Cilomilast Ameliorates Renal Tubulointerstitial Fibrosis by Inhibiting the TGF-β1-Smad2/3 Signaling Pathway

Background: Renal tubulointerstitial fibrosis is the key pathological feature in chronic kidney diseases (CKDs) with no satisfactory therapies in clinic. Cilomilast is a second-generation, selective phosphodiesterase-4 inhibitor, but its role in renal tubulointerstitial fibrosis in CKD remains unclear. Material and Methods: Cilomilast was applied to the mice with unilateral ureteric obstruction (UUO) and renal fibroblast cells (NRK-49F) stimulated by TGF-β1. Renal tubulointerstitial fibrosis and inflammation after UUO or TGF-β1 stimulation were examined by histology, Western blotting, real-time PCR and immunohistochemistry. KIM-1 and NGAL were detected to evaluate tubular injury in UUO mice. Results: In vivo, immunohistochemistry and western blot data demonstrated that cilomilast treatment inhibited extracellular matrix deposition, profibrotic gene expression, and the inflammatory response. Furthermore, cilomilast prevented tubular injury in UUO mice, as manifested by reduced expression of KIM-1 and NGAL in the kidney. In vitro, cilomilast attenuated the activation of fibroblast cells stimulated by TGF-β1, as shown by the reduced expression of fibronectin, α-SMA, collagen I, and collagen III. Cilomilast also inhibited the activation of TGF-β1-Smad2/3 signaling in TGF-β1-treated fibroblast cells. Conclusion: The findings of this study suggest that cilomilast is protective against renal tubulointerstitial fibrosis in CKD, possibly through the inhibition of TGF-β1-Smad2/3 signaling, indicating the translational potential of this drug in treating CKD.


INTRODUCTION
Chronic kidney disease (CKD) has become a major public health problem in many countries. Almost all forms of chronic renal diseases can ultimately result in end-stage renal diseases (ESRDs), leading to a significant impact on quality of life as well as a substantial social burden (1). Renal tubulointerstitial fibrosis is characterized by the deposition of extracellular matrix (ECM), excessive accumulation of activated myofibroblasts, and infiltration of inflammatory cells (2,3). Unfortunately, no satisfactory therapeutic strategies for inhibiting or reversing renal tubulointerstitia fibrosis are clinically available. Therefore, there is an urgent need to find new effective therapeutic drugs for renal tubulointerstitial fibrosis.
Phosphodiesterase 4 (PDE4) isozymes belong to the PDE superfamily and selectively hydrolyse 3 ′ ,5 ′ -cyclic adenosine monophosphate (cAMP) with high affinity (4). In recent years, PDE4 inhibition has been applied to study its probable therapeutic value in the nervous system (5), respiratory system (6) and immune system (7). Furthermore, PDE4 inhibition has been found to attenuate lung fibrosis (8) and dermal fibrosis (9). Previous studies have shown that PDE4 is widely expressed in renal tubules (4). The inhibition of PDE4 has suppressive effect on tubular damage in acute renal failure (10)(11)(12). Recently, a report showed that one PDE4 inhibitor, rolipram, played an antifibrotic role in CKD possibly via acting on C/EBPβ and PGC-1α in tubular epithelial cells (13). Cilomilast is another PDE4 inhibitor that is currently being investigated in a phase III clinical trial for the treatment of chronic obstructive pulmonary disease (COPD). It has beneficial effects on COPD (14), tumors (15), acute lung injury (16), and acute kidney injury (12). Furthermore, cilomilast has been reported to attenuate pulmonary fibrosis (17). However, the effect of cilomilast on renal tubulointerstitial fibrosis has not been studied.
Transforming growth factor-β1 (TGF-β1) is an essential fibrogenic factor that plays a crucial role in the renal fibrotic process (18). Emerging evidence suggests that TGF-β1 initiates renal tubular epithelial cell transdifferentiation to myofibroblasts, enhancing collagen and fibronectin (FN) synthesis and extracellular matrix deposition (19)(20)(21). TGF-β1 receptor activation stimulates the translocation of decapentaplegic homolog 3 (Smad3) to the nucleus, where it regulates the transcription of target genes (22). Smad7 has an inhibitory effect on TGF-β1, Smad2, and Smad3 (23). The activation of TGFβ1-Smad2/3 signaling or the loss of inhibitory Smad7 triggers fibrotic cascades (24). In this study, we investigated the therapeutic effects of cilomilast on renal tubulointerstitial fibrosis using a mouse model of obstructive uropathy. Additionally, we further explored the antifibrotic action of cilomilast and its effect on regulating the TGFβ1-Smad2/3 pathway in renal fibroblasts. We found that cilomilast attenuated the development of renal tubulointerstitial fibrosis possibly by inhibiting the TGFβ1-Smad2/3 signaling pathway.

Histological Analysis
Kidney tissues were fixed in 4% PFA, embedded in paraffin, and cut into sections (4-µm-thick), which were stained with Masson's trichrome. Masson's trichrome staining was used to assess collagen deposition in the obstructed kidney tissue. Next, 8-10 randomly selected fields were observed under the microscope, and then, each mouse kidney tissue was evaluated in a blinded manner.

Immunofluorescence Staining
The cells were fixed with PBS containing 4% PFA for 30 min. After blocking with 5% BSA for 1 h, the slides were incubated overnight at 4 • C with an anti-FN antibody (#26836, CST, Danvers, MA, USA, 1:250), which was diluted with 5% BSA overnight at 4 • C. Subsequently, the cells were incubated with anti-rabbit secondary antibodies (ab150077, Abcam, Cambridge, MA, USA, 1:250) for 1 h at room temperature and were then stained with the nuclear-specific stain DAPI (Beyotime Institute of Biotechnology) for 3 min at room temperature. Then, the cells were washed three times in PBS and imaged. The slides were viewed with a Carl Zeiss LSM 5 PASCAL laser scanning confocal microscope.

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted from kidney cortexes and cells by TRIzol (Invitrogen, Carlsbad, CA) based on the manufacturer's protocol (28). We reverse transcribed total RNA (1 µg) into cDNA using a PrimeScript TM Reverse Transcriptase System. Quantitative real-time PCR was subsequently carried out with SYBR Green Master Mix (Vazyme) on a QuantStudio 3 Real-time PCR System (Applied Biosystems, Foster City, CA). The cycling programme consisted of a preliminary denaturation (95 • C for 10 min) followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min. Relative mRNA levels were normalized to the levels of GAPDH and calculated with the comparative threshold cycle ( Ct) method. The primer sequences are shown in Table 1.

ELISA for TGF-β1
Mouse TGF-β1 in kidney tissue homogenates was evaluated by an ELISA kit (E-EL-M0044c, Elascience, China) according to the manufacturer's protocol.

Statistical Analysis
All data are presented as the means ± standard errors of the mean (SEMs). Differences between 2 groups were analyzed using two-tailed Student's t-test and incorporated into GraphPad Prism 6 software (GraphPad Software). ANOVA was used for comparisons among multiple groups. P < 0.05 was considered significant.

Cilomilast Treatment Attenuates UUO-Induced Renal Tubulointerstitial Fibrosis
First, we used a UUO model to explore the effect of cilomilast treatment on renal tubulointerstitial fibrosis ( Figure 1A). Firstly, Masson's trichrome staining showed that cilomilast treatment led to a remarkable reduction in collagen deposition. In addition, HE staining showed that cilomilast significantly reduced tubule brush border disruption and tubular atrophy, indicating the attenuation of UUO-induced tubular injury ( Figure 1B). Then, western blot and qRT-PCR analysis confirmed the deceased expression of FN, α-SMA (a marker of myofibroblasts) and Col-I and a significant increase in protein levels of E-cadherin after cilomilast treatment (Figures 1C-E). Furthermore, by using immunohistochemistry, we further found cilomilast treatment markedly inhibited the UUO-induced upregulation of FN and α-SMA expression ( Figure 1F). These results demonstrated that cilomilast improved renal tubulointerstitial fibrosis.

Cilomilast Treatment Reduced TGF-β1 in the Kidneys of UUO Mice
TGF-β1 is involved in renal tubulointerstitial fibrosis and is produced in large quantities during renal fibrogenesis. Therefore, we examined whether the decreased fibrosis in obstructed kidneys in cilomilast-treated mice was associated with change of TGF-β1 production. By qRT-PCR assays, we found that the enhanced expression of TGF-β1 in UUO mice was significantly blunted after cilomilast treatment The data are presented as the mean ± SEM. Statistically significant differences were determined by one-way ANOVA. ## P < 0.01, #### P < 0.0001, *P < 0.05, ***P < 0.001, ****P < 0.0001. Representative images and quantitative assessment of the expression and distribution of NGAL in kidney tissues using immunohistochemical staining. Scale bar =20 µm. The data are presented as the mean ± SEM. Statistically significant differences were determined by two-way ANOVA. #### P < 0.0001, ****P < 0.0001. (Figure 2A). Furthermore, immunohistochemistry staining and ELISA also showed that the expression of TGF-β1 was significantly reduced in cilomilast-treated UUO mice (Figures 2B-D).

Cilomilast Treatment Suppressed Renal Tubular Injury Induced by UUO
To evaluate the extent of renal damage, the expression of KIM-1 and NGAL, both markers of tubular damage, was Representative images and quantitative assessment of the expression and distribution of F4/80 and TNF-α in kidney tissues using immunohistochemical staining. Scale bar = 20 µm. The data are presented as the mean ± SEM. Statistically significant differences were determined by two-way ANOVA. ## P < 0.01, #### P < 0.0001, *P < 0.05, ***P < 0.001, ****P < 0.0001. measured in the kidney. As shown in Figure 3, KIM-1 and NGAL expression was markedly increased in the UUO group according to western blot and qRT-PCR analyses, which was attenuated by cilomilast treatment (Figures 3A,B). The trend of NGAL change measured by immunohistochemistry was consistent with that of the protein and mRNA levels ( Figure 3C). These data suggested that the amelioration of tubular injury after cilomilast treatment could protect tubular integrity and attenuate subsequent pathological events.

Cilomilast Treatment Attenuated Renal Inflammation Induced by UUO
Inflammation plays a key role in the progression of renal tubulointerstitial fibrosis (29). Thus, we evaluated the inflammatory status (inflammatory cell infiltration and proinflammatory cytokine expression) in obstructed kidneys with or without cilomilast. As expected, the enhanced mRNA expression of Il-6, Il-18, Tnf-α, Icam-1, and Mcp-1 was significantly blunted after cilomilast treatment ( Figure 4A). Immunohistochemistry analysis also showed reductions in macrophage infiltration (F4/80 + ) and TNF-α expression in the obstructed kidney after cilomilast therapy (Figure 4B). These results revealed that cilomilast could ameliorate renal inflammation in UUO.

Cilomilast Treatment Reduced the TGF-β1-Induced Profibrotic Response and Inflammation in NRK-49F Cells
To examine the cytotoxicity of cilomilast, cell viability assay was performed in cultured NRK-49F cells using a CCK8 assay kit.
the expression of Fn, α-SMA, Col-I, and Col-III (Figures 5B-D). Immunofluorescence analysis demonstrated that the expression of FN was significantly reduced after treatment with cilomilast ( Figure 5E). Similarly, the inflammatory response induced by TGF-β1 was also attenuated by cilomilast treatment (Figure 5F). These data revealed an antifibrotic role of cilomilast via the inhibition of renal fibroblast activation.
Cilomilast Treatment Inhibited the Activation of TGF-β1-Smad2/3 Signaling TGF-β1-Smad2/3 signaling plays a critical role in renal interstitial fibrosis and inflammation (30). In vivo, we found cilomilast treatment inhibited the activation of Smad2/3 pathway in obstructed kidney tissues, as evidenced by decreased protein expression of TGF-β1, p-Smad2 and p-Smad3 and a significant increase in protein levels of Smad7 (Figures 6A,B). In vitro, western blot results showed that cilomilast could markedly decrease the expression of p-Smad2 and p-Smad3 in NRK-49F cells treated with TGF-β1 (Figures 6C,D). Furthermore, we used Smad2/3 inhibitor SB-431542 (1 µM) to explore whether cilomilast protected against TGF-β1-induced fibroblast activation through suppressing Smad2/3 pathway in this study. As shown in Figure 6E, when NRK-49F cells were pretreated with SB-431542 (1 µM), cilomilast failed to further ameliorate TGF-β1-induced fibroblast activation. Collectively, these results suggested that cilomilast may exert its antifibrotic effect by inhibiting the activation of TGF-β1-Smad2/3 signaling.

DISCUSSION
Renal tubulointerstitial fibrosis is the final outcome for all CKDs, leading to progression to end-stage renal failure (ESRD). To date, there are no effective therapeutic approaches in clinic, which results in a heavy socioeconomic burden. Thus, it is necessary to develop effective drugs for treating renal tubulointerstitial fibrosis. Based on the data from our study, cilomilast, a selective phosphodiesterase-4 inhibitor and phase III clinical drug, can remarkably reduce UUO-induced renal tubulointerstitial fibrosis and renal inflammation in vivo and in vitro. To the best of our knowledge, this is the first study to report that cilomilast may The data are presented as the mean ± SEM. Statistically significant differences were determined by one-way ANOVA. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant.
serve as a potent therapeutic agent for preventing the progression of renal tubulointerstitial fibrosis. Factors that contribute to CKD progression include parenchymal cell loss, chronic inflammation, fibrosis and reduced regenerative capacity of the kidney (31). PDE4, which is a member of the PDE family, has four subtypes (PDE4A-PDE4D), and PDE4B plays an important role in inflammation (32). Cilomilast has been reported to treat cisplatin nephrotoxicity by antagonizing inflammation (12). Inflammation can lead to the progression of renal tubulointerstitial fibrosis (29). In the present study, the mRNA expression of proinflammatory factors was significantly reduced after cilomilast treatment. Immunohistology results revealed that cilomilast dramatically decreased the F4/80 + and TNF-α expression in vivo. Meanwhile, we confirmed that cilomilast downregulated the expression of fibrotic markers. Thus, cilomilast, as a selective phosphodiesterase-4 inhibitor, could block the inflammation to relieve renal tubulointerstitial fibrosis to some extent. KIM-1 and NGAL are considered to be important markers for evaluating renal tubular damage (33,34). Recent studies indicated that the expression of KIM-1 and NGAL was significantly increased in UUO mice (35,36). Our results revealed that cilomilast reversed renal tubular injury, which could contribute to improved tubular integrity and reduced subsequent pathology, such as inflammation and fibrosis.
Excessive deposition of ECM components in the renal interstitium could result in renal tubulointerstitial fibrosis (37). Smad2/3 signaling is closely associated with renal tubulointerstitial fibrosis (38), which can promote the fibrotic response by directly facilitating the production of ECM through its binding to specific promoter regions of collagen genes and the inhibition of ECM degradation (39,40). TGF-β1 is a potent pathogenic factor of renal fibrosis, which could promote ECM production and renal tubulointerstitial fibrosis (41). The activation of TGF-β1 triggers the nuclear localization of Smad2/3 in tubular epithelial cells and fibroblasts (42). To exert its profibrotic role in kidney disease, TGF-β1 can act by stimulating Smad2/3 to positively or negatively regulate microRNAs (43,44). Inhibiting Smad2/3 phosphorylation during TGF-β1-mediated epithelial-mesenchymal transition attenuated kidney fibrosis (45). In the contrast, up-regulating Smad2/3related signaling pathway may enhance the progression of CKD (46). Indeed, Smad3-deficient mice were protected from UUOinduced renal tubulointerstitial fibrosis (47). In this study, we found that cilomilast could inhibit the expression of TGF-β1 induced by UUO and downregulated the phosphorylation levels of p-Smad2 and p-Smad3. The present study provided evidence that cilomilast has a protective effect on renal tubulointerstitial fibrosis possibly by downregulating the expression of TGF-β1, further inhibiting Smad2/3 phosphorylation.
In summary, we found that cilomilast remarkably attenuated renal tubulointerstitial fibrosis and inflammation in a CKD model of UUO. Cilomilast could decrease the expression of collagen, fibronectin and α-SMA, possibly by inhibiting TGF-β1-Smad2/3 pathway activation. Cilomilast is a selective phosphodiesterase-4 inhibitor that is currently in clinical trial for the treatment of COPD. Thus, the present study provided the rationale for further clinical trials to evaluate cilomilast in treating CKD.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Nanjing Medical University Institutional Animal Care and Use Committee (registration number: IACUC-1809017).

AUTHOR CONTRIBUTIONS
XY and ZJ designed the experiment. MX, SL, XY, and JW performed the experiments and data analyses. XY, ZJ, and MX drafted the manuscript. ZJ, XY, YZ, SH, WG, and AZ revised and approved the manuscript. All authors contributed to the article and approved the submitted version.