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Keywords:

  • male;
  • nerve growth factor;
  • prostaglandins;
  • urine;
  • urinary tract

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Abstract  Aim:  Nerve growth factor (NGF) and prostaglandins (PG) in the urinary bladder can be affected by pathology of bladder, and this change can be noted in the urine. This study was performed to investigate the changes in urinary NGF and PG in male patient with overactive bladder (OAB) symptoms.

Methods:  The study group included 75 male patients with OAB symptoms and 20 males without bladder symptoms as controls. Evaluation included history-taking, urinalysis, International Prostate Symptom Score (IPSS) and urodynamic study. The NGF, PGE2, PGF and PGI2 levels in voided urine were analyzed by enzyme linked immunosorbent assay and these results were compared in control and OAB patients. Also, the urinary levels of NGF and PG were correlated with IPSS score and urodynamic parameters in OAB patients.

Results:  The urinary levels of NGF and PGE2 were significantly increased in patients with OAB compared with control (P < 0.05). The urodynamic study in OAB patients showed that more than half of the patients had detrusor overactivity and bladder outlet obstruction. The incidence of detrusor underactivity was noted in seven patients in the OAB group. The urinary level of PGE2 was decreased in patients with detrusor underactivity compared with patients without detrusor underactivity (P < 0.05), and negatively correlated with maximum bladder capacity in OAB patients (P < 0.05).

Conclusions:  NGF and PG may have important role in male patients with OAB, and the urinary level of PGE2 can change according to detrusor function. Therefore, these results may be used as urinary markers to evaluate the OAB symptoms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Lower urinary tract symptoms (LUTS) are highly prevalent in elderly men, and the prevalence of this condition rises with age. LUTS can be categorized into storage, voiding, and postmicturition symptoms. Although often associated with bladder outlet obstruction (BOO), they can be caused by several factors. Overactive bladder (OAB) is a symptomatic diagnosis based on the presence of urgency, with or without incontinence, and it is usually accompanied by frequency and nocturia. There have been many studies about the overactive bladder, but the cause and mechanism are still poorly understood. OAB symptoms can be affected by neurotransmitter release or inflammatory mediators; nerve growth factor (NGF) and prostaglandins (PG) in particular can contribute to these symptoms.

Nerve growth factor is a secretory protein which plays a critical role in the development of the peripheral nervous system. Increased expression of NGF in the urinary bladder may contribute to irritative (storage) symptoms in patients with LUTS.1,2 PG play an important role in regulating lower urinary tract function.3 PG are produced by cyclooxygenase-2 (COX-2), whose expression in the tissue is promoted by the expansion of detrusor muscle at the time of filling, bladder inflammation, damage to the bladder mucosa, and the stimulation of muscarinic receptors. PG are involved in micturition reflex by decreasing the threshold of stimuli necessary to trigger the bladder contraction through the activation of capsaicin-sensitive afferent nerves.4–6 Therefore, those with irritative bladder symptoms such as urinary frequency and urgency can be characterized by alterations in NGF and PG release. And it is assumed that the alterations of these chemical transmitter levels can eventually be detected in the urine. This study was performed to evaluate the alterations of urinary NGF and PG level in the male patients with lower urinary tract symptoms accompanied by OAB, and to identify the effects of these substances on their symptoms and the possible diagnostic value for evaluating OAB.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patients

A total of 75 OAB male patients with urgency, frequency and/or urge incontinence were evaluated. Patients who had an International Prostate Symptom Score (IPSS) of greater than eight were enrolled to this study. Subjects in the OAB group, with an average age of 62.5 years (range 21–82 years), had not suffered from any neurological disease and showed no abnormality in their urine analysis. The control group included 20 male healthy volunteers with an average age of 56.8 years (range 25–70 years). They showed no bladder symptom (based on IPSS and history) and no abnormality in their urine analysis.

Urine collection

Voided urine was collected, with permission, from all subjects when they felt a full sensation, and the urine samples were centrifuged at 5000 g. for 10 min. The supernatant was separated into aliquots in a 1.5 mL tube and preserved in a −20°C refrigerator.

Patient evaluation

The IPSS voiding symptom (questionnaire number 1, 3, 5, 6) and IPSS storage symptom (questionnaire number 2, 4, 7) scores were evaluated and urodynamic study including uroflowmetry, filling cytometry, and pressure flow study was performed in OAB patients. The pressure flow study was assessed with the Schäfer nomogram, which depends on the linear passive urethral resistance relation. Based on the Schäfer nomogram, obstruction grade II or greater was considered to indicate BOO. The inclusion of detrusor underactivity required weak or very weak contractility on this nomogram. The urodynamic parameters such as maximum flow rate, post void residual, first sensation volume, maximum cystometric capacity, presence of detrusor overactivity and underactivity, and presence of BOO were assessed.

Enzyme linked immunosorbent assay for NGF and PG

The levels of NGF, PGE2, PGF2α and PGI2 in the urine were measured by enzyme-linked immunosorbent assay (ELISA). The Emax ImmunoAssay System (Promega, Madison, WI, USA) was used to assess NGF level in the urine. To coat 96 well plates with the antibody, anti-NGF pAb diluted in carbonate coating buffer (pH 9.7) was pipetted into each plate in a drop of 100 µL per well and incubated overnight at 4°C. The wells were washed once with TBST washing buffer and reacted with 200 µL of 1× Block & Sample buffer for 1 h at room temperature to prevent non-specific reactions. The standard NGF and 100 µL of urine were added to the wells. After 6 h of reaction, the wells were washed five times and incubated overnight at 4°C after 100 µL of the secondary antibody (2.5 µL anti-NGF mAb + 10 mL Block & Sample 1× buffer) was seeded into each well. After washing five times, each well was reacted with 100 µL of antirat IgG HRP-conjugate for 2.5 h at room temperature. The wells were again washed five times and reacted with 100 µL of TMB substrate solution for 10 min at room temperature. To terminate reactions, 100 µL of 1 mol/L hydrochloric acid was added. The amount of NGF was measured using an ELISA reader (SpectraMax 250, Molecular Devices, Sunnyvela, CA, USA). To measure the amount of PGE2, PGF2α and PGI2, the High Sensitivity ELISA kit (R & D systems, Inc., Minneapolis, MN, USA) was used. Total activity (TA), non-specific binding (NSB), maximum binding, and substrate blank wells were separately marked among those antibody-coated wells for comparison. Then, assay buffer (buffered protein base) was added to zero standard (B0; 100 µL) and NSB (150 µL) wells. The standard PG and diluted urine samples were pipetted into the rest of the wells in drops of 100 µL each. 50 µL of HS conjugate was added to each well except for TA and substrate blank wells. Again, 50 µL of HS antibody solution was dispensed into all wells of plates except for NSB, TA and substrate blank wells. The wells were covered with an adhesive strip and reacted. After three washes with 200 µL of washing buffer, all liquid was removed. 5 µL of conjugate was added to TA well and 200 µL of pNPP to each well. 50 µL of stop solution (trisodium phosphate solution) was pipetted into each well and the concentration of PG was measured by using the ELISA reader.

The urinary levels of NGF and PG were compared in control and OAB groups. The voiding and storage symptom scores based on IPSS assessment and urodynamic parameters were also compared in OAB groups.

Statistics

Results were expressed by mean ± SEM. SigmaStat for Windows (Rackware, Golden CO, USA) was used for statistical analysis. Statistical significance was tested by using the Pearson product moment correlation for voiding and storage symptom score, maximum flow rate, post void residual, first sensation volume and maximum cystometric capacity, and the Student's t-test for presence  of  detrusor  overactivity,  detrusor  underactivity, and BOO. A P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

IPSS and urodynamics

IPSS revealed that the voiding and storage symptom scores were 12.5 ± 5.8 and 9.6 ± 3.3, respectively. Based on urodynamic study, the first sensation volume and maximum cystometric capacity were 163.2 ± 82.2 mL and 336.8 ± 125.0 mL, respectively. The presence of detrusor overactivity was detected in 38 subjects (50.7%) and BOO was found in 47 subjects (62.7%). The incidence of detrusor underactivity was noted in seven subjects (9%) in the OAB group (Table 1).

Table 1.  Characteristics of patients with overactive bladder symptom (n = 75)
CharacteristicValue
International Prostate Symptom Score [mean ± SEM]
 Voiding symptom score 12.5 ± 5.8
 Storage symptom score 9.6 ± 3.3
Urodynamic results [mean ± SEM]
 Maximum flow rate (mL/s) 14.7 ± 8.5
 Post void residual (mL) 73.8 ± 107.5
 First sensation volume (mL)163.2 ± 82.2
 Maximum cystometric capacity (mL)336.8 ± 125.0
Urodynaic parameters [no. patients (%)]
 Detrusor overactivity38/75 (50.7%)
 Bladder outlet obstruction47/75 (62.7%)
 Detrusor underactivity 7/75 (9%)

Urinary NGF and PG

The levels of NGF and PGE2 in the urine were significantly increased in subjects in the OAB group compared with the control group (P < 0.05; Table 2). However the levels of PGF and PGI2 in the urine were not significantly different between OAB and control groups (Table 2).

Table 2.  The urinary level of NGF and prostaglandins in control subjects and in patients with overactive bladder (OAB)
GroupNGF (ng/mL)PGE2 (ng/mL)PGF (ng/mL)PGI2 (ng/mL)
  • *

    P < 0.05 as compared with control. NGF, nerve growth factor; PG, prostaglandin.

Control2.03 ± 0.431.15 ± 0.200.99 ± 0.181.83 ± 0.52
OAB patients7.67 ± 7.50*1.91 ± 0.18*1.40 ± 0.221.49 ± 0.18

In the OAB group, the urinary levels of PGE2 revealed 0.44 ± 0.22 ng/mL in patients with detrusor underactivity, which is significantly lower than 2.00 ± 0.18 ng/mL found in patients without detrusor underactivity (P < 0.05) (Fig. 1). Also, the urinary level of PGE2 was correlated negatively with maximum cystometric capacity (P < 0.05) (Fig. 2). However, there was no significant relationship between PGE2 and IPSS values as well as the other urodynamic parameters, including maximum flow rate, post void residual, first sensation volume, presence of detrusor overactivity and BOO. The urinary levels of NGF, PGF2α and PGI2 were not correlated with IPSS values and urodynamic parameters in OAB patients.

image

Figure 1. Urinary PGE2 level in OAB patients with and without detrusor underactivity (DU). Urinary PGE2 level in patients with detrusor underactivity was significantly decreased compared with control. OAB, overactive bladder; DU(–), without detrusor underactivity; DU(+), with detrusor underactivity. *P < 0.05 compared with DU(–).

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image

Figure 2. Correlation between urinary PGE2 level and maximum cystometric capacity in OAB patients. The urinary level of PGE2 was negatively correlated with maximum cystometric capacity in OAB patients. r = −0.321; P < 0.05. OAB, overactive bladder.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Our results clearly demonstrate that the urinary levels of NGF and PG vary in association with OAB. LUTS in male patients can be caused by various factors, and OAB symptoms such as urinary frequency and urgency are partially caused by afferent nerve changes, in which NGF and PG play an important role.

This study confirms a significant increase in urinary NGF levels in patients with OAB. NGF regulates nerve cell growth and survival, and induces hyperalgesia and proliferation of afferent sensory neurons.7 The increased NGF levels seem to lead to irritative bladder symptoms by overly activating afferent neurons in the sensory system. A recent study has reported that NGF levels are increased in the bladder of animals with hyperactive voiding.8 BOO stimulates an increase in bladder NGF, which in turn triggers morphologic and physiological alterations in both afferent and efferent neurons.2,9,10 Although animal-based research on the effects of BOO on bladder functions may not be enough to define the pathophysiological mechanism of clinical LUTS, these results suggest that NGF expression in the bladder is associated with clinical symptoms of prostatic hypertrophy or abnormal bladder functions. Indeed, those with sensory urgency and interstitial cystitis showed increased NGF protein levels in the bladder, and in particular those with sensory urgency had increased NGF expression in the urothelium as shown by immunohistochemical staining.1 Research efforts have recently been stepped up to assess bladder functions or the presence of a bladder lesion through urine analysis. Recent studies have shown that urinary NGF changes according to bladder function after BOO in rat,11 and urinary NGF, tryptase, neurotrophin-3 and glial cell line-derived neurotrophic factor increase in patients with interstitial cystitis and bladder cancer.12 Therefore, it is likely that NGF associates with the activation of afferent neurons, which in turn leads to irritative bladder symptoms. Also, increased NGF levels can eventually be founded in urine, raising the possibility of detecting changes in bladder function using urine analysis for diagnosis.

The current study examined significantly increased urinary PGE2 levels and insignificantly increased urinary PGF level in patients with OAB. PG are usually produced on urothelium or bladder muscle,13–15 and it is known that the PG are produced directly in the tissue by cyclooxygenase (COX) from arachidonic acid. The COX-1 isoform is constitutively expressed in many cell lines, whereas the COX-2 isoform is induced by various physiological stimuli, including stretching of the detrusor muscle, injuries to the vesical mucosa, nerve stimulation, and by agents such as ATP and mediators of inflammation. PG have been suggested to play a physiological role in contributing to the basal tone of the detrusor and modulating activity of bladder nerves.4 PG may affect bladder activity directly by effects on the smooth muscle and/or indirectly via effects on neurotransmission.16 The latter action is most likely mediated by capsaicin-sensitive afferent neurons, because pretreatment with capsaicin or with a tachykinin receptor antagonist blocks this stimulation.4,17 It has previously been suggested that PG play a physiological role in lower urinary tract function. PG released by urinary bladder distension during filling may regulate the threshold for activating the micturition reflex through activation of capsaicin-sensitive bladder afferent nerves.4,17,18 Thus, it is possible that BOO, which is commonly observed in patients with LUTS, and/or other changes within the bladder trigger to increase PG in bladder. These increased PG result in development of lower urinary tract symptoms, especially OAB symptoms such as frequency, urgency, and urge incontinence. In addition, increased PG in the bladder can be reflected in the urine because the PG may diffuse into the urine through the bladder mucosa. That is, alteration in PG level in the bladder can be detected by analysis of urine. In the current study, the urinary PGE2 level increased significantly in patients with OAB. However, urinary PGF2α and PGI2 levels were not changed significantly between OAB and control patients. It has been reported that the generation of PGE2 is significantly higher in rabbit urinary bladder mucosa than PGF and PGI2.19 Generally, the urinary PG level reflects the mucosal PG more than muscular PG because the PG in the mucosal layer can be released easily into the urine. Therefore, it is likely that the urinary levels of PGF and PGI2 were not changed between OAB and control patients and the PGE2 has a more important role in the lower urinary tract function.

The results of this study raise the possibility that the elevated level of urinary NGF and PGE2 could be associated with OAB. However, it is not enough to explain exactly which type of LUTS correspond to the elevations of urinary NGF and PGE2 because all patients have voiding symptoms as well as storage symptoms. Although the NGF and PGE2 is usually related to overactivity, it is possible that the voiding symptoms can be associated with the changes of NGF and PGE2. Therefore, it is necessary for changes of urinary factors to be evaluated in patients with pure storage symptoms in the future.

The current study analyzed the relationship between urodynamic changes and the urinary levels of PG and found that those with detrusor underactivity showed decreased PGE2 level in the urine. Also, the urinary level of PGE2 was correlated negatively with maximum cystometric capacity. These results suggest that the detrusor contraction and bladder capacity is likely to associate with the changes of PGE2 level. The detrusor underactivity can be developed by detrusor decompensation due to prolonged BOO or neural injury. As a result of these findings, it is likely that PG, especially PGE2, levels decreased in the bladder according to loss of stimulants for PG production when the bladder has detrusor underactivity. It has been reported that PG induce slow tonic contractions of detrusor muscle in vitro4–6 and the application of PGE2 and PGF into the bladder results in a decrease in bladder capacity by lowering the threshold of stimuli necessary to trigger the bladder contraction.6,20 Therefore, it is likely that an increase in PGE2 levels is followed by a decrease in the bladder capacity as in the current study.

In summary, the results of the current study showed that the urinary NGF and PGE2 levels increased in patients with OAB and demonstrate the relationship between urodynamic changes and PGE2 levels. Thus, the results underscore the roles of NGF and PGE2 in the bladder in developing OAB symptoms. Also, these factors may be used as non-invasive diagnostic urinary markers for OAB symptoms.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by a Korea Research Foundation Grant (KRF-2002-041-E00179).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References