Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney.

The extracellular matrix (ECM) is composed of collagens, ECM glycoproteins, and proteoglycans (also named core matrisome proteins) that are critical for tissue structure and function, and matrisome-associated proteins that balance the production and degradation of the ECM proteins. The identification and quantification of core matrisome proteins using mass spectrometry is often hindered by their low abundance and their propensity to form macromolecular insoluble structures. In this study, we aimed to investigate the added value of decellularization in identifying and quantifying core matrisome proteins in mouse kidney. The decellularization strategy combined freeze-thaw cycles and sodium dodecyl sulphate treatment. We found that decellularization preserved 95% of the core matrisome proteins detected in non-decellularized kidney and revealed few additional ones. Decellularization also led to an average of 59 times enrichment of 96% of the core matrisome proteins as the result of the successful removal of cellular and matrisome-associated proteins. However, the enrichment varied greatly among core matrisome proteins, resulting in a misrepresentation of the native ECM composition in decellularized kidney. This should be brought to the attention of the matrisome research community, as it highlights the need for caution when interpreting proteomic data obtained from a decellularized organ.

Improved prenatal assessment of kidney disease using multiple ultrasound features.

BACKGROUND AND HYPOTHESIS: Congenital anomalies of the kidney and the urinary tract (CAKUT), often discovered in utero, cover a wide spectrum of outcomes ranging from normal postnatal kidney function to fetal death. The current ultrasound workup does not allow for an accurate assessment of the outcome. The present study aimed to significantly improve the ultrasound-based prediction of postnatal kidney survival in CAKUT. METHODS: Histological analysis of kidneys of 15 CAKUT fetuses was performed to better standardize the ultrasound interpretation of dysplasia and cysts. Ultrasound images of 140 CAKUT fetuses with 2-year postnatal follow-up were annotated for amniotic fluid volume and kidney number, size, dysplasia and/or cysts using standardized ultrasound readout. Association of ultrasound features and clinical data (sex and age at diagnosis) with postnatal kidney function was studied using logistic regression. Amniotic fluid proteome associated to kidney dysplasia or cysts was characterized by mass spectrometry. RESULTS: Histologically, poor ultrasound corticomedullary differentiation was associated to dysplastic lesions and ultrasound hyperechogenicity was associated to the presence of microcysts. Of all ultrasound and clinical parameters, reduced amniotic volume, dysplasia and cysts were the best predictors of poor outcome (Odd ratio = 57 [95%CI: 11-481], 20 [3-225] and 7 [1-100], respectively). Their combination into an algorithm improved prediction of postnatal kidney function compared to amniotic volume alone (area under the ROC curve = 0.92 [0.86-0.98] in a 10-fold cross validation). Dysplasia and cysts were correlated (Cramer's V coefficient = 0.44, p<0.0001), but amniotic fluid proteome analysis revealed that they had distinct molecular origin (extracellular matrix and cell contacts versus cellular death, respectively), probably explaining the additivity of their predictive performances. CONCLUSION: Antenatal clinical advice for CAKUT pregnancies can be improved by a more standardized and combined interpretation of ultrasound data.

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.

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.

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.

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.

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.

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.

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.

Pro-fibrotic activity of lysophosphatidic acid in adipose tissue: in vivo and in vitro evidence.

Lysophosphatidic acid (LPA) is a pro-fibrotic mediator acting via specific receptors (LPARs) and is synthesized by autotaxin, that increases with obesity. We tested whether LPA could play a role in adipose tissue (AT)-fibrosis associated with obesity. Fibrosis [type I, III, and IV collagens (COL), fibronectin (FN), TGFß, CTGF and αSMA] and inflammation (MCP1 and F4/80) markers were quantified: (i) in vivo in inguinal (IAT) and perigonadic (PGAT) AT from obese-diabetic db/db mice treated with the LPAR antagonist Ki16425 (5mg/kg/day ip for 7 weeks); and (ii) in vitro in human AT explants in primary culture for 72h in the presence of oleoyl-LPA (10µM) and/or Ki16425 (10µM) and/or the HIF-1α inhibitor YC-1 (100µM). Treatment of db/db mice with Ki16425 reduced Col I and IV mRNAs in IAT and PGAT while Col III mRNAs were only reduced in IAT. This was associated with reduction of COL protein staining in both IAT and PGAT. AT explants showed a spontaneous and time-dependent increase in ATX expression and production of LPA in the culture medium, along with increased levels of Col I and III, TGFß and αSMA mRNAs and of COL protein staining. In vitro fibrosis was blocked by Ki16425 and was further amplified by oleoyl-LPA. LPA-dependent in vitro fibrosis was blocked by co-treatment with YC1. Our results show that endogenous and exogenous LPA exert a pro-fibrotic activity in AT in vivo and in vitro. This activity could be mediated by an LPA1R-dependent pathway and could involve HIF-1α.

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.

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.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation.

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with ß-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

A hypomorphic mutation in Lpin1 induces progressively improving neuropathy and lipodystrophy in the rat.

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular analyses revealed that these lesions improve in older Lpin1(1Hubr) rats as compared with young Lpin1(1Hubr) rats and Lpin1(fld/fld) mice. We observed activation of compensatory biochemical pathways substituting for missing PAP1 activity that, in combination with a possible non-enzymatic Lipin 1 function residing outside of its PAP1 domain, may contribute to the less severe phenotypes observed in Lpin1(1Hubr) rats as compared with Lpin1(fld/fld) mice. Although we are cautious in making a direct parallel between the presented rodent model and human disease, our data may provide new insight into the pathogenicity of recently identified human LPIN1 mutations.

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.

Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA). ATX is secreted by adipose tissue and its expression is enhanced in obese/insulin-resistant individuals. Here, we analyzed the specific contribution of adipose-ATX to fat expansion associated with nutritional obesity and its consequences on plasma LPA levels. We established ATX(F/F)/aP2-Cre (FATX-KO) transgenic mice carrying a null ATX allele specifically in adipose tissue. FATX-KO mice and their control littermates were fed either a normal or a high-fat diet (HFD) (45% fat) for 13 weeks. FATX-KO mice showed a strong decrease (up to 90%) in ATX expression in white and brown adipose tissue, but not in other ATX-expressing organs. This was associated with a 38% reduction in plasma LPA levels. When fed an HFD, FATX-KO mice showed a higher fat mass and a higher adipocyte size than control mice although food intake was unchanged. This was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)γ2 and of PPAR-sensitive genes (aP2, adiponectin, leptin, glut-1) in subcutaneous white adipose tissue, as well as in an increased tolerance to glucose. These results show that adipose-ATX is a negative regulator of fat mass expansion in response to an HFD and contributes to plasma LPA levels.

Atherosclerotic lesion progression changes lysophosphatidic acid homeostasis to favor its accumulation.

Lysophosphatidic acid (LPA) accumulates in the central atheroma of human atherosclerotic plaques and is the primary platelet-activating lipid constituent of plaques. Here, we investigated the enzymatic regulation of LPA homeostasis in atherosclerotic lesions at various stages of disease progression. Atherosclerotic lesions were induced in carotid arteries of low-density lipoprotein receptor-deficient mice by semiconstrictive collar placement. At 2-week intervals after collar placement, lipids and RNA were extracted from the vessel segments carrying the plaque. Enzymatic-and liquid chromatography-mass spectrometry-based lipid profiling revealed progressive accumulation of LPA species in atherosclerotic tissue preceded by an increase in lysophosphatidylcholine, a precursor in LPA synthesis. Plaque expression of LPA-generating enzymes cytoplasmic phospholipase A(2)IVA (cPLA(2)IVA) and calcium-independent PLA(2)VIA (iPLA(2)VIA) was gradually increased, whereas that of the LPA-hydrolyzing enzyme LPA acyltransferase alpha was quenched. Increased expression of cPLA(2)IVA and iPLA(2)VIA in advanced lesions was confirmed by immunohistochemistry. Moreover, LPA receptors 1 and 2 were 50% decreased and sevenfold upregulated, respectively. Therefore, key proteins in LPA homeostasis are increasingly dysregulated in the plaque during atherogenesis, favoring intracellular LPA production. This might at least partly explain the observed progressive accumulation of this thrombogenic proinflammatory lipid in human and mouse plaques. Thus, intervention in the enzymatic LPA production may be an attractive measure to lower intraplaque LPA content, thereby reducing plaque progression and thrombogenicity.

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.

Lysophosphatidic acid and renal fibrosis.

The development of fibrosis involves a multitude of events and molecules. Until now the majority of these molecules were found to be proteins or peptides. But recent data show significant involvement of the phospholipid lysophosphatidic acid (LPA) in the development of pulmonary, liver and renal fibrosis. The latest data on the role of LPA and the G-protein-coupled LPA1 receptor in the development of renal fibrosis will be discussed. LPA1-receptor activation was found to be associated with increased vascular leakage and increased fibroblast recruitment in pulmonary fibrosis. Furthermore, in renal fibrosis LPA1-receptor activation stimulates macrophage recruitment and connective tissue growth factor expression. The observations make this receptor an interesting alternative and new therapeutic target in fibrotic diseases.

Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development.

Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the Galpha13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Galpha13.