The Effects Of Physiological Intrauterine Hypoxia On The Development Of Mammal Metanephric Kidney In Ex Vivo

Part oneEstablishment of the model of kidney development in ex vivo with two-dimension culture systemObjective:To establish the model of kidney development in ex vivo system via two-dimension （2D） culturing of E13.5 metanephric kidneys.Methods:Metanephric kidneys were microdissected from the embryos of timed pregnant SD rats on day 13.5 of gestation （embryonic days 13.5）. Embryonic kidneys were placed on a 0.4μm pore size transwell（?） membrane, cultured in DMEM/F-12 medium（10%FBS,1% penicillin and 1% streptomycin） at a medium gas interphase and incubated at 37 ℃ with fully humidified 5% CO2. Embryonic kidneys were cultured for 3 to 5 days with medium changed every 1 to 2 days. Inverted microscope was used to monitor kidneys’growth and immunostaining was performed to label kidney ureteric bud and metanephric mesenchyme and pictures were taken with confocal microscope. For kidneys cultured for 5 days, histological analysis was performed to evaluate the structure integrity.Results:Embryonic kidneys grew under culture with ureteric bud branching and size expanding rapidly. With immunostaining we can clearly noticed the ureteric bud branching （E-cadherin labeling） and metanephric mesenchyme transforming into epithelial structures （Wt-1 labeling）. We confirmed histologically that condensation and aggregation of the metanephric mesenchyme at the tips of the ureteric bud were observed, including the formation of immature and mature nephrons and glomeruli.Conclusion:Our kidney culture system was capable of maintaining kidney development in ex vivo for at least 5 days with good structure generation, which was enough for our following research.Part TwoEffects of hypoxic microenvironment on the morphology of developing kidneys in ex vivo modelObjective:To explore the effects of hypoxic microenvironment on the morphology of mammal kidney development in ex vivo model.Methods:Kidney rudiments were microdissected from E13.5 SD rat embryos. All the kidneys were pooled and assigned randomly to control group （normoxia,21%O2） and experimental groups（hypoxia,3%-5% O2） with 8 to 10 in each group. For conventional culture, the rudiments were placed on 0.4μm pore-size polycarbonate filters at the bottom of a well insert in a six well plate and incubated at 5% CO2 at 37℃. The hypoxia micro-environment was created as 1% to 5% oxygen tension in an incubator through injecting N₂. Immunostaining was carried out to label the structure of developing kidney, pictures were taken by a laser confocal microscope and analysis was performed to evaluate the difference between two groups.Results:Kidney development was significantly suppressed under hypoxia condition with decreased ureteric buds as well as developing nephrons compared with that under normoxia condition （P<0.05）.Conclusion:Physiological intrauterine hypoxia as an important microenvironment factor may suppress mammal kidney development in ex vivo model.Part threePossible mechanism of hypoxia-mediated suppression of kidney development in ex vivo modelObjective:To investigate the mechanism of physiological intrauterine hypoxia mediated suppression of mammal kidney development in ex vivo model.Methods:Kidney rudiments were micro-dissected, pooled and assigned randomly to control group （normoxia,21% O₂,n=8～10） versus experimental groups（hypoxia, 5% O2, n=8～10） and cultured for 3 to 5 days as preciously described. The hypoxia micro-environment was created as 5% oxygen tension in an incubator through injecting N2 continuously. Immunostaining was carried out to label the structure of developing kidney and pictures were taken by a laser confocal microscope. TdT-mediated dUTP nick end labeling was used to detect cell apoptosis while EdU （5-Ethynyl-2’-deoxyuridine） was added to the medium for cell proliferation detection during kidney development. At the end of culturing kidneys of both groups were processed and the changes of gene expression were measured by Real Time PCR.Results:The proliferation of embryonic kidney cells was significantly inhibited while the apoptosis reduced in hypoxia groups compared with normoxia groups （P<0.05）. Six2-positive progenitors were better maintained within kidneys cultured with lower O2 tension （P<0.05）. Moreover, genes regulating metanephric mesenchymal to epithelial transformation such as Wnt9b and Wnt4 were down-regulated while genes for metanephric mesenchymal cell maintenance such as Six2, Bmp7, WT-1 and Pax2 were up-regulated under hypoxia condition.Conclusion:Physiological intrauterine hypoxia as an important microenvironment factor may suppress mammal kidney development through inhibiting the proliferation and differentiation of embryonic kidney progenitors while maintain their undifferentiated status （or self-renewal）, thus contributing to the balance between differentiation and maintenance of mesenchymal progenitor cell pool which determines the final number of adult kidney nephrons.