**3.2 Urinary cytokines**

The presence of cytokines in the urine has been addressed in various bladder and kidney disorders, including chronic renal disease, interstitial cystitis and vesicoureteral reflux (Ninan et al., 1999; Abdel-Mageed et al., 2003). It has been suggested that OAB may result from an underlying inflammation of the bladder, a hypothesis supported by recent studies reporting the presence of histological signs of inflammation in biopsies from OAB patients (Comperat et al., 2006; Apostolidis et al., 2008). However, confirming the presence of inflammation through biopsies is certainly an invasive procedure not exempt of morbidities. The detection of signs of inflammation in the urine of OAB patients is a more attractive alternative. Tyagi and co-workers have recently collected urine samples from patients and determined the levels of various cytokines, chemokines, growth factors and soluble receptors (Tyagi et al., 2010). Using a luminometry-based assay, they found a significant increase, when compared with controls, in the concentration of various elements, including the monocyte chemotatic protein-1 (MCP-1), the soluble fraction of the CD40 ligand, the macrophage inflammatory protein (MIP-1β) and interleukins 10, 5 and 12. Another group of researchers also analysed of urine samples from healthy individuals and OAB patients using a proteomic approach (Ghoniem et al., 2011). Interestingly, their results indicate that while the concentration of certain elements increases (such as interleukin 16), the concentration of others decreases (such as interleukin 7). Thus, the actual role of all of these cytokines in OAB is far from being well understood, undermining its utility as biomarkers.

#### **3.3 Neurotrophins**

192 Urinary Incontinence

Clearly, the measurement of BWT or DT by ultrasound faces some drawbacks that are not yet overcome. Intra- and inter-operator variability in ultrasound measurements is probably the most important one. The use of different ultrasound probes, as well as in the resolutions of ultrasound-generated images (Kuo, 2009), is another limiting factor to the use of BWT/DT as a biomarker. Although clinically appealing, more studies are necessary before it

Prostanoids (prostaglandins and thromboxanes) are synthesized by cyclo-oxygenase (COX), present in several tissues including the bladder wall (Khan et al., 1998; Lecci et al., 2000; Azadzoi et al., 2003; Andersson & Wein, 2004). Two COX isoforms exist. COX-1 is expressed constitutively and participates in normal bladder function whereas COX-2 is activated during cystitis (Lecci et al., 2000; Tramontana et al., 2000). Prostanoid synthesis can be induced by physiological stimuli (for example, detrusor muscle stretching), injuries of the mucosa, nerve stimulation, ATP and inflammatory mediators such as bradykinin and the chemotactic peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (Khan et al., 1998;

Prostaglandin (PG) E2 is one the most abundant prostanoid in the bladder (Jeremy et al., 1987; Khan et al., 1998). In rats, intravesical administration of PGE2 facilitates micturition, increases basal intravesical pressure and induces bladder hyperactivity (Ishizuka et al., 1995). In the urethra, topical application of PGE2 causes relaxation of the sphincter (Yokoyama et al., 2007). Likewise, intravesical instillation of an inhibitor of the COX-2 enzyme improved bladder function in an animal model of OAB (Jang et al., 2006). This supports a role for this prostanoid during bladder dysfunction and has justified a few inconclusive clinical trials regarding the use of non-steroidal anti-inflammatory drugs in the treatment of OAB. So, expectations were raised when Kim and co-workers found that levels of PGE2 and PGF2α were significantly higher in male and female OAB patients that in healthy controls (Kim et al., 2005; Kim et al., 2006). However, PG concentrations were not corrected for urine concentration. After correction for creatinine values, Liu and co-workers found no significant differences in PGE2 content in patients with OAB wet, OAB dry and

Thus, at this moment the role of urinary PGs, most notably PGE2, as a putative tool for OAB diagnosis and follow-up is highly debatable. Moreover, the measuring methods rely on labour-intensive and expensive laboratory procedures. In addition, although it is relatively consensual that prostanoids participate in the mechanisms of OAB, it is not known if the

The presence of cytokines in the urine has been addressed in various bladder and kidney disorders, including chronic renal disease, interstitial cystitis and vesicoureteral reflux (Ninan et al., 1999; Abdel-Mageed et al., 2003). It has been suggested that OAB may result from an underlying inflammation of the bladder, a hypothesis supported by recent studies

recommended OAB therapies effectively reduce the PGs levels.

becomes a tool for daily practice.

**3. Urinary biomarkers** 

Andersson & Wein, 2004).

controls (Liu et al., 2010).

**3.2 Urinary cytokines** 

**3.1 Prostaglandins** 

Neurotrophins are tissue-derived trophic factors necessary for the embryonic differentiation, survival and maintenance of neuronal cells both in the peripheral and central nervous system (Pezet & McMahon, 2006). The most well studied neurotrophins are Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF). They exert their effects via their specific tyrosine kinase (Trk) receptors. NGF binds to TrkA while TrkB is the receptor of BDNF. Both TrkA and TrkB are present in the bladder urothelium and sensory afferents innervating the organ (Qiao & M.A. Vizzard, 2002; Murray et al., 2004).

#### **3.3.1 Nerve Growth Factor (NGF)**

NGF has attracted considerable attention in the Urology field. It is accepted that NGF is produced by detrusor muscle cells and by the urothelium (Steers et al., 1991; Steers et al., 1996; Clemow et al., 1997; Clemow et al., 2000; Steers & Tuttle, 2006). In humans and in rodents, the production of NGF in the lower urinary tract and in the neuronal circuits regulating bladder function is increased in pathological conditions, including cystitis and spinal cord injury (Lowe et al., 1997; Vizzard, 2000; Murray et al., 2004). In addition, exogenous NGF is known to induce bladder overactivity, irrespective of the route of delivery (Lamb et al., 2004; Yoshimura et al., 2006; Zvara & Vizzard, 2007). Likewise, manipulation of NGF levels improves bladder function and referred pain in rats with cystitis (Hu et al., 2005; Frias et al., 2009).

Recent studies have demonstrated the presence of NGF in the urine of OAB patients (Kim et al., 2005; Kim et al., 2006; Liu & Kuo, 2008; Liu et al., 2009a, b; Liu et al., 2009c; Jacobs et al., 2010; Liu et al., 2011a). Levels were significantly higher than in healthy individuals and subsided after successful treatment with antimuscarinics (Liu et al., 2009b) or botulinum toxin-A (Liu et al., 2009a), in parallel with a decrease in the USS score. Based on these results, some authors have forwarded the use of NGF as presumed biomarker for OAB (Kuo et al., 2010a). Nevertheless, caution should be advised as most studies have not been placebo controlled which may hamper the interpretation of results.

Biomarkers in the Overactive Bladder Syndrome 195

stability of urine samples. Studies show that sequential freeze/thaw cycles may affect the concentration of certain proteins. Schaub and co-workers showed that there were no significant differences in the protein profile between samples analysed before freezing and after 1 to 4 freeze-thaw cycles (Schaub et al., 2004). These results have been confirmed in subsequent studies by non-related groups (Fiedler et al., 2007; Thongboonkerd, 2007). No

CRP is a highly conserved plasma protein. It was identified in the 1930's in the sera of patients in the acute phase of pneumonia by Tillet and co-workers (Tillett & Francis, 1930; Black et al., 2004). Further studies demonstrated that the concentration of CRP in plasma is significantly increased during inflammatory states, a characteristic that has often been used for diagnostic purposes. In what concerns the urinary tract, plasma CRP has been used to monitor the progression of bladder cancer (Hilmy et al., 2006; Gakis et al., 2011a; Gakis et al., 2011b). As far as we are aware, CRP has been addressed in OAB only in a recent pilot study. Chung and co-workers observed higher levels of serum CRP in OAB patients than in controls, particularly in the group of OAB wet patients (Chung et al., 2011). Both urinary CRP and the amount of CRP mRNA present in the bladder wall were very low, indicating that the serum is the body fluid of choice to measure this protein. However, one should be aware that serum CRP would most likely reflect the presence of any inflammatory condition

data is available regarding the proteomic analysis of the urine of OAB patients.

(Black et al., 2004), making its use as a putative biomarker in OAB is very modest.

Like in urine, the presence of NGF in the serum of OAB patients has also been investigated (Liu et al., 2011b). The authors found a positive correlation between urinary and serum NGF contents. Interestingly, serum NGF remained elevated in OAB patients not responding to antimuscarinic treatment, suggesting that increased circulating NGF may be a factor in refractory OAB. This is, however, the only available study regarding serum NGF levels in OAB. It is presently not clear if the high content of NGF in the serum was strictly associated with OAB or dependent on associated comorbidities (hypertension, diabetes, coronary arterial disease, etc.) (Brown et al., 2000). More studies are needed in order to fully

Clinicians and researchers are still far from having a tool to efficiently detect and monitor OAB. Several attempts have been made to identify specific bladder parameters, serum and urinary proteins that could fulfil such purpose. In all cases, researchers have come across issues. Due to several problems, the real clinical relevance of each proposed biomarker is still very much unclear, most of the publications reflecting more the willingness of the investigators rather than the true scientific value of findings. More studies are clearly necessary. In the near future, it is likely that some of these issues are overcome, as researchers are increasingly aware of the need to standardize methodologies and are already proposing basic protocols that could be easily adopted by most laboratories. Multicentre

**5. Serum biomarkers** 

**5.2 Serum NGF** 

**6. Conclusion** 

understand the relevance of serum NGF in OAB.

**5.1 C- reactive protein (CRP)** 

#### **3.3.2 Brain Derived Neurotrophic Factor (BDNF)**

The presence and role of BDNF in the bladder has been scarcely analysed and available results mostly refer to rodent models of bladder dysfunction. Like NGF, BDNF can be synthesized by bladder cells, most notably the urothelium during cystitis (Pinto et al., 2010a) or spinal cord injury (Vizzard, 2000). The expression of TrkB is also abundant in sensory neurons innervating the bladder wall (Qiao & Vizzard, 2002; Murray et al., 2004). Like in somatic tissue (Kerr et al., 1999; Thompson et al., 1999), BDNF expression in the bladder seems to be under the control of NGF (Schnegelsberg et al., 2010; Girard et al., 2011). BDNF appears to be a key participant in bladder dysfunction in an animal model of cystitis as its sequestration improved both bladder reflex activity and peripheral hypersensitivity (Frias et al., 2009; Pinto et al., 2010a).

In humans, it has been reported that urinary BDNF is elevated in patients suffering from bladder pain syndrome/interstitial cystitis (Pinto et al., 2010b). In OAB, a recent study demonstrated that urinary BDNF was also elevated and significantly decreased after therapeutic intervention (Antunes-Lopes et al., 2011). In addition, the concentration of urinary BDNF was shown to be decreased to normal values after successful OAB treatment (Antunes-Lopes et al., unpublished observations). This may indicate that, like NGF (Kuo et al., 2010a), urinary BDNF may serve as an OAB biomarker. However, further studies are necessary to fully understand the importance of BDNF in OAB, particularly how it can influence the OAB outcome.
