Edema is the main symptom of nephrotic syndrome associated to minimal change disease (MCD). Besides the increase in glomerular permeability, possibly induced by a circulating permeability factor,1Maas R.J. Deegens J.K. Wetzels J.F. Permeability factors in idiopathic nephrotic syndrome: historical perspectives and lessons for the future.Nephrol Dial Transplant. 2014; 1: 2207-2216Crossref Scopus (62) Google Scholar an increase in systemic vascular permeability could participate in the constitution of edema. This study was conducted to evaluate the permeability of endothelial cells (EC) exposed to serum from nephrotic patients with MCD, to low- or high-molecular-weight molecules, by trans- or paracellular transport. First, we evaluated the effect of MCD serum on the global permeability of human umbilical vein endothelial cells (HUVECs) via the xCELLigence Real Time Cell Analyzer system (ACEA Biosciences, Inc., San Diego, CA) (Figure 1a). We observed a significant decrease in the impedance, reflecting an increased permeability, with sera from MCD patients compared to sera from healthy volunteers (HVs) (Figure 1b). Second, we compared the endothelial permeability, via the Transwell system (Thermo Fisher Scientific, Waltham, MA), for FITC-Dextran, a high-molecular-weight (HMW) molecule (40kDa), and for sodium fluoresceine (NaF), a low-molecular-weight (LMW) molecule (276 Da) (Figure 2a). No difference was observed between MCD patient and HV sera in terms of passage of FITC-Dextran (Supplementary Figure S1). Conversely, the passage of a NaF through the HUVEC monolayer was significantly greater at 15, 45, and 60 minutes after stimulation with MCD patient sera compared to HV sera (Figure 2b). We therefore demonstrated an increased permeability of HUVEC in vitro after stimulation by sera of patients with MCD, probably restricted to LMW molecules such as sodium. We evaluated in vitro, by western blot analysis, the quantity of vascular endothelial (VE)−cadherin (total and phosphorylated), reflecting paracellular permeability (tight junctions). No difference was observed between MCD patient and HV sera (Supplementary Figure S2A and B). We then evaluated in vivo the paracellular permeability induced by the sera of patients, injected in the skin of mice, using the Miles Assay technique. The extravasation of Evans blue dye, a molecule strongly bound to albumin, did not differ between skin zones injected with MCD patient and HV sera (Supplementary Figure S2C). The quantity of Caveolin 1 was not different in lysates of HUVECs stimulated with MCD patient or HV sera in western blot analysis (Supplementary Figure S3A and B). The transfection of HUVEC with small interfering ribonucleic acids (siRNAs) targeting Caveolin 1 decreased the expression of this protein compared to control siRNA (Supplementary Figure S3C) but did not modify the increased permeability to NaF after stimulation with MCD patient sera. We evaluated the role of endothelial sodium channels in the increased permeability due to MCD patient sera. The HUVECs were incubated in the Transwell system with MCD or HV sera with or without amiloride, an inhibitor of sodium channels such as ENaC (Figure 3a). The increase in NaF permeability induced by MCD patient sera was significantly reversed by amiloride (P = 0.0499). We then evaluated the role of serine proteases in the increased endothelial permeability due to MCD patient sera. The HUVECs were incubated with MCD patient or HV sera with or without aprotinin, an inhibitor of serine proteases (Figure 3b). The increase in NaF permeability induced by MCD patient sera was reversed by aprotinin (P = 0.0465). In addition, the baseline endothelial permeability of endothelium with HV sera was reduced with aprotinin. To determine whether the channel involved in this increased endothelial permeability to sodium was the endothelial ENaC, we studied ENaC subunits using western blot analyses. We did not detect any differences with MCD patient or HV sera in the cleavage of the ENaC-α, -β, or -γ subunits (Supplementary Figure S4A−C). In the present study, we show that serum from MCD patients increases the permeability of cultured EC to sodium through a transcellular pathway involving a sodium channel inhibited by amiloride and aprotinin. Edema is a major clinical symptom in patients with MCD, favored both by decreased oncotic pressure (hypoalbuminemia) and by increased renal sodium reabsorption (activation of tubular ENaC in the collecting duct), and possibly by still-unidentified factors.2Ellis D. Pathophysiology, evaluation, and management of edema in childhood nephrotic syndrome.Front Pediatr. 2016; 3: 111Crossref PubMed Scopus (11) Google Scholar Indeed, a permeability factor in MCD has been sought for decades,1Maas R.J. Deegens J.K. Wetzels J.F. Permeability factors in idiopathic nephrotic syndrome: historical perspectives and lessons for the future.Nephrol Dial Transplant. 2014; 1: 2207-2216Crossref Scopus (62) Google Scholar but mainly to determine the cause of albumin leakage through the glomerular filtration barrier,1Maas R.J. Deegens J.K. Wetzels J.F. Permeability factors in idiopathic nephrotic syndrome: historical perspectives and lessons for the future.Nephrol Dial Transplant. 2014; 1: 2207-2216Crossref Scopus (62) Google Scholar,3Warnock D.G. Kusche-Vihrog K. Tarjus A. et al.Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells.Nat Rev Nephrol. 2014; 10: 146-157Crossref PubMed Scopus (80) Google Scholar whereas we propose here a role of systemic endothelial permeability to explain the brutal constitution of edema. In fact, we demonstrate that MCD patient sera increase endothelial permeability to sodium, thus allowing a large passage of sodium and fluid in the interstitial space. One of the major roles of the vascular endothelium is to provide a selective barrier between blood circulation and tissue. This selective permeability is controlled by intercellular junctions (tight and adherent junctions, regulating paracellular permeability), transport through the endothelium (transcellular permeability), and interactions between EC and pericytes.4Goddard L.M. Iruela-Arispe M.L. Cellular and molecular regulation of vascular permeability.Thromb Haemost. 2013; 109: 407-415Crossref PubMed Scopus (104) Google Scholar The transcellular permeability involves caveolae, which are invaginations of the plasma membrane structured mainly around caveolin-1; transcellular pores or channels allowing passage of LMW solutes; and vesiculovacuolar organelles.5Sowa G. Caveolae, caveolins, cavins, and endothelial cell function: new insights.Front Physiol. 2012; 2: 120Crossref PubMed Scopus (134) Google Scholar In this work, we show that only transcellular permeability is affected by MCD patient serum, and exclude a role of caveolin 1. We demonstrate an increase in vascular permeability to sodium, which is reversed by amiloride, and by aprotinin. Some animal and human data support the use of amiloride, as an inhibitor of ENaC, in nephrotic patients.6Lourdel S. Loffing J. Favre G. et al.Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis.J Am Soc Nephrol. 2005; 16: 3642-3650Crossref PubMed Scopus (56) Google Scholar, 7Svenningsen P. Bistrup C. Friis U.G. et al.Plasmin in nephrotic urine activates the epithelial sodium channel.J Am Soc Nephrol. 2009; 20: 299-310Crossref PubMed Scopus (193) Google Scholar, 8Artunc F. Wörn M. Schork A. Bohnert B.N. Proteasuria—the impact of active urinary proteases on sodium retention in nephrotic syndrome.Acta Physiol. 2019; 225: e13249Crossref PubMed Scopus (31) Google Scholar ENaC is a sodium channel mainly expressed by the distal and collecting tubule, which is activated after the cleavage of its α- and γ-subunits by serine proteases such as plasmin.8Artunc F. Wörn M. Schork A. Bohnert B.N. Proteasuria—the impact of active urinary proteases on sodium retention in nephrotic syndrome.Acta Physiol. 2019; 225: e13249Crossref PubMed Scopus (31) Google Scholar In nephrotic syndrome, an aberrant filtration of blood serine proteases leads to an increased urinary excretion of these proteases and exposure of ENaC to their proteolytic properties, with increased ENaC activation and sodium reabsorption.8Artunc F. Wörn M. Schork A. Bohnert B.N. Proteasuria—the impact of active urinary proteases on sodium retention in nephrotic syndrome.Acta Physiol. 2019; 225: e13249Crossref PubMed Scopus (31) Google Scholar A high serine protease activity is observed in the urine of nephrotic patients,8Artunc F. Wörn M. Schork A. Bohnert B.N. Proteasuria—the impact of active urinary proteases on sodium retention in nephrotic syndrome.Acta Physiol. 2019; 225: e13249Crossref PubMed Scopus (31) Google Scholar which resolves after remission, and the fluid overload of nephrotic patients is correlated with their urinary excretion of serine proteases.S1 Moreover, the systemic administration of aprotinin, a protease inhibitor, prevents the formation of edema in nephrotic mice, despite a similar level of proteinuria.9Bohnert B.N. Menacher M. Janessa A. et al.Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome.Kidney Int. 2018; 93: 159-172Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar ENaC, in addition to its renal localization, is also expressed in the endothelium, where it is involved in morphological and mechanical properties.3Warnock D.G. Kusche-Vihrog K. Tarjus A. et al.Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells.Nat Rev Nephrol. 2014; 10: 146-157Crossref PubMed Scopus (80) Google Scholar,S2,S3 Here, although we show that both amiloride and aprotinin reverse the increase in salt permeability of ECs exposed to nephrotic serum, we observe no differential cleavage of ENaC by western blot. These results do not rule out a role for ENaC in the endothelial permeability to sodium induced by MCD patient serum. In particular, the use of undiluted serum (instead of 1/10 diluted serum) would have been closer to the in vivo conditions and may have revealed a differential cleavage of ENaC by western blot. However, ENaC is not the only sodium channel inhibited by amiloride.S4 Another hypothesis is that another endothelial sodium channel, also inhibited by amiloride and activated by a serine protease present in the serum of healthy controls and increased in the serum of MCD patients, could be implicated in this increased endothelial permeability. In nephrotic patients, the benefit of amiloride could be related not only to its diuretic properties but also to the reduction of endothelial salt permeability and fluid leakage. Our work also suggests that the baseline physiological activity of endothelial sodium channels could be maintained by serine proteases, as HUVEC permeability to sodium was reduced in the presence of aprotinin. There are several limitations to our study. First, because of the limited amount of serum available for each patient, we used diluted sera in all experiments, and this may have blunted the effect of MCD patient serum. Future research in the field may benefit from the use of undiluted, or less diluted, serum. Second, our experiment model based on HUVECs does not include the pericytes or vascular wall, which also participate in vascular permeability. Yet, because edema probably arise from capillary leaks, a monolayer of HUVECs may be a relevant model in this setting. Third, only MCD patient sera were tested, without nephrotic or non-nephrotic controls from patients with other kidney diseases, and the specificity of this endothelial permeability cannot be asserted. Finally, we have not identified the permeability factor of MCD, or the sodium channel that it activates. However, this work highlights the fact that the permeability factor of MCD may not be solely a glomerular one (allowing the leakage of albumin through the glomerular filtration barrier), but also a systemic one, allowing the leakage of sodium in the interstitial space, possibly through the activation of an endothelial sodium channel by proteases. In conclusion, this original work shows, for the first time, an increased endothelial permeability induced by sera from patients with MCD, allowing the passage of sodium through the endothelium, probably participating in the constitution of edema, and reversed by amiloride and by aprotinin. All the authors declared no competing interests. Download .docx (2.71 MB) Help with docx files Supplementary File (Word)

Aims: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration suggests an involvement for this adhesion molecule in the inflammatory response. In this study, we investigated the role of CD146 in leukocyte recruitment by using an experimental model of atherogenesis. Methods and results: The role of CD146 was explored in atherosclerosis by crossing CD146 -/- mice with ApoE -/- mice. CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall was significantly thicker in CD146-deficient mice. After 24 weeks of Western diet, a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146-null mice was observed. In addition, atherosclerotic lesions were more inflammatory since plaques from CD146-deficient mice contained more neutrophils and macrophages. This was due to up-regulation of RANTES secretion by macrophages in CD146-deficient atherosclerotic arteries. This prompted us to further address the function of CD146 in leukocyte recruitment during acute inflammation by using a second experimental model of peritonitis induced by thioglycollate. Neutrophil recruitment was significantly increased in CD146-deficient mice 12 h after peritonitis induction and associated with higher RANTES levels in the peritoneal cavity. In CD146-null macrophages, we also showed that increased RANTES production was dependent on constitutive inhibition of the p38-MAPK signaling pathway. Finally, Maraviroc, a RANTES receptor antagonist, was able to reduce atherosclerotic lesions and neutrophilia in CD146-deficient mice to the same level as that found in ApoE -/- mice. Conclusions: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES production and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 agonists as potential new therapeutic candidates for atherosclerosis treatment.

Rationale: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration in vitro suggests a role for this endothelial junction molecule in the pathogenesis of atherosclerosis. However, its involvement in atherosclerotic plaques formation has never been investigated. Objective: We evaluated the role of CD146 in atherogenesis. Methods and Results: CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall thickness was significantly higher in CD146 deficient mice. We evidenced a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146 deficient mice. In addition, atherosclerotic lesions were more inflammatory since CD146 deficient plaques contained more neutrophils and more macrophages. During atherosclerosis, circulating neutrophils were significantly increased in the absence of CD146. Consistent with the higher recruitment of inflammatory cells to the atheroma, we demonstrated that RANTES was up-regulated in CD146 deficient atherosclerotic arteries. In addition, CD146 deficient mice presented significant higher levels of circulating RANTES during atherosclerosis. Finally, we showed that macrophages were the source of RANTES and its production by CD146 null macrophages was also significantly increased through a mechanism dependent of p38-MAPK signaling pathway. Conclusions: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 as a potential new target for atherosclerosis treatment.

Atherosclerosis is a chronic inflammatory disease of the large vessels and its progression is based in part on the continued recruitment of macrophages in the inflammatory wall. The well described role of CD146 in leukocyte infiltration in vitro suggests a role for this endothelial junction molecule in the early stages of the formation of atherosclerotic plaques. However, the involvement of CD146 in atherogenesis has not been elucidated. To investigate the role of CD146 deficiency in atherosclerosis, CD146 knocked-out (KO) mice were bred with ApoE KO mice. Mice were fed a high-fat Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. After 24 weeks of diet, the arterial wall thickness was significantly higher in CD146 deficient mice (0.19±0.01mm vs 0.27±0.01mm; p=0.012) and there was a significant 47 % increase (p=0.015) in total aortic lesion area, evaluated by histological oil red O coloration. However, mice weights and total cholesterol plasma levels were similar between the two groups of mice. We detected a 2-fold increase in macrophage recruitment to the atherosclerotic plaque of the double KO mice compared with ApoE KO mice, suggesting that the atherosclerotic lesions in these mice were more inflammatory. In addition, CD146 deficient plaques contained significantly less smooth muscle cells (p=0.036) and could be more vulnerable. We observed a maladaptive immune response in double KO mice since the circulating neutrophils and monocytes were significantly increased (p=0.008 and p=0.015 respectively) in the absence of CD146 whereas circulating T lymphocytes were significantly decreased (p=0.026). In a similar way, splenocytes isolated from double KO mice contained significantly lower levels of T cells (p=0.002), suggesting a default of adaptive immunity in the absence of CD146 during atherosclerosis. Our results demonstrated the involvement of CD146 in the progression of atherosclerosis in mice. Our data also suggest that CD146 is involved in the regulation of adaptive immune response and its absence lead to an inflammatory phenotype, which could influence atherosclerosis plaque fate. Thus, CD146 should be considered as a potential target for protection against atherosclerosis.

In CKD, uremic solutes may induce endothelial dysfunction, inflammation, and oxidative stress, leading to increased cardiovascular risk. We investigated whether the uremic solute indole-3 acetic acid (IAA) predicts clinical outcomes in patients with CKD and has prooxidant and proinflammatory effects. We studied 120 patients with CKD. During the median study period of 966 days, 29 patients died and 35 experienced a major cardiovascular event. Kaplan-Meier analysis revealed that mortality and cardiovascular events were significantly higher in the higher IAA group (IAA>3.73 µM) than in the lower IAA group (IAA<3.73 µM). Multivariate Cox regression analysis demonstrated that serum IAA was a significant predictor of mortality and cardiovascular events after adjustments for age and sex; cholesterol, systolic BP, and smoking; C-reactive protein, phosphate, body mass index, and albumin; diastolic BP and history of cardiovascular disease; and uremic toxins p-cresyl sulfate and indoxyl sulfate. Notably, IAA level remained predictive of mortality when adjusted for CKD stage. IAA levels were positively correlated with markers of inflammation and oxidative stress: C-reactive protein and malondialdehyde, respectively. In cultured human endothelial cells, IAA activated an inflammatory nongenomic aryl hydrocarbon receptor (AhR)/p38MAPK/NF-κB pathway that induced the proinflammatory enzyme cyclooxygenase-2. Additionally, IAA increased production of endothelial reactive oxygen species. In conclusion, serum IAA may be an independent predictor of mortality and cardiovascular events in patients with CKD. In vitro, IAA induces endothelial inflammation and oxidative stress and activates an inflammatory AhR/p38MAPK/NF-κB pathway.

CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146‐deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor‐induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF‐induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR‐2/Ve‐cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve‐cadherin network in melanoma extravasation across the vessel barrier that identifies CD146‐targeted therapy as a potential strategy for the treatment of melanoma metastasis.

CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.