Structural analysis of 5'-flanking regions of rat, mouse and human renin genes reveals the presence of a transposable-like element in the two mouse genes

The two renin genes of the mouse (Ren1 and Ren2) are expressed at different levels in the submaxillary gland (SMG). In contrast to mice, there is no detectable renin gene expression in the rat SMG. To determine the molecular basis for these different levels of renin expression, we have compared the 5'-flanking regions of the rat and mouse genes. The sequence of mouse, but not rat, genes reveals the presence in Ren1 and Ren2 of a large insertion, probably a new class of transposable elements. A second, apparently unrelated shorter insertion is present only in Ren2. Otherwise, the mouse and rat 5'-flanking sequences are well conserved and resemble the corresponding region of the human Ren gene, indicating that the insertions occurred after the separation of the rat and mouse species but before the duplication of the mouse Ren gene. We suggest that these structural differences may have a role in the differential expression of the Ren genes in mice and other animals.     

Differential roles of renin and angiotensinogen in the feto-maternal interface in the development of complications of pregnancy

We previously identified a transgenic mouse model that developed pregnancy-associated hypertension (PAH) and intrauterine growth restriction (IUGR) by mating females expressing human angiotensinogen (hANG) with males expressing human renin (hRN). These phenotypic defects were not observed in the opposite type of mating combination, despite the feto-placental overexpression of hRN and hANG detected in both types of crossbreeding. Detailed analysis of transgene localization in the labyrinth and its permeability to the maternal circulation revealed that hRN produced in trophoblast giant cells was secreted into the maternal circulation, whereas hANG, produced in chorionic trophoblasts and trophoblastic epithelium, was undetectable in the maternal plasma, probably due to their distinct spatial and temporal expression in labyrinth. These results demonstrated that PAH and IUGR could be mediated by feto-placental hRN through its permeability to the maternal circulation, not by feto-placental hANG production. Furthermore, overexpression of maternally derived hANG in decidua and spiral arteries of pregnant females with PAH and IUGR raises the possibility of local activation of the renin-angiotensin system and its pathophysiological effects on placental hypoperfusion in complications of pregnancy. This study provides in vivo evidence that the cell-specific expression of RN and ANG in the feto-maternal interface impacts their differential roles in pregnancy.     

Endothelial-derived nitric oxide and angiotensinogen: blood pressure and metabolism during mouse pregnancy

The regulation of blood pressure during pregnancy involves several biological pathways. Candidate genes implicated in hypertensive diseases during pregnancy include those of the renin-angiotensin system and nitric oxide synthase (NOS). We evaluated blood pressure and metabolic characteristics during pregnancy in mutant mice. These included mice with a null mutation in the endothelial NOS (eNOS) gene (Nos3(-/-)), four copies of the angiotensinogen gene (Agt(2/2)), and mutations in both genes [four copies of Agt and heterozygous deficient for eNOS (Agt(2/2)Nos3(+/-)), four copies of Agt and homozygous deficient for eNOS (Agt(2/2)Nos3(-/-))]. Blood pressure measurements of nulliparous females from mutant strains were compared with two common laboratory strains C57Bl6/J and SV129 throughout their first pregnancy. Serum and urine analysis for the evaluation of renal and liver physiology were measured in the prepregnant state and during the third trimester of pregnancy. Throughout pregnancy blood pressures in all mutant strains were higher compared with controls. Agt(2/2)Nos3(-/-) showed the highest blood pressures and C57Bl6/J the lowest. Control mice, but not mutant mice, showed a second trimester decline in blood pressure. No immediate differences were noted regarding behavioral characteristics, renal or liver function parameters. Mice deficient for eNOS, mice with overexpression of Agt, and mice with mutations in both genes demonstrated higher blood pressure throughout pregnancy. There was no evidence of renal dysfunction, liver dysfunction, or hemolysis among any of the strains studied. We conclude that Nos3 and Agt are important genes in the regulation of blood pressure during pregnancy.     

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.     

Kidney and submaxillary gland renins are encoded by two non-allelic genes in Swiss mice.

Two distinct phenotypic groups of inbred strains of mice, with different amounts of submaxillary gland (SMG) renin have been described. We have previously shown that strains with high levels of SMG renin, such as Swiss or AKR mice, have two renin genes, Rn1 and Rn2, per haploid genome, while strains with low levels of SMG, such as BALB/c or C57Bl/6, have only one renin gene. We now report the molecular cloning of cDNA copies of Swiss mouse kidney renin mRNA and present nucleotide sequence data of the recombinant clones. Comparison of these sequences with the sequence of Swiss mouse SMG renin mRNA we have previously reported, demonstrates that Swiss mice express the two non-allelic genes, Rn1 and Rn2.     

The mouse Rn locus: S allele of the renin regulator gene results from a single structural gene duplication.

Inbred strains of mice have been divided into two distinct phenotypic groups having different levels of renin activity regulated by androgen in the submaxillary gland (SMG). Strains carrying the Rnrs allele of the renin gene regulator, located on chromosome 1, have a high level of renin activity; strains carrying the Rnrb allele have a low level of renin activity. The level of SMG renin activity correlates with the level of renin mRNA. We have analyzed, by Southern blot hybridization, the organization of renin genes in both strains. Strains carrying the Rnrb allele, such as BALB/c or C57 Bl/6, or CH3 mice, have one renin structural gene per haploid genome, while those having the Rnrs allele, such as AKR or Swiss mice, have two renin genes. We have also identified renin genes in mice belonging to different biochemical groups: Mus spretus has one renin gene while M. vrania and M. musculus brevirostris have two renin genes.     

Lasting effects of early life stress in mice: interaction of maternal environment and infant genes

In the mouse, a powerful paradigm of early life stress, infant maternal separation (IMS), can trigger emotional and cognitive dysfunctions in adulthood similar to those found in humans with a history of childhood adversity. The magnitude of IMS effects differs among diverse inbred strains suggesting an interaction between the genetic background of pups and the maternal care they received. Here, we investigated this interaction with studies on reciprocal F1 hybrid mice of the stress‐susceptible Balb/c and the resilient C57Bl/6 strains that were either raised by Balb/c mothers (low maternal care) or by C57Bl/6 mothers (higher maternal care) with or without IMS exposure. The ultrasonic vocalization response to isolation was recorded from infant F1 pups, and their emotional, executive cognitive and epigenetic phenotypes were assessed in adulthood. These studies showed that, regardless of the maternal care received, the emotional phenotype of F1 hybrids was not significantly affected by IMS exposure. However, F1 pups raised by Balb/c (but not C57Bl/6) mothers during IMS exposure exhibit deficits in working memory and attention‐set‐shifting in adulthood. They also exhibit reduced histone deacetylase 1 levels at promotors of brain‐derived neurotrophic factor and early growth response 2 genes, and abnormally high induction of expression of these genes during cognitive testing. As one of affected genes was previously shown to associate with the Balb/c and the other with the C57Bl/6 genetic background, these findings indicate that both parental alleles interact with the maternal environment to modulate the cognitive and epigenetic phenotypes of F1 mice exposed to the IMS.     

Homeostasis in mice with genetically decreased angiotensinogen is primarily by an increased number of renin-producing cells.

Here we investigate the biochemical, molecular, and cellular changes directed toward blood pressure homeostasis that occur in the endocrine branch of the renin-angiotensin system of mice having one angiotensinogen gene inactivated. No compensatory up-regulation of the remaining normal allele occurs in the liver, the main tissue of angiotensinogen synthesis. No significant changes occur in expression of the genes coding for the angiotensin converting enzyme or the major pressor-mediating receptor for angiotensin, but plasma renin concentration in the mice having only one copy of the angiotensinogen gene is greater than twice wild-type. This increase is mediated primarily by a modest increase in the proportion of renal glomeruli producing renin in their juxtaglomerular apparatus and by four times wild-type numbers of renin-producing cells along afferent arterioles of the glomeruli rather than by up-regulating renin production in cells already committed to its synthesis.     

A targeted approach to developing environmental enrichment for two strains of laboratory mice

Previous studies on environmental enrichment have generally placed a purported enrichment in the cage and observed changes in behavioural and physiological indicators of welfare. However, many of these ‘enrichments’ are not purposely designed, or appear to be designed with anthropomorphic or anthropocentric concerns, which have little biological relevance. The aim of this study was to screen the behavioural responses of mice to a range of 24 potential enrichments (PEs) considered by an expert panel to have characteristics most biologically relevant to mice. Female mice (64 C57/Bl/6 and 64 ICR(CD-1)) were housed in groups of four and observed three days per week for 12 weeks whilst provided with a different PE weekly. The overall time-budgets of the two strains differed significantly. ICR(CD-1) mice performed more bar chewing, digging, manipulation of PEs, and rearing in the cage. They were also more likely to be hidden in the cage. C57/Bl/6 mice performed more drinking, cage sniffing and social interaction, and showed three times more bar climbing. The two strains responded differently to the PEs, with the ICR(CD-1) mice making more use of PEs, particularly those that could be manipulated or which provided additional hiding places. The higher trait anxiety of the C57/Bl/6 mice may have reduced their utilisation of novel PEs. We conclude that, whilst enrichment can significantly improve animal welfare, those types of enrichments that work well for all strains need to be identified, or strain-specific enrichment policies devised.     

Differential thiol status in blood of different mouse strains exposed to cigarette smoke

C57Bl/6J, DBA/2 and ICR mouse strains are known to possess different susceptibilities to developing emphysema after exposure to cigarette smoke with DBA/2 and C57Bl/6J strains being significantly more susceptible to pulmonary damage than the ICR strain. This study was aimed at analysing the occurrence of systemic oxidative stress in the blood of these different mouse strains after exposure to cigarette smoke. This study did not observe a significant decrease in glutathione in erythrocytes or in plasma cysteine, cysteinylglycine, homocysteine and glutathione in C57Bl/6J or DBA/2 mice, whereas a significant increase in the corresponding oxidized forms was observed in plasma. However, the ICR strain showed a significant increase in glutathione in erythrocytes and a significant decrease in most of the oxidized forms of cysteine, cysteinylglycine, homocysteine and glutathione in plasma after the same exposition. These experiments demonstrate that exposure to cigarette smoking induces systemic oxidative stress only in some mouse strains which are susceptible to developing emphysema.     

Divergent effects of leptin in mice susceptible or resistant to obesity.

Consumption of a high-fat diet decreases hypothalamic neuropeptide Y (NPY) and increases proopiomelanocortin (POMC) and brown adipose uncoupling protein (UCP)-1 mRNA in obesity-resistant SWR/J but not obesity-prone C57Bl/6J mice. Although leptin was elevated in both strains in response to a high-fat diet, its role in the development of diet-induced obesity has remained unclear since insulin and other factors that affect similar tissue targets are altered. Thus, we administered recombinant leptin by subcutaneous infusion to chow-fed mice to mimic the changes in plasma leptin across its broad physiologic range. We observed strain differences in responsiveness to reduced and elevated leptin levels. A reduction in leptin during fasting evoked a greater response in C57Bl/6J mice by decreasing energy expenditure and thyroxin, increasing corticosterone and stimulating food intake and weight gain during refeeding. However, C57Bl/6J mice were less responsive to an increase in leptin in the fed state. Conversely, the leptin-mediated response to fasting was blunted in SWR/J mice, whereas an increase in leptin profoundly reduced food intake and body weight in SWR/J mice fed ad libitum. Sensitivity to fasting in C57Bl/6J mice was associated with higher hypothalamic NPY mRNA and reduced POMC and UCP-1 mRNA expression, while the robust response to high leptin levels in SWR/J mice was associated with suppression of NPY mRNA. These results indicate that differences in leptin responsiveness between strains might occur centrally or peripherally, leading to alteration in the patterns of food intake, thermogenesis and energy storage.     

Midkine, a newly discovered regulator of the renin-angiotensin pathway in mouse aorta: significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling

We previously demonstrated that pleiotrophin (PTN the protein, Ptn the gene) highly regulates the levels of expression of the genes encoding the proteins of the renin-angiotensin pathway in mouse aorta. We now demonstrate that the levels of expression of these same genes are significantly regulated in mouse aorta by the PTN family member midkine (MK the protein, Mk the gene); a 3-fold increase in expression of renin, an 82-fold increase in angiotensinogen, a 6-fold decrease in the angiotensin converting enzyme, and a 6.5-fold increase in the angiotensin II type 1 and a 9-fold increase in the angiotensin II type 2 receptor mRNAs were found in Mk-/- mouse aorta in comparison with the wild type (WT, +/+). The results in Mk-/- mice are remarkably similar to those previously reported in Ptn-/- mouse aorta, with the single exception of that the levels of the angiotensinogen gene expression in Ptn-/- mice are equal to those in WT+/+ mouse aorta, and thus, in contrast to Mk gene expression unaffected by levels of Ptn gene expression. The data indicate that MK and PTN share striking but not complete functional redundancy. These data support potentially high levels importance of MK and the MK/PTN developmental gene family in downstream signals initiated by angiotensin II either in development or in the many pathological conditions in which MK expression levels are increased, such as atherosclerosis and many human neoplasms that acquire constitutive endogenous Mk gene expression by mutation during tumor progression and potentially provide a target through the renin-angiotensin pathway to treat advanced malignancies.     

Cell cycle inhibitor p57 expression in normal and diabetic rat placentas during some stages of pregnancy

Placentomegaly, an abnormal increase in the size of the placenta, is commonly seen in human diabetic pregnancies and diabetic animal experimental models. Proper placental development depends on the proliferation and differentiation of trophoblasts. However, our knowledge about the mitotic regulators that play key roles in synchronizing these events is limited. p57 is a cyclin-dependent kinase (CDK) inhibitor acting in the G1/S transition of the cell cycle. There is no data regarding p57 expression in either rat or human diabetic placentas. The purpose of this study was to investigate p57 expression in control and diabetic rat placentas at different stages of pregnancy. Diabetes was induced by streptozotocin on the first day of pregnancy, and placentas were taken on days 11, 13, 17, and 21 of pregnancy. Our results showed that on day 11, p57 immunostaining intensity was stronger in control group placentas compared to the diabetic group. On day 13, p57 immunostaining intensity increased in both groups, but increased more in the diabetic group. On day 17, p57 immunostaining intensity decreased in both the control and diabetic groups compared to day 13, yet the intensity remained higher in control placentas compared to diabetic placentas. On day 21 of pregnancy, p57 immunostaining intensity increased in the control group and it decreased from the day 17 level in the diabetic group. Western blot results showed consistency with immunohistochemistry results. Our study shows different expression patterns of p57 between control and diabetic rat placentas, which indicate p57 may play a role in abnormal placental formation resulting in placentomegaly arising from diabetes.     

Effects of non-24-hour days on reproductive efficacy and embryonic development in mice

ICR female mice were exposed to either 22 (L11, D11) or 26 hour day (L13, D13) light/dark cycles for at least 2 weeks before mating and/or during pregnancy. The mating rates of these animals decreased considerably. When pregnant females were examined at gestation days 12.0 or 17.5, resorption rates were increased, the embryos weighed less, and development was retarded in the experimental groups with preconceptional exposure to non-24-hour days. We speculate that in mice maternal and paternal pre- and periconceptional environment of daily light/dark cycles is important for normal reproductive efficacy and normal embryonic development during pregnancy.     

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-gamma) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-gamma gene (IFN-gamma KO) to address this issue. We found that IFN-gamma KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-gamma KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-gamma KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-gamma is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essential for the development of a protective Th1 type immune response in the later stages.     

The biosynthesis of tumor necrosis factor during pregnancy: studies with a CAT reporter transgene and TNF inhibitors.

Tumor necrosis factor (TNF) is a protein hormone which mediates diverse inflammatory conditions, but which also may be involved in physiologic processes. To detect the expression of TNF as it might occur in normal tissues we developed a transgenic mouse line bearing a reporter gene construct in which the INF coding sequence and introns are replaced by a chloramphenicol acetyl transferase (CAT) coding sequence. In these animals, expression of CAT within tissues has been shown to reflect TNF production. We now report upon the pattern of CAT expression that is observed during normal pregnancy. CAT is constitutively expressed in both the fetal and maternal thymuses, and in the placenta, but in no other tissues. Placental CAT activity first becomes measurable at day 13 of gestation, peaks at day 16, and is maintained at high levels until parturition. Crosses between transgenic and non-transgenic mice clearly indicate that the trophoblast, rather than the decidua or uterus, is the source of CAT activity. A soluble TNF receptor/IgG heavy chain chimeric protein, which strongly inhibits TNF activity in vitro and in vivo, was shown to cross the placenta, gaining access to the fetal circulation when administered on the maternal side. However, the chimeric protein did not interrupt pregnancy, and had no obvious effect on fetal development, suggesting that TNF may not be required for completion of a normal gestation.     

Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice

Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57Bl or BDF1 (C57Bl x DBA2) mice with caffeine (1 or 3 x 50 mg/kg and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57Bl and BDF1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57Bl compared to BDF1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57Bl mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57Bl and BDF1 mice but not in fetal mouse livers.     

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.