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Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut. ispartof: VIRULENCE vol:12 issue:1 pages:329-345 ispartof: location:United States status: published

BACKGROUND: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available. METHODS: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661. FINDINGS: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

In this paper an efficient numerical tool is proposed to investigate delamination type failure in multi-layered composite shells. In the current contribution the extended finite element method (XFEM), the mixed-mode cohesive zone model, the contact formulation, and the damage criterion are incorporated into a new algorithm to study the interfacial delamination initiation and growth with less computational effort. A flat-shell formulation is developed in the geometrically non-linear regime to study the response of shells in small strains and moderate rotations. In addition, the equivalent single layer theory (ESLT) is applied to simulate the multi-layered laminates. This formulation is enhanced through the XFEM topology to be able to model discontinuous domains and a mixed-mode bilinear cohesive formulation to track the delamination growth. In the current study, the simulation can be initiated in an intact laminate. Thus, unlike formulations in existing finite element models, incorporating cohesive zone model at all available interfaces is not necessary. The interlaminar stresses are calculated during post-processing and they are being used in the delamination onset criterion. As soon as the criterion is satisfied at a specific layer and location, the formulation of that corresponding element is locally changed to XFEM and the cohesive behaviour. Consequently, the possibility to track delamination growth is locally provided; and hence, the computational cost is reduced.

Myofibroblasts are the key players in extracellular matrix remodeling, a core phenomenon in numerous devastating fibrotic diseases. Not only in organ fibrosis, but also the pivotal role of myofibroblasts in tumor progression, invasion and metastasis has recently been highlighted. Myofibroblast targeting has gained tremendous attention in order to inhibit the progression of incurable fibrotic diseases, or to limit the myofibroblast-induced tumor progression and metastasis. In this review, we outline the origin of myofibroblasts, their general characteristics and functions during fibrosis progression in three major organs: liver, kidneys and lungs as well as in cancer. We will then discuss the state-of-the art drug targeting technologies to myofibroblasts in context of the above-mentioned organs and tumor microenvironment. The overall objective of this review is therefore to advance our understanding in drug targeting to myofibroblasts, and concurrently identify opportunities and challenges for designing new strategies to develop novel diagnostics and therapeutics against fibrosis and cancer.

Background: Immune cells have been implicated in the graft failure after kidney transplantation. However, the best therapeutic target(s) among immune cells subtypes have not been identified yet. We aimed therefore to identify which immune cell subtypes possess the highest prognostic value for graft failure. Methods: We recently described the role of activated NK cell transcripts in ABMR diagnosis and activity in BIOMARGIN. Here, we applied an innovative deconvolution algorithm to estimate the relative fraction of immune cells subsets, using RNA transcripts expression of 547 genes in a dataset of 282 indication biopsy samples. These estimates were corrected for the global inflammation in each biopsy. Results: Analysis of estimated immune cells subsets infiltration, based on deconvolution of transcriptomic data, showed that activated NK cells have the highest discriminative power in predicting graft failure at both 1 and 2 years post-biopsy in all biopsies; irrespective of rejection (AUC = 0.74; p < 0.0001 and AUC = 0.67; p = 0.0006, respectively), and within biopsies with rejection (AUC = 0.79 (p = 0.002) and AUC = 0.68 (p = 0.03), respectively). Cox proportional hazard analyses confirmed that activated NK cells were the cell type most robustly associated with graft failure (HR 3.60; p < 0.0001), especially in biopsies with acute rejection (HR 3.89; p = 0.002). We then evaluated individual NK cell transcripts expression in relation to graft failure. Using both the differentially expressed genes from the deconvolution algorithm and a literature-based NK-cell related transcript set, the prognostic performance of individual activated NK cell genes for post-biopsy kidney allograft failure was confirmed. Conclusion: These findings, in addition the emerging role of NK cells infiltration in ABMR diagnosis and activity, offer promising new avenues for development of NK-cell targeted prognostic biomarkers and therapeutic strategies to prevent or attenuate graft failure.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation. ispartof: Kidney International vol:95 issue:1 pages:188-198 ispartof: location:United States status: published

Renal fibrosis cannot be adequately treated since anti-fibrotic treatment is lacking. Interferon-gamma is a pro-inflammatory cytokine with anti-fibrotic properties. Clinical use of interferon-gamma is hampered due to inflammation-mediated systemic side effects. We used an interferon-gamma peptidomimetic (mim gamma) lacking the extracellular IFN gamma Receptor recognition domain, and coupled it to the PDGF beta R-recognizing peptide BiPPB. Here we tested the efficacy of mim gamma-BiPPB (referred to as "Fibroferon") targeted to PDGF beta R-overexpressing interstitial myofibroblasts to attenuate renal fibrosis without inducing inflammation-mediated side effects in the mouse unilateral ureter obstruction model.Unilateral ureter obstruction induced renal fibrosis characterized by significantly increased alpha-SMA, TGF beta 1, fibronectin, and collagens I and III protein and/or mRNA expression. Fibroferon treatment significantly reduced expression of these fibrotic markers. Compared to full-length IFN gamma, anti-fibrotic effects of Fibroferon were more pronounced. Unilateral ureter obstruction-induced lymphangiogenesis was significantly reduced by Fibroferon but not full-length IFN gamma. In contrast to full-length IFN gamma, Fibroferon did not induce IFN gamma-related side-effects as evidenced by preserved low-level brain MHC II expression (similar to vehicle), lowered plasma triglyceride levels, and improved weight gain after unilateral ureter obstruction.In conclusion, compared to full-length IFN gamma, the IFN gamma-peptidomimetic Fibroferon targeted to PDGF beta R-overexpressing myofibroblasts attenuates renal fibrosis in the absence of IFN gamma-mediated adverse effects.

Summary The production of new composite laminates with variable stiffness within the surface of plies was enabled by tow-placement machines. Because of the variation of stiffness, these materials are called variable stiffness composite laminates (VSCL). Recently, many attempts were made to investigate their structural behaviour. In this contribution, a first-order shear deformation theory is selected to model the multilayered composite laminates. The adopted theory is enhanced by the extended finite element method (XFEM) to describe discontinuities at element level of any interface of interest. To predict the location of the delamination onset, a traction–separation law is developed that is consistent with the XFEM topology. An exponential softening behaviour is implemented within the interface to model the delamination growth in a mixed-mode direction. In order to solve the non-linear equations of the delamination propagation, an arc-length method is applied. The effect of the curvilinear fibre orientation on the location of the delamination onset is investigated. Subsequently, the structural response of the laminates is computed. According to the simplicity of the new approach using the XFEM; and based on the computational cost for calculating the stiffness of VSCL, the method is able to determine structural response of VSCL with less computational effort. Copyright © 2016 John Wiley & Sons, Ltd.

A First-order Shear Deformation Theory (FSDT) is chosen to simulate composite laminates in the linear and the geometrically non-linear regimes. The formulation is based on the Equivalent Single Layer (ESL) theory that fails to predict the delamination onset in composite laminates. The lack of resolving three-dimensional states and correct transverse stresses in this model is principally improved using post-processing. In order to precisely compute interlaminar stresses, a non-frictional linear adhesive contact model is applied in the context of the eXtended Finite Element Method (XFEM). The discontinuities are imposed within any arbitrary interface by enriching the displacement field. Thus two sub-domains define the plane of the discontinuity. Thereafter, the aforementioned adhesive contact can be formulated at the discontinued interface. Stress values are retrieved at nodal points using the interface constitutive equation. Consequently, the interface formulations are extended into the softening regime to model the delamination growth as a mixed-mode cohesive effect. The accuracy of the proposed method in predicting the interlaminar stresses and the delamination propagation is demonstrated by comparing the results with the ones available in literature. By combining the lower-order plate theory and the novel XFEM technique, the model is able to accurately calculate the delamination onset and the propagation with less computational effort.

Herein, modes of vibration of a novel type of composite laminates, manufactured by tow-placement machines and which are called Variable Stiffness Composite Laminates (VSCL), are of interest. Layerwise theory is chosen, because it leads to an accurate prediction of displacements through the thickness of laminates, even when these are thick. Furthermore, it naturally has the capability of modelling the bending-membrane coupling that occurs on free vibration of VSCL plates with unsymmetric stacking sequences. The continuity of displacements at layer interfaces is imposed and in-plane displacements vary in a zig-zag fashion along the thickness. The accuracy of the model is confirmed by testing Constant Stiffness Composite Laminates (CSCL) analysed in the literature by others, who employed either Equivalent Single Layer (ESL) or layerwise theories. Abaqus commercial finite element software is as well employed in order to test the present approach. Finally, published natural frequencies of VSCLs, which were studied by an ESL theory, are compared with frequencies resulting from the present layerwise theory. The verification of the proposed model is followed by an investigation on the effect of curvilinear fibre orientation parameters on natural frequencies of thin to thick composite plates, with symmetric and unsymmetric lay-ups.

BackgroundHigh dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in normotensive rats converts renal HS into a pro-inflammatory phenotype, able to bind more sodium and orchestrate inflammation, fibrosis and lymphangiogenesis.MethodsWistar rats received a normal diet for 4 weeks, or 8% NaCl diet for 2 or 4 weeks. Blood pressure was monitored, and plasma, urine and tissue collected. Tissue sodium was measured by flame spectroscopy. Renal HS and tubulo-interstitial remodeling were studied by biochemical, immunohistochemical and qRT-PCR approaches.ResultsHigh sodium rats showed a transient increase in blood pressure (week 1; p ConclusionWe show that high salt intake by healthy normotensive rats convert renal HS into high sulfated pro-inflammatory glycans involved in tissue remodeling events, but not in increased sodium storage.

Background: Renal fibrogenesis is associated with increased ECM remodeling and release of collagen fragments in urine in progressive renal disease. We investigated the diagnostic value of urinary collagen degradation products in a proteinuria-driven fibrosis rat model with and without anti-fibrotic S1P-receptor modulator FTY720 treatment.Methods: Proteinuria was induced in male Wistar rats by Adriamycin (ADR) injection (n = 16). Healthy rats served as controls (n = 12). Six weeks post-injection, all underwent renal biopsy, and FTY720-treatment started in ADR-rats (n = 8) and controls (n = 6). Others remained untreated. Rats were sacrificed after 12 weeks. Collagen type I (C1M) and III (C3M) degradation fragments were measured in blood and urine using ELISA. Kidneys were stained for various inflammatory and fibrotic markers.Results: Six weeks post-injection proteinuria increased (versus controls, P Conclusions: These data displayed urinary collagen breakdown products as sensitive early markers of interstitial fibrosis, preceding histological fibrotic changes, which might replace the invasive renal biopsy procedure to assess fibrosis. Anti-fibrotic FTY720 intervention reduced some fibrotic markers without affecting collagen type III metabolism.

Excessive lymphangiogenesis is associated with cancer progression and renal disease. Attenuation of lymphangiogenesis might represent a novel strategy to target disease progression although clinically approved anti-lymphangiogenic drugs are not available yet. VitaminD(VitD)-deficiency is associated with increased cancer risk and chronic kidney disease. Presently, effects of VitD on lymphangiogenesis are unknown. Given the apparently protective effects of VitD and the deleterious associations of lymphangiogenesis with renal disease, we here tested the hypothesis that VitD has direct anti-lymphangiogenic effects in vitro and is able to attenuate lymphangiogenesis in vivo. In vitro cultured mouse lymphatic endothelial cells (LECs) expressed VitD Receptor (VDR), both on mRNA and protein levels. Active VitD (calcitriol) blocked LEC tube formation, reduced LEC proliferation, and induced LEC apoptosis. siRNA-mediated VDR knock-down reversed the inhibitory effect of calcitriol on LEC tube formation, demonstrating how such inhibition is VDR-dependent. In vivo, proteinuric rats were treated with vehicle or paricalcitol for 6 consecutive weeks. Compared with vehicle-treated proteinuric rats, paricalcitol showed markedly reduced renal lymphangiogenesis. In conclusion, our data show that VitD is anti-lymphangiogenic through VDR-dependent anti-proliferative and pro-apoptotic mechanisms. Our findings highlight an important novel function of VitD demonstrating how it may have therapeutic value in diseases accompanied by pathological lymphangiogenesis.

This paper presents a layerwise theory, p -version finite element method for static analysis of unsymmetric laminates with curvilinear fibres. A displacement field that varies linearly along the thickness of each layer is considered and its continuity between the layers is imposed, in this way representing the zig-zag variation of displacements through the thickness. Von Karman non-linear strain–displacement relationships are applied to take into account the effect of large displacements. Several test problems are examined to verify the accuracy of the proposed model for both symmetric and unsymmetric constant stiffness composite laminates (CSCL), in the linear and non-linear regimes. Deflections and stresses computed with the model proposed here are compared with results available in the literature and with results from Abaqus and Ansys commercial software. Then, static analysis of unsymmetric variable stiffness composite laminated plates (VSCL) are investigated in the geometrically non-linear regime and the effect of unsymmetric stacking sequences and fibre orientations are studied.

Nephrotoxicity is usually thought of as a common invariable consequence of hemodynamic toxicity whose effects, including oliguria and dysuria, has largely limited the clinical use of cisplatin. In this study, we investigated the protective effects of low and high dose of vitamin C against cisplatin-induced rat nephrotoxicity. Hence, 50 adult male Swiss albino rats were randomly divided into five equal groups to receive a corresponding dose of either normal saline as control, vitamin C (600 mg/kg/BW, i.v.), or cisplatin alone (7 mg/kg/BW, i.p.) or in combination with vitamin C at low dose (200 mg/kg/BW, i.v.) and high dose (600 mg/kg/BW, i.v.) for 9 days. Daily administration of cisplatin at a dose of 7 mg/kg/BW resulted in a significant increase in oxidative stress in renal tissues and plasma and a concomitant decrease in the creatinine clearance and renal blood flow as a result of early hemodynamic toxicity. Histopathological examination revealed acute tubular necrosis with hyaline cast formation triggered by cisplatin over 9 days of experiment. Further biochemical studies showed protecting effects of supplemented vitamin C at a high dose, illustrated by slowdown in the urinary enzyme activity, a significant decrease in plasma lipid peroxidation, and an increased tissue superoxide dismutase activity with recovery in the glomerular hemodynamicity and the ATPase activity up to 50 % when compared to controls and rats receiving low-dose. In high-dose animals, normal glomerular and tubular function on recovery from toxic renal failure led us to conclude that antioxidant property of vitamin C increases with dose, and, therefore, high dose of vitamin C prevents both functional and histological renal changes induced by cisplatin in rats, more efficient than low dose of the vitamin.

A new p-version finite element based on a zig-zag layerwise theory is developed. With the proposed model, one can accurately determine the behaviour of laminated composites with different material properties, straight or curvilinear fibres. Comparative studies with published results and with results from finite element software Abaqus are performed to verify the new model. Deflections in the linear and non-linear regimes are then calculated for several constant and variable stiffness composite laminates (VSCL), in which the fibre orientation angle varies linearly in each layer. In a few cases, VSCL plates show better performance in comparison to CSCL plates both in the linear and non-linear regimes. It is also found that it is possible to take advantage of VSCL by varying fibre orientations only in some plies of a laminate, hence reducing manufacturing costs, as well as the amount of gaps and overlaps.

The sphingosine-1-phosphate (S1P) analog FTY720 exerts pleiotropic effects on the cardiovascular system and causes down-regulation of S1P receptors. Myogenic constriction is an important mechanism regulating resistance vessel function and is known to be modulated by S1P. Here we investigated myogenic constriction and vascular function of mesenteric arteries of rats chronically treated with FTY720. Wistar rats received FTY720 1mg/kg/daily for six weeks. At termination, blood pressure was recorded and small mesenteric arteries collected for vascular studies in a perfusion set up. Myogenic constriction to increased intraluminal pressure was low, but a sub-threshold dose of S1P profoundly augmented myogenic constriction in arteries of both controls and animals chronically treated with FTY720. Interestingly, endothelial denudation blocked the response to S1P in arteries of FTY720-treated animals, but not in control rats. In acute experiments, presence of FTY720 significantly augmented the contractile response to S1P, an effect that was partially abolished after the inhibition of cyclooxygenase (COX-)-derived prostaglandins. FTY720 down regulated S1P1 but not S1P2 in renal resistance arteries and in cultured human endothelial cells. This study therefore demonstrates the endothelium is able to compensate for the complete loss of responsiveness of the smooth muscle layer to S1P after long term FTY720 treatment through a mechanism that most likely involves enhanced production of contractile prostaglandins by the endothelium.

Delamination analysis of laminated composite shells is studied. For this purpose, a fournoded flat shell element for linear and geometrically non-linear analyses is developed. The formulation is based on a first-order shear deformation theory (FSDT) and is valid for large displacements but moderate rotations. The extended finite element method (XFEM) is used to impose the discontinuous domain at an arbitrary through-the-thickness location. The progressive failure at the interface region is simulated through a mixed-mode cohesive zone model based on an exponential softening behavior. In addition, a simple non-frictional contact formulation is utilized to avoid the penetration of the discontinuous subdomains. To diminish the instability problem of the interface formulation, different integration rules are taken into account. Furthermore, the arc-length method with full Newton-Raphson iteration technique is applied to solve the non-linear governing equations. In order to verify the accuracy of the predictions, standard benchmark tests are carried out for analyzing shells, delamination, and the delamination buckling problems. The proposed model can be effectively used to model the delamination onset and its propagation in shell structures with less computational cost.

The Rho kinase pathway plays an important role in epithelial dedifferentiation and inflammatory cell infiltration. Recent studies suggest that inflammation promotes lymphangiogenesis, which has been associated with renal allograft rejection. We investigated whether targeted inhibition of the Rho kinase pathway in proximal tubular cells reduces inflammation and lymphangiogenesis in acute renal allograft rejection. The Rho kinase inhibitor Y27632 was coupled to lysozyme (Y27632-lysozyme), providing a kidney-specific conjugate that can release its drug in proximal tubular cells. Isogenic (Fisher-Fisher, n=18), or allogenic (Fisher-Lewis, n=24) kidney transplantations were performed, with the contralateral kidney remaining in situ. To elicit acute rejection, no immunosuppressive treatment was given. Animals were treated daily with Y27632-lysozyme (10 mg/kg/day i.v.) or vehicle (saline i.v.) until sacrifice (1 or 4 days post-transplantation). After allogenic transplantation, interstitial macrophage accumulation was strongly reduced by Y27632-lysozyme at day 4 after transplantation. Interstitial lymphangiogenesis, which was induced in allografts as compared to control kidney, was also reduced by renal Rho kinase inhibition at day 4 after transplantation. The increase of vimentin and procollagen-1alpha1 gene expression in renal allografts from day 1 to day 4 after transplantation was significantly reduced by Y27632-lysozyme. Y27632-lysozyme did not affect systolic blood pressure in isogenic or allogenic transplantation groups. In cultured tubular epithelial cells (NRK-52E), Rho kinase inhibition dose-dependently reduced IL-1β-induced MCP-1 gene expression. Renal inhibition of Rho kinase causes a marked reduction in renal inflammation and renal lymphangiogenesis during acute transplant rejection, suggesting that this treatment regimen is a valuable future treatment in renal transplantation.

Nephrotoxicity results from an abrupt decline in glomerular functions of the kidney and is regarded as a major cause of concern for limiting therapeutic uses of gentamicin in hospital settings. Recent evidences suggest that both endothelial nitric oxide synthesis (eNOS) and inducible NOS (iNOS)-derived nitric oxide (NO) vasodilators reduce renal toxicity, restoring normal glomerular functions and hemodynamic stability, but this has not been adequately investigated in gentamicin nephrotoxicity. To determine the isoform of NOS that plays a predominant role in the control of glomerular vasodilation under the oxidative stress of nephrotoxicity, we conducted a controlled, randomized study in five equal groups of ten Sprague–Dawley rats. All animals were given a corresponding dose of either normal saline, only l-arginine, only gentamicin, or a combination thereof with and without N-3-aminomethyl benzylacetamidine (a selective iNOS inhibitor, 1400W) for 9 days. After 6 days, gentamicin-treated rats developed nephrotoxic alterations evident by a significant fall in renal blood flow and glomerular filtration rate, increased urinary excretion of gamma glutamyl transpeptidase and serum concentration of inflammatory interleukins (IL-6 and IL-8) as well as a decrease in the tissue antioxidant activity of superoxide dismutase. Changes in these biochemical variables were completely inhibited by supplemented l-arginine up to 9 days. However, a combination of 1400W partially removed protective effects of l-arginine on the glomerular hemodynamics of the kidney. Our results suggest that at irradiation of eNOS developed by increased oxidative process, administration of l-arginine increases iNOS-derived NO production and glomeruli infiltration on its direct antioxidant effects counteracting the undesirable effects of peroxynitrite formation on the kidney.

Background. Recent experimental findings demonstrate vascular endothelial growth factor C (VEGF-C)-mediated water-free storage of salt in the interstitium, which prevents a salt-sensitive blood pressure state. It is unknown whether this mechanism plays a role in salt homeostasis and regulation of blood pressure in humans as well. Therefore, we investigated circulating VEGF-C levels and blood pressure during different well-controlled salt intake in chronic kidney disease (CKD) patients and in healthy subjects.Methods. In two crossover studies, non-diabetic proteinuric CKD patients (n = 32) and healthy subjects (n = 31) were treated with consecutively a low-sodium diet (LS, aim 50 mmol Na+/day) and a high-sodium diet (HS, aim 200 mmol Na+/day) in random order, during two 6-week (CKD patients) and two 1-week periods (healthy subjects).Results. We found that VEGF-C levels are higher during HS than during LS in CKD patients (P = 0.034) with a trend towards higher VEGF-C in healthy subjects as well (P = 0.070). In CKD patients, HS was associated with higher NT-proBNP levels (P = 0.005) and body weight (P = 0.013), consistent with extracellular volume (ECV) expansion and with higher blood pressure (P Discussion. Our findings support a role for VEGF-C-mediated salt homeostasis in humans. Considering the salt sensitivity of blood pressure, this buffering mechanism appears to be insufficient in proteinuric CKD patients. Future studies are needed to provide causality and to substantiate the clinical and therapeutic relevance of this VEGF-C regulatory mechanism in humans.

Some diseases associated with a temporary deterioration in kidney function and/or development of proteinuria show an apparently complete functional remission once the initiating trigger is removed. While it was earlier thought that a transient impairment of kidney function is harmless, accumulating evidence now suggests that these patients are more prone to developing renal failure later in life. We therefore sought to investigate to what extent renal functional changes, inflammation and collagen deposition are reversible after cessation of disease induction, potentially explaining residual sensitivity to damage. Using a rat model of Angiotensin II (Ang II)-induced hypertensive renal disease we show the development of severe hypertension (212 ± 10.43 vs. 146 ± 1.4 mmHg, p

Renal fibrosis leads to end-stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN-γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN-γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell-specific delivery of IFN-γ to platelet-derived growth factor receptor β (PDGFRβ)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-γ conjugated to PDGFRβ-recognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-γ. PDGFRβ expression was >3-fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α-smooth muscle actin-positive (SMA(+)) myofibroblasts. In vitro, PPB-PEG-IFN-γ significantly inhibited col1a1, col1a2, and α-SMA mRNA expression in TGF-β-activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB-PEG-IFN-γ specifically accumulated in PDGFRβ-positive myofibroblasts. PPB-PEG-IFN-γ treatment significantly reduced renal collagen I, fibronectin, and α-SMA mRNA and protein expression. Compared with vehicle treatment, PPB-PEG-IFN-γ preserved tubular morphology, reduced interstitial T-cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB-PEG-IFN-γ reduced IFN-γ-related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN-γ). Our findings demonstrate that specific targeting of IFN-γ to PDGFRβ-expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects.-Poosti, F., Bansal, R., Yazdani, S., Prakash, J., Post, E., Klok, P., van den Born, J., de Borst, M. H., van Goor, H., Poelstra, K., and Hillebrands, J.-L. Selective delivery of IFN-γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis.

Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not clear yet. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in three-month old male Wistar rats by adriamycin injection. After 6 weeks, when proteinuria has developed, rats were treated for another 6 weeks by anti-VEGFR3 antibody, clodronate liposomes, and FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs. week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however without significant effects on inflammatory and fibrotic markers, and proteinuria. Clodronate liposomes inhibited macrophage influx, partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation and T cell influx and interstitial fibrosis, partially declined macrophage number and proteinuria; however, it did not influence significantly on lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or influx of macrophages. On the other hand, FTY720 treatment could prevent T-cells influx, myofibroblasts accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions.

Tissue fibrosis, characterized by excessive accumulation of aberrant extracellular matrix (ECM) produced by myofibroblasts, is a growing cause of mortality worldwide. Understanding the factors that induce myofibroblastic differentiation is paramount to prevent or reverse the fibrogenic process. Integrin-mediated interaction between the ECM and cytoskeleton promotes myofibroblast differentiation. In the present study, we explored the significance of integrin alpha 11 (ITGA11), the integrin alpha subunit that selectively binds to type I collagen during tissue fibrosis in the liver, lungs and kidneys. We showed that ITGA11 was co-localized with α-smooth muscle actin-positive myofibroblasts and was correlatively induced with increasing fibrogenesis in mouse models and human fibrotic organs. Furthermore, transcriptome and protein expression analysis revealed that ITGA11 knockdown in hepatic stellate cells (liver-specific myofibroblasts) markedly reduced transforming growth factor β-induced differentiation and fibrotic parameters. Moreover, ITGA11 knockdown dramatically altered the myofibroblast phenotype, as indicated by the loss of protrusions, attenuated adhesion and migration, and impaired contractility of collagen I matrices. Furthermore, we demonstrated that ITGA11 was regulated by the hedgehog signaling pathway, and inhibition of the hedgehog pathway reduced ITGA11 expression and fibrotic parameters in human hepatic stellate cells in vitro, in liver fibrosis mouse model in vivo and in human liver slices ex vivo. Therefore, we speculated that ITGA11 might be involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway. These findings highlight the significance of the ITGA11 receptor as a highly promising therapeutic target in organ fibrosis.

Background Chronic renal transplant dysfunction is characterized by loss of renal function and tissue remodeling, including chronic inflammation and lymph vessel formation. Proteoglycans are known for their chemokine presenting capacity. We hypothesize that interruption of the lymphatic chemokine–proteoglycan interaction interferes with the lymphatic outflow of leukocytes from the renal graft and might decrease the anti-graft allo-immune response. Methods In a rat renal chronic transplant dysfunction model (female Dark-Agouti to male Wistar Furth), chemokines were profiled by qRT-PCR in microdissected tubulo-interstitial tissue. Disruption of lymphatic chemokine–proteoglycan interaction was studied by (non-anticoagulant) heparin-derived polysaccharides in vitro and in renal allografts. The renal allograft function was assessed by rise in plasma creatinine and urea. Results Within newly-formed lymph vessels of transplanted kidneys, numerous CD45+ leukocytes were found, mainly MHCII+, ED-1-, IDO-, HIS14-, CD103- antigen presenting cells, most likely representing a subset of dendritic cells. Treatment of transplanted rats with regular heparin and two different (non-)anticoagulant heparin derivatives revealed worsening of kidney function only in the glycol-split heparin treated group despite a two-fold reduction of tubulo-interstitial leukocytes (p

Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not yet clear. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately in a rat model to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in 3-month old male Wistar rats by adriamycin injection. After 6 weeks, when proteinuria has developed, rats were treated for another 6 weeks by anti-VEGFR3 antibody, clodronate liposomes or FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however, without significant effects on inflammatory and fibrotic markers or proteinuria. Clodronate liposomes inhibited macrophage influx and partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation, T-cell influx and interstitial fibrosis, and partially reduced macrophage number and proteinuria; however, it did not significantly influence lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or the influx of macrophages. On the other hand, FTY720 treatment did prevent T-cell influx, myofibroblast accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions., Summary: Targeting lymphangiogenesis, inflammation or fibrosis separately in a rat model of proteinuric nephropathy showed that treating any of these changes alone is not effective in treating the disease.

Lymphatic vessels (LVs) are involved in a number of physiological and pathophysiological processes such as fluid homoeostasis, immune surveillance, and resolution of inflammation and wound healing. Lymphangiogenesis, the outgrowth of existing LVs and the formation of new ones, has received increasing attention over the past decade on account of its prominence in organ physiology and pathology, which has been enabled by the development of specific tools to study lymph vessel functions. Several studies have been devoted to renal lymphatic vasculature and lymphangiogenesis in kidney diseases, such as chronic renal transplant dysfunction, primary renal fibrotic disorders, proteinuria, diabetic nephropathy and renal inflammation. This review describes the most recent findings on lymphangiogenesis, with a specific focus on renal lymphangiogenesis and its impact on renal diseases. We suggest renal lymphatics as a possible target for therapeutic interventions in renal medicine to dampen tubulointerstitial tissue remodelling and improve renal functioning.

In this paper, the experimental and numerical failure analysis of Fiber Metal Laminates (FML) with dif-ferent types of cutouts, were investigated. Fiber metal laminates are types of materials which are consist of com-bination of metal sheets, especially aluminum ones, with fiber reinforced epoxy layers. At first, specimens were manufactured and elliptical and circular cutouts were cre-ated in them. Then subjected to in-plane tensional loading to carry out the behaviors, also, finite element analysis was carried out with ABAQUS commercial software. RIKS method was used in this analysis. Finally, experimental and finite element results were compared with results of elastic compensation method. DOI: http://dx.doi.org/10.5755/j01.mech.20.1.3530

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10(-137)) in male mice; Ctnnbip1 (P = 6.42×10(-267)) and Tnfrsf8 (P = 5.42×10(-245)) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.

Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for these collagens, namely a supportive role in leukocyte recruitment. We subjected mice deficient in collagen XV or collagen XVIII, and their compound mutant, as well as the wild-type control mice to bilateral renal ischemia/reperfusion, and evaluated renal function, tubular injury, and neutrophil and macrophage influx at different time points after ischemia/reperfusion. Five days after ischemia/reperfusion, the collagen XV, collagen XVIII and the compound mutant mice showed diminished serum urea levels compared to wild-type mice (all p

In the present study, the experimental and numerical stress analysis of Fiber Metal Laminates (FML) with different types of cutouts was investigated. FMLs consist of combination of metal sheets, especially aluminium ones, with fiber reinforced epoxy layers. Mechanical properties of FML were investigated by standard tensile tests. Strain gauges were used to measure the strain values near the cutout and then stresses were estimated according to the mechanical properties which obtained in the experiments. Moreover, finite element analysis was applied as the second method. Finally, experimental and finite element results were compared. DOI: http://dx.doi.org/10.5755/j01.mech.19.2.4152

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet. Therefore, we evaluated the time course of lymph vessel (LV) formation in relation to proteinuria and interstitial damage in a rat model of chronic unilateral adriamycin nephrosis. Proteinuria and kidneys were evaluated up to 30 weeks after induction of nephrosis. LVs were identified by podoplanin/VEGFR3 double staining. After 6 weeks proteinuria was well-established, without influx of interstitial macrophages and myofibroblasts, collagen deposition, osteopontin expression (tubular activation) or LV formation. At 12 weeks, a ∼3-fold increase in cortical LV density was found (p