Hypothesis / aims of study
The urine samples from a cohort of female patients with overactive bladder syndrome (OAB) has displayed low levels of the neurotrophin nerve growth factor NGF and stable levels of its precursor proNGF. The urine samples were also characterized by elevated levels of nitric oxide (NO) which was associated with insulin resistance, a potential causative factor of OAB. The aim of this study is to determine how elevated glycemia might regulate NO levels and in turn, controls NGF and proNGF synthesis and secretion in bladder cells in culture.
Study design, materials and methods
Primary cultures of urothelial (URO) and smooth muscle cells (SMC) were extracted from rat bladders by collagenase digestion. Using ELISA kits, levels of NGF, proNGF and cyclic GMP (cGMP) were measured. NGF,proNGF and MMP-9 mRNA expression was quantified by RT-qPCR. The NO secretion levels were measured using the Greiss reaction. MMP-9 intracellular content was semi-quantified by immunoblotting. Enzymatic kits were used to assess activity of proteases involved in NGF metabolism (i.e. MMP-9, MMP-7, plasmin). Genomic knockout of MMP-9 was done by Crispr-cas9.
Results
Cells were incubated in hyperglycemic conditions which resulted in an increased secretion of NO and decreased levels of NGF in the medium for both UROs and SMCs. Preincubation with L-NAME (1 mM), the NOS inhibitor, prevented the increase of NO in hyperglycemic conditions and partially reversed the decrease in NGF secretion. Treating the cells with the NO generator, sodium nitroprusside (SNP, 300 µM, 24 hours) resulted in decreased NGF secretions and stable proNGF levels, mimicking our observations in hyperglycemic conditions. SNP treatment led to increased levels of cGMP in SMCs but decreased in UROs. Stable permeable analogs of cGMP, 8-(4-Chlorophenylthio)-cGMP (3 mM) and N2,2'-O-Dibutyryl-cGMP (1 mM), and the cGMP synthetase inhibitor, ODQ (100 µM) mimicked the changes observed in cGMP and NGF by SNP in SMCs and UROs, respectively. NGF and MMP-9 mRNA expression levels increased in SNP-treated UROs but remained stable in SMCs. In MMP-9 Crispr-cas9 transfected SMCs, NGF decrease was unaffected. This implies that the change in NGF by SNP is independent of MMP-9. Alternatively, in UROs, the transfection with MMP-9 Crispr-cas9 partially inhibited the effect of SNP on NGF secretion. This implies that MMP-9 is essential in the SNP-mediated changes in NGF in UROs. Finally, the activity of MMP-7 decreased in SNP-treated SMCs, while plasmin levels remained unchanged, supporting the observed decrease of NGF by SNP
Interpretation of results
Clinical data obtained from urine samples were confirmed at the cellular and molecular levels using bladder cells [1]. The decrease in NGF observed in urine samples could be explained by enhanced MMP-9 activity resulting from increased nitric oxide production.
Concluding message
The increase in NO secretion in urothelial and smooth muscle cells is directly linked to hyperglycemic conditions, and this subsequently affects the secretion of NGF. Though the regulation patterns differ between cell types (UROs vs SMCs), the control implicates cGMP, MMP-9, and MMP-7. The obtained results are consistent with our previous clinical observations. This suggests that an increased glycemia, leading to an increase in NO levels, could be part of the pathological process of OAB.