Histone deacetylase 6 inhibition mitigates renal fibrosis by suppressing TGF-β and EGFR signaling pathways in obstructive nephropathy

Abstract

We have recently shown that histone deacetylase 6 (HDAC6) is critically involved in the pathogenesis of acute kidney injury. Its role in renal fibrosis, however, remains unclear. In this study, we examined the effect of ricolinostat (ACY-1215), a selective inhibitor of HDAC6, on the development of renal fibrosis in a murine model induced by unilateral ureteral obstruction (UUO). HDAC6 was highly expressed in the kidney following UUO injury, which was coincident with deposition of collagen fibrils and expression of α-smooth muscle actin, fibronectin, and collagen type III. Administration of ACY-1215 reduced these fibrotic changes and inhibited UUO-induced expression of transforming growth factor-β1 and phosphorylation of Smad3 while increasing expression of Smad7. ACY-1215 treatment also suppressed phosphorylation of epidermal growth factor receptor (EGFR) and several signaling molecules associated with renal fibrogenesis, including AKT, STAT3, and NF-κB in the injured kidney. Furthermore, ACY-1215 was effective in inhibiting dedifferentiation of renal fibroblasts to myofibroblasts and the fibrotic change of renal tubular epithelial cells in culture. Collectively, these results indicate that HDAC6 inhibition can attenuate development of renal fibrosis by suppression of transforming growth factor-β1 and EGFR signaling and suggest that HDAC6 would be a potential therapeutic target for the treatment of renal fibrosis.

INTRODUCTION

Chronic kidney disease is a major public health problem, affecting nearly 10% of the world’s population (24). Tubular interstitial fibrosis is considered to be the most common pathway leading to end-stage renal disease (23). The pathogenesis of renal fibrosis is characterized by renal interstitial fibroblast activation and abnormal accumulation of the extracellular matrix (ECM) (23, 24). So far, there are no effective approaches to prevent and halt the progression of chronic kidney disease to end-stage renal disease. Understanding the molecular basis of renal fibrosis will aid in the development of therapeutic strategies to treat kidney diseases.

Renal fibrosis is a complicated process associated with the activation of multiple signaling pathways and numerous genes. Transforming growth factor-β (TGF-β)/Smad signaling is considered the key regulator in renal fibrosis. Upon TGF-β1 binding to the TGF-β receptor, Smad3 is recruited and phosphorylated. Phosphorylated Smad3 is translocated to the nucleus, where it drives the expression of profibrotic genes like collagen type I (12, 24). Smad7 counters the activation of TGF-β receptor and Smad3 to inhibit renal fibrosis (24). In addition to TGF-β/Smad signaling, the epidermal growth factor receptor (EGFR) is also a critical mediator of profibrotic signals initiated by its ligands and other biological substances (14). The activation of EGFR by substances other than its own ligands is called transactivation, which mediates the profibrotic responses induced by many cytokines and vascular substances such as TGF-β1, angiotensin II, and endothelin (19). Activation of EGFR and other cellular membrane receptors can induce phosphorylation of multiple intracellular signaling molecules such as AKT, STAT3, and NF-κB, which act as mediators in gene expression (14, 19).

Increasing evidence indicates that epigenetic modification plays an important role in the regulation of gene expression (33, 34). Among several types of epigenetic modifications, histone acetylation has been widely studied. Histone acetylation is positively regulated by histone acetyltransferases and negatively regulated by histone deacetylases (HDACs) (33). At present, 18 histone deacetylases (HDACs) have been identified in mammals and divided into four categories: class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), class II HDACs, subdivided into class IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and class IIb (HDAC6 and HDAC10), class III HDACs (sirtuin 1–7), and class IV HDACs (HDAC11). Unlike other HDAC isoforms, whose deletion in mice leads either to death in utero or severe developmental defects, HDAC6 can be deleted in mice, which still develop normally without major organ dysfunction. This unique feature of HDAC6 may have important implications for the safety of potential therapeutic inhibition of HDAC6.

In the past several years, HDAC6 inhibitors have been developed and used in preclinical and clinical studies. Specific HDAC6 inhibitors have shown anticancer properties in several tumors including multiple myeloma (3), chronic lymphocytic leukemia (3), and acute myeloid leukemia (13). Tubastatin A was also effective in improving polycystic kidney disease (17), hypertensive nephropathy (7), acute kidney injury (30, 32), and peritoneal fibrosis (38) in animal models. However, tubastatin A may have limited success in clinical trials because of its poor pharmacokinetic properties and potential genotoxicity (4, 38). Recently, other HDAC6 inhibitors have been developed, and ACY-1215 (ricolinostat) and ACY-241 have reached clinical trial to treat tumors (27, 35). Studies have shown that ACY-1215 is a potent and selective HDAC6 inhibitor with an IC₅₀ value of 5 nM (29) and can attenuate several diseases, including neurodegenerative diseases, acute liver injury, and tumor diseases in animal models (36, 39–41). However, ACY-1215 has not been studied to treat renal fibrosis yet.

In this study, we assessed the effect of ACY-1215 on renal fibrosis and the mechanism involved in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO) to provide evidence for future clinical trials in chronic fibrotic kidney disease.

MATERIALS AND METHODS

Chemical and antibodies.

ACY-1215 (ricolinostat) and aristolochic acid were purchased from Selleckchem (Houston, TX). Antibodies to Smad3, phosporylated (p-)Smad3, Smad7, acetyl-H3, acetyl-α-tubulin, GAPDH, EGFR, p-EGFR, p-NF-κB, NF-κB, p-STAT3, STAT3, p-AKT, and AKT were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies to collagen type III were purchased from Servicebio (Wuhan, China). Antibodies to α-smooth muscle actin (α-SMA), fibronectin, and HDAC6 were purchased from Absin Bioscience (Shanghai, China).

Cell culture and treatments.

Rat renal interstitial fibroblasts (NRK-49F) were obtained from the American Type Culture Collection (Manassas, VA). Murine renal tubular epithelial cells (mTECs) were a gift from Dr. Jeffrey B. Kopp (National Institutes of Health, Bethesda, MD), which were shown to be of proximal tubular origin by a combination of morphological, biochemical, and transport characteristics (15). NRK-49F cells and mTECs were cultured in DMEM containing 5% FBS and 1% penicillin in an atmosphere of 5% CO₂ and 95% air at 37°C. To determine the effect of HDAC6 inhibition on renal fibroblast activation induced by serum, ACY-1215 was directly cultured on NRK-49F cells with 5% FBS at different concentrations. To determine the effect of HDAC6 inhibition on TGF-β1-induced renal fibroblast activation, NRK-49F cells cultured with DMEM containing 5% FBS were exposed to TGF-β1 (5 ng/mL) for 36 h in the presence or absence of ACY-1215. To determine the effect of HDAC6 inhibition on the expression of TGF-β1 and fibrotic responses to injury, mTECs were cultured for 24 h in DMEM without FBS and then exposed to TGF-β1 (5 ng/mL) or aristolochic acid (10 μM) or for an additional 24 h before cells were harvested for immunoblot analysis.

UUO model and ACY-1215 treatment.

The UUO model was established in male C57BL/6J mice that weighed 20–25 g (Shanghai SLAC Laboratory Animal Company), as described in our previous study (26). Briefly, the abdominal cavity was exposed via a midline incision, and the left ureter was isolated and ligated. The contralateral kidney was used as a control. To examine the effects of ACY-1215 on renal fibrosis after UUO injury, 25 mg/kg ACY-1215 in 50 µL DMSO was intraperitoneally administered immediately and then given every day at the same dose for 6 days. Selection of this dose of 25 mg/kg was based on a previous report (42). For the UUO-alone group, mice were injected with an equivalent amount of DMSO. Five mice were used in each group. Animals were euthanized, and the kidneys were removed at day 7 for protein analysis and histological examination. All experiments were conducted in accordance with the animal experimentation guidelines of Tongji University School of Medicine.

Immunoblot analysis.

Immunoblot analysis of kidney tissue samples was conducted as previously described (26). Densitometry analysis of immunoblot results was conducted using ImageJ software (National Institutes of Health, Bethesda, MD).

Immunofluorescent and histochemical staining.

Immunofluorescent and immunohistochemical staining was performed according to the procedures described in our previous study (21). Renal tissue was fixed in 4.5% buffered formalin, dehydrated, and embedded in paraffin. For immunofluorescent staining, primary antibodies and fluorescent-conjugated secondary antibodies were applied to the sections. For assessment of renal fibrosis, Masson trichrome staining was performed according to the protocol provided by the manufacturer (Sigma, St. Louis, MO). The collagen tissue area (blue color) was quantitatively measured using Image Pro-Plus software (Media-Cybernetics, Silver Spring, MD) by drawing a line around the perimeter of positive staining area. The average ratio to each microscopic field (×200) was calculated and graphed.

Statistical analysis.

All experiments were conducted at least three times. Data depicted in graphs represent means ± SD for each group. Intergroup comparison was made using one-way ANOVA. Multiple means were compared using Tukey’s test. Differences between two groups were determined by Student’s t test. The statistically significant difference between mean values was marked in each graph. P < 0.05 was considered significant. Statistical analyses were conducted using IBM SPSS Statistics 20.0 (Beijing, China).

RESULTS

Administration of ACY-1215 inhibits HDAC6 expression and renal fibrosis in a murine model induced by UUO.

To demonstrate the role of HDAC6 in renal fibrosis, we established a murine model of renal fibrosis induced by UUO and then administered ACY-1215, a highly selective HDAC6 inhibitor (28), immediately after UUO. At 7 days after injection, we collected the kidney tissue to analyze renal fibrosis by Masson staining. As shown in Fig. 1A, UUO injury resulted in renal fibrosis (blue area), which was significantly attenuated by administration of ACY-1215, but no renal fibrosis was seen in sham-operated mice with and without drug treatment (Fig. 1, A and B). In parallel with the fibrotic changes, expression levels of HDAC6 were upregulated in the kidney after injury, and ACY-1215 treatment reduced this response (Fig. 1, C and D). In contrast, ACY-1215 increased expression levels of acetyl-histone H3 and acetyl α-tubulin in sham-operated and injured kidneys, indicating the effectiveness of this inhibitor (Fig. 1, C–F). These data suggest that HDAC6 can induce acetylation of proteins located in both the nucleus and cytosol of kidney cells after UUO injury but can only induce acetylation of its substrates in the cytosol of sham-operated kidneys.

Fig. 1.

Inhibition of histone deacetylase 6 (HDAC6) with ACY-1215 attenuates renal fibrosis. Mice were subjected to unilateral ureteral obstruction (UUO) and treated daily with ACY-1215 for 7 days before samples were harvested analysis. A: photomicrographs illustrating Masson trichrome staining of kidney tissue. B: the percentage of Masson trichrome-positive tubulointerstitial area (blue) relative to the whole area was quantified (original magnification: ×200). Scale bar = 50 μm. C: kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against HDAC6, acetyl-H3, acetyl-α-tubulin, or GAPDH. D–F: protein expression levels of HDAC6 (D), acetyl-H3 (E), or acetyl-α-tubulin (F) were qualified by densitometry and normalized with GAPDH. Values are means ± SD of at least three independent experiments. Bars with different letters (a–c) for each molecule were significantly different from one other (P < 0.05).

HDAC6 is expressed in renal tubules in the UUO model.

To examine the distribution of HDAC6 in the injured kidney, we conducted immunofluorescent staining. Figure 2 shows that the expression of HDAC6 in the UUO group was significantly higher than that in the sham group, and HDAC6 was mainly expressed in the cytoplasm of the renal tubules. Expression of α-SMA in the kidney of the UUO model was also significantly increased compared with the sham group, but HDAC6 rarely costained with α-SMA. Since α-SMA is mainly expressed in myofibroblasts and HDAC6 is expressed in renal tubular epithelial cells, these results suggested that HDAC6 may act in renal tubular cells to mediate the development of renal fibrosis after UUO injury.

Fig. 2.

Expression of histone deacetylase 6 (HDAC6) in the kidney. Mice were subjected to unilateral ureteral obstruction (UUO) and treated daily with ACY-1215 for 7 days before samples were harvested for analysis. Photomicrographs show protein expression of HDAC6 (red) and α-smooth muscle actin (α-SMA; green) after immunofluorescent costaining and counterstaining with DAPI (blue) (original magnification: ×400). In the injured kidney, HDAC6 was most abundant in the cytoplasm of renal tubular cells but was also observed in the nucleus of this cell type. Scale bar = 50 μm.

Inhibition of HDAC6 reduces fibroblast activation and ECM deposition in renal fibrosis induced by UUO.

Deposition of excessive ECM and renal myofibroblast activation are two major pathological processes of renal fibrosis (23). To investigate the effect of ACY-1215 in the UUO model, we examined, by immunoblot analysis, the expression of α-SMA, a hallmark of myofibroblasts (active fibroblasts) as well as the expression of ECM proteins collagen type III and fibronectin. As shown in Fig. 3, A–D, α-SMA, collagen type III, and fibronectin were detected in sham-operated kidneys with and without ACY-1215 administration; their expression levels were dramatically increased, however, in the kidneys of mice subjected to UUO. Administration of ACY-1215 largely blocked UUO-induced α-SMA, collagen type III, and fibronectin expression. These results suggest that pharmacological targeting of HDAC6 can prevent the development of renal fibrosis and inhibit the differentiation of renal interstitial fibroblasts into myofibroblasts.

Fig. 3.

Inhibition of histone deacetylase 6 (HDAC6) with ACY-1215 reduces renal fibroblast activation and extracellular matrix (ECM) protein deposition in the renal interstitium. Mice were subjected to unilateral ureteral obstruction (UUO) and treated daily with ACY-1215 for 7 days before samples were harvested for analysis. A: whole kidney tissue lysates from obstructed (UUO) and contralateral nonobstructed (sham) ureters were processed for immunoblot analysis with antibodies specific to α-smooth muscle actin (α-SMA), fibronectin, collagen type III, and GAPDH. Expression levels of α-SMA (B), fibronectin (C), and collagen type III (D) were qualified by densitometry and normalized with GAPDH. Values are means ± SD of at least three independent experiments. Bars with different letters (a–c) for each molecule were significantly different from one other (P < 0.05).

HDAC6 is required for activation of the TGF-β/Smad3 signaling pathway in the kidney after UUO injury.

The TGF-β1 signaling pathway plays a predominant role in promoting development of renal interstitial fibrosis (12). To explore whether HDAC6 is involved in the activation of the TGF-β1/Smad signaling pathway, we examined the effect of ACY-1215 on the phosphorylation of Smad3 (p-Smad3) and expression of TGF-β1 and Smad7 in UUO-injured kidneys. As shown in Fig. 4, A–D, a small amount of TGF-β1 was expressed in sham-operated kidneys; it was increased after UUO. p-Smad3 is minimally detectable in normal kidneys, but UUO damage significantly increased its phosphorylation. Smad7 is abundantly expressed in normal kidneys, but its levels declined in UUO injured kidneys. ACY-1215 treatment significantly reduced TGF-β1 expression and Smad3 phosphorylation while partially restored Smad7 expression in the injured kidney. These results show that ACY-1215 may alleviate renal fibrosis by inhibiting the TGF-β1/Smad3 signaling pathway through a mechanism associated with preservation of Smad7 expression.

Fig. 4.

Histone deacetylase 6 (HDAC6) blockade inhibits unilateral ureteral obstruction (UUO)-induced activation of transforming growth factor-β (TGF-β)/Smad3 signaling in the kidney. Mice were subjected to UUO and treated daily with ACY-1215 for 7 days before samples were harvested for analysis. A: Whole kidney tissue lysates from obstructed (UUO) and contralateral nonobstructed (sham) were processed for immunoblot analysis with antibodies specific to TGF-β1, phosphorylated (p-)Smad3, Smad3, Smad7, and GAPDH. Expression levels of TGF-β1 (B), p-Smad3 (C), Smda3 (D), and Smad7 (E) were qualified by densitometry and normalized with GAPDH. Values are means ± SD of at least three independent experiments. Bars with different letters (a–d) for each molecule were significantly different from one other (P < 0.05).

Inhibition of HDAC6 suppresses phosphorylation of EGFR and AKT in the kidney after UUO injury.

Activation of the EGFR/AKT signaling pathway promotes the progression of renal fibrosis. In neuronal cells, HDAC6 is involved in the activation of the AKT signaling pathway (44). As shown in Fig. 5, A and B, the expression of p-EGFR in the injured kidney was increased, but largely suppressed by ACY-1215, while the expression level of total EGFR remained the same in all groups. Corresponding to this observation, AKT phosphorylation also increased in the injured kidney, while ACY-1215 treatment reduced this response. The expression level of total AKT was the same in the injured kidney and in the control kidney. Thus, these data suggest that HDAC6 may also contribute to renal fibrosis by activation of the EGFR/AKT signaling pathway.

Fig. 5.

Histone deacetylase 6 (HDAC6) blockade inhibits unilateral ureteral obstruction (UUO)-induced activation of the epidermal growth factor receptor (EGFR)/AKT signaling pathway in the kidney. Mice were subjected to UUO and treated daily with ACY-1215 for 7 days before samples were harvested for analysis. A: whole kidney tissue lysates from obstructed (UUO) and contralateral nonobstructed ureters (sham) were processed for immunoblot analysis with antibodies specific to phosphorylated (p-)EGFR, EGFR, p-AKT, and AKT. B: p-EGFR expression levels were qualified by densitometry and normalized to EGFR. C: p-AKT expression levels were qualified by densitometry and normalized to AKT. Values are means ± SD of at least three independent experiments. Bars with different letters (a–c) for each molecule were significantly different from one other (P < 0.05).

Blockade of HDAC6 inhibits activation of NF-κB and STAT3 signaling pathway in the kidney after UUO injury.

NF-κB is a key transcription factor involved in the inflammatory response. Its activation can trigger the release of various inflammatory factors. Activation of the STAT3 signaling pathway is also related to the inflammatory response (25). To investigate the effect of the NF-κB and STAT3 signaling pathway on renal fibrosis, we examined the protein expression of p-NF-κB (p65), NF-κB (p65), p-STAT3, and STAT3. Figure 6, A–C, shows that in the injured kidney, p-NF/κB (p65) and p-STAT3 increased; administration of ACY-1215 significantly decreased phosphorylation of NF-κB (p65) and STAT3 but did not affect expression of their total levels. Taken together, these results indicate that blockade of HDAC6 partially inhibits activation of NF-κB and STAT3 signaling pathways in the kidney after UUO injury.

Fig. 6.

Histone deacetylase 6 (HDAC6) blockade inhibits unilateral ureteral obstruction (UUO)-induced activation of the STAT3/NF-κB (p65) signaling pathway in the kidney. Mice were subjected to UUO and treated daily with ACY-1215 for 7 days before samples were harvested for analysis. A: whole kidney tissue lysates from obstructed (UUO) and contralateral nonobstructed (sham) groups were processed for immunoblot analysis with antibodies specific to phosphorylated (p-)NF-κB (p65), NF-κB (p65), p-STAT3, STAT3, and GAPDH. B: p-NF-κB (p65) expression levels were qualified by densitometry and normalized to NF-κB (p65). C: p-STAT3 expression levels were qualified by densitometry and normalized to STAT3. Values are means ± SD of at least three independent experiments. Bars with different letters (a–d) for each molecule were significantly different from one another (P < 0.05).

ACY-1215 inhibits activation of renal interstitial fibroblasts in culture.

It has been previously reported that HDAC6 is expressed in fibroblasts (43). To understand whether HDAC6 mediates renal fibroblast activation, we examined the effect of ACY-1215 on the expression of α-SMA in renal interstitial fibroblasts (NRK-49F) cultured with 5% FBS. We demonstrated that ACY-1215 reduced the expression of α-SMA in a dose-dependent manner with a maximum at 50 μM (Fig. 7, A and B). As the concentration of ACY-1215 increased, the expression level of HDAC6 gradually decreased. In contrast, the expression level of acetylated histone 3 was gradually increased with increasing doses of ACY-1215, indicative of its effective inhibition of HDAC6. These inhibitory effects were not significantly different at 25 and 50 μM (Fig. 7, A, C, and D). We thus suggest that HDAC6 mediates dedifferentiation of renal fibroblasts to myofibroblasts. Given that 25 μM ACY-1215 reached the maximum inhibitory effect on HDAC6 expression and activation, this dose of ACY-1215 was used in the following in vitro experiments.

Fig. 7.

Inhibition of histone deacetylase 6 (HDAC6) with ACY-1215 reduces activation of renal interstitial fibroblasts in cultured NRK-49F cells. NRK-49F were cultured with 5% FBS and treated with various concentrations of ACY-1215 (0–50 μM) for 36 h. A: Western blot analysis of cell lysates with various antibodies as indicated. Expression levels of α-smooth muscle actin (α-SMA; B), HDAC6 (C), and acetyl-H3 (D) were qualified by densitometry and normalized with GAPDH. Values shown in the graph are means ± SD of at least three independent experiments. Each letter (a–d) indicates that different bars were significantly different from each other (P < 0.05).

ACY-1215 inhibits TGF-β1-induced activation of renal interstitial fibroblasts.

TGF-β1 is a major cytokine/growth factor, and serum is a mixture of growth factors, both of which can induce renal interstitial fibroblast activation and renal fibrosis. As such, we asked whether TGF-β1 would further stimulate activation of renal fibroblasts in the presence of 5% serum and whether ACY-1215 would affect activation of renal fibroblasts. To do this, we added TGF-β1 (5 ng/mL) to the culture of NRK-49F cells with 5% FBS in the presence or absence of 25 μM ACY-1215 and then continued culturing for 36 h. The collected cell lysates were subjected to immunoblot analysis. Figure 8, A–G, shows that compared with the control group (5% FBS), TGF-β1 addition further increased the expression level of α-SMA, which was accompanied by a slight increase of HDAC6. Treatment with ACY-1215 suppressed the expression of α-SMA and HDAC6 in cells treated with and without TGF-β1, which was coincident with the increased expression of acetylated histone 3. These data suggest that combined treatment with serum and TGF-β1 has an additive effect on renal fibroblast activation and that HDAC6 also mediates this process.

Fig. 8.

Inhibition of histone deacetylase 6 (HDAC6) with ACY-1215 reduces activation of renal interstitial fibroblasts in cultured NRK-49F cells. Normally cultured NRK-49F was exposed to 5 ng/mL transforming growth factor-β1 (TGF-β1) and then cultured for 36 h in the absence or presence of ACY-1215 (25 μM). A and E: Western blot analysis of cell lysates with various antibodies as indicated. Protein expression levels of fibronectin (B), collagen type III (C), α-smooth muscle actin (α-SMA; D), HDAC6 (F), and acetyl-H3 (G) were qualified by densitometry and normalized with GAPDH. Values shown in the graphs are means ± SD of at least three independent experiments. Each letter (a–c) indicates that different bars were significantly different from each other (P < 0.05).

ACY-1215 inhibits TGF-β1-induced profibrotic phenotype changes of cultured renal epithelial cells.

It has been previously reported that upon injury or stimulation with growth factors or cytokines such as TGF-β1, renal tubular epithelial cells display a profibrotic phenotype that expresses α-SMA and ECM proteins (10, 23). Given that HDAC6 is highly expressed in renal tubular epithelial cells, we further examined the effect of ACY-1215 on the transition of renal epithelial cells to a profibrotic phenotype by examining expression of α-SMA, fibronectin, and collagen type III in cultured mTECs. As shown in Fig. 9, A–D, basal levels of α-SMA, fibronectin, and collagen type III were detected in mTECs, and ACY-1215 treatment did not significantly alter their expression. Exposure of cells to TGF-β1 resulted in increased expression of these three proteins, and the presence of ACY-1215 markedly reduced their expression; this was coincident with the downregulation of HDAC6 and upregulation of acetyl-histone H3 (Fig. 9, E–G). These data suggest that HDAC6 also mediates TGF-β1-induced profibrotic phenotype changes of renal epithelial cells.

Fig. 9.

ACY-1215 inhibits profibrotic phenotype changes of renal epithelial cells. Serum-starved murine renal tubular epithelial cells (mTECs) were treated with transforming growth factor-β1 (TGF-β1; 5 ng/mL) in the presence or absence of ACY-1215 (25 μM) for 24 h and then harvested. A and E: Western blot analysis of cell lysates with various antibodies as indicated. Protein expression levels of fibronectin (B), collagen type III (C), α-smooth muscle actin (α-SMA; D), histone deacetylase 6 (HDAC6; F), and acetyl-histone H3 (G) were qualified by densitometry and normalized with GAPDH. Values shown in the graphs are means ± SD of at least three independent experiments. Each letter (a–c) indicates that different bars were significantly different from each other (P < 0.05).

ACY-1215 inhibits expression of TGF-β1 in the kidney after UUO and in cultured renal epithelial cells after aristolochic acid exposure.

Recent research has demonstrated that the injury-induced profibrotic phenotype of renal tubular cells acquires the ability to produce a variety of profibrotic factors and cytokines, including TGF-β1 (10, 23). Given that ACY-1215 treatment reduced the expression of TGF-β1, as shown in Fig. 4, A and B, we proceeded to examine specifically whether HDAC6 would mediate the expression of TGF-β1 in renal epithelial cells. Immunohistochemical staining indicated that TGF-β1 was abundantly expressed in renal tubular cells of UUO-injured kidneys and significantly declined after treatment with ACY-1215 (Fig. 10, A and B). Notably, TGF-β1 was minimally expressed in this cell type in sham-operated kidneys. Similar to this observation, TGF-β1 expression levels were also increased in mTECs upon exposure to aristolochic acid compared with the control culture; ACY-1215 treatment also reduced this response (Fig. 10, C and D). As expected, ACY-1215 was effective in the inactivation of HDAC6 as indicated by the increased expression of acetyl-histone H3 (Fig. 10, C and E). This inhibitor also slightly reduced HDAC6 expression (Fig. 10, C and F). On this basis, we suggest that HDAC6 contributes to the expression and production of TGF-β1 in renal epithelial cells after injury.

Fig. 10.

ACY-1215 inhibits expression of transforming growth factor-β1 (TGF-β1) in the kidney after unilateral ureteral obstruction (UUO) and in cultured renal tubular epithelial cells after aristolochic acid (AA) exposure. Mice were subjected to UUO and daily treatment with ACY-1215 for 7 days before samples were harvested for analysis. A: photomicrographs illustrating TGF-β1 staining of kidney tissue. B: the percentage of TGF-β1-positive area (yellow) relative to the whole area was quantified (original magnification: ×200). Scale bar = 100 μm. C: murine renal tubular epithelial cells were treated as indicated in MATERIALS AND METHODS. The prepared cell lysates were subjected to immunoblot analysis using antibodies against TGF-β1, acetyl-histone H3, or histone deacetylase 6 (HDAC6). Protein expression levels of TGF-β1 (D), acetyl histone H3 (E), or HDAC6 (F) were qualified by densitometry and normalized with GAPDH. Values shown in the graphs are means ± SD of at least three independent experiments. Each letter (a–c) indicates that different bars were significantly different from each other (P < 0.05).

DISCUSSION

Our recent studies have demonstrated that HDAC6 plays a critical role in acute kidney injury (30, 32) and peritoneal fibrosis (38) in animal models. In the current study, we found that inhibition of HDAC6 with ACY-1215 also reduced the accumulation of ECM components and inhibited TGF-β/Smad3 and EGFR, two key signaling pathways associated with fibrosis in the kidney after UUO injury. Moreover, ACY-1215 was effective in inhibiting activation of renal interstitial fibroblasts in culture. These data indicate that HDAC6 is a critical mediator in renal fibrosis and suggest that pharmacological inactivation of HDAC6 could offer therapeutic effects for renal fibrosis.

Unlike most HDAC isoforms, which are located in the nucleus, HDAC6 contains a cytoplasmic retention signal and a nuclear localization signal (2). This structural feature enables it to shuttle between the nucleus and the cytoplasm and deacetylate proteins in both the nucleus (i.e., histone H3) and cytoplasm (i.e., α-tubulin) (17). In this study, we observed that UUO injury resulted in increased expression of renal HDAC6, which was mainly expressed in the cytosol of renal tubular cells in the injured kidney. This suggests that profibrotic actions of HDAC6 may be primarily initiated in renal epithelial cells. Although it remains controversial whether renal epithelial cells become renal fibroblasts through a complete or partial process of epithelial-mesenchymal transition (EMT), recent studies have shown that partial EMT can occur in tubular epithelial cells that then arrests at the G₂/M phase of the cell cycle (10, 23). This type of cells acquires the ability to produce profibrotic factors leading to renal fibrosis (10, 23). In support of this hypothesis, the present study found that blockade of HDAC6 with ACY-1215 inhibited the transition of renal tubular epithelial cells to a profibrotic phenotype in cultured mTECs and injury-induced expression of TGF-β1 in renal epithelial cells in vitro and in vivo. Our previous study also demonstrated that HDAC6 is involved in the EMT response of peritoneal mesothelial cells (38). Moreover, Gu et al. have shown that HDAC6 activation is essential for the induction of EMT in lung cancer cell lines (A549) and breast epithelial cells (11). Nevertheless, we cannot exclude the possibility that HDAC6 may also contribute to renal fibrosis through direct activation of renal interstitial fibroblasts. This is evident by our observations that increased HDAC6 in cultured renal interstitial fibroblasts exposed to serum and TGF-β1 and inhibition of HDAC6 significantly reduced the expression levels of α-SMA, a hallmark of myofibroblasts in vivo and in vitro.

The mechanisms by which HDAC6 mediates renal fibrosis remain elusive but may be associated with the activation of TGF-β1/Smad3 signaling. In the UUO-injured kidney, treatment with ACY-1215 reduced TGF-β1 expression and Smad3 phosphorylation levels, suggesting that HDAC6 is required for the activation of TGF-β1/Smad3 signaling. How HDAC6 promotes Smad3 activation remains unclear, but it may be related to the regulation of Smad7. As Smad7 is a negative regulator of the TGF-β1/Smad3 pathway, its downregulation can reciprocally promote the recruitment of Smad3 to phosphorylated TGF-β1 receptor to induce its phosphorylation (8). Thus, HDAC6-induced upregulation of Smad7 may counteract the action of TGF-β1/Smad3. Indeed, our results show that UUO injury increased the expression level of TGF-β1 and p-Smad3 and reduced Smad7 expression, whereas administration of ACY-1215 significantly inhibited the expression of TGF-β1 and p-Smad3 while partially restoring Smad7 expression. Similarly, ACY-1215 also effectively reduced the expression of TGF-β1 in cultured renal epithelial cells stimulated by aristolochic acid. Since HDAC6 is mainly distributed in the cytoplasm and can acetylate many cytoplasmic proteins (16, 29), it is also possible that HDAC6 may directly modify Smad3 and then change its phosphorylation levels. Further work is needed to test this hypothesis.

HDAC6 may also attenuate renal fibrosis by inhibiting the EGFR signaling pathway. Increasingly, studies have revealed that activation of the EGFR signaling pathway not only regulates kidney development and regeneration but that the pathway also participates in chronic kidney disease caused by different etiologies such as diabetic nephropathy (18), uric acid nephropathy (22), and obstructive nephropathy (20). The primary pathological change of these various forms of kidney disease is renal interstitial fibrosis. Our previous research indicates that the fibrotic kidney contains persistently high expression of p-EGFR (31), suggesting excessive activation of EGFR. Excessive activation of EGFR signaling can promote the expression of TGF-β1, Smad3 activation, epithelial cell arrest in the G₂/M stage of the cell cycle, and inflammatory cytokine release (37). In addition, EGFR also plays a key role in mediating angiotensin II-induced renal fibrosis (5). Therefore, EGFR can be used as a convergent point of signaling pathways to promote the occurrence and development of fibrosis (37). In this study, we found that blockade of HDAC6 significantly reduced levels of UUO-induced phosphorylated EGFR and also inhibited the phosphorylation of its downstream signaling protein molecule AKT. As such, HDAC6 may also promote renal fibrosis by regulating activation of the EGFR signaling pathway.

HDAC6 activation may also be required for the inflammatory response during the process of fibrogenesis in the kidney. Renal inflammation is characterized by the expression of cytokines/chemokines and macrophage infiltration, and STAT3 and NF-κB are two major transcription factors involved in promoting the release of proinflammatory cytokines and chemokines (1, 6). We found that targeted inhibition of HDAC6 significantly reduced the phosphorylation level of STAT3 and NF-κB (p65), suggesting that HDAC6 intervention can reduce the expression of various inflammatory cytokines/chemokines by inhibiting STAT3 and NF-κB and other inflammation-related transcription factors, thus alleviating renal fibrosis. In line with this speculation, our recent study revealed that dephosphorylation of STAT3 and NF-κB as a result of HDAC6 inhibition is coincident with the suppression of multiple proinflammatory cytokines (38).

HDACs are overexpressed in many kidney diseases, in particular renal fibrosis, and are thus proposed as promising therapeutic targets. Although pan HDAC inhibitors have shown excellent efficacy in the treatment of many forms of kidney diseases, including diabetic nephropathy, polycystic kidney disease, and lupus nephritis (17), their significant adverse effects largely limited their clinical application in chronic indications. On this basis, development of HDAC isoform-selective inhibitors may have more clinical value than pan HDAC inhibitors. Only HDAC6 knockout mice develop normally and have no life-limiting defects, suggesting that HDAC6 inhibitors could exert therapeutic effects with no apparent toxicity (43). Currently, ACY-1215 is undergoing phase I/II clinical evaluation for the treatment of multiple myeloma and lymphoid malignancies (9). Our results showed that ACY-1215 effectively reduced renal fibrosis, thus providing a theoretical basis for future clinical trials of that HDAC6 inhibitor to prevent and treat renal fibrosis.

In conclusion, we used HDAC6 inhibitors for the first time to successfully alleviate the occurrence and development of renal fibrosis. The antifibrotic effects of HDAC6 inhibition are related to the inactivation of TGF-β1/Smad3, EGFR/AKT, NF-κB, and STAT3 signaling pathways. These results provide evidence that HDAC6 could be a feasible target for the prevention and treatment of renal fibrosis.

GRANTS

This work was supported by the National Natural Science Foundation of China [Grants 81670623 and 81830021 (to S.Z.)], National Key R&D Program of China [Grant 2018YFA0108802 (to S.Z.)], and National Institute of Diabetes and Digestive and Kidney Diseases [Grant 2R01DK08506505A1 (to S.Z.)].

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

X.C. and S.Z. conceived and designed research; X.C., C.Y., X.H., J.L., and T.L. performed experiments; X.C. analyzed data; X.C., A.Q., and N.L. interpreted results of experiments; X.C. prepared figures; X.C. and S.Z. drafted manuscript; S.Z. edited and revised manuscript; X.C., C.Y., X.H., J.L., T.L., A.Q., N.L., and S.Z. approved final version of manuscript.