• Users Online: 624
  • Print this page
  • Email this page


 
 
Table of Contents
CLASSROOM READING
Year : 2020  |  Volume : 6  |  Issue : 3  |  Page : 70-73

Gut microbiota dysbiosis and chronic kidney disease


1 Department of Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, Uttar Pradesh, India
2 Department of Nephrology and Renal Transplant, Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow, Uttar Pradesh, India

Date of Submission05-Oct-2020
Date of Acceptance07-Nov-2020
Date of Web Publication13-Apr-2021

Correspondence Address:
Dr. Brijesh Yadav
Department of Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrnm.jrnm_25_20

Get Permissions

  Abstract 


Chronic kidney disease (CKD) is an irreversible progressive health problem often associated with cardiovascular complication, bone mineral metabolism disorder, uremic toxin deposition, and immune dysregulation. The gut microbiome is an important modulator of immune function and performs a plethora of functions inside the host body. Several factors such as diet, antibiotic use, environmental pollutant modulates the composition of Gut microbiota. An intervention with dietary substances may be a therapeutic strategy to modulate the healthy Gut microbiota composition and slowing the progression of CKD.

Keywords: Chronic kidney disease, dysbiosis, gut microbiota, immune cell, short-chain fatty acids


How to cite this article:
Yadav B, Prasad N, Saxena A. Gut microbiota dysbiosis and chronic kidney disease. J Renal Nutr Metab 2020;6:70-3

How to cite this URL:
Yadav B, Prasad N, Saxena A. Gut microbiota dysbiosis and chronic kidney disease. J Renal Nutr Metab [serial online] 2020 [cited 2021 Jun 13];6:70-3. Available from: http://www.jrnm.in/text.asp?2020/6/3/70/313630




  Chronic Kidney Disease Top


Chronic kidney disease (CKD) is an irreversible progressive problem associated with structural and functional damage in the kidney. It affects approximately 10%–13% of the world population[1] and 17.2% of the Indian population[2] and progress toward end-stage renal failure over the years. It is often associated with cardiovascular risk,[3] dysregulated immune response, bone mineral metabolic disorder,[4] accumulation of uremic toxin, and metabolites.[5] Dysregulated inflammatory response often worsens the kidney function. Results of Vitamin-D supplementation and probiotics studies show positive impacts in inhibiting the progression of CKD, suggesting an intervention through dietary supplementation may be an effective strategy in the management of CKD patient,[6],[7] Composition of diet impacts the quality of microbiota and overall health of the host.[8] Recent study has shown a dysbiosis of Gut microbiota in CKD patients.[9] However, it is the subject of debate, whether dysbiosis of Gut microbiota is the cause or effects of CKD. In this review, we will discuss Gut microbiota composition dysbiosis, function, and factors influencing in Gut microbiota composition and the mechanism how Gut microbiota may mediate CKD.


  Gut Microbiota Top


The human gut microbiota comprises a complex niche of 100 trillion microbiota comprising a total of 1100 bacterial species[10] of which at least 160 common bacterial species, which remained shared among the other members in the community,[10] Gut microbiota mostly (90%) colonized in the distal gut. The total human microbiota counts is 10 times that of the total human cell and having a total gene 3.3 million genes, mostly of uncharacterized function.[10]

An estimated total count of adult healthy human bacterial number is of the range 10^14, belongs to five major phyla, Firmicutes (80%), Bacteroidetes(20%), Actinobacteria(3%), Proteobacteria (1%), and Verrucomicrobia (0.1%) of total Gut microbiota[11],[12] The Bacteroidetes (Gram-negative anaerobes), Firmicutes (Gram-positive anaerobes) are two predominating bacterial phyla in Gut, make approximately 90% of the total gut microbiota. The key member of phyla Firmicutes includes genera Roseburi, Clostridium, Eubacterium, Lactobacillus, Enterococcus, Ruminicoccus, Fecalibacterium. The member of phyla Bacteroidetes belongs to the family Bacteroidaceae, Prevotellaceae and Rikenellaceae, Xylanibacter. Low levels of Bifidobacteriaceae, Lactobacillaceae and higher levels of Enterobacteriaceae are associated with CKD.[9]


  Gut Epithelial Integrity Barrier Top


Gut intestinal epithelial cells (IECs) forms an intact continuous physical and biochemical barrier between gut lumen and inner mucosal tissues, requires for preventing the leakage of microbial components in blood stream.[13] It is made of a single layer of columnar epithelial cells. Bifidobacterium of Phyla Actinobacteria is used as a major prebiotic in the treatment of many inflammatory diseases.[14] Bifidobacterium increases the strength of intestinal integrity barrier by inducing increased ß-defensin-2, secretion and junctional protein ZO-1, Occludin and decreasing Claudin 2 synthesis in intestine epithelial cell.[15]


  Gut Microbiota in Immune Cell Development Top


Gut microbiota and the immune system have bidirectional relationship. Gut microbiota control immune cell development and later, immune cell regulate the colonization of specific set of microbiota,[16],[17] Gut microbiota has microbial-associated molecular pattern, molecules such as flagellin, peptidoglycan, lipo-polysaccharides, teichoic acid, formyl peptides, polysaccharide-A (PSA), D-glycero-β-D-manno-heptose, that bind to pattern recognition receptors of the host (toll-like receptor 2 [TLR2], nod-like receptors, rig-1 like receptors) cell and prime immune cell for maturation and activation.

Studies have shown that Germfree mice had poor immune cell development and reduced number of CD4+ T, Th17 cell, reduced intraepithelial CD8+ T-cell and reduced secretory immunoglobulin-A and increased Th2 cell-specific signature molecule,[18],[19],[20] Similarly, specific pathogen-free mice lead hyper-activation of B-cell activation factor (APRIL) together with increased hypogalctosylated IgA formation.[21] Hypogalactosylated, antibody lead induction of immune response in many inflammatory diseases.[22],[23] The presence of capsular PSA of Bacteroides fragilis leads to Th1 cell development, and germfree mice lead to Th2 and B-cell development,[18] Similarly, segmented filamentous bacteria, cytophaga-flavobacter-bacteroidetes and Helicobacter hepaticus induce Th17 development from naive CD4+ T cell,[24],[25] suggesting a dysbiosis in a specific set of Gut microbiota might lead to an inflammatory immune response in CKD patients.

Gut microbiota plays an important role in orchestrating the immune function. Candida albicans colonization in the human gut leads to the secretion of pro-inflammatory cytokines interleukin (IL)-22 for the elimination of the Candida and other pathogens in the intestine.[26] Members of Clostridium, and Akkermansia muciniphilla, Roseburi, Lactobacillus, Fecalibacterium facilitates degradation of dietary mucopolysaccharides and other dietary substances that generate short-chain fatty acids such as Butyrate, propionate, and Acetate, which induces anti-inflammatory Treg cell differentiation and proliferation from naïve CD4+ T cell and induces secretion of IL-10, IL-35 that inhibit inflammatory cell activation in colon and circulation. Member of the genus Clostridium and Fusobacterium are potent inducers of transforming growth factor (TGF)-ß1 from the IEC, which provides a milieu to differentiate intestinal naïve CD4+ T cell into T-regulatory cell,[27],[28] Furthermore, Clostridium butyricum induces TLR2-dependent TGF-ß1 production from the lamina propria and Dendritic cell and facilitates induced Treg cell development in lamina propria which prevents Gut inflammation and leakage from gut into circulation.


  Microbiota Role in Vitamin Synthesis Top


Vitamins works as co-enzymes for many enzymes required for the normal physiological homeostasis, like cellular metabolism and energy generation for cell division, blood clotting in host, signal transduction, etc., Inside the host, gut microbiota performs many vitamins synthesis from dietary substances, like folate, Biotin, Vitamin K, B12,.[29] Lactobacillus spp. are found to be associated with Vitamin B12 production.[29] Vitamins B12 is required for Red blood cells formation. A deficiency of B12 leads to diminish the immune function of CD8+ T and NK cell required for protection from pathogen infections.[30] Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum produce folate, required for the blood cell formation[31] a common problem in CKD patients.


  Gut Microbiota in Biotransformation Top


Gut microbiota bio-transform environmental pollutant polycyclic aromatic hydrocarbon compounds into estrogenic intermediates, thus mediates secondary toxicity.[32] Gut microbiota transform bile acid by deconjugation, dihydroxylation. An unconjugated and hydroxylated bile acid inhibit the efflux activity of the multidrug-resistant protein and permeable glycoprotein and increase the bio-availability of steroid medicine to the cell.[33] Furthermore, microbiota ferments dietary protein and generates a myriad amount of uremic toxins like indoxyl sulfate, p-cresol, trimethyl-n-oxide, ammonia in the gut. Indoxyl-sulphate activates macrophage and induces inflammatory cytokines expression,[34] while ammonia increases the gut permeability and allowing translocation of microbial components, food metabolite in circulation, aggravating the inflammation by activating the inflammatory cell,[34],[35] Tri-methyl-n-oxide (TMAO) generated from the gut microbiota by fermenting meat and egg phosphocholine, and lecithin.[36] TMAO is reported to be associated with cardiovascular risk; one of the common problems in CKD patients. Lactobacilli catabolize Tryptophan into indole-3-aldehyde (I3A), which binds to aryl-hydrocarbon receptors (AHR) on innate lymphoid cells-3, an inflammatory innate counterpart of Th17 cell.[37] I3A mediates AHR signaling pathway-dependent secretion of cytokines, IL-22 from colon lymphocytes and increased expression of Gap junction protein, Claudin, Junctional adhesion molecules in the epithelial cell, these molecules are required for epithelial integrity barrier protection, thus reduces local and systemic inflammation.[38]


  Factors Affecting Gut Microbiota Top


Probiotics

Prebiotics are live microbial organisms given as a dietary supplement to boost the pre-existing microbiota in gut. Prebiotics work on gut microbiota, leading to the reduction of oxidative stress and inflammation in the gut cell, decreasing gut epithelial barrier permeability,[38],[40] Further, they induce the increased secretion of IgA and anti-inflammatory cytokines from the mucosal plasma B-cell and helps in maintaining the intestinal permeability barrier and prevents translocation of microbial components from lumen to circulation.

Prebiotics

Prebiotics are food substances that enhance the growth and activity of the intestinal gut microbiota like Bifidobacteria. They are nonabsorbable to the intestine, but their fermentation generates substances which are utilized by the colonocyte for their energy source. Common prebiotics is inulin-type fructans and short-chain fructooligosaccharides, found to be associated with decreased endotoxin and homeostasis model assessment.[41]

Synbiotics

Synbiotic is the combination of both prebiotics and probiotics. Synbiotics are used to promote bacterial growth and its functional activity, where either pro or prebiotics are not much effective alone. Synbiotics have shown an increased population of probiotics bacteria, Bifidobacteria and Lactobacillus.[42]

Use of antibiotics

An intervention with antibiotics leads to gut microbiota dysbiosis and consequently aberrant immune cell development and leading to increase susceptibility to inflammatory diseases in CKD patients.[43],[44]

Environmental pollutants

It has been found that increasing environmental pollutant leads to gut microbiota dysbiosis in the general population.[45],[46] Carbon nanotube is found to be linked with increasing intestinal permeability, gut microbiota dysbiosis, and inflammatory gene expression in the intestine,[45],[47] which may allow translocation of microbial components in circulation leading to systemic inflammatory effect.


  Conclusion Top


Gut microbiota performs pleiotropic function inside the host cell, of which one of the important functions is the modulation of the immune system. A manipulation of Gut microbiota with dietary substances may be an effective strategy in reducing inflammation and slowing the progression of CKD in patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, et al. Prevalence of chronic kidney disease in the United States. JAMA 2007;298:2038-47.  Back to cited text no. 1
    
2.
Varma PP. Prevalence of chronic kidney disease in India – Where are we heading? Indian J Nephrol 2015;25:133-5.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Sarnak MJ, Amann K, Bangalore S, Cavalcante JL, Charytan DM, Craig JC, et al. Chronic kidney disease and coronary artery disease: JACC state-of-the-art review. J Am Coll Cardiol 2019;74:1823-38.  Back to cited text no. 3
    
4.
Massy ZA, Drueke TB. Gut microbiota orchestrates PTH action in bone: Role of butyrate and T cells. Kidney Int 2020;98:269-72.  Back to cited text no. 4
    
5.
Castillo-Rodriguez E, Fernandez-Prado R, Esteras R, Perez-Gomez MV, Gracia-Iguacel C, Fernandez-Fernandez B, et al. Impact of Altered Intestinal Microbiota on Chronic Kidney Disease Progression. Toxins (Basel). 2018;10:1-21.  Back to cited text no. 5
    
6.
Jean G, Souberbielle JC, Chazot C. Vitamin D in chronic kidney disease and dialysis patients. Nutrients 2017;9:328.  Back to cited text no. 6
    
7.
Jia L, Jia Q, Yang J, Jia R, Zhang H. Efficacy of probiotics supplementation on chronic kidney disease: A systematic review and meta-analysis. Kidney Blood Press Res 2018;43:1623-35.  Back to cited text no. 7
    
8.
Zmora N, Suez J, Elinav, E. You are what you eat: Diet, health and the gut microbiota. Nat Rev Gastroenterol Hepatol 2019;1635-56.  Back to cited text no. 8
    
9.
Sampaio-Maia B, Simμes-Silva L, Pestana M, Araujo R, Soares-Silva IJ. The role of the gut microbiome on chronic kidney disease. Adv Appl Microbiol 2016;96:65-94.  Back to cited text no. 9
    
10.
Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010;464:59-65.  Back to cited text no. 10
    
11.
Schroeder BO, Bäckhed F. Signals from the gut microbiota to distant organs in physiology and disease. Nat Med 2016;22:1079-89.  Back to cited text no. 11
    
12.
Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol 2016;14:e1002533.  Back to cited text no. 12
    
13.
Andersen K, Kesper MS, Marschner JA, Konrad L, Ryu M, Kumar Vr S, et al. Intestinal dysbiosis, barrier dysfunction, and bacterial translocation account for CKD-related systemic inflammation. J Am Soc Nephrol 2017;28:76-83.  Back to cited text no. 13
    
14.
Ewaschuk JB, Diaz H, Meddings L, Diederichs B, Dmytrash A, Backer J, et al. Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function. Am J Physiol Gastrointest Liver Physiol 2018;295:G1025-34.  Back to cited text no. 14
    
15.
Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol 2000;1:113-8.  Back to cited text no. 15
    
16.
Zhang H, Sparks JB, Karyala SV, Settlage R, Luo XM. Host adaptive immunity alters gut microbiota. ISME J 2015;9:770-81.  Back to cited text no. 16
    
17.
Fung TC, Artis D, Sonnenberg GF. Anatomical localization of commensal bacteria in immune cell homeostasis and disease. Immunol Rev 2014;260:35-49.  Back to cited text no. 17
    
18.
Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005;122:107-18.  Back to cited text no. 18
    
19.
Kamada N, Seo SU, Chen GY, Núñez G. Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol 2013;13:321-35.  Back to cited text no. 19
    
20.
Ivanov II, Frutos Rde L, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 2008;4:337-49.  Back to cited text no. 20
    
21.
McCarthy DD, Kujawa J, Wilson C, Papandile A, Poreci U, Porfilio EA, et al. Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy. J Clin Invest 2o11;121:3991-4002.  Back to cited text no. 21
    
22.
Kemna MJ, Plomp R, van Paassen P, Koeleman CAM, Jansen BC, Damoiseaux JGMC, et al. Galactosylation and sialylation levels of IgG predict relapse in patients with PR3-ANCA associated vasculitis. EBioMedicine 2017;17:108-18.  Back to cited text no. 22
    
23.
Ząbczyńska M, Polak K, Kozłowska K, Sokołowski G, Pocheć, E. The contribution of IgG glycosylation to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in hashimoto's thyroiditis: An in vitro model of thyroid autoimmunity. Biomolecules 2020;10:171.  Back to cited text no. 23
    
24.
Yi J, Jung J, Han D, Surh CD, Lee YJ. Segmented filamentous bacteria induce divergent populations of antigen-specific CD4 T cells in the small intestine. Mol Cells 2019;42:228-36.  Back to cited text no. 24
    
25.
Morrison PJ, Bending D, Fouser LA, Wright JF, Stockinger B, Cooke A, et al. Th17-cell plasticity in Helicobacter hepaticus-induced intestinal inflammation. Mucosal Immunol 2013;6:1143-56.  Back to cited text no. 25
    
26.
Puel A, Döffinger R, Natividad A, Chrabieh M, Barcenas-Morales G, Picard C, et al. Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I. J Exp Med 2010;207:291-7.  Back to cited text no. 26
    
27.
Martin-Gallausiaux C, Béguet-Crespel F, Marinelli L, Jamet A, Ledue F, Blottière HM, et al. Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells. Sci Rep 2018;8:9742.  Back to cited text no. 27
    
28.
Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013;504:446-50.  Back to cited text no. 28
    
29.
Masuda M, Ide M, Utsumi H, Niiro T, Shimamura Y, Murata M. Production potency of folate, vitamin B (12), and thiamine by lactic acid bacteria isolated from Japanese pickles. Biosci Biotechnol Biochem 2012;76:2061-7.  Back to cited text no. 29
    
30.
Tamura J, Kubota K, Murakami H, Sawamura M, Matsushima T, Tamura T, et al. Immunomodulation by vitamin B12: augmentation of CD8+T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment. Clin Exp Immunol 1990;116:28-32.  Back to cited text no. 30
    
31.
Rossi M, Amaretti A, Raimondi S. Folate production by probiotic bacteria. Nutrients 2011;3:118-34.  Back to cited text no. 31
    
32.
Van de Wiele T, Vanhaecke L, Boeckaert C, Peru K, Headley J, Verstraete W, et al. Human colon microbiota transform polycyclic aromatic hydrocarbons to estrogenic metabolites. Environ Health Perspect 2005;113:6-10.  Back to cited text no. 32
    
33.
Enright EF, Govindarajan K, Darrer R, MacSharry J, Joyce SA, Gahan CG. Gut Microbiota-mediated bile acid transformations alter the cellular response to multidrug resistant transporter substrates in vitro: Focus on P-glycoprotein. Mol Pharm 2018;15:5711-27.  Back to cited text no. 33
    
34.
Adesso S, Popolo A, Bianco G, Sorrentino R, Pinto A, Autore G, et al. The uremic toxin indoxyl sulphate enhances macrophage response to LPS. PLoS One 2013;8:e76778.  Back to cited text no. 34
    
35.
Cummings JH. Fermentation in the human large intestine: Evidence and implications for health. Lancet 1983;1:1206-9.  Back to cited text no. 35
    
36.
Li DY, Tang WH. Contributory role of gut microbiota and their metabolites toward cardiovascular complications in chronic kidney disease. Semin Nephrol 2018;38:193-205.  Back to cited text no. 36
    
37.
Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pieraccini G, et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 2013;39:372-85.  Back to cited text no. 37
    
38.
Teng Y, Ren Y, Sayed M, Hu X, Lei C, Kumar A, et al. Plant-derived exosomal MicroRNAs shape the gut microbiota. Cell Host Microbe 2018;24:637-5200000000.  Back to cited text no. 38
    
39.
Schlee M, Harder J, Köten B, Stange EF, Wehkamp J, Fellermann K. Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol 2008;151:528-35.  Back to cited text no. 39
    
40.
Sherman PM, Johnson-Henry KC, Yeung HP, Ngo PS, Goulet J, Tompkins TA. Probiotics reduce enterohemorrhagic Escherichia coli O157:H7- and enteropathogenic E. coli O127:H6-induced changes in polarized T84 epithelial cell monolayers by reducing bacterial adhesion and cytoskeletal rearrangements. Infect Immun 2005;73:5183-8.  Back to cited text no. 40
    
41.
Salazar N, Dewulf EM, Neyrinck AM, Bindels LB, Cani PD, Mahillon J, et al. Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal short-chain fatty acids in obese women. Clin Nutr 2015;34:501-7.  Back to cited text no. 41
    
42.
Likotrafiti E, Tuohy KM, Gibson GR, Rastall RA. An in vitro study of the effect of probiotics, prebiotics and synbiotics on the elderly faecal microbiota. Anaerobe 2014;27:50-5.  Back to cited text no. 42
    
43.
Noverr MC, Noggle RM, Toews GB, Huffnagle GB. Role of antibiotics and fungal microbiota in driving pulmonary allergic responses. Infect Immun 2004;72:4996-5003.  Back to cited text no. 43
    
44.
Russell SL, Gold MJ, Hartmann M, Willing BP, Thorson L, Wlodarska M, et al. Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma. EMBO Rep 2012;13:440-7.  Back to cited text no. 44
    
45.
Chen H, Zhao R, Wang B, Zheng L, Ouyang H, Wang H, et al. Acute oral administration of single-walled carbon nanotubes increases intestinal permeability and inflammatory responses: Association with the changes in gut microbiota in mice. Adv Healthc Mater 2018;7:e1701313.  Back to cited text no. 45
    
46.
Li R, Yang J, Saffari A, Jacobs J, Baek KI, Hough G, et al. Ambient ultrafine particle ingestion alters gut microbiota in association with increased atherogenic lipid metabolites. Sci Rep 2017;7:42906.  Back to cited text no. 46
    
47.
Lahiani MH, Khare S, Cerniglia CE, Boy R, Ivanov IN, Khodakovskaya M. The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition. Nanoscale 2019;11:3639-55.  Back to cited text no. 47
    




 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Chronic Kidney D...
Gut Microbiota
Gut Epithelial I...
Gut Microbiota i...
Microbiota Role ...
Gut Microbiota i...
Factors Affectin...
Conclusion
References

 Article Access Statistics
    Viewed389    
    Printed10    
    Emailed0    
    PDF Downloaded52    
    Comments [Add]    

Recommend this journal