Effective Dose and Duration in Administration of L. Plantarum AD3 on Experimentally Induced Uremic Rats

Transcription

1 Reserh Artile Effetive Dose nd Durtion in Administrtion of L. Plntrum AD3 on Experimentlly Indued Uremi Rts Arpit Ptr, Animesh Smnt, Shrey Mondl, Shrni Prdhn, Dilip Kumr Nndi * Deprtment of Miroiology, Nutrition, nd Humn Physiology, Rj N.L. Khns Women s College, Pshim Medinipur, West Bengl, Indi. *Corresponding uthor s E-mil: rpitptr28@gmil.om Reeived: ; Revised: ; Aepted: ABSTRACT Proiotis re defined s live miroorgnisms whih when dministered in dequte mounts onfer helth enefit on the host (FAO/WHO, 22). Effets of proiotis re strin speifi, wht tht mount should e is not indited. The miniml effetive dose or the level of vile ells of the proioti strin in terms of CFU/mL/dy tht demonstrtes generl helth promoting funtions. Thirty six mle lino rts were divided into six groups (6 rts /dose /week). Group I served s negtive ontrol- norml diet, groups II uremi ontrol - exposed etminophen interperitonilly t the dose of 5mg/kg ody weight /dy for 1 dys, group III, IV, V nd VI were different doses group, where 1 7, 1 8, 1 9 nd 1 1 CFU/mL of L.plntrum AD3 strin were o-dministered with etminophen (5mg/kg ody weight /dy for 1 dys) t different durtion (for 1 week, 2 week nd 3 week). Experimentl findings suggested tht etminophen exposure deresed the levels of totl ount of RBC nd H% signifintly (p<.5) fter 2nd week. Co-dministrtion of L. plntrum AD3 resulted in lowered the level of BUN nd retinine when dosed t 1 7 nd 1 8 CFU /ml/1g ody weight/dy, ut hd no signifint differene etween the group I, group II nd group III. However, signifint protetion ws oserved t dose of 1 9 CFU /ml/1g ody weight. However, L. plntrum AD3 signifintly llevited the kidney dmge in APAP-exposed rts when treted with t minimum dose t 1 9 CFU/mL/1 g of odyweight/dy for 14 dys. No ovious differene ws oserved in the kidneys in etween Group IV nd Group V smples. No signifint hnge in iohemil nd histologil prmeters ws found etween 14 dys nd 21 dys exposure with L.plntrum AD3. Similrly tking the minimum effetive dose, 1 9 CFU /kg ody dose optimum for L. plntrumad3for drug indued uremi mle rts. Keywords: Proioti, uremi, optimum dose. INTRODUCTION A Populr nlgesi nd ntipyreti drug, espeilly pretmol nd etminophen (APAP), nd nonsteroidl nti-inflmmtory drugs (NSAIDs) re widely used through the world. Pretmol nd APAP re nephrotoxi drugs. Severl in vitro nd in vivo studies showed tht nlgesis nephrotoxiity is used y inresed ROS in kidney 1,2. On the other hnd, APAP lso undergoes ytohrome P45 medited tivtion to the toxi N-etylp- enzoquinone imine (NAPQI) oth in liver nd kidney 3. At therpeuti doses, the quntity of NAPQI formed is reltively smll nd is detoxified y onjugtion with redued glutthione (GSH) 4,5. APAP overdose produes exessive NAPQI nd only prt of it n form GSH onjugte with resulting redution of ellulr GSH. The residul prt of NAPQI inds to ellulr proteins nd indues oxidtive stress, leding to renl injury. Therefore, serhing of defensive pthwy tht would offer mximum protetion ginst APAP indued renl dmges re required. Enteri dilysis is n lterntive pproh for solute removl in uremi is sed on the relity tht the intestinl rrier funtions s semipermele memrne. Conentrtion grdient mke solutes disperse from plsm into the lumen when onentrtion of uremi solute eome lrger in plsm thn lumen nd mjor prts of uremi solutes eome distriuted throughout the intestine y inding to ingestile solute-speifi sorents within the gut 6,7. This pproh requires lrge quntities of speifi sorents to e ingested dily. Thus to mitigte uremi solutes using live teri whih degrde uremi toxins within the gut hs een preferle pproh of todynd is further investigted in the present study 8, 9, 1. Proiotis re defined s live miroorgnisms whih when dministered in dequte mounts onfer helth enefit on the host (FAO/WHO, 22). Effets of proiotis re strin speifi. The tivity of ny strin in the gut would only e expressed fter its olonil estlishment. Potentility of teriotherpy to llevite uremi sttus ws lso noted y other investigtors. Mndl et l. (213) 11 reported tht orl dministrtion of Ltoillus spp effetively redued the levels of uremi toxins in dilysis ptients. Generlly dose of proioti is very importnt (s these re dynmi in nture) for exerting ny therpeuti effet. It ws estimted tht 1 9 CFU/mL is n effetive dose tht n rete soile environment t intestinl lumen nd exert enefiil effet to the host. But effetive dose my vry for different ilments. The miniml effetive dose or the level of vile ells of the proioti strin in terms of CFU/mL/dy tht demonstrtes generl helth promoting funtions or well eing or speifi helth lims in trget popultion should e lerly indited efore using it s phrmeutil vehiles s. Keeping this in view the im of the present study ws to evlute the optimum dose nd durtion of ntiuremi tivities of L. plntrum AD3 ginst etminophen indued uremi in rts. 163

2 MATERIAL AND METHODS Seletion of Animls nd Cre The study ws onduted on helthy, dult, mle lino rts of Wister strin (Supplied from Ghosh niml, niml foods nd niml ges Supplier, Kolkt, Indi) hving ody weight of 1 ± 15 g. They were limtized to lortory ondition for 2 weeks prior to experimenttion. Animls were housed six per ge in temperture-ontrolled room (22 ± 2 ºC) with h drk-light yle (8.-2. h light: h drk) t humidity of 5 ± 1 %. They were provided with stndrd food (pellet diet) nd wter d liitum. The priniple of lortory niml re Ntionl Institute of Helth USA Grouping of nimls & experimentl proedure The rts were divided into four equl groups s follows Groups Tle 1: Experimentl proedure Tretment proedure (NIH, 1985) guideline ws followed throughout the durtion of experiment 8 nd our Institute Ethis ommittee pproved the experimentl protool ording to the CPCSEA guidelines (CPCSEA Registrtion No- 195/PO/Re/S/216/CPCSEA). Grouping of nimls nd experimentl proedure Aute toxiity study ws performed ording to Orgniztion of Eonomi Co-opertion nd Development (OECD) guidelines-423 (ute toxi lss method). Experiments of etminophen injetion were done t the dose of 5mg/kg ody weight /dy for1dys ws hosen for studying iohemil ltertions in etminophen indued uremi rts. Durtion of L. plntrum AD3 tretment (weeks) GroupI-NC (Negtive Control) norml diet GroupII- UC (Uremi Control) Aetminophen indued Uremi GroupIII-D 1 D 7,D 1 D 14,D 1 D 21 GroupIV- D 2 D 7,D 2 D 14,D 2 D 21 GroupV- D 3 D 7,D 3 D 14,D 3 D 21 GroupVI-D 4 D 7,D 4 D 14,D 4 D 21 AD3 t 1 7 CFU /ml/1 g of ody wt /dy AD CFU /ml/1 g of ody wt /dy AD CFU /ml/1 g of ody wt /dy AD CFU /ml/1 g of ody wt /dy Aetminophen indued uremi - etminophen exposure (5mg/kg ody weight, for 1 dy), D 1, D 2, D 3 7, 18,19 nd D 4- for speifi dose of L. plntrumad3 (1 nd 1 1 CFU /ml/1 g of ody wt /dy respetively). D 7 D 14 D 21- for different durtion (1 st week-7 dys, 2 nd week-14 dys nd 3 rd week- 21 dys respetively). Hemtologil study After 3 weeks, nimls were srified y diethyl ether nesthesi. Blood smples were olleted y hepti rtery punhe under diethyl ether nesthesi, using 21 guge (21 G) needles mounted on 5mL syringe (Hindustn syringes nd medil devies ltd, Fridd, Indi.) into heprin oted smple ottles for nlyzed Hemtologil prmeters like RBC ount y hemoytometer nd hemogloin (H) y stndrd kit method (Merk, Jpn). Blood Uremi Profile Biohemil estimtion of Blood Ure The olleted lood ws entrifuged nd plsm frtion ws seprted. Ure level of plsm ws mesured y ommerilly ville stndrd Blood Ure Kit (Merk, Jpn) y Semi utonlyser (Merk, Jpn) y stndrd protool for phtotometri determintion of ure ording to the Urese GLDH method 12. Biohemil estimtion of Blood Cretinine The olleted lood ws entrifuged nd plsm frtion ws seprted. Cretinine level of plsm ws mesured y ommerilly ville stndrd retinine Kit (Merk, Jpn) y Semi utonlyser (Merk, Jpn) y stndrd protool for phtotometri determintion of retinine sed on Jffe kineti method without deproteiniztion 13. Sttistil nlysis All the vlues re expressed s men ± SE (n = 6). Signifint differenes etween the groups were determined with SPSS 1. softwre (SPSS In., Chigo, IL, USA) for Windows using one-wy nlysis of vrine (ANOVA) nd the group mens were ompred y Dunn s Multiple Rnge Test (DMRT). A differene ws onsidered signifint t the P <.5 level. RESULT AND DISCUSSION Blood uremi profile Aording to the result we found tht etminophen exposure ws signifintly deresed the levels of totl ount of RBC nd H% fter 2 nd week omprison to 164

3 negtive ontrol (Group I). It ws oserved tht totl ount of RBC nd H% were signifintly ltered when L.plntrum AD3 ws dosed t1 9 nd 1 1 CFU /ml/1g ody weight/ dy in omprison to uremi group (Group II). However, there ws no signifint differene in etween GroupV nd Group VI (t1 9 nd 1 1 CFU /ml/1g ody weight/ dy dose respetively) (in Fig. 1.1IIInd IV). In time dependent study t the effetive dose of 1 9 CFU//mL/1g ody weight/ dy of L.plntrum AD3, mximum protetion ws oserved fter the tretment for 2 nd week (14 dys). No signifint hnge ws oserved even with inresing the time of L. plntrum AD3 tretment for 3 rd week (21 dys) respet to 2 nd week (Fig 1.1 V nd VI). Aute exposure of etminophen, widely used nlgesi nd ntipyreti drug, uses severe renl dmge. No speifi gent hs een reported so fr tht plys ny enefiil role in the orgn pthophysiology. These findings let us to investigte the pproprite dose nd durtion involved in the nephro intoxitive effet of isolted urese positive proioti LAB strin, ginst etminophen indued renl pthophysiology. Aetminophen nephrotoxiity results from its highly retive nd toxi metolite, N-etyl-p-enzoquinone imine (NAPQI), whih destroy proteins in the S3 segment of the proximl tuule nd tht in turn, initites the dmge of renl tuulr 14.The fundmentl funtion tht lood ells perform together with the suseptiility of this highly prolifertive tissue to intoxition y xenoiotis mkes the hemtopoieti system unique s trget orgn. Erythroytes, leukoytes nd pltelets were produed t turnover rte of out 1-3 million per seond in helthy humn dult, nd these vlues n e distorted in ertin physiologil or pthologil sttes during hemolytinemi or suppressive inflmmtion 15. Certin drugs inluding lkylting ytotoxi gents n lso ffet lood ell development rtes nd the norml rnge of hemtologil prmeters. In the present study, it ws reveled tht the high dose of etminophen intrperitonelly for 1 dys were signifintly deresed H levels nd lso lowered of totl RBC ounts (Figure 1.1). The deresed H level ws supposed due to e destrution of RBCs nd lower totl RBC ount due to hemolytinemi or suppressive inflmmtion. Codministrtion of L. plntrum AD3 with etminophen for minimum 2 weeks, t dose of 1 9 CFU /ml/1g ody weight/dy, restored the H levels s well s totl RBC ounts thn the etminophen-indued uremi group (II). However, this study showed tht the teri ould reverse the hemtotoxi effet of etminophen, with resulting improvement of hemtopoiesis. Experimentl findings suggested tht etminophen exposure (5mg/kg ody weight, for 1 dy) ws signifintly inresed the levels of lood ure nitrogen (BUN) nd retinine fter 14 dys in uremi group (group II) ompred to negtive ontrol (group I) (Figure 1.1 III nd IV). Co-dministrtion of L. plntrum AD3 resulted in lowered the levelof BUN nd retinine when dosed t 1 7 nd 1 8 CFU /ml/1g ody weight/dy, ut hd no signifnt differene etween the group I, group II nd group III (Fig 1.1). However, signifint protetion ws oserved t dose of 1 9 CFU /ml/1g ody weight (s presented in the Fig. 1.1). Similrly the dose of 1 9 CFU /ml/1g ody weight/dy optimum for oth teri in time dependent study. In this study the mximum elevtion ws oserved fter the tretment for 14 dys for L. plntrum AD3 nd the result did not hnge muh even with inresing the time of teri supplementtion. This drug-indued nephrotoxiity is frequently ssoited with mrked elevtions in lood ure nitrogen, serum retinine nd ute tuulr nerosis 16, I 8 B.W (%) Cr(mg/dL) NC UC D1D7 D2D7 D3D7 D4D7 III NC UC D1D14 D2D14 D3D14 D4D14 V B.W (%) Cr(mg/dL) B.W (%) Cr(mg/dL) NC UC D1D21 D2D21 D3D21 D4D II Ure (mg/dl) H% RBC(U) NC UC D1D7 D2D7 D3D7 D4D7 IV Ure (mg/dl) H% RBC(U) NC UC D1D14 D2D14 D3D14 D4D14 VI Ure(mg/dL)H H% RBC(U) NC UC D1D21 D2D21 D3D21 D4D21 Effet of L. Plntrum AD3 fter 7 dys Effet of L. Plntrum AD3 fter 14 dys Effet of L. Plntrum AD3 fter 21 dys Different dose (CFU/mL/1g ody weight/dy) Figure 1.1: Dose nd time dependent effet of L.plntrum AD3 on uremi prmeters in APAP indued toxiity in the lood of the experimentl rts. In this figure-i nd II show effet of 7 dys dministrtion of L.plntrum AD3 on uremi rts. In figure-iii nd IV show effet of 14 dys dministrtion of L.plntrum AD3 on uremi rts. NC negtive ontrol 165

4 group, UC- uremi group, In the groups D1, D2, D3 nd D4- for speifi dose of L.plntrum AD3(1 7,1 8,1 9 nd 1 1 CFU /ml/1 g of ody wt /dy respetively. D7D14D21- for different durtion (7 dys, 14 dys nd 21 dys respetively. In ll ses dt re expressed s Men±SE (n=6). ANOVA followed y multiple two til t-test. Brs with different supersripts (,, ) differ from eh other signifintly (p <.5) In the present study, dministrtion of nephrotoxi dose of etminophen to rts resulted in progression of oxidtive stress hrmful to renl tissues. Aetminophenindued nephrotoxiity showed signifint (p<.5) inrese in the plsm ure nd retinine onentrtions in Group II (etminophen indued uremi) rts when ompred with the negtive ontrol group (Group I) (Fig 1.1). Moreover, orl dministrtion of L. plntrum AD3 for 2 weeks, in the dose of 1 9 CFU /ml/1g ody weight/dy signifintly (p<.5) deresed plsm ure nd retinine, when ompred with Group II. The intestinl muosl surfe funtions s semi-permele memrne. Driven y the onentrtion grdient, solutes with higher onentrtion in irulting lood diffuse from plsm into the intestine, nd lrge portion of uremi solutes re differentilly distriuted within the owel. Histologil prmeter Kidney histologil study ws used to determine the protetive effet of L.plntrum AD3 on APAP-indued injury (Fig.1.2) t different dose for 14 dys. APAP tretment (UC) used severl visile histologil hnges. The renl setions showed extensive tuulr dmge y presene of neroti epithelil ells. Tuulr degenertion, nerosis, ell swelling, mononuler ell infiltrtion nd degenerted orgnelles were lso oserved in the kidney following the APAP exposure. Some epithelil ells were found dmged in the tuulr lumen (Fig. 1.2). However, L.plntrum AD3 signifintly llevited the kidney dmge in APAP-exposed rts when treted with t minimum dose t 1 9 CFU/mL/1 g of odyweight/dy for 14 dys. No ovious differene ws oserved in the kidneys in etween Group IV nd Group V smples (Fig. 1.2 E nd F). Figure 1.2 Histologil study of L. plntrum AD3 tretment group t different dose for 14 dys omprison to ontrol groups.(a): Showing norml histology of kidney of negtive ontrol group of rts (NC). Norml Glomerulus (NG) nd renl tuules (NT) re well orgnized in historhiteture. (B) Showing severe disorgniztion of rt kidney fter etminophen injetion of 5mg/kg ody weight to uremi ontrol group rts (UC). Dmged Glomerulus (DG) nd dilted renl tuules (DT) re seen. (C) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 7 CFU/mL/1 g of odyweight/dy for 14 dys to group III rts showing dmged glomerulus (DG) nd dilted tuule (DT).nd.(D) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 8 CFU/mL/1 g of odyweight/dy for 14 dys to group IV rts showing dmged glomerulus (DG) nd dilted renl tuule (DT).nd (E) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 9 CFU/mL/1 g of odyweight/dy for 14 dys to group V rts showing ontrol like histology with reorgnized glomerulus (RG) nd renl tuule (RT).nd (F) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 1 CFU/mL/1 g of odyweight/dy for 21 dys to group VI rts showing orgnized glumerulus (RG) nd renl tuule (RT). 166

5 Tetment y L.plntrum AD3 in Group IV t minimum dose of 1 9 CFU/mL/1 g of odyweight/dy for 14 dys showed n improvement nd rerrngement of these ells omprison to Group III nd Group IV (Fig 1.2). Orlly dministrtion of urese positive proioti L.plntrumAD3 t high dose showed ontrol likes historhiteture of kidney. No signifint hnge in iohemil nd histologil prmeters ws found etween 14 dys nd 21 dys exposure withl.plntrumad3 (Fig 1.3). Figure 1.3 Histologil study of L. plntrum AD3 tretment group t 1 9 CFU/mL/1 g of odyweight/dy for different durtion omprison to ontrol groups. (A) Showing norml histology of kidney of ontrol group of rts. Norml Glomerulus (NG) nd renl tuules (NT) re well orgnized in historhiteture. (B) Showing severe disorgniztion of rt kidney fter etminophen injetion of 5mg/kg ody weight to Group II rts. Dmged Glomerulus (DG) nd dilted renl tuules (DT) re seen. (C) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of 1 9 CFU/mL/1 g of odyweight/dy for 7 dys to group III rts showing ontrol like histology with norml glomerulus (NG) nd renl tuule (NT). (D) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 9 CFU/mL/1 g of odyweight/dy for 14 dys to group III rts showing ontrol like histology with norml glumerulus (NG) nd renl tuule (NT) nd (E) Showing etminophen indued uremi rt kidney (5mg/kg ody weight) with tretment y L. plntrum AD3 t the dose of1 9 CFU/mL/1 g of odyweight/dy for 21 dys to group III rts showing norml glumerulus (NG) nd renl tuule (NT) like negtive ontrol. 167

6 CONCLUSION In this study iohemil nlysis of uremi mrkers were mesured to onfirm the nephrotoxiity. The histology of kidney lso showed severe disorgniztion fter tretment of APAP (5mg/Kg ody weight for 1 dys). And o- tretment with L.plntrumAD3 strin t 1 9 CFU/mL/1 g of odyweight/dy for 14 dys ould restore the dmge. Control like historhiteture of kidney ws lso oserved (Fig 4.2d). Thus the lower dose (1 1 9 CFU /ml/1g ody weight/ dy for 14 dys) ws suggested for isolted proioti strin thn the higher dose (1 1 1 CFU /ml/1g ody weight/ dy) s effetive therpeuti dose ording to ICMR-DBT guide line 211. It ws evident tht the teri supplementtion t dose of 1 9 CFU /ml/1g ody weight/ dy/ 14 dys hs urtive role ginst etminophen indued uremi. The mode of tion of uremi prevention my proly due to its high urese enzyme prodution property s well s ntioxidnt property (disussed in hpter2). In the se of group treted with L. plntrum AD3 t dose of 1 9 CFU /ml/1g ody weight/ dy durtion for 2 weeks showed signifint redution of uremi profiles. REFERENCES 1. Ozek E. Indution of Oxidtive Stress in Kidney. Int J Nephrol Volume 212, Artile ID , 9 pges. 2. Roy S, Prdhn S, Ds K, Mndl S, Mndl A, Ptr A,Smnt A, Sinh BD, Nndi DK, Aetminophen indued kidney filure in rts: A dose response study, J Biosienes, 15, 215, Arhm P, Vimin C my e enefiil the prevention of pritmol induedrenl dmge, ClinExpNephrol, 9, 25, 9, Mutsers H A, Strios E G, Glorieux G, Vnholder R nd Oling P. Chroni kidney disese nd firosis: the role of uremi retention solutes, Frontiers in mediine, 215, Pndy D, Ngrjpp A K, Rvi K S. Assessment nd Correltion of Ure nd Cretinine Levels in Sliv nd Serum of Ptients with Chroni Kidney Disese, Dietes nd Hypertension A Reserh Study. J Clini Dignosti Res, 1, 216, ZC58 6. Iley Y O, Ozek E, Cekmen M, Somy A, Ozn L otuntemur A, Simsek A, Mete F., Melotonin prevents etminophen-indued nephrotoxiity in rts, IntUrolNephrol, 41, 29, Sprks RE, Review of gstrointestinl perfusion in the tretment of uremi, ClinNephrol 11, 1979, Hid M, Ai Y, Swmur S, Suzuki N, Stoh T, Kog Y, Inhiition of the umultion of uremi toxins in the lood nd their preursors in the fees fter orl dministrtion of leenin, lti id teri preprtion, to uremi ptients undergoing hemodilysis, Nephron,74,1996, Rngnthn N, Rngnthn P, Friedmn EA, Joseph A, Delno B, Goldfr D S, Tm P, Ro AV, Anteyi E, Musso CG, Pilot study of proioti dietry supplementtion for promoting helthy kidney funtion in ptients with hroni kidney disese, Adv Ther, 27, 21, Vitett L, Goe G, Uremi nd hroni kidney disese: The role of the gut miro flor nd therpies with pro-nd preiotis. Mol Nut Food Res, 57, 213, Mndl A, Roy S, Ds K, Mondl K & Nndi DK, In vivo ssessment of teriotherpy on etminophen-indued uremi rts, J Nephrol, 26, 213, Burtis CA & Ash wood ER, Tietz textook of linil hemistry, 3rd edn (W B Sunders, Phildelphi, USA) Sgh M, Rik W, Shneider S, A kineti method for the diret determintion of retinine in serum with 3, 5- dinitroenzoi id without deproteiniztion, J ClinChemClinBiohem, 26, 1988, Guyton AC, Red lood ells, nemi, nd polyythemi. In: Textook of Medil Physiology. 8th ed. Phildelphi: W.B. Sunders, 1991, pp Prdhn S, Roy S, Mndl S, Smnt A, Ptr A, Ds K, Ds S, Nndi DK, Alph-Lipoi Aid A Cholesterol Reduer for Trgeting Aetminophen Indued Uremi Mle Alino Rt Models, Interntionl Journl of Phrmeutil Sienes Review nd Reserh, 29(1), 214, Ptr A, Mndl A, Smnt A, Roy S, Mndl S, Prdhn S, Ds Mhptr T, Mondl KC, Nndi DK, Therpeuti potentil effet of Streptoous thermophillus(mtcc 1938) on etminophen indued uremi mle rts, Ind J Biotehnol, 13, 214, Verpooten GA, Tulkens PM, Bennett WM, Aminoglyosides nd vnomyin. In: De BroeME, Porter GA, Bennett AM, Verpooten GA, editors, Clinil nephrotoxints, renl injury from drugs nd hemils. Netherlnd: Kluwer, 1998, pp Soure of Support: Nil, Conflit of Interest: None. 168