The Role Of TRPC6 In Overactive Bladder

Objective:OAB is a common urodynamic dysfunction that is characterized by symptoms of urgency,frequency,and nocturia.The BOO is one of the main causes of OAB.Urodynamic assessments typically show detrusor instability in patients with OAB.Because a variety of receptors and ion channels play pivotal roles in regulating both contraction and relaxation of bladder smooth muscle tissue,and the immunosuppressant agent FK506?Tacrolimus?,have also been found to improve some OAB voiding symptoms?i.e.,reductions inurinary frequency and increases in the inter contraction interval?in both clinical cases and animal models.Whether the FK506 act through TRPC6 channel during this process,however,remains largely unknown.So,we want to prove that whether the increased expression of TRPC6 may be a potential cause of OAB,and downregulation of TRPC6 expression is a critical molecular event during FK506 treatment for OAB.Methods:1.FK506 was injected intraperitoneally into rats in which OAB was induced via BOO,and urodynamic indices and muscle contraction test were recorded.2.HE staining was used to observe the structure characteristics of bladder smooth muscle.3.Transmission electron microscope was used to observe the ultrastructural characteristics of bladder smooth muscle.4.The location and expression of TRPC6 in bladder smooth muscle were examined by IHC staining,IF staining,western blot,RT-PCR and q-PCR.5.Cell growth was determined by haemocytometer cell counting method,and cell cycle analysis was determined by PI staining.6.The contractility of BSMCs and TRPC6 channel function were examined by collagen gel contraction,whole-cell patch clamp and time-lapse Ca2+imaging.7.Co-IP were performed to test the potential molecular mechanisms of FK506 action on TRPC6 activity.Results:1.The HE staining were found that,compared with normal,the smooth muscle of OAB were hyperplasia,hypertrophy,and arranged disorder,normal muscle bundle structure was less,mesenchyma and collagen fiber was more.The TEM shows,there are a large number of collagen fibers and GJ between the muscle cells,intracellular myofilament arrangement disorder even dissolved,mitochondrial vacuolar degeneration,nuclear week has dilated rough endoplasmic reticulum.2.The IHC and IF were found that,the positive staining of TRPC6 appeared at cell membrane of bladder smooth muscle,as the similar results with IHC and IF,the expression of TRPC6 protein and mRNA in OAB was significantly more than normal.3.The urodynamic test shows,urinary dynamics of OAB rats appeared DI,and the results of ICI,Pmax,PVR and bladder weight in OAB were higher than those in normal rats,but the results of MCC and BC in OAB were lower than normal.The muscle contraction test shows,the bladder smooth muscle of OAB could be induced voluntary contraction by a lower front load.And in the same high front load,the bladder smooth muscle of OAB could produce voluntary contraction with faster frequency and shorter mSTI than those in normal rats.4.The results found that,after used FK506 to block the TRPC6 of OAB,the positive expression rate of TRPC6 in FK506 was lower than OAB,and the expression of TRPC6protein and mRNA in FK506 was less than OAB.Meanwhile,after reduced the expression of TRPC6,the results of involuntary contraction,ICI,PVR and bladder weight were lower than before in OAB rats,but the results of MCC,Pmax and BC were higher than before.The muscle contraction test shows,after reduced the expression of TRPC6,the smooth muscle contraction of OAB in the environment of low,normal and high calcium ion concentration has different degree of improvement.5.PDGF-induced upregulation of TRPC6 was significantly abolished by FK506 in a dose-dependent manner,and qPCR tests showed similar results.IF results also showed that the intensity of the immunostaining for TRPC6 in BSMCs was increased after PDGF treatment,and this effect was blocked by FK506.Cell numbers was significantly increased after PDGF treatment for more than 24h,and FK506 significantly inhibited PDGF-induced increase in cell proliferation.Flow cytometric analysis showed that PDGF treatment for 24h resulted an increase of cells percentage in the S phase with a concomitant decrease in the G0/G1 phase and this effect of PDGF was blocked by FK506.6.OAG activated significantly larger currents in PDGF-treated BSMCs than control,and the OAG-induced currents were inhibited by the TRPC6-specific inhibitor SAR7334.SAR7334 treatment accelerated inactivation of TRPC6 currents,resulting in a shorter time required for the currents to return to the baseline.PDGF treatment increased Ca²⁺influx of BSMCs,and this effect was significantly inhibited by SAR7334.Similar to the TRPC6-specific inhibitor SAR7334,FK506 inhibited Ca²⁺influx with a shorter time to the peak in BSMCs induced by PDGF.Similarly,PDGF enhanced contractility of BSMCs in collagen gel contraction experiments,and this effect was significantly inhibited by both SAR7334 and FK506.7.PDGF treatment increased the nuclear expression of NFATc4and TRPC6 in BSMCs,and this effect was blocked by FK506.Co-IP results showed that TRPC6 directly interacted with FKBP12 in BSMCs.The immunocomplexes were incubated with increasing concentrations of FK506 after immunoprecipitation with anti-TRPC6.FKBP12 was completely associated with the agarose beads in the absence of FK506,and was partially or completely displaced from the beads and appeared in the supernatant in the presence of 0.5 and 10?M FK506.Conclusion:1.Compared with the normal bladder smooth muscle,the structure and ultrastructure of OAB had obvious changes.2.The TRPC6 appeared at cell membrane of bladder smooth muscle,andthe expression of TRPC6 protein and mRNA in OAB was significantly more than normal.3.OAB could obviously affect the bladder function and smooth muscle contraction in rats.4.Reduced the expression of TRPC6 can improve bladder function and contraction of smooth muscle in OAB rats.5.FK506 inhibited the upregulation of TRPC6 expression and activity induced by PDGF in BSMCs,which by inhibiting the NFAT translocation to the nucleus and disrupting the interaction of TRPC6with FKBP12.