Regulation of CMIP by WT1 and impact on hereditary and acquired glomerulopathies

Le syndrome néphrotique idiopathique (SNI) est défini cliniquement par une protéinurie abondante associée à une hypoalbuminémie et histologiquement par des lésions glomérulaires minimes ou une hyalinose segmentaire et focale. Il traduit une maladie du podocyte (podocytopathie) qui peut résulter soit d’une anomalie structurale, soit d’une altération des signaux cellulaires, aboutissant à une perte de l’intégrité fonctionnelle.A côté des podocytopathies dites idiopathiques (SNI), nous avons observé que les mutations inactivatrices de WT1 (syndrome de Denys Drash et syndrome de Frasier) étaient associées à une forte induction de CMIP tandis que que WT1 était peu ou pas exprimé. En revanche, à l’état physiologique, l’expression constitutive de WT1 dans le podocyte était associée à une expression faible ou indétectable de CMIP. Ces observations nous ont amenés à nous intéresser à la régulation de CMIP par WT1.Nous avons identifié respectivement un et deux éléments de réponse à WT1 dans la partie proximale des promoteurs murin et humain CMIP. Nous avons démontré in vitro que WT1 se lie au promoteur humain et murin CMIP par la technique d’immunoprécipitation de la chromatine sur la lignée de cellules humaines leucémiques K562 et sur une lignée de podocytes murins exprimant WT1. Nous avons ensuite montré que WT1 inhibe l’activité luciférase dépendante du promoteur CMIP. Dans la lignée M15 derivant du mésonephros murin, la dégradation de WT1 par les SiRNA spécifiques induit une nette augmentation de l’expression protéique de CMIP.Ce travail identifie un nouveau gène cible de WT1 dans le podocyte qui est réprimé à l’état basal. L’induction de CMIP dans le podocyte entraine une perte de l’intégrité podocytaire avec désorganisation du cytosquelette, apoptose et inhibition de la signalisation proximale. Ces résultats suggèrent que la répression de CMIP par WT1 est nécessaire à l’homéostasie podocytaire.La glomerulopathie lupique revêt des aspects histologiques très divers, reflétant possiblement différents mecanismes physiopatologiques sous jacents. Les podocytopathies au cours des néphropathies lupiques restent une entité peu connue. Nous avons recherché si CMIP pouvait être un des biomarqueurs de cette entité. Nous avons observé une expression podocytaire de CMIP dans les glomerulonéphrites lupiques de classe V (glomerulonephrite extramembraneuse) et dans les glomerulopathies lupiques de classe II avec syndrome néphrotique. En revanche, CMIP n’était pas detecté dans les podocytes au cours des glomérulopathies lupiques prolifératives (classe III/IV). CMIP pourrait être un marqueur de podocytopathie lupique.Collectivement, ces résultats mettent en lumière le rôle central de CMIP dans la physiopathologie des podocytopathiesIdiopathic nephrotic syndrome (INS) is a primary glomerular disease which mainly includes two histological variants, minimal change nephrotic syndrome (MCNS) and focal and segmental glomerulosclerosis (FSGS). The underlying mechanisms involve structural and/or functional disturbances of epithelial cell lining the outer side of glomerular basement membrane so called podocyte.The Wilm’s tumor suppressor gene, WT1, is expressed throughout life in podocytes and is essential for its functional integrity and survival. This constitutive expression contrasts with a weak or virtual absence of CMIP expression at physiological state. On the other hand, we found that Denys Drash syndrome and Frasier syndrome, two glomerular diseases resulting from a primary defect of WT1 were associated with a high induction of CMIP in podocyte, while WT1 was barely detected. These observations led us to investigate the regulation of CMIP by WT1.We identified one and two potential WT1 response elements in the proximal region of mouse and human CMIP promoters respectively. We took advantage of WT1 expression in human leukemia cell line K562 and mouse podocyte cell line to demonstrate in vitro that WT1 binds to human and mouse CMIP promoter by chromatine immunoprecipitation assay. We then showed that overexpression of WT1 in HEK cell line inhibits the luciferase activity driven by CMIP promoter. We demonstrated that knockdown of endogenous WT1 by transient siRNA transfection in M15 cell line, induced CMIP expression, suggesting that WT1 silencing facilitates the expression of CMIP.Altogether, these results suggest that CMIP could be considered as a new target gene of WT1 in podocyte, which acts as transcriptional repressor. The induction of CMIP compromises podocyte integrity through disorganization of the cytoskeleton, apoptosis and inhibition of proximal signaling. These results suggest that repression of CMIP by WT1 is necessary to podocyte homeostasis.In another part of this thesis, I investigated the expression of CMIP in lupus nephritis. Lupus glomerulopathy are classified into various histological patterns, which probably result from different pathophysiological origins. Podocytopathy during lupus nephritis remains an unclear entity. We looked whether CMIP might be a relevant marker of lupus podocytopathy. Immunohistochemistry analyses of kidney biopsies showed that CMIP was expressed in podocytes during class V (membranous nephropathy) and class II glomerulopathies associated with nephrotic syndrome. By contrast, CMIP was not detected in podocyte during proliferative glomerulopathies (classes III and IV). These data suggest that CMIP may be a marker of lupus podocytopathy

Rejet humoral aigu en transplantation rénale (étude anatomo-clinique monocentrique rétrospective de 35 patients)

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF

Renal Diseases Associated with Hematologic Malignancies and Thymoma in the Absence of Renal Monoclonal Immunoglobulin Deposits

International audienceIn addition to kidney diseases characterized by the precipitation and deposition of overproduced monoclonal immunoglobulin and kidney damage due to chemotherapy agents, a broad spectrum of renal lesions may be found in patients with hematologic malignancies. Glomerular diseases, in the form of paraneoplastic glomerulopathies and acute kidney injury with various degrees of proteinuria due to specific lymphomatous interstitial and/or glomerular infiltration, are two major renal complications observed in the lymphoid disorder setting. However, other hematologic neoplasms, including chronic lymphocytic leukemia, thymoma, myeloproliferative disorders, Castleman disease and hemophagocytic syndrome, have also been associated with the development of kidney lesions. These renal disorders require prompt recognition by the clinician, due to the need to implement specific treatment, depending on the chemotherapy regimen, to decrease the risk of subsequent chronic kidney disease. In the context of renal disease related to hematologic malignancies, renal biopsy remains crucial for accurate pathological diagnosis, with the aim of optimizing medical care for these patients. In this review, we provide an update on the epidemiology, clinical presentation, pathophysiological processes and diagnostic strategy for kidney diseases associated with hematologic malignancies outside the spectrum of monoclonal gammopathy of renal significance

Belatacept inhibit human B cell germinal center development in immunodeficient mice

Abstract The humoral response mediated by alloantibodies directed against donor HLA molecules (DSAs) is one of the main causes of graft loss in kidney transplantation. Understanding the pathophysiology leading to humoral kidney rejection as the development of therapeutic tools is therefore a main objective in the field of solid organ transplantation and necessitate adapted experimental models. Among the immunosuppressive agents used in renal transplantation, belatacept, a fusion protein targeting T costimulatory molecules has shown its ability to prevent more efficiently the secretion of DSA by different mechanisms including a direct action on plasma cells but also on B lymphocytes and follicular helper T lymphocytes (Tfh) cooperation. This cellular cooperation occurs within germinal centers (GC), the seat of B lymphocytes differentiation. Here, we aimed to develop a dedicated mouse model in which human GC would be functional to study the effect of belatacept on GC formation and the ability of B lymphocytes to secrete immunoglobulin. We next demonstrate that belatacept inhibits the formation of these GCs, by inhibiting the frequency of Tfh and B lymphocytes. This alters the B maturation and therefore the generation of plasma cells and consequently, immunoglobulin secretion

Podocyte Injury in Lupus Nephritis

Systemic lupus erythematosus (SLE) is characterized by a broad spectrum of renal lesions. In lupus glomerulonephritis, histological classifications are based on immune-complex (IC) deposits and hypercellularity lesions (mesangial and/or endocapillary) in the glomeruli. However, there is compelling evidence to suggest that glomerular epithelial cells, and podocytes in particular, are also involved in glomerular injury in patients with SLE. Podocytes now appear to be not only subject to collateral damage due to glomerular capillary lesions secondary to IC and inflammatory processes, but they are also a potential direct target in lupus nephritis. Improvements in our understanding of podocyte injury could improve the classification of lupus glomerulonephritis. Indeed, podocyte injury may be prominent in two major presentations: lupus podocytopathy and glomerular crescent formation, in which glomerular parietal epithelial cells play also a key role. We review here the contribution of podocyte impairment to different presentations of lupus nephritis, focusing on the podocyte signaling pathways involved in these lesions

Rituximab and Fibrillary Glomerulonephritis: Interest of B Cell Reconstitution Monitoring

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease characterized by glomerular deposition of randomly arranged non-amyloid fibrils. FGN has a poor renal prognosis and its optimal treatment is a medical challenge. Rituximab therapy has recently emerged as a promising approach even though its mechanism of action remains hypothetical. We describe the case of a 55-year-old woman with FGN successfully treated by rituximab. During the 36-month follow-up, she had three relapses of FGN, occurring each time in the context of B cell recovery. Investigation of the distribution of B cell subpopulations at the time of the third relapse showed, as previously described for some immunological diseases, an increase in the proportion of switched memory B cells relative to healthy subjects, whereas global memory B cell pool was not yet recovered. This case suggests that B cell reconstitution should be carefully monitored in the management of FGN treated with rituximab

Expression of CMIP in podocytes is restricted to specific classes of lupus nephritis.

Lupus glomerulopathies are classified into various histological patterns, which probably result from different pathophysiological origins. Podocyte injury can be demonstrated in lupus nephritis but its clinical relevance is far little appreciated and is often masked by proliferative lesions and inflammatory cell infiltrations. Two patterns of podocyte lesions may be considered, either occurring in the context of renal inflammation or reflecting podocyte dysfunction in non-proliferative and non-inflammatory glomerulopathies. This distinction remains elusive since no reliable biomarker discriminates between both entities. CMIP was recently found induced in some glomerular disease but its expression in different lupus nephritis classes has not been investigated. Twenty-four adult patients with lupus nephritis, including non-proliferative (n = 11) and proliferative (n = 13) glomerulopathies were analyzed. Clinical, biological and immunological data were compared with immunomorphological findings. We analyzed by quantitative and qualitative methods the expression of CMIP in different histological classes. We found CMIP abundance selectively increased in podocytes in class II and class V glomerulopathies, while in proliferative forms (class III and class IV), CMIP was rarely detected. CMIP was not expressed in cellular crescents, endothelial cells or mesangial cells. CMIP colocalized with some subsets of B and T cells within glomerular or interstitial mononuclear cell infiltrates but never with macrophages. Hematuria is rarely present in lupus glomerulopathies expressing CMIP. There was no correlation between classical immunological markers and CMIP expression. Thus, CMIP induction in lupus nephritis seems restricted to non-proliferative glomerulopathies and may define a specific pattern of podocyte injury

Three-Year Outcomes in Kidney Transplant Recipients Switched From Calcineurin Inhibitor-Based Regimens to Belatacept as a Rescue Therapy

International audienceBackground: The long-term benefits of conversion from calcineurin inhibitors (CNIs) to belatacept in kidney transplant recipients (KTr) are poorly documented .Methods: A single-center retrospective work to study first-time CNI to belatacept conversion as a rescue therapy [eGFR <30 ml/min/1.73 m 2 , chronic histological lesions, or CNI-induced thrombotic microangiopathy (TMA)]. Patient and kidney allograft survivals, eGFR, severe adverse events, donor-specific antibodies (DSA), and histological data were recorded over 36 months after conversion.Results: We included N = 115 KTr. The leading cause for switching was chronic histological lesions with non-optimal eGFR (56.5%). Three years after conversion, patient, and death-censored kidney allograft survivals were 88% and 92%, respectively, eGFR increased significantly from 31.5 ± 17.5 to 36.7 ± 15.7 ml/min/1.73 m 2 ( p < 0.01), the rejection rate was 10.4%, OI incidence was 5.2 (2.9–7.6) per 100 person-years. Older age was associated with death, eGFR was not associated with death nor allograft loss. No patient developed dn DSA at M36 after conversion. CNI-induced TMA disappeared in all cases without eculizumab use. Microvascular inflammation and chronic lesions remained stable.Conclusion: Post-KT conversion from CNIs to belatacept, as rescue therapy, is safe and beneficial irrespective of the switch timing and could represent a good compromise facing organ shortage. Age and eGFR at conversion should be considered in the decision whether to switch