Pathophysiology of Peritoneal Transport

Michael F. Flessner, MD, PhD Bethesda, Maryland, USA

No Conflicts of Interest

Major Points I • There is no single “peritoneal membrane”. The

peritoneal barrier is made up of a microvasculature distributed within the cell-interstitial matrix of the tissue surrounding the peritoneal cavity.

• Trans-peritoneal transport is directly proportional to the area of peritoneum in contact with the solution.

• Solute transport occurs via diffusion and convection across endothelia and through interstitial matrix.

• Solute-free water transports from both blood capillaries and cells in peritoneal tissue via specialized water channels or aquaporins (AQP-1, AQP-3,-4?). Water transport also depends on the structure of the cell-interstitial matrix and lymphatics.

Major Points II • The endothelial glycocalyx lines the inter-endothelial

clefts and limits solute transport between plasma and interstitium and affects Starling Forces.

• The glycocalyx is sensitive to inflammatory cytokines and hyperglycemia, which alter the trans-endothelial permeability and may explain D/P changes with time on dialysis.

• Inflammation alters transport by angiogenesis and peritoneal sclerosis, often limiting fluid removal by altering the primary structures of the barrier: the endothelial surface area, the cell-interstitial matrix, and the peritoneal surface area of transfer.

From: The Visible Female

Peritoneal Cavity is a potential space, surrounded by a multitude of different tissues and cells.

From: Hepinstall’s Textbook of Anatomy

Where is the barrier?

Peritoneal Barrier of Abdominal Wall (HE; 200x)

Transport of solute and water

• Anatomy and Physiology of the peritoneal barrier to water and solutes • Importance of surface contact area • Net Ultrafiltration, lymph flow, and “wrong-way flow” • Role of Mesothelium • Interstitium: distributed osmosis • Blood Capillary: role of the aquaporin and endothelial

glycocalyx • Response of the glycocalyx to inflammation • Effect of angiogenesis on transport • Sclerosis of the peritoneum limits the surface area and water

transport but not solute transport

Topics

Peritoneal surface area in contact with the solution is an important variable in solute transport.

Rate of diffusion = -Deff x Area x dC/dx

≈ P x Area (Cblood - CPC)

Diffusion Equation

If the solution does not touch the tissue, transport does not occur across the peritoneum!!

Effect of Increased Dialysate Volume on Peritoneal Surface among Peritoneal

Dialysis Patients, Chagnac JASN 13:2554, 2002

• Measured the area of contact in 2 and 3 L dwells in 10 adult patients by infusion of contrast in the dialysate and multiple CT scans and a special stereologic technique

• With ~50% increase in volume, contact area increased by ~20% and MTACcreatinine increased by ~25%

Conclusion: Contact area, which is determined by the volume instilled, is a major determinant in rate of mass transfer across the peritoneum.

WRONG-WAY FLOW:

Flow from the cavity to the body: why does it occur?

volume drained volumeinNet Ultrafiltrationdialysis duration

−=

Fluid in the cavity increases pressure, which causes flow into the local tissues.

Flessner, AJP 1996

Durand Adv Perit Dial 8:22, 1992

Fluid loss during peritoneal dialysis (flow back to the patient) can amount to ~1.5-2 L/day.

Increases in PD dwell volume will increase IP pressure and may lead to a decrease in net UF.

• Anatomy and Physiology of the peritoneal barrier to water and solutes • Importance of surface contact area • Net Ultrafiltration, lymph flow, and “wrong-way flow”

• Role of Mesothelium • Interstitium: distributed osmosis • Blood Capillary: role of the aquaporin and endothelial glycocalyx

• Response of the glycocalyx to inflammation • Effect of angiogenesis on transport • Sclerosis of the peritoneum limits the surface area and water

transport but not solute transport

Topics

Dialysate fluid

Dialysate fluid

Blood flow

Capillary (3-pore) Model of Peritoneal Transport

Solute – water transport

Pore Theory cannot adequately model the peritoneal barrier!

Transport of solute and water

Distributed Concept of PD Transport

Intact peritoneum No peritoneum

Is the peritoneum a barrier to small solutes and water?

Flessner, PDI 23:542, 2003

0.50

1.00

mass

Flessner, PDI 23:542, 2003

Elimination of peritoneum does not alter water or solute transport between cavity and tissue

Conclusion:

The anatomic peritoneum is not a significant barrier to

small solutes.

But the peritoneum is important for the integrity of

the barrier.

Distributed Concept of PD Transport

Does the interstitium alter solute transport?

Flessner, AJP, 1985

What is the role for the Cell-Extracellular Matrix in osmotic filtration?

Distributed modeling of glucose induced osmotic flow Waniewski, Stachowska-Pietka et al AJP 296:H1960-68, 2009

Theorizes an Osmotic Resistance in the Interstitial-Cell Matrix`

c

• AQP1 plays an essential role in water permeability and ultrafiltration during PD Ni KI 69: 1518-1525, 2006.

• AQP1 is found in endothelial cells

• AQP4 is present in the entire plasma membrane of fast muscle fibers. AQP4 expression is associated with high water permeability and changes in muscle fiber volume. Frigeri Faseb J 18:905; 2004

Which Aquaporin play a role in water transport?

AQP1

AQP1 A

QP1

AQP4

KO

WT

WT WT

Yang and Verkman showed that AQP1 and AQP4 knockout mice decrease osmotic filtration by 60%. AQP1 was located primarily in endothelial cells, while AQP4 was located in the membrane of the underlying muscle cells.

Yang and Verkman AJP 276:C76, 1999

} Note Location

Hypothesized Cell-Extracellular Matrix Mechanism of Filtration Flow

CD31 stain - inflamed peritoneum

Water Flow AQP1

AQP?

Yang and Verkman AJP 276:C76, 1999

Yang and Verkman detected AQP3 in the peritoneum AJP 276:C76, 1999.

AQP1 AQP3 Anti-AQP3 MAb

Lai KN KI 62:1431-39, 2002

AQP3 is present in mesothelial cells and some underlying parenchymal cells in humans

HPMC respond to increasing concentrations of glucose by increasing mRNA for AQP3

Mechanism of Filtration Flow: upregulated AQP3 with interstitium?

CD31 stain - inflamed peritoneum

Water Flow

AQP3 ?

Interstitial-cell matrix presents a

significant barrier to the transport of solutes and water between plasma in distributed microvessels and the solution in the peritoneal cavity and results in far less efficient transport and dialysis.

The mechanism of water transport from the capillary to the cavity is still unknown.

• Anatomy and Physiology of the peritoneal barrier to water and solutes • Importance of surface contact area • Net Ultrafiltration, lymph flow, and “wrong-way flow” • Role of Mesothelium • Interstitium: distributed osmosis

• Blood Capillary: roles of the aquaporin and of the endothelial glycocalyx

• Response of the glycocalyx to inflammation • Effect of angiogenesis on transport • Sclerosis of the peritoneum limits the surface area and water

transport but not solute transport

Topics

Inter-Endothelial Cleft-Matrix Concept of Transport

Vink, Duling Circ Res 79:581, 1996.

• AQP-1 discovered Peter Agre Science 256:385, 1992.

• Trans-peritoneal UF in AQP1-KO mice demon-strated a decrease of 60% Yang AJP 276:C76, 1999.

• AQP-1 plays an essential role in water permeability and ultrafiltration during PD Ni KI 69: 1518-1525, 2006.

• Aquaporin-1 are transendothelial pores, but data over the last 10 years provides extensive evidence to support an additional barrier in the inter-endothelial cleft.

Aquaporin-1

Re-Discovery of Luminal Endothelial Glycocalyx

• Extracellular coating of anionic polysaccharides discovered on luminal surface of endothelia. Bennet J Histochem Cytochem 11:14, 1963

• Endothelial glycocalyx excluded blood from a layer 1.2 µm on the luminal surface and was suspected to influence transcapillary transport. Klitzman, Duling AJP 237:H481, 1979

Vink, Duling AJP 278:H285; 2000

Why change from pore theory to the science of the glycocalyx? • Glycocalyx limits permeation of

dextrans in a molecular size- and charge-dependent manner. Vink, Duling AJP 278:H285, 2000

• Damage of the glycocalyx leads to increases in capillary permeability. Vink AJP 290:H2174; 2006.

Revision of Starling’s Law

JR Levick J Physiol 557.3:704, 2004.

S Weinbaum, AJP Heart 291:2950, 2006.

Decreased Glycocalyx in angiogenic vessels in chronically exercised muscle

Brown et al Experimental Physiol 81:1043; 1996

• Examined sections of rat striated muscle stained with ruthenium red to examine glycocalyx before and after 2-4 days of electrical stimulation

• Before stimulation: glycocalyx continuous on 63%, absent on 13% capillaries

• After stimulation: glycocalyx continuous on 10%, absent on 44-58% of angiogenic capillaries

• Angiogenic vessels have less glycocalyx and therefore are more permeable. This would dissipate the glucose more rapidly.

100% >50%

<50% absent

EM: muscle capillaries (% coverage of Endothelium) Exp Physiol 81:1043, 1996

1 µm

Endothelial Glycocalyx

Glycocalyx may decrease the effective osmotic pressure driving ultrafiltration.

• Anatomy and Physiology of the peritoneal barrier to water and solutes • Importance of surface contact area • Net Ultrafiltration, lymph flow, and “wrong-way flow” • Role of Mesothelium • Interstitium: distributed osmosis • Blood Capillary: role of the aquaporin and endothelial glycocalyx

• Response of the glycocalyx to inflammation • Effect of angiogenesis on transport • Sclerosis of the peritoneum limits the surface area and water

transport but not solute transport

Topics

Can endothelial glycocalyx explain observed increased transport during inflammatory states or peritonitis?

• Damage of the glycocalyx due to: oxidized lipoproteins, heparitinase, fluid shear stress, adhesion of WBCs and platelets, cytokines, and ischemia-reperfusion leads to increases in capillary permeability.

• Vink AJP 290:H2174; 2006.

Alteration of Glycocalyx Increases Microvascular Permeability

Acute or chronic increase of glucose to 25 mM in mice (6 x normal) results in marked increase in permeability to 70 kDa dextran and is correlated with glycocalyx alterations.

Zuurbier J Appl Physiol 99:1471, 2005

Damage to Glycocalyx in Clinical Hyperglycemia

• Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and rapid loss of a macromolecular marker in 10 healthy males. Nieuwdorp Diabetes 55:480; 2006

• Endothelial glycocalyx damage coincides with microalbuminuria in Type I DM Nieuwdorp Diabetes 55:1127, 2006

Angiogenic vessels have less glycocalyx and therefore are more permeable. This would dissipate the glucose more rapidly in chronically-inflamed peritoneum, leading ultimately to poor ultrafiltration.

After 8 weeks of exposure to a glucose-based solution

Entire Cohort Low Glucose

High Glucose

Icodextrin

No Icodextrin

Exposure to Glucose increases D/P Cr and decreases UF over time

Davies et al. KI 67:1609, 2005

Mechanism for the observed increase in D/P after years on hypertonic dialysis?

• Evidence from basic research demonstrates the

importance of the glycocalyx to trans-endothelial transport.

• Inflammation, ischemia-reperfusion, hyperglycemia, and angiogenesis alter the glycocalyx and increase endothelial permeability.

• The effect of hyperglycemia on endothelial permeability could be the mechanism for increase in D/P creatinine and decrease of D/D0 for glucose with time on dialysis.

Dark brown = CD31

100 µm

Does inflammation result in a loss of Aquaporin?

Are all of the new vessels in the sub-compact zone perfused?

Do they contain aquaporin?

Angiogenesis resulting from chronic inflammation

Water channels play a fundamental role in cell migration. Saadoun Nature 434:786792, 2005

• Aortic endothelia, harvested from wild-type and from AQP-1 deficient mice, were grown in primary cultures.

• Cell adhesion and proliferation were similar • Cell migration was severely impaired in

AQP-1 deficient cells. • Transfection of AQP-1 into non-

endothelial cells accelerates cell migration and wound healing, in vitro.

Aquaporins in Endothelia Verkman KI 69:1120-3, 2006

Conclusion from Studies of Endothelial Proliferation

• Angiogenesis resulting from inflammation absolutely depends on the presence of AQP1. Therefore AQP1 deficiency is unlikely in chronic inflammation in the peritoneum.

Normal Expression of Aquaporin-1 in a Long-Term Peritoneal Dialysis Patient with Impaired

Transcellular Water Transport Goffin AJKD 33:383, 1999

AQP1-Staining Avascular “Tanned” Peritoneum

~500 µm

Note fibrotic layer

Expression of Aquaporin-1 in a Long-Term Peritoneal Dialysis Patient with Impaired

Transcellular Water Transport Goffin AJKD 33:383, 1999

controls case peritonitis

• Anatomy and Physiology of the peritoneal barrier to water and solutes • Importance of surface contact area • Net Ultrafiltration, lymph flow, and “wrong-way flow” • Role of Mesothelium • Interstitium: distributed osmosis • Blood Capillary: role of the aquaporin and endothelial glycocalyx

• Response of the glycocalyx to inflammation • Effect of angiogenesis on transport

• Sclerosis of the peritoneum limits the surface area and water transport but not solute transport

Topics

Normal Control After 9 years of PD Compact mesothelial

zone

500 µm 500 µm

Williams JD et al, JASN 13:470, 2002.

Long-Term Effects of PD

How does the avascular, acellular scar alter transport of solute and water?

Abnormal interstitial-cell matrix does not transport water to the cavity

• Avascular submesothelial compact zone markedly decreases the effective osmotic pressure near the exchange microvessels

• Increased perfused vascular area, which may be hyper-permeable, exacerbates the UF rate by dissipating the osmotic gradient rapidly in the vicinity of the exchange microvessels

Major Points I • There is no single “peritoneal membrane”. The

peritoneal barrier is made up of a microvasculature distributed within the cell-interstitial matrix of the tissue surrounding the peritoneal cavity.

• Trans-peritoneal transport is directly proportional to the area of peritoneum in contact with the solution.

• Solute transport occurs via diffusion and convection across endothelia and through interstitial matrix.

• Solute-free water transports from both blood capillaries and cells in peritoneal tissue via specialized water channels or aquaporins (AQP-1, AQP-3,-4?). Water transport also depends on the structure of the cell-interstitial matrix and lymphatics.

Major Points II • The endothelial glycocalyx lines the inter-endothelial

clefts and limits solute transport between plasma and interstitium and affects Starling Forces.

• The glycocalyx is sensitive to inflammatory cytokines and hyperglycemia, which alter the trans-endothelial permeability and may explain D/P changes with time on dialysis.

• Inflammation alters transport by angiogenesis and peritoneal sclerosis, often limiting fluid removal by altering the primary structures of the barrier: the endothelial surface area, the cell-interstitial matrix, and the peritoneal surface area of transfer.

Thank you for your attention!

Questions?

Glycocalyx-Endothelial Cleft Theory of Trans-Capillary Transport

Vink, Duling Circ Res 79:581, 1996.

(dense glycocalyx)

(less dense glycocalyx)