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.

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.

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of a-smooth muscle actin (a-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-b1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-b1-or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of a-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.