Recent approaches to the pathogenesis of minimal-change nephrotic ...

Nephrol Dial Transplant (2003) 18: Editorial Comments

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Nephrol Dial Transplant (2003) 18: 245–248

Recent approaches to the pathogenesis of minimal-change nephrotic syndrome Philippe Grimbert, Vincent Audard, Philippe Remy, Philippe Lang and Djillali Sahali INSERM U99 and Service de Ne´phrologie, Hoˆpital Henri Mondor, Cre´teil, France

Introduction Minimal-change nephrotic syndrome (MCNS) is a clinical and pathological entity defined by selective proteinuria and hypoalbuminaemia that occurs in the absence of cellular glomerular infiltrates or immunoglobulin deposits. The only detectable abnormalities involve the epithelial visceral cells with effacement of foot processes. These morphological alterations are typical of the nephrotic syndrome but not specific of the MCNS disease [1]. Whereas recent genetic approaches to familial idiopathic nephrotic syndromes have been determining factors in elucidating several molecular aspects of focal glomerular sclerosis [2–4], our knowledge about the pathogenesis of MCNS is inchoate, despite arguments suggesting a disorder of the immune function. In support of this view, occurrence of the disease in the context of immune challenge initiated by infectious or allergic stimuli, as well as relapse sensitivity to drugs known to inhibit the immune system (glucocorticoids, cyclosporin and cyclophosphamide) are particularly relevant [5]. The possible link between abnormal T-cell response and glomerular disease was postulated 30 years ago. First, Hoyer et al. [6] described early recurrences of nephrotic syndrome following renal transplantation. In two of the three cases reported, early renal biopsies on the native kidney did not detect histological signs of focal glomerular sclerosis although they were prominent during the progression of the disease. Secondly, Lagrue et al. [7] showed that systemic infusion of supernatants of T lymphocytes from patients with MCNS relapse induced proteinuria in rats. These Correspondence and offprint requests to: D. Sahali, Unite´ INSERM 99, Hoˆpital Henri Mondor, 51, avenue du Mal de Lattre-deTassigny, 94010, Cre´teil, France. Email: [email protected] #

observations have suggested that peripheral immune cells produce a circulating factor, which impairs the glomerular filtration barrier. Here, we would like to integrate some data deduced from recent molecular approaches on the immunopathogenesis of MCNS in the light of clinical characteristics and immunological and experimental findings.

T cells in MCNS Activation of T cells at the initial step of the immune response is dependent on the interaction with dendritic cells through cell-surface receptors. An expansion of CD4q and CD8q T-cell populations, identified by immunophenotyping of peripheral lymphocytes, is easily demonstrated during the nephrotic phase in most cases. Interestingly, it has been reported that an increase of the CD4q T cells expressing the CD25 marker (IL-2 receptor a chain) occurred during the relapse [8]. The CD25 antigen is expressed by at least two subsets of CD4q T cells: a large population (;50%) that express CD25 upon activation by an immunogen stimulus and a minor population, consisting of natural CD4q CD25q T cells (;10%), which plays a regulatory role in the immune response [9]. Experimental studies have shown that natural CD4q CD25q T cells, which exit the thymus very early in life, display a suppressor function and are expanded in the context of autoimmune organ specific disease, to circumvent the autoreactive cells. The CD4q CD 25q natural T cells do not exhibit a particular cytokine profile and their suppressive function is associated with a down-regulation of IL-2. The role of the CD4q CD 25q regulatory T cells in MCNS remains to be clarified. Activated CD4q helper T cells (Th) can be differentiated into two functionally and mutually exclusive distinct subsets: type 1 Th (Th1) and type 2 Th (Th2), on the basis of their cytokine profiles [10]. Polarization of uncommitted Th (Th0) into Th1 or Th2 cells involves different mechanisms, of which the

2003 European Renal Association–European Dialysis and Transplant Association

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Keywords: c-maf; minimal-change nephrotic syndrome; NFkB; pathogenesis; proteasome; T cells

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cytokine field at the site of antigen encounter plays a driving role. While interferons aub (IFN-aub) and IL-12 are critical mediators of Th1 development, IL-4 was shown to be a potent inducer of Th2 cells from naive T cells. Th1 cells express the IL-12 receptor, and secrete IFN-c, TNF-a and lymphotoxin-a (LT-a, also called TNF-b). Unlike TNF-a, which is produced primarily by activated macrophages, LT-a is synthesized by activated T and B cells and its biological role has not been clearly established in vivo. Th1 cytokines initiate inflammatory reactions, enhance cell immunity response, and mediate delayedtype hypersensitivity. Th2 cells are prone to synthesize IL-4, IL-5, IL-13, which together contribute to anti-inflammatory responses, help B cells to produce antibodies and protect against extracellular pathogens. We have recently shown that T lymphocytes from MCNS display a down-regulation of the IL-12 receptor b2 subunit (IL-12R b2) during relapse, while the second component of IL-12R, the b1 chain, was normally expressed [11]. The IL-12R b2 is selectively

expressed by Th1 cells and plays a key role in the transduction of IL-12 signalling through the JakuStat pathway. The down-regulation of the IL-12R b2 is compatible with a lack of IL-12 production during relapse, as reported by Stefanovic et al. [12], and suggests that activated T cells of MCNS were early driven toward Th2 phenotype. Additional evidence came from recent isolation by subtractive cloning of a Th2 specific factor, c-maf, which is strongly induced during relapse [11]; Valanciute A et al., submitted for publication]. These results are in agreement with early reports by Kimata et al. [13] and Yap et al. [14], who found an increased production of IL-13 Th2 cytokine during relapse. IL-13 regulates the switching of immunoglobulin production towards IgE, which is frequently increased in serum of patients with MCNS [15]. Interestingly, Ho et al. [16] have demonstrated that c-maf promotes Th2 and attenuates Th1 differentiation. Patients with MCNS often exhibit a defect in delayed-type hypersensitivity response, suggesting inhibition of Th1-dependent cellular immunity [17]. The inability to mount an effective Th1 response might


Fig. 1. Plausible immunological sequence of events leading to abnormal T-cell response.


account for the susceptibility to pneumococcal and other pathogen agents in this disease, known from the outset of the last century [18].

Transcription factors and proteasome activity in MCNS

glucocorticoids and cyclosporin induce a remission in MCNS might not be similar but rather complementary. This hypothesis is based on experimental findings showing that NFkB activation, in PBMC and T cells from MCNS relapse, is inhibited upon ex vivo addition of the proteasomal inhibitor MG132, which blocks the degradation of phosphorylated form of IkBa [19]. Cyclosporin A exerts an effect similar to MG132 [23,24]. On the other hand, glucocorticoids induce a transactivation of the IkBa gene, which might contribute to increased IkBa expression in remission, the highest levels being observed in patients on steroids. Thus, the glucocorticoids and cyclosporin A act by distinct mechanisms on the stabilization of IkBa and the inhibition of NFkB activation. Conceptually, it is hard to draw a simple model for T-cell activation in MCNS, given our current knowledge, but a working scheme may be proposed (Figure 1), which will undoubtedly be corrected and completed in the near future.

Alteration of other immune cell subsets in MCNS Immune dysfunction does not seem to be restricted to CD4q T cells, but our knowledge concerning the contribution of other T-cell subsets, monocytes, and even B cells, is very limited. Inasmuch that the immune system functions as a complex network, there could well exist other abnormalities that need characterization. As a matter of fact, the bulk of NFkB activation detected in relapse is not restricted to CD4q T cells, but appears more striking in the non-CD4q mononuclear cell fraction [19]. Frank et al. [25], analysing the genetic polymorphism of the variable region of the TCR b-chain, found a selective recruitment of some Vb families in peripheral non-CD4qT cells from patients with frequent relapse, suggesting a clonal expansion of CD8qT cells in long-lasting active disease.

Contribution of subtractive and differential cloning to study of MCNS pathogenesis Initial analysis of subtracted transcripts revealed two important and unexpected findings. First, several transcripts that are selectively up-regulated during the relapse are produced by unusual splicing, given that these forms are undetected in normal subjects. Preliminary data suggest that the protein forms encoded by these new transcripts display functional characteristics distinct from normal spliced proteins (Grimbert P et al., manuscript submitted). Secondly, some genes expressed but not translated in normal subjects exhibit a high protein level during the relapse (Audard V et al., work in progress). These results suggest that both transcriptional and post-transcriptional mechanisms are involved in immunopathogenesis of MCNS, which appears to be a complex disease in which


We recently reported abnormal activities of transcriptionfactors in MCNS, along the same line as Sahali et al. [19] and Cao et al. [20]. We detected high NFkB activation in CD4q T cells during the relapse. NFkB is rapidly activated by a wide variety of pathogenic signals such as viral and bacterial agents, T cell and B cell mitogens, cytokines (TNF-a, LT-a) and oxidative stress [21]. NFkB is involved to various extents in the transcriptional activation of a large set of genes including those encoding IL-1, IL-6, IL-2, IL-8, TNF-a and LT-a, many of them being increased in MCNS relapse. Interestingly, Das et al. [22] identified two NFkB-responsive elements on the IL-13 promoter, thus including this cytokine as a potential downstream target of NFkB. The transcriptional activation of NFkB is tightly regulated by inhibitory proteins called IkB proteins of which IkBa is the most important inhibitor. Following T-cell activation by NFkB inducers such as TNF-a, IkBa is rapidly phosphorylated, then degraded by the proteasome, allowing the nuclear translocation of NFkB, which activates its target’s genes, including its proper inhibitor IkBa. In return, IkBa is rapidly re-synthesized and sequestrates NFkB in the cytoplasm, thus switching off the NFkB activity. The fact that NFkB activation is sustained during the relapse, before the initiation of steroid therapy, suggests a break in the NFkB feedback loop in the active phase of the MCNS [19]. The upregulation of IkBa expression, in T cells and monocytes, following glucocorticoid therapy probably contributes to inhibition of the NFkB activation and the down-regulation of cytokines during the remission. In three patients who developed a steroid-resistant but cyclosporin-sensitive MCNS, we observed a strong NFkB activation during steroid therapy, which diminished upon addition of cyclosporin (Sahali D et al., unpublished data). The break in NFkB autoregulatory loop during relapses, and in some patients with resistance to steroids, is apparently not due to genetic alterations of IkBa, since increased levels of the inhibitor were found in remission. Moreover, we did not detect mutations or deletions in the IkBa promoter in seven patients analysed, including, the three patients described above (Valanciute A, Sobrier ML et al. unpublished data). Although a close relationship between NFkB activation and MCNS relapse seems to prevail, the fact that NFkB activation remains increased in some patients, despite steroid therapy, suggests that additional factors are involved in these transcriptional alterations and might contribute to steroid resistance. These data also suggest that the mechanisms by which

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several genes may contribute to immunological disorders. Perspectives Evidence can be gathered supporting the hypothesis that T cells, and most probably other immune cells as well, are involved in MCNS disease. Recent data provide one of the first molecular analyses of the signalling pathways preferentially recruited by T cells during the relapse. Although the molecular mechanisms underlying MCNS have yet to be clarified, the strategy developed in our laboratory may allow identification of the genes that play a central role in the immunopathogenesis of this disease. This work opens new perspectives in the molecular characterization of lymphocyte activation pathways in MCNS.

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Acknowledgements. We are indebted to Gabriel Richet, Michel Broyer, Patrick Niaudet and Pierre Ronco, who have provided much encouragement throughout the course of this project. We also thank Remy Salomon, Chantal Loirat and Albert Bensman, who have been essential to the research conducted in our laboratory.
