Complement Cascade Proteins Correlate with Fibrosis and Inflammation in Early-Stage Type 1 Diabetic Kidney Disease in the Ins2Akita Mouse Model.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses' results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = -0.58 to -0.842 and range for IGHM rs = -0.6 to -0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

Single-cell RNA sequencing identifies senescence as therapeutic target in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: The role of macrophages in the development of rhabdomyolysis-induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells. METHODS: Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2 days after injury. Public datasets from murine AKI models were reanalysed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI. RESULTS: Unsupervised clustering of nearly 17 000 single-cell transcriptomes identified seven known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells revealed nine distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one major histocompatibility complex class IIhigh (MHCIIhigh) cluster only present in Control to two MHCIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI. CONCLUSIONS: Single-cell RNA sequencing unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation.

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.

Proteomic Analysis of Mouse Kidney Tissue Associates Peroxisomal Dysfunction with Early Diabetic Kidney Disease.

BACKGROUND: The absence of efficient inhibitors for diabetic kidney disease (DKD) progression reflects the gaps in our understanding of DKD molecular pathogenesis. METHODS: A comprehensive proteomic analysis was performed on the glomeruli and kidney cortex of diabetic mice with the subsequent validation of findings in human biopsies and omics datasets, aiming to better understand the underlying molecular biology of early DKD development and progression. RESULTS: LC-MS/MS was employed to analyze the kidney proteome of 2 DKD models: Ins2Akita (early and late DKD) and db/db mice (late DKD). The abundance of detected proteins was defined. Pathway analysis of differentially expressed proteins in the early and late DKD versus the respective controls predicted dysregulation in DKD hallmarks (peroxisomal lipid metabolism and ß-oxidation), supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, the consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal and upregulated mitochondrial proteins. Tissue sections from 16 DKD patients were analyzed by IHC confirming our results. CONCLUSION: Our study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity, providing new perspectives and potential markers of diabetic kidney dysfunction.

Hepatocyte nuclear factor-1ß shapes the energetic homeostasis of kidney tubule cells.

Energetic metabolism controls key steps of kidney development, homeostasis, and epithelial repair following acute kidney injury (AKI). Hepatocyte nuclear factor-1ß (HNF-1ß) is a master transcription factor that controls mitochondrial function in proximal tubule (PT) cells. Patients with HNF1B pathogenic variant display a wide range of kidney developmental abnormalities and progressive kidney fibrosis. Characterizing the metabolic changes in PT cells with HNF-1ß deficiency may help to identify new targetable molecular hubs involved in HNF1B-related kidney phenotypes and AKI. Here, we combined 1 H-NMR-based metabolomic analysis in a murine PT cell line with CrispR/Cas9-induced Hnf1b invalidation (Hnf1b-/- ), clustering analysis, targeted metabolic assays, and datamining of published RNA-seq and ChIP-seq dataset to identify the role of HNF-1ß in metabolism. Hnf1b-/- cells grown in normoxic conditions display intracellular ATP depletion, increased cytosolic lactate concentration, increased lipid droplet content, failure to use pyruvate for energetic purposes, increased levels of tricarboxylic acid (TCA) cycle intermediates and oxidized glutathione, and a reduction of TCA cycle byproducts, all features consistent with mitochondrial dysfunction and an irreversible switch toward glycolysis. Unsupervised clustering analysis showed that Hnf1b-/- cells mimic a hypoxic signature and that they cannot furthermore increase glycolysis-dependent energetic supply during hypoxic challenge. Metabolome analysis also showed alteration of phospholipid biosynthesis in Hnf1b-/- cells leading to the identification of Chka, the gene coding for choline kinase α, as a new putative target of HNF-1ß. HNF-1ß shapes the energetic metabolism of PT cells and HNF1B deficiency in patients could lead to a hypoxia-like metabolic state precluding further adaptation to ATP depletion following AKI.

Metabolic and cardiovascular adaptations to an 8-wk lifestyle weight loss intervention in younger and older obese men.

The number of older obese adults is increasing worldwide. Whether obese adults show similar health benefits in response to lifestyle interventions at different ages is unknown. The study enrolled 25 obese men (body mass index: 31-39 kg/m2) in two arms according to age (30-40 and 60-70 yr old). Participants underwent an 8-wk intervention with moderate calorie restriction (∼20% below individual energy requirements) and supervised endurance training resulting in ∼5% weight loss. Body composition was measured using dual energy X-ray absorptiometry. Insulin sensitivity was assessed during a hypersinsulinemic-euglycemic clamp. Cardiometabolic profile was derived from blood parameters. Subcutaneous fat and vastus lateralis muscle biopsies were used for ex vivo analyses. Two-way repeated-measure ANOVA and linear mixed models were used to evaluate the response to lifestyle intervention and comparison between the two groups. Fat mass was decreased and bone mass was preserved in the two groups after intervention. Muscle mass decreased significantly in older obese men. Cardiovascular risk (Framingham risk score, plasma triglyceride, and cholesterol) and insulin sensitivity were greatly improved to a similar extent in the two age groups after intervention. Changes in adipose tissue and skeletal muscle transcriptomes were marginal. Analysis of the differential response to the lifestyle intervention showed tenuous differences between age groups. These data suggest that lifestyle intervention combining calorie restriction and exercise shows similar beneficial effects on cardiometabolic risk and insulin sensitivity in younger and older obese men. However, attention must be paid to potential loss of muscle mass in response to weight loss in older obese men.NEW & NOTEWORTHY Rise in obesity and aging worldwide are major trends of critical importance in public health. This study addresses a current challenge in obesity management. Do older obese adults respond differently to a lifestyle intervention composed of moderate calorie restriction and supervised physical activity than younger ones? The main conclusion of the study is that older and younger obese men similarly benefit from the intervention in terms of cardiometabolic risk.

Mapping of the amniotic fluid proteome of fetuses with congenital anomalies of the kidney and urinary tract identifies plastin 3 as a protein involved in glomerular integrity.

Congenital anomalies of the kidney and the urinary tract (CAKUT) are the first cause of chronic kidney disease in childhood. Several genetic and environmental origins are associated with CAKUT, but most pathogenic pathways remain elusive. Considering the amniotic fluid (AF) composition as a proxy for fetal kidney development, we analyzed the AF proteome from non-severe CAKUT (n = 19), severe CAKUT (n = 14), and healthy control (n = 22) fetuses using LC-MS/MS. We identified 471 significant proteins that discriminated the three AF groups with 81% precision. Among them, eight proteins independent of gestational age (CSPG4, LMAN2, ENDOD1, ANGPTL2, PRSS8, NGFR, ROBO4, PLS3) were associated with both the presence and the severity of CAKUT. Among those, five were part of a protein-protein interaction network involving proteins previously identified as being potentially associated with CAKUT. The actin-bundling protein PLS3 (plastin 3) was the only protein displaying a gradually increased AF abundance from control, via non-severe, to severe CAKUT. Immunohistochemistry experiments showed that PLS3 was expressed in the human fetal as well as in both the fetal and the postnatal mouse kidney. In zebrafish embryos, depletion of PLS3 led to a general disruption of embryonic growth including reduced pronephros development. In postnatal Pls3-knockout mice, kidneys were macroscopically normal, but the glomerular ultrastructure showed thickening of the basement membrane and fusion of podocyte foot processes. These structural changes were associated with albuminuria and decreased expression of podocyte markers including Wilms' tumor-1 protein, nephrin, and podocalyxin. In conclusion, we provide the first map of the CAKUT AF proteome that will serve as a reference for future studies. Among the proteins strongly associated with CAKUT, PLS3 did surprisingly not specifically affect nephrogenesis but was found as a new contributor in the maintenance of normal kidney function, at least in part through the control of glomerular integrity. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The low affinity p75 neurotrophin receptor is down-regulated in congenital anomalies of the kidney and the urinary tract: Possible involvement in early nephrogenesis.

Congenital Anomalies of the Kidney and of the Urinary Tract (CAKUT) cover a broad range of disorders including abnormal kidney development caused by defective nephrogenesis. Here we explored the possible involvement of the low affinity p75 neurotrophin receptor (p75NTR) in CAKUT and nephrogenesis. In mouse, p75NTR was highly expressed in fetal kidney, located within cortical early nephrogenic bodies, and decreased rapidly after birth. In human control fetal kidney, p75NTR was also located within the early nephrogenic bodies as well as in the mature glomeruli, presumably in the mesangium. In CAKUT fetal kidneys, the kidney cortical structure and the localization of p75NTR were often disorganized, and quantification of p75NTR in amniotic fluid revealed a significant reduction in CAKUT compared to control. Finally, invalidation of p75NTR in zebrafish embryo with an antisense morpholino significantly altered pronephros development. Our results indicate that renal p75NTR is altered in CAKUT fetuses, and could participate to early nephrogenesis.

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.

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].

The CKD plasma lipidome varies with disease severity and outcome.

BACKGROUND: Various alterations in lipid metabolism have been observed in patients with chronic kidney disease (CKD). OBJECTIVES: To determine the levels of lipid species in plasma from CKD and hemodialysis (HD) patients and test their association with CKD severity and patient outcome. METHODS: Seventy-seven patients with CKD stage 2 to HD were grouped into classes of CKD severity at baseline and followed-up for 3.5 years for the occurrence of transition to HD or death (combined outcome). Plasma levels of phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), sphingomyelins (SMs), and fatty acids were analyzed by flow-injection analysis coupled to tandem mass spectrometry or gas chromatography coupled with mass spectrometry. Kruskal Wallis rank tests and Cox regressions were used to analyze the association of lipids with CKD severity and the risk of combined outcome, respectively. RESULTS: The plasma level of PCs, LPCs, and SMs was decreased in HD patients compared with nondialyzed CKD patients (all P < .05), whereas esterified and/or nonesterified fatty acids level did not change. Thirty-four lipids displayed significantly lower abundance in plasma of HD patients, whereas elaidic acid (C18:1ω9t) level was increased (P < .001). The total amount of LPCs and individual LPCs were associated with better outcome (P < .05). In particular, LPC 18:2 and LPC 20:3 were statistically associated with outcome in adjusted models (P < .05). DISCUSSION: In HD patients, a reduction in plasma lipids is observed. Some of the alterations, namely reduced LPCs, were associated with the risk of adverse outcome. These changes could be related to metabolic dysfunctions.

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.

Lysophosphatidic Acid Protects Against Endotoxin-Induced Acute Kidney Injury.

Septic shock is the most common cause of acute kidney injury (AKI), but the underlying mechanisms remain unclear and no targeted therapies exist. Lysophosphatidic acid (LPA) is a bioactive lipid which in vivo administration was reported to mitigate inflammation and injuries caused by bacterial endotoxemia in the liver and lung. The objective of the present study was to determine whether LPA can protect against sepsis-associated AKI. C57BL/6 mice were treated with LPA 18:1 (5 mg/kg, i.p.) 1 h before being injected with the endotoxin lipopolysaccharide (LPS), and AKI was evaluated after 24 h. LPA significantly decreased the elevation of plasma urea and creatinine caused by LPS. In the kidney, LPA pretreatment significantly reduced the upregulation of inflammatory cytokines (IL-6, TNFα, monocyte chemoattractant protein-1 (MCP-1)), and completely prevented downregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and upregulation of heme oxygenase-1 caused by LPS. LPA also prevented LPS-mediated alterations of the renal mitochondrial ultrastructure. In vitro pretreatment with LPA 18:1 significantly attenuated LPS-induced upregulation of the inflammatory cytokines (TNFα and MCP-1) in RAW264 macrophages. Moreover, in vivo LPS treatment lowered urinary LPA concentration and reduced LPA anabolic enzymes (autotaxin and acylglycerol kinase), and increased the LPA catalytic enzyme (lipid phosphate phosphatase 2) expression in the kidney cortex. In conclusion, exogenous LPA exerted a protective action against renal inflammation and injuries caused by bacterial endotoxemia. Moreover, LPS reduces the renal production of LPA suggesting that sepsis-associated AKI could be mediated, at least in part, by alleviation of the protective action of endogenous LPA.

Urinary lysophopholipids are increased in diabetic patients with nephropathy.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease that frequently leads to end stage renal failure. Lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are lysophospholipid mediators shown to accumulate in kidney and to promote renal inflammation and tubulo-interstitial fibrosis in diabetic rodent models. Here we assessed whether LPA and LPC were associated to the development of nephropathy in diabetic human patients. Several molecular species of LPA and LPC were quantified by LC/MS-MS in urine and plasma from type 2 diabetic patients with (cases; n=41) or without (controls, n=41) nephropathy symptoms (micro/macro-albuminuria and eGFR<60ml/min/1.73m2). Cases and controls were matched for sex, age and diabetes duration. Six species were detected in urine for both LPA and LPC, LPA16:0, LPA20:4, LPC16:0, LPC18:0, LPC18:1, and LPC18:2 that were significantly more concentrated in cases than in controls. Total LPC and LPA (sum of detected species) were significantly and exclusively associated with albuminuria (P<0.0001 and P=0.0009 respectively) and were significantly higher in the 3rd when compared to the 1st albuminuria tertile in cases. Plasma lysophospholipids showed a different species profile urine and their concentrations were not different between cases and controls. In conclusion, urine concentration of lysophospholipids increases in diabetic patients with DN as the likely result of their co-excretion with albumin combined with possible local production by kidney. Because LPA and LPC are known to promote renal inflammation and tubulo-interstitial fibrosis, their increased production in DN could participate to the development of kidney damage associated with diabetes.

Increased urine acylcarnitines in diabetic ApoE-/- mice: Hydroxytetradecadienoylcarnitine (C14:2-OH) reflects diabetic nephropathy in a context of hyperlipidemia.

Hyperlipidemia is a risk factor for initiation and progression of diabetic nephropathy but the metabolic pathways altered in the diabetic kidney in a context of hyperlipidemia remain incompletely described. Assuming that changes in urine composition reflect the alteration of renal metabolism and function, we analyzed the urine metabolite composition of diabetic (streptozotocin-treatment) and control (non diabetic) ApoE-/- mice fed a high cholesterol diet using targeted quantitative metabolomics. Urine metabolome was also compared to the plasma metabolome of the same animals. As previously shown, urine albuminuria/urine creatinine ratio (uACR) and glomerular area and plasma lipids (cholesterol, triglycerides) were more elevated in diabetic mice compared to control. After adjustment to urine creatinine, the abundance of 52 urine metabolites was significantly different in diabetic mice compared to control. Among them was a unique metabolite, C14:2-OH (3-hydroxytetradecadienoylcarnitine) that, in diabetic mice, was positively and significantly correlated with uACR, glomerular hypertrophy, blood glucose and plasma lipids. That metabolite was not detected in plasma. C14:2-OH is a long-chain acylcarnitine reminiscent of altered fatty acid beta oxidation. Other acylcarnitines, particularly the short chains C3-OH, C3-DC, C4:1, C5-DC, C5-M-DC, C5-OH that are reminiscent of altered oxidation of branched and aromatic amino acids were also exclusively detected in urine but were only correlated with plasma lipids. Finally, the renal gene expression of several enzymes involved in fatty acid and/or amino acid oxidation was significantly reduced in diabetic mice compared to control. This included the bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA (Ehhadh) that might play a central role in C14:2-OH production. This study indicate that the development of diabetes in a context of hyperlipidemia is associated with a reduced capacity of kidney to oxidize fatty acids and amino acids with the consequence of an elevation of urinary acetylcarnitines including C14:2-OH that specifically reflects diabetic nephropathy.

Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease

Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment (AU)

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Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease.

Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment.

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, ß-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and ß-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and ß-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

Autotaxin downregulates LPS-induced microglia activation and pro-inflammatory cytokines production.

Inflammation is essential in defense against infection or injury. It is tightly regulated, as over-response can be detrimental, especially in immune-privileged organs such as the central nervous system (CNS). Microglia constitutes the major source of inflammatory factors, but are also involved in the regulation of the inflammation and in the reparation. Autotaxin (ATX), a phospholipase D, converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and is upregulated in several CNS injuries. LPA, a pleiotropic immunomodulatory factor, can induce multiple cellular processes including morphological changes, proliferation, death, and survival. We investigated ATX effects on microglia inflammatory response to lipopolysaccharide (LPS), mimicking gram-negative infection. Murine BV-2 microglia and stable transfected, overexpressing ATX-BV-2 (A +) microglia were treated with LPS. Tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-10 mRNA and proteins levels were examined by qRT-PCR and ELISA, respectively. Secreted LPA was quantified by a radioenzymatic assay and microglial activation markers (CD11b, CD14, B7.1, and B7.2) were determined by flow cytometry. ATX expression and LPA production were significantly enhanced in LPS treated BV-2 cells. LPS induction of mRNA and protein level for TNFα and IL-6 were inhibited in A+ cells, while IL-10 was increased. CD11b, CD14, and B7.1, and B7.2 expressions were reduced in A+ cells. Our results strongly suggest deactivation of microglia and an IL-10 inhibitory of ATX with LPS induced microglia activation.