Extracellular Microenvironment and Cytokine Profile of the Ureterovesical Junction in Children With Vesicoureteral Reflux

Extracellular Microenvironment and Cytokine Profile of the Ureterovesical Junction in Children With Vesicoureteral Reflux C. Schwentner, J. Oswald, A. Lunacek, A. E. Pelzer, H. Fritsch, B. Schlenck, A. Karatzas, G. Bartsch and C. Radmayr* From the Department of Pediatric Urology (CS, JO, AL, AEP, BS, GB, CR), and Institute of Anatomy and Histology (HF), Medical University Innsbruck, Innsbruck, Austria, and Department of Urology, University Hospital Larissa, Larissa, Greece (AK)

Purpose: Vesicoureteral reflux is caused by a defective valve mechanism of the ureterovesical junction. Previous studies have revealed structural and metabolic changes in the intravesical ureter, impairing its contractile properties. Smooth musculature and nerves are replaced by collagen, while matrix degrading enzymes are over expressed. We investigated the presence of regulating cytokines and the extracellular matrix composition to elucidate further the pathophysiology of vesicoureteral reflux. Materials and Methods: Ureteral endings were obtained from 28 children during antireflux surgery, and 14 age matched autopsy specimens served as controls. Routine histological sections were immunostained for insulin-like growth factor-1, nerve growth factor, transforming growth factor-␤1, tumor necrosis factor-␣ and vascular endothelial growth factor. Smooth muscle staining was supplemented by tenascin C, tetranectin and fibronectin detection. Staining patterns were investigated using computer assisted, high power field magnification analyses. Results: Tumor necrosis factor-␣ and transforming growth factor-␤1 were significantly more abundant in vesicoureteral reflux samples, whereas insulin-like growth factor-1, nerve growth factor and vascular endothelial growth factor were more prevalent in healthy controls. Fibronectin was intensely expressed in refluxing ureters, while it was scarce in healthy children. Tenascin C was notable within the urothelium of both groups. Only vesicoureteral reflux samples displayed tenascin C in the musculature and connective tissue. Tetranectin staining was only detected in vesicoureteral reflux. Conclusions: Several cytokines are differentially expressed in primary refluxing ureters, indicating an ongoing tissue remodeling process in the ureterovesical junction region. Additionally, the smooth muscle coat is widely lacking, while extracellular matrix proteins typical for tissue shrinkage and reorganization are over expressed. These alterations are likely to contribute to the malfunctioning active ureteral valve mechanism in primary vesicoureteral reflux. Key Words: vesico-ureteral reflux; extracellular matrix; cytokines; fibrosis; muscle, smooth

elements, ganglionic cells and gap junctions within the ureteral wall further compromises the coordinated action of the ureterotrigonal unit, permitting VUR.7 The degree of structural derangement correlates with intraluminal pressures at the UVJ, underlining the functional relevance.2 However, the nature of these changes and their pathophysiological role remain unclear. The physiological UVJ also undergoes complex prenatal and postnatal maturational development.8 Ultrastructural studies suggest cellular dysfunction of ureteral SMCs even in low grade VUR. Cellular damage increases with reflux grade, explaining low resolution rates. Vacuoles appear adjacent to the mitochondria, yielding dysfunction of cellular energy production and apoptosis.4 Regarding the neuromuscular development of the UVJ, smooth muscle loss due to apoptosis has to be considered detrimental, impeding normal valve function. During childhood, smooth muscle mass and neural elements disproportionately increase in the intravesical ureter, representing physiological maturation.8 The interaction of SMCs and neurons with the surrounding connective tissue in the developing ureter is poorly understood. Organ development depends on the extracellular microenvironment, which consists of connective tissue pro-

he UVJ is of utmost importance in VUR. The structural integrity and functional integrity of the UVJ are required to allow for a unidirectional transport of urine, while protecting against reflux during storage and voiding. Vesicoureteral reflux is a risk factor for febrile urinary tract infections and kidney damage, although its etiology and natural history remain unclear. Passive antireflux mechanisms have been shown to be of minor importance under physiological circumstances.1 Therefore, most investigators focus on the active part of protection against VUR, the ureterotrigonal unit.2– 4 Refluxing ureteral endings display distinct structural and functional changes. The smooth musculature is replaced by collagenous matrix, while the remaining bundles are disorganized, impeding the active closure of the UVJ.3 Metabolic changes such as activation of matrix degrading enzymes, accumulation of phagocytes and impaired vascularization indicate a progressive process.5,6 Loss of neural

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Submitted for publication October 22, 2007. * Correspondence: Department of Pediatric Urology, Medical University, Anichstr. 35, 6020 Innsbruck, Austria (telephone: 43-512504-24811; FAX: 43-512-504-28365; e-mail: christian.radmayr@ i-med.ac.at).

0022-5347/08/1802-0694/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION

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Vol. 180, 694-700, August 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.04.048

EXTRACELLULAR MICROENVIRONMENT OF URETEROVESICAL JUNCTION teins and associated cytokines.9 Evolving cells interact with ECM and cytokines to coordinate growth and differentiation. Appropriate ECM prevents cells from apoptosis, dedifferentiation and invasion.9 There is a lack of knowledge about the extracellular microenvironment of the normal and pathological UVJ. Given the aforementioned changes of the UVJ, we focused on the role of cytokines promoting fibrosis and tissue destruction (TNF-␣, TGF-␤1), as well as on growth promoting factors regulating muscular and neuronal development (IGF-1, NGF, VEGF). Furthermore, ECM proteins suggestive of active tissue remodeling (tenascin, tetranectin, fibronectin) were studied to improve our understanding of VUR pathophysiology. MATERIALS AND METHODS Specimens of the UVJ (ultimate 10 mm of the ureter) were taken from 14 girls and 14 boys (mean age 52.25 months, range 10 to 110) with primary VUR undergoing ureteroneocystostomy. Reflux was grade II in 11 ureterorenal units, grade III in 9, grade IV in 7 and grade V in 1. After parental informed consent was obtained, specimens were donated to the department of anatomy for research and teaching purposes, according to the regulations of the National Ethical Review Board. Reflux persistency, parental preference, reflux nephropathy and complications due to antibiotic prophylaxis were indications for surgery. Patients with recurrent infections were excluded from the study. No child had neuropathic bladder or voiding dysfunction. Ureters of 14 age matched autopsies without evidence of urological disease served as controls. Immunohistochemical Analysis Samples were fixed in 4% formaldehyde and embedded in paraffin according to routine histology. Sections (ultimate 5 to 7 mm) were cut transversely at 4 ␮m, dried, dewaxed and rehydrated. Antigen retrieval included heat induced epitope unmasking in citrate buffer. Immunohistochemical analysis was initiated using an automated device. A peroxidase/diaminobenzidine kit was used for detection, while hematoxylin was administered for counterstaining. Specimens were dehydrated and mounted permanently in xylene based medium. Smooth muscle cell staining was done to assess ureteral morphology. A ready to use anti-␣ actin antibody was applied. Chromogen reaction, counterstaining and mounting procedure were performed as described previously, with colon as positive control. A prediluted anti-VEGF antibody was used to investigate VEGF distribution, with angiosarcoma as positive control. Insulin-like growth factor-1 staining was performed applying a prefabricated antibody, with pancreas as positive control. Tumor necrosis factor-␣ and TGF-␤1 staining were carried out using prediluted antibodies (mAb 1096 for TNF-␣, mAb 1032 for TGF-␤1), with colon as positive control for TNF-␣ and tonsil as positive control for TGF-␤1. Anti-NGF antibody was used in a dilution of 1:100, while neurilemmoma served as positive control. Tetranectin (neomarkers, 11F1), Fn (mAb 88904, 1:100) and Te (CBL 213, 1:100) were also investigated. The residual procedure was done as described previously. Positive controls were kidney for Fn, breast cancer for Te and tonsil

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for Tn. Negative controls were achieved omitting the primary antibody. Analysis Sections were investigated using a computer assisted microscope. Photomicrographs were analyzed using AxioVision AutoMeasure® software, allowing single cell labeling and automatic counting. Two observers concordantly scored all specimens. Ureteral smooth musculature was studied regarding structural anomalies and architectural derangement, as described previously.3,5,7 Cytokines Labeled cells were counted in all specimens in 10 randomly captured HPFs (⫻400 magnification ⫽ 0.152 mm2) with regard to the distribution within the refluxing and healthy ureteral wall.6 Urothelial and intraluminal cells were not counted. ECM Due to difficulties with objective quantification, matrix composition was morphologically described, scoring relative staining intensity and cellular and topographical distribution. Furthermore, the ECM profile was correlated with architectural abnormalities within the refluxing ureters. Descriptive statistics were applied, analyzing all semiquantitative results. The Spearman-Rho correlation test was used to evaluate the association between cytokine pattern and age as well as reflux grades. Differences in cytokine content between the 2 groups and different ureteral wall areas were investigated applying the 2-tailed Student t test, using SPSS®, version 11.0. Data are expressed as mean ⫾ SD, or mean and range, with statistical significance at p ⬍0.05. RESULTS In VUR the smooth muscle coat was widely absent, being replaced by connective tissue, while endomysial and perimysial fibrosis was noted in residual bundles.3,5,6 Controls exhibited a regular morphology. There was no correlation between VUR grade and age, as demonstrated previously.3 The lack of specific T or B-lymphocytes precludes chronic inflammation. Cytokines There was a significant difference in the cytokine patterns between patients with VUR and controls (fig. 1). Some were pronounced in VUR (TNF-␣, TGF-␤1), whereas others (IGF-1, NGF, VEGF) predominated in controls. For example TNF-␣ and TGF-␤1 were more abundant in VUR (14.96 ⫾ 6.35 vs 3.45 ⫾ 1.75 cells per HPF and 105.64 ⫾ 53.11 vs 55.6 ⫾ 23.84 cells per HPF, respectively; p ⬍0.0001; fig. 2). Those cytokines promoting muscular development were diminished in reflux. Conversely, IGF-1, NGF and VEGF were more prevalent in controls (144.76 ⫾ 46.07 vs 36.30 ⫾ 20.26 cells per HPF, 89.61 ⫾ 34.58 vs 41.41 ⫾ 11.15 cells per HPF and 169.89 ⫾ 48.52 vs 58.76 ⫾ 29.07 cells per HPF, respectively; p ⬍0.0001; fig. 3). No marker was correlated with age or VUR grade.

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EXTRACELLULAR MICROENVIRONMENT OF URETEROVESICAL JUNCTION 9.38 cells per HPF and for VEGF 40.05 ⫾ 19.78 vs 77.43 ⫾ 25.42 cells per HPF (p ⬍0.0001 for all markers). Even macroscopically well preserved regions displayed diminished growth factor production (p ⬍0.0001 for all markers). In contrast, TNF-␣ and TGF-␤1 content was more intense in regions lacking SMCs compared to intact areas (18.37 ⫾ 7.5 vs 11.91 ⫾ 2.52 cells per HPF and 134.82 ⫾ 47.99 vs 72.94 ⫾ 37.2 cells per HPF, respectively). Even within relatively healthy parts cytokine concentration was notably increased (11.91 ⫾ 2.52 vs 3.45 ⫾ 1.75 cells per HPF and 72.94 ⫾ 37.2 vs 55.6 ⫾ 23.84 cells per HPF, respectively; p ⬍0.0001). The TNF-␣ and TGF-␤1 production and growth factor content negatively correlated with each other (correlation coefficient – 0.421, p ⫽ 0.029).

FIG. 1. Cytokine profile of UVJ in healthy controls and patients with VUR (mean ⫾ SD), based on Student’s t test. Asterisk indicates statistical significance.

Differences were observed in the cytokine profile in conjunction with architectural alterations of the refluxing ureter (fig. 4). Those areas consisting of connective tissue exhibited the lowest rates of VEGF, IGF-1 and NGF production, whereas regions with relatively intact musculature yielded higher values. The respective differences for IGF-1 were 22.49 ⫾ 12.17 vs 50.13 ⫾ 17.06 cells per HPF, for NGF 35.24 ⫾ 8.85 vs 48.05 ⫾

ECM Fibronectin was prominently detected in VUR, while it was weaker in controls. In VUR Fn was mainly found in regions lacking musculature. Fibronectin was specifically expressed in a cell bound fashion, yielding tissue contraction (fig. 5, A and B). Fibronectin was also noted in regions of endomysial and perimysial fibrosis. In controls Fn was located in the connective tissue separating the muscle bundles. In VUR Tn was expressed in the whole connective tissue space (fig. 5, C and D). Tetranectin was detected in SMCs (cytoplasm and membrane), as well as in areas of endomysial and perimysial fibrosis. Tetranectin content was low in controls, and was limited to intramuscular and subepithelial connective tissue. Tetranectin appears to be specific for VUR.

FIG. 2. Tissue distribution of TGF-␤1 and TNF-␣ in normal and refluxing ureters. Expression of TGF-␤1 is more abundant in VUR (note significant expression in smooth musculature, A) compared to normal controls (B). Expression of TNF-␣ is mainly found in connective tissue, being more pronounced in VUR (C) than in healthy individuals (D). Peroxidase/diaminobenzidine-hematoxylin stain, reduced from ⫻400.

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FIG. 3. Important SMC growth factors in healthy individuals and children with primary VUR. Insulin-like growth factor-1 (A), NGF (C) and VEGF (E) are significantly reduced in VUR, while their content is normal in controls (B, D and F, respectively). Peroxidase/diaminobenzidinehematoxylin stain, reduced from ⫻400.

Tenascin was noted within the urothelium of both groups. In SMCs (cytoplasm and membrane) and connective tissue Te was only demonstrable in VUR (fig. 5, E and F). Tenascin positive cell accumulations were a specific feature of regions lacking musculature. Extracellular matrix proteins being typical for shrinkage (Fn) and active remodeling (Tn and Te) are over expressed in primary VUR. DISCUSSION Functional integrity of the UVJ is essential for VUR prevention and upper tract protection.2– 4 There is evidence for a pivotal role of active VUR protection based on coordinated ureteral and trigonal contraction.2,4,8 In primary VUR functional changes have been described at the UVJ. Smooth musculature is absent, being replaced by connective tissue. Macrophages and tissue degrading enzymes document a progressive process. Neural and vascular elements are decreased, compromising contractility. Ultrastructural studies have revealed subcellular dysfunction in

SMCs.2–7 However, the etiology and time course of these changes remain unknown. To our knowledge we report for the first time preliminary data on ECM pattern and cytokine profile in primary VUR. While TNF-␣ and TGF-␤1 predominate, VEGF and IGF-1 are less abundant in VUR. Transforming growth factor-␤1 is a potent growth inhibitor for SMCs.10 Since SMCs are diminished in VUR, TGF-␤1 may be important in understanding this phenomenon. Segmental up-regulation of TGF-␤1 has been observed in primary megaureters, and is attributed to a segmental developmental delay.11 Similar abnormalities may account for primary VUR, explaining its occasional spontaneous resolution. Conversely, TGF-␤1 induces collagen production in refluxing ureters. Hence, we cannot rule out progressive SMC loss and fibrosis in primary VUR. Matrix protein receptors that act as chemoattractants for macrophages and fibroblasts are up-regulated, perpetuating fibrosis.10

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FIG. 4. Topography of cytokine expression within refluxing ureteral wall (mean ⫾ SD), based on Student’s t test. Asterisk indicates statistical significance.

Tumor necrosis factor-␣ also derives from macrophages, up-regulating its own expression and other mediators, contributing to tissue destruction.12 Tumor necrosis factor-␣ and TGF-␤1 induce apoptosis in SMCs, leading to contractile dysfunction.10,12 Ischemia provokes TNF-␣ synthesis. Microperfusion is diminished in VUR, likely enhancing apoptosis.6 Ureteral SMCs exhibit mitochondrial changes, resulting in subcellular dysfunction.4 Vascular endothelial growth factor production is reduced, impairing angiogenesis, nerve sprouting and gap junctions.5,7 Those cytokines promoting SMC growth and neuronal guidance (IGF-1, NGF) are also less abundant in VUR. Insulin-like growth factor-1 is a mitogen for SMCs during embryogenesis and postnatally.13 Insulin-like growth factor-1 modifies the phenotype of SMCs, promoting contractile behavior. A relative lack of IGF-1 creates a synthetic SMC phenotype, leading to increased ECM deposition and fibrosis, as in primary VUR.3–5,13 Additionally, IGF-1 stimulates glucose uptake, protein synthesis and contractility.14 Reduced glucose uptake leads to mitochondrial dysfunction, energy depletion and apoptosis.4,14 Insulin-like growth factor-1 generally stimulates neoangiogenesis.14 Reduced IGF-1 production will consequently impair ureteral blood supply and SMC function. Insulin-like growth factor-1 stabilizes the mitochondria, protecting SMCs from apoptosis due to hypoxia.14 Insulin-like growth factor-1 regulates neuronal growth and energy housekeeping.15 A lack of IGF-1 will create aberrant innervation, as shown in primary VUR.2,5,7 Insulin-like growth factor-1 may be pivotal for the development and integrity of the neuromuscular network of the UVJ. Nerve growth factor is a key factor in neuronal physiology that is required for axonal guidance and differentiation.16 Particularly cholinergic neurons are dependent on NGF for survival.17 A reduction of NGF in VUR may attenuate cho-

linergic activities, implying contractile dysfunction. Deprivation of NGF causes neuronal death, a reduction of nerve fibers and ganglia, as detected in VUR.16 Vascular endothelial growth factor and NGF are interrelated, regulating vascularization and innervation.18 While VEGF is neurotrophic and neuroprotective, NGF stimulates angiogenesis. Correspondingly, microvessel density and nerve supply are diminished at the UVJ in VUR.6 Vesicoureteral reflux is considered congenital due to a dysfunctional UVJ. Poorly developed tissues display defects in their growth factor metabolism, while concomitant alterations of the ECM suggest tissue remodeling.6 Tetranectin, Te and Fn were found in VUR, with Tn and Te being restricted to reflux. These ECM proteins indicate growth and remodeling. In addition to their structural role, these molecules interact directly with SMCs.9 Moreover, cytokine effects are modified and adapted by ECM components. Tetranectin is a marker of myogenesis, and muscle regeneration and differentiation.19 Tetranectin activates plasmin, initiating proteolysis via metalloproteinases. Tetranectin is not present in healthy adult tissues.19 Tetranectin was observed in the cytoplasm and membrane of SMCs in VUR, indicating muscle development or regeneration. Hence, there may be either a defect in healing or delayed muscular differentiation in VUR. Unfortunately, immunohistochemical analysis does not allow us to distinguish between these 2 processes. Like Tn, Te is also restricted to embryogenesis and remodeling.20 Tenascin acts on tyrosine kinase receptors via growth factor-like domains, regulating growth and ECM production. Tenascin is critical during wound healing, providing migratory tracks for fibroblasts, while cell motility and mitogenic capacities are enhanced.9 Tenascin also promotes collagen synthesis and fibrosis. Ureteral SMCs are connected via gap junctions, acting as a functional syncytium. In VUR Te is mainly found in the cytoplasm and on the cell membrane, counteracting cellular adhesion mechanisms and promoting a migratory phenotype. Therefore, Te is capable of disorganizing functional syncytia.20 Refluxing ureters lack gap junctions, reflecting a disorganization of the SMC syncytium.3,6 Hence, Te may be a key molecule in understanding the architectural derangement occurring in VUR. Fibronectin content is increased in VUR, particularly in a cell bound manner, indicating tissue contraction via myofibroblasts, which is typical for wound healing and fibrosis.9 Fibronectin enhances the potential of cytokines, promoting ECM remodeling. Additionally, Fn polymers regulate collagen deposition, contributing to ureteral rigidity.9 Transforming growth factor-␤1 further stimulates Fn production, and has an essential role in the recruitment and phenotypic differentiation of myofibroblasts.10 The UVJ exhibits a distinct microenvironment, indicating remodeling and fibrosis, although immunocompetent cells that are lacking inflammation and secondary changes due to VUR itself cannot be completely ruled out. Therefore, we cannot finally determine the nature of these mechanisms. Still, we hypothesize that defective growth regulation occurs due to developmental disturbances in the UVJ area. Since even normally appearing ureteral musculature is affected in VUR, ECM remodeling may

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FIG. 5. Topographical distribution of ECM proteins in primary VUR and normal controls. Cell bound Fn is typical feature of primary VUR (A). Fibronectin production is lower in healthy individuals (B). Tetranectin is found only in VUR (C) and is absent in physiological UVJ tissue (D). Tenascin is produced by SMCs and interstitial cells in refluxing ureters (E), while there is virtually no expression in healthy children (F). Peroxidase/diaminobenzidine-hematoxylin stain, reduced from ⫻400.

represent an attempt to reestablish a functional UVJ. Due to many similarities with scar formation, this finding may also reflect defect healing.10,12,19,20 Increased rigidity and loss of contractility will further hamper the valve mechanism, leading to VUR.2 These assumptions currently are hypothetical, and are further complicated by the fact that we only investigated patients with persistent VUR. Given the limits of immunohistochemical analysis, our data have to be considered preliminary, requiring further investigation based on molecular biological techniques. Additional studies focusing on SMC metabolism and growth as well as ECM interaction are needed to clarify the pathophysiology of primary VUR. CONCLUSIONS The UVJ exhibits distinct architectural changes in primary VUR, including loss of SMCs, and neural and vascular ele-

ments.2–7 These structural modifications preclude adequate closure of the intravesical ureter, permitting VUR.2,3 Furthermore, the UVJ possesses a particular extracellular microenvironment, reflecting active remodeling. While profibrotic cytokines and ECM proteins are up-regulated, important SMC related growth factors are diminished in the refluxing ureteral wall. These metabolic abnormalities may lead to increased collagen deposition, further disorganization of the smooth muscle coat and extensive fibrosis. Hence, the active valve mechanism of the UVJ is impaired, resulting in persistence of VUR. These developmental abnormalities may occur secondary to a congenital disorganization of the UVJ, leading to defect healing and fibrosis instead of functional maturation. Due to the inherent limits of immunohistochemical analysis, further studies on the proteome and gene expression of ureteral SMCs are needed to delineate normal and dysfunctional development of the UVJ.

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Abbreviations and Acronyms ECM Fn HPF IGF-1 NGF SMC Te TGF-␤1 Tn TNF-␣ UVJ VEGF VUR

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

extracellular matrix fibronectin high power field insulin-like growth factor-1 nerve growth factor smooth muscle cell tenascin transforming growth factor-␤1 tetranectin tumor necrosis factor-␣ ureterovesical junction vascular endothelial growth factor vesicoureteral reflux

8.

9.

10.

11.

12.

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