Background
Chronic kidney disease (CKD) is a major health problem and its incidence and prevalence are increasing worldwide [
1]. Regardless of disease etiology, fibrosis is a final common pathogenic process for CKD leading to end stage renal diseases (ESRD) [
2]. Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM) in the tubulointerstitium. Reactive oxygen species (ROS) play an important pathogenic role in the development of renal fibrosis by inducing epithelial-mesenchymal transition (EMT) of tubular epithelial cells and by inducing macrophage infiltration [
3‐
5]. Intermedin [IMD, also known as adrenomedullin-2 (ADM-2)] is a novel member of the calcitonin/calcitonin gene-related peptide family [
6]. Our previous study demonstrated that kidney-specific IMD gene transfer significantly ameliorated unilateral ureteral obstruction (UUO)-mediated fibronectin up-regulation and the fibrotic changes, and the anti-fibrotic effect is achieved by inhibition of oxidative stress [
7]. However, the precise mechanisms remain unknown.
Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme [
8]. It converts heme to biliverdin via a reaction that produces carbon monoxide and liberates iron [
9]. There are two isozymes for HO, an inducible form (HO-1) and a constitutive one (HO-2) [
10]. HO-1 is strongly induced by oxidative stress and shows cytoprotective effects by the anti-inflammatory, anti-apoptotic, and anti-proliferative actions [
11]. Its expression is upregulated in renal fibrosis [
12], and furthermore, recent study demonstrated that induction of HO-1 prevented renal fibrosis induced by UUO [
13].
ADM is reported to induce HO-1 gene expression in rat vascular smooth muscle cells [
14]. Whether IMD has any effect on HO-1 has not been investigated yet. Since IMD and ADM have similar biological actions, we hypothesized that IMD can also induce HO-1 expression and that may explain its anti-oxidant and anti-fibrotic effects. To test this hypothesis, we investigated the effects of IMD overexpression on HO-1 expression and HO activity in the obstructed kidney of UUO rat and in rhTGF-β1-stimulated rat renal proximal tubular epithelial cells (NRK-52E). To evaluate the role of HO-1 induction in the protective effects of IMD on renal fibrosis, zinc protoporphyrin (ZnPP), an HO activity inhibitor [
15], was used. Furthermore, NRK-52E were analyzed for the effect of IMD on EMT.
Methods
Animals and UUO-induced renal fibrosis model
Ethics Committee for Animals of Shanxi Medical University approved all animal protocols. Male Wistar rats (180 to 200 g) were purchased from the Experimental Animal Center of Shanxi Medical University (Taiyuan, China) and maintained in a specific pathogen-free environment in our facility. All animal were fed with standard chow and had free access to water. Renal fibrosis was induced by ligation of left ureter, UUO, as we previously reported [
7]. 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.
The eukaryotic expression plasmid pcDNA3.1-IMD containing fulllength complementary DNA (cDNA) sequence of rat IMD was successfully constructed in our previous study [
16]. Before the ureter was obstructed, pcDNA3.1-IMD plasmid or control empty vector was transfected into the left kidney via the renal artery using an ultrasound-microbubble mediated system as we previously described [
16]. Rats were killed at 7 days after UUO. The transfection rate was detected by quantitative RT-PCR and Western blot analysis, respectively.
Experiment design
Animal study
Rats were randomly divided into the following five groups: sham, UUO, UUO + empty plasmid, UUO + IMD and UUO + IMD + ZnPP. Rats in UUO + empty plasmid, UUO + IMD and UUO + IMD + ZnPP groups were treated with ultrasound as described above before UUO operation. In UUO + IMD + ZnPP group, ZnPP (50 μg/kg body wt, Sigma-Aldrich, St. Louis, MO, prepared in the dark) was administered intraperitoneally with a single dose 48 h before UUO operation as previously reported [
17]. Animals were killed at 7 days after UUO. Obstructed kidneys and contralateral kidneys were harvested for further analysis.
Cell culture and treatments
Rat proximal tubular epithelial cell line (NRK-52E) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM/Ham’s F-12 (2:1) containing with 10% fetal bovine serum and incubated at 37 °C, room air with 5% CO
2. Cells were then cultured in fibronectin pre-coated flasks until they reached approximately 80% confluence. The over-expression of IMD in the NRK-52E cells that were transfected with pcDNA3.1-IMD was accomplished according to the methods described in our previous study [
18]. The model of EMT in the NRK-52E cells was established by stimulating with recombinant human TGF-β1 (rhTGF-β1, 10 ng/ml, Gibco) for 72 h. The cells were assigned to 1 of 5 groups: control (untreated NRK-52E cells); TGF-β1 (NRK-52E cells were incubated with rhTGF-β1); TGF-β1 + empty vector (NRK-52E cells were transfected with the empty plasmid pcDNA3.1 and incubated with rhTGF-β1); TGF-β1 + IMD (NRK-52E cells were transfected with pcDNA3.1-IMD and incubated with rhTGF-β1); and TGF-β1 + IMD+ ZnPP [NRK-52E cells were transfected with pcDNA3.1-IMD and incubated with rhTGF-β1 plus 2 μM ZnPP (prepared in the dark)].
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′-GGCCCAGTTGCTGATGGT-3′ and antisense: 5′-TGCCCGGGAGCAGGTA-3′), HO-1 (sense: 5′-AGGTGCACATCCGTGCAGAG -3′ and antisense: 5′-CTTCCAGGGCCGTATAGATATGGTA-3′) and β-actin (sense: 5′-CCCATCTATGAGGGTTACGC-3′ and antisense: 5′-TTTAATGTCACGCAC GATTTC-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 [
16]. Immunoblot analysis was performed with rabbit polyclonal anti-IMD (Beijing Biosynthesis Biotechnology, Beijing, China), goat polyclonal anti-HO-1 (Santa Cruz Biotechnology), mouse monoclonal anti-collagen I (Col-I, Santa Cruz Biotechnology), mouse monoclonal anti-α-SMA (Sigma-Aldrich), or rabbit moloclonal anti-E-cadherin (Abcam, Cambridge, UK) and then peroxidase conjugated AffiniPure secondary antibodies (Santa Cruz Biotechnology), respectively. The ECL Western blotting system (Santa Cruz Biotechnology) was used for detection. Rabbit polyclonal anti-β-actin (Santa Cruz Biotechnology) was used as the control for each sample.
HO enzymatic activity assay
HO activity was estimated by determining the level of bilirubin generated in isolated microsomes as described earlier [
19]. In brief, homogenized samples from the obstructed kidneys, contralateral kidneys or cultured NRK-52E cells were incubated in medium consisting of heme (0.25 mmol/L), rat liver cytosol (5 mg/mL) as a source of biliverdin reductase, MgCl
2(1 mmol/L), glucose-6-phosphate dehydrogenase (1 unit), glucose-6-phosphate (0.8 mmol/L) and NADPH (0.8 mmol/L) in 0.5 mL of 0.1 mol/L phosphate buffer saline (pH 7.4) for 1 h at 37 °C in the dark. Reactions were terminated by ice and then the bilirubin product was extracted with chloroform. Absorbance was measured using a spectrometer at wavelengths 464 and 530 nm. HO activity was calculated by the difference in absorbance between the two wavelengths, and expressed as picomoles of bilirubin per milligram of protein per h.
Immunohistochemical analysis
Rat kidneys were fixed in 10% formaldehyde and embedded in paraffin. Paraffin sections (4 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 goat polyclonal anti-HO-1 (1:200, Santa Cruz Biotechnology), mouse monoclonal anti-ED-1 (1:200, Santa Cruz Biotechnology) or mouse moloclonal anti-IL-6 (1:200, Abcam, Cambridge, UK). This was followed by biotinylated secondary antibodies (Santa Cruz Biotechnology) and finally by avidin conjugated horseradish peroxidase. All slides were counterstained with haematoxylin. HO-1 or IL-6 positive stained areas were assessed and expressed as integrated optical density (IOD) and area. Interstitial macrophages were expressed as the percentage of ED-1 positive interstitial area. Three sections of each rat kidney were measured, and 10 random fields were chosen and calculated under magnification of 400×. The IOD and positive area were acquired by the Image-Pro Plus 6.0 program (Media Cybernetics, Bethesda, MD, USA).
Histological examinations
Renal histological changes were assessed at days 7 after UUO. Paraffin-embedded transverse kidney slices were sectioned at 4 μm. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome. Twenty cortical tubulointerstitial fields that were randomly selected at ×200 magnifications 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 color of tone was counted in each section, and then automatically converted into dimensions.
Immunofluorescence
NRK-52E monolayers were fixed in methanol (20 min, −20 °C), and blocked with 2% bovine serum albumin (BSA) in PBS (1 h, room temperature). Cells were then incubated with primary antibodies against epithelial cell marker rabbit moloclonal anti-E-cadherin (1:50, Abcam, Cambridge, UK) and mesenchymal marker mouse monoclonal anti-α-SMA (1:100; Sigma-Aldrich) with 2% BSA overnight at 4 °C). Secondary fluorescent conjugated anti-rabbit Alexa Fluor®647 (1:400; Invitrogen) was used for E-cadherin and anti-mouse Alexa Fluor®546 (1:400; Invitrogen) was used for α-SMA for 40 min at room temperature in dark. Slides were mounted using ProLong® Gold Antifade reagent with DAPI (Life technologies). Images were photographed using Olympus FV1000 Confocal microscope at ×40 magnification.
Statistical analysis
The data are expressed as means ± standard deviation (SD). Significant differences between groups were assessed by one-way ANOVA with Student-Newman-Keuls post hoc tests. P values <0.05 were considered statistically significant.
Discussion
Tubulointerstitial fibrosis is a common pathway of all CKD leading to ESRD. Furthermore, it remains the best predictor of disease progression [
20]. In the present study, we analyzed the protective effect of IMD on renal fibrosis in rat UUO model, a well-established in vivo model of renal fibrosis. Our results showed that IMD reduced parameters of fibrosis such as elevated collagen deposition measured by Masson’s trichrome staining, and Col-I production. In addition, IMD inhibited macrophages infiltration, pro-inflammatory cytokine IL-6 expression and EMT in UUO-induced kidneys. In vitro studies demonstrated that TGF-β1-stimulated EMT in proximal tubular epithelial cells was reduced by IMD. Furthermore, all the above beneficial effects of IMD were abolished by inhibition of HO-1 enzyme activity with ZnPP.
It is commonly accepted that ROS have been implicated in the pathophysiology of renal fibrosis [
21]. Our previous studies demonstrated that IMD attenuated renal fibrosis by inhibition of oxidative stress [
7] and the precise mechanisms warrant further studies. HO is a microsomal enzyme that exists in two isoforms: the inducible HO-1 and the constitutive HO-2. HO-1 is a stress response protein with antioxidative properties [
22]. It is reported that induction of HO-1 is an adaptive and protective response against renal fibrosis [
17]. Whether IMD has any effect on HO-1 expression and HO activity has not been investigated yet. In this study, we observed a significant amplification of HO-1 mRNA and protein as well as HO activity in IMD-treated animals (Fig.
2a–d). Immunohistology reveals that IMD-induced HO-1 protein expressions were mainly in tubular epithelial cells (Fig.
2c). We reached the same results in TGF-β1-stimulated tubular cells (Fig.
3). Importantly, this upregulation of HO-1 and induction of HO activity by IMD was paralleled with suppression of tubulointerstitial fibrosis (Fig.
4).
To verify if the anti-fibrotic effect of IMD is achieved by induction of HO-1, we used ZnPP. It has been reported that ZnPP inhibited HO-1 enzyme activity, although it increased HO-1 protein and mRNA expression [
23]. Although HO-2 activity is also inhibited by ZnPP, the constitutive nature of HO-2 makes it less attractive as a drug target [
10]. In line with previous reports, our results indicated that ZnPP markedly decreased HO activity in obstructed kidney and cultured tubular cells. HO activity in contralateral kidney of UUO rat was also inhibited by ZnPP, indicating that the effect of ZnPP on HO activity was systemic. We showed that inhibition of HO enzyme activity abrogated the protective effect of IMD on interstitial fibrosis. Thus, the antifibrotic effect of IMD in our model is dependent on induction of HO-1.
A number of evidence demonstrated that oxidative stress could trigger the inflammatory response in renal fibrosis [
24]. Inflammation plays an important role in the development and progression of renal fibrosis [
25]. Macrophages, a major type of inflammatory cell, are recruited during tubulointerstitial fibrosis [
26,
27]. IL-6 is a well-known pro-inflammatory cytokine that is also correlated with increased fibrosis [
28]. In this study, we showed the number of macrophages and expression of IL-6 in the obstructed kidneys of UUO rats was significantly higher than that of the sham-operated rats. Although the recruitment of macrophages may contribute to kidney repair at the early stages, this repair can ultimately cause renal fibrosis [
27,
29]. In our study, in line with the infiltration of macrophages and up-regulation of IL-6, renal fibrosis is associated enhanced (Fig.
4). IMD overexpression obviously inhibited the infiltration of macrophages and IL-6 expression following UUO, which effect could subsequently be blocked by the HO-1 inhibitor ZnPP. This indicates that the protection against macrophage infiltration and IL-6 production afforded by IMD was due to induction of HO-1.
EMT plays a pivotal role in the pathogenesis of renal tubulointerstitial fibrosis [
30]. EMT is recognized as a loss of epithelial cell phenotype, decrease of intercellular epithelial adhesion molecules, such as E-cadherin, with a concomitant development of mesenchymal phenotype, including expression of α-SMA [
31]. In this study, we showed that α-SMA expression significantly increased, while E-cadherin expression markedly decreased, after UUO operation, indicating extensive EMT (Fig.
6). It has been suggested that EMT is mediated, at least in part, through ROS [
32]. Thus, IMD, through its antioxidant properties, may reduce EMT. Our results demonstrated that IMD gene delivery attenuated UUO-induced EMT, which was abolished by HO-1 activity inhibition.
For further clarification of the effects of IMD on TGF-β1-mediated EMT in proximal tubules, in vitro studies were performed to eliminate the confounding effects of infiltrating cells. These in vitro studies, using rat proximal tubular cells (NRK-52E), indicated that IMD inhibited TGF-β1-mediated EMT as indicated by up-regulation of E-cadherin and down-regulation of α-SMA measured by immunofluorescence (Fig.
7). ZnPP abrogated the beneficial effects of IMD on EMT. These results, along with the in vivo evidence, demonstrate that IMD inhibits EMT by induction of HO-1.
Although our current study provided significant results, there are still some limitations. The primary one is that we did not use HO-1-deficient mice or HO-1-deficient tubular cells to investigate the effect of IMD on renal fibrosis. ZnPP is commonly used as a HO-1 inhibitor [
15], however, its specificity is still a matter of debate [
33,
34]. The second limitation is the use of UUO as an animal model CKD. Although UUO is a very well-accepted renal fibrosis model, some experts criticized it as a model of CKD because of the compensation by the normal contralateral kidney that precludes meaningful renal functional measures [
35]. More CKD models that provide us with an opportunity to include renal functional study end-points together with quantitative measures of fibrosis severity are needed. The last one is we didn’t investigate the transcription pathways by which IMD activates HO-1. Examining some transcription factors involved in HO-1 modulation are required to provide further mechanistic insight into the mechanism of IMD induced HO-1 activation pathway.