pNaKtide ameliorates renal interstitial fibrosis through inhibition of sodium-potassium adenosine triphosphatase-mediated signaling pathways in unilateral ureteral obstruction mice

pNaKtide ameliorates renal interstitial fibrosis through inhibition of sodium-potassium adenosine... Abstract Background Sodium-potassium adenosine triphosphatase (Na/K-ATPase) has been shown to regulate Src activity by combining with Src to keep it in an inactive form. We previously reported that Na/K-ATPase was downregulated in unilateral ureteral obstruction (UUO) animals. In this study, we examined whether inhibition of Na/K-ATPase-mediated Src signaling pathways ameliorated renal interstitial fibrosis induced by UUO. Methods UUO was performed on male C57BL/6J mice. pNaKtide, a mimic of Na/K-ATPase, was administered by intraperitoneal injection on Day 0 and Day 4 after ureteral ligation. Markers of interstitial fibrosis, inflammation and oxidative stress and transforming growth factor-β1 (TGF-β1) expression were examined after the mice were sacrificed on Day 7. Activation of Src and its downstream signaling effectors, including extracellular regulated protein kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (AKT), were evaluated. Results pNaKtide administration markedly attenuated myofibroblast accumulation and extracellular matrix deposition in obstructed kidneys. Also, pNaKtide significantly reduced the increased expression of 8-iso-prostaglandin F2α, TGF-β1, interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), as well as reduced macrophage infiltration, in UUO animals. All these changes were obtained, along with inhibition of Src and its downstream effector activity. Conclusions Na/K-ATPase-mediated signaling pathways contribute to fibrogenesis and could represent a potential target in the treatment of renal fibrosis. fibrosis, Na/K-ATPase, pNaKtide, Src, UUO INTRODUCTION Interstitial fibrosis is the common hallmark of pathological change underlying chronic kidney disease (CKD) induced by various nephropathies [1]. Numerous cytokines and growth factors are involved in the fibrosis process by activating multiple intracellular signaling pathways [2]. These complex signaling pathways share points of cross-talk and amalgamation. Targeting these points could provide greater benefits by blocking upstream signaling cascades, rather than blocking a single downstream pathway, in the treatment of interstitial fibrosis. Src is a member of the Src tyrosine kinase family and is capable of activating intracellular signaling cascades upon its activation [3]. Accumulating data have revealed that Src activation is involved in the development of chronic fibrotic lesions, including renal interstitial fibrosis [4]. Sodium-potassium adenosine triphosphatase (Na/K-ATPase) is a member of the P-type ATPase family, which localizes to the basolateral membrane of renal tubular epithelial cells and plays an important role in sodium reabsorption. In recent years, it has been shown that Na/K-ATPase also serves as a signaling regulator by combining with other signaling molecules to form a signaling complex [5]. Notably, the α1 subunit of Na/K-ATPase combines with Src and regulates Src activation in two ways. First, the Na/K-ATPase/Src complex functions as a receptor for cardiotonic steroids. When combined with the ligand, the Na/K-ATPase changes its conformation from E1 to E2, which results in Src activation. Fedorova et al. found that marinobufagenin induced transdifferentiation of renal tubular epithelial cells through the Na/K-ATPase/Src complex [6]. In our previous study, we also demonstrated that Na/K-ATPase mediated reactive oxygen species (ROS)-induced extracellular regulated protein kinase 1/2 (ERK1/2) activation by activating Src in LLC-PK1 cells [7]. Second, changes in Na/K-ATPase expression alter the interaction equilibrium between Na/K-ATPase and Src, resulting in Src activation, since the interactions maintain Src in an inactive state. In our previous work, we found that the expression of Na/K-ATPase was markedly decreased in CKD patients and unilateral ureteral obstruction (UUO) models [8, 9]. Extending these findings, we reasoned that Na/K-ATPase could be implicated in fibrogenesis, as a potential regulator of the Src signaling pathway in CKD. pNaKtide is a polypeptide, designed and constructed according to the sequence from the nucleotide-binding (N) domain of the α1 subunit of Na/K-ATPase. It binds with the kinase domain of Src and specifically inhibits Src activation without affecting the pumping function of Na/K-ATPase [10, 11]. pNaKtide readily crosses the cell membrane and resides in intracellular membrane compartments. It has been shown to attenuate the development of adiposity [12] and uremic cardiomyopathy [13] in vivo by inhibiting Na/K-ATPase-modulated oxidation amplification, which involves Src activation. Given the potential importance of the Src-regulated signaling cascade in the development of renal fibrosis, we hypothesized that pNaKtide could attenuate renal fibrosis through inhibition of Na/K-ATPase-modulated Src signaling cascade activation. MATERIALS AND METHODS Experimental animals This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Experimentation Ethics Committee of Peking University First Hospital. Male C57BL/6J mice (23 ± 3 g) were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China), and housed at 25°C in 40% humidity and 12/12-h light/dark cycle. Mice were allowed free access to water and standard chow until the night before surgery. Animals were randomly divided into the following groups: sham surgery, pNaKtide alone, UUO alone or UUO with pNaKtide treatment (UUO + pNaKtide). An extra group was set as UUO with PP2 (UUO + PP2). pNaKtide (25 mg/kg) was administered by intraperitoneal injection on Day 0 and Day 4 after surgery. PP2 (2 mg/kg/day) was given daily by intraperitoneal injection after surgery. All surgeries were performed under anesthesia with sodium pentobarbital, and all efforts were made to minimize suffering. The left ureter was exposed using a mid-abdominal incision and ligated with 3-0 silk sutures in the UUO groups. Sham surgery involved mobilization, but not ligation, of the ureter. All animals were sacrificed on Day 7 after surgery. The left kidneys were decapsulated, washed with ice-cold normal saline and then rapidly dissected. Coronal sections of 2- to 3-mm thickness through the mid-portion of the kidney were embedded in paraffin after fixation in 10% neutral buffered formalin, and the remaining kidney tissue was snap-frozen in liquid nitrogen for further study. Reagents and antibodies The following antibodies were used for immunoblotting and immunohistochemical staining: monoclonal anti-Na/K-ATPase, α1 (clone C464.6) (Upstate, Billerica, MA, USA); monoclonal anti-α1, α6f (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA); monoclonal anti-pERK, and polyclonal anti-ERK and anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-collagen I (COL-1), polyclonal anti-CD68 and polyclonal anti-fibronectin (FN) (Abcam, Cambridge, MA, USA); monoclonal anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich Chemicals, St Louis, MO, USA); monoclonal anti-cSrc, p-p38 mitogen-activated protein kinase (MAPK) and p38 MAPK, pAKT and AKT and polyclonal anti-pSrc (Cell Signaling Technology, Danvers, MA, USA). The enhanced chemiluminescence (ECL) kit was purchased from PerkinElmer (Rockford, IL, USA). PP2 was purchased from Merck Millipore (Darmstadt, Germany). pNaKtide was obtained from HD Bioscience (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kit for matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1) was purchased from Elabscience Biotechnology (Wuhan, China). The interleukin-6 (IL-6) ELISA assay kit was purchased from Abcam (Cambridge, MA, USA). The assay kit for 8-iso-prostaglandin F2α (8-iso-PGF2α) was purchased from Cayman (Ann Arbor, MI, USA). All chemicals used in the experiments, unless otherwise stated, were purchased from Sigma-Aldrich Chemicals. Immunocytochemistry staining Following formalin fixation and paraffin embedding, 4-μm sections were deparaffinized in xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was suppressed by exposing slide-mounted tissue to 0.3% hydrogen peroxide after antigen retrieval by microwave heating. Sections were then incubated with the indicated primary antibody overnight at 4°C. Control sections were incubated without the primary antibody. After washing in phosphate-buffered saline, slides were incubated with biotinylated goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG antibody, then peroxidase conjugated to streptavidin (Zymed Laboratories, San Francisco, CA, USA). Sections were stained with diaminobenzidine tetrahydrochloride substrate and counterstained with hematoxylin. The number of interstitial CD68+ cells was counted in 10 consecutive fields (200×) and expressed as cells per high-power field. Immunoblot Levels of Na/K-ATPase, α-SMA, FN, phosphor- and total-Src, ERK1/2, p38 MAPK and AKT in tissue homogenates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then electrophoretically transferred to nitrocellulose membranes. Immunoblotting was performed as previously described [9]. Briefly, membranes were blocked with 5% skimmed milk in tris-buffered saline–Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the indicated antibodies at 4°C overnight. Blots were incubated in TBS-T 30 min before incubation with HRP-conjugated anti-rabbit or anti-mouse IgG for 1 h at room temperature. Staining was detected using the chemiluminescence ECL kit, and developed with Image Quant LAS 4000mini (GE, Boston, MA, USA). The membrane was then stripped and probed with mouse anti-GAPDH antibody (Sigma-Aldrich Chemicals) to confirm and estimate loading and transfer, except for phosphor-Src, ERK, p38 MAPK and AKT, which were probed with an antibody to the indicated total kinase for adjustment, respectively. The bands were quantified using ImageJ software (an open source Java image processing program inspired by NIH Image). ELISA assay MMP-9, TIMP-1, MCP-1, IL-6, TGF-β1 and 8-iso-PGF2α levels were determined in renal tissue homogenates according to the ELISA kit manufacturer’s instructions. The results were adjusted according to the protein concentration of the renal tissue homogenates. Statistical analysis Data were expressed as mean ± standard error (SE). SPSS software 13.0 (Chicago, IL, USA) was used for all analyses. Groups were compared using Student’s t-test, and one-way analysis of variance when more than two groups were compared. P-values of <0.05 were considered significant. Each presented immunoblot is representative of the results of at least three separate experiments. RESULTS Effect of pNaKtide on Na/K-ATPase expression in UUO mice The expression levels of α1 Na/K-ATPase were first examined by immunocytochemical staining. α1 Na/K-ATPase was quantitatively expressed on the basolateral plasma membrane of tubules in mice that received sham surgery with or without pNaKtide treatment (Figure 1A and B), a mimic of Na/K-ATPase. By contrast, the α1 Na/K-ATPase level was substantially reduced in tubules of obstructed kidneys 7 days after surgery (Figure 1C), which was not substantially rescued by the administration of pNaKtide (Figure 1D). To further validate this phenomenon, the α1 Na/K-ATPase content of whole kidney homogenates was assessed by immunoblotting. Administration of pNaKtide did not enhance the renal levels of α1 Na/K-ATPase in UUO mice (Figure 1E). These results suggested that pNaKtide had no direct effect on renal Na/K-ATPase expression. FIGURE 1 View largeDownload slide Expression of Na/K-ATPase in UUO mice with pNaKtide treatment. UUO was induced in C57BL/6J mice. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of Na/K-ATPase expression in kidney homogenates. ***P < 0.001 versus sham alone. FIGURE 1 View largeDownload slide Expression of Na/K-ATPase in UUO mice with pNaKtide treatment. UUO was induced in C57BL/6J mice. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of Na/K-ATPase expression in kidney homogenates. ***P < 0.001 versus sham alone. pNaKtide attenuates renal interstitial myofibroblast accumulation and the development of renal fibrosis in the obstructed kidney Renal fibrosis induced by UUO is characterized by activation of myofibroblasts and accumulation of excessive amount of extracellular matrix (ECM) proteins. As shown by sirius red staining (Figure 2A–D), there was significant deposition of collagens in UUO mice on Day 7, which was markedly reduced by pNaKtide treatment. To confirm this finding, the deposition of collagen type I, a key ECM component, was further examined by immunocytochemistry. The result was in agreement with that obtained using sirius red staining (Figure 2E–H). In addition, the expression of FN was examined by immunoblot analysis. As shown in Figure 2I, pNaKtide treatment significantly inhibited the increased expression of FN in UUO mice on Day 7. FIGURE 2 View largeDownload slide Effect of pNaKtide on UUO-induced renal fibrosis. (A–D) Representative images of sirius red renal histology; (E–H) representative images of collagen type I expression; (I) representative western blot and data analysis of FN expression in kidney homogenates (n = 6–8 mice per group). ***P <0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 2 View largeDownload slide Effect of pNaKtide on UUO-induced renal fibrosis. (A–D) Representative images of sirius red renal histology; (E–H) representative images of collagen type I expression; (I) representative western blot and data analysis of FN expression in kidney homogenates (n = 6–8 mice per group). ***P <0.001 versus sham alone; ###P < 0.001 versus UUO alone. α-SMA is a marker of myofibroblasts, the major contributor to ECM production. We examined the expression of α-SMA both by immunocytochemistry staining (Figure 3A–D) and by immunoblot analysis (Figure 3E). There was increased expression of α-SMA in the obstructed kidney, indicating an accumulation of myofibroblasts. pNaKtide treatment significantly inhibited α-SMA expression in UUO mice. Taken together, these data supported that activation of Na/K-ATPase-mediated signaling contributed to accumulation of renal fibroblasts and the development of renal fibrosis induced by ureteral obstruction. FIGURE 3 View largeDownload slide Effect of pNaKtide on myofibroblast accumulation in UUO mice. Representative images of α-SMA immunohistochemical staining shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of α-SMA expression in kidney homogenates (n = 6–8 mice per group). ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 3 View largeDownload slide Effect of pNaKtide on myofibroblast accumulation in UUO mice. Representative images of α-SMA immunohistochemical staining shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of α-SMA expression in kidney homogenates (n = 6–8 mice per group). ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Effect of pNaKtide on the degradation of ECM in the obstructed kidney The amount of ECM is generally kept stable through a balance between its production and degradation. MMP-9 and TIMP-1 are major players in the ECM degradation system. We examined the effect of pNaKtide treatment on MMP-9 and TIMP-1. As shown in Table 1, MMP-9 and TIMP-1 expression levels significantly increased in UUO mice, as previously reported. However, pNaKtide treatment showed no significant inhibitory effect on either MMP-9 or TIMP-1 expression. Taken together, these data supported that Na/K-ATPase-mediated signaling contributes to renal fibrogenesis in UUO by inducing accumulation of ECM without affecting its degradation. Table 1 Expression of MMP-9 and TIMP-1 Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** ** P < 0.01 and ***P < 0.001 versus sham. Table 1 Expression of MMP-9 and TIMP-1 Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** ** P < 0.01 and ***P < 0.001 versus sham. pNaKtide contributes to decreased expression of TGF-β1 in UUO mice TGF-β1 is the predominant cytokine stimulating the differentiation of renal fibroblasts and inducing renal fibrosis. Studies by Yan et al. showed that activation of Src played an important role in mediating TGF-β1 signaling in UUO injury [4]. We examined whether pNaKtide affected TGF-β1 expression in UUO mice. The expression of TGF-β1 significantly increased in the obstructed kidney in UUO mice, compared with the sham group (1928 ± 132.9 versus 1087 ± 76.93 pg/mg, P < 0.001), which was partly, but significantly, decreased by pNaKtide treatment (1521 ± 58.1 versus 1928 ± 132.9 pg/mg, P < 0.01). These data suggest that Na/K-ATPase-mediated signaling contributed to renal fibrosis in UUO, at least partly, through modulating TGF-β1 expression. pNaKtide attenuates inflammation in the obstructed kidney Inflammation is also a characteristic feature of UUO that contributes to the development of renal fibrosis. Increased secretion of inflammatory factors and chemokines induces the subsequent infiltration of macrophages and lymphocytes into the obstructed kidneys. To determine whether Na/K-ATPase-mediated signaling contributed to renal fibrosis through inducing inflammation in the kidney after UUO injury, we examined the effect of pNaKtide on the expression of MCP-1 and IL-6. Both MCP-1 and IL-6 levels were significantly elevated in UUO mice, and were partially reduced by pNaKtide treatment (Table 2). Accordingly, we also found less infiltration of macrophages in pNaKtide-treated UUO mice on immunocytochemistry staining analysis (Figure 4). These results demonstrated pNaKtide alleviated inflammation in UUO mice. Table 2 Expression of MCP-1 and IL-6 Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# ** P < 0.01 and ***P < 0.001 versus sham; #P < 0.05 and ##P < 0.01 versus UUO. Table 2 Expression of MCP-1 and IL-6 Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# ** P < 0.01 and ***P < 0.001 versus sham; #P < 0.05 and ##P < 0.01 versus UUO. FIGURE 4 View largeDownload slide pNaKtide reduced UUO-induced infiltration of macrophages. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Macrophage infiltration was analyzed by CD68 staining. Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) semi-quantitative analysis of immunohistochemical staining of CD68. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 4 View largeDownload slide pNaKtide reduced UUO-induced infiltration of macrophages. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Macrophage infiltration was analyzed by CD68 staining. Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) semi-quantitative analysis of immunohistochemical staining of CD68. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Inhibitory effects of pNaKtide on oxidative stress in UUO mice 8-iso-PGF2α is an interaction product between O2− and esterified or free arachidonate. It is a well-established marker used to reflect oxidative stress. In the present study, there was a significantly increased level of 8-iso-PGF2α in UUO mice, which was markedly suppressed by pNaKtide treatment (Figure 5). This result indicated pNaKtide alleviated oxidative stress in UUO mice. FIGURE 5 View largeDownload slide Analysis of renal 8-iso-PGF2α content. The level of 8-iso-PGF2α in renal tissue homogenates was assessed and adjusted according to the total protein concentration in the renal tissue homogenates. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 5 View largeDownload slide Analysis of renal 8-iso-PGF2α content. The level of 8-iso-PGF2α in renal tissue homogenates was assessed and adjusted according to the total protein concentration in the renal tissue homogenates. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. pNaKtide blocks Src-regulated signaling cascade activation in the obstructed kidney A previous study showed that activation of Src plays an important role in UUO injury. We showed, in a series of previous studies, that Na/K-ATPase acted as a regulator of Src activation. In this present study, PP2, the direct inhibitor of Src activation, was used as control to evaluate the effect of pNaKtide on Src and its regulated downstream effectors. With significant downregulation of α1 Na/K-ATPase expression, we found the level of phosphorylated Src, the active form of Src, was markedly elevated in the obstructed kidney collected on Day 7, indicating that Src was constitutively activated in UUO mice (Figure 6). The total Src level was not altered in UUO mice. Administration of pNaKtide significantly reduced Src activation without affecting the expression level of Src in the obstructed kidney (Figure 6). FIGURE 6 View largeDownload slide Effect of pNaKtide and PP2 on UUO-induced Src activation. Representative western blot and data analysis of Src activation in kidney homogenates (n = 6–8 mice per group). Src activation was expressed as pY418 Src/total Src (p-Src/t-Src) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 6 View largeDownload slide Effect of pNaKtide and PP2 on UUO-induced Src activation. Representative western blot and data analysis of Src activation in kidney homogenates (n = 6–8 mice per group). Src activation was expressed as pY418 Src/total Src (p-Src/t-Src) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Src is known to activate several intracellular signal transduction pathways upon its activation. We investigated the effect of pNaKtide on Src-regulated downstream effectors in UUO mice. As members of the MAPK family, ERK1/2 and p38 MAPK have been reported, in several studies, to be activated by Src activation, contributing to inflammation and cell differentiation [14, 15]. We found increased activation of both p38 MAPK and ERK1/2 in UUO mice, which was partially, but significantly, inhibited by pNaKtide treatment (Figure 7A and B). Previous work also showed that inhibition of AKT decreased the expression of α-SMA and accumulation of ECM in UUO mice [16]. To determine whether Src activation transduced signals to the AKT pathway in the kidney after UUO injury, we examined the effect of pNaKtide on AKT phosphorylation in the present study. As shown in Figure 7C, the phosphorylation level of AKT was increased in UUO-injured kidneys, and pNaKtide administration resulted in a significant reduction of AKT phosphorylation, suggesting activation of the AKT signaling pathway is under Src regulation in kidneys undergoing fibrosis. Notably, activation of all these downstream effectors was inhibited, to a comparable degree, by PP2 administration. Taken together, these data indicated that pNaKtide inhibited UUO-induced activation of Src and its downstream signaling cascades in the development of renal fibrosis. FIGURE 7 View largeDownload slide Effect of pNaKtide and PP2 on Src-mediated signaling cascade activation. Representative western blot and analysis of kinase activation in renal tissue homogenates (n = 6–8 mice per group). (A) ERK1/2 activation expressed as phosphor-ERK/total ERK (p-ERK/t-ERK) ratio. (B) p38 MAPK activation expressed as phosphor-p38 MAPK/total p38 MAPK (p-38 MAPK/t-p38 MAPK) ratio. (C) AKT activation expressed as phosphor-AKT/total AKT (p-AKT/t-AKT) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 7 View largeDownload slide Effect of pNaKtide and PP2 on Src-mediated signaling cascade activation. Representative western blot and analysis of kinase activation in renal tissue homogenates (n = 6–8 mice per group). (A) ERK1/2 activation expressed as phosphor-ERK/total ERK (p-ERK/t-ERK) ratio. (B) p38 MAPK activation expressed as phosphor-p38 MAPK/total p38 MAPK (p-38 MAPK/t-p38 MAPK) ratio. (C) AKT activation expressed as phosphor-AKT/total AKT (p-AKT/t-AKT) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. DISCUSSION CKD is characterized by an irreversible deterioration of renal function, with renal fibrosis as the final common pathologic change. There is a great need to identify treatment targets for slowing CKD development. In the present study, we demonstrated that pNaKtide, an analog of Na/K-ATPase, suppressed Src and downstream p38 MAPK, ERK1/2 and AKT activation in UUO kidneys. We showed that pNaKtide administration also attenuated myofibroblast accumulation and ECM deposition, renal inflammation, oxidative stress and TGF-β1 production in the injured kidney. Collectively, these data suggested that Na/K-ATPase acts as a key mediator of Src signaling transduction in the process of renal fibrogenesis. Na/K-ATPase is heavily expressed in tubular cells in normal kidneys. In addition to its function as a pump, Na/K-ATPase has been found to have scaffolding and signaling function, in numerous studies on the Na/K-ATPase/Src complex. This signaling pathway has been shown to be involved in cardiovascular diseases, hypertension, salt balance and renal diseases [17–23]. We found a significant decrease in Na/K-ATPase expression in injured kidneys from our previous studies [8, 9]. In addition, Src has been shown to play important roles in renal fibrosis in UUO. On this background, we sought to examine whether blockade of Na/K-ATPase-mediated signaling transduction by pNaKtide could effectively ameliorate fibrosis in UUO animals. As an analog of Na/K-ATPase, pNaKtide binds to the kinase domain of Src to specifically inhibit Src activation without affecting the pumping function of the Na/K-ATPase. Our data clearly showed that pNaKtide administration significantly ameliorated interstitial fibrosis induced by UUO. The mechanism of pNaKtide effect on renal fibrogenesis may be associated with its various cellular functions. First, exposure to pNaKtide resulted in lower accumulation of myofibroblasts in UUO mice. Myofibroblasts are a major contributor to renal fibrosis. A decrease in myofibroblast accumulation resulted in reduced production and deposition of ECM in UUO kidneys. Second, inflammation is an important feature of UUO. Patients with CKD are exposed to persistent low-grade inflammation, which supposedly acts as a pre-conditioning event in the development of renal fibrosis, as observed, for example, in diabetic nephropathy and cardiovascular comorbidities in CKD [24–26]. For instance, hyperhomocysteinemia is a well-known cardiovascular risk factor in CKD patients. It was found that homocysteinylated albumin could upregulate MCP-1, inducing a proinflammatory state by promoting monocyte adhesion onto endothelial cells, which is directly related to the pathogenesis of atherosclerosis [27]. Inflammation also plays a detrimental role in the development of renal fibrosis in UUO [28]. Infiltration of macrophages and monocytes contribute to the inflammatory response in obstructive kidney disease [29, 30]. To understand the role of Na/K-ATPase-mediated signaling in the induction of inflammation in UUO, we examined the expression of the inflammatory cytokines MCP-1 and IL-6. Increased expression of both MCP-1 and IL-6 in UUO mice was significantly reduced by administration of pNaKtide, along with less macrophage infiltration, indicating that pNaKtide exerted anti-fibrotic effects, at least partly, through anti-inflammation. Furthermore, it has been suggested that oxidative stress is associated with CKD progression and comorbidity. For instance, hydrogen sulfide (H2S), the third gasotransmitter, possesses beneficial anti-oxidative effects [31]. Low plasma H2S levels have been detected in uremia patients, contributing to the development of hyperhomocysteinemia, in turn leading to increased cardiovascular risk [32]. In addition, Song et al. demonstrated that H2S exhibited anti-fibrotic effects in obstructed nephropathy and inhibited the proliferation and differentiation of renal fibroblasts both in vitro and in vivo [33]. We also found, in our previous work, that renal fibrosis was alleviated with the suppression of nicotinamide adenine dinucleotide phosphate oxidase (NOXs) activity and subsequent oxidative stress [34], suggesting oxidative stress is an important contributor in the pathogenesis of renal fibrosis in UUO. There is evidence suggesting that Na/K-ATPase-mediated signaling is involved in oxidation amplification [12, 13]. Hence, in the present study, we examined the oxidative stress marker 8-iso-PGF2α to understand the role of Na/K-ATPase-mediated signaling in the induction of oxidative stress in UUO. As shown in Figure 5, the UUO-induced increased level of 8-iso-PGF2α was significantly reduced by pNaKtide treatment, suggesting pNaKtide alleviates oxidative stress in UUO mice. The exact mechanism of Na/K-ATPase-mediated signaling in oxidative stress induction in UUO, such as whether it involves impaired H2S production converging into the same signaling pathway, needs further study. Finally, it has been well documented that TGF-β1 is central to fibroblast activation and fibrogenesis in UUO [35]. Src has been suggested to upregulate TGF-β1 signaling through overproduction of TGF-β1 [36]. We found pNaKtide treatment significantly reduced TGF-β1 overexpression in UUO mice. Taken together, these data clearly demonstrated that blockade of Na/K-ATPase-mediated signaling by pNaKtide alleviates renal fibrosis through multiple mechanisms, as schematically summarized in Figure 8. FIGURE 8 View largeDownload slide Schematic illustration of the mechanisms of Na/K-ATPase-regulated signaling pathways in the mediation of renal fibrosis in UUO. FIGURE 8 View largeDownload slide Schematic illustration of the mechanisms of Na/K-ATPase-regulated signaling pathways in the mediation of renal fibrosis in UUO. We found that these anti-fibrotic changes with pNaKtide treatment in injured kidneys were associated with inhibition of Src activation and its downstream effectors. Src can be activated by multiple profibrotic growth factors/cytokines and transduces signals initiated from diverse membrane receptors. Evidence indicates that Src activation is involved in renal lesions in animal models of, for example, diabetic nephropathy, renal cyst formation and proliferative glomerulonephritis [37–39]. Specifically, Yan et al. provided strong evidence for Src involvement in renal fibroblast activation and renal fibrosis in their study [4]. Thus, it is possible Src represents a converging point for integrating diverse profibrotic signals in the process of renal fibrosis. We have previously demonstrated that Na/K-ATPase serves as a regulator of Src by binding to Src to form a signaling complex, and thus keeping Src in an inactive form. In the present study, pNaKtide administration significantly inhibited Src activation induced by UUO to a comparable degree as that of PP2 administration, indicating that the anti-fibrotic effect of pNaKtide could be attributed to its effects on Na/K-ATPase-mediated Src signaling transduction. As a non-receptor tyrosine kinase, Src activates several downstream intracellular signaling cascades upon its activation, such as serine/threonine protein kinase ERK1/2 and p38 MAPK, EGFR and protein kinase B/phosphatidylinositol 3 kinase (AKT/PI3K). ERK1/2 plays an important role in renal cell proliferation and differentiation in nephropathy. In our previous study, we found ERK1/2 was involved in ROS-induced myofibroblast accumulation in UUO rats [34]. As a member of the MAPK family, p38 MAPK is activated in response to a variety of stress stimuli, thus contributing to inflammation and cell differentiation in several pathological conditions, including nephropathy. For instance, inhibition of p38 MAPK activity markedly decreases the secretion of IL-1β, tumor necrosis factor-α and MCP-1, and therefore ameliorates renal injury in mice [40]. Specifically, Src has been reported to enhance p38 MAPK activity [41]. In addition, activation of the AKT pathway has also been implicated in renal fibrogenesis in the UUO model. Previous work showed inhibition of AKT decreased the expression of α-SMA and the accumulation of ECM in UUO mice [16]. TGF-β1 plays a critical role in renal fibrosis. Src-induced activation of AKT has been suggested to be associated with TGF-β1 receptor expression in UUO. In the present study, our results clearly showed that blockade of Src with pNaKtide markedly inhibited the activation of ERK1/2, p38 MAPK and AKT, in line with the attenuation of myofibroblast accumulation, inflammation and TGF-β1 expression induced by UUO. Collectively, these findings strongly indicate that these pathways are closely involved in renal fibrogenesis as the downstream effectors of Src. In summary, we demonstrated in this report that pNaKtide effectively attenuates UUO-induced renal fibrogenesis. The anti-fibrotic effect of pNaKtide involves suppression of Src activation and its downstream ERK1/2, p38 MAPK and AKT signaling pathways. Thus, targeting Na/K-ATPase-mediated signaling transduction could represent a novel therapeutic approach for the prevention and treatment of renal fibrosis. FUNDING This work was supported by grants from the National Natural Science Foundation of China (81270796 and 81770671). AUTHORS’ CONTRIBUTIONS C.X. and S.Y. performed the experiments and analysis of the data. W.Y. designed the experiments, interpreted the data and wrote the manuscript. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. 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Administration of FR167653, a new anti-inflammatory compound, prevents renal ischaemia/reperfusion injury in mice . Nephrol Dial Transplant 2002 ; 17 : 399 – 407 Google Scholar CrossRef Search ADS PubMed 41 Wu H , Shi Y , Deng X et al. Inhibition of c-Src/p38 MAPK pathway ameliorates renal tubular epithelial cells apoptosis in db/dbmice . Mol Cell Endocrinol 2015 ; 417 : 27 – 35 Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

pNaKtide ameliorates renal interstitial fibrosis through inhibition of sodium-potassium adenosine triphosphatase-mediated signaling pathways in unilateral ureteral obstruction mice

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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0931-0509
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Abstract

Abstract Background Sodium-potassium adenosine triphosphatase (Na/K-ATPase) has been shown to regulate Src activity by combining with Src to keep it in an inactive form. We previously reported that Na/K-ATPase was downregulated in unilateral ureteral obstruction (UUO) animals. In this study, we examined whether inhibition of Na/K-ATPase-mediated Src signaling pathways ameliorated renal interstitial fibrosis induced by UUO. Methods UUO was performed on male C57BL/6J mice. pNaKtide, a mimic of Na/K-ATPase, was administered by intraperitoneal injection on Day 0 and Day 4 after ureteral ligation. Markers of interstitial fibrosis, inflammation and oxidative stress and transforming growth factor-β1 (TGF-β1) expression were examined after the mice were sacrificed on Day 7. Activation of Src and its downstream signaling effectors, including extracellular regulated protein kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (AKT), were evaluated. Results pNaKtide administration markedly attenuated myofibroblast accumulation and extracellular matrix deposition in obstructed kidneys. Also, pNaKtide significantly reduced the increased expression of 8-iso-prostaglandin F2α, TGF-β1, interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), as well as reduced macrophage infiltration, in UUO animals. All these changes were obtained, along with inhibition of Src and its downstream effector activity. Conclusions Na/K-ATPase-mediated signaling pathways contribute to fibrogenesis and could represent a potential target in the treatment of renal fibrosis. fibrosis, Na/K-ATPase, pNaKtide, Src, UUO INTRODUCTION Interstitial fibrosis is the common hallmark of pathological change underlying chronic kidney disease (CKD) induced by various nephropathies [1]. Numerous cytokines and growth factors are involved in the fibrosis process by activating multiple intracellular signaling pathways [2]. These complex signaling pathways share points of cross-talk and amalgamation. Targeting these points could provide greater benefits by blocking upstream signaling cascades, rather than blocking a single downstream pathway, in the treatment of interstitial fibrosis. Src is a member of the Src tyrosine kinase family and is capable of activating intracellular signaling cascades upon its activation [3]. Accumulating data have revealed that Src activation is involved in the development of chronic fibrotic lesions, including renal interstitial fibrosis [4]. Sodium-potassium adenosine triphosphatase (Na/K-ATPase) is a member of the P-type ATPase family, which localizes to the basolateral membrane of renal tubular epithelial cells and plays an important role in sodium reabsorption. In recent years, it has been shown that Na/K-ATPase also serves as a signaling regulator by combining with other signaling molecules to form a signaling complex [5]. Notably, the α1 subunit of Na/K-ATPase combines with Src and regulates Src activation in two ways. First, the Na/K-ATPase/Src complex functions as a receptor for cardiotonic steroids. When combined with the ligand, the Na/K-ATPase changes its conformation from E1 to E2, which results in Src activation. Fedorova et al. found that marinobufagenin induced transdifferentiation of renal tubular epithelial cells through the Na/K-ATPase/Src complex [6]. In our previous study, we also demonstrated that Na/K-ATPase mediated reactive oxygen species (ROS)-induced extracellular regulated protein kinase 1/2 (ERK1/2) activation by activating Src in LLC-PK1 cells [7]. Second, changes in Na/K-ATPase expression alter the interaction equilibrium between Na/K-ATPase and Src, resulting in Src activation, since the interactions maintain Src in an inactive state. In our previous work, we found that the expression of Na/K-ATPase was markedly decreased in CKD patients and unilateral ureteral obstruction (UUO) models [8, 9]. Extending these findings, we reasoned that Na/K-ATPase could be implicated in fibrogenesis, as a potential regulator of the Src signaling pathway in CKD. pNaKtide is a polypeptide, designed and constructed according to the sequence from the nucleotide-binding (N) domain of the α1 subunit of Na/K-ATPase. It binds with the kinase domain of Src and specifically inhibits Src activation without affecting the pumping function of Na/K-ATPase [10, 11]. pNaKtide readily crosses the cell membrane and resides in intracellular membrane compartments. It has been shown to attenuate the development of adiposity [12] and uremic cardiomyopathy [13] in vivo by inhibiting Na/K-ATPase-modulated oxidation amplification, which involves Src activation. Given the potential importance of the Src-regulated signaling cascade in the development of renal fibrosis, we hypothesized that pNaKtide could attenuate renal fibrosis through inhibition of Na/K-ATPase-modulated Src signaling cascade activation. MATERIALS AND METHODS Experimental animals This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Experimentation Ethics Committee of Peking University First Hospital. Male C57BL/6J mice (23 ± 3 g) were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China), and housed at 25°C in 40% humidity and 12/12-h light/dark cycle. Mice were allowed free access to water and standard chow until the night before surgery. Animals were randomly divided into the following groups: sham surgery, pNaKtide alone, UUO alone or UUO with pNaKtide treatment (UUO + pNaKtide). An extra group was set as UUO with PP2 (UUO + PP2). pNaKtide (25 mg/kg) was administered by intraperitoneal injection on Day 0 and Day 4 after surgery. PP2 (2 mg/kg/day) was given daily by intraperitoneal injection after surgery. All surgeries were performed under anesthesia with sodium pentobarbital, and all efforts were made to minimize suffering. The left ureter was exposed using a mid-abdominal incision and ligated with 3-0 silk sutures in the UUO groups. Sham surgery involved mobilization, but not ligation, of the ureter. All animals were sacrificed on Day 7 after surgery. The left kidneys were decapsulated, washed with ice-cold normal saline and then rapidly dissected. Coronal sections of 2- to 3-mm thickness through the mid-portion of the kidney were embedded in paraffin after fixation in 10% neutral buffered formalin, and the remaining kidney tissue was snap-frozen in liquid nitrogen for further study. Reagents and antibodies The following antibodies were used for immunoblotting and immunohistochemical staining: monoclonal anti-Na/K-ATPase, α1 (clone C464.6) (Upstate, Billerica, MA, USA); monoclonal anti-α1, α6f (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA); monoclonal anti-pERK, and polyclonal anti-ERK and anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-collagen I (COL-1), polyclonal anti-CD68 and polyclonal anti-fibronectin (FN) (Abcam, Cambridge, MA, USA); monoclonal anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich Chemicals, St Louis, MO, USA); monoclonal anti-cSrc, p-p38 mitogen-activated protein kinase (MAPK) and p38 MAPK, pAKT and AKT and polyclonal anti-pSrc (Cell Signaling Technology, Danvers, MA, USA). The enhanced chemiluminescence (ECL) kit was purchased from PerkinElmer (Rockford, IL, USA). PP2 was purchased from Merck Millipore (Darmstadt, Germany). pNaKtide was obtained from HD Bioscience (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kit for matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1) was purchased from Elabscience Biotechnology (Wuhan, China). The interleukin-6 (IL-6) ELISA assay kit was purchased from Abcam (Cambridge, MA, USA). The assay kit for 8-iso-prostaglandin F2α (8-iso-PGF2α) was purchased from Cayman (Ann Arbor, MI, USA). All chemicals used in the experiments, unless otherwise stated, were purchased from Sigma-Aldrich Chemicals. Immunocytochemistry staining Following formalin fixation and paraffin embedding, 4-μm sections were deparaffinized in xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was suppressed by exposing slide-mounted tissue to 0.3% hydrogen peroxide after antigen retrieval by microwave heating. Sections were then incubated with the indicated primary antibody overnight at 4°C. Control sections were incubated without the primary antibody. After washing in phosphate-buffered saline, slides were incubated with biotinylated goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG antibody, then peroxidase conjugated to streptavidin (Zymed Laboratories, San Francisco, CA, USA). Sections were stained with diaminobenzidine tetrahydrochloride substrate and counterstained with hematoxylin. The number of interstitial CD68+ cells was counted in 10 consecutive fields (200×) and expressed as cells per high-power field. Immunoblot Levels of Na/K-ATPase, α-SMA, FN, phosphor- and total-Src, ERK1/2, p38 MAPK and AKT in tissue homogenates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then electrophoretically transferred to nitrocellulose membranes. Immunoblotting was performed as previously described [9]. Briefly, membranes were blocked with 5% skimmed milk in tris-buffered saline–Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the indicated antibodies at 4°C overnight. Blots were incubated in TBS-T 30 min before incubation with HRP-conjugated anti-rabbit or anti-mouse IgG for 1 h at room temperature. Staining was detected using the chemiluminescence ECL kit, and developed with Image Quant LAS 4000mini (GE, Boston, MA, USA). The membrane was then stripped and probed with mouse anti-GAPDH antibody (Sigma-Aldrich Chemicals) to confirm and estimate loading and transfer, except for phosphor-Src, ERK, p38 MAPK and AKT, which were probed with an antibody to the indicated total kinase for adjustment, respectively. The bands were quantified using ImageJ software (an open source Java image processing program inspired by NIH Image). ELISA assay MMP-9, TIMP-1, MCP-1, IL-6, TGF-β1 and 8-iso-PGF2α levels were determined in renal tissue homogenates according to the ELISA kit manufacturer’s instructions. The results were adjusted according to the protein concentration of the renal tissue homogenates. Statistical analysis Data were expressed as mean ± standard error (SE). SPSS software 13.0 (Chicago, IL, USA) was used for all analyses. Groups were compared using Student’s t-test, and one-way analysis of variance when more than two groups were compared. P-values of <0.05 were considered significant. Each presented immunoblot is representative of the results of at least three separate experiments. RESULTS Effect of pNaKtide on Na/K-ATPase expression in UUO mice The expression levels of α1 Na/K-ATPase were first examined by immunocytochemical staining. α1 Na/K-ATPase was quantitatively expressed on the basolateral plasma membrane of tubules in mice that received sham surgery with or without pNaKtide treatment (Figure 1A and B), a mimic of Na/K-ATPase. By contrast, the α1 Na/K-ATPase level was substantially reduced in tubules of obstructed kidneys 7 days after surgery (Figure 1C), which was not substantially rescued by the administration of pNaKtide (Figure 1D). To further validate this phenomenon, the α1 Na/K-ATPase content of whole kidney homogenates was assessed by immunoblotting. Administration of pNaKtide did not enhance the renal levels of α1 Na/K-ATPase in UUO mice (Figure 1E). These results suggested that pNaKtide had no direct effect on renal Na/K-ATPase expression. FIGURE 1 View largeDownload slide Expression of Na/K-ATPase in UUO mice with pNaKtide treatment. UUO was induced in C57BL/6J mice. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of Na/K-ATPase expression in kidney homogenates. ***P < 0.001 versus sham alone. FIGURE 1 View largeDownload slide Expression of Na/K-ATPase in UUO mice with pNaKtide treatment. UUO was induced in C57BL/6J mice. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of Na/K-ATPase expression in kidney homogenates. ***P < 0.001 versus sham alone. pNaKtide attenuates renal interstitial myofibroblast accumulation and the development of renal fibrosis in the obstructed kidney Renal fibrosis induced by UUO is characterized by activation of myofibroblasts and accumulation of excessive amount of extracellular matrix (ECM) proteins. As shown by sirius red staining (Figure 2A–D), there was significant deposition of collagens in UUO mice on Day 7, which was markedly reduced by pNaKtide treatment. To confirm this finding, the deposition of collagen type I, a key ECM component, was further examined by immunocytochemistry. The result was in agreement with that obtained using sirius red staining (Figure 2E–H). In addition, the expression of FN was examined by immunoblot analysis. As shown in Figure 2I, pNaKtide treatment significantly inhibited the increased expression of FN in UUO mice on Day 7. FIGURE 2 View largeDownload slide Effect of pNaKtide on UUO-induced renal fibrosis. (A–D) Representative images of sirius red renal histology; (E–H) representative images of collagen type I expression; (I) representative western blot and data analysis of FN expression in kidney homogenates (n = 6–8 mice per group). ***P <0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 2 View largeDownload slide Effect of pNaKtide on UUO-induced renal fibrosis. (A–D) Representative images of sirius red renal histology; (E–H) representative images of collagen type I expression; (I) representative western blot and data analysis of FN expression in kidney homogenates (n = 6–8 mice per group). ***P <0.001 versus sham alone; ###P < 0.001 versus UUO alone. α-SMA is a marker of myofibroblasts, the major contributor to ECM production. We examined the expression of α-SMA both by immunocytochemistry staining (Figure 3A–D) and by immunoblot analysis (Figure 3E). There was increased expression of α-SMA in the obstructed kidney, indicating an accumulation of myofibroblasts. pNaKtide treatment significantly inhibited α-SMA expression in UUO mice. Taken together, these data supported that activation of Na/K-ATPase-mediated signaling contributed to accumulation of renal fibroblasts and the development of renal fibrosis induced by ureteral obstruction. FIGURE 3 View largeDownload slide Effect of pNaKtide on myofibroblast accumulation in UUO mice. Representative images of α-SMA immunohistochemical staining shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of α-SMA expression in kidney homogenates (n = 6–8 mice per group). ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 3 View largeDownload slide Effect of pNaKtide on myofibroblast accumulation in UUO mice. Representative images of α-SMA immunohistochemical staining shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) representative western blot and data analysis of α-SMA expression in kidney homogenates (n = 6–8 mice per group). ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Effect of pNaKtide on the degradation of ECM in the obstructed kidney The amount of ECM is generally kept stable through a balance between its production and degradation. MMP-9 and TIMP-1 are major players in the ECM degradation system. We examined the effect of pNaKtide treatment on MMP-9 and TIMP-1. As shown in Table 1, MMP-9 and TIMP-1 expression levels significantly increased in UUO mice, as previously reported. However, pNaKtide treatment showed no significant inhibitory effect on either MMP-9 or TIMP-1 expression. Taken together, these data supported that Na/K-ATPase-mediated signaling contributes to renal fibrogenesis in UUO by inducing accumulation of ECM without affecting its degradation. Table 1 Expression of MMP-9 and TIMP-1 Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** ** P < 0.01 and ***P < 0.001 versus sham. Table 1 Expression of MMP-9 and TIMP-1 Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** Sham Sham + pNaKtide UUO UUO + pNaKtide MMP-9 (pg/mg) 6913 ± 342.4 6529 ± 240.8 21 110 ± 2661*** 18 332 ± 2421** TIMP-1 (ng/mg) 15.16 ± 0.81 13.61 ± 0.56 38.66 ± 2.44*** 32.83 ± 2.56*** ** P < 0.01 and ***P < 0.001 versus sham. pNaKtide contributes to decreased expression of TGF-β1 in UUO mice TGF-β1 is the predominant cytokine stimulating the differentiation of renal fibroblasts and inducing renal fibrosis. Studies by Yan et al. showed that activation of Src played an important role in mediating TGF-β1 signaling in UUO injury [4]. We examined whether pNaKtide affected TGF-β1 expression in UUO mice. The expression of TGF-β1 significantly increased in the obstructed kidney in UUO mice, compared with the sham group (1928 ± 132.9 versus 1087 ± 76.93 pg/mg, P < 0.001), which was partly, but significantly, decreased by pNaKtide treatment (1521 ± 58.1 versus 1928 ± 132.9 pg/mg, P < 0.01). These data suggest that Na/K-ATPase-mediated signaling contributed to renal fibrosis in UUO, at least partly, through modulating TGF-β1 expression. pNaKtide attenuates inflammation in the obstructed kidney Inflammation is also a characteristic feature of UUO that contributes to the development of renal fibrosis. Increased secretion of inflammatory factors and chemokines induces the subsequent infiltration of macrophages and lymphocytes into the obstructed kidneys. To determine whether Na/K-ATPase-mediated signaling contributed to renal fibrosis through inducing inflammation in the kidney after UUO injury, we examined the effect of pNaKtide on the expression of MCP-1 and IL-6. Both MCP-1 and IL-6 levels were significantly elevated in UUO mice, and were partially reduced by pNaKtide treatment (Table 2). Accordingly, we also found less infiltration of macrophages in pNaKtide-treated UUO mice on immunocytochemistry staining analysis (Figure 4). These results demonstrated pNaKtide alleviated inflammation in UUO mice. Table 2 Expression of MCP-1 and IL-6 Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# ** P < 0.01 and ***P < 0.001 versus sham; #P < 0.05 and ##P < 0.01 versus UUO. Table 2 Expression of MCP-1 and IL-6 Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# Sham Sham + pNaKtide UUO UUO + pNaKtide MCP-1 (pg/mg) 154.8 ± 6.27 145.0 ± 10.75 522.2 ± 18.29*** 403.0 ± 31.6***,## IL-6 (pg/mg) 736.8 ± 61.08 756.4 ± 55.01 1933.0 ± 119.2*** 1409.9 ± 102.6**,# ** P < 0.01 and ***P < 0.001 versus sham; #P < 0.05 and ##P < 0.01 versus UUO. FIGURE 4 View largeDownload slide pNaKtide reduced UUO-induced infiltration of macrophages. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Macrophage infiltration was analyzed by CD68 staining. Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) semi-quantitative analysis of immunohistochemical staining of CD68. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 4 View largeDownload slide pNaKtide reduced UUO-induced infiltration of macrophages. pNaKtide (25 mg/kg) was given by intraperitoneal injection on Day 1 and Day 4 after ureteral ligation (n = 6–8 mice for each group). Macrophage infiltration was analyzed by CD68 staining. Representative images of immunohistochemical staining are shown in (A–D). (A) Sham; (B) sham with pNaKtide; (C) UUO mice; (D) UUO with pNaKtide; (E) semi-quantitative analysis of immunohistochemical staining of CD68. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Inhibitory effects of pNaKtide on oxidative stress in UUO mice 8-iso-PGF2α is an interaction product between O2− and esterified or free arachidonate. It is a well-established marker used to reflect oxidative stress. In the present study, there was a significantly increased level of 8-iso-PGF2α in UUO mice, which was markedly suppressed by pNaKtide treatment (Figure 5). This result indicated pNaKtide alleviated oxidative stress in UUO mice. FIGURE 5 View largeDownload slide Analysis of renal 8-iso-PGF2α content. The level of 8-iso-PGF2α in renal tissue homogenates was assessed and adjusted according to the total protein concentration in the renal tissue homogenates. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 5 View largeDownload slide Analysis of renal 8-iso-PGF2α content. The level of 8-iso-PGF2α in renal tissue homogenates was assessed and adjusted according to the total protein concentration in the renal tissue homogenates. Data are expressed as mean ± SEM. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. pNaKtide blocks Src-regulated signaling cascade activation in the obstructed kidney A previous study showed that activation of Src plays an important role in UUO injury. We showed, in a series of previous studies, that Na/K-ATPase acted as a regulator of Src activation. In this present study, PP2, the direct inhibitor of Src activation, was used as control to evaluate the effect of pNaKtide on Src and its regulated downstream effectors. With significant downregulation of α1 Na/K-ATPase expression, we found the level of phosphorylated Src, the active form of Src, was markedly elevated in the obstructed kidney collected on Day 7, indicating that Src was constitutively activated in UUO mice (Figure 6). The total Src level was not altered in UUO mice. Administration of pNaKtide significantly reduced Src activation without affecting the expression level of Src in the obstructed kidney (Figure 6). FIGURE 6 View largeDownload slide Effect of pNaKtide and PP2 on UUO-induced Src activation. Representative western blot and data analysis of Src activation in kidney homogenates (n = 6–8 mice per group). Src activation was expressed as pY418 Src/total Src (p-Src/t-Src) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 6 View largeDownload slide Effect of pNaKtide and PP2 on UUO-induced Src activation. Representative western blot and data analysis of Src activation in kidney homogenates (n = 6–8 mice per group). Src activation was expressed as pY418 Src/total Src (p-Src/t-Src) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. Src is known to activate several intracellular signal transduction pathways upon its activation. We investigated the effect of pNaKtide on Src-regulated downstream effectors in UUO mice. As members of the MAPK family, ERK1/2 and p38 MAPK have been reported, in several studies, to be activated by Src activation, contributing to inflammation and cell differentiation [14, 15]. We found increased activation of both p38 MAPK and ERK1/2 in UUO mice, which was partially, but significantly, inhibited by pNaKtide treatment (Figure 7A and B). Previous work also showed that inhibition of AKT decreased the expression of α-SMA and accumulation of ECM in UUO mice [16]. To determine whether Src activation transduced signals to the AKT pathway in the kidney after UUO injury, we examined the effect of pNaKtide on AKT phosphorylation in the present study. As shown in Figure 7C, the phosphorylation level of AKT was increased in UUO-injured kidneys, and pNaKtide administration resulted in a significant reduction of AKT phosphorylation, suggesting activation of the AKT signaling pathway is under Src regulation in kidneys undergoing fibrosis. Notably, activation of all these downstream effectors was inhibited, to a comparable degree, by PP2 administration. Taken together, these data indicated that pNaKtide inhibited UUO-induced activation of Src and its downstream signaling cascades in the development of renal fibrosis. FIGURE 7 View largeDownload slide Effect of pNaKtide and PP2 on Src-mediated signaling cascade activation. Representative western blot and analysis of kinase activation in renal tissue homogenates (n = 6–8 mice per group). (A) ERK1/2 activation expressed as phosphor-ERK/total ERK (p-ERK/t-ERK) ratio. (B) p38 MAPK activation expressed as phosphor-p38 MAPK/total p38 MAPK (p-38 MAPK/t-p38 MAPK) ratio. (C) AKT activation expressed as phosphor-AKT/total AKT (p-AKT/t-AKT) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. FIGURE 7 View largeDownload slide Effect of pNaKtide and PP2 on Src-mediated signaling cascade activation. Representative western blot and analysis of kinase activation in renal tissue homogenates (n = 6–8 mice per group). (A) ERK1/2 activation expressed as phosphor-ERK/total ERK (p-ERK/t-ERK) ratio. (B) p38 MAPK activation expressed as phosphor-p38 MAPK/total p38 MAPK (p-38 MAPK/t-p38 MAPK) ratio. (C) AKT activation expressed as phosphor-AKT/total AKT (p-AKT/t-AKT) ratio. ***P < 0.001 versus sham alone; ###P < 0.001 versus UUO alone. DISCUSSION CKD is characterized by an irreversible deterioration of renal function, with renal fibrosis as the final common pathologic change. There is a great need to identify treatment targets for slowing CKD development. In the present study, we demonstrated that pNaKtide, an analog of Na/K-ATPase, suppressed Src and downstream p38 MAPK, ERK1/2 and AKT activation in UUO kidneys. We showed that pNaKtide administration also attenuated myofibroblast accumulation and ECM deposition, renal inflammation, oxidative stress and TGF-β1 production in the injured kidney. Collectively, these data suggested that Na/K-ATPase acts as a key mediator of Src signaling transduction in the process of renal fibrogenesis. Na/K-ATPase is heavily expressed in tubular cells in normal kidneys. In addition to its function as a pump, Na/K-ATPase has been found to have scaffolding and signaling function, in numerous studies on the Na/K-ATPase/Src complex. This signaling pathway has been shown to be involved in cardiovascular diseases, hypertension, salt balance and renal diseases [17–23]. We found a significant decrease in Na/K-ATPase expression in injured kidneys from our previous studies [8, 9]. In addition, Src has been shown to play important roles in renal fibrosis in UUO. On this background, we sought to examine whether blockade of Na/K-ATPase-mediated signaling transduction by pNaKtide could effectively ameliorate fibrosis in UUO animals. As an analog of Na/K-ATPase, pNaKtide binds to the kinase domain of Src to specifically inhibit Src activation without affecting the pumping function of the Na/K-ATPase. Our data clearly showed that pNaKtide administration significantly ameliorated interstitial fibrosis induced by UUO. The mechanism of pNaKtide effect on renal fibrogenesis may be associated with its various cellular functions. First, exposure to pNaKtide resulted in lower accumulation of myofibroblasts in UUO mice. Myofibroblasts are a major contributor to renal fibrosis. A decrease in myofibroblast accumulation resulted in reduced production and deposition of ECM in UUO kidneys. Second, inflammation is an important feature of UUO. Patients with CKD are exposed to persistent low-grade inflammation, which supposedly acts as a pre-conditioning event in the development of renal fibrosis, as observed, for example, in diabetic nephropathy and cardiovascular comorbidities in CKD [24–26]. For instance, hyperhomocysteinemia is a well-known cardiovascular risk factor in CKD patients. It was found that homocysteinylated albumin could upregulate MCP-1, inducing a proinflammatory state by promoting monocyte adhesion onto endothelial cells, which is directly related to the pathogenesis of atherosclerosis [27]. Inflammation also plays a detrimental role in the development of renal fibrosis in UUO [28]. Infiltration of macrophages and monocytes contribute to the inflammatory response in obstructive kidney disease [29, 30]. To understand the role of Na/K-ATPase-mediated signaling in the induction of inflammation in UUO, we examined the expression of the inflammatory cytokines MCP-1 and IL-6. Increased expression of both MCP-1 and IL-6 in UUO mice was significantly reduced by administration of pNaKtide, along with less macrophage infiltration, indicating that pNaKtide exerted anti-fibrotic effects, at least partly, through anti-inflammation. Furthermore, it has been suggested that oxidative stress is associated with CKD progression and comorbidity. For instance, hydrogen sulfide (H2S), the third gasotransmitter, possesses beneficial anti-oxidative effects [31]. Low plasma H2S levels have been detected in uremia patients, contributing to the development of hyperhomocysteinemia, in turn leading to increased cardiovascular risk [32]. In addition, Song et al. demonstrated that H2S exhibited anti-fibrotic effects in obstructed nephropathy and inhibited the proliferation and differentiation of renal fibroblasts both in vitro and in vivo [33]. We also found, in our previous work, that renal fibrosis was alleviated with the suppression of nicotinamide adenine dinucleotide phosphate oxidase (NOXs) activity and subsequent oxidative stress [34], suggesting oxidative stress is an important contributor in the pathogenesis of renal fibrosis in UUO. There is evidence suggesting that Na/K-ATPase-mediated signaling is involved in oxidation amplification [12, 13]. Hence, in the present study, we examined the oxidative stress marker 8-iso-PGF2α to understand the role of Na/K-ATPase-mediated signaling in the induction of oxidative stress in UUO. As shown in Figure 5, the UUO-induced increased level of 8-iso-PGF2α was significantly reduced by pNaKtide treatment, suggesting pNaKtide alleviates oxidative stress in UUO mice. The exact mechanism of Na/K-ATPase-mediated signaling in oxidative stress induction in UUO, such as whether it involves impaired H2S production converging into the same signaling pathway, needs further study. Finally, it has been well documented that TGF-β1 is central to fibroblast activation and fibrogenesis in UUO [35]. Src has been suggested to upregulate TGF-β1 signaling through overproduction of TGF-β1 [36]. We found pNaKtide treatment significantly reduced TGF-β1 overexpression in UUO mice. Taken together, these data clearly demonstrated that blockade of Na/K-ATPase-mediated signaling by pNaKtide alleviates renal fibrosis through multiple mechanisms, as schematically summarized in Figure 8. FIGURE 8 View largeDownload slide Schematic illustration of the mechanisms of Na/K-ATPase-regulated signaling pathways in the mediation of renal fibrosis in UUO. FIGURE 8 View largeDownload slide Schematic illustration of the mechanisms of Na/K-ATPase-regulated signaling pathways in the mediation of renal fibrosis in UUO. We found that these anti-fibrotic changes with pNaKtide treatment in injured kidneys were associated with inhibition of Src activation and its downstream effectors. Src can be activated by multiple profibrotic growth factors/cytokines and transduces signals initiated from diverse membrane receptors. Evidence indicates that Src activation is involved in renal lesions in animal models of, for example, diabetic nephropathy, renal cyst formation and proliferative glomerulonephritis [37–39]. Specifically, Yan et al. provided strong evidence for Src involvement in renal fibroblast activation and renal fibrosis in their study [4]. Thus, it is possible Src represents a converging point for integrating diverse profibrotic signals in the process of renal fibrosis. We have previously demonstrated that Na/K-ATPase serves as a regulator of Src by binding to Src to form a signaling complex, and thus keeping Src in an inactive form. In the present study, pNaKtide administration significantly inhibited Src activation induced by UUO to a comparable degree as that of PP2 administration, indicating that the anti-fibrotic effect of pNaKtide could be attributed to its effects on Na/K-ATPase-mediated Src signaling transduction. As a non-receptor tyrosine kinase, Src activates several downstream intracellular signaling cascades upon its activation, such as serine/threonine protein kinase ERK1/2 and p38 MAPK, EGFR and protein kinase B/phosphatidylinositol 3 kinase (AKT/PI3K). ERK1/2 plays an important role in renal cell proliferation and differentiation in nephropathy. In our previous study, we found ERK1/2 was involved in ROS-induced myofibroblast accumulation in UUO rats [34]. As a member of the MAPK family, p38 MAPK is activated in response to a variety of stress stimuli, thus contributing to inflammation and cell differentiation in several pathological conditions, including nephropathy. For instance, inhibition of p38 MAPK activity markedly decreases the secretion of IL-1β, tumor necrosis factor-α and MCP-1, and therefore ameliorates renal injury in mice [40]. Specifically, Src has been reported to enhance p38 MAPK activity [41]. In addition, activation of the AKT pathway has also been implicated in renal fibrogenesis in the UUO model. Previous work showed inhibition of AKT decreased the expression of α-SMA and the accumulation of ECM in UUO mice [16]. TGF-β1 plays a critical role in renal fibrosis. Src-induced activation of AKT has been suggested to be associated with TGF-β1 receptor expression in UUO. In the present study, our results clearly showed that blockade of Src with pNaKtide markedly inhibited the activation of ERK1/2, p38 MAPK and AKT, in line with the attenuation of myofibroblast accumulation, inflammation and TGF-β1 expression induced by UUO. Collectively, these findings strongly indicate that these pathways are closely involved in renal fibrogenesis as the downstream effectors of Src. In summary, we demonstrated in this report that pNaKtide effectively attenuates UUO-induced renal fibrogenesis. The anti-fibrotic effect of pNaKtide involves suppression of Src activation and its downstream ERK1/2, p38 MAPK and AKT signaling pathways. Thus, targeting Na/K-ATPase-mediated signaling transduction could represent a novel therapeutic approach for the prevention and treatment of renal fibrosis. FUNDING This work was supported by grants from the National Natural Science Foundation of China (81270796 and 81770671). AUTHORS’ CONTRIBUTIONS C.X. and S.Y. performed the experiments and analysis of the data. W.Y. designed the experiments, interpreted the data and wrote the manuscript. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. 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Journal

Nephrology Dialysis TransplantationOxford University Press

Published: May 17, 2018

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