Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.
INTRODUCTION
Even though various classes of anticancer drugs are available, 60% of cancer patients receive radiation therapy. High-energy radiation, such as X and γ rays can cause serious damage to cancer cells, regardless of whether they are drug-resistant or not. However, this procedure inevitably affects normal cells in the tumor environment (1). X and γ rays are categorized as ionizing radiation and reportedly induce atom ionization causing serious cellular damage, including DNA damage, lipid peroxidation, and protein oxidation (2). In humans, exposure to radiation doses higher than 0.5 Gy leads to acute radiation syndrome (ARS), including hematopoietic, gastrointestinal, and neurovascular sub-syndromes (3). Therefore, animal models of ARS have been developed to identify effective drugs to alleviate ARS-associated symptoms (3–6). On the other hand, several previous clinical and preclinical studies applying bone marrow transfusion suggested that total-body irradiation (TBI) induced late biological effects, such as pulmonary and cardiac fibrosis, renal dysfunction, malfunction of the immune system, and tumorigenesis (7–11).
Amphiregulin (AREG) is an epidermal growth factor receptor (EGFR) ligand synthesized as a transmembrane precursor. Its activation occurs via juxtacrine signaling during wound healing and fibrotic processes (12), after which AREG is generated from pro-AREG by disintegrin and metalloproteinase (ADAM) 17-mediated cleavage. This results in the release of a mature EGFR ligand, which is known to act in an autocrine, paracrine, or endocrine fashion (12, 13). Previous studies have focused on the role of AREG in managing inflammation and tissue repair (14–16). AREG is also known to be involved in fibrotic tissue formation mediated by transforming growth factor-β1 (TGF-β1), as uncontrolled and exaggerated wound healing processes can lead to tissue fibrosis (17–21). Importantly, AREG silencing using small interfering RNA (siRNA) is sufficient to inhibit fibroblast proliferation and to reduce collagen accumulation in the lungs of TGF-β1 transgenic mice (22).
The role of AREG in TBI-mediated processes has not been studied extensively in the past. Shao et al. suggested that AREG plays a critical role in intestinal epithelial growth after 12 Gy TBI. Accordingly, expression of Areg messenger (m)RNA was elevated in the intestines of C57BL/6 mice after irradiation, whereas that of other EGFR ligands, including Egf and Tgfa mRNAs, was not significantly altered (23). However, whether AREG function is linked to radiation-induced wound healing processes or fibrosis is yet to be investigated. We thus aimed to investigate the changes in AREG expression in different organs after TBI, as well as the role of AREG in radiation-induced fibrosis.
RNA interference (RNAi) technology is employed to inhibit translation of mRNAs into proteins using complementary sequences to certain mRNA segments (24). By targeting key proteins involved in disease development, nanomedicine-based approaches employing RNAi technology are considered emerging therapeutic strategies (25). However, unintended innate immune stimulation and off-target effects are challenges to RNAi-based approaches (26, 27). To overcome these limitations, we developed a novel siRNA modality conjugated with a hydrophobic hydrocarbon and hydrophilic polymer at the 5′ and 3′ ends of the siRNA sense strand, respectively. As the modified siRNA molecules form micelles in physiological solution without any additives or processing steps, they are referred to as self-assembled-micelle inhibitory RNA (SAMiRNA). In contrast to naked siRNA or encapsulated liposomes, SAMiRNA was shown not to elicit innate immune responses (19). Moreover, SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) inhibited the expression of Areg mRNA and alleviated pulmonary or renal fibrosis development upon successful delivery to target organs (19, 28).
Several previous studies utilized RNAi technology to alleviate radiation-induced organ damage. Knockdown of Apc mRNA, which is a negative regulator of Wnt, resulted in Wnt pathway activation and mitigation of gastrointestinal syndrome induced by whole-abdomen irradiation (29). Delivery of TGF-β1 or Smad3 siRNA to the lungs or skin, respectively, reduced radiation-induced injuries (30, 31). Regulating expression of specific genes involved in radiation response or fibrosis development has recently gained increasing research interest (32, 33).
The current study was conducted to investigate whether SAMiRNA-mAREG is applicable to radiation-induced fibrosis treatment. For this, mRNA expression levels of Areg and fibrosis markers were measured in various organs at different time points after TBI. We also examined possible correlations between AREG and fibrotic marker expression levels using intravenous (i.v.) injection of SAMiRNA-mAREG in 6 Gy TBI mice. After administration of SAMiRNA-mAREG, we investigated the expression levels of AREG and α-smooth muscle actin (α-SMA), as well as collagen deposition. We examined the role of AREG in TBI-induced fibrosis and the potential of SAMiRNA-mAREG as a novel therapeutic agent for the treatment of radiation-induced kidney fibrosis.
MATERIALS AND METHODS
Animal Experiments and Irradiation
All experimental methods and protocols were approved by the Committee on the Ethics of Animal Experiments of the siRNAgen Therapeutics Corporation (Daejeon, Republic of Korea, siRNAgen Therapeutics IACUC, AEC-20200319-0002). The siRNAgen Therapeutics-IACUC was approved by the Animal and Plant Quarantine Agency (Gimcheon-si, Gyeongsangbuk-do, Republic of Korea). We used 7-week-old male C57BL/6 mice for the TBI studies, which were maintained in individual cages. Five mice were included in each experimental group. Single-dose TBI at a dose rate of 1 Gy/min was performed using a Gammacell 3000 Elan irradiator (Nordion, Canada) provided by the KAIST Analysis Center for Research Advancement (KARA) at the Korea Advanced Institute of Science and Technology (KAIST, Daejeon, Republic of Korea). The mice were euthanized at the times indicated, after which the organs were harvested for quantitative reverse transcription (qRT)-PCR and histological analyses.
SAMiRNA Design and Treatment
SAMiRNA was manufactured as described previously (19). SAMiRNA-mAREG (30 mg/mL) was diluted to a final concentration of 5 mg/kg for injections using Dulbecco's phosphate-buffered saline (DPBS; Welgene, Gyeongsan, Republic of Korea). The molecular weights of the SAMiRNA-mAREG sense and antisense strands were measured using the Axima LNR MALDI-TOF MS system (Kratos Analytical, Shimadzu, UK). The purity of SAMiRNA-mAREG single strand preparations was analyzed by high-performance liquid chromatography (Shimadzu, Kyoto, Japan), and was determined to be ≥40% of sense and antisense strands, respectively. The osmolality of SAMiRNA-mAREG was in the range of 320 ± 30 mOsmol/kg, which was measured using an Osmomat 3000 (Gonotec, Berlin, Germany). The size of SAMiRNA-mAREG was analyzed using Zetasizer Nano-ZS (Malvern Panalytical, UK) and qNano Gold instruments (Izon Science Ltd., Christchurch, New Zealand), which was determined to be ≤200 nm. For 6 Gy TBI experiments, SAMiRNA-mAREG at 5 mg/kg in 100 µL of DPBS or 100 µL of DPBS were administered intravenously biweekly (from weeks 4 to 12 after TBI) or weekly (from weeks 13 to 23 after TBI) via the tail veins of the mice. The dosing schedule was determined on the basis of results from 4 Gy TBI experiments and weeks 4 and 8 after TBI of 6 Gy models.
qRT-PCR
Total RNA was extracted using the AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) or the AccuPrep Blood RNA Extraction Kit (Bioneer) according to the manufacturer's protocol. After determining the RNA concentration based on the absorbance at 260 nm (Tecan, Groedig, Austria), cDNA was synthesized from 1 µg of total RNA using the AccuPower Rocket-Script Cycle RT PreMix (Bioneer) and AllInOneCycler PCR system (Bioneer). AccuPower 2× GreenStar qPCR MasterMix (Bioneer) and an Exicycler 96 (Bioneer) were employed to quantify cDNA targets. Expression levels of mRNAs encoding fibrotic markers, inflammatory regulators, and reference genes were quantitatively analyzed using qRT-PCR. Primer sequences are listed in Supplementary Table S1 (471_rare-197-05-07_s01.docx) ( https://doi.org/10.1667/RADE-21-00220.1.S1). β-actin (Actb) and hypoxanthine phosphoribosyltransferase 1 (Hprt) were used as housekeeping genes. Data were analyzed using the 2–ΔΔCt method.
Immunofluorescence (IF) Staining
Paraffin-embedded tissue sections were deparaffinized, submerged in 1 mM ethylenediaminetetraacetic acid at pH 8.0 and 90°C, and allowed to cool for 30 min for antigen retrieval. The sections were incubated in 0.3% Triton X-100 in PBS for 5 min at 20°C for permeabilization. Nonspecific antibody-binding sites were blocked with 5% normal goat serum (Cell Signaling Technology, Danvers, MA) in PBS for 30 min at 20 °C. Next, fluorescence-conjugated anti-AREG and anti-α-SMA primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; 1:200 dilutions) were added to the tissue sections, which were subsequently incubated for 16 h at 4°C. After three sequential washing steps with PBS, the tissue sections were mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Burlingame, CA). Confocal laser scanning images were obtained using a Dragonfly confocal microscopy system (Andor, Belfast, Northern Ireland).
Masson's Trichrome Staining
Kidney tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. The samples were then sectioned and subjected to Masson's trichrome staining to detect collagen fibers following standard procedures. Images were acquired using a Nikon Eclipse Ts2 microscope (Nikon, Tokyo, Japan). Fibrotic areas were quantified by measuring the blue areas using Fiji software (45).
Measurements of BUN and sCr Levels
To measure BUN and sCr levels, whole blood was collected in BD Microtainer blood collection tubes (BD Biosciences, Franklin Lakes, NJ) as the mice were sacrificed, and blood samples were incubated at 20°C for 15 min to promote clotting. Whole blood samples were then centrifuged at 3,000 rpm and 4°C for 10 min, and the supernatants were used to measure BUN and sCr levels using an automatic analyzer (Hitachi 7180; Hitachi High-Technologies Co., Tokyo, Japan).
RESULTS
Fibrotic Phenotypes are not Induced at 12 Weeks after 4 Gy TBI
Previous reports have suggested that fibrosis could develop in several organs as a side effect of radiation therapy or in irradiated experimental animals (7, 34, 35). To determine the molecular mechanisms of radiation-induced fibrosis and to develop potential treatment strategies, we initially used 4 Gy TBI to establish a fibrosis model using C57BL/6 mice. Based on this model, we investigated Areg, Acta2, and Col1a1 expression in various organs at 1, 2, 4, 8, and 12 weeks after 4 Gy TBI (Fig. 1). Areg expression was increased in the kidney, intestine, and spleen at 8 and 12, 1, and 12 weeks after 4 Gy TBI, respectively (Fig. 1A). However, increased Acta2 and Col1a1 expression was not observed in the kidney, lung, liver, intestine, and spleen at 12 weeks after 4 Gy TBI (Fig. 1B and C). Moreover, we discovered that the basal mRNA expression level of Areg was very low in the heart, blood, and bone marrow of C57BL/6 mice. Collectively, these data suggest that 4 Gy TBI led to increased Areg expression levels in the kidney, intestine, and spleen, which did not lead to fibrosis formation in C57BL/6 mice.
FIG. 1
Gene expression analysis of the kidney, lung, liver, intestine, and spleen of a 4 Gy total-body irradiation (TBI) mouse model. Panel A: Amphiregulin (Areg) expression in the kidney, lung, liver, intestine, and spleen of C57BL/6 mice subjected to 4 Gy TBI was examined by quantitative reverse transcription (qRT)-PCR. Panels B and C: Expression levels of the fibrosis markers α-smooth muscle actin (Acta2) and collagen type I alpha 1 (Col1a1) were evaluated in various organs isolated from 4 Gy TBI model mice. Relative expression levels were calculated for each time point, with the values obtained using non-irradiated mice set to 100. For quantifications, data (mean ± SEM) were analyzed by t-tests. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to non-irradiated mice.
Kidney Fibrosis and Inflammation Develop at 24 weeks after 6 Gy TBI and can be Inhibited by SAMiRNA-mAREG Treatment
Following gene expression analysis after 4 Gy TBI, we investigated whether an elevated dose (6 Gy) and an extended post-TBI period (24 weeks) could elicit fibrosis in C57BL/6 mice. Areg expression in the kidney was elevated at 4, 8, 12, and 16 weeks after 6 Gy TBI, but the expression of Tgfb1, which leads to reciprocal activation of AREG signaling (16, 17), and fibrosis markers was not increased (Fig. 2A and B). However, we found that the expression levels of Areg and Tgfb1 were increased at 24 weeks after 6 Gy TBI (Fig. 2D). Furthermore, expression of the fibrotic markers, Acta2, Fn1, Col1a1, and Col3a1 was also increased at 24 weeks (Fig. 2E). Taken together, these results suggest that kidney fibrosis had developed over the course of 24 weeks in response to 6 Gy TBI.
FIG. 2
Expression of renal fibrosis and inflammation markers is induced at 24 weeks after 6 Gy total-body irradiation (TBI), which can be attenuated by self-assembled-micelle inhibitory RNA targeting mouse amphiregulin mRNA (SAMiRNA-mAREG) injection. Panels A and B: Renal expression levels of Areg, transforming growth factor-β1 (Tgfb1), α-smooth muscle actin (Acta2), and collagen type I alpha 1 (Col1a1) were analyzed by quantitative reverse transcription (qRT)-PCR at 4, 8, 12, and 16 weeks after 6 Gy TBI. *p < 0.05 and ***p < 0.001 compared to non-irradiated mice. Panel C: A graphic of self-assembled-micelle inhibitory RNA (SAMiRNA) structure. Panels D-F: At 24 weeks post-TBI, expression levels of Areg, Tgfb1, fibrosis-related genes (Acta2, Fn1, Col1a1, and Col3a1), and inflammation regulators (nuclear factor-κB subunit 1; Nfkb1, tumor necrosis factor-α; Tnf, connective tissue growth factor; Ccn2, and vascular cell adhesion molecule 1; Vcam1) after TBI with or without SAMiRNA-mAREG treatment were analyzed by qRT-PCR. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to non-irradiated or TBI-only mice. Expression of Tnf was not significantly (ns) reduced by SAMiRNA-mAREG treatments due to large variation between mice in the same group. Relative gene expression levels were calculated for each time point, with the values obtained using non-irradiated mice set to 100. For quantifications, data (mean ± SEM) were analyzed by t-tests.
Our previous studies demonstrated that SAMiRNA-mAREG, the structure of which is depicted in Fig. 2C, exert therapeutic effects on pulmonary fibrosis induced by bleomycin or in TGF-β-transgenic mice, as well as on UUO- or adenine diet-induced renal fibrosis and inflammation (19, 28). Therefore, we analyzed the effects of SAMiRNA-mAREG on TBI-induced renal fibrosis and inflammation. TBI-dependent upregulation of Areg expression was significantly inhibited by SAMiRNA-mAREG treatment, and Tgfb1 expression was concomitantly decreased (Fig. 2D). We also confirmed that SAMiRNA-mAREG inhibited TBI-induced Acta2, Fn1, Col1a1, and Col3a1 expression (Fig. 2E). Overexpression of inflammatory mediators such as Nfkb1, Tnf, Ccn2, and Vcam1 was induced by TBI, which was reversed by SAMiRNA-mAREG (Fig. 2F). Expression of Il1b and Icam1, which are other inflammatory markers, was not increased by TBI ( Supplementary Fig. S1 (471_rare-197-05-07_s01.docx); https://doi.org/10.1667/RADE-21-00220.1.S1), and that of Il10, Il17a, and Ifng in the kidneys was very low. These results demonstrated that i.v. injection of SAMiRNA-mAREG ameliorated renal fibrosis and inflammation induced by 6 Gy TBI via knockdown of Areg expression.
Fibrosis is not Induced in the Lung, Liver, Intestine, and Spleen at 24 Weeks after 6 Gy TBI
After fibrosis formation in the kidney in response to 6 Gy TBI had been confirmed, we also evaluated Areg and fibrosis marker expression levels in the lung, liver, intestine, and spleen. The pulmonary expression levels of Tgfb1 and Col1a1 were increased at 8 weeks after TBI, but the observed overexpression was not sustained at later time points (Fig. 3A and B). In the lung, Areg or Acta2 overexpression after TBI was not observed at any of the time points analyzed. In the liver, upregulation of Areg and fibrosis marker expression was detected at 8 weeks after 6 Gy TBI (Fig. 3C and D). However, Areg, Tgfb1, Acta2, and Col1a1 expression was not increased in the liver of 6 Gy TBI mice compared to non-irradiated mice at 24 weeks after TBI.
FIG. 3
No significant activation of amphiregulin (AREG) signaling and fibrosis development is observed in the lung and liver of 6 Gy total-body irradiation (TBI) model mice. Panels A and B: Expression levels of Areg, transforming growth factor-β1 (Tgfb1), α-smooth muscle actin (Acta2), and collagen type I alpha 1 (Col1a1) in the lung at 24 weeks post-TBI. Panels C and D: mRNA expression levels of AREG signaling components and fibrosis markers in the liver of 6 Gy TBI model mice were assessed by quantitative reverse transcription (qRT)-PCR. Relative expression levels were calculated for each time point, with the values obtained using non-irradiated mice set to 100. For quantifications, data (mean ± SEM) were analyzed by t-tests. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to non-irradiated mice.
In accordance with a previous report (23), Areg expression in the intestine was increased by TBI at an early time point (Fig. 4A). Nonetheless, expression levels of Acta2 and Col1a1 in the intestine were not significantly elevated by 6 Gy TBI at the times analyzed (Fig. 4B). Although Areg expression was increased in the spleen at 16 and 24 weeks after TBI, fibrosis was not detected at 24 weeks after TBI (Fig. 4C and D). These results suggest that 6 Gy TBI C57BL/6 mice did not lead to fibrosis formation in the lung, liver, intestine, and spleen at 24 weeks postirradiation.
FIG. 4
Analysis of amphiregulin (AREG) signaling and fibrosis marker expression in the intestine and spleen of C57BL/6 mice in response to 6 Gy total-body irradiation (TBI). Panels A and B: Analysis of Areg, transforming growth factor-β1 (Tgfb1), α-smooth muscle actin (Acta2), and collagen type I alpha 1 (Col1a1) expression in the intestine after TBI. Panels C and D: Expression of Areg, Tgfb1, Acta2, and Col1a1 mRNAs in the spleen of 6 Gy TBI model mice were confirmed by quantitative reverse transcription (qRT)-PCR. Relative expression levels were calculated for each time point, with the values obtained using non-irradiated mice set to 100. For quantifications, data (mean ± SEM) were analyzed by t-tests. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to non-irradiated mice.
SAMiRNA-mAREG Treatment Attenuates 6 Gy TBI-Induced AREG and α-SMA Protein Expression and Collagen Deposition in the Kidney
After confirming the overexpression of mRNAs encoding AREG and fibrosis markers in the kidney in response to TBI, we assessed the protein levels and localization of AREG and α-SMA by IF staining. The results showed that the expression of AREG and α-SMA proteins was upregulated by 6 Gy TBI. Moreover, the proteins were found to be predominantly expressed in the proximal and distal tubules of the kidney (Fig. 5A). We also confirmed that i.v. injection of SAMiRNA-mAREG inhibited overexpression of AREG and α-SMA at the protein level (Fig. 5A and B).
FIG. 5
Confocal microscopy images of kidney tissues isolated from mice at 24 weeks after 6 Gy total-body irradiation (TBI). Panel A: Effects of TBI and self-assembled-micelle inhibitory RNA targeting mouse amphiregulin mRNA (SAMiRNA-mAREG) treatment on AREG and α-smooth muscle actin (α-SMA) expression levels were assessed by immunofluorescence staining. The samples were co-stained for AREG (green) and α-SMA (red), as well as with DAPI to identify the nuclei (blue). Scale bar, 50 µm. Panel B: Fluorescence intensities of the signals in panel A were quantified using Fusion software. *p < 0.05 and **p < 0.01.
Masson's trichrome staining is widely used to characterize renal fibrosis formation and to verify the deposition of collagen, which stains blue (28). As shown in Fig. 6A, the kidneys of TBI mice displayed significant interstitial collagen deposition compared to those of non-irradiated control mice. Masson's trichrome staining also revealed that SAMiRNA-mAREG treatment reduced the TBI-induced collagen deposition (Fig. 6A and B). Collectively, these results demonstrated that SAMiRNA-mAREG treatment led to reduced AREG and α-SMA protein expression and collagen deposition in the kidneys of 6 Gy TBI mice.
FIG. 6
Histological changes in the kidney of total-body irradiation (TBI) model mice. Panel A: Representative Masson's trichrome staining of renal cortex and medulla sections from control, TBI-only, and self-assembled-micelle inhibitory RNA targeting mouse amphiregulin mRNA (SAMiRNA-mAREG)-administered TBI mice at week 24 post-TBI. Scale bar, 100 µm. Panel B: Areas of collagen deposition were analyzed by Fiji software. *p < 0.05.
Kidney Function is not Significantly Affected by 6 Gy TBI
Next, we investigated whether 6 Gy TBI resulted in altered blood urea nitrogen (BUN) and serum creatinine (sCr) levels in C57BL/6 mice. Increased BUN and sCr levels often indicate renal failure (36). We found that BUN levels were decreased by 25% in response to TBI, which slightly recovered after i.v. injection of SAMiRNA- mAREG ( Supplementary Fig. S2A (471_rare-197-05-07_s01.docx); https://doi.org/10.1667/RADE- 21-00220.1.S1). In contrast, neither TBI nor SAMiRNA-mAREG treatment resulted in significantly altered sCr levels ( Supplementary Fig. S2B (471_rare-197-05-07_s01.docx); https://doi.org/10.1667/RADE-21-00220.1.S1). These results suggest that kidney function was not significantly affected in the 6 Gy TBI mouse model.
DISCUSSION
Controlling and preventing radiation-induced side effects is important for patients receiving radiation therapy or for individuals who are subjected to occupational radiation exposure. However, the key proteins involved in radiation-induced kidney fibrosis have not been thoroughly investigated in the past. The results obtained in this study suggest that AREG is a prime candidate for inducing renal fibrosis in TBI. It was previously shown that 4 Gy TBI eliminated the ability of hematopoietic stem cells to develop into T cells in C57BL/6 mice (10). However, 4 Gy TBI did not cause fibrosis in any of the organs analyzed in the course of this study. We demonstrated that AREG expression was elevated in the kidneys of 6 Gy TBI mice, and renal fibrosis and inflammation were observed 24 weeks after TBI. Furthermore, our gene expression and histological analyses confirmed that i.v. injection of SAMiRNA-mAREG was able to diminish AREG and α-SMA expression, as well as collagen deposition. To the best of our knowledge, this study provides empirical evidence that downregulation of AREG expression might be an effective way to mitigate radiation-induced kidney fibrosis.
This study highlights fibrotic changes caused by 6 Gy TBI that were particularly prominent in the kidney, but absent from other organs. The comparably high-expression levels of AREG in the kidney likely triggered fibrotic signaling (AREG↑→TGF-β↑→AREG↑→. . .) after 6 Gy TBI. It is noteworthy that the expression of fibrotic markers, such as Acta2 and Col1a1 started to increase at 24 weeks after TBI, while that of Areg was significantly increased at all time points analyzed, i.e., 4 to 24 weeks after TBI (Fig. 2). As Areg expression in the kidneys of 4 Gy-irradiated mice started to increase at 8 weeks after TBI (Fig. 1A), AREG seems to be overexpressed early and in a radiation dose-dependent manner. It is thus speculated that there may be a threshold level of AREG that triggers the continuous activation of AREG signaling in response to TBI, which induces kidney fibrosis as a late effect. However, BUN and sCr levels, which reflect renal function, did not increase after 6 Gy TBI, and glomerular injury was not observed after 6 Gy TBI (Fig. 6 and Supplementary Fig. S2 (471_rare-197-05-07_s01.docx); https://doi.org/10.1667/RADE-21-00220.1.S1). This could be due to the low dose applied and the short post-TBI period examined compared to other studies (7, 10, 37). Higher radiation doses or prolonged post-TBI periods could possibly lead to kidney malfunction.
Several previous studies revealed that the kidney is a vulnerable and radiation-sensitive abdominal organ, leading to the development of kidney disease in response to radiation (38, 39). It seems that kidney damage by radiation negatively affects other organs. Accordingly, protecting the kidney benefits all other organs as well. For example, atomic bomb survivors with radiation-associated chronic kidney disease are more likely to develop cardiovascular disease (40). Lenarczyk et al. demonstrated that WAG/RijCmcr rats receiving 10 Gy TBI with shielded kidneys showed a decrease in risk factors contributing to the development of kidney dysfunction and heart damage (41). In contrast, shielding of the intestines could not prevent heart disease. These results suggest that therapeutic agents that improve TBI-induced kidney damage, such as SAMiRNA-mAREG, not only protect the kidneys, but also hold the potential to prevent radiation-induced heart disease or other side effects caused by radiation.
In response to 6 Gy TBI, one out of five mice in the TBI-only and TBI with SAMiRNA-mAREG treatment groups died one week before the end of the experiment. The mice displayed approximately 30% body weight loss at the time of death compared to 4 weeks earlier. Meanwhile, the body weight of one of the four remaining mice in the TBI-only group was reduced by 20% on the day of sacrifice compared to 2 weeks earlier. Multi-organ tumorigenesis likely accounted for the significant weight loss and lethality in irradiated mice, as radiation is known to cause an accumulation of DNA damage during replication, ultimately leading to abnormal cellular signal transduction. We observed tumor-like multicellular lesions in the kidney tubules and liver veins of the TBI-only mouse with a marked decrease in body weight ( Supplementary Fig. S3 (471_rare-197-05-07_s01.docx); https://doi.org/10.1667/RADE-21-00220.1.S1). Additionally, a significant increase in fibrosis marker mRNA expression levels detected in this particular mouse compared to non-irradiated control mice (Acta2: 2.4-fold increase in the kidney and 5-fold increase in the liver; Col1a1: 14.5-fold increase in the kidney and 12-fold increase in the liver) led us to exclude those values in Figs. 2 and 3, as they were considered outliers. It has been demonstrated previously that AREG is involved in tumorigenesis, cancer invasion, and therapeutic resistance (42–44). Future studies investigating the beneficial effects of SAMiRNA-mAREG on cancer progression and survival after TBI are expected to prove useful for novel treatment strategies.
Unavoidable exposure of normal cells to radiation during radiation therapy or nuclear accidents contributes to the development of organ fibrosis. The results of this study indicate that TBI increases AREG expression in the kidney, which eventually activates fibrotic signaling. Treatment with SAMiRNA-mAREG significantly inhibited AREG and fibrosis marker expression in the kidneys. Moreover, the reduction of AREG expression led to decreased myofibroblast formation, as evidenced by the expression of α-SMA. Collectively, these findings suggest that AREG regulates the development of radiation-induced renal fibrosis. The present study provides insights into novel strategies to alleviate radiation-induced fibrosis based on SAMiRNA-mAREG-mediated downregulation of AREG expression during radiation therapy or after accidental radiation exposure.
ACKNOWLEDGMENTS
Han-Oh Park is a patent holder of SAMiRNA system. We would like to thank Editage ( www.editage.co.kr) for English language editing.
