Abstract
Tubulointerstitial fibrosis is an inevitable consequence of all
progressive chronic kidney disease (CKD) and contributes to a
substantial health burden worldwide. Icariin, an active flavonoid
glycoside obtained from Epimedium species, exerts potential
antifibrotic effect. The study aimed to explore the protective effects
of icariin against tubulointerstitial fibrosis in unilateral ureteral
obstruction (UUO)-induced CKD mice and TGF-β1-treated HK-2 cells, and
furthermore, to elucidate the underlying mechanisms. The results
demonstrated that icariin significantly improved renal function,
alleviated tubular injuries, and reduced fibrotic lesions in UUO mice.
Furthermore, icariin suppressed renal inflammation, reduced oxidative
stress as evidenced by elevated SOD activity and decreased MDA level.
Additionally, TOMM20 immunofluorescence staining and transmission
electron microscope revealed that mitochondrial mass and morphology of
tubular epithelial cells in UUO mice was improved by icariin.
In
HK-2 cells treated with TGF-β1, icariin markedly decreased profibrotic
proteins expression, inhibited inflammatory factors, and protected
mitochondria along with improving mitochondrial morphology, reducing
reactive oxygen species (ROS) and mitochondrial ROS (mtROS)
overproduction, and preserving membrane potential. Further
investigations demonstrated that icariin could activate Nrf2/HO-1
pathway both in vivo and in vitro , whereas inhibition of
Nrf2 by ML385 counteracted the protective effects of icariin on
TGF-β1-induced HK-2 cells. In conclusion, icariin protects against renal
inflammation and tubulointerstitial fibrosis at least partly through
Nrf2-mediated attenuation of mitochondrial dysfunction, which suggests
that icariin could be developed as a promising therapeutic candidate for
the treatment of CKD.
Keywords: renal tubulointerstitial fibrosis, icariin, oxidative
stress, mitochondrial dysfunction, Nrf2 pathway
Introduction
In
recent decades, chronic kidney disease (CKD) has garnered widespread
concern due to its high prevalence, severe complications and tremendous
healthcare costs. It is estimated to affect 10% to 15% of the global
population1. Regardless of the various etiologies of
CKD, renal tubulointerstitial fibrosis (TIF) is considered as the final
shared pathway of CKD and the most reliable indicator of renal
survival2. Importantly, recent researches have
revealed that tubulointerstitial fibrosis serves not just as the
histological feature of CKD, but also as a catalyst of CKD advancement
to end-stage renal disease (ESRD)3,4. Therefore,
prevention and treatment of TIF can delay the development of CKD.
Unfortunately, to date, there are limited effective therapeutic drugs
that can inhibit or reverse TIF.
Despite the pathogenesis of renal tubulointerstitial fibrosis being
complicated, emerging evidences from clinical and experimental studies
have shown that oxidative stress and inflammation are closely involved
in the initiation and advancement of TIF5.
Mitochondria serve as the primary generator of reactive oxygen species
(ROS) and take the center stage in orchestrating oxidative stress and
subsequent inflammation response6,7. Mitochondrial
damage leads to ROS accumulation, mitochondrial fragmentation, and
membrane potential depolarization, resulting in deregulated inflammatory
responses and secretion of profibrotic cytokines, and eventually
contributes to the fibrotic remodeling observed in
TIF8. In particular, renal tubular epithelial cells
(RTECs) possess abundant mitochondria, rendering them more susceptible
to mitochondrial dysfunction. Of note, increasing studies have indicated
that RTECs serve as both targets and active contributors in kidney
injury, as they have the ability to secrete numerous inflammatory
factors, profibrotic molecules and extracellular matrix in kidney
tissues9. Therefore, targeting mitochondria
homeostasis may function as an efficacious approach for preventing and
treating renal fibrosis.
Recently, natural products have emerged as promising source of novel
drugs owing to their distinctive advantages, such as multi-target
activities and low adverse effects. Icariin is a pleiotropic flavonoid
extracted from Epimedium ( in Chinses: Yin yang huo ), a renowned
traditional Chinese medicinal formula that has undergone extensive
clinical validation over the years as a highly effective remedy for
patients suffering from kidney or bone diseases. It has attracted much
attention in modern pharmacological research because of its multiple
properties, including anti-inflammatory, antioxidant and antifibrotic
activities. For example, icariin has been reported to reduce live
fibrosis in various mouse models by inhibiting epithelial-mesenchymal
transition10 or autophagy11.
Furthermore, icariin ameliorated STZ-induced diabetic nephropathy by
suppressing NF-κB signaling pathway12. Intriguingly,
recent studies have connected its therapeutic effect with its potential
to maintain mitochondrial homeostasis13-15. However,
prior studies have provided suggestive but restricted evidence regarding
the potential role of icariin against tubulointerstitial fibrosis in
CKD. In particular, there is a paucity of data on the protective effects
of icariin on mitochondrial abnormalities in CKD.
Therefore, the current study was designed to investigate the
ameliorative effects of icariin on renal inflammation, oxidative stress
and tubulointerstitial fibrosis induced by unilateral ureteral
obstruction (UUO). We particularly focused on the protective effects of
icariin on mitochondrial homeostasis. Considering that nuclear factor
erythroid 2-related factor 2 (Nrf2) /heme oxygenase-1 (HO-1) are
extensively involved in the regulation of oxidative stress and
mitochondrial homeostasis, we further investigated the influence of
icariin on Nrf2-related signaling pathway both in vivo andin vitro , which aimed at gaining fresh perspectives on the
underlying protective mechanism of icariin against renal fibrosis.
Materials and methods
Materials and reagents
Icariin (purity≥ 98%, I107343) was obtained from Aladdin. Recombinant
TGF-β1 protein (100-21C) was obtained from PeproTech. ML385 (SML1833)
was purchased from Sigma-Aldrich. Collagen I (72026, 1:1000), Nrf2
(12721, 1:1000) and F4/80 (70076, 1:500) antibodies were purchased from
Cell Signaling Technology. TOMM20 (ab186735, 1:250) antibody and Alexa
Fluor 594-conjugated secondary antibody (ab150076, 1:500) were purchased
from Abcam. HO-1 (10701-1-AP, 1:3000), Lamin B1 (12987-1-AP, 1:5000),
α-SMA (67735-1-Ig, 1:20000) and β-tubulin (10094-1-AP, 1:5000)
antibodies were purchased from Proteintech. Nuclear and cytoplasmic
protein extraction kit (P0027) was purchased from Beyotime.
Animal studies
All animal experiments were conducted following approval from the Animal
Care and Use Committee of Shandong Provincial Hospital Affiliated to
Shandong First Medical University (NO.2023-020) and in strict accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. C57BL/6J mice underwent UUO or sham operation as
previously described16. In brief, the UUO group
underwent ligation of left ureter with 4-0 silk under general
anesthesia, while the sham operation followed a similar procedure but
without the ureteral ligation. The mice were randomized into three
groups (n = 6 in each group): (1) Sham, mice that underwent sham
surgery, (2) UUO + vehicle, mice with UUO that treated with vehicle, (3)
UUO + icariin, mice with UUO that treated with icariin (50 mg/kg/day) by
gavage for 14 consecutive days. The dose of icariin administration was
chosen based on published literatures14,17 and
preliminary experiments.
Biochemical analysis
The levels of serum creatinine (Cr) and blood urea nitrogen (BUN) were
examined by an automatic biochemical analyzer using commercial kits.
Histopathological analysis
Kidney tissues were immersed in 4% paraformaldehyde solution for
fixation, followed by dehydration and embedding in paraffin.
Subsequently, the tissues were sectioned into 4 μm slices and subjected
to H&E staining for morphological analysis or Masson’s trichrome
staining for fibrotic analysis. Tubular injury was characterized by the
presence of sloughed tubules, formation of casts, tubular dilation,
degeneration, and atrophy. The severity of the injury was assessed using
a scoring system based on the proportion of tubular involvement (0, no
injury; 1, < 25%; 2, 25%–50%; 3, 50%–75%, and 4,
> 75%). Renal fibrosis was quantitatively assessed by
analyzing the percentage of stained area in randomly chosen fields
(400×) using Image J software. All samples were examined in a blinded
fashion.
Immunofluorescence and immunohistochemistry
Immunofluorescent and immunohistochemistry analyses were conducted on
paraffin-embedded kidney tissues sections. After standard
deparaffinization, rehydration, and blocking steps, the sections were
incubated with a primary antibody overnight at 4°C. Subsequently, they
were washed with PBS and exposed to either fluorescence-conjugated or
HRP-conjugated secondary antibodies for 1.5 hours at room temperature.
Images were captured by either fluorescent or optical microscope.
Transmission electron microscope (TEM)
The TEM was used to visualize the ultrastructural characteristics of the
mitochondria. The kidney tissues were fixed in fresh 2.5%
glutaraldehyde solution at 4°C for 2 hours, followed by dehydration,
embedding, and sectioning before observing the mitochondrial
ultrastructure. The percentage of swollen mitochondria was determined as
previously described18.
Malondialdehyde (MDA) content and superoxide dismutase
(SOD)activity assays
Kidney tissues were collected, homogenized, and then centrifuged at
10,000× g for 30 minutes. The resultant supernatant was subjected to MDA
assay kit analysis following the manufacturer’s protocol (A003-1-2,
Jiancheng). The activity of SOD enzyme was also evaluated following the
user’s recommendations (A001-3-2, Jiancheng).
Cell culture and viability assay
The human renal proximal tubular epithelial (HK-2) cells were cultured
in DMEM/F12 medium with 10% fetal bovine serum (FBS) under 37°C, 5%
CO2 conditions. The cells were subjected to serum deprivation for 12
hours, followed by treatment with TGF-β1 (10 ng/ml) in the presence or
absence of icariin (50μМ) for 24 hours. For authenticate reverse, the
cells were pretreated with ML385 (Nrf-2 inhibitor, 5μM) for 1 hour,
before being incubated with icariin and TGF-β1 for the specified
duration.
The viability of HK-2 cells was assessed using a CCK-8 assay. The cells
were plated in 96-well plates and exposed to icariin (10, 25, 50, 100,
200 and 400 μM) for 24 hours, followed by incubation with CCK-8 solution
at 37°C for 2 hours. The absorbance at 450 nm was then determined using
a microplate reader.
Protein extraction and western blot analysis
Total or nuclear proteins were extracted following the respective
protocols and then separated by SDS-PAGE gel, subsequently transferred
to PVDF membrane (Merck Millipore, Germany). After blocking with 5% BSA
for 1 hour, the membranes were subjected to overnight incubation with
primary antibodies at 4°C, and subsequently incubated with HRP-secondary
antibodies for 2 hours at room temperature. The ECL system was utilized
to identify specific protein bands, and the grayscale intensity of the
bands was subsequently quantified by Image J software.
RNA extraction and quantitative real-time PCR
The total RNA was extracted from tissues or cells using Trizol reagent
(Takara, Japan) and then reverse transcribed into cDNA using reverse
transcription kits (Takara, Japan). Amplification was conducted using
SYBR Green real-time quantitative PCR system. Ct values were used to
determine the relative expression level of target mRNAs that were
normalized to
β-actin.
Primers were designed as follows:
GACGTGGAACTGGCAGAAGAG (forward) and TTGGTGGTTTGTGAGTGTGAG (reverse) for
TNF-α;
GCAACTGTTCCTGAACTCAACT (forward) and ATCTTTTGGGGTCCGTCAACT (reverse) for
IL-1β;
TCCATCTGCCCTTCAGGAACA (forward) and GGAAGGCAGTGGCTGTCAAC (reverse) for
IL-6;
TGACGTGGACATCCGCAAAG (forward) and CTGGAAGGTGGACAGCGAGG (reverse) for
β-actin.
Measurement of ROS and mitochondrial ROS (mtROS)
To observe intracellular ROS or mtROS, HK-2 cells were stained by
DCFH-DA (S0033S, Beyotime) or MitoSOX Red (40778ES50, Yeasean)
fluorescent probes for 30 minutes at 37 °C. After staining, the cells
were washed and examined under a fluorescence microscope.
Measurement of mitochondrial membrane potential (MMP)
The mitochondrial membrane potential of HK-2 cells was assessed by JC-1
staining following the protocol of the detection kit (C2006, Beyotime).
The fluorescence intensity was detected by fluorescence microscope. At
elevated membrane potential (polarized mitochondria), JC-1 exists as an
aggregated state and emits red fluorescence. Conversely, at decreased
membrane potential (depolarized mitochondria), JC-1 exists as a
monomeric state and emits green fluorescence. The ratio of red to green
fluorescence intensity was used to represent the mitochondrial membrane
potential.
MitoTracker Red Staining
The mitochondria in HK2 cells was visualized using MitoTracker Red dye
(40741ES50, Yeasean) following the manufacturer’s protocol, and the
nuclei were visualized using Hoechst staining (C1028, Beyotime). All
images were viewed under a confocal microscope. The length of
mitochondria was determined by measuring 30 randomly selected cells in
each experiment using Image J software.
RNA Sequencing
RNA was extracted from kidney tissues using the Trizol reagent and then
transferred to Hangzhou KAITAI Biotechnology Co. for library
construction and sequencing. Gene expression was quantified using the
FPKM value. Genes meeting the criteria of p<0.05 and log2(fold
change) >1 were differentially expressed genes. Gene
Ontology (GO) analysis was conducted with a public online database.
Statistical analysis
Data are expressed as mean ± SEM and analyzed using GraphPad Prism.
Comparisons between two groups were performed using the two-tailed
unpaired Student’s t-tests or Mann-Whitney U tests. One-way ANOVA test
was used for comparisons among multiple groups. P < 0.05 was
considered statistically significant.
Results:
Icariin ameliorated renal function and pathological
lesions in UUO mice.
To explore the effect of icariin against tubulointerstitial fibrosis, an
experimental UUO-induced fibrosis mouse model was employed, followed by
the treatment of icariin (50 mg/kg/day) for 14 days post-surgery. Body
weight was assessed on day 1 and again before sacrifice. As depicted in
Figure 1C, icariin reduced the weight loss induced by UUO. Compared with
the sham-operated group, UUO mice displayed a notable increase in the
levels of serum BUN and Cr, while icariin effectively reduced BUN and Cr
levels (Figure 1D,E). In terms of histopathological features (Figure
1F-H), H&E staining revealed that UUO mice developed typical
pathological lesions, including extensive renal tubular atrophy or
expansion, interstitial cell proliferation and inflammatory cells
infiltration. Masson’s staining revealed a heavy deposition of collagen
in the obstructed kidneys of UUO mice. In parallel with histological
changes, western blot further confirmed more severe fibrosis and renal
injury in UUO mice, as demonstrated by remarkably enhanced expression of
a-SMA and collagen I compared with the sham-operated mice (Figure 1I).
After icariin treatment, renal tubular damage and extracellular matrix
deposition were remarkedly alleviated, which were consistent with the
changes in serum markers mentioned above. Collectively, these results
indicated that icariin exerts a protective effect in TIF induced by UUO.