Ischemia-reperfusion (I/R) injury of the kidney, which occurs during renal transplantation, surgical revascularization of the renal artery, or severe burn with delay resuscitation, is a complex pathophysiological process that initiates a complex sequence of events leading to a major cause of acute renal failure (1-4). Although the pathophysiology of I/R-induced acute renal failure is not completely understood, some key events leading to tissue injury and renal failure have been identified. It has been shown to be related to inflammatory responses and activation of apoptotic pathways (5). Inhibition of certain elements of inflammatory responses and apoptotic pathway seemed to ameliorate renal I/R injury (6).
The initial pathological events in the kidney after renal I/R injury include vasoconstriction, calcium dyshomeostasis, endothelial-cell activation, tubular swelling and desquamation, depolarization, and necrosis of the tubular epithelium and interstitial edema (7, 8). Organ reperfusion increases endothelial expression of adhesion molecules and promotes leukocyte recruitment and activation, giving way to increased reactive oxygen species (ROS) production and the development of the inflammatory process (9, 10). In many ways, the inflammatory reaction to this injury resembles that seen during ascending urinary infection, and it may represent a general response of the tubular epithelial cells to stress or injury.
Apoptosis is characterized by distinct morphologic and biochemical alterations of the cell, such as mitochondrial dysfunction, activation of caspases, chromatin condensation, and DNA fragmentation, and has been implicated in I/R-induced cell injury in many different organ systems, including kidney (11, 12). Although ischemic events alone may lead to necrosis and apoptosis in the kidney, reperfusion occurs upon restoration of blood flow and is associated with increased apoptotic cell death (6).
Panax notoginseng, a famous traditional Chinese herb medicine, has long been used in treating various cardiovascular diseases in China, Korea, and Japan and is now a popular and worldwide-used natural medicine. Reports show that it can reduce inflammatory reactions in patients and animal models and can ameliorate the symptoms of experimental disseminated intravascular coagulation (13, 14). It is proved that the main ingredient of P. notoginseng with cardiovascular activity is NR1, molecular formula C47H80O18. As an effective fraction of P. notoginseng, NR1 could reduce endotoxin-induced lethality in mice in vivo (15). More recently, reports show that NR1 could inhibit TNF-α-induced fibronectin accumulation via the ROS/ERK pathway and directly scavenges ROS (16). Panax notoginseng could reduce leukocyte adhesion in venules under the inhibitory effect on the expression of adhesion molecules (CD11b and CD18) on neutrophils (17). In general, antioxidant, anti-inflammatory, antiapoptotic, and immune-stimulatory activities are mostly underlying the possible effective fractions of NR1-mediated protective mechanisms (16, 18, 19).
The purpose of the present study was to determine the effects of NR1 on renal function, apoptosis and signaling, and the inflammatory response of the kidney after I/R injury.
MATERIALS AND METHODS
Healthy adult male Sprague-Dawley rats weighing 230 to 250 g purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences were used in the present studies. Animals were acclimated to their surroundings for at least 7 days before experimentation. The animals were housed in individual cages under light-controlled conditions and at room temperature. All animals received postoperative care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. All experimental protocols were approved by the Second Military Medical University Ethics Committee in Shanghai, People's Republic of China.
Rats were anesthetized by i.p. injection of pentobarbital sodium 50 mg·kg−1. After medial laparotomy, both left renal artery and vein were clamped by microaneurysm clamps for 45 min (20). After the renal clamps were removed, the kidney was observed for a further 5 min to ensure blood reflow, and the incision was sutured in 2 layers. Rats were then returned to their cages where they were allowed to recover from anesthesia and were allowed free access to food and water. The right kidney was removed 5 min before the end of ischemia period. Rats with a delayed recovery from anesthesia or with signs of hemorrhage were excluded from the study.
Rats were randomly allocated into the following four groups: (1) sham group(sham: n = 6), rats underwent identical surgical procedures to I/R rats except that microaneurysm clamps were not applied; (2) I/R control group (I/R: n = 6), rats received i.p. injection of 1 mL isotonic saline as vehicle 1 h before renal vessel clamp was applied; (3) NR1-1 group (NR1-1: n = 6), rats received i.p. injection of NR1 20 mg·kg−1 in 1 mL isotonic saline 1 h before renal vessel clamp was applied; and (4) NR1-2 group(NR1-2: n = 6), rats received i.p. injection of NR1 40 mg·kg−1 in 1 mL isotonic saline 1 h before renal vessel clamp was applied.
NR1 (purity >99%) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
Further injections of isotonic saline or NR1 with the same dosages were performed once a day until the animals were killed 3 days after reperfusion.
Sample collection and renal function measurement
At several time points after reperfusion, peripheral blood samples were drawn from the rats in each group; plasma was separated by centrifugation at 1,000g for 10 min and stored at −20°C until measurements were performed. Serum creatinine levels were measured biochemically and determined as an indicator of renal dysfunction. Three days after I/R or sham operation, kidneys of the animals were removed under anesthesia. A piece of renal tissue including cortex and medulla was trimmed down and fixed by 10% buffered formalin for 24 h and embedded in paraffin for histological studies. Sections in 3- to 4-μm thickness were cut, mounted on glass slides, and stained with hematoxylin-eosin. Another portion of the kidney specimen was snap frozen in liquid nitrogen and conserved at −80°C for biochemical analyses.
Renal myeloperoxidase activity
Renal myeloperoxidase (MPO) activity was used as an index of organ neutrophil infiltration (20, 21). Tissue samples were pulverized using pestle and mortar under liquid nitrogen and then homogenized in 10% hexadecyltrimethyl ammonium bromide buffer (0.5% hexadecyltrimethyl ammonium bromide in 50 mM phosphate buffer, pH 6.0) with a Polytron homogenizer. The homogenate was sonicated on ice for 15 s, frozen at −80°C, thawed three times, and then centrifuged at 40,000g at 4°C for 15 min. The supernatant was collected and was measured from the optical density (at 460 nm) changes resulting from decomposition of H2O2 in the presence of dianisidine. Results were expressed as units per gram of protein.
Serial sections (4 μm in thickness) of paraffin-embedded renal tissue were prepared. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) apoptosis assay was performed with an in situ apoptosis detection kit (Roche Diagnostics, Mannheim, Germany). The nuclei of TUNEL-positive cells were stained brownish. Positive cells were identified, counted in 10 nonoverlapping ×400 microscopic fields of sections and averaged for the value of one section. The apoptotic index was calculated with the following equation: apoptotic index = (number of apoptotic cells / total cells) × 100%.
Northern blot analysis
Rat complementary DNA fragments for TNF-α, bcl-2, bax, and 18S rRNA were obtained by reverse transcriptase-polymerase chain reaction as described previously (22). The following oligonucleotide primers were used: for TNF-α, sense 5′-TAC TGA ACT TCG GGG TGA TTG GTC C-3′ and antisense 5′-CAG CCT TGT CCC TTG AAG AGA ACC-3′; for bcl-2, sense 5′-CCT TCC AGC CTG AGA GCA AC-3′ and antisense 5′-ATC CCA GCC TCC GTT ATC CT-3′; for bax sense 5′-CAT GGA GCT GCA GAG GAT GAT TG-3′ and antisense 5′-CAG TGT CCA GCC CAT GAT GG-3′; and for 18S, sense 5′-TCA AGA ACG AAA GTC GGA GG-3′ and antisense 5′-GGA CAT CTA AGG GCA TCA CA-3′. Total RNA was isolated from renal tissue using the Trizol (Invitrogen, Carlsbad, Calif) separation method as described in the manufacturer's protocol. RNA concentration and purity were determined by spectrophotometry (A260/A280 ratio). Integrity of the RNA samples was verified by electrophoresis on 2% agarose gels. RNA was electrophoresed and transferred onto nylon membranes. The blots were hybridized separately with [α-32P]dCTP (3,000 Ci · mmol−1; Amersham Biosciences, Little Chalfont, UK)-labeled rat TNF-α, bcl-2, and bax cDNA probes; subsequently stripped; and reprobed with 32P-labeled rat 18S rRNA cDNA as internal control, as described previously (22). Densitometric analysis was performed using a Phosphor-Imager system (STORM 820; Molecular Dynamics, Sunnyvale, Calif).
Immunohistochemical detection of bcl-2 protein expression
Renal bcl-2 protein expression was immunohistochemically detected. Briefly, fixed rat kidney sections were deparaffinized in xylene and rehydrated through a graded ethanol series to water. After blockage with 10% normal horse serum in phosphate-buffered saline, the slides were stained for bcl-2 in sequential incubations with rabbit polyclonal bcl-2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) for 30 min, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:60 dilution) for 30 min, and diaminobenzidine reagent (Vector Laboratories, Burlingame, Calif) for 10 min. Expression levels were determined by immunohistochemical localization and intensity in paraffin sections.
Western blot analysis of activated p38 expression
The whole-cell protein extracts from frozen renal tissues were prepared as described previously (23). Briefly, the frozen samples were ground in liquid nitrogen into a fine powder and Dounce homogenized in ice-cold lysis buffer containing 20 mmol·L−1 Tris-HCl (pH 7.4), 25 mmol·L−1 NaCl, 1 mmol·L−1 EDTA, 1 mmol·L−1 EGTA, 1 mmol·L−1 sodium orthovanadate, 10 mmol·L−1 sodium fluoride, 10 mmol·L−1 sodium pyrophosphate, 1 mmol·L−1 phenylmethylsulfonyl fluoride, 1 μg·mL−1 leupeptin, 10 μg·mL−1 aprotinin, and 10 mg·mL−1 p-nitrophenylphosphate. Cell debris and nuclei were removed by centrifugation at 12,000g for 10 min at 4°C, and the supernatants obtained were used as the whole-cell lysates. Protein concentrations were measured using the BCA Protein Assay Reagent kit (Biotechnology Inc, Rockford, Ill) with bovine serum albumin as standard, according to the manufacturer's instructions. The activation of p38 kinase was examined as its phosphorylated form detected by Western blot analysis. In brief, equal amounts of proteins (50 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was probed overnight at 4°C with the rabbit polyclonal antibody to phospho-p38 (Thr180/Tyr182) (Cell Signaling Technology, Beverly, Mass), followed by horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Pierce Biotechnology Inc, Rockford, Ill). Membranes were subsequently stripped (62.5 mM Tris-HCl pH 6.8, 20% sodium dodecyl sulfate, and 100 mM β-mercaptoethanol) and reprobed for total p38 MAPK antibody (Cell Signaling Technology). Relative quantities of protein were determined using a gel documentation system (Fluor-S-MultiImager and Quantity one analysis software; Bio-Rad, Hercules, Calif) and presented in comparison to total p38 MAPK expression.
Electromobility gel shift assay
The nuclear protein extracts from frozen renal tissues were prepared as described previously (22, 23). Double-stranded nuclear factor κB (NF-κB) consensus oligonucleotide (Promega, Madison, Wis) were end labeled with [γ-32P]ATP (3,000 Ci·mmol−1; Amersham Biosciences) and purified over a Sephadex G-25 column. Nuclear extracts (10 μg) were incubated with labeled probe at room temperature for 20 min in a binding buffer containing poly(dI-dC) as described previously (22). The specificity of the binding reaction was determined by preincubation with 100-fold molar excess of unlabeled NF-κB probes. DNA-protein complexes were separated in 5% nondenaturing polyacrylamide gel at 90 V for 2 to 3 h. The autoradiographs were quantified by density analysis using phosphorimager analysis.
All values in the text and figures are expressed as mean ± SEM. The data were analyzed with one-way ANOVA plus the Dunnett post hoc multiple-comparisons test, for comparisons of mean values among multiple treatment groups. P < 0.05 was considered statistically significant.
Effects of NR1 on renal dysfunction caused by I/R: serum creatinine and histological evaluation
Serum creatinine increased and reached similar peak levels 24 h after I/R injury irrespective of whether the animals were treated with vehicle or with NR1 1 h before ischemia (Fig. 1A). When compared with sham-operated rats (24 ± 0.53 mmol·L−1), I/R caused a significant increase in the serum creatinine levels in control rats 72 h after I/R injury (28.7 ± 0.37 mmol·L−1), suggesting a significant degree of renal dysfunction by I/R injury. However, rats that had been treated with NR1 20 mg·kg−1·d−1 (25.4 ± 0.47 mmol·L−1) and 40 mg·kg−1·d−1 (25.3 ± 0.44 mmol·L−1) demonstrated a significant decrease in serum creatinine levels 72 h after I/R injury (Fig. 1B).
Three days after I/R, kidneys from the untreated I/R rats showed a significant degree of renal injury (Fig. 2B). Specifically, kidneys obtained from these animals exhibited degeneration of tubular structure, tubular dilatation, swelling and necrosis, and luminal congestion. In most cases, lesions appeared to be circumscribed to the deep renal cortex; however, the whole cortex was affected in some cases. This feature appeared mainly in the corticomedullary interface, but it was also detected in the outer cortex and inner medulla. Moderate lesions were observed in kidneys from the NR1-1 group rats (Fig. 2C). Kidneys from the NR1-2 group rats showed no alterations in most areas of tissue. Some scarce areas of tubular necrosis were circumscribed to the deep cortex, usually accompanied by congestion (Fig. 2D). No alterations were detected in the sham animals (Fig. 2A).
NR1 affects renal inflammation after I/R injury
Determination of MPO levels in the prepared tissue homogenates was used to assess the activation of infiltrating neutrophils during reperfusion of ischemic kidneys. The renal tissue MPO activity in the sham group (19.9 ± 0.72 U·g−1) was elevated by I/R injury (50.6 ± 1.3 U·g−1; P < 0.01); however, both 20 and 40 mg·kg−1·d−1 administration of NR1 significantly decreased the I/R-induced elevation in renal MPO level (32.67 ± 1.08 U·g−1 in the NR1-1 group, 24.5 ± 0.87 U·g−1 in the NR1-2 group; both P < 0.01) compared with control group (Fig. 3).
As shown in Figure 4, NR1 treatment significantly decreased expression of TNF-α mRNA 3 days compared with the control group. In the I/R group treated with saline, renal expression of TNF-α mRNA level showed a dramatic increase (1.28 ± 0.036 OD) as compared with the sham group (0.76 ± 0.015 OD; P < 0.05), and both the 20 and 40 mg·kg−1·d−1 administration of NR1 reduced the concentration of this inflammatory cytokine significantly (0.96 ± 0.014 OD in the NR1-1 group, 0.87 ± 0.01 in the NR1-2 group; both P < 0.05) compared with the control group (Fig. 4).
Effects of NR1 on I/R-induced apoptosis
To further compare the extent of tissue damage in ischemic kidneys, TUNEL assay was conducted to examine whether NR1 treatment has an antiapoptotic effect on renal I/R injury. At 3 days after reperfusion, quantitative analysis revealed a significant increase in the number of TUNEL-positive cells in the I/R kidneys (32% ± 7%) compared with nonischemic kidneys (2% ± 0.7%; P < 0.05; Fig. 5). Administration of NR1 caused significant attenuation of I/R-induced kidney apoptosis; the number of TUNEL-positive cells was markedly decreased in the NR1 treatment group (21% ± 2.1% in the NR1-1 group, 16 ± 2.2% in the NR1-2 group; both P < 0.05) compared with the I/R group treated with saline (32% ± 7%).
NR1 increased the expression of bcl-2 after I/R injury
Bcl-2, an antiapoptitic protein, is known to inhibit various caspases and one among many key regulators of apoptosis after ischemic injury (24). Therefore, we thus proceeded to determine whether the antiapoptotic effects of NR1 also involved bcl-2. In the I/R group treated with saline, renal expression of bcl-2 mRNA level showed a dramatic decrease (0.61 ± 0.05 OD) as compared with sham group (0.92 ± 0.02 OD; P < 0.05) 72 h after I/R injury. However, administration of NR1 increased the expression of bcl-2 mRNA level significantly (1.09 ± 0.01 OD in the NR1-1 group, 1.22 ± 0.04 in the NR1-2 group; both P < 0.05) as compared with the I/R control group. In contrast, levels of proapoptotic bax were unchanged in the NR1-treated group, as compared with saline controls and other groups, 72 h after I/R injury (Fig. 6).
These results were further confirmed by using immunohistochemistry to determine locality and level of expression of the bcl-2 protein (Fig. 7). The pattern of staining for bcl-2 after renal I/R injury correlated closely with the pattern of staining for bcl-2 mRNA seen with Northern blot analysis. Expression of bcl-2 was modestly in distal and proximal tubular epithelial cells in sham animals. Ischemia-reperfusion injury markedly decreased bcl-2 expression levels seen in distal tubule. Low levels of expression of bcl-2 were occasionally seen in the proximal tubule. Administration of NR1 markedly enhanced bcl-2 expression levels compared with I/R group rats both in distal and proximal tubule epithelial cells.
Phosphorylation of p38 MAPK was reduced by NR1 treatment after I/R injury
A change in expression of proinflammatory and apoptosis-related p38MAPK activation during the course of I/R was analyzed by Western blotting 3 days after reperfusion. Phosphorylation of p38MAPK protein significantly increased after I/R injury compared with the sham group (Fig. 8). Administration of NR1 caused significant decrease of phosphorylation of p38 MAPK protein compared with the I/R group treated with saline.
Administration of NR1 decreased NF-κB activation in renal tissue after I/R injury
To assess whether attenuation of I/R-induced kidney injury and apoptosis were associated with NF-κB pathway, an electromobility gel shift assay analysis of nuclear NF-κB activity in rat's kidney was performed. The NF-κB activity was markedly increased by I/R-induced kidney injury treated with saline (17.68 ± 1.48 OD) compared with the sham group (7.11 ± 0.45 OD; P < 0.05; Fig. 9). Administration of NR1 20 mg·kg−1·d−1 (9.45 ± 0.63 OD; P < 0.05) or 40 mg·kg−1·d−1 (7.68 ± 0.82 OD; P < 0.05) caused further significant decreases of NF-κB activity compared with the I/R group treated with saline alone. These results suggested that the attenuation of I/R-induced kidney apoptosis by NR1 administration is associated with the decreases of NF-κB activity.
This is a preliminary report that evaluates the effects of administration NR1 on renal dysfunction and inflammatory reaction, necrosis, and apoptosis after experimental I/R injury in rats. Using an in vivo I/R injury model, we demonstrated that NR1 administration caused significant attenuation of I/R-induced renal apoptosis, neutrophil accumulation (MPO assay), and characteristic histological signs of marked tubular injury. All these data confirmed a well-known pattern of renal dysfunction and injury caused by I/R of the kidney (5). Although the precise mechanism for the inhibition of I/R injury remains unclear, it appears from the present study that the anti-inflammatory and antiapoptotic function of NR1 plays a key role in improving the recovery of renal function.
Inflammation is recognized as an important pathophysiological component of renal I/R injury (25). Our data also reveal that proinflammatory cytokine TNF-α is upregulated after renal I/R, consistent with previous studies. TNF-α has the ability of initiating upregulation of other cytokines and chemokines that are likely responsible for the accumulation of neutrophils. Increasing evidence suggests that TNF-α is an important mediator of renal I/R injury (26-28). The reversal of TNF-α and neutrophil accumulation by NR1 treatment suggests that the mechanism of the protective effect of NR1 involves the inhibition of TNF-α release and inflammatory cell infiltration.
Apoptosis has been recorded previously in both the proximal and distal tubular cell populations in ischemic renal injury (24). Bcl-2/Bax and NF-κB axis are the 2 main signaling pathways regulating cell survival and inflammation. Bcl-2, an antiapoptitic protein, is known to inhibit various caspases. Overexpression of bcl-2 by adenoviral transfection was considered associated with strong protection against reperfusion injury (29). Not all bcl-2 family members are inhibitors of apoptosis, however; some are proapoptotic. The first proapoptotic bcl-2-associated protein identified was bax, which has partial amino acid sequence homology to bcl-2 and coimmunoprecipitates with bcl-2 in cell extracts and in vivo. In functional assays, bax suppresses the ability of bcl-2 to block apoptosis through the formation of heterodimers with bcl-2, leading to its inactivation (24). Because the ratio of bcl-2 to bax is an important regulator of apoptosis, the expression of bax was also examined in the present study. Renal expression of Bcl-2/Bax mRNA level showed a dramatic increase after NR1 treatment, indicating that enhancing the expression of bcl-2 may be one of the antiapoptotic mechanisms of NR1.
To study in depth the mechanisms responsible for the functional protection afforded by administration of NR1 after I/R injury, we analyzed activation of p38MAPK and NF-κB. NF-κB-inducing kinase-NF-κB axis is one of the main signaling pathways regulating cell survival and inflammation (30). Several studies support the involvement of NF-κB in different organs including the kidney in I/R injury, although very little is known about the mechanism underlying its activation in this setting (31). For both apoptosis and the production of proinflammatory cytokines, p38MAPK is an important mediator, and it has been shown that p38MAPK inhibitors can decrease TNF-α production and apoptosis and provide protection from renal I/R injury and glomerular damage (31-34). Specific inhibitors of p38 kinase significantly inhibited bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells (35). In the present study, we have shown that the decreased p38MAPK activation and the decreased NF-κB activation by NR1 may be responsible for the decreased proinflammatory cytokines and the decreased apoptosis through both Bcl-2/Bax and NF-κB axis signaling pathways.
Once renal injury reduces the number of functioning nephrons, choric changes develop at an accelerated rate. Ischemia-reperfusion injury amplifies innate immune-mediated intragraft inflammation and necrosis (10). Short- and long-term adverse outcomes including decreased graft function continue to plague renal transplantation (36-38). In the current study, we found that administration of NR1 can attenuate renal dysfunction caused by I/R injury, suggesting that it may probably prevent a progression of chronic changes. Long-term follow-up is therefore needed to see whether NR1 is able to protect the nephrons from these chronic changes.
Our preliminary research has demonstrated that NR1 significantly reduces renal injury and dysfunction by attenuation of apoptosis and inflammation in rats. Further systematic studies are warranted. Is the effect of NR1 dose-dependent? Is NR1 also effective with treatment starting after I/R injury? More importantly, what is the exact mechanism of how NR1 works? Intensive investigation on NR1 may lead to advances in the clinical management of I/R injury.
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