Anatomy and physiology of the peritoneal membrane.

Scarpioni LL, Ballocchi S (eds): Evolution and Trends in Peritoneal Dialysis. Contrib Nephrol. Basel, Karger, 1990, vol 84, pp 10-26

Anatomy and Physiology of the Peritoneal Membrane N. Di Paolo a, G. Sacchi b aNephrology and Dialysis Department, Regional Hospital of Siena; bInstitute of Anatomy, University of Siena, Italy

The first descriptions of peritoneal structure date back to 1863 when Von Recklinghausen [62] observed, among other things, the lubricating capacity of this membrane. Although the literature has been much concerned with the peritoneum, especially in relation to infection in certain pathologies, little was known about its morphology until about 10 years ago. The advent of continuous ambulatory peritoneal dialysis (CAPD) brought peritoneal structure to the attention of researchers [19, 21, 23, 35, 66]. Studies currently underway have provided a detailed ultrastructural description of the peritoneum and its pathology during dialysis. They have also demonstrated [8, 16,24] that the mesothelia secrete various substances and can modulate (or perhaps even regulate) the transport of solutes and water from the peritoneal cavity to blood and lymph vessels. These findings open a completely new chapter on the physiology of the peritoneal membrane. The peritoneum is similar in experimental animals and man. Under the light microscope it is seen to consist of a single cell layer of mesothelium over a continuous basal membrane. This overlies the connective tissue which consists of collagen fibers in layers with fibroblasts, macrophages, adipocytes and mast cells, supplied by blood and lymph vessels (fig. 1). The mesothelial cell has a regular shape and measures 20-35Ilm. It tends to be polygonal in shape and forms a continuous layer with the adjacent cells. The nucleus is oval and located in the center of the cell, but sometimes it protrudes on the free side and contains clearly visible nucleoles (fig. 2). The cytoplasmic organelles are quite well developed but resemble those of a common lining cell in a normal human or animal peritoneum. The mesothe-

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Anatomy of the Peritoneum

Anatomy and Physiology of the Peritoneal Membrane

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lial cell also has vesicles, microvilli and characteristic intercellular junctions. The vesicles are roundish and 0.717 ± 0.4551lm in diameter [34]; they are in contact with the plasmalemma and may be open on the peritoneal side, on the interstitial side, or in intercellular junctions. The junctions are still a subject of discussion: some authors [36] have described intercellular stomata and others deny their existence. These junctions are of the closed type on the luminal side and underneath there are usually zonulae adherens [36]. Mesothelial cell microvilli are observed throughout the peritoneum but their density depends upon the particular abdominal organ. For example, in bladder peritoneum there are about 230 microvilli/mm. In the peritoneum enveloping the stomach there are twice as many and the spleen has even more [2, 52]. The microvillus has no particular internal structures and tends to ramify.


Fig. 1. Scheme of the peritoneal membrane.

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Fig. 2. Human mesothelial cells under normal conditions and without dialysis. TEM.

The mesothelial cells overlie a continuous single basal membrane. The underlying connective tissue is characterized by a small cell population [3] immersed in a matrix of fibers and gel containing macromolecules [23]. There are also adipocytes (very numerous in the mesenteric peritoneum), fibroblasts, mast cells, macro phages and occasionally monocytes [3, 15]. Near the surface of the connective tissue there are large lymph vessels. Deeper down (at least 100 !lm from the mesothelial basal membrane) there are blood vessels [15, 35, 51]. The most frequent blood microvessels are capillaries and postcapillary venules. The former have a layer of endothelial cells over a basal lamina which defines a lumen of 7-10 !lm, whereas in the postcapillary venules the lumen reaches 20 !lm [34].


x2,350.


13

The lymphatics attract more attention because of the vast area they cover [15]. They are generally very large (up to more than 20 11m) and often give rise to lymphatic lacunae that reach 3-6 mm [47] but have a very fine endothelium and a discontinuous basal membrane. The large number of lymph vessels, their large diameter, the absence of occlusive junctions and the discontinuity ofthe basal membrane make it possible for these vessels to exchange substances. They probably playa part in the transport of materials during peritoneal dialysis [15, 49,53]. In the cytoplasm of the endothelium of blood and lymph vessels there are many vesicles which resemble those of the mesothelium. They occupy about 7% of the cellular volume. The surface area of the adult human peritoneum has long been considered [65] to vary between 1.7 and 2.0 m 2• Various formulae have been proposed based on body weight, body surface area, sternum-pubis distance, etc. [27, 38, 54]. However, recent studies in man, children and animals [29, 55] throw doubt upon these long-accepted ideas. The area of animal and human autopsy peritoneal membrane has been measured and quite different values were found. For example, in the rat the mean surface area found was 595 cm 2 and in man 7,792 cm 2 [55]. In children the area seems to be even greater [29]. In rabbits it is about 820 cm 2 • The figures for man are much lower than hitherto believed, and those for animals are much higher. Hence, the peritoneal surface area is not correlated to body area, weight or xiphoid-pubic symphysis distance [55]. In our study of cultured mesothelial cells, we observed that at confluence, they have the same dimensions and morphology as in vivo. In 75 cm 2 flasks there are 7,500,000 cells at confluence and hence we can roughly calculate the number of mesothelial cells which cover the whole peritoneum of animals and man [11].

In dialysis patients with an integral peritoneum at the time of catheter placement, various modifications occur as dialysis proceeds. These changes depend on the time in dialysis and the number of bouts of peritonitis [19, 20, 25, 31, 35]. During peritoneal dialysis, the mesothelium is continually damaged by commercial dialysis solutions [16, 30, 35]. There is morphological evidence of exaggerated turnover (fig. 3) with continuous degeneration of damaged mesothelial cells. After only a few days of dialysis can the cytoplas-


Histological Modifications during Peritoneal Dialysis

Di Paolo/Sacchi

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mic organelles of these cells be seen to be intensely active and develop to such a degree that the cells become unrecognizable (fig. 4). The progressive changes include the gradual reduction and finally complete disappearance of microvilli followed by the opening of cell junctions [13, 23, 35]. Extraneous material (blood residues, salts?) are visible inside some vesicles [4, 35] (fig. 4). After several months of CAPD, the mesothelial basal membrane shows clear


Fig. 3. Desquamation of human mesothelial cells during peritoneal dialysis. TEM. x2,300.


15

signs of replication [18, 35]. In our opinion, this is an important sign of iatrogenic damage caused by the glucose in the dialysis solutions. The replication of the basal membrane of the capillaries has been observed in diabetes, various other pathologies, and uremia [33. 40], but as far as we know there are no data on the replication of the mesothelial basal membrane. A plausible explanation is that the glucose of the solutions acts as


Fig. 4. Human mesothelial cell during peritoneal dialysis. There are morphological changes and highly developed organelles. Note the vacuoles containing extraneous material. TEM. x5,300.

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stimulus just as it acts in the vessels of the diabetic [18]. During peritonitis, the most evident damage is that to the Il}esothelium. SEM shows dramatic exfoliation of the mesothelium, baring the underlying connective tissue [12, 13, 19]. We have observed many variants: detachment of the mesothelial cell leaving the basal membrane in place, detachment also of the latter together with part of the connective tissue, partial detachment of cells, etc. (fig. 5). Necrosis of mesothelial tissue is the most severe picture but all the other manifestations of inflammation are no less striking: edema of the connective tissue, cellular infiltrate, evident mobilization ofleukocytes in the blood and lymph. One to 4 months after peritonitis, anatomical damage is still visible: the mesothelium is missing in places and replaced by fibrous tissue. In some places the peritoneum is difficult to evaluate because it is obscured by fibrin. Peritoneal fibrosis or sclerosis is frequent after several years of peritoneal dialysis. The area and severity are certainly related to the number of episodes of peritonitis. Peritoneal fibrosis should be distinguished from sclerosing


Fig. 5. Scheme representing peritonitis of varying severity.


17

peritonitis [7,31,57] which is a dramatic condition of fibrotic villous proliferation of the peritoneum, a known etiological agent of which is the acetate contained in certain peritoneal dialysis formulations. Peritoneal sclerosis is usually only a few micrometers in thickness, reaching a maximum of 1-2 mm, whereas the villus of sclerosing peritonitis is always several millimeters thick [41].

When it was discovered that the mesothelial cell secretes phospholipids [14, 24, 39]. the need was felt for a better understanding of mesothelial physiology. The simplest way to achieve this seemed to be to study mesothelial cells in culture as performed for other mesothelia [1,9,32,42,46, 50, 56, 63, 64]. Peritoneal mesothelial cells can be sampled by two original techniques devised by our group [11]. The first may be performed during the insertion of the permanent Tenckhoff catheter. The animal is anesthetized, a small median laparotomy performed (5 cm), a corner of omentum (2-3 cm) brought out with tweezers, tied twice and cut between the ligatures. The sample is placed in buffered 0.1 % trypsin for 10-12 min. The second technique is for animals who already have an in situ catheter. A buffered 0.1 % solution of trypsin is introduced into the abdominal cavity for 15 min. Neither method causes damage to the animal. Trypsin at these concentrations does not have systemic effects. In man obviously only the first technique can be used during the surgical insertion or removal of a peritoneal catheter [11]. Mesothelial cells grow well in common media and can be resown up to 8 times, after which they lose their reproductive capacity. After the eighth replication chromosome abnormalities were noticed [42]. The cultivated cells can be frozen. The characterization of the mesothelial cells already provides much information about their physiology. The techniques for characterization include flowcytometry [49, 60, 61], morphological evaluation [56], biochemical evaluation (study of the protein cytoskeleton including factor VIII as antigen) [6, 11, 45, 56, 58, 59], cell lipid content [11], prostaglandin production [11, 56] and phospholipid production [11]. Before confluence, mesothelial cells in culture resemble fibroblasts under the microscope. At confluence they resemble the mesothelial cells described in vivo during peritoneal dialysis (fig. 6) [11, 19, 34] and in the drainage liquid [5, 10]. The cytoplasm is rich in organelles and the surface is covered in microvilli.


The Culture of Mesothelial Cells

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Fig. 6. Cultured mesothelial cells at confluence. TEM. x3,600.

Purposely ignoring general capillary physiology, it can justly be claimed that this is a completely new chapter in medicine. In our opinion, peritoneal physiology should not be confused with general capillary physiology, or worse still, the exchange kinetics of peritoneal dialysis. With the term 'peritoneal physiology' we only mean to indicate the peculiar functional characteristics of the peritoneal membrane as a biological membrane. These characteristics distinguish it from other lining tissues. Because of the lack of ultrastructural knowledge of the peritoneum and the total absence of data on the secretory capacity of mesothelial cells, until the present nobody thought that peritoneal physiology could play any part in peritoneal dialysis. The peritoneum has been treated as a physical barrier, and mathematical and hydrodynamic models were used to devise treatment schemes, which turned out to be very efficient. The chapter on peritoneal physiology opened when Hills et al. [39] demonstrated the phospholipid character of the liquid that normally bathes mesothelial surfaces. Later it was proved that surface-active material (SAM) is secreted by the mesothelial cells [11, 24]. It was then found that in peritoneal dialysis, the mesothelial cells secrete very actively [11, 14]. We propose the following classification of the different secretory products:


Physiology o/the Peritoneal Membrane


19

(1) Dialysate material (DIM) consists of all the material contained in a

dialysate. If the content of a 2,000 to 2,500-ml drainage bag is freeze dried after dialysis to remove salts, we obtain 2.5-3.0 g of substances, mostly proteins and lipids. These come from the interstitium, blood, lymph and mesothelial secretions. (2) Mesothelial material (MEM) is the group of substances secreted by the mesothelial cells. So far we only know a few, but it is reasonable to suppose that there are many. (3) SAM is a component ofMEM. It is the surfactant discovered by Hills et al. [39] and consists of a mixture of phospholipids with lubricating properties. It is the first substance found to be secreted by the mesothelium [8, 11, 16,24]. There are 30-40 mg in every liter of dialysate under normal conditions. (4) Transport material (TAM) = DIM - MEM. It consists of substances transported from the peritoneum towards the blood and lymph vessels. Besides the transport of exogenous material there is probably also reabsorption of material secreted by the mesothelial cells [11].

Phospholipids As far as we know at the moment, the animal and human peritoneum normally secretes SAM consisting of as mixture of phospholipids [16, 24, 37]. This mixture has particular chemicophysical properties: it is highly lubricant and surfactant and repels water. Positively charged molecules of choline, ethanolamine, serine and inositol form a continuous phospholipid coat on the negatively charged mesothelium. Under normal conditions, the peritoneum is covered in dense microvilli which are coated in phospholipid molecules with their double chain facing the peritoneal cavity. They create a very efficient lubricating system, even more efficient than would be obtainable, for example, with olive oil [14]. The lubrication system is of the type known as 'boundary lubrication' and is suitable for slowly moving surfaces as distinct from the 'fast hydrodynamic' type found in joints [39]. The study of the phospholipid composition of SAM has been carried out on peritoneal dialysate and cultured peritoneal mesothelial cells [11]. In dialysis (4-hour exchanges) the lipid material is extracted with chloroformmethanol and then run on chromatrographic plates of silica gel. A quantity of 25.32 ± 18.48 mg/l of phospholipids with the following composition was


Peritoneal Mesothelial Cell Secretions

20

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found: lysophosphatidylcholine 13%, phosphatidylcholine 56%, sphingomyelin 16%, phosphatidylethanolamine 9%, and phosaphatidylinositol 6% [11]. Seventy-five cm 2 of cultured human mesothelial cells at confluence, without stimulation, produced 5.14 mg of phospholipids per liter of supernatant [11] in 6 h in a lipid-free culture medium. The peritonea of our patients which have an average area of 8,000 cm 2 of mesothelial cells at confluence, should theoretically produce about 500 g of phospholipids per liter in 6 h. If we take the same mesothelial cells in culture and add commercial dialysis solution with lactate containing 1.5% glucose (50% dialysis liquid and 50% culture medium), the phospholipid concentration drops to 1.50 mg/I. Hence, it may be supposed that commercial dialysis solutions, already known to be cytotoxic [30] decrease peritoneal phospholipid production to less than one-third the in vitro value (fig. 7). Although much reduced, peritoneal secretion during dialysis should be much greater than that actually found in dialysis patients. This suggests that the human peritoneum partly reabsorbs phospholipids secreted by the mesothelial cells, at least during peritoneal dialysis [11]. In any case the demonstration that the mesothelium secretes much larger quantities of


Fig. 7. Phospholipid content of cell culture supernatant. Mesothelial cells secrete different phospholipids and in greater quantity than the other cells.

Anatomy and Physiology of the Peritoneal Membrane 100

21

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Fig. 8. Cultured peritoneal mesothelial cell production of 6-keto-PGFln, the main metabolite of prostacyclin 12, stimulating with the agonists calcium ionophore and bradykinin. Peritoneal dialysis solution also stimulates prostacyclin 12 production.

Prostaglandins Prior studies [49] showed thast endogenous prostaglandins are not found in the regular peritoneal blood supply. However, it was recently shown that the pericardium produces prostacydin 12 and other arachidonic acid derivatives [56] and that during pericardial perfusion in animals in vivo after angiotensin II or bradykinin stimulation, there is a vasodilatory effect [28] accompanied by an increase in 6-keto-F I (1, a stable metabolite of prostacydin 12. Our rabbit and human mesothelial cells in culture also produced prostacyclin 12 [11] after stimulation with bradykinin or calcium ionophore, but commercial dialysis solutions used as agonist were also found to stimulate prostacyclin 12 production (fig. 8). This leads us to suspect that the vasodila-


phospholipids in vitro than the endothelium confirms all the previous studies which predicted this fact [19, 24, 26].

Di Paolo/Sacchi

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tory effect of commercial dialysis solutions is due to increased secretion of prostacyclin 12, a powerful vasodilator, by the mesothelial cells. So far there has been no explanation of the vasodilatory effect of commercial dialysis solutions. Many years ago our group found that dialysis solution caused vasodilatation ofthe abdominal organs in guinea pigs [17] leading to the suspicion that the peritoneal mesothelium modulates the microcirculation of the abdominal organs. More recently we observed that our cultured mesothelia secrete an even larger quantity of prostaglandin E2 than endothelial cells, even in the absence of an agonist (fig. 9). The metabolic activity of the mesothelial cell does not end here. We know, for example, that cultured rat pleural mesothelia synthesize elastin and proteoglycans [33] which are fundamental components of connective tissue. Lanfrancone [45] observed that mesothelial cells secrete interleukins 1, 6 and 8 and cell growth factors G-CSF, G M-CSF and CSF 1. The moderate quantity of total proteins produced by the cell supernatant of mesothelia in culture media [11], suggests that many other unidentified proteic substances are secreted by these cells. The first notions of peritoneal physiology emerge from the functional study of the mesothelial cell in culture, revealing that this organ has unsuspected properties. Dobbie [22] showed that the peritoneum


Fig. 9. Secretion ofprostag!andin E2 by rabbit mesothelium (RMS), human mesothelium (HMS) and human endothelium (HAEL) before and after bradykinin stimulation.


23

of certain fish is an excretory organ which communicates with the outside [22]. The application of the peritoneal catheter and filling of the abdominal cavity with liquid may reproduce ancestral functional conditions. Correct study will certainly soon enable the peritoneum to be more efficiently utilized and will benefit the vaster fields of biology and medicine. Recently, for example, our group succeeded in performing an autologous implant of peritoneal mesothelium in animals and man [11].

2 2 3 4 5 6 7 8 9 10 11 12 13

14 15

Akedo H, Shinkai K, Mukai M, Mori Y, Tateishi R, Tanaka K, Yamamoto R, Morishita T: Interaction of rat ascites hepatoma cells with cultured mesothelial cell layers. A model for tumor invasion. Cancer Res 1986;46:2416-2422. Baraldi F, Rao SN: A scanning electron microscope study of mouse peritoneal mesothelium. Tissue Cell 1976;8: 159-162. Baron MA: Structure of intestinal peritoneum in man. Am J Anat 1941;69:439497. Bartlett JR: Phosphorus assay in column chromatography. J Bioi Chern 1959;234: 466-468. Bercovici B, Gallily R: The cytology of the human peritoneal fluid. Cytology 1978; 22:124-127. Boraschi D, Censini S, Bartalini M, Tagliabue A: Regulation of arachidonic acid metabolism in macrophages by immune and nonimmnune interferons. J Immun 1985; 135:502-505. Bradley JA, McWhinnie DL, Hamilton DNH, StarnefF, MacPherson SG, Seywright M, Briggs JD, Junor BJ: Sclerosing obstructive peritonitis after CAPD. Lancet 1983; i:113-116. Breborowicz A, Sombolos K, Rodella H: Mechanism of phosphatidylcholine action during peritoneal dialysis. Nephron 1986;44:365-368. Cantor JO, Willhite M, Bray BA, Keller S, Mandl I, Turino GM: Synthesis of crosslinked elastin by mesothelial cell culture. Proc Soc Exp Bioi Med 1986;181 :387-391. Cichoki T, Hanicki Z, Sulowicz W, Smolen sky 0, Kopec J, Zembala M: Output of peritoneal cells into peritoneal dialysate. Nephron 1983;35:175-182. Di Paolo N, Sacchi G, Vanni L, Corazzi S, Pallini V, Rossi P, Gaggiotti E, Buoncristiani U: Implant of autologous mesothelium in animals and a peritoneal dialysis patient. Int J Artif Organs 1989; 12:485-50 1. Di Paolo N: CAPD. Milano, Wichtig, 1987, pp 3-20. Di Paolo N: The morphology ofthe peritoneal membrane during peritoneal dialysis; in La Greca, Chiaromonte, Fabris (eds): Peritoneal Dialysis. Milan, Wichtig, 1989, pp 3-6. Di Paolo N: The peritoneal mesothelium. An excretory organ. Periton Dial Int 1989; 9. Di Paolo N, Alessandrini C, Gerli R, Sacchi G: Trasporto peritoneale e cinetica degli scambi; in D'Amico, Vendemmia, Sorgato (eds): Attualita nefrologiche e dialitiche. II Pensiero Scientifico, Roma, 1979, pp 79-91.


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Rubin BJ, Clawson BS, Planch A, Jones BS: Measurements of peritoneal surface area in man and rat. Am J Med Sci 1988;295:453-458. Satoh K, Prescott SM: Culture of mesothelial cells from bovine pericardium and characterization of their arachidonate metabolism. Biochim Biophys Acta 1987;930: 283-286. Schmidt RW, Blumenkrantz M: Peritoneal sclerosis. A sword of Damocles for peritoneal dialysis? Arch Intern Med 1981;141:1264-1267. Starger JM, Brown WE, Goldman AE, Goldman RA: Biochemical and immunological analysis of rapidly purified 1O-nm filaments from baby hamster kidney (BHK-21) cells. J Cell Bioi 1978;78:93-109. Towbin H, Staehlin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedures and some applications. Proc Natl Acad Sci USA 1979;76:4350-4354. Traganos F: Flow cytometry: principles and applications. Part I. Cancer Invest 1984; 2: 149-163. Traganos F: Flow cytometry: principles and applications. Part II. Cancer Invest 1984; 2:239-258. Von Recklinghausen F: Zur Fettresorption. Virchows Arch Path Anat Physiol1863; 26:172. Wagner JC, Johnson NF, Brown DG, Wagner MMF: Histology and ultrastructure of serially transplanted rat mesotheliomas. Br J Cancer 1982;46:294-299. Wang SN, Jaurand MC, Magne L, Kheuang L, Pinchon MC, Bignon J: The interactions between asbestos fibres and metaphase chromosomes of rat pleural mesothelial cells in culture. Am J Path 1986;126:343-349. Wegner G: Chirurgische Bemerkungen iiber die Peritonealhbhle, mit besonderer Beriicksichtigung der Ovariotomie. Arch Klin Chir 1876;20:96-145. Werger C, Brunschvicg 0, Le Charpentier Y, Lavergne A, Vanttelon J: Structural and ultrastructural peritoneal membrane changes and permeability alterations during CAPD. Proc EDTA 1981;18:199-205.

Prof. N. Di Paolo, MD, Divisione di Nefrologia e Dialisi, Ospedale Regionale, 1-53100 Siena (Italy)


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Connecting the Dots? Linking Anatomy, Connectivity, and Physiology in Epilepsy.

Anatomy and physiology of velopharyngeal closure and insufficiency.

Self-testing promotes superior retention of anatomy and physiology information.

Gastrointestinal anatomy and physiology of select exotic companion mammals.

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology.

Physiology of the peritoneum. Implications for peritoneal dialysis.

Pointing and tracing gestures may enhance anatomy and physiology learning.