*2.2. Preferential Accumulation of M1 and M2a Macrophages in Obstructed Kidneys*

As macrophage plasticity is critical for regulating renal fibrosis [8], next, we analyzed the phenotypes of macrophages at different time points of UUO. Immunohistochemistry showed that both iNOS-positive M1 macrophages and Arg1-positive M2 macrophages progressively accumulated in the day 14 obstructed kidneys. Thereafter, we explored the M2 macrophage subpopulation in UUO. Interestingly, CLEC7A-positive M2a macrophages predominantly expressed in the day 14 obstructed kidneys. By contrast, the number of SLAM-positive M2c macrophages was only slightly increased over time (Figure 2A,B). Western blot analyses also showed the consistent results that the expression of M1 and M2a markers progressively increased with the time of UUO (Figure 2C,D). These data suggest that M1 and M2a macrophages are involved in the development of renal fibrosis.

*Int. J. Mol. Sci.* **2020**, *21*, 5966

**Figure 1.** Macrophage infiltration correlates with renal interstitial fibrosis following a unilateral ureteral obstruction (UUO) injury. (**A**) Representative images of the Masson trichrome staining showed that the extent of fibrosis (blue staining) progressively increased in day 14 UUO kidneys as compared to day 7 ones. Immunohistochemistry demonstrated that more α-smooth muscle actin (α-SMA)-positive myofibroblasts and F4/80-positive macrophages accumulated in day 14 UUO kidneys. Scale bar = 100 μm. (**B**) Quantification of the fibrosis extent, α-SMA-positive and F4/80-positive areas. \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group. (**C**) Western blot analysis of fibronectin and α-SMA expression in day 7 and day 14 obstructed kidneys. β-actin served as the loading control. (**D**) Quantification of the Western blot analyses. \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group.

**Figure 2.** M1 and M2a macrophage infiltrates predominate in the late stage of unilateral ureteral obstruction (UUO). (**A**) Representative immunohistochemical photomicrographs of inducible nitric oxide synthase (iNOS), arginase-1 (Arg1), C-type lectin domain family 7 member A (CLEC7A) and signaling lymphocytic activation molecule (SLAM) in day 7 and day 14 obstructed kidneys. Scale bar = 25 μm. (**B**) Quantification of the iNOS-, Arg1-, CLEC7A- and SLAM-positive interstitial cells in day 7 and day 14 obstructed kidneys. \* *p* < 0.05 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group. (**C**) Western blot analyses of iNOS, Arg1, CLEC7A and SLAM expressions in day 7 and day 14 obstructed kidneys. β-actin served as the loading control. (**D**) Quantification of the Western blot analyses. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group.

#### *2.3. HDAC Inhibition Represses Renal Fibrosis and Macrophage Infiltration in UUO*

Given that increased histone deacetylation may be associated with renal fibrogenesis [17], we then investigated the therapeutic effect of TSA in UUO. The administration of TSA significantly reduced the extent of α-SMA-positive myofibroblasts and the area of renal interstitial fibrosis in UUO. Moreover, TSA also decreased the extent of interstitial macrophage infiltrate (Figure 3A,B). Western blot analysis also found that TSA reduced the expression of the extracellular matrix (fibronectin) and α-SMA in obstructed kidneys (Figure 3C,D).

**Figure 3.** Trichostatin A (TSA) ameliorates renal inflammation and fibrosis in unilateral ureteral obstruction (UUO). (**A**) Representative images of Masson trichrome staining of obstructed kidneys at 7 days and 14 days following UUO in the vehicle and TSA groups. The TSA treatment markedly reduced the extent of interstitial fibrosis (blue area). Immunohistochemical staining for α-smooth muscle actin (α-SMA, myofibroblast marker) and F4/80 (pan-macrophage marker) in day 7 and day 14 obstructed kidneys. Scale bar = 100 μm. (**B**) Quantification of the fibrosis extent, α-SMA-positive and F4/80-positive areas. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group. (**C**) Western blots of fibronectin and α-SMA in day 7 and day 14 UUO kidneys treated with the vehicle or TSA. β-actin served as the loading control. (**D**) Quantification of fibronectin and α-SMA expression levels. \*\* *p* < 0.01 by the unpaired Student's *t*-test; *n* = 5 for each group.
