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.