Aristolochic acids (AAs) are well-known nephrotoxins, while information regarding the attenuation of AAs-induced toxicity is scarce. AAs primarily damage renal tubulointerstitium, culminating in profound tubulointerstitial fibrosis (TIF), end-stage kidney disease (ESKD) and fatal urothelial cancer [1
]. TIF is the outcome of multiple forms of chronic kidney disease (CKD), which is commonly caused by diabetes, hypertension and nephrotoxins [2
]. The irreversible renal fibrosis and sustained retention of uremic toxins from the dietary-protein intake play a pivotal role in CKD pathogenesis [3
]. In parallel with a decline in renal function, a myriad of uremic retention solutes exhibit pro-oxidant, pro-inflammation and pro-fibrotic effects on renal injury, leading to a vicious cycle [4
]. That is, an accumulation of uremic toxins following impaired renal clearance inhibits renal metabolic capacity and induces progressive TIF [6
]. Emerging evidences indicate the protein-bound non-dialyzable uremic toxins such as p
-cresyl sulfate (PCS) and indoxyl sulfate (IS) are intricately associated with oxidative injury and fibrogenesis in CKD and diverse organ systems [8
]. Furthermore, TIF is reminiscent of epithelial mesenchymal transition (EMT), excess deposition of extracellular matrix (ECM) components, collagens, SMAD 2/3-dependent and SMAD-independent JNK/ERK pathways in transforming growth factor-β (TGF-β) family signaling [10
]. Nonetheless, a comprehensive treatment to inhibit AAs-induced TIF signaling cascades and PCS/IS accumulation is still lacking.
Propolis, a natural product rich in prenylated flavonoids, has been reported to exert versatile biological activities in our prior research, including anti-inflammasome [12
], anti-oxidant [12
], anti-diabetes [13
] and anti-cancer properties [15
]. Our recent rodent model of metabolic endotoxemia with obesity demonstrated that elimination of reactive oxygen species (ROS) using antioxidant therapy ameliorates kidney fibrosis as well as inflammation [16
]. Considering pro-oxidant and pro-fibrotic effects of uremic retention solutes on human organ systems, the natural antioxidant PE may serve as a potential breakthrough treatment for AAs-induced nephropathy (AAN). Despite previously documented implications, the therapeutic effect of PE on TIF and PCS/IS retention remains elusive. Thus, we developed an experimental mouse model to explore therapeutic targets in AAN. We hypothesized that PE treatment in AAN model attenuated not only SMAD 2/3-dependent pathways but also SMAD-independent JNK/ERK activation in the signaling cascades of TGF-β family, contributing to a reversal of TIF and uremic burden.
CKD is a critical health concern and economic burden globally [2
]. As a result of the current huge unmet need for new and reliable therapies, CKD prevalence is expected to increase. The reasons why CKD is so difficult to treat are intricate. Irreversibility of renal fibrosis, decline in glomerular filtration rate, and retention of uremic solutes reflect the trilogy of CKD progression. A progressive TIF due to various etiologies is an irreversible common process of CKD, ultimately resulting in renal atrophy, ESKD, uremic toxin-related morbidities and mortality [4
]. Here, we provided a comprehensive treatment for AAI induced TIF and uremic solute retention through the investigation of AAN model. Conventionally, organ fibrosis has been considered potentially irreversible. However, compelling evidences from animal models and human studies have demonstrated that if the injury is removed, early stages of organ fibrosis may be reversible [23
]. There is a clear need for safe and effective therapeutic regimens focusing on TIF to timely preserve renal function in patients with AAs intoxication, preventing from ESKD and fatal urothelial cancer in clinical medicine. PE, a potent natural antioxidant with multi-components, serves as a multi-targeted regimen in inflammation [12
], diabetes [13
], and cancers [15
]. Our recent study unraveled that the scavenging of ROS by antioxidants ameliorates obesity-related kidney fibrosis [16
]. Considering pro-oxidant and pro-fibrotic effects of AAs and uremic toxins on human organ systems, PE could be a potential breakthrough treatment for AAs toxicity and CKD progression. Through testing the therapeutic effects of PE on TIF and PCS/IS retention in our AAN model, major breakthroughs were achieved and novel findings markedly advance our understanding of irreversible process in CKD (Figure 5
). Several important findings in this work deserve a more in-depth discussion.
To date, AAN remains regularly reported all over the world [24
]. The incidence of AAN is probably highly underestimated due to the presence of AAs in traditional herbal remedies worldwide and the poor awareness of the disease. The majority of AAN patients presented BW loss, anemia, mild proteinuria, hypertension, a rapid decline of renal function and reached ESKD at the end of the seven-year follow up [25
]. The progression of CKD can continue despite removing exposure to AAI [21
]. Furthermore, AAI also suppressed expression of epidermal growth factor in tubular epithelial cells, indicating lack of tubular regeneration [21
]. Concerning this critical issue, typical manifestations of AAI induced progressive CKD are increasingly recognized through both human and animal models. Macroscopically, intoxicated kidneys were shrunken, asymmetrical and irregular in cortical outline. Biopsies of human cases revealed interstitial inflammatory infiltrate and evidence of tubular necrosis. Microscopically, kidneys with AAN depicted extensive paucicellular interstitial fibrosis with atrophy and loss of tubules initiating from the peripheral cortex and progressing towards the deep cortex [26
]. Progressive renal fibrosis featured with prominent tissue scarring, glomerulosclerosis, tubular atrophy and TIF is considered as a final common process of CKD leading to ESKD. Irrespective of the nature of the initial renal injury, the degree of TIF correlates well with the decline of the renal function and long-term prognosis. To mimic typical findings of TIF in humans with advanced CKD, we developed an experimental mouse model of AAN. Our data pointed out that plasma Cr was positively correlated with BUN (r
= 0.59; p
< 0.05), PCS (r
= 0.51; p
< 0.05) and IS (r
= 0.55; p
< 0.05), and negatively correlated with UCr (r
= −0.51; p
< 0.05) and UUN (r
= −0.61; p
< 0.01) (Figure 1
B). The above results demonstrated that AAN induced renal function decline led to the accumulation of various uremic toxins in the circulation and impaired urinary excretion of waste products (Figure 1
). It is a vicious cycle that uremic retention solutes following impaired renal clearance could exhibit pro-oxidant [28
], pro-inflammation [29
] and pro-fibrotic effects [30
] on renal tubulointerstitium, leading to progressive TIF [31
]. As expected, mice in AAN group exerted the most prominent TIF and the highest expressions of α-SMA, collagen IaI/IV, SMAD 2/3 and JNK/ERK than the other groups (Figure 4
). Moreover, BW of AAN mice is negatively correlated with BUN (r
= −0.71; p
< 0.01), Cr (r
= −0.52; p
< 0.05), PCS (r
= −0.78; p
< 0.01) and IS (r
= −0.84; p
< 0.01), respectively (Figure 1
C). Our findings imply that cachexia is present in the uremic milieu, corresponding with previous clinical observation and basic research [17
Molecular mechanisms involved in TGF-β overexpression remain to be fully elucidated. Pozdzik et al. reported that vimentin and α-SMA-positive cells accumulated in the renal interstitium, along with an overexpression of TGF-β in rats with AAN [32
]. TGF-β1 upregulation has emerged as the key driver of matrix synthesis, inhibition of matrix degradation and stimulator of myofibroblast activation [33
]. It has become more evident that profound reduction of peritubular capillaries in AAN resulting in hypoxia and tubular cell death, resulting in further progression to fibrogenesis [1
]. TGF-β1 is a multi-functional cytokine that plays a fundamental role in regulating inflammation, cellular behaviors, fibrotic EMT, fibroblast viability and collagen degradation [35
]. Smad2 and Smad3 serve as the two major downstream regulators that promote TGF-β1-mediated tissue fibrosis [37
]. Deletion of Smad3 has been demonstrated to protect against several kidney disease, including AAN [38
]. It is now well accepted that TGF-β/Smad signaling is the major pathway for renal fibrogenesis. Nevertheless, TGF-β1 can induce renal fibrosis in both canonical (SMAD-dependent) and non-canonical (SMAD-independent) signaling pathways, resulting in activation of myofibroblasts, excessive production of ECM and inhibition of ECM degradation [39
]. TGF-β1-mediated non-SMAD pathways were involved in various branches of mitogen-activated protein kinase (MAPK) pathways, including the JNK and ERK signaling cascades [41
]. In response to TGF-β1, the co-operation between SMAD and non-SMAD signaling pathways determines cell fate of fibrotic EMT. In our study, PE treatment did ameliorate TIF in Masson’s trichrome stained tissue (Figure 4
A). In addition, PE treatment suppressed α-SMA expression and deposition of collagen IaI and IV in the process of fibrotic EMT (Figure 4
B). Furthermore, PE treatment attenuated not only SMAD 2/3-dependent pathways, but also SMAD-independent JNK/ERK activation in the signaling cascades of TGF-β family (Figure 4
C–E). In light of this, PE is considered to be a multi-targeted agent that exerts therapeutic effects on progressive CKD.
Our study has several limitations. To begin with, the amount of food intake and metabolic efficiency were not measured. Next, further parameters were not evaluated for renal function decline, e.g., proteinuria, cystatin C, β-trace protein, inulin and iohexol. In addition, expressions of picrosirius red staining and TGF-Beta proteins in the kidneys were not determined in our AAN models.
In conclusion, our research elucidates AAI damages tubular epithelium by TGF-β family signaling pathways and fibrotic EMT, leading to TIF, ECM deposition and PCS/IS retention. Notably, the natural product PE exerts nephroprotetive effects through the regression of renal fibrosis, thereby improving renal clearance of uremic toxins. Furthermore, PE treatment attenuates AA toxicity through disrupting not only SMAD 2/3-dependent pathways but also SMAD-independent JNK/ERK activation in the intricate cascades of TGF-β family. In light of multi-faced toxicity of AAs, PE may be capable of developing a new potential drug to treat CKD patients exposed to AAs in the future.
4. Materials and Methods
4.1. Creating Animal Models to Mimic TIF in Humans with Progressive CKD
To mimic TIF in humans with progressive CKD, we developed an AAN model using seven-week-old C57BL/6 mice by intraperitoneal (IP) injection of aristolochic acid I (AAI, Sigma Aldrich, Wuxi, China) 3 mg/kg once every 3 days for 6 weeks. The details of the study protocol and materials for AAN were also mentioned previously [8
]. All animals were randomly divided into four groups of six mice each: vehicle-treated control group (normal renal function), PE-treated group (normal renal function), AAN group and PE-treated AAN group. PE was collected in Taiwan using propolis collectors as described previously [12
]. The dosage and experimental administration were presented here: Group I (control; IP injection of vehicle (DMSO, Sigma Aldrich, China) once every 3 days for 6 weeks and orally administered with vehicle (distilled water, 200 μL) everyday, 12 weeks; n
= 6). Group II (PE alone; IP injection of vehicle and orally administered with PE (0.2 mg/kg in 200 μL vehicle), 12 weeks; n
= 6). Group III (AAI treatment; IP injection of AAI and orally administered with vehicle (200 μL) everyday, 12 weeks; n
= 6) and Group IV (PE + AAI treatment; IP injection of AAI and orally administered with PE, 12 weeks; n
= 6). All mice were maintained at temperature (23 ± 3 °C) and relative humidity (40–60%) on a 12 h light/dark cycle and allowed free access to standard rodent chow and tap water. Animal experiments were handled according to the guidelines of the Institutional Animal Care and Use Committee of the National Ilan University (approval number: 107-17; date: 16 December 2018) and NIH Guides for the Care and Use of Laboratory Mouse body weight was measured at least once a week throughout the 12 weeks. Kidneys were removed at termination and directly compared.
4.2. Tissue Preparation for Histopathological Evaluation of H&E Stain
Mice were anaesthetized via the inhalation of isoflurane and euthanized by cervical dislocation. Kidneys were removed and fixed in 10% formalin. Specimens were embedded in paraffin and sliced into 2–3 μm in thickness. Subsequently, the kidney tissues were stained with Hematoxylin-eosin (H&E stain). The images were captured using a Nikon Digital Camera Microscope (Nikon, Tokyo, Japan).
4.3. Masson’s Trichrome Staining Method
Masson’s trichrome staining method was used to determine the extent of collagen deposition and fibrosis in mouse kidney tissues. In the corresponding area, H&E staining of the adjacent paraffin section was performed for comparisons of tissue morphology. The experiments were conducted as follows: sections were first deparaffinized and rehydrated in ethanol/water solutions then post-fixed with Bouin’s solution for 1 h at room temperature. The fixation buffer was removed, and slides were stained with iron hematoxylin, Biebrich scarlet-acid fuchsin, and phosphomolybdic-phosphotungstic acid sequentially for 10 min per stain. Slides were then stained with Aniline blue. Finally, slides were washed in 10% acetate solution for 3–5 min and mounted in the mounting medium for observations to be made. The slices were visualized by an Olympus BX-41 microscope.
4.4. Biochemical Assays of Urea nitrogen, Creatinine (Cr), PCS and IS
Plasma and urine concentrations of urea nitrogen and Cr were determined by MeDiPro CREA (BC-0017) and MeDiPro BUN (BC-0012). PCS and IS in plasma samples were analyzed by a Mass Spectrometer Analytical System (Thermo Fisher Scientific Inc., Waltham, MA, USA) and UHPLC analytical system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Xcalibur software (version 2.2, Thermo-Finnigan Inc., San Jose, CA, USA) was used for method setup and data processing.
4.5. Western Blot Analysis
The kidney tissues were homogenized in radioimmunoprecipitation assay buffer (Millipore # 20-188) and incubated at 4 °C for 30 min. The supernatant was collected after centrifugation for 20 min at 14,000 rpm, 4 °C. Protein concentration was assessed by a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Sixty micrograms of protein were electrophoresed on SDS/PAGE gels. The gels were transferred to a PVDF membrane (Millipore, #IPVH00010) and incubated in 1% skim milk blocking buffer overnight at 4 °C. The antibodies used included anti-α-smooth muscle actin (SMA) (GeneTex, GTX100034, 1:1000, 4 °C, o/n), Col IaI (OriGene, TA309097, 1:1000, 4 °C, o/n), Col IV (Abcam, ab6586, 1:1000, 4 °C, o/n), p-Smad2/3 (Cell Signaling, 8828S, 1:1000, 4 °C, o/n), Smad2/3 (Cell Signaling, 5678S, 1:1000, 4 °C, o/n), p-JNK (Cell Signaling, 9255s, 1:1000, 4 °C, o/n), JNK (Cell Signaling, 9252, 1:1000, 4 °C, o/n), p-ERK1/2 (Cell Signaling, 9106S, 1:1000, 4 °C, o/n), ERK1/2 (Signaling, 9102, 1:1000, 4 °C, o/n) and β-actin (Santa Cruz, sc-47778, 1:2000, 4 °C, o/n). Appropriate horseradish peroxidase-conjugated secondary antibody was incubated for 60 min at RT. Positive immunostaining was detected with the Super Signal West Pico enhanced chemiluminescence substrate (Thermo Fisher Scientific, Hudson, NH, USA). Images were digitally acquired by LAS-3000 Plus instrument (Fuji Film, Tokyo, Japan). The reactions were quantified and normalized to the β-actin control band using ImageJ 1.48q (National Institutes of Health, Bethesda, MD, USA).
4.6. Statistical Analysis of Data
All data are expressed as the mean ± SEM using the GraphPad Prism Program (GraphPad, San Diego, CA, USA) or SPSS version 22.0 (IBM, Armonk, NY, USA). Spearman’s test with linear regression analysis was used for correlation data. Quantitative data were analyzed with Kruskal and Wallis test. A p-value < 0.05 was considered statistically significant for each of the experiments.