Decrease of urinary nerve growth factor levels after antimuscarinic therapy in patients with overactive bladder

Authors

  • Hsin-Tzu Liu,

    1. Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan, and Department of Urology, William Beaumont Hospital, Royal Oak, MI, USA
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  • Michael B. Chancellor,

    1. Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan, and Department of Urology, William Beaumont Hospital, Royal Oak, MI, USA
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  • Hann-Chorng Kuo

    1. Department of Urology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan, and Department of Urology, William Beaumont Hospital, Royal Oak, MI, USA
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Dr Hann-Chorng Kuo, Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan.
e-mail: hck@tzuchi.com.tw

Abstract

OBJECTIVE

To determine urinary nerve growth factor (NGF) levels in patients with overactive bladder (OAB) and after treatment with antimuscarinics.

PATIENTS AND METHODS

Urinary NGF levels were measured in 38 ‘normal’ controls and 70 patients with OAB. Patients were treated with tolterodine 4 mg once daily. Urinary NGF levels were measured by enzyme-linked immunosorbent assay method and normalized by urinary creatinine levels (NGF/Cr). The urinary NGF/Cr levels and urgency severity scale (USS) were compared at baseline, 1, 2 and 3 months after antimuscarinics, and 1 month after discontinuing treatment.

RESULTS

The urinary NGF/Cr level was very low in normal controls with a mean (sem) of 0.005 (0.003). Patients with OAB had significantly higher baseline urinary NGF/Cr levels than the controls. Urinary NGF/Cr levels were significantly reduced at 3 months in 50 responders (1.10 [0.26] before vs 0.41 [0.09] after, P = 0.008) but not in the 20 non-responders (1.38 [0.54] before vs 1.30 [0.46] after, P = 0.879). However, after discontinuing antimuscarinic treatment for 1 month, the urinary NGF/Cr level was elevated in 23 responders at 0.83 (0.33) and in five non-responders at 2.72 (1.41). The USS scores significantly changed with the change of urinary NGF/Cr levels in responders at different time points. The voided volume increased but maximum urinary flow rate and postvoid residual volume did not increase in responders after 3-months of antimuscarinic treatment. The limitation of this study was the lack of a control arm for comparison.

CONCLUSIONS

Changes in the urinary NGF levels were associated with the changes of the USS scores after antimuscarinic treatment and discontinued medication. The urinary NGF level could be a potential biomarker for evaluating therapeutic results of antimuscarinics therapy.

Abbreviations
NGF

nerve growth factor

OAB

overactive bladder

Cr

creatinine

USS

urgency severity scale

DO

detrusor overactivity

Qmax

maximum urinary flow rate

PVR

postvoid residual urine volume

DC-1M

discontinuing antimuscarinics for 1 month.

INTRODUCTION

Overactive bladder (OAB) is a condition of urgency with or without urge incontinence and is usually accompanied by frequency and nocturia. Urgency is the core symptom for the presence of OAB [1]. Currently, antimuscarinics are the most commonly used treatment for OAB. However, OAB is a condition related to bladder filling during the period when the sacral parasympathetic outflow is absent. Control of the urgency sensation or detrusor overactivity (DO) contractility might not occur through motor actions [2,3].

In the human bladder, non-neuronal acetylcholine release has been reported from urothelium during stretching of muscle strips [4]. Muscarinic receptors are present on urothelial cells, suburothelial interstitial cells and on suburothelial nerves [5]. The mechanism responsible for the effectiveness of intravesical treatment of OAB with antimuscarinics has been postulated to occur by blocking of muscarinic receptors in the bladder afferent pathways [6].

Nerve growth factor (NGF) levels in urine were reported to increase in patients with OAB [7,8]. Effective antimuscarinic treatment of OAB might act mainly on the muscarinic receptors in sensory pathways and alter urinary NGF production, which in turn reduces the urgency sensation during bladder filling. If urinary NGF can be shown to be reduced in patients with OAB with symptomatic improvement after antimuscarinic treatment, it would support the existence of a link between NGF production and muscarinic receptor activation in OAB. The urinary NGF level could therefore be used as an objective tool to assess the therapeutic outcome of antimuscarinic treatment. The present study was designed to determine urinary NGF levels in patients with OAB and after antimuscarinic treatment.

PATIENTS AND METHODS

Urinary NGF levels were measured in 70 patients who presented with OAB symptoms of urgency and frequency with or without urge incontinence, and in 38 subjects with no LUTS. All patients with OAB were verified by 3-day voiding diary and only patients with a daily frequency of >8 episodes of urgency or urgency incontinence ≥1 per day were diagnosed to have OAB.

All patients were free of neurogenic bladder, BOO, and upper urinary tract diseases such as stone, tumour or UTI at enrolment. These patients had not been treated with any medication for their LUTS. All patients had been investigated by pressure-flow study and confirmed free of BOO in men and stress urinary incontinence in women. Control subjects were recruited from volunteers of the department employees and patients with urogenital diseases such as inguinal hernia, phimosis or lower back pain and free of LUTS.

This study was approved by the Institution Review Board and Ethics Committee of this hospital. Informed consent was obtained from all participants before the collection of urine samples.

All urine samples from ‘normal’ controls and patients with OAB were obtained at full bladder. The patients with OAB received antimuscarinic treatment (tolterodine SR 4 mg once daily) for 3 months that was then discontinued for 1 month. No medication that might affect lower urinary tract function was prescribed concomitantly. If patients OAB symptoms returned to baseline levels after discontinuing medication, recurrence of OAB was considered. All patients were instructed to take medication as prescribed and visit at scheduled time-points. Urine samples were collected before treatment, at 1, 2 and 3 months after starting antimuscarinic treatment, and at 1 month after discontinuing following the 3-month treatment period (DC-1 month).

The urgency severity scale (USS) was measured using a modification of the Indevus Urgency Severity Scale [9], where urgency severity was rated by circling 0, 1, 2, or 3, which were defined as none, mild, moderate and severe urgency, respectively. In addition to scores of 0–3, we defined urgency with urinary incontinence as a score of 4. Uroflowmetry was performed at each visit. The voided volume, maximum urinary flow rate (Qmax) were recorded and postvoid residual urine volume (PVR) was measured by transabdominal ultrasonography. The total bladder capacity was calculated from voided volume plus PVR.

The presence of OAB was defined according to the recommendations of the ICS as the presence of urgency with or without urge incontinence as the core symptom [10]. Patients with successful treatment of OAB (responders) were free of urgency or urge incontinence, or had a ≥2 point reduction of USS score after treatment. Patients with no improvement of the USS score or with a 1-point reduction in the USS score were considered to have failed treatment (non-responder). The USS score was recorded at each visit and the results were compared between responders and non-responders at different treatment stages.

Urinary NGF levels were measured by the ELISA method. Voided urine was put on ice immediately and centrifuged at 3000g for 10 min at 4 °C. The supernatant was separated into aliquots in 1.5 mL tubes and preserved in a −80 °C freezer. At the same time, 3 mL urine was taken to measure urinary creatinine (Cr) level.

Urinary NGF concentration was determined using the Emax® ImmunoAssay System (Promega, Madison, WI, USA) with a specific and highly sensitive ELISA kit, which had a minimum sensitivity of 7.8 pg/mL. Assays were performed according to the manufacturer’s instructions. The detailed procedure had been described in a previous report [7]. Generally, urine samples were not diluted in the ELISA assay. When the urinary NGF concentration was higher than the upper detection limit (250 pg/mL) the urine samples were diluted to fit the detection limit. For urine samples with NGF concentrations lower than the detectable limit but above zero, a concentration method was used by using a column-protein concentrate kit (Amicon Ultra-15, Millipore, USA) to measure the NGF value. All samples were run in triplicate, urinary NGF levels with no consistent value in three measures were repeated and the values were averaged. The total urinary NGF levels were further normalized by the concentration of urinary Cr (NGF/Cr level).

Data of urinary NGF levels were compared between controls and patients with OAB at different treatment stages. The differences in urinary NGF levels, USS scores and urine flow variables were also compared between responders and non-responders. The paired t-test was used for intra-group statistical analysis. A repeated measures analysis was used for statistical analysis of patients with OAB who had repeated measurements of NGF levels at baseline, 1, 2 and 3 months. A P < 0.05 was considered to indicate statistical significance.

RESULTS

There were 48 men and 22 women in the OAB group with a mean (sd, range) age of 68 (13, 37–88) years and seven men and 31 women in the control group with a mean (sd, range) age of 41 (15, 21–79) years. At 3 months of treatment 50 (71.4%) patients were responders and 20 (28.6%) were non-responders. There were no significant differences in gender distribution and mean age between responders and non-responders.

There was no significant difference in NGF/Cr levels between the age group and gender in the controls. The baseline urinary NGF/Cr levels in patients with untreated OAB were significantly higher than in controls in both responders and non-responders (Table 1). The baseline NGF/Cr levels were not significantly different between responders and non-responders (P = 0.613), however, 14 (28%) responders and seven (35%) non-responders had zero baseline NGF levels.

Table 1.  The levels of urinary NGF in the controls and patients with OAB at baseline and after antimuscarinics treatment
Bladder dysfunctionNo. of urinesamplesMean (sem) urinary: P
total NGF, pg/mLNGF/Cr
  • *

    NGF/Cr level controls vs different OAB conditions,

  • NGF/Cr level baseline vs 3 months,

  • NGF/Cr level 3 months vs DC 1 month.

Normal control38  0.46 (0.35)0.005 (0.003) 
OAB-responder
 Baseline50 33.3 (6.13)1.10 (0.26)<0.001*
 3 months50 17.8 (4.31)0.41 (0.09)  0.008
 DC-1 month23 36.3 (14.0)0.83 (0.33)  0.181
OAB-non-responder
 Baseline20 56.9 (19.4)1.38 (0.54)<0.001*
 3 months20 70.57 (25.0)1.30 (0.46)  0.879
 DC-1 month 5131.3 (77.5)2.72 (1.41)  0.105

After 3 months of treatment, urinary NGF/Cr levels were significantly decreased in responders. The NGF/Cr levels remained elevated similar to their baseline levels in non-responders after 3 months of treatment. In all, 23 responders and five non-responders discontinued treatment at 3 months. Among the 23 responders at 3 months, 13 (56%) had a sustained response but 10 (44%) had recurrence of OAB symptoms at DC-1 month. Among the five non-responders at 3 months, one had symptom improvement but four had continued poor response at DC-1 month. The NGF/Cr levels elevated at DC-1 month in responders and non-responders were not significantly different compared with the 3-month data.

When analysing the urinary NGF levels according to USS scores in both responders and non-responders, there were significantly higher NGF levels in patients with USS scores of 4 and 3 compared with those in patients with a USS score of 2 or 1 at baseline and at 3 months (P < 0.001). However, there was no difference in NGF levels between patients with a USS score of 3 and 4 (P = 0.852) and between a USS score of 2 and 1 (P = 0.165) (Table 2).

Table 2.  The mean (sem) urinary NGF levels (pg/mL) in patients with different USS scores at baseline and after antimuscarinic therapy
Patient groupUSS score1
432
Responders (n = 50)
 Baseline1.09 ( 0.33)1.12 (0.45)  
(n = 32)(n = 18)  
 3 months  0.29 (0.09)0.55 (0.16)
  (n = 26)(n = 24)
Non-responders (n = 20)    
 Baseline1.38 (0.54)   
(n = 20)   
 3 months1.23 (1.18)1.33 (0.46)  
(n = 6)(n = 14)  
Overall1.21 (0.28)1.21 (0.31)0.29 (0.09)0.55 (0.16)
(n = 58)(n = 32)(n = 26)(n = 24)

In all, 34 patients completed regular visits, urine collections and other examinations at baseline, 1, 2 and 3 months. Among them 28 were responders and six were non-responders. The differences in urinary NGF/Cr levels between baseline and follow-up time-points are shown in Fig. 1. In responders, the USS scores significantly decreased from baseline to each time-point and within each time point (P < 0.001). However, urinary NGF/Cr levels did not significantly decrease from baseline (mean [sem] 1.46 [0.43]) to 1 month (1.37 [0.67], P = 0.860) or 2 months (1.12 [0.41], P = 0.525), and within 1, 2 and 3 months (0.36 [0.11], P = 0.249), but were significantly decreased from baseline to 3 months (P = 0.010) and rebounded at DC-1 month (0.82 [0.40], P = 0.628). By contrast, there was no significant change in the USS score and NGF/Cr levels at any time-point in non-responders.

Figure 1.

The changes of mean NGF/Cr levels and USS scores in (A) 28 responders and (B) six non-responders at baseline, 1, 2 and 3 months after treatment and at 1 month after discontinuing medication.

Among the 17 responders who discontinued antimuscarinics 11 remained in ‘well-treated’ condition at DC-1 month but the other six had OAB symptoms recurrence. The urinary NGF/Cr level remained low in 11 patients with no OAB symptoms at DC-1 month (0.25 [0.095], P = 0.043); however, the NGF/Cr levels were elevated in the other six with OAB symptoms at DC-1 month (2.87 [0.84], P = 0.28) compared with their baseline levels.

Table 3 shows the differences in the USS scores, total bladder capacity, Qmax and PVR in the patients with OAB at baseline and 3 months after antimuscarinic treatment. The baseline USS scores of responders and non-responders were not significantly different (P = 0.26); however, the reduction of USS scores of responders were significantly greater than those of non-responders (P < 0.001). Bladder capacity was significantly increased at 3 months in responders but not in non-responders. There was no significant difference in Qmax and PVR at 3 months after treatment in both responders and non-responders.

Table 3.  The mean (sd) changes of USS score, bladder capacity, Qmax and PVR in patients with OAB after antimuscarinics treatment
Patient groupModified USS score, 0–4Bladder capacity, mLQmax, mL/sPVR, mL
Responders,n = 50
 Baseline 3.64 (0.86)334.7 (190)17.5 (11.8)68.7 (66.4)
 3 months 1.52 (0.71)404.8 (237.9)19.5 (11.2)77.3 (81.3)
 P<0.001  0.036 0.091 0.436
Non-responders,n = 20    
 Baseline 4.0 (0.0)257.5 (143.9)12.7 (8.1)91.2 (66.1)
 3 months 3.30 (0.11) 271.1 (147.0)13.2 (6.7)92.2 (70.8)
 P 0.001  0.720 0.757 0.958

DISCUSSION

In the present study, urinary NGF levels were significantly higher in patients with OAB than in the controls. Patients with OAB who responded to antimuscarinic treatment had significantly reduced urinary NGF levels in association with a decreased USS score at 3 months, which remained low in those with sustained response after discontinuing medication for 1 month. By contrast, there was an elevated urinary NGF level in patients with failed antimuscarinic treatment and in those who had recurrent OAB symptoms at DC-1 month. These results suggest that urinary NGF level could be a potential biomarker for assessing the therapeutic effects of treatment.

NGF is produced from the urothelium and bladder muscles. Urethral obstruction in rats produced increased bladder weight and significantly more NGF, which were only partially reversed after relief of obstruction [11]. Patients with idiopathic DO, neurogenic bladder or inflammatory bladder diseases such as in interstitial cystitis were reported to have increased urinary and bladder NGF levels [12,13]. In the diagnosis of patients with urgency frequency symptoms, determination of the NGF level in a urine sample may allow a more objective diagnosis compared with that based on the subjective symptoms alone [8,14].

Although the NGF in bladder tissue was reported to not be significantly associated with patients with idiopathic DO [15], patients with neurogenic DO who were successfully treated with detrusor botulinum toxin-type A injections had reduced NGF bladder tissue levels as well as decreased levels of sensory receptors P2X3 and TRPV1 [16,17]. These data suggest that NGF is associated with activation of sensory receptors that lead to OAB symptoms.

Recently, the effect of antimuscarinics on OAB has been considered to occur mainly via blocking of muscarinic receptors in bladder sensory pathways [18]. Detrusor contractility is not affected when antimuscarinic treatment is given at the dose recommended for treatment of OAB [19]. Therefore, antimuscarinics can be used to treat urgency during the storage phase without reducing detrusor pressure or urine flow during the voiding phase. The results of the present study also confirm this effect as well as the association between the reduction of USS score and the reduction in urinary NGF levels in successfully treated patients with OAB.

Interestingly, 30% of the patients with OAB in the present study had a zero urinary NGF level at baseline. The reason for this finding remains unclear but could be due to different causes of OAB. Another interesting point is that patients who responded to antimuscarinic treatment had urinary NGF/Cr levels after 3 months of treatment and at DC-1 month significantly higher than those of the controls despite their USS scores being decreased significantly from baseline. This result might be due to incomplete resolution of the underlying pathophysiology for OAB after 3-months antimuscarinic therapy. Furthermore, because muscarinic receptor over-expression is not the sole cause of OAB, some bladder aetiologies responsible for OAB that do not involve the muscarinic receptor pathways might not be affected by antimuscarinic therapy. Measurement of other sensory proteins in the urine or comparison of urinary NGF levels over longer therapeutic durations may clarify these questions.

Previous studies have shown that urinary NGF is a sensitive biomarker for the diagnosis of OAB [7,8,14]. It is possible that NGF is taken up by sensory nerves and transported through the CNS in a retrograde fashion. Therefore, NGF production could be a biomarker for neuroplasticity via some common pathway involved in the pathogenesis of OAB [20]. The present study further showed that urinary NGF levels decreased in association with the reduction of urgency severity and increased when OAB symptoms recurred. Interestingly, there was a lag response time between changes in the USS score and NGF in responders. The mechanism for this difference could be due to a subjective report of the USS and a time lag of NGF production decreases after antimuscarinic treatment. Patients with improved USS scores might still have incompletely solved underlying pathophysiology of OAB. After antimuscarinic treatment for 3 months, the USS scores had not decreased to zero and urinary NGF levels also remained significantly higher than those of controls. The elevated urinary NGF level might imply the existence of a residual inflammation in the CNS.

Analysis of bladder capacity and urinary flow variables at baseline and after antimuscarinic therapy in the present study showed that treatment with tolterodine for 3 months did not alter voiding efficiency in patients with OAB. Improvement of the USS scores after antimuscarinic treatment was highly associated with an increase in bladder capacity in responders but not in non-responders, suggesting the increase of bladder capacity and reduction of the USS scores after treatment was caused by the desensitization of sensory nerves. This result implies that the mechanism responsible for a successful outcome of antimuscarinic therapy in OAB involves the sensory pathway.

The limitation of the present study was the absence of a control arm of patients with OAB given placebo. Because the present study aimed at investigating the changes of urinary NGF levels after antimuscarinics, we did not include the control arm for study. Clinical trials for OAB usually have a 20–30% placebo effect. Whether this placebo effect would decrease urinary NGF levels deserves further investigation, which might reveal different pathophysiologies of OAB.

In conclusion, urinary NGF levels were increased in patients with OAB and these were decreased after successful antimuscarinic treatment for 3 months in association with a reduction in USS scores. The urinary NGF level could be a potential biomarker for evaluating therapeutic results of antimuscarinic treatment.

CONFLICT OF INTEREST

None declared.

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