Histone demethylase JMJD3 protects against renal fibrosis by suppressing TGFβ and Notch signaling and preserving PTEN expression.

Rationale: The Jumonji domain containing-3 (JMJD3), a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3), is associated with the pathogenesis of many diseases, but its role in renal fibrosis remains unexplored. Here we examined the role of JMJD3 and mechanisms involved in the activation of renal fibroblasts and development of renal fibrosis. Methods: Murine models of 5/6 surgical nephrectomy (SNx) and ureteral unilateral obstruction (UUO) were used to assess the effect of a specific JMJD3 inhibitor, GSKJ4, and genetic deletion of JMJD3 from FOXD1 stroma-derived renal interstitial cells on the development of renal fibrosis and activation of renal interstitial fibroblasts. Cultured rat renal interstitial fibroblasts (NRK-49F) and mouse renal tubular epithelial cells (mTECs) were also used to examine JMJD3-mediated activation of profibrotic signaling. Results: JMJD3 and H3K27me3 expression levels were upregulated in the kidney of mice subjected to SNx 5/6 and UUO. Pharmacological inhibition of JMJD3 with GSKJ4 or genetic deletion of JMJD3 led to worsening of renal dysfunction as well as increased deposition of extracellular matrix proteins and activation of renal interstitial fibroblasts in the injured kidney. This was coincident with decreased expression of Smad7 and enhanced expression of H3K27me3, transforming growth factor β1 (TGFβ1), Smad3, Notch1, Notch3 and Jagged1. Inhibition of JMJD3 by GSK J4 or its specific siRNA also resulted in the similar responses in cultured NRK-49F and mTECs exposed to serum or TGFβ1. Moreover, JMJD3 inhibition augmented phosphorylation of AKT and ERK1/2 in vivo and in vitro. Conclusion: These results indicate that JMJD3 confers anti-fibrotic effects by limiting activation of multiple profibrotic signaling pathways and suggest that JMJD3 modulation may have therapeutic effects for chronic kidney disease.

Yu C ，Xiong C ，Tang J ，Hou X ，Liu N ，Bayliss G ，Zhuang S ... -  《Theranostics》

Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis.

SET and MYND domain protein 2 (SMYD2) is a lysine methyltransferase that mediates histone H3 lysine 36 trimethylation (H3K36me3) and acts as a regulator of tumorgenesis and cystic growth. However, its role in renal fibrosis remains unknown. In this study, we found that SMYD2 was highly expressed in the murine kidney of renal fibrosis induced by unilateral ureteral obstruction, and primarily located in interstitial fibroblasts and renal tubular epithelial cells. Pharmacological inhibition of SMYD2 with AZ505, a highly selective inhibitor of SMYD2, protected against renal fibrosis and inhibited activation/proliferation of renal interstitial fibroblasts and conversion of epithelial cells to a profibrotic phenotype in this model. In cultured renal interstitial fibroblasts, treatment with AZ505 or silencing of SMYD2 by specific siRNA also inhibited serum- or TGF-β1-induced activation and proliferation of renal interstitial fibroblasts. Mechanistic studies showed that SMYD2 inhibition reduced phosphorylation of several profibrotic signaling molecules, including Smad3, extracellular signal-regulated kinase 1/2, AKT, signal transducer and activator of transcription-3 and nuclear factor-κB in both injured kidney and cultured renal fibroblasts. AZ505 was also effective in suppressing renal expression of Snail and Twist, two transcriptional factors that mediate renal partial epithelial-mesenchymal transition and fibrosis. Conversely, AZ505 treatment prevented downregulation of Smad7, a renoprotective factor in vivo and in vitro. These results indicate that SMYD2 plays a critical role in mediating conversion of epithelial cells to a profibrotic phenotype, renal fibroblast activation and renal fibrogenesis, and suggest that SMYD2 may be a potential target for the treatment of chronic fibrosis in kidney disease.

Liu L ，Liu F ，Guan Y ，Zou J ，Zhang C ，Xiong C ，Zhao TC ，Bayliss G ，Li X ，Zhuang S ... -  《-》

Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys.

Rationale: Ureteral obstruction-induced hydronephrosis is associated with renal fibrosis and progressive chronic kidney disease (CKD). Exosome-mediated cell-cell communication has been suggested to be involved in various diseases, including renal fibrosis. However, little is known regarding how exosomes regulate renal fibrosis in obstructed kidneys. Methods: We first examined the secretion of exosomes in UUO (unilateral ureteral obstruction) mouse kidneys and TGF-β1-stimulated tubular epithelial cells (NRK-52E). Exosomes from NRK-52E cells were subsequently harvested and incubated with fibroblasts (NRK-49F) or injected into UUO mice via the tail vein. We next constructed Rab27a knockout mice to further confirm the role of exosome-mediated epithelial-fibroblast communication relevant to renal fibrosis in UUO mice. High-throughput miRNA sequencing was performed to detect the miRNA profiles of TGFβ1-Exos. The roles of candidate miRNAs, their target genes and relevant pathways were predicted and assessed in vitro and in vivo by setting specific miRNA mimic, miRNA inhibitor, siRNA or miRNA LNA groups. Results: Increased renal fibrosis was associated with prolonged UUO days, and the secretion of exosomes was markedly increased in UUO kidneys and TGF-β1-stimulated NRK-52E cells. Purified exosomes from TGF-β1-stimulated NRK-52E cells could activate fibroblasts and aggravate renal fibrosis in vitro and in vivo. In addition, the inhibition of exosome secretion by Rab27a knockout or GW4869 treatment abolished fibroblast activation and ameliorated renal fibrosis. Exosomal miR-21 was significantly increased in TGFβ1-Exos compared with Ctrl-Exos, and PTEN is a certain target of miR-21. The promotion or inhibition of epithelial exosomal miR-21 correspondingly accelerated or abolished fibroblast activation in vitro, and renal fibrosis after UUO was alleviated by miR-21-deficient exosomes in vivo through the PTEN/Akt pathway. Conclusion: Our findings reveal that exosomal miR-21 from tubular epithelial cells may accelerate the development of renal fibrosis by activating fibroblasts via the miR-21/PTEN/Akt pathway in obstructed kidneys.

Zhao S ，Li W ，Yu W ，Rao T ，Li H ，Ruan Y ，Yuan R ，Li C ，Ning J ，Li S ，Chen W ，Cheng F ，Zhou X ... -  《Theranostics》

Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. However, the role of EZH2 in renal fibrogenesis remains unexplored. In this study, we found high expression of EZH2 and H3K27me3 in cultured renal fibroblasts and fibrotic kidneys from mice with unilateral ureteral obstruction and humans with CKD. Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) or GSK126 or siRNA-mediated silencing of EZH2 inhibited serum- and TGFβ1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of α-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFβ receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFβ receptors and downstream signaling molecules signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 after injury. Moreover, EZH2 inhibition increased the expression of phosphatase and tensin homolog (PTEN), a protein previously associated with dephosphorylation of tyrosine kinase receptors in the injured kidney and serum-stimulated renal interstitial fibroblasts. Finally, blocking PTEN with SF1670 largely diminished the inhibitory effect of 3-DZNeP on renal myofibroblast activation. These results uncovered the important role of EZH2 in mediating the development of renal fibrosis by downregulating expression of Smad7 and PTEN, thus activating profibrotic signaling pathways. Targeted inhibition of EZH2, therefore, could be a novel therapy for treating CKD.

Zhou X ，Zang X ，Ponnusamy M ，Masucci MV ，Tolbert E ，Gong R ，Zhao TC ，Liu N ，Bayliss G ，Dworkin LD ，Zhuang S ... -  《-》

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

Bergmann C ，Brandt A ，Merlevede B ，Hallenberger L ，Dees C ，Wohlfahrt T ，Pötter S ，Zhang Y ，Chen CW ，Mallano T ，Liang R ，Kagwiria R ，Kreuter A ，Pantelaki I ，Bozec A ，Abraham D ，Rieker R ，Ramming A ，Distler O ，Schett G ，Distler JHW ... -  《-》