In the present study, treatment with oleanolic acid attenuated renal inflammation and fibrosis in UUO mice. This study also demonstrated that oleanolic acid suppressed oxidative stress and cellular apoptosis, and promoted antioxidant enzyme expression which may be induced by enhancement of nuclear translocation of cytosolic Nrf2.
Oxidative stress plays a significant role in the progression of tubulointerstitial damage in UUO-induced renal injury . Chronic kidney disease characterized by fibrosis is related to decreased expression of superoxide dismutase and increased expression of NADPH oxidase . UUO also increased lipid peroxidation, shown as the levels of MDA, in the obstructed kidneys, and pretreatment with a superoxide dismutase mimetic reduced the increase in tissue lipid peroxidation after UUO . Excess ROS production from damaged tubular cells or infiltrated leukocytes such as ED-1-positive macrophages leads to renal tubular apoptosis in kidneys after UUO . In the present study, oleanolic acid significantly reduced renal tissue lipid peroxidation and tubular apoptosis, suggesting that oleanolic acid would play a preventative role in the oxidative stress pathway induced by UUO.
Oleanolic acid is a natural triterpenoid that is a constituent of the leaves of Olea europaea, Viscum album L. and other plants , and has been used as a traditional medicine . It is also found in promace olive oil and is the principal source of fat in the Mediterranean diet, supporting the hypothesis that some components of olive oil may have beneficial effects on human health . Recently, several studies have shown the therapeutic properties of oleanolic acid. One study demonstrated an inhibitory effect of oleanolic acid on the production of advanced glycation end-products in the kidneys of streptozotocin-induced diabetic mice . A previous study reported that oleanolic acid improved neurological symptoms in mice with experimental autoimmune encephalomyelitis . The effects of oleanolic acid are thought to result from its anti-inflammatory or anti-oxidant activity . For example, a recent study reported that treatment with oleanolic acid had beneficial effects on the development of atherosclerosis in apolipoprotein E knockout mice, which may be due to its antioxidant properties . Another study demonstrated that oleanolic acid treatment reduced blood pressure in hypertensive animals via endothelium-dependent vasodilatation mediated by nitric oxide . In our study, oleanolic acid reduced total collagen accumulation and interstitial macrophage infiltration in the obstructed kidneys. It also attenuated UUO-induced renal oxidative stress, possibly by up-regulating HO-1, NQO1 and Hsp70 expression and reducing lipid peroxidation. This suggests that oleanolic acid alleviates renal inflammation and fibrosis by decreasing oxidative stress in mice with UUO.
Activation of Nrf2 has been shown to be responsible for oleanolic acid-mediated protection against various insults [8, 15]. In a recent study, the synthetic triterpenoid analog of oleanolic acid, CDDO-Im, was used in mice fed a high-fat diet. Treatment with CDDO-Im prevented high-fat diet-induced obesity in wild-type mice, but not in Nrf2-disrupted mice . This finding suggests that oleanolic acid targets Nrf2 signaling. Keap1, a cysteine-rich protein, plays an important role in the inhibition of nuclear translocation of Nrf2 as well as facilitating proteasomal degradation of Nrf2 . Under normal conditions, Nrf2 is sequestered in the cytoplasm via binding to its repressor molecule, Keap1 . Stressful conditions such as oxidative or ER stress induce the dissociation of the Nrf2-Keap1 complex, and subsequent nuclear translocation of Nrf2. This is thought to be the mechanism of Nrf2 activation. An earlier study reported that oleanolic acid facilitated Nrf2 nuclear accumulation, which contributed to protection from acetaminophen hepatotoxicity . Compatible with the study, our results showed that oleanolic acid enhanced the expression of nuclear Nrf2, resulting in an increased nuclear Nrf2/total Nrf2 ratio in the obstructed kidneys. In the present study, UUO or oleanolic acid treatment did not change the renal expression of Keap1 and total Nrf2 in the obstructed kidneys. On the other hand, a previous study demonstrated a paradoxical reduction of inactivated Nrf2, accompanied by a significant elevation of Keap1 in cases of severe oxidative stress or inflammation . Although the reason for this discrepancy remains unclear, the changes in Keap1 and total Nrf2 levels may differ according to experimental models of oxidative stress and the severity of renal injury. Taken together, our results may suggest that oleanolic acid facilitates nuclear translocation of cytosolic Nrf2 rather than directly inducing dissociation of the Nrf2-Keap1 complex.
Within the nucleus, Nrf2 binds to regulatory sequences called antioxidant response elements or electrophile response elements, which are located in the promoter region of genes encoding the antioxidant and phase 2 detoxifying enzymes such as glutathione-S-transferases, NQO1, glutamate-cysteine ligase catalytic subunit, catalase, thioredoxin, and HO-1 [8, 16]. The present study showed that oleanolic acid markedly increased the expression of HO-1, an important antioxidant enzyme, in the obstructed kidneys of oleanolic acid-treated mice. In general, HO-1 metabolizes heme that accumulates in tissues as a by-product of red blood cell turnover. Metabolites produced by such degradation reactions have been known to trigger signaling cascades that aid antioxidant defenses and protect against pathological increases in oxidative stress . Unlike HO-1, in our results, expression of NQO1 upregulated by oleanolic acid was more prominent on day 7 than on day 3. There appears to be some differences in the expression of phase 2 enzymes according to the experimental model: the regulatory mechanisms of phase 2 enzymes in relation to the Nrf2 pathway may be tissue- or cell-specific . Our results suggest that HO-1, rather than NQO1, plays a major role in the attenuation of renal inflammation and fibrosis in this UUO model. We also observed that oleanolic acid treatment increased Hsp70 protein expression in renal tissue on UUO day 7. This finding accords with previous results that activation of Nrf2 upregulates its downstream gene products and the Hsp70 gene . Our results demonstrated that oleanolic acid improved interstitial fibrosis and the total collagen content of renal tissue on day 7 after UUO, although attenuation of tubular injury and interstitial inflammation by oleanolic acid was evident even in day 3. Considering that renal fibrosis in our study was further attenuated on day 7 than it was on day 3, late upregulation of NQO1 and Hsp70 might have synergistic effects on the suppression of progressive fibrosis resulting from UUO. On the other hand, there was no difference in the expression of catalase, which is responsible for H2O2 neutralization, in obstructed kidneys with or without OA treatment. It is unclear why oleanolic acid did not change the expression of catalase together with the levels of tissue H2O2 in this study. In addition, MnSOD protein level showed no differences with administration of oleanolic acid. Taking findings of previous studies into consideration [6, 12], we cautiously speculate that the type of oxidative stress may influence the detailed action of Nrf2 that regulates induction of genes encoding specific antioxidants.
The present study demonstrated that renal apoptosis was attenuated by treatment with oleanolic acid. This finding is consistent with a recent study showed that the up-regulation of HO-1 significantly decreased cellular apoptosis . Our study also showed that although oleanolic acid had no effect on the levels of the pro-apoptotic enzyme Bax it increased the expression of the anti-apoptotic enzyme Bcl-2, resulting in a decreased Bax/Bcl-2 ratio. It has been suggested that Nrf2 targets the anti-apoptotic Bcl-2 protein, and that antioxidant treatment releases Nrf2 and increases Bcl-xL heterodimerization with Bax, resulting in reduced cellular apoptosis . Our results suggest that HO-1 and Bcl-2 may serve as key players in Nrf2-upregulated cell survival under oxidative stress induced by UUO. Furthermore, the interaction between Hsp70 and Bcl-2 is considered to contribute to renoprotection in this study. It has been known that Hsp70 can inhibit apoptosis by antagonizing the apoptosis-inducing factor .
The current study has some limitations. First, we did not measure the blood urea nitrogen (BUN) and serum creatinine as renal functional parameters. However, many previous studies have reported that BUN or serum creatinine was not significantly affected by UUO because of the presence of a contralateral kidney with good renal function [33, 34], suggesting that BUN and serum creatinine are not good indicators of renal function in an animal model of UUO. Second, we could not evaluate the molecular mechanism through which oleanolic acid modulates nuclear translocation of Nrf2. Third, we did not investigate the possible Nrf2-independent mechanisms that could contribute to renoprotection in the UUO model. Like a previous report , we found that oleanolic acid treatment increased the expression of Hsp70 protein that may contribute to Nrf2-independent renoprotection. A previous study used microarray to compare the overall gene expression signatures modulated by pharmacologic or genetic activation of Nrf2 signaling in liver tissue and found several Keap1-Nrf2-independent genes . Another report indicated that oleanolic acid protected against hepatotoxicity in wild type mice, but did so to a lesser degree in Nrf2-null mice, suggesting that oleanolic acid could activate Nrf2-independent protective mechanisms . Further investigation is needed to elucidate the exact mechanisms of the Nrf2-independent effects of oleanolic acid.