Transcriptional and Post-Transcriptional Alterations of IkBa in Active Minimal-Change Nephrotic Syndrome

Minimal-change nephrotic syndrome (MCNS) is a renal disease characterized by heavy glomerular proteinuria and increased production of cytokines by immune cells. Because of the central role of nuclear factor-kB (NF-kB) in the regulation of cytokine expression, its activity during the relapse and remission phases of steroid-sensitive MCNS was analyzed. During relapse, nuclear extracts from peripheral blood mononuclear cells displayed high levels of NF-kB DNAbinding activity, consisting primarily of p50/RelA (p65) complexes. NF-kB p65 and IkBa proteins were barely detected or not detected in cytosolic fractions during relapse, in contrast to remission. The lack of expression of IkBa protein was associated with downregulation of IkBa mRNA and increases in the levels of the mRNA encoding the proteasome a2 subunit proteolytic pathway. In addition, inhibition of proteasome activity induced cytosolic accumulation of phosphorylated IkBa and significant reductions in the NF-kB binding activity in nuclear extracts from peripheral blood mononuclear cells from patients experiencing relapses. These results suggest that alterations in the NF-kB/IkBa regulatory feedback loop may contribute to the immunologic abnormalities that occur in steroidsensitive MCNS. Minimal-change nephrotic syndrome (MCNS) is a kidney disease defined by selective proteinuria and hypoalbuminemia occurring in the absence of cellular glomerular infiltrates or Ig deposits. Electron microscopy reveals changes focused on glomerular epithelial cells, in the form of foot process effacement (1). Most children with primary nephrotic syndrome respond to steroid therapy, but the disease is often characterized by a relapsing course. For such patients, prolonged remission can be obtained with the addition of cyclosporin A or cyclophosphamide. However, a minority of patients fail to respond to this treatment and may develop chronic renal failure (2). Because the relapses occur concurrently with immune alterations, it has been suggested that the proteinuria is a consequence of a putative circulating factor produced by immune cells. Indirect evidence is derived from experiments demonstrating that systemic infusion of supernatants of cultured peripheral blood mononuclear cells (PBMC) from patients with MCNS relapses induced proteinuria in rats (3–5). Recent studies of T cell compartments in MCNS demonstrated expansion of CD4 and CD8 T cell populations, including those with the CD45RO memory phenotype (6,7). T cell expansion was often associated with increased synthesis of several cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin-13 (IL-13) (8,9). Clinical observations and experimental data also suggest that T cells with Th2-like phenotypes are involved in the pathophysiologic processes of MCNS. First, the production of IgE is often increased during relapses, independent of previous atopic manifestations. Second, both cell-mediated immunity and delayed hypersensitivity, which are characteristic of Th1 cell function, seem to be defective in relapses (10). Immune dysfunction seems not to be restricted to T cells, because levels of cytokines produced primarily by monocytes, such as IL-1 and IL-8, were increased in relapses. In contrast, remissions were characterized by downregulation of these cytokines (11). These findings suggest that molecular events upstream from cytokine production may be impaired in MCNS. Nuclear factor-kB (NF-kB) plays a key role in the regulation of cytokine expression, through association with other transcription factors and protein-protein interactions with coactivator proteins (12). Because most cytokines whose levels are increased during relapses and downregulated during remissions are partly or predominantly regulated by NF-kB, we postulated possible involvement of this regulatory pathway in MCNS. The human NF-kB/Rel family includes five members, i.e., NF-kB1 (p50), NF-kB2 (p52), RelA (p65), cRel, and RelB, which form various homoand heterodimers. Their activity is Received March 14, 2000. Accepted February 8, 2001. Correspondence to Dr. Djillali Sahali, INSERM Unité 99, Hôpital Henri Mondor, AP-HP, 51, avenue du Mal de Lattre-de-Tassigny, 94010, Créteil, France. Phone: 33-1-49-81-35-30; Fax: 33-1-48-98-09-08; E-mail: sahali@