Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation)

Cannabinoid Receptors : a New Therapeutic Target of Renal Fibrosis

L’insuffisance rénale chronique et la dysfonction chronique de l’allogreffe (DCA) sont associées à la fibrogenèse rénale, qui représente un enjeu majeur en santé publique et nécessite l’exploration de nouvelles cibles thérapeutiques. Dans ce contexte, nous avons étudié l’expression des gènes modulés au cours d’un modèle reconnu de fibrogenèse chez la souris (le modèle d’obstruction urétérale unilatéral, ou OUU). L'expression du gène codant pour le récepteur cannabinoïde apparait sept fois augmentée dans les reins pathologiques comparée à leurs contrôles internes. L’expression du récepteur est également significativement augmentée dans plusieurs types de néphropathies et au cours de la DCA chez l’homme. Le système cannabinoïde se compose de deux types de récepteurs, le type 1 (CB1) et le type 2 (CB2). Ceux-ci jouent des rôles opposés au cours de la fibrose hépatique, suggérant que l’inactivation de CB1 et l’activation de CB2 pourraient constituer une thérapeutique anti-fibrosante d’intérêt, indépendamment de leur implication dans le syndrome métabolique. Ainsi, nous avons montré pour la première fois que le blocage de CB1 par invalidation génétique ou par inhibition pharmacologique permet une réduction significative de la fibrose rénale induite par OUU. La potentialisation de cet effet par l’administration d’un agoniste sélectif de CB2 ne semble en revanche pas relevante, illustrant le rôle prédominant de CB1 dans ce modèle. L’étude du mécanisme réalisée in vitro sur des myofibroblastes primaires activés au TGF-β1 révèle une expression de CB1 augmentée, associée à une synthèse de collagènes significativement bloquée après un traitement par un antagoniste sélectif de CB1. Ceci suggère que l’effet anti-fibrosant dépendant de CB1 agit directement sur le myofibroblaste. Ainsi, nos travaux montrent pour la première fois l’effet anti-fibrosant de l’inactivation de CB1, avec une action directe sur la cellule effectrice de la fibrose, suggérant que CB1 représente une nouvelle cible thérapeutique et un médiateur majeur de la fibrose rénale. Son expression dans les néphropathies humaines des reins natifs présente une forte corrélation avec le taux de créatinine et pourrait constituer un nouveau biomarqueur d’atteinte rénale.Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. In the present study, we show that the cannabinoid 1 receptor (CB1) may be a new pathway in renal fibrogenesis, independently of its involvement in metabolic disease. We found that CB1 expression was highly expressed in kidney biopsies of patients suffering from IgA nephropathy, diabetes, and acute interstitial nephritis. We also used an experimental model of renal fibrosis, the unilateral ureteral-obstruction model, in mice. Both genetic and pharmacological invalidation of CB1 induced a profound reduction in renal fibrosis, showing its prominent role in renal fibrosis. Cannabinoid receptor 2 is also involved in renal fibrogenesis but does not potentialize the role of CB1. CB1 expression is drastically increased in myofibroblasts upon TGFß-1 stimulation. The decrease in renal fibrosis during CB1 invalidation is explained by a direct action on myofibroblasts: CB1 blockade reduced collagen expression in vitro. In addition, CB1 also modulates the macrophage infiltrate responsible for renal fibrosis in unilateral ureteral obstruction through a decrease in MCP1 synthesis, a major chemoattractant cytokine. Our study strongly suggests a major role for CB1 in the activation of myofibroblasts, which are the main effector cells in renal fibrogenesis, and suggests that CB1 may represent a major new target for treating chronic kidney disease

The cannabinoid receptor 1 is involved in renal fibrosis during chronic allograft dysfunction: Proof of concept

International audienceChronic allograft dysfunction (CAD), defined as the replacement of functional renal tissue by extracellular matrix proteins, remains the first cause of graft loss. The aim of our study was to explore the potential role of the cannabinoid receptor 1 (CB1) during CAD. We retrospectively quantified CB1 expression and correlated it with renal fibrosis in 26 kidney-transplanted patients who underwent serial routine kidney biopsies. Whereas CB1 expression was low in normal kidney grafts, it was highly expressed during CAD, especially in tubular cells. CB1 expression significantly increased early on after transplantation, from day 0 (D0) to month 3 post-transplant (M3) (22.5% ± 15.4% vs 33.4% ± 13.8%, P < .01), and it remained stable thereafter. CB1 expression correlated with renal fibrosis at M3 (P = .04). In an in vitro model of tacrolimus-mediated fibrogenesis by tubular cells, we found that tacrolimus treatment significantly induced mRNA and protein expression of CB1 concomitantly to col3a1 and col4a3 up regulation. Administration of rimonabant, a CB1 antagonist, blunted collagen synthesis by tubular cells (P < .05). Overall, our study strongly suggests an involvement of the cannabinoid system in the progression of fibrosis during CAD and indicates the therapeutic potential of CB1 antagonists in this pathology

Circulating CASK is associated with recurrent focal segmental glomerulosclerosis after transplantation

International audienceINTRODUCTION:Focal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption.RESULTS:To identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes.CONCLUSION:Our results suggest that CASK can induce the recurrence of FSGS after renal transplantation

Cannabinoid receptor 1 is a major mediator of renal fibrosis

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-β1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Reestablishment of IL-2Rγ chain-dependent signal transduction pathway in RCC interferes with the rhIL-15-induced E-cadherin down-regulation.

<p>RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. <b>A</b>) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. <b>B</b>) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. <b>C</b>) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.</p

E-cadherin modulation by soluble IL-15 controls epithelial-mesenchymal transition on renal epithelial cells.

<p><b>A</b>) Immunofluorescence of cell–cell adhesion molecules show that IL-15 favors epithelial-mesenchymal transition (EMT) on RCC7, whereas it preserves the EMT commitment of RPTEC. The medium culture of RPTEC was not changed in order to induce the EMT process. Cells stimulated or not with 10 pg/ml of rhIL-15 for 5 days, were fixed and stained using standard immunofluorescence procedures with Abs against epithelial (cytokeratins and ZO-1) and mesenchymal markers (F-actin, ASO2 and vimentin). Similar results were obtained using different RCC (RCC5, RCC8) and RPTEC batches. <b>B</b>) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating the epithelial (cytokeratins) and mesenchymal (vimentin) markers expression by flow cytometry. RhIL-15 induced EMT was counterbalanced only in IL-2Rγ/JAK3 co-transfected RCC. Mean fluorescence intensity values for each marker are shown in each histogram. Results are representative of three experiments.</p

Up-regulation of E-cadherin expression by rhIL-15 on RPTEC is dependent of the βc-dependent signaling pathway.

<p>The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.</p

Soluble IL-15, at physiologic concentration, differently controls E-cadherin expression in RCC and RPTEC.

<p>Immunofluorescence analysis (<b>A</b>) and immunoblot (<b>B</b>) show that 5 days rhIL-15 treatment (10 pg/mL) preserves membrane E-cadherin expression on primary normal epithelial cells RPTEC, whereas it induces its down-regulation on RCC7. The medium culture of RPTEC was not changed in order to induce the decrease of E-cadherin expression. Treatment with rhIL-15 was renewed at day 3. Histograms represent densitometry comparison of E-cadherin immunoblots normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) cells and 3 RPTEC batches. * P<0.05.</p