**Wei-Cheng Tseng 1,2,3,4, Ming-Tsun Tsai 1,2,3, Nien-Jung Chen <sup>5</sup> and Der-Cherng Tarng 1,2,3,4,6,7,\***


Received: 13 August 2020; Accepted: 16 August 2020; Published: 19 August 2020

**Abstract:** Mounting evidence indicates that an increase in histone deacetylation contributes to renal fibrosis. Although inhibition of histone deacetylase (HDAC) can reduce the extent of fibrosis, whether HDAC inhibitors exert the antifibrotic effect through modulating the phenotypes of macrophages, the key regulator of renal fibrosis, remains unknown. Moreover, the functional roles of the M2 macrophage subpopulation in fibrotic kidney diseases remain incompletely understood. Herein, we investigated the role of HDAC inhibitors on renal fibrogenesis and macrophage plasticity. We found that HDAC inhibition by trichostatin A (TSA) reduced the accumulation of interstitial macrophages, suppressed the activation of myofibroblasts and attenuated the extent of fibrosis in obstructive nephropathy. Moreover, TSA inhibited M1 macrophages and augmented M2 macrophage infiltration in fibrotic kidney tissue. Interestingly, TSA preferentially upregulated M2c macrophages and suppressed M2a macrophages in the obstructed kidneys, which was correlated with a reduction of interstitial fibrosis. TSA also repressed the expression of proinflammatory and profibrotic molecules in cultured M2a macrophages and inhibited the activation of renal myofibroblasts. In conclusion, our study was the first to show that HDAC inhibition by TSA alleviates renal fibrosis in obstructed kidneys through facilitating an M1 to M2c macrophage transition.

**Keywords:** macrophage subpopulation; renal fibrosis; trichostatin A

## **1. Introduction**

Chronic kidney disease (CKD) is an emerging global public health issue with a prevalence rate of 10% to 12% worldwide [1]. Regardless of the etiologies of renal diseases, unresolved renal insult engages an excessive deposition of extracellular matrix in the tubulointerstitium, thereby bringing about end-stage renal disease [2]. Tubulointerstitial fibrosis is the final common pathway of all kidney diseases and, also, represents the major determinant of renal function decline [2]. A wealth of studies has indicated that renal function decline correlates well with the increasing risks for all-cause mortality, cardiovascular events and hospitalization [1,3,4]. Despite that current available therapies have targeted traditional risk factors for renal function decline—namely, hypertension, hyperglycemia and hyperlipidemia, nearly half of CKD patients still experience progressive renal function decline and eventual end-stage renal disease [5]. Hence, a novel treatment modality to tackle renal fibrosis and halt the progression of CKD is urgently needed.

Infiltrated renal macrophages play pivotal roles in the homeostasis of renal fibrogenesis following initial renal insult, either ischemic, immunologic, mechanical or toxic damage [6]. Macrophages are highly plastic and differentiate into different phenotypes in response to the local environments. Macrophage phenotypes can be broadly categorized into the proinflammatory "M1" macrophages (characterized by inducible nitric oxide synthase (iNOS)) and the anti-inflammatory, reparative "M2" macrophages (characterized by arginase-1 (Arg1)) [6]. M2 macrophages can be further classified into M2a and M2c subpopulations by the presence of C-type lectin domain family 7 member A (CLEC7A) and signaling lymphocytic activation molecule (SLAM), respectively [7]. These macrophage subsets play distinct roles in the wound-healing process following tissue injury [8]. During the normal wound-healing process, the initial proinflammatory milieu recruits M1 macrophages to induce apoptosis and eliminate the pathogen and necrotic tissue. Thereafter, anti-inflammatory M2 macrophages predominate in the later tissue repair stage to activate re-epithelialization and neoangiogenesis in the injured area. Finally, a resolution stage ends the whole healing process by promoting the apoptosis of recruited immune cells, suppression of inflammation and tissue remodeling [8]. Nonetheless, dysregulation of the M1-to-M2 transition in a normal wound-healing process would lead to pathologic fibrosis and tissue scarring [8]. Recent studies further suggest that the excessive activation of M1 macrophages and certain profibrotic M2 macrophages both contribute to the development of fibrosis formation [9].

M2 macrophages may function as a double-edged sword in regulating renal fibrosis. M2 macrophages help control inflammation through releasing interleukin (IL)-10, Arg1, transforming growth factor-β (TGF-β) and heme oxygenase-1 [9]. On the other hand, the chronic activation of M2 macrophages can activate resident fibroblasts through the release of TGF-β, platelet-derived growth factor, vascular endothelium growth factor, insulin-like growth factor-1 and galactin-3 [9]. In this regard, M2 macrophages are proposed to be profibrotic in the renal fibrosis model of unilateral ureteral obstruction (UUO), and depletion of these M2 macrophages in UUO should be beneficial [9,10]. However, not all macrophage depletion strategies result in a reduction in fibrosis in UUO [10]. Inhibition of the c-fms kinase almost suppresses all infiltrating macrophage numbers in day 14 obstructed kidneys but does not change the course of fibrosis, suggesting some antifibrotic M2 macrophages are also depleted [11]. Until now, the functional roles of the M2 macrophage subpopulation in renal fibrogenesis remained unclear and conflicting. M2a and M2c macrophages are initially found to be anti-inflammatory and reparative in murine Adriamycin nephrosis [12]. Nonetheless, two recent studies indicate that M2a macrophages are upregulated in endometrial fibrosis and skeletal muscle fibrosis [13,14], suggesting M2a macrophages may exhibit a profibrotic feature in chronic fibrotic diseases. Therefore, further elucidating the roles of the M2a and M2c subsets may help delineate the complex fibrogenic process in UUO.

Emerging evidence indicates that epigenetic modulation of the chromatin state is crucial in determining the progression of CKD and macrophage polarization [15,16]. The histone acetylation status has recently been found to associate with certain kidney diseases and renal fibrogenesis [17]. Histone deacetylases (HDACs) are a group of enzymes that exert epigenetic effects by altering the acetylation status of histone and nonhistone proteins [18]. Although potential favorable effects of HDAC inhibitors have been found in animal models of acute kidney injury [19], diabetic nephropathy [20] and Adriamycin nephropathy [21], the roles of HDAC inhibitors in UUO and macrophage plasticity remain incompletely understood. Marumo et al. found that the expression of HDAC1 and HDAC2 are upregulated in obstructed kidneys and contribute to proinflammatory and fibrotic responses [22]. Nonetheless, whether HDAC inhibition regulates the phenotypic change of renal interstitial macrophages in UUO is still unclear. Previously, HDAC3-deficient bone marrow-derived macrophages displayed an M2-polarized IL-4-induced alternatively activated phenotype [11], implying HDAC inhibition may contribute to M2 macrophage polarization. Currently, there is no study exploring the interaction between histone acetylation and macrophage subsets in kidney diseases. Hence, we aimed to investigate whether HDAC inhibition attenuates renal fibrosis through modulating the phenotype of renal interstitial macrophages.

In this study, we found the distinct expression of M2a and M2c subset macrophages in obstructed kidneys. An increased M2a macrophage infiltrate correlated with a higher extent of renal interstitial fibrosis. Interestingly, the administration of trichostatin A (TSA), an HDAC inhibitor, suppressed M2a macrophage infiltration, enhanced M2c macrophage expression and attenuated renal fibrosis in UUO. TSA also repressed the expression of proinflammatory and profibrotic molecules in cultured M2a macrophages and inhibited the activation of renal myofibroblasts. Our study is the first to demonstrate that TSA modulates the renal macrophage M2 subpopulation to inhibit renal fibrosis.
