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Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice
Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice Shaojiang Tian, Chenxiao Li, Ran Ran, Shi-You Chen PII: DOI: Reference:
S0925-4439(16)30326-X doi:10.1016/j.bbadis.2016.11.032 BBADIS 64627
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
20 August 2016 20 November 2016 30 November 2016
Please cite this article as: Shaojiang Tian, Chenxiao Li, Ran Ran, Shi-You Chen, Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice, BBA - Molecular Basis of Disease (2016), doi:10.1016/j.bbadis.2016.11.032
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ACCEPTED MANUSCRIPT Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice
Department of Physiology & Pharmacology, University of Georgia, Athens, GA, USA; and 2Department of Nephrology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China;
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Shaojiang Tian1,2†, Chenxiao Li1†, Ran Ran1,2, and Shi-You Chen1,2
† These authors contributed equally to this work.
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Running Title: SP-A in renal fibrosis Correspondence Author:
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Shi-You Chen, PhD Department of Physiology & Pharmacology The University of Georgia 501 D.W. Brooks Drive Athens, GA 30602 Tel: 706-542-8284 Fax: 706-542-3015 Email: [email protected]
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Abbreviations:
SP-A: surfactant protein A UUO: unilateral ureter obstruction HMGB1: high mobility group box 1 TGF-β: transforming growth factor β BMM: Bone marrow derived-macrophages HPTC: Human proximal tubular cell qRT-PCR: Reverse Transcription and quantitative RT-PCR Co-IP: co-immunoprecipitation α-SMA: smooth muscle α-actin FSP-1: fibroblast specific protein 1 IL-6: Interleukin 6
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ACCEPTED MANUSCRIPT Abstract
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Renal interstitial fibrosis is an inevitable consequence of virtually every type of chronic kidney disease. The underlying mechanisms, however, are not completely understood. In the present study, we identified surfactant protein A (SP-A) as a novel protein factor involved in the renal fibrosis induced by unilateral ureter obstruction (UUO). UUO induced SP-A expression in mouse kidney epithelium, likely due to the increased acidic stress and inflammation. Interestingly, SP-A deficiency aggravated UUO-prompted kidney structural damage, macrophage accumulation, and tubulointerstitial fibrosis. SP-A deficiency appeared to worsen the fibrosis by enhancing interstitial myofibroblast accumulation. Moreover, SP-A deficiency increased the expression of TGF-β1, the major regulator of kidney fibrosis, particularly in the interstitial cells. Mechanistically, SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1), a factor regulating TGF-β expression/signaling and implicated in renal fibrosis. SP-A also blocked HMGB1 activities in inducing TGF-β1 expression and myofibroblast transdifferentiation from kidney fibroblasts, demonstrating that SP-A protects kidney by impeding both the expression and fibrogenic function of HMGB1. Since SP-A physically interacted with HMGB1 both in vitro and in kidney tissue in vivo, SP-A may exert its protective role by binding to HMGB1 and thus titrating its activity during UUO-induced renal fibrosis.
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Key words: Kidney fibrosis, unilateral ureter obstruction, myofibroblast activation, surfactant protein A, high mobility group box 1, TGF-β
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ACCEPTED MANUSCRIPT 1. Introduction
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Surfactant protein A (SP-A) is a member of the C-type lectin family that play an important role in host defense and regulating inflammation in the lung. It can bind to its target ligands on pathogens, allergens, and apoptotic cells to facilitate the clearance process. SP-A can also interact with cell surface receptors to modulate cytokine production and regulate the inflammatory response.1 In addition to its role in infectious respiratory diseases, emerging evidence suggests that SP-A is also involved in a number of non-infectious lung disease such as cancer and fibrosis.2 Although SP-A is considered as a lung disease marker,3 numerous studies have demonstrated that SP-A is also expressed in many other organs including skin,4 middle ear,5 vagina,6 and gastric intestine.7 Moreover, this ubiquitous protein also exhibits a varying pattern of expression in response to different stimuli. In the lung, SP-A expression is affected by environmental insults and its gene polymorphism.8,9 In chronic rhinosinusitis, SP-A expression is up-regulated by the inflammation and tissue injury.10 Recent studies indicate that SP-A is expressed in human renal tubular epithelial cells, and the expression can be stimulated by lipopolysaccharide.11 In addition, SP-A and SP-D attenuate kidney injury by modulating inflammation and apoptosis in sepsis-induced acute kidney injury 12. However, whether or not SP-A plays a role in the process of non-infectious renal diseases, especially the chronic kidney diseases, the major cause of renal failure, remains unknown. In this study, we explored the expression patterns of SP-A in mouse kidney, and determined if SP-A affects the process of kidney fibrosis induced by unilateral ureteral obstruction (UUO). By taking advantage of SP-A knock-out mice, we demonstrated that SP-A deficiency worsens UUO-induced kidney interstitial fibrosis, suggesting a protective role of SP-A in the kidney. SP-A appeared to attenuate high mobility group box 1 (HMGB1)-induced TGF-β1 expression in interstitial cells, thus inhibit myofibroblast activation from fibroblasts. 2. Materials and Methods 2.1 Animals and UUO mouse model: SP-A knock-out (SPA-/-) mice with 129 genetic background were purchased from the Jackson Laboratory (STOCK Sftpa1tm1Kor/J). The mice were crossed with C57BL/6J for three generation, and its C57BL/6J×129 wild type littermates were used as controls. Wild type (WT) or SPA-/- male mice were either sham-operated or subjected to unilateral ureteral obstructive ligation (UUO). Briefly, mice weighing 20-25 g were anesthetized with isoflurane inhalation, and UUO was achieved by double-ligating the left ureter with 3-0 silk through a left abdominal lateral incision. Sham operation was performed similarly but without ureteral ligation. Mice were sacrificed at 3, 5, 7, 10 or 14 days after the surgery. Urine, blood and kidneys were collected and subjected to the studies described below. For mice with UUO, the retained urine in left ureters and pelvis were collected using 1 ml syringe. For sham-operated animals, metabolic 3
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cages were used to collect the urine. All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) of The University of Georgia.
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2.2 Isolation and culture of mouse renal primary fibroblasts: Normal (WT) mouse kidneys were minced to 1 mm3 cubes. The cubes were then transferred to a 10 cm cell culture dish, and digested in 5 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mg/ml collagenase Ⅳ at 37 ℃ for 30 min. The digestion was repeated for three times, and the suspension was filtered after each digestion through a 70 μm sterile filter. Cells were then centrifuged at 1200 rpm for 5 min and washed twice with phosphate buffer saline (PBS). Cells were cultured with complete DMEM medium containing 10% FBS at 37 ℃ with 5% v/v CO2 in a humidified atmosphere. One hour after plating, the medium was replaced to remove non-adherent cells. The adherent fibroblasts were verified by vimentin staining.
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2.3 Isolation and culture of mouse macrophage: Bone marrow derived-macrophages (BMM) were obtained as described previously.13 Briefly, femur and tibia were dissected in DMEM containing 10% FBS, and bone marrow cells were flushed from the femurs and tibias. After red cells were lysed, the remaining cells were counted and plated in a T-25 flask with 10 ng/ml of macrophage colony stimulating factor (M-CSF, Sigma) added in the medium. After culturing overnight, non-adherent cells were collected, washed, and plated in 60-mm petri dishes with 10 ng/ml of M-CSF in DMEM containing 10% FBS. After 7 days, cells were washed, and the adherent cells were released and removed with 0.1% EDTA. The resulting BMMs were verified to be >98% pure based on F4/80 staining. 2.4 Human proximal tubular cell (HPTC) culture: HPTCs were cultured in DMEM/F12 medium (Invitrogen) with supplements as described previously.14 1N HCI was used to adjust medium PH for the preparation of the acidified medium. 2.5 Human kidney specimens: The human kidney specimens were collected from hospitalized patients in Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China. The patients underwent nephrectomy due to severe hydronephrosis with ureteral stricture. Kidney cortex adjacent to the hydronephrosis were chosen for examining the pathological alterations. All patients provided written informed consent, and the study protocol was approved by the Scientific-Ethical Committee of Hubei University of Medicine. 2.6 Reverse Transcription and quantitative RT-PCR (qRT-PCR): Total RNA was extracted using TRIzol reagent (Invitrogen) by following the manufacturer’s 4
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instruction. cDNA was synthesized from 1 μg of total RNA using iScript cDNA Synthesized Kit (Bio-Rad). PCR reaction was performed as described previously.14 To avoid potential genomic DNA contamination, RNA samples were treated with DNase I followed by incubation at 65°C for 15 min. In addition, PCR reactions with samples without RT were used as negative controls. The PCR cycles ranged from 25 to 30. mRNA expression of pertinent genes was normalized to cyclophilin level. The following primers were used for each individual gene: mouse HMGB1 (forward: GCT GAC AAG GCT CGT TAT GAA; reverse: CCT TTG ATT TTG GGG CGG TA), SP-A (forward: TCC TGG AGA CTT CCA CTA CCT; reverse: CAG GCA GCC CTT ATC ATT CC), TGF-β1 (forward: CGC CAT CTA TGA GAA AAC C; reverse: GTA ACG CCA GGA ATT GT), and cyclophilin (forward: TGC AGC CAT GGT CAA CCC C; reverse: CCC AAG GGC TCG TCA).
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2.7 Western blot analysis: Western blot was performed as described previously.14 Proteins in kidney or cultured cells were extracted using RIPA lysis buffer containing protease inhibitors. Protein concentration was determined using a BCA kit (Pierce). Protein expressions were detected with an enhanced chemiluminescence kit (Millipore).
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2.8 Histopathological analysis: A half of left kidney tissues from sham or UUO mice were fixed in buffered 4% paraformaldehyde for 24 h and then embedded in paraffin wax. For assessment of tubulointerstitial injury and fibrosis, 5 μm sections were stained with H&E stain kit (MasterTech Lab) or Masson’s Trichrome 2000 Stain Kit (MasterTech Lab), respectively. Tubular injury index, characterized by tubular dilation and epithelial desquamation with interstitial expansions, was graded according to the extent of cortical involvement on a scale from 0 to 4 assessed using a semi-quantitative method previously described.15 Interstitial fibrosis was assessed by collagen deposition using a point-counting approach.16 2.9 Immunohistochemistry staining: For assessing SP-A and TGF-β1 expression, kidney sections were incubated with SP-A (Santa Cruz, sc-13977) or TGF-β1 antibody (Santa Cruz, sc-146) at a 1:100 dilution overnight followed by incubation with a HRP-conjugated goat anti-rabbit second antibody (Abcam, ab6721, 1:200 dilution). The bound peroxidase was developed using diaminobenzidine (DAB) as a substrate to produce a brown color followed by a blue nuclear haematoxylin counterstain. 2.10 Immunofluorescent staining: For assessing protein expression in kidney tissues or cultured cells, sections or cells adhered to coverslips were incubated with the primary FSP-1 (Abcam, ab197896), α-SMA (Sigma, A2547), HMGB1 (Cell Signaling, #3935), F4/80 (Abcam, ab16911), SP-A (Santa Cruz), or TGF-β1 antibody (Santa Cruz) at a 1:100 dilution overnight. Corresponding FITC-conjugated secondary antibodies (Sigma, F0257-anti-mouse or F0382-anti-rabbit) were applied for each individual primary antibody at a 1:100 dilution, and fluorescent 5
ACCEPTED MANUSCRIPT photomicrographs were obtained at x100 or 200 magnification.
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2.11 Measurement of urine and blood HMGB1: HMGB1 level in urine and serum were measured using a HMGB1 ELISA kit (MyBioSource) by following manufacturer’s instructions.
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2.12 SPA-HMGB1 binding and co-immunoprecipitation (Co-IP) assay: For examining the SP-A binding with HMGB1 in vitro, SP-A (10 μg/ml) and HMGB1 (125 ng/ml) were added into the fibroblast culture medium (DMEM containing 10% FBS) and incubated at 37℃ for 2 h. For examining SP-A binding with HMGB1 in vivo, kidney tissues were lysed with ice-cold lysis buffer with protease inhibitor mix (Thermo Scientific). The mediums or lysates were incubated with IgG (negative control), SP-A, or HMGB1 antibodies immobilized on agarose beads following Instruction provided in the Co-IP kit (Thermo Scientific). The immunoprecipitates were pelleted, washed, and subjected to immunoblotting using SP-A or HMGB1 antibodies according to the manufacturer’s instructions.
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2.13 Statistical analysis: All data are reported as means ± SD. Data were analyzed using ANOVA followed by q test using the SPSS for windows 10.0. P values < 0.05 were considered as a statistically significant difference. 3. Results
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3.1 SP-A was induced in mouse kidney with UUO. Since SP-A has been reported to express in renal tubular epithelial cells,11 we sought to determine if SP-A is involved in the progression of renal fibrosis using the UUO model. We first detected if SP-A is induced in mouse kidney with UUO using an antibody that detects SPA in the lungs of wild type, but not SP-A-/- mice (Supplemental Figure sFig 1). As shown in Fig 1A, SP-A was barely expressed in sham-operated kidney. However, strong SP-A expression was observed in tubular epithelial cells and interstitium of kidneys with UUO (Fig 1, A). In fact, UUO injury rapidly induced both SP-A mRNA and protein expression as early as 3 days following UUO (Fig 1, A-C). The expression increased along with the progression of the injury. The high level of SP-A expression was maintained up to 14 days after the surgery (Fig 1, A-C). The tubular SP-A appeared to locate in both nuclei and cytoplasm of tubule epithelial cells (Supplemental sFig 2). Importantly, SP-A was expressed in human kidney with interstitial fibrosis due to hydronephrosis (Fig 1, D-F), indicating that SP-A may play a role in the development of human obstructive kidney diseases. A urine analysis indicated that the retained urine in the left ureter following UUO had a mean pH value of 5.52, and thus tubular epithelial cells exposed directly to the acidified urine. We therefore tested if acidic stress affects SP-A induction in tubular cells. As shown in Supplemental sFig 3, A-C, acidified medium (PH 5.5) markedly increased both SP-A mRNA and protein expression in human proximal tubule cells (HPTCs), suggesting that acidic stress is an important factor inducing 6
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SP-A expression in the kidney with UUO. In addition to acidic stress, pro-inflammatory cytokine such as IL-6 is also produced and secreted in kidney during the early stages of UUO.17 We therefore tested if IL-6 influences the SP-A expression in HPTCs and found that IL-6 induced SP-A mRNA and protein expression in a dose-dependent manner (0-200 ng/ml, Supplemental sFig 3, D-F), suggesting that inflammation is another factor inducing SP-A expression in mouse kidney with UUO.
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3.2 SP-A deficiency exacerbated UUO-induced kidney injury, macrophage accumulation, and tubulointerstitial fibrosis. To determine if SP-A plays a role in the progression of renal fibrosis, we performed UUO in both wild type and SP-A knockout (SP-A-/-) mice. As shown in Fig 2, A-B and Supplemental sFig 4, UUO caused obvious tubulointerstitial damages and epithelial cell transdifferentiation. Surprisingly, SP-A-/- resulted in a more severe damage compared to the wild type mice. In addition, more macrophages were observed in SP-A-/- mouse kidney, as shown by immunostaining of macrophage marker F4/80 in the sections of kidney with UUO for 5 days (Supplemental sFig 5A), suggesting an increased inflammation in SP-A-/- kidneys. Indeed, the IL6 level was significantly increased in SP-A-/mouse kidney with UUO compared to the control (Supplemental sFig 5B). Moreover, UUO injury led to a significant amount of collagen deposition in tubulointerstitium. SP-A-/- aggravated the collagen accumulation (Fig 2, C). Quantitative analysis confirmed that SP-A deficiency enhanced type I collagen expression in kidney with UUO (Fig 2, D). These data indicate that SP-A inhibits the development of renal fibrosis.
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3.3 SP-A deficiency increased interstitial myofibroblast accumulation. Myofibroblasts are the main source of collagen production in kidney fibrosis. Therefore, we examined if SP-A-/- affects myofibroblast accumulation. Two myofibroblast markers, smooth muscle α-actin (α-SMA) and fibroblast specific protein 1 (FSP-1), were used to label myofibroblasts. Immunostaining showed that UUO caused the accumulation of α-SMA- or FSP-1-positive cells in kidney interstitium. SP-A-/- increased the numbers of α-SMA- or FSP-1-expressing cells (Fig 3, A). Since FSP-1 also stains macrophage, we performed a co-immunostaining of FSP-1 and F4/80. As shown in sFig 5C, although a small portion of FSP-1-positive cells appeared to be macrophages, the majority of FSP-1+ cells were F4/80 negative, confirm the increased myofibroblast accumulation due to SP-A deficiency. Moreover, both α-SMA and FSP-1 protein expression was markedly increased in SP-A-/- mouse kidney with UUO injury compared to the wild type (Fig 3, B-C). These data demonstrate that SP-A inhibited renal fibrosis by limiting myofibroblast activation. 3.4 SP-A deficiency enhanced TGF-β1 expression in kidney tissue. It is well established that TGF-β1 is a key player in myofibroblast activation and the progression of renal fibrosis.18 Therefore, we investigated if SP-A-/- influences 7
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TGF-β1 expression. We found that UUO induced strong expression of TGF-β1 in mouse kidney (Fig 4), consistent with previous report.18 SP-A-/- further increased both the mRNA and protein expression of TGF-β1 (Fig 4). Interestingly, it appeared that UUO induced TGF-β1 expression mainly in tubular epithelial cells with very limited TGF-β1-expressing cells in the interstitium. However, SP-A-/- significantly increased the numbers of interstitial cells that expressed TGF-β1 (Fig 4, A-B, arrows in the enlarged image), suggesting that SP-A may mainly affect the interstitial cells such as inflammatory cells or residential fibroblasts.
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3.5 SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1) in kidney tissue. Our previous studies have found that HMGB1 plays an important role in initiating tubulointerstitial injury, interstitial inflammation, and renal fibrosis.19 In addition, HMGB1 has been shown to regulate TGF-β expression/signaling in different cells including kidney tubule epithelial cells.20 Therefore, we detected HMGB1 expression and its release in wild type and SP-A-/kidney with or without UUO injury. As shown in Fig 5, A, HMGB1 expression was observed in tubular epithelial cells. UUO significantly increased the HMGB1 mRNA and protein expression, and SP-A-/- further enhanced its expression (Fig 5, B-C). Since HMGB1 is a secreted protein, we detected serum and urine HMGB1 levels. As shown in Fig 5, D-E, serum and urine HMGB1 concentrations were markedly increased in UUO injured kidney compared to sham kidneys. SP-A-/- caused a further elevation of HMGB1 release to serum and urine from mouse kidneys with UUO. These data suggest that HMGB1 may be involved in SP-A deficiency-caused exacerbation of renal fibrosis.
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3.6 SP-A blocked HMGB1-induced fibroblast activation. To determine how SP-A affects the progression of renal fibrosis, we tested if HMGB1 induces fibroblast activation and if SP-A regulates HMGB1 function. As shown in Fig 6, A, HMGB1 induced the activation of kidney primary fibroblasts, as indicated by the induction of myofibroblast markers α-SMA and FSP-1. In fact, HMGB1 induced kidney fibroblasts to become myofibroblasts in a dose-dependent manner (Fig 6, B). However, SP-A dose-dependently inhibited the HMGB1-induced expression of α-SMA and FSP-1 (Fig 6, A and C), indicating a blockade of fibroblast activation. Since HMGB1 or combination of SP-A and HMGB1 did not significantly alter fibroblast proliferation or survival (Supplemental sFig 6), SP-A may lessen the severity of renal fibrosis mainly by blocking HMGB1-induced myofibroblast differentiation. 3.7 SP-A blocked HMGB1-induced TGF-β expression. Since HMGB1 has been shown to regulate TGF-β expression in renal tubular epithelial cells, 20 and SP-A deficiency induced TGF-β1 expression in interstitial cells (Fig 4), we sought to determine if HMGB1 induces TGF-β1 expression in kidney fibroblasts and if SP-A affects HMGB1 function. As shown in Fig 7, A, HMGB1 indeed induced TGF-β1 expression in kidney fibroblasts, suggesting that HMGB1 may promote fibroblast to 8
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myofibroblast transition through up-regulating TGF-β1 expression. Actually, HMGB1 induced TGF-β1 expression in a dose-dependent manner (Fig 7, B). However, SP-A dose-dependently blocked HMGB1-induced TGF-β1 expression in renal fibroblasts (Fig 7, A and C), indicating that SP-A may attenuate the fibroblast-to-myofibroblast transition through disturbing HMGB1-induced TGF-β1 expression.
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3.8 SP-A bound to HMGB1 both in vitro and in vivo. To explore how SP-A blocks HMGB1 functions, we tested if SP-A physically interacts with HMGB1. As shown in Fig 8, A-B, SP-A bound to HMGB1 in fibroblast culture medium, suggesting that SP-A may bind to HMGB1 and thus interfere HMGB1 binding to its receptors in fibroblasts. In mouse kidney tissue, SP-A also bound to HMGB1 (Fig 8, C), indicating that SP-A secreted from tubular epithelial cells may inhibit fibroblast activation through binding to HMGB1 in interstitium, thus protect kidney from the more severe fibrosis or damage. 4. Discussion
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Tubulointerstitial fibrosis is considered as a final common pathway for chronic kidney disease progressing to renal failure.21 Although numerous factors are identified to be involved in renal fibrosis, the mechanisms underlying this complex process are not completely understood. Our study demonstrated for the first time that SP-A is a new protein factor involved in the progression of renal fibrosis following the obstructive injury. We found that a high level of SP-A is expressed in tubular epithelial cells in the injured mouse kidney, and SP-A deficiency causes more severe tubular injury, interstitial inflammation, and interstitial fibrosis. It appears that SP-A inhibits fibroblast to myofibroblast differentiation induced by HMGB1, which is expressed and released from kidney due to UUO injury. Therefore, SP-A expression in tubular epithelial cells following UUO is a protective response to the kidney injury. SP-A protects kidney from the potential more severe damage by attenuating HMGB1 function in inducing TGF-β expression, fibroblast activation and consequently the fibrosis. Although SP-A is clearly important for the obstruction-induced renal fibrosis, whether or not SP-A is expressed in kidney has been a debate. There are reports showing that neither SP-A mRNA or protein is detectable in kidney,22,23 while other studies suggest that SP-A protein is expressed in kidney tissue.5 Recent studies find that lipopolysaccharide induces SP-A expression in human renal tubular epithelial cells.11 Our current study reveals that a low level of SP-A may be present in normal mouse kidney. However, SP-A is significantly induced in kidney by UUO injury. Consistently, SP-A expression is significantly increased in HPTCs when they are treated with acidified medium or pro-inflammatory cytokine, further demonstrating that SP-A can be induced by pathological insults. Two possible mechanisms may be involved in the role of SP-A regulating HMGB1 function. Firstly, SP-A regulates HMGB1 expression because SP-A 9
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deficiency increases both the mRNA and protein levels of HMGB1 in kidney with UUO, mainly in epithelial cells (Fig 5). Secondly, SP-A attenuates HMGB1 function through physically interacting with HMGB1, which may block HMGB1 binding to its receptors. Indeed, SP-A can bind with HMGB1 in culture medium in vitro and in kidney with UUO in vivo (Fig 8). The SP-A inhibition of HMGB1-induced fibroblast activation and TGF-β expression (Fig 6-7) supports that SP-A may block HMGB1 activities through interfering HMGB1 interaction with its receptor. Interestingly, SP-A deficiency significantly increases the number of TGF-β-expressing cells in kidney interstitium following UUO. The two possible cells in the interstitium that may produce TGF-β1 are macrophages and fibroblasts. Our data show that HMGB1 induces TGF-β1 expression in fibroblasts (Fig 7), which is blocked by SP-A. However, HMGB1 does not induce macrophage to express TGF-β1. Rather, HMGB1 blocks TGF-β1 expression in macrophages (Supplemental sFig 7). Therefore, SP-A is likely to only block HMGB1-mediated TGF-β expression in fibroblasts. SP-A may regulate macrophage function via a different mechanism, which could be studied in the future. One limitation of this study is that UUO is not an ideal model for chronic kidney disease because it is not a common cause of adult human renal diseases. In addition, the UUO mouse exhibits only modest change in serum creatinine without proteinuria and hypertension. However, SP-A is expressed in human kidney with hydronephrosis, underscoring its association/importance in the development of human renal fibrosis due to ureter obstruction. Another limitation of this study is that the role of acidic stress and pro-inflammatory cytokines in SP-A release are not established in vivo. 5. Conclusion
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Our study has identified a novel factor involved in renal fibrosis and provided evidence that SP-A lessens the UUO-induced kidney damage and fibrosis via inhibiting HMGB1-mediated TGF-β expression and fibroblast activation. Acknowledgements This work was supported by grants from National Institutes of Health (grant numbers HL123302 and HL119053) and National Natural Science Foundation of China (grant number 81328002).
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Figure 1. SP-A expression in injured mouse and human kidneys. (A) Immunohistochemistry staining showed SP-A expression in tubular epithelium and interstitium of mouse kidney with UUO for the days (D3-D14) indicated. (B) SP-A mRNA expression was detected by qRT-PCR in mouse kidney following UUO injury. *P<0.05 compared with sham group (D0), #P<0.05 compared to day 3, $ P<0.05 compared to day 5 (n=5). (C) SP-A protein expression was detected by Western blot and quantified by normalizing to α-Tubulin. *P<0.05 compared with sham group (D0), #P<0.05 compared to day 3, $P<0.05 compared to day 5 (n=8). (D-F) SP-A expression in human kidney tubules along with tubulointerstitial fibrosis due to ureter obstruction-caused hydronephrosis. Healthy (normal) and hydronephrotic human kidney specimen sections were stained with Masson’s trichrome (D, upper panel) or SP-A antibody (D, lower panel). Collagen deposition was measured by the intensity of collagen deposition and normalized to the level in normal kidney (E). SP-A expression was quantified by measuring the staining intensity and normalized to the normal kidney staining (F). *P<0.01 compared with the normal kidney in E and F, respectively (n=5).
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Figure 2. SP-A deficiency exacerbated UUO-caused tubular injury and renal fibrosis. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 14 days. (A) SPA-/- worsen tubular injury as shown by H&E staining. (B) Kidney tubular injury index calculated using a semiquantitative method based on the H&E-stained kidney structures as shown in A. (C) SPA-/- caused more severe renal fibrosis in mice with UUO as shown by Masson’s staining. (D) Western blot analysis to quantify SP-A and type I collagen I (CoI-I) expression. *P<0.01 compared to sham group; #P<0.01 compared to WT mice with UUO; n=8. Figure 3. SP-A deficiency promoted interstitial fibroblast activation. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) SPA-/increased the numbers of α-SMA and FSP1-positive cells as shown by immunostaining. (B) α-SMA and FSP-1 protein expression in mouse kidneys was examined by western blot. (C) α-SMA and FSP-1 protein expression shown in B was quantified by normalizing to α-Tubulin level. *P<0.01 compared with sham group for each corresponding protein; #P<0.05 compared with WT mice with UUO for each corresponding protein; n=8. Figure 4. SP-A deficiency enhanced TGF-β1 expression in mouse kidney with UUO. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) TGF-β1 expression was detected by immunostaining. TGF-β1+ interstitial cells in the SP-A-/- kidney with UUO injury was significantly more than that in the WT kidney, as shown in the image enlarged from the red square-enclosed section. (B) Quantification of TGF-β-expressing interstitial cells by counting TGF-β-positive cells in 8 different sections and normalized to the sham-operated WT kidney. (C-D) 13
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TGF-β1 mRNA and protein expression was quantified by qRT-PCR (C) and western blot (D), respectively. Protein expression was normalized to the α-Tubulin level. *P<0.01 compared with sham group; #P<0.01 compared with WT mice with UUO (n=8 for B, C and D).
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Figure 5. SP-A deficiency increased UUO-induced HMGB1 expression. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) HMGB1 expression was detected by immunostaining. Arrows indicate the strong HMGB1 expression in epithelial cells of structurally-damaged tubules. (B-C) HMGB1 mRNA and protein expression was quantified by qRT-PCR (B) and western blot (C), respectively. Protein expression was normalized to α-Tubulin level. (D-E) SPA-/- increased UUO-induced HMGB1 secretion in serum (D) and urine (E) as measured by ELISA. *P<0.01 compared with sham group; #P<0.05 compared with WT mice with UUO; n=8.
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Figure 6. SP-A inhibited HMGB1-mediated fibroblast activation. Mouse kidney primary fibroblasts were cultured as described in Methods and treated with (+) or without (-) HMGB1 or SP-A as indicated. (A) HMGB1-induced myofibroblast marker α-SMA and FSP-1 was dose-dependently inhibited by SP-A, as detected by immunostaining. (B) Western blot showed a dose-dependent induction of α-SMA and FSP-1 by HMGB1. (C) SP-A dose-dependently inhibited the HMGB1-induced α-SMA and FSP-1 protein expression (50 ng/ml), as examined by Western blot. Protein expression (B and C) was normalized to α-Tubulin level. *P<0.01 compared with vehicle-treated cells (0) for FSP-1; #P<0.01 compared with vehicle-treated cells (0) for α-SMA; n=3.
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Figure 7. SP-A inhibited HMGB1-induced TGF-β1 expression in kidney fibroblasts. Primary kidney fibroblasts were treated similarly as in Figure 6. TGF-β1 expression was examined. (A) SP-A dose-dependently inhibited HMGB1 (50 ng/ml)-induced TGF-β1 expression as detected by immunostaining. (B) HMGB1 dose-dependently induced TGF-β1 expression as measured by Western blot. (C) SP-A dose-dependently inhibited TGF-β1 expression induced by HMGB1 (50 ng/ml) as examined by Western blot. Protein expression (B and C) was normalized to α-Tubulin level. *P<0.01 compared with vehicle-treated cells (0) for HMGB1 (B) or SP-A (C); n=3. Figure 8. SP-A interaction with HMGB1 both in vitro and in vivo. (A-B) SP-A interacted with HMGB1 in fibroblast culture medium. Medium containing SP-A and HMGB1 was immunoprecipitated (IP) with normal IgG, SP-A (A), or HMGB1 antibody (B). The immunoprecipitates were immunoblotted (IB) with SP-A or HMGB1 antibody as indicated. HMGB1 was found in complexes pulled down by SP-A antibody and vice versa. (C) Tissue lysates from kidneys of WT and SP-A-/mice with sham or UUO were immunoprecipitated (IP) with normal IgG, SP-A, or HMGB1 antibody followed by immunoblotting (IB) with SP-A or HMGB1 antibody 14
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as indicated. HMGB1 interacted with SP-A, which was enhanced by UUO.
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Highlights: SP-A is up-regulated in mouse and human kidney epithelium with ureter
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obstruction.
SP-A deficiency exacerbates UUO-caused kidney structural damage and fibrosis
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in mice.
SP-A inhibits HMGB1-mediated myofibroblast activation.
SP-A attenuates HMGB1-induced TGF-β expression.
SP-A physically interacts with HMGB1.
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S0925-4439(16)30326-X doi:10.1016/j.bbadis.2016.11.032 BBADIS 64627
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
20 August 2016 20 November 2016 30 November 2016
Please cite this article as: Shaojiang Tian, Chenxiao Li, Ran Ran, Shi-You Chen, Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice, BBA - Molecular Basis of Disease (2016), doi:10.1016/j.bbadis.2016.11.032
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice
Department of Physiology & Pharmacology, University of Georgia, Athens, GA, USA; and 2Department of Nephrology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China;
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Shaojiang Tian1,2†, Chenxiao Li1†, Ran Ran1,2, and Shi-You Chen1,2
† These authors contributed equally to this work.
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Running Title: SP-A in renal fibrosis Correspondence Author:
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Shi-You Chen, PhD Department of Physiology & Pharmacology The University of Georgia 501 D.W. Brooks Drive Athens, GA 30602 Tel: 706-542-8284 Fax: 706-542-3015 Email: [email protected]
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Abbreviations:
SP-A: surfactant protein A UUO: unilateral ureter obstruction HMGB1: high mobility group box 1 TGF-β: transforming growth factor β BMM: Bone marrow derived-macrophages HPTC: Human proximal tubular cell qRT-PCR: Reverse Transcription and quantitative RT-PCR Co-IP: co-immunoprecipitation α-SMA: smooth muscle α-actin FSP-1: fibroblast specific protein 1 IL-6: Interleukin 6
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ACCEPTED MANUSCRIPT Abstract
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Renal interstitial fibrosis is an inevitable consequence of virtually every type of chronic kidney disease. The underlying mechanisms, however, are not completely understood. In the present study, we identified surfactant protein A (SP-A) as a novel protein factor involved in the renal fibrosis induced by unilateral ureter obstruction (UUO). UUO induced SP-A expression in mouse kidney epithelium, likely due to the increased acidic stress and inflammation. Interestingly, SP-A deficiency aggravated UUO-prompted kidney structural damage, macrophage accumulation, and tubulointerstitial fibrosis. SP-A deficiency appeared to worsen the fibrosis by enhancing interstitial myofibroblast accumulation. Moreover, SP-A deficiency increased the expression of TGF-β1, the major regulator of kidney fibrosis, particularly in the interstitial cells. Mechanistically, SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1), a factor regulating TGF-β expression/signaling and implicated in renal fibrosis. SP-A also blocked HMGB1 activities in inducing TGF-β1 expression and myofibroblast transdifferentiation from kidney fibroblasts, demonstrating that SP-A protects kidney by impeding both the expression and fibrogenic function of HMGB1. Since SP-A physically interacted with HMGB1 both in vitro and in kidney tissue in vivo, SP-A may exert its protective role by binding to HMGB1 and thus titrating its activity during UUO-induced renal fibrosis.
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Key words: Kidney fibrosis, unilateral ureter obstruction, myofibroblast activation, surfactant protein A, high mobility group box 1, TGF-β
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ACCEPTED MANUSCRIPT 1. Introduction
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Surfactant protein A (SP-A) is a member of the C-type lectin family that play an important role in host defense and regulating inflammation in the lung. It can bind to its target ligands on pathogens, allergens, and apoptotic cells to facilitate the clearance process. SP-A can also interact with cell surface receptors to modulate cytokine production and regulate the inflammatory response.1 In addition to its role in infectious respiratory diseases, emerging evidence suggests that SP-A is also involved in a number of non-infectious lung disease such as cancer and fibrosis.2 Although SP-A is considered as a lung disease marker,3 numerous studies have demonstrated that SP-A is also expressed in many other organs including skin,4 middle ear,5 vagina,6 and gastric intestine.7 Moreover, this ubiquitous protein also exhibits a varying pattern of expression in response to different stimuli. In the lung, SP-A expression is affected by environmental insults and its gene polymorphism.8,9 In chronic rhinosinusitis, SP-A expression is up-regulated by the inflammation and tissue injury.10 Recent studies indicate that SP-A is expressed in human renal tubular epithelial cells, and the expression can be stimulated by lipopolysaccharide.11 In addition, SP-A and SP-D attenuate kidney injury by modulating inflammation and apoptosis in sepsis-induced acute kidney injury 12. However, whether or not SP-A plays a role in the process of non-infectious renal diseases, especially the chronic kidney diseases, the major cause of renal failure, remains unknown. In this study, we explored the expression patterns of SP-A in mouse kidney, and determined if SP-A affects the process of kidney fibrosis induced by unilateral ureteral obstruction (UUO). By taking advantage of SP-A knock-out mice, we demonstrated that SP-A deficiency worsens UUO-induced kidney interstitial fibrosis, suggesting a protective role of SP-A in the kidney. SP-A appeared to attenuate high mobility group box 1 (HMGB1)-induced TGF-β1 expression in interstitial cells, thus inhibit myofibroblast activation from fibroblasts. 2. Materials and Methods 2.1 Animals and UUO mouse model: SP-A knock-out (SPA-/-) mice with 129 genetic background were purchased from the Jackson Laboratory (STOCK Sftpa1tm1Kor/J). The mice were crossed with C57BL/6J for three generation, and its C57BL/6J×129 wild type littermates were used as controls. Wild type (WT) or SPA-/- male mice were either sham-operated or subjected to unilateral ureteral obstructive ligation (UUO). Briefly, mice weighing 20-25 g were anesthetized with isoflurane inhalation, and UUO was achieved by double-ligating the left ureter with 3-0 silk through a left abdominal lateral incision. Sham operation was performed similarly but without ureteral ligation. Mice were sacrificed at 3, 5, 7, 10 or 14 days after the surgery. Urine, blood and kidneys were collected and subjected to the studies described below. For mice with UUO, the retained urine in left ureters and pelvis were collected using 1 ml syringe. For sham-operated animals, metabolic 3
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cages were used to collect the urine. All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) of The University of Georgia.
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2.2 Isolation and culture of mouse renal primary fibroblasts: Normal (WT) mouse kidneys were minced to 1 mm3 cubes. The cubes were then transferred to a 10 cm cell culture dish, and digested in 5 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mg/ml collagenase Ⅳ at 37 ℃ for 30 min. The digestion was repeated for three times, and the suspension was filtered after each digestion through a 70 μm sterile filter. Cells were then centrifuged at 1200 rpm for 5 min and washed twice with phosphate buffer saline (PBS). Cells were cultured with complete DMEM medium containing 10% FBS at 37 ℃ with 5% v/v CO2 in a humidified atmosphere. One hour after plating, the medium was replaced to remove non-adherent cells. The adherent fibroblasts were verified by vimentin staining.
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2.3 Isolation and culture of mouse macrophage: Bone marrow derived-macrophages (BMM) were obtained as described previously.13 Briefly, femur and tibia were dissected in DMEM containing 10% FBS, and bone marrow cells were flushed from the femurs and tibias. After red cells were lysed, the remaining cells were counted and plated in a T-25 flask with 10 ng/ml of macrophage colony stimulating factor (M-CSF, Sigma) added in the medium. After culturing overnight, non-adherent cells were collected, washed, and plated in 60-mm petri dishes with 10 ng/ml of M-CSF in DMEM containing 10% FBS. After 7 days, cells were washed, and the adherent cells were released and removed with 0.1% EDTA. The resulting BMMs were verified to be >98% pure based on F4/80 staining. 2.4 Human proximal tubular cell (HPTC) culture: HPTCs were cultured in DMEM/F12 medium (Invitrogen) with supplements as described previously.14 1N HCI was used to adjust medium PH for the preparation of the acidified medium. 2.5 Human kidney specimens: The human kidney specimens were collected from hospitalized patients in Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China. The patients underwent nephrectomy due to severe hydronephrosis with ureteral stricture. Kidney cortex adjacent to the hydronephrosis were chosen for examining the pathological alterations. All patients provided written informed consent, and the study protocol was approved by the Scientific-Ethical Committee of Hubei University of Medicine. 2.6 Reverse Transcription and quantitative RT-PCR (qRT-PCR): Total RNA was extracted using TRIzol reagent (Invitrogen) by following the manufacturer’s 4
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instruction. cDNA was synthesized from 1 μg of total RNA using iScript cDNA Synthesized Kit (Bio-Rad). PCR reaction was performed as described previously.14 To avoid potential genomic DNA contamination, RNA samples were treated with DNase I followed by incubation at 65°C for 15 min. In addition, PCR reactions with samples without RT were used as negative controls. The PCR cycles ranged from 25 to 30. mRNA expression of pertinent genes was normalized to cyclophilin level. The following primers were used for each individual gene: mouse HMGB1 (forward: GCT GAC AAG GCT CGT TAT GAA; reverse: CCT TTG ATT TTG GGG CGG TA), SP-A (forward: TCC TGG AGA CTT CCA CTA CCT; reverse: CAG GCA GCC CTT ATC ATT CC), TGF-β1 (forward: CGC CAT CTA TGA GAA AAC C; reverse: GTA ACG CCA GGA ATT GT), and cyclophilin (forward: TGC AGC CAT GGT CAA CCC C; reverse: CCC AAG GGC TCG TCA).
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2.7 Western blot analysis: Western blot was performed as described previously.14 Proteins in kidney or cultured cells were extracted using RIPA lysis buffer containing protease inhibitors. Protein concentration was determined using a BCA kit (Pierce). Protein expressions were detected with an enhanced chemiluminescence kit (Millipore).
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2.8 Histopathological analysis: A half of left kidney tissues from sham or UUO mice were fixed in buffered 4% paraformaldehyde for 24 h and then embedded in paraffin wax. For assessment of tubulointerstitial injury and fibrosis, 5 μm sections were stained with H&E stain kit (MasterTech Lab) or Masson’s Trichrome 2000 Stain Kit (MasterTech Lab), respectively. Tubular injury index, characterized by tubular dilation and epithelial desquamation with interstitial expansions, was graded according to the extent of cortical involvement on a scale from 0 to 4 assessed using a semi-quantitative method previously described.15 Interstitial fibrosis was assessed by collagen deposition using a point-counting approach.16 2.9 Immunohistochemistry staining: For assessing SP-A and TGF-β1 expression, kidney sections were incubated with SP-A (Santa Cruz, sc-13977) or TGF-β1 antibody (Santa Cruz, sc-146) at a 1:100 dilution overnight followed by incubation with a HRP-conjugated goat anti-rabbit second antibody (Abcam, ab6721, 1:200 dilution). The bound peroxidase was developed using diaminobenzidine (DAB) as a substrate to produce a brown color followed by a blue nuclear haematoxylin counterstain. 2.10 Immunofluorescent staining: For assessing protein expression in kidney tissues or cultured cells, sections or cells adhered to coverslips were incubated with the primary FSP-1 (Abcam, ab197896), α-SMA (Sigma, A2547), HMGB1 (Cell Signaling, #3935), F4/80 (Abcam, ab16911), SP-A (Santa Cruz), or TGF-β1 antibody (Santa Cruz) at a 1:100 dilution overnight. Corresponding FITC-conjugated secondary antibodies (Sigma, F0257-anti-mouse or F0382-anti-rabbit) were applied for each individual primary antibody at a 1:100 dilution, and fluorescent 5
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2.11 Measurement of urine and blood HMGB1: HMGB1 level in urine and serum were measured using a HMGB1 ELISA kit (MyBioSource) by following manufacturer’s instructions.
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2.12 SPA-HMGB1 binding and co-immunoprecipitation (Co-IP) assay: For examining the SP-A binding with HMGB1 in vitro, SP-A (10 μg/ml) and HMGB1 (125 ng/ml) were added into the fibroblast culture medium (DMEM containing 10% FBS) and incubated at 37℃ for 2 h. For examining SP-A binding with HMGB1 in vivo, kidney tissues were lysed with ice-cold lysis buffer with protease inhibitor mix (Thermo Scientific). The mediums or lysates were incubated with IgG (negative control), SP-A, or HMGB1 antibodies immobilized on agarose beads following Instruction provided in the Co-IP kit (Thermo Scientific). The immunoprecipitates were pelleted, washed, and subjected to immunoblotting using SP-A or HMGB1 antibodies according to the manufacturer’s instructions.
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2.13 Statistical analysis: All data are reported as means ± SD. Data were analyzed using ANOVA followed by q test using the SPSS for windows 10.0. P values < 0.05 were considered as a statistically significant difference. 3. Results
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3.1 SP-A was induced in mouse kidney with UUO. Since SP-A has been reported to express in renal tubular epithelial cells,11 we sought to determine if SP-A is involved in the progression of renal fibrosis using the UUO model. We first detected if SP-A is induced in mouse kidney with UUO using an antibody that detects SPA in the lungs of wild type, but not SP-A-/- mice (Supplemental Figure sFig 1). As shown in Fig 1A, SP-A was barely expressed in sham-operated kidney. However, strong SP-A expression was observed in tubular epithelial cells and interstitium of kidneys with UUO (Fig 1, A). In fact, UUO injury rapidly induced both SP-A mRNA and protein expression as early as 3 days following UUO (Fig 1, A-C). The expression increased along with the progression of the injury. The high level of SP-A expression was maintained up to 14 days after the surgery (Fig 1, A-C). The tubular SP-A appeared to locate in both nuclei and cytoplasm of tubule epithelial cells (Supplemental sFig 2). Importantly, SP-A was expressed in human kidney with interstitial fibrosis due to hydronephrosis (Fig 1, D-F), indicating that SP-A may play a role in the development of human obstructive kidney diseases. A urine analysis indicated that the retained urine in the left ureter following UUO had a mean pH value of 5.52, and thus tubular epithelial cells exposed directly to the acidified urine. We therefore tested if acidic stress affects SP-A induction in tubular cells. As shown in Supplemental sFig 3, A-C, acidified medium (PH 5.5) markedly increased both SP-A mRNA and protein expression in human proximal tubule cells (HPTCs), suggesting that acidic stress is an important factor inducing 6
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SP-A expression in the kidney with UUO. In addition to acidic stress, pro-inflammatory cytokine such as IL-6 is also produced and secreted in kidney during the early stages of UUO.17 We therefore tested if IL-6 influences the SP-A expression in HPTCs and found that IL-6 induced SP-A mRNA and protein expression in a dose-dependent manner (0-200 ng/ml, Supplemental sFig 3, D-F), suggesting that inflammation is another factor inducing SP-A expression in mouse kidney with UUO.
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3.2 SP-A deficiency exacerbated UUO-induced kidney injury, macrophage accumulation, and tubulointerstitial fibrosis. To determine if SP-A plays a role in the progression of renal fibrosis, we performed UUO in both wild type and SP-A knockout (SP-A-/-) mice. As shown in Fig 2, A-B and Supplemental sFig 4, UUO caused obvious tubulointerstitial damages and epithelial cell transdifferentiation. Surprisingly, SP-A-/- resulted in a more severe damage compared to the wild type mice. In addition, more macrophages were observed in SP-A-/- mouse kidney, as shown by immunostaining of macrophage marker F4/80 in the sections of kidney with UUO for 5 days (Supplemental sFig 5A), suggesting an increased inflammation in SP-A-/- kidneys. Indeed, the IL6 level was significantly increased in SP-A-/mouse kidney with UUO compared to the control (Supplemental sFig 5B). Moreover, UUO injury led to a significant amount of collagen deposition in tubulointerstitium. SP-A-/- aggravated the collagen accumulation (Fig 2, C). Quantitative analysis confirmed that SP-A deficiency enhanced type I collagen expression in kidney with UUO (Fig 2, D). These data indicate that SP-A inhibits the development of renal fibrosis.
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3.3 SP-A deficiency increased interstitial myofibroblast accumulation. Myofibroblasts are the main source of collagen production in kidney fibrosis. Therefore, we examined if SP-A-/- affects myofibroblast accumulation. Two myofibroblast markers, smooth muscle α-actin (α-SMA) and fibroblast specific protein 1 (FSP-1), were used to label myofibroblasts. Immunostaining showed that UUO caused the accumulation of α-SMA- or FSP-1-positive cells in kidney interstitium. SP-A-/- increased the numbers of α-SMA- or FSP-1-expressing cells (Fig 3, A). Since FSP-1 also stains macrophage, we performed a co-immunostaining of FSP-1 and F4/80. As shown in sFig 5C, although a small portion of FSP-1-positive cells appeared to be macrophages, the majority of FSP-1+ cells were F4/80 negative, confirm the increased myofibroblast accumulation due to SP-A deficiency. Moreover, both α-SMA and FSP-1 protein expression was markedly increased in SP-A-/- mouse kidney with UUO injury compared to the wild type (Fig 3, B-C). These data demonstrate that SP-A inhibited renal fibrosis by limiting myofibroblast activation. 3.4 SP-A deficiency enhanced TGF-β1 expression in kidney tissue. It is well established that TGF-β1 is a key player in myofibroblast activation and the progression of renal fibrosis.18 Therefore, we investigated if SP-A-/- influences 7
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TGF-β1 expression. We found that UUO induced strong expression of TGF-β1 in mouse kidney (Fig 4), consistent with previous report.18 SP-A-/- further increased both the mRNA and protein expression of TGF-β1 (Fig 4). Interestingly, it appeared that UUO induced TGF-β1 expression mainly in tubular epithelial cells with very limited TGF-β1-expressing cells in the interstitium. However, SP-A-/- significantly increased the numbers of interstitial cells that expressed TGF-β1 (Fig 4, A-B, arrows in the enlarged image), suggesting that SP-A may mainly affect the interstitial cells such as inflammatory cells or residential fibroblasts.
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3.5 SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1) in kidney tissue. Our previous studies have found that HMGB1 plays an important role in initiating tubulointerstitial injury, interstitial inflammation, and renal fibrosis.19 In addition, HMGB1 has been shown to regulate TGF-β expression/signaling in different cells including kidney tubule epithelial cells.20 Therefore, we detected HMGB1 expression and its release in wild type and SP-A-/kidney with or without UUO injury. As shown in Fig 5, A, HMGB1 expression was observed in tubular epithelial cells. UUO significantly increased the HMGB1 mRNA and protein expression, and SP-A-/- further enhanced its expression (Fig 5, B-C). Since HMGB1 is a secreted protein, we detected serum and urine HMGB1 levels. As shown in Fig 5, D-E, serum and urine HMGB1 concentrations were markedly increased in UUO injured kidney compared to sham kidneys. SP-A-/- caused a further elevation of HMGB1 release to serum and urine from mouse kidneys with UUO. These data suggest that HMGB1 may be involved in SP-A deficiency-caused exacerbation of renal fibrosis.
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3.6 SP-A blocked HMGB1-induced fibroblast activation. To determine how SP-A affects the progression of renal fibrosis, we tested if HMGB1 induces fibroblast activation and if SP-A regulates HMGB1 function. As shown in Fig 6, A, HMGB1 induced the activation of kidney primary fibroblasts, as indicated by the induction of myofibroblast markers α-SMA and FSP-1. In fact, HMGB1 induced kidney fibroblasts to become myofibroblasts in a dose-dependent manner (Fig 6, B). However, SP-A dose-dependently inhibited the HMGB1-induced expression of α-SMA and FSP-1 (Fig 6, A and C), indicating a blockade of fibroblast activation. Since HMGB1 or combination of SP-A and HMGB1 did not significantly alter fibroblast proliferation or survival (Supplemental sFig 6), SP-A may lessen the severity of renal fibrosis mainly by blocking HMGB1-induced myofibroblast differentiation. 3.7 SP-A blocked HMGB1-induced TGF-β expression. Since HMGB1 has been shown to regulate TGF-β expression in renal tubular epithelial cells, 20 and SP-A deficiency induced TGF-β1 expression in interstitial cells (Fig 4), we sought to determine if HMGB1 induces TGF-β1 expression in kidney fibroblasts and if SP-A affects HMGB1 function. As shown in Fig 7, A, HMGB1 indeed induced TGF-β1 expression in kidney fibroblasts, suggesting that HMGB1 may promote fibroblast to 8
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myofibroblast transition through up-regulating TGF-β1 expression. Actually, HMGB1 induced TGF-β1 expression in a dose-dependent manner (Fig 7, B). However, SP-A dose-dependently blocked HMGB1-induced TGF-β1 expression in renal fibroblasts (Fig 7, A and C), indicating that SP-A may attenuate the fibroblast-to-myofibroblast transition through disturbing HMGB1-induced TGF-β1 expression.
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3.8 SP-A bound to HMGB1 both in vitro and in vivo. To explore how SP-A blocks HMGB1 functions, we tested if SP-A physically interacts with HMGB1. As shown in Fig 8, A-B, SP-A bound to HMGB1 in fibroblast culture medium, suggesting that SP-A may bind to HMGB1 and thus interfere HMGB1 binding to its receptors in fibroblasts. In mouse kidney tissue, SP-A also bound to HMGB1 (Fig 8, C), indicating that SP-A secreted from tubular epithelial cells may inhibit fibroblast activation through binding to HMGB1 in interstitium, thus protect kidney from the more severe fibrosis or damage. 4. Discussion
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Tubulointerstitial fibrosis is considered as a final common pathway for chronic kidney disease progressing to renal failure.21 Although numerous factors are identified to be involved in renal fibrosis, the mechanisms underlying this complex process are not completely understood. Our study demonstrated for the first time that SP-A is a new protein factor involved in the progression of renal fibrosis following the obstructive injury. We found that a high level of SP-A is expressed in tubular epithelial cells in the injured mouse kidney, and SP-A deficiency causes more severe tubular injury, interstitial inflammation, and interstitial fibrosis. It appears that SP-A inhibits fibroblast to myofibroblast differentiation induced by HMGB1, which is expressed and released from kidney due to UUO injury. Therefore, SP-A expression in tubular epithelial cells following UUO is a protective response to the kidney injury. SP-A protects kidney from the potential more severe damage by attenuating HMGB1 function in inducing TGF-β expression, fibroblast activation and consequently the fibrosis. Although SP-A is clearly important for the obstruction-induced renal fibrosis, whether or not SP-A is expressed in kidney has been a debate. There are reports showing that neither SP-A mRNA or protein is detectable in kidney,22,23 while other studies suggest that SP-A protein is expressed in kidney tissue.5 Recent studies find that lipopolysaccharide induces SP-A expression in human renal tubular epithelial cells.11 Our current study reveals that a low level of SP-A may be present in normal mouse kidney. However, SP-A is significantly induced in kidney by UUO injury. Consistently, SP-A expression is significantly increased in HPTCs when they are treated with acidified medium or pro-inflammatory cytokine, further demonstrating that SP-A can be induced by pathological insults. Two possible mechanisms may be involved in the role of SP-A regulating HMGB1 function. Firstly, SP-A regulates HMGB1 expression because SP-A 9
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deficiency increases both the mRNA and protein levels of HMGB1 in kidney with UUO, mainly in epithelial cells (Fig 5). Secondly, SP-A attenuates HMGB1 function through physically interacting with HMGB1, which may block HMGB1 binding to its receptors. Indeed, SP-A can bind with HMGB1 in culture medium in vitro and in kidney with UUO in vivo (Fig 8). The SP-A inhibition of HMGB1-induced fibroblast activation and TGF-β expression (Fig 6-7) supports that SP-A may block HMGB1 activities through interfering HMGB1 interaction with its receptor. Interestingly, SP-A deficiency significantly increases the number of TGF-β-expressing cells in kidney interstitium following UUO. The two possible cells in the interstitium that may produce TGF-β1 are macrophages and fibroblasts. Our data show that HMGB1 induces TGF-β1 expression in fibroblasts (Fig 7), which is blocked by SP-A. However, HMGB1 does not induce macrophage to express TGF-β1. Rather, HMGB1 blocks TGF-β1 expression in macrophages (Supplemental sFig 7). Therefore, SP-A is likely to only block HMGB1-mediated TGF-β expression in fibroblasts. SP-A may regulate macrophage function via a different mechanism, which could be studied in the future. One limitation of this study is that UUO is not an ideal model for chronic kidney disease because it is not a common cause of adult human renal diseases. In addition, the UUO mouse exhibits only modest change in serum creatinine without proteinuria and hypertension. However, SP-A is expressed in human kidney with hydronephrosis, underscoring its association/importance in the development of human renal fibrosis due to ureter obstruction. Another limitation of this study is that the role of acidic stress and pro-inflammatory cytokines in SP-A release are not established in vivo. 5. Conclusion
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Our study has identified a novel factor involved in renal fibrosis and provided evidence that SP-A lessens the UUO-induced kidney damage and fibrosis via inhibiting HMGB1-mediated TGF-β expression and fibroblast activation. Acknowledgements This work was supported by grants from National Institutes of Health (grant numbers HL123302 and HL119053) and National Natural Science Foundation of China (grant number 81328002).
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fibrosis through facilitating M1 macrophage phenotype at early stage of obstructive injury. Am J Physiol Renal Physiol. 2015; 308:F69-75. 20. Cheng M, Liu H, Zhang D, et al. HMGB1 Enhances the AGE-Induced Expression of CTGF and TGF-β via RAGE-Dependent Signaling in Renal Tubular Epithelial Cells. Am J Nephrol. 2015; 41:257-266. 21. Zeisberg M, Neilson EG. Mechanisms of tubulointerstitial fibrosis. J Am Soc Nephrol. 2010; 21:1819-1834. 22. Akiyama J, Hoffman A, Brown C, et al. Tissue distribution of surfactant proteins A and D in the mouse. J Histochem Cytochem. 2002; 50:993-996. 23. Madsen J, Tornoe I, Nielsen O, et al. Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol. 2003; 29:591-597.
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Figure 1. SP-A expression in injured mouse and human kidneys. (A) Immunohistochemistry staining showed SP-A expression in tubular epithelium and interstitium of mouse kidney with UUO for the days (D3-D14) indicated. (B) SP-A mRNA expression was detected by qRT-PCR in mouse kidney following UUO injury. *P<0.05 compared with sham group (D0), #P<0.05 compared to day 3, $ P<0.05 compared to day 5 (n=5). (C) SP-A protein expression was detected by Western blot and quantified by normalizing to α-Tubulin. *P<0.05 compared with sham group (D0), #P<0.05 compared to day 3, $P<0.05 compared to day 5 (n=8). (D-F) SP-A expression in human kidney tubules along with tubulointerstitial fibrosis due to ureter obstruction-caused hydronephrosis. Healthy (normal) and hydronephrotic human kidney specimen sections were stained with Masson’s trichrome (D, upper panel) or SP-A antibody (D, lower panel). Collagen deposition was measured by the intensity of collagen deposition and normalized to the level in normal kidney (E). SP-A expression was quantified by measuring the staining intensity and normalized to the normal kidney staining (F). *P<0.01 compared with the normal kidney in E and F, respectively (n=5).
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Figure 2. SP-A deficiency exacerbated UUO-caused tubular injury and renal fibrosis. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 14 days. (A) SPA-/- worsen tubular injury as shown by H&E staining. (B) Kidney tubular injury index calculated using a semiquantitative method based on the H&E-stained kidney structures as shown in A. (C) SPA-/- caused more severe renal fibrosis in mice with UUO as shown by Masson’s staining. (D) Western blot analysis to quantify SP-A and type I collagen I (CoI-I) expression. *P<0.01 compared to sham group; #P<0.01 compared to WT mice with UUO; n=8. Figure 3. SP-A deficiency promoted interstitial fibroblast activation. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) SPA-/increased the numbers of α-SMA and FSP1-positive cells as shown by immunostaining. (B) α-SMA and FSP-1 protein expression in mouse kidneys was examined by western blot. (C) α-SMA and FSP-1 protein expression shown in B was quantified by normalizing to α-Tubulin level. *P<0.01 compared with sham group for each corresponding protein; #P<0.05 compared with WT mice with UUO for each corresponding protein; n=8. Figure 4. SP-A deficiency enhanced TGF-β1 expression in mouse kidney with UUO. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) TGF-β1 expression was detected by immunostaining. TGF-β1+ interstitial cells in the SP-A-/- kidney with UUO injury was significantly more than that in the WT kidney, as shown in the image enlarged from the red square-enclosed section. (B) Quantification of TGF-β-expressing interstitial cells by counting TGF-β-positive cells in 8 different sections and normalized to the sham-operated WT kidney. (C-D) 13
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TGF-β1 mRNA and protein expression was quantified by qRT-PCR (C) and western blot (D), respectively. Protein expression was normalized to the α-Tubulin level. *P<0.01 compared with sham group; #P<0.01 compared with WT mice with UUO (n=8 for B, C and D).
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Figure 5. SP-A deficiency increased UUO-induced HMGB1 expression. Wild type (WT) and SPA-/- mice underwent sham operation or UUO for 7 days. (A) HMGB1 expression was detected by immunostaining. Arrows indicate the strong HMGB1 expression in epithelial cells of structurally-damaged tubules. (B-C) HMGB1 mRNA and protein expression was quantified by qRT-PCR (B) and western blot (C), respectively. Protein expression was normalized to α-Tubulin level. (D-E) SPA-/- increased UUO-induced HMGB1 secretion in serum (D) and urine (E) as measured by ELISA. *P<0.01 compared with sham group; #P<0.05 compared with WT mice with UUO; n=8.
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Figure 6. SP-A inhibited HMGB1-mediated fibroblast activation. Mouse kidney primary fibroblasts were cultured as described in Methods and treated with (+) or without (-) HMGB1 or SP-A as indicated. (A) HMGB1-induced myofibroblast marker α-SMA and FSP-1 was dose-dependently inhibited by SP-A, as detected by immunostaining. (B) Western blot showed a dose-dependent induction of α-SMA and FSP-1 by HMGB1. (C) SP-A dose-dependently inhibited the HMGB1-induced α-SMA and FSP-1 protein expression (50 ng/ml), as examined by Western blot. Protein expression (B and C) was normalized to α-Tubulin level. *P<0.01 compared with vehicle-treated cells (0) for FSP-1; #P<0.01 compared with vehicle-treated cells (0) for α-SMA; n=3.
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Figure 7. SP-A inhibited HMGB1-induced TGF-β1 expression in kidney fibroblasts. Primary kidney fibroblasts were treated similarly as in Figure 6. TGF-β1 expression was examined. (A) SP-A dose-dependently inhibited HMGB1 (50 ng/ml)-induced TGF-β1 expression as detected by immunostaining. (B) HMGB1 dose-dependently induced TGF-β1 expression as measured by Western blot. (C) SP-A dose-dependently inhibited TGF-β1 expression induced by HMGB1 (50 ng/ml) as examined by Western blot. Protein expression (B and C) was normalized to α-Tubulin level. *P<0.01 compared with vehicle-treated cells (0) for HMGB1 (B) or SP-A (C); n=3. Figure 8. SP-A interaction with HMGB1 both in vitro and in vivo. (A-B) SP-A interacted with HMGB1 in fibroblast culture medium. Medium containing SP-A and HMGB1 was immunoprecipitated (IP) with normal IgG, SP-A (A), or HMGB1 antibody (B). The immunoprecipitates were immunoblotted (IB) with SP-A or HMGB1 antibody as indicated. HMGB1 was found in complexes pulled down by SP-A antibody and vice versa. (C) Tissue lysates from kidneys of WT and SP-A-/mice with sham or UUO were immunoprecipitated (IP) with normal IgG, SP-A, or HMGB1 antibody followed by immunoblotting (IB) with SP-A or HMGB1 antibody 14
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as indicated. HMGB1 interacted with SP-A, which was enhanced by UUO.
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Highlights: SP-A is up-regulated in mouse and human kidney epithelium with ureter
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obstruction.
SP-A deficiency exacerbates UUO-caused kidney structural damage and fibrosis
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in mice.
SP-A inhibits HMGB1-mediated myofibroblast activation.
SP-A attenuates HMGB1-induced TGF-β expression.
SP-A physically interacts with HMGB1.
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