Transcription of nephrin-Neph3 gene pair is synergistically activated by WT1 and NF- B and silenced by DNA methylation

Abstract

Background. Nephrin and Neph3 are homologous molecules expressed in the podocyte slit diaphragms that are essential for normal glomerular ultrafiltration. Nephrin and Neph3 genes form a bidirectional gene pair suggesting that they may share key features in their regulation. We investigated if nephrin and Neph3 genes have similar mechanisms in their transcriptional regulation focussing on transcription factor Wilms' tumour 1 (WT1) and nuclear factor-jB (NF-jB) and DNA methylation. Methods. Transcriptional regulation of nephrin and Neph3 by WT1 and NF-jB was analysed by overexpression studies, reporter gene assay and chromatin immunoprecipitation using A293 cells and cultured podocytes. The interaction between WT1 and NF-jB was studied by coimmunoprecipitation. The effect of NF-jB activator tumour necrosis factor-a (TNF-a) with or without NF-jB pathway inhibitor (BAY 11-7082) on nephrin and Neph3 messenger RNA (mRNA) expression and on cellular distribution of NF-jB was determined by quantitative polymerase chain reaction (PCR) and immunostaining, respectively. The role of DNA methylation in regulating nephrin and Neph3 genes was studied by demethylating agent (5-aza-2#-deoxycytidine) treatment and quantitative PCR. Results. WT1 and NF-jB interact with nephrin and Neph3 promoter and cooperatively regulate nephrin and Neph3. The cooperation was further supported by the physical interaction between WT1 and NF-jB. TNF-a increased nephrin and Neph3 mRNA expression and this effect was mediated by NF-jB. Furthermore, DNA methylation played a role in silencing nephrin and Neph3 expression in a cell-type and differentiation stage-dependent manner. Conclusion. These results provide novel insights into the transcriptional regulation of nephrin and Neph3 genes and indicate that nephrin and Neph3 share the same mechanisms in their regulation.

α-Actinin 4 Potentiates Nuclear Factor κ-Light-chain-enhancer of Activated B-cell (NF-κB) Activity in Podocytes Independent of Its Cytoplasmic Actin Binding Function

“…2A). The nephrin gene NPHS1 has been shown to be an NF-B target gene (25). Indeed, NPHS1 mRNA expression was induced by TNF␣, and knockdown of ACTN4 abolished TNF␣-mediated induction of NPHS1 mRNA.…”

TGF-β–Activated Kinase 1 Is Crucial in Podocyte Differentiation and Glomerular Capillary Formation

“…Recent evidence indicates that WT1 and NF-kB physically interact with the promoters for two homologous proteins, nephrin and Neph3 (filtrin), expressed in the podocyte slit diaphragm, and they cooperatively regulate the expression of nephrin and Neph3. 46 Thus, these results present a plausible explanation that the reduction of nephrin expression occurring in the Tak1 Δ/Δ kidney might be caused by decreased WT1 expression and failed NF-kB activation resulting from Tak1 deletion. Null mutations in the nephrin gene nphs1 cause congenital nephrotic syndrome of the Finnish type characterized by podocyte foot process effacement and absence of slit diaphragms.…”

“…Although it has been previously known that WT1, 42,43 Sp1, 44 and Snail 45 are implicated in the regulation of Nphs1 expression, recent studies have shown that NF-kB and WT1 cooperatively regulate Nphs1 transcription. 46 Here, we show that podocyte-specific deletion of Tak1 in mice resulted in high incidence of perinatal lethality, proteinuria, delayed glomerulogenesis, and reduced expression of WT1 and nephrin in the podocytes. Tak1 deletion in podocytes caused disruption of the podocyte architecture and foot process effacement.…”

Untargeted DNA-Demethylation Therapy Neither Prevents Nor Attenuates Ischemia-Reperfusion-Induced Renal Fibrosis

“…It was hypothesized by Bechtel et al [11] that fibroblast activation in fibrotic kidney disease is the result of epigenetic alterations, more precisely DNA-methylation, which renders them terminally activated. It is becoming clear that DNA-methylation, in addition to cancer pathologies, also plays an important role in renal pathologies, since independent investigators found that aberrant DNA-methylation is associated with inflammation [12,13] , development of renal fibrosis [11,[14][15][16], and that it occurs during ischemia in transplantation [17,18] . Importantly, aberrant DNA-methylation is thought to influence allograft survival, since advanced donor age, differentiation, and polarization of immune cells as well as ischemia are associated with alterations in the DNA-methylation pattern [5,19,20] .…”