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(Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

Background & Aims Activation of the (pro)renin receptor (PRR) up-regulates the expression of profibrotic genes in the kidney and heart. We aimed to investigate the role of PRR in hepatic fibrogenesis. Methods Human hepatic PRR levels were measured in patients with or without liver fibrosis. PRR expression was analyzed in primary mouse hepatic stellate cells (HSCs). Experimental fibrosis was studied in thioacetamide (TAA)-treated or methionine choline-deficient (MCD) diet-fed C57BL/6 mice. Lentivirus-mediated PRR short hairpin RNA was used to knockdown hepatic PRR expression. Lentiviral vectors expressing PRR short hairpin RNA or complementary DNA from the α-smooth muscle actin promoter were used for myofibroblast-specific gene knockdown or overexpression. Results PRR is up-regulated in human and mouse fibrotic livers, and in activated HSCs. Hepatic PRR knockdown reduced liver fibrosis by suppressing the activation of HSCs and expression of profibrotic genes in TAA or MCD diet–injured mice without significant changes in hepatic inflammation. Renin and prorenin increased the expression of PRR and production of TGF-β1 in human activated HSC Lieming Xu-2 cells, and knockdown of PRR inactivated Lieming Xu-2 cells with decreased production of transforming growth factor (TGF)-β1 and Mothers against decapentaplegic homolog 3 (Smad3) phosphorylation. Myofibroblast-specific PRR knockdown also attenuated liver fibrosis in TAA or MCD diet–injured mice. Mice with both myofibroblast-specific and whole-liver PRR knockdown showed down-regulation of the hepatic extracellular signal-regulated kinase (ERK)/TGF-β1/Smad3 pathway. Myofibroblast-specific PRR overexpression worsened TAA-induced liver fibrosis by up-regulating the ERK/TGF-β1/Smad3 pathway. Conclusions PRR contributes to liver fibrosis and HSC activation, and its down-regulation attenuates liver fibrosis through inactivation of the ERK/TGF-β1/Smad3 pathway. Therefore, PRR is a promising therapeutic target for liver fibrosis.

H epatic fibrosis results from chronic liver injury caused by viral infection, alcohol abuse, metabolic disorder, or immune attack. Hepatic fibrosis is a critical pre-stage in the development of cirrhosis, which causes severe morbidity and mortality. 1 After chronic injury, the liver undergoes a repair process accompanied by inflammation and accumulation of extracellular matrix. Hepatic stellate cells (HSCs), the major collagenproducing cells, are responsible for liver fibrosis. 2 Damaged hepatocytes release inflammatory cytokines that activate Kupffer cells (KCs) and recruit inflammatory immune cells, which in turn secrete cytokines, chemokines, and reactive oxygen species to stimulate the activation of resident HSCs from a quiescent state into myofibroblast-like cells, thereby acquiring proliferative, proinflammatory, and fibrogenic properties. 3,4 In addition, the hepatocytic apoptotic bodies resulting from various injuries are engulfed by KCs and HSCs, thereby promoting the expression of profibrogenic proteins. 5 Activated HSCs up-regulate interstitial collagen production, increase synthesis of tissue inhibitor of metalloproteinase-1 (TIMP1), and decrease the production of fibrolytic metalloproteinases, leading to liver fibrosis. 3,4 Besides the treatment of underlying liver diseases, new antifibrotic drugs are being developed to target liver metabolism, apoptosis, inflammation, and fibrosis remodeling. Nevertheless, only a minority of patients achieve treatment response. 6 Therefore, novel effective and safe antifibrotic treatments are needed urgently.
(Pro)renin receptor (PRR), also known as adenosine triphosphatase H(þ)-transporting lysosomal accessory protein 2, is a newly identified single-pass transmembrane protein encoded by the ATP6AP2 gene in human beings, which is expressed in a variety of organs, including the heart, brain, placenta, kidneys, and liver. 7,8 Binding of PRR to renin or prorenin increases the catalytic efficiency for angiotensin II generation 7,8 and directly triggers intracellular signal transduction, leading to activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) by a angiotensin II-independent mechanism, thereby upregulating the expression of profibrotic genes such as transforming growth factor (TGF)-b1, fibronectin, collagen I, and plasminogen activator inhibitor 1 (PAI-1). 9,10 PRR has been shown to contribute to fibrosis in the heart and kidney. 11,12 Levels of serum-soluble PRR correlate with the severity of glomerulosclerosis in patients with nephritis. 12 In in vitro studies, renin increased mesangial cell TGF-b1 and matrix protein production through a PRR-mediated mechanism. 10 These findings provide new insights into the role of PRR in tissue remodeling.
Although PRR is expressed in the liver, 7 its role in hepatic fibrogenesis has not been investigated. In this study, we investigated the role of PRR in liver fibrosis by targeting PRR in the liver, specifically in the HSCs. Our data suggest that PRR contributes to hepatic fibrogenesis, and inhibition of PRR signaling represents a potentially novel therapeutic strategy in the regression of liver fibrosis.

(Pro)renin Receptor Is Up-regulated in Human and Mouse Fibrotic Livers and in Activated HSCs
To examine whether PRR has a role in liver fibrosis, we first measured the PRR expression in the livers of patients with chronic fibrosis. As shown in Figures 1A and 2A, hepatic PRR protein levels were significantly higher in patients with fibrosis and increased with fibrosis progression. Plasma renin levels also were higher in patients with liver fibrosis ( Figure 1B). PRR expression was barely detectable in nonfibrotic human liver, but was more abundant in fibrotic areas in the fibrotic human livers, and PRR immunostaining co-localized predominantly with that of asmooth muscle actin (a-SMA) ( Figure 1C).
To further confirm the link between PRR and liver fibrosis, we used a fibrosis animal model. In the liver of mice treated with thioacetamide (TAA) for 8 weeks, PRR expression level was up-regulated significantly ( Figure 1D). Given that the expression of PRR has not been studied in liver cells, we isolated primary cells from normal mouse livers and found that PRR was expressed in hepatocytes and nonparenchymal cells ( Figure 2B). To specify the location of PRR up-regulated in response to fibrotic stimuli, hepatic PRR expression was examined by immunofluorescence; the fluorescence intensity of PRR was augmented markedly in fibrotic liver, and found to mostly co-localize with a-SMA ( Figure 1E), as a positive control, PRR immunofluorescence in mouse kidney is shown in Figure 2C. Because activated HSCs play a key role in the development and progression of liver fibrosis, we analyzed the transcriptional changes of PRR that occur during HSC activation in mouse-derived primary cultures. A significant increase in the messenger RNA (mRNA) expression of PRR was detected in cultureactivated and in vivo-activated HSCs isolated from TAAtreated mice compared with quiescent HSCs ( Figure 1F and G).
These data indicate that the expression of PRR is upregulated in fibrotic liver and highly expressed in activated HSCs.

PRR Knockdown Attenuated TAA-Induced Liver Fibrosis
To investigate whether PRR knockdown plays a therapeutic role in liver fibrosis, the effects of PRR knockdown on liver fibrosis were examined. Lentivirus-delivered PRRspecific short hairpin RNA (shRNA) was administered intravenously to inhibit hepatic PRR expression, and a scrambled sequence shRNA was used as a control. PRR shRNA virus significantly suppressed PRR mRNA expression in all isolated primary liver cells ( Figure 3).
PRR and scramble-shRNA virus-treated mice showed similar alanine aminotransferase (ALT) levels and severity of liver necroinflammation ( Figure 4A and 4B), as well as hepatic expression of proinflammatory cytokines ( Figure 4C) after TAA injury. In addition, no significant alternations were observed in hepatic infiltration of Kupffer cells (KCs), neutrophils, natural killer cells, or invariant nature killer T cells, which were determined by F4/80, myeloperoxidase, Natural killer cell p46-related protein immunostaining, and hepatic Va14Ja18 mRNA expression, respectively ( Figure 4D-G).
The effect of PRR knockdown in activated macrophages was investigated using small interference RNA (siRNA) targeting PRR and lipopolysaccharide (LPS) supplementation in the mouse macrophage cell line, RAW 264.7. PRR siRNA significantly inhibited PRR expression in RAW 264.7 cells ( Figure 5A). However, the expression of interleukin (IL)1b, IL6, tumor necrosis factor (TNF)-a, and C-C Motif Chemokine Ligand 2 did not decrease after PRR knockdown in these cells ( Figure 5B). Representative Western blot of hepatic PRR from patients without liver fibrosis (normal), and with mild and severe fibrosis (Ishak scores 1~3 and 4~6, respectively). The Western blots of hepatic PRR from other patients are shown in Figure 2A. Right: Quantification of hepatic PRR expression of the total patients (normal, n ¼ 12; mild fibrosis, n ¼ 15; severe fibrosis, n ¼ 14). *P < .05 vs normal group; # P < .05 vs mild fibrosis group. (B) Plasma renin levels in patients without liver fibrosis, and with mild and severe fibrosis. *P < .05 vs normal group. (C) Immunohistochemical staining for PRR in normal and fibrotic human livers with various etiologies, and dual immunofluorescence staining in normal and fibrotic human livers (PRR, red; a-SMA, green; diamidino-2-phenylindole [DAPI] nuclear stain, blue). (D) Western blot analysis of PRR level in the livers of mice treated with TAA (n ¼ 6) or vehicle (n ¼ 5). (E) Hepatic dual immunofluorescence in TAA mouse fibrosis model (PRR, red; a-SMA, green; DAPI, blue). (F) mRNA expression of PRR in quiescent (n ¼ 5) and culture-activated (n ¼ 8) HSCs. *P < .05 vs quiescent HSC group. (G) mRNA expression of PRR in quiescent HSCs from normal mice treated with vehicle (NV) (n ¼ 6) and in vivo-activated HSCs from TAA-injured mice (n ¼ 5). *P < .05 vs NV group. Scale bars: 100 mm. a-HSC, activated hepatic stellate cell; CHB, chronic hepatitis B; CHC, chronic hepatitis C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NASH, nonalcoholic steatohepatitis; q-HSC, quiescent hepatic stellate cell. PRR shRNA virus successfully knocked down hepatic PRR expression in fibrotic livers ( Figure 6A) and reduced PRR co-localization with a-SMA ( Figure 7A). Hepatic renin transcript levels increased after TAA injury, and PRR knockdown modestly reduced the up-regulation of hepatic renin induced by TAA ( Figure 6A). Notably, knockdown of PRR attenuated liver fibrosis in TAA-treated mice, as determined by Sirius red staining ( Figure 6B) and liver hydroxyproline levels ( Figure 6C). The antifibrotic effect of PRR knockdown was supported by the reduction in hepatic a-SMA expression ( Figures 6D and 7B), and transcript levels of collagen 1a1, TIMP1, and PAI-1 ( Figure 6E).
These data suggest that the antifibrotic effect of PRR knockdown may be in part through inactivation of HSCs via the down-regulation of the ERK/TGF-b1/Smad3 pathway, and less likely through inactivation of KCs or decreased liver inflammation.

PRR Knockdown Attenuated Methionine Choline-Deficient Diet-Induced Liver Fibrosis
The antifibrotic effect of PRR knockdown was validated further in a methionine choline-deficient (MCD) diet mouse model. A significant increase in PRR mRNA levels was detected in the in vivo-activated HSCs isolated from 12-week MCD diet-fed mice compared with quiescent HSCs ( Figure 8A). Hepatic PRR expression was upregulated and co-localized with a-SMA ( Figure 8B) in the mice fed with MCD diet. PRR shRNA virus reduced hepatic PRR expression in fibrotic livers ( Figure 8C), as well as liver fibrosis ( Figure 8D and E). PRR knockdown also modestly decreased the up-regulation of hepatic renin induced by an MCD diet ( Figure 8C). As in the TAA model, PRR shRNA virus reduced hepatic a-SMA expression ( Figure 8F) and transcript levels of collagen 1a1 and TIMP1 ( Figure 8G). In addition, PRR knockdown attenuated the ERK/TGF-b1/Smad3 pathway in MCD mice ( Figure 8H) without significantly altering liver inflammation and steatosis ( Figure 9).

Myofibroblast-Specific Knockdown of PRR Ameliorated Liver Fibrosis in Mice Treated With TAA or MCD Diet
Given that suppressed PRR expression was observed in all isolated primary liver cells in the hepatic PRR knockdown experiment, we cannot exclude that the antifibrotic effect of PRR knockdown may be mediated partially by hepatocytes, KCs, or sinusoidal endothelial cells (SECs). To focus on the effects of PRR knockdown in activated HSCs/ myofibroblasts on liver fibrosis, we further generated a lentiviral construct comprising the gene encoding PRRspecific shRNA within an optimized shRNA-microRNA system, 13 downstream of the a-SMA promoter ( Figure 11A), and the packaged virus vectors were administered to mice. Successful delivery to the myofibroblasts was confirmed in vivo by co-localization of green fluorescent protein (GFP) with a-SMA-positive cells ( Figure 11B) and was verified by GFP expression in isolated HSCs with decreased PRR levels ( Figure 11C). Isolated hepatocytes, KCs, and SECs did not show decreased PRR expression levels, indicating myofibroblast-specific knockdown ( Figure 12A). In Sirius red staining and hepatic hydroxyproline assay, knockdown of PRR, specifically in myofibroblasts, significantly attenuated TAA-induced collagen deposition ( Figure 11D and E). ALT levels (SMA-GFP microRNA [miR]-scramble group: 232.2 ± 70.5 vs SMA-miR-shPRR: 196.4 ± 101.0 U/L; P ¼ .18) and hepatic renin mRNA levels ( Figure 12B) were similar between the groups.

Myofibroblast-Specific Overexpression of PRR Exacerbated TAA-Induced Liver Fibrosis
Conversely, we examined whether myofibroblastspecific overexpression of PRR aggravates fibrosis in vivo. A lentiviral construct expressing PRR downstream from the a-SMA promoter was constructed ( Figure 14A) and injected into mice. Successful delivery of the histidine (His)tagged PRR vector to the activated isolated HSCs with the overexpression of PRR was shown ( Figure 14B). Hepatic PRR expression was increased after the administration of the SMA-PRR-His virus ( Figure 14C). Myofibroblast-specific overexpression of PRR modestly increased hepatic renin transcript levels in TAA-injured mice ( Figure 14C). Similar PRR mRNA levels were observed in isolated hepatocytes, KCs, and SECs from SMA-PRR-His and SMA-scramble mice ( Figure 12D). Myofibroblast-specific overexpression of PRR significantly aggravated TAA-induced liver fibrosis ( Figure 14D and E), with an increase in hepatic collagen 1a1, PAI-1, and fibronectin transcript levels ( Figure 14F), as well as a-SMA levels ( Figure 14G). In addition, overexpression of PRR in myofibroblasts up-regulated the ERK/ TGF-b1/Smad3 pathway in TAA-injured livers ( Figure 14G).

Discussion
In this study, we first showed that PRR expression was increased notably in human and mouse fibrotic livers, and in activated HSCs, and knockdown of PRR induced a marked antifibrotic effect in TAA-and MCD diet-induced mouse models of liver fibrosis with attenuation of HSC activation. The role of the PRR pathway in liver fibrosis, through the activation of HSCs, was shown further in in vitro experiments, and myofibroblast-specific PRR knockdown/overexpression models. These results suggest that therapeutic modulation of PRR is therefore a promising approach for liver fibrosis regression ( Figure 15).
Currently, a few novel antifibrotic drugs are being tested in clinical trials targeting liver metabolism, apoptosis, or inflammation with modest efficacy. 6 Because of the complex  networks contributing to liver fibrosis, therapeutics against PRR combined with other promising drugs targeting different mechanisms may achieve a synergistic response with better efficacy. Recently, in vivo inhibition of type 1 collagen synthesis in HSCs was achieved using collagen 1 alpha 1 (Col1a1) siRNA complexed in lipoplexes. 14 Another study using a vitamin A-coupled liposome to deliver siRNA against a collagen-specific chaperone, heat shock protein-47, showed remarkable specificity in animal models. 15 In our study, the promising results observed with the myofibroblast-specific PRR knockdown in liver fibrosis may be translated into clinical therapeutics through these impressive HSC-targeted approaches in the future. In this study, knockdown of PRR expression in the whole liver also improved fibrosis without detrimental effects, suggesting the therapeutic potential of PRR antagonists in clinical practice. However, no reliable PRR antagonists currently are available. 16 The first PRR blockers, handle region peptides, 17 have been shown to improve diabetic nephropathy and cardiac fibrosis 11,17 ; however, other studies showed conflicting results 18,19 and partial PRR agonistic properties. 20 Another new competitive PRR antagonist, the first 20 amino acids of the prorenin prosegment (PRO20), has been shown to block proreninmediated PRR function; however, the effect of PRO20 on blocking renin-mediated PRR activation is unknown. 16 Therefore, a reliable PRR antagonist needs to be developed soon to serve as a possible antifibrotic agent.
PRR was found to be up-regulated in activated HSCs in this study. PRR specifically binds renin and prorenin, and activation of PRR triggers intracellular signal transduction, which activates the ERK1/2 pathway, leading to upregulation of profibrotic genes, including TGF-b1, PAI-1, collagen, and fibronectin. 9,10 Previous studies have found that intracellular signaling requires minimal binding of PRR, and ERK-1/2 activation was observed with renin and prorenin concentrations as low as 1 pmol/L. 21,22 Bataller et al 23 reported that activation of HSCs in vivo and in vitro is associated with increased expression of renin. Therefore, in activated HSCs with up-regulated PRR expression, the activation of intracellular PRR signaling, either by circulating renin 24 or locally increased renin production, 23 could be relevant and participate in hepatic tissue remodeling.
In this study, PRR knockdown in LX-2 cells resulted in significant down-regulation of profibrotic genes and decreased TGF-b1 production. TGF-b1 is a potent fibrogenic cytokine, and HSCs respond to fibrogenic stimuli by producing TGF-b1, which induces phosphorylation of the downstream SMAD proteins, predominantly Smad3, leading to collagen production. 25,26 Previous studies have shown that PRR induces the expression of TGF-b1 and other profibrogenic markers through an ERK-dependent pathway in various renal disease models. 9,10,27,28 The addition of an ERK inhibitor and a neutralizing TGF-b1 antibody, in the current study, confirmed that knockdown of PRR in LX-2 cells led to HSC inactivation by impeding the ERK/TGF-b1/Smad3 pathway. To further confirm the relevance of PRR signaling in HSCs in hepatic fibrogenesis, we generated lentiviral vectors to target PRR in myofibroblasts in vivo. Interestingly, myofibroblast-specific PRR knockdown significantly attenuated liver fibrosis in both TAA and MCD diet models with reduced profibrotic gene expression and suppressed the ERK/TGF-b1/Smad3 pathway. These findings were consistent with those of the in vitro study. On the other hand, myofibroblast-specific overexpression of PRR aggravated liver fibrosis by up-regulating the ERK/TGF-b1/ Smad3 pathway. These findings indicate that PRR in myofibroblasts contributes to the progression of liver fibrosis.
Although PRR also is expressed in KCs and knockdown of PRR in rat mesangial cells attenuated the expression of proinflammatory cytokines, 29 and intravitreal knockdown of PRR with shRNA reduced retinal mRNA expression of IL6, C-C Motif Chemokine Ligand 2, and TNF-a in mice with ocular inflammation, 30 hepatic inflammation was not altered significantly in PRR knockdown mice after TAA or MCD diet-induced injury in the present study. In the in vitro experiment, similar expression levels of proinflammatory cytokines and chemokines were noted in PRR or control-siRNA-treated macrophages. Therefore, the results supported the notion that the antifibrotic effects of hepatic PRR knockdown are contributed mainly through the attenuation of HSC activation.
A few studies have shown that PRR inhibition has protective effects for nonalcoholic fatty liver disease (NAFLD), 31,32 and our study further elucidates the role of PRR in mice with NAFLD and liver fibrosis. Ren et al 31 administered N-acetylgalactosamine-modified antisense oligonucleotides in C57BL/6 mice fed a high-fat diet for 14 weeks and found that PRR silencing in hepatocytes for 14 weeks attenuated hepatosteatosis without worsening liver inflammation. In another study, Gayban et al 32 administered PRO-20 for 4 weeks in C57BL/6 mice fed a high-fat diet for  6 weeks and reported that competitive PRR antagonism attenuated the development of hepatosteatosis and portal vein collagen deposition. However, liver inflammation and fibrosis in C57BL/6 mice after being fed a short period of high-fat diet are minimal. 33 Our study administered lentivirus expressing PRR shRNA into C57BL/6 mice after 6 weeks of MCD diet feeding and down-regulated PRR in hepatocytes, HSCs, KCs, and SECs for 6 weeks and found that hepatic PRR knockdown did not reduce hepatosteatosis and liver inflammation; however, it significantly ameliorated nonalcoholic steatohepatitis-related fibrosis, which is the most important prognostic indicator for patients with NAFLD. 34 Our study further showed that myofibroblastspecific PRR knockdown significantly attenuated liver fibrosis in MCD-fed mice and the hepatic PRR expression was up-regulated in patients with nonalcoholic steatohepatitis. The discrepancy in the antihepatic steatosis effect among the studies may result from different animal models and different methods of PRR inhibition used. Taken together, these studies highlight the different aspects of therapeutic potential of PRR inhibition for NAFLD.
Our study had some limitations. First, global PRR knockout mice or HSC-specific PRR knockout mice are not available, therefore, the effects of PRR modulation for the prevention of liver fibrosis cannot be assessed. However, our models simulate the clinical setting in patients diagnosed with established liver fibrosis, and our results suggest that modulation of PRR is a promising therapeutic approach for fibrosis regression. Second, the effects of pharmacologic inhibition of PRR on liver fibrosis was not investigated in our study. A reliable PRR blocker and HSC-targeted pharmacologic therapeutics for PRR inhibition need to be developed.
In conclusion, our results show the pathophysiology of PRR in liver fibrosis. The promising effect of PRR knockdown in myofibroblasts on liver fibrosis provides evidence for developing a new myofibroblast-targeted therapeutic, which may be combined with other potential antifibrotic drugs for treatment of chronic liver injury.

Human Samples
Nontumorous liver and plasma samples were obtained from 41 patients who underwent surgical liver resection for hepatocellular carcinoma and were stored at -80 C until use. Histologic samples were analyzed and classified as nonfibrosis (normal; Ishak score; 0), mild fibrosis (Ishak score, 1À3), or severe fibrosis (Ishak score, 4À6) by the pathologists at Taipei Veterans General Hospital. Demographic data pertaining to the patients are presented in Table 1. Immunohistochemistry and Western blot analyses were performed to analyze the expression of PRR expression as well as the ERK/ TGF-b1/SMAD3 pathway. Plasma renin levels were measured using an enzyme-linked immunosorbent assay (Human Renin Enzyme-Linked Immunosorbent Assay Kit, catalog E4719-100; BioVision, Inc, Milpitas, CA). The study protocol was approved by the Institutional Research Board of Taipei Veterans General Hospital (no. 2018-01-015CC).

Animals
Eight-week-old adult male C57BL/6 mice (National Laboratory Animal Center, Taipei, Taiwan) were used in each experiment. The mice were caged at 22 C with a 12hour light-dark cycle and allowed free access to food. The study was approved by the Animal Experiment Committee of Taipei Veterans General Hospital and National Yang-Ming University, and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences.

Lentivirus Preparation
A shRNA was designed to target the mRNA encoding mouse PRR at the TAATCACCTCTTACAACATTT (NM-027439.3-1002s21c1) sequence. Target and scrambled sequences were ligated and cloned into a lentiviral vector under the control of the U6 promoter. For specific PRR knockdown in myofibroblasts, the (a-SMA) promoter was amplified from the the mouse smooth muscle cell alphaactin promoter fragment (pSMP8) plasmid, a gift from Dr J. A. Fagin (Memorial Sloan Kettering Cancer Center, New York, NY), using the forward primer 5'-GATCATGATC-GAATTCACACCATAAAACAAGTGCATGAG-3' and the reverse primer 5'-GATCATGATCGAATTCAGCTGCACCAGCGTCT-CAGG-3'. 35 The polymerase chain reaction (PCR) product was cloned into a lentiviral vector with a downstream enhanced GFP. shRNAs target mouse PRR and the scrambled sequence was constructed within a miR-E backbone 13 and cloned downstream of the a-SMA promoter. For specific PRR overexpression in myofibroblasts, mouse PRR complementary DNA open reading frame clone with C-terminal His tag (Sino Biological, Inc, Beijing, China) was extracted and cloned downstream of the a-SMA promoter. The viral construct encoding a scrambled sequence under the a-SMA promoter served as a control. The constructs were sequenced to verify the orientation and integrity of the inserts. All vectors were constructed and packaged at the RNAi Core Lab (Academia Sinica).

Experimental Design
In the hepatic PRR knockdown experiment, liver fibrosis was induced in mice by intraperitoneal injection of TAA (100 mg/kg body weight) 3 times a week for 8 weeks 36 or by feeding the mice a MCD diet for 12 weeks. In the TAA group, lentivirus expressing PRR shRNA or scrambled sequences (100 mL of 1.5 Â 10 7 transducing units) was administered via tail vein injection after 4 weeks of TAA or vehicle (saline) treatment. 37 All the mice were killed 4 weeks after administration of the lentivirus. In the MCD group, lentivirus with PRR shRNA or scrambled sequences (100 mL of 1.5 Â 10 7 transducing units) was administered via tail vein injection after 6 weeks of MCD diet feeding. Mice that were fed normal chow diet and received lentivirus with scrambled sequence were served as controls. All mice were killed 6 weeks after the administration of lentivirus.
For the myofibroblast-targeted PRR knockdown experiments, liver fibrosis was induced by intraperitoneal injection of TAA (100 mg/kg body weight) 3 times a week for 8   Figure 15. Schematic of the proposed mechanism of PRR in liver fibrosis. PRR expression is increased in activated HSCs during chronic liver injury. The binding of renin or prorenin to PRR leads to the activation of the ERK/TGFb1/Smad3 pathway, promoting liver fibrosis. The knockdown of PRR in activated HSCs suppresses the ERK/TGFb1/Smad3 pathway, leading to reduction of liver fibrosis. p-ERK, phosphorylated extracellular signal regulated kinase; p-SMAD3, phosphorylated SMAD3.
weeks or by feeding the mice an MCD diet for 12 weeks. The mice randomly received lentivirus-expressing PRR shRNA or scrambled sequences under the a-SMA promoter (labeled as SMA-miR-shPRR or SMA-miR-scramble, respectively) at a dose of 100 mL of 1.5 Â 10 7 transducing units by tail vein injection after 4 weeks of TAA treatment or 6 weeks of MCD diet. The mice were killed after 4 more weeks of TAA treatment or 6 more weeks of MCD diet, respectively. In the myofibroblast-specific PRR overexpression experiments, the mice randomly received lentivirus expressing PRR complementary DNA or scrambled sequences under the a-SMA promoter (labeled as SMA-PRR-His or SMAscramble, respectively) at a dose of 100 mL of 1.5 Â 10 7 transducing units via tail vein injection after 4 weeks of TAA or vehicle treatment. The mice were killed after 4 weeks of lentivirus administration.
All the lentivirus vectors themselves did not cause liver inflammation or fibrosis in mouse livers ( Figure 16).

PRR Knockdown in Human Activated HSC LX-2 Cells and In Vitro Experiments
Lentivirus expressing shRNA targeting human PRR mRNA at the sequence GGAACGAGTTTAGTATATTAA (NM-005765.2-149s21c1), under the control of a U6 promoter, were constructed by the RNAi Core Lab (Academia Sinica).
To perform lentiviral infection, 3 Â 10 5 of LX-2 cells (Merck Millipore, Burlington, MA) were plated in 6-cm plates and incubated overnight at 37 C in 5% CO 2 . On day 2, the medium was replaced with fresh medium containing polybrene (8 mg/mL) to enhance lentiviral transfection, 38 and incubated for 30 minutes. Then, the constructed viruses (with a final multiplicity of infection of 3) were added to the medium. After incubation for 24 hours, puromycin (3 mg/mL) was added for selection of stable clones and the infected cells were incubated for 72 hours. Protein lysates from the stable clones of PRR knockdown cells and scrambled shRNA-transfected cells were quantified by Western blot to confirm the effect of RNA interference. Cells also were harvested for RNA analysis.

Isolation and Culture of Primary Cells
Mouse HSCs, Kupffer cells, and sinusoidal endothelial cells were isolated from male C57BL/6 mice by collagenase-pronase perfusion and subsequent density centrifugation on Nycodenz (Alere Technologies AS, Oslo, Norway) gradient as described previously. [40][41][42] The purity of the HSCs was tested by retinoid autofluorescence 42 and exceeded 95% in all isolations ( Figure 17A). HSCs isolated from normal liver were cultured for 12 hours (quiescent HSCs) or 9 days (culture-activated HSCs) in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), and antibiotics at 37 C in a humidified atmosphere of 95% air and 5% CO 2 . For in vivo HSC activation, primary HSCs were isolated from mice that were administered intraperitoneal injections of TAA (Sigma, St Louis, MO) (100 mg/kg body weight) for 8 weeks or maintained on a MCD diet for 12 weeks, and cultured in DMEM with 10% FBS and antibiotics for 20 hours (in vivo-activated HSCs). For RNA and protein extraction, 3 Â 10 5 HSCs/well were plated in 6-well plates and cultured for the indicated times. To measure mRNA expression of PRR after exposure to PRR ligands, in vivo-activated HSCs from TAA mice were treated with vehicle, renin, or prorenin (10 -8 mol/L) for 24 hours. 9,10  Kupffer cells were positively selected as F4/80expressing cells using magnetic-activated cell sorting (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany). 43 The procedure led to the isolation of KCs with 95% purity that was confirmed by flow cytometry for F4/80 expression ( Figure 17B). To investigate the effect of LPS on PRR expression, 3 Â 10 5 KCs were plated in 6-well plates in RPMI-1640 in the presence of LPS (100 ng/mL; Sigma-Aldrich, Inc, St. Louis, MO) or vehicle for 24 hours. Sinusoidal endothelial cells were isolated using magneticactivated cell sorting-based positive selection using CD31specific antibodies (Miltenyi Biotec).
Primary hepatocytes were isolated from male C57BL/6 mice by modifying a previously described collagenase perfusion method. 44 The viability of hepatocytes after 24 hours in culture was >95%, as assessed by flow cytometry after propidium iodide staining ( Figure 17C). Cell culture plates were coated with 0.1% rat tail collagen (Sigma-Aldrich) 24 hours before plating the cells. Collagenases D and P were purchased from Roche (Mannheim, Germany). Primary hepatocytes were plated in 6-well plates (2 Â 10 5 cells/well) and cultured in DMEM containing 10% FBS and antibiotics at 37 C in a humidified atmosphere of 95% air and 5% CO 2 . RNA was extracted after 24 hours of culture.

Measurement of Blood Biochemistry
Serum ALT levels were measured with a standard auto-SMAC analyzer (Cobas 8000; Roche Diagnostics GmbH, Mannheim, Germany).

Real-Time Quantitative Reverse Transcriptase PCR
Nucleotide sequences of the primers used in this study are shown in Table 2. Gene expression levels were measured quantitatively using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Inc, Foster City, CA) using SYBR Green. The specificity of each PCR product was evaluated by melting curve analysis, followed by agarose gel electrophoresis.
Nested PCR analysis was used to detect hepatic Va14-Ja18 mRNA. 45 Briefly, first-round PCR was performed with the following primers: 5'-ATGAAAAAGCGCCTGAGTGCC-3' and 5'-CAGGAGGATTCGGAGTCCCA-3' using the following cycling conditions: 40 cycles of 94 C for 1 minute, 53 C for 1 minute, and 72 C for 2 minutes. Five microliters of firstround PCR product were used to run in the nested PCR with the following primers: 5'-TAAGCACAGCACGTGCACAT-3' and 5'-CAATCAGCTGAGTCCCAGCT-3' using the following cycling conditions: 40 cycles of 94 C for 1 minute, 52 C for 1 minute, and 72 C for 2 minutes. The products were visualized and quantified on a 3% agarose gel.

Western Blot Analysis
The blots were incubated with the primary antibodies shown in Table 3. After washing, the membranes were incubated with horseradish-peroxidase-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for the rabbit primary antibodies for 1 hour. Subsequently, the blots were developed by enhanced chemiluminescence (ECL Western Blotting Analysis System, Amersham, Buckinghamshire, UK). The intensities of the bands of interest were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Immunohistochemical Staining
Slides were incubated at 4 C overnight with the primary antibodies as shown in Table 3. After overnight incubation, the slides were incubated with the corresponding secondary antibody for 30 minutes. The slides were stained with a super-sensitive polymer-horseradish peroxidase immunohistochemistry detection system (BioGenex Laboratories, Inc, Fremont, CA) and then counterstained with Mayer's hematoxylin. Images were captured with a microscope (Olympus, AX-80, Tokyo, Japan) and Olympus Cell Sens imaging software.

Immunofluorescence Staining
Liver sections were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Abcam, Cambridge, MA). On the first day of double staining, the sections were incubated with PRR and a-SMA antibodies or GFP and a-SMA antibodies (Table 3) for 16 hours at 4 C. Then, the sections were incubated with Alexa Fluor 488 donkey antisheep IgG H&L (ab150177; Abcam, Cambridge, MA) at a 1/ 1000 dilution for a-SMA and Alexa Fluor 647 donkey antirabbit IgG H&L (ab150075; Abcam) at a 1/1000 dilution for PRR and GFP for 120 minutes at 37 C on day 2 and counterstained with 4', 6-diamidino-2-phenylindole (Cell Signaling).

Sirius Red Staining
Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Measurement of Hepatic Hydroxyproline and Triglyceride Levels
Hydroxyproline level was determined using a Hydroxyproline Colorimetric Assay Kit (BioVision, Inc). Liver tissue (20 mg) was homogenized and hydrolyzed with 200 mL of 12 N HCl in a pressure-tight, Teflon-capped vial (Scientific Specialties, Inc, CA) at 120 C for 3 hours. After transferring 10 mL of each hydrolyzed sample to a 96-well plate to dry at 65 C, 100 mL of the chloramine T reagent was added to each sample, and the plate was incubated at 22 CÀ28 C for 5 minutes. After adding 100 mL of the 4-(dimethylamino) benzaldehyde (DMAB) reagent to each well and incubating for 90 minutes at 60 C, the absorbance of all samples was measured at 560 nm using a microplate reader. Hepatic levels of triglyceride were measured using the Triglyceride Liquid Reagents Kit (Pointe Scientific, Canton, MI).

Flow Cytometry
The mouse-specific antibodies used are shown in Table 3. Mouse fragment crystallizable (Fc) block (eBioscience, San Diego, CA) was used to block binding of aggregated immunoglobulins to Fc receptors. Flow cytometry was performed on a FACSCanto II cytometer (BD Biosciences, San Jose, CA).

In Vitro siRNA Transfection
To silence PRR expression in RAW 264.7 cells, specific mouse siRNAs (Santa Cruz Biotechnology, Inc, Paso Robles, CA) were used for transfection using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) following the manufacturer's instructions. Briefly, siRNA and Lipofectamine RNAiMAX were mixed and incubated at  22 CÀ28 C for 5 minutes in Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA). Then, the mixture was transferred to a 6-well plate at a final concentration of 20 nmol/L (siRNA) and 7.5 mL RNAiMAX per well. Cells were assayed 48 hours after siRNA transfection, and the protein levels of PRR were evaluated using b-actin as a loading control. To test the effect of PRR knockdown on LPSinduced proinflammatory cytokines and chemokine expression, cells were treated with LPS (100 ng/mL; Sigma) for 24 hours for analysis of mRNA at 48 hours after siRNA transfection.