| BackgroundPrevention and treatment of Chronic kidney disease (CKD) have become a global health problem, and the morbidity of CKD in recent years has significantly increased. Progressive CKD often causes renal tissue scarring that contributes to irreversible renal dysfunction and end-stage renal failure following dialysis and renal replacement. However, there are still no current satisfactory treatments that can effectively arrest the progressive loss of kidney functions.Renal interstitial fibrosis (RIF) is the final common pathway for all CKD leading to end-stage renal disease (ESRD). Many factors play an important role in the development and progression of RIF, including increased expression of pro-fibrotic cytokine, myofibroblast proliferation and extracellular matrix (ECM) excessive deposition, of which the proliferation of myofibroblast play a vital role in the pathogenesis of RIF. Under pathological conditions, a large number of myofibroblasts cell production can promote the excessive deposition of ECM in renal interstitial. Tubular epithelial cells-mesenchymal transdifferentiation (EMT), characterized by loss of epithelial proteins (E-cadherin, zonula occludens-1, etc.) and acquisition.of new mesenchymal markers (a-smooth muscle actin (α-SMA), vimentin, etc.), is regarded as a pivotal pathway in matrix-producing myofibroblast generation.Of the numerous growth factors involved in the pathophysiology of renal fibrosis, transforming growth factor β1 (TGF-β1) is considered the crucial mediator of initiation arid progression of interstitial fibrosis. TGF-β1 signal is transduced via its cell membrane type I and type II serine/threonine kinase receptors. Receptor activation induces the phosphorylation and activation of its downstream signaling molecules, Smad2 and Smad3. Phosphorylated-Smad2/3 binds to common partner Smad4, and subsequently translocates into the nuclei, where they regulate the transcription of genes response to TGF-β1. TGF-β1 signaling plays a pivotal role in the process of renal tubulointerstitial fibrosis, driving EMT progression and excess deposition of ECM.Inflammation including infiltration of inflammatory cells, predominantly are macrophages, and the expression of pro-inflammatory cytokines such as TNF-a, IL-1β, is considered the initial factor of RIF. In addition, NF-kB activation is a common pathway of many pro-fibrotic cytokines contribute to RIF. During the process of RIF, NF-κB activation can further induce a variety of pro-inflammatory, pro-fibrotic cytokine expression and extracellular matrix proteins.Unilateral ureteral obstruction (UUO) is an acute mechanical injury, it can induced the obstructed kidney injury in a short time. It is characterized by tubular cell injury, interstitial inflammation and fibrosis, and has been recognized as a classic animal model in studying RIF.Rutin is a natural flavonoids found in many foods and traditional Chinese medicine. It has a wide range of pharmacological properties, including antioxidant, anti-inflammatory, anti-allergic, anti-cancer, anti-bacterial, anti-viral, cytoprotective effects. In recent years, many studies have shown that rutin can ameliorate liver or cardie fibrosis in animal experiments, In addition, several studies also demonstrated that rutin has significant beneficial kidney protective effects in ischemia/reperfusion renal injury, drug-induced renal injury, and diabetic nephropathy. To date, many studies suggest that the mechanism of fibrosis is similar in many organs, but effects of rutin in rat kidney interstitial fibrosis induced by UUO and its mechanism is not clear. Therefore, the present study was conducted to examine the protective effects of rutin in unilateral ureteral obstruction (UUO)-inflicted rats and to identify possible=underlying mechanisms.Objectives:To observe the influence of rutin on histopathology and expression of relevant profibrotic factors in UUO rats, and to explore protective benifits and underlying mechanism of rutin on renal interstitial fibrosis.Method:1. Establishment of UUO model and Grouping24 adult male Wistar rats (SPF grade), weighing 200-250g. After 1 week of acclimatization, the rats were randomly divided into 4 groups:sham operated group (Sham+Vehicle), sham operated group with rutin treatment (Sham+Rutin), UUO group (UUO+Vechicle), UUO group with rutin treatment (UUO+Rutin). After intraperitoneal anesthesia with pentobarbital sodium (50 mg/kg), the abdominal cavity was exposed via a midline incision. The left proximal ureter was ligated at two locations with 4-0 silk sutures and cut between the knots. Rats subjected to sham surgery underwent the same procedure, but without ureter ligation. After recovering from general anesthesia, each rat received oral gavage of rutin (100 mg/kg) dissolved in 1% carboxymethycellulose-Na (CMC-Na) buffer daily for 13 days. CMC-Na buffer (1%) was used as a vehicle. Two weeks after surgery, the rats were euthanized and the left kidneys were harvested. One part of the left kidney was fixed in 4% paraformaldehyde for histological examination and the remaining tissue was rapidly frozen in liquid nitrogen and stored at -80℃ for protein extraction.2. Histopathological ExaminationKidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm. Deparaffinized sections of kidney tissues were stained with hematoxylin and eosin (H&E) and Masson’s trichrome according to routine methods for histopathological examination. Ten randomly selected fields were observed and analyzed at 200× magnification in each section.3. Immunohistochemical stainingDAB immunohistochemistry staining was processed employing 2-step plus(?) System (PV-9000, ZSGB) for renal immunohistochemical assay. Sections obtained from paraffin-embedded tissue were dewaxed and washed. After antigen retrieval with microwave irradiation in citrate buffer for 20 min, the sections were incubated in a solution of 3% H2O2 for 15 min to inhibit endogenous peroxidase activity. Sections were incubated with primary antibodies (overnight at 4℃), reagent 1 (37 ℃, 20 min) and reagent 2 (37℃,20-30 min). After washing with PBS, sections were incubated with diaminobenzidine.10 randomly selected fields in each section were observed at 200 × magnification and semiquantitatively calculated using Image-Pro Plus, version 6.0.4. Western BlotProtein samples (50 μg) were separated by 5%-10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes, which were blocked for 1 h with 1X Tris-buffered saline buffer containing 5% skim milk and individually incubated overnight at 4℃ with primary antibodies against proteins of interest, including collagen I and III, a-SMA, fibronectin, E-cadherin, p-Smad3, TGF-β1, CD68, TNF-α, IL-1β, p-NF-κB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (each 1:1,000). After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at 1:1000 for 1 h at room temperature. Blots were detected by enhanced chemiluminescence and band density was quantified using ImageJ software (http://imagej.nih.gov/ij/).Statistical analysisStatistical software SPSS (?) 19.0 is used to analysis data. All experimental data were expressed as mean±standeard deviation (x ±s). One-way ANOVA was used in Intergroup comparisons. LSD test was used in multiple comparisons between groups. Differences between 2 groups were determined by t test. P＜0.05 is considered statistically significant.Results(1) Histopathological ExaminationH&E staining showed normal kidney histology in sham group. The UUO group showed severe tubulointerstitial injury, including tubular dilatation, atrophy and vacuolization, interstitial inflammatory cells infiltrates, and interstitial fibrosis. Compared with the sham-operated group, score of tubulointerstitial injury in UUO group was significantly increased (P＜0.001). Compared with UUO group, score of tubulointerstitial injury in UUO rats with rutin treatment was reduced (P＜0.05).Tubulointerstitial fibrosis was assessed by Masson staining. Collagen was significantly deposited in the interstitial space in UUO group, which had a significantly higher collagen deposition area than sham-operated rats (P<0.001). Rutin treatment reduced the development of interstitial fibrosis in UUO rats, and the percentage area of the fibrous area was significantly decreased (P<0.05).(2) Immunohistochemical staining resultsAs compared to sham-operated rats, UUO rats exhibited a dramatic decrease of E-cadherin (P<0.05) and increase of collagen I, collagen III, fibronectin, a-SMA, and CD68 expression in their renal tissue (P＜0.001). Rutin treatment not only inhibited the up-regulation of collagen I, collagen III, fibronectin, a-SMA, and CD68 expression (P<0.05), but also prevented the decrease in E-cadherin expression (P<0.05).(3) Western Blot resultsAs compared to sham-operated rats, UUO rats exhibited a dramatic decrease of E-cadherin (P＜0.05) and increase of collagen I, collagen III, fibronectin, a-SMA, TGF-β1, p-Smad3, CD68, TNF-α、 IL-1β and p-NF-κB expression in their renal tissue (P<0.001). Rutin treatment not only inhibited the increase of c collagen I, collagen III, fibronectin, a-SMA, TGF-β1, p-Smad3, CD68, TNF-α、IL-1β and p-NF-κB (P<0.05), but also prevented the decrease in E-cadherin expression (p＜0.05).Conclusions(1) Rutin can significantly ameliorate renal interstitial injury and fibrosis in UUO rats.(2) Rutin can significantly inhibit EMT and excessive deposition of ECM in UUO rats.(3) Rutin can inhibit inflammation response in UUO rats.(4)The antifibrotic effects of rutin might be correlated well with the inhibition of NF-κB activation and TGF-β1/Smad3 signaling... |
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