Accumulation of both interstitial myofibroblasts and excessive production of extracellular matrix proteins is a common pathway contributing to chronic kidney disease. In a number of tissues, activation of STAT3 (signal transducer and activator of transcription 3) increases expression of multiple profibrotic genes. Here, we examined the effect of a STAT3 inhibitor, S3I-201, on activation of renal interstitial fibroblasts and progression of renal fibrosis. Treatment of cultured rat renal interstitial fibroblasts with S3I-201 inhibited their activation, as evidenced by dose- and time-dependent blockade of alpha-smooth muscle actin and fibronectin expression. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction, STAT3 was activated, and administration of S3I-201 attenuated both this activation and extracellular matrix protein deposition following injury. S3I-201 reduced infiltration of the injured kidney by inflammatory cells and suppressed the injury-induced expression of fibronectin, alpha-smooth muscle actin, and collagen type-1 proteins, as well as the expression of multiple cytokines. Furthermore, S3I-201 inhibited proliferation and induced apoptosis preferentially in renal interstitial fibroblasts of the obstructed kidney. Thus, our results suggest that increased STAT3 activity mediates activation of renal interstitial fibroblasts and the progression of renal fibrosis. Inhibition of STAT3 signaling with S3I-201 may hold therapeutic potential for fibrotic kidney diseases.

Activation of renal interstitial fibroblasts is critically involved in the development of tubulointerstitial fibrosis in chronic kidney diseases. In this study, we investigated the effect of trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, on the activation of renal interstitial fibroblasts in a rat renal interstitial fibroblast line (NRK-49F) and the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO) . α-Smooth muscle actin (α-SMA) and fibronectin, two hallmarks of fibroblast activation, were highly expressed in cultured NRK-49F cells, and their expression was inhibited in the presence of TSA. Similarly, administration of TSA suppressed the expression of α-SMA and fibronectin and attenuated the accumulation of renal interstitial fibroblasts in the kidney after the obstructive injury. Activation of renal interstitial fibroblasts was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3), and TSA treatment also abolished these responses. Furthermore, inhibition of the STAT3 pathway with AG490 inhibited expression of α-SMA and fibronectin in NRK-49F cells. Finally, TSA treatment inhibited tubular cell apoptosis and caspase-3 activation in the obstructive kidney. Collectively, we suggest that pharmacological HDAC inhibition may induce antifibrotic activity by inactivation of renal interstitial fibroblasts and inhibition of renal tubular cell death. STAT3 may mediate those actions of HDACs.

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 siRNAmediated silencing of EZH2 inhibited serum-and TGFb1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of a-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFb receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFb 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.