consistent association was transport and IL-6 gene polymorphisms

MicroRNA Expression Profiling in Peritoneal Fibrosis Morishita Y et al Transl Res 2015

• Screened tissue from rat peritoneum for miRNAs • Profiling of PF rat peritoneal tissue 6 miRNAs • Examined expression levels of miRNAs in serum and

drained dialysate and correlated these with PD transport in 33 patients

• Serum levels of 3 miRNAs correlated with PD transport • Drained dialysate levels of 2 miRNAs correlated as well • Anti-miRNA-21-LNA significantly inhibited peritoneal

fibrosis thickening in the PF mouse model • Results suggest miRNAs may be novel diagnostic

biomarkers and possible therapeutic targets

Genomics • Small studies (N = 50-200 participants) of

individual SNPs will not likely yield useful results to classify at-risk patients.

• We will likely know a lot more about genetic predisposition in a few years: PDOPPS (N=1000s)

• Expression profiling (miRNA) of tissue biopsies, serum or drained dialysate levels and their correlation with peritoneal fibrosis or transport may provide new diagnostic tools as well as new therapeutic targets

Topics


Can we use additives to current solutions to decrease inflammation and damage to

the peritoneum?

Agent

EMT

Fibrosis

Histology Angiogenesis

AGEs

Small-solute transport rate

Inflammation

Disodium cromoglycate

—

↓

↓

—

—

↓

PPARG agonists ↓ ↓ ↓ ↓ ↓ ↓

Aminoguanidines — ↓ ↓ ↓ ↓ —

Alagebrium — — — ↓ ↓ —

Zopolrestat — ↓ ↓ — — —

Pyridoxamine — — ↓ ↓ ↓ —

Benfotiamine — — ↓ ↓ ↓ ↓

ACE inhibitor or ARB — ↓b ↓ — ↓b ↓

Statins — ↓ — — ↓ —

COX2 inhibitors — ↓ ↓ — ↓ ↓

Vitamin D or VDR activators — — — — ↓b —

Bone morphogenic protein 7 ↓ ↓ ↓ — — —

Sunitinib — — ↓ — = —

Pharmacologic Targets and Peritoneal Remodeling (Farhat et al PDI 2014; 34:114)

Novel Additives to Current PD Solutions

• Decorin (proteoglycan inactivates TGF-β) via gold nanoparticles or adenovirus decreases peritoneal fibrosis in an in vivo rodent model. Chaudhary K AJP 307:F777; 2014

Additives (cont’d) • Astragalus membranaceus inhibits

peritoneal fibrosis via MCP-1 and TGF- β1 pathway in rats undergoing PD. Li Z, et al Intl J Mol Sci 15:12959, 2014

control PD PD+Astragalus

Additives (cont’d)

• MicroRNA-29b inhibits peritoneal fibrosis in mouse model of PD Yu JW et al Lab Invest 94:978;2014

UT = untreated EV = empty vector miR-29b-vector

While these pre-clinical studies are promising, each of these

agents require clinical confirmation in FDA-qualified

trials.

Take-Home Points • Currently used dialysis solutions, even so-called

“biocompatible” ones do not promote peritoneal health. Are there alternatives to glucose?

• The PD catheter acts as a foreign body and elicits significant inflammation within hours after insertion. Needed: better biomaterials that preclude biofilm formation.

• Patient genetics affect peritoneal function, opening the door for a “personalized medical approach” to the selection of PD as a dialysis modality and future care of a specific patient (PDOPPS). Expression profiling (miRNA) may provide new diagnostic and therapeutic targets.

• Novel pharmacologic measures to preserve the peritoneum need clinical evaluation in patients.

Thank you for your attention!

Questions?

protecting the peritoneal membrane: future...

Documents