Ruxolitinib Alleviates Renal Interstitial Fibrosis in UUO Mice

Ruxolitinib is a selective inhibitor of Jak1/2. Downstream signaling pathways of Jak, such as Stat3 and Akt/mTOR, are overactivated and contribute to renal interstitial fibrosis. Therefore, we explored the effect of Ruxolitinib on this pathological process. Unilateral ureteral obstruction (UUO) models and TGF-β1-treated fibroblasts and renal tubular epithelial cells were adopted in this study. Ruxolitinib was administered to UUO mice and TGF-β1-treated cells. Kidneys from UUO mice with Ruxolitinib treatment displayed less tubular injuries compared with those without Ruxolitinib treatment. Ruxolitinib treatment suppressed fibroblast activation and extracellular matrix (ECM) production in UUO kidneys and TGF-β1-treated fibroblasts. Ruxolitinib treatment also blocked epithelial-mesenchymal transition (EMT) in UUO kidneys and TGF-β 1-treated renal tubular epithelial cells. Moreover, Ruxolitinib treatment alleviated UUO-induced inflammation, oxidative stress and apoptosis. Mechanistically, Ruxolitinib treatment attenuated activation of both Stat3 and Akt/mTOR/Yap pathways. In conclusion, Ruxolitinib treatment can ameliorate UUO-induced renal interstitial fibrosis, suggesting that Ruxolitinib may be potentially used to treat fibrotic kidney disease.


Introduction
Renal fibrosis is a hallmark of most forms of progressive renal disease [1]. The unilateral ureteral obstruction (UUO) method, involving ligation of the ureter, most commonly of the left one, has been widely adopted to establish animal models with interstitial fibrosis. The kidney of the ligated ureter is conventionally termed as the obstructed kidney, which exhibits several important events such as mechanical stretching, activation of reninangiotensin-aldosterone system (RAS), loss of renal epithelial cells, inflammation, oxidative stress, macrophage infiltration and fibroblast activation, together leading to the final fibrosis [2]. Histologically, the obstructed kidney is characterized by tubular dilation, interstitial expansion, loss of renal parenchyma, infiltration of inflammatory cells, and accumulation of extracellular matrix (ECM) [2]. Some molecular mechanisms have been identified involved in renal interstitial fibrosis. Signaling pathways such as Notch, Wnt and Hedgehog, which are crucial for kidney development, were shown overactivated and to play positive roles in UUO-induced fibrosis [3,4,5,6]. Moreover, Erk and Akt signaling pathways were revealed to have a pro-proliferative effect on myofibroblasts by affecting molecules involved in cell cycle such as c-Myc, cyclin D1 and p21 [7,8]. Recently, the mammalian target of rapamycin (mTOR) was found implicated in fibroblast proliferation and activation [9,10,11,12]. Activation of Stat3 signaling may be another mechanism underlying renal interstitial fibrosis. Expression of phosphorylated Stat3 was enhanced in both tubular epithelial cells and interstitial fibroblasts after UUO [13]. Furthermore,

Ivyspring
International Publisher blockade of Stat3 pathway was shown to inhibit fibroblast activation in obstructive nephropathy [14,15].
Ruxolitinib is a potent and selective inhibitor of Jak1/2. It has been approved for the treatment of myelofibrosis, a neoplasm characterized by bone marrow fibrosis, and in which Jak/Stat pathway is over-activated. Ruxolitinib ameliorates diseaserelated symptoms, improves health-related quality of life, and increases patient survival [16,17,18]. Notably, a phase 3 study demonstrated that 15.8% patients with Ruxolitinib treatment had improved fibrosis [19].
Based on the above findings, in the present study we explored the effect of Ruxolitinib on UUO-induced renal interstitial fibrosis. We found that Ruxolitinib treatment suppressed fibroblast activation and reduced ECM deposition. Mechanistically, Ruxolitinib treatment attenuated Stat3, Akt, and mTOR signaling pathways in both obstructed kidneys and in vitro activated fibroblasts.

Ruxolitinib alleviates renal damage
UUO was used to establish mouse models of obstructive nephropathy. After two weeks, PAS and Masson′s trichrome staining were used to evaluate renal damage and fibrosis. The obstructed kidneys from UUO mice without Ruxolitinib treatment (later called UUO kidneys) exhibited severe structural disorders, characterized by tubular dilation and atrophy, intratubular cast formation, inflammatory cell infiltration, and ECM deposition ( Figure 1A-D). However, kidneys from UUO mice with Ruxolitinib treatment displayed remarkably less tubular injuries and ECM deposition, indicating Ruxolitinib treatment alleviated UUO-induced renal damage ( Figure 1A-D).

Ruxolitinib ameliorates ECM deposition
To evaluate the effect of Ruxolitinib on UUO-induced fibrosis, we analyzed the deposition of several ECM including Collagen I, Collagen III and Fibronectin. Immunohistochemistry staining showed that UUO kidneys expressed much higher levels of Collagen I (Figure 2A  UUO kidneys ( Figure 2G-H). Furthermore, Western blot detection indicated that Ruxolitinib treatment suppressed UUO-induced metallopeptidase inhibitor 1 (Timp-1) upregulation ( Figure 2G-H), suggesting that Ruxolitinib treatment may not only reduce ECM production, but also promote ECM degradation.

Ruxolitinib blocks renal fibroblast activation
Fibroblast activation is crucial for renal interstitial fibrosis. Activated fibroblasts or myofibroblasts are sources of ECM. We assessed the effect of Ruxolitinib on fibroblast activation using UUO models and TGF-β1 -treated NRK-49F cells. Staining of α-SMA by Immunohistochemistry revealed much more activated fibroblasts in UUO kidneys compared with normal control. Ruxolitinib treatment notably blocked UUO-induced fibroblast activation, which was indicated as a decrease in α-SMA expression ( Figure 3A-B). This finding was further confirmed by Western blot detection ( Figure 3C-D). TGF-β1 was used to activate NRK-49F cells. MTT measurement showed that Ruxolitinib treatment suppressed TGF-β1 -induced NRK-49F cell proliferation ( Figure  3E). Furthermore, Ruxolitinib treatment down-regulated TGF-β1 -induced α-SMA expression ( Figure  3F-G). Consistently, Ruxolitinib treatment counteracted TGF-β1 -induced Collagen I and Fibronectin expression in NRK-49F cells ( Figure 3F-G).

Ruxolitinib suppresses renal tubular cell EMT
Epithelial-mesenchymal transition (EMT) endows tubular epithelial cells a mesenchymal phenotype, with increased migratory capacity and ECM production [2]. We next investigated the effect of Ruxolitinib on renal tubular cell EMT using UUO models and TGF-β1 -treated NRK-52E cells. Both Immunohistochemistry and Western blot detection showed that E-cadherin, an epithelial marker, was remarkable downregulated in UUO kidneys, while Ruxolitinib treatment effectively restored its expression ( Figure

Ruxolitinib inhibits inflammatory responses
Inflammation is a hallmark of obstructed kidney characterized by inflammatory cell infiltration and inflammatory cytokine production. We examined F4/80 expression to assess macrophage infiltration. As shown by Immunohistochemistry, F4/80 expression level is higher in UUO kidneys compared with normal control. Ruxolitinib treatment reduced F4/80 expression in UUO kidneys significantly, indicating macrophage infiltration was effectively inhibited ( Figure 5A-B). NFκB signaling was activated in UUO kidneys, whereas Ruxolitinib treatment suppressed p-65 phosphorylation ( Figure 5C-D). We further evaluated the effect of Ruxolitinib on the expression of several inflammatory cytokines. Real-time PCR detection revealed that Ruxolitinib treatment reduced levels of TNF-α, IL-1β, IL-6 and MCP-1/CCL2 mRNA ( Figure 5E-H).

Ruxolitinib reduces renal tubular epithelial cell apoptosis
Renal tubular epithelial cell apoptosis is an important event in obstructed kidney. TUNEL staining was used to determine cell apoptosis. It was shown that apoptotic cells increased in UUO kidneys. Ruxolitinib treatment ameliorated the situation ( Figure 6A-B). Cleaved caspase-3 was subsequently examined. As shown by both Immunohistochemistry and Western blot, cleaved caspase-3 expression increased in UUO kidneys, while decreased after Ruxolitinib treatment ( Figure 6C-F). As oxidative stress is one mechanism underlying cell apoptosis, we thereafter investigated levels of Malondialdehyde (MDA) and total Superoxide dismutase (T-SOD). As expected, UUO resulted in an increase in MDA, and a reduction in T-SOD; Ruxolitinib treatment restored T-SOD and MDA towards normal level ( Figure  6G-H).

Discussion
Activated fibroblasts expressing α-SMA are conventionally called myofibroblasts [1]. Myofibroblasts produce ECM; therefore they are crucial for organ fibrosis. Study by LeBleu and colleagues demonstrated that in UUO kidneys, 50% of myofibroblasts originate from local resident fibroblasts through proliferation, and 35%, 10% and 5% of myofibroblasts arise from bone marrow differentiation, endothelial-to-mesenchymal transition and EMT, respectively [21]. Our study revealed that Ruxolitinib treatment reduced α-SMA expression in both UUO kidneys and TGF-β1 -treated NRK-49F cells, and consistently, Ruxolitinib treatment resulted in a reduced ECM production; Moreover, Ruxolitinib treatment interfered with TGF-β1 -induced NRK-49F cell proliferation. Together, these findings indicate that Ruxolitinib has a potentiality to suppress fibroblast activation or myofibroblast generation.
As renal tubular epithelial cell EMT contributes to UUO-induced renal interstitial fibrosis, we observed the effect of Ruxolitinib on E-cadherin expression. Just as expected, UUO resulted in a decreased E-cadherin expression, which was partially recovered by Ruxolitinib treatment. Furthermore, Ruxolitinib treatment blocked TGF-β1 -induced E-cadherin downregulation in NRK-52E cells. Transcription factors snail and twist have been shown involved in renal tubular epithelial cell EMT [22,23]. Our study demonstrated that Ruxolitinib treatment suppressed snail and twist upregulation in both UUO kidneys and TGF-β1 -treated NRK-52E cells. However, study by Pang and colleagues indicated that a specific Stat3 inhibitor, S3I-201, has no effect on UUO-induced snail overexpression [15]. These findings suggest that Ruxolitinib inhibits snail upregulation independent on Stat3.
UUO kidneys overproduce chemokines and their receptors [24,25,26]. These chemokines are responsible for the recruitment of inflammatory cells, which, when activated, produce more cytokines to sustain and enhance inflammation. MCP-1 recruits macrophages into renal interstitium [27,28,29]. Our study showed that Ruxolitinib treatment reduced MCP-1 expression as well as macrophage infiltration in the obstructed kidneys. In contrast, blockade of Stat3 by S3I-201 failed to suppress UUO-induced MCP-1 upregulation, but still reduced macrophage infiltration, suggesting that other cytokines were involved in macrophage recruitment [15]. Besides growth factor such as TGF-β1, cytokine such as IL-6 also contributes to fibroblast activation and fibrosis [30]. A recent study showed that blockade of IL-6 improved UUO-induced fibrosis [31].
Several factors have been identified to induce tubular epithelial cell apoptosis, including mechanical stretch, Angiotensin II, TGF-β1, Fas/FasL and oxidative stress [32,33]. In our study, Ruxolitinib treatment reduced expression of TNF-α, which was shown to stimulate apoptosis in UUO kidneys [34,35]. Moreover, Ruxolitinib treatment attenuated the oxidative stress response. It has been shown that UUO impairs renal antioxidant enzyme activation [36]. Ruxolitinib treatment recovered T-SOD level in the obstructed kidneys, and consistently, alleviated UUO-induced apoptosis. Our study finally identified that Ruxolitinib treatment attenuated Akt/mTOR pathway. Both mTOR complex 1 (mTORC1) and mTORC2 were activated in UUO kidneys [9,10]. Furthermore, Yap was revealed to mediate mTORC2-induced renal interstitial fibrosis [12]. Increasing evidence indicated that Yap functioned as a pro-fibrotic factor in kidneys, and targeting Yap improved renal interstitial fibrosis [20,37,38,39]. Ruxolitinib treatment inhibited Yap expression in UUO kidneys, further suggesting that Akt/mTOR/Yap is a potential target signaling by Ruxolitinib.
In summary, Ruxolitinib treatment alleviated inflammation and oxidative stress in UUO kidneys, and suppressed fibroblast activation, tubular cell EMT and ECM production in both UUO kidneys and in vitro cultured cells. Mechanistically, Ruxolitinib treatment blocked UUO or TGF-β1 -induced activation of both Stat3 and Akt/mTOR/Yap pathways. These findings indicate that Ruxolitinib treatment can ameliorate UUO-induced renal interstitial fibrosis, and suggest that Ruxolitinib could be potentially used to treat fibrotic kidney disease.

UUO models and Ruxolitinib treatment
Male C57BL/6 mice (Beijing Huafukang Biotechnology, China) that weighed 22-24g were randomly assigned to three groups with 5 mice in each group as follows: (1) Sham-operated mice with vehicle (Sham); (2) UUO mice with vehicle (UUO); (3) UUO mice treated with Ruxolitinib (UUO+RUX). To establish UUO model, mice were given general anesthesia by intraperitoneal injection of pentobarbital (50mg/kg body weight). The left ureter was exposed via a left flank incision, ligated with 4-0 silk at two points, and cut between the 2 ligation points. The Sham-operated group had no ligation. For in vivo experiments, Ruxolitinib was dissolved in PEG300/dextrose 5% in a ratio of 1:3 (PEG/dex) and administered to mice by oral gavage at a dosage of 30 mg/kg twice daily for 14 days immediately after UUO or Sham-operation. The Sham and UUO group received PEG/dex alone as vehicle. The mice were sacrificed, and the left kidneys were collected at days 14 after surgery. All procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of China Medical University.

Cell culture and treatment
Rat fibroblast NRK-49F and rat renal tubular epithelial cell NRK-52E were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37°C with 5% CO 2 . 2ng/ml TGF-β1 was used to activate NRK-49F cells or induce NRK-52E cell EMT. These cells were starved for 12 h and then exposed to TGF-β1 with or without 5μM Ruxolitinib for 24 hours.

Immunohistochemistry
Tissue sections were deparaffinized, hydrated, and incubated with 3% H 2 O 2 to remove endogenous peroxidase. Then the sections were incubated with primary antibody overnight at 4 ℃, and next with biotinylated secondary antibody for 30 min at 37 °C. Subsequently the sections were stained with DAB, re-stained in hematoxylin, dehydrated, and sealed with cover slides. Ten non-repeating images of each sample were acquired using microscope image system. Positive signals and numbers of positive cells were quantified using Image-Pro Plus software.

Western blot
Equal amount of protein was isolated by SDS-PAGE, and transferred to PVDF membranes which was blocked by 5% bovine serum albumin or skimmed milk for 2 h at room temperature, and incubated with primary antibody overnight at 4°C.
The membrane was subsequently incubated with peroxidase-conjugated goat secondary antibody (1:5000, ZSGB Bio) for 2 h at room temperature. Protein content was determined by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).

TUNEL
The apoptotic cells in kidney tissue sections were detected using a TUNEL apoptosis detection kit (KeyGEN BioTECH, China, KGA7061) according to the instructions provided by the manufacture. Briefly, paraffin-embedded sections were treated with fresh diluted proteinase K for 20 min at 37 ℃ after deparaffinage and rehydration. TdT enzyme reaction mixture was applied to each slide at 37 ℃ for 60 min. Then, Streptavidin-TRITC was added into the slides at 37 ℃ for 30 min. At last, the slides were reacted with DAPI solution for 10 min at room temperature. The number of TUNEL-positive nuclei per field was counted in 10 non-repeating micrographs for each sample.

MTT
Cell viability was measured at 48 h using an MTT assay kit (Beyotime, China, C0009). 2,000 Cells in 96-well plate were washed and incubated with MTT staining solution for 4 h. Then Formazan solving solution was added. The cell viability was indicated by the absolution value at 570nm.

T-SOD and MDA measurement
T-SOD (A001-1) and MDA (A003-1) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Briefly, kidney tissues were prepared into 10% homogenates which were centrifuged at 3,000 rpm for 10 min. The supernatants were collected to measure levels of T-SOD and MDA according to the instructions.

Statistical analysis
All the experiments were conducted at least three times. GraphPad Prism 7.0 was used to analyze the data, and all data were expressed as the Mean ± SEM. Inter-group comparisons were performed using One-way analysis of variance (ANOVA). Multiple means were compared by Tukey′s test. P<0.05 is considered as significant.