Matilde Alique

Retinoids as a potential treatment for experimental puromycin‐induced nephrosis

Puromycin aminonucleoside (PAN)‐induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic‐range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all‐trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti‐inflammatory effect of tRA was evidenced by the inhibition of PAN‐induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein‐1. TUNEL assays showed that tRA inhibited the PAN‐induced apoptosis of cultured differentiated mouse podocytes. We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

All-trans retinoic acid induces COX-2 and prostaglandin E2 synthesis in SH-SY5Y human neuroblastoma cells: involvement of retinoic acid receptors and extracellular-regulated kinase 1/2

BackgroundOur recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity.MethodsCultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test.ResultsATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-β expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells.ConclusionThese results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.

GSK3, Snail, and Adhesion Molecule Regulation by Cyclosporine A in Renal Tubular Cells

Tubular cell injury and fibrosis are key features of calcineurin inhibitor nephrotoxicity, but the molecular processes involved are not fully understood. In cultured murine MCT and human kidney 2 proximal tubular cells, gene expression and protein levels were studied by real-time polymerase chain reaction, Western blot, and confocal microscopy. Protein function was evaluated by pharmacological inhibitors and confirmed by small interfering RNA (siRNA) gene targeting. In renal tubular cells, cytotoxic concentrations of cyclosporine A (CsA) inhibited both gene and protein expression of adherent and tight junction proteins (E-cadherin, ZO-1, claudin-1, and β-catenin) and increased vimentin expression, without involvement of transforming growth factor β1 or caspase activity. CsA upregulated transcriptional repressors (Snail, Slug, and Twist) of the adherent and tight junction proteins were studied. Snail siRNA targeting prevented the downregulation of E-cadherin by CsA. CsA promoted glycogen synthase kinase 3 (GSK3) phosphorylation and increased Snail half-life. The GSK3 inhibitor lithium upregulated Snail and decreased E-cadherin expression in a Snail-dependent manner. Moreover, targeting GSK3 activity by siRNA also upregulated Snail. Furthermore, GSK3 siRNA had a negative impact on CsA-induced upregulation of Snail. Tacrolimus also inhibited GSK3 and mimicked CsA responses in tubular cells. We conclude that calcineurin inhibitors may directly decrease the expression of epithelial adhesion molecules by repressing GSK3 and stabilizing Snail. This offers potential pharmacological targets for prevention of nephrotoxicity.

Connective tissue growth factor is a new ligand of epidermal growth factor receptor

Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-β-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.

Angiotensin II Contributes to Renal Fibrosis Independently of Notch Pathway Activation

Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-β1 (TGF-β1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-β1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-β1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.

The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and RORγt) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-β and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.

Integrin-linked kinase plays a key role in the regulation of angiotensin II-induced renal inflammation

ILK (integrin-linked kinase) is an intracellular serine/threonine kinase involved in cell-matrix interactions. ILK dysregulation has been described in chronic renal disease and modulates podocyte function and fibrosis, whereas data about its role in inflammation are scarce. AngII (angiotensin II) is a pro-inflammatory cytokine that promotes renal inflammation. AngII blockers are renoprotective and down-regulate ILK in experimental kidney disease, but the involvement of ILK in the actions of AngII in the kidney has not been addressed. Therefore we have investigated whether ILK signalling modulates the kidney response to systemic AngII infusion in wild-type and ILK-conditional knockout mice. In wild-type mice, AngII induced an inflammatory response, characterized by infiltration of monocytes/macrophages and lymphocytes, and up-regulation of pro-inflammatory factors (chemokines, adhesion molecules and cytokines). AngII activated several intracellular signalling mechanisms, such as the NF-κB (nuclear factor κB) transcription factor, Akt and production of ROS (reactive oxygen species). All these responses were prevented in AngII-infused ILK-deficient mice. In vitro studies characterized further the mechanisms regulating the inflammatory response modulated by ILK. In cultured tubular epithelial cells ILK blockade, by siRNA, inhibited AngII-induced NF-κB subunit p65 phosphorylation and its nuclear translocation. Moreover, ILK gene silencing prevented NF-κB-related pro-inflammatory gene up-regulation. The results of the present study demonstrate that ILK plays a key role in the regulation of renal inflammation by modulating the canonical NF-κB pathway, and suggest a potential therapeutic target for inflammatory renal diseases.

Gremlin regulates renal inflammation via the vascular endothelial growth factor receptor 2 pathway

Inflammation is a main feature of progressive kidney disease. Gremlin binds to bone morphogenetic proteins (BMPs), acting as an antagonist and regulating nephrogenesis and fibrosis among other processes. Gremlin also binds to vascular endothelial growth factor receptor‐2 (VEGFR2) in endothelial cells to induce angiogenesis. In renal cells, gremlin regulates proliferation and fibrosis, but there are no data about inflammatory‐related events. We have investigated the direct effects of gremlin in the kidney, evaluating whether VEGFR2 is a functional gremlin receptor. Administration of recombinant gremlin to murine kidneys induced rapid and sustained activation of VEGFR2 signalling, located in proximal tubular epithelial cells. Gremlin bound to VEGFR2 in these cells in vitro, activating this signalling pathway independently of its action as an antagonist of BMPs. In vivo, gremlin caused early renal damage, characterized by activation of the nuclear factor (NF)‐κB pathway linked to up‐regulation of pro‐inflammatory factors and infiltration of immune inflammatory cells. VEGFR2 blockade diminished gremlin‐induced renal inflammatory responses. The link between gremlin/VEGFR2 and NF‐κB/inflammation was confirmed in vitro. Gremlin overexpression was associated with VEGFR2 activation in human renal disease and in the unilateral ureteral obstruction experimental model, where VEGFR2 kinase inhibition diminished renal inflammation. Our data show that a gremlin/VEGFR2 axis participates in renal inflammation and could be a novel target for kidney disease. Copyright

Vitamin A active metabolite, all‐trans retinoic acid, induces spinal cord sensitization. I. Effects after oral administration

Retinoic acid is an active metabolite of vitamin A involved in the modulation of the inflammatory and nociceptive responses. The aim of the present study was to analyze the properties of spinal cord neuronal responses of male Wistar rats treated with all‐trans retinoic acid (ATRA) p.o. in the normal situation and under carrageenan‐induced inflammation. We also studied the expression and distribution of cyclooxygenases (COX) in the spinal cord.

Angiotensin receptors and β -catenin regulate brain endothelial integrity in malaria

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum-infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter-endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin-induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC-induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised.