Anemia In Adenine-Induced Chronic Renal Failure And The .
https://doi.org/10.33549/physiolres.932685Physiol. Res. 63: 351-358, 2014Anemia in Adenine-Induced Chronic Renal Failure and the Influenceof Treatment With Gum Acacia ThereonB. H. ALI1, M. AL ZA’ABI1, A. RAMKUMAR1, J. YASIN2, A. NEMMAR31Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences,Sultan Qaboos University, Al Khod, Oman, 2Department of Medicine and 3Departmentof Physiology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain,United Arab EmiratesReceived October 18, 2013Accepted January 17, 2014On-line February 24, 2014SummaryUniversity, P. O. Box 35, Al Khod, Postal code 123, Oman. E-mail:Anemia frequently complicates chronic kidney disease (CKD). Wealibadreldin@hotmail.com and akthmali@squ.edu.ominvestigated here the effect of adenine-induced CKD in rats onerythrocyte count (EC), hematocrit (PCV) and hemoglobin (Hb)Introductionconcentration, as well as on the activity of L-γ-glutamyltransferase (GGT) and the concentrations of iron (Fe), transferrin(Tf), ferritin (F), total iron binding capacity (TIBC) / unsaturatediron binding capacity (UIBC) and hepcidin (Hp) in serum anderythropoietin (Epo) in renal tissue. Renal damage was erumconcentrations of the uremic toxin indoxyl sulfate (IS), creatinine,and urea, and by creatinine clearance. We also assessed theinfluence of concomitant treatment with gum acacia (GA) on theabove analytes. Adenine feeding induced CKD, accompanied bysignificant decreases (P 0.05) in EC, PCV, and Hb, and in theserum concentrations of Fe, Tf, TIBC, UIBC and Epo. It alsoincreased Hp and F levels. GA significantly ameliorated thesechanges in rats with CKD. A general improvement in the renalstatus of rats with CKD after GA is shown due to its antiinflammatory and anti-oxidant actions, and reduction of theuremic toxin IS, which is known to suppress Epo production, andthis may be a reason for its ameliorative actions on the indices ofanemia studied.Key wordsRats Anemia Iron Gum acacia Adenine Chronic renalfailureCorresponding authorB. H. Ali, Department of Pharmacology and Clinical Pharmacy,College of Medicine and Health Sciences, Sultan QaboosAnemia is known to be an early and inevitablesign in patients with chronic kidney disease (CKD), andcan confer significant multiple adverse clinicalconsequences, morbidity and mortality (Shah et al. 2006)and its management is a core component of nephrologycare (Atkinson and Furth 2011). The occurrence ofcardiovascular and renal diseases with anemia is termed‘cardio-renal-anemia syndrome’ (Jürgensen et al. 2010).It is caused by a relative shortage of erythropoietin (Epo)and can also be associated with disordered ironhomeostasis caused by reduced iron absorption, occultblood loss and impaired iron mobilization (Ruedin et al.2012), as well as chronic inflammation (Nangaku andEckardt 2006). Among the most important factors in thepathogenesis of iron metabolism defects is hepcidin (Hp),as it maintains mammalian iron homeostasis (Pantopoulos et al. 2012). Various methods for treating theanemia associated with CKD in humans and chronic renalfailure (CRF) in laboratory animals have been used.These include iron (Liles 2012), Epo (Gianella et al.2013, Silverberg et al. 2010), recombinant human EPO(Nichols et al. 2011, Teixeira et al. 2010) erythropoietingene electrotransfer (Ataka et al. 2003), and some newerythropoiesis-stimulating agents such as peginesatide(Graul 2012, Locatelli and Del Vecchio 2011).The adenine-induced CRF model in rats is aPHYSIOLOGICAL RESEARCH ISSN 0862-8408 (print) ISSN 1802-9973 (online) 2014 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech RepublicFax 420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres
352Ali et al.standard method for inducing a metabolic abnormality,similar to that which occurs in humans, in which adenineis given to rats in the feed at a concentration of 0.75 %,w/w, for 4 weeks (Ali et al. 2013b, Yokozawa et al.1986). The excretion of nitrogenous compounds inadenine-treated rats is suppressed by renal tubularocclusion because of the formation of 2,8dihydroxyadenine crystals, leading to accumulation ofvarious guanidino compounds (such as methylguanidineand guanidinosuric acid) and urea nitrogen in blood(Yokozawa et al. 1986). As far as we are aware, there isonly limited published work about anemia in adenineinduced CRF in rats and its pathogenesis, or possibletreatment (Hamada et al. 2008, Okada et al. 1999, Sun etal. 2013). In this work the aim was to verify the effect ofadenine-induced CRF on anemia, and further, toinvestigate the status of several factors involved in thepathogenesis of anemia in adenine-induced CRF such asiron (Fe), ferritin (F), transferrin (Tf), Epo and Hp in ratswith the experimental disease, and further, to test theusefulness of treatment with the natural product gumacacia (GA) thereon. The salutary effect of GA inhumans with CKD (Ali et al. 2008, Bliss et al. 1996), andin rat adenine-induced CRF, and some of itsconsequences have been previously reported (Ali et al.2010, 2011a, b, 2013a). As far as we are aware, there areno reports in the literature describing the use of naturalproducts to ameliorate the anemia induced by adenineinduced CRF in the rat except for two publications (bothin Chinese) using two local medicinal plants (Ma et al.2007, Wang et al. 2012).MethodsAnimalsMale Wistar rats (9-10 weeks old, weighing249 10 g) were housed in a room at a temperature of22 2 C, relative humidity of about 60 %, with a 12 hlight-dark cycle (lights on 6:00), and free access tostandard pellet chow diet containing 0.85 % phosphorus,1.12 % calcium, 0.35 % magnesium, 25.3 % crudeprotein and 2.5 IU/g vitamin D3 (Oman Flour Mills,Muscat, Oman) and water. Ethical clearance was obtainedfrom our University Animal Ethics Committee and allprocedures involving animals and their care were carriedout in accordance with international laws and policies(EEC Council directives 86/609, OJL 358, 1 December,12, 1987; NIH Guide for the Care and Use of LaboratoryAnimals, NIH Publications No. 85-23, 1985).Vol. 63Experimental designAfter an acclimatization period of a week, rats(n 24) were randomly divided into four equal groups andtreated for four consecutive weeks. The first groupcontinued to receive the same diet without treatment untilthe end of the study (control group).The second group was switched to a powder dietcontaining adenine (0.75 %, w/w, in feed). The third groupwas given normal food and GA (SUPERGUMTM EM 10)in the drinking water at a concentration of 15 %, w/v. Thefourth group was given adenine in the feed as in group two,plus GA in drinking water at a concentration of 15 %, w/v.During the treatment period, the rats wereweighed weekly and one day before the last day oftreatment were placed individually in metabolic cages tocollect the urine voided in the last 24 h. Twenty-fourhours after the end of the treatment the rats wereanesthetized with ketamine (75 mg/kg) and xylazine(5 mg/kg) intraperitoneally, and blood (about 3-4 ml) wascollected from the anterior vena cava and placed intoplain tubes (about 3 ml) and in heparinized tubes (1 ml).The first aliquot of blood and urine were centrifuged at900 g at 4 C for 15 min. The serum obtained, togetherwith the urine specimens, were stored at 80 C to awaitanalysis. The two kidneys were excised, blotted on filterpaper and weighed. A small piece of the right kidney wasplaced in 10 % neutral buffered formalin for subsequenthistopathology, and the rest of the kidneys were keptfrozen at 80 C for pending measurement of Epo withina week.Hematological methodsIn the blood collected in heparinized tubeserythrocyte count (EC), hemoglobin (Hb) concentrationand hematocrit (Packed cell volume, PCV) were analyzedusing automated methods (COBAS MICROS, Roche,Palo Alto, CA, USA).Biochemical methodsThe concentrations of creatinine and urea, aswell as that of iron (Fe), ferritin (F), transferrin (Tf) andL-γ-glutamyl transferase (GGT) in serum were measuredusing kits from Human GmbH (Mannheim, Germany).Creatinine clearance (CCr) was calculated as reportedbefore (Duarte and Preuss 1993). Total iron bindingcapacity (TIBC) / Unsaturated iron binding capacity(UIBC) concentrations were measured in serum usingcommercial kits in an automated machine (Cobas Integra,Roche Diagnostics, Basel, Switzerland). Renal Epo
2014Anemia in Adenine-Induced Chronic Renal Failure353Fig. 1. Percentage body weightchange (between final and initialbody weight), and kidney weight (aspercentage of final body weight) ofcontrol rats, rats treated withadenine (0.75 %, w/w, in feed for 28days), and in rats treated with GA indrinking water at concentration of15 %, w/v, with or without adeninefor 28 days. Each column andvertical bar represent the mean SEM(n 6rats).Statisticaldifferences between the groups areshown.concentration was measured by an ELISA method usingcommercial kits from R&D Systems, Inc. (Minneapolis,MN). Concentration of serum Hp was measured by anELISA method using kit from Novateinbio (Woburn,MA, USA) and, plasma indoxyl sulfate (IS) concentrationwas measured using a validated HPLC method (Al Za’abiet al. 2013).Histopathological methodsThe kidneys were fixed in 10 % neutral-bufferedformalin, dehydrated in increasing concentrations of ethylalcohol, cleared with xylene and embedded in paraffin.Three micrometer sections were prepared from kidneyparaffin blocks and stained with hematoxylin and eosin(H & E). The microscopic scoring of the kidney sectionswas carried out in a blinded fashion by a pathologist whowas unaware of the treatment groups.ChemicalsAll chemicals used in this work were of thehighest possible commercial grade available. Adenine wasobtained from Sigma (St. Louis, MO, USA). GA(SUPERGUMTM EM 10, Lot 101008, 1.1.11) was obtainedfrom Sanwa Cho, Toyonaka, Osaka, Japan. The chemicalproperties of GA have been fully reviewed before (Ali etal. 2013a), and according to the manufacturer’s data.SUPERGUMTM EM 10 was characterized by sizefractionation followed by multiple angle laser lightscattering (GPC-MALLS) to give its molecular profile.The average molecular weight was 3.436 106, and thecontent of the arabinogalactan protein (AGP) was 26.4 %.Aqueous solutions of both adenine and GA wereprepared freshly every day just before use.Statistical analysisStatistical analysis was carried out usingGraphPad Prism 5.0 (GraphPad Software, San Diego,CA, USA). Each group consisted of at least six animals.All data are shown as means S.E.M. Group means werecompared with an analysis of variance (ANOVA)followed by Tukey’s multiple comparison test. Values ofP 0.05 were regarded as significant.ResultsAs shown in Figure 1, adenine feeding (0.75 %,w/w, for 4 weeks) caused significant decrease in bodyweight and a significant increase in relative kidney
354Vol. 63Ali et al.Table 1. Effect of Gum Arabic (GA) on some biochemical parameters in serum of rats treated with adenine (0.75 %, w/w, 4 weeks).Parameters/GroupUrea (μmol/l)Creatinine (μmol/l)Creatinine clearance (ml/min)Indoxyl sulfate (μmol)L-γ-glutamyl transferase (U/l)ControlAdenineGA6.4 0.863.1 4.81.1 0.21.8 0.84.2 0.8150.7 2.9*205.6 12.5*0.3 0.0*159.6 13.5*12.2 0.3*14.0 1.254.5 3.00.9 0.20.0 0.05.4 1.0Adenine GA26.1 1.0#89.7 11.3#0.6 0.11.5 0.7#11.5 0.3*Values in the table are mean SEM (n 6). Adenine was added to the feed at a concentration of 0.75 %, w/w, for 4 weeks, and GA(either alone or with adenine) was given in drinking water at 15 %, w/v. * P 0.05 (Control vs. all groups). # P 0.05 (Adenine vs.Adenine gum)Table 2. Adenine-induced changes in erythrocyte count, hematocrit and hemoglobin concentration in rats, and influence of gum acaciathereon (GA).Parameters/GroupErythrocyte count (1012/l)Hematocrit (%)Hemoglobin (g/l)ControlAdenineGAAdenine GA7.0 0.239.7 0.8127.2 2.76.4 0.2*29.5 0.7*85.4 2.9*7.2 0.240.1 0.9129.2 3.06.9 0.237.7 0.9121.2 6.1Values in the table are mean SEM (n 6-12 rats). * Significant (P 0.05) difference from the control and the three other groups.No significant difference was noted between the control and GA and adenine GA groups.weight and in water intake and urine output (P 0.05).These changes were significantly but not completelyantagonized by GA treatment.As reported in the previous work on thehistopathology of renal tissues (Ali et al. 2010, 2013a, b),here we have found that the control and the GA-treatedgroups of rats showed normal kidney histology (damagescore of zero). The adenine-treated group showed diffuseacute tubular necrosis in about 70 % of the examinedtissue areas (damage score of 4), and exhibited tubulardistention with necrotic material involving loss of brushborder of proximal tubules, dilatation of large number oftubules, mixed inflammatory cells infiltration of theinterstitium, and focal tubular atrophy. The rats givenadenine plus GA concomitantly showed improvement inthe histological appearance when compared with theadenine-treated groups. There were focal areas of acutetubular necrosis involving about 30 % of examined areas,and there was also less dilatation of the tubules, lessinterstitial inflammatory cell infiltration, and less tubularatrophy (damage score of 1).Adenine treatment significantly increased theconcentrations of serum urea and creatinine, andsignificantly decreased the creatinine clearance. It caused asignificant (P 0.05) increase in IS concentration and GGTactivity (Table 1). Treatment with GA significantly abatedthese adenine induced actions. Adenine-induced CRFcaused significant decreases (P 0.05) in EC, PCV, and Hb(Table 2), and in the serum concentrations of Fe, Tf, TIBC,UIBC and Hp (Table 3). It also increased Hp and Fconcentrations in serum. GA significantly amelioratedthese changes in the adenine-treated rats. Renal Epoconcentration was significantly decreased in adeninetreated rats, compared with control rats (P 0.05), with GAtreatment having no effect on this parameter (Fig. 2).DiscussionThe global incidence of CKD is on the rise(Couser et al. 2011), but access to renal replacementtherapy, by either transplantation or dialysis is limited inmany parts of the world because of lack of financial andmedical resources (Aviles-Gomez et al. 2006, Jha 2009).Strategies aiming at delaying the onset of dialysis or toameliorate uremia often rely on dietary supplements(Cheu et al. 2013, Holden et al. 2012).In the present study, we assessed the effects ofadenine-induced CRF on several hematologicalparameters in rats, and the influence of GA thereon. Theresults indicated that adenine induces anemia, and thatconcomitant treatment with GA significantly abrogatedthis action.
2014Anemia in Adenine-Induced Chronic Renal Failure355Table 3. Effect of Gum Arabic (GA) on some serum constituents in rats treated with adenine (0.75 %, w/w, 4 weeks).Parameters/ GroupFerritin (ng/ml)Iron (μg/dl)Total iron binding capacity(μmol/l)Unsaturated iron binding capacity(μmol/l)Transferrin (mg/dl)Hepcidin (ng/ml)ControlAdenineGAAdenine GA108.0 33.1209.6 19.1245.0 53.8*191.1 17.9117.8 16.4215.6 21.5160.2 6.6201.5 15.5100.1 2.272.1 1.52*99.28 7.582.9 3.3*73.4 4.141.42 3.76*73.82 8.655.3 3.5115.4 12.812.4 0.081.9 12.416.2 1.6*105.4 19.012.5 2.3114.1 19.710.3 1.4#Values in the table are mean SEM (n 6). Adenine was added to the feed at a concentration of 0.75 %, w/w, for 4 weeks, and GA(either alone or with adenine) was given in drinking water at 15 %, w/v. * P 0.05 (Control vs. all groups). # P 0.05 (Adenine vs.adenine gum).Fig. 2. Renal erythropoietin concentration (% control) of controlrats, and in rats treated with adenine (0.75 %, w/w, in feed for28 days), or GA in drinking water at concentration of 15 %, w/v,with or without adenine for 28 days. Each column and verticalbar represent the mean SEM (n 6 rats). Statistical differencesbetween the groups are shown.Insufficient production of the glycoproteinhormone Epo is one of the main causes of uremic anemia(Nangaku and Eckardt 2006). It has also been shown thatIS impairs oxygen metabolism in tubular Epo-producingcells in vitro, and that its administration to rats suppressesrenal Epo mRNA expression and plasma Epoconcentrations (Chiang et al. 2011). This suggests apossible connection between the uremic toxin IS and thedesensitization of the oxygen-sensing mechanism in Epoproducing cells, which may, at least, partly explaininadequate Epo production in hypoxic kidneys of CKDpatients. In this work we have found that adenine feedingcauses a significant rise in the concentration of the uremictoxin IS, confirming earlier work on adenine-inducedCRF (Ali et al. 2010) and in rats with CRF induced by7/8 nephrectomy (Ali and Ahmed 2006). However, wehave found here that treatment with GA in adeninetreated rats significantly decreased the adenine-inducedrise in IS, but this was not accompanied by anysignificant alteration in the renal Epo concentration whencompared to that in rats treated with adenine alone(Fig. 2). Therefore, it is not possible from this work toassociate the renal Epo concentration in adenine-treatedrats to their serum level of IS. Further work to investigatethe relationship of plasma IS and renal Epo levels, andthe effect of treatment with Epo-recombinant drugs onthe adenine-induced anemia are warranted. Results fromprevious work using Epo-recombinant drugs on anemiain rats with CKD were not conclusive (Teixeira et al.2012).GA has been reported before to increase fecalnitrogen excretion and to lower serum urea nitrogenconcentration (Ali et al. 2009), and has since then beenused in treating CKD in several developing countriessuch as Sudan and Iraq (Ali et al. 2008, Al Mosawi2009). In addition to the increased clearance of nitrogenin CKD, GA has an additional beneficial effect on kidneyfunction, which is related to its anti-inflammatory andantioxidative effects (Ali et al. 2013a). Both oxidativestress and inflammation may be involved in CRF-inducedanemia (Cachofeiro et al. 2008), and treatment with GAmay also abate the anemia through these two actions.The liver-derived peptide hormone Hp in the
356Vol. 63Ali et al.kidney has an iron-regulatory role in the renal tubularsystem, involving the iron transporter divalent metaltransporter-1 (Kulaksiz et al. 2005). The concentration ofhepatic Hp was shown to be increased following CRFinduced by 5/6 nephrectomy (Srai et al. 2010) andadenine treatment (Hamada et al. 2008). As far as we areaware, there are no data on Hp in serum from rats withCRF, but it has been shown that in humans with CRF, Hpis elevated (Srai et al. 2010). Here, we have found thatadenine induces a significant rise in Hp concentration inserum, and that was significantly reversed byconcomitant GA treatment. The rise in serum Hpconcentration was probably due to the decrease in renalEpo concentration, as addition of Epo has been shown todownregulate Hp (Babitt and Lin 2012).In this work, serum GGT activity was measuredto assess the status of the liver. The significant increase inGGT activity following adenine feeding was suggestiveof hepatic damage. Although total hepatic Fequantification has not been conducted in the work, thehepatic damage might suggest that Fe stores werereduced, and this may be a factor in adenine-inducedanemia, although our current data does not support a rolefor the enzyme GGT in the regulation of hepatic Fe. Itshould also be mentioned that there are reports suggestingthe implication of GGT in the development of a disturbedredox status in the kidney cortex in rats with CKD(Ceyssens et al. 2004), and it is also established that thepathogenesis of adenine-induced CKD involves oxidativestress in the renal tissues (Ali et al. 2013a). Furtherstudies on this aspect are warranted.In conclusion, we have s
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Cooley's Anemia) - Blackfan-Diamond Anemia - Pure Red Cell Aplasia - Sickle Cell Disease Other Disorders of Blood Cell Proliferation Anemias Anemias are deficiencies or malformations of red cells. - severe Aplastic Anemia - Congenital Dyserythropoietic Anemia - Fanconi Anemia (Note: the first cord blood transplant in 1988 was for this disease)
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