Abstract

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson’s trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

Introduction

Epidemiological studies have demonstrated that the prevalence of chronic kidney disease (CKD) is approximately 8–16% worldwide [1,2]. Renal fibrosis, especially tubulointerstitial fibrosis, is the inevitable outcome for all progressive CKDs [3]. It is also the pathological hallmark and predictor of the prognosis of CKDs [3]. Renal fibrosis is a complicated process characterized by the deposition of the extracellular matrix by activated fibroblasts [3,4]. It is an orchestration of several fundamental cellular events. In response to the pro-fibrotic stimuli, the unresolved and persistent inflammation serves as the trigger of fibrosis. Then the fibrogenic genes were up-regulated in the injured tubular epithelial cells [3]. Recently, the pathogenic role of metabolic reprogramming in energy metabolism, amino acid catabolism, and oxidative stress was highlighted in the injured tubular epithelial cells in fibrosis [5,6]. It is widely accepted that tubular epithelial cells play an instrumental role in the orchestration of the renal fibrosis. Targeting these cellular events may effectively prevent tubulointerstitial fibrosis [4,7].

The Rho-associated kinase (ROCK) is a serine/threonine kinase activated by Rho and phosphorylates several downstream targets to regulate numerous biological processes, including actin cytoskeleton organization, cytokinesis, differentiation, apoptosis, glucose metabolism, cell adhesion/motility, and inflammation [8]. Extensive studies have demonstrated that ROCK inhibitors are promising potential therapies for cardiovascular diseases [9], neurological disorders [10], metabolic disorders [11], and kidney dysfunction [12]. In particular, in response to fibrogenic signals, such as the transforming growth factor-β1 (TGF-β1) [13], Rho/ROCK signaling regulates the progression of fibrosis of the heart [14], the liver [13], the urethra [15] and the kidneys [16]. Inhibition of ROCK by fasudil [16], a moderate ROCK inhibitor that has been approved for clinical use in Japan and China, or Y-27632 [12] prevented tubulointerstitial fibrosis in the unilateral ureteral obstruction (UUO) model, indicating the importance of ROCK activity in the modulation of renal fibrosis. There are two isoforms in the ROCK family, i.e., ROCK1 and ROCK2. Although they share 65% of homology in their amino sequence and are similar in the kinase activity and affinity to Rho kinases in vitro, their regulatory roles in the progression of tubulointerstitial fibrosis may be divergent [17]. Further studies used genetic knockdown/knockout mice models to decipher the specific roles of ROCK1 and ROCK2. Deletion of ROCK1 did not affect renal fibrosis in the UUO model [18]. Hence ROCK2 is considered to be more critical in renal fibrosis. Knockdown of ROCK2 failed to affect the renal fibrosis in the UUO model because the activity of ROCK2 was compensatorily restored [19]. Therefore, selective inhibition of ROCK2 activity is vital in elucidating the role of ROCK2 in the progression of renal tubulointerstitial fibrosis and may be a potential therapy against it.

KD025, formerly named as SLx-2119, is one of the first-in-class ATP competitive ROCK2 selective inhibitor [20]. Almost all the existing ROCK inhibitors lack selectivity among ROCK1 and ROCK2 due to the structural homology in the kinase domain (92% amino acid homology) of the ROCK1 and ROCK2 isoforms [20,21]. The affinity of KD025 to ROCK2 is approximately 200-fold higher than ROCK1 [22] and showed no significant activity against 300 other intracellular kinases and surface receptors [23], including those that are known to be inhibited by fasudil [21]. It is currently under Phase II clinical trial for immune diseases including psoriasis, graft vs. host disease, and systemic sclerosis, and fibrotic disease, i.e., idiopathic pulmonary fibrosis (IPF). Accumulating experimental studies also demonstrate that KD025 has potent therapeutic effects in inflammatory disorders [24], adipogenesis [25], and focal cerebral ischemia [26], indicating KD025 regulates inflammation, metabolism and is protective to cellular injury. The effect of KD025 on the progression of tubulointerstitial fibrosis is unknown.

In the present study, we found that the ROCK2 level was positively correlated with the severity of the tubulointerstitial fibrosis in the kidneys of CKD patients and was time-dependently increased in the UUO model. Inhibition of ROCK2 by KD025 alleviated the pathology of tubulointerstitial fibrosis, the expression of the fibrotic markers, and inflammation in the renal cortex of the UUO model. KD025 or ROCK2 knockdown/knockout directly prevented the fibrotic response in the mouse renal proximal tubular epithelial cells (mPTCs) induced by TGF-β1. KD025 also corrected metabolic disturbance, decreased oxidative stress, and increased the nuclear content of nuclear erythroid 2-related factor 2 (Nrf2) in the fibrotic model. Besides, KD025 inhibited the M1-activation of macrophages. These results collectively show that inhibition of ROCK2 by KD025 prevented the renal tubulointerstitial fibrosis, possibly by correcting the metabolic disturbance and inhibiting inflammation.

Materials and methods

Materials

KD025 (purity > 99%, purchased from Selleck, U.S.A.) was dissolved in DMSO and diluted with vehicle (5% DMSO, 35% PEG300, 2% Tween 80, 58% sterile saline) before administration to mice. Cell culture reagents, including DMEM/F-12 medium, fetal bovine serum (FBS), phosphate buffer saline (PBS), were supplied by Gibco, U.S.A. Human recombinant TGF-β1 (240-B, R&D company) and reconstituted at 5 μg/ml in sterile 4 mM HCl containing 1 mg/ml bovine serum albumin as stock, which was aliquoted and stored in −80°C. Lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma, U.S.A.) was dissolved in sterile PBS and were diluted for 1000 times to achieve the working concentration, i.e., 100 ng/ml. Primary antibodies included fibronectin (ab2413, Abcam, U.S.A.), ROCK2 (sc-398519, Santa Cruz, U.S.A.), collagen I (ab34710, Abcam, U.S.A.), F4/80 (ab111101, Abcam, U.S.A.), p-Smad2 (Ser465/467) (3108S, Cell Signaling Technology, U.S.A.), Smad2 (5339S, Cell Signaling Technology, U.S.A.), β-actin (3700, Cell Signaling Technology, U.S.A.), Nrf2 (16396-1-AP, Proteintech, U.S.A.), lamin B1 (12987-1-AP, Proteintech, U.S.A.), and GAPDH (10494-1-AP, Proteintech, U.S.A.).

Patients

The renal biopsy samples were obtained from CKD patients under diagnostic evaluation at the Department of Nephrology, Children’s Hospital of Nanjing Medical University. The control samples were nephrectomized tissue collected from renal carcinoma patients that were age and sex-matched to the CKD patients and were not diagnosed with renal fibrosis. The pathology of each patient was evaluated by a blinded pathologist and quantified as Interstitial Fibrosis Score. The Interstitial Fibrosis Score measures the summary of tubular atrophy, infiltration of inflammatory cells, and interstitial fibrosis as 0, none; 1, ≤25%; 2, >25%, <50%; 3, ≥50%. The details are shown in Table 1. The protocol for using these biopsy samples and nephrectomized tissues from patients was approved by the local committee on human subjects at the Children’s Hospital of Nanjing Medical University. Written informed consent was provided by each patient.

Table 1
Renal interstitial fibrosis score of CKD patients
Age (years)GenderDiagnosisInfiltration of inflammatory cellsTubular atrophyInterstitial fibrosisInterstitial fibrosis score
12 Female IgA nephropathy 
Female Crescent glomerulonephritis 
13 Female LN IV-G(A/C)+V 
Female FSGS 
13 Male HSPN III-a 
Male Subacute tubulointerstitial nephropathy 
11 Male Glomerulonephritis 
Female CKD-III 
11 Female FSGS 
14 Female LN 
Male Glomerulonephritis 
Male Glomerulonephritis 
10 Female ANCA-associated vasculitis 
Female Refractory nephrotic syndrome 
Female LN III-a 
10 Female Mesangial proliferative glomerulonephritis 
Age (years)GenderDiagnosisInfiltration of inflammatory cellsTubular atrophyInterstitial fibrosisInterstitial fibrosis score
12 Female IgA nephropathy 
Female Crescent glomerulonephritis 
13 Female LN IV-G(A/C)+V 
Female FSGS 
13 Male HSPN III-a 
Male Subacute tubulointerstitial nephropathy 
11 Male Glomerulonephritis 
Female CKD-III 
11 Female FSGS 
14 Female LN 
Male Glomerulonephritis 
Male Glomerulonephritis 
10 Female ANCA-associated vasculitis 
Female Refractory nephrotic syndrome 
Female LN III-a 
10 Female Mesangial proliferative glomerulonephritis 

Abbreviations: ANCA, antineutrophil cytoplasmic antibody; FSGS, focal segmental glomerular sclerosis; HSPN, Henoch–Schonlein purpura nephritis; LN, lupus nephritis.

Animals

Eighteen 8-week-old male C57BL6/J mice were purchased from the Animal Core Facility of Nanjing Medical University. Upon arrival, the mice were randomly grouped by their body weights and were group-housed in cages with free access to food and water in the specific pathogen-free animal room. The mice were habituated to the housing environment for a week. Afterward, half of the mice were administrated with either vehicle (5% DMSO, 35% PEG300, 2% Tween 80, 58% sterile saline) or KD025 (50 mg/kg/day) via intraperitoneal injection once per day for 8 days in total. Two hours after the second injection, all the mice were anesthetized for UUO modeling. Seven days after the surgery, the mice were killed and renal cortex from both kidneys was dissected for sampling. The animal work was performed in the Animal Core Facility of Nanjing Medical University. The usage of the mice was approved by the IACUC in Nanjing Medical University.

UUO model

The UUO model was the same as described in previous reports [27]. Briefly, an incision was made on the shaved back of the mouse anesthetized by isoflurane to expose the left kidney. The urethra was ligated near the kidney, and the ligation site remained similar in all the mice. After suturing, the mice were returned to home cages for recovery.

Cell culture, transfection, and administration

mPTCs

The mPTCs is an immortalized cell line of mouse proximal tubular epithelial cells, which were acquired from ATCC and cultured in DMEM-F12 medium supplemented with 10% FBS [28].

Genetic knockdown

Twenty-four hours after seeding cells, the ROCK2 siRNA cocktail purchased from Ribobio (siG140829115758, Guangzhou, China) was transfected into mPTCs by Lipofectamine 2000 (11668027, Thermo Fisher) according to the manufacturer’s protocol. Twenty-four hours later, the cells were treated with KD025 in DMEM/F12 medium for 2 h before TGF-β1 stimulation (5 ng/ml) for 24 h before harvest for RNA or protein or performing immunocytochemical analysis. Since the potency of KD025 was estimated to be 1–10 μM against ROCK2 and above 200 μM against ROCK1 in the cells [22], we treated the cells at the concentration of 2.5 μM in most of our experiments unless otherwise indicated.

Genetic knockout

CRISPR/Cas 9 technique was used to knock out ROCK2 in the mPTCs similar as previously described [29]. Briefly, the sgRNAs target mouse ROCK2 were designed and synthesized by Genebay Biotech (Nanjing, China) and the sequence was 5′-TCGTCATAAGGCATCACAGA-3′. The oligonucleotides were annealed into double chains after phosphorylation and cloned into pSpCas9(BB)-2A-Puro (PX459) v2.0, which was a gift from Feng Zhang (Addgene plasmid # 62988). The plasmid was transfected into mPTCs using PolyJet In Vitro DNA Transfection Reagent (SL100688, SignaGen Laboratories, U.S.A.). Positive cells were selected using puromycin (2 μg/ml) for 2 days prior to clonal expansion. The empty vector was used as control. The cells were treated with TGF-β1 same as mentioned above.

RAW264.7 cells

RAW264.7 cells were cultured in DMEM supplemented with 10% FBS. Cells were pretreated with KD025 for 2 h before LPS exposure (100 ng/ml). Twenty-four hours after LPS stimulation, the supernatant was collected for ELISA to quantify the release of interleukin-1β (IL-1β) according to the manufacturer’s instructions (DKW12-2012-096, Dakewe Bioengineering, China). The mRNA of the cells was extracted for RT-PCR analysis. The cell proliferation was examined by CCK8 assay kit (KGA317, KeyGEN Biotech, China).

RNA extraction and RT-PCR

The RNA extraction, the reverse transcription, and the RT-PCR were performed according to the manufacturers’ instructions. The RNA in the renal cortical tissue or mPTCs was extracted by RNAiso reagent (9108, Takara, Japan), and reverse transcription was performed using a kit according to the manufacturer’s protocols (PR036A, Takara, Japan). The primers for mouse IL-1β, IL-6, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), GAPDH were the same as previously reported [30]. The sequence of primers were as follows: mouse fibronectin: Forward 5′-CGTGGAGCAAGAAGGACAA-3′ and reverse 5′-GTGAGTCTGAGGTTGGTAAA-3′; mouse α-sma: Forward 5′-CCCTGAAGAGCATCCGACA-3′ and reverse 5′-CCAGAGTCCAGCACAATACC-3′; mouse collagen I: Forward 5′-CCGGCTCCTCTT-3′ and reverse 5′-TTGCACGTCATCGCACAC-3′; mouse collagen III: Forward 5′-CAGGACCTAAGGGCGAAGATG-3′ and reverse 5′- TCCGGGCATACCCCGTATC-3′; mouse CD206: Forward 5′-CTCTGTTCAGCTATTGGACGC-3′ and reverse 5′-TGGCACTCCCAAACATAATTTGA-3′; mouse Ym-1: Forward 5′- ATGAGTGGGTTGGTTATG-3′ and reverse 5′-AAAGTAGATGTCAGAGGGA-3′; mouse Arg: Forward 5′- CTCCAAGCCAAAGTCCTTAGAG-3′ and reverse 5′-AGGAGCTGTCATTAGGGACATC-3′; mouse glutathione synthetase (GSS): Forward 5′-CAAAGCAGGCCATAGACAGGG-3′ and reverse 5′-AAAAGCGTGAATGGGGCATAC-3′; and mouse SOD-1: Forward 5′-AGCCCGGCGGATGAAG-3′ and reverse 5′-CCTTTCCAGCAGTCACATTGC-3′. The RT-PCR was performed using SyBR Green Supermix in ABL Q3 PCR machine. The data were normalized to GAPDH as an internal reference gene and compared with the intact control group.

Western blot

The western blot was performed as previously described [28]. The renal cortex was homogenated by RIPA reagent (P0013, Beyotime, China). The nuclear fraction was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, China). The buffers were all supplemented with proteinase inhibitor (4693116001, Roche, U.S.A.) and phosphotase inhibitor (4906837001, Roche, U.S.A.). The samples were quantified by BCA protein quantification kit (P0012, Beyotime, China). Twenty micrograms/lane of protein were subjected to electrophoresis in SDS/PAGE gel and were transferred to PVDF membrane (Bio-Rad, U.S.A.). After blockade with 5% skim milk, the membrane was probed with primary antibodies and secondary antibodies (A0208, A0216, Beyotime, China). The band was developed with ECL illuminance kit in Imagelab (Bio-Rad, U.S.A.). The grayscale of the bands was quantified by Chemi software and was normalized to the internal reference gene and compared with the control group.

Tissue fixation and staining

Immunohistochemical analysis

The protocol of sample processing and immunohistochemical analysis was similar as previously described [29]. The mice kidney samples were sagittally dissected and fixed in 4% PFA for 24 h at 4 degree before processed using Leica automatic dehydration machine. Both the mice and human samples were embedded in paraffin wax and sectioned into 4 microns. After deparaffinization, rehydration, antigen retrieval with citrate buffer (P0083, Beyotime, China), blockade of endogenous peroxidase with 3% H2O2, and blockade by 5% normal goat serum, the sections were stained with primary antibody overnight. The signal detection of mice and human sections were performed with DAB kit (ZLI-9018, zsbio, China) and DAKO REAL Envision DAB detection system (DAKO, Danmark), respectively. IgG control (mouse: 53484, Cell Signaling Technology, U.S.A.; rabbit: A7016, Beyotime, China) were used as negative control for each trial.

Masson’s trichrome staining

After deparaffinization and rehydration, the section of mouse kidneys was stained for Masson’s trichrome staining with a commercially available kit (D026, Jiangcheng, Nanjing) as previously described [31].

Imaging

For each section from the mice sample, at least five non-overlapping images were taken by light microscopy (Olympus BX51, Japan). The quantification of the positive area was performed by a blinded experimenter using the Image-Pro Plus software. After opening an image in the software, a positive staining color was manually selected and quantified for positive staining area. The data were normalized to images of the control group.

For each human sample, the non-overlapping images were taken for the entire section and quantified by a blinded experimenter in the same way as mentioned above. The expression of ROCK2 was normalized to controls and was correlated with the Interstitial Fibrosis Score shown in Table 1.

Immunofluorescence

The mPTCs were seeded on coverslips. Twenty-four hours after stimulation with TGF-β1, the cells were fixed with 4% PFA and stained with fibronectin (ab2413, Abcam, U.S.A.) while the nuclei were counterstained with DAPI. The cells were then imaged with confocal microscopy (LSM710, Carl Zeiss, Germany). At least ten cells were imaged.

The fluorescent intensity of each integral cell in the image was analyzed by ImageJ software [32]. First, areas of interest were manually chosen for each cell by a blinded experimenter. Then the fluorescent intensity of the area of interest was measured by clicking ‘measure’ in the software. The fluorescent intensity was normalized to the average fluorescent intensity of control group.

Metabolome analysis of mouse proximal tubular cells

For each sample, approximately 1× 107 cells were harvested with a cell scraper for metabolome analysis. The cell pellets were treated with 1 ml of the mixture of acetonitrile, methanol, and dd water in the volume ratio of 2:2:1 under vortex for 30 s. Then the samples were snap-frozen in liquid nitrogen for 5 min and thawed at room temperature before grinding at 70 Hz for 2 min. This step was repeated twice before centrifuging the samples at 13000 rpm for 10 min at 4ºC. The supernatant was transferred and vacuum-dried to concentrate the samples. Afterward, the samples were re-dissolved with 300 μl of 2-chlorobenzalanine solution (4 ppm) prepared with acetonitrile: 0.1% FA (1:9 v/v) (−20°C), and the supernatant was filtered through 0.22-μm membrane to obtain the prepared samples for LC-MS. Chromatographic separation was accomplished in an Thermo Ultimate 3000 system equipped with an ACQUITY UPLC® HSS T3 (150 × 2.1 mm, 1.8 μm, Waters) column maintained at 40°C. The temperature of the autosampler was 8°C. Gradient elution of analytes was carried out with 0.1% formic acid in water (C) and 0.1% formic acid in acetonitrile (D) or 5 mM ammonium formate in water (A) and acetonitrile (B) at a flow rate of 0.25 ml/min. Injection of 2 μl of each sample was done after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0–1 min, 2% B/D; 1–9 min, 2–50% B/D; 9–12 min, 50–98% B/D; 12–13.5 min, 98% B/D; 13.5–14 min, 98–2% B/D; 14–20 min, 2% D-positive model (14–17 min, 2% B-negative model). The ESI-MSn experiments were executed on the Thermo Q Exactive mass spectrometer with the spray voltage of 3.8 and –2.5 kV in positive and negative modes, respectively. Sheath gas and auxiliary gas were set at 30 and 10 arbitrary units, respectively. The capillary temperature was 325°C. The analyzer scanned over a mass range of m/z 81–1000 for full scan at a mass resolution of 70000. Data-dependent acquisition (DDA) MS/MS experiments were performed with HCD scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented to remove some unnecessary information in MS/MS.

Statistical analysis

The Pearson correlation analysis was used to study the correlation between the ROCK2 level and the interstitial fibrosis score in the patient samples. A linear regression curve was plotted. In the animal study and cell culture studies, the data were normalized to the control group, and the statistical significance was examined by two-way ANOVA followed by Sidak post hoc analysis if applicable in Prism 6.0 software. In all bar charts, mean + SEM was plotted. P<0.05 was considered to be statistically significant.

Results

ROCK2 increased in the fibrotic kidneys in patients and the UUO model

To study the relevance of ROCK2 expression level in the human fibrotic kidneys, we enrolled 16 CKD patients with different degrees of interstitial fibrosis in pathology and stained for ROCK2 using immunohistochemical analysis. The ROCK2 level in patients’ kidneys was positively correlated with the Interstitial Fibrosis Score, i.e., the summary of tubular atrophy, infiltration of inflammatory cells, and fibrosis (Figure 1A). Consistently, PCR array data of adult CKD patients [33] sorted on Nephroseq (www.Nephroseq.org, <May 2020 of use>, University of Michigan, Ann Arbor, MI), the online database, showed that ROCK2 expression was significantly up-regulated in the CKD kidneys (Figure 1B), which showed moderate to severe tubulointerstitial fibrosis and tubular cell damage [33]. These results from both pediatric and adult human fibrotic kidneys indicated that ROCK2 may be implicated in the progression of renal fibrosis. Noteworthily, the up-regulation of ROCK2 was predominant in the proximal tubules (Figure 1A), indicating ROCK2 was implicated in the injury of the tubular epithelial cells during renal fibrosis. We then evaluated the protein level of ROCK2 in the UUO model that was obstructed for a different period of time, i.e., 1, 3, and 7 days. The protein level of ROCK2 was time-dependently increased during the progression of the obstruction. The increase in ROCK2 protein was statistically significant in the 3- and 7-day UUO model comparing to the intact kidney (Figure 1C). Similarly, by using immunohistochemical analysis, we found that ROCK2 increased in the 7-day UUO kidney and was more abundant in the renal proximal tubules in the UUO kidney (Figure 1D).

ROCK2 was up-regulated in the fibrotic kidneys in patients and UUO model

Figure 1
ROCK2 was up-regulated in the fibrotic kidneys in patients and UUO model

The biopsy samples from patients were stained with ROCK2 using the immunohistochemical analysis to study the correlation of ROCK2 level with the severity of interstitial fibrosis. Microarray data of 48 adult CKD patients with moderate to severe fibrosis and tubular injury and 5 matching controls [33] were sorted and analyzed on the online database, Nephroseq (www.Nephroseq.org, University of Michigan, Ann Arbor, MI). The left kidneys of 8-week-old C57BL/6J mice were obstructed for 1, 3, or 7 days and the expression of ROCK2 was studied by Western blot analysis. The distribution of ROCK2 was examined by immunohistochemical analysis in both the intact kidney and UUO kidney after 7 days of obstruction. (A) Representative images of ROCK2 expression in human fibrotic kidneys. ROCK2 level was positively correlated with the severity of interstitial fibrosis scores in the patients. Scale bar: 50 μm. (B) ROCK2 was significantly up-regulated in the adult CKD patients with moderate to severe tubulointerstitial fibrosis and tubular injury comparing with control according to the published microarray data sorted and analyzed on Nephroseq. Information of the patients was published in literature [33]. (C) ROCK2 protein level increased with the elongation of obstruction. In the 3- and 7-day UUO model, the ROCK2 protein level was significantly higher than intact control, n=3. ** P<0.01 comparing to intact kidney tissue. (D) Immunohistochemical analysis revealed that ROCK2 protein was mainly up-regulated in the tubular epithelial cells in the UUO kidney. Scale bar: 20 μm. Negative controls in (A,C): IgG negative control.

Figure 1
ROCK2 was up-regulated in the fibrotic kidneys in patients and UUO model

The biopsy samples from patients were stained with ROCK2 using the immunohistochemical analysis to study the correlation of ROCK2 level with the severity of interstitial fibrosis. Microarray data of 48 adult CKD patients with moderate to severe fibrosis and tubular injury and 5 matching controls [33] were sorted and analyzed on the online database, Nephroseq (www.Nephroseq.org, University of Michigan, Ann Arbor, MI). The left kidneys of 8-week-old C57BL/6J mice were obstructed for 1, 3, or 7 days and the expression of ROCK2 was studied by Western blot analysis. The distribution of ROCK2 was examined by immunohistochemical analysis in both the intact kidney and UUO kidney after 7 days of obstruction. (A) Representative images of ROCK2 expression in human fibrotic kidneys. ROCK2 level was positively correlated with the severity of interstitial fibrosis scores in the patients. Scale bar: 50 μm. (B) ROCK2 was significantly up-regulated in the adult CKD patients with moderate to severe tubulointerstitial fibrosis and tubular injury comparing with control according to the published microarray data sorted and analyzed on Nephroseq. Information of the patients was published in literature [33]. (C) ROCK2 protein level increased with the elongation of obstruction. In the 3- and 7-day UUO model, the ROCK2 protein level was significantly higher than intact control, n=3. ** P<0.01 comparing to intact kidney tissue. (D) Immunohistochemical analysis revealed that ROCK2 protein was mainly up-regulated in the tubular epithelial cells in the UUO kidney. Scale bar: 20 μm. Negative controls in (A,C): IgG negative control.

Inhibition of ROCK2 by KD025 attenuated tubulointerstitial fibrosis in the UUO model

ROCK2 was significantly up-regulated in the fibrotic kidneys in human and mouse UUO model, hence it may contribute to the tubulointerstitial fibrosis. Inhibiting ROCK2 may prevent tubulointerstitial fibrosis. We used KD025, a first-in-class ROCK2 inhibitor, to investigate the effect of ROCK2 inhibition in the 7-day UUO kidney. The obstructed kidney in the control group showed profound pathological changes of fibrosis in Masson’s trichrome staining (Figure 2A). KD025-treatment showed significantly less severe fibrotic pathology (Figure 2A). Consistently, immunohistochemical analysis revealed that KD025 profoundly decreased the collagen I in the UUO kidney (Figure 2B,C). The mRNA expression of fibronectin, α-sma, collagen I, collagen III, and vimentin was also significantly decreased by KD025 treatment in the renal cortex of the UUO model (Figure 2D–H). Furthermore, KD025 significantly inhibited the phosphorylation of coffilin, the downstream substrate of ROCK signaling [25] (Figure 2I,J), indicating KD025 effectively inhibited the activation of ROCK signaling. These data demonstrated that inhibition of ROCK2 via KD025 effectively alleviated the tubulointerstitial fibrosis in the UUO model.

KD025 alleviated the renal tubulointerstitial fibrosis in the UUO model

Figure 2
KD025 alleviated the renal tubulointerstitial fibrosis in the UUO model

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and fixed with 4% PFA for histological examination or homogenated for Western blotting analysis or RT-PCR. (A) Masson’s trichrome staining revealed profound fibrosis in the UUO kidney. In the kidney of KD025-treated mice, the tubulointerstitial fibrosis was less severe than the UUO model. Left: 100×, scale bar: 100 μm; Right: 200×, scale bar: 50 μm. (B,C) Immunohistochemical analysis demonstrated that KD025 decreased the deposition of collagen I in the UUO kidney. (B) Representative images, scale bar: 20 μm; (C) quantification of collagen I. (D–H) RT-PCR demonstrated a significant increase in the mRNA level of fibronectin (D), α-sma (E), collagen I (F), collagen III (G), and vimentin (H) in the UUO model comparing with control. KD025 significantly decreased the mRNA level of these markers. n=9. (I,J) Western blotting analysis showed KD025 significantly decreased the protein level of p-coffilin in UUO kidneys. (I) Representative blots and (J) semi-quantification. n=9. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. **, *** means P<0.01, 0.001 comparing to control, respectively. # and ### means P<0.05, 0.001 comparing to UUO model, respectively.

Figure 2
KD025 alleviated the renal tubulointerstitial fibrosis in the UUO model

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and fixed with 4% PFA for histological examination or homogenated for Western blotting analysis or RT-PCR. (A) Masson’s trichrome staining revealed profound fibrosis in the UUO kidney. In the kidney of KD025-treated mice, the tubulointerstitial fibrosis was less severe than the UUO model. Left: 100×, scale bar: 100 μm; Right: 200×, scale bar: 50 μm. (B,C) Immunohistochemical analysis demonstrated that KD025 decreased the deposition of collagen I in the UUO kidney. (B) Representative images, scale bar: 20 μm; (C) quantification of collagen I. (D–H) RT-PCR demonstrated a significant increase in the mRNA level of fibronectin (D), α-sma (E), collagen I (F), collagen III (G), and vimentin (H) in the UUO model comparing with control. KD025 significantly decreased the mRNA level of these markers. n=9. (I,J) Western blotting analysis showed KD025 significantly decreased the protein level of p-coffilin in UUO kidneys. (I) Representative blots and (J) semi-quantification. n=9. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. **, *** means P<0.01, 0.001 comparing to control, respectively. # and ### means P<0.05, 0.001 comparing to UUO model, respectively.

Inhibition of ROCK2 by KD025 inhibited inflammatory response in the UUO model

Inflammation is both the trigger and the key pathological changes in the tubulointerstitial fibrosis [3]. Following UUO, the obstructed kidney exhibited drastic macrophage infiltration into the interstitium of the cortex, as shown in the F4/80 staining (Figure 3A,B). KD025 treatment profoundly prevented the infiltration of the macrophages (Figure 3A,B). This finding was supported by the results of RT-PCR, which demonstrated that KD025 significantly decreased the up-regulation of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β (Figure 3C–E), and pro-inflammatory mediator iNOS (Figure 3F). It has been reported that the mRNA expression of M2 macrophage markers or enriched genes was correlated with renal dysfunction and fibrosis [34]. We found that CD206, the M2 macrophages marker, together with the M2 macrophage-enriched genes including Arg and YM-1 drastically increased in the UUO kidney comparing to control. Such elevation was suppressed in the renal cortex of KD025-treated mice (Figure 3G–I). These data demonstrated that KD025 strikingly decreased the activated macrophages and prevented inflammation in the UUO kidney.

KD025 showed a profound anti-inflammatory effect in the UUO model

Figure 3
KD025 showed a profound anti-inflammatory effect in the UUO model

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and homogenated for RT-PCR analysis of the mRNA expression of cytokines or fixed with 4% PFA. (A) The F4/80 primary antibody was used to detect the infiltration of the macrophages in the interstitium of the renal cortex. Profound macrophages infiltration was found in the interstitium of the kidney in the UUO model group comparing with control. Comparing with UUO group, KD025 treated mice showed a drastic reduction in the F4/80 positive cells after UUO. Red arrows demonstrate the representative staining. Scale bar: 20 μm. (B) Quantification of the images from the F4/80 immunohistochemical analysis demonstrated that KD025 significantly decreased the F4/80 staining area in the renal cortex in the UUO model. (C–I) The UUO model showed a drastic increase in the mRNA expression of pro-inflammatory cytokines, i.e., TNF-α (C), IL-6 (D) and IL-1β (E), the pro-inflammatory mediator iNOS (F), and CD206 (G), the cell marker of the M2 macrophages, and genes that were enriched in M2 macrophage, i.e., Arg (H), and YM-1 (I). n=9. The expression of these markers was significantly decreased in the KD025-treated mice kidney after UUO modeling. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *, *** means P<0.05, 0.001 comparing to control, respectively. #, ##, and ### means P<0.05, 0.01, and 0.001 comparing to UUO model, respectively.

Figure 3
KD025 showed a profound anti-inflammatory effect in the UUO model

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and homogenated for RT-PCR analysis of the mRNA expression of cytokines or fixed with 4% PFA. (A) The F4/80 primary antibody was used to detect the infiltration of the macrophages in the interstitium of the renal cortex. Profound macrophages infiltration was found in the interstitium of the kidney in the UUO model group comparing with control. Comparing with UUO group, KD025 treated mice showed a drastic reduction in the F4/80 positive cells after UUO. Red arrows demonstrate the representative staining. Scale bar: 20 μm. (B) Quantification of the images from the F4/80 immunohistochemical analysis demonstrated that KD025 significantly decreased the F4/80 staining area in the renal cortex in the UUO model. (C–I) The UUO model showed a drastic increase in the mRNA expression of pro-inflammatory cytokines, i.e., TNF-α (C), IL-6 (D) and IL-1β (E), the pro-inflammatory mediator iNOS (F), and CD206 (G), the cell marker of the M2 macrophages, and genes that were enriched in M2 macrophage, i.e., Arg (H), and YM-1 (I). n=9. The expression of these markers was significantly decreased in the KD025-treated mice kidney after UUO modeling. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *, *** means P<0.05, 0.001 comparing to control, respectively. #, ##, and ### means P<0.05, 0.01, and 0.001 comparing to UUO model, respectively.

ROCK2 inhibition by KD025 or knockdown/knockout prevented the pro-fibrotic response in the TGF-β1-stimulated renal tubular epithelial cells

Since KD025 profoundly inhibited the fibrotic response in the UUO model, we then confirmed the direct inhibitory effect of KD025 on the fibrotic response in tubular epithelial cells. TGF-β1 triggers the expression of fibrotic genes and leads to dysfunction of tubular epithelial cells in tubulointerstitial fibrosis [35]. We treated KD025 to the TGF-β1-stimulated mPTCs. According to the results in the immunofluorescence, TGF-β1 significantly increased the protein level of fibronectin in the mPTCs while KD025 significantly decreased it (Figure 4A,B). The mRNA levels of α-sma, collagen I, collagen III, and vimentin were also significantly reduced in the presence of KD025 when the mPTCs were stimulated with TGF-β1 (Figure 4C–F). These data suggested that KD025 prevented the pro-fibrotic response induced by TGF-β1 in tubular epithelial cells.

KD025, ROCK2 knockdown or knockout inhibited pro-fibrotic response in TGF-β1-stimulated mPTCs

Figure 4
KD025, ROCK2 knockdown or knockout inhibited pro-fibrotic response in TGF-β1-stimulated mPTCs

mPTCs were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) for 24 h. mPTCs were ROCK2 knocked down with ROCK2 siRNA, or ROCK2 knockout were stimulated by TGF-β1 (5 ng/ml) for 24 h. (A,B) Immunofluorescent analysis combined with confocal microscopy demonstrated that KD025 significantly suppressed the protein level of fibronectin induced by TGF-β1. (A) Representative images of fibronectin (green) merged with DAPI (blue), scale bar: 50 μm. (B) Quantification of the fluorescent intensity of fibronectin. (CF) The results in the RT-PCR demonstrated that KD025 significantly decreased the mRNA expression of α-sma (C), collagen I (D), collagen III (E), and vimentin (F) in the TGF-β1-stimulated mPTCs. (GI) ROCK2 knockdown by siRNA significantly decreased the expression of fibronectin in the TGF-β1-stimulated mPTCs. (G) Representative blots of ROCK2 and fibronection. (H,I) quantification results. (JM) ROCK2 knockout mPTCs (J) showed significantly decreased mRNA expression of α-sma (K) and protein level of fibronectin (L,M) when stimulated by TGF-β1. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. n=3. *, *** means P<0.05, or 0.001 comparing to control, respectively. #, ##, and ### means P<0.05, 0.01, and 0.001 comparing to TGF-β1 model, respectively.

Figure 4
KD025, ROCK2 knockdown or knockout inhibited pro-fibrotic response in TGF-β1-stimulated mPTCs

mPTCs were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) for 24 h. mPTCs were ROCK2 knocked down with ROCK2 siRNA, or ROCK2 knockout were stimulated by TGF-β1 (5 ng/ml) for 24 h. (A,B) Immunofluorescent analysis combined with confocal microscopy demonstrated that KD025 significantly suppressed the protein level of fibronectin induced by TGF-β1. (A) Representative images of fibronectin (green) merged with DAPI (blue), scale bar: 50 μm. (B) Quantification of the fluorescent intensity of fibronectin. (CF) The results in the RT-PCR demonstrated that KD025 significantly decreased the mRNA expression of α-sma (C), collagen I (D), collagen III (E), and vimentin (F) in the TGF-β1-stimulated mPTCs. (GI) ROCK2 knockdown by siRNA significantly decreased the expression of fibronectin in the TGF-β1-stimulated mPTCs. (G) Representative blots of ROCK2 and fibronection. (H,I) quantification results. (JM) ROCK2 knockout mPTCs (J) showed significantly decreased mRNA expression of α-sma (K) and protein level of fibronectin (L,M) when stimulated by TGF-β1. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. n=3. *, *** means P<0.05, or 0.001 comparing to control, respectively. #, ##, and ### means P<0.05, 0.01, and 0.001 comparing to TGF-β1 model, respectively.

Next, we studied the role of ROCK2 in the pro-fibrotic response in the tubular epithelial cells by using genetic knockdown and knockout techniques. Consistent with the finding in the CKD patients’ kidneys and the UUO model (Figure 1), ROCK2 protein level was significantly increased in the TGF-β1-stimulated mPTCs (Figure 4G,H). By using ROCK2 siRNA, we found that knockdown of ROCK2 significantly decreased the protein level of fibronectin after exposure to TGF-β1 (Figure 4G–I). To further confirm the protective effect of ROCK2 on the fibrotic response in TGF-β1-stimulated mPTCs, we used CRISPR/Cas 9 technique to delete ROCK2 in the mPTCs (Figure 4J). Consistently, ROCK2 knockout also effectively decreased the mRNA expression of α-sma (Figure 4K) and the protein expression of fibronectin (Figure 4L,M) in the TGF-β1-stimulated mPTCs. These data demonstrated that targeting ROCK2 could prevent the fibrotic response of the tubular epithelial cells stimulated by TGF-β1.

To study whether the antifibrotic effect of KD025 in the TGF-β1-stimulated mPTCs was attributed to ROCK2 inhibition or another mechanism, we combined KD025 treatment and genetic knockdown of ROCK2. The results proved that ROCK2 was up-regulated in the TGF-β1-stimulated mPTCs (Figure 5A,B). Although KD025 did not significantly affect the protein level of ROCK2 in the mPTCs in the control group, it decreased ROCK2 protein level in the TGF-β1-stimulated mPTCs (Figure 5A,B). KD025 and knockdown of ROCK2 alone or together significantly decreased the protein level of fibronectin in the TGF-β1-stimulated mPTCs (Figure 5A,C). Co-treatment of KD025 and ROCK2 siRNA did not offer an additive antifibrotic effect in the TGF-β1-stimulated mPTCs comparing to ROCK2 siRNA (Figure 5A,C). These data indicated that KD025 might elicit its antifibrotic effect on the TGF-β1-stimulated tubular epithelial cells via inhibition of ROCK2 activity.

ROCK2 knockdown and KD025 inhibited pro-fibrotic response and Smad2 activation in TGF-β1-stimulated mPTCs

Figure 5
ROCK2 knockdown and KD025 inhibited pro-fibrotic response and Smad2 activation in TGF-β1-stimulated mPTCs

mPTCs that were knocked down with ROCK2 siRNA were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) with the presence of KD025 for 24 h. The protein levels of ROCK2, fibronectin, p-Smad2, and Smad2 were studied by Western blot analysis. (AC) ROCK2 knockdown and KD025 significantly decreased the protein level of ROCK2 and fibronectin in the TGF-β1-stimulated mPTCs. (A) Representative blots of ROCK2 and fibronectin. (B,C) Quantification of protein levels of ROCK2 (B) and fibronectin (C). (DF) KD025 and ROCK2 knockdown inhibited the phosphorylation of Smad2 in the TGF-β1-stimulated mPTCs. (D) Representative blots of p-Smad2, and Smad2. (E,F) quantification results. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. n=3. *, **, *** means P<0.05, 0.01, or 0.001 comparing to control, respectively. # and ### means P<0.05, 0.001 comparing to TGF-β1 model, respectively.

Figure 5
ROCK2 knockdown and KD025 inhibited pro-fibrotic response and Smad2 activation in TGF-β1-stimulated mPTCs

mPTCs that were knocked down with ROCK2 siRNA were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) with the presence of KD025 for 24 h. The protein levels of ROCK2, fibronectin, p-Smad2, and Smad2 were studied by Western blot analysis. (AC) ROCK2 knockdown and KD025 significantly decreased the protein level of ROCK2 and fibronectin in the TGF-β1-stimulated mPTCs. (A) Representative blots of ROCK2 and fibronectin. (B,C) Quantification of protein levels of ROCK2 (B) and fibronectin (C). (DF) KD025 and ROCK2 knockdown inhibited the phosphorylation of Smad2 in the TGF-β1-stimulated mPTCs. (D) Representative blots of p-Smad2, and Smad2. (E,F) quantification results. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. n=3. *, **, *** means P<0.05, 0.01, or 0.001 comparing to control, respectively. # and ### means P<0.05, 0.001 comparing to TGF-β1 model, respectively.

The phosphorylation of Smad2 demonstrated the activation of TGF-β/Smads signaling in fibrosis, leading to the transcription of the fibrogenic genes [36]. Activation of Smad2 has been found in UUO kidneys [36,37]. As shown in Figure 5D–F, TGF-β1 significantly increased the phosphorylation of Smad2 in the mPTCs. KD025 and ROCK2 knockdown effectively decreased the phosphorylation level of Smad2 (Figure 5D–F). These data demonstrated that KD025 and ROCK2 knockdown prevented the phosphorylation of Smad2 in the mPTCs.

Inhibition of ROCK2 by KD025 corrected the metabolic reprogramming in the TGF-β1-stimulated renal tubular epithelial cells

Metabolic reprogramming in renal tubular epithelial cells contributes to the pathogenesis of renal fibrosis [35]. Metabolism is one of the top dysregulated pathways in the fibrotic human kidney tubules according to a genome-wide transcriptome study on a large cohort of human samples [6]. As the key modulator during fibrosis, TGF-β1 induces Warburg-like metabolic reprogramming [35], disturbs the metabolism of amino acid, carbohydrate and lipid [6], and causes oxidative stress [38] in the tubular epithelial cells, contributing to the cellular injury and fibrogenic process in CKD. Since regulation of the metabolic pathway is also a major effect of KD025 in human cell lines [22], we hypothesized that KD025 might modulate the metabolic reprogramming of tubular epithelial cells stimulated by TGF-β1. Therefore, we used an LC-MS-based metabolomics to study the metabolic changes in the KD025-treated mPTCs stimulated with or without TGF-β1 and found 77 affected metabolites (Figure 6A). As shown by the data, glutathione metabolism was primarily altered in the TGF-β1-treated tubular epithelial cells (Figure 6B), indicating induction in the oxidative stress. TGF-β1 induced a drastic reduction in the level of glutathione (Figure 6A) and an accumulation of γ-glutamylcysteine (Figure 6A,D), the intermediate of glutathione synthesis [39], suggesting impairment in the biogenesis of glutathione. Strikingly, treatment of KD025 effectively restored the metabolic disturbance in glutathione metabolism (Figure 6C) as shown by significantly reduced γ-glutamylcysteine, which was accumulated in the TGF-β1-stimulated tubular epithelial cells (Figure 6D). This finding indicated that KD025 corrected the glutathione metabolism in the TGF-β1-stimulated tubular epithelial cells.

KD025 restored the metabolic disturbance in the TGF-β1-stimulated tubular epithelial cells

Figure 6
KD025 restored the metabolic disturbance in the TGF-β1-stimulated tubular epithelial cells

mPTCs were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) with the presence of KD025 for 24 h before harvesting for metabolical analysis (n=4). (A) Red: control, (B) green: TGF-β1, (C) blue: KD025, (D) yellow: KD025+TGF-β1. (A) Heat map of the relative levels of metabolites in the tubular epithelial cells in each group. Metabolic pathways that were affected in the TGF-β1 comparing to control group (B) and in the TGF-β1-stimulated mPTCs treated with or without KD025 (C). (D) The level of γ-glutamylcysteine, a representative metabolite in glutathione metabolism, was increased in the TGF-β1-stimulated mPTCs and was decreased by KD025. KD025 decreased the levels of representative metabolites, including homovanillic acid (E), the acidic end product in tyrosine metabolism, and increased the pantothenic acid, whose reduction was related to tubular injury (F). * means P<0.05 comparing to control. # means P<0.05 comparing to the TGF-β1 model.

Figure 6
KD025 restored the metabolic disturbance in the TGF-β1-stimulated tubular epithelial cells

mPTCs were pretreated with KD025 at the concentration of 2.5 μM for 2 h before stimulated by TGF-β1 (5 ng/ml) with the presence of KD025 for 24 h before harvesting for metabolical analysis (n=4). (A) Red: control, (B) green: TGF-β1, (C) blue: KD025, (D) yellow: KD025+TGF-β1. (A) Heat map of the relative levels of metabolites in the tubular epithelial cells in each group. Metabolic pathways that were affected in the TGF-β1 comparing to control group (B) and in the TGF-β1-stimulated mPTCs treated with or without KD025 (C). (D) The level of γ-glutamylcysteine, a representative metabolite in glutathione metabolism, was increased in the TGF-β1-stimulated mPTCs and was decreased by KD025. KD025 decreased the levels of representative metabolites, including homovanillic acid (E), the acidic end product in tyrosine metabolism, and increased the pantothenic acid, whose reduction was related to tubular injury (F). * means P<0.05 comparing to control. # means P<0.05 comparing to the TGF-β1 model.

Besides glutathione metabolism, we also found that pentose and glucuronate interconversions, starch and sucrose metabolism, and TCA cycle were changed in the TGF-β1-treated tubular epithelial cells (Figure 6B), indicating TGF-β1 disturbed the energy metabolism in the cells. Pyrimidine metabolism and amino sugar and nucleotide sugar metabolism were also affected in the TGF-β1-stimulated tubular epithelial cells. TGF-β1 also profoundly interfered with the amino acid metabolism, including alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism; valine, leucine and isoleucine degradation and tyrosine metabolism (Figure 6B). Treatment of KD025 effectively modulated the metabolic disturbance in glycolysis or gluconeogenesis, alanine, aspartate and glutamate metabolism; fructose and mannose metabolism; citrate cycle; pentose phosphate pathway; starch and sucrose metabolism; pentose and glucuronate interconversions (Figure 6C). Energy metabolism was also corrected by KD025 in the TGF-β1-stimulated cells as shown by the level of oxoglutaric acid in the TCA cycle and erythritol in the starch metabolism (Figure 6A). It should also be noted that TGF-β1 induced the accumulation of homovallinic acid, the acidic end product in the tyrosine metabolic pathway, and was corrected by KD025 (Figure 6E). It was reported that pantothenic acid was significantly reduced in the urine samples of patients with acute kidney injury [40], indicating the reduction in pantothenic acid may be correlated with tubular injury. We found that TGF-β1 induced a significant reduction in pantothenic acid in the mPTCs while KD025 restored the level of pantothenic acid (Figure 6F), suggesting KD025 protected the tubular epithelial cells from TGF-β1.

Together, these results in the metabolomics indicated that KD025 restored the metabolic disturbance in the amino acid metabolism, especially glutathione metabolism and energy metabolism induced by TGF-β1 in the mPTCs. These effects may contribute to the protection against cellular injury in the TGF-β1-stimulated tubular epithelial cells.

Inhibition of ROCK2 by KD025 alleviated oxidative stress and promoted the nuclear translocation of Nrf2

Since KD025 strikingly corrected the TGF-β1-induced disturbance of glutathione metabolism in mPTCs (Figure 6A,D), we further validated this finding in the animal model by measuring the mRNA expression of GSS, the enzyme that catalyzes γ-glutamylcysteine to synthesize glutathione [39]. We found that the GSS mRNA expression was significantly decreased in the UUO kidney (Figure 7A). KD025 significantly increased the mRNA expression of GSS in the control kidneys and the UUO kidneys comparing to the vehicle groups (Figure 7A). This finding was consistent with our metabolomics results of glutathione and γ-glutamylcysteine in the mPTCs, indicating KD025 regulated glutathione metabolism and alleviated the oxidative stress in UUO kidneys. Further, we examined the mRNA levels of superoxide dismutase 1 (SOD1), an antioxidative enzyme, in the UUO kidneys and found that SOD1 was significantly decreased in the UUO kidneys, while KD025 significantly increased it (Figure 7B). Nrf2 was the transcriptional factor regulating components in the antioxidative stress. We extracted the nuclear fraction of the mPTCs and found that KD025 significantly increased the protein level of Nrf2 in the nuclear fraction of the cells (Figure 7C,D), indicating KD025 may alleviate the oxidative stress possibly through promoting the nuclear translocation of Nrf2 to some extent.

KD025 increased the antioxidative stress enzymes in UUO model and increased the Nrf2 content in nuclear fraction of the TGF-β1-stimulated mPTCs

Figure 7
KD025 increased the antioxidative stress enzymes in UUO model and increased the Nrf2 content in nuclear fraction of the TGF-β1-stimulated mPTCs

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and homogenated for RT-PCR analysis of the mRNA expression of GSS and SOD-1. GSS (A) and SOD-1 (B) were both decreased in the UUO kidneys and significantly increased after KD025 treatment. n=9 (C,D) KD025 increased the nuclear fraction of Nrf2 in the TGF-β1-stimulated mPTCs. (C) Representative blots. (D) Quantification of Nrf2 in the nuclear fraction. n=3. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *, *** means P<0.05, 0.001 comparing to control, respectively. # means P<0.05 comparing to the UUO or TGF-β1 models.

Figure 7
KD025 increased the antioxidative stress enzymes in UUO model and increased the Nrf2 content in nuclear fraction of the TGF-β1-stimulated mPTCs

Eight-week-old C57Bl/6J mice were treated with KD025 via intraperitoneal injection once per day from the day before UUO surgery till the 7th day of UUO. The cortex of the kidney was dissected and homogenated for RT-PCR analysis of the mRNA expression of GSS and SOD-1. GSS (A) and SOD-1 (B) were both decreased in the UUO kidneys and significantly increased after KD025 treatment. n=9 (C,D) KD025 increased the nuclear fraction of Nrf2 in the TGF-β1-stimulated mPTCs. (C) Representative blots. (D) Quantification of Nrf2 in the nuclear fraction. n=3. The data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *, *** means P<0.05, 0.001 comparing to control, respectively. # means P<0.05 comparing to the UUO or TGF-β1 models.

Inhibition of ROCK2 by KD025 inhibited the pro-inflammatory response in the LPS-activated macrophages

The infiltration and activation of inflammatory cells, such as macrophages, followed by tissue damage, is an early event and a major driving force for fibrogenesis [3]. Pro-inflammatory macrophages, or M1 macrophages, are known to mediate inflammatory response and promote renal fibrosis [41]. Previous studies have found that almost all the infiltrating cells at the early stage of UUO were M1 macrophages [41]. In the present study, we also observed that KD025 attenuated the infiltration of macrophages in UUO kidneys. Thus, we studied the direct effect of KD025 on the inflammatory response in the macrophages. We activated macrophages (RAW264.7 cells) with LPS for 24 h. LPS induced profound inflammation in the macrophages, characterized by significant cell proliferation (Figure 8A) and drastic mRNA expression and the release of pro-inflammatory cytokines such as IL-1β (Figure 8B,C) in the macrophages. At the concentration of 2.5 μM, KD025 significantly prevented the cell proliferation induced by LPS without showing the cytotoxicity (Figure 8A), indicating 2.5 μM of KD025 inhibited the inflammatory response induced by LPS. Consistently, KD025 at the concentration of 2.5 μM also significantly decreased the release of IL-1β induced by LPS. These data from the macrophages demonstrated the direct anti-inflammatory effect of KD025, which was consistent with the inhibited inflammatory response found in KD025-treated UUO kidneys.

KD025 inhibited the inflammatory response in the LPS-activated macrophages

Figure 8
KD025 inhibited the inflammatory response in the LPS-activated macrophages

The RAW264.7 macrophages were treated with KD025 at the concentration of 2.5 μM for 2 h before stimulated with LPS (100 ng/ml) for 24 h with the presence of KD025. The cell proliferation was studied by CCK8 assay and the release of IL-1β was studied by ELISA. (A) The proliferation of RAW264.7 cells was significantly increased after 24 h of LPS exposure. KD025 at 2.5 μM significantly prevented the cell proliferation induced by LPS without showing significant cytotoxicity in the naïve macrophages. n=6. KD025 significantly decreased the mRNA expression (B) and the release (C) of IL-1β induced by LPS. n=3. All the data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *** means P<0.001 comparing to control. ## means P<0.01 comparing to the LPS model.

Figure 8
KD025 inhibited the inflammatory response in the LPS-activated macrophages

The RAW264.7 macrophages were treated with KD025 at the concentration of 2.5 μM for 2 h before stimulated with LPS (100 ng/ml) for 24 h with the presence of KD025. The cell proliferation was studied by CCK8 assay and the release of IL-1β was studied by ELISA. (A) The proliferation of RAW264.7 cells was significantly increased after 24 h of LPS exposure. KD025 at 2.5 μM significantly prevented the cell proliferation induced by LPS without showing significant cytotoxicity in the naïve macrophages. n=6. KD025 significantly decreased the mRNA expression (B) and the release (C) of IL-1β induced by LPS. n=3. All the data were examined by two-way ANOVA followed by Sidak post hoc test and presented as mean + SEM in the bar chart. *** means P<0.001 comparing to control. ## means P<0.01 comparing to the LPS model.

Discussion

Renal tubulointerstitial fibrosis contributes to the pathological progression of CKD and is a clinical predictor of end-stage kidney disease. However, this progressive, usually irreversible histological change lacks effective therapeutic intervention or drug targets. Although pan-ROCK inhibition shows a protective effect in mice model [16], ROCK1 knockout mice are not protected from it [18]. Since ROCK2 knockout mice mostly die in utero [9], deciphering the implication of ROCK2 in renal interstitial fibrosis could benefit from the selective inhibitor. KD025 is a first-in-class selective ROCK2 inhibitor that showed no off-target effect on 300 other kinases or receptors, including ROCK1 and those that are known to be affected by pan-ROCK inhibitors [23]. It is safe for humans in Phase I clinical trial and is currently in Phase II clinical trials for autoimmune disorders and IPF [42]. In the present study, we found that ROCK2 was significantly up-regulated in the fibrotic kidneys from both the pediatric and adult patients and in the mice UUO model. The protein level of ROCK2 was positively correlated with the severity of the interstitial fibrosis. Inhibition of ROCK2 by KD025 effectively alleviated the tubulointerstitial fibrosis in the mice UUO model. Since ROCK2 was promptly increased in the proximal tubules in the fibrotic kidneys in both the patient samples and the UUO kidneys, we studied the direct effect of ROCK2 on the pro-fibrotic response in the tubular epithelial cells. Knockdown and knockout of ROCK2 and KD025 prevented the pro-fibrotic effect of the tubular epithelial cells stimulated by TGF-β1, the fibrogenic stimulus. KD025 showed putative effect in correcting the metabolic disturbance in glutathione metabolism and energy metabolism in the TGF-β1-stimulated tubular epithelial cells, which may contribute to the inhibitory effect on the pro-fibrotic response induced by TGF-β1. KD025 significantly increased the expression of GSS and increased SOD1 in the fibrotic kidneys possibly via activating the nuclear translocation of Nrf2. Besides, KD025 showed potent anti-inflammatory activity in the UUO model and the LPS-activated macrophages. These data collectively suggest the therapeutic potential of KD025 in renal tubulointerstitial fibrosis and indicated that ROCK2 may be a candidate of drug target in treating tubulointerstitial fibrosis.

Tubulointerstitial fibrosis is a dynamic and convergent process, featured with dysfunction of tubular epithelial cells and deposition of the extracellular matrix [3,7]. Sustained inflammation, either primary or subsequently triggered by tissue injury, promotes the progression of fibrosis [3]. UUO model exhibits typical renal fibrosis independent of hypertension or systemic immune disorder, characterized by the fibrotic pathology in Masson’s trichrome staining, the up-regulation of fibrogenic markers, and inflammation in the obstructed kidney [41]. We found that ROCK2 protein level was positively correlated with the tubulointerstitial fibrosis in the pediatric and adult CKD patient samples and was time-dependently increased in the UUO model, indicating that ROCK2 may be implicated in the tubulointerstitial fibrosis. In the renal cortex of mice treated with 50 mg/kg KD025 via intraperitoneal injection, the phosphorylation of coffilin was significantly inhibited, indicating suppression of ROCK signaling. Further analysis demonstrated that inhibition of ROCK2 by KD025 significantly prevented the renal fibrosis in the UUO model as shown by improved fibrotic pathology in Masson’s trichrome staining, decreased collagen I deposition in the renal cortex, and the reduced levels of fibrotic genes, including fibronectin, α-sma, collagen I, collagen III, and vimentin. These findings suggest that targeting ROCK2 via pharmacological inhibition by KD025 is a potential therapy for renal fibrosis in CKD. The inhibition of ROCK, composed by ROCK1 and ROCK2, has shown the potential to be a therapy against tubulointerstitial fibrosis in abundant studies. The non-selective ROCK inhibitors such as fasudil and Y27632 demonstrated their therapeutic efficacy in renal tubulointerstitial fibrosis in the UUO model, shown by the alleviated the fibrosis pathology and decreased the expression of fibrotic markers and the production of extracellular matrix [12,16]. ROCK1 may not be fundamentally involved in the TGF-β1-mediated renal fibrosis, according to the finding using ROCK1 knockout mice [18]. The implication of ROCK2 in tubulointerstitial fibrosis was unknown because neither the global knockout nor knockdown mouse model was feasible to clarify the contribution of ROCK2 to the pathological progression of renal interstitial fibrosis. The ROCK2 knockout fetuses were 90% dead in utero due to a perturbed embryo–placenta interaction unless they were bred on to a CD-1 (C57BL/6 × Dba) background [9]. Partial deletion of ROCK2 was not able to affect its activity due to the compensatory effect [19]. KD025 is a first-in-class ROCK2 selective inhibitor, which is highly selective to ROCK2 among 300 other intracellular kinases and surface receptors, including ROCK1 and those inhibited by fasudil [23]. Therefore, proving the efficacy of KD025 against renal fibrosis also helps to determine the implication of ROCK2 in pathogenesis of renal fibrosis. Recently, it was found that KD025 protected the glomerulosclerosis and the fibrosis of the mesengial cells in the db/db mice [43], indicating the therapeutic potential of KD025 on diabetic nephropathy. We showed the protective effect of KD025 on the renal tubulointerstitial fibrosis, an inevitable pathological outcome for almost all the progressive CKDs. Therefore, KD025 is a potential therapeutic candidate for the renal tubulointerstitial fibrosis.

In the fibrotic kidneys from patient samples and UUO model, our immunohistochemical analysis demonstrated that ROCK2 was mainly up-regulated in the tubular epithelial cells, which suggests that ROCK2 might be implicated in the fibrosis of the tubular epithelial cells during renal fibrosis. Therefore, we further studied the direct effect of ROCK2 and KD025 in the tubular epithelial cells by stimulating mPTCs cells with TGF-β1 and explored the underlying mechanism. Consistently, we found that TGF-β1 significantly increased the protein level of ROCK2 in the mPTCs. The results from the Western blotting, RT-PCR, and immunofluorescent analysis showed that ROCK2 knockdown, knockout, or inhibition via KD025 significantly inhibited the pro-fibrotic response, including the expression of fibrotic genes and the activation of Smad2, the fibrotic signaling, in the tubular epithelial cells triggered by TGF-β1. These data indicated that ROCK2 was implicated in the pro-fibrotic response in the tubular epithelial cells, and KD025 alleviated the renal fibrosis in the UUO model by directly preventing the pro-fibrotic effect in the tubular epithelial cells. Consistently, previous studies revealed that fasudil protected the tubular epithelial cells NRK-52E from the stimulation of TGF-β1 by significantly decreasing the mRNA expression of collagen I and α-sma [16]. Fasudil also inhibited the epithelial–mesangial transition (EMT) of human tubular epithelial cells HK-2 cells induced by high glucose [44]. Inhibiting Rho, the upstream kinase that directly activates ROCK, has been shown to inhibit the EMT of renal tubular epithelial cells [45]. Therefore, our results revealed that KD025 alleviated the renal fibrosis in the UUO model through protecting the tubular epithelial cells.

Our data also indicated that KD025 exerted antifibrotic action mainly via inhibiting ROCK2. First, in the UUO kidneys, KD025 significantly decreased the phosphorylation of coffilin, the downstream substrate of ROCK signaling [25]. KD025 was selective to ROCK2, rather than ROCK1, in cell-free assay [23]. Its inhibitory potency was 1–10 μM and over 200 μM against ROCK2 and ROCK1, respectively [22]. The inhibitory effect on the phosphorylation of coffilin was likely due to ROCK2 inhibition. KD025 also significantly decreased the protein level of ROCK2 in the TGF-β1-stimulated mPTCs. This result was consistent with the publication showing reduced ROCK2 level in the skin sample of psoriatic patients treated with KD025 for 12 weeks [42]. Second, we found that KD025 plus ROCK2 siRNA did not offer an additive effect on antagonizing the TGF-β1-induced fibrotic response in mPTCs. This result indicated that KD025 prevented the fibrotic response in mPTCs induced by TGF-β1 via inhibiting ROCK2.

The fibrotic renal tissue is highlighted with disturbed cellular metabolism in β-oxidation, amino acid catabolism, carbohydrate metabolism, and fatty acid metabolism [6]. In the urine samples of UUO rats, glycine, serine and threonine metabolism, retinol metabolism, arginine and proline metabolism, and fructose and mannose metabolism are primarily affected [46]. Metabolic rearrangement in the tubular epithelial cells is one of the most representative pathological changes during renal fibrosis, according to a genome-wide transcriptome study using a large cohort of human fibrotic samples [6]. A microarray study demonstrated that KD025 modulated 122 genes in metabolism in the primary culture of human smooth muscle cells [22]. Therefore, KD025 may regulate cellular metabolism in the TGF-β1-stimulated cells. We employed metabolomics to investigate the effect of KD025 in the TGF-β1-stimulated mPTCs and found changes in the amino acid metabolism and glucose metabolism of the TGF-β1-stimulated tubular epithelial cells. Noteworthy, glutathione metabolism was profoundly altered by TGF-β1 and restored by KD025. Among the affected metabolites in the glutathione metabolism pathway, TGF-β1 induced a significant accumulation of γ-glutamylcysteine, the intermediate for glutathione synthesis [39] a significant decrease in the glutathione, indicating that TGF-β1 provoked oxidative stress in the tubular epithelial cells. KD025 corrected the changes in the glutathione metabolism pathway by decreasing the level of γ-glutamylcysteine in the TGF-β1-stimulated cells. To confirm the effect of KD025 on oxidative stress represented by disturbed glutathione metabolism, we performed additional examination and found that KD025 significantly increased the mRNA expression of GSS, the enzyme that catalyzes γ-glutamylcysteine to glutathione, which was significantly decreased in the UUO kidney. Meanwhile, KD025 also up-regulated the mRNA expression of SOD-1 in UUO kidney and increased the nuclear content of Nrf2 in TGF-β1-stimulated mPTCs. These data in the animal model and cell cultures supported the finding in metabolomics, demonstrating that KD025 corrected the glutathione metabolism and oxidative stress in the TGF-β1-stimulated tubular epithelial cells. Our metabolomics study also revealed that KD025 significantly increased the pantothenic acid in mPTCs, while TGF-β1 significantly decreased it in a pattern similar to acute kidney injury [40]. This result indicated that KD025 might protect mPTCs from cellular injury. Pathway analysis revealed that the energy metabolism was also impaired by TGF-β1 and corrected by KD025, indicating the KD025 restored the homeostasis of the energy metabolism in the tubular epithelial cells. These results demonstrated that KD025 corrected the metabolic disturbance in the TGF-β1-stimulated tubular epithelial cells.

The infiltration of activated inflammatory cells, including the M1 macrophages, was triggered by tissue damage in CKD. It is an early event of fibrogenesis because the nonresolved and persistent inflammation may further damage the cells and tissue [3]. UUO model is featured with the infiltration of activated macrophages. The M1 macrophage, which expresses a high level of iNOS in response to the activation of Toll-like receptor, is the key factor for the induction of inflammatory injury during the acute phase of renal injury, which subsequently promotes the progress of tubulointerstitial fibrosis by inducing an inflammatory response [41]. The M2 macrophage is implicated in tissue repairment and wound healing. It has been found that M2 enriched gene expression was correlated with the progression of renal fibrosis [34]. Indeed, we found that UUO model manifested with drastic inflammatory response, demonstrated by significant infiltration of macrophages in the tubulointerstitium as indicated by F4/80 staining in the immunohistochemical analysis, elevated mRNA expression of pro-inflammatory cytokines and mediators, IL-6, IL-1β, TNF-α and iNOS, and the increased mRNA level of M2 macrophages marker, CD206, and M2 macrophage-enriched genes Arg and YM-1. To further study the anti-inflammatory effect of KD025 in the activated macrophages, we treated the LPS-activated macrophages with KD025 and found that KD025 significantly inhibited the proliferation of the cells and the release of IL-1β in the LPS-activated RAW 264.7 macrophages. ROCK2 is a crucial regulator of the immune response. Abundant studies have revealed that KD025 effectively balanced the pro-inflammatory and immunosuppressive subtypes of T cells and suppressed the release of pro-inflammatory cytokines in patients with autoimmune diseases [23], including systemic lupus erythematosus [24] and psoriasis vulgaris [23,42]. Inhibition of ROCK by fasudil significantly decreased the macrophage infiltration to UUO kidney [16]. Selective inhibition of ROCK2 is more effective in preventing the macrophage infiltration to the injured macular tissue comparing to fasudil [47]. Therefore, the anti-inflammatory effect of KD025 is beneficial for preventing renal interstitial fibrosis since it decreased the fibrogenic stimulation.

Taken together, our data collectively demonstrated that KD025 may be a potential therapy for renal tubulointerstitial fibrosis probably through correcting the metabolic disturbance and inhibiting inflammation.

Clinical perspectives

  • Pan-ROCK inhibitors showed their therapeutic potential in the tubulointerstitial fibrosis of CKD. However, the renal fibrosis in ROCK1 knockout mice was not affected by ROCK1 deletion. Since ROCK2 knockout mice mostly die in utero, whether ROCK2 is a proper target for the treatment of tubulointerstitial fibrosis needs to be investigated.

  • KD025 is a novel selective ROCK2 inhibitor. Here we showed that ROCK2 was up-regulated in fibrotic kidneys of CKD patients and animals, and inhibition of ROCK2 by KD025 or genetic approaches effectively alleviated tubulointerstitial fibrosis, corrected the metabolic disturbance in the tubule epithelial cells, and inhibited the pro-inflammatory effect of activated macrophages.

  • The findings from patient samples, animal models, and cell cultures collectively indicated that KD025 which is safe to human in the Phase I clinical trial may be an effective therapeutic drug for renal tubulointerstitial fibrosis.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Key Research and Development Program [grant number 2016YFC0906103]; the National Natural Science Foundation of China [grant numbers 81800598, 81530023, 81830020, 81873599, 81670647]; the Jiangsu High-Level Innovation and Entrepreneurship Talent Introduction Program [2019SCBS001]; the Nanjing Merit-based Funding for Science and Technology Innovation Projects for Overseas Scholars (to R.Y.); and the Postdoctoral Funding [grant number 2019M651906].

Author Contribution

Z.J., A.Z., and R.Y. designed the experiments, analyzed the data, and wrote the manuscript text. R.Y., W.Z., and Y.L performed the experiments and prepared the figures. Y.Z. and S.H. contributed to the protocols and technical advice. Z.J., A.Z., and S.H. helped to craft the final manuscript, and all authors reviewed the manuscript.

Abbreviations

     
  • Cas9

    CRISPR-associated protein 9

  •  
  • CKD

    chronic kidney disease

  •  
  • CRISPR

    clustered regularly interspaced short palindromic repeats

  •  
  • dd water

    double distilled water

  •  
  • DMEM/F12

    dulbcco's modified eagle's medium/nutrient mixture F-12

  •  
  • EMT

    epithelial–mesangial transition

  •  
  • ESI-MSn

    electrospray ionization tandem mass spectrometry

  •  
  • FBS

    fetal bovine serum

  •  
  • GSS

    glutathione synthetase

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HCD

    higher energy collision dissociation

  •  
  • IL-1β

    interleukin-1β

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • IPF

    idiopathic pulmonary fibrosis

  •  
  • LPS

    lipopolysaccharide

  •  
  • mPTC

    mouse renal proximal tubular epithelial cell

  •  
  • m/z

    Mass-to-Charge ratio

  •  
  • Nrf2

    nuclear erythroid 2-related factor 2

  •  
  • PBS

    phosphate buffer saline

  •  
  • PFA

    paraformaldehyde

  •  
  • ROCK

    Rho-associated kinase

  •  
  • RIPA

    radio immunoprecipitation assay

  •  
  • sgRNA

    small guide RNA

  •  
  • SOD1

    superoxide dismutase 1

  •  
  • TGF-β1

    transforming growth factor-β1

  •  
  • TNF-α

    tumor necrosis factor-α

  •  
  • UUO

    unilateral ureteral obstruction

  •  
  • α-sma

    alpha-smooth muscle actin

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Author notes

*

These authors contributed equally to this work.