| A number of studies showed that undernutrition in utero can permanently change the body’s structure, function and metabolism, leading to intrauterine growth restriction (IUGR), which is a risk factor lead to long-term disease risk, such as cardiovascular disease, insulin resistance, diabetes mellitus, obesity, and hypertension, as well as renal disease in adulthood. Kidney is an important target organ of the "programming". Epidemiological data indicate that, the IUGR fetal kidney has less volume than the normal fetal kidney, with a significant reduction in the number of glomeruli. In addition, long-term follow-up after birth shows that compared to the control group, IUGR group has significantly lower glomerular filtration and greater incidence of proteinuria. Our preliminary animal studies also found that, glomerular number was significantly reduced in IUGR newborn rats. But the exact molecular mechanism of "programming"of kidney disease has not yet been elucidated.Wilms’tumor gene (WT1) and insulin-like growth factor2(IGF2) are two important genes affecting kidney development, which play an important role in nephrogenesis and glomerular filtration function. Our previous studies have confirmed a significantly down-regulated expression of WT1in IUGR newborn rat. Therefore, we speculate that WT1and IGF2may be the key molecules in the process of "programming" of kidney disease. Since epigenetic regulation of gene promoters which is established during development and is retained throughout the life span of the organism confers patterns of transcriptional expression and silencing, perturbations to such processes represent one possible molecular mechanism for induction of an altered phenotype. During embryonic development, DNA methylation state is particularly sensitive to environmental factors. While in the process of "programming", the intrauterine environment can affect the DNA methylation, to participate in the occurrence of diseases. Therefore, whether DNA methylation has changed in the IUGR rat kidney? Studies on this point will contribute to a better understanding of pathogenesis of "programming", and to look for the possible warning signs and treatment targets early in the disease.In the first part of this experiment, a low-protein isocaloric diet consisting of6%protein was provided to the study group throughout the entire pregnancy period until natural labor. The resulting newborn rats with body weight2standard deviations below the average were assigned to the neonatal IUGR rats (IUGR group). The normal control group was supplied during the gestation period with conventional feed (22%protein) until natural labor. The neonatal period,4weeks and12weeks after birth were selected as research nodes, and6kidneys of each group were sacrificed as the objects. Real-time PCR was used to test the mRNA level of WT1and IGF2, as well as the methyltransferases, such as DNMT1, DNMT3a and DNMT3b. MassARRAY was used to analysis the quantitative methylation of WT1and IGF2gene promoter region.24-hour urine protein creatinine ratio, liver and renal function, blood lipids and glucose levels of rats in each group were detected at the age of4weeks and12weeks. Renal tissue morphology was observed at12weeks of age using light and electron microscopy, and glomerular number was count with the light micoscopy. The results showed that, body weight of IUGR newborn rat was significantly lower than normal rats, and the glomerular number was also significantly lower. At the same time, the thickness of nephrogenic zone in IUGR group was significantly larger than that in control group, indicating a development delay in IUGR rat’s kidney. WT1expression was significantly lower in neonatal IUGR rat kidney than the normal group, whereas the expression of IGF2was increasing in IUGR group. Quantitative methylation results show that, at this time the WT1promoter methylation levels significantly increased, and significantly associated with its mRNA expression. Methylation level of IGF2gene did not show significant changes. And we also found the mRNA expression of DNMT1and DNMT3a increased significantly, and had a linearly correlation with WT1gene DNA methylation level. So we speculate that the reduction of WT1expression likely to be directly responsible for, or through the weakening of the inhibition of IGF2expression increased the concentration to intervention mesenchymal cells and mesenchymal epithelial transdifferentiation, makes the ureteral buds branch abnormally, and influence the metanephron’s differentiation, and eventually lead to reduce renal unit. And DNMTl and DNMT3a mediated WT1promoter methylation levels increase is the important mechanism of the decreased expression of WT1. When IUGR rat into adulthood, the expression of WT1and IGF2in kidney were significantly higher than normal group, and the IUGR rats also appeared abnormal kidney function, mainly as urine protein creatinine ratio significantly higher than the control group, with increased in urea nitrogen, uric acid and Cystatin C. Podocyte foot process fusion can also be observed by the electron microscope. Quantitative methylation analysis showed that the WT1promoter methylation levels significantly reduced, and the mRNA expression of DNMT1and DNMT3b also appeared obviously reduced. But the methylation status of the IGF2promoter region was still no noticeable change.In previous studying, IUGR children suffer from insufficient muscle strength and high energy demanding. To achieve the catch-up growth, high protein diet should be provided, but the fast situation may also increase the risk of metabolic diseases in adulthood, such as obesity. In our group’s intervening health studying of the IUGR neonatal rat protein diet, we found that high protein diet not only did not correct the reduction trend of glomerular, it increased the severity of hypertension and proteinuria. Therefore, it has been suggested that diet restrictions after birth can reduce the incidence of obesity. Studies have shown that low protein diet could increase the early postnatal stage IUGR rats’glucose tolerance and insulin sensitivity. Then, postnatal protein restriction, to maintain their consistent with the intrauterine environment, will it improve the IUGR fetal kidney injury? There is still lack of relevant research reports. Appropriate nutritional intervention of postnatal IUGR fetuses and its effect on kidney and possible mechanism has not fully clarified.In order to identify the impact of the DNA methylation status of WT1and IGF2genes in different developmental stages of kidney, as well as the renal function with different nutrition interventions on the IUGR rats after birth, we conducted the second part of the study. IUGR pups were divided into three groups, fed with a conventional diet (containing22%protein, IUGR+N group), a high-protein diet (containing30%protein, IUGR+H group) or a low-protein diet (containing6%protein, IUGR+L group) respectively until12weeks after birth. The normal control group was fed with a conventional pregnancy diet (22%protein) until natural delivery, and the newborn rats were fed with a conventional diet (22%protein, control group) until12weeks after birth. Testing WT1and IGF2mRNA level of kidney at the new period,4weeks and12weeks after birth of each group, analysis the two genes’DNA methylation in promoter region, as well as the DNMT1, DNMT3a and DNMT3b mRNA levels, while testing24-hour urine protein creatinine ratio, blood lipids and glucose levels, liver and renal function at4weeks and12weeks after birth, the method was the same as the first part. The results showed that weight gain of the high-protein fed rats was not ideal, although catch-growth also appeared after4weeks, but adulthood weight was significantly lower than control group and normal feed group. At the same time, we found that a urine protein creatinine ratio was significantly increased at4weeks of age in high-protein fed group, and at12weeks, other than the higher urine protein creatinine ratio, the blood urea nitrogen and uric acid levels was also significantly increased. And there was no catch-up growth after birth in the low-protein fed group, indicating a limited growth and development. Although the total protein and albumin in blood were lower than the other groups, there were no significant difference in the urine protein creatinine ratio and renal function between IUGR+L group and control group, better than IUGR+N and IUGR+H group. Real-time PCR results showed that to adulthood the expression of WT1and IGF2of the two groups with nutrition intervention has noticeably increased compared with that of the control group, among them, IGF2expression of low-protein group increased very significant. Different from the first part, this time the DNA methylation levels of WT1and IGF2genes of the two nutrition intervention groups were all significantly lower, and this change lasted from4weeks to12weeks of age. And at12weeks, methylation levels of the two groups has significantly lower than IUGR+N group, and this was a significant linear relationship with the high expression of WT1and IGF2mRNA. DNMT1expression of high-protein fed rats was significantly reduced in the12weeks of age, showing a significant correlation with the low methylation state of the two genes, while the three kinds of methyltransferase in the low-protein fed group did not change significantly.To sum up, poor environment may affect the methylation state of WT1, then causes abnormal expression of it. The abnormal expressions of WT1and IGF2in different stages participate in reducing the glomerular amount of IUGR rats and also their proteinuria in adulthood. High protein feeding to postnatal IUGRs rat could adjust the abnormal expression of WT1and IGF2through altering the condition of methylation in promoter region, which results in kidney damage. Although protein feeding consistent with the intrauterine environment could reduce the burden on kidneys, it also increases the anomalous expression level of WT1and IGF2, by changing the methylation condition in promoter area, leading to ascending risk of kidney damage, and against their growth and development. Thus based on the existing research results, we should explore new nutritional interventions ways that ensures the growth and development of IUGR children without increasing the burden of kidney, so as to better protecting the renal function. |
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