Abstract
GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.
Highlights
The members of Gadd[45] gene family, Gadd45a, Gadd45b and Gadd45r have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA damage repair, cell survival, senescence and apoptosis.[1]
We further showed that podocyte-specific overexpression of zebrafish orthologs of gadd45b predisposed podocytes to injury, whereas inhibition of gadd45b expression in zebrafish larvae ameliorated podocyte injury and reduced proteinuria
We found that gadd45ba expression was more abundant than gadd45bb in normal zebrafish glomeruli and the induction of gadd45ba by podocyte injury was more prominent than that of gadd45bb (Figures 2c and d), suggesting gadd45ba may have a major role in zebrafish podocytes compared with gadd45bb
Summary
Gadd45ba/b expression is upregulated in zebrafish with podocyte injury. We isolated podocytes from kidney of Tg (pod:Gal4;UAS:NTR-mcherry) zebrafish and confirmed the isolated podocytes had enriched podocin expression (Figure 1a). gadd45ba/bb mRNA expression were detected on the isolated podocytes (Figure 1b). We refined this model of podocyte injury using a double transgenic zebrafish Tg(pod:Gal4;UAS:NTRmCherry), in which transcription factor Gal[4] was expressed in podocytes, and NTR-mCherry was under the control of UAS promoter In this model, renal glomeruli could be conveniently isolated based on the mCherry fluorescence and podocyte injury induced by MTZ treatment (Figure 2a). Without MTZ treatment, Tg(pod:Gal4;UAS:NTR-mCherry;UAS:gadd45ba/b;lfabp: VDBP-GFP) did not show any significant excretion of GFP either, indicating that gadd45ba/b overexpression alone was not sufficient to impair the glomerular filtration barrier These proteinuria data were consistent with the tubular accumulation of GFP and the edema phenotype. We observed that apoptosis was significantly increased following MTZ treatment in the pronephric glomeruli of gadd45ba/b overexpression fish than those of the control group expressing NTR-mCherry alone in podocytes (Figure 5).
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