Calprotectin is a contributor to and potential therapeutic target for vascular calcification in chronic kidney disease.

Vascular calcification is an important risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). It is also a complex process involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. In an observational, multicenter European study, including 112 patients with CKD from Spain and 171 patients on dialysis from France, we used serum proteome analysis and further validation by ELISA to identify calprotectin, a circulating damage-associated molecular pattern protein, as being independently associated with CV outcome and mortality. This was confirmed in an additional cohort of 170 patients with CKD from Sweden, where increased serum calprotectin concentrations correlated with increased vascular calcification. In primary human VSMCs and mouse aortic rings, calprotectin exacerbated calcification. Treatment with paquinimod, a calprotectin inhibitor, as well as pharmacological inhibition of the receptor for advanced glycation end products and Toll-like receptor 4 inhibited the procalcifying effect of calprotectin. Paquinimod also ameliorated calcification induced by the sera of uremic patients in primary human VSMCs. Treatment with paquinimod prevented vascular calcification in mice with chronic renal failure induced by subtotal nephrectomy and in aged apolipoprotein E-deficient mice as well. These observations identified calprotectin as a key contributor of vascular calcification, and increased circulating calprotectin was strongly and independently associated with calcification, CV outcome, and mortality in patients with CKD. Inhibition of calprotectin might therefore be a promising strategy to prevent vascular calcification in patients with CKD.

Insulin signaling promotes neurogenesis in the brain of adult zebrafish.

Insulin is a peptide hormone that plays a central role in the regulation of circulating blood glucose in vertebrates, including zebrafish. Increasing evidence has demonstrated the important role of insulin in many brain functions. In zebrafish, two insulin receptor genes (insra and insrb) have been identified. However, their biodistribution in the adult brain as well as their cell-specific expression pattern has not been well described. Using gene expression analysis, in situ hybridization and transgenic fish, we confirmed the expression of insra, insrb, and irs1 (insulin receptor substrate 1, the downstream effector of insulin receptor) in the brain of adult zebrafish and characterized their specific expression in neurons and neural stem cells (radial glia). After demonstrating that intracerebroventricular (ICV) injection resulted in the diffusion of the injected solution within the ventricular system, we analyzed the effect of insulin ICV injection on neurogenesis. We showed that insulin promotes ventricular cell proliferation 24 h postinjection. This neurogenic effect appeared to be independent of neuroinflammatory processes. Also, after a mechanical telencephalic stab-wound injury, we highlighted the overexpression of irs1 gene 5 days postlesion notably in the ventricular zone where radial glial cells (RGCs) are localized, suggesting key roles of insulin signaling in regenerative processes. Finally, our results reinforced the expression of insulin-related proteins in the brain of adult zebrafish, highlighting the potential role of insulin signaling on neurogenesis.

Aqueous Extract of Psiloxylon mauritianum, Rich in Gallic Acid, Prevents Obesity and Associated Deleterious Effects in Zebrafish.

Obesity has reached epidemic proportions, and its prevalence tripled worldwide between 1975 and 2016, especially in Reunion Island, a French overseas region. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island registered in the French pharmacopeia, has recently gained interest in combating metabolic disorders because of its traditional lipid-lowering and "anti-diabetic" use. However, scientific data are lacking regarding its toxicity and its real benefits on metabolic diseases. In this study, we aim to determine the toxicity of an aqueous extract of P. mauritianum on zebrafish eleutheroembryos following the OECD toxicity assay (Organization for Economic Cooperation and Development, guidelines 36). After defining a non-toxic dose, we determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) that this extract is rich in gallic acid but contains also caffeoylquinic acid, kaempferol and quercetin, as well as their respective derivatives. We also showed that the non-toxic dose exhibits lipid-lowering effects in a high-fat-diet zebrafish larvae model. In a next step, we demonstrated its preventive effects on body weight gain, hyperglycemia and liver steatosis in a diet-induced obesity model (DIO) performed in adults. It also limited the deleterious effects of overfeeding on the central nervous system (i.e., cerebral oxidative stress, blood-brain barrier breakdown, neuro-inflammation and blunted neurogenesis). Interestingly, adult DIO fish treated with P. mauritianum display normal feeding behavior but higher feces production. This indicates that the "anti-weight-gain" effect is probably due to the action of P. mauritianum on the intestinal lipid absorption and/or on the microbiota, leading to the increase in feces production. Therefore, in our experimental conditions, the aqueous extract of P. mauritianum exhibited "anti-weight-gain" properties, which prevented the development of obesity and its deleterious effects at the peripheral and central levels. These effects should be further investigated in preclinical models of obese/diabetic mice, as well as the impact of P. mauritianum on the gut microbiota.

High Glucose Induces in HK2 Kidney Cells an IFN-Dependent ZIKV Antiviral Status Fueled by Viperin.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that rapidly became a major medical concern worldwide. We have recently reported that a high glucose level decreases the rate of Zika virus (ZIKV) replication with an impact on human kidney HK-2 cell survival. However, the mechanisms by which cells cultured in a high glucose medium inhibit ZIKV growth remain unclear. Viperin belongs to interferon-stimulated genes (ISG) and its expression is highly up-regulated upon viral infection, leading to antiviral activity against a variety of viruses, including flaviviruses. As such, viperin has been shown to be a major actor involved in the innate immune response against Zika virus (ZIKV). Our present study aims to further characterize the involvement of viperin in ZIKV growth inhibition under high glucose concentration (HK-2HGC). We show for the first time that endogenous viperin is over-expressed in HK-2 cells cultured under high glucose concentration (HK-2HGC), which is associated with ZIKV growth inhibition. Viperin knockdown in HK-2HGC rescues ZIKV growth. In addition, our results emphasize that up-regulated viperin in HK-2HGC leads to ZIKV growth inhibition through the stimulation of IFN-ß production. In summary, our work provides new insights into the ZIKV growth inhibition mechanism observed in HK-2 cells cultured in a high glucose environment.

Hypericum lanceolatum Lam. Medicinal Plant: Potential Toxicity and Therapeutic Effects Based on a Zebrafish Model.

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.

Zika Virus Growth in Human Kidney Cells Is Restricted by an Elevated Glucose Level.

Mosquito-borne Zika virus (ZIKV) became a real threat to human health due to the lack of vaccine and effective antiviral treatment. The virus has recently been responsible for a global outbreak leading to millions of infected cases. ZIKV complications were highlighted in adults with Guillain-Barré syndrome and in newborns with increasing numbers of congenital disorders ranging from mild developmental delays to fatal conditions. The ability of ZIKV to establish a long-term infection in diverse organs including the kidneys has been recently documented but the consequences of such a viral infection are still debated. Our study aimed to determine whether the efficiency of ZIKV growth in kidney cells relates to glucose concentration. Human kidney HK-2 cells were infected with different ZIKV strains in presence of normal and high glucose concentrations. Virological assays showed a decrease in viral replication without modifying entry steps (viral binding, internalization, fusion) under high glucose conditions. This decrease replication was associated with a lower virus progeny and increased cell viability when compared to ZIKV-infected HK-2 cells in normal glucose concentration. In conclusion, we showed for the first time that an elevated glucose level influences ZIKV replication level with an effect on kidney cell survival.

Urinary Peptides as Potential Non-Invasive Biomarkers for Lupus Nephritis: Results of the Peptidu-LUP Study.

Background: Lupus nephritis (LN) is a severe manifestation of Systemic Lupus Erythematosus (SLE). The therapeutic strategy relies on kidney biopsy (KB) results. We tested whether urinary peptidome analysis could non-invasively differentiate active from non-active LN. Design: Urinary samples were collected from 93 patients (55 with active LN and 38 with non-active LN), forming a discovery (n = 42) and an independent validation (n = 51) cohort. Clinical characteristics were collected at inclusion and prospectively for 24 months. The urinary peptidome was analyzed by capillary-electrophoresis coupled to mass-spectrometry, comparing active LN to non-active LN, and assessing chronic lesions and response to therapy. The value of previously validated prognostic (CKD273) and differential diagnostic (LN172) signatures was evaluated. Results: Urinary peptides could not discriminate between active and non-active LN or predict early response to therapy. Tubulo-interstitial fibrosis was correlated to the CKD273. The LN172 score identified 92.5% of samples as LN. Few patients developed new-onset CKD. Conclusions: We validated the CKD273 and LN172 classifiers but did not identify a robust signature that could predict active LN and replace KB. The value of urinary peptidome to predict long-term CKD, or renal flares in SLE, remains to be evaluated.

Caffeic Acid, One of the Major Phenolic Acids of the Medicinal Plant Antirhea borbonica, Reduces Renal Tubulointerstitial Fibrosis.

The renal fibrotic process is characterized by a chronic inflammatory state and oxidative stress. Antirhea borbonica (A. borbonica) is a French medicinal plant found in Reunion Island and known for its antioxidant and anti-inflammatory activities mostly related to its high polyphenols content. We investigated whether oral administration of polyphenol-rich extract from A. borbonica could exert in vivo a curative anti-renal fibrosis effect. To this aim, three days after unilateral ureteral obstruction (UUO), mice were daily orally treated either with a non-toxic dose of polyphenol-rich extract from A. borbonica or with caffeic acid (CA) for 5 days. The polyphenol-rich extract from A. borbonica, as well as CA, the predominant phenolic acid of this medicinal plant, exerted a nephroprotective effect through the reduction in the three phases of the fibrotic process: (i) macrophage infiltration, (ii) myofibroblast appearance and (iii) extracellular matrix accumulation. These effects were associated with the mRNA down-regulation of Tgf-ß, Tnf-α, Mcp1 and NfkB, as well as the upregulation of Nrf2. Importantly, we observed an increased antioxidant enzyme activity for GPX and Cu/ZnSOD. Last but not least, desorption electrospray ionization-high resolution/mass spectrometry (DESI-HR/MS) imaging allowed us to visualize, for the first time, CA in the kidney tissue. The present study demonstrates that polyphenol-rich extract from A. borbonica significantly improves, in a curative way, renal tubulointerstitial fibrosis progression in the UUO mouse model.

Phenolic Profile of Herbal Infusion and Polyphenol-Rich Extract from Leaves of the Medicinal Plant Antirhea borbonica: Toxicity Assay Determination in Zebrafish Embryos and Larvae.

Antirhea borbonica (A. borbonica) is an endemic plant from the Mascarene archipelago in the Indian Ocean commonly used in traditional medicine for its health benefits. This study aims (1) at exploring polyphenols profiles from two types of extracts-aqueous (herbal infusion) and acetonic (polyphenol rich) extracts from A. borbonica leaves-and (2) at evaluating their potential toxicity in vivo for the first time. We first demonstrated that, whatever type of extraction is used, both extracts displayed significant antioxidant properties and acid phenolic and flavonoid contents. By using selective liquid chromatography-tandem mass spectrometry, we performed polyphenol identification and quantification. Among the 19 identified polyphenols, we reported that the main ones were caffeic acid derivatives and quercetin-3-O-rutinoside. Then, we performed a Fish Embryo Acute Toxicity test to assess the toxicity of both extracts following the Organisation for Economic Cooperation and Development (OECD) guidelines. In both zebrafish embryos and larvae, the polyphenols-rich extract obtained by acetonic extraction followed by evaporation and resuspension in water exhibits a higher toxic effect with a median lethal concentration (LC50: 5.6 g/L) compared to the aqueous extract (LC50: 20.3 g/L). Our data also reveal that at non-lethal concentrations of 2.3 and 7.2 g/L for the polyphenol-rich extract and herbal infusion, respectively, morphological malformations such as spinal curvature, pericardial edema, and developmental delay may occur. In conclusion, our study strongly suggests that the evaluation of the toxicity of medicinal plants should be systematically carried out and considered when studying therapeutic effects on living organisms.

Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease.

While blocking the renin angiotensin aldosterone system (RAAS) has been the main therapeutic strategy to control diabetic kidney disease (DKD) for many years, 25-30% of diabetic patients still develop the disease. In the present work we adopted a systems biology strategy to analyze glomerular protein signatures to identify drugs with potential therapeutic properties in DKD acting through a RAAS-independent mechanism. Glomeruli were isolated from wild type and type 1 diabetic (Ins2Akita) mice treated or not with the angiotensin-converting enzyme inhibitor (ACEi) ramipril. Ramipril efficiently reduced the urinary albumin/creatine ratio (ACR) of Ins2Akita mice without modifying DKD-associated renal-injuries. Large scale quantitative proteomics was used to identify the DKD-associated glomerular proteins (DKD-GPs) that were ramipril-insensitive (RI-DKD-GPs). The raw data are publicly available via ProteomeXchange with identifier PXD018728. We then applied an in silico drug repurposing approach using a pattern-matching algorithm (Connectivity Mapping) to compare the RI-DKD-GPs's signature with a collection of thousands of transcriptional signatures of bioactive compounds. The sesquiterpene lactone parthenolide was identified as one of the top compounds predicted to reverse the RI-DKD-GPs's signature. Oral treatment of 2 months old Ins2Akita mice with dimethylaminoparthenolide (DMAPT, a water-soluble analogue of parthenolide) for two months at 10 mg/kg/d by gavage significantly reduced urinary ACR. However, in contrast to ramipril, DMAPT also significantly reduced glomerulosclerosis and tubulointerstitial fibrosis. Using a system biology approach, we identified DMAPT, as a compound with a potential add-on value to standard-of-care ACEi-treatment in DKD.

Impaired brain homeostasis and neurogenesis in diet-induced overweight zebrafish: a preventive role from A. borbonica extract.

Overweight and obesity are worldwide health concerns leading to many physiological disorders. Recent data highlighted their deleterious effects on brain homeostasis and plasticity, but the mechanisms underlying such disruptions are still not well understood. In this study, we developed and characterized a fast and reliable diet-induced overweight (DIO) model in zebrafish, for (1) studying the effects of overfeeding on brain homeostasis and for (2) testing different preventive and/or therapeutic strategies. By overfeeding zebrafish for 4 weeks, we report the disruption of many metabolic parameters reproducing human overweight features including increased body weight, body mass index, fasting blood glucose levels and liver steatosis. Furthermore, DIO fish displayed blood-brain barrier leakage, cerebral oxidative stress, neuroinflammation and decreased neurogenesis. Finally, we investigated the preventive beneficial effects of A. borbonica, an endogenous plant from Reunion Island. Overnight treatment with A. borbonica aqueous extract during the 4 weeks of overfeeding limited some detrimental central effects of DIO. In conclusion, we established a relevant DIO model in zebrafish demonstrating that overfeeding impairs peripheral and central homeostasis. This work also highlights the preventive protective effects of A. borbonica aqueous extracts in DIO, and opens a way to easily screen drugs aiming at limiting overweight and associated neurological disorders.

Pathogenic Leptospira and their animal reservoirs: testing host specificity through experimental infection.

Leptospirosis is caused by pathogenic Leptospira transmitted through contact with contaminated environments. Most mammalian species are infectable by Leptospira but only few act as efficient reservoir being capable of establishing long term kidney colonization and shedding Leptospira in urine. In Madagascar, a large diversity of pathogenic Leptospira display a tight specificity towards their endemic volant or terrestrial mammalian hosts. The basis of this specificity is unknown: it may indicate some genetically determined compatibility between host cells and bacteria or only reflect ecological constraints preventing contacts between specific hosts. In this study, Rattus norvegicus was experimentally infected with either Leptospira interrogans, Leptospira borgpetersenii or Leptospira mayottensis isolated from rats, bats or tenrecs, respectively. Leptospira borgpetersenii and L. mayottensis do not support renal colonization as featured by no shedding of live bacteria in urine and low level and sporadic detection of Leptospira DNA in kidneys. In contrast 2 out of the 7 R. norvegicus challenged with L. interrogans developed renal colonization and intense Leptospira shedding in urine throughout the 3 months of experimental infection. These data suggest that host-Leptospira specificity in this biodiversity hotspot is driven at least in part by genetic determinants likely resulting from long-term co-diversification processes.

Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation.

BACKGROUND: The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation. METHODS: Complement deposition on endothelial microvesicles was assayed in vasculitis patient plasma samples and controls. Plasma was perfused over glomerular endothelial cells and complement deposition assayed by flow cytometry. The effect of the kinin system was assessed using kinin receptor antagonists and C1-inhibitor. The in vivo effect was assessed in kidney sections from mice with nephrotoxic serum-induced glomerulonephritis treated with a kinin receptor antagonist. FINDINGS: Vasculitis patient plasma had significantly more C3- and C9-positive endothelial microvesicles than controls. Perfusion of patient acute-phase plasma samples over glomerular endothelial cells induced the release of significantly more complement-positive microvesicles, in comparison to remission or control plasma. Complement activation on endothelial microvesicles was reduced by kinin B1- and B2-receptor antagonists or by C1-inhibitor (the main inhibitor of the classical pathway and the KKS). Likewise, perfusion of glomerular endothelial cells with C1-inhibitor-depleted plasma induced the release of complement-positive microvesicles, which was significantly reduced by kinin-receptor antagonists or C1-inhibitor. Mice with nephrotoxic serum-induced glomerulonephritis exhibited significantly reduced glomerular C3 deposition when treated with a B1-receptor antagonist. INTERPRETATION: Excessive complement deposition on the endothelium will promote endothelial injury and the release of endothelial microvesicles. This study demonstrates that blockade of the KKS can reduce complement activation and thereby the inflammatory response on the endothelium. FUNDING: Full details are provided in the Acknowledgements/Funding section.

Apelin affects the mouse aging urinary peptidome with minimal effects on kidney.

Kidney function is altered by age together with a declined filtration capacity of 5-10% per decade after 35 years. Renal aging shares many characteristics with chronic kidney disease. Plasma levels of the bioactive peptide apelin also decline with age and apelin has been shown to be protective in chronic kidney disease. Therefore we evaluated whether apelin could also improve aging-induced renal lesions and function in mice. Since urine is for the major part composed of proteins and peptides originating from the kidney, we first studied apelin-induced changes, in the aging urinary peptidome. Despite the recently published age-associated plasma decrease of apelin, expression of the peptide and its receptor was increased in the kidneys of 24 months old mice. Twenty-eight days treatment with apelin significantly modified the urinary peptidome of 3 and 24 months old mice towards a signature suggesting more advanced age at 3 months, and a younger age at 24 months. The latter was accompanied by a decreased staining of collagen (Sirius red staining) in 24 months old apelin-treated mice, without changing aging-induced glomerular hypertrophy. In addition, apelin was without effect on aging-induced renal autophagy, apoptosis, inflammation and reduced renal function. In conclusion, treatment of aged mice with apelin had a limited effect on kidney lesions although modifying the urinary peptidome towards a younger signature. This supports evidence of apelin inducing more general beneficial effects on other aging organs, muscles in particular, as recently shown for sarcopenia, markers of which end up via the glomerular filtration in urine.

Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment.

Although cardiovascular disease (CVD) is the leading cause of morbimortality worldwide, promising new drug candidates are lacking. We compared the arterial high-resolution proteome of patients with advanced versus early-stage CVD to predict, from a library of small bioactive molecules, drug candidates able to reverse this disease signature. Of the approximately 4000 identified proteins, 100 proteins were upregulated and 52 were downregulated in advanced-stage CVD. Arachidonyl trifluoromethyl ketone (AACOCF3), a cytosolic phospholipase A2 (cPLA2) inhibitor was predicted as the top drug able to reverse the advanced-stage CVD signature. Vascular cPLA2 expression was increased in patients with advanced-stage CVD. Treatment with AACOCF3 significantly reduced vascular calcification in a cholecalciferol-overload mouse model and inhibited osteoinductive signaling in vivo and in vitro in human aortic smooth muscle cells. In conclusion, using a systems biology approach, we have identified a potentially new compound that prevented typical vascular calcification in CVD in vivo. Apart from the clear effect of this approach in CVD, such strategy should also be able to generate novel drug candidates in other complex diseases.

Proteomics based identification of KDM5 histone demethylases associated with cardiovascular disease.

BACKGROUND: The increased prevalence of cardiovascular disease (CVD) indicates a demand for novel therapeutic approaches. Proteome analysis of vascular tissues from animal models and humans with CVD could lead to the identification of novel druggable targets. METHODS: LC-MS/MS analysis of thoracic aortas from three mouse models of non-diabetic and diabetic (streptozotocin (STZ)-induced) atherosclerosis followed by bioinformatics/pathway analysis was performed. Selected findings were confirmed by proteomics analysis of human vessels from patients with CVD as well as in vitro studies (migration, proliferation, angiogenesis assays) using endothelial (HUVEC) cells. FINDINGS: Comparative tissue proteomics of low density lipoprotein receptor deficient (Ldlr-/-) and diabetic Ldlr-/- (Ldlr-/-STZ) with wild type (WT) animals led to the identification of 284 differentially expressed proteins in both models. Among them, 177 proteins were also differentially expressed in diabetic apolipoprotein E deficient (ApoE-/-STZ) mice, suggesting expression changes associated with atherosclerosis independent of the model used. These proteins recapitulated the hallmarks of atherosclerosis. Comparison of these findings with differentially expressed proteins in human vessels with CVD enabled shortlisting of six commonly dysregulated proteins. Among them, lysine-specific demethylase 5D (KDM5D) exhibited pronounced overexpression accompanied by a reduction in the protein levels of its substrate, the trimethylated lysine 4 of histone H3 (H3K4me3), in patients with CVD. Functional interference studies applying a KDM5 inhibitor on HUVEC reduced cell proliferation, migration and tube-forming ability in vitro. INTERPRETATION: This high-throughput proteomics strategy identified KDM5 histone demethylases being potentially involved in CVD, possibly by affecting H3K4 methylation. FUND: [SysVasc, HEALTH-2013 603288], [ERA-CVD PROACT: ANR-17-ECVD-0006, 01KL1805], [FRM, DEQ20170336759].

Three Leptospira Strains From Western Indian Ocean Wildlife Show Highly Distinct Virulence Phenotypes Through Hamster Experimental Infection.

Leptospirosis is one of the most widespread zoonoses worldwide, with highest incidence reported on tropical islands. Recent investigations carried out in a One-Health framework have revealed a wide diversity of pathogenic Leptospira lineages on the different islands of Western Indian Ocean carried out by a large diversity of mammal reservoirs, including domestic and wild fauna. Using golden Syrian hamsters as a model of acute infection, we studied the virulence of Leptospira interrogans, L. mayottensis, and L. borgpetersenii isolates obtained from rats, tenrecs, and bats, respectively. Hamsters were inoculated with 2.108 bacterial cells and monitored for 1 month. The L. interrogans isolate proved to be the most pathogenic while L. mayottensis and L. borgpetersenii isolates induced no clinical symptoms in the infected hamsters. High leptospiral DNA amounts were also detected in the urine and organs of hamsters infected with the L. interrogans isolate while L. mayottensis and L. borgpetersenii isolates mostly failed to disseminate into the organism. In addition, histological damage was more pronounced in the kidneys and lungs of hamsters infected with the L. interrogans isolate. Altogether, these data support that Leptospira strains shed by mammals endemic to this insular ecosystem (L. mayottensis and L. borgpetersenii isolates) are less pathogenic than the L. interrogans rat-borne isolate. These results may provide a relevant framework for understanding the contrasting epidemiology of human leptospirosis observed among Western Indian Ocean islands.

Ldlr-/- and ApoE-/- mice better mimic the human metabolite signature of increased carotid intima media thickness compared to other animal models of cardiovascular disease.

BACKGROUND AND AIMS: Preclinical experiments on animal models are essential to understand the mechanisms of cardiovascular disease (CVD). Metabolomics allows access to the metabolic perturbations associated with CVD in heart and vessels. Here we assessed which potential animal CVD model most closely mimics the serum metabolite signature of increased carotid intima-media thickness (cIMT) in humans, a clinical parameter widely accepted as a surrogate of CVD. METHODS: A targeted mass spectrometry assay was used to quantify and compare a series of blood metabolites between 1362 individuals (KORA F4 cohort) and 5 animal CVD models: ApoE-/-, Ldlr-/-, and klotho-hypomorphic mice (kl/kl) and SHRSP rats with or without salt feeding. The metabolite signatures were obtained using linear regressions adjusted for various co-variates. RESULTS: In human, increased cIMT [quartile Q4 vs. Q1] was associated with 26 metabolites (9 acylcarnitines, 2 lysophosphatidylcholines, 9 phosphatidylcholines and 6 sphingomyelins). Acylcarnitines correlated preferentially with serum glucose and creatinine. Phospholipids correlated preferentially with cholesterol (total and LDL). The human signature correlated positively and significantly with Ldlr-/- and ApoE-/- mice, while correlation with kl/kl mice and SHRP rats was either negative and non-significant. Human and Ldlr-/- mice shared 11 significant metabolites displaying the same direction of regulation: 5 phosphatidylcholines, 1 lysophosphatidylcholines, 5 sphingomyelins; ApoE-/- mice shared 10. CONCLUSIONS: The human cIMT signature was partially mimicked by Ldlr-/- and ApoE-/- mice. These animal models might help better understand the biochemical and molecular mechanisms involved in the vessel metabolic perturbations associated with, and contributing to metabolic disorders in CVD.

Combination of the fetal urinary metabolome and peptidome for the prediction of postnatal renal outcome in fetuses with PUV.

Most of biomarker panels, extracted from single omics traits, still need improvement since they display a gray zone where prediction is uncertain. Here we verified whether a combination of omics traits, fetal urinary metabolites and peptides analyzed in the same sample, improved prediction of postnatal renal function in fetuses with posterior urethral valves (PUV) compared to individual omics traits. Using CE-MS, we explored the urinary metabolome of 13 PUV fetuses with end stage renal disease (ESRD) and 12 PUV fetuses without postnatal ESRD at 2 years postnatally. This allowed the selection of 24 differentially abundant metabolite features which were modelled into predictive classifiers, alone or in combination with 12 peptides previously identified as predictive of ESRD. Validation in 35 new fetuses showed that the combination of peptides and metabolites significantly outperformed the 24 metabolite features with increased AUC (0.987 vs 0.905), net reclassification improvement (36%) and better sensitivity accuracy (86% vs 60%). In addition, the two trait combination tended to improve, but without reaching statistical significance, the already high performances of the 12 peptide biomarkers (AUC 0.967, accuracy 80%). In conclusion, this study demonstrates the potential of cumulating different omics traits in biomarker research where single omics traits fall short. SIGNIFICANCE: Although increasingly proposed in disease-diagnosis and -prognosis because of their improved efficacy over single markers, panels of body fluid biomarkers based on single omics analysis still fail to display perfect accuracy, probably due to biological variability. Here, we hypothesized that combination of different omics traits allowed to better capture this biological variability. As proof of concept, we studied the added value of fetal urine metabolites and peptides using CE-MS, starting from the same urine sample, to predict postnatal renal outcome in fetuses with posterior urethral valves. We observed that the prognostic power of combined metabolite and peptide markers was clearly higher than that of metabolites alone and slightly, but non-significantly, improved compared to the peptides alone. To our knowledge, this report is the first to demonstrate that combining multiomics traits extracted from (fetal) urine samples displays clear promise for kidney disease stratification.

Urinary peptidomics analysis reveals proteases involved in diabetic nephropathy.

Mechanisms underlying the onset and progression of nephropathy in diabetic patients are not fully elucidated. Deregulation of proteolytic systems is a known path leading to disease manifestation, therefore we hypothesized that proteases aberrantly expressed in diabetic nephropathy (DN) may be involved in the generation of DN-associated peptides in urine. We compared urinary peptide profiles of DN patients (macroalbuminuric, n = 121) to diabetic patients with no evidence of DN (normoalbuminuric, n = 118). 302 sequenced, differentially expressed peptides (adjusted p-value < 0.05) were analysed with the Proteasix tool predicting proteases potentially involved in their generation. Activity change was estimated based on the change in abundance of the investigated peptides. Predictions were correlated with transcriptomics (Nephroseq) and relevant protein expression data from the literature. This analysis yielded seventeen proteases, including multiple forms of MMPs, cathepsin D and K, kallikrein 4 and proprotein convertases. The activity of MMP-2 and MMP-9, predicted to be decreased in DN, was investigated using zymography in a DN mouse model confirming the predictions. Collectively, this proof-of-concept study links urine peptidomics to molecular changes at the tissue level, building hypotheses for further investigation in DN and providing a workflow with potential applications to other diseases.