Urine storage and bladder voiding are under control of the peripheral and central nervous systems. Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), controls neuroregeneration while their respective precursor proNGF and proBDNF trigger inflammatory processes and apoptosis. The ratio between neurotrophins to proneurotrophins must therefore be tighly controlled. Several studies have demonstrated that these hormones constitute viable biomarkers for overactive bladder syndrome (OAB). On the other hand, natriuretic peptide A (ANP) and B (BNP) are linked to bladder voiding. We here examine how ANP and BNP might be involved in the secretion of neurotrophins by cells of the bladder.

Smooth muscle (SMC) and urothelial cells (URO) were grown from rat bladder after collagenase digestion. After confluency, they were incubated for 24 hours with ANP (100 nM) or BNP (1 µM). NGF, proNGF, BDNF, proBDNF and matrix metalloproteinase-9 (MMP-9) were measured using specific kits. Natriuretic receptors NPR1, 2 and 3 mRNA were detected by classic RT-PCR. RT-qPCR was used to quantify mRNAs

We found that both cell types are major source of NGF and proNGF while BDNF and proBDNF were mainly released by SMC. The 3 natriuretic receptors were found expressed in both cells. ANP decreases the ratio NGF/proNGF and increased the BDNF/proBDNF one in SMC, mimicking the observations obtained in urine samples of a female cohort of patients diagnosed with OAB. Interestingly, ANP also increased MMP-9 activity in the same cell culture medium. BNP only decreased NGF secretion. In UROs, no changes in the secretion of neurotrophins or proneurotrophins could be observed neither by ANP or BNP, however, the secretion of active MMP-9 was found potently enhanced by ANP with final levels around 20 times higher than the ones found in SMC medium. Levels of mRNA for NGF, BDNF and MMP-9 were unchanged by neurotrophins, which was confirmed by the absence of NFkB translocation into the nuclei. On the other hand, ANP and BNP elicited a rise in cyclic GMP and did not affect cyclic AMP, in both cell types, suggesting other intracellular pathways explaining the differences between each hormone.Finally, using CrisprCas9 to knockdown MMP-9 gene, we confirmed the essential role of this enzyme in the proteolysis of NGF into peptides and in the conversion of proBDNF to BDNF.

We found increased levels of ANP, similar concentrations of BNP and decreased levels of NGF and proBDNF in the urine from a cohort of female patients with overactive bladder. Nitric oxide, also increased during OAB, that also increases cyclic GMP, could only partially explained these data. Our data suggest that ANP coordinates the secretion of neurotrophins together with nitric oxide.

Bladder cells exhibit unique pattern of neurotrophin secretions. Both are affected by ANP, SMC mostly at the level of neurotrophin secretions and urothelial cells in the amount of MMP-9 released in the extracellular medium. We suggest that the imbalance in neutrophin ratios observed in the urine of patients with OAB could result, at least in part, from a direct action of ANP on bladder cells.

Continence 7S1 (2023) 100809
DOI: 10.1016/j.cont.2023.100809