| Background: The morbidity and mortality of hypertension and kidney diseaseshowed a highly prevalent and increasing tendency in recent decades. It has long beenrecognized that genetic and lifestyle factors play an important role in these diseases.However, epidemiologic studies have demonstrated that the suboptimal intrauterineenvironment can induce fetal development programming, leading to increased risk ofhypertension and kidney disease. Low birth weight (LBW) infers.Infant birth weight lessthan2500grams are known as. Preterm birth (PB) or intrauterine growth retardation(IUGR) often leads to LBW. According to statistics, the incidence of LBW is about7-15%.In1989, a professor, named David Barker, firstly demonstrated that cardiovascular diseasewas associated with birth weight, and the viewpoint was put forth the provocativehypothesis of fetal programming or developmental plasticity of adult disease: LBW is oneof the independent risk factors for cardiovascular disease, and affects adulthood on renaldisease and high blood pressure. LBW infants, caused by PB or IUGR, are oftenaccompanied with kidney and nephron dysplasia, which is a risk factor for kidney diseasein the future. Adequate oxygen is necessary for the fetal growth and hypoxia can affectdevelopment of fetuses. Hypoxia is a common phenomenon in embryonic development.During pregnancy, a number of maternal physiologic or pathologic factors, includingplateau-hypoxia, gestational hypertension, heart failure, anaemia, preeclampsia, pathologicand umbilical cord factors, which induced maternal hypoxia and lead to fetal poorintrauterine fetal growth (PIFG) because of nutrient substance insufficent. Many studiesreported that prenatal hypoxia (PH) not only caused litter size reduction and small bodysize at birth, but also induced fetal programming of abnormality in many important organssuch as kidney. It is unknown how prenatal hypoxia affects the fetal renal development. Alarge amount of research shows that some molecules which are high expression in maturepathological kidney as well as in fetal kidney, and moreover, the changes of cellularphenotype are similar to the inverse process of cellular differentiation from metanephronMSCs to epithelial cell. These results indicate that cellular regeneration after renal damage closely relates to embryonic development of kidney. Therefore, the study on renaldevelopment helps to understand the pathophysiology of renal diseases. Sufficient oxygenis necessary for fetal development, and in utero hypoxia has been demonstrated adverseeffects on fetal development. In the study of possible mechanisms underlying the reducednumber of nephron by insults during pregnancy, previous work demonstrated the roles ofapoptosis which is known as type I programmed cell death. Apoptosis, which is known astype I programmed cell death (PCD), plays an important role in nephrogenesis. Recentstudies showed that apoptosis was adversely involved in renal development. Autophagyand apoptosis play an important role in the development of embryo and the differentiationof organ and tissue. Whether autophagy, type II PCD, also contributes to impairment ofnephrogenesis remains unknown. Furthermore, there is no information on whetherintrauterine hypoxia may affect renal autophagy in the fetal kidney. Thus, the present studyinvestigated whether and how autophagy occurred in the fetal kidney following hypoxia.Autophagy is an evolutionarily conserved pathway which involves in degradation of theaging and damaged organelles or macromolecular proteins and maintains cellarhomeostasis when cells undergoing stress. In autophagy, double-membraneautophagosomes envelop and sequester intracellular components and then fuse withlysosomes to form autolysosomes which degrade their contents to regenerate nutrients.Autophagy often coexist with apoptosis. The relationship between autophagy andapoptosis is complicated but interesting. A groundbreaking research shows that apoptosiscan affect autophagy by BNIP3competeing with Beclin-1for binding to BCL2andthereby increasing the levels of free Beclin-1, which triggers autophagy. Whether and howhypoxia during pregnancy impacts on fetal renal development as well as renal functions isstill unclear. Thus, the present study investigated whether and how autophagy occurred inthe fetal kidney following hypoxia.There are two important signaling pathways in the regulation of autophagy induced byhypoxia. One is HIF-1α/BNIP3/Beclin-1pathway, the other PI3K/Akt/mTOR. The presentstudy examined the key elements in both signaling pathways associated with renaldevelopmental problems. Together, the present study tested the hypothesis that chronichypoxia during pregnancy may adversely affect renal development related to renalautophagy in the rat fetus. In order to clarify the function of autophahy on the fetal kidneywith hypoxia, we employ the glomerular endothelial cells of rats to analyze the harmful orbeneficial effect on cells by active autophagy which was induced by hypoxia. The presentstudy aimed at:1) to determine what fetal pathophysiological changes in vivo in the kidney of term fetal rats following hypoxia; and2) to investigate whether prenatal chronic hypoxiainfluence apoptosis or autophagy in the kidney of the fetus; and3) to explore the possiblemechanisms or pathways of autophagy in the fetal kidney. The data gained provided novelinformation on hypoxia-induced renal developmental risks and the related mechanisms forkidney health problems in fetal origins.Part1The effects of intrauterine hypoxia on morphology, bloodgas indexs, and renal function of fetusObjective: To determine the effects of the intrauterine hypoxia on morphology, bloodgas indexes, and renal function of fetus. Methods: Pregnant rats exposed to hypoxia (O210.5±0.5%) from day4to day21of pregnancy (GD4-21). The fetus (GD21) were studied.Physiological and histopathological examinations (with light microscope and withtransmission electron microscope) of the fetal kidney were evaluated. Food intake andbody weight of the pregnant rats were measured during gestation period; Fetal body weight,heart weight, kidney weight, brain weight, lung weight, liver weight, stem length and taillength were measured at gestation21day; Blood gases and electrolytes were determinedwith a Nova analyzer. Blood samples were centrifuged and serum was used formeasurements of albumin (ALB), uric acid (UA), creatinine (CRE), and urea nitrogen(BUN) using the enzyme colorimetric method. For histological analysis, the slices of fetalkidney were stained with HE, and then analyzed with microscope. The ultrastructure wasobserved under electron microscopy. Results: Hypoxia during gestation briefly influencethe food intake during GD4-7, and during other times there was no influence on the foodintake of pregnant. Compared with the control, body weight gain of the pregnant ratremained unchanged. In hypoxia group, the survival rate of fetal rat decreased, and theincidence of IUGR increased significantly. Except brain, hypoxia significantly decreasedweight of the fetal body, heart, kidney, lung and liver, accompanied with increased BUN.Intrauterine hypoxia reduced fetal stem and tail lengths, also decreased plasma pO2andSO2%level of fetus, others blood gas index (pH、Na+、K+、Osm、pCO2、Hb、Hct、Glu and Lac) did not changed. Intrauterine hypoxia significantly decreased viscera indexessuch as kidney, liver, and lung index, while the brain/body weight ratio apparentlyincreased, and the heart/body weight ratio remained unchanged. There was no difference infetal ALB, UA and CRE between the normoxia and hypoxia groups. Meanwhile, fetal serum BUN was higher in the hypoxia group than that of the control. The notablehistological changes included that the interstitium was much wider, and cells in theinterstitium increased in the hypoxia fetal kidney. In the glomeruli, the Bowman’s spacewas obviously enlarged, while the sizes of the solid parts in the glomeruli were reduced inthe fetuses exposed to hypoxia. Podocyte foot process was flat and decreased in theglomeruli of the hypoxic fetuses. Conclusion: Intrauterine hypoxia adversely affected thedevelopment of fetus and fetal heart, liver, lung, and kidney. Chronic prenatal hypoxia hada negative influence on the fetal renal structure and renal function. Part2The mechanism of autophagy and apoptosis induced byintrauterine hypoxia in fetal rat kidney with IUGRObjective: To study the autophagy and apoptosis induced by intrauterine hypoxia infetal rat kidney with IUGR; To exploit the mechanism of autophagy and apoptosis inducedby intrauterine hypoxia in fetal rat kidney with IUGR. Methods: Pregnant rats exposed tohypoxia (O210.5±0.5%) from day4to day21of pregnancy (GD4-21). The fetus (GD21)with IUGR were studied. Analyzed sFAS and APG5L of the fetal kidneys. Theultrastructure was observed under transmitted electron microscopy (TEM). The proteinswere analyzed by Western-blotting. The TUNEL technique was used to discriminateapoptotic nuclei. Real-time PCR was used to detect the amount of mRNA.Results: Fetal renal BCL-2was decreased accompanied with higher positive TUNELstaining in the hypoxia group. Prenatal hypoxia increased apoptosis related renal APG5Lwithout change of sFAS. Hypoxia also increased autophagic structures, includingautophagosome and autolysosome, in the fetal kidney. There was a significant increase inLC3-II, Beclin-1, p-S6, HIF-1α, ratio of LC3-II/LC3-I, and a decrease in P62, AKT, andp-AKT, while LC3-I unchanged, in the kidney of the hypoxia group. The account of LC3mRNA apparently increase in hypoxia group compared with normoxia group. Conclusion:Intrauterine hypoxia adversely affect renal development in the rat fetus, up-regulated therenal autophagy and the possible mechanism involved may include the Beclin-1pathway.Intrauterine hypoxia also up-regulated apoptosis by inhibiting the BCL-2signaling wayand activating the caspase-3pathway. Part3The impact of hypoxia on autophagy in NRK-52E cellsObjective: To investigate the effects of hypoxia on the growth and cell autophagy inNRK-52E cells; and to investigate the roles of autophagy in cells under hypoxia survival.Methods: Firstly, NRK-52E cells were cultured conventionally, and cell growthtogether with cell counts were observed under an inverted microscope. And then wedrawed growth chart, explored NRK-52E proliferative cells, and found the best time ofNRK-52E cells which drug intervention. Using western-blot assay, we explored the effectsof the concentration of3-MA intervention on the expression levels of marker autophagyproteins, to determine the optimal drug concentration. After finding the optimal timing ofthe best3-MA concentration of drug intervention, the protein levels of LC3, Beclin1andp62wered detected by Western blot, to study the impact of hypoxia on autophagy and itsactivity. Moreover, cell viability and lactate dehydrogenase leakage rate were detected byMTT and LDH assay, to analyze the impact of hypoxia-induced autophagy on cell survival.Using three gas incubators to establish NRK-52E hypoxia model, and using autophagyinhibitor, to study cell growth of NRK-52E cells when autophagy expression wassuppressed. Additionally, we analysised the effects of autophagy on NRK-52E cellsurvival in hypoxic environment.Results: the NRK-52E cells growed exponentially within24hours, and then enteredthe growth plateau after24hours. So that the best time to hypoxia intervention wasabtained. Cell proliferation of NRK-52E cells slowed, cell viability decreased andmortality increased in hypoxic environment. Meanwhile, the rate of cell death alsoincreased with hypoxia time prolonged. The number of cells is still increasing within24h,but the subsequent hypoxia time prolonged, the number of cells gradually declined.Treatment with3-MA could inhibit hypoxia-induced LC3II, Beclin1, activated caspase-3(p20) upregulation and maintained the levels of p62protein expression.Conclusion: Hypoxia could slow NRK-52E cell proliferation and decrease survival rate.Autophagy inhibition may reduce hypoxia-induced cell death (including apoptosis). |
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