The effect of chronic stress on prenatal development of the central nervous system

Abstract

The survival of developing embryos depends on the control and maintenance of homeostasis. Stress caused by chronic immobilization during pregnancy in rats may alter the normal development of the nervous system and increase susceptibility to psychiatric disorders. We investigated the effects of chronic stress on cell proliferation in the forebrains of embryos at 12 days of gestation, and in the hippocampus, dentate gyrus and cortex in embryos at 17 and 21 days of gestation. We examined serial sections of the embryonic brains of control and stressed rats at days 12, 17 and 21 of gestation. Brain sections were immunolabeled with anti-PCNA and stereological analysis was performed on 540 images. The results showed no statistical differences on days 12 and 17 of gestation in the proliferation area of the structures studied, whereas on day 21 of gestation, proliferation decreased in the cortex and dentate gyrus of embryos of the stressed group. These changes were related to decreased prolactin and increased corticosterone concentrations in the plasma.     

β-Casomorphin-7 attenuates the development of nephropathy in type I diabetes via inhibition of epithelial-mesenchymal transition of renal tubular epithelial cells

This study was designed to investigate the putative protective effect of β-casomorphin-7 on diabetic nephropathy in a rat model, and to explore the possible mechanism of this effect. SD rats were randomly divided into the following three groups: control group, diabetes group and β-casomorphin-7-treatment group. All rats were euthanized after 30 days with or without β-casomorphin-7 treatment. Biochemical parameters including blood glucose and renal function were quantified. The concentration of plasma TGF-β1 was measured by ELISA. Histopathological changes to the kidney were studied by Masson and Sirius red staining. Expressions of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, cytokeratin19 and TGF-β1 mRNA in rat renal cortices were analyzed by real-time PCR. Changes in α-SMA and E-cadherin protein expression in rat renal cortices were quantified by Western blot. β-Casomorphin-7 treatment of diabetic rats reduced urinary glucose, urinary protein, serum creatinine, blood urinary nitrogen, plasma TGF-β1 and the ratio of kidney: body weight. Masson and Sirius red staining showed that β-casomorphin-7 treatment attenuated renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats had elevated expressions of α-SMA, vimentin and TGF-β1 mRNA and α -SMA protein and decreased expression of E-cadherin and cytokeratin19 mRNA, and E-cadherin protein. β-Casomorphin-7 treatment of diabetic rats partially normalized these changes. Our results suggest that administration of β-casomorphin-7 attenuates renal interstitial fibrosis caused by diabetes. This protective effect may be associated, in part, with down regulation of epithelial-mesenchymal transition of renal tubular epithelial cells.     

Ontogeny of the Neutral Amino Acid Transporter SNAT1 in the Developing Rat

Summary System A is a highly regulated, Na⁺-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.     

Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF-β-Induced Epithelial-Mesenchymal Transition

ObjectiveThis study was to investigate whether sorafenib can inhibit the progression of renal fibrosis and to study the possible mechanisms of this effect.MethodsEight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were intragastrically administered sorafenib, while control and sham groups were administered vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-β1 and sorafenib for 24 or 48 hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each group. The expression of α-SMA and E-cadherin in kidney tissue and NRK-52E cells were performed using immunohistochemistry and immunofluorescence. The apoptosis rate of NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3 and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis.ResultsHE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib group compared with the vehicle-treated-UUO group (p<0.05). Compared with the vehicle-treated-UUO and TGF-β-stimulated NRK-52E groups, the expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the vehicle-treated UUO and TGF-β-stimulated NRK-52E groups, the ratio of p-Smad3 to Smad3 decreased in the sorafenib-treated groups (p<0.05).ConclusionOur results suggest that sorafenib may useful for the treatment of renal fibrosis through the suppression of TGF-β/Smad3-induced EMT signaling.     

Immunocytochemical localization of gamma-glutamyltranspeptidase during fetal development of mouse kidney

In the fully developed kidney, gamma-glutamyltranspeptidase is localized predominantly to the apical plasma membrane of the proximal tubules. The appearance of this activity during murine fetal nephrogenesis was quantitated using a sensitive fluorometric assay, and development of membrane polarity was assessed by immunocytochemistry. Specific activity of the transpeptidase in 13-day fetal kidney was approximately 1 mU/mg protein. Between 13-21 days of gestation, total transpeptidase activity increased 7500-fold, whereas specific activity increased 50-fold. At 13 days of gestation, gamma-glutamyltranspeptidase immunoreactivity is localized to the apical surfaces of developing renal vesicles and the proximal segment of the S-shaped tubules. The organized cell structures have tight tubular junctions but lack a well-defined brush-border membrane. By 15 days of gestation, immunostaining of the apical surface of developing proximal segments is more prominent, and slight reactivity of the basolateral membrane is evident. By 17 days of gestation, the kidney is organized into discrete zones. The large increase in gamma-glutamyltranspeptidase activity correlates with the appearance of increased immunostaining of the developing brush-border membranes of the proximal tubules contained in the inner cortex. A very similar although somewhat delayed pattern of appearance of transpeptidase activity and immunostaining was observed in metanephric organ culture. Induction of proximal tubular cyst formation had no effect on the increase in transpeptidase activity that occurred during organotypic nephrogenesis.     

Immunohistochemical evidence of Muc1 expression during rat embryonic development

During embryonic development, studies on mouse and human embryos have established that Muc1/MUC1 expression coincides with the onset of epithelial sheet and glandular formation. This study aimed therefore at evaluating the temporal and spatial expression of Muc1 at different stages of rat development. In this experiment, 80 animals were included: 64 rat foetuses at 13, 14, 15, 16, 17, 18, 19 and 20 days of gestation from pregnant females (WKAH/Hok), 8 embryos each stage. Standard immunohistochemistry was performed using anti-MUC1 cytoplasmic tail polyclonal antibody (CT33). The reaction was considered positive when more than 5% of the cells were stained; reaction patterns were: L = linear, membrane, C = cytoplasmic and M = mixed; nuclear staining was also recorded. Intensity was graded as negative (-), low (+), moderate (++) and strong (+++). Muc1 expression was observed with a low intensity on 13th day (13 d) in the stomach, lung and kidney; at 14 d, small intestine and pancreas were also reactive; at 16 d, liver and esophagus and at 18 d, trachea and salivary glands. During the development, intensity increased while the pattern of expression changed: at the first days of gestation, it was predominantly linear and apical while during further development an increase in cytoplasmic expression was observed. Trachea, stomach, kidney and lung epithelia were the more reactive tissues. In specimens belonging to neonates and adults, all tissues analyzed showed similar Muc1 expression. The findings of this study assess that Muc1 is highly expressed in the epithelial rat embryonic development.     

A new family of heparin binding growth/differentiation factors: differential expression of the midkine (MK) and HB-GAM genes during mouse development.

MK (midkine) and HB-GAM (heparin-binding growth-associated molecule) constitute a new family of heparin-binding growth differentiation factors. The modes of expression of MK and HB-GAM during mouse development were quantitatively examined by mRNA hybridization. The following three distinct patterns of expression were observed in the brain/head region. On the 11th-13th days of gestation, MK was intensely, but HB-GAM relatively weakly expressed; on the 15th-19th days, both MK and HB-GAM expression became weaker; and in the neonatal period, HB-GAM was intensely expressed and MK expression increased slightly. The level of HB-GAM expression was lower than that of MK in the whole embryo on the 11th to 13th days of gestation. HB-GAM mRNA was detected in the kidney of newborn and young mice, where MK was more highly expressed. The identity of the weakly expressed MK and HB-GAM signals was confirmed by means of the polymerase chain reaction in the neonatal brain (MK), the head of 13-day embryos (HB-GAM), and the kidney of 7-day-old mice (HB-GAM). In conclusion, MK and HB-GAM are frequently co-expressed in the same cells and anatomic regions of the fetus or new born mouse, while their modes of expression differ.     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.     

Differential expression of alpha and pi isoenzymes of glutathione S-transferase in developing human kidney

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies to determine the developmental expression of these isoforms in human kidney. Before 35 weeks of gestation, both isoenzymes were expressed by the collecting tubules and developing nephrons. After this time, expression of the alpha set was restricted to the proximal tubule and that of the pi set to the distal and collecting tubules and the loop of Henle.     

17-AAG对大鼠颈总动脉球囊损伤后内膜增生的影响及其作用机制

目的:研究17-丙烯胺-17去甲氧格尔德霉素(17-Allylamino-17-emethoxy-geldanamycin, 17-AAG)对球囊损伤后大鼠颈总动脉内膜增生的影响及可能作用机制。方法:将清洁级雄性SD大鼠36只按照随机数字法分为假手术组(Sham组)12只、球囊损伤组(Balloon injury, BI组)12只及17-AAG治疗组(17-AAG组)12只。采用2F Fogarty球囊建立大鼠颈总动脉球囊损伤组模型,17-AAG治疗组大鼠在建模后腹腔注射17-AGG(20 mg/kg 2d)。各组大鼠于球囊损伤3周后取损伤段颈总动脉,通过HE染色观察血管内膜形态学改变并评估内膜增生情况,免疫组化染色(Immunohistochemical staining,IHS)法检测血管壁增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)的表达,评估血管平滑肌细胞的增殖情况。流式细胞术检测血管平滑肌细胞的凋亡情况。结果:BI组、17-AAG组大鼠球囊损伤后颈总动脉内膜出现不同程度增生,内膜/中膜面积比(Intima area/Membrane area,I/M)均较Sham组显著升高(P0.05);17-AAG组的I/M较BI组明显下降(P0.05)。BI组、17-AAG组颈总动脉PCNA表达水平较Sham组明显升高(P0.05),较BI组显著降低(P0.05)。BI组、17-AAG组大鼠血管平滑肌细胞凋亡率较Sham组显著升高(P0.05);17-AAG组大鼠血管平滑肌细胞凋亡程度较BI组明显升高(P0.05)。结论:17-AAG对球囊损伤后颈总动脉内膜增生存在抑制作用,其机制可能是通过提高血管平滑肌细胞凋亡率影响其增殖程度。     

波动性高糖对肾小管上皮细胞Wnt／β-catenin信号途径的影响

肾脏纤维化分为肾小球硬化和肾小管间质纤维化（tubular interstitial fibrosis,TIF）,而TIF过程与肾损伤具有密切的关系。TIF是由于细胞外基质的过度沉积造成的,肌成纤维细胞是TIF发生发展过程中产生细胞外基质的主要细胞,该过程被成纤维细胞激活,涉及上皮细胞向肌成纤维细胞的转分化^[1-2]。Wnt／β-catenin信号途径涉及细胞增殖、肿瘤发生与转移的调控。β-catenin是Wnt信号途径的关键分子,在细胞的生长与分化过程中起着重要的作用,     

The alleviation of acute and chronic kidney injury by human Wharton's jelly-derived mesenchymal stromal cells triggered by ischemia-reperfusion injury via an endocrine mechanism

Background aimsThe effects of human Wharton's jelly-derived mesenchymal stromal cells (WJ-MSC) on acute and chronic kidney injury induced by ischemia-reperfusion injury (IRI) were assessed.MethodsWJ-MSC were injected intravenously immediately after solitary kidney ischemia for 45 min. Cells were labeled with 5-bromo-2′deoxy-uridine (BrdU) for tracing in vivo. At 48 h post-IRI, serum creatinine and blood urea nitrogen (BUN) were measured. Tubular cell proliferation and apoptosis as well as activation of the Akt signal were identified by immunostaining. Real-time polymerase chain reaction (PCR) was employed to determine gene expression of inflammation-related cytokines and hepatocyte growth factor (HGF). Levels of human HGF were assayed by enzyme-linked immunosorbant assay (ELISA). Twenty-two weeks later, renal fibrosis was assessed by Masson's tri-chrome staining, collagen content and α-smooth muscle actin (α-SMA) staining.ResultsThere was no sign of labeled cells residing in the damaged kidney. Acute renal dysfunction elicited by IRI was considerably improved by WJ-MSC, in parallel with a stronger proliferative response and less apoptotic events. Additionally, phosphoAkt staining in injured tubular cells was substantially intensified. Cell treatment also caused a remarkable up-regulation of kidney interleukin (IL)-10, heme oxygenase (HO)-1 and HGF expression. Human HGF was detected in cell supernatants and the serum of cell-infused rats. Moreover, IRI-initiated fibrosis was abrogated by cell therapy, coincident with function amelioration.ConclusionsWJ-MSC alleviate acute kidney injury, thereby rescuing the ensuing fibrotic lesions in an endocrine manner. The Akt signal in impaired tubular cells is reinforced by WJ-MSC, facilitating cell resistance to apoptosis and cell proliferation. HGF, either delivered or induced by WJ-MSC, is an important contributor.     

The protection of selenium on adriamycin-induced mitochondrial damage in rat

Although adriamycin (ADR) exhibits high anti-tumor efficacy in vitro, its clinical use in cancer chemotherapy is limited due to its high renal toxicity. This study investigated the mechanism of ADR nephropathy and the protective effect of selenium on ADR-induced kidney damage by analyzing of the relationship between selenium and mitochondria. Rats were divided into four groups. The first group was injected with saline i.p. for 21 days, the second group received the 4 mg/kg i.p. ADR every alternate day for 8 days, the third group received the 50 μg/kg i.p. Se for 21 days, and the fourth group received the Se. ADR co-administration i.p. blood pressures were assessed, the mitochondrial membrane potential (MMP) was assessed, and the adenosine triphosphate (ATP) levels were determined. The total antioxidant (TAS) and oxidant status (TOS) in cytosol, the mitochondria of kidney cells, and plasma were measured. Mitochondrial TAS decreased and TOS increased in the ADR group compared to the Se group. ADR-treated rats showed significantly lower MMP than did the control and Se groups. MMP was significantly restored in the Se + ADR group through selenium treatment compared to the ADR group (p < 0.01). In the ADR group, a reduction in ATP content was seen compared to the control and Se groups (p < 0.01). ATP level was significantly restored through treatment with selenium in the Se + ADR group compared to the ADR group (p < 0.01). We concluded that selenium is effective in vivo against ADR-induced kidney damage via the restoration of TAS and TOS, which prevented mitochondrial damage.     

Changing capacity for DNA excision repair in mouse embryonic cells in vitro

Capacity for excision repair of ultraviolet radiation damage to DNA in primary cultures of mouse embryonic cells is dependent on the gestational stage and the duration of in vitro growth. Fibroblasts of mouse embryos at 13–15 days gestation excise thymine dimers and perform unscheduled DNA synthesis after ultraviolet radiation. After several successive transfers in vitro, concomitantly with a pronounced reduction in growth rate, ability for excision repair decreases. DNA repair capacity is impaired in cells obtained from embryos at late stages of development (17–19 days gestation). Experiments with epithelial kidney cells from 5-day-old mice indicate that capacity for excision repair may depend on cell type and its origin.     

Temporal-spatial expression of ANG-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats

We recently demonstrated that renin-angiotensin system (RAS) overactivity during late gestation in rats is associated with increased kidney and urine levels of ANG-(1-7) and enhanced kidney immunostaining of ANG-(1-7) and angiotensin-converting enzyme 2 (ACE2). To understand the temporal-spatial changes in normal and hypertensive pregnancies, the renal distribution of ANG-(1-7) and ACE2 in association with kidney angiotensin peptides and ACE2 activity was examined in virgin, normal pregnant (NP; gestational days 5, 15, and 19) and reduced uterine perfusion pressure (RUPP at day 19) pregnant Sprague-Dawley rats. ANG-(1-7) and ACE2 immunocytochemical staining increased 1.8- and 1.9-fold and 1.7- and 1.8-fold, respectively, at days 15 and 19 of NP, compared with virgin rats. ANG-(1-7) and ANG II concentrations were increased in the kidney at 19 days of gestation. ACE2 activity measured using a fluorescent substrate was increased 1.9- and 1.9-fold in the cortex and 1.9- and 1.8-fold in the medulla at days 15 and 19 of NP. In the RUPP animals, ANG-(1-7) immunostaining and concentration were significantly decreased compared with 19-day NP rats. ACE2 activity was unchanged in the cortex and medulla of RUPP rats. In conclusion, during NP, the concurrent changes of ACE2 and ANG-(1-7) suggest that ACE2 plays an important role in regulating the renal levels of ANG-(1-7) at mid to late gestation. However, the decrease in renal ANG-(1-7) content in the absence of a concomitant decrease in ACE2 implicates the participation of other ANG-(1-7) forming or degrading enzymes during hypertensive pregnancy.     

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.