Effects of dietary protein restriction and oil type on the early progression of murine polycystic kidney disease

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1 Kiney Interntionl, Vol. 42 (1992), pp LABORATORY INVESTIGATION Effects of ietry protein restriction n oil type on the erly progression of murine polycystic kiney isese HAROLD M. AUKEMA, MALCOLM R. OGBORN, Koi TOMOBE, HIsAHIDE TAKAHASHI, TSUTOMU HIBIN0, n BRUCE J. HOLUB Deprtment of Nutritionl Sciences, University of Guelph, Guelph, Ontrio n Section of Nephrology, Deprtment of Peitrics, University of Mnitob, Winnipeg, Mnitob, Cn; Deprtment of Pthology, College of Meicl Technology, n Lbortory Animl Centre, Fujit-Helth University, Toyoke, Aichi, Jpn Effects of ietry protein restriction n oil type on the erly progression of murine polycystic kiney isese. A pucity of reserch t exists on the potentil for erly ietry moifiction to irectly retr cystic growth n prolifertion in polycystic kiney isese (PKD). We hve therefore exmine the reltive effects of ietry protein levels n oil type on the progression of isese in murine moel of PKD. In the first stuy, wenling DBAI2FG-pcy (pcy) mice were fe either norml (NP), 25%, or low (LP), 6%, csein iet with 10% of either sunflower see oil (SO) (contining n-6 ftty cis), or fish oil (FO) (contining n-3 ftty cis), in 2 x 2 esign. At the en of the ietry tretment, kiney weight reltive to boy weight ws higher in mice on the NP iets. In ition, kiney phospholipi to kiney weight (prnol/g) ws lower in pcy mice on NP iets, inicting tht the increse kiney size ws lrgely ue to increse cyst evelopment. Replcement of ietry SO with FO resulte in ltertions in renl phospholipi ftty ci compositions: 18:2 n-6, 20:4 n-6, n 22:5 n-6 were lower, n 20:5 n-3, 22:5 n-3, n 22:6 n-3 were higher in FO-fe nimls. No effect of ietry lipi type on isese progression ws note, however. In secon stuy, morphometric nlysis revele n 11% lower percentge cyst re n 46% lower totl cyst re (mm2) in kiney sections erive from mice on LP iets compre to NP iets. These results inicte tht erly ietry protein restriction in PKD prior to clinicl mnifesttion of symptoms of the isese my hve significnt impct on the pthogenesis of PKD. There is growing boy of evience which suggests tht ietry protein restriction my hve protective effect in estblishe, lte renl isese [reviewe in 1 4]. Severl lrge prospective trils testing this hypothesis in ptients with estblishe chronic renl filure re presently in progress. With respect to the efficcy of low protein (LP) iets in polycystic kiney isese (PKD) specificlly, Olrizzi et! [5] stuie ptients with vnce PKD on LP iets for n verge of three n one-hlf yers. They clculte tht compre to controls with PKD, the slope of reciprocl serum cretinine over time for the ptients on LP iets ws significntly lower thn for those on control iets. Gretz, Korb n Struch [6] stuie smll number of PKD ptients n foun mrkely slower increse in serum cretinine in ptients on LP iets supple- Receive for publiction November 26, 1991 n in revise form April 27, 1992 Accepte for publiction April 30, by the Interntionl Society of Nephrology mente with keto cis compre to controls. Recently, Loctelli et l [7] stuie renl ptients on LP iets for up to two yers n foun no evience for protective effect of protein restriction on lte chronic renl insufficiency, incluing ptients with PKD. In PKD, cyst enlrgement is consiere to cuse estruction of surrouning nephrons [8, 9], resulting in hyperfiltrtion in the remining nephrons. Dietry protein my ccelerte isese progression by incresing the hyperfiltrtion of remining helthy nephrons, s occurs in renl bltion moels of isese [10, 11, 12]. Diets enriche in fish oil (FO) contining n-3 ftty cis in humn n niml forms of renl isese hve lso been shown to be beneficil in some, but not ll, renl isorers [reviewe in 13 15]. Recently, cytokines n eicosnois [erive from tissue rchionic ci (20:4 n-6)] hve been implicte in cystic kiney isese [16]. The formtions n ctions of these hve been foun in other stuies to be ffecte by ietry FO contining n-3 ftty cis [13, 15, 17, 18]. No reserch t exist on the potentil for erly mnipultion of ietry protein level to moify cyst growth n prolifertion in PKD, prticulrly before the presenttion of clinicl symptoms or bnorml mesures of renl functioning. In the present stuy, we hve use murine moel of utosoml ominnt PKD [19, 20] to stuy the effects of ietry mnipultion [protein level n oil type (n-6 vs. n-3 ftty cis)] on the erly progression of PKD. We hve mesure kiney weight to boy weight rtios; this hs been use to inicte cyst evelopment in PKD [20 23] since kiney enlrgement reflects erly chnges in PKD progression in humns [24, 25] n in this mouse moel [23] of the isese. For exmple, espite consierble renl enlrgement inpcy mice t 18 weeks of ge, bloo ure nitrogen is not elevte in these mice. Progressive renl insufficiency, however, evelops in pcy mice fter 18 weeks of ge [23]. In ition, we hve mesure renl phospholipi composition (level n ftty ci profiles), s well s phospholipi to kiney rtios (mol/g), which pper to be inversely relte to the progression of PKD. To further stuy the effects of protein restriction on cyst growth, morphometric nlysis of kineys erive from mice on NP n LP iets were lso performe. The results presente herein provie supportive evience for significnt beneficil effect of ietry protein restriction on the erly progression of PKD, regrless of oil type. 837

2 838 Aukem et!: Dietry protein restriction in murine PKD Diet ingreient Csein, vitmin-free Cornstrch Sucrose Sunflower see oil MxEPA oil Minerl mix Choline chorie Inositol DL-methionine Vitmins Solk-floc(flber) Tble 1. Composition of experimentl iets % Of iet by weight NP-SO NP-FO LP-SO LP-FO Methos Animls n iets The experimentl niml use ws the DBA/2FG-pcy (pcy) mouse, moel of utosoml ominnt PKD [19, 20). Twentyeight mle pcy mice were wene t 30 ys of ge n rnomly ivie into four groups; one mouse ie of unknown cuses uring the course of the experiment. Mice were house iniviully in temperture (23 C), humiity (60 to 65% R.H.), n light (12 hours light: 12 hours rk) controlle environment. Wter n iet were provie libitum. Stuy 1. Mice were fe semi-purifie iets contining either norml (NP, 25% csein) or low (LP, 6% csein) level of protein, n either sunflower see oil (SO) rich in the n-6 ftty ci, linoleic ci (18:2 n-6), or FO concentrte (MxEPA, R.P. Scherer, Cn) rich in n-3 ftty cis (eicospentenoic ci, 20:5 n-3, plus ocoshexenoic ci, 22:6 n-3), in 2 x 2 esign. The FO enriche iet contine 1% SO to supply equte mounts of the essentil ftty, 18:2 n-6. Ethoxyquin ws e s n nti-oxint t level of 0.05% [26]. The composition of these iets is given in Tble 1. All mice were weighe t the beginning n en of the stuy, n fee consumption ws monitore for seven y spn uring the stuy. Bloo smples tken from the orbitl sinus plexus fter eight weeks on stuy were poole for serum lbumin nlysis. When the mice were 120 ys of ge, they were scrifice fter CO2 nesthesi. Stuy 2. For morphometric stuies, wenling mice (3 per group) were fe LP n NP iets exctly s bove except tht corn oil (rich in 18:2 n-6) ws use s the lipi source for both groups such tht these iets iffere only in protein level. Mice were weighe t the beginning n en of the stuy, n scrifice t 120 ys of ge. Biochemicl nlysis In stuy I, the kineys n livers were immeitely remove t scrifice, frozen in liqui nitrogen, n store t 80 C until further nlysis. Kiney lipis were extrcte [27] n totl phospholipi frctions were purifie by thin-lyer chromtogrphy using heptne/isopropyl ether/cetic ci (60/40/3, by volume) s the mobile phse [28]. Ftty ci methyl ester erivtives were forme from the isolte phospholipi frctions n nlyze by gs-liqui chromtogrphy to etermine their ftty ci content s previously escribe [29]. Morphometric nlysis Kineys from mice in stuy 2 were fixe in 10% formlin. Prior to embeing in prffin, the kineys were bisecte in the coronl plne through the hilum of the kiney. Five micron sections were stine with hemtoxylin n eosin using stnr techniques. Morphometric nlysis ws performe on Nikon Lbplot microscope equippe with Cohu high resolution blck n white vieo cmer with computer interfce vi Visionplus bor. Imge nlysis ws performe using the ensitometry n fluorometry moule (1M4 100) of the Imgemesure progrm (Phoenix Biotechnology Inc., Feerl Wy, Wshington, USA). At object to screen mgnifiction of 142 x, tubulr luminl re ws mesure fluorometriclly over the entire kiney excluing the ppill. The screen ws ivie into gri of 12 sections n fluorometric mesurements tken of ech section. Mesurements were repete in non overlpping sections of the kiney until the entire orgn h been covere. Mesurements were performe in uplicte from two lternte five micron sections tken from ner the centre of the kiney. A minimum of 400 mesurements were tken from ech kiney. Sttistics All t re expresse s the men stnr error. The t from the stuy 1 were nlyze by nlysis of vrince followe by the protecte lest significnt ifferences test when interctions were present. The moel inclue effects for protein level, oil type, n litter. Stuent's t-test ws use for the stuy 2, using iniviul mesurements s the bse unit of stuy. Differences were consiere significnt t P 0.05, interctions t P 0.10 [30]. Results Stuy I The verge initil boy weight for mice in ll groups ws g (men SE, N = 27), with no significnt ifferences between the four groups. At the en of the stuy, the overll boy weights of mice on the NP iets were higher thn for those on the LP iets (Tble 2). FO-feeing lso resulte in higher boy weights compre to SO-feeing. The mice on the LP iets consume g of iet per y, s compre to g/y for the mice on the NP iets (P < 0.001). There ws no ifference in foo consumption between mice fe SO versus FO ( vs g/y). The lower finl boy weight n increse fee consumption by the mice on the LP iets inicte tht these mice my hve been mrginlly protein eficient. Serum lbumin levels, however, were in the norml rnge for mice on ll iets ( n g/l, for mice on NP n LP iets, respectively). When kiney n liver weights were expresse in bsolute vlues or s proportion of boy weight, both were elevte in mice on the NP iets, but oil type h no effect on these prmeters (Tble 2). Kineys were enlrge to much greter extent thn livers in the mice on the NP iets s evience by their significntly higher kiney weight to liver weight rtios. Totl renl phospholipi n cholesterol were both elevte in mice on the NP iets; renl phospholipi ws lso higher in the FO-fe mice (Tble 3). When renl phospholipi n cholesterol were expresse per grm of kiney, however, only ietry protein level h n effect on these two prmeters. The totl phospholipi to kiney weight rtio (mol/g) ws 48%

3 Aukem et!: Dietry protein restriction in mrine PKD 839 Tble 2. Effect of ietry protein level n oil type on boy n orgn weights n rtios of pcy mice NP-SO NP-FO LP-SO LP-FO Effects Boy wt. g ,c Kiney wt. g (totl) Kiney wt./boy wt. g/100 g Liverwt. g Liver wt./boy wt. g/100 g Kiney wt./liver wt. g/g b Ech vlue represents men SE for 6 to 7 nimls per group. p < b P < 0.01, min effects of protein level P < 0.05, min effects of oil type Tble 3. Effect of ietry protein level n oil type on phospholipi n cholesterol in pcy mouse kineys (both) on ifferent iets NP-SO NP-FO LP-SO LP-FO Effects Phospholipi pinol ,b Cholesterol mol Phospholipi/kiney wt. pinollg Cholesterol/kiney wt. pinol/g Cholesterol/phospholipi molr rtio NS p < 0.001, min effects of protein level; b p < 0.05, min effects of oil type; NS, not significnt. Tble 4. Effect of ietry protein level n oil type on renl phospholipi ftty ci composition in pcy mouse kineys Ftty ci NP-SO NP-FO LP-SO LP-FO Effects 16: c. 16: : : , NS 18:2(n-6) b, 20:3(n-6) :4(n-6) :5(n-3) tr tr g 22: :4(n-6) :5(n-6) o.o g :5(n-3) :6(n-3) I O.8' 24: O.l mt 24: Vlues represent mol percent of totl ftty cis; tr, trce. Other minor ftty cis hve been omitte from the tble. Abbrevitions re: NS, not significnt; int, interction between protein level n oil type. P < 0.001, b p < 0.01, C P < 0.05, min effects of protein level; P < 0.001, P < 0.05, min effects of oil type Oil effect within tht level of protein, P < 0.05 (NP-FO vs. NP-SO, or LP-FO vs. LP-SO) g Protein effect within tht oil type, P < 0.05 (NP-FO vs. LP-FO, or NP-SO vs. LP-SO) mt c mt mt higher (overll) on the LP versus NP iets. The renl cholesterol to phospholipi rtio ws not ltere by the ietry tretments. In generl greement with previous stuies of this moel [31], replcement of ietry SO with FO resulte in mrke chnges in renl phospholipi ftty ci composition (Tble 4). FOfeeing resulte in significntly lower levels of n-6 ftty cis (incluing 18:2 n-6, 20:4 n-6, n ocospentenoic ci, 22:5 n-6) n higher mounts of n-3 ftty cis (incluing 20:5 n-3 n 22:6 n-3) in renl phospholipi. Stuy 2 In greement with stuy 1, kineys from mice on the LP iets were mrkely less enlrge thn kineys from mice on NP iets. Kiney weights were g versus g (men SE, P < 0.001), n kiney weights s percentge of boy weight were versus (P < 0.005), for mice on LP n NP iets, respectively. Morphometric nlysis of sections obtine from these kineys (Figs. 1 n 2) revele tht the percent cyst re ws lower in kineys obtine from mice on LP versus NP iets ( % vs %, respectively, P < 0.001). The totl cyst re per section ws 46% lower in mice on LP iets compre to mice on NP iets ( mm2 vs mm2, P <0.005). Discussion In previous stuies (unpublishe observtions), we compre norml control DBA/2J mice with isese pcy mice on chow

4 840 Aukem et l: Dietry protein restriction in murine PKD -I cm Fig. 1. Light microgrph of kiney section obtine from pcy mouse on NP iet (mgn(fiction, X45). iets contining 17% protein. Norml n isese mice of 120 ys of ge h kiney weight to boy weight rtios of n , respectively, while kiney weight to liver weight rtios were n , respectively. Another prmeter which ppers to reflect (inversely) the extent of cyst growth n cyst flui ccumultion is the kiney phospholipi to kiney weight (prnol/g) rtio. In norml versus isese chow-fe mice of 120 ys of ge, kiney phospholipi to kiney weight rtios were versus mol/g, respectively. This rtio vries inversely with cystic isese progression; s cysts enlrge, greter proportion of the kiney is tken up by cyst flui, thus ecresing the phospholipi (cellulr membrne component) to kiney weight rtio. In the present stuy, we use these prmeters to evlute the progression of cyst growth in pcy mice, since clinicl mesures of renl function (serum bloo ure nitrogen n cretinine) o not revel ny bnormlities t this erly stge of the isese, both in this moel [23] n in humns [24, 25]. Dt from the morphometric nlysis (showing n 11% lower percent cyst re n 46% lower totl cyst re in kiney sections from mice on LP iets compre to NP iets) confirms the vliity of these prmeters in pcy mice. Totl kiney weights n kiney weights s percentge of boy weight were lower in the pcy mice on the LP iets. The level of ietry protein, however, lso h similr, but lesser effect on liver weights n liver weights expresse s percentge of boy weight, espite the bsence of cysts in the livers. This higher liver weight (n presumbly prt of the increse kiney weight) on the NP iets is probbly ue to the fct tht ietry protein hs hypertrophic effect on liver n kiney prt from isese [32]. To ccount for this effect, in stuy 1 we mesure kiney weight in reltion to liver weight, thus using liver weight s n internl stnr. The higher kiney weight to liver weight rtios on the NP iets confirms tht, in ition to incresing norml tissue growth, NP iets (compre to LP iets) lso ccelerte cystic growth n isese progression. Kineys from the mice on LP iets lso h significntly higher phospholipi to kiney weight rtios, confirming tht these kineys contine smller proportion of cysts n cyst flui reltive to norml tissue (ssocite with membrne phospholipi). Consistent with cholesterol being cellulr membrne component, higher kiney cholesterol to kiney weight rtio ws lso foun in pcy mice on the LP iets. Compre to LP iets, NP iets my versely ffect PKD by incresing renl hyperfiltrtion n perfusion. Since the number of functioning nephrons in PKD my lrey be reuce by cyst encrochment on surrouning renl tissue [8, 9], the increse worklo inuce by NP iets my shorten the functionl lifespn of the remining helthy nephrons [10, 11]. The reuction of functioning nephrons in PKD is nlogous to the renl bltion experimentl niml moel, in which it hs been shown tht lthough iets with higher levels of protein result in renl

5 Aukem et!: Dietry protein restriction in murine PKD 841 Fig. 2. Light microgrph of kiney section obtine from pcy mouse on LP iet (mgnifiction, x45). hypertrophy, the increse worklo is ssocite with structurl lesions, suggesting tht this my be mlptive response in the long term. NP iets my lso exert their eleterious effects on PKD by incresing renin prouction, since bnormlities in the renin-ngiotensin-losterone system hve been ocumente in PKD [33 35]. High protein iets hve been reporte to increse plsm renin ctivity in stuies of norml experimentl rts n in stuies of ptients with renl iseses [36 38]. Another bnormlity which my be excerbte by NP iets in PKD is the increse ctivity n/or reverse polrity of NK-ATPse [39, 40], since high protein iets hve been shown to increse NK-ATPse ctivity in norml rts [41, 42]. We foun no protective effect of ietry FO on the erly progression of PKD in stuy 1. An erlier stuy with pcymice in which ietry tretment ws initite month lter n for shorter time perio inicte tht P0 iets my hve some moerte beneficil effects [31]. Replcing ietry SO with FO from wening to eth, however, i not improve survivl in pcy mice [43]. These stuies suggest tht if there is ny beneficil effect of ietry FO in pcy mice, it my be limite to smll time perio in the course of the isese. With respect to the ftty ci ltertions in renl phospholipi, of note ws the rmtic replcement of the 22-crbon n-6 ftty cis, renic ci (22:4 n-6) n ocospentenoic ci (22:5 n-6), n the reuction by more thn 50% of 18:2 n-6 n 20:4 n-6, by the n-3 ftty cis, 20:5 n-3, ocospentenoic ci (22:5 n-3), n 22:6 n-3, with ietry P0 reltive to SO. The role of eicosnois in the evelopment of PKD in pcy mice ppers not to be importnt since protein restriction ltere isese progression, but i not ffect the level of vilble eicosnoi precursor s 20:4 n-6, while oil type i ffect 20:4 n-6 levels, but not the erly progression of PKD. Increse levels of PGE2 hve been reporte in renl cyst flui erive from ptients with PKD [16], but the reltive formtion of eicosnois (thromboxnes, prostglnins, leukotrienes) remins to be stuie in this isese. In conclusion, erly ietry protein restriction ppers to retr cyst evelopment n isese progression in this murine moel of PKD. It is possible to mrkely lter renl phospholipi ftty ci compositions by replcing ietry SO with FO, but these ltertions i not ffect the erly progression of cystic isese in pcy mice. These results inicte tht very erly ietry protein restriction in PKD prior to clinicl mnifesttion of the isese my hve significnt impct on the pthogenesis of PKD. Acknowlegements This reserch ws presente t the 24th nnul meeting of the Americn Society of Nephrology, n publishe in bstrct form (JAm Soc Nephro/ 2:254, 1991). This reserch ws supporte by grnt (to BJH) from the Nturl Sciences n Engineering Reserch Council of Cn. The imge nlysis equipment ws supporte by grnts (to MRO) from the Chilren's Hospitl Reserch Fountion of Winnipeg n the Mnitob brnch of the Kiney Fountion of Cn. We re grteful to Dr. D.J. Philbrick for her technicl ssistnce. Reprint requests to Dr. Bruce J. Holub, Deprtment of Nutritionl Sciences, University of Guelph, Guelph, Ontrio, Cn NIG 2 WI.

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