Next Article in Journal
Laparoscopic Laser Speckle Contrast Imaging Can Visualize Anastomotic Perfusion: A Demonstration in a Porcine Model
Next Article in Special Issue
Brassica oleracea L. var. botrytis Leaf Extract Alleviates Gentamicin-Induced Hepatorenal Injury in Rats—Possible Modulation of IL-1β and NF-κB Activity Assisted with Computational Approach
Previous Article in Journal
Unmasking the ‘Asymptomatic’ COVID-19: A Nose Question
Previous Article in Special Issue
Docking and Molecular Dynamics Study to Identify Novel Phytobiologics from Dracaena trifasciata against Metabolic Reprogramming in Rheumatoid Arthritis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Life
Volume 12
Issue 8
10.3390/life12081250
life-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model

by
Szu-Ju Chen
1,
Sunderiya Dalanbaatar
2,3,
Huey-Yi Chen
3,4,
Shih-Jing Wang
3,
Wei-Yong Lin
3,4,
Po-Len Liu
5,
Ming-Yen Tsai
6,
Der-Cherng Chen
3,4,
Yung-Hsiang Chen
3,4,7,* and
Wen-Chi Chen
3,4,*
1
Division of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung 40705, Taiwan
2
Department of Family Medicine, Second State Central Hospital, Ulaanbaatar 15160, Mongolia
3
Graduate Institute of Integrated Medicine, School of Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
4
Department of Obstetrics and Gynecology, Department of Medical Research, Department of Neurosurgery, Department of Urology, China Medical University Hospital, Taichung 40447, Taiwan
5
Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
6
Department of Chinese Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan
7
Department of Psychology, College of Medical and Health Science, Asia University, Taichung 41354, Taiwan
*
Authors to whom correspondence should be addressed.
Life 2022, 12(8), 1250; https://doi.org/10.3390/life12081250
Submission received: 29 June 2022 / Revised: 4 August 2022 / Accepted: 15 August 2022 / Published: 16 August 2022
(This article belongs to the Collection The Role of Bioactive Natural Compounds in the Treatment of Diseases)
Browse Figures
Versions Notes

Abstract

:
Approximately 1 in 20 people develops kidney stones at some point in their life. Although the surgical removal of stones is common, the recurrence rate remains high and it is therefore important to prevent the occurrence of kidney stones. We chose Astragalus membranaceus (AM), which is a traditional Chinese medicine, to study the prevention of urolithiasis using a Drosophila model based on our previous screening of traditional Chinese herbs. Wild-type Drosophila melanogaster Canton-S adult fruit flies were used in this study. Ethylene glycol (EG, 0.5%) was added to food as a lithogenic agent. The positive control agent (2% potassium citrate (K-citrate)) was then compared with AM (2, 8, and 16 mg/mL). After 21 days, the fruit flies were sacrificed under carbon dioxide narcotization, and the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination to observe calcium oxalate (CaOx) crystallization. Then, the ex vivo dissolution of crystals in the Malpighian tubules was compared between K-citrate and AM. Survival analysis of the EG, K-citrate, and AM groups was also performed. Both 2% K-citrate and AM (16 mg/mL) significantly inhibited EG-induced CaOx crystal formation. Mean lifespan was significantly reduced by the administration of EG, and the results were significantly reversed in the AM (8 and 16 mg/mL) groups. However, AM extract did not directly dissolve CaOx crystals in Drosophila Malpighian tubules ex vivo. In conclusion, AM extract decreased the ratio of CaOx crystallization in the Malpighian tubules and significantly ameliorated EG-induced reduction of lifespan. AM prevented CaOx crystal formation in the Drosophila model.

1. Introduction

A urinary stone is a crystal concretion generally formed within the kidneys. Urinary stone disease or urolithiasis is common, painful, and associated with significant medical expenditure [1]. It is a highly prevalent urological disorder occurring in 8–19% of males and 3–5% of females worldwide [2,3]. In addition, the prevalence is increasing globally, irrespective of age, sex, and race, with rates ranging from 7% to 13% in North America, 5% to 9% in Europe, and 1% to 5% in Asia [4]. The etiology of kidney stones is multifactorial; the risk factors include age, gender, geographic variation, climate and seasonal factors, diet, fluids, occupational factors, inheritance and genetics, and numerous associated diseases (such as obesity, diabetes mellitus, hypertension, gout, and metabolic syndrome). General dietary recommendations of increased fluid, decreased salt, and moderate intake of protein have not changed. Understanding the impact of associated conditions will improve the prevention of stone disease [5]. It is associated with an increased risk of end-stage renal failure [6].
Lifestyle and dietary habits (such as high salt and protein consumption and low fluid intake) can induce urinary metabolic abnormalities, which undoubtedly play an important role in the risk of urinary stone disease [5,7,8]. The symptoms of urolithiasis include flank pain (the pain can be quite severe) and blood in the urine (hematuria). Calcium oxalate (CaOx) constitutes the majority of stones formed in the human kidney. Nonsteroidal anti-inflammatory drugs were the preferred analgesic option for renal colic patients presenting to the emergency department. Medical treatment is crucial for patients suffering from urolithiasis to palliate pain, enhance spontaneous stone passage, and prevent stone regrowth and recurrences [9]. Unfortunately, since the 1980s, when potassium citrate (K-citrate) was introduced, new drugs have not been developed for stone prevention, perhaps because the long observation time needed to demonstrate efficacy discourages researchers from undertaking clinical studies [10]. In addition, extracorporeal shock wave lithotripsy (ESWL) and minimally invasive approaches such as percutaneous nephrolithotomy and retrograde intrarenal surgery are considered as the mainstay for the treatment of kidney stones. More recently, laser devices have been used for lithotripsy during these procedures [11]. Although surgical treatment of stone disease has greatly advanced, there have been no improvements in the prevention strategies. Recently, Wu et al. evaluated the outcomes of patients with active surveillance or prophylactic intervention for asymptomatic kidney stones and found that patients who underwent prophylactic intervention had lower risks of stone-related events and future intervention [12]. Therefore, there is an urgent need to identify the potential antilithic effects of medicines to prevent stone disease.
Astragalus membranaceus (Fisch.) Bunge (AM; Chinese name: Huangqi/Huang-Chi) is a herb of traditional Chinese medicine (TCM) that has been used for thousands of years in Taiwan, China, and East Asia to treat kidney disease [13]. AM is a perennial herbaceous plant; Astragalus belongs to the Leguminosae family. In TCM, the root of AM is believed to tonify Qi, induce diuresis, relieve edema, and nourish blood. It may also replenish body fluids and relieve pain [13]. Our earlier screening of 80 herbs of TCM using an animal model of Drosophila indicated that AM has a potential antilithic effect for urolithiasis [14]. However, screening studies of TCM are limited; further, there is a new study model to dissolve CaOx crystals ex vivo [15]. In this study, we aimed to investigate the antilithic effect of different concentrations of AM extract by increasing the number of animals in the study along with the addition of this new model.

2. Materials and Methods

2.1. Fruit Fly Stocks and Rearing Conditions

We used wild-type adult male fruit flies, Drosophila melanogaster Canton-S, in this study. About 200 fruit flies (3 independent experiments were performed) in each group were bred in plastic vials containing standard fly medium at 25 °C and 60% humidity, with a 12 h light−dark cycle [16]. The formula of the standard fly medium consisted of 0.67 g of agar, 2.17 g of yeast, 1.31 g of sugar, and 6.66 g of corn powder with the addition of water to a final amount of 100 mL. The solution was put in a microwave to boil. Then, 1.33 mL of 99% alcohol and 0.34 g of β-hydroxybenzoic acid methylester were added after cooling to 65 °C. Next, 10 ml of the medium was decanted into a 50 mL test tube and stored in a 4 °C freezer after the medium returned to room temperature (ready for use only within a 21-day interval).

2.2. Lithogenesis of Drosophila

We used 0.5% EG as lithogenic agent added to the nutritional medium. The 2% K-citrate (Gentle Pharma Co. Ltd., Yunlin, Taiwan) was used as a positive control. The extracts of AM were purchased from Sun Ten Pharmaceutical Co., Ltd. (Taichung, Taiwan). Since a herbal extract of TCM, AM, was used for the study, the high-performance liquid chromatography (HPLC) analysis for the main components’ fingerprints was provided as a reference for the quality control of the medicinal herb. The quality control of AM with composition ratio of each component and elucidation of the mixture components is shown in Supplementary Materials Figures S1–S4. The study groups of crystal formation by AM were 2, 8, and 16 mg/mL. All groups of fruit flies were fed with 0.5% EG from the beginning to the end of experiment (exception: control group). After 3 weeks, the fruit flies were sacrificed under carbon dioxide (CO2) narcotization, and the Malpighian tubules were microscopically dissected, removed, and processed for polarized light microscopy (Olympus BX51) examination. The different degrees (Grade 0, 1, 2, and 3) of CaOx crystal deposition in the Malpighian tubules were rated. Each blinded specimen was evaluated by three investigators who assessed crystal formation using a crystal score of 0 = none, 1 = weak, 2 = moderate, and 3 = strong [16]. The number of each grade crystal formation among the groups was calculated and labelled as N0 × 0, N1 × 1, N2 × 2, and N3 × 3. The average score of crystal formation in each group was calculated as (N0 × 0 + N1 × 1 + N2 × 2 + N3 × 3)/total number of fruit flies.

2.3. Lifespan Assay

To conduct the lifespan experiment, new sprung fruit flies were collected under light by CO2 using anesthesia. Foam plugs were used by researchers because the food vials were used horizontally to prevent weaker fruit flies accidentally being adhered to food or cotton plugs. About 200 fruit flies (3 independent experiments were performed) in each group. Each survivor in the vials needed to be counted and dead fruit flies were taken out of the vials daily. Survival was compared and checked for significance with log-rank tests. Lifespan curves were from pooled counts of a huge number of vials. An adult needs nearly 2 kg of food and water per day, totaling 2000 kcal or 8400 kJ [17]. The package insert (Off-Label Use) of the TCM concentrated herbal extract recommends the directions for adults are 1.2 to 3 g daily. We chose 2 g/day as a standard human dosage to calculate the fruit fly dosage. Human dosage: 2 g (daily drug weight)/2000 g (daily human food weight) = fruit fly dosage: 0.1% (w/w; in fly medium). Based on our previous experience [14], we used 2×, 8×, and 16× (2, 8, and 16 mg/mL, respectively) to evaluate the potential dose-dependent effects of AM on lithogenesis and lifespan tests.

2.4. CaOx Crystal Dissolution Assay Ex Vivo

Methods of the dissolution of CaOx crystal ex vivo was modified from Fan et al. [18]. Briefly, intact Malpighian tubules were dissected in phosphate buffered saline (PBS) under dissection microscope and transferred onto a slide from 3 weeks after lithogenesis. We added 100 μL PBS solution to completely cover the Malpighian tubule tissue and subjected it to live-imaging under polarized light with an Olympus BX51 optical microscope without a coverslip. Images were taken every 40 min to 2 h and overnight (0 min, 40 min, 80 min, 120 min, and ~16 h). The total area of Malpighian tubules was photographed in both light and dark fields. The grades of crystal dissolution were evaluated in each interval. Grading of the final crystal dissolution was as 0: no dissolution, 1: partial dissolution, and 2: complete dissolution. Three independent experiments were performed in each group. The crystal dissolution rate was calculated by the sum of the grade divided by the total number of Malpighian tubules in each group. The rate of dissolution was calculated in time series.

2.5. Statistical Analyses

One-way analysis of variance was used to analyze overall differences between the groups. Bonferroni correction was applied for all multiple comparisons. For the ratio between two lifespan curves, we determined the p value in the log-rank test. All statistics were produced using the Statistical Package for Social Sciences (SPSS for Windows, release 28.0, SPSS Inc., Chicago, IL, USA). Statistically significant difference is defined as p value below 0.05.

3. Results

3.1. Crystal Formation

The crystallization grade was measured after completion of 3 weeks of breeding. Figure 1 shows a typical morphological pattern of EG-induced CaOx crystal deposition and K-citrate/AM-treated groups in the Malpighian tubules. Table 1 shows the number and average grades of CaOx crystallization in the control, 0.5% EG, 2% K-citrate, and 2, 8, and 16 mg/mL AM groups. K-citrate had a protective effect on CaOx crystallization (43.9 ± 4.1%), which reduced the grades of crystallization compared to the EG group (91.9 ± 8.2%). Only high-dose AM (16 mg/mL) reduced crystallization (53.5 ± 4.8%), similar to K-citrate (Figure 2).

3.2. Lifespan Assay

To test whether the effects of AM were associated with a decreased mortality rate under treatment with the lithogenic agent EG, the lifespan of Drosophila was measured after the flies’ food was supplemented with K-citrate and various concentrations of AM (Table 2). Figure 3 shows the survival survey results of each group. EG has a toxic effect on the lifespan of Drosophila, with the shortest mean survival time. The K-citrate and AM (8 and 16 mg/mL, respectively) groups had a protective effect with longer mean survival days than the EG group (Figure 4).

3.3. Ex Vivo Dissolution of CaOx Crystals in Malphigian Tubules

To explore the mechanism through which AM inhibits CaOx crystal formation, we examined the dissolution of CaOx crystals in Malpighian tubules ex vivo. The rate of dissolution in each group was calculated at different times. K-citrate directly dissolved the CaOx crystals in the Malpighian tubules ex vivo in a time-dependent manner. During the observation period, no CaOx crystals were excreted from the Malpighian tubule. Almost 100% of the CaOx crystals were dissolved in a 2% K-citrate solution after overnight (~16 h) treatment. In contrast, PBS and AM extracts had no effect on the dissolution of CaOx crystals preformed in the Malpighian tubule of Drosophila (Figure 5 and Figure 6).

4. Discussion

In this study, we examined the antilithogenic effects of different concentrations of AM extract and compared them with those of K-citrate. The inhibitory effect of AM, comparable to that of K-citrate, was observed only at a high dose of 16 mg/mL. The control group had a mean and maximum lifespan of 42.3 ± 4.1 and 62 days, respectively. The mean lifespan was significantly reduced by the administration of EG, while the administration of AM resulted in decreased CaOx crystal formation and increased lifespan. Regarding ex vivo dissolution, K-citrate had the highest rate of dissolution, while AM extract had no direct effect on the dissolution of CaOx crystals.
Despite advances in diagnosis and treatment, urolithiasis is still a disease with high prevalence and recurrence. The recurrence rate of urinary tract stones is approximately 10% per year. Half of the patients experienced recurrence within 10 years. Recurrence has been reported to occur more than once in 80% of the patients. Therefore, it is important to prevent the development of urolithiasis [19].
Landry et al. used a fruit fly model to study the homolog of Slc26a6 (a nephrolithiasis-related gene involved in oxalate transportation) related to Drosophila (Slc26a5/6 and dPrestin), which found that thiosulfate decreased CaOx stone formation in Malpighian tubules [20]. This result indicates that thiosulfate or oxalate mimics may act as competitive therapeutic inhibitors for the crystallization of CaOx. Fan et al. used fruit flies to perform RNAi knockdown of nephrolithiasis-related genes in the principal cells of Malpighian tubules, and studied the effects of antilithic agents on CaOx crystal formation [18]. Therefore, genetic studies of stone disease in fruit flies may have future potential [21,22,23]. The pathogenesis of CaOx formation is a multistep process and essentially includes nucleation, crystal growth, crystal aggregation, and crystal retention. Many inorganic and organic substances are known to modulate stone formation. A comprehensive account of the mechanisms of renal stone formation does not yet exist, and the role of promoters/inhibitors in CaOx crystallization is still unclear [24]. Nevertheless, we speculate that these nephrolithiasis-related genes associated with calcium and oxalate transportation or metabolization might possibly be involved in the molecular mechanisms of AM treatment for kidney stones.
Chen et al. first observed the formation of CaOx crystals in Malpighian tubules within weeks after the addition of lithogenic agents in fly food and directly examined them under polarized light microscopy [8]. Since then, Drosophila models have been used to screen potential antilithic traditional Chinese medicinal plants with 80 candidate herbs [14]. Wu et al. reported that the use of certain tested herbs of TCM, including Salviae miltiorrhizae, Paeonia lactiflora, Carthami flos, and Scutellaria baicalensis resulted in a crystal formation rate of approximately 0.0%. However, the actual mechanism causing this remains unknown, and related studies lack the adequate number of animals required to establish statistical significance [25,26]. Thus, Salviae miltiorrhizae and Carthami flos were further studied using rat animal models, as well as with additional clinical analyses [14,27,28,29,30]. Drosophila models, therefore, provide a fast-breeding and large population, which assists in the screening of potential antilithic drugs. Chen et al. developed a new observation method in which a whole fruit fly can be viewed using micro-computerized tomography, which makes this model more beneficial [31].
AM is a well-known “Qi-tonifying” or adaptogenic herb used in TCM. Its Chinese name, “Huangqi/Huang-Chi”, means “yellow leader”. It is a long yellow tap root and is deservedly popular because it helps the body in a variety of ways. AM was listed in the first order of the traditional herb drug book, Ben Cao Bei Yao (“Essentials of materia medica”), as one of the most important drugs among herbs of TCM. It has been prescribed for centuries for general debilitation and chronic illnesses, and to increase overall vitality. It offers several health benefits, such as boosting the immune system [32], promoting natural urination, supporting normal body fluids, and benefiting the respiratory system. AM is often used in liver and blood tonics. It possesses antiviral, antioxidant, antibacterial, and detoxifying properties. According to a meta-analysis of studies from China, AM was found to have renal protective effects in 1804 diabetic patients with nephropathy [33,34]. In TCM [35,36], Astragalus is used either alone or in combination with other herbs in various doses and forms [37]. For example, AM combined with Chuan Xiong (Ligusticum chuanxiong Hort., a major component of tetramethylpyrazine) was widely used in TCM for the treatment of abdominal pain and cardiovascular disease [38].
Recently, Chung et al. reported in the journal Nature that hydroxycitric acid (HCA), a component of the extract from the fruit Garcinia cambogia, can dissolve CaOx crystals in vitro [39]. This tropical fruit found in Southeast Asia, which is rich in HCA, is traditionally used for cooking purposes in India and may be a potential antilithogenic herb. Fan et al. and Chen et al. further studied HCA to analyze its preventative effects on CaOx formation in Drosophila models [15,18,40]. However, the ex vivo effect of HCA on the dissolution of CaOx crystals differed from that of both these groups when compared with that of K-citrate. Fan et al. reported that 1% HCA completely dissolved CaOx crystals (induced by sodium oxalate) within 2 h in an ex vivo study [15,18]. Chen et al. reported that the effect of HCA was only 39% for in vivo dissolution of CaOx (induced by EG) in Drosophila [40]. Our results indicated that K-citrate was the most effective agent for the prevention of CaOx crystal formation in the Drosophila model. In contrast, most of the studies focus on the antioxidative and anti-inflammatory effects of AM. The main ingredients of AM include polysaccharides, astragalosides, saponins, and flavonoids, with strong effects on scavenging oxidative free radicals and anti-inflammation [41]. It has been reported in a clinical study that herbs (containing AM) of TCM could protect against ESWL-induced renal damage [42]. However, to the best of our knowledge, none of the main components of AM has been reported to have the same ability to dissolve CaOx crystals as HCA.
Nevertheless, Astragalus polysaccharides (APS) have been shown to inhibit the adhesion and promote the endocytosis of CaOx dihydrate nanocrystals to damaged renal proximal tubular epithelial cells [43]. APS could inhibit CaOx monohydrate growth, induce CaOx dihydrate formation, and increase the absolute zeta potential of the crystals to inhibit crystal aggregation [44]. Moreover, after being repaired by APS, the amount of adherent crystals on the cell surface decreased, but the amount of endocytic crystals increased [45]. Thus, these results suggest that APS may serve as potential drugs for preventing kidney stones. On the other hand, AM is a major medicinal herb that has been commonly used in many herbal formulations in the practice of TCM to treat a wide variety of diseases and body disorders, or marketed as life-prolonging extracts for human use. The major components of AM are polysaccharides, flavonoids, and saponins. Pharmacological research indicates that some of the extract components of AM can increase telomerase activity, and has various antioxidative and anti-inflammatory effects. Hence, targeting these pathological changes could reverse aging and treat age-associated diseases [46].
One of the advantages of using Drosophila models is their lifespan [47,48]. Chemical agents that are potentially hazardous to human health can be rapidly tested through lifespan studies because toxic agents may shorten life cycles. Drosophila has a short life cycle of approximately 8–9 weeks and is easy to breed in large numbers over a short period of time [48,49]. Therefore, fruit flies are an ideal model for studying potentially toxic agents. Melamine (sometimes illegally added to food products to increase their apparent protein content) and ractopamine (an animal feed additive used to promote leanness and increase food conversion efficiency) have been reported to have potentially hazardous effects on humans in Taiwan [50]. Chen et al. used a Drosophila model to study their effects on toxicity. Melamine significantly reduced the lifespan of Drosophila in a dose-dependent manner [19], while ractopamine (10 ppb added to food) significantly reduced the lifespan of fruit flies [50]. Lifespan studies can also be used to test other agents by following the aforementioned method [48,51].
The disadvantages of using a fruit fly model include the difficulty of studying the mechanisms of tubular fluid formation and the absence of serum or urine biochemistry [52]. Further, this invertebrate lacks many solid organs, including the kidneys. However, Malpighian tubules of the fruit fly have a similar genetic composition, physiology, and anatomy to those of the human kidney [22]. Miller et al. proposed that dietary manipulation, environmental alteration, and genetic variations affect stone formation and can be rapidly observed and quantified in Drosophila within a few days [22].

5. Conclusions

In summary, we demonstrated that TCM AM extract efficiently prevented CaOx crystal formation in the Malpighian tubule of Drosophila in vivo, but the AM extract did not directly dissolve or excrete CaOx crystals from the Malpighian tubule ex vivo. Since urolithiasis is still a disease with high recurrence, these data suggest that AM can be used as a prophylactic intervention in bodies that have the tendency to form crystals. Further clinical studies are warranted to confirm its effects on the prevention of lithiasis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/life12081250/s1, Figure S1. HPLC analysis for Astragalus membranaceus extract. Figure S2. 2D-HPLC chromatogram of Astragalus membranaceus extract analyzed at 250 nm by photodiode array detector. Figure S3. 2D-HPLC chromatogram of Astragalus membranaceus extract analyzed by evaporative light scattering detector. Figure S4. 3D-HPLC chromatogram of Astragalus membranaceus extract.

Author Contributions

Conceptualization, W.-C.C.; methodology, S.-J.C., S.D., H.-Y.C., S.-J.W., W.-Y.L., P.-L.L., M.-Y.T., D.-C.C., Y.-H.C. and W.-C.C.; software, S.-J.C., S.D., S.-J.W. and Y.-H.C.; validation, S.-J.C., S.D., H.-Y.C., S.-J.W., W.-Y.L., P.-L.L., M.-Y.T., D.-C.C., Y.-H.C. and W.-C.C.; writing—original draft preparation, S.D. and W.-C.C.; writing—review and editing, Y.-H.C. and W.-C.C.; supervision, W.-C.C.; project administration, W.-C.C.; funding acquisition, Y.-H.C. and W.-C.C. S.-J.C. and S.D. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Funding

This study is supported China Medical University (DMR-111-080), Taichung, Taiwan and Ministry of Science and Technology (MOST 110-2320-B-039-032 and MOST 111-2320-B-039-022), Taiwan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be provided on request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Tinti, F.; Umbro, I.; D’Alessandro, M.; Lai, S.; Merli, M.; Noce, A.; Di Daniele, N.; Mazzaferro, S.; Mitterhofer, A.P. Cholemic Nephropathy as Cause of Acute and Chronic Kidney Disease. Update on an Under-Diagnosed Disease. Life 2021, 11, 1200. [Google Scholar] [CrossRef] [PubMed]
  2. Romero, V.; Akpinar, H.; Assimos, D.G. Kidney stones: A global picture of prevalence, incidence, and associated risk factors. Rev. Urol. 2010, 12, e86–e96. [Google Scholar] [PubMed]
  3. Trinchieri, A. Epidemiology of urolithiasis: An update. Clin. Cases Miner. Bone Metab. 2008, 5, 101–106. [Google Scholar] [PubMed]
  4. Kasper, D.; Fauci, A.; Hauser, S.; Longo, D.; Jameson, J.; Loscalzo, J. (Eds.) Harrison’s Principles of Internal Medicine, 19th ed.; McGraw Hill: New York, NY, USA, 2015. [Google Scholar]
  5. Sorokin, I.; Mamoulakis, C.; Miyazawa, K.; Rodgers, A.; Talati, J.; Lotan, Y. Epidemiology of stone disease across the world. World J. Urol. 2017, 35, 1301–1320. [Google Scholar] [CrossRef]
  6. Keller, J.J.; Chen, Y.K.; Lin, H.C. Association between chronic kidney disease and urinary calculus by stone location: A population-based study. BJU Int. 2012, 110, E1074–E1078. [Google Scholar] [CrossRef]
  7. Ho, C.Y.; Chen, Y.H.; Wu, P.Y.; Chang, C.H.; Chen, H.Y.; Man, K.M.; Shen, J.L.; Tsai, F.J.; Lin, W.Y.; Lee, Y.J.; et al. Effects of commercial citrate-containing juices on urolithiasis in a Drosophila model. Kaohsiung J. Med. Sci. 2013, 29, 488–493. [Google Scholar] [CrossRef]
  8. Huang, W.C.; Chen, Y.J.; Hung, M.C. Implication of nuclear EGFR in the development of resistance to anticancer therapies. Biomedicine 2011, 1, 2–10. [Google Scholar] [CrossRef]
  9. Skolarikos, A. Medical treatment of urinary stones. Curr. Opin. Urol. 2018, 28, 403–407. [Google Scholar] [CrossRef]
  10. Morgan, M.S.; Pearle, M.S. Medical management of renal stones. BMJ 2016, 352, i52. [Google Scholar] [CrossRef]
  11. Kallidonis, P.; Vagionis, A. The future of laser technology in kidney stones. Curr. Opin. Urol. 2022, 32, 411–414. [Google Scholar] [CrossRef]
  12. Wu, T.; Liu, Z.; Ma, S.; Xue, W.; Jiang, X.; Ma, J. Should we support prophylactic intervention for asymptomatic kidney stones? A retrospective cohort study with long-term follow-up. Urolithiasis 2022, 50, 431–437. [Google Scholar] [CrossRef]
  13. Li, M.; Wang, W.; Xue, J.; Gu, Y.; Lin, S. Meta-analysis of the clinical value of Astragalus membranaceus in diabetic nephropathy. J. Ethnopharmacol. 2011, 133, 412–419. [Google Scholar] [CrossRef]
  14. Wu, S.Y.; Shen, J.L.; Man, K.M.; Lee, Y.J.; Chen, H.Y.; Chen, Y.H.; Tsai, K.S.; Tsai, F.J.; Lin, W.Y.; Chen, W.C. An emerging translational model to screen potential medicinal plants for nephrolithiasis, an independent risk factor for chronic kidney disease. Evid. Based Complement. Altern. Med. 2014, 2014, 972958. [Google Scholar] [CrossRef]
  15. Fan, Q.X.; Gong, S.Q.; Hong, X.Z.; Feng, X.M.; Zhang, F.J. Clinical-grade Garcinia cambogia extract dissolves calcium oxalate crystals in Drosophila kidney stone models. Eur. Rev. Med. Pharmacol. Sci. 2020, 24, 6434–6445. [Google Scholar]
  16. Hirata, T.; Cabrero, P.; Berkholz, D.S.; Bondeson, D.P.; Ritman, E.L.; Thompson, J.R.; Dow, J.A.; Romero, M.F. In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis. Am. J. Physiol. Ren. Physiol. 2012, 303, F1555–F1562. [Google Scholar] [CrossRef]
  17. Levitsky, D.A.; Raea Limb, J.E.; Wilkinson, L.; Sewall, A.; Zhong, Y.; Olabi, A.; Hunter, J. Lack of negative autocorrelations of daily food intake on successive days challenges the concept of the regulation of body weight in humans. Appetite 2017, 116, 277–283. [Google Scholar] [CrossRef]
  18. Fan, Q.; Feng, X.; Hong, X.; Gong, S.; Tian, J.; Hou, F.; Zhang, F. Garcinia cambogia extract removes calcium oxalate kidney stones in both genetic and non-genetic Drosophila models of nephrolithiasis. bioRxiv 2018, arXiv:10.1101/477570. [Google Scholar]
  19. Chen, W.C.; Lin, W.Y.; Chen, H.Y.; Chang, C.H.; Tsai, F.J.; Man, K.M.; Shen, J.L.; Chen, Y.H. Melamine-induced urolithiasis in a Drosophila model. J. Agric. Food Chem. 2012, 60, 2753–2757. [Google Scholar] [CrossRef]
  20. Landry, G.M.; Hirata, T.; Anderson, J.B.; Cabrero, P.; Gallo, C.J.; Dow, J.A.; Romero, M.F. Sulfate and thiosulfate inhibit oxalate transport via a dPrestin (Slc26a6)-dependent mechanism in an insect model of calcium oxalate nephrolithiasis. Am. J. Physiol. Renal Physiol. 2016, 310, F152–F159. [Google Scholar] [CrossRef]
  21. Wang, S.; Ju, Y.; Gao, L.; Miao, Y.; Qiao, H.; Wang, Y. The fruit fly kidney stone models and their application in drug development. Heliyon 2022, 8, e09232. [Google Scholar] [CrossRef]
  22. Miller, J.; Chi, T.; Kapahi, P.; Kahn, A.J.; Kim, M.S.; Hirata, T.; Romero, M.F.; Dow, J.A.T.; Stoller, M.L. Drosophila melanogaster as an emerging translational model of human nephrolithiasis. J. Urol. 2013, 190, 1648–1656. [Google Scholar] [CrossRef]
  23. Dow, J.A.T.; Simons, M.; Romero, M.F. Drosophila melanogaster: A simple genetic model of kidney structure, function and disease. Nat. Rev. Nephrol. 2022, 18, 417–434. [Google Scholar] [CrossRef]
  24. Aggarwal, K.P.; Narula, S.; Kakkar, M.; Tandon, C. Nephrolithiasis: Molecular mechanism of renal stone formation and the critical role played by modulators. Biomed. Res. Int. 2013, 2013, 292953. [Google Scholar] [CrossRef]
  25. Liao, W.L.; Tsai, F.J. Personalized medicine: A paradigm shift in healthcare. Biomedicine 2013, 3, 66–72. [Google Scholar] [CrossRef]
  26. Hsu, S.C.; Lin, J.H.; Weng, S.W.; Chueh, F.S. Crude extract of Rheum palmatum inhibits migration and invasion of U-2 OS human osteosarcoma cells by suppression of matrix metalloproteinase-2 and -9. Biomedicine 2013, 3, 120–129. [Google Scholar] [CrossRef]
  27. Lin, W.C.; Lai, M.T.; Chen, H.Y.; Ho, C.Y.; Man, K.M.; Shen, J.L.; Lee, Y.J.; Tsai, F.J.; Chen, Y.H.; Chen, W.C. Protective effect of Flos carthami extract against ethylene glycol-induced urolithiasis in rats. Urol. Res. 2012, 40, 655–661. [Google Scholar] [CrossRef]
  28. Chen, W.C.; Wu, S.Y.; Liao, P.C.; Chou, T.Y.; Chen, H.Y.; Chiang, J.H.; Su, Y.C.; Man, K.M.; Tsai, M.Y.; Chen, Y.H. Treatment of Urolithiasis with Medicinal Plant Salvia miltiorrhiza: A Nationwide Cohort Study. Evid. Based Complement. Altern. Med. 2018, 2018, 8403648. [Google Scholar] [CrossRef]
  29. Chen, W.C.; Chou, T.Y.; Chen, H.Y.; Yang, Y.R.; Man, K.M.; Tsai, M.Y.; Chen, Y.H. Salvia miltiorrhiza Bunge (Danshen) for Treatment and Prevention of Urolithiasis: A Drosophila Animal Study. Evid. Based Complement. Altern. Med. 2019, 2019, 1408979. [Google Scholar] [CrossRef]
  30. Lin, L.-H.; Chiang, J.-H.; Chou, P.-C.; Liao, P.-C.; Wu, S.-Y.; Tsai, K.-S.; Chen, H.-Y.; Chen, Y.-H.; Chen, W.-C. Chinese Herbal Medicine Safflower (Flos carthami) Does Not Increase Bleeding Complications: A Population-based Cohort Study. J. Food Nutr. Res. 2016, 4, 108–114. [Google Scholar]
  31. Chen, W.C.; Chen, H.Y.; Liao, P.C.; Wang, S.J.; Tsai, M.Y.; Chen, Y.H.; Lin, W.Y. Toward a new insight of calcium oxalate stones in Drosophila by micro-computerized tomography. Urolithiasis 2018, 46, 149–155. [Google Scholar] [CrossRef]
  32. Zhao, K.S.; Mancini, C.; Doria, G. Enhancement of the immune response in mice by Astragalus membranaceus extracts. Immunopharmacology 1990, 20, 225–233. [Google Scholar] [CrossRef]
  33. Juan, Y.C.; Kuo, Y.H.; Chang, C.C.; Zhang, L.J.; Lin, Y.Y.; Hsu, C.Y.; Liu, H.K. Administration of a decoction of sucrose- and polysaccharide-rich radix astragali (huang qi) ameliorated insulin resistance and Fatty liver but affected Beta-cell function in type 2 diabetic rats. Evid. Based Complement. Altern. Med. 2011, 2011, 349807. [Google Scholar] [CrossRef] [PubMed]
  34. Zhang, J.; Xie, X.; Li, C.; Fu, P. Systematic review of the renal protective effect of Astragalus membranaceus (root) on diabetic nephropathy in animal models. J. Ethnopharmacol. 2009, 126, 189–196. [Google Scholar] [CrossRef] [PubMed]
  35. Lin, F.Y.; Chen, Y.H.; Chen, Y.L.; Wu, T.C.; Li, C.Y.; Chen, J.W.; Lin, S.J. Ginkgo biloba extract inhibits endotoxin-induced human aortic smooth muscle cell proliferation via suppression of toll-like receptor 4 expression and NADPH oxidase activation. J. Agric. Food Chem. 2007, 55, 1977–1984. [Google Scholar] [CrossRef]
  36. Su, H.F.; Samsamshariat, A.; Fu, J.; Shan, Y.X.; Chen, Y.H.; Piomelli, D.; Wang, P.H. Oleylethanolamide activates Ras-Erk pathway and improves myocardial function in doxorubicin-induced heart failure. Endocrinology 2006, 147, 827–834. [Google Scholar] [CrossRef]
  37. Litbarg, N.O. Integrative Medicine; Elsevier: Amsterdam, The Netherlands, 2017; p. 411. [Google Scholar]
  38. Liu, S.P.; Shibu, M.A.; Tsai, F.J.; Hsu, Y.M.; Tsai, C.H.; Chung, J.G.; Yang, J.S.; Tang, C.H.; Wang, S.; Li, Q.; et al. Tetramethylpyrazine reverses high-glucose induced hypoxic effects by negatively regulating HIF-1α induced BNIP3 expression to ameliorate H9c2 cardiomyoblast apoptosis. Nutr. Metab. 2020, 17, 12. [Google Scholar] [CrossRef]
  39. Chung, J.; Granja, I.; Taylor, M.G.; Mpourmpakis, G.; Asplin, J.R.; Rimer, J.D. Molecular modifiers reveal a mechanism of pathological crystal growth inhibition. Nature 2016, 536, 446–450. [Google Scholar] [CrossRef]
  40. Chen, W.C.; Chen, H.Y.; Lin, W.Y.; Yang, Y.R.; Tsai, M.Y.; Chen, Y.H. Inhibitory Effect of Hydroxycitrate on Calcium Oxalate Crystal Formation in a Drosophila Model. J. Food Nutr. Res. 2018, 6, 706–709. [Google Scholar] [CrossRef]
  41. Sheng, B.W.; Chen, X.F.; Zhao, J.; He, D.L.; Nan, X.Y. Astragalus membranaceus reduces free radical-mediated injury to renal tubules in rabbits receiving high-energy shock waves. Chin. Med. J. 2005, 118, 43–49. [Google Scholar]
  42. Sheng, B.; He, D.; Zhao, J.; Chen, X.; Nan, X. The protective effects of the traditional Chinese herbs against renal damage induced by extracorporeal shock wave lithotripsy: A clinical study. Urol. Res. 2011, 39, 89–97. [Google Scholar] [CrossRef]
  43. Han, J.; Guo, D.; Sun, X.Y.; Wang, J.M.; Ouyang, J.M.; Gui, B.S. Comparison of the adhesion and endocytosis of calcium oxalate dihydrate to HK-2 cells before and after repair by Astragalus polysaccharide. Sci. Technol. Adv. Mater. 2019, 20, 1164–1177. [Google Scholar] [CrossRef]
  44. Huang, F.; Sun, X.Y.; Ouyang, J.M. Preparation and characterization of selenized Astragalus polysaccharide and its inhibitory effect on kidney stones. Mater. Sci. Eng. C Mater. Biol. Appl. 2020, 110, 110732. [Google Scholar] [CrossRef]
  45. Huang, F.; Sun, X.Y.; Chen, X.W.; Ouyang, J.M. Effects of Selenized Astragalus Polysaccharide on the Adhesion and Endocytosis of Nanocalcium Oxalate Dihydrate after the Repair of Damaged HK-2 Cells. ACS Biomater. Sci. Eng. 2021, 7, 739–751. [Google Scholar] [CrossRef]
  46. Liu, P.; Zhao, H.; Luo, Y. Anti-Aging Implications of Astragalus Membranaceus (Huangqi): A Well-Known Chinese Tonic. Aging Dis. 2017, 8, 868–886. [Google Scholar] [CrossRef]
  47. Huang, W.T.; Chong, I.W.; Chen, H.L.; Li, C.Y.; Hsieh, C.C.; Kuo, H.F.; Chang, C.Y.; Chen, Y.H.; Liu, Y.P.; Lu, C.Y.; et al. Pigment epithelium-derived factor inhibits lung cancer migration and invasion by upregulating exosomal thrombospondin 1. Cancer Lett. 2019, 442, 287–298. [Google Scholar] [CrossRef]
  48. Arias-Rojas, A.; Iatsenko, I. The Role of Microbiota in Drosophila melanogaster Aging. Front. Aging 2022, 3, 909509. [Google Scholar] [CrossRef]
  49. Li, T.; Chen, Y.H.; Liu, T.J.; Jia, J.; Hampson, S.; Shan, Y.X.; Kibler, D.; Wang, P.H. Using DNA microarray to identify Sp1 as a transcriptional regulatory element of insulin-like growth factor 1 in cardiac muscle cells. Circ. Res. 2003, 93, 1202–1209. [Google Scholar] [CrossRef]
  50. Chen, W.C.; Wang, Y.C.; Shen, J.L.; Chen, H.Y.; Chang, C.H.; Tsai, F.J.; Lin, W.Y.; Chen, Y.H. Potential genitourinary toxicity and lithogenic effect of ractopamine. J. Food Nutr. Res. 2015, 3, 670–674. [Google Scholar]
  51. Liu, J.T.; Chen, Y.L.; Chen, W.C.; Chen, H.Y.; Lin, Y.W.; Wang, S.H.; Man, K.M.; Wan, H.M.; Yin, W.H.; Liu, P.L.; et al. Role of pigment epithelium-derived factor in stem/progenitor cell-associated neovascularization. J. Biomed. Biotechnol. 2012, 2012, 871272. [Google Scholar] [CrossRef]
  52. Knauf, F.; Preisig, P.A. Drosophila: A fruitful model for calcium oxalate nephrolithiasis? Kidney Int. 2011, 80, 327–329. [Google Scholar] [CrossRef]
Life 12 01250 g001 550
Figure 1. Effects of K-citrate (2%) and AM (16 mg/mL) on EG-induced crystal deposition in the Malpighian tubules of Drosophila. The images show representative polarized microscopy for the Drosophila with 0.5% EG-induced crystal formation in Malpighian tubules. Scale bar = 500 μm.
Figure 1. Effects of K-citrate (2%) and AM (16 mg/mL) on EG-induced crystal deposition in the Malpighian tubules of Drosophila. The images show representative polarized microscopy for the Drosophila with 0.5% EG-induced crystal formation in Malpighian tubules. Scale bar = 500 μm.
Life 12 01250 g001
Life 12 01250 g002 550
Figure 2. Rates of CaOx crystal formation in the in vivo study. Crystal formation in 0.5% EG, 2% K-citrate, and 2, 8, and 16 mg/mL AM-treated Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). Data are expressed as mean ± S.D. * p < 0.05, compared to the control. # p < 0.05, compared to the 0.5% EG-treated group.
Figure 2. Rates of CaOx crystal formation in the in vivo study. Crystal formation in 0.5% EG, 2% K-citrate, and 2, 8, and 16 mg/mL AM-treated Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). Data are expressed as mean ± S.D. * p < 0.05, compared to the control. # p < 0.05, compared to the 0.5% EG-treated group.
Life 12 01250 g002
Life 12 01250 g003 550
Figure 3. Survival curve of lifespan for control, 0.5% EG, EG + 2% K-citrate, and EG + 2, 8, and 16 mg/mL AM-treated Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). For ratio between two lifespan curves, we determined the p value in the log-rank test.
Figure 3. Survival curve of lifespan for control, 0.5% EG, EG + 2% K-citrate, and EG + 2, 8, and 16 mg/mL AM-treated Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). For ratio between two lifespan curves, we determined the p value in the log-rank test.
Life 12 01250 g003
Life 12 01250 g004 550
Figure 4. Effects of K-citrate and AM extract on lifespan. Effect of EG, EG + K-citrate, and EG + 2, 8, and 16 mg/mL AM on lifespan of Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). Data are expressed as mean ± S.D. * p < 0.05 compared to the control; # p < 0.05 compared to the 0.5% EG-treated group.
Figure 4. Effects of K-citrate and AM extract on lifespan. Effect of EG, EG + K-citrate, and EG + 2, 8, and 16 mg/mL AM on lifespan of Drosophila (n ≅ 200 for each group; three independent experiments were performed in each group). Data are expressed as mean ± S.D. * p < 0.05 compared to the control; # p < 0.05 compared to the 0.5% EG-treated group.
Life 12 01250 g004
Life 12 01250 g005 550
Figure 5. K-citrate, but not AM, extract directly dissolves CaOx crystals in Drosophila Malpighian tubules ex vivo. Represented images of CaOx crystal dissolution in intact Malpighian tubules treated with 2% K-citrate and AM (2, 8, and 16 mg/mL) solutions ex vivo at different time points.
Figure 5. K-citrate, but not AM, extract directly dissolves CaOx crystals in Drosophila Malpighian tubules ex vivo. Represented images of CaOx crystal dissolution in intact Malpighian tubules treated with 2% K-citrate and AM (2, 8, and 16 mg/mL) solutions ex vivo at different time points.
Life 12 01250 g005
Life 12 01250 g006 550
Figure 6. K-citrate, but not AM, extract directly dissolves CaOx crystals in Drosophila Malpighian tubules ex vivo. Wild-type Drosophila was reared in fly food containing 0.5% EG for 3 weeks. Intact Malpighian tubules were dissected from Drosophila and treated with PBS, 2% K-citrate, and AM solutions. CaOx crystal dissolution was monitored using live-imaging. Renal CaOx crystal dissolution rate was calculated. Data are expressed as mean ± S.D. (three independent experiments were performed in each group). * p < 0.05 compared to the control.
Figure 6. K-citrate, but not AM, extract directly dissolves CaOx crystals in Drosophila Malpighian tubules ex vivo. Wild-type Drosophila was reared in fly food containing 0.5% EG for 3 weeks. Intact Malpighian tubules were dissected from Drosophila and treated with PBS, 2% K-citrate, and AM solutions. CaOx crystal dissolution was monitored using live-imaging. Renal CaOx crystal dissolution rate was calculated. Data are expressed as mean ± S.D. (three independent experiments were performed in each group). * p < 0.05 compared to the control.
Life 12 01250 g006
Table
Table 1. Overall CaOx crystal formation in each group of Drosophila (n ≅ 200).
Table 1. Overall CaOx crystal formation in each group of Drosophila (n ≅ 200).
GroupTotalGrade 0Grade 1Grade 2Grade 3Average Score
Control17014426000.15
EG173146465301.64 *
EG + 2% K-citrate15587421970.65 #
EG + AM (2 mg/mL) 166235462271.56
EG + AM (8 mg/mL)168286252261.45
EG + AM (16 mg/mL)170794932100.84 #
CaOx: calcium oxalate, EG: ethylene glycol (0.5%), K-citrate: potassium citrate, AM: Astragalus membranaceus. * p < 0.05, compared to the control. # p < 0.05, compared to the EG-treated group.
Table
Table 2. Lifespan assay in each group of Drosophila (n ≅ 200).
Table 2. Lifespan assay in each group of Drosophila (n ≅ 200).
GroupDays Until Last Fly DiedMedian Survival DaysMean Survival Days
Control624042.3 ± 4.1
EG411820.2 ± 1.9 *
EG + 2% K-citrate482832.6 ± 3.4
EG + AM (2 mg/mL) 411721.1 ± 1.8
EG + AM (8 mg/mL)482230.8 ± 2.1 #
EG + AM (16 mg/mL)512735.7 ± 4.5 #
EG: ethylene glycol (0.5%), K-citrate: potassium citrate, AM: Astragalus membranaceus. * p < 0.05, compared to the control. # p < 0.05, compared to the EG-treated group.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Chen, S.-J.; Dalanbaatar, S.; Chen, H.-Y.; Wang, S.-J.; Lin, W.-Y.; Liu, P.-L.; Tsai, M.-Y.; Chen, D.-C.; Chen, Y.-H.; Chen, W.-C. Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model. Life 2022, 12, 1250. https://doi.org/10.3390/life12081250

AMA Style

Chen S-J, Dalanbaatar S, Chen H-Y, Wang S-J, Lin W-Y, Liu P-L, Tsai M-Y, Chen D-C, Chen Y-H, Chen W-C. Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model. Life. 2022; 12(8):1250. https://doi.org/10.3390/life12081250

Chicago/Turabian Style

Chen, Szu-Ju, Sunderiya Dalanbaatar, Huey-Yi Chen, Shih-Jing Wang, Wei-Yong Lin, Po-Len Liu, Ming-Yen Tsai, Der-Cherng Chen, Yung-Hsiang Chen, and Wen-Chi Chen. 2022. "Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model" Life 12, no. 8: 1250. https://doi.org/10.3390/life12081250

APA Style

Chen, S.-J., Dalanbaatar, S., Chen, H.-Y., Wang, S.-J., Lin, W.-Y., Liu, P.-L., Tsai, M.-Y., Chen, D.-C., Chen, Y.-H., & Chen, W.-C. (2022). Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model. Life, 12(8), 1250. https://doi.org/10.3390/life12081250

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop