The Offspring Renal Developmental Toxicity By Perinatal Dehp Exposure And The Possible Mechanisms In Rats

Embryonic and lactation are critical for organism development. From the origin to formation, the life undergoes cell proliferation, migration, differentiation, procedural apoptosis and other complex processes. During the late1990s, the British scholar put forward "the intrauterine origins of adult disease" hypothesis, and then this view had aroused worldwide attention from scholars. The researchers believed that conditions experienced during perinatal played powerful roles in later life, and leading to certain chronic diseases in adulthood, including cardiovascular disease, metabolic disease and chronic kidney disease. Some studies indicate that placental insufficiency, maternal low protein diet, glucocorticoid and high-fat diet are all known to affect kidney development, which would cause reduced fetal and neonatal nephron number, leading to enhanced adult disease burden.Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer in many types of polyvinyl chloride consumer products, continually enters into human body via food, water, the atmosphere and medical devices. Both DEHP and Mono (2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP in vivo, were translocated across the placenta of pregnant rodents and distributed into the milk of lactating rat dams, leading to the fetus and neonate to be at risk. Previous animal studies have demonstrated short-term exposure to high-dosage DEHP induced hepatotoxicity, testicular toxicity, renal toxicity, developmental disturbance, reproductive toxicity, and teratogenicity. MEHP is believed as a peroxisome proliferator-activated receptor a (PPARa) activator, and PPARa is verified to be required for the metabolic toxicity of DEHP in the fetuses and pups. Interestingly, it can also transactivate PPARy which correlates with the ability to induce fibroblasts and adipocyte differentiation. Moreover, PPAR could influence the renin-angiotensin system (RAS) and glucocorticoid, which could directly and indirectly induce ureteric bud (UB) branching during nephrogenesis, which determines the nephron numbers. Therefore, we hypothesize that perinatal exposure to DEHP might affect kidney development in rat offspring.In this study, we have exposed maternal to DEHP during the embryos and lactating, and examine the effect of perinatal DEHP exposure on nephron formation, adult renal disease and blood pressure in offspring. To further confirm this damage, we gave parts of them high-fat diet (HFD) after weaning and examined the effects of maternal DEHP exposure combined with HFD on the development of kidney damage in the offspring. Otherwise, in order to explore the possible mechanisms of perinatal DEHP exposure on nephron formation, we detected the gene expression profiles of embryonic kidneys during critical periods in development, and analyzed the gene otology and pathway of the differentially expressed genes.Part1:Perinatal exposure to DEHP affects kidney development in offspringObjective:This study investigated the consequences of perinatal exposure to DEHP on nephron formation, examined the programming of renal function and blood pressure and explored the mechanism in offspringMethods:Two mature females and one male were placed together in one cage for breeding. Mating was confirmed by the appearance of a vaginal plug in the following morning. And then, maternal rats were treated with vehicle,0.25and6.25mg/kg body weight/day DEHP respectively from gestation day0to postnatal day21. Body weight and kidney weight were carefully recorded at birth day and week3,15and21. The H&E staining, PAS staining and Masson staining were used to detect the histopathological changes. The ELISA and Griss method were used to detect the concentration of serum rennin, angiotensin Ⅱ, ET-1and NO. Moreover, Q-PCR, western blotting and immunohistochemistry were used to detect the expression of RAS, PPARs and other kidney development-related genes.Results:Maternal DEHP exposure resulted in lower number of nephrons, higher glomerular volume and smaller Bowman’s capsule in the DEHP-treated offspring at weaning, as well as glomerulosclerosis, interstitial fibrosis and effacement of podocyte foot processes in adulthood. In the DEHP-treated offspring, the renal function was lower and the blood pressure was higher. The renal protein expression of renin and angiotensin Ⅱ was reduced at birth day and increased at weaning. Maternal DEHP exposure also led to reduced mRNA expression of some renal development involved genes at birth day, including Foxdl, Gdnf, Pax2and Wntll. While, the mRNA expression of some genes was raised, including Bmp4, Cdhll, Calml and Ywhab.Conclution:These data show that maternal DEHP exposure impairs the offspring renal development, resulting in a nephron deficit, and subsequently elevated blood pressure later in life. Our findings suggest that DEHP exposure in developmental periods may affect the development of nephrons and adult renal disease through inhibition of the renin-angiotensin system. Part2:Perinatal exposure to DEHP exacerbates high fat diet-induced kidney damage in offspringObjective:The first part suggested that perinatal exposure to DEHP induced renal toxicity in offspring rats. And, high-fat diet induced obesity has emerged as a strong independent risk factor for kidney disease. In this study, we investigated the effects of perinatal DEHP exposure on the kidney development of the offspring and confirm whether the effects could exacerbate high fat diet-induced renal damage in offspring.Methods:Pregnant Wistar rats were exposed to vehicle or1.25mg/kg body weight/day DEHP throughout gestation and lactation by oral gavage, and then the offspring were randomLy assigned to a normal diet (ND) or a high-fat diet (HFD) after weaning. Body weight, blood pressure, renal morphology and renal function were evaluated in the offspring. The H&E staining, PAS staining and Masson staining were used to detect the histopathological changes. Moreover, Q-PCR, western blotting and immunohistochemistry were used to detect the expression of RAS, PPARs and other kidney development-related genes.Results:Maternal DEHP exposure decreased the number of nephrons, increased renal protein expression of peroxisome roliferator-activated receptors (PPARs) and reduced renin-angiotensin system (RAS) expression in the offspring at birth day. In adulthood, as expected, HFD significantly increased weight and blood pressure. In addition, DEHP or HFD induced renal dysfunction and glomerulosclerosis, and the DEHP/HFD offspring had the most serious phenomenon. Moreover, maternal DEHP exposure reduced PPARs expression, increased RAS expression and TGFβ1expression in kidney of HFD-fed offspring.Conclution:Our data suggest that the maternal DEHP exposure induces congenital dysplasia of the kidney in the offspring, which exacerbates HFD-induced kidney damage in offspring. The mechanism probably involves reducing nephron numbers and impairing PPARs expression. Part3:The influence on offspring gene expression profile in kidney developmental window by prenatal DEHP exposureObjective:Analyze the influence of offspring expression profile in kidney developmental window induced by prenatal DEHP exposure, to further explore the mechanism of the early life DEHP exposure on kidney development.Methods:Pregnant Wistar rats were exposed to vehicle or0.25mg/kg body weight/day DEHP respectively from gestation day0. Pregnant rats were scarified in embryonic14.5days (E14.5) and16.5days (E16.5) respectively. The H&E staining was used to detect the histopathological changes of embryonic kidney. Moreover, we separated the embryonic kidney and extraction of kidney RNA, and used gene chips to detect gene expression profiling. The differentially expressed genes (DEGs), cluster analysis, the Gene Ontology features significant enrichment and Pathway significant enrichment were analyzed.Results:Exposure to DEHP in embryonic period caused kidney dysphasia. Comparing with the control and the exposure group, the DEGs of E14.5were significant higher than E16.5. And, the DEGs of E14.5-D vs E14.5-C were similar with E16.5-C vs E14.5-C, while the DEGs of E16.5-D vs E16.5-C were similar with E16.5-D vs E16.5-C. Moreover, the DEGs of E14.5-D vs E14.5-C mainly concentrated in the cell, the DEGs of E16.5-D vs E14.5-D mainly concentrated in the extracellular region, the DEGs of E16.5-C vs E14.5-C also mainly concentrated in the cell, and the DEGs of E16.5-D vs E14.5-D mainly concentrated in the contractile fibers. Additionally, the DEGs of E14.5-D vs E14.5-C and E16.5-C vs E16.5-D were related with PPAR signaling pathways, Wnt signaling pathways, metabolic pathways and others. While, the DEGs of E16.5-D vs E16.5-C were related with apoptosis, adhesion, etc. and the DEGs of E16.5-D vs E14.5-D were related with protein digestion and absorption.Conclution:We believe that kidney development in E14.5is more sensitive than in E16.5. Prenatal DEHP exposure could affect kidney development by influencing the PPARs pathway, Wnt signaling pathway, glucose and lipid metabolism, apoptosis and other related signaling pathways. And thus, these changes could reduce the sensitivity of the genes and affect the development process of cells, organs and systems, resulting in accelerated programing of kidney development, leading to nephron hyperplasia.