The Mechanism And Function Of Ouabain Rescuing Embryonic Rat Kidney Development During Intrauterine Growth Restriction

Purpose:Intrauterine growth restriction(IUGR)is associated with reduction of nephron number at birth,which increases the risk of hypertension,end-stage renal disease and cardiovascular disease.Low dose of ouabain could rescue the development of embryonic rat kidney during IUGR,partially restore the nephron number.To investigate the molecular mechanism of the rescuing effect and evaluate the nephric function of the offspring treated with ouabain,we conducted the high throughput sequencing technology to analyze the differentially expressed genes influenced by ouabain.Meanwhile,we performed a following-up study on the IUGR offspring treated with or without ouabain to assess the nephric function.Methods:(1)Rat IUGR model was induced by 75%caloric low protein diet during pregnancy,and low dose of ouabain was delivered using mini osmotic pump from gestational day 2.The offspring were received normal diet or 8%high salt diet after weaning.The blood pressure and urinary protein excretion were measured.At the age of 40 weeks,all the offspring were sacrificed.The pathological features of kidney and ultrastructure of glomeruli were studied.(2)Rat IUGR model was induced by 75%caloric low protein diet during pregnancy,and low dose of ouabain was delivered using mini osmotic pump from gestational day 2.At gestational day 16,pregnant rats were sacrificed.Total RNA was extracted from isolated embryonic kidneys,and was subjected to Illumina HiSeq2000 for sequencing analysis.The differentially expressed genes(DEGs)were identified,and the sequencing result was confirmed using quantitative real time PCR.Gene ontology and KEGG biological pathway analysis were conducted to explore the potential pathways.Results:(1)Maternal malnutrition resulted in significant reduction of birth weight.The nephron number of IUGR offspring was significantly decreased,while ouabain could partially restore the nephron number.The systolic and diastolic blood pressure and urinary protein excretion were significantly increased in IUGR group,and even higher in the offspring fed with 8%high salt diet.The results of HE staining showed that more glomeruli of kidney in male offspring of IUGR group presented atrophic or dilated.The electron microscopy reveals apparent alteration of podocyte ultrastructure in IUGR offspring with foot process effacement.While these unfavorable effect did not present in the offspring received ouabain treatment.(2)Ouabain partially restored the maternal body weight gain which was significantly decreased by protein restriction.A total of 322(177 up-regulated,145 down-regulated)DEGs were detected between Control and IUGR group,meanwhile,318 DEGs were found expressed differentially(180 increased,138 decreased)among IUGR and Ouabain group.KEGG pathway analysis indicated that the complement and coagulation cascades and calcium signaling pathway were the common and prominent pathways which were disturbed by maternal undernutrition and reversed by ouabain treatment.Conclusions:The kidney of IUGR offspring treated with ouabain could function well,mainttaining the blood pressure at normal level.IUGR disturbed complement and coagulation cascades and calcjum signaling pathway to impair the development of the embryonic kidney,which may be counterbalanced by low concentration of ouabain.