Simplified quantification of urinary protein excretion in children with nephrotic syndrome

Objective: To assess the value of single voided random (spot) urinary protein to creatinine ratio in accurately predicting the 24-hour urinary protein excretion in Pakistani pediatric population with nephrotic syndrome. Design: Cross-sectional, analytical study. Place and Duration of Study: The Pediatric Department, Nishtar Hospital/ Children Complex, Multan, from January 2003 to November, 2005. Patients and Methods: Fifty seven children between 1-18 years with nephrotic syndrome were included. Seventy pairs of spot urine (5 milliliter) and 24-hour urine were collected in different phases of their disease e.g. initial, induction and remission. The protein to creatinine ratio was determined in spot urine samples and total protein content in 24-hour urine samples. The correlation between the ratio and 24-hour urinary protein excreted was determined using Pearson's coefficient (r) linear regression analysis. Results: The protein to creatinine ratio in a spot urine sample was significantly correlated with the 24-hour urinary protein. The correlation coefficient (least square method) was found to be significant (r=0.9444). A random (spot) urinary protein to creatinine ratio of greater than 2 correlated well with the massive proteinuria (i.e. nephrotic syndrome), between 2 to 0.2 indicated glomerulopathy while a ratio of less than 0.2 was suggestive of physiological values. Conclusion: The random spot urinary protein to creatinine ratio can reliably be used to assess the degree of proteinuria in children with nephrotic syndrome and can replace the 24-hour urinary protein excretion/collection.     

Simplified quantification of urinary protein excretion using a novel dipstick in children

Although the quantification of protein excretion is valuable for diagnosing and monitoring renal disease, accurate, timed, urine collection entails practical difficulties in children. Several authors have shown that the random urine protein/creatinine ratio (UP/UC) correlates well with timed protein excretion. A novel dipstick, Multistix PRO, has recently enabled us to analyze concentrations of both urinary protein and creatinine, semi-quantitatively, in 60 s. The aim of this study was to investigate whether the UP/UC values obtained by Multistix PRO correlate well with those obtained by quantitative methods and daily urinary protein excretion. In order to obtain the UP/UC values, we measured urinary protein and creatinine concentrations both semi-quantitatively by Multistix PRO and quantitatively by conventional methods. The relationship between the semi-quantitative UP/UC by Multistix PRO and the quantitative UP/UC by conventional methods was analyzed. Similarly, the relationship between the semi-quantitative UP/UC and daily urinary protein excretion was studied. Semi-quantitative UP/UC by Multistix PRO correlated closely with both quantitative UP/UC and daily urinary protein excretion (r=0.86 and r=0.91, respectively). A cut-off level of heavy proteinuria, i.e., nephrotic range of proteinuria (>3.5 g/day) corresponded to 3.0, assessed by UP/UC by Multistix PRO. The semi-quantitative UP/UC by Multistix PRO correlated well with both quantitative UP/UC and daily urinary protein excretion, and use of the Multistix PRO would avoid errors and difficulties associated with timed urine collection. It is, therefore, a useful tool to monitor the urinary protein excretion in children with renal diseases at outpatient clinic.     

Glycosaminoglycans: urinary excretion in children with myelomeningocele

BACKGROUND: The main consequences of neurogenic bladder dysfunction are renal damage related to high intravesical pressure, vesicoureteral reflux (VUR) and urinary tract infections (UTIs). Neurologic impairment, UTIs and VUR are known to be linked with a potential for renal scarring. Of paramount importance as predisposing conditions for UTIs in neurogenic bladder are poor bladder drainage and detrusor-sphincter dyssynergy which cause further abnormalities on the internal bladder surface and, consequently, a bladder wall rich in glycosaminoglycans (GAGs). MATERIALS and METHODS: The aim of this study is to investigate the correlation between GAG excretion and bladder wall degeneration in 43 patients affected by spina bifida (SB) and 40 healthy age-matched control children. RESULTS: The amounts of GAGs excreted vary greatly in SB patients aged from 0 to 5 years, and values are comparable to those observed in normal controls. They are significantly higher in children over 5 years of age. CONCLUSION: The increased excretion of GAGs in older SB patients is an important parameter in the evaluation of the physiopathological condition of the bladder wall and hence may be considered a possible marker for monitoring the beginning of bladder damage.     

Orthostatic proteinuria and the spectrum of diurnal variability of urinary protein excretion in healthy children

The aim of this study was to characterize the 24-h and diurnal variability of urinary protein excretion and identify the prevalence of orthostatic proteinuria (OP) in healthy children. Upright, supine, and 24-h total urinary protein (UrTP) and creatinine clearance (CrCl) were measured in 91 healthy children ages 6–19 years. Urinary protein and creatinine excretions were calculated and examined by gender, age, Tanner stage, and body mass index (BMI). Orthostatic proteinuria (OP) was defined as a 24-h UrTP >100 mg/m² with a normal supine UrTP (<4 mg/m²/h). There exists a marked diurnal variability in UrTP. The upright UrTP rate was three to four-times greater than the supine rate. UrTP, adjusted for body surface area, is higher in boys than girls and increases with age and BMI. There is a similar increase in upright CrCl compared with supine. Urinary protein to creatinine ratio (UPcr) is strongly correlated with UrTP. OP is common, being found in 20% of children in this cohort, and is more common in boys and associated with age >10 years and BMI >85%. In children with OP, a first morning UPcr shows a value in the normal range, whereas a random daytime UPcr is elevated. There exists a diurnal variability in urinary protein excretion that is exaggerated in participants with OP. UPcr reliably estimates 24-h UrTP. Using current pediatric criteria, OP is very common, particularly in boys. A normal first morning UPcr ratio indicates that a child with elevated random urinary protein has OP.     

Urinary protein excretion in healthy children

The urinary total protein excretion was determined in 270, 18-24 hr urine samples from 130 healthy children of different age groups using the tannic acid-Fe3+-method of Yatzidis [1977]. The daily protein excretion of premature infants in the first month of life varies between 14-60 mg, with a mean of 29 mg, and that of fullterm newborn infants between 15-68 mg, with a mean of 32 mg. Protein excretion increases with age and amounts to 29-238 mg (mean 83 mg) in 10-16 year old children. Thus, the urinary protein concentration during the neonatal period is high when compared to adult values. This explains the "trace" and "positive" reactions frequently obtained in this period of life with Albustix. In 92 urine samples proteins were fractionated by sodium dodecyl sulphate gel discelectrophoresis. Hemoglobinuria was found during the first weeks of life and tubular type proteinuria was found in newborns and infants. The present data suggest that the proteinuria is due to ineffective proximal tubular reabsorption of low molecular weight microproteins as a result of glomerulo-tubular imblance in early life.     

Diurnal variation in urinary protein excretion in diabetic nephropathy

We examined the diurnal variation in urinary excretion rate of albumin, IgG and beta 2-Microglobulin (beta 2-M) in healthy volunteers (n = 24), and in patients with type I diabetes mellitus having normal albumin excretion rate (less than 20 micrograms/min; n = 16), incipient diabetic nephropathy (albumin excretion rate 20-200 micrograms/min; n = 12) and clinical diabetic nephropathy (albumin excretion rate greater than 200 micrograms/min; n = 12). Diurnal variation was defined as [(overnight minus daytime): daytime excretion rate] times 100%. Median diurnal variation in albumin excretion rate in the various groups varied from -32 to -57%, and in IgG excretion rate from -42 to -65%, being not significantly different between the proteins or between the groups. Diurnal variation in beta 2-M excretion rate was similar in healthy volunteers and in patients with normal albumin excretion rate or incipient diabetic nephropathy (median -36 to -43%), but significantly reduced in patients with clinical diabetic nephropathy (median 0%; P less than 0.005), nine of whom had elevated beta 2-M excretion rates, suggesting tubular dysfunction. Except for beta 2-M excretion rate in patients with clinical diabetic nephropathy, the diurnal variations in albumin excretion rate, IgG excretion rate and beta 2-M excretion rate were larger than the diurnal variation in creatinine excretion rate (median -7 to -11%, P less than 0.005). Diurnal variations in albumin excretion rate and IgG excretion rate were highly correlated (r = 0.89, P less than 0.00001). These data suggest that similar mechanisms may account for diurnal variations in albumin excretion rate and IgG excretion rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased urinary adrenomedullin excretion in children with urinary-tract infection

Background: Adrenomedullin (AM), a smooth-muscle relaxant peptide, is stimulated by cytokines and bacterial endotoxins. We hypothesized that urinary-tract infections may be associated with elevated urinary AM excretion. Methods: AM in urine was quantified in eleven children with urinary-tract infection and 11 age- and sex-matched controls by radioimmunoassay. RT-PCR was used to demonstrate local AM mRNA expression in the urinary tract. Results: In healthy controls but not in diseased children there was a significant correlation between AM and creatinine in urine (r-0.91, P<0.001). AM levels in children with urinary tract infection were significantly higher than in controls (0.6±0.41 vs 0.15±0.14 ng/&mgr;mol creatinine; P<0.001; (means±SD)). There was a significant correlation between white cell count and AM in urine (r-0.78, P<0.001). AM mRNA was expressed in renal tissue, renal pelvis, ureter, bladder, and urethra. Conclusion: The smooth-muscle relaxant peptide adrenomedullin that is synthesized in tissue of the human urinary tract is elevated in urine of patients with urinary-tract infections. A possible consequence might be the interference with the ureteral anti-reflux mechanisms.     

Assessment of retinol-binding protein excretion in normal children

Retinol-binding protein (RBP) is a low molecular weight protein freely filtered at the glomerulus. The fractional tubular reabsorption of RBP is 99.97% and increased excretion is therefore a sensitive marker of tubular dysfunction. We obtained early-morning urine specimens from 151 well children, from newborn to 16 years of age. RBP was measured using an enzyme-linked immunosorbent assay, albumin by a radioimmunoassay and creatinine by a modified Jaffé reaction. Protein excretion was assessed by calculating the protein: creatinine ratio for early-morning urine samples. We found a fall in both RBP and albumin excretion with increasing age, particularly in the 1st year of life, with a much wider variation in values from the infants studied. The mean excretion of RBP for children aged 0–6 months [51.4 (0.6–4,719) g/mmol] was significantly higher (P<0.001) than the mean for children aged 6 months to 16-years [15.0 (3.8–60) g/mmol]. It has been shown that measurement of tubular proteinuria using the RBP: creatinine ratio is useful in the assessment of children with renal disease and we propose a value two standard deviations above the geometric mean for the age of the patient as an upper limit of normal.     

Nocturnal urinary protein excretion rates in patients with sleep apnea

We observed nocturnal urinary protein excretion to be 16.2 +/- 5.5 micrograms/min (mean +/- SE) in 9 healthy control subjects (group I), 29.3 +/- 9.5 micrograms/min in 12 obese patients suspected to have obstructive sleep apnea syndrome (OSAS) but with negative polysomnographic studies (group II), and 94.0 +/- 31.8 micrograms/min in 14 patients with documented OSAS (group III) (II vs. I, NS; III vs. I, p less than 0.05; III vs. II, p less than 0.05). The frequency of abnormal proteinuria, defined as protein excretion greater than the highest rate observed in group I (46 micrograms/min), was 14% in group II and 64% in group III (p less than 0.05). There were no significant differences in age, body weight, body surface area, blood pressure, or indices of sleep apnea between OSAS patients with and without proteinuria. Although the mechanism is unclear, this study shows that nocturnal protein excretion rates are commonly elevated in patients with OSAS.     

Proteomic profiling of urinary protein excretion in the factor H-deficient mouse

BACKGROUND: Since the 1970s a variety of experimental techniques have been employed in an attempt to identify urinary biomarkers of renal injury. While these approaches have met with some success, modern proteomic tools now permit broad based high-throughput analysis of the urinary proteome. METHODS: Using the ICAT isotopic labeling based LC/MS/MS approach, comparative urinary protein profiling was performed in a murine model of membranoproliferative glomerulonephritis. Paired samples were analyzed mice with a targeted deletion of the complement regulatory protein factor H (FH-/-) and control mice. RESULTS: 25 distinct urinary proteins were identified of which 7 were differentially expressed in the FH-/- mice. Two proteins were markedly altered in the urine of FH-/- mice compared to controls: uromodulin (5.5-fold lower) and the MHC class II molecule H2e (8.6-fold higher). Differential expression was confirmed by Western blot and RT-PCR. Immunofluorescent staining demonstrated a marked increased expression of H2e and a reduction of uromodulin expression in the tubular epithelium of FH-/- mice. CONCLUSIONS: These findings provide insight into early complement-dependent alterations in tubular protein expression which may play critical roles in the development of tubulointerstitial disease, and provide experimental support for the use of urinary proteomic profiling in murine models of renal injury.     

Mutations in the uromodulin gene decrease urinary excretion of Tamm-Horsfall protein

BACKGROUND: Mutations in the uromodulin (UMOD) gene that encodes Tamm-Horsfall protein (THP) cause an autosomal-dominant form of chronic renal failure. We have now investigated effects of UMOD gene mutations on protein expression by quantitatively measuring THP excretion. METHODS: THP excretion was determined by enzyme-linked immunosorbent assay (ELISA) of urine collections obtained from 16 related individuals with a 27 bp deletion in the UMOD gene and seven individuals with other UMOD mutations. THP excretion of 22 control subjects (18 genetically related individuals and four spouses in the UMOD deletion family) was also determined. RESULTS: The 16 individuals carrying the deletion mutation excreted 5.8 +/- 6.3 mg THP/g creatinine into their urine. The 18 unaffected relatives from the same family excreted 40.8 +/- 9.7 mg THP/g creatinine (P < 0.0001) and the four spouses excreted 43.9 +/- 25.1 mg THP/g creatinine (P < 0.0001 vs. individuals with the deletion mutation). THP excretion of seven individuals with other UMOD gene mutations was also extremely low (range of 0.14 to 5.9 mg THP/g creatinine). All individuals with UMOD mutations had low THP excretion, irrespective of gender, glomerular filtration rate (GFR), or age. CONCLUSION: These studies quantitatively show that the autosomal-dominant gene mutations responsible for UMOD-associated kidney disease cause a profound reduction of THP excretion. We speculate that this suppression of normal THP excretion reflects deleterious effects of mutated THP within the kidney. Such effects may also play an important role in the pathogenesis of the progressive renal failure observed in patients with UMOD gene mutations.     

Increased urinary albumin excretion in children from families with Balkan nephropathy

Balkan nephropathy (BN) has not been described in children. However, some previous studies have revealed abnormalities of the urinary tract in children from families with BN. In the present study, urinary excretion of albumin was studied in 703 healthy children, age 9-13 years, from endemic and non-endemic settlements around the South Morava River. Since BN is an environmentally induced disease, with possible seasonal variation of toxicant(s), children were studied three times a year: spring, autumn, and winter. After a water load of 15 ml/kg body weight, a 3-h urine sample was collected, from 7 to 10 a.m. Albumin excretion in urine was highest in children from families with BN in all three periods investigated. It was significantly different from excretion in children from the city, and in autumn it was also different ( P<0.01) from children in non-endemic families. Correlation analysis of albumin excretion with some urinary markers of tubular nephrotoxicity shows the highest correlation with both beta(2)-microglobulin and N-acetyl-beta-D-glucosaminidase in endemic villages in autumn. If the upper limit of albumin excretion is set at 8.5 mg/mmol creatinine, then in autumn increased albumin excretion was found in 15 of 229 children from endemic settlements and in only 5 of 454 children from non-endemic areas ( P<0.0001). Evidence is presented that in autumn children from families with BN excreted significantly more albumin than those from non-endemic families but living in the same settlements, or from children living outside of the endemic region in the city of Nis.     

Follow-up of renal function and urinary protein excretion in childhood IgA nephropathy

Renal haemodynamics and urinary protein excretion (UPE) were investigated in 36 patients with IgA nephropathy more than 3 years after renal biopsy (mean interval 6.3±0.5 years). At follow-up, 39% of patients had a reduced glornerular filtration rate (GFR) and 11% end-stage renal failure. Twenty-five percent had albuminuria, and a further 25% microalbuminuria. All albuminuric patients had GFRs below the mean, and 78% of the albuminurics had a reduced GFR. However, non-albuminurics also had decreased GFRs and GFR tended to fall with the duration of the disease in this group of patients. On comparing the histological changes in the biopsies with haemodynamic and UPE studies performed 6 years later, we found significant correlations between the extent of segmental glomerular sclerosis and GFR, effective renal plasma flow, urinary albumin and IgG excretion, respectively. Histological grading correlated with the same variables. Of the 4 uraemic patients, 2 were nephrotic at presentation, while the other 2 had a nephritic onset of disease and later developed heavy proteinuria. Three of their biopsies showed 10% segmental glomerulosclerosis. Juvenile IgA nephropathy is not a harmless disease. Our results indicate that these children should be carefully monitored with adequate GFR measurements and urine protein analyses.     

Urinary excretion of Tamm-Horsfall protein in women with recurrent urinary tract infections

Since MS-fimbriated bacteria adhere to Tamm-Horsfall protein, it has been suggested that Tamm-Horsfall protein may trap urinary pathogens and prevent them from colonizing the mucosal surfaces of the urinary tract. To test the hypothesis that low urinary Tamm-Horsfall protein excretion rates predispose to urinary tract infection we obtained serial urine samples from 17 women with and 18 without a history of recurrent urinary tract infection. None of the women had known structural abnormalities of the urinary tract. Concentrations of Tamm-Horsfall protein in urine were measured with a sensitive enzyme-linked immunosorbent assay method. On the average, 3 urine samples per person collected within 3 to 6 months were analyzed. The mean Tamm-Horsfall protein excretion of women with recurrent urinary tract infection was 57.0 mg./l. and that of controls was 66.3 mg./l.; this difference was not statistically significant. The mean coefficient of variation was 44.2 and 62.1%, respectively. We conclude that urinary Tamm-Horsfall protein concentration is not significantly decreased in women with recurrent urinary tract infection compared with controls, and that excretion varies widely in repeat samples obtained from the same individual.     

Quantitation of urinary Tamm-Horsfall protein in children with urinary tract infection.

It has been suggested that urinary Tamm-Horsfall protein (THP) prevents colonization of the urinary tract by binding uropathogens. We tested the hypothesis that low urinary THP levels may predispose to urinary tract infection (UTI) by measuring THP levels in children. We studied a cohort of 35 girls with uncomplicated recurrent UTI (group 1) that was compared with 27 patients with myelomeningoceles undergoing clean intermittent catheterization (group 2) and 16 female controls (group 3). We measured urinary THP in both aggregated (aTHP) and disaggregated form (dTHP), leukocyte esterase activity, urine chemistries and culture. No significant differences in dTHP or aTHP levels were seen between groups 1 and 3, but group-1 patients had higher dTHP levels than group-2 patients (p < 0.008). History of reflux or the presence of bacteriuria or pyuria at the time of urine collection did not affect dTHP levels; in contrast, pyuria or bacteriuria at the time of sampling was associated with markedly lower aTHP levels when compared with sterile samples (p < 0.0001). For all groups, measured quantities of dTHP did not correlate with aTHP levels. We conclude that excretion of dTHP in children with history of recurrent UTI is not reduced. In contrast, concentrations of aTHP are profoundly depressed in children during times of UTI, suggesting a role for THP in the pathogenesis of UTI. Assaying THP in its aggregated form may prove valuable when studying its physiologic function and merits further investigation.     

Urinary excretion of retinol-binding protein in healthy children and adolescents

Retinol-binding protein (RBP) is a marker of tubular reabsorption in the kidneys. The aim of our study was to investigate urinary RBP excretion in healthy children to obtain reference values related to age and pubertal stage. Overnight samples from 143 subjects (73 girls, 70 boys) aged 10–18 years were investigated. RBP was quantified by a solid-phase sandwich enzyme immunoassay. Both the RBP excretion rate and the RBP/ creatinine ratio (RBP/Cr) showed a skewed distribution. The medians and the 5th–95th percentiles were 38 ng/min (15–127) and 9 μg/mmol (4–23), respectively. The RBP excretion rate and RBP/Cr ratio were similar in both sexes, and linear multiple regression analysis showed no association with age or pubertal stage, although a weak relationship (r = 0.27) was found between RBP excretion rate and age in boys and RBP/Cr ratio and age (r = -0.28) in girls by simple correlation analysis. The correlation between RBP excretion rate and RBP/Cr ratio was 0.76; the RBP excretion rate and RBP/Cr ratio measured on 2 consecutive days, showed a correlation coefficient of 0.84 and 0.88, respectively. We conclude that overnight RBP excretion in children over 10 years shows a low day-to-day variation and, in practical terms, is independent of age, gender and pubertal stage.     

Estimation of the 24-h urinary protein excretion based on the estimated urinary creatinine output

Background

The urinary protein/creatinine ratio [Up/Ucr (g/gCr)] has been used in the clinical management of patients with chronic kidney disease (CKD). However, a discrepancy is often noted between the Up/Ucr and 24-h urinary protein excretion [24hUp (g/day)] in patients with extremes of muscle mass. We examined devised a method for precise estimation of the 24-h urinary protein excretion (E-24hUp) based on estimation of 24-h urinary creatinine output (E-24hCr).

Methods

Three parameters, spot Up/Ucr, 24hUP and E-24hUp (=Up/Ucr × E-24hCr), were determined in 116 adult patients with CKD. The correlations among the groups were analyzed.

Results

There was a significant correlation between the Up/Ucr and 24hUp (p < 0.001). We divided the patients into three groups according to the 24hUp; the low urinary protein group (<1.0 g/day), the intermediate urinary protein group (1.0–3.5 g/day), and the high urinary protein group (>3.5 g/day). There was a significant correlation between the Up/Ucr and 24hUp in the low (p = 0.04) and high urinary protein (p = 0.01) groups, whereas the correlation coefficient was lower in the intermediate urinary protein (p = 0.07) group. Thus, we found a significant correlation between 24hUp and E-24hUp in the study population overall (p < 0.001), in the low (p = 0.01), in the intermediate (p < 0.001), and in the high urinary protein group (p < 0.001).

Conclusion

We conclude that a poor correlation exists between the Up/Ucr and 24hUp in patients with intermediate urinary protein excretion levels. The recommended parameter for monitoring proteinuria in such patients may be the E-24hUp, which is calculated using the E-24hCr.

     

Differential excretion of urinary proteins in children with vesicoureteric reflux and reflux nephropathy

We studied 40 children with a history of vesicoureteric reflux (VUR) without evidence of renal scarring, 93 children with a history of VUR and renal scarring and 10 children with previous urinary tract infections in whom the urinary tract was radiologically normal. Urine retinol-binding protein (RBP), albumin andN-acetyl--d-glucosaminidase (NAG) were measured in each child. All were free from infection at the time of the analysis. Urinary RBP and NAG levels were significantly elevated (P<0.001) in the group of children with renal scarring. Elevated RBP levels were detected in 51% of children with bilateral renal scarring compared with 7% of children with unilateral scarring. Urine RBP excretion increased progressively according to the type of scarring, best determined by the type of scarring of the less affected kidney. In children with renal scarring, elevated NAG levels were seen mostly in the 65 children with bilateral scarring and severe reflux. Urine albumin excretion was elevated in 10 children, 9 with bilateral scarring, all of whom had elevated RBP excretion. Urine protein excretion was unaffected by the presence or absence of persisting VUR. There was a strong negative correlation between glomerular filtration rate and RBP excretion (r=–0.69). We conclude that evidence of tubular dysfunction is common in children with bilateral renal scarring and usually precedes any glomerular protein leak. Tubular dysfunction may be the consequence of relative nephron hyperperfusion in the presence of bilateral scarring.