تلاالحا نم اهيلع لوصلحا تم يتلا ةيضرلما تانيعلا ةنراقم :فادهلأا يضيولحا يبلالحا لصولما دادسنلإ ةيحارج ةيلمعل تعضخ يتلا .تانيعلا صحف للاخ اهيلع لوصلحا تم يتلا تانيعلا عم )UPJ( اوعضخ نيذلا ىضرلما 42 يعجر رثأب ةساردلا هذه تلمش :ةقيرطلا مييقت ىرجو . )1 ةعومجلما( UPJ دادسنا ببسب ةيحارج ةيلمعل و SHH، TBX18 نم لًاكل ةيعانلماو ةيضرلما ةيجيسنلا صئاصلخا اهصحف ىرج ةلاح 20 عم جئاتنلا ةنراقم تتمو UPJ نم TSHZ3 .)2 ةعومجلما( نم ىلولأا ةعومجلما يف ريثكب ىلعأ اًيئاصحا جئاتنلا تناك:جئاتنلا cytoplasmic SHH, nuclear TBX18, cytoplasmic ثيح رمعلا ينب ةقلاع يأ ىلع رثعيٌ ملو .TSHZ3 يوونلا غبصتلاو ، يف ةثلاثلا ةداضلما ماسجلأا هذه ىلإ ينمتنملل ةيغبصلا جئاتنلاو و فثكلما باهتللأا ينب طابترا كانه ناك.2 ةعومجلماو 1 ةعومجلما .TBX18 يوونلا غبصتلاو و SHH، TBX18 نم لًاكل ةينيلجا تاريبعتلا جئاتن :ةتمالخا ،UPJ دادسنا نم نوناعي نيذلا ىضرلما يف ايئاصحإ ىلعأ TSHZ3 .مكحتلا ةعومجم عم ةنراقلماب Objectives: To compare pathological samples obtained from cases that underwent surgery for ureteropelvic junction (UPJ) obstruction with samples obtained during autopsies of subjects. Methods: Retrospectively, 42 patients who had undergone surgery due to UPJ obstruction (group 1) were included in the study. Histopathological and immunohistochemical features for sonic hedgehog (SHH), TBX18, and TSHZ3 of UPJ were evaluated and findings were compared with 20 autopsy cases (group 2). Original Articles Results: In group 1, the scores were statistically significantly higher in terms of cytoplasmic SHH, nuclear TBX18, cytoplasmic and nuclear TSHZ3 staining. Statistically, no correlation was found between age and the staining scores belonging to these 3 antibodies in group 1 and group 2. Intense inflammation was found to be related with nuclear staining for TBX18. Conclusion: Gene product expressions of SHH, TBX18 and TSHZ3 are statistically higher in patients with UPJ obstruction, when compared with control group. The explanation may be the reactivation of the processes, which had shown their effects in the embryological period, due to the chronic inflammation and long-term micro-trauma created by the disease. Saudi Med J 2016; Vol. 37 (7): 737-743 doi:10.15537/smj.2016.7.14789 From the Departments of Pediatric Surgery (Yilmaz, Genc, Taneli, Gunsar, Sencan, Cayirli), Pathology (Nese, Isisag), Faculty of Medicine, Celal Bayar University, Manisa, and the Council of Forensic Medicine (Dalgic, Kesici), Izmir, Turkey. Received 10th February 2016. Accepted 16th May 2016. Address correspondence and reprint request to: Dr. Omer Yilmaz, Department of Pediatric Surgery, Faculty of Medicine, Celal Bayar University, Manisa, Turkey. E-mail: firstname.lastname@example.org OPEN ACCESS 737 www.smj.org.sa Saudi Med J 2016; Vol. 37 (7) Disclosure. Authors have no conflict of interest, and the work was not supported or funded by any drug company. The study is granted by the Scientific Research Project Coordination Unit of Celal Bayar University, Manisa, Turkey (Grant # 2014/012). SHH, TBX18, TSHZ3 proteins in UPJ obstructions ... Yilmaz et al 738 Saudi Med J 2016; Vol. 37 (7) www.smj.org.sa U junction (UPJ) obstruction is the most frequent cause of urinary obstructions in children.1 So far, the studies have failed to show how UPJ obstructions develop, with conclusive evidence. Histopathologically, it has been found that there are findings, such as inadequately or irregularly structured smooth muscle fibers, abnormal collagen deposition, increased muscle cell apoptosis, abnormal innervation, and disordered distribution of Cajal cells in UPJ samples obtained during surgery.2-5 Studies that would explain the genetic mechanisms of the UPJ obstructions have been carried out experimentally; the studies performed on humans are very limited in English medical literature. In experimental animal studies, the gene expressions of BMP4,6 sonic hedgehog (SHH),7 TBX18,8 and TSZH39 have been shown to play a primary role in the development of UPJ, particularly in smooth muscle formation. However, studies related to the effects of the productions of these genes on UPJ obstruction in humans are very limited. The aim of this study is to investigate the roles of the products of these genes in the pathogenesis of UPJ obstructions by comparing pathological samples obtained from cases who underwent surgery for UPJ obstruction with samples obtained during legal autopsies of subjects who had died due to legal causes and had no known disease provided by the District Forensic Medicine Institute. Methods. Search of related articles. Searching of the prior articles related to the study idea has been made by using PubMed. Patients and control group selection. Retrospectively, a total of 42 patients who had undergone surgery for the diagnosis of UPJ obstruction due to the symptoms and signs like more than 10% decrease in split renal function, pain, and stone formation in our department between January 2009 and December 2014 were included in the study. Grade III or IV hydronephrosis was ultrasonographically detected in all of the patients before the operation. Only patients who had intrinsic obstruction were selected as group 1, excluding cases that had extrinsic obstructions or associated renal disorders. Twenty seven of the cases were boys and 15 were girls in ages ranging between one and 15 (mean 6.72 ± 4.13) years. Twenty autopsy cases of 14 boys and 6 girls, prospectively collected in the Forensic Medicine Institute, Izmir District, Turkey within similar age range (2-15; mean 6.09 ± 4.74), had no known disease and who did not undergo autolysis were selected as control group (group 2); in this group, the cause of death was traffic accident in 8, drowning in 4, and falling off in 3 cases, while no identifiable cause of death were detected in 5 cases. Autopsy cases with renal or another medical disorders identified during autopsy, were excluded from the study. All procedures were in accordance with the Helsinki Declaration, and approvals of Local Ethics Committee and District Forensic Medicine Institute were obtained before the study. Sampling and microscopic evaluation. The ureteropelvic junctions containing the stenotic area (for group 1) and a small renal parenchymal area (for group 2) were sampled for histopathological examination. After fixing in formalin, embedding in paraffin and obtaining 5μ sections, the slides were stained with hematoxylin and eosin and evaluated for chronic inflammatory cellular infiltration, smooth muscle atrophy or hypertrophy, and subepithelial fibrosis, as well as to rule out presence of any other pathology. Inflammatory cellular infiltration in lamina propria, muscular and serosal layers togetherly and subepithelial fibrosis were scored semi-quantitatively as: absent or mild (0); moderate (1); and intense (2); whereas smooth muscle atrophy and hypertrophy were numbered as atrophy (1) and hypertrophy (2) to facilitate statistical analysis. All slides were evaluated by 2 pathologists in a double-blinded fashion. Immunohistochemical assessment. After routine tissue processing, selected slides were stained with BMP4, SHH, TBX18, and TSHZ3 antibodies (Table 1) in fully automatic immuno-staining device (Ventana®, Benchmark XT, Ventana Medical Systems, Tucson, Arizona) with standard streptavidin-biotin immunoperoxidase method with 3% hydrogen peroxide for inhibiting endogenous peroxidase activity, Ultra V Block to inhibite non-spesific staining, diaminobenzidine (DAB, DAKO, K4011) as chromogen, Mayer’s hematoxylin for counter-staining, and Entellan® (Merck and Co., Berlin) for sealing. Staining properties were assessed under standard light microscope by the same pathologists in a semiquantitatively manner in mesenchymal cells (namely, smooth muscle cells and fibroblasts). Since only cytoplasmic staining for SHH and both cytoplasmic and nuclear staining was observed for TBX18 and TSHZ3 in control tissues, staining in respective sites was assessed in group 1 and 2 cases. For the latter 2, antibodies, cytoplasmic, and nuclear staining were separately evaluated. As for BMP4, this antibody was not found to be reliable to assess and excluded from the study, since diffuse and non-specific staining in extracellular matrix areas and in cytoplasm in control tissues, as well as in both of the study groups. SHH, TBX18, TSHZ3 proteins in UPJ obstructions ... Yilmaz et al 739 www.smj.org.sa Saudi Med J 2016; Vol. 37 (7) Either nuclear or cytoplasmic staining of the 3 antibodies was scored as follows: no staining (0); positive staining cells <5% (1); 5%≤ positive staining cells <50% (2), and positive staining cells ≥50% (3). For cytoplasmic staining, intensity of staining was also considered as no staining (0), weak staining (1), moderate staining (2), and intense staining (3). For antibodies staining both nuclear and cytoplasmic (namely, TBX18, and TSHZ3), the average of 2 scores were taken into consideration. Final staining scores were obtained by getting the average score of the scores of the 2 pathologists. Statistical evaluation. Spearman’s Rho test, KruskalWallis variance analysis test, and Mann-Whitney U test were performed using the Statistical Package for the Social Sciences version 15 (SPSS Inc., Chicago, ILL, USA). A value of p<0.05 was considered statistically significant. Results. Histopathological findings. In histopathological examination, chronic inflammation was observed in all cases of group 1, which was mild in 2, moderate in 27, and intense in 13 cases with an average inflammation score of 1.21 ± 0.56. No signs of inflammation was found in cases of group 2. In group 1, fibrosis was absent in 2, moderate in 17, and intense in 23 cases with an average fibrosis score of 1.50 ± 0.59, whilst no fibrosis was identified in the subjects of group 2. In all cases of group 1, atrophy (n=29) or hypertrophy (n=13) was observed in ureteral muscular layer, contrary to group 2 carrying none of these findings. In terms of these 3 parameters (namely, inflammation, fibrosis, and athrophy/hiperthrophy), there were statistically significant differences between the 2 groups (p=0.01, p=0.01, p=0.01, Mann Whitney U test). Immunohistochemical findings. Detailed staining scores of group 1 and group 2 were given in Table 1. Immunohistochemical staining could be evaluated in 39 cases for SHH, 38 cases for TBX18, 42 cases for TSHZ3 of 42 cases (group 1), and all autopsy cases (group 2; n=20). As shown in Table 2, there were statistically significant differences between final average staining scores of the 2 groups, in terms of cytoplasmic SHH (p=0.01), nuclear TBX18 (p=0.01) and cytoplasmic TSHZ3 (p=0.01) staining, as well as a borderline significance in nuclear TSHZ3 (p=0.05) staining. Only cytoplasmic staining of TBX18 was found to be statistically significance. Reclassifying cases of group 1 according to severity of inflammatory cell infiltrate as group 1a (cases with moderate inflammatory reaction) and group 1b (cases with intense inflammatory reaction), and comparing the final average staining scores of the 3 antibodies, only statistically significant difference was obtained for TBX18 nuclear staining between the 2 subgroups (p=0.01, Kruskal-Wallis variance analysis test), as shown in Table 3. This datum indicates that nuclei of the stromal cells in cases with intense inflammatory reaction are more likely to be stained with TBX18. Two cases with mild inflammatory reaction were excluded because of inadequacy for statistical tests. The patients in group 1 were subsequently divided into 2 subgroups according to the presence of atrophy (group α) and hypertrophy (group β). Analysis of these subgroups according to the final average staining scores of the 3 antibodies revealed no statistically significant differences except in SHH cytoplasmic staining, indicating that stromal cells in hypertrophic cases are more prone to SHH staining (p=0.03, Mann Whitney U test, Table 4). Comparison of the subgroups according to the degree of fibrosis revealed no relationship between subgroups showing no or mild (n=2), moderate (n=17) and intense (n=23) fibrosis, in terms of expression of SHH, TBX18 (cytoplasmic-nuclear), and TSHZ3 (cytoplasmic-nuclear) gene products (p=0.08, p=0.28, p=0.25, p=0.06, 0.20, Kruskal-Wallis variance analysis Table 1 Staining scores for sonic hedgehog (SHH), TBX18, and TSHZ3 in group 1 and 2.