Hypothesis / aims of study
Peripheral and central nervous systems control urine storage and voiding by inducing relaxation and contraction of the bladder wall. Neurotrophins are hormones released by cells of the urinary tract and essential in the maintenance and activity of nerves irrigating the bladder. Among them, Nerve Growth Factor (NGF) and Brain-derived neurotrophic factor (BDNF) promotes neuroregeneration by binding receptor TrkA and TrkB respectively while their precursor proNGF and proBDNF trigger inflammation and apoptosis through receptor p75NTR. Imbalance in the urinary ratio of mature to pro-neurotrophins was found to be a viable biomarker for female overactive bladder syndrome (OAB). This state was shown to promote an inflammatory profile in the bladder tissue itself. On the other hand, rodent models of diabetes (type 1 and 2) are characterized by voiding dysfunction and present a similar neurotrophin imbalance. Chronic treatment of these mice with THX-B, a p75NTR antagonist, improves bladder parameters through reduction of matrix metalloproteinase-9 (MMP-9), the major protease involved in neurotrophin proteolysis. In the present study, we used a Crispr-Cas9 plasmid in vivo to target MMP-9 gene specifically in the urothelium, in order to improve bladder contraction in a murine model of moderate insulin resistance, the TallyHo mice.
Study design, materials and methods
A sgDNA sequence targeting MMP-9 gene was inserted in Crispr-Cas9 plasmids. Empty plasmids (sham) were synthesized in parallel. Bladders of female Tally Ho mice (age 5 months) in prediabetic state (glycemia between 7 and 14 mM) were transfected by electroporation after vesical insertion of 30 ng of plasmid. Voiding spot assays were performed weekly to measure urine volume, spot number and volume/spot. Bladders were taken and urothelium scraped for analysis of NGF and proNGF by ELISA, and MMP-9 and markers of nerve endings,Vacht and pgp9.5 by semi-quantitative immunoblotting. Bladder contraction was measured by assessing muscle tension in organ baths. Histology was carried out to observe bladder wall morphology. T-test and ANOVA were used for statistics.
Results
Two weeks after transfection, glycemia, body weight and ratio bladder mass/body weight were similar between groups (respectively 11.1 ± 2.1 vs 9.7 ± 1.2 mM, 37.8 ± 3.0 vs 40.6 ± 2.2 g, 0.533 ± 0.077 vs 0.437 ± 0.046). Looking at VSA, urine volume increased in the sham group by 195% while it decreased by 39% with MMP-9 knockdown (P<0.01). Number of spots were increased in sham group (+27%) and decreased in Crispr group (-21%) (P<0.05). Volume/spot were not statistically different between groups. Immunoblotting showed that MMP-9 urothelial content was decreased by 63% (P<0.001) after transfection, which leads to an increase in NGF (22.1 ± 4.2 vs 42.4 ± 5.9 pg/mg protein, compared to sham, P<0.01). ProNGF was unchanged (16.1 ± 2.2 vs 12.3 ± 2.4 ng/mg protein) and the ratio NGF/proNGF increased (0.0018 ± 0.0004 vs 0.0045 ± 0.0006 mol/mol, P<0.01). Regarding markers of nerve endings, Vacht expression was decreased by 55% while pgp9.5 was unaffected. Contractions of bladder strips elicited by KCl (120 mM), EFS (1 to 32 Hz) and carbachol (3 nM to 100 microM) were decreased respectively as follows: 179 ± 21.4 vs 101 ± 10.9, 275 ± 39 vs 86 ± 13 and 380 ±75 vs 234 ± 35 g/g tissue. NGF plasma levels were unaffected by the transfection. Finally, histologic examination of tissues did not reveal signs of inflammation or changes in bladder wall structure and thickness.
Interpretation of results
MMP-9 is an enzyme involved in the maturation and proteolysis of neurotrophins. Enhanced activity of this protease is a common characteristic of OAB patients and animal models of diabetic bladder dysfunction, at least in females. Our data suggest a direct link between MMP-9 and the contraction of the bladder muscles, possibly through control of neurogenesis or activity of the peripheral nervous system surrounding the bladder muscle.