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Supplementary Figure Legend from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

Supplementary Figure 1 from Angiotensinogen Delays Angiogenesis and Tumor Growth of Hepatocarcinoma in Transgenic Mice

Brain tumors, benign or malignant, are characterized by a very high degree of vascularization. Recent accumulating evidence suggests that during development the neuronal wiring follows the same routes as the vasculature and that these two systems may share some of the same factors for guidance. Thus, expression of dual angiogenic/neurogenic growth factors was evaluated by in situ hybridization in human primary brain tumors of three different types, i.e., astrocytomas, oligodendrogliomas, and ependymomas, of increasing grades, in relation with the grade and type of the tumor. For this evaluation we selected vascular endothelial growth factor (VEGF-A) and its receptors VEGF-R1 and VEGF-R2 and the neuropilins 1 and 2 (NRP-1 and NRP-2), which have proangiogenic properties, platelet-derived growth factor (PDGF) receptor-beta (PDGF-Rβ), which is required for the functional maturation of blood vessels, the ephrins and their Eph receptors, angiotensinogen (AGT) and thrombospondin-2 (TSP-2), which have potential antiangiogenic properties, and netrin-1 (Net-1), which regulates vascular architecture. We show that the expression of the VEGF-NRP system, PDGF-Rβ, TSP-2, AGT, and Net-1 are differentially regulated, either increased or decreased, in relation with the type and grade of the tumor, whereas regulation of the ephrinB system does not seem to be relevant in these human brain tumors.

Nutrition plays a critical role in programming and shaping linear growth during early postnatal life through direct action on the development of the neuroendocrine somatotropic (GH/IGF-1) axis. IGF-1 is a key factor in modulating the programming of linear growth during this period. Notably, IGF-1 preferentially stimulates axonal growth of GHRH neurons in the arcuate nucleus of the hypothalamus (Arc), which is crucial for the proliferation of somatotroph progenitors in the pituitary, thus influencing later GH secretory capacity. However, other nutrition-related hormones may also be involved. Among them, insulin shares several structural and functional similarities with IGF-1, as well as downstream signaling effectors. We investigated the role of insulin in the control of Arc axonal growth using an in vitro model of arcuate explants culture and a cell-type specific approach (GHRH-eGFP mice) under both physiological conditions (normally fed pups) and those of dietary restriction (underfed pups). Our data suggest that insulin failed to directly control axonal growth of Arc neurons or influence specific IGF-1-mediated effects on GHRH neurons. Insulin may act on neuronal welfare, which appears to be dependent on neuronal sub-populations and is influenced by the nutritional status of pups in which Arc neurons develop.

International audience; The enteric nervous system originates from neural crest cells that migrate in chains as they colonize the embryonic gut, eventually forming the myenteric and submucosal plexus. Failure of the neural crest cells to colonize the gut leads to aganglionosis in the terminal gut, a pathological condition called Hirschsprung disease (HSCR) in humans, also known as congenital megacolon or intestinal aganglionosis. One of the characteristics of the human HSCR is its variable penetrance, which may be attributable to the interaction between genetic factors, such as the endothelin-3/endothelin receptor B pathway, and non-genetic modulators, although the role of the latter has not well been established. We have created a novel HSCR model in the chick embryo allowing to test the ability of non-genetic modifiers to alter the HSCR phenotype. Chick embryos treated by phosphoramidon, which blocks the generation of endothelin-3, failed to develop enteric ganglia in the very distal bowel, characteristic of an HSCR-like phenotype. Administration of dexamethasone influenced the phenotype, suggesting that glucocorticoids may be environmental modulators of the penetrance of the aganglionosis in HSCR disease.

International audience; Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postna-tal period with programming of the growth trajectory.

International audience; Organism development is controlled by both genetic programs and the environment to insure a reproductive success as adults. Linear growth is an important part of the development and is mostly controlled by genetic factors. However, the variability of height in a given species does not seem to be specifically associated with SNP. This suggests that environment may play a crucial role. In agreement, an important part of height-related genes present CpG island in their proximal promoter, indicating potential involvement of epigenetic mechanisms. In mammals, the linear growth is regulated by the IGF system, with IGF-I and IGF-II during the fetal period, and IGF-I being included within the somatotropic axis during the postnatal period. Nutrition during the lactating period programs linear growth and adult size through a modulation of the somatotropic axis development and of the setting of its activity later on. The study of underlying mechanisms suggest two waves of programming, which involve both structural adaptation during the early postnatal period and permanent functional adaptation in adulthood. The former may involve a direct stimulation of axon growth of GHRH neurons by IGF-I in first weeks of life while the latter could involve permanent epigenetic modifications in adulthood.; Le développement de l’organisme est contrôlé à la fois par des programmes génétiques et par l’environnement afin d’assurer un succès reproductif à l’âge adulte. La croissance staturale est une partie importante du développement et est principalement contrôlée par des facteurs génétiques. Cependant, la variabilité de la taille chez une espèce donnée ne semble pas être entièrement sous la dépendance du background génétique. Ceci suggère que l’environnement pourrait jouer un rôle crucial. En accord avec cela, une proportion importante des gènes liés à la croissance présente un îlot CpG dans leur promoteur proximal, ce qui indique l’implication potentielle de mécanismes épigénétiques. Chez les mammifères, la croissance staturale est régulée par le système IGF, avec l’IGF-I et IGF-II durant la période fœtale et l’IGF-I étant inclus dans l’axe somatotrope lors la période postnatale. La nutrition pendant la période transitoire qu’est l’allaitement programme la croissance staturale et la taille adulte par une modulation du développement de l’axe somatotrope et de son activité à l’âge adulte. L’étude des mécanismes sous-jacents suggère deux vagues de programmation, qui impliquent à la fois une adaptation structurelle en période postnatale précoce et une adaptation fonctionnelle permanente chez l’adulte. La première semble impliquer une stimulation directe de la croissance axonale des neurones GHRH par l’IGF-I dans les premières semaines de vie, alors que la seconde pourrait impliquer des modifications épigénétiques permanentes à l’âge adulte.

[This corrects the article DOI: 10.1371/journal.pone.0170083.].

Epidemiologic studies of rare diseases may produce surprising findings and raise ethical issues. This is illustrated in this study performed in 171,977 consecutive Taiwanese newborns (including 90,288 boys) from July 2006 through June 2008 by measuring dry blood spot and then leukocyte -galactosidase A ( -Gal A) activities and finally by detecting mutations in the GLA gene involved in Fabry disease. Schematically, two phenotypes of this disease are known: the classic form, with systemic involvement and very low -Gal activity in males; and the later-onset form ( 40 years of age), with some residual -Gal A activity, which is dominated by cardiac involvement. All 11 newborns who had 5% of normal mean -Gal A activity were boys who had GLA mutations. In the group of 66 newborns (64 boys and 2 girls) with -Gal A activities between 5% and 30%, 61 hemizygous boys and 2 heterozygous girls had GLA mutations. Among the group of 12 newborns (11 boys and 1 girl) with -Gal A 30%, only 1 boy had a previously reported mutation, identified in a family with later-onset renal disease. In total, 72 male and 2 female newborns had GLA mutations, an overall frequency of approximately 1 in 1250 boys and approximately 1 in 40,840 girls. Four boys were “predicted” to have the classic phenotype, a frequency of about 1 in 22,570 newborn boys. In contrast, the estimated frequency of the lateronset phenotype is approximately 1 in 1390 male newborns mutations. Three families provided information on other members of the kindreds, two with classic and one with later-onset phenotype. All three families had previously undiagnosed symptomatic family members, including one heterozygous female with ESRD and two males with renal involvement. This is undoubtedly the positive side of such studies. A second study was performed more recently in 110,027 Taiwanese newborns between January 2008 and January 2009 by using a similar protocol (plasma -Gal A activity was measured) (1). The results of this study confirmed those of the previous screening. A high prevalence of the cardiac variant Fabry mutation IVS4 919G3A, first discovered in Japanese patients, was found among newborns (approximately 1 in 1600 boys) in both studies. This splicing mutation was most common (82% of patients). The alternatively spliced transcript was normally present in small amount ( 5% of normal transcript) in most human tissues. However, the G3A transversion enhanced the percent expression of the alternatively spliced -Gal A variant and included a 57-nucleotide intronic sequence that caused a frameshift mutation, resulting in a truncated enzyme polypeptide that had no detectable enzyme activity. The clinical significance of this splicing mutation remains to be fully clarified. Of interest, Lin et al. (1) have investigated 9 grandfathers and 11 grandmothers carrying this mutation, as do their respective grandsons. Among the 9 maternal grandfathers, only 3 had hypertrophic cardiomyopathy, compared with none of the 11 grandmothers. These results should be compared with those reported in 2006 from Torino, Piedmont, Italy, by Spada et al. (2). They screened 37,000 consecutive newborns with similar methods and identified 12 infants with GLA mutations, including 11 who had molecular lesions that expressed residual activity consistent with the later-onset phenotype. The overall frequency of Fabry mutations was approximately 1 in 3100 Caucasian boys. Mutation analysis predicted that one of the newborns had the classic phenotype (1 in approximately 37,000), whereas 11 of the newborns were predicted to have the later-onset phenotype (1 in approximately 3400). The prevalence of the classic form is close to that found in previous estimations, whereas that of the later-onset phenotype seems to be higher than commonly thought. In Taiwan, the frequency was 2.5 times more frequent than in the Italian population. These studies raise many ethical and clinical issues. What can be said to the parents of neonates harboring a GLA mutation suggestive of a later-onset disease? The clinical consequences of some mutations, if any, cannot be predicted. The ethnic background should be taken into account. If clinical consequences can be expected, when to start evaluating the cardiac condition and when to consider enzyme replacement therapy, if necessary? What are the psychologic consequences of the screening on the mutation carrier and his/her family? The ethical issues raised by early detection and prediction of later-onset genetic Published online ahead of print. Publication date available at www.cjasn.org.

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

Summary Objective It is unknown why some patients with biochemical evidence of primary aldosteronism (PA) do not develop hypertension. We aimed to compare clinical and biochemical characteristics of normotensive and hypertensive patients with PA. Design and patients Retrospective comparison of 10 normotensive and 168 hypertensive patients with PA for office or ambulatory blood pressure, serum potassium, plasma aldosterone and renin concentrations; the aldosterone : renin ratio, and tumour size. Comparison of initial hormonal pattern and drop in blood pressure following adrenalectomy in five normotensive and nine hypertensive patients matched for age, sex and body mass index. Results The 10 normotensive patients were women and presented with hypokalemia or an adrenal mass. Age, plasma aldosterone and renin concentrations were similar in normotensive and hypertensive cases, but kalemia and body mass index were significantly lower in the normotensive patients. Mean tumour diameter was larger in the normotensive patients than in the hypertensive matched patients with an adenoma (P

Les mutations d’AIP predisposent aux adenomes hypophysaires, majoritairement somatotropes et chez le sujet jeune. Le role d’AIP dans le fonctionnement de l’axe somatotrope et la tumorigenese hypophysaire reste toutefois mal connu. Les souris Aip+/– constituent le seul modele physiopathologique in vivo decrit mais, en dehors de l’identification d’adenomes hypophysaires a l’histologie, leur phenotype a ete peu caracterise. Nous avons compare la croissance somatique, la composition corporelle et la secretion de GH de souris mâles sauvages et Aip+/– suivies longitudinalement de 3 a 12 mois. Le poids des jeunes souris Aip+/– est superieur a celui des souris sauvages, sans difference de taille naso-anale. Les animaux Aip+/– de 3 et 12 mois presentent une diminution de la masse grasse et une augmentation de la masse maigre suggerant un effet de la GH. L’analyse de la pulsatilite de GH sur 6 h a montre une secretion accrue chez les souris Aip+/– de 12 mois par rapport aux souris sauvages, sans difference entre les taux d’IGF-I. La secretion de GH induite par la GHRH ex vivo ainsi que l’expression des transcrits hypophysaires de GH et des transcrits hypothalamiques de GHRH sont plus elevees chez les souris Aip+/– de 3 mois que chez les souris sauvages. En conclusion, les souris Aip+/– ne presentent pas de gigantisme ni d’acromegalie evidents aux âges etudies. Contrairement a la pathologie humaine, le developpement tumoral est tardif. La reponse accrue a la GHRH ainsi que sa surexpression hypothalamique suggerent toutefois une hyperplasie somatotrope precedant l’apparition des adenomes, actuellement recherchee par immunohistochimie.

Aims/hypothesis Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. Methods 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1–10 twice/week. Results In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1–10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. Conclusions This work highlighted an important role of Ang2 in pancreatic vascular defects induced by hyperglycaemia.

Background Insulin-like growth factors (IGF-I and -II) are pleiotropic regulators of somatic growth and development in vertebrate species. Endocrine and paracrine effects of both hormones are mediated by a common IGF type 1 receptor (IGF-1R). Lethal respiratory failure in neonatal IGF-1R knockout mice suggested a particular role for this receptor in pulmonary development, and we therefore investigated the consequences of IGF-1R inactivation in lung tissue. Methods and findings We first generated compound heterozygous mutant mice harboring a hypomorphic (Igf1r(neo)) and a null (Igf1r(-)) allele. These IGF-1R(neo/-) mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1R(neo/-) mice, we assessed lung morphology and respiratory physiology and found normal histomorphometric characteristics and normal breathing response to hypercapnia. We then generated homozygous IGF-1R knockout mutants (IGF-1R(-/-)) and analyzed their lung development during late gestation using histomorphometric and immunohistochemical methods. IGF-1R(-/-) embryos displayed severe lung hypoplasia and markedly underdeveloped diaphragms, leading to lethal neonatal respiratory distress. Importantly, IGF-1R(-/-) lungs from late gestation embryos were four times smaller than control lungs and showed markedly thickened intersaccular mesenchyme, indicating strongly delayed lung maturation. Cell proliferation and apoptosis were significantly increased in IGF-1R(-/-) lung tissue as compared with IGF-1R(+/+) controls. Immunohistochemistry using pro-SP-C, NKX2-1, CD31 and vWF as markers revealed a delay in cell differentiation and arrest in the canalicular stage of prenatal respiratory organ development in IGF-1R(-/-) mutant mice. Conclusions/significance We found that low levels of IGF-1R were sufficient to ensure normal lung development in mice. In contrast, complete absence of IGF-1R significantly delayed end-gestational lung maturation. Results indicate that IGF-1R plays essential roles in cell proliferation and timing of cell differentiation during fetal lung development.

Objective. To determine the skin and fibroblast expression of ephrins (EphB4 and EphrinB2) and thrombospondins (TSPs: TSP1 and TSP2) in patients with SSc. Methods. All experiments were performed in skin sections and dermal fibroblasts issued from control and clinically involved/non-involved SSc skin biopsies. Dermal fibroblasts were stimulated with hypoxia or TGF-b, or treated with TGF-b-neutralizing antibodies. Ephrin and TSP mRNA levels were assessed in skin tissue and dermal fibroblasts by in situ hybridization and quantitative RTPCR, respectively, and protein levels were assessed by immunohistochemistry and western blots, respectively. Results. Enhanced ephrin and TSP mRNA and protein levels were observed in clinically involved SSc skin. EphrinB2, TSP1 and TSP2 mRNA and protein levels were also up-regulated in non-involved SSc skin. Similar mRNA and protein levels of ephrinB2 and EphB4 were detected in unstimulated and stimulated control and SSc dermal fibroblasts. TSP1 and TSP2 mRNA and protein levels were significantly increased in fibroblasts issued from involved and non-involved SSc skin. This up-regulation was not modified by hypoxic exposure, but was markedly reduced by the addition of TGF-b-neutralizing antibodies. Stimulation of healthy fibroblasts with TGF-b significantly increased TSP1 and TSP2 mRNA and protein levels. Conclusion. EphB4 and EphrinB2 are up-regulated in clinically involved skin of SSc patients, suggesting their participation in SSc-perturbed angiogenesis. TSP1 and TSP2 are up-regulated in both clinically involved and non-involved SSc skin and are constitutively overexpressed in a TGF-b-dependent and hypoxia-independent manner in SSc dermal fibroblasts, suggesting their potential early contribution in SSc pathogenesis.

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized by persistent fetal anuria and perinatal death. During the systematic screening of mutations of the different genes of the renin-angiotensin system associated with RTD, two missense mutations in the renin gene were previously identified, the first affects one of the two catalytic aspartates (D38N) of renin, and the second, S69Y, is located upstream of the 'flap', a mobile β-hairpin structure which covers the substrate-binding site of renin. Here we report a novel renin mutation leading to the duplication of the tyrosine residue Y15dup, homologous to Y9 in some other aspartyl proteases, which seems to play a crucial role along the activation pathway. The biochemical and cellular mechanisms underlying renin inactivation were investigated. We expressed prorenin constructs harboring the identified point mutations in two established cell lines, able (AtT-20 cells) or unable (CHO cells) to process prorenin to renin and we evaluated the cellular localization of renin mutants and their functional properties. All three mutants were misfolded to different levels, were enzymatically inactive and exhibited abnormal intracellular trafficking. We suggest a misfolding of Y15dup renin, a partial misfolding of D38N prorenin and a misfolding of S69Y prorenin leading to complete absence of secretion. The structural consequences of the renin mutations were estimated by molecular modeling, which suggested some important structural alterations. This is the first characterization of the mechanisms underlying loss of renin function in RTD.

The mineralocorticoid receptor (MR) is a major regulator of renal sodium reabsorption and body fluid homeostasis. However, little is known about glucocorticoid receptor (GR)-dependent renal effects. Glucocorticoids may activate both receptors, so it is difficult to distinguish between MR- and GR-mediated effects in vivo. To overcome this complexity, we used a transgenic mouse model allowing conditional GR overexpression (doxycycline inducible TetON system, Hoxb7 promoter) in the renal collecting duct (CD) to identify GR-regulated genes involved in sodium transport in the CD. In microdissected cortical CD, induction of GR expression led (after 2 d of doxycycline) to increased α-epithelial sodium channel and glucocorticoid-induced leucine zipper and decreased abundance of with-no-lysine kinase 4 transcripts, without modification of Na,K-ATPase, serum- and glucocorticoid-kinase-1, or MR expression. No changes occurred in the upstream distal and connecting tubules [distal convoluted tubule (DCT), connecting tubule (CNT)]. Sodium excretion was unaltered, but the urinary aldosterone concentration was reduced, suggesting compensation of transitory extracellular volume expansion that subsequently disappeared. At steady state, i.e. after 15 d of doxycycline administration, transcript abundance remained altered in the CD, whereas mirror changes appeared in the DCT and CNT. Plasma aldosterone or glucocorticoids and blood pressure were all unaffected. These experiments show that: 1) GR, in addition to MR, controls epithelial sodium channel- and glucocorticoid-induced leucine zipper expression in vivo in the CD; 2) with-no-lysine kinase 4 is negatively controlled by GR; and 3) the DCT and CNT compensate for these alterations to maintain normal sodium reabsorption and blood pressure. These results suggest that enhanced GR expression may contribute to enhanced sodium retention in some pathological situations.

The mineralocorticoid receptor (MR) is a transcription factor of the nuclear receptor family, activation of which by aldosterone enhances salt reabsorption in the kidney. The MR is also expressed in nonclassical aldosterone target cells (brain, heart, and skin), in which its functions are incompletely understood. To explore the functional importance of MR in mammalian skin, we have generated a conditional doxycycline-inducible model of MR overexpression, resulting in double-transgenic (DT) mice [keratin 5-tTa/tetO-human MR (hMR)], targeting the human MR specifically to keratinocytes of the epidermis and hair follicle (HF). Expression of hMR throughout gestation resulted in early postnatal death that could be prevented by antagonizing MR signaling. DT mice exhibited premature epidermal barrier formation at embryonic day 16.5, reduced HF density and epidermal atrophy, increased keratinocyte apoptosis at embryonic day 18.5, and premature eye opening. When hMR expression was initiated after birth to overcome mortality, DT mice developed progressive alopecia and HF cysts, starting 4 months after hMR induction, preceded by dystrophy and cycling abnormalities of pelage HF. In contrast, interfollicular epidermis, vibrissae, and footpad sweat glands in DT mice were normal. This new mouse model reveals novel biological roles of MR signaling and offers an instructive tool for dissecting nonclassical functions of MR signaling in epidermal, hair follicle, and ocular physiology.

Large deletions in WNK1 are associated with inherited arterial hypertension. WNK1 encodes two types of protein: a kidney-specific isoform (KS-WNK1) lacking kinase activity and a ubiquitously expressed full-length isoform (L-WNK1) with serine threonine kinase activity. Disease is thought to result from hypermorphic mutations increasing the production of one or both isoforms. However, the pattern of L-WNK1 expression remains poorly characterized. We generated transgenic mice bearing a murine WNK1 BAC containing the nlacZ reporter gene for monitoring L-WNK1 expression during development and adulthood. We observed previously unsuspected early expression in the vessels and primitive heart during embryogenesis, consistent with the early death of WNK1(-/-) mice. The generalized cardiovascular expression observed in adulthood may also suggest a possible kidney-independent role in blood pressure regulation. The second unsuspected site of L-WNK1 expression was the granular layer and Purkinje cells of the cerebellum, suggesting a role in local ion balance or cell trafficking. In the kidney, discordance between endogenous L-WNK1 and transgene expression suggests that either cis-regulatory elements important for physiological renal expression lie outside the BAC sequence or that illegitimate interactions occur between promoters. Despite this limitation, this transgenic model is a potentially valuable tool for the analysis of spatial and temporal aspects of WNK1 expression and regulation.

Introduction Au cours du diabete de type 2, nous suggerons qu’une alteration de la vascularisation pancreatique puisse participer au defaut d’augmentation compensatoire de la masse des cellules b. Nous avons precedemment mis en evidence que l’hyperglycemie provoque des anomalies de la vascularisation pancreatique, associees a une surexpression de l’angiopoietine 2 (Ang2). Pour determiner le role de cette augmentation d’Ang2, nous avons bloque son action en situation ou non d’hyperglycemie et etudie la vascularisation pancreatique. Materiels et Methodes Nous travaillons dans un modele de greffe interspecifique : du pancreas d’embryon de poulet E14 est greffe sous la capsule renale de souris SCID normoglycemiques (NG) ou de souris rendues hyperglycemiques par injection de streptozotocine (HG), puis est recupere apres 2 semaines. L’inhibition de l’Ang2 a ete obtenue par injection de L1-10 (2x/sem, s.c. ; proteine de fusion, don de AMGEN). Resultats En NG, l’expression d’Ang2 est quasi inexistante, bloquer son action n’altere pas l’architecture pancreatique, ni insulaire ni vasculaire. En HG, l’expression d’Ang2 est fortement augmentee (x2,5 p Conclusion Ainsi, nous mettons en evidence un role majeur de l’expression de l’Ang2 dans les alterations de la vascularisation du pancreas diabetique dans ce modele. En bloquant l’action de l’Ang2, nous avons corrige la desorganisation vasculaire pancreatique induite par l’hyperglycemie et augmente la densite de cellules b.