| Objective In order to further study the influence of paternal poor nutritional status on the renal function of offspring,especially on F2 offspring,our study examined the effect of feeding male rats before mating with a high-fat-sucrose-salt diet(HFSSD)over two generations(F0 and F1)on the renal function of their second offspring(F2).Methods 1.Establishment of animal model and animal experiments: F0 male SD rats were randomly divided into two groups:(1)control diet group(CD,n=15),feeding with CD for 9 weeks;(2)high-fat-sucrose-salt diet group(HFSSD,n=15),feeding with HFSSD for 9 weeks.Male rats of two groups mated with CD-fed female rats to produce F1 generation.The first stage of F1 offspring: from the pregnant period of female F0 to the 15 th week of F1 offspring,all the rats were fed with control diet.The second stage: from 15 th week to 24 th week of F1 offspring,2 male rats,randomly taken from both CD and HFSSD group,were fed with HFSSD by the second times exposure for 9 weeks.At the same time,2 male rats,randomly taken from the two groups,were fed with CD.At 24 th weeks,the above 8 male rats were mated with 12 CD-fed F1 female rats to produce F2 generation.F2 offspring were divided into 4 groups:(1)F0CD+ F1 CD group: F2 offspring of F0 and F1 male rats fed with CD;(2)F0CD+ F1 HD group: F2 offspring of F0 male founders fed with CD and F1 male rats fed with HFSSD;(3)F0HD+ F1 CD group: F2 offspring of F0 male founders fed with HFSSD and F1 male rats fed with CD;(4)F0HD+ F1 HD group: F2 offspring of F0 and F1 male rats fed with HFSSD.All of F2 rats were fed with normal diet.Body weight of F2 rats were measured at 0,2nd,3rd,6th,9th,12 th,15th,18 th,22nd and 25 th weeks,respectively.At the 15 th week,24 hours of urine were collected by metabolic cages,and at the 25 th week,all blood and kidney tissues were collected.2.Detection of F2 serological and urine indicators related to renal function: Blood was collected from the abdominal aorta of F2 offspring and serum was separated.Serum cystatin C was detected by ELISA,and blood urea nitrogen(BUN),creatinine(Cr)and uric acid were measured by automatic biochemical analyzer.The 24 h urine of F2 offspring was collected by metabolic cage.The volume of urine was measured.After centrifugation,the supernatant was taken to detect the urine microalbumin,total urine protein and urine creatinine content by an automatic biochemical analyzer.3.Detection of F2 renal stereological indicators: Renal cortical sections were prepared after paraffin embedding of the F2 kidney tissues.The number of glomeruli and glomerular size were measured by PAS staining,and the glomerular sclerosis index was calculated.Renal interstitial fibrosis was detected using Sirius red staining.4.Microarray analysis of mRNA expression profiles of F2 kidney tissue: 7 cases of the kidney tissue of every female F0CD+F1CD group and every female F0HD+F1HD group were analyzed by Affymetrix gene chip technology for detecting the differential expression of mRNA.Functional analysis of differential mRNAs was performed using GO analysis to predict the highly enriched biological processes,cellular components,and molecular functions of differentially expressed mRNA.KEGG passway analysis was used to annotate differential mRNA signaling pathways and to predict differentially expressed mRNAs that are highly enriched in biological signaling pathways.Real-time quantitative PCR was used to further verify mRNA expression levels of target genes in kidney tissues of F2 offspring.5.Statistical methods: The experimental data were analyzed by SPSS 22.0 statistical software and expressed as Mean±SD.Two independent samples t-test were used for comparison between the two groups.One-way analysis of variance was used for comparison between groups.If the variance is uniform,the LSD method is used.If the variance is not uniform,the multiple comparison Dunnett’s T3 method is used.P < 0.05 indicated that the difference was statistically significant.Results 1.The result of body weight and kidney weight in F2 offspring: Compared with the control group(F0CD+F1CD),the F0HD+F1HD group in F2 female offspring showed a significant increase in body weight.There were no significant differences in left kidney weight,right kidney weight and the ratio of kidney weight to body weight among the four groups of both sexes.2.The result of F2 renal function-related serological index showed that: compared with F0CD+F1CD group,BUN of F2 female rats in F0CD+F1HD group decreased significantly.The content of serum Cr,uric acid and cystatin C were significantly higher in the female F0HD+F1CD group.The serum uric acid content was significantly higher in the male F0CD+F1HD group.The content of cystatin C in the female and male F0HD+F1HD group showed an increasing trend,but there was no statistical difference.3.The result of F2 renal function-related urinary index showed that: compared with F0CD+F1CD group,the ratio of microalbuminuria to urinary creatinine was significantly higher in the female F0HD+F1HD group,and the ratio of GFR to 25th-week body weight was significantly decreased in the F0HD+F1HD group of both sexes.4.The result of F2 renal stereological indicators showed that no significant differences in number of glomeruli and glomerular size were observed between the F0CD+F1CD and F0HD+F1HD group of both sexes.However,glomerular sclerosis index of the F2 offspring was significantly higher in F0HD+F1HD group than F0CD+F1CD group of both sexes.And a significant increase in the renal interstitial fibrosis was oberseved in the F0HD+F1HD group of F2 male offspring.5.The mRNA expression profile of the F2 kidney tissues showed that: compared with the F0CD+F1CD group,there were 385 genes abnormally expressed in the kidneys of the F0HD+F1HD group in F2 female offspring(FC≥1.5and P<0.05).Among them,218 genes(56.6%)were up-regulated,and 167 genes(43.4%)were down-regulated.GO analysis showed that the differentially expressed genes were mainly concentrated in MHC class I protein complexes,membranes,Golgi,etc(cellular components),affecting antigen binding,receptor binding and other molecular functions as well as antigen processing and presentation,immune response and other biological processes.KEGG pathway analysis showed that differentially expressed genes were mainly enriched in antigen processing and presentation,graft versus host disease,transplant rejection,viral myocarditis,autoimmune thyroid disease,type 1 diabetes and other pathways.Real-time quantitative PCR was used to further verify the expression of the target genes.The result showed that in F2 female offspring,compared with the F0CD+F1CD group,the expression level of Enpp6 and Tmem144 mRNA in the kidney tissues of F0HD+F1HD group was significantly down-regulated,and the expression level of Actr3 b mRNA in the kidney tissues of F0CD+F1HD group was significantly up-regulated.In F2 male offspring,the expression level of Ifi27 and Acadsb mRNA in the kidney tissues of the F0HD+F1HD group was significantly up-regulated and the expression level of Cd300 lf mRNA in the kidney tissues of the F0CD+F1HD group and F0HD+F1CD group was significantly down-regulated compared with the control group.Conclusions 1.Two times exposures of the HFSSD in male rats resulted in an increase in body weight and abnormal renal function in F2 offspring,which is more obvious in females.2.Two times exposures of the HFSSD in male rats resulted in glomerular sclerosis,renal interstitial fibrosis and significant changes in the expression of genes(Enpp6,Tmem144,Actr3 b,Cd300lf,Ifi27,and Acadsb)in F2 offspring kidney.3.The glomerular sclerosis,renal fibrosis and abnormal expression of above genes may be an important cause of renal dysfunction in F2 offspring after two times exposures of the HFSSD. |
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