UNIVERSITY OF NIŠ The scientific journal FACTA UNIVERSITATIS

Series: Medicine and Biology Vol.7, No 1, 2000 pp. 97 – 101 Editor of Series: Vladisav Stefanović, e-mail: factacivil@medfak.medfak.ni.ac.yu

Adress: Univerzitetski trg 2, 18000 Niš, YU, Tel: +381 18 547-095 Fax: +381 18 547-950 http://ni.ac.yu/Facta

UC 616.38

PERITONEAL LINING CELL CHANGES IN END STAGE KIDNEY DISEASE

Biljana Stojimirović 1, Mirjana Obradović 3, Dušan Trpinac 3, Drago Milutinović 2, Vidosava Nešić 1

1Institute of Nephrology and 2Urology, Clinical Center of Serbia, 3Institute of Histology, School of Medicine, University of Belgrade, Yugoslavia

Summary. Certain changes, known as uremic serositis, appear in morphology of serous membranes in end stage kid-ney disease (ESKD). They have been recognized on the pleura and pericardium for a long time. But uremic serositis affects, as it has been found in recent years, the peritoneum as well. The aim of our investigation was to examine morphology of peritoneal lining cells in ESKD patients prior to the be-ginning of peritoneal dialysis (PD), to compare it with normal morphology of these structures and to point out the possible advantages of semi-thin sections of samples embedded in Epon resin stained with Toluidin Blue (TB) in com-parison with Hematoxylin-Eosin (HE) stained sections of samples embedded in Paraplast. Biopsies of the peritoneum in 12 ESKD patients were taken during the catheter implantation prior to the first PD, so the changes of the peritoneal lining cells result from the pathophysiology of uremia. Biopsies of the peritoneum in 10 healthy volunteers were taken during the kidney donation. The samples were prepared in the standard way for study-ing on the light microscopy (LM) and transmission electron microscopy (TEM) levels. One layer mesothelium of the cuboidal or flattened lining cells of healthy subjects showed some more subtle structural features using the semi-thin TB stained sections. In uremic patients, routine LM examination of HE stained sections evidenced epithelial stratification differentiating them from peritoneum of healthy persons. Unusual bulging of the mesothelial apical surface, deformation of mesothelial cells and their detachment from the basement membrane were observed using semi-thin TB stained sections analyzed by LM. Intracytoplasmic inclusions were visible only under TEM. Our findings comply with reports of other authors. It should be stressed that EM examination is necessary only for detecting intracytoplasmic mesothelial cell inclusions and other characteristic ultrastructural changes in uremic sero-sitis. Some characteristic changes of peritoneal morphology mentioned above could be identified by LM of TB stained semi-thin sections. This is of particular importance in those institutions which are not equipped with modern technology.

Key words: Mesothelium, uremia, peritoneal biopsy, peritoneal dialysis.

Introduction

The end stage kidney disease (ESKD) is followed by significant metabolic disturbances. Accumulation of uremic toxins, development of metabolic acidosis and electrolyte level changes affect the morphology and function of numerous systems and organs. Along with renal failure anemia and osteodystrophy, uremic serosi-tis is also present. Until recent years clinical signs of uremic pleuritis and pericarditis were well recognized. But, uremic serositis affects, as it has been found, the peritoneum as well.

Some thirty years ago peritoneal dialysis (PD) be-came a respectable modality of renal replacement ther-apy. That is why the peritoneal membrane, a part of the peritoneal dialysis system, has attracted interest of in-vestigators.

The peritoneal barrier is clinically important because

peritoneal dialysis takes place through it (1), and ex-change of substances between the peritoneal cavity and blood capillary lumen occurs. This barrier includes: a stagnant fluid layer within the peritoneal capillary, the capillary endothelium, the capillary basement mem-brane, connective tissue of lamina propria, the mesothe-lium basement membrane, the peritoneal mesothelium and a stagnant fluid film within the peritoneal cavity (2). Peritoneal morphology and function, notably morphol-ogy and function of peritoneal lining cells in healthy persons, uremic and patients on peritoneal dialysis have become a focus of interest for scientists (3, 4).

The aim of the study

The aim of this study was: 1. To examine morphology of peritoneal lining cells

98 B. Stojimirović, M. Obradović, D. Trpinac et al.

in end stage kidney disease patients prior to the beginning of peritoneal dialysis

2. To compare morphology of peritoneal lining cells in end stage kidney disease patients prior to the beginning of peritoneal dialysis with normal morphology of these structures

3. To point out the possible advantages of semi-thin sections of samples embedded in Epon resin stained with Toluidin Blue (TB) in comparison with routine Hematoxylin-Eosin (HE) dyeing of samples embedded in Paraplast.

Patients and methods

We examined 12 end stage kidney disease patients. Biopsies were taken from the parietal peritoneum of the freshly opened anterior abdominal wall, prior to PD catheter implantation.

Ten healthy persons who were kidney donors, repre-sented our control group. Small samples of the perito-neal tissue, taken during the surgical operation for kid-ney extirpation, were treated in the same way as the biopsies of the end stage kidney disease patients.

Special care was taken in getting appropriate sample without artificial damage because of the extreme fragil-ity of the peritoneal tissue. It is necessary to take the sample immediately after the peritoneum is opened and to put it in the fixative prepared before, very gently and carefully. The temperature of 4°C is recommended over all fixation period. The preparing procedure was stan-dard for routine HE staining and for plastic embedded semi-thin sections studying.

Buffered formalin (0.1M Sörensen phosphate buffer pH 7.4) was used for fixation and after a routine proce-dure the samples were subsequently embedded in Para-plast. Sections made on microtome were stained by HE and studied by light microscopy (LM).

For semi-thin sections the tissue was fixed in 2.5% glutar aldehyde in 0.1M cacodylate buffer pH 7.4, post-fixed in 1% osmium tetroxyde in the same buffer and, after dehydration in alcohols and propylene oxide (5) embedded in Epon resin. The semifine sections were made on ultramicrotome, stained with TB and studied by LM.

Results

Epithelial part of the peritoneum, mesothelium, is known as a site of notable changes in uremic serositis. We compared the mesothelium of uremic patients to the mesothelium taken from the control group of healthy subjects.

Peritoneal lining cells of healthy subjects Using routine LM method of HE stained sections,

we could only see the thin region of simple (usually squamous) epithelium (mesothelium) and connective tissue of lamina propria. One layer epithelium was

composed of discoid or flattened cell type in the region of the abdominal wall (Fig. 1A, 1B), or cuboidal cell type in the region of the diaphragmatic and pelvic peri-toneum (Fig. 1C). Both cell types had similar structural appearance. The nuclei were situated in the center of the cells producing prominence of the apical cell surface (Fig. 1A).

Fig. 1. Peritoneum of healthy person

A) Flattened mesothelial cells (HE, under immersion)

B) Flattened mesothelial cells (TB, semi-thin section, under immersion)

C) Cuboidal mesothelial cells (TB, semi-thin section, under immersion);

M-mesothelium, Ct-connective tissue, BV- blood vessel, E-endothelium, mv-microvilli, N-nucleus, n-nucleolus.

Although analyzed by LM, semi-thin TB stained sections showed some more subtle structural features. So we could see that apical plasmalemma extended a number of microvilli, thin and short cytoplasmic pro-jections (Fig. 1C). The nuclear surface was usually ir-regular, with small protrusions and indentations (Fig. 1C). The main part of the nucleus was euchromatic ex-cept for a ring of heterochromatin situated immediately under nuclear envelope. The nucleoli were well devel-oped (Fig. 1C).

PERITONEAL LINING CELL CHANGES IN END STAGE KIDNEY DISEASE 99

Peritoneal lining cells of uremic patients In biopsies of patients with uremia, using routine

LM method of HE stained sections we could only see epithelial stratification in some peritoneal areas (Fig. 2A) differentiating them from healthy persons.

Using TB stained semi-thin sections, also for LM, in addition to the already mentioned features of the perito-neum of healthy subjects and epithelial stratification, we recognized unusual bulging of mesothelial apical sur-faces (Fig. 2B, 2C). We could also see the irregular de-formations of mesothelial cells in these patients (Fig. 2C) and their detachment from the basement membrane (Fig. 2B). Like in controls, in chronic renal failure patients, using TB staining of semi-thin sections, it was easier to identify fine cytological characteristics, and especially mesothelial detachment from the basement membrane.

Fig. 2. Uremic peritoneum:

A) Flattened mesothelial cells (HE, under immersion) B) Flattened mesothelial cells

(TB, semi-thin section, under immersion) C) Cuboidal mesothelial cells

(TB, semi-thin section, under immersion); M-mesothelium, Ct-connective tissue, BV- blood vessel, E- endothelium, mv-microvilli, N-nucleus, n-nucleolus, arrow-detachment from the basement membrane, arrowhead-bulging of the apical surface, S-stratification of mesothelium.

In our patients, using both LM methods we did not

observe any intracytoplasmic mesothelial cell inclu-sions.

These inclusions were identified on our samples ex-amined by TEM (Fig. 3)

Fig. 3. Uremic peritoneum:

A) Mesothelial cell (TEM, bar - 1µm) B) A part of mesothelial cell from fig.3A

(TEM, bar – 300nm) M–mesothelial cell, mv-microvilli, N-nucleus, n-nucleolus, arrow-paracrystaline inclusions, Ct-connective tissue, arrowhead-bulging of the apical surface

Discussion

Our report on peritoneal LM morphology in end stage kidney disease patients, as well as in a control group of healthy subjects is generally in agreement with findings of other investigators (6-9, 10).

Although in our patients, using both LM methods, we did not observe any intracytoplasmic mesothelial cell inclusions, which were observed using electron microscope (references and our personal data), TB staining of semi-thin sections enabled identification of fine cytological features, especially mesothelial detach-ment from the basement membrane and bulging of api-cal mesothelial membrane. These changes could be a direct consequence of the existence of cytoplasmic in-clusions in mesothelial cells.

100 B. Stojimirović, M. Obradović, D. Trpinac et al.

The fact that we failed to observe intracytoplasmic mesothelial cell inclusions using LM in our patients resulted from the lower resolution of LM compared to TEM (9, 10).

According to our results in controls and in patients with terminal renal insufficiency, it was easier to iden-tify fine cytological characteristics, and especially mesothelial detachment from the basement membrane using TB staining of semi-thin sections, than routine HE method.

The exact reasons for changes in uremic serositis are still unknown. The final results of this process visible on the biopsies comprise deformed mesothelial cells with inclusions, detached from the basement membranes (9), which is in agreement with findings in our patients. Comparable changes were found in the pericardial and pleural mesothelium of patients with uremic pericarditis (11, 12). It seems that this exfoliative mesothelial dis-turbance of the peritoneum, pericardium, and possibly

pleura, can explain up to date unexplored causes of uremic serositis (9). The origin and nature of these in-clusions are not known. However, since these changes were found neither in patients on PD, nor in healthy controls, it is assumed that they represent specific signs of uremic serositis (9).

Finally, it should be stressed that EM examination is necessary only for detecting intracytoplasmic mesothe-lial cell inclusions and, of course, other characteristic ultrastructural changes in uremic serositis (degeneration of rough endoplasmic reticulum cisternae as sacular dilatation, accumulation of electron dense granular ma-terial and disruption of cisternae membranes). Some characteristic changes of peritoneal morphology men-tioned above could be identified by LM of TB stained semi-thin sections. This is of particular importance in institutions which are not equipped with modern tech-nology.

References

1. Rubin J, Jones Q, Andrew M. An analysis of ultrafiltration during acute peritoneal dialysis in rats. Am J Med Sci 1989; 298: 383-389.

2. White R, Khorthuis R, Granger DN. The peritoneal microcirculation in peritoneal dialysis. In: Gokal R, Nolph KD (eds),The textbook of peritoneal dialysis. Kluwer Academic Publishers, Dordrecht, 1994: 45-68.

3. Neutra M. The gastrointestinal tract. In: Weis L (ed), Cell and tissue biology. A textbook of histology, 6th ed., Urban & Schwarzenberg, Baltimore, 1988: 646.

4. Hall JC, Heel KA, Papadimitriou JM, Platell C. The pathobiology of peritonitis. Gastroenterology 1998; 114: 185-196.

5. Hayat MA. Basic Techniques for Transmission Electron Microscopy. Academic Press, N.Y., 1986.

6. Li J-C, Yu S-M. Study on the ultrastructure of the peritoneal stomata in humans. Acta Anat 1991; 141:26-30.

7. Li J, Zhou J, Gao Y. The ultrastructure and computer imaging

of the lymphatic stomata in the human pelvic peritoneum. Anat Anz 1997; 179(3):215-220.

8. Gotloib L, Shostak A. Peritoneal ultrastructure. In: Nolph KD (ed), Peritoneal dialysis. Martinus Nijhoff Publishers, Boston, 1989: 67-95.

9. Dobbie JW. Ultrastructure and pathology of the peritoneum in peritoneal dialysis. In: Gokal R, Nolph KD (eds), The textbook of peritoneal dialysis, Kluwer Academic Publishers, Dordrecht, 1994: 17-44.

10. Dobbie JW, Lloyd JK, Gall CA. Categorization of ultrastructural changes in peritoneal mesothelium, stroma, and blood vessels in uremia and CAPD patients. In: Khana R, Nolph KD, Prowant B (eds), Advances in continuous ambulatory peritoneal dialysis, University of Toronto Press, Toronto, 1990: 3-12.

11. Rutski EA, Rostand SG. Treatment of uremic pericarditis and pericardial effusion. Am J Kidney Dis 1987; 10: 2-8.

12. Maher JF. Uremic pleuritis. Am J Kidney Dis 1987; 10: 19-22.

MORFOLOŠKE PROMENE ĆELIJA KOJE OBLAŽU PERITONEUM U TERMINALNOJ HRONIČNOJ BUBREŽNOJ BOLESTI

Biljana Stojimirović, Mirjana Obradović, Dušan Trpinac, Drago Milutinović, Vidosava Nesić

Institut za nefrologiju i urologiju, Klinički centar Srbije, Institut za histologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd

Kratak sadržaj: Promene, poznate kao uremijski serozitis, pojavljuju se na seroznim membranama u terminalnoj insuficijenciji bubrega. Za njih se već duže vreme zna na pleuri i perikardu. Poslednjih godina pokazano je da uremijski serozitis zahvata i peritoneum. Cilj našeg istraživanja bio je da ispita morfologiju ćelija peritoneuma kod uremičnih bolesnika neposredno pre započinjanja peritoneumske dijalize (PD), da je uporedi sa normalnom morfologijom ovih struktura i da podvuče moguće prednosti metoda kalupljenja u Eponu i korišćenja polu tankih isečaka bojenih Toluidinom (TB), u odnosu na rutinski metod kalupljenja u Paraplastu i bojenja Hematoksilinom i Eozinom (HE). Biopsije peritoneuma 12 uremičnih bolesnika uzete su tokom plasiranja katetera pre prve PD, pa su promene epitela

PERITONEAL LINING CELL CHANGES IN END STAGE KIDNEY DISEASE 101

peritoneuma posledica patofiziologije uremije. Biopsije peritoneuma 10 zdravih osoba uzete su tokom davanja bubrega. Uzorci su standardno pripremljeni za posmatranje svetlosnim mikroskopom (LM) i transmisionim elektronskim mikroskopom (TEM). Kod zdravih osoba, jednoslojni mezotelijum sa kockastim i ljuspastim ćelijama, pokazao je nešto suptilnije strukturne odlike na polutankim isečcima bojenim Toluidinom. Kod uremičnih bolesnika, rutinska LM ispitivanja isečaka bojenih HE, za razliku od zdravih, pokazala su stratifikaciju epitela. Kod ovih bolesnika, neuobičajena ispupčenja mezotelne apikalne površine, deformacija mezotelnih ćelija i njihovo odlubljivanje od bazalne membrane uočeni su upotrebom LM i polutankih isečaka bojenih TB. Intracitoplazmatske inkluzije vidjene su samo korišćenjem TEM. Naši rezultati saglasni su sa nalazima drugih autora. Treba naglasiti da je TEM potrebna samo za otkrivanje intracitoplazmatskih inkluzija i drugih ultrastrukturnih promena u mezotelnim ćelijama. Neke karakteristične promene morfologije peritoneuma, pomenute ranije, mogu se uočiti i upotrebom LM i proučavanjem TB obojenih polutankih isečaka. To je naročito važno u onim institucijama koje nisu opremljene modernom tehnologijom.

Ključne reči: Mezotel, uremija, biopsija peritoneuma, peritoneumska dijaliza

Received: August 20,1999