Maud Clemessy

Dominant Renin Gene Mutations Associated with Early-Onset Hyperuricemia, Anemia, and Chronic Kidney Failure

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 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.

Knockout of Insulin-Like Growth Factor-1 Receptor Impairs Distal Lung Morphogenesis

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 (Igf1rneo) and a null (Igf1r−) allele. These IGF-1Rneo/− mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1Rneo/− 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.

Clinical and biochemical characteristics of normotensive patients with primary aldosteronism: a comparison with hypertensive cases

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.

Angiopoietin 2 Alters Pancreatic Vascularization in Diabetic Conditions

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.

IGF-1 Induces GHRH Neuronal Axon Elongation during Early Postnatal Life in Mice

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 postnatal period with programming of the growth trajectory.

Impact of insulin on primary arcuate neurons culture is dependent on early-postnatal nutritional status and neuronal subpopulation

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.

Regulation of growth: Epigenetic mechanisms?

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.

More on Clinical Renal Genetics

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.