Urinary NGF, TGF-b1, TIMP-2 and Bladder WallThickness Predict Neurourological Findings in Childrenwith Myelodysplasia

Cagri Akin Sekerci, Banu _Isbilen, Ferruh _Isman, Cem Akbal,

Ferruh Simsek and Tufan Tarcan*,†

From the Department of Urology, Division of Pediatric Urology, Marmara University School of Medicine (CAS, CA, FS, TT)

and Department of Biochemistry, G€oztepe Training and Research Hospital (B _I, F _I), Istanbul, Turkey

Abbreviations

and Acronyms

BoNT-A ¼ botulinum neurotoxin A

BWT ¼ bladder wall thickness

CIC ¼ clean intermittentcatheterization

DLPP ¼ detrusor leak pointpressure

DMSA ¼ dimercapto-succinicacid

MMP ¼ matrix metalloproteinase

NGF ¼ nerve growth factor

TGF-b1 ¼ transforming growthfactor-b1

TIMP ¼ tissue inhibitor ofmetalloproteinase

VUR ¼ vesicoureteral reflux

Accepted for publication August 7, 2013.Supported by Marmara University, Scientific

Research Projects Fund (Project No. SAG-C-TUP-031110-0251).

Study received local ethical committeeapproval.

* Financial interest and/or other relationshipwith Pfizer, Astellas and Allergan.

† Correspondence: Department of Urology,Division of Pediatric Urology, Marmara UniversitySchool of Medicine, Uskudar, Istanbul, Turkey(e-mail: tufan@marmara.edu.tr).

Purpose: Dimercapto-succinic acid scintigraphy and urodynamic studies are goldstandards to evaluate renal scarring and neurogenic bladder dysfunction,respectively. We sought to establish the value of bladder wall thickness togetherwith urine NGF, TGF-b1 and TIMP-2 to predict the urodynamic profile andupper urinary tract damage in children with myelodysplasia.

Materials and Methods: A total of 80 children with myelodysplasia underwenturodynamic investigation, bladder wall thickness measurement and dimercapto-succinic acid scintigraphy with basic neurourological evaluation. Two study and2 control groups were created according to presence or absence of renal scarringon dimercapto-succinic acid scan (study and control groups 1) and according todetrusor leak point pressure greater or less than 40 cm H2O (study and controlgroups 2). Urine samples were analyzed with ELISA.

Results: The study population consisted of 44 girls and 36 boys with a median �SD age of 7.2 � 3.6 years (range 2 to 17). Study and control groups 1 consisted of35 and 45 children with abnormal and normal dimercapto-succinic acid scanfindings, respectively. Study and control groups 2 included 30 and 50 childrenwith detrusor leak point pressure greater and less than 40 cm H2O, respectively.Bladder wall thickness and urinary levels of TGF-b1, NGF and TIMP-2 weresignificantly increased in both study groups compared to controls.

Conclusions: Urine markers and bladder wall thickness measurement maypredict urinary tract impairment in children with myelodysplasia. Such markersmay differentiate at risk patients with either renal scarring or high detrusor leakpoint pressure, and decrease the need for urodynamics and renal scintigraphy.

Key Words: nerve growth factor; neural tube defects;

tissue inhibitor of metalloproteinase-2; transforming growth factor beta1;

urinary bladder, neurogenic

NEUROGENIC bladder dysfunctionaccompanying myelodysplasia maylead to recurrent urinary tract in-fections, vesicoureteral reflux, loss ofrenal parenchyma, scar formation andchronic renal failure.1 An initial riskanalysis by urodynamic classification

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helps to differentiate at risk in-dividuals from relatively lower riskcases and plan neurourological man-agement accordingly.2 Monitoringthe upper and lower urinary tractduring followup by radiological andurodynamic tests is mandatory, since

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200 URINE MARKERS FOR NEUROGENIC BLADDER DYSFUNCTION

myelodyplasia is a dynamic disease. However, uro-dynamic studies are not always easy to performor interpret in children. Dimercapto-succinic acidscintigraphy is more sensitive to assess renal scardevelopment but it carries the disadvantage ofradiation exposure, since even low dose radiationmay increase the lifetime risk of cancer.3,4 Thus, lessinvasive and less morbid tests are needed to monitorthe urinary tract.

To our knowledge there is no study in childrenwith myelodysplasia regarding the prognostic roleof the cytokines as urine markers, ie NGF, whoseurinary concentration is known to increase in thepresence of detrusor overactivity,5e7 TGF-b18 andTIMP-2, which have a role in the breakdown ofmetalloproteinases involved especially in thedevelopment of fibrosis and tumor invasion in theextracellular compartment.9

MATERIALS AND METHODSWe sought to establish the value of bladder wall thick-ness, which is predictive of upper urinary tract damageand related urodynamic findings,10,11 and urine NGF,TGF-b1 and TIMP-2 levels to predict urodynamic profileand upper urinary tract damage in children withmyelodysplasia.

Study Population and DesignA total of 80 children with myelodysplasia were includedin the study after local ethical committee approval.Written consent was obtained from the parents before thestart of the study via patient consent and patient infor-mation forms. All children were evaluated with physicalexamination, urinalysis, urine culture, renal/bladderultrasound, voiding cystourethrogram, urodynamic testsand renal DMSA scintigraphy. Two study and 2 controlgroupsdall with myelodysplasiadwere created accordingto the presence or absence of renal scarring on DMSAscan (study and control groups 1) and according to DLPPgreater or less than 40 cm H2O (study and control groups2). Study group 1 consisted of 35 children with unilateralor bilateral renal scarring on DMSA scan. Control group 1included 45 children without renal scarring. Study group2 consisted of 30 children with DLPP greater than 40 cmH2O. Control group 2 included 50 children with DLPP lessthan 40 cm H2O. Thus, 5 children with a low DLPP hadrenal scarring.

Bladder Wall Thickness MeasurementBWT measurements by ultrasound were performed usingthe standardized method described by M€uller et al.12,13

The relatively hypoechogenic muscular middle layer ofthe 3 strata imaged for the bladder wall was measured.Measurements were performed in the anterior wall at apoint between the median umbilical ligament and thestart of the lateral walls, and in the posterior wall at thelateral side of the rectal contour, both in the transverseplane. Measurements of the anterior and posteriorbladder wall were averaged. The bladder was full at thetime of measurement. Bladder fullness was ascertained

by 1 of 4 criteria, which included reaching a pressure of40 cm H2O during bladder filling through a urodynamiccatheter, onset of continuous urine leak during cystom-etry, strong urge to void during cystometry and reaching90% of the lower expected bladder capacity. Expectedbladder capacity was calculated using the formula, age �30 þ 30 ml.

Urodynamic EvaluationUrodynamic assessment was conducted simultaneouslywith the upper tract evaluation. DLPP or end fillingpressure (cm H2O) was evaluated. Standard fluid cys-tometry was done with patients in the supine positionusing a 6Fr double lumen urethral cystometry catheterand a rectal balloon catheter, filling at a rate of less than10% of predicted bladder capacity per minute.

Detrusor Leak Point Pressure and End FillingPressureDLPP is defined as the lowest detrusor pressure at whichurine leakage occurs in the absence of either a detrusorcontraction or increased abdominal pressure. The major-ity of children with myelodysplasia do not leak duringurodynamics, and the only available value is the endfilling pressure.

Urine Marker AnalysisUrine samples were collected before urodynamic studywhen the urine was sterile, from a full bladder. They werecentrifuged for 4 minutes at 3,000 rpm at 4C and frozenat �80C after discarding the supernatant. Urine sampleswere taken to the biochemistry clinics by refrigeratedtransportation system and analyzed in a single batchby ELISA, using the RayBio� recombinant humanTIMP-2, recombinant human TGF beta 1 and recombi-nant human NGF (beta) ELISA kits. Results werecorrected to urine creatinine.

Statistical MethodsFrequency, proportion, median and standard deviationswere calculated for descriptive statistics. Data distribu-tion was tested by the Kolmogorov-Smirnov method.Parametric interval data were evaluated by t-test andANOVA, while nonparametric data were subjected tothe Mann-Whitney U and Kruskal-Wallis tests. SPSS�,version 1.0 was used for the analyses.

RESULTSThe study population consisted of 44 girls and36 boys with a median � SD age of 7.2 � 3.6 years(range 2 to 17). Primary repair was performedwithin the first 72 hours in 33 patients and later inthe remaining 47. Of the 80 patients 30 had a ven-triculoperitoneal shunt. A secondary untetheringsurgery had been performed in 14 children.

The spinal lesion was a myelomeningocele in54 children, tethered spinal cord in 8 and menin-gocele in 5. The remaining 13 children had lesscommon lesions, such as diastematomyelia, lipo-myelomeningocele, dermal sinus, hydromyelia,syringomyelia and split cord malformation. Study

Table 2. Comparison of bladder wall thickness and urinarylevels of b-NGF, TGF-b1 and TIMP-2 in group 2

Controls Pts p Value*

Median � SD mm BWT (range) 4.2 (2.4e6.4) 4.7 (2.5e8.3) 0.007Median � SD urine b-NGF (range)† 0.6 (0.1e24) 1.85 (0.2e36) <0.001Median � SD urine TGF-b1 (range)† 1.8 (0.1e55) 4.1 (0.3e101.6) 0.008Median � SD urine TIMP-2 (range)† 10.4 (2.9e45.8) 17.9 (2.3e67.5) 0.000

* t-test/Mann-Whitney U test, 95% CI.†Based on urinary ng/mg creatinine.

URINE MARKERS FOR NEUROGENIC BLADDER DYSFUNCTION 201

groups 1 and 2 included 5 children and controlgroups 1 and 2 included 8 children with less com-mon lesions. The lesions were lumbar in 57 patients,sacral in 14, lumbosacral in 2, thoracic in 2 andthoracolumbar in 4.

A total of 80 patients had received neuro-urological treatment beginning at diagnosis. Cleanintermittent catheterization was being performed in47 children, and the remaining 33 used the Cred�emaneuver. Anticholinergic treatment was used in49 patients and antibiotic prophylaxis in 50. Atinitiation of the study all 35 children in study group1 were using CIC and anticholinergic therapy, and23 of 45 in control group 1 were using CIC. All 30children in study group 2 were using CIC andanticholinergic therapy, and 28 of 50 in controlgroup 2 were using CIC. Later in followup 22 morechildren were started on CIC. BoNT-A injection wasperformed in 3 patients.

Mean age in group 1 was 7.56 years in those withrenal scarring (patients) and 6.98 years in thosewithout scarring (controls). Patients were comparedto controls for bladder wall thickness and urinarylevels of b-NGF, TGF-b1 and TIMP-2 (table 1). Allparameters in controls were significantly lower thanin patients. BWT in controls was significantly lessthan in patients with unilateral or bilateral scar-ring. However, BWT was not significantly differentbetween groups with unilateral and bilateral scar-ring. Urinary b-NGF, TIMP-2 and TGF-b1 levelswere significantly lower in controls compared topatients with unilateral and bilateral scarring,while no significant difference was demonstratedbetween children with unilateral and bilateralscarring.

Mean age in group 2 was 6.86 years in those withDLPP greater than 40 cm H2O (patients) and 7.42years in those with DLPP less than 40 cm H2O(controls). Patients were compared to controls forBWT and urinary levels of b-NGF, TGF-b1 andTIMP-2 (table 2). BWT measurements were signif-icantly less in controls than in patients. b-NGF,TGF-b1 and TIMP-2 levels were also significantlyless in controls.

The sensitivity of urine markers in predictingDLPP greater than 40 cm H2O is described in

Table 1. Comparison of bladder wall thickness and urinarylevels of b-NGF, TGF-b1 and TIMP-2 in group 1

Controls Pts

Median � SD mm BWT (range) 4 (2.5e6.1) 4.8 (2.4e8.3)Median � SD urine b-NGF (range)* 0.6 (0.1e11.7) 2 (0.2e36)Median � SD urine TGF-b1 (range)* 1.6 (0.2e11.3) 4.7 (0.1e101.6)Median � SD urine TIMP-2 (range)* 10.4 (2.3e34) 17.9 (3.2e67.5)

p <0.0001 between patients and controls for all values compared (t-test/Mann-Whitney U test, 95% CI).*Based on urinary ng/mg creatinine.

table 3. ROC curves and cutoff values of urinemarkers for predicting renal scarring and DLPPgreater than 40 cm H2O are shown in figures 1and 2, and tables 4 and 5.

In the entire study group 15 children had uni-lateral or bilateral VUR. Study group 1 included10 children with VUR, whereas control group 1included 5 children with VUR. Study group 2included 8 children with VUR, whereas controlgroup 2 included 7 children with VUR. All childrenwith VUR were treated with CIC and anticholin-ergic therapy, and 2 children further received sub-ureteral injection. BWT and urine markers inchildren with VUR were significantly greater thanin those without VUR (table 6).

DISCUSSIONOur study reveals that bladder wall thicknesstogether with urinary NGF, TGF-b1 and TIMP-2levels may predict upper urinary tract damageand related urodynamic findings in children withmyelodysplasia. NGF, a trophic protein, is a retro-grade messenger between the peripheral effectortissue and its nerves.14 Smooth muscle cells, fibro-blasts, astrocytes and other cells synthesize NGF inculture media.15 Denervation, inflammation andmechanical tension increase NGF in the lower uri-nary system.16 NGF is produced in the urotheliumand the smooth muscle cells of the urinary tract.17

Chronic prostatitis and interstitial cystitis increaseNGF levels in bladder tissue and urine.18

An experimental study demonstrated thatchronic urothelial NGF over expression in trans-genic mice led to neuronal hyperinnervation, pelvicsensitivity and changes in bladder function.19

Urinary NGF increases in patients with overactivebladder and decreases significantly after anticho-linergic treatment.20 Post-BoNT-A therapy levels of

Table 3. Sensitivity of urinary markers in predicting DLPPgreater than 40 cm H2O

ROC (95% CI)

Urine TGF-b1 60.9 (45.9e76.1)Urine TIMP-2 68.3 (54.1e82.5)Urine b-NGF 70.6 (56.4e84.8)

Figure 1. Sensitivity of urine markers in predicting DLPP greater than 40 cm H2O

202 URINE MARKERS FOR NEUROGENIC BLADDER DYSFUNCTION

NGF may be predictive of treatment response.21

Urinary NGF was significantly increased in pa-tients with neurogenic/idiopathic detrusor over-activity.22 The latter study further indicated thaturinary NGF levels are significantly decreased inpatients responding to anticholinergic drugscompared to nonresponders, and BoNT-A adminis-tration decreased urinary NGF/creatinine ratio. Inaddition to DLPP levels, we also investigated thepreviously unexamined relationship between thepresence of renal scarring and urinary NGF levels.The latter were increased in children with myelo-dysplasia and renal scarring.

TGF-b1 has a substantial role in the renin-aldosterone-angiotensin system, which is part ofthe pathogenesis of tubulointerstitial and renalfibrosis.8 TGF-b1 is a potent fibrogenic cytokinesecreted by a group of cells including renal cells.Taha et al reported increased levels of urinaryTGF-b1 in children with ureteropelvic junctionobstruction compared to healthy subjects.23 Theselevels decreased following surgery, indicating a rolefor TGF-b1 levels in followup. Increased preopera-tive urinary TGF-b1 was also found in 19 cases ofureteropelvic junction obstruction without VUR.24

A significant decrease in urinary TGF-b1 levelsfollowing dismembered pyeloplasty was noted in

these patients. This marker was also significantlyincreased in children with obstructive uropathycompared to healthy subjects.25 DLPP greater than40 cm H2O in our study is associated with a signif-icantly higher concentration of TGF-b1, suggestinga role in predicting renal scarring.

The MMP family is an important part of theextracellular proteinases. Their most relevant func-tion seems to be the breakdown of extracellular ma-trix. These enzymes occupy a fairly significant placein tissue restructuring, angiogenesis, morphogen-esis, development and apoptosis.26 MMPs can beconsidered in 4 groups according to their substratespecificity. One of these groups, the collagenases,comprises MMP-1. The substrate for this proteaseconsists of type 1 and type 2 collagen, the essentialcollagens of bladder extracellular matrix. MMPactivity is inhibited by TIMP specific tissue in-hibitors.27 Numerous studies regarding the rela-tionship of MMP and atherosclerosis, lung cancerand chronic obstructive pulmonary disease areavailable. However, we are not aware of a similarpublication on their relationship with renal scarring.

In our study urine markers and BWT measure-ment were sensitive in children with VUR (table 6).Markers subject to this study did not significantlydiffer between patients with unilateral and bilateral

Figure 2. Sensitivity of urine markers in predicting unilateral or bilateral renal scarring

URINE MARKERS FOR NEUROGENIC BLADDER DYSFUNCTION 203

renal scarring. Therefore, the informative value ofthese markers, which were determined to be sig-nificant in predicting the presence or absence ofscarring, remains uncertain regarding severity ofscarring. Measurement of these markers in childrenwith myelodysplasia in whom no renal scar has yetdeveloped will be useful in later stages of followup.We believe the number of invasive examinations,such as DMSA scan and urodynamics, during fol-lowup might be reduced as long as the marker levelsremain low. As shown on ROC curve, each of theseurine markers was quite sensitive for high DLPP,which leads us to believe that using these 3 markerstogether may improve diagnostic specificity for highrisk cases.

Although Cvitkovi�c-Kuzmi�c et al found a statis-tically significant difference in mean detrusorthickness between children with normal andabnormal urodynamics, there was a substantial

Table 4. Cutoff values of urinary markers for predicting DLPPgreater than 40 cm H2O

Urine b-NGF Urine TGF-b1 Urine TIMP-2

80% Sensitivity 0.40 1.15 6.9560% Sensitivity 0.95 1.95 10.90AUC 0.71 0.61 0.68p Value 0.008 0.159 0.019

Urinary markers based on ng/mg creatinine.

overlap of measured values.11 M€uller et al dividedtheir patients with myelomeningocele into 3 groupsaccording to bladder urodynamics, ie mild, moder-ate and severe (DLPP less than 20, 20 to 40 andgreater than 40 cm H2O, respectively), andmeasured BWT.12 No relationship between grade ofbladder dysfunction, renal function and anticholin-ergic therapy was observed. According to Tanakaet al, in children with myelomeningocele, DLPPgreater than 40 cm H2O and detrusor sphincterdyssynergia mean � SD bladder wall thickness was3.9 � 1.0 mm, compared to 2.4 � 0.7 mm in urody-namically stable children.10 In our study BWTwas significantly increased in children with renalscarring or high DLPP compared to those with noscars or low DLPP. To decrease the subjectivity ofBWT measurement, all measurements were per-formed by the first author of the study and themeasurement protocol was further standardized as

Table 5. Cutoff values of urinary markers for predictingrenal scarring

Urine b-NGF Urine TGF-b1 Urine TIMP-2

80% Sensitivity 0.85 2.05 10.6060% Sensitivity 1.65 3.75 14.80% AUC 80 78 75

Urinary markers based on ng/mg creatinine.p <0.001 for 80% vs 60% sensitivity for all values compared.

Table 6. BWT and urine markers in children with andwithout VUR

Pts without VUR Pts with VUR p Value

Median � SD mm BWT (range) 4.0 (2.4e6.5) 4.6 (2.5e8.3) 0.005Median � SD urine b-NGF (range)* 1.2 (0.1e28) 2.2 (0.6e36) 0.003Median � SD urine TGF-b1 (range)* 2.3 (0.1e45) 6.7 (2.2e101.6) <0.0001Median � SD urine TIMP-2 (range)* 12.3 (2.3e54.6) 23.4 (3.7e67.5) <0.0001

*Based on urinary ng/mg creatinine.

204 URINE MARKERS FOR NEUROGENIC BLADDER DYSFUNCTION

mentioned previously. Despite these efforts, webelieve BWT should not be used as the soleparameter, but should be combined with otherclinical and urodynamic findings in clinical deci-sion making.

Our study has some limitations. We did notmeasure urinary cytokine levels and BWT innormal children. Thus, levels in patients at low riskwere not compared to levels in normal children. Wedid not investigate urinary marker levels before andafter treatment. Therefore, our study did not yieldany data about using these biomarkers for treat-ment during followup. At the beginning of the studyour data set was planned to use DLPP values, sobladder compliance measures were not compared to

urinary biomarker values. No power calculation wasdone for this nonrandomized study. No data areavailable regarding status of the bladder neck, sincethe study group did not undergo videourodynamics,but underwent urodynamics and voiding cysto-urethrogram separately.We did not calculate patientglomerular filtration rate, which could partly affectthe results. It is also noteworthy that combinationof urine marker cutoffs allowed us to better definethe subset of patients at increased risk who wouldrequire further statistical analysis.

CONCLUSIONSBladder wall thickness and urinary levels of TGF-b1,b-NGF and TIMP-2 are increased in children withrenal scarring and detrusor leak point pressuregreater than 40 cm H2O. To our knowledge thepresent study is the first to evaluate these 3 urinemarkers and bladder wall thickness in children withmyelodysplasia. Clinical use of these markers maydecrease the need for urodynamics and DMSAscintigraphy in the followup of children withmyelodysplasia.

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EDITORIAL COMMENT

The authors present an early experience with follow subtle yet cumulative changes in the urinary

noninvasive biomarker analysis in children withneurogenic bladder. They should be congratulatedfor this effort. For those who routinely care forchildren with neural tube defects it is clear thatbetter translational tools are needed to bridge thegap between standard urological tests and themolecular processes taking place in the bladder. Inthe future urinary tract management in cases ofneurogenic bladder will require more sophisticatedand individualized care. From a clinical andresearch perspective it is plausible that the urinarybiomarkers described could one day be used to

tract much the same way that hemoglobin A1c isused in patients with diabetes. While the use ofurinary biomarkers will not replace quality urody-namic studies and careful clinical followup, nonin-vasive tests will improve care and allow for moretailored clinical care protocols.

Douglass B. ClaytonDepartment of Urology

Division of Pediatric Urology

Vanderbilt University

Nashville, Tennessee