Hypothesis / aims of study
Urine contains components that reflect the patient metabolic and physiologic states. 70% of these biomarkers originate from the urogenital tract and are therefore of prime importance for urology diagnostics. Urinary proteins and miRNA are quite resistant to thaw/freezing cycles and present the advantage of being available in great amounts. Nerve Growth Factor (NGF) is considered a biomarker for OAB, but its precursor (proNGF) has been found to accumulate in metabolic stress conditions due to inhibition of its processing enzymes. In addition, proNGF can cause degenerative changes through its receptor p75NTR. We measured here the ratio of NGF and proNGF in the urine of patients suffering from overactive bladder disease (OAB) in an aging female population and correlated them to disturbances in protein convertase activities.
Study design, materials and methods
After ethical approval and patients' consent obtained, early morning urine samples from 40 female subjects, aged between 50 and 80 years old (20 controls and 20 OAB), were collected and frozen until use. Blood samples were taken in parallel for standard medical care. Blood parameters and osmolality were analyzed at the Montreal Jewish General Hospital as part of a routine. Urine samples were processed using specific ELISA kits for the analysis of NGF and proNGF (Biosensis, Australia), PGE2 (Cayman, USA), and TIMP-1 (Sino Biological inc, USA). Creatinine was measured by the classic picric acid method.
Subjects in the OAB group had a significant higher mean age, (56.3 years ± 5.2 control vs 68.9 ± 11.4 OAB, p<0.001, t-test). Serum analysis showed a higher insulin resistance index (HOMA-IR) in the OAB group when controlled for age [2.13 (95% CI: 1.48-2.8) in control vs 3.13 (95%CI: 2.47-1.770 in OAB patients, p<0.05, ANCOVA) despite the absence of diabetes diagnosis at the time of the study. Urine osmolality and creatinine levels did not show significant differences between control and OAB groups (results not shown). Validation of NGF ELISA kit specificity: Quality of ELISA kits for the measurement of NGF has recently been a matter of debate (1,2). The specificity of NGF Elisa kit assessed by performing a standard curve in the presence or absence of high concentrations of proNGF. The two standard curves were plotted and analyzed by linear correlation, one curve with NGF (from to 3.9 to 250 pg/mL) alone (red dots) and the other with addition of 500 pg/mL of proNGF (blue dots). Plotting of the curves revealed that proNGF, even present in large amounts compared to NGF, does not interfere with the detection of NGF. (Figure 1)
Analysis of the ratio NGF and proNGF in urine samples: Subsequently, levels of NGF and proNGF were measured in urine samples of 40 subjects. Ratio of proNGF/creatinine (ng/mg), of NGF/creatinine (pg/mg), of NGF/proNGF and proNGF/NGF were reported. Plotting of osmolality as a function of creatinine yielded a correlation of 0.79 (Pearson test) and results compared to osmolality compared to creatinine provided similar results (data not shown). Values of NGF and proNGF reported to creatinine did not yield significant differences between control and OAB groups. However, the mean of the ratio of (NGF/proNGF and proNGF/NGF) revealed a significant decrease in NGF at the expense of an increase in proNGF in the OAB group when we consider the relative concentrations of each protein in individual urine samples. (Table 1)
Levels of PGE2 and TIMP-1 in urine: ProNGF is converted to NGF by plasmin/MMP7 (matrix metalloprotease 7) and NGF is hydrolyzed into inactive peptides by MMP9 (matrix metalloprotease 9). PGE2 decreases the activity of metalloproteases, such as MMP7, through its receptors EP1-4. TIMP-1 is an inhibitor of metalloprotease, particularly MMP7, therefore inhibiting the conversion of proNGF to NGF. Analysis of urine revealed that PGE2 and TIMP-1 are increased in subjects suffering from OAB. This might be interpreted as a decrease in the conversion of proNGF which explains the increase in proNGF at the expense of NGF in the urine samples of OAB patients. (Table 2) Furthermore, correlation analysis showed that the urine levels of TIMP-1 was negatively correlated with the NGF/proNGF ratio in the total cohort including control and OAB groups, (r =-0.453, p value =0.003, Pearson correlation)(table 3).
Figure 1: Validation of NGF Elisa kit specificity: The specificity of NGF Elisa kit assessed by performing a standard curve in the presence or absence of high concentrations of proNGF. The two standard curves were plotted and analyzed by linear correlation, one curve with NGF (from to 3.9 to 250 pg/mL) alone (red dots) and the other with addition of 500 pg/mL of proNGF (blue dots). Plotting of the curves revealed that proNGF, even present in large amounts compared to NGF, Does not interfere with the detection of NGF. Both linear equations are parallel and overlapping. Samples were carried out in triplicates.
Interpretation of results
OAB patients with higher glucose intolerance indicators reflects more metabolic stress which can affect enzymatic activity and bioavailability. Other studies have previously reported higher NGF levels in OAB patients. Our findings suggest that the NGF/proNGF ratio presents more clinical interests. The negative correlation found between TIMP-1 and NGF/proNGF ratio reiterates the involvement of these tissue factors in conditions of metabolic stress by disturbing the NGF/proNGF balance. Screening of urine samples might provide insight into the metabolic disturbances affecting patients with OAB, namely disrupted activities of protein convertases and increased concentrations of their inhibitors.