Insulin-like growth factor-1 expression in reflux nephropathy

Background Reflux nephropathy (RN) is recognised as a major cause of end-stage renal failure in children and young adults. Insulin-like growth factor-1 (IGF-1), a peptide growth factor produced by collecting ducts, and its receptor, insulin-like growth factor-1 receptor (IGF-1R), are present in the glomeruli and basolateral membrane of renal proximal tubular cells. Exogenous IGF-1 has been shown to enhance proliferation and reduce apoptosis of tubular cells following renal injury.Methods We designed this study to investigate the expression of IGF-1 in RN. The kidney specimens from 15 children with RN were obtained at the time of nephrectomy. Control material included normal kidney specimens obtained from adult patients during partial nephrectomy for an incidentaloma. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to IGF-1 and IGF-1R employing laser scanning confocal microscopy. Double-label immunofluorescence histochemistry was carried out using monoclonal antibodies to vimentin and clusterin to assess tubulointerstitial fibrosis. IGF-1 and IGF-1R gene expression were evaluated by in situ hybridisation (ISH). The TUNEL method was utilised to assess tubular apoptosis.Results In the normal kidney there was strong IGF-1 and IGF-1R immunoreactivity in the proximal tubules, whereas IGF-1 and IGF-1R immunoreactivity was markedly reduced in RN specimens. Strong IGF-1 and IGF-1R mRNA expression was observed in the proximal tubules in normal kidneys, whereas IGF-1 and IGF-1R mRNA expression was undetectable in RN. Renal tubulointerstitial expression of vimentin and clusterin was markedly increased in RN kidneys. Decreased IGF-1 and IGF-1R expression in RN strongly correlated with severity of tubular apoptosis in RN compared with controls.Conclusion These data suggest that the downregulation of IGF-1 and IGF-1R may play an important role in the pathogenesis of RN, at least in part by increasing interstitial collagen deposition and tubular apoptosis.     

Vesicoureteric reflux and reflux nephropathy

Reflux nephropathy i.e. renal scarring associated with vesico-ureteric reflux (VUR) and urinary tract infection (UTI) was originally considered an acquired disease. The renal scarring seems to get worse with recurrent urine infections especially in the young. Therefore, in the past much effort was undertaken to correct the VUR surgically and minimize the number of recurrent urinary tract infections by antibiotic prophylaxis with the hope of reducing if not arresting the onset of complications that follow i.e. hypertension and renal failure. However, it is now becoming clear that reflux nephropathy encompass at least two major categories of disease; “acquired” renal scarring secondary to UTI and VUR predominantly affecting females and “congenital” scarring with dysplastic features associated with prenatal VUR but with no infection and predominantly affecting boys. The latter is much less common but is disproportionately represented in the group of patients with reflux nephropathy that go on to develop renal failure. Unfortunately, the susceptibility to renal scarring, the onset of hypertension and progression to renal failure seems to be significantly influenced by genetic factors and hence measures undertaken to prevent recurrence of UTI may not change the ultimate outcome although it will certainly improve the comfort of the individual. Therefore, the extensive investigation and management routines adopted today in these children may not be cost-effective in preventing end stage renal disease in VUR. The progression to renal failure, however, can be delayed but not halted with adequate control of high blood pressure and hence the need for life long follow-up.     

Serum cytokine profile in reflux nephropathy

Cytokines are small regulatory peptides with diverse functions. They regulate the immune system and modulate the inflammatory response, both of which are implicated in vesico-ureteric reflux (VUR) and associated reflux nephropathy (RN). The cytokine profile in VUR and RN has yet to be fully investigated. Blood was obtained from three subject groups immediately after induction of anaesthesia: group A [subjects with VUR and established RN, (N=9)]; group B [VUR alone but no associated RN, (N=6)]; and group C [age- and sex-matched controls with no history of urinary sepsis, (N=14)]. Serum cytokine levels of tumour-necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), soluble TNF receptor-1 (sTNF-R1), and interleukin-8 (IL-8) were measured using standard ELISA technique. Serum levels of IL-6 were higher in group A subjects (1.798–4.638 pg/ml, median 3.253 pg/ml) than controls (1.531–2.078 pg/ml, median 1.798 pg/ml). There was no significant difference in levels in group B subjects (1.498–3.048 pg/ml, median 1.948 pg/ml) and controls. These same relationships were observed for levels of TNF-α (group A: 8.501–14.471 pg/ml, median 13.483 pg/ml; group B: 7.088–10.650 pg/ml, median 8.886 pg/ml; group C: 6.746–13.344 pg/ml, median 7.671 pg/ml) and sTNF-R1 (group A: 690.34–5780.74 pg/ml, median 1197.38 pg/ml; group B: 366.65–1401.62 pg/ml, median 592.82 pg/ml; C: 313.49–636.33 pg/ml, median 504.17 pg/ml). IL-8 was not significantly elevated in any of the study groups (A or B) compared with control group C (group A: 27.08–56.38 pg/ml, median 31.35 pg/ml; group B: 29.90–35.87 pg/ml, median 31.35 pg/ml; group C: 25.05–30.22 pg/ml, median 29.90 pg/ml). These results suggest there may be an immunological basis to RN. Accepted: 21 December 1998     

Hypothyroidism mimicking chronic renal failure in reflux nephropathy

An adolescent with a history of pyelonephritis and renal scarring had antireflux surgery at the age of 2.5 years. His serum creatinine was high at the age of 14 years (133 micromol/l; glomerular filtration rate (GFR) 56 ml/min x 1.73 m(2)), and reflux nephropathy with chronic renal failure was diagnosed. Because of a fall in height velocity, endocrinological investigations were performed six months later which showed hypothyroidism caused by autoimmune thyroiditis. Substitution with thyroxine was started; renal function improved to normal six months later (GFR 108 ml/min x 1.73 m(2)). Metabolic changes of hypothyroidism led to a reduction of GFR in this patient and mimicked chronic renal failure.     

The role of nitric oxide in reflux nephropathy

Reflux nephropathy (RN) is recognized as a major cause of end-stage renal failure in children and young adults. Inhibition of nitric oxide (NO) exacerbates and enhanced production ameliorates tubulointerstitial fibrosis (TIF) in experimental obstructive uropathy. NO is synthesised by NO synthase (NOS), three distinct isoforms of which have been identified: inducible (iNOS), endothelial (eNOS), and neuronal (nNOS). It has been reported that iNOS induces immunologic injury to glomerular cells and enhances accumulation of extracellular matrix in the glomerulus and tubulointerstitial space. Furthermore, it has been suggested that nNOS and eNOS have beneficial effects in ameliorating TIF. We investigated the expression of different isoforms of NOS in severe refluxing kidneys in order to further understand the pathogenesis of RN in kidney specimens from nine children with severe RN obtained at nephrectomy. Control material included normal kidney specimens from three adult patients undergoing partial nephrectomy for small kidney tumours. Histochemistry for NO was performed using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to nNOS, iNOS, eNOS, and transforming growth factor (TGF)-beta 1 employing laser-scanning confocal microscopy. The TUNEL method was used to assess tubular apoptosis. Strong NADPH staining was observed in the proximal tubules of RN kidneys compared to controls, where there was weak staining. Control kidneys demonstrated weak immunoreactivity for iNOS in the proximal tubules and a lack of immunoreactivity for nNOS and eNOS. RN kidneys demonstrated strong immunoreactivity for nNOS in the tubulointerstitial space, for eNOS in the glomerulus, and for iNOS in the glomerulus and proximal tubules. Strong immunoreactivity for TGF beta 1 was seen in the glomerulus and proximal tubules identical to iNOS. Increased immunoreactivity for iNOS and TGF-beta 1 strongly correlated with the severity of apoptosis in RN. Our data demonstrate that NO derived from nNOS, iNOS, and eNOS is strongly expressed in RN. The selective shunting of NO via iNOS may induce renal fibrosis in RN. The upregulation of nNOS and eNOS in RN appears to be a compensatory mechanism of ameliorating TIF.     

Serum soluble vascular cell adhesion molecule-1 (VCAM-1) concentrations in children with reflux nephropathy

Systemic inflammatory disorders causing renal tissue damage do so by the adherence of polymorphonuclear leukocytes to endothelium, a process that is mediated by cell surface adhesion molecules. We determined the circulating levels of serum vascular cell adhesion molecule-1 (VCAM-1) in vesicoureteric reflux (VUR) patients and investigated the relationship between serum VCAM-1, grade of VUR, and secondary renal scarring. Serum levels of VCAM-1 were measured in 53 children aged between 3 months and 15 years with VUR (13 had grade III, 29 had grade IV, and 11 had grade V) and 25 controls using ELISA. Radionuclide scanning was used to assess renal scarring. Renal scarring was found in 29 of the 53 subjects. Serum VCAM-1 was significantly higher in subjects with high grades of VUR without renal scarring (grade IV: 715.9±121.0 ng/ml; grade V: 778.5±33.2ng/ml) compared with subjects with grade III VUR without renal scarring (609.8±64.3ng/ml, p<0.01). Serum VCAM-1 was also significantly higher in subjects with high grades of VUR with renal scarring (grade IV: 791.2±131.9ng/ml; grade V: 1171.8±235.6 ng/ml) compared with subjects with grade III VUR with renal scarring (687.3±163.4 ng/ml, p<0.001).     

Hypothyroidism mimicking chronic renal failure in reflux nephropathy.

An adolescent with a history of pyelonephritis and renal scarring had antireflux surgery at the age of 2.5 years. His serum creatinine was high at the age of 14 years (133 micromol/l; glomerular filtration rate (GFR) 56 ml/min x 1.73 m(2)), and reflux nephropathy with chronic renal failure was diagnosed. Because of a fall in height velocity, endocrinological investigations were performed six months later which showed hypothyroidism caused by autoimmune thyroiditis. Substitution with thyroxine was started; renal function improved to normal six months later (GFR 108 ml/min x 1.73 m(2)). Metabolic changes of hypothyroidism led to a reduction of GFR in this patient and mimicked chronic renal failure.     

The association of cytokine gene polymorphism with reflux nephropathy

ObjectiveTo identify genetic risk factors for the progression of vesicoureteral reflux (VUR) to reflux nephropathy, we examined polymorphisms of multiple cytokine genes among VUR patients with or without renal scarring.MethodsA total of 238 VUR patients aged between 1 and 18 years with (n = 113) or without renal scarring (n = 125) were included. The presence of renal scarring was demonstrated by renal parenchymal examination using Technetium-99m dimercaptosuccinate scintigraphy. Sera of the patients were examined for tumor necrosis factor-alpha (TNF-α, −308), transforming growth factor-beta1 (TGF-β1, +869, +915), interleukin-6 (IL-6, −174), interleukin-10 (IL-10, −1082, −819, −592) and interferon-gamma (IFN-γ, +874) gene polymorphisms using the polymerase chain reaction sequence-specific primer method.ResultsAmong patients with renal scarring, frequencies for the T/T G/C and C/C G/C genotypes of TGF-β1 gene (p = 0.003), GCC/GCC genotype of IL-10 gene (p = 0.015), GC phenotype of IL-6 gene (p = 0.001) and T/T genotype of IFN-γ gene (p = 0.001) were higher compared to patients without renal scarring. Regarding the TNF-α gene, among patients with low grade VUR only, the G/G genotype was associated with an increased risk.ConclusionsCertain genotypes of cytokine gene polymorphisms seem to be associated with an increased or decreased susceptibility to reflux nephropathy, which may explain why only a proportion of VUR patients progress to reflux nephropathy. This information may aid in prediction of prognosis and implementing more aggressive management strategies at earlier stages. Further immunogenetic studies may identify novel targets for the management and prevention of the condition.     

Evaluation of reflux nephropathy, pyelonephritis and renal dysplasia

MR urography has the potential to significantly improve our understanding of the relationship between reflux nephropathy, pyelonephritis, vesicoureteric reflux and renal dysplasia. MR urography utilizes multiple parameters to assess both renal anatomy and function and provides a more complete characterization of acquired and congenital disease. Pyelonephritis and renal scarring can be distinguished by assessing the parenchymal contours and signal intensity. Characteristic imaging features of renal dysplasia include small size, subcortical cysts, disorganized architecture, decreased and patchy contrast enhancement as well as a dysmorphic pelvicalyceal system. Because of its ability to subdivide and categorize this heterogeneous group of disorders, it seems inevitable that MR urography will replace DMSA renal scintigraphy as the gold standard for assessment of pyelonephritis and renal scarring. MR urography will contribute to our understanding of renal dysplasia and its relationship to reflux nephropathy.     

Increased expression of ICAM-1 and VCAM-1 in the lung of nitrofen-induced congenital diaphragmatic hernia in rats

Recently, increased expression of inflammatory cytokine, tumor necrosis factor (TNF)-alpha, has been reported in both humans and animal models with CDH and the decreased TNF-alpha expression in CDH lung after antenatal dexamethasone (Dex) treatment. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 are induced by several inflammatory cytokines such as TNF-alpha. The aim of this study was to investigate pulmonary ICAM-1 and VCAM-1 expression in CDH lung in rats and to determine the effect of antenatal glucocorticoid. CDH model was induced in pregnant rats following administration of nitrofen on day 9.5 of gestation. In control animals, the same dose of olive oil was given without nitrofen. Dex (0.25 mg/kg) was given on day 18.5 and 19.5 of gestation. RT-PCR was performed to evaluate the relative amount of ICAM-1 and VCAM-1 mRNA expression. Fluorescein immunohistochemistry using anti-ICAM-1 and anti-VCAM-1 antibody was performed using light and confocal microscopy. ICAM-1 and VCAM-1 mRNA expression and ICAM-1 and VCAM-1 immunoreactivity were markedly increased in CDH lung compared to controls. Dex downregulated the expression of both adhesion molecules in the hypoplastic lung. Increased ICAM-1 and VCAM-1 mRNA expression in hypoplastic lungs would suggest that the increased local synthesis of pulmonary adhesion molecules may induce respiratory distress in CDH. Decreased expression of adhesion molecules in CDH lungs after Dex treatment suggests that antenatal glucocorticoids therapy may improve pulmonary immaturity and associated respiratory distress in nitrofen-induced CDH lung.     

Prediction of blood pressure from plasma renin activity in reflux nephropathy.

As there is a 10% risk of hypertension developing in children with reflux nephropathy and the renin-angiotensin system has been implicated in its aetiology, a long term prospective study has been undertaken to explore the relationship between plasma renin activity (PRA) and blood pressure in such patients. In 1978, of 100 normotensive children with reflux nephropathy 8% were shown to have PRA above normal. Five years later of 85 subjects suitable for analysis 13% had increased PRA and it was shown that PRA and blood pressure SD scores significantly increased. The present study refers to the 10 year follow up in which 95 of the original group were traced but eight of these were unavailable for study and 28 others were excluded from analysis because of extraneous factors that might influence blood pressure or PRA. Results therefore on 59 have been analysed. PRA was above normal in 13/59 (20%) subjects, and PRA and blood pressure SD scores had further increased. The data continue to support the role of the renin-angiotensin system in the observed rise of blood pressure in reflux nephropathy, but individual PRA measurements do not appear so far to predict reliably the onset of hypertension in affected patients.     

Assessment of cystatin C and cystatin C-based GFR formulas in reflux nephropathy

ObjectiveEarly identification of reflux nephropathy (RN) could reduce the frequency of chronic kidney disease (CKD) caused by vesicoureteral reflux (VUR). We aimed to assess whether cystatin C has value for determining RN in children with VUR.Materials and methodsNinety-three children with VUR were classified into two groups according to the presence of renal parenchymal scarring (RS). Patients with RS were divided into three subgroups according to scar grade. Serum cystatin C, serum creatinine (Scr) and urine creatinine were measured. eGFR values of the patients were calculated with Scr-based, cystatin C-based and combined formulas.ResultsCystatin C was significantly higher in patients with RS than patients without RS and declined in parallel with grade of RS (p = 0.01). Scr was not significant in patients with and without RS. It was only significant between mild and severe scar subgroups (p < 0.05). All eGFR values were lower in RS (+) patients compared with RS (−) patients. All eGFR equations were negatively correlated with grade of RS (p < 0.05).ConclusionCystatin C could be a useful marker for identifying the risk and severity of RN in patients with VUR. Renal functions could be more accurately determined with Scr-cystatin C combined eGFR equations.     

Duodenogastric bile reflux is common in cystic fibrosis

BACKGROUND: Patients with cystic fibrosis (CF) have a high incidence of gastroesophageal reflux disease, but few cases of mucosal injury are reported. Duodenogastric reflux has not been studied in CF but has been suggested to have a pathogenic role in producing alkaline injury to the esophageal mucosa. The aim of this study was to analyze the presence of duodenogastric reflux in patients with CF. PATIENTS AND METHODS: Ten patients with CF and 7 healthy volunteers participated in the study. Gastroduodenal manometry and intragastric perfusion were performed in all subjects. Gastric perfusate was analyzed for bilirubin and bile acids. Only patients and controls exhibiting normal migrating motor complexes were evaluated. RESULTS: Eight patients with CF had normal motility recordings and had significantly higher gastric bilirubin levels compared with healthy subjects (P = 0.003). The bilirubin concentration was associated with bile acid regurgitation in five patients with CF. All bile acids were conjugated with a high glycine/taurine ratio and low levels of secondary bile acids. Small amounts of keto bile acids were found in two patients. CONCLUSION: The patients with CF had an increased incidence of duodenogastric reflux compared with healthy subjects. The bile acid composition was typical for CF with low levels of secondary bile acids. Although high bile acid concentration was found in the duodenogastric reflux in most patients with CF, the less toxic profile of the bile acids might possibly contribute to the low frequency of Barrett's esophagus in CF.     

Physical growth in children with reflux nephropathy with normal or mildly impaired renal function

Ten children aged 11 months to 10 years (means 5.7 years) with reflux nephropathy, vesicoureteric reflux (VUR) and normal or mildly impaired renal function having GFR more than 50 ml/min/1.72 m², were included in the study. The hematological and biochemical parameters were within normal limits. Height standard deviation score (HZ score) was reduced at entry and, decreased further during follow-up (−2.2 and −2.6 at 0 and 12 months, respectively). Weight for height index (WHI) improved significantly (p=0.0004) during follow-up. The basal and stimulated peak growth hormone levels of these patients were found to be elevated, 18.53 ± 11.36 μg/L and 34.20 ± 5.86 μg/L, respectively. The IGF-1 levels were low ranging from 45.00 to 84.40 ng/dl (mean ± SD 61.54 ± 10.21 ng/dl) compared to 51.80 to 247.50 ng/dl (mean ± SD111.20 ± 70.24 ng/dl) in age and sex matched controls, indicating partial insensitivity to growth hormone.