Accepted Manuscript Neuronal defects an etiological factor in congenital pelviureteric junction obstruction? Guo Yuan How, Kenneth Tou En Chang, Anette Sundfor Jacobsen, Te-Lu Yap, Caroline Choo Phaik Ong, Yee Low, John Carson Allen, Chik Hong Kuick, Malcolm Zhun Leong Lim, Narasimhan Kannan Laksmi PII:
S1477-5131(17)30305-4
DOI:
10.1016/j.jpurol.2017.07.014
Reference:
JPUROL 2621
To appear in:
Journal of Pediatric Urology
Received Date: 6 March 2017 Revised Date:
1477-5131 1477-5131
Accepted Date: 26 July 2017
Please cite this article as: How GY, Chang KTE, Jacobsen AS, Yap T-L, Ong CCP, Low Y, Allen JC, Kuick CH, Lim MZL, Laksmi NK, Neuronal defects an etiological factor in congenital pelviureteric junction obstruction?, Journal of Pediatric Urology (2017), doi: 10.1016/j.jpurol.2017.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Neuronal defects an etiological factor in congenital pelviureteric junction obstruction? Guo Yuan Howa,*, Kenneth Tou En Changb, Anette Sundfor Jacobsenc, Te-Lu Yapc, Caroline Choo Phaik Ongc, Yee Lowc, John Carson Allend, Chik Hong Kuickb, Malcolm Zhun Leong Limb, and Narasimhan Kannan Laksmic Yong Loo Lin School of Medicine, National University of Singapore, Singapore Department of Pathology and Laboratory Medicine, KK Women’s and Children’s
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b
Hospital, Singapore c
Department of Paediatric Surgery, KK Women’s and Children’s Hospital, Singapore
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Centre for Quantitative Medicine, Duke-NUS Medical School, Singapore
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a
* Corresponding author. Yong Loo Lin School of Medicine, 1E Kent Ridge Road, Singapore 119228, NUHS Tower Block, Level 11
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E-mail address: [email protected]
Summary Introduction: Congenital pelviureteric junction obstruction (PUJO) is one of the most frequent causes of neonatal hydronephrosis. Obstruction at the PUJ has potential severe adverse outcomes, such as renal damage. While pyeloplasty has been
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established as the definitive treatment, the exact pathophysiology of congenital PUJO remains unknown. Recent research has proposed neuronal innervation defects as an etiological factor in congenital PUJO. We aim to study the expression of various neuronal markers in PUJO specimens compared with controls, and evaluate whether
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severity of renal disease or dysfunction pre-operatively is related to expression of neuronal markers in resected PUJO specimens. Materials and methods: All consecutive patients who underwent dismembered
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pyeloplasty at KK Women’s and Children’s Hospital, Singapore, for intrinsic PUJO from 2008 to 2012 were included. Patients with other co-occurring renal pathologies were excluded. Controls were obtained from nephrectomy patients with Wilm’s tumor or other benign renal conditions during the same period. Specimens were stained immunohistochemically with neuronal markers protein gene product 9.5 (PGP9.5), synaptophysin, and S-100, and with CD-117, a marker for interstitial cells of Cajal (Table). Levels of expression of the markers were assessed semiquantitatively (decreased, increased or no change) in comparison with controls by two independent observers. Pre-operative data of patients’ renal anatomical (ultrasonography
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ACCEPTED MANUSCRIPT measurements of renal pelvis size) and functional parameters (differential renal function measured using MAG-3 renal scans) were obtained. Discussion: Thirty-eight PUJO specimens (38 renal units) and 20 controls were studied. Mean patient age at pyeloplasty was 25.3 months (2.9-167.6 months). Median preoperative pelvic size was 25.0 mm (17.0-50.0 mm). Both PUJO specimens and controls
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showed great heterogeneity in distribution of innervation. All four immunohistochemical markers were not predictive of significant pre-operative renal pelvis dilation or pre-operative diminished renal function of the operated kidney. Conclusions: There exists marked variability in expression of neuronal markers
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synaptophysin, PGP9.5, and S-100, and CD-117 in PUJO specimens compared with
controls. Our results show no clinical significance of the expression of neuronal markers in predicting degree of pre-operative renal pelvis dilation or differential renal function.
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The heterogeneity of expression of neuronal markers in PUJO specimens and controls in our population is at variance with prior studies. The etiology of PUJO is likely to be complex and multifactorial.
KEYWORDS
Neuronal;
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Pelviureteric;
Etiology
Function - Most abundant integral membrane protein constituent of synaptic vesicles - Ubiquity in synapses allows for quantification of synapses - Function unknown - Deubiquitinating enzyme in neurons and nerve fibers - Neuron-specific cytoplasmic marker - Transmembrane tyrosine kinase growth factor receptor - Expressed in interstitial cells of Cajal, which generate and propagate electrical slow waves causing phasic contractions in gastrointestinal tract - Interstitial cells of Cajal present in renal pelvis and ureter - Function unknown - Specific marker for localizing cells of neural origin especially Schwann cells
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Neuronal marker
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Immunohistochemistry;
Synaptophysin PGP9.5
CD-117
S-100
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ACCEPTED MANUSCRIPT Introduction Congenital pelviureteric junction obstruction (PUJO) is one of the most frequent causes of neonatal hydronephrosis, occurring in 1/1,000 to 1/2,000 newborns [1]. Boys are affected more commonly than girls [2,3], with a higher incidence in the left kidney. The
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pelviureteric junction (PUJ) is defined as the junction between the renal pelvis and proximal ureter. However, this junction exists as a concept rather than a readily
identifiable landmark because of the lack of a clear anatomical definition [4]. The PUJ is well established as a common site of obstruction causing pelvicalyceal dilatation.
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Obstruction at the PUJ is believed to result in renal parenchymal damage, hypertension, and potential renal failure with bilateral PUJO. Although the exact pathophysiology of PUJO is unknown, underlying causes can be classified into structural (e.g. intraluminal,
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luminal, and extraluminal) and/or functional (e.g. abnormal smooth muscle function or impaired motility secondary to abnormal expression of interstitial cells of Cajal, ICC) disorders [3,4].
The definitive treatment for PUJO is surgery [5], which involves resection of the PUJ (dismembered pyeloplasty) or refashioning of the PUJ (non-dismembered
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technique). While surgery is the established treatment for PUJO, the etiology of PUJO remains unknown. It is unclear if pyeloplasty works by improving peristalsis or just mechanically opens the passage to allow dependent urinary drainage. Histological changes observed in some PUJ specimens include smooth muscle hypertrophy [6] and
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abnormal collagen deposition [7]. In recent years, some studies have suggested that PUJO may be caused by defective innervation or abnormalities in smooth muscle arrangement [7,8]; however, these findings have not been confirmed unanimously. To
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investigate neuronal innervation defects in PUJO we have identified four specific neuronal markers based on recent literature [9-11]: protein gene product 9.5 (PGP9.5), synaptophysin, S-100, and CD-117. PGP9.5 is a protease found in neurons of the central and peripheral nervous
systems. This neuron-specific cytoplasmic marker is also present in neuronal networks between smooth muscle fibers in the ureteral wall and the renal pelvis. Synaptophysin is the most abundant integral membrane protein constituent of synaptic vesicles of neurons. Although the function of synaptophysin is currently unknown, its ubiquity in synapses allows it to be used in the quantification of neuronal synapses. S-100 functions
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ACCEPTED MANUSCRIPT as a calcium flux regulator and is present in nerves innervating the ureter and renal cells. CD-117 is a transmembrane tyrosine kinase receptor that is expressed in ICC. ICC are involved in generation and propagation of electrical slow waves that cause phasic contractions in the gastrointestinal tract. ICC are also present in the ureter and renal pelvis and are thought to be involved in ureteral urinary drainage.
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Our study aimed to evaluate the expression of the four neuronal markers: PGP9.5, synaptophysin, S-100, and CD-117, in excised PUJO specimens compared
with controls to investigate whether neuronal abnormalities are associated with intrinsic PUJO. Our secondary aim was to look for any association between severity of renal
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impairment with neuronal marker expression levels at the site of obstruction. Methodology Patients and samples
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All patients who underwent dismembered pyeloplasty from 2008 to 2012 at our institution with an operative diagnosis of intrinsic PUJO were included. All PUJO specimens were examined histologically. Patients with extrinsic PUJO (e.g. crossing vessel) and other co-existing renal pathologies (e.g. posterior urethral valve or multicystic kidney disease) were excluded. Pre-operative and post-operative
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ultrasonographic measurements of the affected renal pelves, and differential renal function from Tc99m-MAG3 (mercaptoacetyltriglycine) scan results were reviewed. A significantly dilated renal pelvis was defined as a transverse intrarenal pelvis size above the median in our study population. Diminished renal function was defined as
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differential function of the operated kidney of less than 35%. Control PUJ specimens were obtained from children who underwent nephrectomy in our institution in the same period for Wilms’ tumor or for benign renal
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conditions not involving the PUJ such as duplex renal units and extrinsic PUJO secondary to crossing vessels. All control specimens were histologically confirmed to be free of tumor cell invasion. Immunohistochemistry
Immunohistochemical studies on PUJ specimens at the PUJO level were performed using antibodies specific for the neuronal markers. We studied expression of four neuronal markers: PGP9.5 (neuron specific protein), synaptophysin (synaptic vesicle membrane protein), S-100 (nerve cell fiber marker), and CD-117 (receptor with tyrosine kinase activity).
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ACCEPTED MANUSCRIPT Immunohistochemistry staining was performed for all specimens on 4 µm-thick sections using the Ventana Benchmark Ultra (Ventana Medical Systems) and ultraView detection kit (Ventana Medical Systems). The primary antibodies used were synaptophysin, rabbit monoclonal antibody (MRQ-40; Cell Marque), PGP9.5, rabbit polyclonal antibody (Dako), CD-117, rabbit polyclonal antibody (Dako), and S-100,
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rabbit polyclonal antibody (Dako). Tests were performed and interpreted according to the manufacturers’ protocols.
The immunohistochemical sections were examined under ordinary light
microscopy by two independent observers and graded according to level of expression
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of the neuronal markers. High (×200) and low (×100) powered fields of PUJO
specimens and controls were reviewed by both observers. As the range of histological innervation in normal PUJ has not been previously defined, we standardized our
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evaluation as follows. Expression levels of neuronal markers were classified relative to normal controls as increased, decreased, and unchanged. Classifications were assigned based on a visual semiquantitative scale (Fig. 1). Both the pattern and quantity of staining were taken into consideration when quantifying neuronal marker expression. Discrepancies in grading expression level were resolved between both observers by
Statistical analysis
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discussion.
Statistical analysis of the data was performed using Fisher’s exact test to study associations between neuronal marker expression and pre-operative renal
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ultrasonographic measurements, differential function, and patient demographics. The Kruskal-Wallis test was performed to investigate association of neuronal marker expression with age at first pyeloplasty. A p value of < 0.05 was considered to be
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statistically significant. Results
PUJO specimens from 38 patients (17 male (71%), 11 female (29%)) were studied. The mean age at pyeloplasty was 25.3 months (range 2.9-167.6 months). Median preoperative mid-pole transverse pelvic diameter was 25.0 mm (17.0-50.0 mm). Of 38 renal units, 17 (44.7%) exhibited a significantly dilated renal pelvis and seven (18.4%) diminished renal function. We obtained 20 control specimens from children with a mean age at operation of 47.8 months (range 0.8-187.7 months). Thirteen control specimens (65.0%) were taken
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ACCEPTED MANUSCRIPT from nephrectomies for renal tumors, two specimens (10.0%) resulted from an aberrant crossing vessel, three specimens (15.0%) were duplex renal units, one specimen (5.0%) was from a non-functioning left kidney, and one specimen (5.0%) was a Y ureter. The control specimens displayed considerable heterogeneity in innervation, with substantial variation in expression and distribution of neuronal immunohistochemical staining and
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non-uniformity in neuronal staining. The staining pattern in controls was similar to that found in the PUJO specimens (Fig. 2). Seventeen PUJO specimens (44.7%) exhibited no significant difference in expression from immunohistochemical staining with
PGP9.5 and synaptophysin compared with controls. Twenty-two PUJO specimens
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showed no differences compared with control specimens in staining with S-100 (57.9%) and 30 PUJO specimens showed no differences in staining with CD-117 compared with controls (78.9%). Results of immunohistochemical staining are summarized in Table 1.
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We found no statistically significant association between expression levels of the four immunohistochemical markers and pre-operative renal pelvis dilation or preoperative renal function of the operated kidney (Table 3). Likewise, we found no association between neuronal marker expression and gender, race, side of PUJO, age at diagnosis of hydronephrosis or age at first pyeloplasty (Table 2).
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The control specimens displayed considerable heterogeneity with substantial variation in expression and distribution of neuronal immunohistochemical staining for each of the four markers. The PUJO specimens showed considerable variation in immunohistochemical staining in different regions of the PUJ as demonstrated by
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PGP9.5 (Fig. 2). In most PUJO specimens, there was no difference in expression of all four markers when compared with controls, ranging from 44.7% of the specimens (PGP9.5 and synaptophysin) to 78.9% (CD-117) (Table 1).
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We also did not find any significant difference when we compared the
expression levels of the four immunohistochemical markers and the clinical presentation in terms of the pre-operative renal pelvis dilation or pre-operative renal function of the operated kidney (Table 3). There was no association between neuronal marker expression and gender, race, side of PUJO, age at diagnosis of hydronephrosis or age at first pyeloplasty (Table 2). Discussion Wang et al. in 1995 [1] described variability in the presence of several neuronal markers in PUJO. They demonstrated abundant staining with PGP9.5, synaptophysin, and nerve
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ACCEPTED MANUSCRIPT growth factor receptor-positive nerve fibers in normal PUJ, but markedly decreased staining within musculature of PUJO specimens. They postulated that PUJO could be caused by defective innervation with diminished synaptic vesicle expression, resulting in failure of transmission of peristaltic waves across the PUJ, and/or decreased nerve growth factor mRNA expression. Since then, several other studies on
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immunohistochemistry of neuronal markers in PUJO specimens have been published. However, there exists great variation among findings in these studies as summarized in Table 4. Two studies report reduced staining of PGP9.5 in PUJO specimens [1,6], two studies reduced CD-117 [12,13], while there are conflicting reports with regard to
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staining of synaptophysin and S-100 [1,9,10].
Differing methodologies in preparation of specimens could account for differences in findings. Wang et al. graded expression of neuronal markers on
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immunohistochemistry by examining four visual fields enumerated in three sections [1], whereas in our study, we studied levels of expression of neuronal markers in the entire section.
It is possible that varying methods in quantifying expression of neuronal markers have contributed to the inconsistent findings of different studies. Roman et al.
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evaluated the number of CD-117 positive cells by counting positively stained cells per high power field at ×200, and three high power fields were counted and averaged for each slide [14]. Gunduz et al. classified staining with synaptophysin, S-100, PGP9.5, and CD-117 as “0” (no staining), “1” (minimal staining), and “2” (intensive staining)
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[10]. Solari et al. obtained histological micrographs using an Axioscope and processed these digitally using an IPS computer-assisted image analyzer. Density of CD-117 positive cells was graded after evaluating 10 well stained and orientated high-powered
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fields (×400). Levels of neuronal expression were graded: sparse, few, moderate, and many (0 or 1, 2 or 3, 4-8, and greater than 8 cell bodies per high powered field, respectively) [12].
In our study, expression of immunohistochemical markers was non-uniform in
both control and PUJO specimens, which made it challenging to define a normal PUJ innervation pattern. We chose to use a histopathological semi-quantitative method to grade intensity of immunohistochemical staining that was easily reproducible. We recognized that present conventional methods of studying immunohistochemical expression and quantification could represent a limitation of our study findings.
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ACCEPTED MANUSCRIPT Given the variation in staining patterns of neuronal innervation even for the same stain, defective neuronal innervation as a possible etiology of PUJO was not demonstrated in our study. Our findings are at variance from several previous studies that hypothesize decreased or increased innervation as a cause of PUJO [1,10]. The lack of correlation between clinical severity of obstruction and innervation markers shown in
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our findings also does not support a neuronal cause for PUJO. Our findings seem to support the traditional view of PUJO etiology being a
mechanical obstruction. Variability in neuronal marker expression could be related to
secondary changes resulting from mechanical obstruction. As there is no evidence of a
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defective neuronal segment causing abnormal peristalsis, clinical judgment is needed regarding operative need, timing, and the technical aims of pyeloplasty to achieve dependent gravitational urinary drainage remains key.
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Given the relatively small sample size of our study, and heterogeneity of neuronal expression in both PUJO and control specimens, our findings do not support a neuronal cause for PUJO. We suggest further multicenter studies involving collaboration between various pediatric surgical institutions to conduct more research on the etiology of PUJO.
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Conclusion
We found no consistent pattern for immunoreactivity of four neuronal markers, PGP9.5, synaptophysin, S-100, and CD-117, between PUJO specimens and controls in our population. There was no correlation between expression levels of the four markers
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used and severity of renal disease. Our study findings do not support a primary neuronal cause for PUJO.
Conflict of interest
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None.
Funding
This work was supported by the KKH Health Endowment Fund [grant number: KKHHEF/2013/15]. References 1.
Wang Y, Puri P, Hassan J, Miyakita H, Reen DJ. Abnormal innervation and
altered nerve growth factor messenger ribonucleic acid expression in ureteropelvic junction obstruction. J Urol, 1995. 154(2 Pt 2): p. 679-83.
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ACCEPTED MANUSCRIPT 2.
Duong HP, Piepsz A, Collier F, Khelif K, Christophe C, Cassart M, et al.,
Predicting the clinical outcome of antenatally detected unilateral pelviureteric junction stenosis. Urology, 2013. 82(3): p. 691-6. 3.
Morin L, Cendron M, Crombleholme TM, Garmel SH, Klauber GT, D'Alton
ME. Minimal hydronephrosis in the fetus: clinical significance and implications for
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Stringer MD, Yassaie S. Is the pelviureteric junction an anatomical entity? J
Pediatr Urol, 2013. 9(2): p. 123-8. 5.
Gonzalez R, Schimke CM. Ureteropelvic junction obstruction in infants and
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children. Pediatr Clin North Am, 2001. 48(6): p. 1505-18. 6.
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management. J Urol, 1996. 155(6): p. 2047-9.
Murakumo M, Nonomura K, Yamashita T, Ushiki T, Abe K, Koyanagi T.
Structural changes of collagen components and diminution of nerves in congenital
7.
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ureteropelvic junction obstruction. J Urol, 1997. 157(5): p. 1963-8.
Richstone L, Seideman CA, Reggio E, Bluebond-Langner R, Pinto PA, Trock B,
Kavoussi LR. Pathologic findings in patients with ureteropelvic junction obstruction and crossing vessels. Urology, 2009. 73(4): p. 716-9; discussion 719. 8.
Kajbafzadeh AM, Payabvash S, Salmasi AH, Monajemzadeh M, Tavangar SM.
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Smooth muscle cell apoptosis and defective neural development in congenital ureteropelvic junction obstruction. J Urol, 2006. 176(2): p. 718-23; discussion 723. 9.
Demirbilek S, Edali MN, Gürünlüoğlu K, Türkmen E, Taş E, Karaman A, et al.,
Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral
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junction obstruction. Urology, 2006. 67(2): p. 400-5. Gündüz M, Yurtçu M, Toy H, Abasiyanik A, Demirci S. Immunohistochemical
and morphometric evaluation of neuronal dysfunction in pelviureteral junction
11.
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obstruction. J Pediatr Urol, 2013. 9(3): p. 359-63. . 12.
Solari V, Piotrowska AP, Puri P. Altered expression of interstitial cells of Cajal
in congenital ureteropelvic junction obstruction. J Urol, 2003. 170(6 Pt 1): p. 2420-2. 13.
Yang X, Zhang Y, Hu J. The expression of Cajal cells at the obstruction site of
congenital pelviureteric junction obstruction and quantitative image analysis. J Pediatr Surg, 2009. 44(12): p. 2339-42.
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ACCEPTED MANUSCRIPT 14.
Metzger R, Schuster T, Till H, Stehr M, Franke FE, Dietz HG. Cajal-like cells in
the human upper urinary tract. J Urol, 2004. 172(2): p. 769-72.
Figure 1. Immunohistochemistry of neuronal marker expression in controls. (A), (B)
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High and low staining with CD-117, respectively. (C), (D) High and low staining with PGP9.5, respectively. (E), (F) High and low staining with S-100, respectively. (G), (H) High and low staining with synaptophysin, respectively.
Figure 2. Immunohistochemical stains for PGP9.5 in separate PUJO specimens
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(circled). Note the heterogeneity in staining patterns, limiting ability to objectively compare levels of expression of the stains.
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Table 1. Results of immunohistochemical staining in comparison with controls (in 38 patients)
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No difference Increased Decreased PGP9.5 17 (44.7%) 12 (31.6%) 9 (23.7%) Synap 17 (44.7%) 14 (36.8%) 7 (18.4%) S-100 22 (57.9%) 15 (39.5%) 1 (2.6%) CD-117 30 (78.9%) 8 (21.1%) 0 (0.0%) 0 represents no difference between PUJO and controls. 1 represents an increased expression of the neuronal marker compared with controls. 2 represents a decreased expression of the neuronal marker compared with controls.
Neuronal marker S-100 0 (n=22)
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Patient demographic
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Table 2. Association of neuronal marker expression and patient demographics
1 (n=15)
2 (n=1)
PGP9.5 0 (n=17)
1 (n=12)
2 (n=9)
Synaptophysin 0 1 (n=17) (n=14)
Gender
Male Female
77.3 (17) 22.7 (5)
60.0 (9) 40.0 (6) 0.4994
100.0 (1) 0.0 (0)
64.7 (11) 35.3 (6)
75.0 (9) 25.0 (3) 0.7311
77.8 (7) 22.2 (2)
70.6 (12) 29.4 (5)
78.6 (11 21.4 (3 0.5167
Chinese NonChinese p-value Side of PUJO Left
68.2 (15)
53.3 (8)
100.0 (1)
47.1 (8)
66.7 (8)
88.9 (8)
70.6 (12)
50.0 (7
31.8 (7)
46.7 (7) 0.6800
0.0 (0)
52.9 (9)
33.3 (4) 0.1097
11.1 (1)
29.4 (5)
50.0 (7 0.5545
86.4 (19)
46.7 (7)
100.0 (1)
58.8 (10)
66.7 (8)
100.0 (9)
76.5 (13)
64.3 (9
p-value Race
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ACCEPTED MANUSCRIPT 0.0 (0) 13.6 (3)
40.0 (6) 13.3 (2) p-value 0.0057 Age at diagnosis of hydronephrosis Prenatal 77.3 (17) 46.7 (7) Postnata l 22.7 (5) 53.3 (8) p-value 0.0545 Age at first 8.87 7.72 pyeloplasty, (5.45 (3.58, median (IQR) 15.3) 60.1) p-value
0.0 (0) 0.0 (0)
23.5 (4) 17.7 (3)
16.7 (2) 16.7 (2) 0.3174
0.0 (0) 0.0 (0)
11.8 (2) 11.8 (2)
14.3 (2 21.4 (3 0.7209
0.0 (0)
70.6 (12)
41.7 (5)
77.8 (7)
76.5 (13)
50.0 (7
100.0 (1)
29.4 (5)
22.2 (2)
23.5 (4)
46.5 (46.5, 46.5)
9.89 (5.29, 46.5)
58.3 (7) 0.2215 6.14 (3.47, 25.4)
11.4 (7.56, 34.6) 0.617
9.89 (6.28, 46.5)
50.0 (7 0.2784 6.62 (4.40, 13.2)
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Right Bilateral
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Table 3. Association of neuronal marker expression with pre-operative clinical parameters Neuronal marker S-100 0 (n=22)
PGP9.5
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Clinical variables
1 (n=15)
2 (n=1)
0 (n=17)
1 (n=12)
2 (n=9)
0 (n=17)
1 (n=14
4.76 (1)
47.6 (10)
23.8 (5)
28.6 (6)
57.1 (12)
28.6 (6
0.0 (0)
41.2 (7)
41.2 (7) 0.4924
17.6 (3)
29.4 (5)
47.1 (8 0.2499
0.0 (0)
42.9 (3) 45.2 (14)
14.2 (1) 35.5 (11)
42.9 (3)
42.9 (3)
19.3 (4)
45.2 (14)
14.3 (1 41.9 (13)
3.2 (1)
0.7363
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p-value
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Renal pelvis (mm), % (n) ≤25 61.9 (13) 33.3 (7) >25 52.9 (9) 47.1 (8) p-value 0.6203 Differential renal function (%), % (n)
S1477-5131(17)30305-4
DOI:
10.1016/j.jpurol.2017.07.014
Reference:
JPUROL 2621
To appear in:
Journal of Pediatric Urology
Received Date: 6 March 2017 Revised Date:
1477-5131 1477-5131
Accepted Date: 26 July 2017
Please cite this article as: How GY, Chang KTE, Jacobsen AS, Yap T-L, Ong CCP, Low Y, Allen JC, Kuick CH, Lim MZL, Laksmi NK, Neuronal defects an etiological factor in congenital pelviureteric junction obstruction?, Journal of Pediatric Urology (2017), doi: 10.1016/j.jpurol.2017.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Neuronal defects an etiological factor in congenital pelviureteric junction obstruction? Guo Yuan Howa,*, Kenneth Tou En Changb, Anette Sundfor Jacobsenc, Te-Lu Yapc, Caroline Choo Phaik Ongc, Yee Lowc, John Carson Allend, Chik Hong Kuickb, Malcolm Zhun Leong Limb, and Narasimhan Kannan Laksmic Yong Loo Lin School of Medicine, National University of Singapore, Singapore Department of Pathology and Laboratory Medicine, KK Women’s and Children’s
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b
Hospital, Singapore c
Department of Paediatric Surgery, KK Women’s and Children’s Hospital, Singapore
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Centre for Quantitative Medicine, Duke-NUS Medical School, Singapore
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a
* Corresponding author. Yong Loo Lin School of Medicine, 1E Kent Ridge Road, Singapore 119228, NUHS Tower Block, Level 11
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E-mail address: [email protected]
Summary Introduction: Congenital pelviureteric junction obstruction (PUJO) is one of the most frequent causes of neonatal hydronephrosis. Obstruction at the PUJ has potential severe adverse outcomes, such as renal damage. While pyeloplasty has been
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established as the definitive treatment, the exact pathophysiology of congenital PUJO remains unknown. Recent research has proposed neuronal innervation defects as an etiological factor in congenital PUJO. We aim to study the expression of various neuronal markers in PUJO specimens compared with controls, and evaluate whether
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severity of renal disease or dysfunction pre-operatively is related to expression of neuronal markers in resected PUJO specimens. Materials and methods: All consecutive patients who underwent dismembered
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pyeloplasty at KK Women’s and Children’s Hospital, Singapore, for intrinsic PUJO from 2008 to 2012 were included. Patients with other co-occurring renal pathologies were excluded. Controls were obtained from nephrectomy patients with Wilm’s tumor or other benign renal conditions during the same period. Specimens were stained immunohistochemically with neuronal markers protein gene product 9.5 (PGP9.5), synaptophysin, and S-100, and with CD-117, a marker for interstitial cells of Cajal (Table). Levels of expression of the markers were assessed semiquantitatively (decreased, increased or no change) in comparison with controls by two independent observers. Pre-operative data of patients’ renal anatomical (ultrasonography
1
ACCEPTED MANUSCRIPT measurements of renal pelvis size) and functional parameters (differential renal function measured using MAG-3 renal scans) were obtained. Discussion: Thirty-eight PUJO specimens (38 renal units) and 20 controls were studied. Mean patient age at pyeloplasty was 25.3 months (2.9-167.6 months). Median preoperative pelvic size was 25.0 mm (17.0-50.0 mm). Both PUJO specimens and controls
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showed great heterogeneity in distribution of innervation. All four immunohistochemical markers were not predictive of significant pre-operative renal pelvis dilation or pre-operative diminished renal function of the operated kidney. Conclusions: There exists marked variability in expression of neuronal markers
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synaptophysin, PGP9.5, and S-100, and CD-117 in PUJO specimens compared with
controls. Our results show no clinical significance of the expression of neuronal markers in predicting degree of pre-operative renal pelvis dilation or differential renal function.
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The heterogeneity of expression of neuronal markers in PUJO specimens and controls in our population is at variance with prior studies. The etiology of PUJO is likely to be complex and multifactorial.
KEYWORDS
Neuronal;
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Pelviureteric;
Etiology
Function - Most abundant integral membrane protein constituent of synaptic vesicles - Ubiquity in synapses allows for quantification of synapses - Function unknown - Deubiquitinating enzyme in neurons and nerve fibers - Neuron-specific cytoplasmic marker - Transmembrane tyrosine kinase growth factor receptor - Expressed in interstitial cells of Cajal, which generate and propagate electrical slow waves causing phasic contractions in gastrointestinal tract - Interstitial cells of Cajal present in renal pelvis and ureter - Function unknown - Specific marker for localizing cells of neural origin especially Schwann cells
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Neuronal marker
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Immunohistochemistry;
Synaptophysin PGP9.5
CD-117
S-100
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ACCEPTED MANUSCRIPT Introduction Congenital pelviureteric junction obstruction (PUJO) is one of the most frequent causes of neonatal hydronephrosis, occurring in 1/1,000 to 1/2,000 newborns [1]. Boys are affected more commonly than girls [2,3], with a higher incidence in the left kidney. The
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pelviureteric junction (PUJ) is defined as the junction between the renal pelvis and proximal ureter. However, this junction exists as a concept rather than a readily
identifiable landmark because of the lack of a clear anatomical definition [4]. The PUJ is well established as a common site of obstruction causing pelvicalyceal dilatation.
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Obstruction at the PUJ is believed to result in renal parenchymal damage, hypertension, and potential renal failure with bilateral PUJO. Although the exact pathophysiology of PUJO is unknown, underlying causes can be classified into structural (e.g. intraluminal,
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luminal, and extraluminal) and/or functional (e.g. abnormal smooth muscle function or impaired motility secondary to abnormal expression of interstitial cells of Cajal, ICC) disorders [3,4].
The definitive treatment for PUJO is surgery [5], which involves resection of the PUJ (dismembered pyeloplasty) or refashioning of the PUJ (non-dismembered
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technique). While surgery is the established treatment for PUJO, the etiology of PUJO remains unknown. It is unclear if pyeloplasty works by improving peristalsis or just mechanically opens the passage to allow dependent urinary drainage. Histological changes observed in some PUJ specimens include smooth muscle hypertrophy [6] and
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abnormal collagen deposition [7]. In recent years, some studies have suggested that PUJO may be caused by defective innervation or abnormalities in smooth muscle arrangement [7,8]; however, these findings have not been confirmed unanimously. To
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investigate neuronal innervation defects in PUJO we have identified four specific neuronal markers based on recent literature [9-11]: protein gene product 9.5 (PGP9.5), synaptophysin, S-100, and CD-117. PGP9.5 is a protease found in neurons of the central and peripheral nervous
systems. This neuron-specific cytoplasmic marker is also present in neuronal networks between smooth muscle fibers in the ureteral wall and the renal pelvis. Synaptophysin is the most abundant integral membrane protein constituent of synaptic vesicles of neurons. Although the function of synaptophysin is currently unknown, its ubiquity in synapses allows it to be used in the quantification of neuronal synapses. S-100 functions
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ACCEPTED MANUSCRIPT as a calcium flux regulator and is present in nerves innervating the ureter and renal cells. CD-117 is a transmembrane tyrosine kinase receptor that is expressed in ICC. ICC are involved in generation and propagation of electrical slow waves that cause phasic contractions in the gastrointestinal tract. ICC are also present in the ureter and renal pelvis and are thought to be involved in ureteral urinary drainage.
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Our study aimed to evaluate the expression of the four neuronal markers: PGP9.5, synaptophysin, S-100, and CD-117, in excised PUJO specimens compared
with controls to investigate whether neuronal abnormalities are associated with intrinsic PUJO. Our secondary aim was to look for any association between severity of renal
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impairment with neuronal marker expression levels at the site of obstruction. Methodology Patients and samples
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All patients who underwent dismembered pyeloplasty from 2008 to 2012 at our institution with an operative diagnosis of intrinsic PUJO were included. All PUJO specimens were examined histologically. Patients with extrinsic PUJO (e.g. crossing vessel) and other co-existing renal pathologies (e.g. posterior urethral valve or multicystic kidney disease) were excluded. Pre-operative and post-operative
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ultrasonographic measurements of the affected renal pelves, and differential renal function from Tc99m-MAG3 (mercaptoacetyltriglycine) scan results were reviewed. A significantly dilated renal pelvis was defined as a transverse intrarenal pelvis size above the median in our study population. Diminished renal function was defined as
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differential function of the operated kidney of less than 35%. Control PUJ specimens were obtained from children who underwent nephrectomy in our institution in the same period for Wilms’ tumor or for benign renal
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conditions not involving the PUJ such as duplex renal units and extrinsic PUJO secondary to crossing vessels. All control specimens were histologically confirmed to be free of tumor cell invasion. Immunohistochemistry
Immunohistochemical studies on PUJ specimens at the PUJO level were performed using antibodies specific for the neuronal markers. We studied expression of four neuronal markers: PGP9.5 (neuron specific protein), synaptophysin (synaptic vesicle membrane protein), S-100 (nerve cell fiber marker), and CD-117 (receptor with tyrosine kinase activity).
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ACCEPTED MANUSCRIPT Immunohistochemistry staining was performed for all specimens on 4 µm-thick sections using the Ventana Benchmark Ultra (Ventana Medical Systems) and ultraView detection kit (Ventana Medical Systems). The primary antibodies used were synaptophysin, rabbit monoclonal antibody (MRQ-40; Cell Marque), PGP9.5, rabbit polyclonal antibody (Dako), CD-117, rabbit polyclonal antibody (Dako), and S-100,
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rabbit polyclonal antibody (Dako). Tests were performed and interpreted according to the manufacturers’ protocols.
The immunohistochemical sections were examined under ordinary light
microscopy by two independent observers and graded according to level of expression
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of the neuronal markers. High (×200) and low (×100) powered fields of PUJO
specimens and controls were reviewed by both observers. As the range of histological innervation in normal PUJ has not been previously defined, we standardized our
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evaluation as follows. Expression levels of neuronal markers were classified relative to normal controls as increased, decreased, and unchanged. Classifications were assigned based on a visual semiquantitative scale (Fig. 1). Both the pattern and quantity of staining were taken into consideration when quantifying neuronal marker expression. Discrepancies in grading expression level were resolved between both observers by
Statistical analysis
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discussion.
Statistical analysis of the data was performed using Fisher’s exact test to study associations between neuronal marker expression and pre-operative renal
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ultrasonographic measurements, differential function, and patient demographics. The Kruskal-Wallis test was performed to investigate association of neuronal marker expression with age at first pyeloplasty. A p value of < 0.05 was considered to be
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statistically significant. Results
PUJO specimens from 38 patients (17 male (71%), 11 female (29%)) were studied. The mean age at pyeloplasty was 25.3 months (range 2.9-167.6 months). Median preoperative mid-pole transverse pelvic diameter was 25.0 mm (17.0-50.0 mm). Of 38 renal units, 17 (44.7%) exhibited a significantly dilated renal pelvis and seven (18.4%) diminished renal function. We obtained 20 control specimens from children with a mean age at operation of 47.8 months (range 0.8-187.7 months). Thirteen control specimens (65.0%) were taken
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ACCEPTED MANUSCRIPT from nephrectomies for renal tumors, two specimens (10.0%) resulted from an aberrant crossing vessel, three specimens (15.0%) were duplex renal units, one specimen (5.0%) was from a non-functioning left kidney, and one specimen (5.0%) was a Y ureter. The control specimens displayed considerable heterogeneity in innervation, with substantial variation in expression and distribution of neuronal immunohistochemical staining and
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non-uniformity in neuronal staining. The staining pattern in controls was similar to that found in the PUJO specimens (Fig. 2). Seventeen PUJO specimens (44.7%) exhibited no significant difference in expression from immunohistochemical staining with
PGP9.5 and synaptophysin compared with controls. Twenty-two PUJO specimens
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showed no differences compared with control specimens in staining with S-100 (57.9%) and 30 PUJO specimens showed no differences in staining with CD-117 compared with controls (78.9%). Results of immunohistochemical staining are summarized in Table 1.
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We found no statistically significant association between expression levels of the four immunohistochemical markers and pre-operative renal pelvis dilation or preoperative renal function of the operated kidney (Table 3). Likewise, we found no association between neuronal marker expression and gender, race, side of PUJO, age at diagnosis of hydronephrosis or age at first pyeloplasty (Table 2).
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The control specimens displayed considerable heterogeneity with substantial variation in expression and distribution of neuronal immunohistochemical staining for each of the four markers. The PUJO specimens showed considerable variation in immunohistochemical staining in different regions of the PUJ as demonstrated by
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PGP9.5 (Fig. 2). In most PUJO specimens, there was no difference in expression of all four markers when compared with controls, ranging from 44.7% of the specimens (PGP9.5 and synaptophysin) to 78.9% (CD-117) (Table 1).
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We also did not find any significant difference when we compared the
expression levels of the four immunohistochemical markers and the clinical presentation in terms of the pre-operative renal pelvis dilation or pre-operative renal function of the operated kidney (Table 3). There was no association between neuronal marker expression and gender, race, side of PUJO, age at diagnosis of hydronephrosis or age at first pyeloplasty (Table 2). Discussion Wang et al. in 1995 [1] described variability in the presence of several neuronal markers in PUJO. They demonstrated abundant staining with PGP9.5, synaptophysin, and nerve
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ACCEPTED MANUSCRIPT growth factor receptor-positive nerve fibers in normal PUJ, but markedly decreased staining within musculature of PUJO specimens. They postulated that PUJO could be caused by defective innervation with diminished synaptic vesicle expression, resulting in failure of transmission of peristaltic waves across the PUJ, and/or decreased nerve growth factor mRNA expression. Since then, several other studies on
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immunohistochemistry of neuronal markers in PUJO specimens have been published. However, there exists great variation among findings in these studies as summarized in Table 4. Two studies report reduced staining of PGP9.5 in PUJO specimens [1,6], two studies reduced CD-117 [12,13], while there are conflicting reports with regard to
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staining of synaptophysin and S-100 [1,9,10].
Differing methodologies in preparation of specimens could account for differences in findings. Wang et al. graded expression of neuronal markers on
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immunohistochemistry by examining four visual fields enumerated in three sections [1], whereas in our study, we studied levels of expression of neuronal markers in the entire section.
It is possible that varying methods in quantifying expression of neuronal markers have contributed to the inconsistent findings of different studies. Roman et al.
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evaluated the number of CD-117 positive cells by counting positively stained cells per high power field at ×200, and three high power fields were counted and averaged for each slide [14]. Gunduz et al. classified staining with synaptophysin, S-100, PGP9.5, and CD-117 as “0” (no staining), “1” (minimal staining), and “2” (intensive staining)
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[10]. Solari et al. obtained histological micrographs using an Axioscope and processed these digitally using an IPS computer-assisted image analyzer. Density of CD-117 positive cells was graded after evaluating 10 well stained and orientated high-powered
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fields (×400). Levels of neuronal expression were graded: sparse, few, moderate, and many (0 or 1, 2 or 3, 4-8, and greater than 8 cell bodies per high powered field, respectively) [12].
In our study, expression of immunohistochemical markers was non-uniform in
both control and PUJO specimens, which made it challenging to define a normal PUJ innervation pattern. We chose to use a histopathological semi-quantitative method to grade intensity of immunohistochemical staining that was easily reproducible. We recognized that present conventional methods of studying immunohistochemical expression and quantification could represent a limitation of our study findings.
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ACCEPTED MANUSCRIPT Given the variation in staining patterns of neuronal innervation even for the same stain, defective neuronal innervation as a possible etiology of PUJO was not demonstrated in our study. Our findings are at variance from several previous studies that hypothesize decreased or increased innervation as a cause of PUJO [1,10]. The lack of correlation between clinical severity of obstruction and innervation markers shown in
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our findings also does not support a neuronal cause for PUJO. Our findings seem to support the traditional view of PUJO etiology being a
mechanical obstruction. Variability in neuronal marker expression could be related to
secondary changes resulting from mechanical obstruction. As there is no evidence of a
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defective neuronal segment causing abnormal peristalsis, clinical judgment is needed regarding operative need, timing, and the technical aims of pyeloplasty to achieve dependent gravitational urinary drainage remains key.
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Given the relatively small sample size of our study, and heterogeneity of neuronal expression in both PUJO and control specimens, our findings do not support a neuronal cause for PUJO. We suggest further multicenter studies involving collaboration between various pediatric surgical institutions to conduct more research on the etiology of PUJO.
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Conclusion
We found no consistent pattern for immunoreactivity of four neuronal markers, PGP9.5, synaptophysin, S-100, and CD-117, between PUJO specimens and controls in our population. There was no correlation between expression levels of the four markers
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used and severity of renal disease. Our study findings do not support a primary neuronal cause for PUJO.
Conflict of interest
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None.
Funding
This work was supported by the KKH Health Endowment Fund [grant number: KKHHEF/2013/15]. References 1.
Wang Y, Puri P, Hassan J, Miyakita H, Reen DJ. Abnormal innervation and
altered nerve growth factor messenger ribonucleic acid expression in ureteropelvic junction obstruction. J Urol, 1995. 154(2 Pt 2): p. 679-83.
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ACCEPTED MANUSCRIPT 2.
Duong HP, Piepsz A, Collier F, Khelif K, Christophe C, Cassart M, et al.,
Predicting the clinical outcome of antenatally detected unilateral pelviureteric junction stenosis. Urology, 2013. 82(3): p. 691-6. 3.
Morin L, Cendron M, Crombleholme TM, Garmel SH, Klauber GT, D'Alton
ME. Minimal hydronephrosis in the fetus: clinical significance and implications for
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Stringer MD, Yassaie S. Is the pelviureteric junction an anatomical entity? J
Pediatr Urol, 2013. 9(2): p. 123-8. 5.
Gonzalez R, Schimke CM. Ureteropelvic junction obstruction in infants and
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children. Pediatr Clin North Am, 2001. 48(6): p. 1505-18. 6.
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management. J Urol, 1996. 155(6): p. 2047-9.
Murakumo M, Nonomura K, Yamashita T, Ushiki T, Abe K, Koyanagi T.
Structural changes of collagen components and diminution of nerves in congenital
7.
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ureteropelvic junction obstruction. J Urol, 1997. 157(5): p. 1963-8.
Richstone L, Seideman CA, Reggio E, Bluebond-Langner R, Pinto PA, Trock B,
Kavoussi LR. Pathologic findings in patients with ureteropelvic junction obstruction and crossing vessels. Urology, 2009. 73(4): p. 716-9; discussion 719. 8.
Kajbafzadeh AM, Payabvash S, Salmasi AH, Monajemzadeh M, Tavangar SM.
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Smooth muscle cell apoptosis and defective neural development in congenital ureteropelvic junction obstruction. J Urol, 2006. 176(2): p. 718-23; discussion 723. 9.
Demirbilek S, Edali MN, Gürünlüoğlu K, Türkmen E, Taş E, Karaman A, et al.,
Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral
10.
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junction obstruction. Urology, 2006. 67(2): p. 400-5. Gündüz M, Yurtçu M, Toy H, Abasiyanik A, Demirci S. Immunohistochemical
and morphometric evaluation of neuronal dysfunction in pelviureteral junction
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obstruction. J Pediatr Urol, 2013. 9(3): p. 359-63. . 12.
Solari V, Piotrowska AP, Puri P. Altered expression of interstitial cells of Cajal
in congenital ureteropelvic junction obstruction. J Urol, 2003. 170(6 Pt 1): p. 2420-2. 13.
Yang X, Zhang Y, Hu J. The expression of Cajal cells at the obstruction site of
congenital pelviureteric junction obstruction and quantitative image analysis. J Pediatr Surg, 2009. 44(12): p. 2339-42.
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ACCEPTED MANUSCRIPT 14.
Metzger R, Schuster T, Till H, Stehr M, Franke FE, Dietz HG. Cajal-like cells in
the human upper urinary tract. J Urol, 2004. 172(2): p. 769-72.
Figure 1. Immunohistochemistry of neuronal marker expression in controls. (A), (B)
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High and low staining with CD-117, respectively. (C), (D) High and low staining with PGP9.5, respectively. (E), (F) High and low staining with S-100, respectively. (G), (H) High and low staining with synaptophysin, respectively.
Figure 2. Immunohistochemical stains for PGP9.5 in separate PUJO specimens
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(circled). Note the heterogeneity in staining patterns, limiting ability to objectively compare levels of expression of the stains.
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Table 1. Results of immunohistochemical staining in comparison with controls (in 38 patients)
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No difference Increased Decreased PGP9.5 17 (44.7%) 12 (31.6%) 9 (23.7%) Synap 17 (44.7%) 14 (36.8%) 7 (18.4%) S-100 22 (57.9%) 15 (39.5%) 1 (2.6%) CD-117 30 (78.9%) 8 (21.1%) 0 (0.0%) 0 represents no difference between PUJO and controls. 1 represents an increased expression of the neuronal marker compared with controls. 2 represents a decreased expression of the neuronal marker compared with controls.
Neuronal marker S-100 0 (n=22)
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Patient demographic
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Table 2. Association of neuronal marker expression and patient demographics
1 (n=15)
2 (n=1)
PGP9.5 0 (n=17)
1 (n=12)
2 (n=9)
Synaptophysin 0 1 (n=17) (n=14)
Gender
Male Female
77.3 (17) 22.7 (5)
60.0 (9) 40.0 (6) 0.4994
100.0 (1) 0.0 (0)
64.7 (11) 35.3 (6)
75.0 (9) 25.0 (3) 0.7311
77.8 (7) 22.2 (2)
70.6 (12) 29.4 (5)
78.6 (11 21.4 (3 0.5167
Chinese NonChinese p-value Side of PUJO Left
68.2 (15)
53.3 (8)
100.0 (1)
47.1 (8)
66.7 (8)
88.9 (8)
70.6 (12)
50.0 (7
31.8 (7)
46.7 (7) 0.6800
0.0 (0)
52.9 (9)
33.3 (4) 0.1097
11.1 (1)
29.4 (5)
50.0 (7 0.5545
86.4 (19)
46.7 (7)
100.0 (1)
58.8 (10)
66.7 (8)
100.0 (9)
76.5 (13)
64.3 (9
p-value Race
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ACCEPTED MANUSCRIPT 0.0 (0) 13.6 (3)
40.0 (6) 13.3 (2) p-value 0.0057 Age at diagnosis of hydronephrosis Prenatal 77.3 (17) 46.7 (7) Postnata l 22.7 (5) 53.3 (8) p-value 0.0545 Age at first 8.87 7.72 pyeloplasty, (5.45 (3.58, median (IQR) 15.3) 60.1) p-value
0.0 (0) 0.0 (0)
23.5 (4) 17.7 (3)
16.7 (2) 16.7 (2) 0.3174
0.0 (0) 0.0 (0)
11.8 (2) 11.8 (2)
14.3 (2 21.4 (3 0.7209
0.0 (0)
70.6 (12)
41.7 (5)
77.8 (7)
76.5 (13)
50.0 (7
100.0 (1)
29.4 (5)
22.2 (2)
23.5 (4)
46.5 (46.5, 46.5)
9.89 (5.29, 46.5)
58.3 (7) 0.2215 6.14 (3.47, 25.4)
11.4 (7.56, 34.6) 0.617
9.89 (6.28, 46.5)
50.0 (7 0.2784 6.62 (4.40, 13.2)
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Right Bilateral
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Table 3. Association of neuronal marker expression with pre-operative clinical parameters Neuronal marker S-100 0 (n=22)
PGP9.5
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Clinical variables
1 (n=15)
2 (n=1)
0 (n=17)
1 (n=12)
2 (n=9)
0 (n=17)
1 (n=14
4.76 (1)
47.6 (10)
23.8 (5)
28.6 (6)
57.1 (12)
28.6 (6
0.0 (0)
41.2 (7)
41.2 (7) 0.4924
17.6 (3)
29.4 (5)
47.1 (8 0.2499
0.0 (0)
42.9 (3) 45.2 (14)
14.2 (1) 35.5 (11)
42.9 (3)
42.9 (3)
19.3 (4)
45.2 (14)
14.3 (1 41.9 (13)
3.2 (1)
0.7363
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p-value
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Renal pelvis (mm), % (n) ≤25 61.9 (13) 33.3 (7) >25 52.9 (9) 47.1 (8) p-value 0.6203 Differential renal function (%), % (n)
