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Department of Urology, The First People's Hospital Of Yichang, China Three Gorges University, Yichang, Hubei, ChinaDepartment of Microbiology and Immunology, Medical College, China Three Gorges University, Yichang, Hubei, China
Renal ischemia/reperfusion (I/R)-induced acute kidney injury remains to be a troublesome condition in clinical practice. Although the exact molecular mechanisms underlying renal I/R injury are incompletely understood, the deleterious progress of renal I/R injury involves inflammation, apoptosis, and oxidative stress. Diosmetin is a member of the flavonoid glycosides family, which suppresses the inflammatory response and cellular apoptosis and enhances antioxidant activity. The purpose of this study was to investigate the protective effect of diosmetin on I/R-induced renal injury in mice.
Thirty BALB/c mice were randomly divided into five groups. Four groups of mice received diosmetin (0.25, 0.5, and 1 mg/kg) or vehicle (I/R group) before ischemia. Another group received vehicle without ischemia to serve as a negative control (sham-operated group). Twenty-four hours after reperfusion, serum and renal tissues were harvested to evaluate renal function and histopathologic features. In addition, the expression of inflammation-related proteins, apoptotic molecules, and antioxidant enzymes was analyzed.
Compared with sham mice, the I/R group significantly exacerbated renal function and renal tube architecture and increased the inflammatory response and renal tubule apoptosis. Nevertheless, pretreatment with diosmetin reversed these changes. In addition, diosmetin treatment resulted in a marked increase in antioxidant protein expression compared with I/R mice.
The renoprotective effects of diosmetin involved suppression of the nuclear factor-κB and mitochondrial apoptosis pathways, as well as activation of the nuclear factor erythroid 2–related factor 2/heme oxygenase-1 pathway. Diosmetin has significant potential as a therapeutic intervention to ameliorate renal injury after renal I/R.
It has been reported that activation of p65 stimulates downstream genes (interleukin-1β [IL-1β], IL-6 and tumor necrosis factor-α [TNF-α]), which induces leukocytes infiltration and cytokine secretion to further promote the inflammatory response.
Activation of the NF-κB pathway increased expression of several proinflammatory cytokines, including macrophage inflammatory protein-2, IL-1β, and TNF-α, leading to exacerbation of I/R injuries in rat liver transplants.
Moreover, the central subunit of the NF-κB signaling pathway, p65, could induce expression of apoptosis-related proteins and further exacerbate the progression of renal tubule injury and eventual cell death.
Nuclear factor erythroid 2–related factor 2 (Nrf2), a master transcriptional regulator of antioxidant proteins, is normally deactivated by interacting with the Kelch-like ECH-associated protein 1-Cul3 complex (Keap-1) in the cytoplasm.
After cellular insult, it translocates into the nucleus to promote the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase-1 (NQO-1) and glutathione reductase, which encodes an antioxidant involved in the defense against oxidative stress.
reported that Nrf2 protects against lupus nephritis by inhibiting the activation of the NF-κB pathway. After exposure to cerebral, hepatic, and gastric I/R injury, Nrf2 transactivated downstream gene expression, which in turn suppressed the proinflammatory NF-κB pathway.
Diosmetin (3′,5,7-trihydroxy-4′-methoxyflavone, Fig. 1) is extracted from the traditional Chinese herb Galium verum L. and belongs to the flavonoid family. Flavonoids function to protect blood vessel walls.
However, little is known about its renoprotective effects during renal I/R injury.
Based on the aforementioned evidence, we hypothesized that diosmetin may protect the kidney from I/R-induced injury. The present study assessed the effects of diosmetin on murine kidneys after I/R. Moreover, the underlying mechanisms of renal function recovery and morphology were investigated.
Materials and methods
Chemical and animal
Diosmetin (analytical standard) was purchased from Sigma-Aldrich (Hong Kong, China), dissolved in 0.9% sodium chloride containing 2% dimethyl sulfoxide at a concentration of 1 mg/mL and stored at −20°C. Healthy male BALB/c mice (20-25 g) were purchased from the Model Animal Research Center of China Three Gorges University (Yichang, China) and maintained in a specific pathogen-free animal facility at the Experimental Animal Center of China Three Gorges University. All mice involving this study had free access to food and water and were housed in a temperature-controlled facility with 12-h light/dark cycles.
Renal I/R injury was performed as previously described.
Mice were anesthetized with pentobarbital (50 mg/kg) intraperitoneally, and the left kidney pedicle was clamped with an Atraumatic Schwartz microvessel clamp to induce acute ischemia for 45 min, whereas the right was surgically removed. After the clamp was removed, reperfusion was verified visually by restoration of color. Sham mice underwent a similar surgical procedure without clamping the left kidney pedicle. All mice were sacrificed by cervical dislocation after 24 h of reperfusion. Blood and the left kidney were collected for further analysis. The animal procedures were conducted according to the guidelines of the Institutional Animal Care and Use of China Three Gorges University. The study was approved by the animal ethics committee of Medical College of China Three Gorges University.
All animals (n = 30) used in this study were randomly allocated into five groups of six as follows: (1) sham group, (2) I/R group, (3) low-dose group: I/R + diosmetin (0.25 mg/kg), (4) moderate-dose group: I/R + diosmetin (0.5 mg/kg), and (5) high-dose group: I/R + diosmetin (1 mg/kg). The drug was injected intraperitoneally 45 min before the induction of renal ischemia. Mice in groups 1 and 2 were injected intraperitoneally with 300 μL of saline vehicle. Diosmetin stock solution (1 mg/mL) was diluted with saline vehicle and adjusted to a volume of 300 μL before injection into mice in groups 3-5.
Serum was obtained from blood samples to measure blood urea nitrogen (BUN) and serum creatinine (Scr) by an automatic biochemistry analyzer (Hitachi 7060; Tokyo, Japan) after 24 h of reperfusion.
Kidney tissues were fixed with 4% formalin for 24 h, dehydrated. and embedded in paraffin following routine protocols. Then, 4-μm-thick paraffin sections were stained with hematoxylin and eosin and evaluated in a blind manner by a pathologist. The renal scored injury was co-evaluated by Drs J-F.Y. and C.Y. from the Department of Urology, The First People's Hospital Of Yichang, who provide expert guidance for the score calculation. Five randomly selected fields of each sample were quantitated. To guarantee random selection, the fields for each sample were selected by a freshman from the Medical College of China Three Gorges University who was able to operate the microscope. The degree of tubular injury was graded from 0 to 4 according to tubular epithelial cell swelling, interstitial expansion, and intertubular hemorrhaging at 200 × magnification as follows: 0, no damage; 1, <25%; 2, 25% ∼ 50%; 3, 50% ∼ 75%; and 4, >75%. Five randomly selected fields of each sample were quantitated, and the mean score was calculated.
Four-micrometer sections were deparaffinized and boiled for 20 min in citrate buffer (pH = 6.0) for antigen retrieval. Endogenous peroxidases of the sections were blocked with 0.3 % hydrogen peroxide (H2O2). Nonspecific adsorption was blocked by 5% bovine serum albumin in phosphate buffer saline. After incubation with the primary antibody against NF-κB p65 (Abcam, Cambridge, MA) at 4°C overnight, the sections were washed and then incubated with horseradish peroxidase-conjugated antirabbit secondary antibody for 30 min at room temperature. Samples were stained with 3,3-diaminobenzidine tetrahydrochloride (DAB; Maixin Biotech, Fuzhou, China) and counterstained with hematoxylin. Finally, the expression area of NF-κB p65 protein was photographed (Olympus, Tokyo, Japan) at 400 × magnification, and the results were analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD). The results were defined as integrated option densities/total areas.
Apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine nick-end labeling (TUNEL) assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. In brief, samples were incubated in equilibration buffer for 5 min, followed by incubation in the labeling reaction reagent for 1 h at 37°C to mark DNA fragments in apoptotic cells. Cell counting was performed at 400× magnification using five randomly selected fields, and the apoptosis index was expressed as the percentage of positive cells in the field. Data were averaged.
Kidney sample lysates were prepared with RIPA lysis buffer followed by centrifugation. Western blot analysis of IκBɑ (Santa Cruz, Dallas, TX, 1:500), Nrf2 (Santa Cruz, 1:500), HO-1 (Santa Cruz, 1:1000), NQO-1 (Santa Cruz, 1:1000), Bax (Santa Cruz, 1:1000), Bcl-2 (Santa Cruz, 1:1000), poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) (Santa Cruz, 1:1000), β-actin (Abcam, Cambridge, UK, 1:10,000), Histone H3 (Santa Cruz, 1:500), p-NF-κB p65 (Cusabio, Wuhan, China, 1:500), p-IκBɑ (Cusabio, 1:500), NF-κB p65 (Abcam, Cambridge, UK, 1:1500), and GAPDH (Abcam, Cambridge, UK, 1:10,000) was performed according to standard protocols. The blots were detected by the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, MA) followed by exposure to Kodak-X-Omat film (Shanghai, China).
Real-time quantitative polymerase chain reaction
Total RNA was extracted using TRIZOL Reagent (Invitrogen, Carlsbad, CA) and reverse transcribed with ReverTra Ace (Toyobo, Dalian, China) to produce complementary DNA. The sequences of the primers listed in Table were used for quantitative real-time polymerase chain reaction (PCR). Real-time PCR was performed using SYBR Green-based detection in a StepOnePlus (ABI, CA) according to the manufacturer's instructions. The relative messenger RNA (mRNA) levels of specific genes were normalized to 18S rRNA levels.
All data for each group were presented as the mean ± standard error. One-way analysis of variance with a Tukey–Kramer test was performed using Graphpad Prism software, version 5.0 (GraphPad Software Inc, La Jolla, CA), for multiple comparisons. Student's t-test was performed using the statistical package SPSS, version 13.0 (SPSS Inc, Chicago, IL) when two groups were compared. The value of P < 0.05 was considered statistically significant for all tests.
Diosmetin protects the kidney against I/R injury
To assess diosmetin's effect on renoprotection, we detected BUN and blood creatinine (Scr) levels from serum samples. The concentrations of BUN and Scr were significantly increased after renal I/R injury compared with the sham mice (24.44 ± 1.00 versus 5.99 ± 0.65, 135.17 ± 2.34 versus 25 ± 1.91, respectively; Fig. 2A and B). However, treatment with diosmetin before I/R injury elicited a significant dose-dependent decrease in both parameters, implying the beneficial effect of diosmetin on renal function.
Beneficial effects of diosmetin on kidney specimens were evaluated by hematoxylin and eosin staining. Compared with sham mice, the I/R group showed severe damage including extensive renal tube dilation, massive intertubular hemorrhaging, tubular epithelial cell necrosis, and inflammatory cell infiltration. Mice treated with diosmetin before I/R injury had prominently reduced kidney injury compared with the I/R group (Fig. 2C). The tubular injury score results indicated that a high dose of diosmetin (1 mg/kg) before I/R caused significantly decreased tubular dilation and intertubular edema, from 3.67 ± 0.19 to 0.93 ± 0.3 (P < 0.05; Fig. 2D).
Diosmetin inhibits NF-κB signaling and the proinflammatory response
Activation of the NF-κB signaling pathway plays an important role in the proinflammatory response after I/R injury.
Immunohistochemistry was performed to determine whether diosmetin suppressed the expression and subcellular localization of p65 after I/R. The results showed that p65 expression was markedly increased after 24 h of reperfusion. However, p65 expression showed a dose-dependent decrease in the pretreatment groups (Fig. 3A and B). Moreover, p65 nuclear staining indicated that p65 was translocated into the nucleus in response to I/R injury (Fig. 3A). To further explore the NF-κB pathway, p65, phosphorylated-p65, IκBα, and phosphorylated-IκBα were examined by Western blotting (Fig. 3C). I/R increased the protein levels of p65, p-p65, and p-IκBα and decreased the expression of IκBα compared with sham mice, whereas diosmetin reversed these trends (Fig. 3D-G). In addition, to test whether diosmetin suppressed expression of downstream inflammatory cytokines, real-time PCR (RT-PCR) was used to detect the expression of IL-1β, IL-6 and TNF-α in five groups (Fig. 3H). Interestingly, diosmetin did produce markedly change with a significantly dose-dependent decrease in these inflammation mediators when compared with I/R injury mice. Together, these results suggest that diosmetin might be capable of protecting the kidneys from I/R induced injury, in part through inhibiting the NF-κB signaling pathway and attenuating the inflammatory response.
Diosmetin alleviates apoptosis after renal I/R injury
To determine whether diosmetin reduced renal cell apoptosis after I/R injury, a TUNEL assay was used to identify apoptotic cells (Fig. 4A). In the I/R group, an increase in TUNEL-positive cells was observed compared with the sham group, whereas preconditioning with 1 mg/kg diosmetin resulted in a significant reduction in the number of apoptotic cells (5.14 ± 3.33 % versus 60.77 ± 13.52 %, P < 0.05; Fig. 4B). To further explore the potential mechanisms of apoptosis, we performed with Western blotting to test protein expression levels of PARP-1, cleaved caspase 3, Bcl-2, and Bax (Fig. 4C). The results indicated that I/R resulted in significant upregulation of PARP-1 and cleaved caspase 3 expression and downregulation of the ratio of Bcl-2/Bax compared with the sham group. In contrast, preconditioning with diosmetin reversed these changes in a dose-dependent manner (Fig. 4D-F), indicating that diosmetin could have a potential effect on ameliorating I/R-induced cellular apoptosis through suppression of the mitochondrial apoptosis pathway.
Diosmetin prevents renal oxidative stress and activates the Nrf2 signaling pathway after I/R injury
Renal malondialdehyde levels were examined to evaluate the antioxidant effect of diosmetin. The results showed that renal malondialdehyde was significantly increased in the I/R group when compared with the sham group (1.92 ± 0.13 versus 8.02 ± 0.68 nm/mg protein, ∗P < 0.05, Fig. 5A). However, there were significant decreases in the I/R + 0.25 mg/kg diosmetin, I/R + 0. 5 mg/kg diosmetin, and I/R + 1 mg/kg diosmetin groups when compared with the I/R group (5.54 ± 0.43, 4.06 ± 0.54, and 2.28 ± 0.41 nm/mg protein, #P < 0.05). Because Nrf2 plays an important role in regulating the antioxidant response,
Western blotting was performed to identify the effect of diosmetin on nuclear expression levels of Nrf2 and downstream proteins HO-1 and NQO-1 (Fig. 5B). Compared with the sham group, I/R injury mice experienced an increase in Nrf2, HO-1 and NQO-1 expression. In addition, preconditioning with diosmetin significantly augmented the expression of these proteins (Fig. 5C-E). Interestingly, similar results were observed in the mRNA levels of HO-1 and NQO-1 genes, respectively (Fig. 5F and G), which implied that the Nrf2/HO-1 signaling pathway was activated by diosmetin.
Renal I/R injury is a difficult and complex clinical problem. Due to limited treatment options for patients, renal I/R injury has adverse outcomes that increase the risk of mortality.
reported that the protective effects of diosmetin could suppress the translocation of p65, reduce the release of proinflammatory cytokines, and impair the severity of cerulein-induced acute pacreatitis biochemically and morphologically. The properties of antioxidative stress have been shown to decrease the levels of reactive oxidant species and glutathione.
Nevertheless, how diosmetin benefits the kidney has not been reported previously.
In the present study, three doses (0.25, 0.5, and 1 mg/kg) of diosmetin were used to determine its effect on renal I/R injury. Renal morphologic structures displayed tubular hemorrhaging, epithelial cell cataplasia and necrosis from I/R mice, suggesting that renal I/R could result in the destruction of renal tissues. The data showed that diosmetin prevents this destruction in a dose-dependent manner; however, further beneficial effects were not shown with increasing concentrations. We then explored the potential mechanisms of diosmetin protection against renal I/R injury. The results showed that diosmetin alleviated inflammatory response, cellular apoptosis, and promoted potential capacity of antioxidative stress with preconditioning. The underlying mechanisms of renoprotection are mainly due to the repression of NF-κB and apoptosis signaling, as well as activation of Nrf2/HO-1 signaling.
It is well known that members of the NF-κB family play an important role in regulating inflammation.
The crucial subunit p65 of the NF-κB pathway is normally deactivated in the cytoplasm by integration with IκBs. During renal I/R injury, IκBs is phosphorylated and triggers ubiquitin-dependent IκBα degradation, allowing for p65 translocation into the nucleus to upregulate proinflammation-related mediators.
the expression of p-IκBα, p65, and p-p65 was elevated and IκBα levels were reduced in the I/R mice. Similarly, ischemia increased the mRNA expression levels of NF-κB pathway-targeted genes, including IL-1β, IL-6, and TNF-α. Nevertheless, preadministration with diosmetin effectively reversed these changes, demonstrating that activation of the NF-κB pathway correlated with renal I/R injury in mice. Diosmetin had a renoprotective effect through suppression of the NF-κB pathway.
Inflammation is also an important contributor to the progression of cellular apoptosis.
Activated caspase-3 facilitates PARP-1 expression, which can be involved in the recovery of DNA damage. However, excessive PARP-1 activation can result in cell death by depletion of intracellular adenosine triphosphate in tubular cells.
Likewise, data from our present study showed that the expression of TNF-α, cleaved caspase 3, and PARP-1 were markedly increased in the I/R group. However, diosmetin reversed these trends (Fig. 3, Fig. 4), suggesting the effect of antiapoptosis via reducing the mRNA expression levels of TNF-α, subsequently preventing mitochondrial apoptotic pathway regulated by members of the Bcl-2 protein family and then downregulating the downstream protein levels of cleaved capspase 3 and PARP-1.
However, other studies have shown contrary results. Liu et al.
reported that diosmetin was capable of promoting expression of p53 and increasing the ratio of Bcl-2/Bax proteins, leading to hepatocellular carcinoma apoptosis in vitro. Similar results were observed in renal cell carcinoma.
Obviously, p53 is extensively activated in response to strong stress such as oncogenic progression, participating in accelerating tumor cell death through activating mitochondrial apoptotic proteins. By contrast, p53 is slightly activated in response to low stressors, such as I/R, to provide restored function to the injury.
we speculated that the renoprotection of diosmetin might be via inhibiting of mitochondrial apoptotic pathway. Indeed, diosmetin increased the ratio of Bcl-2/Bax after renal I/R and decreased cleaved caspase-3 and PARP-1. Nevertheless, our experiment was performed with mouse model, and a study about the exact mechanism is needed to further research.
Nrf2, a redox-sensitive transcription factor, is responsible for upregulating target genes encoding phase II detoxifying enzymes and antioxidants that maintain cellular redox homeostasis.
reported that knockdown of Nrf2 expression stimulated the NF-κB-mediated inflammatory response compared with normal mouse models of lupus nephritis. Several chemicals were shown to be beneficial by elevating the Nrf2 protein level and repressing the expression of NF-κB and downstream cytokines.
Consistent with previous research, I/R in murine kidneys led to increased Nrf2 accumulation in the nucleus, concomitant with elevation of its downstream target genes HO-1 and NQO-1 at the mRNA and protein levels. Diosmetin augmented these trends dramatically with preconditioning. Accordingly, the results from our study demonstrate the potential renoprotective mechanism of diosmetin through Nrf2/HO-1 pathway activation and suppression of NF-κB signaling.
Inflammation and oxidative stress are two major components involved in the pathogenesis and progression of AKI. NF-κB and Nrf2 are the crucial transcription factors that regulate cellular responses to inflammation and oxidative stress, respectively, after AKI. Nrf2 activation can reduce NF-κB activity, resulting in decreased cytokine production, whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on Nrf2 target gene expression. There is a delicate balance between inflammation-induced cellular apoptosis and enhanced antioxidant defense capacity mediated by Nrf2 in this process. Diosmetin tips the balance in favor of recovering from I/R-induced acute renal injury by suppressing the NF-κB pathway and boosting the Nrf2/HO-1 axis (Fig. 6).
Overall, this study demonstrates that diosmetin can protect mice against renal I/R injury by suppressing inflammation and apoptosis and enhancing antioxidant capabilities. The mechanisms of renoprotection are linked with suppression of NF-κB and the mitochondrial pathways associated with Nrf2/HO-1 signaling. Our results suggest that diosmetin may be a promising therapeutic for preventing renal I/R injury.
This work was supported by the National Natural Science Foundation of China , No. 81100281 , and the Hubei Province health and family planning scientific research project, No. WJ2015XB018.
Authors' contributions: W-F.H. obtained the funding. W-F.H. and K.Y. contributed to the conception and/or design of this study. K.Y. and W-F.L. conducted the experiments. K.Y., W-F.L., J-F.Y., and C.Y. performed the data analysis. K.Y. and W-F.H. wrote and/or revised the article. K.Y., W-F.L., J-F.Y., C.Y., and W-F.H. contributed to final approval of the article.
The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
Acute kidney injury: an increasing global concern.