*2.6. HDAC Inhibition Attenuates TGF-*β*1-Activated Renal Tubular Epithelial Cells and Fibroblasts*

TGF-β1 is a well-known predominant mediator of the activation of renal myofibroblasts and resultant fibrogenesis in UUO [23]. To substantiate the role of HDAC inhibition on TGF-β1-induced renal fibrosis, renal tubular epithelial NRK-52E cells and renal NRK-49F fibroblasts were stimulated with TGF-β1 in the presence or absence of TSA. Western blotting revealed that TGF-β1 upregulated the expression of α-SMA and fibronectin in NRK-52E cells in a time-dependent manner. The treatment with TSA abrogated TGF-β1-induced fibrogenesis in NRK-52E cells (Figure 6A,B). These effects were further confirmed by immunofluorescent staining (Figure 6C,D). In NRK-49F fibroblasts, the TSA treatment also repressed TGF-β1-induced α-SMA expression (Supplementary Figure S1). These data indicate that TSA reduced renal fibrosis through attenuating the activation of myofibroblasts by TGF-β1.

**Figure 6.** The effect of trichostatin A (TSA) on the activation of renal myofibroblasts. (**A**) Western blots of fibronectin and α-smooth muscle actin (a-SMA) expressions in transforming growth factor-β1 (TGF- β1, 20 ng/mL)-stimulated renal tubular epithelial NRK-52E cells at the indicated time. β-actin served as the loading control. (**B**) Quantification for the levels of indicated proteins. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by ANOVA, followed by Tukey's post hoc multiple comparison test. ns, nonsignificance. *n* = 3 for each group. (**C,D**) Immunofluorescent staining of fibronectin and α-SMA in the TGF-β1-treated NRK-52E cells at the indicated time. Scale bar = 50 μm. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by ANOVA, followed by Tukey's post hoc multiple comparison test. ns, nonsignificance. *n* = 3 for each group.

Our findings are summarized schematically in Figure 7. In renal fibrogenesis, the accumulation of M1 and M2a macrophages enhances inflammation and activates myofibroblasts. Trichostatin A suppresses the infiltration of proinflammatory M1 and M2a macrophages and increases the infiltration of anti-inflammatory M2c macrophages, thereby inhibiting the activation of renal myofibroblasts and resultant renal fibrosis.

**Figure 7.** Proposed immunomodulatory and antifibrotic mechanism by trichostatin A in renal fibrosis. In a mice model of unilateral ureteral obstruction, the administration of trichostatin A suppresses the infiltration of proinflammatory M1a and M2a macrophages, as well as promotes the accumulation of anti-inflammatory M2c macrophages, thereby limiting excessive inflammation. Accordingly, the activation of myofibroblasts is limited, and renal fibrosis is attenuated.

## **3. Discussion**

This present study showed that HDAC inhibition by TSA reduced renal fibrosis through modulating the M2 macrophage subpopulation. We found that a TSA treatment significantly decreased the extent of interstitial fibrosis, myofibroblast accumulation and macrophage infiltration in the obstructed kidneys. Interestingly, TSA downregulated iNOS expression and upregulated Arg1 expression in the whole kidney protein expression levels. Consistently, immunochemistry also found that TSA suppressed the infiltration of iNOS-positive M1 macrophages and promoted the accumulation of Arg1-positive M2 macrophages in the obstructed renal interstitial areas. Specifically, TSA suppressed the accumulation of CLEC7A-positive M2a macrophages but increased the number of SLAM-positive M2c macrophages. These data indicated that the increment of M2 macrophages by TSA in UUO was preferentially attributed to the increased accumulation of M2c macrophages. Consistently, we also found that TSA suppressed CLEC7A expression in M2a macrophages and enhanced SLAM expression in M2c macrophages in vitro. Notably, TSA further downregulated the expression of proinflammatory and profibrotic factors in M2a macrophages. Moreover, TSA attenuated the TGF-β1-induced expression of α-SMA and fibronectin in cultured renal tubular epithelial cells and fibroblasts, indicating that TSA also attenuated the activation of renal myofibroblasts through inhibiting the epithelial-mesenchymal transition. Collectively, our findings indicated that TSA suppressed renal fibrogenesis through increasing the anti-inflammatory and antifibrotic M2 subpopulation.

Conflicting results exist regarding the role of M2 macrophages on renal fibrosis in UUO [9], probably related to the heterogeneity of the M2 subpopulation. Previously, M2 macrophages were proposed to be profibrotic, because the macrophage infiltrate in the UUO-kidney has a predominant M2 phenotype and the percentage of M2 macrophages increases along with the duration of obstruction [10]. If this is the case, the depletion of all macrophages in the later stage of UUO should mitigate renal fibrosis. However, not all macrophage ablation strategies result in the improvement of renal fibrosis

in UUO [10]. The depletion of infiltrating macrophages through targeting the c-fms kinase fails to attenuate the extent of renal fibrosis in day 14 obstructed kidneys [11], suggesting certain antifibrotic M2 macrophages are also depleted and also highlighting the complex functional heterogeneity of the M2 macrophage population [24]. Our study found that the TSA treatment resulted in fibrosis reduction, the downregulation of iNOS-positive M1 macrophages and CLEC7A-positive M2a macrophages and the upregulation of SLAM-positive M2c macrophages. In line with our observations, Wang et al. found that M2a macrophages are associated with the accumulation of collagen and muscle fibrosis [14]. Moreover, Duan et al. showed that the adoptive transfer of M2a macrophages significantly increased the extent of fibrosis in endometriosis [13], suggesting that M2a macrophages are profibrotic and critically involved in fibrogenesis. The controversy regarding the profibrotic or antifibrotic roles of M2 macrophages may be reconciled by the balance of distinct M2a and M2c macrophages in chronic fibrotic disorders.

Recent attention has focused on the significance of post-translational histone modifications in determining the progression of CKD and macrophage polarization [15,16]. Nonetheless, the role of epigenetic regulation on macrophage phenotypes in UUO remains elusive. The present study found that HDAC inhibition by TSA significantly alleviated renal inflammation and fibrosis. We also found that TSA inhibited the infiltration of M1 and M2a macrophages, enhanced the infiltration of M2c macrophages and directed an M1-to-M2c switch. The TSA-induced upregulation of M2c macrophages correlated with a reduction of renal interstitial fibrosis, and TSA also enhanced the SLAM expression and suppressed profibrotic and proinflammatory phenotypes in cultured M2c macrophages. These data supported that TSA repressed renal fibrosis through augmenting the expression of antifibrotic M2 macrophages. Although we found that TSA reduced UUO-induced renal fibrosis through modulation of the M2 macrophage subpopulation, our findings may not be extrapolated to renal fibrosis attributed to other insults, such as toxin, diabetes or autoimmune diseases. Clearly, more studies will be required to confirm the immunomodulatory and antifibrotic effects of TSA in other models of kidney diseases. In consistency with our findings, Pang et al. found that TSA can mitigate the extent of renal fibrosis in obstructive nephropathy, but the role of TSA on renal inflammation is undefined in their study [25]. A later study by Marumo et al. also found that TSA suppressed tubulointerstitial fibrosis in obstructed kidneys and further showed that TSA reduced renal inflammation in terms of the downregulation of EMR1 and MCP1 expressions, as well as decreased the number of infiltrated macrophages [22]. However, the effect of HDAC inhibition on the phenotypic transition of macrophages was not examined [22]. Taken together, our study provided novel evidence that TSA attenuated renal fibrosis through suppressing the profibrotic M2a macrophage and promoting anti-inflammatory, antifibrotic M2c macrophages. In conclusion, our study showed that HDAC inhibition by TSA significantly attenuated renal fibrosis through promoting an M1-to-M2c macrophage transition in obstructed kidneys. Our study not only added to the knowledge of antifibrotic M2 macrophages but, also, provided the bench evidence that pharmacological HDAC inhibitors can be applied to clinical treatments of renal fibrosis in the future.

#### **4. Materials and Methods**

#### *4.1. Animals*

Male 6-to-8-week-old C57BL/6J mice weighing 20-25 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed at the Laboratory Animal Center of the National Yang-Ming University (Taipei, Taiwan). The mice were raised in a sound-attenuated, temperature-controlled (22 ± 1 ◦C) room with a 12-h light/dark cycle. Standard rodent chow and drinking water were supplied ad libitum. All experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The study was approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University under the license number 1041244.
