Figure 6 Icariin activates the NRF2 signaling pathway bothin vivo and in vitro. (A) Western blot analysis of Nrf2 and HO-1 expression in the kidney from different groups. (B) Western blot analysis of nuclear and cytoplasmic cell extracts from HK-2 cells with different treatments. ns: not significant. * p < 0.05, ** p < 0.01.
Discussion
Renal tubulointerstitial fibrosis (TIF) is a detrimental progression that inevitably results in renal function deterioration and structural changes, eventually culminating in ESRD. Nevertheless, the clinical management of TIF remains challenging because of the limited availability of effective prevention and treatment options. Novel therapeutic agents against TIF are urgently needed to delay the progression of CKD. In the current study, we confirmed icariin’s protective effect in UUO-induced CKD mouse model and in TGF-β1-treated HK-2 cells. UUO resulted in tubular atrophy, inflammation, oxidative stress and collagen deposition, whereas icariin markedly ameliorated these changes. In particular, icariin significantly improved mitochondrial mass and morphology of tubular epithelial cell in UUO mice. In TGF-β1-treated HK-2 cells, icariin markedly decreased the expression of profibrotic proteins and inflammatory factors, and protected mitochondria along with improving mitochondrial morphology, reducing mtROS accumulation, and preserving membrane potential. Further investigations uncovered that icariin activated Nrf2/HO-1 pathway bothin vivo and in vitro , whereas inhibition of Nrf2 by ML385 counteracted icariin’s protective effects on TGF-β1-treated HK-2 cells. Thus, icariin might exert anti-inflammation and antifibrotic effects, at least partially, through improving Nrf2 activity and subsequent mitochondrial function, and eventually mitigated TIF and preserved renal function (Figure 7).
Epimedium is a renowned traditional Chinese medicine that has been used for many centuries as a potent treatment for individuals with kidney ailments. Icariin has been demonstrated to be the primary bioactive component from Epimedium species. Modern pharmacological researches have showed that icariin exhibits considerable therapeutic capacities including neuroprotective21, cardiovascular protective22, anti-cancer23, as well as improving immune system and reproductive function. Interestingly, recent researches have indicated that icariin could be beneficial in preventing or improving various fibrotic diseases, such as hepatic fibrosis11, pulmonary fibrosis24 and myocardial fibrosis25. Additionally, a prior research has ever reported the protective effect against kidney fibrosis in mouse model26. However, to fully harness the therapeutic benefits of icariin for CKD patients, a deeper comprehension of the underlying mechanisms is essential. In UUO-induced CKD mice, our results showed that icariin effectively reduced BUN and Cr levels. On the other hand, the renal histopathological lesions and elevated fibrotic proteins in UUO group were also markedly mitigated after icariin treatment. As mounting studies have shown that RTECs not only bear the brunt of different insults, but also play a crucial role in TIF by crosstalk with other cells and trans-differentiation into fibrotic phenotype9,27, TGF-β1-treated HK-2 cells were used to verify the antifibrotic effect of icariin in vitro . Consistently, our in vitro experiments identified that icariin inhibited the profibrotic molecules expression, including a-SMA and collagen I. All these results highlight the pharmacological effect of icariin in inhibiting tubulointerstitial fibrosis and protecting renal function.
Oxidative stress and inflammation serve as interconnected players that underlie various causes of TIF and consistently manifest throughout the progression of CKD5. The transcriptomic data also indicated a pronounced enrichment in inflammatory response, mitochondrion, and oxidoreductase activity among the differentially expressed genes in UUO mice as compared to sham mice. Oxidative stress is a condition characterized by an imbalance between the production of ROS and their elimination by antioxidant defense systems, including SOD, glutathione peroxidase (GSH-Px) and catalase, as well as numerous non-enzymatic ROS scavengers. Excessive ROS can trigger renal fibrosis and inflammatory response, leading to substantial tissue lesion through promoting lipid peroxidation, DNA damage, and protein modifications, as well as the activation of profibrotic factors28,29. Icariin has been documented to safeguard vascular endothelial cells against oxidative stress by enhancing the activity of SOD and GSH-Px30. Additionally, icariin has also been shown to shield cardiac cells from oxidative damage through scavenging ROS and stimulating the ERK pathway31. In this study, we confirmed oxidative stress in UUO mice by demonstrating the accumulation of MDA and a decline in the activity of endogenous SOD, which were attenuated after icariin treatment. Accordingly, in TGF-β1-induced HK-2 cells, icariin can significantly inhibit intracellular ROS and mtROS overproduction, and protect HK-2 cells from oxidative stress. These results reinforce the effective anti-oxidation ability of icariin in CKD models.
Oxidative stress triggers deregulated inflammatory responses; while uncontrolled inflammatory responses, in turn, acts as a crucial mediator in the initiation and advancement of TIF, amplifying the deleterious consequences of oxidative stress in a “vicious cycle”. Accumulating studies have implicated anti-inflammatory capacity of icariin in different diseases32,33. Of importance, icariin mitigated renal inflammation in murine lupus nephritis by suppressing NF-κB activation34. In our experiments, icariin treatment markedly reduced macrophages filtration in renal interstitium of UUO mice and downregulated levels of IL-6, TNF-α and IL-1β inflammatory factors both in vivo and in vitro . Therefore, we inferred that icariin might confer protection against renal TIF by inhibiting inflammation and oxidative stress.
Consolidative proposals suggest that mitochondria, apart from being a powerhouse, also serve as central hubs that regulate cellular redox state and pro-inflammation signaling6,35. Mitochondria serve as the primary generator of intracellular ROS; meanwhile, excessive ROS and other damage-associated molecular patterns (DAMPs) originating from mitochondria can stimulate the expression of pro-inflammatory genes, leading to the activation of NF-κB signaling and NLRP3 inflammasome. Growing number of researches have proved the involvement of mitochondrial dysfunction in the development of kidney injury and impaired repair following injury8. Relieving mitochondrial dysfunction through administration of mitochondria-targeted antioxidants has been shown to attenuate kidney injury in animal models of UUO-induced CKD and diabetic kidney disease36,37. In the present investigation, we observed significant impairment in both the quantity and structure of mitochondria in kidneys from UUO mice. Consistent with prior research findings, the defective mitochondria exhibited prominent signs of matrix swelling, cristae depletion, and disruption of mitochondrial membranes. Interestingly, icariin effectively alleviated UUO-induced mitochondrial abnormalities. Consistently, in HK-2 cells treated with TGF-β1, icariin remarkedly protected mitochondrial function along with improving mitochondrial morphology, reducing mtROS accumulation, and preserving membrane potential. Collectively, our results expand the understanding of the renoprotection properties of icariin by maintaining mitochondrial homeostasis.
Mechanistically, we discover that the Nrf2/HO-1 signaling pathway is crucial in connecting icariin to its renoprotection effect. Nrf2 is a central redox-sensitive transcription factor that actively regulates pathways for antioxidant defense. Under normal physiological conditions, Nrf2 is kept at a low level in the cytoplasm by its inhibitor protein, Kelch-like ECH-associated protein 1 (Keap1), which facilitates the degradation of Nrf2 through a ubiquitin-proteosome pathway38. Upon oxidative stress, Keap1 undergoes modified that render it incapable of promoting Nrf2 degradation, leading to the accumulation of Nrf2 protein and its translocation to the nucleus. Once in the nucleus, Nrf2 binds to the antioxidant response element (ARE) and activates downstream antioxidant genes, such as HO-1, NAD(P)H quinone oxidoreductase 1 (NQO1) and SOD39. Besides, Nrf2 contributes to the anti-inflammatory process via multiple different mechanisms, especially negatively regulating NF-κB signaling pathway40. Several experimental models of CKD have shown an upregulation of Keap1 expression and a decrease in Nrf2 nuclear translocation, resulting in the suppression of antioxidant enzymes39,41, which were consistent with our findings. Indeed, recent researches have demonstrated that the activation of Nrf2 contributes to the improvement of renal fibrosis42-44. Icariin has been demonstrated to alleviate extracellular matrix accumulation and oxidative stress by activating Nrf2 in experimental diabetic kidney disease45. Moreover, another study has revealed that icariin enhances mitophagy to suppress the activation of NLRP3 inflammasome through the Keap1-Nrf2/HO-1 axis in diabetic nephropathy rats17. Additionally, further studies have suggested that icariin may modulate acute inflammation via the Nrf2/HO-1 and NF-κB signaling pathways46. These findings collectively support the potential of icariin as a potent activator of Nrf2/HO-1. In our study, further investigations uncovered that icariin treatment restored Nrf2/HO-1 levels both in vivo and in vitro , whereas inhibition of Nrf2 by ML385 counteracted the protective effects of icariin in TGF-β1-treated HK-2 cells, suggesting that the potential renoprotective effect of icariin was mediated by Nrf2 signaling pathway.
In conclusion, our results demonstrate that icariin protects against renal tubulointerstitial fibrosis and inflammation at least partly through Nrf2-mediated attenuation of mitochondrial oxidative damage, strongly suggesting that icariin may be a promising therapeutic strategy to mitigate TIF and preserve renal function.