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Immunohistochemical and electron-microscopic identification of neuroendocrine cells in the stomach of uremic rats
Cell Biology International 28 (2004) 441e447 www.elsevier.com/locate/cellbi
Immunohistochemical and electron-microscopic identification of neuroendocrine cells in the stomach of uremic rats Irena Kasacka) Department of Histology and Embryology, Medical University of Bia1ystok, ul. Kilin´skiego 1, 15-089 Bia1ystok, Poland Received 11 November 2003; revised 2 March 2004; accepted 24 March 2004
Abstract Many disturbances in electrolyte and hormonal balance in the body induced by functional impairment of renal parenchyma may affect the activity of amine precursor uptake and decarboxylation (APUD) cells, which constitute a very important link in the regulation of homeostasis. The aim of the present study was the morphological, immunohistochemical and ultrastructural estimation of enteroendocrine cells in the stomach of uremic rats. Fragments of gastric pylorus were collected 1, 2 and 4 weeks after nephrectomy. Paraffin embedded sections were stained with H C E and by silver impregnation. For identification of neuroendocrine cells, immunohistochemical reactions were performed using specific antibodies against somatostatin, synaptophysin, neuron-specific enolase and anti-calcitonin gene related peptide. The analysis showed an increased number of APUD cells in the stomach of uremic rats compared to control rats, which may be a morphological expression of their hyperfunction in the functional impairment of renal parenchyma. These results suggest that chronic renal failure can modulate the secretory processes of APUD cells. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: APUD cells; Stomach; Chronic renal failure
1. Introduction Chronic renal failure (CRF) is a pathological syndrome caused by progressive damage to renal structures induced by chronic nephropathies, characterized by gradually increasing impairment of function. When the pathological process reduces the active area of renal parenchyma to less than 1/3 its normal size, a very diverse pattern of clinical symptoms originating from most organs of the body gradually develops (Esaian et al., 1997; Kingswood, 1999). Their intensification leads to the phase of uremia characterized by severe metabolic disorders (Hu et al., 2000; Levillain et al. 2001; Noris et al., 2000). Alimentary disorders, such as lack of appetite, vomiting, erosions, bleeding and inflammations
Abbreviations: CRF, chronic renal failure; DNES, diffuse neuroendocrine system; APUD, amine precursor uptake and decarboxylation; SY, synaptophysin; NSE, neuron-specific enolase; ST, somatostatin; CGRP, calcitonin gene related peptide. ) Tel.: C48-85-748-54-55; fax: C48-85-748-54-58. E-mail address: [email protected].
with potential ulceration, are common in patients with CRF and are usually the first to appear (Kingswood, 1999; Wie˛cek and Kokot, 1989). These clinical symptoms observed in patients with CRF can be partly induced by functional disorders of diffuse neuroendocrine system (DNES) cells (Sirinek et al., 1984). This system includes approximately 60 types of neuroendocrine cells, distributed in all organs and tissues, which produce biogenic amines and peptide hormones (Kvetnoy et al., 2001; Panfilov et al., 1989). By the production of these biologically active substances, amine precursor uptake and decarboxylation (APUD) cells are directly involved in the regulation of many metabolic processes and are regulators of homeostasis via neurocrine, endocrine and paracrine mechanisms (Kvetnoy et al., 2000). Any disorder of the APUD system involving excessive production or deficiency of a hormone or biogenic amine may cause dysfunction of body systems, which manifests itself in a complex set of clinical symptoms (Kvetnoi and Iakovlova, 1987; Soehartono et al., 2002). In recent years, many scientists have been concerned
1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.03.012
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with the structure, function and histogenesis of APUD cells, as well as their involvement in various pathological processes. Current opinions based on numerous studies indicate the active involvement of neuroendocrine cells in the processes of pathogenesis and adaptation in various diseases (El-Salhy, 2001; Kvetnoi and Iakovlova, 1987; Kvetnoi and Iuzhakov, 1987; Kvetnoy et al., 2001; Norle´n et al., 2001). Taking into consideration the metabolic disorders observed in CRF and the undoubted involvement of biologically active substances produced by APUD cells in the regulation of homeostasis, I decided to investigate the functional morphology and ultrastructure of DNES cells in the stomach of uremic rats. The aim of this study was to determine the presence, location and changes in functional morphology of APUD cells in the stomach of uremic rats, based on immunohistochemical and ultrastructural investigations. 2. Materials and methods 2.1. Experimental model The study was carried out on 51 young male Wistar rats, 200e250 g body weight (mean 220G10 g) at the beginning of the experiment. The animals were housed at 20 (C with a relative humidity of 40e45% and a 12 h light/dark cycle. The animals had free access to drinking water and standard granulated diet. All experiments were performed at the same time of the day. Procedures involving the animals and their care were conducted in conformity with the institutional guidelines that were in compliance with national and international law and with Guidelines for the Use of Animals in Biomedical Research (Giles, 1987). Study assumptions, aim, schedule and mode of animal treatment were approved by the Senate Committee for the Supervision of Experiments on Humans and Animals at the Medical University of Bia~ystok. The animals were divided into two control groups and one experimental group, the latter being composed of rats in which uremia was induced: Kdcontrol group: 15 rats left intact. SOdcontrol group: 15 rats submitted to sham operation, i.e. decapsulation and removal of the adherent fat from both kidneys. Mduremic group: 26 rats with experimentally induced uremia. Five rats died during the experiment, so 21 were used in the final data. The rats in the control and uremic groups were divided into the following three subgroups: 1. sectioned 1 week after surgery: 5 rats from each control group (K1, SO1) and 7 from the uremic group (M1);
2. sectioned 2 weeks after surgery: 5 rats from K2 and SO2 and 7 from M2; 3. sectioned 4 weeks after surgery: 5 rats from K3 and SO3 and 7 animals from M3. 2.2. Induction of experimental renal insufficiency in rats Experimental uremia was induced by the method described by Azzadin et al. (1999) and Ormrod and Miller (1980). The rats were anaesthetised by pentobarbital (Biowet, Pu~awy), administered intraperitoneally at a dose of 50 mg/kg. Then an incision (2e2.5 cm long) was made on the dorsal side, 1 cm from the lumbar spine, at the level of the kidneys, thus giving a clear approach to the right kidney, followed by its nephrectomy. The approach to the left kidney was obtained in an identical way, followed by removal of 70% of renal cortex, leaving the renal medulla intact. The incised integuments were sutured in layers with catgut. 2.3. Histology: method of experimental material collection and fixation One, 2 and 4 weeks after surgery, the rats were anaesthetised with pentobarbital and blood was collected from their hearts. Then, the animals were sacrificed by decapitation. The stomach was immediately removed. For microscopic analysis, segments of pyloric distal parts of the stomach were used, fixed in Bouin’s fluid and embedded in paraffin in the routine way. For electron microscopy, specimens were embedded in 2.5% purified glutaraldehyde. The specimens were cut in 5 mm slices (Leica 2025 Autocut) and stained by hematoxylineeosin (H C E) for general histological examination, and by Grimelius’ method, revealing neuroendocrine cells following the impregnation of their cytoplasmic granules with silver salts (Grimelius, 1968). 2.4. Histology: identification of DNES cells by immunohistochemical methods 2.4.1. Antibodies Polyclonal rabbit anti-human somatostatin (ST) (1:200), polyclonal rabbit anti-human synaptophysin (SY) (1:10) and monoclonal mouse anti-human neuron-specific enolase (NSE) (1:100) were purchased from DAKO (DAKO Cytomation, Denmark). Anticalcitonin gene related peptide (CGRP) (1:8000) was purchased from SigmaeAldrich (Saint Louis, Missouri 63103, USA). 2.4.2. Immunohistochemical reaction procedure Briefly, using deparaffined and hydrated (with the use of pure, not denatured, alcohol) sections, the activity of endogenous peroxidase was blocked by 3% H2O2 for
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5 min. After washing with distilled water and 0.05 M Tris ( pH 7.4) for 5e10 min, the sections were incubated with antibody for 30 min in a darkroom at room temperature. Then, the sections were washed three times in Tris buffer. The ABC (avidinebiotin peroxidase complex) method was used, according to the manufacturer’s protocol, for the identification of immunohistochemical reactions (Hsu et al., 1981). A secondary, biotinylated antibody (detection kit: KIT DAKO LSAB (C) or LSAB 2) was used for 15 min, then the sections were washed three times in TriseHCl and incubated in streptavidin solution. Mayer’s hematoxylin was used for nuclear staining. Control reactions were simultaneously performed, where the specific antibody was excluded. They all gave negative results. Analysis of the preparations and photographic documentation were performed with an Olympus BX50 light microscope. 2.5. Electron microscopy Specimens were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium phosphate buffer ( pH 7.2) at 4 (C overnight, postfixed for 1 h in 2% osmium tetroxide, dehydrated in graded ethanol solutions and propylene oxide and finally embedded in epon. Ultrathin sections (60 nm) were cut on a Leica Reychard Ultratome Cut S, contrasted with uranyl acetate and lead citrate and examined in an OPTON 900 PC electron microscope. Endocrine cells containing secretory granules were subclassified according to the appearance of secretory granules, as described by Toner and Carr (1971). 2.6. Biochemical blood tests: creatinine and urea assays Cardiac blood was obtained and left at room temperature for 20 min to clot. It was then centrifuged for 15 min at 3000 rpm, and urea and creatinine levels in the serum were determined using a Beckman-CX4 Analyzer (using a ‘Ure´e cinetique UV 800’ bioMe´rieux). 2.7. Statistics Statistical analysis was based on the ANOVA test. Verification of variance analysis assumptions was performed by ShapiroeWilk’s test (normal distribution assumption). The analysis was performed using the SAS ATAT software package. A probability level of p ! 0:05 was considered significant.
3. Results No significant differences were found between the control groups, so only the results from animals subjected to sham operations are discussed. Increased concentrations of urea and creatinine are indicators of renal dysfunction. A significant increase in serum creatinine and urea concentrations was demonstrated in all animals with experimental renal dysfunction, when compared with respective values in the control groups (Table 1). The mucous membrane of the stomach showed cells with a characteristic positive argyrophilic Gomori reactiondcells that typically belong to the APUD system. In control animals, these cells were observed mainly in the fundus of the glands, while in uremic rats they were diffused all over the mucous membrane. In contrast to control rats, the microscopic picture of the sections of uremic animals displayed a considerable increase in the number of neuroendocrine cells, which was revealed due to the argentophilic nature of the cytoplasm. The greatest number of cells was observed 14 days after surgery. All rats showed neuroendocrine cells that were difficult to recognize by light microscopy after routine staining with hematoxylin and eosin. Single cells could sometimes be observed, dispersed between other epithelial cells along the glands, especially in the basal mucous membrane. They were round or pear shaped and had a dark-staining nucleus lying centrally in a light, poorly eosinophilic cytoplasm. The immunohistochemical studies revealed a positive reaction in the cytoplasm of endocrine cells of the stomach in all the animals examined. This indicates that the antibodies reacted against appropriate antigens in DNES cells, recognizing their site in the glandular epithelium of the stomach pylorus. In control rats, the greatest number of endocrine cells was observed in the basal portions of the glands, while they were spread all over the glands of uremic rats. Differences were found between uremic and control rats, not only in the distribution of endocrine cells, but also in their number and reaction intensity. The detecting antibodies SY, NSE, ST and CGRP used in immunohistochemical investigations yielded a distinctly positive reaction in the cytoplasm of APUD cells in the stomach of all the study animals, the reaction intensity and the number of positive cells being markedly intensified in the stomach of uremic rats compared with controls (Fig. 1A, B). Differences in the reaction
Table 1 Serum concentrations of creatinine and urea in control and uremic rats (mg/dl) (data are means G standard deviation)
Creatinine Urea
Control
1 Week
2 Weeks
4 Weeks
*p-Value
0.52 G 0.05 35.33 G 5.98
0.63 G 0.09 52.91 G 16.39
0.94 G 0.06 95.10 G 11.90
0.72 G 0.12 85.58 G 9.77
!0.05 !0.05
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were mostly found in the basal part of the cell; however, they were sometimes so numerous that they could also be seen in the nucleus. Some preparations showed increased transparency of secretory granules and sometimes the surrounding membrane was discontinuous. Ultrastructural examination of endocrine cells in the stomach of uremic rats showed activation of Golgi apparatus structures, with cistern dilation and relatively marked proliferation of smooth endoplasmic reticulum.
4. Discussion
Fig. 1. (A) Immunohistochemical reaction determining SY in the pylorus mucosa of control rat, (B) a significant increase in the number of synaptophysin-immunoreactive cells was seen 4 weeks after nephrectomy. Original magnification !200.
intensity between the groups were most distinct in CGRP-positive cells. In control animals (Fig. 2A), only single cells showed a positive reaction to the antibody, while a marked increase was noted in the number of CGRP-positive enteroendocrine cells in experimental uremic rats (Fig. 2B, C). Results of electron-microscopic pictures supplement and confirm the presence of enteroendocrine cells in the rat stomach. In the preparations of control stomachs, a slight or moderate change in the content of secretory granules distributed in various areas of the cytoplasm was seen (Fig. 3A). These granules, homogenous in content, were usually oval or spherical in shape. Endocrine granules were much more polymorphic in the preparations of uremic rat stomachs (Fig. 3B). A narrow space was sometimes seen to separate a single membrane enclosing the secretory granules from the homogenous contents of varying electron density. They
Laboratory analysis showed a statistically significant increase in the concentration of plasma indices of renal failure: urea and creatinine. The rats in which chronic renal failure was induced had elevated levels of these parameters over the whole 4 week experimental period. Damage to a considerable percentage of nephrons leads to disorders of excretory and endocrine functions and to serious changes in metabolic processes. These disorders, together with compensatory processes in the kidneys and other organs observed during chronic renal failure, have definite clinical and biological consequences (Noris et al., 2000; Sirinek et al., 1984). Histochemical analysis of the preparations of the pyloric part of the stomach confirms previously obtained preliminary data (Kasacka et al., 2001). The data presented here demonstrate that the pyloric part of the stomach contains neuroendocrine amine/peptide producing cells that could play an important role in the endogenous mechanisms of chronic renal failure. Knowledge of the APUD system and basic hormonal activity of the alimentary tract is quite extensive and based on the results of numerous studies conducted in recent years (Bargsten and Grube, 1992; Date et al., 2002; El-Salhy, 2001; Gilon et al., 1990; Hakanson et al., 2001; Huang et al., 2000; Kvetnoi and Iuzhakov, 1987; Raikhlin et al., 1983b). However, mechanisms of mutual relationships and interactions of cells diffused throughout the alimentary tract in the maintenance and regulation of homeostasis have yet to be clarified. Hence, interpretation of the results obtained in the present study is difficult. Analysis of the data shows that enteroendocrine cells, which, in control animals, occur mainly in the basal part of the mucous membrane of the stomach, are seen at various levels of glandular crypts in uremic animals, probably due to changes in activity and hyperplasia. It is reported that more than 50% of the cells of the APUD system are distributed in the alimentary tract (Raikhlin et al., 1983a,b). By producing numerous hormones and biogenic amines, enteroendocrine cells are directly involved in the regulation of physiological processes in normal and pathological conditions (Osadchuk et al., 1989; Panfilov et al., 1989;
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Fig. 2. (A) CGRP-immunoreactive cells in the pyloric mucosa of control rat, (B) of the same rat 1 week, and (C) 2 weeks after nephrectomy. Original magnification !200.
Fig. 3. Electron micrographs of endocrine cells from pyloric mucosa of (A) control rat, and (B) 2 weeks after nephrectomy. Some electron dense neuroendocrine granules (arrows) of slightly varying size can be observed in the cytoplasm, as well as the nucleus (N), mitochondria (m), and cisterna of rough endoplasmic reticulum (rER). Original magnification: (A) !7000, (B) !12,000.
Ratner et al., 1990). Any disturbance in the synchronization of APUD cell activity, which is expressed by an overproduction or deficiency of a particular hormone, involves disorders of both digestive function and general homeostasis (Huang et al. 2000; Kvetnoi and Iuzhakov, 1987; Raikhlin et al., 1983a,b). The increase in functional activity of enteroendocrine cells observed in this study indicates their role not only in normal, but also in pathological, reactions. Numerous DNES cells present in alimentary tract epithelium produce biologically active substances and ensure coordination of all functions of digestive organs. Reports are available indicating some changes in the number of these cells in various pathological conditions, thus confirming the active involvement of enteroendocrine cells in pathological processes of the alimentary tract (Fujimiya et al., 1991; Osadchuk et al., 1989; Panfilov et al., 1989; Ratner et al., 1990; Soehartono et al., 2002). It can be assumed that the increased number of APUD cells and reaction intensity observed in this study to a certain degree compensates for homeostatic disorders associated with the impairment of renal parenchyma functioning. The ultrastructure of enteroendocrine cells observed by light microscopy reflects the changes in activity of these cells. Electron microscopy of the stomach of uremic rats shows symptoms of stimulation of the activity of endocrine cells. Polymorphic neurosecretory granules,
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being the most characteristic element of this type of cell, occur in varying numbers in the analyzed pictures. The electron microscope reveals microvilli on the free area of numerous cells, sometimes quite long, reaching the lumen of the glands where they are located (Rubin and Schwartz, 1981). Due to these and other mechanisms, endocrine cells rapidly and precisely react to environmental changes via enhancement, reduction or inhibition of their secretory function and thus regulate the action of the respective group of parenchymal cells in their vicinity. Previous studies indicate likely changes in the functioning of APUD cells in reaction to the nature of passing food. This shows the high sensitivity of endocrine cells in preserving morphological features of chemoreceptors (Raikhlin et al., 1983a,b). The role of DNES cells in specific conditions of disturbed metabolic processes and electrolyte equilibrium, resulting from the impairment of renal parenchyma functioning, is possible mainly due to the wide range of biologically active substances produced by these cells. The increased quantity of enteroendocrine cells observed in the present study is a likely morphological expression of their hyperfunction in uremia. This seems to be confirmed by the intensification of staining of neurosecretory granules using specific histochemical methods. This study demonstrates that uremia has a significant effect on the endocrine system of the stomach. This suggests that APUD cells, in addition to the endocrine and nervous systems, are a significant element of the internal regulatory system in the stomach, and that their activity may be disturbed by uremia.
Acknowledgements I would like to thank Prof. W. Buczko and Dr. A. Azzadin from the Department of Pharmacodynamics, Medical University of Bia~ystok, for their assistance in performing these experiments.
References Azzadin A, Wollny T, Pawlak R, Malyszko JS, Malyszko J, Mysliwiec M, et al. L-Carnitine effects on anemia in uremic rats treated with erythropoietin. Nephron 1999;83:370e1. Bargsten G, Grube D. Serotonin storage and chromogranins: an experimental study in rat gastric endocrine cells. J Histochem Cytochem 1992;40:1147e55. Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal MS, Hosoda H, et al. Ghrelin is present in pancreatic a-cells of humans and rats and stimulates insulin secretion. Diabetes 2002;51:124e9. El-Salhy M. Gastric emptying in an animal model of human diabetes type 1: relation to endocrine cells. Acta Diabetol 2001;38:139e44. Esaian AM, Titova VA, Shishkina LI, Barabanova VV, Kaiukov IG. The effect of urea on the course of experimental uremia in rats with a subtotal nephrectomy. Patol Fiziol Eksp Ter 1997;2:39e41.
Fujimiya M, Maeda T, Kimura H. Serotonin-containing epithelial cells in rat duodenum. II. Quantitative study of the effect of 5HTP administration. Histochemistry 1991;95:225e9. Giles AR. Guidelines for the use of animals in biomedical research. Tromb Haemost 1987;58:1078e84. Gilon P, Mallefet J, De Vriendt C, Pauwels S, Geffard M, Campistron G, et al. Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach. Histochemistry 1990;93:645e54. Grimelius L. A silver nitrate stain for a2cells in human pancreatic islets. Acta Soc Med Upsal 1968;73:243e94. Hakanson R, Chen D, Lindstrom E, Bernsand M, Norlen P. Control of secretion from rat stomach ECL cells in situ and in primary culture. Scand J Clin Lab Invest Suppl 2001;234:53e60. Hu E, Chen Z, Fredrickson T, Gellai M, Jugus M, Contino L, et al. Identification of a novel kidneydspecific gene downregulated in acute ischemic renal failure. Am J Physiol Renal Physiol 2000;279: 426e39. Huang Y, Lu SJ, Dong JX, Li F. The new proof of neuro-endocrineimmune network-expression of islet amyloid polypeptide in plasma cells in gastric mucosa of peptic ulcer patients. World J Gastroenterol 2000;6:417e8. Hsu SM, Raine L, Fanger H. Use of avidinebiotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577e80. Kasacka I, Azzadin A, Sawicki B, Dadan J, Malla H, Buczko W. Preliminary evaluation of gastric endocrine cells in rats with experimental uremia. Folia Histochem Cytobiol 2001;39:207e8. Kingswood JC. Renal disease. In: Oxford John, Polish 1st ed. Internal disease, vol. 2. Urban & Fisher; 1999. p. 370e514. Kvetnoi IM, Iakovlova ND. Peptidergic innervation and the APUD system in normal conditions and in various pathological states. Arkh Patol 1987;49:85e92. Kvetnoi IM, Iuzhakov VV. APUD and mast cells of the gastrointestinal system: immunohistochemical and ultrastructural identification. Arkh Patol 1987;49:77e80. Kvetnoy I, Popuichiev V, Mikhina L, Anisimov V, Yuzhakov V, Konovalov S, et al. Gut neuroendocrine cells: relationship to the proliferative activity and apoptosis of mucous epitheliocytes in aging. Neuroendocrinol Lett 2001;22:337e41. Kvetnoy IM, Hernandez-Yago J, Hernandez JM, Kvetnaia TV, Reiter RJ, Khavinson VK. Diffuse neuroendocrine system and mitochondrial diseases: molecular and cellular bases of pathogenesis, new approaches to diagnosis and therapy. Neuroendocrinol Lett 2000; 21:83e99. Levillain O, Marescau B, Possemiers I, Al Banchaabouchi M, De Deyn PP. Influence of 72% injury in one kidney on several organs involved in quanidino compound metabolism: a time course study. Pflu¨gers Arch 2001;442:558e69. Noris M, Todeschini M, Zappella S, Bonazzola S, Zoja C, Corna D, et al. 17b-Estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases. Am J Physiol Renal Physiol 2000;279:626e35. Norle´n P, Curry WJ, Bjo¨rkqvist M, Maule A, Cunningham RT, Hogg RB, et al. Cell-specific processing of chromogranin A in endocrine cells of the rat stomach. J Histochem Cytochem 2001;49: 9e18. Ormrod D, Miller T. Experimental uremia. Nephron 1980;26: 249e54. Osadchuk MA, Panfilov Iu.A, Kvetnoi IM, Iakovleva NI, Pogudina NA. Endocrine cells of the gastroduodenal area in duodenal ulcer. Sov Med 1989;3:8e13. Panfilov Iu.A, Osadchuk MA, Kvetnoi IM. Quantitative characteristics of various endocrine cells of the duodenal bulb in the pre-ulcer conditions and duodenal ulcer. Ter Arkh 1989;61: 30e2.
I. Kasacka / Cell Biology International 28 (2004) 441e447 Raikhlin NT, Kvetnoi IM, Solomatina TM. Pathology of the APUDsystem of the digestive organs. Sov Med 1983;7:58e64. Raikhlin NT, Kvetnoi IM, Solomatina TM. The APUD system and the hormonal basis of gastrointestinal activity. Sov Med 1983;6:53e9. Ratner GL, Kvetnoi IM, Bereslavskii MI, Margolis Ia.M, Vaisman BL. Study of gastric APUD cells in gastroduodenal hemorrhage. Khirurgiia (Mosk) 1990;2(Feb):89e93. Rubin W, Schwartz B. An electron microscopic radioautographic identification of the APUD endocrine cells in the rat gastric pyloric glands. Gastroenterology 1981;81:311e20.
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Sirinek KR, O’Dorisio TM, Gaskill HV, Levine BA. Chronic renal failure: effect of hemodialysis on gastrointestinal hormones. Am J Surg 1984;148:732e5. Soehartono RH, Kitamura N, Yamagishi N, Taguchi K, Yamada J, Yamada H. An immunohistochemical study of endocrine cells in the abomasum of vagotomized calf. J Vet Med Sci 2002;64:11e5. Toner H, Carr A. The digestive system on ultrastructural atlas and review. London: Butterwort HS; 1971. Wie˛cek A, Kokot F. Endocrine disorders in patients with chronic renal failure. Post Hig Med Dos´ w 1989;43:423e51.
Immunohistochemical and electron-microscopic identification of neuroendocrine cells in the stomach of uremic rats Irena Kasacka) Department of Histology and Embryology, Medical University of Bia1ystok, ul. Kilin´skiego 1, 15-089 Bia1ystok, Poland Received 11 November 2003; revised 2 March 2004; accepted 24 March 2004
Abstract Many disturbances in electrolyte and hormonal balance in the body induced by functional impairment of renal parenchyma may affect the activity of amine precursor uptake and decarboxylation (APUD) cells, which constitute a very important link in the regulation of homeostasis. The aim of the present study was the morphological, immunohistochemical and ultrastructural estimation of enteroendocrine cells in the stomach of uremic rats. Fragments of gastric pylorus were collected 1, 2 and 4 weeks after nephrectomy. Paraffin embedded sections were stained with H C E and by silver impregnation. For identification of neuroendocrine cells, immunohistochemical reactions were performed using specific antibodies against somatostatin, synaptophysin, neuron-specific enolase and anti-calcitonin gene related peptide. The analysis showed an increased number of APUD cells in the stomach of uremic rats compared to control rats, which may be a morphological expression of their hyperfunction in the functional impairment of renal parenchyma. These results suggest that chronic renal failure can modulate the secretory processes of APUD cells. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: APUD cells; Stomach; Chronic renal failure
1. Introduction Chronic renal failure (CRF) is a pathological syndrome caused by progressive damage to renal structures induced by chronic nephropathies, characterized by gradually increasing impairment of function. When the pathological process reduces the active area of renal parenchyma to less than 1/3 its normal size, a very diverse pattern of clinical symptoms originating from most organs of the body gradually develops (Esaian et al., 1997; Kingswood, 1999). Their intensification leads to the phase of uremia characterized by severe metabolic disorders (Hu et al., 2000; Levillain et al. 2001; Noris et al., 2000). Alimentary disorders, such as lack of appetite, vomiting, erosions, bleeding and inflammations
Abbreviations: CRF, chronic renal failure; DNES, diffuse neuroendocrine system; APUD, amine precursor uptake and decarboxylation; SY, synaptophysin; NSE, neuron-specific enolase; ST, somatostatin; CGRP, calcitonin gene related peptide. ) Tel.: C48-85-748-54-55; fax: C48-85-748-54-58. E-mail address: [email protected].
with potential ulceration, are common in patients with CRF and are usually the first to appear (Kingswood, 1999; Wie˛cek and Kokot, 1989). These clinical symptoms observed in patients with CRF can be partly induced by functional disorders of diffuse neuroendocrine system (DNES) cells (Sirinek et al., 1984). This system includes approximately 60 types of neuroendocrine cells, distributed in all organs and tissues, which produce biogenic amines and peptide hormones (Kvetnoy et al., 2001; Panfilov et al., 1989). By the production of these biologically active substances, amine precursor uptake and decarboxylation (APUD) cells are directly involved in the regulation of many metabolic processes and are regulators of homeostasis via neurocrine, endocrine and paracrine mechanisms (Kvetnoy et al., 2000). Any disorder of the APUD system involving excessive production or deficiency of a hormone or biogenic amine may cause dysfunction of body systems, which manifests itself in a complex set of clinical symptoms (Kvetnoi and Iakovlova, 1987; Soehartono et al., 2002). In recent years, many scientists have been concerned
1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.03.012
442
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with the structure, function and histogenesis of APUD cells, as well as their involvement in various pathological processes. Current opinions based on numerous studies indicate the active involvement of neuroendocrine cells in the processes of pathogenesis and adaptation in various diseases (El-Salhy, 2001; Kvetnoi and Iakovlova, 1987; Kvetnoi and Iuzhakov, 1987; Kvetnoy et al., 2001; Norle´n et al., 2001). Taking into consideration the metabolic disorders observed in CRF and the undoubted involvement of biologically active substances produced by APUD cells in the regulation of homeostasis, I decided to investigate the functional morphology and ultrastructure of DNES cells in the stomach of uremic rats. The aim of this study was to determine the presence, location and changes in functional morphology of APUD cells in the stomach of uremic rats, based on immunohistochemical and ultrastructural investigations. 2. Materials and methods 2.1. Experimental model The study was carried out on 51 young male Wistar rats, 200e250 g body weight (mean 220G10 g) at the beginning of the experiment. The animals were housed at 20 (C with a relative humidity of 40e45% and a 12 h light/dark cycle. The animals had free access to drinking water and standard granulated diet. All experiments were performed at the same time of the day. Procedures involving the animals and their care were conducted in conformity with the institutional guidelines that were in compliance with national and international law and with Guidelines for the Use of Animals in Biomedical Research (Giles, 1987). Study assumptions, aim, schedule and mode of animal treatment were approved by the Senate Committee for the Supervision of Experiments on Humans and Animals at the Medical University of Bia~ystok. The animals were divided into two control groups and one experimental group, the latter being composed of rats in which uremia was induced: Kdcontrol group: 15 rats left intact. SOdcontrol group: 15 rats submitted to sham operation, i.e. decapsulation and removal of the adherent fat from both kidneys. Mduremic group: 26 rats with experimentally induced uremia. Five rats died during the experiment, so 21 were used in the final data. The rats in the control and uremic groups were divided into the following three subgroups: 1. sectioned 1 week after surgery: 5 rats from each control group (K1, SO1) and 7 from the uremic group (M1);
2. sectioned 2 weeks after surgery: 5 rats from K2 and SO2 and 7 from M2; 3. sectioned 4 weeks after surgery: 5 rats from K3 and SO3 and 7 animals from M3. 2.2. Induction of experimental renal insufficiency in rats Experimental uremia was induced by the method described by Azzadin et al. (1999) and Ormrod and Miller (1980). The rats were anaesthetised by pentobarbital (Biowet, Pu~awy), administered intraperitoneally at a dose of 50 mg/kg. Then an incision (2e2.5 cm long) was made on the dorsal side, 1 cm from the lumbar spine, at the level of the kidneys, thus giving a clear approach to the right kidney, followed by its nephrectomy. The approach to the left kidney was obtained in an identical way, followed by removal of 70% of renal cortex, leaving the renal medulla intact. The incised integuments were sutured in layers with catgut. 2.3. Histology: method of experimental material collection and fixation One, 2 and 4 weeks after surgery, the rats were anaesthetised with pentobarbital and blood was collected from their hearts. Then, the animals were sacrificed by decapitation. The stomach was immediately removed. For microscopic analysis, segments of pyloric distal parts of the stomach were used, fixed in Bouin’s fluid and embedded in paraffin in the routine way. For electron microscopy, specimens were embedded in 2.5% purified glutaraldehyde. The specimens were cut in 5 mm slices (Leica 2025 Autocut) and stained by hematoxylineeosin (H C E) for general histological examination, and by Grimelius’ method, revealing neuroendocrine cells following the impregnation of their cytoplasmic granules with silver salts (Grimelius, 1968). 2.4. Histology: identification of DNES cells by immunohistochemical methods 2.4.1. Antibodies Polyclonal rabbit anti-human somatostatin (ST) (1:200), polyclonal rabbit anti-human synaptophysin (SY) (1:10) and monoclonal mouse anti-human neuron-specific enolase (NSE) (1:100) were purchased from DAKO (DAKO Cytomation, Denmark). Anticalcitonin gene related peptide (CGRP) (1:8000) was purchased from SigmaeAldrich (Saint Louis, Missouri 63103, USA). 2.4.2. Immunohistochemical reaction procedure Briefly, using deparaffined and hydrated (with the use of pure, not denatured, alcohol) sections, the activity of endogenous peroxidase was blocked by 3% H2O2 for
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5 min. After washing with distilled water and 0.05 M Tris ( pH 7.4) for 5e10 min, the sections were incubated with antibody for 30 min in a darkroom at room temperature. Then, the sections were washed three times in Tris buffer. The ABC (avidinebiotin peroxidase complex) method was used, according to the manufacturer’s protocol, for the identification of immunohistochemical reactions (Hsu et al., 1981). A secondary, biotinylated antibody (detection kit: KIT DAKO LSAB (C) or LSAB 2) was used for 15 min, then the sections were washed three times in TriseHCl and incubated in streptavidin solution. Mayer’s hematoxylin was used for nuclear staining. Control reactions were simultaneously performed, where the specific antibody was excluded. They all gave negative results. Analysis of the preparations and photographic documentation were performed with an Olympus BX50 light microscope. 2.5. Electron microscopy Specimens were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium phosphate buffer ( pH 7.2) at 4 (C overnight, postfixed for 1 h in 2% osmium tetroxide, dehydrated in graded ethanol solutions and propylene oxide and finally embedded in epon. Ultrathin sections (60 nm) were cut on a Leica Reychard Ultratome Cut S, contrasted with uranyl acetate and lead citrate and examined in an OPTON 900 PC electron microscope. Endocrine cells containing secretory granules were subclassified according to the appearance of secretory granules, as described by Toner and Carr (1971). 2.6. Biochemical blood tests: creatinine and urea assays Cardiac blood was obtained and left at room temperature for 20 min to clot. It was then centrifuged for 15 min at 3000 rpm, and urea and creatinine levels in the serum were determined using a Beckman-CX4 Analyzer (using a ‘Ure´e cinetique UV 800’ bioMe´rieux). 2.7. Statistics Statistical analysis was based on the ANOVA test. Verification of variance analysis assumptions was performed by ShapiroeWilk’s test (normal distribution assumption). The analysis was performed using the SAS ATAT software package. A probability level of p ! 0:05 was considered significant.
3. Results No significant differences were found between the control groups, so only the results from animals subjected to sham operations are discussed. Increased concentrations of urea and creatinine are indicators of renal dysfunction. A significant increase in serum creatinine and urea concentrations was demonstrated in all animals with experimental renal dysfunction, when compared with respective values in the control groups (Table 1). The mucous membrane of the stomach showed cells with a characteristic positive argyrophilic Gomori reactiondcells that typically belong to the APUD system. In control animals, these cells were observed mainly in the fundus of the glands, while in uremic rats they were diffused all over the mucous membrane. In contrast to control rats, the microscopic picture of the sections of uremic animals displayed a considerable increase in the number of neuroendocrine cells, which was revealed due to the argentophilic nature of the cytoplasm. The greatest number of cells was observed 14 days after surgery. All rats showed neuroendocrine cells that were difficult to recognize by light microscopy after routine staining with hematoxylin and eosin. Single cells could sometimes be observed, dispersed between other epithelial cells along the glands, especially in the basal mucous membrane. They were round or pear shaped and had a dark-staining nucleus lying centrally in a light, poorly eosinophilic cytoplasm. The immunohistochemical studies revealed a positive reaction in the cytoplasm of endocrine cells of the stomach in all the animals examined. This indicates that the antibodies reacted against appropriate antigens in DNES cells, recognizing their site in the glandular epithelium of the stomach pylorus. In control rats, the greatest number of endocrine cells was observed in the basal portions of the glands, while they were spread all over the glands of uremic rats. Differences were found between uremic and control rats, not only in the distribution of endocrine cells, but also in their number and reaction intensity. The detecting antibodies SY, NSE, ST and CGRP used in immunohistochemical investigations yielded a distinctly positive reaction in the cytoplasm of APUD cells in the stomach of all the study animals, the reaction intensity and the number of positive cells being markedly intensified in the stomach of uremic rats compared with controls (Fig. 1A, B). Differences in the reaction
Table 1 Serum concentrations of creatinine and urea in control and uremic rats (mg/dl) (data are means G standard deviation)
Creatinine Urea
Control
1 Week
2 Weeks
4 Weeks
*p-Value
0.52 G 0.05 35.33 G 5.98
0.63 G 0.09 52.91 G 16.39
0.94 G 0.06 95.10 G 11.90
0.72 G 0.12 85.58 G 9.77
!0.05 !0.05
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were mostly found in the basal part of the cell; however, they were sometimes so numerous that they could also be seen in the nucleus. Some preparations showed increased transparency of secretory granules and sometimes the surrounding membrane was discontinuous. Ultrastructural examination of endocrine cells in the stomach of uremic rats showed activation of Golgi apparatus structures, with cistern dilation and relatively marked proliferation of smooth endoplasmic reticulum.
4. Discussion
Fig. 1. (A) Immunohistochemical reaction determining SY in the pylorus mucosa of control rat, (B) a significant increase in the number of synaptophysin-immunoreactive cells was seen 4 weeks after nephrectomy. Original magnification !200.
intensity between the groups were most distinct in CGRP-positive cells. In control animals (Fig. 2A), only single cells showed a positive reaction to the antibody, while a marked increase was noted in the number of CGRP-positive enteroendocrine cells in experimental uremic rats (Fig. 2B, C). Results of electron-microscopic pictures supplement and confirm the presence of enteroendocrine cells in the rat stomach. In the preparations of control stomachs, a slight or moderate change in the content of secretory granules distributed in various areas of the cytoplasm was seen (Fig. 3A). These granules, homogenous in content, were usually oval or spherical in shape. Endocrine granules were much more polymorphic in the preparations of uremic rat stomachs (Fig. 3B). A narrow space was sometimes seen to separate a single membrane enclosing the secretory granules from the homogenous contents of varying electron density. They
Laboratory analysis showed a statistically significant increase in the concentration of plasma indices of renal failure: urea and creatinine. The rats in which chronic renal failure was induced had elevated levels of these parameters over the whole 4 week experimental period. Damage to a considerable percentage of nephrons leads to disorders of excretory and endocrine functions and to serious changes in metabolic processes. These disorders, together with compensatory processes in the kidneys and other organs observed during chronic renal failure, have definite clinical and biological consequences (Noris et al., 2000; Sirinek et al., 1984). Histochemical analysis of the preparations of the pyloric part of the stomach confirms previously obtained preliminary data (Kasacka et al., 2001). The data presented here demonstrate that the pyloric part of the stomach contains neuroendocrine amine/peptide producing cells that could play an important role in the endogenous mechanisms of chronic renal failure. Knowledge of the APUD system and basic hormonal activity of the alimentary tract is quite extensive and based on the results of numerous studies conducted in recent years (Bargsten and Grube, 1992; Date et al., 2002; El-Salhy, 2001; Gilon et al., 1990; Hakanson et al., 2001; Huang et al., 2000; Kvetnoi and Iuzhakov, 1987; Raikhlin et al., 1983b). However, mechanisms of mutual relationships and interactions of cells diffused throughout the alimentary tract in the maintenance and regulation of homeostasis have yet to be clarified. Hence, interpretation of the results obtained in the present study is difficult. Analysis of the data shows that enteroendocrine cells, which, in control animals, occur mainly in the basal part of the mucous membrane of the stomach, are seen at various levels of glandular crypts in uremic animals, probably due to changes in activity and hyperplasia. It is reported that more than 50% of the cells of the APUD system are distributed in the alimentary tract (Raikhlin et al., 1983a,b). By producing numerous hormones and biogenic amines, enteroendocrine cells are directly involved in the regulation of physiological processes in normal and pathological conditions (Osadchuk et al., 1989; Panfilov et al., 1989;
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Fig. 2. (A) CGRP-immunoreactive cells in the pyloric mucosa of control rat, (B) of the same rat 1 week, and (C) 2 weeks after nephrectomy. Original magnification !200.
Fig. 3. Electron micrographs of endocrine cells from pyloric mucosa of (A) control rat, and (B) 2 weeks after nephrectomy. Some electron dense neuroendocrine granules (arrows) of slightly varying size can be observed in the cytoplasm, as well as the nucleus (N), mitochondria (m), and cisterna of rough endoplasmic reticulum (rER). Original magnification: (A) !7000, (B) !12,000.
Ratner et al., 1990). Any disturbance in the synchronization of APUD cell activity, which is expressed by an overproduction or deficiency of a particular hormone, involves disorders of both digestive function and general homeostasis (Huang et al. 2000; Kvetnoi and Iuzhakov, 1987; Raikhlin et al., 1983a,b). The increase in functional activity of enteroendocrine cells observed in this study indicates their role not only in normal, but also in pathological, reactions. Numerous DNES cells present in alimentary tract epithelium produce biologically active substances and ensure coordination of all functions of digestive organs. Reports are available indicating some changes in the number of these cells in various pathological conditions, thus confirming the active involvement of enteroendocrine cells in pathological processes of the alimentary tract (Fujimiya et al., 1991; Osadchuk et al., 1989; Panfilov et al., 1989; Ratner et al., 1990; Soehartono et al., 2002). It can be assumed that the increased number of APUD cells and reaction intensity observed in this study to a certain degree compensates for homeostatic disorders associated with the impairment of renal parenchyma functioning. The ultrastructure of enteroendocrine cells observed by light microscopy reflects the changes in activity of these cells. Electron microscopy of the stomach of uremic rats shows symptoms of stimulation of the activity of endocrine cells. Polymorphic neurosecretory granules,
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being the most characteristic element of this type of cell, occur in varying numbers in the analyzed pictures. The electron microscope reveals microvilli on the free area of numerous cells, sometimes quite long, reaching the lumen of the glands where they are located (Rubin and Schwartz, 1981). Due to these and other mechanisms, endocrine cells rapidly and precisely react to environmental changes via enhancement, reduction or inhibition of their secretory function and thus regulate the action of the respective group of parenchymal cells in their vicinity. Previous studies indicate likely changes in the functioning of APUD cells in reaction to the nature of passing food. This shows the high sensitivity of endocrine cells in preserving morphological features of chemoreceptors (Raikhlin et al., 1983a,b). The role of DNES cells in specific conditions of disturbed metabolic processes and electrolyte equilibrium, resulting from the impairment of renal parenchyma functioning, is possible mainly due to the wide range of biologically active substances produced by these cells. The increased quantity of enteroendocrine cells observed in the present study is a likely morphological expression of their hyperfunction in uremia. This seems to be confirmed by the intensification of staining of neurosecretory granules using specific histochemical methods. This study demonstrates that uremia has a significant effect on the endocrine system of the stomach. This suggests that APUD cells, in addition to the endocrine and nervous systems, are a significant element of the internal regulatory system in the stomach, and that their activity may be disturbed by uremia.
Acknowledgements I would like to thank Prof. W. Buczko and Dr. A. Azzadin from the Department of Pharmacodynamics, Medical University of Bia~ystok, for their assistance in performing these experiments.
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