AngiotensinIIType2receptor (AT2R) deficiency in AT2R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2RKO mice were used to assess glomerulogenesis, while wild-type and AT2RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2R deficiency resulted in diminished glomerulogenesisin E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFβ1–Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes.
After social stress, rats become vulnerable to depression, and this state is characterized by persistent low blood levels of brain-derived neurotrophic factor (BDNF). The aim of this study was to determine whether low BDNF levels are associated with long term autonomic changes. Defeated animals were subjected to four daily episodes of social defeats. Twenty five days later, defeated rats with low BDNF levels (Dlow) still displayed elevated sympathetic tone (as indicated by an elevated low frequency to high frequency ratio (LF/HF) in heart rate) and elevated blood pressure, as well as reduced baroreflex sensitivity (BRS). In contrast, those with higher BDNF levels (Dhigh) similar to controls, did not. Dlow animals persistent cardiovascular changes were abolished by acute inhibition of the dorsomedial nucleus of the hypothalamus (DMH). These cardiovascular changes were also prevented by chronic sub-cutaneous osmotic infusion of losartan, an angiotensinIItype 1 receptor (AT 1 ) receptor antagonist, started immediately after social defeat. In conclusion, the results show that greater vulnerability to stress consequences following a traumatic event is associated with an elevated LF/HF ratio, a persistent high blood pressure and a low BRS, all due to an AT 1 receptor activation.
Improved muscle blood flow
An inverse relationship was observed between the blood pressure-lowering effect of captopril and the
improvement in insulin-mediated whole-body glucose disposal . The notion that increased muscular blood flow is associated with increased glucose utilisation has considerable bearing on the proposed haemodynamic theory of insulin resistance [21-23]. Several reviews provide evidence that haemodynamic factors influence insulin sensitivity in subjects with essential hypertension  or with diabetes . Consequently, vasodilatation or a specific increase in blood flow to skeletal muscles would lead to increases in glucose and insulin delivery to insulin-sensitive tissues and cause an increase in glucose utilisation . It has been shown that the ACEI quinapril may significantly improve vascular sensitivity to insulin via global enhancement of endothelial function , and endothelial dysfunction is associated with obesity, insulin resistance, hypertension and type2 diabetes [27, 28]. This haemodynamic hypothesis is further supported by the demonstration that ATII has no direct metabolic effect on skeletal muscle glucose metabolism , thereby implicating haemodynamic factors as causative of the changes in glucose utilisation and insulin sensitivity observed after ATII inhibition . However, it has been shown that ATII induces insulin resistance independent of changes in interstitial insulin , a finding that may suggest that other effects independent of vascular changes could also play a role. ACEIs enhance blood flow through the microcirculation of skeletal muscles although the respective roles of ATII inhibition and bradykinin enhancement remain controversial [32, 33]. Bradykinin seems to play a significant role in arterial blood pressure regulation after RAS inhibition as bradykinin receptor blockade could partially reverse the blood pressure lowering effect of ACEI in normotensive and hypertensive subjects . Infusion of exogenous bradykinin was shown to improve glucose utilisation by forearm muscles and enhance insulin sensitivity in humans . It is now apparent that bradykinin functions as a circulating hormone with a short half-life of a few seconds and as an autocrine-paracrine factor generated and acting locally within various tissues . It is generally accepted that almost all physiologically significant actions of bradykinin are mediated by bradykinin type2 receptors [32, 33, 36]. The increased insulin-mediated glucose uptake by skeletal muscle in response to an ACEI appears to be due to increased bradykinin-mediated nitric oxide production and not to reductions in ATII production or action [37, 38], a finding that may explain the lower effects of ARBs as compared to ACEIs on insulin sensitivity in hypertensive patients observed in some trials [39, 40]. It is
Other key mediators of growth are the extracellular sig- nal-regulated kinases (ERKs) 1/2. ERK1/2 belong to the MAPK family and lie downstream of the cascade of Ras/ Raf/MEK kinases. The nuclear translocation of ERK1/2 is a critical step to transduce cell growth (Brunet et al., 1999). In their phosphorylated and activated forms, ERK1/2 transmit extracellular stimuli from the plasma membrane to the nu- cleus by phosphorylating and activating a variety of tran- scription factors. Among them, Elk-1 is a key element in- volved in the induction of immediate early genes such as cFos (Pearson et al., 2001; Peyssonnaux and Eychene, 2001). The stimulation of both pathways by many common li- gands raises the possibility that cross-talk between the PI3K/Akt and ERK1/2 pathways could play a major role in regulating cell proliferation under particular conditions. Functional interactions between these two pathways have been reported for the regulation of various cellular functions depending on cell types. Indeed, constitutively active Akt can negatively regulate the Ras/Raf/MEF/ERK1/2 cascade via phosphorylation and inactivation of the kinase Raf (Zimmermann and Moelling, 1999), leading to the pheno- type modulation of differentiated myotubes (Rommel et al., 1999) or of vascular smooth muscle cells (Reusch et al., 2001). An additional level of interaction between Akt and the ERK1/2 pathway has been reported downstream of Ras, Raf, and MEK that involves the down-regulation of the
MHz spectrometer at ambient temperature. Spectral data are reported in parts per million (ppm) using residual solvent as a reference. High resolution and accurate mass measurements are acquired in positive mode by flow injection analysis into a Thermo Scientific Q-Exactive Plus Orbitrap Mass Spectrometer (San Jose, CA, USA) interfaced with a heated electrospray ion source. Sep-Pak C18 plus (360 mg, Waters) solid-phase extraction cartridges were pre- conditioned with 10 mL ethanol followed by 20 mL water. Analytical HPLC was performed on a Phenomenex Luna C18 (2) column (250×4.6 mm, 10μm) with a Waters HPLC composed of 1515 isocratic pump, 2487 dual λ absorbance detector and a Raytest Gabi Star radioactivity detector. Two analytical methods were utilized: Method 1 (2 mL/min, A: water and B: CH 3 CN, linear gradient
Figure 1. Endogenous AT1Rs and AT2Rs localize to the nucleus in canine atrial ﬁbroblasts. A, Cultured ﬁbroblasts were fractionated by differential centrifugation and the purity of membrane, cytosolic, and nuclear fractions (50 lg) were validated by immunoblotting for pan- cadherin, HSP70, and lamin B (n=5 separate experiments). The presence of AT1Rs and AT2Rs was determined by immunoblotting. The positions of molecular weight markers are indicated at the right (in kDa). B, Subfractionation of isolated ﬁbroblast nuclei was performed to separate outer (ONM) and inner (INM) nuclear membranes and nucleoplasm (NP). Nesprin, emerin, and HDAC2 served as markers of the ONM, INM/lamina and nucleoplasm, respectively. Immunoblotting detected AT1Rs and AT2Rs in nuclear subfractions. The positions of molecular weight markers are indicated at the right (in kDa). C, Isolated nuclei were plated on laminin-coated 18 mm coverslips and then incubated with 100 nmol/L FITC- Ang-II at room temperature or preincubated with valsartan (1 lmol/L) and/or PD123177 (1 lmol/L) for 30 minutes prior to the addition of FITC-Ang-II. Nuclei were washed and stained with ﬂuorescent DNA dye DRAQ5. Images were acquired with an Olympus FluoView FV1000 confocal microscope. D, Quanti ﬁcation of bound FITC-Ang-II ﬂuorescence, meanSEM. **P<0.01, ***P<0.001, by 1-way ANOVA with Bonferroni-adjusted t test. N=5/condition. Ang-II indicates angiotensin-II; AT1Rs, Ang-IItype 1 receptors; FITC, ﬂuorescein isothiocyanate; HDAC2, histone deacetylase-2; INM, inner nuclear membrane; ONM, outer nuclear membrane.
infusion). Ang II-treated rats received the synthetic com- pound PD98059 at doses of 1 mg/kg (n = 6), 5 mg/kg (n = 7) and 10 mg/kg (n = 6). A group of control rats received 10 mg/kg PD98059 (n = 4). In a second set of experi- ments, rapamycin was injected at doses of 0.1 mg/kg (n = 8), 0.5 mg/kg (n = 6) and 1 mg/kg (n = 3) in Ang II-treated rats. Six control rats received 0.5 mg/kg rapamycin. In a third series, Ang II-treated rats received irbesartan, a selec- tive AT-1 receptor blocker, at doses of 10 mg/kg (n = 9), 30 mg/kg (n = 5) and 40 mg/kg (n = 5), following the same experimental protocol. Additional rats were treated with irbesartan according to a different treatment scheme: Irbesartan was administered subcutaneously at the time of surgery and 12 hours later (10 mg/kg at each occasion) in Ang II-treated (n = 8) or in control rats (n = 6). We used two sets of control and Ang II-treated rats to confirm the reproducibility of the method. The first set (n = 10 and 10, respectively) was studied simultaneously with PD98059 and irbesartan groups. The second set (n = 7 and 9, respec- tively) was studied in parallel with the rapamycin experi- ments. Additional control (n = 3) and Ang II-treated (n = 3) rats were sacrificed 21 hours after the beginning of Ang II administration, to determine ERK-1/2 phosphorylation at the time when PD98059 was normally injected. Finally, in 3 control and 3 rats treated for 5 hours with PD98059, we confirmed the in vivo effectiveness of PD98059 to reduce basal ERK-1/2 phosphorylation (data not shown). Mean arterial pressure (MAP) was continuously measured intra-arterially in freely moving rats 15 minutes before and averaged for the 5 hours following drug administra- tion. The animals were then anesthetized (pentobarital 35 mg/kg i.v.) and exsanguinated. The thoracic aorta and the mesenteric vascular bed were collected and immediately transferred in a modified cold Krebs-Ringer bicarbonate solution (composition in mmol/L: NaCl 118.6; KCl 4.8; CaCl 2 2.5; MgSO 4 1.2; KH 2 PO 4 1.2; NaHCO 3 25.1; Na +
antigen/receptor protein phosphatase mu) et frizzled (figure 1.6). Bien que le rôle du dom aine SEA p ré se n t chez les TTSP n e soit p a s co n n u , ce d o m a in e est g é n é ra le m e n t associé à des p ro té in e s h a u te m e n t glycosylées q u i c o n tie n n e n t des glucides liés p a r d es liaisons O- glycosidiques tel le sulfate d ’h é p aran e (Bork e t Patthy, 1995). Ceux-ci p o u rra ie n t réguler o u assister à la liaison avec les g ro u p es fo n c tio n n e ls d e glucides p ré se n ts su r d es p ro té in e s de le u r voisinage (Bork e t Patthy, 1995). Ces d o m a in e s s o n t aussi retro u v és clivés chez de n o m b re u ses p ro té in e s tra n sm e m b ra n a ire s (Abe, Fukuzaw a et H irose, 2002) p o u v a n t g én érer u n e alliance lig a n d -ré c ep te u r lo rsq u e les so u s-u n ité s clivées se ré a sso c ie n t p o u r conduire à u n e cascade signalétique (W reschner et a l, 2002). Dans le cas d u d o m a in e SRCR, sa fo n ctio n biologique p récise reste à être d é term in é e, m ais des év id ences su g g èren t q u ’il p o u rrait p e rm e ttre des in teractio n s p ro téin e-p ro téin e, h o m o - ou h étéro ty p iq u es, ain si q ue la reco n n aissan ce des m otifs m oléculaires associés aux path o g èn es (PAMP) d an s la défense im m u n itaire (M artinez e t a l, 2011). P o u r ce qu i e st des d o m a in e s LDLRA, ce s o n t des dom aines riches en cystéines d ’environ 40 acides am inés et qu i p ossèden t u n m otif conservé D/NXSDE d a n s lequel les résidus acid e a sp a rtiq u e et a cid e g lu tam iq u e so n t im p liq u és d a n s la liaison d u calcium . Ces d o m a in e s sem b lera ie n t jo u e r d es rôles d a n s la liaison, l’in tern a lisatio n et la m a tu ra tio n des p ro té in e s q u i p o s sè d e n t ces d o m a in e s (H orn et a l , 1998; C am et Bu, 2006; Parkyn e t a l, 2008). Les d o m a in e s CUB so n t p re sq u e exclusivem ent retrouvés chez des p ro téin es extracellulaires o u associées à la m em b ran e cellulaire (Bork et B eckm ann, 1993). Ces d o m ain es so n t im p liq u és d a n s l’ad h ésio n in term o lécu laire (Bork et Beckm ann, 1993). Par exemple, il a été d é m o n tré q u e la cubuline, qu i c o n tie n t 27 d o m ain es CUB, p e u t se lier à la transferrine, à l’h ém o g lo b in e, a u fa c teu r in trin sè q u e d e la v itam ine Bi 2 , à l’ap o lip o p ro téin e Ai et à l’HDL (high-density lipoprotein) (Amsellem e t a l, 2010). Les
Arterial hypertension is intimately associated with type2 diabetes mellitus (T2DM) . Both elevated arterial pressure and impaired glucose tolerance (IGT) are key-components of the metabolic syndrome ("syndrome X" or insulin resistance syndrome), a leading cause of cardiovascular morbidity and mortality [2, 3]. Patients with essential hypertension have been shown to have a higher risk of developing T2DM than non-hypertensive individuals in several large prospective studies on various populations [4, 5]. Antihypertensive agents may exert negative, neutral or even positive metabolic effects that may diversely affect the risk of developing T2DM . The renin-angiotensin system (RAS) is a coordinated hormonal cascade in the control of cardiovascular, renal, and adrenal function. It governs fluid and electrolyte balance and blood pressure regulation, but may also exert various, although still poorly understood, cellular effects . RAS inhibition plays a key-role in cardiovascular pharmacology. Pharmacological agents include angiotensin-converting enzyme inhibitors (ACEIs), that block the conversion of pro-hormone angiotensin I to active hormone angiotensinII, and selective angiotensinIIreceptor-1 blockers (ARBs). Both compounds are now widely used as first-line antihypertensive agents in patients with diabetes mellitus, essentially because of their renal protection effect .
HEK293 cells (as negative controls) were cultured for 2 days in T10 plates. Then, cells were lysed in 1 ml of cold 50 mM Tris (pH 6.8) buffer containing 0.15 M NaCl, 1% (v/v) Triton ×100, 0.1% (v/v) SDS, 1 mM EDTA, 0.1 mM neocuproine and protease inhibitor cock- tail (EDTA ‐free Roche). Briefly, S‐nitrosation was performed at 37°C on a wheel for 1 hr by 1 mM GSNO (or PBS as control). After a 10,000× g centrifugation for 15 min at 4°C, the free thiols in cell lysate supernatants were blocked with 50 mM of N ‐ethylmaleimide for 50 min at 40°C. Samples were precipitated by acetone. Pellets were then incubated with 10 mM sodium ascorbate to liberate thiols from the S –NO bonds, and free new thiols were immediately biotinylated. As negative controls, sodium ascorbate was omitted in one sample treated or not with GSNO. Under such conditions, S –NO bonds are not reduced and S ‐nitrosated proteins cannot be labelled by the biotin ‐HPDP. Excess biotin‐HPDP was removed by acetone precipita- tion. Biotin ‐HPDP‐labelled proteins were purified with High Capacity NeutrAvidin beads overnight on a wheel at 4°C. The following day, NeutrAvidinAgarose beads were washed three times in a high ‐salt containing buffer (50 mM Tris pH 7.5, 0.6 M NaCl, 1% [v/v] Triton ×100, 0.1% [v/v] SDS, 1 mM EDTA, 0.1 mM neocuproine). Beads were suspended in a 2× Laemmli buffer (25 mM Tris pH 6, 8, 3 M urea, 1.5% SDS, 3% [v/v] 2 ‐mercaptoethanol) vortexed for 15 min at room tem- perature, heated for 20 min at 37°C, centrifuged for 3 min at 10,000× g. Biotin ‐HPDP‐labelled proteins eluted from the beads were separated in 10% acrylamide gels then transferred onto PVDF mem- brane for 2 hr. The detection of the EGFP ‐AT 1 receptor proteins
AT1R mRNA Expression
Total RNA was extracted from PBMC using TRIzol reagent (Life technologies) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcripted with moloney murine leukemia virus reverse transcriptase (Invitrogen) and deoxyribonucleic random hexamer as primers (Invitrogen). Real-time gene expres- sion analysis of AT1 receptor was performed with predesigned TaqMan Gene Expression Assay (Hs00258938_m1; Life technolo- gies) using the StepOnePlus Real-Time polymerase chain reaction System (Applied Biosystems). AT1 receptor mRNA expression was normalized to HPRT1 mRNA expression (Hs02800695_1) used as housekeeping control gene. Cycle threshold and relative quantification by the 2 −Ct method values were determined using the
assays. The PEI-mediated sTGFβRII delivery remarkably attenuated TGFβ1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFβRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFβRII overexpression by either method was significantly higher than the naked vector transfection. Fur- thermore, PEI- or Lipofectamine-mediated sTGFβRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2–6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death.
The electronic properties of MoTe 2in the orthorhombic
Pmn2 1 (31) phase have been calculated using DFT with the generalized gradient approximation (GGA) using the full- potential linearized augmented plane-wave method  with local-orbital extensions  in the WIEN 2k implementation . The unit cell parameters have been adjusted and the total energy calculated both with and without spin-orbit coupling; while spin-orbit coupling lowers the total energy, it does not significantly affect the structural refinement. Once the unit cell has been optimized, the atomic fractional coordinates are then relaxed with respect to the total force (spin-orbit coupling is not considered in this step), typically resulting in residual forces of less than 0.2 mRy /a.u. per atom. This procedure is repeated until no further improvement is obtained. The elec- tronic band structure has been calculated from the optimized geometry with GGA and spin-orbit coupling.
Supplementary material for “Low-energy excitations intype-II Weyl semimetal T d -MoTe 2 evidenced through optical conductivity”
D. Santos-Cottin, 1, ∗ E. Martino, 1, 2 F. Le Mardelé, 1 C. Witteveen, 3, 4 F. O. von Rohr, 3, 4 C. C. Homes, 5 Z. Rukelj, 1, 6 and Ana Akrap 1, †
This study has two major strengths. Firstly, it is to our knowledge the first to assess the effect of health behaviours and body mass index on socioeconomic differences intype2 diabetes by using different assessments of current and long term exposure to these factors over the follow-up. This allowed us to examine the possibility that changes in these factors over the study period or a long term exposure might have yielded different results. Secondly, unlike previous studies, we provide a confidence interval around the percentage attenuation of the association between socioeconomic status and type2 diabetes after inclusion of the risk factors examined. Adding a degree of precision to the estimates of the contribution of risk factors to social inequalities greatly helps with the interpretation of these findings.
A magneto-optical imaging (MOI) system capable to resolve single vortices is combined with a focused laser beam to reorganize vortex matter in dense
vortex clusters. The local heating of the superconductor with the laser produces a temperature profile which induces an attraction of the vortices towards the center of the laser spot. We analyze the collective vortex dynamics under high-power laser irradiation. The formation of vortex clusters is described with a model very similar to the one describing the first vortex entry into a type-II superconductor.
Saisir = ECARTYPEP(B2:G2) dans la cellule I2 et recopier vers le bas jusque I4 (et pas plus loin).
Remarque : pour calculer un écart-type, on doit d’abord calculer une variance. La moyenne de Robert-Nadine est : ̅ = ≈ 170,83
La variance correspond à la moyenne des carrés des écarts à la valeur moyenne… C'est-à-dire, pour Robert-Nadine : V 1 = 148 − ̅ + 137 − ̅ + 166 − ̅ + 165 − ̅ + 209 − ̅ + 200 − ̅ ≈
tance between the tip and the sample surface (see Fig. 1).
Note that the current j defined by (1) can be formally repre- sented as the sum of the external uniform current j 0 which does not induce the magnetic field and the deviation dj which has the form of the vortex-antivortex pair [see Fig. 2(b)]. Substituting the expression for dj into the Biot-Savart law and integrating over the sample volume we obtain the analytical expression for the magnetic field component B z . Before
40 iv. Tubular development. At the urinary pole, the Bowman capsule cells of the glomerulus are directly connected to the tubular portion of the nephron.
- Tubular expansion. Tubular expansion starts in the distal and mid limb of the S-shape body (SSB). The distal part of SSB (distal limb and distal part of the mid limb) is characterized by the expression of Brn1, a POU-domain transcription factor, which plays an essential role in the formation of the Henle’s loop (HL), distal convoluted tubule (DCT) and the macula densa. It has been shown that mutant mice for Brn1 present a defect in the HL elongation and a decreased expression of typical genes of this subsegment, such as Umod and Nkcc2 encoding a transporter essential for the reabsorption of NaCl in the TAL of the nephron (Nakai et al., 2003). The mid limb of the SSB is characterized by the expression of the Notch ligands Dll1 and Jag1(Georgas et al., 2008). Activation of Notch pathway starts with the binding of one of the ligands to Notch receptors. This binding induces a first cleavage by a metalloprotease ADAM10 (van Tetering et al., 2009). The cleaved receptor is then internalized and undergoes a second cleavage by the γ secretase enzyme, which releases the Notch intracellar domain (NICD) (De Strooper et al., 1999). The NICD is then ready to translocate into the nucleus and activate its relative target genes. Analysis of mouse mutant for Notch2 and Notch1 has shown that Notch2 plays a predominant role in proximal fate specification whereas Notch1 is more dispensable, although it participates to nephrogenesis (Cheng et al., 2007; Surendran et al., 2010). For example, in Notch2-deficient kidney, specific markers of proximal tubules, such as cadherin6, are not expressed, attesting a defective acquisition of the proximal tubular identity (Cheng et al., 2007). The Notch ligands, Dll1 and Jag1, expressed in the mid limb, have also an important role during proximal specification. The analysis of different models of compound mutant for Dll1 and Jag1 demonstrated that Dll1 deletion in presence of Jag1 leads to a mild defect in proximal tubules development, whereas the absence of Jag1 expression, even in presence of Dll1, leads to a more drastic defect. These results supported the predominant role for Jag1 in the proximal nephron specification (Liu et al., 2013).
OR mRNA EXPRESSION IN THE POSTERIOR SEGMENT
The primer specificity was checked using total RNA isolated from human testis and from endometrial Ishikawa cells and breast adeno- carcinoma MCF-7 cells (data not shown). ORá and ORâ primers amplified a 234 bp and a 258 bp fragment, respectively. OR á and ORâ mRNA expression was detected in ocular tissues from patients of di Verent age and sex, regardless of the region of the sample (inside or outside the macula) (Fig 3). The expression of OR â mRNA was relatively constant between di Verent donors, while there was more varia- tion with OR á. This variation was also observed when RT-PCR was performed using total RNA selectively extracted from neural Figure 2 Human retina contained a band of oestrogen receptor á and â (relative