Effects Of UT-B Gene Knockout On Olfactory Function In Mice

Urea transporter B is a membrane channel protein,which can mediate urea transmembrane transport rapidly. UT-B gene knockout induced urinary concentration dysfunction in kidney of mice, in which the level of urea increased in serum and cerebrospinal fluid. Previous studies found that UT-B m RNA expressed in olfactory system of human and mice, UT-B knockout lead to olfactory dysfunction in mice. Since olfactory bulb has an important role in smell transmission, involved in the pathogenesis of various olfactory dysfunction, this study was to detect the olfactory function of UT-B knockout mice, observe the differences of their olfactory bulb morphology compared with wild-type mice, in order to clear the relevance of UT-B dificiencyt with mouse olfactory dysfunction, study on the mitochondrial function, oxidative stress and apoptosis to explore the pathogenesis of olfactory disorders in UT-B knockout mice, which may offer new clues for prevention and treatment of olfactory disorder.Results and discussion:1. The expression of UT-B gene in olfactory mucosa of human and miceRT-PCR results show that UT-B m RNA is only highly expressed in the olfactory region of human olfactory mucosa, not expressed in other nasal tissues, suggesting that UT-B protein may have important role for olfactory cell physiological function. Further to detect nasal mucosa and olfactorybulb in mice, we found that UT-B m RNA is also highly expressed in olfactory bulb and olfactory mucosa in mice. UT-B m RNA is lack in olfactory bulb and olfactory mucosa in UT-B gene knockout mice.2. Olfactory dysfunction in UT-B knockout mice20-week-old mice were used for buried food pellet trials, in order to detect the olfactory function. We found that wild-type mice can found buried food within 100 seconds, time of UT-B knockout mice in searching of food was significantly longer than wild-type mice(p <0.01), revealed that olfactory function of UT-B knockout mice decreased markedly.3. Olfactory epithelium thinning and reduction of OMP in UT-B knockout miceHE staining of olfactory mucosa in UT-B knockout mice, showed that thickness of mucosal surface to the base layer were markedly thiner and number of cell layers of 20-week lower than wild-type mice.Immunohistochemistry detected the express of functional olfactory neurons labeled olfactory marker protein(olfactory marker protein, OMP), found that olfactory epithelium OMP(+) cells significantly reduced in UT-B knockout mice compared with wild-type mice(p <0.05).4. Small olfactory bulb and reduced olfactory neurons and OMP-positive cells in UT-B knockout miceGenera lanatomy results showed that, olfactory bulb volume and weight in UT-B knockout mice were significantly lower than wild-typemice(p <0.05). HE staining showed that olfactory bulb cell layers remains unchanged, but the number of neurons in the mitral cell layer and subependymal zone also decreased significantly in UT-B knockout mice;immunofluorescence staining and Western blot results also show that, the expression of olfactory marker protein of olfactory bulb in UT-B knockout mice was markedly lower than that of wild-type mice(p <0.05).UT-B knockout lead to significantly reduction of neuronal with function of olfactory bulb.5. Neuronal mitochondrial dysfunction and increased apoptosis of neurons in olfactory bulb of UT-B knockout miceUsing Western blot to detect the expression of apoptosis-related proteins of olfactory bulb in mice, found that apoptosis protein cleaved Caspase-3 in UT-B knockout mice’s olfactory bulb was significantly higher than wild-type mice(p <0.05). The expression of anti-apoptotic protein Bcl-2 in UT-B knockout mice was significantly lower than wild-type mice(p <0.05). Revealed that UT-B gene knockout may induce apoptosis of olfactory bulb neurons, leading to the decrease of olfactory bulb volume and functional neurons, affect the olfactory function.Using Flow cytometry to detect mitochondrial membrane potential(ΔΨm), found that mitochondrial membrane potential of neurons of olfactory bulb cells was significantly decreased(p <0.05), production of ROS increased and the expression of antioxidant enzymes superoxidedismutase SOD2 is significantly lower in UT-B knockout mice, compared with wild-type mice, suggesting that UT-B gene knock-out cause dysfunction of mitochondrial and increased oxidative stress may involved in the pathogenesis of inducing apoptosis and damage of neural cells in olfactory bulb of mice.Conclusion: UT-B knockout lead to mitochondrial dysfunction,elavated oxidative stress and increased levels of apoptosis in olfactory bulb neurons, resulting in decrease of functional olfactory bulb neurons, causing olfactory dysfunction eventually.