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Perit Dial Int 29(Supplement_2): 36-39
2009
© 2009 International Society for Peritoneal Dialysis
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Part 2: Cellular and Molecular Biology of the Peritoneum and Peritoneal Dialysis

HISTOLOGY IN EXPERIMENTAL PERITONEAL DIALYSIS: THE LINK BETWEEN STRUCTURE AND FUNCTION

Lazaro Gotloib

Laboratory for Experimental Nephrology, Ha'Emek Medical Center, Afula, Israel

Correspondence to: L. Gotloib, Department of Nephrology, Ha'Emek Medical Center, Afula 18101, Israel. gotloib{at}012.net.il

Microscopic observation offers the possibility to investigate the relationship between structure and function of the peritoneum as a living dialyzing membrane. The most commonly used complementary techniques are transmission electron microscopy (TEM) and light microscopy, the latter applied to imprints of the mesothelial monolayer and/or to biopsy samples of the peritoneum.

This short review will offer some examples indicative of the relevance and assortment of information that can be acquired using these techniques.


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The high magnification power of TEM makes it appropriate for studying the individual cell and its intracellular organelles, plasmalemma, intercellular junctions, and basement membrane, as well as the interstitial connective tissue where blood and lymphatic microvessels are located. However, a powerful magnification has the shortcoming of covering limited areas of tissue. Therefore, TEM is not suitable for running large enough investigations based on cell population analysis.

The fact that intracellular organelles can be easily visualized with TEM prompted us to investigate the transperitoneal traffic of macromolecular serum albumin (1). Five minutes after intraperitoneal injection of albumin gold to intact mice, mesenteric mesothelial cells showed particles of the tracer in contact with the mesothelial cell luminal plasmalemma and, much more relevant, located in caveolae and transcellular channels. The fact that albumin gold particles were also detected in the abluminal side of the mesothelium in non-junctional areas strongly suggested that a kind of caveolar transport belt was behind the transcellular traffic of the tracer. Similar images were observed in the endothelial layer of continuous and fenestrated blood mesenteric capillaries.

A large body of literature, old as well as recent, supports the concept that caveolae are involved in the transcellular traffic of macromolecules (2,3). This concept has been challenged recently by experiments performed in microvascular endothelium of caveolin-1-deficient mice (4,5). However, it has been shown that reduced expression of caveolin-1 accompanied the diminished expression of the tight junction-associated proteins occludin and zonula occludens-1. Caveolin-1 loss appears as a critical step in the modulation of brain and lung microvascular endothelial cells' junctional protein expression and integrity (6,7). As a consequence, caveolin-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane, leading to abnormally increased capillary permeability to albumin through leaky intercellular junctions (8). Additionally, this specially customized mouse displays a substantially increased production of inflammatory cytokines, chemokines, and other mediators of inflammation (9). Both elements, interendothelial gaps and high concentrations of inflammatory mediators, characterize the acute inflammatory reaction described in experimental animals (10), as well as in peritoneal dialysis patients during peritonitis (11). Consequently, it is unlikely that observations made in caveolin-1-deficient mice can be extrapolated to wild-type mice in a situation of normal physiology.

Additional TEM studies have shown that the peritoneum is a negatively charged membrane (12). Cationic tracers were detected decorating the glycocalyx located on the cavitary aspect of mesothelial cells, in the infundibulum of tight junctions, as well as along the submesothelial basement membrane. A similar distribution was observed in mouse mesenteric, submesothelial, continuous, and fenestrated blood capillaries. The relevance of these charges is still debated. According to Flessner, the microvascular glycocalyx plays a relevant role in peritoneal permeability, limiting solute and water transport between the blood compartment and the interstitial tissue (13). If so, the same criteria should be extended to the mesothelial glycocalyx. Conversely, Rippe (5) invalidates the relevance of the glycocalyx vis-à-vis peritoneal permeability.

The use of cationic tracers applied to investigation of changes in peritoneum derived from experimentally induced long-standing diabetes (6 months) revealed a substantial reduction in mesothelial and microvascular negative charges, which was especially marked along both the submesothelial and the subendothelial basement membranes (14). These alterations coincided with a substantial increase in peritoneal permeability to albumin, evaluated by measuring clearances as well as by determining albumin concentration in the mesenteric interstitial tissue (14).

Additional studies performed in rats exposed to the effect of Escherichia coli acute peritonitis showed similar changes along both basement membranes that concurred with permeability changes comparable to those seen in diabetic animals (15,16).

Not a few studies provide evidence supporting the concept that negative charges placed along the capillary basement membrane modulate the permselectivity of the microvascular wall to macromolecular albumin in continuous and fenestrated microvessels (17,18). The relevance of this classic concept, applied at least to glomerular capillaries, has been questioned recently by some investigators, giving rise to one more controversy (19).


   LIGHT MICROSCOPY
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 LIGHT MICROSCOPY
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The weak point of light microscopy, the substantially lower magnification power than TEM, is counterbalanced by the fact that this technique enables observations of considerably larger areas of tissue. The technique of imprints (20) is the most useful tool for analyzing the life cycle, regeneration, and transplantation of the mesothelial cell population, as well as its reactions to environmental changes.

Using this experimental model and applying techniques of cytochemistry, it has been calculated that mesothelial cells undergo three to four rounds of mitosis before reaching terminal replicative senescence (21). These figures are not far from the average of six replications observed in cultured cells (22). An unbroken sequence of a cell's life cycle is a basic requirement for maintaining the homeostatic population of cells and is critical for setting in motion the mechanisms of repopulation of the monolayer after injury. These cells are particularly sensitive to environmental changes and, when they arise, the cell cycle becomes the main victim. Here lie the foundations of the biocompatibility problem. The use of glucose is associated with a substantial acceleration of the mesothelial cell cycle launched by a degree of oxidative stress (21). The use of icodextrin, in turn, by inducing a higher degree of oxidative injury, results in an underpopulated senescent population of mesothelial cells expressing 8-deoxyguanosin as a clear sign of DNA damage. Coincidentally, both senescence and depopulation lead to replacement of the monolayer by fibrous tissue, the starting point of a chain of tissue reactions, the end of which is membrane failure. This development can be easily evaluated by taking biopsies from the anterior surface of the liver, where the thin mesothelial monolayer (0.6 – 2 µm thickness), including the basement membrane, appears lying on the liver tissue. Biopsies from this part of the visceral peritoneum allow evaluation of early [Figure 1(a)] and late fully developed fibrosis [Figure 1(b)], as well as neovascularization and changes in microvascular walls. Samples taken from the anterior abdominal wall are less suitable for quantitatively assessing the magnitude of fibrosis since the limit between the submesothelial connective tissue and that belonging to the abdominal wall usually appears ill defined.


Figure 1
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  		Figure 1 — In a biopsy of the peritoneum dressing the anterior liver surface, taken from a rat after 30 days' exposure to 4.25% glucose peritoneal dialysis fluid (PDF), the open arrow indicates a small area of early fibrous repair, the black arrows indicate mesothelial cells, and the white star indicates liver tissue (A) [hematoxylin and eosin (H&E); x400 magnification]. In a biopsy of liver peritoneum obtained from a rat that, after focal exfoliation of the mesothelium, was exposed to one daily intraperitoneal injection of high glucose PDF for a period of 30 consecutive days, the open star indicates the thick layer of fibrous tissue that replaced the normally present monolayer and the black star indicates liver tissue (B) (H&E; x400). In a microphotograph taken from rat mesothelial cells cultured for 5 days, the circle surrounds one of the many elongated, fibroblast-like cells positively stained with vimentin (C) (H&E;x400). In an imprint recovered from an intact unexposed rat, the arrow indicates a fibroblast-like cell lying on the peritoneal surface (D) (H&E; x1000).

 
As stated previously, preservation of a normal cell cycle keeps mesothelial regeneration going. This process can be followed sequentially using the technique of imprints. Three complementary mechanisms, the proportional contributions of which remain unknown, have been proposed (11):

  1. Free mesothelial cells wandering in the peritoneal fluid become implanted in depopulated peritoneal areas. The existence of this mechanism finds support from the feasibility of mesothelial cell transplantation pioneered by Di Paolo (23). This intervention can also be sequentially followed using the imprint technique with the monolayer.
  2. Cells limiting depopulated zones replicate and migrate, filling the gaps. Evidence supporting this mechanism has been shown sequentially, following repopulation of the monolayer after experimental localized exfoliation (24).
  3. New cells arising from the submesothelial connective tissue migrate to the peritoneal surface. Evidence in favor of this concept is provided by observation of biopsies from human patients, and by young elongated mesothelial cells, apparently migrating in order to repopulate areas of denuded monolayer, in rat mesothelial cells in culture [Figure 1(c)], as well as on the peritoneal surface of intact unexposed rats [Figure 1(d)]. Morphologically, these fish-like cells appear similar to those described undergoing epithelial-to-mesenchymal transition (25) that appear to be involved, in concert with activated local resident fibroblasts, in the development of peritoneal fibrosis, derived from the use of bioincompatible dialysis solutions. Most likely, the fate of these elongated cells is dictated by the nature, length, and intensity of environmental changes, leading them, in turn, to perform the specifically required task. This interpretation is based on the remarkable plasticity of mesothelial cells, which, being pluripotent, are endowed with the capability of committing themselves in order to generate different cell lines of mesenchymal origin (26). This property justifies the proposed use of mesothelial cells as a potential source of adult stem cells (26).

In summary, all these complementary techniques are useful tools that enable implementation of investigating efforts linking structure with physiology, mesothelial regeneration, biocompatibility of dialysis solutions, as well as mesothelial cell transplantation.


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  1. Gotloib L. Endocytosis and transcytosis of albumin gold through mice peritoneal mesothelium. Kidney Int1995; 47:1274 -84.[Medline]
  2. Palade GE, Simionescu M, Simionescu N. Structural aspects of the permeability of the microvascular endothelium. Acta Physiol Scand 1979; 463:11 -32.
  3. Predescu SA, Predescu DN, Malik AB. Molecular determinants of endothelial transcytosis and their role in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 2007;293 : L823-L842.[Abstract/Free Full Text]
  4. Rosengreen BI, Rippe A, Rippe C, Venturoli D, Sward K, Rippe B. Transvascular protein transport in mice lacking endothelial caveolae. Am J Physiol Heart Circ Physiol 2006;291 : H1371-H1377.[Abstract/Free Full Text]
  5. Rippe B. Does an endothelial surface layer contribute to the size selectivity of the permeable pathways of the three-pore model? Perit Dial Int 2008;28 : 20-4.[Free Full Text]
  6. Song L, Ge S, Pachter JS. Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells. Blood 2007; 109:1515 -23.[Abstract/Free Full Text]
  7. Schubert W, Frank PG, Woodman SE, Hyogo H, Cohen DE, Chow CW, et al. Microvascular permeability in caveolin-1 (-/-) knock-out mice. Treatment with a specific nitricoxide synthase inhibitor, L-NAME, restores normal microvascular permeability in Cav-1 null mice. J Biol Chem 2002; 277:40091 -8.[Abstract/Free Full Text]
  8. Frank PG, Woodman SE, Park DS, Lisanti M. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol 2003; 23:1161 -8.[Abstract/Free Full Text]
  9. Medina FA, de Almeida CJ, Dew E, Li J, Bonuccelli G, Williams TM, et al. Caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection. Infect Immun 2006;74 : 6665-74.[Abstract/Free Full Text]
  10. Majno G, Palade GE. Studies on inflammation: I. The effect of histamine and serotonin on vascular permeability. An electron microscope study. J Biophys Biochem Cytol 1961;11 : 571-605.[Medline]
  11. Gotloib L, Shostak A, Wajsbrot V. Functional structure of the peritoneum as a dialyzing membrane. In: Gokal R, Khanna R, Krediet R, Nolph K, eds. Textbook of Peritoneal Dialysis. 2nd ed. Dordrecht: Kluwer Academic; 1994: 37-106.
  12. Gotloib L, Shostak A, Jaichenko J. Ruthenium-red stained anionic charges of rat and mice mesothelial cells and basal lamina. The peritoneum is a negatively charged dialyzing membrane. Nephron1988; 48:65 -70.[Medline]
  13. Flessner M. Endothelial glycocalyx and the peritoneal barrier. Perit Dial Int 2008;28 : 6-12.[Abstract/Free Full Text]
  14. Shostak A, Gotloib L. Increased peritoneal permeability to albumin in streptozotocin diabetic rats. Kidney Int1996; 49:705 -14.[Medline]
  15. Gotloib L, Shostak A, Jaichenko J. Loss of mesothelial electronegative charges during murine septic peritonitis. Nephron 1989; 51:77 -83.[Medline]
  16. Shostak A, Gotloib L. Increased mesenteric, diaphragmatic, and pancreatic interstitial albumin content in rats with acute abdominal sepsis. Shock 1998; 9:135 -7.[Medline]
  17. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J Cell Biol1980; 86:688 -93.[Abstract/Free Full Text]
  18. Haraldsson B, Nystrom J, Deen WM. Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev2008; 88:451 -87.[Abstract/Free Full Text]
  19. Comper WD, Haraldsson B, Deen WM. Resolved: Normal glomeruli filter nephrotic levels of albumin. J Am Soc Nephrol2008; 19:427 -32.[Abstract/Free Full Text]
  20. Whitaker D, Papadimitriou JM, Walters I. The mesothelium: techniques for investigating the origin, nature and behaviour of mesothelial cells. J Pathol 1980;132 : 263-71.[Medline]
  21. Gotloib L, Shostak A, Wajsbrot V, Kushnier R. High glucose induces a hypertrophic, senescent mesothelial cell phenotype after long in vivo exposure. Nephron 1999;82 : 164-73.[Medline]
  22. Ksiazek K, Piwocka K, Brzezinska A, Sikora E, Zabel M, Breborowicz A, et al. Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence. J Appl Physiol 2006;100 : 988-95.[Abstract/Free Full Text]
  23. Di Paolo N, Vanni L, Sacchi G. Autologous implant of peritoneal mesothelium in rabbits and man. Clin Nephrol1991; 57:323 -31.
  24. Gotloib L, Wajsbrot V, Shostak A, Khrizman V. Repopulation of the mesothelium during long-term experimental peritoneal dialysis. Contrib Nephrol 2006;150 : 54-61.[Medline]
  25. Jimenez-Heffernan JA, Aguilera A, Aroeira RS, Lara-Pezzi E, Bajo MA, del Peso G, et al. Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis induced fibrosis. Virchows Arch2004; 444:247 -56.[Medline]
  26. Gotloib L, Gotloib LC, Khrizman V. The use of peritoneal mesothelium as a potential source of adult stem cells. Int J Artif Organs 2007; 30:501 -12.[Medline]




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