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Nephrology 20 (2015) 820–831
Original Article
Intermedin is upregulated and attenuates renal fibrosis by inhibition of oxidative stress in rats with unilateral ureteral obstruction XI QIAO,1,2 LIHUA WANG,1,2 YANHONG WANG,3 NING ZHAO,1 RUIJING ZHANG,1 WEIXIA HAN1 and ZHIQIANG PENG1,2 1 Department of Nephrology, Second Hospital of Shanxi Medical University, and 2Shanxi Kidney Disease Institute and 3Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan, Shanxi, China
KEY WORDS: calcitonin receptor-like receptor, intermedin, oxidative stress, receptor activity-modifying proteins, renal fibrosis. Correspondence: Dr Xi Qiao, Department of Nephrology, Second Hospital of Shanxi Medical University, Shanxi Kidney Disease Institute, WuYi Road 382, Taiyuan, Shanxi 030001, China. Email: [email protected] Accepted for publication 19 May 2015. Accepted manuscript online 25 May 2015. doi:10.1111/nep.12520 Xi Qiao and Lihua Wang contributed equally to this study.
SUMMARY AT A GLANCE Authors show that treatment of rats with the secreted peptide Intermedin can reduce renal fibrosis and oxidative stress in a model of unilateral ureteral obstruction.
ABSTRACT: Aim: Transforming growth factor-β1 (TGF-β1) plays a pivotal role in the progression of renal fibrosis. Reactive oxygen species mediate profibrotic action of TGF-β1. Intermedin (IMD) has been shown to inhibit oxidative stress, but its role in renal fibrosis remains unclear. Here, we investigated the effects of IMD on renal fibrosis in a rat model of unilateral ureteral obstruction (UUO). Methods: The expression of IMD and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP1/2/3), in the obstructed kidney was detected by real-time polymerase chain reaction (PCR), western blotting and immunohistochemistry. To evaluate the effects of IMD on renal fibrosis, we locally overexpressed exogenous IMD in the obstructed kidney using an ultrasound-microbubble-mediated delivery system. Renal fibrosis was determined by Masson trichrome staining. The expression of TGF-β1, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and fibronectin was measured. Smad2/3 activation and macrophage infiltration were evaluated. We also studied oxidative stress by measuring superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Results: mRNA and protein expression of IMD increased after UUO. CRLR, RAMP1, RAMP2 and RAMP3 were also induced by ureteral obstruction. IMD overexpression remarkably attenuated UUO-induced tubular injury and blunted fibrotic response as shown by decreased interstitial collagen deposition and downregulation of fibronectin. Macrophage infiltration, α-SMA and CTGF upregulation caused by UUO were all relieved by IMD, whereas TGF-β1 upregulation and Smad2/3 activation were not affected. Meanwhile, we noted increased oxidative stress in obstruction, which was also attenuated by IMD gene delivery. Conclusions: Our results indicate that IMD is upregulated after UUO. IMD plays a protective role in renal fibrosis via its antioxidant effects.
Chronic kidney disease is a major health problem with an increasing incidence worldwide.1 Regardless of disease aetiology, fibrosis is the common pathway leading to end stage renal diseases in all progressive chronic kidney diseases and is regarded as a prognostic factor for renal function.2 Therefore, elucidating the aetiological mechanisms underlying renal fibrosis and developing novel therapeutic strategies are 820
of great importance. Transforming growth factor-β1 (TGFβ1) plays a pivotal role in the progression of renal fibrosis.3 TGF-β1 promotes the accumulation of extracellular matrix (ECM), through the enhanced synthesis of ECM proteins, such as fibronectin (FN1), and the inhibition of their degradation, mainly through Smad-dependent pathways.4 Reactive oxygen species (ROS) are important downstream © 2015 Asian Pacific Society of Nephrology
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effectors that mediate the above profibrotic actions of TGFβ1/Smads.5,6 Therapeutic interventions targeting ROS have been successful and well tolerated in several experimental models.5,7 Intermedin (IMD, also known as adrenomedullin-2 (ADM-2)) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family.8 IMD is distributed in a wide variety of tissues including the kidney.9,10 In kidney, IMD mainly expresses in tubular cells of cortex and medulla.9,11 Similar to other peptides in the CGRP family, IMD signals through a type II G protein-coupled receptor complex consisting of the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs).8 Unlike CGRP and ADM, which exhibit a preferential stimulation of CRLR when coexpressed with RAMP1 and RAMP2 or RAMP3, respectively, IMD binds nonselectively to all three CRLR/RAMP complexes: CRLR/RAMP1, CRLR/ RAMP2 and CRLR/RAMP3,8 indicating that it has more potent actions than ADM and any other CGRP. Initial work from our laboratory has demonstrated that IMD gene transfer protects against renal ischaemiareperfusion injury by inhibition of oxidative stress,11 while its role in renal fibrosis is unknown. We hypothesized that IMD can attenuate renal fibrosis by reducing oxidative stress. To test this hypothesis, we examined the expression of IMD and its receptors, CRLR and RAMPs, in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Then, we investigated the effects of kidney-specific IMD gene delivery on renal fibrosis induced by UUO. Furthermore, in order to explain the mechanisms underlying these effects, we studied the oxidative stress in obstructive nephropathy by measuring SOD activity (an important endogenous antioxidant enzyme) and MDA content (a marker of oxidative stress).
METHODS
TGCTGATGGT-3′; antisense: 5′-TGCCCGGGAGCAGGTA-3′), CRLR (sense: 5′-CTCACGGCAGTGGCCAATAA-3′; antisense: 5′-AGCCCAT CAGGTAAAGATGAATGAA-3′), RAMP1 (sense: 5′-AGCTGTGTC TCAGCCGCTTC-3′; antisense: 5′-AATCTTGTTTGCCACGAGTTTG3′), RAMP2 (sense: 5′-GAGTCCCTGAATCAATCTCATCCTA-3′; antisense: 5′-TCAAAGTCCAGTTGCACCAGTC-3′), RAMP3 (sense: 5′-GAAAGCCTTTGCCGAAATGATG-3′; antisense: 5′-GCCCACGA TGTTTGTCTCCA-3′), TGF-β1 (sense: 5′-CATTGCTGTCCCGTG CAGA-3′; antisense: 5′-AGGTAACGCCAGGAATTGTTGCTA-3′), or FN1 (sense: 5′-CATCAGCCCGGATGTCAGAA-3′; antisense: 5′-GGC ATTGTCGTTGAGCGTGTA-3′) and β-actin (sense: 5′-CCCATCTATG AGGGTTACGC-3′; antisense: 5′-TTTAATGTCACGCACGATTTC-3′). The reaction was carried out in a 96-well plate in 25 μL reactions containing 2× SYBR Green Master mix (TAKARA, Dalian, China) – 2 pmol each of sense and antisense primer and the conditions were 95°C for 2 min, followed by 95°C for 10 s, 60°C for 15 s, for 40 cycles. In each assay, a standard curve was determined concurrently with examined samples. Gene expression was quantified using a medication of the 2-ΔΔct method. The amount of PCR products was normalized to the level of β-actin to determine the relative expression ratio for each mRNA.
Western blot analysis Analysis was carried out as previous described.12 Immunoblot analysis was performed with rabbit polyclonal anti-rat IMD antibody, goat polyclonal anti-rat CRLR (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-rat RAMP1, rabbit polyclonal anti-rat RAMP2, rabbit polyclonal anti-rat RAMP3 (all from Beijing Biosynthesis Biotechnology, Beijing, China), goat polyclonal anti-rat fibronectin (FN1) antibody (Santa Cruz Biotechnology), goat polyclonal anti-rat pSmad2/3 or rabbit polyclonal anti-rat Smad2/3 (Santa Cruz Biotechnology) and then peroxidaseconjugated AffiniPure goat anti-rabbit immunoglobulin G or rabbit anti-goat immunoglobulin G (Santa Cruz Biotechnology), respectively. The ECL western blotting system (Santa Cruz Biotechnology) was used for detection. Rabbit polyclonal anti-rat β-actin antibody (Santa Cruz Biotechnology) was used as the control for each sample.
Animal model
Immunohistochemistry
The Experimental Animal Committee approved all animal protocols. Renal fibrosis was induced by ligation of left ureter, UUO, in male Wistar rats (180 to 200 g). Briefly, rats were anaesthetized, laparotomy performed, and the left ureter identified and ligated at two points along the ureter. Sham-operated rats underwent the same surgical procedure except for the ureter ligation. Groups of six animals were killed at 1, 3, 7, and 14 days after UUO. Kidneys were harvested for further analysis.
Rat kidneys were fixed in 10% formaldehyde and embedded in paraffin. Paraffin sections (5 μm) were mounted on glass slides, deparaffinized in xylene, and rehydrated in ethanol with increasing concentrations of water. The rehydrated sections were pretreated with 3% H2O2 for 10 min at room temperature to block the endogenous peroxidase. After boiling in antigen retrieval solution (1 mmol/L tris-HCl, 0.1 mmol/L EDTA, pH = 8.0) for 10 min at high power in a microwave oven, the sections were incubated overnight at 4°C with primary rabbit polyclonal anti-rat IMD antibody (1:200, sc-86272; Santa Cruz Biotechnology), goat polyclonal anti-rat CRLR antibody (1:200, sc-18007; Santa Cruz Biotechnology), rabbit polyclonal anti-rat RAMP1 antibody (1:200, bs-1567R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat RAMP2 antibody (1:200, bs-11971R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat RAMP3 antibody (1:200, bs-11972R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat TGF-β1 antibody (1:200, sc-146; Santa Cruz Biotechnology), mouse monoclonal anti-rat ED-1 antibody (1:200, sc-59103; Santa Cruz
Quantitative RT-PCR Total renal RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then subjected to RT using a first-strand cDNA synthesis system (TAKARA, Dalian, China). Quantitative RT-PCR amplifications were performed in triplicate using the SYBR Green I assay and were carried out using Strategene M3000 Sequence Detection System (Stratagene, Santa Clara, CA, USA). Specific primers were used for IMD (sense: 5′-GGCCCAGT © 2015 Asian Pacific Society of Nephrology
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Biotechnology), rabbit polyclonal anti-rat α-SMA antibody (1:200, bs-10196R; Beijing Biosynthesis Biotechnology), or rabbit polyclonal anti-rat CTGF antibody (1:200, sc-14939; Santa Cruz Biotechnology), respectively. This was followed by biotinylated secondary antibodies (Santa Cruz Biotechnology) and finally by avidinconjugated horseradish peroxidase. All slides were counterstained with haematoxylin. Positive stained area were assessed and expressed as integrated optical density (IOD) and area. Three sections of each rat kidney were measured, and 10 random fields were chosen and calculated under magnification of 100×. The IOD and positive area were acquired by the Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, MD, USA). The number of macrophages was counted as positive staining for ED-1 in a blinded manner from 10 different fields of each section at ×400 magnification.
Ultrasound-mediated gene delivery into the kidney Eukaryotic expression plasmid pcDNA3.1-IMD containing fulllength cDNA sequence of rat IMD was successfully constructed in our previous study.11 Before the ureter was obstructed, pcDNA3.1IMD plasmid or control empty vector was transfected into the left kidney via the renal artery using an ultrasound-microbubblemediated system as we previously described.11 Rats were killed at 7 days after UUO. The transfection rate was detected by quantitative RT-PCR and immunohistochemistry, respectively.
Histological examinations Renal histological changes were assessed at days 7 and 14 after UUO. Paraffin-embedded transverse kidney slices were sectioned at 3 μm, and stained with haematoxylin and eosin. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. Twenty cortical tubulointerstitial fields that were randomly selected at ×100 magnification were assessed in each rat, and the density of trichrome positive signals was analyzed using Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, MD, USA). Briefly, the tonality of the fibrosis area (blue) was determined with control sections as reference. The number of pixels with the predetermined colour of tone was counted in each section, and then automatically converted into dimensions.
Superoxide dismutase activity and malondialdehyde content Rat kidneys were homogenized in ice-cold 20 mM Tris–HCl buffer (pH 7.4). Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were determined with commercially available kits, respectively (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis Results were presented as means ± standard deviation (SD). Student’s t-test was used to determine the significance of differences between two groups, whereas the one way analysis of variance (ANOVA) was used for comparison of multiple groups. P < 0.05 was considered significant. 822
RESULTS Expression of IMD and its receptors, CRLR and RAMPs, was upregulated in UUO kidney We first examined whether the mRNA and protein expression levels of IMD and its receptors, CRLR and RAMPs, in kidney were altered by UUO using quantitative RT-PCR, western blot and immunohistochemistry, respectively. In sham-operated kidneys, IMD was expressed moderately and constantly throughout the experimental period. IMD transcripts in the UUO kidneys increased 1 day after surgery and remained increased at the subsequent time points, reached the highest level at day 7 after UUO, and then declined at day 14 (Fig. S1A). Comparable to the increased transcripts, IMD protein expression was already increased in the UUO kidneys from day 1 after the surgery, and the increased expression levels remained at subsequent time points (Fig. 1A,B). This observation was confirmed further by immunohistochemistry (Fig. 2A–E). At a baseline, endogenous IMD can be detected in tubular cells, endothelial cells of the glomerulus and vasa recta in the kidney of sham-operated rats (Fig. 2A). Immunoreactivity for IMD was increased markedly from day 1 to day 14 in UUO kidneys (Fig. 2B–E). Basal CRLR, RAMP1, RAMP2 and RAMP3 gene and protein expression was detectable in sham-operated kidneys. After ureteral obstruction, the mRNA and protein expression levels of CRLR, RAMP1, RAMP2 and RAMP3 in the kidney increased from day 1, reached the peak at day 3 (RAMP2 and RAMP3) or at day 7 (CRLR and RAMP1), respectively (Fig. S1B–E, Fig. 1A,C–F). Immunohistochemistry for CRLR (Fig. 2F–J), RAMP1 (Fig. 2K–O), RAMP2 (Fig. 2P–T) and RAMP3 (Fig. 2U–Y) also demonstrated the increased immunoreactivity in UUO kidneys.
Efficacy of ultrasound-microbubble-mediated gene transfection We examined the efficacy of ultrasound-microbubblemediated gene transfection in kidney by quantitative RT-PCR. After 7 days of transfection and UUO operation, kidneys from rats treated with pcDNA3.1-IMD plasmid exhibited significant increase in IMD expression compared with kidneys of rats treated with UUO only (Fig. 3A). The expression of IMD showed no significant difference between the kidneys transfected with empty plasmid and nontransfected kidneys when they were subjected to UUO (Fig. 3A). Our results indicated that IMD was transfected into the kidney successfully. To confirm this result, we performed immunohistochemistry. The kidneys transfected with pcDNA3.1-IMD plasmid (Fig. 3B(c),C) exhibited much stronger IMD immunostaining than kidneys treated with UUO only (Fig. 3B(b),C) or kidneys transfected with control empty vector (Fig. 3B(d),C). © 2015 Asian Pacific Society of Nephrology
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Fig. 1 Increased protein expression levels of intermedin (IMD), calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in the kidneys after unilateral ureteral obstruction (UUO) surgery. (A) Representative images of the time-course western blot of whole kidney lysates for IMD, CRLR, RAMP1, RAMP2 and RAMP3 after UUO surgery. β-actin was used as a loading control. (B–F) Bar chart showing the IMD (B), CRLR (C), RAMP1 (D), RAMP2 (E), RAMP3 (F) protein expression time-course normalized by β-actin. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group.
IMD overexpression decreased renal injury and interstitial fibrosis in UUO Renal histologic changes were determined by haematoxylin and eosin staining. No changes were detected in the shamoperated group (Fig. 4A(a),A(e)). In the untreated UUO group, tubular lumens were significantly dilated and the epithelial cells were flattened accompanied by infiltration of inflammatory cells in the interstitium 7 days after UUO (Fig. 4A(b)). At 14 days post-obstruction, all lesions observed at UUO day 7 worsened and increased ECM deposition became a prominent feature. Most tubules appeared markedly atrophic, having lost their original integrity (Fig. 4A(f)). Overexpression of IMD in the kidney considerably decreased © 2015 Asian Pacific Society of Nephrology
renal injury at both 7 and 14 days after UUO (Fig. 4A(c),A(g)). Rats that were treated with the control empty plasmid (Fig. 4A(d),A(h)) revealed no significantly improved renal morphology compared with those in untreated UUO group. The degree of renal fibrosis was determined by Masson trichrome staining. Masson trichrome is most useful to differentiate collagen from other fibres. There was significant interstitial fibrosis that resulted in separation of the tubules at days 7 and 14 in untreated UUO kidneys (Fig. 4Bb,Bf). IMD gene delivery decreased interstitial collagen deposition on days 7 and 14 (Fig. 4Bc,Bg) in comparison with untreated obstructed kidneys. Semiquantitative analysis showed that the fibrotic score of pcDNA3.1-IMD-treated kidneys was 823
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Fig. 2 Localization of intermedin (IMD), calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) expression in the unilateral ureteral obstruction (UUO) kidneys. (A–E) Representative immunohistochemical staining for IMD protein in the sham operation kidneys (A), and the UUO kidneys at day 1 (B), day 3 (C), day 7 (D) and day 14 (E) after surgery. (F–J) Representative immunohistochemical staining for CRLR protein in the sham operation kidneys (F), and the UUO kidneys at day 1 (G), day 3(H), day 7 (I) and day 14 (J) after surgery. (K–O) Representative immunohistochemical staining for RAMP1 protein in the sham operation kidneys (K), and the UUO kidneys at day 1 (L), day 3 (M), day 7 (N) and day 14 (O) after surgery. (P–T) Representative immunohistochemical staining for RAMP2 protein in the sham operation kidneys (P), and the UUO kidneys at day 1 (Q), day 3 (R), day 7 (S) and day 14 (T) after surgery. (U–Y) Representative immunohistochemical staining for RAMP3 protein in the sham operation kidneys (U), and the UUO kidneys at day 1 (V), day 3(W), day 7 (X) and day 14 (Y) after surgery. Scale bar = 100 μm. Magnification: ×100.
significantly lower than that of untreated UUO kidneys (Fig. 4B(i)). Tubulointerstitial fibrosis was not obviously blocked by the addition of control empty vector (Fig. 4Bd,Bh,Bi).
As a control, we did not find that the expression of FN1 was significantly different between the kidneys transfected with empty plasmid and nontransfected kidney when they were subjected to UUO (Fig. 5A,B,C).
IMD overexpression inhibited extracellular matrix accumulation
IMD gene transfer had no effect on the expression of TGF-β1 in UUO kidneys
To evaluate the role of IMD on ECM accumulation, we examined the expression of fibronectin (FN1) after IMD gene transfer by quantitative RT-PCR and western blot analyses. The mRNA and protein expression levels of FN1 were significantly increased at days 7 and 14 after surgery. IMD gene delivery significantly reduced FN1 levels in the UUO kidneys.
We examined the expression of TGF-β1 in the whole kidney on days 7 and 14 after ureteral ligation by quantitative RT-PCR and immunohistochemical analyses. In our study, basal TGF-β1 expression was low in the sham operation kidneys. Both quantitative RT-PCR and immunostaining for TGF-β1 revealed that UUO significantly upregulated renal
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the upregulation of Smad2/3 phosphorylation level but not total Smad2/3 on days 7 and 14 after ureteral ligation. IMD or empty plasmid did not alter either Smad2/3 phosphorylation or total Smad2/3 in the obstructed kidneys (Fig. S2).
IMD gene delivery inhibited CTGF expression in UUO kidneys We examined CTGF, a downstream target of TGF-β1 involved in ECM production, by immunohistochemical analyses. CTGF expression was significantly elevated in the obstructed kidneys on days 7 and 14 after UUO (Fig. 7B,F,I) as compared with the sham-operated kidneys (Fig. 7A,E,I). Introduction of IMD into the kidney (Fig. 7C,G,I) markedly inhibited CTGF expression compared with the untreated obstructed kidney, while CTGF expression was not obviously affected by the addition of control empty vector (Fig. 7D,H,I).
IMD inhibited α-SMA expression in UUO kidneys α-SMA-positive myofibroblasts are responsible for the overproduction of ECM at pathologic conditions. The immunohistochemistry of α-SMA demonstrated that α-SMA expression increased on days 7 and 14 after ureteral ligation (Fig. S3B,F,I). The α-SMA expression levels in the obstructed kidneys of IMD-treated UUO rats (Fig. S3C,G,I) were significantly decreased compared to those of UUO-control rats on both day 7 and day 14. Empty plasmid did not affect the expression levels of α-SMA (Fig. S3D,H,I). Fig. 3 Ultrasound-microbubble-mediated gene transfer into the kidney. Microbubbles loaded with pcDNA3.1-intermedin (IMD) or control empty vector were infused into the kidney via the left renal artery treated with ultrasound before unilateral ureteral obstruction (UUO) surgery. On day 7 after ureter ligation, expression of IMD was assessed. (A) Representative quantitative RT-PCR. (B) Immunohistochemical localization of IMD in the kidney of sham-operated rats (a), untreated UUO rats (b), rats treated with pcDNA3.1IMD plasmid (c), and rats treated with control empty vector (d). (C) Semiquantitative evaluation of IMD expression. Data in bar graphs are means ± standard deviation (SD) (n = 6). O.D., optical density. Scale bar = 100 μm. Magnification: ×100. *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
TGF-β1 mRNA and protein levels (Fig. 6A,B(b),B(f),B(i)). Gene transfer of IMD (Fig. 6A,B(c),B(g),B(i)) or empty plasmid (Fig. 6A,B(d),B(h),B(i)) did not affect the expression level of TGF-β1.
IMD did not affect UUO-induced Smad2/3 activation We evaluated downstream signalling molecules of TGF-β1, Smad2/3. western blot analyses showed that UUO induced © 2015 Asian Pacific Society of Nephrology
IMD reduced oxidative stress in UUO kidneys To understand how overexpression of IMD protects against renal damages caused by UUO, we measured SOD (an important endogenous antioxidant enzyme) activity and MDA content (a marker of oxidative stress) at day 7 after UUO. We found that renal obstruction decreased SOD activity while it increased MDA content in kidney without transfection. Overexpression of IMD significantly increased SOD activity and blocked the increase in MDA content caused by UUO. As a control, we did not find that SOD activity and MDA content had significant differences between the kidneys transfected with empty plasmid and untreated kidneys when they were subjected to renal obstruction (Table 1).
IMD reduced macrophage infiltration in UUO kidneys Macrophage infiltration was assessed using ED-1 immunohistochemical analysis. There was a significant increase in macrophage infiltration into the untreated-obstructed kidneys (Fig. S4B,F,I) compared with that of sham controls (Fig. S4A,E,I) after 7 days and 14 days of UUO. IMD gene 825
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Fig. 4 Intermedin (IMD) overexpression attenuated unilateral ureteral obstruction (UUO)-induced tubular injury and blunted fibrotic response. (A) Representative pictures of haematoxylin and eosin staining (Haematoxylin and eosin, Magnification: ×100). (B) Masson trichrome staining of fibrosis in kidneys (Masson trichrome. Magnification: ×100). Groups were rats in sham-operated group (a and e), UUO group (b and f), treated with pcDNA3.1-IMD plasmid group (c and g), and treated with control empty plasmid group (d and h) at day 7 (a–d) and day 14 (e–h). (i) Semiquantitative score of tubulointerstitial fibrosis in the cortex of 7 days and 14 days obstructed kidneys. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. IMD overexpression ameliorated (c and g) whereas empty vector had no effect (d and h) on tubulointerstitial damage after UUO. Data in bar graphs are means ± standard deviation (SD) (n = 6). Scale bar = 100 μm. *P < 0.05 versus shamoperated group. #P < 0.05 versus untreated UUO group.
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Fig. 5 Quantitative RT-PCR and western blot analyses for fibronectin (FN1) expression in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A) Representative quantitative RT-PCR; (B) representative images of western blot of whole kidney lysates for FN1; (C) bar chart showing the FN1 protein expression normalized by β-actin. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
delivery significantly decreased macrophage infiltration (Fig. S4C,G,I). Macrophage infiltration was not obviously blocked by the addition of control empty vector (Fig. S4D,H,I).
DISCUSSION Previous studies have revealed the presence of mRNA and protein expression of IMD and its receptors (CRLR and RAMP1-3) in the kidney.9,11,13,14 The present study demonstrated for the first time that the levels of IMD transcripts and proteins are upregulated in the obstructed kidney. It has been reported that CRLR, RAMP1 and RAMP2 gene expressions in the obstructed kidney are markedly upregulated, whereas RAMP3 is unchanged after ureteral obstruction.15 However, the change of their expression at protein level in UUO model has not been studied. We examined the mRNA and protein expression of CRLR and RAMPs after ureteral obstruction. The results of our experiment showed significant upregulation of CRLR, RAMP1, RAMP2 and RAMP3, © 2015 Asian Pacific Society of Nephrology
both at mRNA and protein levels, in the kidney during obstructive nephropathy. The upregulation of these proteins in the obstructed kidney suggests that IMD may have a role in regulating renal fibrosis. As a member of the CGRP family, IMD is closely related to ADM.8 IMD and ADM share the CRLR/RAMP receptor system and may, therefore, have similar biological actions. ADM has two receptors formed by respective combination of CRLR and RAMP 2 or 3, while IMD acts non-selectively at all three CRLR/RAMP complexes,8 indicating that it has more potent actions than ADM. It is reported that ADM is induced as an adaptive response to renal fibrosis in UUO model.16 ADM administration or kidney-specific ADM gene delivery inhibits renal fibrosis.13,17 We hypothesized that, like ADM, IMD may also exert an antifibrotic effect on renal damage. Therefore, upregulation of IMD and its relevant receptors might be an adaptive response to renal fibrosis. To address this hypothesis, we investigated the effects of IMD overexpression in the kidney upon UUO. We locally overexpressed IMD in the kidney in obstructed nephropathy 827
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Fig. 6 Quantitative RT-PCR and immunohistochemical analyses for transforming growth factor-β1 (TGF-β1) expression in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A) Representative quantitative RT-PCR. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. (B) Representative immunohistochemical staining for TGF-β1 protein in the sham-operated kidneys (a and e), the untreated UUO kidneys (b and f), pcDNA3.1-intermedin (IMD) treated kidneys (c and g), and empty vector treated kidneys (d and h) at day 7 (a–d) and day 14 (e–h) after surgery. (Ci) Semiquantitative evaluation of TGF-β1 expression. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group.
using an ultrasound-microbubble-mediated system as previously described.11 We have revealed that both mRNA and protein levels of IMD were dramatically increased in the transfected kidneys, and no abnormalities of immunoresponses, histology, and kidney function were found after transfection.11 Furthermore, the overexpression of IMD was confined in the kidney.11 Our results demonstrated that kidney-specific IMD gene delivery significantly ameliorated UUO-mediated increased expression of fibronectin and the fibrotic change. These results indicated that IMD effectively reduced interstitial fibrosis in the model of UUO. Notably, 828
according to our previous studies, ultrasound-microbubblemediated IMD transgene expression peaks at day 7 and declines 2 weeks later. Thus, IMD in the early period after UUO surgery may have a role to prevent kidney fibrosis progression. Unlike systemic delivery of the IMD gene or administration of IMD in previous reports,18,19 we did not observe blood pressure reduction in our model (data not shown), highlighting the importance of direct renal actions of IMD in modulating the pathophysiology of UUO. In the pathogenesis of renal fibrosis, TGF-β1 has been regarded as an extremely important profibrotic © 2015 Asian Pacific Society of Nephrology
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Fig. 7 Effects of intermedin (IMD) on connective tissue growth factor (CTGF) expression in the obstructed kidney on days 7 and 14 after unilateral ureteral obstruction (UUO). (A–H) Representative immunohistochemical staining for CTGF protein in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Semiquantitative evaluation of CTGF expression. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
Table 1 Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in kidneys Sham-operated group
UUO group
UUO + IMD group
UUO + empty vector group
458.17 ± 91.69 11.39 ± 3.28
231.56 ± 67.25* 25.76 ± 5.46*
342.39 ± 85.65# 17.87 ± 4.52#
245.73 ± 89.48 26.35 ± 5.97
SOD activity (U/mg) MDA content (nmol/mg)
*P < 0.05 versus sham-operated group. #P < 0.05 versus untreated unilateral ureteral obstruction (UUO) group. Values are means ± standard deviation (SD); n = 6 per group. IMD, intermedin; UUO, unilateral ureteral obstruction.
mediator.3,20–22 It has been reported that TGF-β1 is increased in chronic ureteral obstruction.23,24 It activates fibroblasts and induces ECM production, mainly through Smad-dependent pathways.4 Blocking TGF-β1 pathway has been shown to reduce tubular fibrosis in several models of chronic kidney injury.20,21,24 Connective tissue growth factor (CTGF) is considered the major downstream effector of TGF-β1 and is important in the production of ECM proteins.25 It is reported that CTGF gene expression is strongly induced by TGF-β1 in a smad3/smad4-dependent manner.25 TGF-β1-induced fibronectin production is reduced with CTGF oligonucleotide © 2015 Asian Pacific Society of Nephrology
antisense transfection of cultured renal fibroblasts.26 In agreement with previous studies, our results revealed that TGF-β1, pSmad2/3 and CTGF expressions were upregulated after UUO. Therefore, we examined whether IMD attenuates renal fibrosis in association with the regulation of TGF-β1/ smads pathway. Interestingly, our results revealed that IMD overexpression had little effect on TGF-β1 and pSmad2/3 expressions, while CTGF expression was reduced with IMD gene transfer and paralleled the reduction in myofibroblast accumulation. Our results demonstrated that the inhibitory effects of IMD on renal fibrosis may not be achieved by 829
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directly inhibiting TGF-β1 expression and smad2/3 activation but by the suppression of downstream steps in the TGF-β1/smads signalling pathway. To elucidate the mechanisms by which IMD inhibits renal fibrosis and ECM accumulation, we examined the effect of IMD on oxidative stress in UUO. Increased concentrations of ROS have been implicated in the pathophysiology of renal fibrosis,27,28 together with decreased activities of the major protective antioxidant enzymes such as SOD, catalase and glutathione peroxidase.29 It has been demonstrated that TGFβ1-induced myofibroblast activation works through a TGF receptor 1 (TGFR1)/Smad/ROS/ERK1/2 signalling cascade.30 In addition, oxidative stress induces macrophage infiltration, which further promotes interstitial fibrosis.31 Thus, ROS are important downstream effectors in mediating the profibrotic action of TGF-β1,5,7,30 and play important roles in TGF-β1induced ECM accumulation in the renal tubulointerstitium.6 Agents that can reduce ROS have received considerable attention.32,33 As a secreted peptide, IMD was shown to inhibit oxidative stress,11,18,19 suggesting that IMD might ameliorate renal fibrosis. We examined alterations of oxidative stress by measuring SOD (an important endogenous antioxidant enzyme) activity and MDA content (a marker of oxidative stress), in kidneys after UUO. Our results revealed that SOD activity significantly reduced and MDA content markedly increased in obstructed kidney. This increased oxidative stress was associated with impaired histological changes, increased macrophage infiltration, enhanced renal fibrosis and increased expression of TGF-β1. However, IMD gene delivery prevented depletion of SOD activity and increase of MDA content, and thus blocked the profibrotic actions of TGF-β1 without affecting its expression renal fibrosis. In summary, our study demonstrates that the expression of IMD as well as its receptors is upregulated in the obstructed kidney of rats with UUO. Locally overexpressed IMD in the kidney suppresses the progression of renal fibrosis in the rat UUO model, apparently by reducing oxidative stress and thus inhibits TGF-β1 signalling. However, further investigations are definitely required to elucidate the exact signalling mechanism whereby this peptide inhibits oxidative stress in the kidney and protects against fibrosis. Targeting IMD could be novel therapeutic target for preventing renal fibrosis.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81100531).
REFERENCES 1. Komenda P, Ferguson TW, Macdonald K et al. Cost-effectiveness of primary screening for CKD: A systematic review. Am. J. Kidney Dis. 2014; 63: 789–97.
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2. Duffield JS. Cellular and molecular mechanisms in kidney fibrosis. J. Clin. Invest. 2014; 124: 2299–306. 3. Yanagita M. Inhibitors/antagonists of TGF-β system in kidney fibrosis. Nephrol. Dial. Transplant. 2012; 27: 3686–91. 4. Wang W, Koka V, Lan HY. Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton) 2005; 10: 48–56. 5. Jiang F, Liu GS, Dusting GJ, Chan EC. NADPH oxidase-dependent redox signaling in TGF-β-mediated fibrotic responses. Redox Biol. 2014; 2: 267–72. 6. Ji X, Naito Y, Weng H et al. Renoprotective mechanisms of pirfenidone in hypertension-induced renal injury: Through anti-fibrotic and anti-oxidative stress pathways. Biomed. Res. 2013; 34: 309–19. 7. Rhyu DY, Yang Y, Ha H et al. Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J. Am. Soc. Nephrol. 2005; 16: 667–75. 8. Roh J, Chang CL, Bhalla A, Klein C, Hsu SY. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes. J. Biol. Chem. 2004; 279: 7264–74. 9. Morimoto R, Satoh F, Murakami O et al. Expression of adrenomedullin2/intermedin in human brain, heart, and kidney. Peptides 2007; 28: 1095–103. 10. Takahashi K, Kikuchi K, Maruyama Y et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney. Peptides 2006; 27: 1383–9. 11. Qiao X, Li RS, Li H et al. Intermedin protects against renal ischemia-reperfusion injury by inhibition of oxidative stress. Am. J. Physiol. Renal Physiol. 2013; 304: F112–9. 12. Qiao X, Chen X, Wu D et al. Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/reperfusion injury. J. Gerontol. A. Biol Sci. Med Sci 2005; 60: 830–39. 13. Nagae T, Mori K, Mukoyama M et al. Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis. Kidney Int. 2008; 74: 70–80. 14. Nishikimi T, Wang X, Akimoto K et al. Alteration of renal adrenomedullin and its receptor system in the severely hypertensive rat: Effect of diuretic. Regul. Pept. 2005; 124: 89–98. 15. Nagae T, Mukoyama M, Sugawara A et al. Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: Cloning and upregulation in obstructive nephropathy. Biochem. Biophys. Res. Commun. 2000; 270: 89–93. 16. Nørregaard R, Bødker T, Jensen BL, Stødkilde L, Nielsen S, Frøkiaer J. Increased renal adrenomedullin expression in rats with ureteral obstruction. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 296: R185–92. 17. Ito K, Yoshii H, Asano T et al. Adrenomedullin increases renal nitric oxide production and ameliorates renal injury in mice with unilateral ureteral obstruction. J. Urol. 2010; 183: 1630–35. 18. Hagiwara M, Bledsoe G, Yang ZR, Smith RS Jr, Chao L, Chao J. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress. Am. J. Physiol. Renal Physiol. 2008; 295: F1735–43. 19. Zhang HY, Jiang W, Liu JY et al. Intermedin is upregulated and has protective roles in a mouse ischemia/reperfusion model. Hypertens. Res. 2009; 32: 861–8.
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20. Chen X, Wang H, Liao HJ et al. Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling. J. Clin. Invest. 2014; 124: 3295–310. 21. Venkatachalam MA, Weinberg JM. New wrinkles in old receptors: Core fucosylation is yet another target to inhibit TGF-β signaling. Kidney Int. 2013; 84: 11–4. 22. Wei X, Xia Y, Li F et al. Kindlin-2 mediates activation of TGF-β/Smad signaling and renal fibrosis. J. Am. Soc. Nephrol. 2013; 24: 1387–98. 23. Ding Y, Kim SL, Lee SY, Koo JK, Wang Z, Choi ME. Autophagy regulates TGF-β expression and suppresses kidney fibrosis induced by unilateral ureteral obstruction. J. Am. Soc. Nephrol. 2014; 25: 2835–46. 24. Kim J, Imig JD, Yang J, Hammock BD, Padanilam BJ. Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation. Am. J. Physiol. Renal Physiol. 2014; 307: F971–80. 25. Yokoi H, Mukoyama M, Nagae T et al. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J. Am. Soc. Nephrol. 2004; 15: 1430–40. 26. Yokoi H1, Mukoyama M, Sugawara A et al. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am. J. Physiol. Renal Physiol. 2002; 282: F933–42. 27. He W, Wang Y, Zhang MZ et al. Sirt1 activation protects the mouse renal medulla from oxidative injury. J. Clin. Invest. 2010; 120: 1056–68. 28. Singh DK, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy: Fueling the fire. Nat. Rev. Endocrinol. 2011; 7: 176–84. 29. Manucha W, Carrizo L, Ruete C, Molina H, Vallés P. Angiotensin II type I antagonist on oxidative stress and heat shock protein 70 (HSP 70) expression in obstructive nephropathy. Cell. Mol. Biol. 2005; 51: 547–55. 30. Bondi CD, Manickam N, Lee DY et al. NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts. J. Am. Soc. Nephrol. 2010; 21: 93–102. 31. Paul-Clark MJ, McMaster SK, Sorrentino R et al. Toll-like receptor 2 is essential for the sensing of oxidants during inflammation. Am. J. Respir. Crit. Care Med. 2009; 179: 299–306. 32. Nlandu Khodo S, Dizin E, Sossauer G et al. NADPH-oxidase 4 protects against kidney fibrosis during chronic renal injury. J. Am. Soc. Nephrol. 2012; 23: 1967–76. 33. Okamura DM, Bahrami NM, Ren S et al. Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD. J. Am. Soc. Nephrol. 2014; 25: 43–54.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
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Fig. S1 Increased mRNA expression levels of IMD, CRLR, and RAMPs in the kidneys after unilateral ureteral obstruction (UUO) surgery. The quantitative RT-PCR time-course for transcripts of whole kidney IMD (A), CRLR (B), RAMP1 (C), RAMP2 (D), RAMP3 (E) following UUO surgery. Expression levels of each mRNA were normalized to the expression level of β-actin. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. Fig. S2 Effects of IMD on the expression of pSmad2/3 in kidney tissues of unilateral ureteral obstruction (UUO) rats. (A) Representative western blot photograph showing expression levels of pSmad2/3 and tSmad2/3 proteins in rat kidneys. (B) Quantitative analysis of the relative ratio of pSmad2/3 to tSmad2/3. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. Fig. S3 Effects of IMD on the expression of α-SMA in kidney tissues of unilateral ureteral obstruction (UUO) rats. (A–H) Representative immunohistochemical staining for α-SMA protein in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Semiquantitative evaluation of α-SMA expression. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group. Fig. S4 Immunohistochemical analyses for macrophages infiltration in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A–H) Representative immunohistochemical staining for ED-1 in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Quantitation of average macrophage number per high-power field. Scale bar = 100 μm. Magnification: ×400. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
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Nephrology 20 (2015) 820–831
Original Article
Intermedin is upregulated and attenuates renal fibrosis by inhibition of oxidative stress in rats with unilateral ureteral obstruction XI QIAO,1,2 LIHUA WANG,1,2 YANHONG WANG,3 NING ZHAO,1 RUIJING ZHANG,1 WEIXIA HAN1 and ZHIQIANG PENG1,2 1 Department of Nephrology, Second Hospital of Shanxi Medical University, and 2Shanxi Kidney Disease Institute and 3Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan, Shanxi, China
KEY WORDS: calcitonin receptor-like receptor, intermedin, oxidative stress, receptor activity-modifying proteins, renal fibrosis. Correspondence: Dr Xi Qiao, Department of Nephrology, Second Hospital of Shanxi Medical University, Shanxi Kidney Disease Institute, WuYi Road 382, Taiyuan, Shanxi 030001, China. Email: [email protected] Accepted for publication 19 May 2015. Accepted manuscript online 25 May 2015. doi:10.1111/nep.12520 Xi Qiao and Lihua Wang contributed equally to this study.
SUMMARY AT A GLANCE Authors show that treatment of rats with the secreted peptide Intermedin can reduce renal fibrosis and oxidative stress in a model of unilateral ureteral obstruction.
ABSTRACT: Aim: Transforming growth factor-β1 (TGF-β1) plays a pivotal role in the progression of renal fibrosis. Reactive oxygen species mediate profibrotic action of TGF-β1. Intermedin (IMD) has been shown to inhibit oxidative stress, but its role in renal fibrosis remains unclear. Here, we investigated the effects of IMD on renal fibrosis in a rat model of unilateral ureteral obstruction (UUO). Methods: The expression of IMD and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP1/2/3), in the obstructed kidney was detected by real-time polymerase chain reaction (PCR), western blotting and immunohistochemistry. To evaluate the effects of IMD on renal fibrosis, we locally overexpressed exogenous IMD in the obstructed kidney using an ultrasound-microbubble-mediated delivery system. Renal fibrosis was determined by Masson trichrome staining. The expression of TGF-β1, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and fibronectin was measured. Smad2/3 activation and macrophage infiltration were evaluated. We also studied oxidative stress by measuring superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Results: mRNA and protein expression of IMD increased after UUO. CRLR, RAMP1, RAMP2 and RAMP3 were also induced by ureteral obstruction. IMD overexpression remarkably attenuated UUO-induced tubular injury and blunted fibrotic response as shown by decreased interstitial collagen deposition and downregulation of fibronectin. Macrophage infiltration, α-SMA and CTGF upregulation caused by UUO were all relieved by IMD, whereas TGF-β1 upregulation and Smad2/3 activation were not affected. Meanwhile, we noted increased oxidative stress in obstruction, which was also attenuated by IMD gene delivery. Conclusions: Our results indicate that IMD is upregulated after UUO. IMD plays a protective role in renal fibrosis via its antioxidant effects.
Chronic kidney disease is a major health problem with an increasing incidence worldwide.1 Regardless of disease aetiology, fibrosis is the common pathway leading to end stage renal diseases in all progressive chronic kidney diseases and is regarded as a prognostic factor for renal function.2 Therefore, elucidating the aetiological mechanisms underlying renal fibrosis and developing novel therapeutic strategies are 820
of great importance. Transforming growth factor-β1 (TGFβ1) plays a pivotal role in the progression of renal fibrosis.3 TGF-β1 promotes the accumulation of extracellular matrix (ECM), through the enhanced synthesis of ECM proteins, such as fibronectin (FN1), and the inhibition of their degradation, mainly through Smad-dependent pathways.4 Reactive oxygen species (ROS) are important downstream © 2015 Asian Pacific Society of Nephrology
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effectors that mediate the above profibrotic actions of TGFβ1/Smads.5,6 Therapeutic interventions targeting ROS have been successful and well tolerated in several experimental models.5,7 Intermedin (IMD, also known as adrenomedullin-2 (ADM-2)) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family.8 IMD is distributed in a wide variety of tissues including the kidney.9,10 In kidney, IMD mainly expresses in tubular cells of cortex and medulla.9,11 Similar to other peptides in the CGRP family, IMD signals through a type II G protein-coupled receptor complex consisting of the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs).8 Unlike CGRP and ADM, which exhibit a preferential stimulation of CRLR when coexpressed with RAMP1 and RAMP2 or RAMP3, respectively, IMD binds nonselectively to all three CRLR/RAMP complexes: CRLR/RAMP1, CRLR/ RAMP2 and CRLR/RAMP3,8 indicating that it has more potent actions than ADM and any other CGRP. Initial work from our laboratory has demonstrated that IMD gene transfer protects against renal ischaemiareperfusion injury by inhibition of oxidative stress,11 while its role in renal fibrosis is unknown. We hypothesized that IMD can attenuate renal fibrosis by reducing oxidative stress. To test this hypothesis, we examined the expression of IMD and its receptors, CRLR and RAMPs, in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Then, we investigated the effects of kidney-specific IMD gene delivery on renal fibrosis induced by UUO. Furthermore, in order to explain the mechanisms underlying these effects, we studied the oxidative stress in obstructive nephropathy by measuring SOD activity (an important endogenous antioxidant enzyme) and MDA content (a marker of oxidative stress).
METHODS
TGCTGATGGT-3′; antisense: 5′-TGCCCGGGAGCAGGTA-3′), CRLR (sense: 5′-CTCACGGCAGTGGCCAATAA-3′; antisense: 5′-AGCCCAT CAGGTAAAGATGAATGAA-3′), RAMP1 (sense: 5′-AGCTGTGTC TCAGCCGCTTC-3′; antisense: 5′-AATCTTGTTTGCCACGAGTTTG3′), RAMP2 (sense: 5′-GAGTCCCTGAATCAATCTCATCCTA-3′; antisense: 5′-TCAAAGTCCAGTTGCACCAGTC-3′), RAMP3 (sense: 5′-GAAAGCCTTTGCCGAAATGATG-3′; antisense: 5′-GCCCACGA TGTTTGTCTCCA-3′), TGF-β1 (sense: 5′-CATTGCTGTCCCGTG CAGA-3′; antisense: 5′-AGGTAACGCCAGGAATTGTTGCTA-3′), or FN1 (sense: 5′-CATCAGCCCGGATGTCAGAA-3′; antisense: 5′-GGC ATTGTCGTTGAGCGTGTA-3′) and β-actin (sense: 5′-CCCATCTATG AGGGTTACGC-3′; antisense: 5′-TTTAATGTCACGCACGATTTC-3′). The reaction was carried out in a 96-well plate in 25 μL reactions containing 2× SYBR Green Master mix (TAKARA, Dalian, China) – 2 pmol each of sense and antisense primer and the conditions were 95°C for 2 min, followed by 95°C for 10 s, 60°C for 15 s, for 40 cycles. In each assay, a standard curve was determined concurrently with examined samples. Gene expression was quantified using a medication of the 2-ΔΔct method. The amount of PCR products was normalized to the level of β-actin to determine the relative expression ratio for each mRNA.
Western blot analysis Analysis was carried out as previous described.12 Immunoblot analysis was performed with rabbit polyclonal anti-rat IMD antibody, goat polyclonal anti-rat CRLR (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-rat RAMP1, rabbit polyclonal anti-rat RAMP2, rabbit polyclonal anti-rat RAMP3 (all from Beijing Biosynthesis Biotechnology, Beijing, China), goat polyclonal anti-rat fibronectin (FN1) antibody (Santa Cruz Biotechnology), goat polyclonal anti-rat pSmad2/3 or rabbit polyclonal anti-rat Smad2/3 (Santa Cruz Biotechnology) and then peroxidaseconjugated AffiniPure goat anti-rabbit immunoglobulin G or rabbit anti-goat immunoglobulin G (Santa Cruz Biotechnology), respectively. The ECL western blotting system (Santa Cruz Biotechnology) was used for detection. Rabbit polyclonal anti-rat β-actin antibody (Santa Cruz Biotechnology) was used as the control for each sample.
Animal model
Immunohistochemistry
The Experimental Animal Committee approved all animal protocols. Renal fibrosis was induced by ligation of left ureter, UUO, in male Wistar rats (180 to 200 g). Briefly, rats were anaesthetized, laparotomy performed, and the left ureter identified and ligated at two points along the ureter. Sham-operated rats underwent the same surgical procedure except for the ureter ligation. Groups of six animals were killed at 1, 3, 7, and 14 days after UUO. Kidneys were harvested for further analysis.
Rat kidneys were fixed in 10% formaldehyde and embedded in paraffin. Paraffin sections (5 μm) were mounted on glass slides, deparaffinized in xylene, and rehydrated in ethanol with increasing concentrations of water. The rehydrated sections were pretreated with 3% H2O2 for 10 min at room temperature to block the endogenous peroxidase. After boiling in antigen retrieval solution (1 mmol/L tris-HCl, 0.1 mmol/L EDTA, pH = 8.0) for 10 min at high power in a microwave oven, the sections were incubated overnight at 4°C with primary rabbit polyclonal anti-rat IMD antibody (1:200, sc-86272; Santa Cruz Biotechnology), goat polyclonal anti-rat CRLR antibody (1:200, sc-18007; Santa Cruz Biotechnology), rabbit polyclonal anti-rat RAMP1 antibody (1:200, bs-1567R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat RAMP2 antibody (1:200, bs-11971R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat RAMP3 antibody (1:200, bs-11972R; Beijing Biosynthesis Biotechnology), rabbit polyclonal anti-rat TGF-β1 antibody (1:200, sc-146; Santa Cruz Biotechnology), mouse monoclonal anti-rat ED-1 antibody (1:200, sc-59103; Santa Cruz
Quantitative RT-PCR Total renal RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then subjected to RT using a first-strand cDNA synthesis system (TAKARA, Dalian, China). Quantitative RT-PCR amplifications were performed in triplicate using the SYBR Green I assay and were carried out using Strategene M3000 Sequence Detection System (Stratagene, Santa Clara, CA, USA). Specific primers were used for IMD (sense: 5′-GGCCCAGT © 2015 Asian Pacific Society of Nephrology
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Biotechnology), rabbit polyclonal anti-rat α-SMA antibody (1:200, bs-10196R; Beijing Biosynthesis Biotechnology), or rabbit polyclonal anti-rat CTGF antibody (1:200, sc-14939; Santa Cruz Biotechnology), respectively. This was followed by biotinylated secondary antibodies (Santa Cruz Biotechnology) and finally by avidinconjugated horseradish peroxidase. All slides were counterstained with haematoxylin. Positive stained area were assessed and expressed as integrated optical density (IOD) and area. Three sections of each rat kidney were measured, and 10 random fields were chosen and calculated under magnification of 100×. The IOD and positive area were acquired by the Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, MD, USA). The number of macrophages was counted as positive staining for ED-1 in a blinded manner from 10 different fields of each section at ×400 magnification.
Ultrasound-mediated gene delivery into the kidney Eukaryotic expression plasmid pcDNA3.1-IMD containing fulllength cDNA sequence of rat IMD was successfully constructed in our previous study.11 Before the ureter was obstructed, pcDNA3.1IMD plasmid or control empty vector was transfected into the left kidney via the renal artery using an ultrasound-microbubblemediated system as we previously described.11 Rats were killed at 7 days after UUO. The transfection rate was detected by quantitative RT-PCR and immunohistochemistry, respectively.
Histological examinations Renal histological changes were assessed at days 7 and 14 after UUO. Paraffin-embedded transverse kidney slices were sectioned at 3 μm, and stained with haematoxylin and eosin. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. Twenty cortical tubulointerstitial fields that were randomly selected at ×100 magnification were assessed in each rat, and the density of trichrome positive signals was analyzed using Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, MD, USA). Briefly, the tonality of the fibrosis area (blue) was determined with control sections as reference. The number of pixels with the predetermined colour of tone was counted in each section, and then automatically converted into dimensions.
Superoxide dismutase activity and malondialdehyde content Rat kidneys were homogenized in ice-cold 20 mM Tris–HCl buffer (pH 7.4). Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were determined with commercially available kits, respectively (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis Results were presented as means ± standard deviation (SD). Student’s t-test was used to determine the significance of differences between two groups, whereas the one way analysis of variance (ANOVA) was used for comparison of multiple groups. P < 0.05 was considered significant. 822
RESULTS Expression of IMD and its receptors, CRLR and RAMPs, was upregulated in UUO kidney We first examined whether the mRNA and protein expression levels of IMD and its receptors, CRLR and RAMPs, in kidney were altered by UUO using quantitative RT-PCR, western blot and immunohistochemistry, respectively. In sham-operated kidneys, IMD was expressed moderately and constantly throughout the experimental period. IMD transcripts in the UUO kidneys increased 1 day after surgery and remained increased at the subsequent time points, reached the highest level at day 7 after UUO, and then declined at day 14 (Fig. S1A). Comparable to the increased transcripts, IMD protein expression was already increased in the UUO kidneys from day 1 after the surgery, and the increased expression levels remained at subsequent time points (Fig. 1A,B). This observation was confirmed further by immunohistochemistry (Fig. 2A–E). At a baseline, endogenous IMD can be detected in tubular cells, endothelial cells of the glomerulus and vasa recta in the kidney of sham-operated rats (Fig. 2A). Immunoreactivity for IMD was increased markedly from day 1 to day 14 in UUO kidneys (Fig. 2B–E). Basal CRLR, RAMP1, RAMP2 and RAMP3 gene and protein expression was detectable in sham-operated kidneys. After ureteral obstruction, the mRNA and protein expression levels of CRLR, RAMP1, RAMP2 and RAMP3 in the kidney increased from day 1, reached the peak at day 3 (RAMP2 and RAMP3) or at day 7 (CRLR and RAMP1), respectively (Fig. S1B–E, Fig. 1A,C–F). Immunohistochemistry for CRLR (Fig. 2F–J), RAMP1 (Fig. 2K–O), RAMP2 (Fig. 2P–T) and RAMP3 (Fig. 2U–Y) also demonstrated the increased immunoreactivity in UUO kidneys.
Efficacy of ultrasound-microbubble-mediated gene transfection We examined the efficacy of ultrasound-microbubblemediated gene transfection in kidney by quantitative RT-PCR. After 7 days of transfection and UUO operation, kidneys from rats treated with pcDNA3.1-IMD plasmid exhibited significant increase in IMD expression compared with kidneys of rats treated with UUO only (Fig. 3A). The expression of IMD showed no significant difference between the kidneys transfected with empty plasmid and nontransfected kidneys when they were subjected to UUO (Fig. 3A). Our results indicated that IMD was transfected into the kidney successfully. To confirm this result, we performed immunohistochemistry. The kidneys transfected with pcDNA3.1-IMD plasmid (Fig. 3B(c),C) exhibited much stronger IMD immunostaining than kidneys treated with UUO only (Fig. 3B(b),C) or kidneys transfected with control empty vector (Fig. 3B(d),C). © 2015 Asian Pacific Society of Nephrology
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Fig. 1 Increased protein expression levels of intermedin (IMD), calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in the kidneys after unilateral ureteral obstruction (UUO) surgery. (A) Representative images of the time-course western blot of whole kidney lysates for IMD, CRLR, RAMP1, RAMP2 and RAMP3 after UUO surgery. β-actin was used as a loading control. (B–F) Bar chart showing the IMD (B), CRLR (C), RAMP1 (D), RAMP2 (E), RAMP3 (F) protein expression time-course normalized by β-actin. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group.
IMD overexpression decreased renal injury and interstitial fibrosis in UUO Renal histologic changes were determined by haematoxylin and eosin staining. No changes were detected in the shamoperated group (Fig. 4A(a),A(e)). In the untreated UUO group, tubular lumens were significantly dilated and the epithelial cells were flattened accompanied by infiltration of inflammatory cells in the interstitium 7 days after UUO (Fig. 4A(b)). At 14 days post-obstruction, all lesions observed at UUO day 7 worsened and increased ECM deposition became a prominent feature. Most tubules appeared markedly atrophic, having lost their original integrity (Fig. 4A(f)). Overexpression of IMD in the kidney considerably decreased © 2015 Asian Pacific Society of Nephrology
renal injury at both 7 and 14 days after UUO (Fig. 4A(c),A(g)). Rats that were treated with the control empty plasmid (Fig. 4A(d),A(h)) revealed no significantly improved renal morphology compared with those in untreated UUO group. The degree of renal fibrosis was determined by Masson trichrome staining. Masson trichrome is most useful to differentiate collagen from other fibres. There was significant interstitial fibrosis that resulted in separation of the tubules at days 7 and 14 in untreated UUO kidneys (Fig. 4Bb,Bf). IMD gene delivery decreased interstitial collagen deposition on days 7 and 14 (Fig. 4Bc,Bg) in comparison with untreated obstructed kidneys. Semiquantitative analysis showed that the fibrotic score of pcDNA3.1-IMD-treated kidneys was 823
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Fig. 2 Localization of intermedin (IMD), calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) expression in the unilateral ureteral obstruction (UUO) kidneys. (A–E) Representative immunohistochemical staining for IMD protein in the sham operation kidneys (A), and the UUO kidneys at day 1 (B), day 3 (C), day 7 (D) and day 14 (E) after surgery. (F–J) Representative immunohistochemical staining for CRLR protein in the sham operation kidneys (F), and the UUO kidneys at day 1 (G), day 3(H), day 7 (I) and day 14 (J) after surgery. (K–O) Representative immunohistochemical staining for RAMP1 protein in the sham operation kidneys (K), and the UUO kidneys at day 1 (L), day 3 (M), day 7 (N) and day 14 (O) after surgery. (P–T) Representative immunohistochemical staining for RAMP2 protein in the sham operation kidneys (P), and the UUO kidneys at day 1 (Q), day 3 (R), day 7 (S) and day 14 (T) after surgery. (U–Y) Representative immunohistochemical staining for RAMP3 protein in the sham operation kidneys (U), and the UUO kidneys at day 1 (V), day 3(W), day 7 (X) and day 14 (Y) after surgery. Scale bar = 100 μm. Magnification: ×100.
significantly lower than that of untreated UUO kidneys (Fig. 4B(i)). Tubulointerstitial fibrosis was not obviously blocked by the addition of control empty vector (Fig. 4Bd,Bh,Bi).
As a control, we did not find that the expression of FN1 was significantly different between the kidneys transfected with empty plasmid and nontransfected kidney when they were subjected to UUO (Fig. 5A,B,C).
IMD overexpression inhibited extracellular matrix accumulation
IMD gene transfer had no effect on the expression of TGF-β1 in UUO kidneys
To evaluate the role of IMD on ECM accumulation, we examined the expression of fibronectin (FN1) after IMD gene transfer by quantitative RT-PCR and western blot analyses. The mRNA and protein expression levels of FN1 were significantly increased at days 7 and 14 after surgery. IMD gene delivery significantly reduced FN1 levels in the UUO kidneys.
We examined the expression of TGF-β1 in the whole kidney on days 7 and 14 after ureteral ligation by quantitative RT-PCR and immunohistochemical analyses. In our study, basal TGF-β1 expression was low in the sham operation kidneys. Both quantitative RT-PCR and immunostaining for TGF-β1 revealed that UUO significantly upregulated renal
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the upregulation of Smad2/3 phosphorylation level but not total Smad2/3 on days 7 and 14 after ureteral ligation. IMD or empty plasmid did not alter either Smad2/3 phosphorylation or total Smad2/3 in the obstructed kidneys (Fig. S2).
IMD gene delivery inhibited CTGF expression in UUO kidneys We examined CTGF, a downstream target of TGF-β1 involved in ECM production, by immunohistochemical analyses. CTGF expression was significantly elevated in the obstructed kidneys on days 7 and 14 after UUO (Fig. 7B,F,I) as compared with the sham-operated kidneys (Fig. 7A,E,I). Introduction of IMD into the kidney (Fig. 7C,G,I) markedly inhibited CTGF expression compared with the untreated obstructed kidney, while CTGF expression was not obviously affected by the addition of control empty vector (Fig. 7D,H,I).
IMD inhibited α-SMA expression in UUO kidneys α-SMA-positive myofibroblasts are responsible for the overproduction of ECM at pathologic conditions. The immunohistochemistry of α-SMA demonstrated that α-SMA expression increased on days 7 and 14 after ureteral ligation (Fig. S3B,F,I). The α-SMA expression levels in the obstructed kidneys of IMD-treated UUO rats (Fig. S3C,G,I) were significantly decreased compared to those of UUO-control rats on both day 7 and day 14. Empty plasmid did not affect the expression levels of α-SMA (Fig. S3D,H,I). Fig. 3 Ultrasound-microbubble-mediated gene transfer into the kidney. Microbubbles loaded with pcDNA3.1-intermedin (IMD) or control empty vector were infused into the kidney via the left renal artery treated with ultrasound before unilateral ureteral obstruction (UUO) surgery. On day 7 after ureter ligation, expression of IMD was assessed. (A) Representative quantitative RT-PCR. (B) Immunohistochemical localization of IMD in the kidney of sham-operated rats (a), untreated UUO rats (b), rats treated with pcDNA3.1IMD plasmid (c), and rats treated with control empty vector (d). (C) Semiquantitative evaluation of IMD expression. Data in bar graphs are means ± standard deviation (SD) (n = 6). O.D., optical density. Scale bar = 100 μm. Magnification: ×100. *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
TGF-β1 mRNA and protein levels (Fig. 6A,B(b),B(f),B(i)). Gene transfer of IMD (Fig. 6A,B(c),B(g),B(i)) or empty plasmid (Fig. 6A,B(d),B(h),B(i)) did not affect the expression level of TGF-β1.
IMD did not affect UUO-induced Smad2/3 activation We evaluated downstream signalling molecules of TGF-β1, Smad2/3. western blot analyses showed that UUO induced © 2015 Asian Pacific Society of Nephrology
IMD reduced oxidative stress in UUO kidneys To understand how overexpression of IMD protects against renal damages caused by UUO, we measured SOD (an important endogenous antioxidant enzyme) activity and MDA content (a marker of oxidative stress) at day 7 after UUO. We found that renal obstruction decreased SOD activity while it increased MDA content in kidney without transfection. Overexpression of IMD significantly increased SOD activity and blocked the increase in MDA content caused by UUO. As a control, we did not find that SOD activity and MDA content had significant differences between the kidneys transfected with empty plasmid and untreated kidneys when they were subjected to renal obstruction (Table 1).
IMD reduced macrophage infiltration in UUO kidneys Macrophage infiltration was assessed using ED-1 immunohistochemical analysis. There was a significant increase in macrophage infiltration into the untreated-obstructed kidneys (Fig. S4B,F,I) compared with that of sham controls (Fig. S4A,E,I) after 7 days and 14 days of UUO. IMD gene 825
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Fig. 4 Intermedin (IMD) overexpression attenuated unilateral ureteral obstruction (UUO)-induced tubular injury and blunted fibrotic response. (A) Representative pictures of haematoxylin and eosin staining (Haematoxylin and eosin, Magnification: ×100). (B) Masson trichrome staining of fibrosis in kidneys (Masson trichrome. Magnification: ×100). Groups were rats in sham-operated group (a and e), UUO group (b and f), treated with pcDNA3.1-IMD plasmid group (c and g), and treated with control empty plasmid group (d and h) at day 7 (a–d) and day 14 (e–h). (i) Semiquantitative score of tubulointerstitial fibrosis in the cortex of 7 days and 14 days obstructed kidneys. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. IMD overexpression ameliorated (c and g) whereas empty vector had no effect (d and h) on tubulointerstitial damage after UUO. Data in bar graphs are means ± standard deviation (SD) (n = 6). Scale bar = 100 μm. *P < 0.05 versus shamoperated group. #P < 0.05 versus untreated UUO group.
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Fig. 5 Quantitative RT-PCR and western blot analyses for fibronectin (FN1) expression in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A) Representative quantitative RT-PCR; (B) representative images of western blot of whole kidney lysates for FN1; (C) bar chart showing the FN1 protein expression normalized by β-actin. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
delivery significantly decreased macrophage infiltration (Fig. S4C,G,I). Macrophage infiltration was not obviously blocked by the addition of control empty vector (Fig. S4D,H,I).
DISCUSSION Previous studies have revealed the presence of mRNA and protein expression of IMD and its receptors (CRLR and RAMP1-3) in the kidney.9,11,13,14 The present study demonstrated for the first time that the levels of IMD transcripts and proteins are upregulated in the obstructed kidney. It has been reported that CRLR, RAMP1 and RAMP2 gene expressions in the obstructed kidney are markedly upregulated, whereas RAMP3 is unchanged after ureteral obstruction.15 However, the change of their expression at protein level in UUO model has not been studied. We examined the mRNA and protein expression of CRLR and RAMPs after ureteral obstruction. The results of our experiment showed significant upregulation of CRLR, RAMP1, RAMP2 and RAMP3, © 2015 Asian Pacific Society of Nephrology
both at mRNA and protein levels, in the kidney during obstructive nephropathy. The upregulation of these proteins in the obstructed kidney suggests that IMD may have a role in regulating renal fibrosis. As a member of the CGRP family, IMD is closely related to ADM.8 IMD and ADM share the CRLR/RAMP receptor system and may, therefore, have similar biological actions. ADM has two receptors formed by respective combination of CRLR and RAMP 2 or 3, while IMD acts non-selectively at all three CRLR/RAMP complexes,8 indicating that it has more potent actions than ADM. It is reported that ADM is induced as an adaptive response to renal fibrosis in UUO model.16 ADM administration or kidney-specific ADM gene delivery inhibits renal fibrosis.13,17 We hypothesized that, like ADM, IMD may also exert an antifibrotic effect on renal damage. Therefore, upregulation of IMD and its relevant receptors might be an adaptive response to renal fibrosis. To address this hypothesis, we investigated the effects of IMD overexpression in the kidney upon UUO. We locally overexpressed IMD in the kidney in obstructed nephropathy 827
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Fig. 6 Quantitative RT-PCR and immunohistochemical analyses for transforming growth factor-β1 (TGF-β1) expression in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A) Representative quantitative RT-PCR. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. (B) Representative immunohistochemical staining for TGF-β1 protein in the sham-operated kidneys (a and e), the untreated UUO kidneys (b and f), pcDNA3.1-intermedin (IMD) treated kidneys (c and g), and empty vector treated kidneys (d and h) at day 7 (a–d) and day 14 (e–h) after surgery. (Ci) Semiquantitative evaluation of TGF-β1 expression. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group.
using an ultrasound-microbubble-mediated system as previously described.11 We have revealed that both mRNA and protein levels of IMD were dramatically increased in the transfected kidneys, and no abnormalities of immunoresponses, histology, and kidney function were found after transfection.11 Furthermore, the overexpression of IMD was confined in the kidney.11 Our results demonstrated that kidney-specific IMD gene delivery significantly ameliorated UUO-mediated increased expression of fibronectin and the fibrotic change. These results indicated that IMD effectively reduced interstitial fibrosis in the model of UUO. Notably, 828
according to our previous studies, ultrasound-microbubblemediated IMD transgene expression peaks at day 7 and declines 2 weeks later. Thus, IMD in the early period after UUO surgery may have a role to prevent kidney fibrosis progression. Unlike systemic delivery of the IMD gene or administration of IMD in previous reports,18,19 we did not observe blood pressure reduction in our model (data not shown), highlighting the importance of direct renal actions of IMD in modulating the pathophysiology of UUO. In the pathogenesis of renal fibrosis, TGF-β1 has been regarded as an extremely important profibrotic © 2015 Asian Pacific Society of Nephrology
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Fig. 7 Effects of intermedin (IMD) on connective tissue growth factor (CTGF) expression in the obstructed kidney on days 7 and 14 after unilateral ureteral obstruction (UUO). (A–H) Representative immunohistochemical staining for CTGF protein in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Semiquantitative evaluation of CTGF expression. (□) sham, (■) UUO, ( ) UUO ± IMD, ( ) UUO ± Empty vector. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
Table 1 Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in kidneys Sham-operated group
UUO group
UUO + IMD group
UUO + empty vector group
458.17 ± 91.69 11.39 ± 3.28
231.56 ± 67.25* 25.76 ± 5.46*
342.39 ± 85.65# 17.87 ± 4.52#
245.73 ± 89.48 26.35 ± 5.97
SOD activity (U/mg) MDA content (nmol/mg)
*P < 0.05 versus sham-operated group. #P < 0.05 versus untreated unilateral ureteral obstruction (UUO) group. Values are means ± standard deviation (SD); n = 6 per group. IMD, intermedin; UUO, unilateral ureteral obstruction.
mediator.3,20–22 It has been reported that TGF-β1 is increased in chronic ureteral obstruction.23,24 It activates fibroblasts and induces ECM production, mainly through Smad-dependent pathways.4 Blocking TGF-β1 pathway has been shown to reduce tubular fibrosis in several models of chronic kidney injury.20,21,24 Connective tissue growth factor (CTGF) is considered the major downstream effector of TGF-β1 and is important in the production of ECM proteins.25 It is reported that CTGF gene expression is strongly induced by TGF-β1 in a smad3/smad4-dependent manner.25 TGF-β1-induced fibronectin production is reduced with CTGF oligonucleotide © 2015 Asian Pacific Society of Nephrology
antisense transfection of cultured renal fibroblasts.26 In agreement with previous studies, our results revealed that TGF-β1, pSmad2/3 and CTGF expressions were upregulated after UUO. Therefore, we examined whether IMD attenuates renal fibrosis in association with the regulation of TGF-β1/ smads pathway. Interestingly, our results revealed that IMD overexpression had little effect on TGF-β1 and pSmad2/3 expressions, while CTGF expression was reduced with IMD gene transfer and paralleled the reduction in myofibroblast accumulation. Our results demonstrated that the inhibitory effects of IMD on renal fibrosis may not be achieved by 829
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directly inhibiting TGF-β1 expression and smad2/3 activation but by the suppression of downstream steps in the TGF-β1/smads signalling pathway. To elucidate the mechanisms by which IMD inhibits renal fibrosis and ECM accumulation, we examined the effect of IMD on oxidative stress in UUO. Increased concentrations of ROS have been implicated in the pathophysiology of renal fibrosis,27,28 together with decreased activities of the major protective antioxidant enzymes such as SOD, catalase and glutathione peroxidase.29 It has been demonstrated that TGFβ1-induced myofibroblast activation works through a TGF receptor 1 (TGFR1)/Smad/ROS/ERK1/2 signalling cascade.30 In addition, oxidative stress induces macrophage infiltration, which further promotes interstitial fibrosis.31 Thus, ROS are important downstream effectors in mediating the profibrotic action of TGF-β1,5,7,30 and play important roles in TGF-β1induced ECM accumulation in the renal tubulointerstitium.6 Agents that can reduce ROS have received considerable attention.32,33 As a secreted peptide, IMD was shown to inhibit oxidative stress,11,18,19 suggesting that IMD might ameliorate renal fibrosis. We examined alterations of oxidative stress by measuring SOD (an important endogenous antioxidant enzyme) activity and MDA content (a marker of oxidative stress), in kidneys after UUO. Our results revealed that SOD activity significantly reduced and MDA content markedly increased in obstructed kidney. This increased oxidative stress was associated with impaired histological changes, increased macrophage infiltration, enhanced renal fibrosis and increased expression of TGF-β1. However, IMD gene delivery prevented depletion of SOD activity and increase of MDA content, and thus blocked the profibrotic actions of TGF-β1 without affecting its expression renal fibrosis. In summary, our study demonstrates that the expression of IMD as well as its receptors is upregulated in the obstructed kidney of rats with UUO. Locally overexpressed IMD in the kidney suppresses the progression of renal fibrosis in the rat UUO model, apparently by reducing oxidative stress and thus inhibits TGF-β1 signalling. However, further investigations are definitely required to elucidate the exact signalling mechanism whereby this peptide inhibits oxidative stress in the kidney and protects against fibrosis. Targeting IMD could be novel therapeutic target for preventing renal fibrosis.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81100531).
REFERENCES 1. Komenda P, Ferguson TW, Macdonald K et al. Cost-effectiveness of primary screening for CKD: A systematic review. Am. J. Kidney Dis. 2014; 63: 789–97.
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2. Duffield JS. Cellular and molecular mechanisms in kidney fibrosis. J. Clin. Invest. 2014; 124: 2299–306. 3. Yanagita M. Inhibitors/antagonists of TGF-β system in kidney fibrosis. Nephrol. Dial. Transplant. 2012; 27: 3686–91. 4. Wang W, Koka V, Lan HY. Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton) 2005; 10: 48–56. 5. Jiang F, Liu GS, Dusting GJ, Chan EC. NADPH oxidase-dependent redox signaling in TGF-β-mediated fibrotic responses. Redox Biol. 2014; 2: 267–72. 6. Ji X, Naito Y, Weng H et al. Renoprotective mechanisms of pirfenidone in hypertension-induced renal injury: Through anti-fibrotic and anti-oxidative stress pathways. Biomed. Res. 2013; 34: 309–19. 7. Rhyu DY, Yang Y, Ha H et al. Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J. Am. Soc. Nephrol. 2005; 16: 667–75. 8. Roh J, Chang CL, Bhalla A, Klein C, Hsu SY. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes. J. Biol. Chem. 2004; 279: 7264–74. 9. Morimoto R, Satoh F, Murakami O et al. Expression of adrenomedullin2/intermedin in human brain, heart, and kidney. Peptides 2007; 28: 1095–103. 10. Takahashi K, Kikuchi K, Maruyama Y et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney. Peptides 2006; 27: 1383–9. 11. Qiao X, Li RS, Li H et al. Intermedin protects against renal ischemia-reperfusion injury by inhibition of oxidative stress. Am. J. Physiol. Renal Physiol. 2013; 304: F112–9. 12. Qiao X, Chen X, Wu D et al. Mitochondrial pathway is responsible for aging-related increase of tubular cell apoptosis in renal ischemia/reperfusion injury. J. Gerontol. A. Biol Sci. Med Sci 2005; 60: 830–39. 13. Nagae T, Mori K, Mukoyama M et al. Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis. Kidney Int. 2008; 74: 70–80. 14. Nishikimi T, Wang X, Akimoto K et al. Alteration of renal adrenomedullin and its receptor system in the severely hypertensive rat: Effect of diuretic. Regul. Pept. 2005; 124: 89–98. 15. Nagae T, Mukoyama M, Sugawara A et al. Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: Cloning and upregulation in obstructive nephropathy. Biochem. Biophys. Res. Commun. 2000; 270: 89–93. 16. Nørregaard R, Bødker T, Jensen BL, Stødkilde L, Nielsen S, Frøkiaer J. Increased renal adrenomedullin expression in rats with ureteral obstruction. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 296: R185–92. 17. Ito K, Yoshii H, Asano T et al. Adrenomedullin increases renal nitric oxide production and ameliorates renal injury in mice with unilateral ureteral obstruction. J. Urol. 2010; 183: 1630–35. 18. Hagiwara M, Bledsoe G, Yang ZR, Smith RS Jr, Chao L, Chao J. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress. Am. J. Physiol. Renal Physiol. 2008; 295: F1735–43. 19. Zhang HY, Jiang W, Liu JY et al. Intermedin is upregulated and has protective roles in a mouse ischemia/reperfusion model. Hypertens. Res. 2009; 32: 861–8.
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20. Chen X, Wang H, Liao HJ et al. Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling. J. Clin. Invest. 2014; 124: 3295–310. 21. Venkatachalam MA, Weinberg JM. New wrinkles in old receptors: Core fucosylation is yet another target to inhibit TGF-β signaling. Kidney Int. 2013; 84: 11–4. 22. Wei X, Xia Y, Li F et al. Kindlin-2 mediates activation of TGF-β/Smad signaling and renal fibrosis. J. Am. Soc. Nephrol. 2013; 24: 1387–98. 23. Ding Y, Kim SL, Lee SY, Koo JK, Wang Z, Choi ME. Autophagy regulates TGF-β expression and suppresses kidney fibrosis induced by unilateral ureteral obstruction. J. Am. Soc. Nephrol. 2014; 25: 2835–46. 24. Kim J, Imig JD, Yang J, Hammock BD, Padanilam BJ. Inhibition of soluble epoxide hydrolase prevents renal interstitial fibrosis and inflammation. Am. J. Physiol. Renal Physiol. 2014; 307: F971–80. 25. Yokoi H, Mukoyama M, Nagae T et al. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J. Am. Soc. Nephrol. 2004; 15: 1430–40. 26. Yokoi H1, Mukoyama M, Sugawara A et al. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am. J. Physiol. Renal Physiol. 2002; 282: F933–42. 27. He W, Wang Y, Zhang MZ et al. Sirt1 activation protects the mouse renal medulla from oxidative injury. J. Clin. Invest. 2010; 120: 1056–68. 28. Singh DK, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy: Fueling the fire. Nat. Rev. Endocrinol. 2011; 7: 176–84. 29. Manucha W, Carrizo L, Ruete C, Molina H, Vallés P. Angiotensin II type I antagonist on oxidative stress and heat shock protein 70 (HSP 70) expression in obstructive nephropathy. Cell. Mol. Biol. 2005; 51: 547–55. 30. Bondi CD, Manickam N, Lee DY et al. NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts. J. Am. Soc. Nephrol. 2010; 21: 93–102. 31. Paul-Clark MJ, McMaster SK, Sorrentino R et al. Toll-like receptor 2 is essential for the sensing of oxidants during inflammation. Am. J. Respir. Crit. Care Med. 2009; 179: 299–306. 32. Nlandu Khodo S, Dizin E, Sossauer G et al. NADPH-oxidase 4 protects against kidney fibrosis during chronic renal injury. J. Am. Soc. Nephrol. 2012; 23: 1967–76. 33. Okamura DM, Bahrami NM, Ren S et al. Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD. J. Am. Soc. Nephrol. 2014; 25: 43–54.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
© 2015 Asian Pacific Society of Nephrology
Fig. S1 Increased mRNA expression levels of IMD, CRLR, and RAMPs in the kidneys after unilateral ureteral obstruction (UUO) surgery. The quantitative RT-PCR time-course for transcripts of whole kidney IMD (A), CRLR (B), RAMP1 (C), RAMP2 (D), RAMP3 (E) following UUO surgery. Expression levels of each mRNA were normalized to the expression level of β-actin. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. Fig. S2 Effects of IMD on the expression of pSmad2/3 in kidney tissues of unilateral ureteral obstruction (UUO) rats. (A) Representative western blot photograph showing expression levels of pSmad2/3 and tSmad2/3 proteins in rat kidneys. (B) Quantitative analysis of the relative ratio of pSmad2/3 to tSmad2/3. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. Fig. S3 Effects of IMD on the expression of α-SMA in kidney tissues of unilateral ureteral obstruction (UUO) rats. (A–H) Representative immunohistochemical staining for α-SMA protein in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Semiquantitative evaluation of α-SMA expression. Scale bar = 100 μm. Magnification: ×100. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group. Fig. S4 Immunohistochemical analyses for macrophages infiltration in the kidneys at days 7 and 14 after unilateral ureteral obstruction (UUO). (A–H) Representative immunohistochemical staining for ED-1 in the sham-operated kidneys (A and E), the untreated UUO kidneys (B and F), pcDNA3.1-intermedin (IMD) treated kidneys (C and G), and empty vector treated kidneys (D and H) at day 7 (A–D) and day 14 (E–H) after surgery. (I) Quantitation of average macrophage number per high-power field. Scale bar = 100 μm. Magnification: ×400. Data in bar graphs are means ± standard deviation (SD) (n = 6). *P < 0.05 versus sham-operated group. #P < 0.05 versus untreated UUO group.
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