Follicular helper T (Tfh) cells are a specialized type of CD4 T-cell subset that support B cells during the germinal center (GC) reaction and determine the quality of the humoral response. Tfh development is a multistep process in which multiple extracellular and intracellular signals mediate CD4 T-cell differentiation, migration to lymphoid follicles and positioning in GC. Here we show that deletion of Cmip, an adaptor protein, in CD4 T cells prevents GC development and alters the humoral immune response after immunization. Deletion of Cmip shapes the differentiation of CD4 T cells toward a Th1 phenotype, while the Th2 and Tfh programs are inhibited. Cmip-deficient CD4 T cells display strong STAT5 activation and produce higher IL-2 both under resting conditions and after immunization, suggesting that Cmip deletion induces constitutive activation of the STAT5/IL-2 axis, while the Tfh program is inhibited at the early steps of differentiation. On the other hand, the frequency of Foxp3+CD4 T-cell subset is increased in Cmip-deficient mice. Collectively, these results suggest that Cmip is required for Tfh generation and inhibits Th1 and Treg differentiation. We found that CMIP is upregulated in circulating Tfh of patients with active idiopathic nephrotic syndrome and repressed in remission, pointing out the role of Tfh in the immunopathogenesis of the disease.

BACKGROUND AND AIMS Tumor angiogenesis is one of the therapeutic targets used in oncology, to limit cancer growth and spreading. The main angiogenesis signalling pathway is mediated by the vascular endothelial growth factor (VEGF) that binds to its tyrosine kinase receptor (VEGFR), mainly expressed on endothelial cells. The antiangiogenic therapy could target the VEGF ligand (anti-VEGF), or its receptors by inhibition of kinase activity (tyrosine kinase inhibitor, TKI) such as sorafenib. The toxicity of antiangiogenic therapies is a growing concern in clinical use. Indeed, nephrotoxicity is a critical side-effect that leads to discontinuation of therapy. Renal histological illustration of this toxicity is minimal change nephropathy/focal segmental glomerulosclerosis (MCNS/FSGS) and thrombotic micro-angiopathy (TMA), involving two distinct cell types, podocytes and endothelial cells respectively. Podocytes are the main source of VEGF and express VEGFR in the glomerulus. Mechanisms linking TKI therapy to podocyte dysfunction and nephrotic level proteinuria are still poorly understood. The working hypothesis is that nephrotoxicity of sorafenib is primarily through its effect on glomerular podocytes. METHOD Based on LC-MS/MS proteomic analysis, we have identified dysregulated cellular pathways on sorafenib-exposed podocytes. We validated these results by western blotting and immunofluorescence staining, on a cultured podocyte line exposed to sorafenib (2.5 µmol/L, 24-h). RESULTS Our results showed that sorafenib inhibits the downstream signalling pathways of VEGFR on podocytes, which induce injury by decreasing the expression of podocyte-specific markers (WT1, podocin). This is associated with podocyte morphology changes with podocyte cytoskeleton damages (actin and microtubules) and focal adhesions loss. We identified a novel podocyte target of sorafenib: the GSK3β. Indeed, sorafenib reduces significantly GSK3β inhibitory phosphorylation. Moreover, GSK3β regulates Tau, microtubule-associated proteins (MAP) and key regulator of microtubules remodelling. We show that tau phosphorylation increases significantly in sorafenib-treated cells. Furthermore, we found that sorafenib impairs the autophagic flux in podocytes, as indicated by an increase in LC3-II/LC3-I ratio in western blot, reflect of autophagosomes accumulation in podocytes. These results indicate that GSK3β overactivity induced by sorafenib participates in the disruption of the microtubules through tau phosphorylation, which interferes with cellular vesicle trafficking and blocks autophagic flux in podocytes. In addition, sorafenib leads to podocyte apoptosis through induction of endoplasmic reticulum (ER) stress (GADD153 protein induction and its nuclear translocation) and mitochondrial dysfunction. ER stress induced a decrease in podocyte protein synthesis with increased eIF2α phosphorylation. Sorafenib also causes mitochondrial membrane potential loss and mitochondrial depolarization. CONCLUSION Thus, in the current study, we show that sorafenib has a direct effect on podocytes, modulating specific signalling pathways to induce podocyte damage and ultimately glomerular lesions. However, we have to confirm the clinical relevance of our results on kidney biopsies from patients diagnosed with nephrotic syndrome following sorafenib treatment.

BACKGROUND AND AIMS Gemcitabine (GEM) is an anticancer drug, indicated in the treatment of several types of solid cancers such as pancreatic, breast, bladder cancer, etc. This molecule is effective in tumor processes but causes adverse effects on other organs, including the haematopoietic system and the kidney. The renal toxicity of gemcitabine is illustrated by thrombotic microangiopathy (TMA), whose onset is dramatic and associated in more than 97% of cases with acute renal failure. This adverse effect induces discontinuation of treatment with negative consequences on tumor control. It is, therefore, essential to understand the pathophysiological mechanisms of gemcitabine nephrotoxicity. TMA at the glomerular level reflects damage to the endothelial cell (ECs). However, previously, data of our team show that other glomerular cells, parietal epithelial cells (PECs), could be involved in these lesions. Thus, in this project, we want: i) to describe the modifications of the PEC and EC proteome exposed to gemcitabine and ii) to analyse PEC and EC secretome exposed to gemcitabine and to study the cell communication between PECs and ECs. METHOD In our in vitro experiments, we used a murine PEC cell line and a murine EC cell line. The PECs are exposed to 100 μM GEM for 24-h and the culture supernatants are recovered. These PEC supernatants are used in conditioned media experiments to expose ECs for 24-h. On the other hand, ECs were directly exposed to 1 μM GEM for 24-h and culture supernatants were also recovered. Analyses of protein expression are carried out by western blot and immunofluorescence staining. RESULTS When PECs are exposed to GEM (100 µM, 24-h), we observe an increase in apoptosis (caspase 3 cleavage) as well as DNA damage (γH2AX). However, PECs specific markers, such as PAX2 and Claudin-1, do not appear to be affected. Our results also show an increase in the expression of the transcription factor CHOP and the chaperone protein BIP, suggesting the presence of endoplasmic reticulum (ER) stress in PECs exposed to gemcitabine. In order to study cell communication between PECs and ECs, we set up conditioned media experiments. We first studied the expression of different endothelial markers on ECs exposed to PEC supernatants. The expression of the endothelial markers such as E-cadherin, CD31 and PDGFRβ decreases compared with control cells when ECs are exposed to PEC supernatants. Actin cytoskeleton reorganization is also observed with an increase in cortical actin. Moreover, the microtubule network seems to be also affected with a tendency to decrease tubulin expression. Finally, there is an increase in γH2AX suggesting the presence of DNA damage in ECs exposed to PEC supernatant. The next experiments aim at identifying the factors responsible for this cell communication (proteins, lipid mediators, non-coding RNA, extracellular vesicles). CONCLUSION Our results show GEM has a direct effect on PECs (apoptosis, DNA damage, ER stress). In addition, we show the effect of PEC supernatants exposed to GEM on ECs (decrease in endothelial markers, cytoskeleton modifications, DNA damage).