Khaled K. Al-Qattan, Martha Thomson, Divya Jayasree, and Muslim Ali

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1 Hindwi Pulishing Corportion Evidence-Bsed Complementry nd Alterntive Medicine Volume 216, Article ID , 11 pges Reserch Article Grlic Attenutes Plsm nd Kidney ACE-1 nd AngII Modultions in Erly Streptozotocin-Induced Dietic Rts: Renl Clernce nd Blood Pressure Implictions Khled K. Al-Qttn, Mrth Thomson, Divy Jysree, nd Muslim Ali Deprtment of Biologicl Sciences, Fculty of Science, Kuwit University, P.O. Box 5969, 136 Sft, Kuwit Correspondence should e ddressed to Mrth Thomson; mmtkuniv@gmil.com Received 21 Mrch 216; Accepted 24 April 216 Acdemic Editor: Khlid Rhmn Copyright 216 Khled K. Al-Qttn et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Rw grlic queous extrct (GE) hs meliortive ctions on the renin-ngiotensin system in type-1 dietes mellitus (DM); however its effects on plsm nd kidney ngiotensin I converting enzyme type-1 (ACE-1) nd ngiotensin II (AngII) require further elucidtion. This study investigted the effect of GE on plsm nd kidney ACE-1 nd AngII concentrtions nd in reltion to systemic nd renl clernce indictors significnt to lood pressure (BP) homeostsis in erly streptozotocin- (STZ-) induced type-1dm.normlrts(n=1) received.5 ml norml sline (NR/NS), dietic rts (n =1) received.5 ml NS (DR/NS), nd treted dietic rts (n =1) received 5 mg/.1 ml/1 g ody weight GE (DR/GE) s dily intrperitonel injections for 8 weeks. Compred to NR/NS, DR/NS showed significnt increse in plsm ACE-1 nd AngII nd conversely decrese in kidney ACE-1 nd AngII. These chnges were ssocited with n increse in BP nd clernce functions. Alterntively nd compred to DR/NS, DR/GE showed normliztion or ttenution in plsm nd kidney ACE-1 nd AngII. These GE induced rectifictions were ssocited with modertion in BP elevtion nd renl clernce functions. Grlic ttenutes modultions in plsm nd kidney ACE-1 nd AngII, in ddition to BP nd renl clernce function in type-1 DM. 1. Introduction The endocrinl renin-ngiotensin system (RAS) ws initilly descried s follows: upon stimultion, renin, protese, is relesed y oth kidneys to the generl circultion. In theplsm,reninctsonngiotensinogen,nα-gloulin synthesized y the liver, to lierte decpeptide known s ngiotensin I (AngI). While pssing through the pulmonry circultion, AngI is cleved y dipeptidyl dipeptidse known s ngiotensin I converting enzyme (ACE-1) to free n octpeptide clled ngiotensin II (AngII) [1]. Recently, it hs een sserted tht the kidneys produce ll components of the RAS [2, 3]. ACE-1 is the second rte limiting enzyme tht controls theliertionofangii:themostctivecomponentofthe RAS [4]. AngII hs numerous iologicl ctivities, including vsoconstriction, ntintriuresis, nd ntidiuresis, ctions which re closely ffilited with renl clernce functions nd BP regultion [5]. It is most prole tht under physiologicl conditions renlly nd systemiclly produced AngII work synergisticlly, where renl AngII cts s the principle prcrine regultor of the kidneys clernce function determinnts, including renl hemodynmics, glomerulr filtrtion rte (GFR), nd tuulr hndling of electrolytes nd wter [6]. Alterntively, systemiclly produced AngII opertes s the min telocrine modultor of, firstly, generl nd, secondly, peripherl vsculr resistnce [7]. The fine tuning of the AngII-medited ctions is chieved y AngII inding to nd ctivting either/or oth of its two mjor receptor types: AT 1 nd AT 2 [8, 9]. Specificlly, AT 1 receptor fcilittes the AngII known ctions of generl nd peripherl vsoconstriction, renl ntintriuresis nd ntidiuresis, nd cell growth nd prolifertion [1], while the AT 2 receptor medites the suggested AngII vsodilttion, ntriuresis nd diuresis, poptosis, nd ntiprolifertion, ctions tht ntgonize those evoked y the AT 1 receptor type [11].

2 2 Evidence-Bsed Complementry nd Alterntive Medicine Dysregultion in the RAS ssocited with AngII AT 1 /AT 2 receptors expression imlnce is mjor fctors in the initition nd progression of tissue remodeling nd refunctioning in iochemicl-physiologicl pthologies [8, 1, 12, 13] including dietes mellitus (DM) [14]. DM is progressive disese tht entils dynmic phse-chnging, structurl-functionl chrcteristics. In n ttempt to elucidte the nture of RAS modultions in insulin-dependent type-1 DM [15], the findings of previous studies led to the formultion of two mjor conflicting views. A group of studies hve suggested n increse in the RAS ctivity nd consequently ACE-1 nd AngII iovilility especilly in renl tissue nd nephronl structures. This view ws supported y findings tht inhiitors of different RAS components nd AngII receptor lockers were effective in prtilly diminishing DM normlities [12, 14]. An ugmented RAS ctivity most likely occurs t lter stges of DM when moleculr/cellulr trnsformtions collectively led to severe renl-nephronl injuries; in prticulr glomerulotuulr sclerosis, which impedes kidney clernce function leding to the end-stge renl filure tht is chrcteristic of dvnced DM [16 18]. A hypoinsulinemichyperglycemic provoked high AngII concentrtion nd stimultion of overexpressed AT 1 receptors led to the following events in the kidneys: sodium trnsport nd retention, vsculr resistnce, glomerulr cpillry pressure, mechnostretch-induced rective oxygen species production, nd tuulointerstitil cell hypertrophy nd hyperplsi ssocited with extrcellulr mesngil mtrix production [19, 2]. This AngII/AT 1 scenrio is excerted y downregultion of intrrenl AT 2 receptor expression [21] nd susequent minimiztion of its medited lleviting responses including inhiition of N + -K + -ATPse ctivity [22], sodium pump ction in renl proximl tuules [23], ntintriuresis [24], vsoconstriction [11], cell hypertrophy, nd renl glomerulr nd tuulr remodeling [8, 1, 25]. Other studies hve reported opposite findings nd vritions in the ctivity of plsm nd renl RAS components, prticulrly ACE-1 nd AngII, in erly DM. In 4 weeks fter streptozotocin- (STZ-) induced type-1 DM rts, it ws reported tht the RAS is downregulted t the level of mrna expression [26]. Furthermore, renl ACE content [27] s well s AngII concentrtion [28] ws reduced. In ddition, further decline in AngII concentrtion ws suggested to result from n increse in degrdtion y the enzyme ngiotensinse A [29]. In review, Copper et l. [3] rgued tht RAS ctivtion is controversil s different nimlmoleculr studies reported conflicting results. In prticulr, it wssuggestedthtinerlydmtheindicesofrasrelower nd the concentrtions of renl AngII nd its receptor AT 1 re reduced leding to hyperfiltrtion. An incresed GFR, which cn e monitored y mesuring cretinine clernce in live sujects [31], with suggested lower AngII type AT 1 receptor-medited ctivtion of sodium retention leds to higher sodium nd wter clernce. This excretory ehvior cuses polyuri, in ddition to luminuri, nd necessry, however futile, polydipsi: myrid of symptoms tht re typicllyoservedttheerlystgesofdm. Grlic hs long een used in trditionl medicine s n esily ville nd ccessile nturl medicine [32] to control BP nd sugr in generl [33] nd when ffilited with DM complictions [34]. Within the pst two decdes, grlic, either s n queous extrct (GE) or isolted orgnosulfur constituents, hs een the focus of intensive studies in the STZ-induced experimentl model of type-1 DM [35] showing severl interesting nd well documented meliortive ctions [36]. Among these ctions is grlic s ility to induce severl iochemicl-physiologicl mesures t the systemic level; prticulrly, serum insulin elevtion [37, 38], lood glucose reduction [38, 39], serum ACE ctivity diminutionndsuggestedreducedangiigenertion[4], nd, therefore, hypotension [36]. The induced hypotension my e medited, in prt, through reduced AngII concentrtion resulting in lower vsoconstrictive ction nd/or indirectly through n increse in the mounts nd ctions of vsodiltory gents [41]. More recently, our group hs lso shown tht GE tretment in erly STZ-DM improved kidney clernce functions [42], in ddition to preserving the norml expression nd lnce of the two AngII receptors types [43, 44]. To further elucidte grlic s meliortive mechnism reltedtorasinerlytype-1dm,thisstudyinvestigted the effect of GE on DM-induced chnges in plsm nd renl ACE-1 nd AngII concentrtions. The effects of GE on ACE- 1 nd AngII were correlted to simultneous modultions in BP nd systemic nd renl indictors of clernce function. 2. Mterils nd Methods 2.1. Mterils. Themterilsusedinthisstudy,unlessotherwise stted, were otined ccordingly: nlysis kits s indictedinthissection,thiopentlsodiumfrommy& Bker (Englnd) nd STZ, chemicls, nd regents from Sigm-Aldrich (USA) Preprtion of Rw Grlic Aqueous Extrct. The GE (5 mg/.1 ml) used in this study ws prepred from loclly purchsed, peeled fresh grlic cloves (Allium stivum L.) s previously descried [45]. The prepred GE ws immeditely stored s 1 ml liquots in 1.5 ml self-cpping, inert-plstic freeze-durle Eppendorf tues t 2 C. The required volume of GE ws thwed dily to mient temperture efore dministrtion nd following use ny remining mount ws discrded. GC-MS comprtive nlysis of the GE fter different times of freeze-storge showed similr composition nd concentrtion of components [42, unpulished dt] Animls. Thenimlsusedinthisstudyweremle Sprgue-Dwley rts (ncestors origin: Hrlnd L, Oxfordshire, Englnd) hving n initil ody weight of 15 2 grms. Before nd during the study, the rts were kept in dequte-sizeseprtecgesndhousedinnanimlcre Fcility under stndrd mient conditions (23±2 C, nturl dy/night cycle). The rts were provided with stndrd rodent diet (17 mmol N + /kg) nd tp wter d liitum.

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 The cre nd use of ll rts used in this study ws in full complince with the Guide for the Cre nd Use of Lortory Animls, Ntionl Reserch Council [46]. 2.4.InductionofType-1Dietes. The type-1 DM rts used in this study were produced y intrperitonelly injecting ech of sufficient numer of overnight fsting rts with single dose of STZ (6 mg/1 g ody weight) dissolved in citrte uffer (.3 ml,.1 M, ph 4.5) s descried previously [39]. After 5 dys, STZ-injected rts with lood glucose concentrtion 16 mmol/l mesured in drop of til-lood (One Touch UltrEsy Glucometer, UK) under mild ether sedtion were deemed dietic (n =2)ndusedinthestudy Rts Groups nd Tretments. At dy 7 fter STZ injection,dmrtsweredividedintotwogroupsndtretedfor 8 weeks with either single dily intrperitonel injection of.1 ml of norml sline/1-grm ody weight (DR/NS, n= 1) or 5 mg/1-grm ody weight/.1 ml of GE (DR/GE, n = 1). For reference, norml rts, injected initilly with.3 ml of only citrte uffer nd hving norml lood glucose 8mmol/L (n=1), received single dily intrperitonel injection of.1 ml/1-grm ody weight of norml sline (NR/NS, n=1) lso for 8 weeks Mesurements of Blood Glucose, Blood Pressure, Wter Intke, nd Urine Output. The following prmeters were mesured for ll rts in ech group s follows: lood glucose eforendtweeks4nd8ofrespectivetretment;bpt weeks 1 nd 8 of respective tretment s n verge of 3 redings for ech rt using the til-cuff method (Hrvrd Apprtus, Englnd); wter intke nd urine output efore nd t weeks 1, 4, nd 8 of respective tretment for 24 h nd clculted for 1 h Collection of Blood nd Preprtion of Plsm nd Serum Smples. At the end of the 8-week tretment period, ech rt ws nesthetized with n intrperitonel injection of Thiopentl Sodium (4 6 mg/1 g). Within 2-3 minutes, lood ws collected vi crdic puncture from ech rt s 3 seprte portions of 2mL ech into 3 15 ml inert-plstic tues (Flcon, USA) nd treted ccordingly: (1) 2 ml lood in tue contining.4 ml of peptidse inhiitor cocktil (.2 ml trisodium citrte (.1 M),.5 ml O-phennthroline (.44 mm),.5 ml pepsttin (.12 mm),.5 ml EDTA (.6 M), nd.5 ml P-hydroxymercurienzoic cid (1 mm)) for plsm preprtion used for AngII concentrtion determintion, which ws done immeditely s descried elow; (2) 2 ml lood in tue contining.2 ml EDTA for plsm preprtion used for ACE-1 concentrtion estimtion; (3) 2 ml lood in tue for serum collection used for insulin, lumin, nd cretinine mesurement. Collected plsm (except for AngII nlysis) nd serum smples were stored s pproximtely.5 ml liquots in Eppendorf tues t 4 C for lter nlysis Preprtion of Kidneys Homogente nd Collection of Superntnt Smples. Following collection of lood nd within 3 45 seconds, the left kidney of ech rt ws excised nd while thing in the peptidse inhiitor cocktil decpsulted cut into 4-5 portions nd plced seprtely in 1 ml, cpped inert-glss vils contining 3 ml of the inhiitor. Also, within 3 45 seconds, the right kidney ws excised nd while thing in Tris-HCl (.5 M, ph = 7.6) uffer decpsulted, cut into 4-5 portions, nd plced seprtely in 1 ml vil contining 3 ml of the uffer. Afterwrds, ech kidney ws homogenized, llowed to stnd on ice for few minutes, nd then centrifuged for 15 minutes t 8 g t 4 C. The superntnt of ech right kidney ws stored seprtelys.5mlliquotsineppendorftuest 4 Cfor lter nlysis, while the superntnt of the left kidney ws ssyed immeditely for AngII nd protein concentrtions s descried elow Determintion of Insulin, Angiotensin Converting Enzyme I, Angiotensin II, Alumin, nd Cretinine Concentrtions. These prmeters were quntitted using nlysis kits supplied s indicted nd following the mnufcturers instructions: serum insulin ws mesured y the ELISA method using kits from SPIio (Frnce); plsm nd kidney ACE-1 concentrtion were lso determined y the ELISA method using kits cquired from Uscn Life Sciences Inc. (Chin); plsm nd kidney AngII were mesured y the immunossy procedure using kits from RyBiotech (USA); serum nd urine lumin were determined y colorimetric technique using kits from BioAssy Systems (USA); nd finlly, serum nd urine cretinine levels were estimted y colorimetric method using kits from Rndox (USA) Numer of Experimenttion Cycles Crried Out. All procedures stted ove were crried out through the necessry numer of experimentl cycles to replce rts tht did not develop typicl symptoms of DM fter STZ injection or died during the different tretment protocols. The rte of success of the first cycle ws very high Sttisticl Anlysis. The dt re presented in r grphs s the men ± SEM of the solute vlues of the mesured nd clculted prmeters. Sttisticl differences etween the 3groupswereclcultedusingOne-Wy ANOVA (SPSS, V 22, IBM) with LSD post hoc test t P <.5 indicting significnce. Differences etween the 3 groups t ech corresponding stge of the experiment re lso presented in percentge vlues in Results. 3. Results 3.1. Effect of Grlic Extrct on Blood Glucose, Body Weight, Wter Intke, Urine Output, nd Blood Pressure. At dy 6 fter STZ injection nd efore commencing the tretment protocol, the lood glucose level of oth dietic rts groups ws significntly higher y 195% compred to tht of the NR/NS group (Figure 1). The elevted lood glucose ws sustined in the DR/NS group during the 8-week tretment period. Alterntively, t week 4 nd week 8 of tretment, the DR/GE group hd lood glucose levels tht were significntly

4 4 Evidence-Bsed Complementry nd Alterntive Medicine Blood glucose (mmole/l) Dy 6 (BT) Week 4 Week 8 NR/NS DR/NS DR/GE Figure 1: GE tretment decresed lood glucose of dietic rts. Blood glucose ws mesured t dy 6 fter STZ injection nd t the end of weeks 4 nd 8 of the tretment period. NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; BT: efore tretment; significntly different compred to NR/NS; significntly different compred to DR/NS. less y 27 nd 54%, respectively, compred to the DR/NS group; however, these levels were significntly higher thn thoseofnr-nsgroupy128%tweek4nd55%tweek 8 (Figure 1). The initil ody weight verge of the rts for ech group ws similr mong the 3 groups. However, s the study proceeded, the verge ody weight strted to shift t dy 6 fter STZ injection, where the NR/NS ody weight strted to increse, while oth dietic groups' ody weight strted, slightly ut significntly, to decline compred to their nd the NR/NS initil weight. As the tretment proceeded, the NR/NS weight incresed stedily, while the DR/NS weight decresed continuously. Alterntively, the DR/GE weight pickedupfromtheinitildropndevenshowedslightgin t the lst 2-3 weeks compred to their strting weight. At the end of the tretment protocol, the 3 groups of rts showed the following chnges in ody weight: NR/NS gined 135%; DR/NS lost 54%; DR/GE gined 18% (Figure 2). Agin, t dy 6 fter STZ injection nd efore strting thetretmentprotocol,themesuredwterintkendurine output of oth dietic groups were significntly higher y n verge of 381 nd 9%, respectively, compred to the wter intke nd urine output mesured for the NR/NS group. The NR/NS group s wter intke nd urine output did not chnge significntly when mesured t week 4 nd week 8. As fr s the DR/NS group is concerned, not only did these rts' elevtedwterintkendurineoutputreminsignificntly higher, ut their wter intke even incresed stedily to higher levels t week 4 nd week 8 of the tretment period. As for the DR/GE group, lthough these rts wter intke nd urine output were still significntly higher thn the mesured vlues of the NR/NS t week 4 nd week 8 y n verge of 323 nd 555%, respectively, the vlues of these prmeters t these sme weeks for the GE-treted dietic group were significntly less y n verge of 29 nd 2%, respectively, compred to the vlues of the DR/NS group (Figure 3). Chnge in ody weight (%) ,,,,,,,, Pre- Dy W-1 W-2 W-3 W-4 W-5 W-6 W-7 W-8 STZ 6 (BT) NR/NS DR/NS DR/GE Figure 2: GE tretment incresed ody weight of dietic rts. GE ws dministered IP to dietic rts for 8 weeks revited t DR/GE. Dietic control rts (DR/NS) nd norml control rts (NR/NS) were given norml sline. The nimls were weighed efore STZ injection (pre-stz), 6 dys fter STZ injection (dy 6 (BT)), nd weekly for the 8-week tretment period. Weights re plotted s percentiles with the strting weights ll stndrdized to 1%. Pre-STZ: efore streptozotocin dministrtion; BT: efore tretment; w: week; significntly different compred to NR/NS; significntly different compred to DR/NS. At week 1 of the tretment period, the NR/NS group BP ws within norml rnge nd remined t tht level when mesured t week 8. Alterntively nd t week 1, oth the DR/NS nd DR/GE groups hd significntly higher BP y n verge of 65% compred to the NR/NS group. Although this elevted BP ws still evident for oth of the DR/NS nd DR/GE groups t week 8, it ws slightly ut significntly less in the DR/GE group y n verge of 1% compred to oth this group s reding t week 1 nd the DR/NS group reding t week 8 (Figure 4) Effect of Grlic Extrct on Insulin, Angiotensin Converting Enzyme-1, Angiotensin II, Alumin, nd Cretinine. At the end of the 8-week tretment period, the serum insulin level for the DR/NS group ws significntly less y 89% compred to the insulin vlue mesured for the NR/NS group. In the DR/GE group, lthough the insulin level ws still less thn for the NR/NS y 54%, it ws significntly higher y 331% compred to the insulin level mesured for the DR/NS group (Figure 5). The plsm nd kidney levels of ACE-1 in the NR/NS group were 55 pg/ml nd 1917 pg/mg protein, respectively. In the DR/NS group, nd compred to the NR/NS, ACE- 1 levels showed opposite, yet significnt chnges, where the plsm ACE-1 ws higher y 37% nd the kidney ACE-1 ws lower y 48%. In the DR/GE group, the plsm nd kidney ACE-1 vlues, with only minor ut significnt difference of 14%, were lmost comprle to those mesured in the NR/NS group (Figure 6). In the DR/NS nd DR/GE rts, the oserved chnges in the plsm nd kidney AngII levels showed prllel ehviors to those quntitted for their respective ACE-1 concentrtions. More precisely, the plsm AngII level ws

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 6 Urine output (ml/h) Dy 6 (BT) Week 4 Week 8 Blood pressure (mm Hg) Week 1 Week 8 Wter intke (ml/h) NR/NS DR/NS DR/GE NR/NS DR/NS DR/GE () Dy 6 (BT) Week 4 Week 8 () Figure 3: GE tretment decresed urine output nd wter intke of dietic rts. () Urine output (ml/h) nd () wter intke (ml/h) were mesured efore tretment (BT) nd fter 4 nd 8 weeks of tretment. NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; BT: efore tretment; significntly different compred to NR/NS; significntly different compred to DR/NS. significntly higher y 25% in the DR/NS group compred to tht in the NR/NS group nd less y 29% in the DR/GE group thn in the DR/NS group. As for the kidney AngII concentrtion, it ws significntly less y 57% in the DR/NS group thn in the NR/NS group nd higher y 92% in the DR/GE group thn in the DR/NS group. Although the plsm nd kidney AngII levels in the DR/GE group were lmost comprle to those in the NR/NS, they were still significntly different y 1 nd 22%, respectively, in mnner lmostsimilrtothtoservedfortheace-1(figure7). The concentrtion of serum lumin ws significntly less y 55% in the DR/NS group thn in the NR/NS group. On the other hnd, in the DR/GE group, the level of serum lumin ws significntly higher y 56% thn in the DR/NS group nd less y 31% compred to tht in the NR/NS group. The urine lumin showed opposite concentrtion ptterns to those mesured for the serum in the three rt groups. The level of urine lumin ws considerly higher y 147% in the DR/NS group compred to the NR/NS group nd ws less y 69% in the DR/GE group thn in the DR/NS group, which ws less y 24% thn in the NR/NS group (Figure 8). NR/NS DR/NS DR/GE Figure 4: GE tretment lowers lood pressure of dietic rts. Systolic lood pressure (mm Hg) ws mesured fter STZ (week 1) ndttheendofthetretmentperiod(week8).nr/ns:norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. Serum insulin (ng/ml) Figure 5: GE tretment incresed serum insulin of dietic rts. Serum insulin ws quntitted t the end of the tretment period (week 8). NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. The mgnitude of serum cretinine ws significntly higher y 41% in the DR/NS group compred to the NR/NS group, while in the DR/GE group the serum cretinine level ws significntly less y 22% thn in the DR/NS group nd higher y 9.6% compred to tht mesured in the NR/NS group (Figure 9()). The urine cretinine levels showed similr pttern of chnge to tht oserved in the serum of the dietic groups. Notly, the urine cretinine level ws considerly higher y 98% in the DR/NS group compred to tht quntified for the NR/NS group nd less y 43% in the DR/GE group compred to tht in the DR/NS group. Furthermore, the urine cretinine level remined higher y 471% in the DR/GE group thn in the NR/NS group (Figure 9()). As for cretinine clernce, it ws significntly higher y 24% in the DR/NS group thn in the NR/NS group. Conversely, in the DR/GE rts, lthough the cretinine clernce ws still higher y 98% thn the level clculted for the NR/NS, it ws less y 42% compred to the mgnitude estimted for the DR/NS group (Figure 9(c)).

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Plsm ACE-1 (pg/ml) Kidney ACE-1 (pg/mg protein) () () Figure 6: GE tretment decresed plsm ACE-1 nd incresed kidney ACE-1 in dietic rts. ACE-1 ws quntitted in oth () plsm (pg/ml) nd () kidney (pg/mg) t the end of the tretment period (week 8). NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. Plsm AngII (pg/ml) Kidney AngII (pg/mg protein) () () Figure 7: GE tretment decresed plsm AngII nd incresed kidney AngII in dietic rts. AngII ws quntitted in oth () plsm (pg/ml) nd () kidney (pg/mg) t the end of the tretment period (week 8). NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. Serum lumin (g/dl) Urine lumin (g/dl) () () Figure 8: GE tretment reversed luminuri of dietic rts. Serum lumin () nd urine lumin () were determined (g/dl) t the end of the tretment period (week 8). NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. 4. Discussion Dietes mellitus is one of the most morid medicl conditionsthtggressivelyfflictgrowingnumerofthe world s popultion [47]. Type-1 DM results from reduction in insulin secretion tht cn vry nd ccordingly determines the severity of this condition. This form of DM cn e induced in rts y chemiclly destroying their pncretic insulinproducing β-cells using the drug STZ. Experimentlly produced STZ-type-1 DM rts develop most of the signture symptoms tht re mnifested in nturlly fflicted dietic humns [48]. It is well known tht type-1 DM is progressive disese tht exhiits chronologiclly vried chrcteristics dependent on the time of commencement nd ggressiveness of the different pthoiochemicl-physiologicl mechnisms. The rguments presented in this section relte to the renl ACE-1 nd AngII findings of this study nd pertin to certin trnsient stge in the life of STZ-induced DM rts. In this study nd t week 8 following the induction of type-1 DM, the DR/NS showed ll the expected symptoms of the erly stges of the condition. Mny of the erly typicl normlities trgeted nd oserved here included severe hypoinsulinemi (Figure 5), hyperglycemi (Figure 1), ody weight loss (Figure 2), wter intke (Figure 3()), urine output (Figure 3()), serum lumin decline (Figure 8()) with

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 Serum cretinine (μmole/l) Urine cretinine (mmole/hr) Cretinine clernce (ml/min) () () (c) Figure 9: GE tretment meliorted chnges in cretinine levels nd cretinine clernce of dietic rts. Cretinine levels in () serum (μmole/l) nd () urine (mmole/hr) were determined t the end of the tretment period (week 8). (c) Cretinine clernce (ml/min) ws clculted from these vlues. NR/NS: norml rts/norml sline treted; DR/NS: dietic rts/norml sline treted; DR/GE: dietic rts/grlic extrct treted; significntly different compred to NR/NS; significntly different compred to DR/NS. luminuri (Figure 8()), nd elevtion in serum nd urine cretinine concentrtion nd cretinine clernce (Figure 9), in ddition to moderte hypertension (Figure 4). Most of thesesymptomshveeenreportedpreviouslyinreview y Elezu et l. [49]. The currently oserved pthologicl rise in wter, lumin, nd cretinine clernce, s indictors of disesed renl functioning, tht is, hyperfiltrtion nd reduced tuulr resorption, cn e tken s evidence of nephronl structurl remodeling including glomerulr nd tuulrinjury,whichhveeenoservedinourlortory s well s others in erly DM [37, 41, 5]. This renl injurious structurl remodeling, hence refunctioning, could e the result of incresed oxidtive stress [51], which, in ddition to cusing mny sic structurl deformities [8, 16, 2], leds to norml receptor expression of dvnced glyction end product (AGEs) [52] tht could e prtly responsile for glomerulr glyction [53]. In ddition, the ugmented renl clernce function could result from reduction in the intrrenl AngII concentrtion, nd therefore decline in this octpeptide stimulted sorptive power, s discussed next. One of the focl ojectives of this study ws investigting the nture of modultions occurring in the levels of plsm nd kidney ACE-1 nd AngII in the erly stges of STZinduced DM. The ACE-1 oservtions of DR/NS (Figure 6) in the current study re in line with the view tht ACE- 1 concentrtion increses in the plsm nd decreses in the kidney [3]. Furthermore, the levels of AngII show tht the chnges in this octpeptide concentrtion re in prllel with those of ACE-1, where the systemiclly mesured ACE-1 nd AngII incresed simultneously (Figures 6() nd 7()), while the renlly mesured ACE-1 nd AngII decresed simultneously (Figures 6() nd 7()). Wht supports the present study s systemic nd renl AngII findings re the following mesured physicl prmeters: first, the detected elevtion in BP (Figure 4), which could e the result of rise in the generl vsculr resistnce induced y n incresed vsoctivity cused y incresed systemtic levels of AngII (Figure 7) [7] nd second, the oserved tremendous increse in renl clernce of wter, lumin, nd cretinine (Figures 3, 8, nd 9), which could possily hve resulted from reduction in the renl conservtion power of the decresed kidney AngII (Figure 7). This possiility is supported y previous studies y our group nd others tht showed n increse in the kidney s AT 1 receptors in DM [16, 44, 54] indicting reductioninrenlangiiconcentrtionndhenceitsinduced iologicl effects. This form of dynmic reciprocl chnge etween AngII nd its AT 1 receptor represents clssicl lignd-receptor reltionship tht strives to mintin proper physiologicl sensitiztion. AsfrsBPisconcerned,exmintionofpreviousstudies crried out on STZ-induced type-1 DM my suggest the existence of two conflicting views. On one hnd, group of studies hve reported no chnge [55] or even decrese [56, 57] in the exhiited BP. Alterntively, more recent studies, in greement with the current oservtion, reported hypertension in the mjority [58 6] or good percentge ofthedieticrts[61].thisviewisevensupportedythe findings of humn study on the sme type of dietes [62]. It is highly conceivle tht these differences in the reported BP findings could hve een due to the fct tht those studies in which oservtions re inconsistent with the current view were crried out on different rt gender [55], time of mesurement nd BP model [56], nd/or method of mesurement [57]. Exploring the GE effect on DM rts plsm nd kidney ACE-1 nd AngII concentrtions reveled further dimension to the corrective ctions of this nturl product. First nd s reported here erlier, DR/GE exhiited significntly higher level of serum insulin (Figure 5), which ws ssocited with more thn 5% lower lood glucose (Figure 1), thus providing further support to the suggested hyperinsulinemichypoglycemic mechnism of the corrective ction of GE reported in the STZ-DM model [37, 39, 41]. Second, nd in reltionship to nother min ojective of this study, the corrections in the levels of insulin nd glucose y GE were ssocited with restortions of plsm nd renl ACE-1 nd AngIIconcentrtions,specificlly,decreseintheplsm

8 8 Evidence-Bsed Complementry nd Alterntive Medicine DR/NS Serum insulin Blood glucose DR/GE Serum insulin Blood glucose Plsm ACE-1 Kidney ACE-1 Plsm ACE-1 Kidney ACE-1 Plsm AngII Kidney AngII Plsm AngII Kidney AngII BP Urine ot pt Serum lm Serum Cr BP Urine ot pt Serum lm Serum Cr Wter in tk Body wt Urine lm Urine Cr ClCr Wter in tk Body wt Urine lm Urine Cr ClCr As result of the rte of protein ctolism to stisfy energy requirements Figure 1: Endocrinl, iochemicl, nd physiologicl chnges in DR/NS (compred to NR/NS) nd DR/GE (compred to DR/NS). The schemtic suggests tht in erly STZ-induced hypoinsulinemi the ensuing hyperglycemi leds to n increse in plsm ACE-1 nd, s result nd simultneously, plsm AngII with concomitnt decrese in kidney ACE-1 nd, hence, kidney AngII. As result of these modultions, BP, wter intke, serum cretinine, nd renl clernce of wter, lumin, nd cretinine incresed significntly; in ddition ody weight nd serum lumin decresed significntly. Conversely nd s seen in the DR/GE group, tretment with GE significntly ttenuted nd countercted the modultions oserved in the DR/NS. DR/NS: dietic rts treted with norml sline; DR/GE: dietic rts treted with grlic extrct; ACE-1: ngiotensin converting enzyme type-1; AngII: ngiotensin II; BP: lood pressure; ody wt: ody weight; urine ot pt: urine output; serum lm: serum lumin; urine Cr: urine cretinine; wter in tk: wter intke; ClCr: clernce of cretinine; :incresein concentrtion or mount; : decrese in concentrtion or mount; / : leds to; stright-lined-rrows: suggestions mde depending on the dt of this study; dotted-lined-rrows: scientific fcts. AngII concentrtion ssocited with reduction in plsm ACE-1 (Figures 6 nd 7) nd, t the sme time, n increse in renl AngII concentrtion ssocited with rise in renl ACE-1 concentrtion (Figures 6 nd 7). The reduction in the systemic AngII genertion correlted nicely to the lower BP mesured in response to GE tretment (Figure 4), while theincreseinrenlangiivililitycorreltedwellwith the documented effect of GE on restoring norml AT 1 /AT 2 lnce tht is distorted in DM [43, 44]. Furthermore, the increse in renl AngII concentrtion, in spite of the reported reduction in AT 1 receptor expression [43], suggests n increse in the ntidiuretic processes nd reduction in renl lumin nd cretinine clernce, which ws oserved here in the DR/GE. The reduction in these clernce vriles lsocouldhveeenmeditedythettenutionofoxidtive stress processes [5], decresed AGE formtion [63], nd most importntly the reduction of glomerulr glyction [53], in ddition to ctions which presumly dely the progression of dietic nephropthy. In this study, the oserved chnges in the concentrtion of plsm nd kidney ACE- 1 nd AngII nd their effects on BP nd renl clernce function re in greement with erlier reports [5 7, 12]. In this study, the oserved concurrent rise in oth serum nd urine cretinine concentrtions presents different perspective regrding the dietic kidney clernce function of this vrile in the erly stges of type-1 DM. Typiclly, it is suggested tht the dietic kidney GFR declines s result of glomerulonephritis leding to fll in cretinine clernce nd consequently rise in its serum concentrtion [64]. This sort of cretinine hndling mterilizes towrds the lte stges of DM nd the eginning of renl filure. However, during the erly onset of DM nd when renl clernce function is exggerted, greter clernce of cretinine isexpectedsoservedinthisstudy.inconnection,the most plusile interprettion for the current rise in urine cretinine, s suggested y the schemtic in Figure 1, is the following metolic cscde: (1) the development of higher stedy-stte rte of protein ctolism s n lterntive source of energy [65] to compenste for the decline in cellulr uptke of glucose due to hypoinsulinemi [66]. This suggestion is supported y the finding in this study tht DR/NS were normlly len nd showed continuous pthologicl decline in ody weight compred to NR/NS, s well s DR/GE. As expected nd consequent to this elevted proteinctolismissurgeinplsmcretininelodnd(2) highly elevted GFR in this erly stge of DM. This prediction is supported y the increse in cretinine excretion oserved in this study, s well s polyuri nd glycosuri reported here nd elsewhere [67], ll of which re clssicl symptoms of n erly dietic kidney. Finlly, it is worth stting the following: ecuse DM is progressive disese, the chnges in its physioiochemicl

9 Evidence-Bsed Complementry nd Alterntive Medicine 9 mechnisms, ssocited with developing structurl modifictions, re lso progressive. Therefore nd in the erly stges, type-1 DM strts with n increse in renl clernce functions permitted y pproprite nephronl nd renl mechnistic nd structurl modifictions, which shift to grdul decline in renl clernce function, gin, fcilitted y pproprite nephronl nd renl mechnistic nd structurl ltertions, ultimtely leding to the clssicl end-stge cse of renl filure. In support, Kikkw et l. [68] suggested tht hypoinsulinemi-induced hyperglycemi cuses iphsic ltertion of RAS due to volume depletion nd lter due to volume expnsion. The control DM rts dt in this study, in prticulr the renl ACE-1 nd AngII chnges, support the excretory ehvior tht is noticed here nd is well known to occurttheerlystgesoftype-1dm.thedtofthisstudy lso suggest strong correltion etween chnges in ACE- 1ndAngIIndinsulinndglucoseconcentrtionsinDM rts. A reltionship etween AngII production nd glucose concentrtion, lthough opposite to wht ws oserved in this study, ws shown y Singh et l. [69] in culture studies. Irrespective of the nture of chnge in AngII genertion, the current oservtions in DR/GE give further support the view tht the GE corrective ctions on the ACE-1 nd AngII concentrtion re closely medited vi the insulin-glucose pthwy. Accordingly, it is highly prole tht, whtever the stge of DM, GE my e effective in slowing down distortion of mechnisms, especilly those relted to ACE-1 nd AngII concentrtion modultion tht ffect renl structure nd function. 5. Conclusion The findings of this study suggest tht the meliortive ction of grlic on the elevted BP nd renl clernce functions in erly STZ-induced type-1 DM my e prtilly medited through ttenuting modultions in plsm nd renl ACE-1 nd AngII concentrtions. Arevitions ACE-1: Angiotensin I converting enzyme type-1 AGE: Advnced glyction end product AngI: Angiotensin I AngII: Angiotensin II AT 1 : Angiotensin type-1 receptor AT 2 : Angiotensin type-2 receptor BP: Blood pressure DM: Dietes mellitus DR/GE: Dietic rts/grlic extrct DR/NS: Dietic rts/norml sline GC-MS: Gs chromtogrphy-mss spectroscopy GE: Grlic extrct GFR: Glomerulr filtrtion rte LSD: Lest significnt difference NR/NS: Norml rts/norml sline RAS: Renin-ngiotensin system SEM: Stndrd error men STZ: Streptozotocin. Competing Interests No competing interests, finncil or otherwise, re declred y the uthors. Acknowledgments The uthors would like to thnk the Reserch Sector t Kuwit University for funding this project (Reserch Grnt no. SL9/1). In prticulr, they would sincerely like to cknowledge the mjor contriution of their lte collegue ndfriendprofessormohmedh.mnsour,whopssed wy during the finl stges of prepring this pper. References [1] M. J. Pech, Renin-ngiotensin system: iochemistry nd mechnisms of ction, Physiologicl Reviews,vol.57,no.2,pp , [2] M. Pul, A. P. Mehr, nd R. Kreutz, Physiology of locl reninngiotensin systems, Physiologicl Reviews, vol. 86, no. 3, pp , 26. [3] H. Koori, M. Nngku, L. G. Nvr, nd A. Nishiym, The intrrenl renin-ngiotensin system: from physiology to the pthoiology of hypertension nd kidney disese, Phrmcologicl Reviews,vol.59,no.3,pp ,27. [4] L. G. Nvr, K. D. Mitchell, L. M. Hrrison-Bernrd, H. Koori, nd A. Nishiym, Intrrenl ngiotensin II levels in norml nd hypertensive sttes, Journl of the Renin-Angiotensin- Aldosterone System,vol.2,no.1,pp.S176 S184,21. [5] P. S. Leung, The peptide hormone ngiotensin II: its new functions in tissues nd orgns, Current Protein nd Peptide Science,vol.5,no.4,pp ,24. [6] L.G.NvrndA.Nishiym, Whyrengiotensinconcentrtions so high in the kidney? Current Opinion in Nephrology nd Hypertension,vol.13,no.1,pp ,24. [7] M. Burnier, Angiotensin II type 1 receptor lockers, Circultion,vol.13,no.6,pp ,21. [8] H. M. Sirgy, AT 1 nd AT 2 receptorinthekidney:roleinhelth nd disese, Seminrs in Nephrology, vol. 24, no. 2, pp. 93 1, 24. [9] M.deGspro,K.J.Ctt,T.Ingmi,J.W.Wright,ndT.Unger, The ngiotensin II receptors, Phrmcologicl Reviews, vol. 52, no.3,pp ,2. [1] E. Kschin nd T. Unger, Angiotensin AT 1 /AT 2 receptors: regultion, signlling nd function, Blood Pressure,vol.12,no. 2, pp. 7 88, 23. [11] R. M. Crey, Updte on the role of the AT 2 receptor, Current Opinion in Nephrology nd Hypertension, vol.14,no.1,pp.67 71, 25. [12] U. C. Brewster nd M. A. Perzell, The renin-ngiotensinldosterone system nd the kidney: effects on kidney disese, Americn Journl of Medicine,vol.116,no.4,pp ,24. [13] G. Wolf, Moleculr mechnisms of ngiotensin II in the kidney: emerging role in the progression of renl disese: eyond hemodynmics, Nephrology Dilysis Trnsplnttion, vol. 13, no. 5, pp , [14] A. Rieiro-Oliveir Jr., A. I. Nogueir, R. M. Pereir, W. W. Vils Bos,R.A.SouzdosSntos,ndA.C.Simões e Silv, The renin-ngiotensin system nd dietes: n updte, Vsculr Helth nd Risk Mngement,vol.4,no.4,pp ,28.

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