Identification of novel non-invasive biomarkers of urinary chronic pelvic pain syndrome: findings from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network.

The idiopathic nature of Urological Chronic Pelvic Pain Syndrome (UCPPS), which encompasses interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), has prompted an intense search for clinical biomarkers. Identification of clinically relevant, validated biologic markers of UCPPS has great potential to inform our understanding of UCPPS pathophysiology, development, and progression, and ultimately clinical management. To date, the cause of this syndrome is unknown and there is no ability to determine who is at risk of progression or who will respond to specific therapy. Accurate noninvasive tests, based on objective, specific, and definable levels of validated biomarkers, are essential to improve and standardized clinical guidelines and for more targeted management strategies based on patient profiles. The MAPP Research Network was established by the NIDDK of the NIH and represents a comprehensive approach to the study of UCPPS 1, 2. A major goal of this initiative is to better understand pathophysiology and identify potential therapeutic targets. In this MAPP Network study, we have utilized a biologically-driven, candidate biomarker discovery strategy, grounded in the basic biochemistry and physiology of this syndrome, to evaluate candidate non-invasive biomarkers of UCPPS. Reviews of the literature have revealed a number of candidate protein biomarker targets. Increased VEGF levels have been detected in bladder biopsy samples from patients with IC compared to controls, with levels correlating with pain severity 3, 4. Prior studies also suggest an important connection between neovascularization and IC, in that new blood vessels in biopsy samples of IC patients showed significantly lower levels of pericyte coverage of the nascent endothelium, evidence consistent with the known association between high levels of VEGF and immature vessel formation 3. Moreover, VEGF originally discovered as VPF (Vascular Permeability Factor), 5, 6 is the most potent endogenous vascular permeability factor and may mediate the dysregulated vascular permeability postulated to be important in UCPPS 7–9. Given these associations, we chose to include VEGF and its receptor VEGFR1, as biomarker candidates. Inflammation has been suggested to be an underlying pathophysiologic mechanism in UCPPS 10 and increased concentrations of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8) have been reported in patients with UCPPS as compared with controls 11, 12. In this respect, MMPs are of particular interest. Originally thought to be associated exclusively with tissue remodeling and destruction, it is now widely appreciated that MMPs play a role in a variety of biologic processes including cytokine and growth factor release, tumor growth and progression, angiogenesis and a number of inflammatory conditions 13–17. During the inflammatory response, MMPs and their complexes are released from connective tissue cells in response to pro-inflammatory cytokines. Upregulation of MMP activity results in the recruitment of pro-inflammatory cells to the site of tissue injury. MMPs have also been implicated in the regulation of the immune response because of their ability to cleave inflammatory mediators and stimulate the clearance of inflammatory cells 18–21. Given that MMPs may play an important role in UCPPS as a function of the inflammatory phenotype, abnormal vasculature and the dysregulation of ECM (extracellular matrix) turnover present in this and other potentially related diseases we included these proteins in our study. NGAL, also known as Lipocalin-2 (Lcn2), is upregulated in numerous chronic and acute inflammatory conditions, including chronic kidney disease, ulcerative colitis and myocardial infarction 22, 23. There exists an intricate interplay between NGAL and other inflammatory cytokines as well, such that NGAL upregulation can be triggered by multiple inflammatory cytokines, including TNF-α, IL-1β and IL-17 22, 23. Moreover, NGAL itself is a modulator of the levels of other inflammatory cytokines, as well as of the behavior of inflammatory leukocytes 24–26. In our laboratory, we have demonstrated that NGAL is a significant stimulator of VEGF levels in breast tumor cells and can also independently induce the epithelial to mesenchymal transition in breast cancer, as well as being a noninvasive biomarker of this disease 27–29. NGAL can also form a complex with MMP-9, representing a distinct protein complex referred to as MMP-9/NGAL complex. We have previously shown that when NGAL complexes with MMP-9, it protects MMP-9 from degradation preserving its enzyme activity 16, 30. The MMP-9/NGAL complex has also been detected in urine samples from patients with various diseases. Based on these findings, we chose to measure NGAL as well as MMP-9/NGAL complex in the urine of individuals with UCPPS and correlate them with clinical symptoms. Here, we report sex-specific comparisons of candidate biomarkers among UCPPS and control cohorts to gain new insights into underlying pathophysiology. In addition, associations of biomarkers with symptom severity within the UCPPS cohort are examined. We anticipate that the identified biological markers may serve as potential candidates for further clinical evaluation, including in the development of new treatment strategies directed toward novel targets, as objective measures for patient classification schemes, and evaluation of clinical outcomes, as well as further characterization of underlying mechanism.