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Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral junction obstruction
PEDIATRIC UROLOGY
GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR AND SYNAPTOPHYSIN EXPRESSION IN PELVIURETERAL JUNCTION OBSTRUCTION ˘ SAVAS¸ DEMIRBILEK, MEHMET NACI EDALI, KUBILAY GÜRÜNLÜOGLU, EMINE TÜRKMEN, ˘ ERKAN TAS¸, ABDURRAHMAN KARAMAN, MELIH AKıN, RAUF TUGRUL AKSOY, ˙ OSMAN CELBIS, AND IBRAHIM ÜZÜN
ABSTRACT Objectives. To examine the expression of neuronal markers in congenital pelviureteral junction (PUJ) obstruction as a causative factor. The findings from some investigations have suggested that defective neuronal innervation may play an important role in the pathogenesis of PUJ obstruction. Methods. Using specific antibodies, we studied the neuronal markers of specimens from 12 cases of PUJ obstruction and 10 normal PUJs by immunohistochemistry using glial cell line-derived neurotrophic factor (GDNF), synaptophysin, S-100, and neurofilament. Results. In the PUJ obstruction specimens, staining with hematoxylin-eosin and Masson’s trichrome revealed muscular hypertrophy and an increase in collagen tissue and fibrosis in the lamina propria and tunica muscularis. The most striking finding on immunohistochemistry was the marked nuclear staining of cells with synaptophysin in all layers of the PUJ obstruction specimens that was totally absent in the normal PUJ specimens. In addition, significantly less intense staining for GDNF was found in the PUJ obstruction specimens compared with the normal PUJ specimens. The underexpression of GDNF in PUJ obstruction specimens was localized in the muscular layer especially. Immunohistochemical staining for S-100 and neurofilament showed no differences in the expression level of these neuronal markers in normal and PUJ obstruction specimens. Conclusions. Because GDNF is a survival factor for central and peripheral neurons, defective expression of GDNF could play an important role in the defective neuronal innervation of PUJ obstruction. Intense nuclear expression of synaptophysin in all layers of obstructed PUJ specimens suggested that obstructed PUJs have a serious structural abnormality. UROLOGY 67: 400–405, 2006. © 2006 Elsevier Inc.
C
ongenital obstructive nephropathy is the most frequent cause of renal failure in infants and children. Antenatal screening detects fetal hydronephrosis in 1 of 100 births, with about 20% clinically significant.1 Pelviureteral junction (PUJ) obstruction is found in 40% to 50% of these clinically significant cases, with an estimated incidence of 1 in 1000 to 1500.1 Currently, the adynamic dysplasia of this stenotic segment is considered the main pathologic change.2 Some investigators have reported abnormalities of smooth muscle or collagen in PUJ obstruction From the Departments of Pediatric Surgery, Pathology, and Forensic Medicine, I˙nönü University School of Medicine, Malatya, Turkey; and Council of Forensic Medicine, I˙stanbul, Turkey Reprint requests: Savas¸ Demirbilek, M.D., I˙nönü U¨niversitesi Tıp Fakültesi, Turgut Özal Tıp Merkezi, Çocuk Cerrahisi A.D., Malatya 44280, Turkey. E-mail: [email protected] Submitted: March 1, 2005, accepted (with revisions): August 25, 2005 © 2006 ELSEVIER INC. 400
ALL RIGHTS RESERVED
as revealed by light and electron microscopy.3,4 The abnormalities described include absent or deficient muscle at the PUJ, abnormal muscle orientation, and replacement of muscle by collagen.5–7 Some investigators have suggested that defective neuronal innervation may have an important role in the pathogenesis of PUJ obstruction. However, little information is available about the innervation patterns in PUJ obstruction.8 Glial cell line-derived neurotrophic factor (GDNF) and related molecules, neurturin, artemin, and persephin signal through a unique multicomponent receptor system consisting of RET tyrosine kinase and a glycosyl-phosphatidylinositol-anchored co-receptor.9 These neurotrophic factors promote the survival of various neurons, including peripheral autonomic and sensory neurons and central motor and dopamine neurons. Therefore, the first aim of this study was to test the hypothesis that GDNF expression 0090-4295/06/$32.00 doi:10.1016/j.urology.2005.08.056
FIGURE 1. (A) Masson’s trichrome staining in normal PUJ segment. (B) Obstructed PUJ segment showing massive collagen accumulation in muscular wall.
may be altered in the stenotic tissue of patients with PUJ obstruction and that this could be responsible for the defective neural innervation of the PUJ. We first examined the expression of GDNF in PUJ obstruction. We also examined the expression of various neuronal markers, including synaptophysin (major integral protein of small synaptic vesicles), S-100 (Schwann cell marker), and neurofilament (neuronal protein) using immunohistochemistry. MATERIAL AND METHODS PATIENTS AND METHODS The specimens (1.5 to 3 cm) of stenotic tissue from the PUJ were obtained from 12 patients 2 months to 6 years old (9 boys and 3 girls, average 29.66 ⫾ 25.06 months) with congenital PUJ obstruction at operation. All patients had an intrinsic anomaly, and no patients had an extrinsic anomaly, such as crossing vessels (or aberrant renal vessels). The diagnosis was based on ultrasound, radiologic, and scintigraphic findings and confirmed by the histologic findings of the obstructed segment (eg, muscle hypertrophy/hyperplasia and collagen deposition). The furosemide clearance half-time in diuretic renography was used to define whether obstruction was present. A drainage clearance half-time greater than 20 minutes identified obstruction. Patients were selected for corrective surgery using the Anderson-Hynes procedure if they had reduced (less than 40%) split function. In addition, 10 control ureters were obtained from children 1 day to 6 years old (9 boys and 1 girl, average 34.90 ⫾ 26.49 months) who were autopsied, and the tissues were confirmed histologically to be unaffected. Histologic and immunohistochemical analyses were performed in all cases on formalin-fixed, paraffin-embedded tissues. Serial sections (5 to 6 ) from the PUJ region were cut and used for hematoxylin-eosin and Masson’s trichrome stains and immunohistochemistry. A master pathologist at the University Hospital analyzed all specimens.
IMMUNOHISTOCHEMICAL LABELING Glial Cell Line-Derived Neurotrophic Factor. Sections on poly-L-lysine-coated slides were used after drying in an oven for 1 hour at 60°C. The sections were dewaxed in xylene, rehydrated in ethanol, and then incubated for 7 minutes in 3% hydrogen peroxide to block endogenous peroxidase. After washing in phosphate-buffered saline (PBS), the sections inUROLOGY 67 (2), 2006
cubated 5 minutes at ultraviolet block. The immunoreaction was performed for 25 minutes with 1:100 diluted anti-rabbit polyclonal antibodies against a peptide mapping within the carboxyl terminal domain of GDNF of human origin (Santa Cruz Biotechnology, Santa Cruz, Calif, code No. sc-328). After washing in PBS, the slides were incubated for 13 minutes with biotinylated goat anti-rabbit. After washing in PBS, the slides were incubated for 13 minutes with large-volume streptavidin peroxidase. Finally, the preparations were developed in AEC chromogen, counterstained with hematoxylin, and mounted with Aqueous-Mount. Synaptophysin. For immunohistochemistry, sections were transferred onto poly-L-lysine-coated slides and placed in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. Sections were then incubated in a microwave oven for 20 minutes and were kept at room temperature for 20 minutes. After washing in PBS (pH 7.6), the sections incubated 5 minutes at ultraviolet block. Each slide was then incubated for 30 minutes at room temperature with the primary polyclonal rabbit anti-human antibody to synaptophysin (Dako, Carpinteria, Calif) at appropriate dilutions (1:150). The slides were then soaked in PBS for 5 minutes, and excess fluid was removed. After soaking in PBS, the slides were incubated with biotinylated goat antipolyvalent for 13 minutes. Finally, the preparations were developed in AEC chromogen, counterstained with hematoxylin, dehydrated, and mounted with Aqueous-Mount. S-100 and Neurofilament. The antibodies used were rabbit polyclonal antiserum to S-100 (Novocastra Laboratories, United Kingdom), at 1:150; mouse monoclonal antibody 2F11 (Dako), against the 70-kD and 200-kD components of neurofilament protein, at 1:400.
RESULTS In the PUJ obstruction specimens, staining with hematoxylin-eosin and Masson’s trichrome revealed muscular hypertrophy and an increase in collagen tissue and fibrosis in the lamina propria and tunica muscularis (Fig. 1). In the normal PUJ specimens, GDNF expression within the muscle cells was strong. In contrast, GDNF expression was markedly reduced within the muscle cells of PUJ obstruction specimens (Fig. 2). Because the specimens obtained from those with PUJ obstruction were limited by con401
FIGURE 2. Immunohistochemistry for GDNF. (A,B) Controls in which GDNF expression was strongly positive in muscular layer. (C–F) Immunohistochemical staining with GDNF antibodies was mildly positive (C,D,F) or negative (E) in obstructed PUJ segments.
sisting of only stenotic tissue, we could not study the extension of GDNF expression in the tissue proximal to the PUJ obstruction. The most striking finding on immunohistochemistry was the marked nuclear staining of cells with polyclonal rabbit anti-human antibody to synaptophysin in all the layers of the PUJ obstruc402
tion specimens that was totally absent in the normal PUJ specimens (Fig. 3). The expression of S-100 (Fig. 4) and neurofilament (Fig. 5) showed no differences and was moderately positive in normal and obstructed PUJ specimens; it was mainly localized in the peripheral neuronal tracks. UROLOGY 67 (2), 2006
FIGURE 3. (A,B) Expression of synaptophysin protein against polyclonal rabbit anti-rat antibody to synaptophysin was negative in control specimens. (C-F) Expression of synaptophysin was strongly positive in nuclear cells of stenotic tissue and was localized in all layers.
No variability was found in any of the histologic findings in each group of patients. COMMENT The exact nature of the histopathologic abnormality is controversial in PUJ obstruction. There UROLOGY 67 (2), 2006
are proponents of both myogenic and neurogenic theories of peristalsis. Notley3 and Gosling and Dixon10 examined these obstructive segments and concluded that the muscle cells were normal and that the excessive collagen fibers in the submucosa and between the muscle cells ac403
FIGURE 4. Histochemical staining with S-100 protein in (A) normal and (B) stenotic tissue. No difference was noted in immunoreactivity findings. Original magnification ⫻40.
FIGURE 5. Neurofilament immunostaining showing no difference in (A) normal and (B) stenotic tissue specimen.
counted for the impairment in urine drainage. Kaneto et al.11 did a computer-assisted analysis of three-dimensional muscular architecture at the PUJ and concluded that various growth-related changes were ambiguous or lacking. Starr et al.12 showed major abnormalities of the pelvic microanatomy in the hydronephrotic kidneys of asymptomatic infants. Tainio et al.13 showed a dense innervation of neuropeptide Y and vasoactive intestinal peptide in PUJ obstruction. Wang et al.8 found marked reduction of protein gene product 9.5, synaptophysin, and nerve growth factor receptor staining positive nerve fibers in the muscle layers of the PUJ segment compared with normal. They concluded that defective neuronal innervation may have an important role in the pathogenesis of PUJ obstruction. Murakuma et al.14 proposed a combined neurogenic and myogenic theory. They concluded that in intrinsic obstruction, nerve fibers were depleted in the muscle layers in the ureteral walls, resulting in dysfunction and atrophy of the muscle fibers and an in404
crease in collagen fibers in the muscle layers with abnormal accumulation of intercellular and interstitial collagen. They suggested that these changes may disrupt the motility at the PUJ and lead to mechanical and functional obstruction.15 GDNF, a recently discovered growth and trophic (survival) factor, acts on a remarkable array of target cells throughout the body. The molecule, distantly related transforming growth factor-beta, is the most potent known survival factor for substantia nigra dopaminergic neurons, which degenerate in patients with Parkinson’s disease, and for motor spinal neurons, which die in patients with Lou Gehrig’s disease.16,17 However, GDNF actions are not restricted to the nervous system. Targeted deletion of the GDNF gene results in pleomorphic deficits in mice. The animals die shortly after birth with renal dysgenesis and abnormal development of the gastrointestinal tract, associated with severe deficits in the enteric nervous system.18 Clearly, GDNF acts on multiple somatic and neural cells of tissues. We examined the expression of GDNF in PUJ obUROLOGY 67 (2), 2006
struction. Our studies showed that GDNF expression in the PUJ obstructed segment was decreased, and the decrease in GDNF expression was located mainly in the muscular layer. This finding suggests that the lower expression of GDNF could play an important role in the defective neuronal innervation of PUJ obstruction. The weak expression of GDNF might also be related to the fibromuscular dysplasia changes in PUJ obstruction, perhaps through induction of apoptosis of smooth muscle. The mechanism needs additional investigation. Synaptophysin, also called protein p38, is a glycoprotein that was first identified in small presynaptic vesicle membranes of neurons and in the chromaffin cells in the adrenal medulla.19 This integral membrane glycoprotein has also been demonstrated in all cell types of the endocrine pancreas.20 Synaptophysin, a 38-kD synaptic vesicle protein, is present in the neuronal synaptic vesicle membrane and is responsible for neurotransmission.21 By using a polyclonal antibody to this protein, we have found, by immunohistochemistry, that an identical or similar protein is intensely expressed in the nucleus of the cells in all layers of the PUJ obstruction specimens. Our data were different from the finding of Wang et al.8 In their study, they found a reduced expression of synaptophysin in PUJ obstruction specimens. This difference could be explained by the use of different antibodies to synaptophysin (polyclonal antibodies versus monoclonal) in these studies. In the PUJ obstructed specimens, overexpression of synaptophysin immunoreactivity throughout the nucleus, as demonstrated in our study, is additional evidence of structural abnormalities in PUJ obstruction. Supporting nerve cell fibers (S-100) and neurofilament were preserved in cases of PUJ obstruction and normal PUJ specimens and were moderately positive. CONCLUSIONS These findings suggest that GDNF expression is decreased in the stenotic tissue after clinical PUJ obstruction. The alteration of GDNF expression may be involved in the pathogenesis of PUJ obstruction. The presence of an amino acid sequence to polyclonal rabbit anti-synaptophysin in the nuclear cells of pelviureteral tissue could provide an important criterion or marker (together with GDNF expression) to define PUJ obstruction. REFERENCES 1. Ransley PG, Dhillon HK, Gordon I, et al: The postnatal management of hydronephrosis diagnosed by prenatal ultrasound. J Urol 144: 584 –587, 1990.
UROLOGY 67 (2), 2006
2. Bernard MC, and Waldo CF: Ureteropelvic junction anomalies: congenital UPJ problems in children, in John PG, Richard CR, and Pierre DEM (Eds): Pediatric Urology. Philadelphia, WB Saunders, 2000, pp 318 –346. 3. Notley RG: Electron microscopy of the upper ureter and the pelvi-ureteric junction. Br J Urol 40: 37–52, 1968. 4. Gosling JA, and Dixon JS: Functional obstruction of the ureter and renal pelvis: a histological and electron microscopic study. Br J Urol 50: 145–152, 1978. 5. Foote JW, Blennerhassett JB, Wiglesworth FW, et al: Observations on the ureteropelvic junction. J Urol 104: 252– 257, 1970. 6. Antonakopoulos GN, Fuggle WJ, Newman J, et al: Idiopathic hydronephrosis: light microscopic features and pathogenesis. Arch Path Lab Med 109: 1097–1101, 1985. 7. D’Aquila RT, Bechtel LJ, Videler JA, et al: Maximizing sensitivity and specificity of PCR by pre-amplication heating. Nucleic Acids Res 19: 3749, 1991. 8. Wang Y, Puri P, Hassan J, et al: Abnormal innervation and altered nerve growth factor messenger ribonucleleic acid expression in ureteropelvic junction obstruction. J Urol 154: 679 – 683, 1995. 9. Kawakami T, Wakabayashi Y, and Aima Y: Developmental expression of glial cell-line derived neurotrophic factor, neurturin, and their receptor mRNA in the rat urinary bladder. Neurol Urodyn 22: 83– 88, 2003. 10. Gosling JA, and Dixon JS: The structure of the normal and hydronephrotic upper urinary tract, in O’Reilly PH, and Gosling JA (Eds): Idiopathic Hydronephrosis. Berlin, SpringerVerlag, 1982, pp 1–15. 11. Kaneto H, Orikasa S, Chiba T, et al: Three-D muscular arrangement at the ureteropelvic junction and its changes in congenital hydronephrosis: a stereo-morphometric study. J Urol 146: 909 –914, 1991. 12. Starr NT, Maizels M, Chou P, et al: Micro anatomy and morphometry of the hydronephrotic “obstructed” pelvis in asymptomatic infants. J Urol 148: 519 –524, 1992. 13. Tainio H, Kylmalo T, and Heikkiren A: Peptidergic innervation of normal and obstructed human pelvi-ureteric junction obstruction. Urol Int 48: 31–34, 1992. 14. Murakuma M, Nonmura K, Yamashita T, et al: Structural changes of collagen components and diminution of nerves in congenital ureteropelvic junction obstruction. J Urol 157: 1963–1968, 1997. 15. Shafik A, and Al-Sherif A: Ureteropelvic junction: a study of its anatomical structure and function— uretropelvic junction sphincter? Eur Urol 36: 150 –157, 1999. 16. Henderson CE, Phillips RA, Pollock AM, et al: GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266: 970 –972, 1994. 17. Lin LF, Doherty DH, Lile JD, et al: GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130 –1132, 1993. 18. Moore MW, Klein RD, Farinas I, et al: Renal and neuronal abnormalities in mice lacking GDNF. Nature 382: 76 – 79, 1996. 19. John R, Schber W, Oimet CH, et al: 38,000-dalton membrane protein (P38) present in synaptic vesicles. Proc Natl Acad Sci USA 82: 4137– 4141, 1985. 20. Wiedenmann B, Franke WW, Kuhn C, et al: Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci USA 83: 3500 –3504, 1986. 21. Navone F, Jahn R, Di Gioia G, et al: Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol 103: 2511–2527, 1986.
405
GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR AND SYNAPTOPHYSIN EXPRESSION IN PELVIURETERAL JUNCTION OBSTRUCTION ˘ SAVAS¸ DEMIRBILEK, MEHMET NACI EDALI, KUBILAY GÜRÜNLÜOGLU, EMINE TÜRKMEN, ˘ ERKAN TAS¸, ABDURRAHMAN KARAMAN, MELIH AKıN, RAUF TUGRUL AKSOY, ˙ OSMAN CELBIS, AND IBRAHIM ÜZÜN
ABSTRACT Objectives. To examine the expression of neuronal markers in congenital pelviureteral junction (PUJ) obstruction as a causative factor. The findings from some investigations have suggested that defective neuronal innervation may play an important role in the pathogenesis of PUJ obstruction. Methods. Using specific antibodies, we studied the neuronal markers of specimens from 12 cases of PUJ obstruction and 10 normal PUJs by immunohistochemistry using glial cell line-derived neurotrophic factor (GDNF), synaptophysin, S-100, and neurofilament. Results. In the PUJ obstruction specimens, staining with hematoxylin-eosin and Masson’s trichrome revealed muscular hypertrophy and an increase in collagen tissue and fibrosis in the lamina propria and tunica muscularis. The most striking finding on immunohistochemistry was the marked nuclear staining of cells with synaptophysin in all layers of the PUJ obstruction specimens that was totally absent in the normal PUJ specimens. In addition, significantly less intense staining for GDNF was found in the PUJ obstruction specimens compared with the normal PUJ specimens. The underexpression of GDNF in PUJ obstruction specimens was localized in the muscular layer especially. Immunohistochemical staining for S-100 and neurofilament showed no differences in the expression level of these neuronal markers in normal and PUJ obstruction specimens. Conclusions. Because GDNF is a survival factor for central and peripheral neurons, defective expression of GDNF could play an important role in the defective neuronal innervation of PUJ obstruction. Intense nuclear expression of synaptophysin in all layers of obstructed PUJ specimens suggested that obstructed PUJs have a serious structural abnormality. UROLOGY 67: 400–405, 2006. © 2006 Elsevier Inc.
C
ongenital obstructive nephropathy is the most frequent cause of renal failure in infants and children. Antenatal screening detects fetal hydronephrosis in 1 of 100 births, with about 20% clinically significant.1 Pelviureteral junction (PUJ) obstruction is found in 40% to 50% of these clinically significant cases, with an estimated incidence of 1 in 1000 to 1500.1 Currently, the adynamic dysplasia of this stenotic segment is considered the main pathologic change.2 Some investigators have reported abnormalities of smooth muscle or collagen in PUJ obstruction From the Departments of Pediatric Surgery, Pathology, and Forensic Medicine, I˙nönü University School of Medicine, Malatya, Turkey; and Council of Forensic Medicine, I˙stanbul, Turkey Reprint requests: Savas¸ Demirbilek, M.D., I˙nönü U¨niversitesi Tıp Fakültesi, Turgut Özal Tıp Merkezi, Çocuk Cerrahisi A.D., Malatya 44280, Turkey. E-mail: [email protected] Submitted: March 1, 2005, accepted (with revisions): August 25, 2005 © 2006 ELSEVIER INC. 400
ALL RIGHTS RESERVED
as revealed by light and electron microscopy.3,4 The abnormalities described include absent or deficient muscle at the PUJ, abnormal muscle orientation, and replacement of muscle by collagen.5–7 Some investigators have suggested that defective neuronal innervation may have an important role in the pathogenesis of PUJ obstruction. However, little information is available about the innervation patterns in PUJ obstruction.8 Glial cell line-derived neurotrophic factor (GDNF) and related molecules, neurturin, artemin, and persephin signal through a unique multicomponent receptor system consisting of RET tyrosine kinase and a glycosyl-phosphatidylinositol-anchored co-receptor.9 These neurotrophic factors promote the survival of various neurons, including peripheral autonomic and sensory neurons and central motor and dopamine neurons. Therefore, the first aim of this study was to test the hypothesis that GDNF expression 0090-4295/06/$32.00 doi:10.1016/j.urology.2005.08.056
FIGURE 1. (A) Masson’s trichrome staining in normal PUJ segment. (B) Obstructed PUJ segment showing massive collagen accumulation in muscular wall.
may be altered in the stenotic tissue of patients with PUJ obstruction and that this could be responsible for the defective neural innervation of the PUJ. We first examined the expression of GDNF in PUJ obstruction. We also examined the expression of various neuronal markers, including synaptophysin (major integral protein of small synaptic vesicles), S-100 (Schwann cell marker), and neurofilament (neuronal protein) using immunohistochemistry. MATERIAL AND METHODS PATIENTS AND METHODS The specimens (1.5 to 3 cm) of stenotic tissue from the PUJ were obtained from 12 patients 2 months to 6 years old (9 boys and 3 girls, average 29.66 ⫾ 25.06 months) with congenital PUJ obstruction at operation. All patients had an intrinsic anomaly, and no patients had an extrinsic anomaly, such as crossing vessels (or aberrant renal vessels). The diagnosis was based on ultrasound, radiologic, and scintigraphic findings and confirmed by the histologic findings of the obstructed segment (eg, muscle hypertrophy/hyperplasia and collagen deposition). The furosemide clearance half-time in diuretic renography was used to define whether obstruction was present. A drainage clearance half-time greater than 20 minutes identified obstruction. Patients were selected for corrective surgery using the Anderson-Hynes procedure if they had reduced (less than 40%) split function. In addition, 10 control ureters were obtained from children 1 day to 6 years old (9 boys and 1 girl, average 34.90 ⫾ 26.49 months) who were autopsied, and the tissues were confirmed histologically to be unaffected. Histologic and immunohistochemical analyses were performed in all cases on formalin-fixed, paraffin-embedded tissues. Serial sections (5 to 6 ) from the PUJ region were cut and used for hematoxylin-eosin and Masson’s trichrome stains and immunohistochemistry. A master pathologist at the University Hospital analyzed all specimens.
IMMUNOHISTOCHEMICAL LABELING Glial Cell Line-Derived Neurotrophic Factor. Sections on poly-L-lysine-coated slides were used after drying in an oven for 1 hour at 60°C. The sections were dewaxed in xylene, rehydrated in ethanol, and then incubated for 7 minutes in 3% hydrogen peroxide to block endogenous peroxidase. After washing in phosphate-buffered saline (PBS), the sections inUROLOGY 67 (2), 2006
cubated 5 minutes at ultraviolet block. The immunoreaction was performed for 25 minutes with 1:100 diluted anti-rabbit polyclonal antibodies against a peptide mapping within the carboxyl terminal domain of GDNF of human origin (Santa Cruz Biotechnology, Santa Cruz, Calif, code No. sc-328). After washing in PBS, the slides were incubated for 13 minutes with biotinylated goat anti-rabbit. After washing in PBS, the slides were incubated for 13 minutes with large-volume streptavidin peroxidase. Finally, the preparations were developed in AEC chromogen, counterstained with hematoxylin, and mounted with Aqueous-Mount. Synaptophysin. For immunohistochemistry, sections were transferred onto poly-L-lysine-coated slides and placed in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. Sections were then incubated in a microwave oven for 20 minutes and were kept at room temperature for 20 minutes. After washing in PBS (pH 7.6), the sections incubated 5 minutes at ultraviolet block. Each slide was then incubated for 30 minutes at room temperature with the primary polyclonal rabbit anti-human antibody to synaptophysin (Dako, Carpinteria, Calif) at appropriate dilutions (1:150). The slides were then soaked in PBS for 5 minutes, and excess fluid was removed. After soaking in PBS, the slides were incubated with biotinylated goat antipolyvalent for 13 minutes. Finally, the preparations were developed in AEC chromogen, counterstained with hematoxylin, dehydrated, and mounted with Aqueous-Mount. S-100 and Neurofilament. The antibodies used were rabbit polyclonal antiserum to S-100 (Novocastra Laboratories, United Kingdom), at 1:150; mouse monoclonal antibody 2F11 (Dako), against the 70-kD and 200-kD components of neurofilament protein, at 1:400.
RESULTS In the PUJ obstruction specimens, staining with hematoxylin-eosin and Masson’s trichrome revealed muscular hypertrophy and an increase in collagen tissue and fibrosis in the lamina propria and tunica muscularis (Fig. 1). In the normal PUJ specimens, GDNF expression within the muscle cells was strong. In contrast, GDNF expression was markedly reduced within the muscle cells of PUJ obstruction specimens (Fig. 2). Because the specimens obtained from those with PUJ obstruction were limited by con401
FIGURE 2. Immunohistochemistry for GDNF. (A,B) Controls in which GDNF expression was strongly positive in muscular layer. (C–F) Immunohistochemical staining with GDNF antibodies was mildly positive (C,D,F) or negative (E) in obstructed PUJ segments.
sisting of only stenotic tissue, we could not study the extension of GDNF expression in the tissue proximal to the PUJ obstruction. The most striking finding on immunohistochemistry was the marked nuclear staining of cells with polyclonal rabbit anti-human antibody to synaptophysin in all the layers of the PUJ obstruc402
tion specimens that was totally absent in the normal PUJ specimens (Fig. 3). The expression of S-100 (Fig. 4) and neurofilament (Fig. 5) showed no differences and was moderately positive in normal and obstructed PUJ specimens; it was mainly localized in the peripheral neuronal tracks. UROLOGY 67 (2), 2006
FIGURE 3. (A,B) Expression of synaptophysin protein against polyclonal rabbit anti-rat antibody to synaptophysin was negative in control specimens. (C-F) Expression of synaptophysin was strongly positive in nuclear cells of stenotic tissue and was localized in all layers.
No variability was found in any of the histologic findings in each group of patients. COMMENT The exact nature of the histopathologic abnormality is controversial in PUJ obstruction. There UROLOGY 67 (2), 2006
are proponents of both myogenic and neurogenic theories of peristalsis. Notley3 and Gosling and Dixon10 examined these obstructive segments and concluded that the muscle cells were normal and that the excessive collagen fibers in the submucosa and between the muscle cells ac403
FIGURE 4. Histochemical staining with S-100 protein in (A) normal and (B) stenotic tissue. No difference was noted in immunoreactivity findings. Original magnification ⫻40.
FIGURE 5. Neurofilament immunostaining showing no difference in (A) normal and (B) stenotic tissue specimen.
counted for the impairment in urine drainage. Kaneto et al.11 did a computer-assisted analysis of three-dimensional muscular architecture at the PUJ and concluded that various growth-related changes were ambiguous or lacking. Starr et al.12 showed major abnormalities of the pelvic microanatomy in the hydronephrotic kidneys of asymptomatic infants. Tainio et al.13 showed a dense innervation of neuropeptide Y and vasoactive intestinal peptide in PUJ obstruction. Wang et al.8 found marked reduction of protein gene product 9.5, synaptophysin, and nerve growth factor receptor staining positive nerve fibers in the muscle layers of the PUJ segment compared with normal. They concluded that defective neuronal innervation may have an important role in the pathogenesis of PUJ obstruction. Murakuma et al.14 proposed a combined neurogenic and myogenic theory. They concluded that in intrinsic obstruction, nerve fibers were depleted in the muscle layers in the ureteral walls, resulting in dysfunction and atrophy of the muscle fibers and an in404
crease in collagen fibers in the muscle layers with abnormal accumulation of intercellular and interstitial collagen. They suggested that these changes may disrupt the motility at the PUJ and lead to mechanical and functional obstruction.15 GDNF, a recently discovered growth and trophic (survival) factor, acts on a remarkable array of target cells throughout the body. The molecule, distantly related transforming growth factor-beta, is the most potent known survival factor for substantia nigra dopaminergic neurons, which degenerate in patients with Parkinson’s disease, and for motor spinal neurons, which die in patients with Lou Gehrig’s disease.16,17 However, GDNF actions are not restricted to the nervous system. Targeted deletion of the GDNF gene results in pleomorphic deficits in mice. The animals die shortly after birth with renal dysgenesis and abnormal development of the gastrointestinal tract, associated with severe deficits in the enteric nervous system.18 Clearly, GDNF acts on multiple somatic and neural cells of tissues. We examined the expression of GDNF in PUJ obUROLOGY 67 (2), 2006
struction. Our studies showed that GDNF expression in the PUJ obstructed segment was decreased, and the decrease in GDNF expression was located mainly in the muscular layer. This finding suggests that the lower expression of GDNF could play an important role in the defective neuronal innervation of PUJ obstruction. The weak expression of GDNF might also be related to the fibromuscular dysplasia changes in PUJ obstruction, perhaps through induction of apoptosis of smooth muscle. The mechanism needs additional investigation. Synaptophysin, also called protein p38, is a glycoprotein that was first identified in small presynaptic vesicle membranes of neurons and in the chromaffin cells in the adrenal medulla.19 This integral membrane glycoprotein has also been demonstrated in all cell types of the endocrine pancreas.20 Synaptophysin, a 38-kD synaptic vesicle protein, is present in the neuronal synaptic vesicle membrane and is responsible for neurotransmission.21 By using a polyclonal antibody to this protein, we have found, by immunohistochemistry, that an identical or similar protein is intensely expressed in the nucleus of the cells in all layers of the PUJ obstruction specimens. Our data were different from the finding of Wang et al.8 In their study, they found a reduced expression of synaptophysin in PUJ obstruction specimens. This difference could be explained by the use of different antibodies to synaptophysin (polyclonal antibodies versus monoclonal) in these studies. In the PUJ obstructed specimens, overexpression of synaptophysin immunoreactivity throughout the nucleus, as demonstrated in our study, is additional evidence of structural abnormalities in PUJ obstruction. Supporting nerve cell fibers (S-100) and neurofilament were preserved in cases of PUJ obstruction and normal PUJ specimens and were moderately positive. CONCLUSIONS These findings suggest that GDNF expression is decreased in the stenotic tissue after clinical PUJ obstruction. The alteration of GDNF expression may be involved in the pathogenesis of PUJ obstruction. The presence of an amino acid sequence to polyclonal rabbit anti-synaptophysin in the nuclear cells of pelviureteral tissue could provide an important criterion or marker (together with GDNF expression) to define PUJ obstruction. REFERENCES 1. Ransley PG, Dhillon HK, Gordon I, et al: The postnatal management of hydronephrosis diagnosed by prenatal ultrasound. J Urol 144: 584 –587, 1990.
UROLOGY 67 (2), 2006
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