Glial-derived growth factor signaling pathway in infantile hypertrophic pyloric stenosis

BACKGROUND/PURPOSE: Glial-derived growth factor (GDNF), which is the ligand of RET is reported to be essential for the development of enteric nervous system. A GDNF knockout mouse model has shown that the gastric region is a critical passing site between GDNF-RET-independent neuroblasts (colonizing the esophagus) and GDNF-RET-dependent neuroblasts (colonizing the small and large bowel). The earliest GDNF site of production is the mesenchyme and the outer smooth muscle cell (SMC) layer of the developing bowel. In the mature gastrointestinal tract the presence of GDNF is restricted to enteric glial cells. The aim of this study was to investigate the expression of GDNF and RET in infantile hypertrophic pyloric stenosis (IHPS). METHODS: Full-thickness muscle biopsy specimens were obtained from 8 IHPS patients at pyloromyotomy and from 8 age-matched controls without gastrointestinal disease. Indirect immunohistochemistry was performed using avidin-biotin-peroxidase complex method with anti-GDNF and anti-RET antibodies. Quantitative analysis was performed using sandwich-type enzyme-linked immunosorbent assay (ELISA) for GDNF. RESULTS: GDNF- and RET-positive nerve fibers were absent or markedly reduced in IHPS compared with controls. GDNF was expressed strongly by smooth muscle cells of both muscular layers in IHPS, whereas no GDNF expression was detected in pyloric muscle of controls. The quantity of total GDNF in IHPS was significantly higher than in controls (P < .01). CONCLUSIONS: The lack or markedly decreased number of GDNF-positive nerve fibers in IHPS supports the hypothesis of a selective immaturity of the enteric glia in the muscular layers in IHPS. The strong expression of GDNF in smooth muscle cells in IHPS and the increased levels of GDNF in IHPS suggest a compensatory mechanism by which the smooth muscle cells continue to produce GDNF until maturation of the enteric glial cells occurs.     

Immunolocalization of the gap junction protein Connexin43 in the interstitial cells of Cajal in the normal and Hirschsprung's disease bowel

BACKGROUND: Interstitial cells of Cajal (ICC) are pacemaker cells between gastrointestinal smooth muscles; they generate spontaneous slow waves of the smooth muscle layers and mediate neurotransmission. The cellular network of ICC is connected by Gap junctions to each other and to the smooth muscle cells. Although there have been several studies reporting distribution of ICC in the normal bowel and pathological conditions such as Hirschsprung's disease, there is little information on the crucial role of Gap junctions in the intercellular communication in the gut musculature. The aim of this study was to investigate the immunolocalization of the Gap junction protein Connexin43 in the normal and Hirschsprung's disease (HD) bowel using whole-mount preparation technique and confocal laser scanning microscopy. METHODS: Full-thickness bowel specimens were collected at pull-through operation from 8 patients diagnosed as having HD. Normal control large bowel specimens were collected from 12 patients during bladder augmentation operation. Whole-mount preparation was performed on all specimens and double immunostaining was carried out using anti c-kit and antiConnexin43 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. RESULTS: Connexin43 immunoreactivity appeared in and between the c-kit-positive cells and along the smooth muscle fibers of the normal bowel and ganglionic part of HD bowel. In the aganglionic part of HD bowel there was no expression of Connexin43. In the transitional zone of HD the Connexin43 staining was weak and colocalized only in the processes of the c-kit-positive Cajal cells. CONCLUSIONS: Results of this study show for the first time that Gap junctional protein Connexin43 is present in the ICCs, which form a 3-dimensional network in the normal bowel wall. The lack of expression of Connexin43 in the aganglionic bowel and reduced expression in the transitional zone of HD suggest that the impaired intercellular communication between ICCs and smooth muscle cells may partly be responsible for the motility dysfunction in HD.     

Mucosal neuroendocrine cell abnormalities in the colon of patients with Hirschsprung's disease.

We studied the distribution of mucosal neuroendocrine (NE) cells in the colon from 13 patients with Hirschsprung's disease (HD) and from 8 controls. Immunohistochemical studies were carried out using monoclonal and polyclonal antibodies against chromogranin A and synaptophysin (general markers of NE cells), 5-hydroxytryptamine (5-HT) (a marker of amine), peptide YY (PYY), and somatostatin (markers of neuropeptides). Chromogranin A immunoreactive cells were significantly increased in the aganglionic bowel compared with ganglionic bowel and controls (P less than .05). There was an increase in the number of synaptophysin immunoreactive cells in the aganglionic bowel compared with ganglionic bowel and controls but the results were not statistically significant. 5-HT immunoreactive cells were also significantly increased in the aganglionic bowel compared with ganglionic bowel and controls (P less than .05). The immunostaining for PYY demonstrated abundance of this NE cell type in the aganglionic bowel and this was highly significant compared with ganglionic bowel and controls (P less than .001). There was a significant increase in somatostatin immunoreactive cells in the aganglionic bowel compared with ganglionic bowel (P less than .01). The increase in neuroendocrine cells was found over the entire length of the aganglionic segment in rectosigmoid HD as well as in long-segment HD. These results demonstrating the increased levels of NE cells in the mucosa of aganglionic colon suggest that the NE cells may have a role in regulating the sustained contraction of the aganglionic intestine in HD.     

Mast cells and gut nerve development: implications for Hirschsprung's disease and intestinal neuronal dysplasia

BACKGROUND/PURPOSE: In Hirschsprung's disease (HD), the aganglionic bowel is characterized by the presence of hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic nerve fibers. Intestinal neuronal dysplasia (IND), if associated with HD, occurs proximal to the aganglionic segment in HD, and is characterized by dysplasia of parasympathetic nerves, hyperganglionosis, and giant ganglia. However, the cause of such abnormalities in HD and IND is unclear. Recent reports that mast cells (MC) have been observed in direct contact with nerve fibers generally, suggest that MC are essential for nerve growth and repair. MC synthesize, store, and release nerve growth factor (NGF). NGF supports the development and functional maintenance of sympathetic and cholinergic neurons. The aim of this study was to examine the colonic distribution of MC with respect to nerves in HD and HD associated with IND. METHODS: MC and NGF were examined immunohistochemically in ganglionic, transitional, and aganglionic segments of colon from 20 patients with HD (five patients associated with IND) and 15 age-matched controls. MC were counted in each of five random fields using light microscopy (x100). RESULTS: Interestingly, aganglionic and IND segments had large numbers of MC in all layers compared with ganglionic segments in HD patients and controls (P< .0001). The number of MC in transitional segments was significantly less compared with ganglionic segments in HD patients and controls (P< .01). MC stained positively for NGF, and some were found in contact with abnormal hypertrophic nerve trunks in HD and giant ganglia in IND. CONCLUSIONS: MC may cause hypertrophic nerve trunks and giant ganglia by releasing NGF and also may be an important factor in the excessive development of cholinergic and adrenergic nerve fibers in HD and IND.     

Expression of glial cell line-derived neurotrophic factor family members and their receptors in pancreatic cancers

BACKGROUND: The glial cell line-derived neurotrophic factor (GDNF) is a member of neurotrophic polypeptide family, which promotes survival and rescue of various neural cells in the central and peripheral nerve systems. We previously reported that GDNF promotes tumor cell invasion in pancreatic cancer cell lines. The purpose of this study was to investigate GDNF family expression and the status of related receptors in actual cancer tissues, and assess correlations with clinicopathologic behavior. METHODS: Immunohistochemical assessment of GDNF, neurturin, persephin, artemin, GDNF family receptor alpha-1 and alpha-2, and RET was performed for 51 cases of surgically resected pancreatic cancer. RESULTS: In all intrapancreatic nerves, GDNF and artermin were expressed strongly. In pancreatic cancer tissues. The expression of RET was stronger than that seen in normal ductal cells and was significantly related to the survival rate after resection (P = .026) and lymphatic invasion (P = .014). Intrapancreatic neural invasion was significantly related to the expression of GDNF (P = .047). CONCLUSIONS: We conclude that the expression of RET in pancreatic cancer tissues may be a useful prognostic marker and GDNF may play an important role in neural invasion.     

The role of nitrergic system on the contractility of colonic circular smooth muscle in Hirschsprung's disease.

The contribution of nitrergic tone on the contractility of colonic smooth muscle in Hirschsprung's disease (HD) was investigated. METHODS: Ganglionic and aganglionic bowel specimens were taken from 8 patients with HD during pull-through operations and electrical field stimulation (EFS)-induced isometric contractions of the circular smooth muscle were recorded in vitro. Isolated circular muscle strips prepared from colonic segments of sex- and age-matched patients (n = 3) who underwent surgery for nonmotility-related colonic diseases formed the control group. Statistical analysis was performed by two way analysis of variance and unpaired Student's ttest. RESULTS: The amplitude of spontaneous rhythmic activity was lower in aganglionic segments than in ganglionic ones. The amplitudes of contractile responses were significantly greater in aganglionic segments. In ganglionic preparations, N(omega)-nitro-L-arginine (L-NNA) addition into the medium increased the contractile responses to the level of aganglionic preparations. This increase was completely blocked by L-arginine application. Neither L-NNA nor L-arginine produced any change in aganglionic segments. A relaxation phase was detected in both ganglionic and aganglionic segments. In ganglionic preparations, this relaxation phase was completely inhibited by L-NNA and restored by L-arginine, whereas no effect was detected in aganglionic ones. Responses obtained from the control group were similar to the ganglionic segments of HD patients. CONCLUSIONS: In normal colon and as well as in ganglionic segments of HD, the evoked contractile activity and relaxations are under the tonic influence of the nitrergic system. Aganglionic segments totally lack the nitrergic activity in both evoked contraction and relaxation responses, while still maintaining an inefficient relaxation capacity under unknown mechanisms.     

Does Abnormal Expression of Acetylcholine Receptors in Hirschsprung's Disease Induce Acetylcholine Esterase Positive Nerve Fibres?

OBJECTIVE: Alpha bungarotoxin (alpha-BTX) is a neurotoxin isolated from the venom of Bungarus multicinctus that binds specifically to the beta-subunits of nicotinic acetylcholine receptors (nAChR) on myotube membranes. The purpose of the present study was to investigate the distribution of alpha-BTX-sensitive nAChR in Hirschsprung's disease (HD) to understand the histopathological features of HD, especially the increase in acetylcholine esterase (AChE) positive nerve fibres. METHODS: Confocal microscopy was used to study the expression of FITC (fluorescein isothiocyanate)-alpha-BTX, anti-synaptophysin (A-SY) antibody, and anti-neurofilament (A-NF) antibody to determine the distribution of nAChR and ganglion cell and nerve fibres in colon specimens from five cases of HD and three normal controls. RESULTS: Quantitative assessment of the immunoreactivity of colonic muscle and colonic mucosal epithelium from an aganglionic segment of HD bowel demonstrated markedly increased nAChR compared with colonic muscle and colonic mucosal epithelium from a ganglionic segment of HD bowel and normal bowel (p < 0.0001, respectively), both of which have only a few positive nAChR. In colonic muscle from aganglionic and transitional segments of HD, there were many nAChR around hypertrophic nerve trunks identified by A-NF and A-SY staining. CONCLUSION: We suggest that abnormal expression of nAChR in HD might be implicated in causing gastrointestinal dysmotility because of their localization around hypertrophic nerve trunks.     

细胞黏附分子在先天性巨结肠症中的表达

目的　观察先天性巨结肠症 (HD)患者细胞黏附分子 (CAMs)在神经节正常肠段和神经节缺如肠段的表达情况。探讨CAM 成纤维细胞生长因子 (FGFR)信号传导在HD致病机制中的作用。方法　应用链酶抗生素蛋白 生物素 过氧化酶复合体法 (SABC) ,检测 16例HD患者中神经节正常和缺如肠段中CAMs的表达。结果　神经节细胞黏附分子 (NCAM )和神经元细胞钙黏素(N cadherin)在神经组织和平滑肌组织中都有表达 ,但在神经节正常肠段 (NG )肠组织中NCAM和N cadherin染色的神经纤维的数量明显多于神经节缺如肠段 (AG)。结论　CAMs在AG肠段的明显减少说明HD患者中CAM FGFR信号传导发生了异常 ,可能是导致肠神经母细胞移行障碍的原因之一。     

Persistent bowel dysfunction after surgery for Hirschsprung’s disease: A neuropathological perspective

Hirschsprung’s disease (HD) is a congenital disorder, characterized by aganglionosis in the distal part of the gastrointestinal tract. Despite complete surgical resection of the aganglionic segment, both constipation and fecal incontinence persist in a considerable number of patients with limited treatment options. There is growing evidence for structural abnormalities in the ganglionic bowel proximal to the aganglionosis in both humans and animals with HD, which may play a role in persistent bowel dysfunction. These abnormalities include: (1) Histopathological abnormalities of enteric neural cells; (2) Imbalanced expression of neurotransmitters and neuroproteins; (3) Abnormal expression of enteric pacemaker cells; (4) Abnormalities of smooth muscle cells; and (5) Abnormalities within the extracellular matrix. Hence, a better understanding of these previously unrecognized neuropathological abnormalities may improve follow-up and treatment in patients with HD suffering from persistent bowel dysfunction following surgical correction. In the long term, further combination of clinical and neuropathological data will hopefully enable a translational step towards more individual treatment for HD.     

Persistent bowel dysfunction after surgery for Hirschsprung’s disease: A neuropathological perspective

Hirschsprung’s disease (HD) is a congenital disorder, characterized by aganglionosis in the distal part of the gastrointestinal tract. Despite complete surgical resection of the aganglionic segment, both constipation and fecal incontinence persist in a considerable number of patients with limited treatment options. There is growing evidence for structural abnormalities in the ganglionic bowel proximal to the aganglionosis in both humans and animals with HD, which may play a role in persistent bowel dysfunction. These abnormalities include: (1) Histopathological abnormalities of enteric neural cells; (2) Imbalanced expression of neurotransmitters and neuroproteins; (3) Abnormal expression of enteric pacemaker cells; (4) Abnormalities of smooth muscle cells; and (5) Abnormalities within the extracellular matrix. Hence, a better understanding of these previously unrecognized neuropathological abnormalities may improve follow-up and treatment in patients with HD suffering from persistent bowel dysfunction following surgical correction. In the long term, further combination of clinical and neuropathological data will hopefully enable a translational step towards more individual treatment for HD.     

RET基因多态性与中国湖北地区汉族人群先天性巨结肠的相关性研究

目的建立中国汉族人群BET基因位于exon2（密码子45）、exon13（密码子769）、exon11（密码子691）和exon15（密码子904）的基因多态性的基因型和等位基因频率的分布背景，探讨其基因多态性与先天性巨结肠的关系。方法应用PCR-RFLP在中国湖北地区汉族人群中检测正常对照组（122例）及散发性先天性巨结肠组（HD组，94例）G45A（GCG→GCA）、T769G（CTT→CTG）、G691A（GGT→AGT）和C904G（TCC→TCG）的单核苷酸多态性（single nucleotide polymorphisms,SNPs）。结果G45A、T769G和G691A在对照组中均存在多态性，但未发现C904G存在基因多态性。均为CC型。G45A在对照组中的基因型频率分别为AA 0．17、AG 0．72和GG 0．11，突变型A和野生型G等位基因的频率为0．53和0．47；而G45A在HD组则分别为从0．61、AG 0．35和GG 0．04，突变型A和野生型G等位基因的频率为0．78和0．22。T769G在对照组中基因型频率分布分别为GG 0．30、GT 0．52和TT 0．18，G和T等位基因的频率为0．56和0．44；而T769G在HD组则分别为GG 0．49、GT 0．36和TT 0．15，突变型G和野生型T等位基因的频率为0．67和0．33。两组间的两个位点的基因型和等位基因的分布频率差异均有统计学意义（x^2=28．64，P〈0．001；x^2=5．27，P=0．022）。G691A在对照组中基因型频率分别为AA 0．05、AG 0．16和GG 0．79，突变型A和野生型G等位基因的频率为0．13和0．87；而在HD组则分别为AA 0．02、AG 0．14和GG 0．84，突变型A和野生型G等位基因的频率为0．09和0．91，两组间比较差异无统计学意义（x^2=1．232，P=0．267）。结论在中国湖北地区汉族人群中，RET密码子904可能不存在基因多态性，未发现G691A与先天性巨结肠存在相关性，而G45A和T769G的基因多态性可能与中国湖北地区汉族人群先天性巨结肠相关。     

Effects of uraemia and haemodialysis on neutrophil apoptosis and expression of apoptosis-related proteins.

BACKGROUND: In haemodialysis (HD) patients, it is unclear whether increased apoptosis of neutrophils is due to uraemia or HD itself. The purpose of the current study was to assess the effect of uraemia and HD on the rate of apoptosis and apoptosis-related protein expression in whole blood neutrophils. METHODS: We employed a whole-blood micromethod to test spontaneous apoptosis and expression of apoptosis-regulating proteins in cultured neutrophils from uraemic patients (pre-HD), HD patients and healthy controls. Blood samples were drawn before, after 20 min and after 4 h of haemodialysis, and were then cultured for 20 h. We evaluated the rate of apoptosis from annexin V and propidium iodide staining, and examined bcl-2, Fas/Apo-1 and p53 expression in the cultured neutrophils. RESULTS: Fas/APO-1 expression and total percentage of apoptotic whole blood neutrophils of pre-HD and HD patients before HD were significantly higher than controls. There was a transient but significant decrease in the percentage of apoptotic neutrophils and Fas/APO-1 expression after 20 min of dialysis. The expression of bcl-2 protein was significantly lower from neutrophils in HD patients compared with controls, and HD significantly downregulated bcl-2 expression. The p53 protein content in HD patients before HD was significantly higher than in pre-HD patients. CONCLUSIONS: These findings suggest that uraemia accelerates neutrophil apoptosis by increasing Fas/Apo-1, and that HD does not affect neutrophil apoptosis more than uraemia. In addition, HD produces only in a transient sequestration of potentially apoptotic neutrophils.     

Ultra high frequency ultrasonography to distinguish ganglionic from aganglionic bowel wall in Hirschsprung disease: A first report

Background/PurposeIn Hirschsprung disease (HD) surgery, confirming ganglionic bowel is essential. A faster diagnostic method than the current frozen biopsy is desirable. This study investigated whether aganglionic and ganglionic intestinal wall can be distinguished from each other by ultra high frequency ultrasound (UHF ultrasound).MethodsIn an HD center during 2019, intestinal walls of recto-sigmoid specimens from HD patients were examined ex vivo with a 70 MHz UHF ultrasound transducer. Data from four sites were described. Histopathologic analysis was compared to the ultrasonography outcome at each site. Each patient's specimen served as its own control.Results11 resected recto-sigmoid specimens (median 20 cm long [range 6.5–33]) with transition zones of 5 cm (2–11 cm) were taken from children aged 22 days (13–48) weighing 3668 g (3500–5508); 44 key sites were analyzed. There was full concordance for 42/44 (95%) key sites and 10 of 11 (91%) specimens. The specimen with discordance of two key sites contained a segment of aganglionosis (3 cm) and a transition zone (1 cm): the site discordance was limited to the transition zone ends.ConclusionsThis first report on UHF ultrasound in recto-sigmoid HD shows promising results in identifying aganglionosis, transition zones and ganglionic bowel. Further in vivo studies are required.     

Clinical analysis of thrombocytopenia in chronic dialysis patients

Thrombopoietic status in dialysis patients is controversial. This study addressed this issue by analyzing factors associated with thrombopoiesis. One hundred and fifty-one dialysis patients(119 HD and 32 CAPD) and 41 age-matched control subjects were studied. Thrombocytopenia was defined as a platelet count less than 150 x 10(9)/l. Reticulated platelets(RET), a marker for marrow megakaryopoiesis, were measured using thiazole orange dye by flowcytometry. Serum thrombopoietin (TPO) level was measured by ELISA. Hepatitis C virus(HCV) antibody, platelet-associated IgG(PAIgG), and other clinical parameters were examined in the patients. The platelet counts in the HD patients were significantly lower than those in the CAPD or controls. The incidence of thrombocytopenia was 30.0% in the HD patients. Thrombocytopenia was more prominent in the HCV-infected HD patients in whom PAIgG was mostly positive(81.8%) and its titer was remarkably high(102.9 +/- 92.7 pg/ml). There were significant differences in the RET counts between patients with and without thrombocytopenia. None of the other parameters, such as iPTH, beta 2 microglobulin, and Kt/V, nor any prescribed drugs were related to thrombocytopenia. Serum TPO was significantly higher in HD patients(135.9 +/- 60.1 pg/ml) than in the controls(97.0 +/- 53.4 pg/ml). In conclusion, thrombocytopenia is frequent in HD patients, especially in HCV-infected HD patients. Reduced marrow magakaryopoiesis is mostly responsible for thrombocytopenia and peripheral destruction of platelets could be, in part, involved. Mild elevation of serum TPO possibly indicates a counter-response to decreased mass of megakaryocyte in bone marrow.     

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the human kidney and is a growth factor for human mesangial cells.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, is a potent neurotrophic factor in vitro and in vivo. GDNF is essential for nephrogenesis and the highest expression of GDNF is found in the developing kidney. Increased plasma GDNF levels have recently been documented in patients with chronic renal failure; the source and role of this increase, however, remain unclear. No data are available about the expression of GDNF in human adult kidney or human adult mesangial cell (HMC) cultures. We hypothesized that GDNF, similar to other members of the TGF-beta superfamily, might play a role as a growth factor in the pathogenesis of glomerulosclerosis. METHODS: To address this hypothesis, we first investigated (by RT-PCR) the expression of GDNF mRNA and the mRNAs of the GDNF receptors Ret and GFRalpha-1 in (i) adult human renal cortex and medulla and (ii) in HMC in culture. The results were compared to the expression of these molecules in different developmental stages of the rat kidney. We found that both GDNF and its receptors were expressed in human adult kidney and HMC. Since this finding implicates a role for GDNF beyond nephrogenesis, i.e. in renal physiology/pathophysiology, we investigated the effect of GDNF on HMC growth, i.e. (i) cellular protein synthesis as an index of hypertrophy ([(3)H]methionine incorporation), (ii) DNA synthesis ([(3)H]thymidine incorporation) and cell proliferation (cell numbers) as indices of hyperplasia, and (iii) extracellular matrix synthesis, i.e. collagenous and non-collagenous extracellular proteins ([(3)H]proline incorporation into the collagenase-sensitive and -insensitive fraction). HMC cultures were used as a surrogate model for the development of glomerulosclerosis. RESULTS: GDNF induced a biphasic growth stimulatory effect in HMC with stimulation at the lowest concentration used (2 ng/ml) but had no effect at higher concentrations (20 and 50 ng/ml). In contrast, cellular protein synthesis and extracellular matrix synthesis were significantly and dose-dependently increased by GDNF. CONCLUSIONS: These results suggest that GDNF, similar to other members of the TGF-beta superfamily, might play a role as a growth factor for mesangial cells and might thus be a player in the pathogenesis of glomerulosclerosis.     

Protein expression in vesicoureteral reflux: What about children?

PurposePathogenesis of vesicoureteral reflux (VUR) which concerns improper embryonal ureteric bud development still remains controversial, despite current studies have revealed several candidate genes. In this study, we aimed to determine the protein expression of certain genes which might play role in the pathogenesis of VUR, in the resected ureterovesical junction segments.MethodsThe study group consisted of 19 children; 12(63%) girls, 7(37%) boys who had ureteroneocystostomy (UNC) operation; 3(15.7%) right sided, 7(36.8%) left sided, 9(47.3%) bilateral due to VUR. As a total, 28 ureterovesical junction segments were available for analysis of protein expressions of GDNF/RET, PAX2 and FGFR2 genes by their Western Blot analysis.ResultsProtein based expressions of FGFR2, PAX2 and RET were significantly lower than β-Actin (p = 0.001, for all proteins). Correlation analyses between grade of reflux and protein expressions revealed no significant relations (p>0.05, for all proteins). When we grouped the patients into 2 groups as high grade (grade 4–5) and low grade reflux (grade 1–3) for convenient analyses, no statistically significant difference was found between groups (p>0.05, for all proteins). Renal units were also grouped according to differential functions (≥40% and <40%) obtained by renal scintigraphy and compared in terms of proteins’ expressions. There was also no significant difference between two groups regarding FGFR2, PAX2 and RET band areas (p>0.05, for all proteins).ConclusionOur study revealed decreased protein expressions of GDNF/RET, PAX2 and FGFR2 genes in the patients with VUR. Relation between clinical parameters and expression levels were statistically uncorrelated. Prospective studies of larger sample size are necessary in order to delineate the impact of certain proteins in the etiopathogenesis of VUR.