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The identification of patients at increased risk for chronic kidney disease offers the potential to prevent or delay end-stage renal disease and the associated cardiovascular events. Data from recently completed controlled clinical trials of endothelin (ET) receptor blockers confirmed their potent antiproteinuric effect after a number of preclinical studies. A spectrum of proteinuric glomerular diseases results from podocyte abnormalities and, in return, impact podocyte structure and function. Because podocytes are cells in the glomerulus that form a crucial component of the glomerular filtration barrier, contributing to size selectivity and maintaining a large filtration surface, we focus on evidence that suggest ET-1 may promote podocyte injury thereby aggravating albumin urinary loss and alteration of the glomerular microvasculature. Systematic confrontation of animal models and studies in human subjects should help decipher pathophysiological mechanisms whereby the local renal ET system promotes podocyte injury and chronic kidney disease in specific pathophysiological contexts. Current evidence suggests that more experimental and clinical attention should be paid to conditions with increased vascular or endocapillary ET-1 production on the one hand, and in diseases with altered podocyte phenotype and survival such as focal segmental glomerulosclerosis and crescentic glomerulonephritis on the other. These conclusions may assist clinicians in creating optimal clinical trials for patients at increased risk for or with overt chronic kidney disease. more...

Sickle cell disease (SCD), the first 'molecular disease' to be identified, has been well characterized as a single amino acid molecular disorder of hemoglobin leading to its pathological polymerization, with resulting red cell rigidity that causes poor microvascular blood flow, with consequent tissue ischemia and infarction. Thus, the manifestations of SCD chronic renal disease have long been considered clinical manifestations of an obstructive vasculopathy of the arterial and capillary microcirculation. Recently, accumulating evidence have indicated that blood vessel functions are affected by SCD, involving abnormal vascular tone and activated endothelium. These abnormalities are particularly prominent in the kidney where specific biochemical conditions in the medulla and papilla favor change in endothelial phenotype and in tubular phenotype that, in turn, may promote dysfunction and destruction of this organ through active endothelin (ET)-1 production and signals. High ET-1 urinary output in SCD subjects at steady state may reflect increased tubular activation of ET-1 production acting on the collecting duct thereby favoring the constant hyposthenuria. Chronically, augmented ET-1 concentrations in the SCD kidney would further aggravate ischemia and sickling through actions on vasa recta and red blood cells. The kidneys suffer multiple ischemic hits during SCD as consequences of vasos-occlusive crisis (VOC). Blockade of ET receptors unraveled the major vasoconstrictive role of ET-1 in the pathophysiology of VOC, stressing the pivotal role of abnormal endothelial phenotype in this hemoglobinopathy and opening potential new therapeutic options. At last, indirect evidence suggest that ET-1 may be involved in the progression of chronic glomerulosclerosis affecting a number of patients. In fact, sickle cell nephropathy is an emerging severe disease that requires pathophysiological studies and development of specific therapies. more...

We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by varying the oxygen partial pressure within arrays of microdroplets in flowing oil. By etching holes into the channel's top surface, droplets can be held stationary while maintaining an external flow of oil. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous microdroplet ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated within drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers (Abbyad et al., Lab Chip, 2010, 10, 2513). We demonstrate in particular how the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological blood flow. Droplet content is varied to study the effect of different biochemical or therapeutic agents on sickling. more...

Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN. more...

The identification of patients at increased risk for chronic kidney disease offers the potential to prevent or delay end-stage renal disease and the associated cardiovascular events. Data from recently completed controlled clinical trials of endothelin (ET) receptor blockers confirmed their potent antiproteinuric effect after a number of preclinical studies. A spectrum of proteinuric glomerular diseases results from podocyte abnormalities and, in return, impact podocyte structure and function. Because podocytes are cells in the glomerulus that form a crucial component of the glomerular filtration barrier, contributing to size selectivity and maintaining a large filtration surface, we focus on evidence that suggest ET-1 may promote podocyte injury thereby aggravating albumin urinary loss and alteration of the glomerular microvasculature. Systematic confrontation of animal models and studies in human subjects should help decipher pathophysiological mechanisms whereby the local renal ET system promotes podocyte injury and chronic kidney disease in specific pathophysiological contexts. Current evidence suggests that more experimental and clinical attention should be paid to conditions with increased vascular or endocapillary ET-1 production on the one hand, and in diseases with altered podocyte phenotype and survival such as focal segmental glomerulosclerosis and crescentic glomerulonephritis on the other. These conclusions may assist clinicians in creating optimal clinical trials for patients at increased risk for or with overt chronic kidney disease. more...

Sickle cell disease (SCD), the first 'molecular disease' to be identified, has been well characterized as a single amino acid molecular disorder of hemoglobin leading to its pathological polymerization, with resulting red cell rigidity that causes poor microvascular blood flow, with consequent tissue ischemia and infarction. Thus, the manifestations of SCD chronic renal disease have long been considered clinical manifestations of an obstructive vasculopathy of the arterial and capillary microcirculation. Recently, accumulating evidence have indicated that blood vessel functions are affected by SCD, involving abnormal vascular tone and activated endothelium. These abnormalities are particularly prominent in the kidney where specific biochemical conditions in the medulla and papilla favor change in endothelial phenotype and in tubular phenotype that, in turn, may promote dysfunction and destruction of this organ through active endothelin (ET)-1 production and signals. High ET-1 urinary output in SCD subjects at steady state may reflect increased tubular activation of ET-1 production acting on the collecting duct thereby favoring the constant hyposthenuria. Chronically, augmented ET-1 concentrations in the SCD kidney would further aggravate ischemia and sickling through actions on vasa recta and red blood cells. The kidneys suffer multiple ischemic hits during SCD as consequences of vasos-occlusive crisis (VOC). Blockade of ET receptors unraveled the major vasoconstrictive role of ET-1 in the pathophysiology of VOC, stressing the pivotal role of abnormal endothelial phenotype in this hemoglobinopathy and opening potential new therapeutic options. At last, indirect evidence suggest that ET-1 may be involved in the progression of chronic glomerulosclerosis affecting a number of patients. In fact, sickle cell nephropathy is an emerging severe disease that requires pathophysiological studies and development of specific therapies. more...

We thank Nahmod et al1 for their interest in our studies and for their intriguing experiments with dendritic cells that corroborate and complement our findings. One limitation of the bone marrow transfer strategy is that this approach does not elucidate which specific mononuclear cell populations are mediating blood pressure elevation and/or tissue damage. Therefore, experiments like those conducted by Nahmod et al1 will be crucial in defining the precise roles of angiotensin II type 1 (AT1) receptors on individual immune cell lineages in regulating inflammation. Reconciling the results of the diverse studies exploring the functions of AT1 receptors on immune cells is indeed challenging, … more...

Activation of type 1 angiotensin (AT1) receptors causes hypertension, leading to progressive kidney injury. AT1receptors are expressed on immune cells, and previous studies have identified a role for immune cells in angiotensin II–dependent hypertension. We, therefore, examined the role of AT1receptors on immune cells in the pathogenesis of hypertension by generating bone marrow chimeras with wild-type donors or donors lacking AT1Areceptors (BMKO). The 2 groups had virtually identical blood pressures at baseline, suggesting that AT1receptors on immune cells do not make a unique contribution to the determination of baseline blood pressure. By contrast, in response to chronic angiotensin II infusion, the BMKOs had an augmented hypertensive response, suggesting a protective effect of AT1receptors on immune cells with respect to blood pressure elevation. The BMKOs had 50% more albuminuria after 4 weeks of angiotensin II–dependent hypertension. Angiotensin II–induced pathological injury to the kidney was similar in the experimental groups. However, there was exaggerated renal expression of the macrophage chemokine monocyte chemoattractant protein 1 in the BMKO group, leading to persistent accumulation of macrophages in the kidney. This enhanced mononuclear cell infiltration into the BMKO kidneys was associated with exaggerated renal expression of the vasoactive mediators interleukin-1β and interleukin-6. Thus, in angiotensin II–induced hypertension, bone marrow–derived AT1receptors limited mononuclear cell accumulation in the kidney and mitigated the chronic hypertensive response, possibly through the regulation of vasoactive cytokines. more...

Collagen is a major protein of the extracellular matrix that is characterized by triple helical domains. It plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) so that SHG microscopy proved to be a sensitive tool to probe the three-dimensional architecture of fibrillar collagen and to assess the progression of fibrotic pathologies. We obtained sensitive and reproducible measurements of the fibrosis extent, but we needed quantitative data at the molecular level to further process SHG images. We therefore performed Hyper- Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its aminoacid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro- Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagenous biomimetic matrices. more...

Low concentrations of reactive oxygen species, notably hydrogen peroxide (H2O2), mediate various signalling processes in the cell1,2. Production of these signals is highly regulated3 and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H2O2 generation in living cells. The Y0.6Eu0.4VO4 nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H2O2 with a 30-s temporal resolution and a dynamic range of 1–45 µM. The differences in the timing of intracellular H2O2 production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H2O2, in many signalling processes H2O2 is, however, the dominant oxidant3,4,5,6. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species. The dynamic production of oxidants inside cells can be quantified by a probe based on a single nanoparticle, offering a tool to monitor and understand how cells regulate their signalling responses. more...

International audience; Interstitial fibrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I fibrillar collagen, a protein scarce in normal interstitium. However, assessment of fibrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation SHG microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial fibrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of fibrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from fibrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonfibrillar collagens, our work supports pathophysiological differences between interstitial fibrosis and glomerulosclerosis, a clearly nonfibrotic process. © 2008 Society of Photo-Optical Instrumentation Engineers more...

Activation of the renin-angiotensin system contributes to the progression of chronic kidney disease. Based on the known cellular effects of ANG II to promote inflammation, we posited that stimulation of lymphocyte responses by ANG II might contribute to the pathogenesis of hypertensive kidney injury. We therefore examined the effects of the immunosuppressive agent mycophenolate mofetil (MMF) on the course of hypertension and kidney disease induced by chronic infusion of ANG II in 129/SvEv mice. Although it had no effect on the severity of hypertension or cardiac hypertrophy, treatment with MMF significantly reduced albuminuria and ameliorated kidney injury, decreasing glomerulosclerosis and reducing lymphocyte infiltration into the renal interstitium. Attenuation of renal pathology with MMF was associated with reduced expression of mRNAs for the proinflammatory cytokines interferon-γ and tumor necrosis factor-α and the profibrotic cytokine transforming growth factor-β. As infiltration of the kidney by T lymphocytes was a prominent feature of ANG II-dependent renal injury, we carried out experiments examining the effects of ANG II on lymphocytes in vitro. We find that exposure of splenic lymphocytes to ANG II causes prominent rearrangements of the actin cytoskeleton. These actions require the activity of Rho kinase. Thus, ANG II exaggerates hypertensive kidney injury by stimulating lymphocyte responses. These proinflammatory actions of ANG II seem to have a proclivity for inducing kidney injury while having negligible actions in the pathogenesis of cardiac hypertrophy. more...

Patients with sickle-cell disease (SCD) suffer from tissue damage and life-threatening complications caused by vasoocclusive crisis (VOC). Endothelin receptors (ETRs) are mediators of one of the most potent vasoconstrictor pathways in mammals, but the relationship between vasoconstriction and VOC is not well understood. We report here that pharmacological inhibition of ETRs prevented hypoxia-induced acute VOC and organ damage in a mouse model of SCD. An in vivo ultrasonographic study of renal hemodynamics showed a substantial increase in endothelin-mediated vascular resistance during hypoxia/reoxygenation-induced VOC. This increase was reversed by administration of the dual ETR antagonist (ETRA) bosentan, which had pleiotropic beneficial effects in vivo. It prevented renal and pulmonary microvascular congestion, systemic inflammation, dense rbc formation, and infiltration of activated neutrophils into tissues with subsequent nitrative stress. Bosentan also prevented death of sickle-cell mice exposed to a severe hypoxic challenge. These findings in mice suggest that ETRA could be a potential new therapy for SCD, as it may prevent acute VOC and limit organ damage in sickle-cell patients. more...

One of the major clinical manifestations of sickle cell disease (SCD) is vaso-occlusive crisis in response to hypoxic exposure, leading to acute and chronic organ damages, especially in kidneys. In a SCD transgenic murine model, ultrasound imaging allowed us to characterize the circulatory changes in renal arteries during vaso-occlusive crisis. Cardiac output, heart rate and renal blood flow velocities (BFV) were measured in 10 male transgenic and 10 male wild-type (WT) mice with a conventional echograph (Vivid 7, GE Medical), before and after hypoxic exposure (8%O(2), 18h). To assess entrapment of red cells, histologic study of the kidneys was performed in both groups. Hypoxic exposure decreased heart rates in both groups (-17%, p0.001). Cardiac output remained stable in WT, and decreased in transgenic (-26%, p0.01). Peak systolic BFV in the renal artery was not modified in both groups. End-diastolic and mean BFV remained stable in WT, but decreased in sickle transgenic (-56%, p0.01 and -47%, p0.001, respectively). Transgenic mice displayed marked congestion in peritubular capillaries and glomerular abnormalities with trapped sickle red cells, whereas WT did not present any histologic injury. Five hours after hypoxic exposure, blood flow velocities returned to basal values in both groups. Decrease in end-diastolic and mean BFV in absence of peak systolic BFV after hypoxic exposure strongly indicated that the increase in vascular resistance in kidneys related to sickling of red cells. Thus, ultrasound imaging of the renal artery in mouse is a powerful, noninvasive, easy-to-repeat method to evidence circulatory changes in murine models of vascular renal human diseases. more...

International audience; We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals. © 2007 Optical Society of America more...

International audience; We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice. more...

Introduction La glomerulonephrite rapidement progressive avec proliferation extra-capillaire (GNRP) est l’expression morphologique d’une atteinte glomerulaire severe de mecanismes etiopathogeniques multiples (auto-immuns, infectieux…), caracterisee par l’afflux de cellules inflammatoires et la formation d’un croissant epithelial. Cette formation resulte en partie d’un changement du phenotype normal des podocytes. Nous avons montre une perte d’expression et d’activite transcriptionnelle du facteur de transcription Peroxisome proliferator-activated receptor gamma (PPARγ) dans les podocytes au cours de la GNRP chez la souris et chez les patients. Materiels et methodes Nous avons examine l’expression de PPARγ par immunohistochimie dans les biopsies de patients atteints de GNRP. Nous avons utilise un mode murin de nephrite nephrotoxique (NTN) par injection de serum nephrotoxique (NTS) chez des souris normales ou dont le gene Pparγ etait selectivement inactive dans les podocytes. L’activation de PPARγ a ete obtenue, in vitro et in vivo, par l’utilisation de thiazolidinediones (TZD, rosiglitazone ou pioglitazone). Resultats Dans le modele experimental de NTN, les souris deficientes en PPARγ developpent une insuffisance renale plus severe comparativement aux souris sauvages. L’analyse des mecanismes cellulaires et moleculaires associes nous ont permis d’identifier : – que l’activation specifique de PPARγ par les TZD permettait de prevenir l’apparition de lesions dans notre modele de NTN indiquant le role protecteur majeur de PPARγ ; – l’implication du facteur NRF2 ( nuclear factor erythroid 2-related factor 2 ) dans les processus impliques dans la survenue de la GNRP. Discussion L’expression constitutive de PPARγ dans les podocytes est un facteur cytoprotecteur local au moins par un effet antioxidant probable. Conclusion Ce travail demontre le role cle d’une voie impliquant les facteurs PPARγ-NRF2 dans la tolerance du glomerule a l’agression inflammatoire et pourraient ouvrir de nouvelles perspectives therapeutiques dans le traitement de la GNRP. more...

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension. more...

Mechanical factors regulate both blood vessel growth and the development and progression of vascular disease. Acting on apoptotic and inflammatory signaling, the transcription factor nuclear factor kappaB (NF-kappaB) is a likely mediator of these processes. Nevertheless, pressure-dependent NF-kappaB activation pathways remain mostly unknown. Here we report that high intraluminal pressure induces reactive oxygen species (ROS) in arteries and that inhibition of NADPH oxidase prevents both the generation of ROS and the activation of NF-kappaB associated with high pressure. We also identify the epidermal growth factor receptor (EGFR) as a ROS-dependent signaling intermediate. In arteries from EGFR mutant mice (waved-2), pressure fails to activate NF-kappaB. Moreover, using vessels from EGFR ligand-deficient mice, we show that transforming growth factor (TGF)-alpha, but neither heparin-binding EGF-like growth factor nor epiregulin, transduces NF-kappaB activation by high pressure. Preventing the release of the active form of TGF-alpha also abolishes NF-kappaB induction by strain. The role of TGF-alpha signaling in vascular remodeling is substantiated in vivo; angiotensin II-induced activation of NF-kappaB and associated cell proliferation and wall thickening are much reduced in TGF-alpha-mutant mice compared with wild-type, despite equivalent hypertension in both groups. Conversely, apoptotic cells are detected only in vessels from hypertensive TGF-alpha-mutant mice, outlining the role of NF-kappaB in cell survival. Finally, the NF-kappaB activation pathway contrasts with that of extracellular signal-regulated kinase 1/2, which is activated by stretch through the EGFR but does not implicate TGF-alpha. Hence, our data identify TGF-alpha as a potential specific target to modulate mechanosensitive NF-kappaB activation and associated vascular remodeling. more...

To assess the cardiac consequences of successful respiratory weaning using the variations of circulating B-type and atrial natriuretic peptides (BNP, ANP) and Doppler mitral flow. A prospective preliminary observational study. A 14-bed medical ICU in a French university hospital. Thirty-one patients undergoing a spontaneous breathing trial on a T-tube. Circulating BNP and ANP levels and Doppler-derived E/A ratio and deceleration time of the E wave were measured before and 1 h after disconnection. BNP levels increased from 299 pg/ml (range 56–1079) to 412 pg/ml (147–1324) (p = 0.02) in patients with systolic left ventricular dysfunction, decreased from 98 pg/ml (25–337) to 45 pg/ml (38–180) (p = 0.04) in patients with right ventricular dilation and remained unchanged in patients with neither of these cardiac abnormalities. Overall ANP levels increased from 33 pg/ml to 67 pg/ml (p more...

Vaso-occlusive (VOC) crisis is a major manifestation of sickle cell disease (SCD) leading to acute organ damages. Increased levels of endothelin-1 (ET-1), a potent vasoconstrictor peptide, have been reported in SCD patients. In addition, ETB receptor has been identified in red blood cells (RBCs) membrane and in vitro, ET-1 increases the activity of Gardos channel in sickle RBCs, which is important in generation of dehydrated sickle RBCs. First, we evaluated the effects of the dual-endothelin-receptor (ETR) antagonist bosentan in vitro on mouse RBCs from transgenic SAD mice and in vivo on hypoxic model of acute sickle VOCs. ET-1 (500 nM) significantly increased Gardos channel activity in SAD mouse RBCs, this was prevented by bosentan (10 μM). We next evaluated the in vivo effects of bosentan (100 mg/kg/d by gavage for 14 days) in both control and SAD mice. Under normoxic condition, bosentan did not modify either RBCs density profiles or RBCs K+ content in SAD mice, suggesting that ET-1 may not contribute to sickle cell dehydration in the steady state. The bosentan-treated mice were then exposed to hypoxia (8%, 18 hrs). We first evaluated the cardiac output and renal artery blood flow (RBF) by ultrasound color imaging. In SAD mice only, hypoxia induced a marked reduction in RBF (− 40%; p RBCs dehydration; the reduction in RBF; congestion of the renal microcirculation and injury; lung injury and pulmonary vessel constriction; renal and pulmonary leukocytic infiltrates. In conclusion, ETR blockade was beneficial by preventing RBC dehydration in vivo with protection from kidney and lung injury. In addition, we provide the first evidence that VOC is due not only to cell entrapment within the microvasculature but also to marked ET-dependent vasoconstriction. These data provide new insights into the possible use of bosentan in the treatment of acute VOCs in SCD. more...

BACKGROUND: Sickle cell disease (SCD) affects the kidney by acute mechanisms as well as by insidious renal medullary/papillary necrosis, resulting in tubular defects, which increase the risk of dehydration and subsequent sickle crisis. Hypoxia has been reported to stimulate endothelin-1 (ET-1) synthesis by endothelial cells and also in the renal tubule. METHODS: This case-control study measured ET-1 in urine as a marker of its renal synthesis in asymptomatic SCD patients. Baseline plasma and urinary ET-1 levels were measured and followed during a water deprivation study and a subsequent administration of desmopressin. RESULTS: Urine and plasma levels of ET-1 were elevated in patients with SCD, compared with carefully matched African-French and African controls, and urine ET-1 excretion was associated with a marked urine-concentrating defect. Moreover, urinary ET-1 output was correlated with microalbuminuria in SCD patients. CONCLUSIONS: ET-1 is known to antagonize the tubular effects of vasopressin and to promote renal scarring; increased renal production of ET-1 could produce nephrogenic diabetes insipidus and dehydration in SCD patients through a combination of fibrosis and functional resistance to vasopressin. This study provides a rationale for trials with endothelin receptor antagonists in sickle cell disease nephropathy. more...

The renin-angiotensin system (RAS) modulates a diverse set of physiological processes including development, blood pressure, renal function and inflammation. The principal effector molecule of this system, angiotensin II, mediates most of these actions. The classically recognized functions of the RAS are triggered via the type 1 (AT(1)) class of angiotensin receptors. Pharmacological blockade of the AT(1) receptor lowers blood pressure and slows the progression of cardiovascular and renal diseases. Gene-targeting technology provides an experimental approach for precisely dissecting the physiological functions of the RAS. Here, we review how gene-targeting experiments have elucidated AT(1) receptor functions. more...

Renovascular hypertension is characterised by stenosis of the renal artery and high plasma renin levels due to the recruitment of renin-producing cells along the afferent arterioles. This increase in myoepithelioid cells is mainly a result of the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the model of renovascular hypertension known as the two-kidney, one-clip model in the Lewis rat. Renal arterioles were isolated using magnetised iron suspension. Differential proteomic analysis was performed using two-dimensional electrophoresis gel followed by mass spectrometry for identification. The most striking protein revealed by proteomics is troponin T, which is down-regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific to the smooth muscle phenotype and absent in the myoepithelioid phenotype. more...

End-stage renal disease (ESRD) is characterized by the development of fibrotic lesions in the glomerular, interstitial and vascular compartments. Renal fibrogenesis, a common complication of diabetes and hypertension, is a complex dynamic process involving several players such as inflammatory agents, cytokines, vasoactive agents and enzymes participating in extracellular matrix assembly, anchoring or degradation. The only available treatment today against chronic renal failure is dialysis or kidney transplantation, making thus ESRD one of the most expensive diseases to treat on a per-patient basis. An emerging challenge for clinicians, maybe the nephrologist's Holy Grail in the 21st century, is to stop definitively the decline of renal function and, if possible, to achieve regression of renal fibrosis and restoration of renal structure. Over the last 5 years, different approaches have been tested in experimental models of nephropathy with variable degree of success. In this review, we will focus on the mechanisms of the hypertension-associated fibrosis and the few recent studies that gave promising results for a therapeutic intervention. more...

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling. more...

Vasoactive peptides are implied in the development of renal sclerosis as evidenced by the efficiency of their antagonists in preventing glomerulosclerosis of experimental and human nephropathies. Genetically engineered models provide a new approach to investigate the mechanisms of the renal profibrotic actions of angiotensin II and endothelin. Overexpression of the human angiotensinogen and renin genes in rats induces renal sclerosis independently of changes in systemic hemodynamics. The same results are observed when the endothelin-1 gene is overexpressed in mice. Transgenic mice harboring the luciferase gene under the control of the collagen I-alpha 2 chain promoter (procol alpha 2[1]) and made hypertensive by induction of nitric oxide (NO) deficiency were used to study the renal profibrotic actions of vasoactive peptides. In this strain of mice, luciferase activity is an early index of renal fibrosis. Luciferase activity was increased in preglomerular arterioles and glomeruli when mice were deficient in NO. The pharmacological blockade of angiotensin II and endothelin prevented the development of renal sclerosis without modifying blood pressure. Moreover, when the endothelin receptor antagonist was administered after the development of renal fibrosis, preformed glomerulosclerosis partially regressed. Acute administration of vasoactive peptides and TGF-beta in transgenic procol alpha 2[1] mice showed that the angiotensin II activation of collagen I gene requires participation and/or cooperation of endothelin and TGF-beta. Recent data suggest that the profibrotic actions of vasoactive peptides also need the activation of EGF receptor, ERK and rho kinase pathways in renal and vascular cells. more...

Homozygous sickle cell anaemia (SS disease) involves a high prevalence of skin ulcerations, and background experience concerning the cutaneous microcirculatory flux and reactivity in this disease is very limited. We investigated, by laser-Doppler velocimetry, the microcirculatory cutaneous blood flow and vasoreactivity in 17 patients with SS disease but no cutaneous trophic changes, vs. the corresponding values in 18 normal matched controls. The laser-Doppler probe was placed on the foot dorsum, and recordings were made in the supine and dependent positions, and after post-ischaemic hyperaemia. The venoarteriolar reflex was calculated as the difference between the fluxes in the supine and dependent positions. In both positions, patients with SS disease exhibited clear vasodilation, with larger cutaneous fluxes than those of the controls (P=0.024 and 0.0009, respectively). The venoarteriolar reflex, expressed as a percentage of the resting supine flux, was lower in the patients (P=0.0004). These impairments of the microcirculatory fluxes, which combine a vasodilated state with abnormal vasoreactivity, resemble those observed in patients with chronic venous insufficiency and might be crucial in determining the pathogenesis of the skin ulcerations that occur in SS disease. Laser-Doppler velocimetry seems a suitable non-invasive technique for investigating such cutaneous microangiopathy. more...

Because atrial natriuretic peptide (ANP) is considered to play a role in lung physiology and pathology, our aim was to characterize natriuretic peptide receptors in cultured rat alveolar type II (ATII) cells. Guanylate cyclase A- and B-receptor but not clearance-receptor mRNAs were detected by reverse transcription-polymerase chain reaction. The absence of clearance-receptor expression in ATII cells was confirmed by competitive inhibition of ANP binding; ANP (0.1–100 nM) decreased the binding of125I-ANP, whereas C-ANP-(4—23), a specific ligand of clearance receptors, was ineffective. ANP induced a dose-dependent increase in guanosine 3′,5′-cyclic monophosphate (cGMP) production, with a threshold of 0.1 nM, whereas the response to C-type natriuretic peptide was weak and was observed only at high concentrations (100 nM). In ATII cells cultured on filters, 1) ANP receptors were present on both the apical and basolateral surfaces and 2) cGMP egression was polarized, as indicated by the greater ANP-induced cGMP accumulation in the basolateral medium, and was partially inhibited by probenecid, an organic acid transport inhibitor. Influx studies demonstrated that ANP decreased the amiloride-sensitive component of22Na influx but did not change ouabain-sensitive86Rb influx. In conclusion, ATII cells behave as a target for ANP. ANP activation of guanylate cyclase A receptors produces cGMP, which is preferentially extruded on the basolateral side of the cells and inhibits the amiloride-sensitive Na-channel activity. more...

Role of α v integrins in mesangial cells adhesion to vitronectin and von Willebrand factor. This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to α v , β 3 , and α v β 3 and α v β 5 complexes) that α v β 3 , the vitronectin receptor, and α v β 5 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to 8th passages. Antibodies to α v , β 3 and α v β 3 respectively precipitated an αβ heterodimer with molecular weights of 140 and 97kDa. We analyzed the role of the various integrins in HMC interactions with vitronectin, and with fibronectin and von Willebrand factor (vWf), which are synthetized respectively by mesangial and endothelial cells. Cell adhesion increased in a dose dependent manner with the concentration of plastic-coated matrix protein and vWf. Inhibition of cell attachment with monoclonal antibodies to integrins indicated that HMC adhesion to vWf primarily involves α v β 3 , and that α v β 5 may also contribute to cell binding to vWf. Adhesion to vitronectin involves both α v β 3 and α v β 5 complexes. In contrast, adhesion to fibronectin was not affected by monoclonal antibodies to α v β 3 and α v β 5 complexes. We propose that integrins α v β 3 and α v β 5 , present on HMC, could mediate an interaction between mesangial and endothelial cells by binding to vWf, released at the basal site of endothelial cells. more...

Introduction Sickle cell disease (SCD) is not only responsible for acute vaso-occlusive events but also for chronic vasculopathy that affects many organs including kidneys. Animal studies have suggested that SCD vasculopathy mainly results from chronic hemolysis. However hemolysis markers have not been consistently associated with clinical vascular complications. Moreover, SCD vasculopathy events have been almost exclusively studied in the USA or in Europe, although more than 80% of SCD patients are born and living in sub-Saharan Africa, a very different environment. We have settled the first multinational African SCD cohort to measure the incidence of SCD vascular complications and looked for their predictive factors in sub-Saharan Africa. We present here our first results, focusing on SCD nephropathy with glomerular involvement and urinary loss of albumin. Methods CADRE is an ongoing cohort of SCD patients in five African countries: Cameroon, Gabon, Ivory Coast, Mali, and Senegal. Included subjects are enrolled at steady state and undergo clinical exam, blood sampling for hemoglobin electrophoresis, blood count, creatinine, lactate dehydrogenase (LDH) and bilirubin levels, and urine sample for albumin and creatinine levels, as well as echocardiography. Main outcome for the present study was micro or macroalbuminuria defined as urine albumin/creatinine ratio >30 mg/g, that has been shown to be predictive of chronic renal insufficiency in SCD. We differentiated two main SCD groups: 1) SS and Sβ0 and 2) SC and Sβ+. Frequency of albuminuria at inclusion was calculated in both groups, with further stratification according to age. We looked for clinical and biological correlates to albuminuria in both groups using multivariate logistic regression. Results In June 2013, 3850 SCD patients have been recruited, among them 3032 (2290 SS, 445 SC, 202 Sβ+, 95 Sβ0) had available complete inclusion data. Median age was 15 and 20 years in SS/Sβ0 and SC/Sβ+ group, respectively, with similar female/male ratio of 1.2. Frequency of albuminuria was 36.9% [34.4-38.7] in SS/Sβ0 and 16.1% [13.2-19.9] in SC/Sβ+ patients, and increased significantly with age in SS/Sβ0 patients (Fig 1). Albuminuria remained significantly higher in SS/Sβ0 group than in SC/Sβ+ group, after adjustment for age, sex, country, blood pressure and hemoglobin level (p Conclusion SCD glomerulopathy is frequent in sub Saharan Africa affecting more a third of patients. Children ( Disclosures: No relevant conflicts of interest to declare. more...

Various in vitro preparations have been utilized to study the cellular activity of vasoactive agents on renal cortical microvessels and on mesangial cells. The receptors and the transduction pathways of bradykinin and atrial natriuretic factor were characterized on cultured cortical vascular smooth muscle cells from the rabbit kidney. A preparation of afferent arterioles that had been freshly isolated from the rat kidney was used to study the NO-dependent regulation of renin release. The influence of endothelin and angiotensin II on mesangial cell proliferation was evaluated, using cocultures of human endothelial and mesangial cells. These appropriate in vitro preparations have provided new insights on renal vascular endocrinology. However, extrapolation of in vitro data to in vivo physiology must be cautious because the phenotype of vascular cells often changes in culture conditions. more...

General anaesthesia is associated with hypothermia, oxidative stress, and immune depression. Uncoupling Protein (UCP2) is a member of the mitochondrial carrier family present in many organs including the spleen, the lung and the brain. A role of UCP2 in the activation of the inflammatory/immune cells, in the secretion of hormones, and in the excitability of neurons by regulating the production of reactive oxygen species has been discussed. Because of the side effects of anaesthesia listed above, we aimed to question the expression and the function of UCP2 during anaesthesia. Induction of anaesthesia with ketamine (20 mg/kg) or isoflurane (3.6%) and induction of sedation with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg) stimulated infiltration of immune cells in the lung and increased UCP2 protein content in the lung, in both immune and non-immune cells. UCP2 content in the lung inversely correlated with body temperature decrease induced by medetomidine treatment. Challenge of the Ucp2(-/-) mice with isoflurane and medetomidine revealed an earlier behavioral recovery phenotype. Transponder analysis of body temperature and activity showed no difference between Ucp2(-/-) and control mice in basal conditions. However, upon an acute decrease of body temperature induced by medetomidine, Ucp2(-/-) mice exhibited increased locomotion activity. Together, these results show that UCP2 is rapidly mobilized during anaesthesia and sedation in immune cells, and suggest a role of UCP2 in locomotion. more...

1. In order to appreciate the effect of changes in left atrial pressure on plasma brain natriuretic peptide, 20 patients with mitral stenosis treated by percutaneous valvulotomy were studied 10 min before and 15 min after the first balloon inflation. They were also studied 24 h before and 48 h after the valvulotomy. At these times the effect of postural changes on brain natriuretic peptide secretion was examined. A group of 10 control subjects was also studied under basal conditions. In each case, plasma atrial natriuretic peptide was measured in parallel with plasma brain natriuretic peptide. 2. Similarly to plasma atrial natriuretic peptide, plasma brain natriuretic peptide was elevated in patients with mitral stenosis (32 ± 2.9 and 32 ± 2.8 pg/ml in the upright and supine position respectively versus 13.5 ± 0.5 and 13.8 ± 1.8 pg/ml in controls; P < 0.01). Changing from standing to lying did not modify plasma brain natriuretic peptide, whereas it produced an increase in plasma atrial natriuretic peptide in controls (13.3 ± 1.6 versus 24.8 ± 5.2 pg/ml; P < 0.01) and in patients 48 h after valvulotomy (52.5 ± 4.6 versus 66.9 ± 6.6 pg/ml; P < 0.01). Plasma brain natriuretic peptide also fell at this time (18.8 ± 1.1 and 19.1 ± 1.1 pg/ml in the upright and supine position respectively; P < 0.01) similarly to plasma atrial natriuretic peptide and cyclic GMP (P < 0.01). The acute left atrial mean pressure variation was significantly correlated with the parallel change in plasma atrial natriuretic peptide (P < 0.001) but not in plasma brain natriuretic peptide. Plasma brain natriuretic peptide measured 24 h before and 48 h after valvulotomy was not correlated with plasma cyclic GMP, contrary to plasma atrial natriuretic peptide (P < 0.001). 3. The results of the present study indicate that plasma brain natriuretic peptide depends on long-term but not on acute changes in left atrial pressure. This difference from atrial natriuretic peptide may result from both its preferential ventricular site of synthesis and its longer biological half-life. more...

International audience; We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers. Cop. 2010 The Royal Society of Chemistry. more...

In sickle cell disease (SCD), the expression of mutated hemoglobin allows the formation of rigid, sickle-shaped and dense red blood cell (RBC). In patients, hypoxia, exercise or cold lead to vaso-occlusive crisis (VOC), with hypoperfusion in microcirculatory beds occluded by sickled, dense erythrocytes, causing ischemic tissue injury with intense pain. The renal medulla has substantial metabolic activity, with high oxygen demand, hyperosmolarity and low pH. Kidneys are thus particularly vulnerable to hypoxia and acute VOC. Platelet TSP1 released during thrombosis, and its RBC surface receptor CD47 have previously been involved in increased adhesion of RBC to endothelium in SCD. Here, we investigated other effects of TSP1 and CD47 on RBC in connection with VOC. We used SAD transgenic mice expressing mutated human hemoglobin (α2β2SAD). We found that circulating TSP1 levels increased 4 fold in SAD mice under hypoxia (8 % O2) for 16 hours, whereas TSP1 levels were strongly depressed in wild type mice (−70 %). Electron microscopy and ektacytometry revealed that TSP1-derived CD47 agonist peptides in synergy with hypoxia triggered RBC echinocytosis in healthy RBC (+600 %). Wild type echinocytes however conserved constant numbers of short round spicules. SAD RBC also responded with increased echinocytosis (+100 %), coupled to a doubling in the aberrantly rigid, fine and elongated spicules they carried, compared to normoxia. No true sickling was observed. Intravenous injection of CD47 agonist to anesthetized wild type mice significantly reduced renal blood flow by 17 %, as assessed by echo-Doppler, indicating increased renal microvascular resistance. Renal blood flow returned to normal values within 5 minutes. In contrast, administration of CD47 agonist peptides to SAD mice reduced renal blood flow by 48 %, for more than 20 minutes. Mutated control peptides had no effects, and we observed no significant modulation of heart beat rates, or cardiac output. We concluded that the release of TSP1 may synergize with hypoxia to mediate echinocytosis and contribute to lasting vasoocclusive crises. more...

Reactive oxygen species (ROS) in low concentrations mediate a variety of physiological processes. In the vascular system, Endothelin-1 (ET-1) and Platelet Derived Growth Factor (PDGF) regulate contraction and migration, respectively, by producing intracellular H2O2. How these signaling cascades sharing the same second messenger can lead to different physiological effects is still an open question. It possibly relies on a fine regulation of amplitude, timing and location of H2O2 production and thus requires an adequate sensor.We here propose a novel method based on lanthanide nanoparticles, Y1-xEuxVO4, for the quantitative, dynamic and local detection of H2O2 generation in living cells. Y1-xEuxVO4 nanoparticles are photostable probes presenting a continuous emission due to fluorescence of Eu3+ ions. We demonstrated in vitro that photoreduction and chemical oxidation by H2O2 causes a fluorescence modulation. We identified the temporal response of these nanoprobes submitted to an oxidative signal and proposed a method to determine the H2O2 concentration based on the particle fluorescence for concentrations down to 1 μM, relevant to cell physiology, with temporal resolution down to 10-30 s. In addition, the capability of single-particle detection allows spatial resolution.Imaging of these nanoparticles loaded in vascular smooth muscle cells by pinocytic influx revealed the production of H2O2 under stimulation (CPDGF=7 μM, CET1=13 μM) and a notable timing difference between the two pathways. This points to a method for the cell to integrate distinct signals sharing secondary messengers. Pharmacological treatments, moreover, revealed that H2O2 production is partly due to rapid transactivation of EGF receptors. Such cross-talk between pathways is essential for the signal transduction.These results constitute the first quantitative, time-resolved monitoring of H2O2 production and open new perspectives for the deciphering of complex signaling pathways in a variety of biological systems. more...