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.