Immune Expression in Children With Vesicoureteral Reflux: A Pilot Study.

OBJECTIVE: To perform an exploratory, descriptive pilot study of the systemic and local immune environment in patients with vesicoureteral reflux (VUR) and bladder-bowel dysfunction (BBD). METHODS: Consecutive children with VUR undergoing intravesical ureteral reimplantation were enrolled. Patients were assessed for presence of BBD by reported patient history and validated questionnaire. Fresh blood and bladder tissue, collected at the time of surgery, were immediately processed for analysis. Immune cell compositions were determined via flow cytometry. Immune cell activation was also defined at the time of analysis. LegendPlex assay analysis was utilized to define levels of circulating chemokines and cytokines. RESULTS: A total of 7 patients were enrolled. Although percentages of circulating immune cells in the blood of those with VUR/BBD and VUR alone were similar, within bladder tissue, VUR/BBD demonstrated increased immune infiltrates compared to VUR alone. Bladder sample analysis showed that B cells, and Effector Memory and Naïve T cell percentages were significantly increased in VUR/BBD patients compared to VUR patients. T cell expression of PD1 was increased in bladder tissues of BBD/VUR. Additionally, analysis of circulating neutrophils displayed significantly increased upregulation of PDL-1 in patients with VUR/BBD vs those with VUR only. CONCLUSION: These pilot data suggest an immune-rich microenvironment is present within VUR. Severity of inflammation appeared to correlate with presence of BBD. This implies that targeting pelvic inflammation may be a novel therapy for children with VUR- or non-VUR-related BBD. Follow-up studies are currently underway.

The pathogenesis and management of renal scarring in children with vesicoureteric reflux and pyelonephritis.

Bacterial urinary tract infections (UTIs) are one of the most common reasons for children to be admitted to hospital. Bacteria infect and invade the bladder (the lower urinary tract) and if the infection disseminates to the upper urinary tract, significant inflammation in the kidneys may arise. Inflammation is a double-edged sword: it is needed to clear bacteria, but if excessive, kidney tissue is injured. During injury, nephrons are destroyed and replaced with deposition of extracellular matrix and a renal scar. In this review, we explore the pathogenesis of UTIs and discuss the risk factors that result in dissemination of bladder infection to the kidneys. Three major risk factors predispose to kidney infections: the presence of vesicoureteric reflux, the presence of bladder and bowel dysfunction, and defects in the ability of the host immune response to clear bacteria. In this review, we will discuss these factors, their relationship to renal scarring, and potential treatments that might be beneficial to prevent renal scar formation in children.

Inflammation drives renal scarring in experimental pyelonephritis.

Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.

Cytokines in chronic kidney disease: potential link of MCP-1 and dyslipidemia in glomerular diseases.

BACKGROUND: Many studies have indicated a role for cytokines in chronic kidney disease (CKD). The aim of this study was to evaluate plasma and urinary levels of monocyte chemoattractant protein-1 (MCP-1/CCL2), transforming growth factor-beta1 (TGF-ß1), and interleukin-8 (IL-8/CXCL8) in pediatric patients with CKD stages 2-4. METHODS: Cytokines were measured in 37 healthy controls and in 42 CKD patients by enzyme-linked immunoassay. Patients were divided into groups according to CKD etiology: glomerular disease (group 1, n = 11) and congenital anomalies of the kidney and urinary tract (group 2, n = 31). Urinary cytokine measurements were standardized for creatinine. RESULTS: Plasma and urinary levels of MCP-1/CCL2 were significantly higher in both CKD groups compared to the control group. Between the two CKD groups, only urinary MCP-1/CCL2 levels were significantly different, with MCP-1/CCL2 levels higher in group 1 patients. Plasma and urinary levels of IL-8/CXCL8 and TGF-ß1 were undetectable in the control group but comparable between the two CKD groups. In group 1 patients, urinary MCP-1/CCL2 levels were negatively correlated to serum albumin levels and positively correlated to the levels of total cholesterol and triglycerides. In group 2 patients, urinary levels of IL-8/CXCL8 were negatively correlated with the estimated glomerular filtration rate and positively correlated with body mass index. CONCLUSIONS: Differences in cytokine profiles may be related to CKD etiology and other disease-associated alterations.

IL-19 and IL-20 genes polymorphisms and haplotype analysis in a vesicoureteral reflux population.

UNLABELLED: Vesicoureteral reflux (VUR) is a common childhood problem, causing renal wounds and escalating the risk of renal deficiency and hypertension. A vast literature exists suggesting that genetic variations play a significant role in the pathogenesis of VUR. The aim of the present study was to estimate whether genetic polymorphisms of IL-19 (GC rs2243158, AT rs2243158) and IL-20 (AG rs2981573, TG rs2981572) genes are involved in the development of VUR. MATERIALS AND METHODS: The tetra amplification mutation refractory system-polymerase chain reaction (Tetra-ARMS PCR) was applied for analyzing four polymorphic sites of IL-19 (GC rs2243158, AT rs2243158) and IL-20 (AG rs2981573, TG rs2981572) genes in 110 healthy controls and 124 VUR children. RESULTS: A significant association was found between the combined genotypes of IL19GC+CC and IL20TG+GG and increased risk of VUR (OR = 1.90, 95% CL, 1.06-3.41; OR=1.87, 95% CL, 1.06-3.29, respectively). The frequency of allele G in both sites of IL-20 (IL20AG rs2981573 and IL20TG, rs2981572) showed a statistically significant difference (p = 0.01) between cases and controls in comparison with the wild type. The combined haplotype analysis of IL-19 and IL-20 polymorphic sites revealed that HT2, HT3 and HT5 haplotypes marginally increased the risk of VUR, but not statistically significantly. Gene-gene interaction data of IL-19 (GC rs2243158, AT rs2243158) and IL-20 (AG rs2981573, TG rs2981572) in various genotype patterns highlighted the fact that most of the genotype combinations increased the risk of disease insignificantly. CONCLUSION: This is the first evidence regarding IL-19 and IL-20 cytokine genes polymorphism and risk of VUR, suggesting the need for further study with large sample size and in different populations to confirm the presented data.

B cell infiltration and lymphonodular hyperplasia in bladder submucosa of patients with persistent bacteriuria and recurrent urinary tract infections.

PURPOSE: We defined chronic inflammatory cell types in bladder submucosa and the presence of umbrella cells on the surface of bladder epithelium in patients 5 to 21 years old with persistent bacteriuria due to neurogenic bladder and recurrent urinary tract infections associated with vesicoureteral reflux. MATERIALS AND METHODS: Bladder mucosa biopsies from 12 patients and 6 controls were fixed in Carnoy's solution and examined for T cells (CD3, CD4, CD8), B cells (CD79) and plasma cells (CD138). The number of cells in a defined area of submucosa was determined by counting all nuclei in the area. A contiguous section was also stained for uroplakin expression with a monoclonal antibody against uroplakin III to ascertain the integrity of bladder umbrella cells. RESULTS: B cells, plasma cells and lymphoid nodules were found only in patient biopsies. T cell expression was evident in patient and control biopsies. Uroplakin staining of surface epithelium was uniform from control biopsies but spotty or entirely absent from patient biopsies. CONCLUSIONS: Patients with persistent bacteriuria or recurrent urinary tract infections had significant B cell infiltration in the submucosa, including lymphoid nodules. These inflammatory changes are likely due to antigenic stimulation from repeated exposure to bacteria. These changes are associated with frequent absence of uroplakin on surface epithelium.

Urinary levels of interleukin-6 and interleukin-8 in patients with vesicoureteral reflux and renal parenchymal scar.

The objective of this study was to assess the urine levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) as noninvasive markers of vesicoureteral reflux (VUR) and renal parenchymal scarring (RPS) in children in the absence of a recent urinary tract infection (UTI) episode. Urine concentrations of IL-6 and IL-8 in 114 children aged 1 month to 16 years were evaluated. The children were divided into four groups: group 1, 26 children with VUR and RPS; group 2, 27 children with VUR without RPS; group 3, 34 children with RPS without VUR, group 4, 27 children without VUR and RPS, as the control group. After the first assessment, the children were divided into four larger groups for comparison purposes: group A (groups 1+2), 53 children with VUR; group B (groups 3+4), 61 children without VUR; group C (groups 1+3), 60 children with RPS; group D (groups 2+4), 54 children without RPS. Urinary IL-6 and IL-8 concentrations were determined. To avoid dilution effects and to the standardize samples, urinary levels of IL-6 and IL-8 were expressed as the ratio of cytokine to urinary creatinine (pg/mg). The median urine IL-6/creatinine was significantly higher in patients with VUR than in those without VUR (5.72 vs. 3.73). In patients with VUR, there was a significant but rather weak correlation between IL-6/creatinine concentrations and there flux grade (p<0.05, R=0.305). The median urine IL-8/creatinine was significantly higher in patients with RPS than in those without RPS (43.12 vs. 16.36). In patients with RPS, there was a significant but rather weak correlation between IL-8/creatinine concentrations and the renal scar grade (p<0.05, R=0.251). The results of this study provide preliminary evidence that children with VUR have a high urine IL-6 concentration, whereas children with RPS have a high urine IL-8 concentration.

Urinary evaluation of the balance between the soluble interferon-gamma receptor (IFN-gammaR1) and the interleukin-4 receptor (IL-4Ralpha) in children with vesicoureteral reflux.

We planned to investigate the urinary soluble cytokine receptor profile in patients with vesico-ureteric reflux (VUR). The urine levels of soluble interferon-gamma receptor R1 (sIFN-gammaR) and soluble interleukin-4 receptor alpha (sIL-4R) were measured using an ELISA technique. The urine levels of sIFN-gammaR in the patients with VUR were significantly higher than those in the healthy controls (p < 0.001). On the other hand, although the urine sIL-4R levels in the patients with VUR were also higher than those in the controls, there were no significant differences between them. The urinary soluble receptor levels did not correlate with the clinical severity of VUR. These results suggest that there may be an immunological basis to VUR complicatedly.

Intraoperative anaphylactic shock in a child with no history of type I hypersensitivity.

Natural rubber latex is the second most implicated agent in intraoperative anaphylactic reactions. This report describes a case of intraoperative anaphylaxis occurring in a non-atopic fourteen-year-old girl undergoing multiple surgical procedures, but without spina bifida, in which latex surgical gloves were the main culprit for the anaphylactic reactions. Clinical manifestations of an anaphylactic reaction were also experienced during the examination of the possible cause of intraoperative anaphylaxis by skin prick testing with a latex allergen extract. Skin tests with anesthetics were negative. Specific IgE to latex was positive at 92.9 kUA/L (class 5). The molecular basis for the reported intraoperative anaphylaxis was ascribed to three low-molecular mass latex allergens (10-15 kD) detected in the brand of latex surgical gloves used during the operation. Given the potential of a dramatic outcome, latex allergy testing as a regular preoperative measure may contribute to the reduction of anaphylactic reactions during surgical interventions.

Serum and urine levels of interleukin-6 and interleukin-8 in children with acute pyelonephritis.

Urinary tract infection (UTI) is a common clinical disorder in younger infants and children and may result in permanent renal damage. The inflammatory cytokines interleukin (IL)-6 and IL-8 play an important role in response to bacterial infection. This prospective study investigated the association between serum and urine IL-6 and IL-8 levels and acute pyelonephritis confirmed by (99m)Tc-dimercaptosuccinic acid (DMSA) scan. A total of 78 children aged 1-121 months with a diagnosis of first-time febrile UTI were included. The following inflammatory markers were assessed: fever; white blood cells count (WBC); C-reactive protein (CRP); and serum and urine IL-6 and IL-8. The patients were divided into the acute pyelonephritis group (n=42) and the lower UTI group (n=36) according to the results of DMSA scan. Fever, WBC and CRP levels were significantly higher in children with acute pyelonephritis than in those with lower UTI (all p <0.001). Significantly, higher initial serum and urine IL-6 and IL-8 levels were found in children with acute pyelonephritis than in those with lower UTI (all p <0.001). Serum and urine IL-6 in children with acute pyelonephritis were positively correlated with fever, CRP and leucocyturia. These results indicate that both serum and urine IL-6 and IL-8 levels, particularly IL-6, are useful diagnostic tools for early recognition of acute pyelonephritis in febrile children.

ICAM-1 expression is upregulated in reflux nephropathy.

Reflux nephropathy (RN) is a major cause of end-stage renal failure in children and young adults. Intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein, has a role in the regulation of interaction among immune cells. It has been demonstrated that increased levels of tubular ICAM-1 correlate with an extent of tubular damage in diabetic nephropathy. We hypothesized that ICAM-1 local synthesis is altered in reflux nephropathy and therefore designated this study to investigate ICAM-1 expression in RN. The kidney specimens from six patients with severe reflux nephropathy secondary to primary vesicoureteral reflux were obtained at the time of nephrectomy. Control materials included normal kidney specimens obtained from three adult patients during partial nephrectomy for an incidentaloma. Fluorescent immunohistochemistry was carried out using monoclonal antibodies to ICAM-1 utilizing confocal laser scanning microscopy. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of ICAM-1. In the control kidneys, there was lack of ICAM-1 immunoreactivity in the interstitium and proximal tubules and moderate immunoreactivity in the glomerulus. In the refluxing kidney there was strong ICAM-1 immunoreactivity in the glomerulus, interstitium and proximal tubules. The RT-PCR showed strong ICAM-1 mRNA expression in the refluxing kidneys and absent or weak ICAM-1 expression in the controls. Our findings of increased expression of ICAM-1 in the severe reflux nephropathy kidney suggests that ICAM-1 may play a role in the pathogenesis of renal parenchymal damage associated with RN.

Evaluation of antibody class in response to endoscopic subureteral collagen injection in patients with vesicoureteral reflux.

PURPOSE: Clinical problems after glutaraldehyde cross-linked collagen injection for vesicoureteral reflux are the appearance of anticollagen antibodies and a decrease in collagen with time. We evaluated whether antibody production affects reflux recurrence and implanted collagen absorption. MATERIALS AND METHODS: We treated 27 patients (39 ureters) who had vesicoureteral reflux with endoscopic subureteral glutaraldehyde cross-linked collagen injection. The maximum diameter of the elevated ureteral orifice was measured 3-dimensionally and the numerical value calculated by multiplying each diameter by approximately pi/6 was used as the ultrasound estimate of injected collagen volume. The 1-to-6-month collagen volume ratio was used as an index of the decrease in implant collagen volume. The antibody class against bovine collagen was characterized by indirect enzyme-linked immunosorbent assay. RESULTS: Seroconversion in 6 patients was noted a mean 6.8 months after the first injection. In these patients the antibody class was IgG dominant and IgA or IgM was not detected. There was no significant difference in the total injected collagen volume, total number of injections or collagen volume ratio in the seropositive and seronegative groups. Reflux recurred in 4 patients and the curve of the reflux-free rate was similar regardless of antibody appearance. CONCLUSIONS: The immune response to bovine collagen injection for vesicoureteral reflux does not depend on injected collagen volume or an increased number of treatments. Antibody production had no effects on absorption of the implanted collagen or reflux recurrence.

[Studies on the behavior of some immunologic parameters after local, endoscopic autohemotherapy in children treated for vesicoureteral reflux]. / Badania nad zachowaniem sie okreslonych parametrów immunologicznych po miejscowej, endoskopowej autohemoterapii u dzieci leczonych z powodu odplywu pecherzowo-moczowodowego.

Evaluation of the effect of autologous blood injection into muscular layer on the immune system in children who were treated endoscopically for vesicoureteral reflux. There were 29 children examined after before and endoscopic autohemoinjection. The examination included determination of basic immunoglobulin levels in serum (IgA, IgG, IgM). Also the human lymphocyte subpopulations were determined. Initial and final values were compared, average values and standard deviations for respective age groups were determined. Autologous blood used for injections in endoscopic treatment of vesicoureteral reflux stimulates immune system, especially in young children.

Urinary excretion of brush border antigens and other proteins in children with vesico-ureteric reflux.

This study was designed to evaluate the occurrence and the type of proteinuria in 82 children with vesico-ureteric reflux (VUR) with or without renal scars. The urinary excretion of the high molecular weight protein albumin was taken as an index of glomerular alterations and the excretion of retinol-binding protein (RBP), beta 2-microglobulin and brush border antigens (BBA) (measured by monoclonal antibody-based enzyme-linked immunosorbent assay) was taken as an index of tubular alterations. All such markers were increased in children with VUR and were related to the degree of renal function. Patients showing reduced creatinine clearance had very high levels of albuminuria, microproteinuria and BBA, with all these variables reciprocally correlated. In children with normal renal function however, only microproteins (not albumin or BBA) were slightly increased, thus indicating an isolated tubular defect without involvement of the proximal segment of the tubule. However, microprotein excretion did not correlate with the grade of scarring (99mtechnetium-dimercaptosuccinic acid scan), both RBP and beta 2-microglobulin excretion being normal in 75% of children with radioisotopic signs of renal lesions but increased in 17% of children without scars. Therefore, tubular proteinuria identifies different groups of children with VUR but is not related to renal scarring. Prospective studies will define the usefulness of proteinuria as a reliable indicator of renal outcome.

HLA linkage with familial vesicoureteral reflux and familial pelvi-ureteric junction obstruction.

The inheritance of HLA haplotypes has been looked at in a family with pelvi-ureteric junction obstruction (PUJO) and two families with vesicoureteric reflux (VUR). The data have been combined with those of other reported families and lod scores calculated for both these urinary tract anomalies. There seems no doubt that VUR is linked to HLA whilst the case for PUJO is equivocal.

Relevance of vesico-ureteric reflux in development of lipid A antibodies in recurrent urinary tract infections in children--a preliminary study.

The serum titres of IgG and IgM antibodies to lipid A were measured in 24 children with chronic pyelonephritis (PN), 55 with recurrent lower urinary tract infections (LUTI), 13 with gram-negative sepsis (S), and in 50 control children using an enzyme-linked immunosorbent assay (ELISA). Children ranged in age from 1 month-17 years. Patients with PN were differentiated by the presence or absence of an acute infectious episode and/or vesico-ureteric reflux (VUR). During an acute episode in PN and LUTI, IgG titres were significantly higher than in controls, but only PN patients with an acute infectious episode also had significantly elevated IgM titres. Overall, children with LUTI showed a significantly lower frequency of detectable IgG lipid A antibodies (27%) than in PN (63%). In PN children with VUR not accompanied by an infectious episode, lipid A antibody was found at relatively low titres, while an episode not accompanied by VUR displayed significantly elevated IgG titres, and an episode accompanied by VUR showed elevation of both IgG and IgM anti-lipid A antibody titres.

Human leukocyte antigens and vesicoureteral reflux in Greek children.

Human leukocyte antigens (HLAs) were determined in 43 Greek children suffering from vesicoureteral reflux. Children carrying HLA AW19 + 29 were found to have a higher risk of developing the disease. It is suggested that the HLA system may play a role in the pathogenesis of vesicoureteral reflux.

The role of Tamm-Horsfall protein in the pathogenesis of reflux nephropathy and chronic pyelonephritis.

Recurrent bacterial infection of the kidney was previously thought to be responsible for the renal scarring typical of chronic pyelonephritis until recent studies suggested that recurrent bacteriuria rarely produces chronic pyelonephritis in the absence of obstructive uropathy. In contrast, the association between vesicoureteral reflux (VUR) and chronic pyelonephritis has been observed frequently in the absence of urinary infection. Although the mechanism by which VUR injures the kidney has not been defined, recent observations have suggested that some component of urine might serve as an antigenic determinant involved in the immunopathogenesis of renal scarring in VUR. Therefore, the present studies investigated the immunopathogenic role of Tamm-Horsfall protein (THP) in (1) a rabbit model of tubulointerstitial nephritis; (2) a swine model of reflux nephropathy; and (3) patients with recurrent nephrolithiasis. The antigenic similarities between THP and uropathic bacteria were also studied. Our observations indicate that autoimmune responses to THP may occur after exposure to THP by intravenous challenge in rabbits, by urinary reflux in pigs, and in recurrent nephrolithiasis in man. Also, extracts of uropathic coliforms competitively inhibit the binding of human THP to its antibody. These studies suggest that autoimmune responses to THP may be the pathogenetic mechanism by which these factors, including bacteriuria, contribute to "chronic pyelonephritis."