Histological and immunohistochemical insights into pelviureteric junction obstruction in children: A case-control study

INTRODUCTION

A pelviureteric junction (PUJ) obstruction, characterized by impaired urine flow from the renal pelvis to the proximal ureter, is the most common congenital cause of hydronephrosis. With an incidence of approximately 1/750-1500 live births, the condition is often asymptomatic, and many cases spontaneously resolve without renal compromise. However, severe obstruction may lead to irreversible renal damage.^[1] The PUJ complex involves smooth muscle fibers, connective tissue, and neural elements, all crucial for maintaining urinary flow. Histopathological studies utilizing light and electron microscopy have identified structural abnormalities in the PUJ, including the uneven presence of longitudinal muscle and abnormal neural innervations.^[2,3] Although the precise mechanisms leading to PUJ obstruction are unknown, excessive collagen deposition coupled with smooth muscle hypertrophy has been reported to cause the obstruction.^[4]

Immunohistochemistry (IHC) and morphometric analysis offer insights into the cellular and structural changes underlying PUJ dysfunction.

Interstitial cells of Cajal (ICCs), identified by c-Kit (CD117) immunoreactivity, act as pacemaker cells that coordinate peristaltic activity in hollow viscera and are increasingly recognized in the urinary tract as “Cajal-like cells.” Alterations in ICC density or distribution can impair coordinated ureteral contractions and have been implicated in the pathogenesis of PUJ obstruction. Prior studies report ICC reduction at the obstruction site, with several series showing significantly lower ICC counts in PUJ obstruction than in controls, aligning with a neuromuscular mechanism for outflow impairment.^[5,6] While these cells are hypothesized to control urinary tract peristalsis, the exact pathophysiological mechanisms of PUJ obstruction remain unclear.

Neural elements at the PUJ can be assessed with S100, which labels Schwann cells and peripheral nerve fibers within the ureteric wall. Reduced S100-positive fibers may reflect axonal loss or disordered innervation, whereas thickened or clustered fibers may indicate aberrant remodeling. Evidence across pediatric cohorts is mixed, but several reports support a reduction or redistribution of S100-positive fibers in obstructed segments, consistent with a neural contribution to dysmotility.^[7,8]

The knowledge of histological changes and innervations at PUJ is required for a proper understanding of its etiology and pathogenesis, which will help in the better management of these patients. The study investigates histological and molecular features of the PUJ in children with hydronephrosis, focusing on smooth muscle integrity, neural distribution, and extracellular matrix alterations.

MATERIALS AND METHODS

This observational case-control study was conducted in a tertiary center in central India from May 2022 to January 2025. The Institutional Review Board approved the study (IHEC-LOP/2022/IL021), and informed consent was obtained from parents/guardians.

A convenient sample size of 40 cases of PUJ obstruction and 20 cases of controls (children and adults who underwent nephrectomy not related to PUJ obstruction, such as trauma and tumors) was considered. PUJ tissue samples were obtained from children aged 1 month to 15 years undergoing surgery for hydronephrosis. A PUJ sample measuring 7-8 mm was taken each time. During the procedure, the PUJ was surgically removed, along with 3-4 mm of healthy tissue from both above and below the PUJ. The specimens were fixed in 10% formaldehyde. Fixed tissue specimens were dehydrated through graded alcohol and subsequently processed based on standard tissue processing guidelines. The tissues were embedded in paraffin, and sections measuring 4-5 mm in thickness were cut. Sections were stained with hematoxylin and eosin stain, and elastic von Gieson stain for muscles, fibrous tissue, and elastic tissue. Masson’s trichrome was used to visualize collagen deposition.

Formalin-fixed paraffin-embedded PUJ segments were sectioned at 4-5 mm. Antigen retrieval and indirect immunoperoxidase staining were performed for: (i) CD117 (c-Kit) to quantify ICCs (Rabbit Monoclonal, Clone EP10, Pathnsitu); positivity was scored as ICC count per HPF and (ii) S100 to highlight neural elements (Rabbit monoclonal, Clone Beta EP32, Pathnsitu); cytoplasmic ± nuclear staining in nerve fibers was counted per HPF.

The data were recorded and analyzed in Microsoft Excel. The statistical analysis was done using Statistical Package for Social Sciences 23.0 (Armonk, NY, USA). Pearson’s Chi-square test was used to conclude the association between the categorical variables. A two-tailed P < 0.05 was considered statistically significant.

RESULTS

This study evaluated the expression of S100 for neuronal cells and CD117 for ICC in 40 cases and 20 control samples using IHC.

Histopathological findings

On gross examination, the excised PUJ segments from cases were visibly thickened and showed a narrowed lumen. Microscopically, hematoxylin-eosin staining confirmed that all case specimens had an intact urothelial lining without dysplasia. Beneath the urothelium, however, marked differences were noted between case and control tissues. The muscularis propria of the ureter at the PUJ in obstructed cases often appeared hypertrophic with an irregular arrangement of muscle fibers.

Instead of the normal circumferential and longitudinal muscle bundle layering, haphazardly oriented muscle fascicles were observed in 28/40 cases (70%). In some areas, muscle bundles were split by intervening streaks of collagen or appeared whorled. In contrast, control PUJ samples displayed the expected organized two-layered smooth muscle architecture with no hypertrophy. Some case samples showed muscle bundle thinning in focal areas [Figure 1]. No specific inflammatory infiltrate was seen in most cases; a mild chronic inflammatory infiltrate (scattered lymphocytes) was present in eight cases (20%), and two cases (5%) showed focal lymphoid aggregates. These inflammatory changes were absent in controls.

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Figure 1:

Photomicrograph showing muscle layer measurements in a case of PUJO.

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IHC

In the control group PUJ sections, S100 highlighted a rich plexus of nerve fibers in the submucosa and muscular layer of the ureter. The mean S100-positive fiber count in the case group was 4.05 ± 1.7 per HPF, significantly lower than the 8.15 ± 2.3 per HPF in controls (P < 0.001, t-test). In cases, the few fibers present were often thickened and clustered near blood vessels [Figure 2]. In the analysis, CD117 (ICC Marker), small spindle-shaped ICCs with membranous c-Kit positivity were readily identified, especially at the interface of the circular and longitudinal muscle layers in control tissues. The average ICC count in controls was 4.25 ± 1.8 cells per HPF. In the PUJ obstruction cases, CD117-positive cells were markedly fewer. Many high-power fields in case specimens showed zero or one ICC visible. The mean ICC count in cases was only 0.68 ± 0.9 per HPF, representing an 80% reduction compared to controls. This difference was highly significant (P = 0.003, Mann-Whitney U-test). Moreover, 30 out of 40 cases (75%) had an ICC count of ≤1 per HPF [Figure 3]. A summary of the IHC results has been provided in Table 1. In brief, the case group showed significantly lower expression of both S100 and CD117 relative to controls [Table 1]. A more intense staining for S-100 was observed in samples with PUJ obstruction due to thickened nerve fibers. In CD117, the number of ICCs appeared to be reduced in PUJ obstruction samples.

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Figure 2:

Photomicrograph showing S100 highlighting thickened nerve fibers (black arrows) in PUJO cases. (S100, DAB, ×200).

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Figure 3:

Photomicrograph showing CD117 with (a-b) no Interstitial cells of Cajal (ICC) only highlighting mast cell in PUJO (asterisk) (Di-amino Benzidine (DAB), ×200), (c) CD117 highlighting many ICCs in control sample (black arrows) (DAB, ×400), (d) CD117 highlighting ICCs (black arrows) and mast cells in PUJO case (asterisk) (CD117, DAB, ×400).

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Table 1: Summary of immunohistochemical findings.

Marker	Mean±SD (Cases)	Mean±SD (Controls)	P-value*
S100 (nerve fibers/HPF)	4.05±1.7	8.15±2.3	P<0.001
CD117 (ICCs/HPF)	0.68±0.9	4.25±1.8	P=0.003

*P-value was calculated using Mann Whitney test and p value of less than 0.05 is considered significant. SD: Standard deviation, ICC: Interstitial cells of Cajal

DISCUSSION

The study aimed to determine if there are significant differences in the staining patterns of these markers between the case and control groups, which could provide insights into potential differences in cellular structures or pathophysiological changes. The results for S100 staining showed that the case group had significantly lower expression of S100 compared to the control group (P = 0.001).

This suggests that the neuronal cells in the case group have reduced or altered S100 expression, which may reflect underlying changes in neuronal function or structure. S100 is often used as a marker for neurons, and its reduced expression could indicate neurodegeneration, neuronal injury, or an altered cellular environment in the pathological conditions of the case group.^[8,9] The analysis of CD117 staining, which identifies ICC, exhibited a similar trend. The case group significantly decreased in expression of CD117 (P = 0.003). This suggests that the ICCs, crucial for motility, may be diminished or dysfunctional in the case group. Reduced ICC expression has been implicated in various gastrointestinal disorders, including gastroparesis and intestinal dysmotility, which are characterized by disrupted peristalsis and impaired motility.^[5] These results are important for understanding the pathogenesis of diseases or conditions that affect both neuronal cells and ICCs. The findings suggest that there may be multisystem involvement in the cases studied, particularly affecting both the nervous system and musculature.

Numerous studies have observed conflicting patterns, with some reporting significant differences and some proving otherwise. Asati et al.^[10] observed that the difference in mean positivity was statistically significant (P = 0.0001) and the presence of three or fewer ICCs was conclusively associated with PUJ obstruction. The study reported no significant differences in the levels of S100 proteins between controls and cases.^[10] A similar study by How et al.^[7] noted that in 22 PUJ obstruction specimens, no differences were observed compared to control specimens in S-100 (57.9%) staining, and in CD-117 staining, 30 PUJ obstruction specimens also showed no differences (78.9%).^[7]

It is challenging to conclude the impact of these markers in predicting PUJ obstruction, as the results seem to vary in multiple studies, but collagen deposition appears to be consistent.^[11-13] The staining for S-100 was found in the PUJ obstruction specimens compared with the normal PUJ specimens, and was more intense due to elevated neural innervations.^[12] In a study by Yang et al.,^[14] ICC was observed to be lower in PUJ obstruction, consistent with the study.^[14] Mehrazma et al.^[15] also noted a significant reduction (P > 0.00) in ICC in PUJ obstruction patients and an elevated collagen deposition.^[15]

While the results of this study provide valuable insights, some limitations should be considered, such as the relatively small sample sizes (40 cases and 20 controls), which may limit the generalizability of the findings. Larger sample sizes would help confirm the robustness of these results. Age-matched controls were not available due to limited cases requiring nephrectomies in children. The control group may not fully account for all variables influencing the staining results, such as age, sex, or other underlying health conditions. Future longitudinal studies with more markers would be beneficial in exploring the relationship between these markers and disease progression with functional outcomes. Controlling these factors in future studies could enhance the accuracy of the results.

CONCLUSIONS

This study shows significantly reduced expression of S-100 and CD117 in PUJ specimens compared to controls, along with changes in muscular architecture. These findings suggest that the development of PUJ obstruction involves neuromuscular damage and a decreased ICC population. Future studies using larger, age-matched cohorts and standardized ICC/neuronal quantification could clarify prognostic and therapeutic implications.