THE EFFECTS OF INCREASED PROTEIN INTAKE ON KIDNEY SIZE AND FUNCTION

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1 The Journl of Experimentl iology 21, (1998) Printed in Gret ritin The Compny of iologists Limited 1998 JE THE EFFECTS OF INCRESED PROTEIN INTKE ON KIDNEY SIZE ND FUNCTION KIMERLY. HMMOND ND DONLD N. JNES Deprtment of iology, University of Cliforni, Riverside, C 92521, US e-mil: [email protected] ccepted 23 pril; pulished on WWW 11 June 1998 In endothermic vertertes, long-term increses in metolic energy demnd re often ssocited with increses in food intke nd ccompnied y increses in orgn mss. Wide-scle increses in orgn mss hve often een ttriuted to metolic response to incresed energy intke nd utiliztion. On constnt diet, however, incresed food intke is lso ssocited with incresed protein intke. We hypothesized tht, while incresed food intke itself my e responsile for increses in digestive trct mss, the consequent incresed protein intke would e the fctor responsile for incresed kidney mss nd function. Thus, we exposed mle nd femle mice to diets differing in protein level (7 %, 15 % or 46 % csein y mss) t different cclimtion tempertures (5 C or 23 C). Within n cclimtion temperture, food intke rte remined constnt over the entire rnge of dietry protein level, nd protein intke rte incresed s dietry content incresed. The mice in the cold-cclimtion group Summry incresed food intke rte y % over those in the wrm-cclimtion group. Liver, kidney nd stomch mss incresed with protein intke rte, while digestive trct nd other vitl orgn msses incresed only in response to incresed energy intke rte. lood ure nitrogen levels incresed with protein intke rte. Glomerulr filtrtion rtes incresed with increses in dietry protein level in mle mice ut not femle mice. Finlly nitrogen filtrtion rte incresed with protein intke rte for mice on the high-protein diet. We suggest tht it is primrily the incresed protein intke rte rther thn the incresed food intke rte tht results in the chnges in kidney nd liver mss nd kidney function oserved to occur in situtions of high energy demnd. Key words: kidney, protein intke, orgn mss, nitrogen filtrtion rte, mouse. Introduction Mny niml orgn systems possess functionl cpcity tht is 1 1 times the verge lod plced upon them y norml physiologicl demnds (lexnder, 1981; Weiel et l. 1991). This multiple of verge lod is often referred to s sfety fctor. The dvntge of mintining lrge sfety fctor is tht the excess or spre cpcity cn e used in periods when demnds on the system increse. The disdvntge of mintining sustntil sfety fctor is presumly relted to the costs of mintining nd operting excess tissue in times when tht tissue is not operting t full cpcity. etter understnding of how nd why nimls mintin specific sfety fctors might e gined y exmining the sfety fctors of specific orgns y themselves; however, these orgns often operte in concert with other orgns or orgn systems. Thus, it is often not cler whether lrge sfety fctor in one orgn is importnt for cooperting with other orgns tht my lso hve incresed metolic demnds or whether tht orgn lone is responsile for mintining whole-ody physiologicl output. For exmple, in mice mintined t room temperture, the smll intestinl cpcity for nutrient uptke is 3 5 times the physiologicl lod plced upon it y the verge dily intke of nutrients (Hmmond et l. 1994). When mice increse their sustined metolic rte (SusMR) in response to cold cclimtion nd/or lcttion, however, food intke (nutrient lod) increses y up to five times over control vlues, the mss of the smll intestine increses y up to three times nd totl smll intestinl uptke cpcity increses y up to 1.5 times (Hmmond et l. 1994). Thus, the sfety fctor is diminished from 3 5 to pproximtely 1. In these mice, the exhustion of the smll intestinl sfety fctor for glucose is coincident with the highest mesured SusMR for cold nd lcttion. These dt hve shown tht smll intestinl size nd cpcity re highly responsive to chnges in energy input or ssimiltion (e.g. Hmmond et l. 1994). In ddition, other orgns such s the kidney lso increse in mss with elevted energy demnds in white mice (Toloz et l. 1991; Hmmond nd Dimond, 1992, 1997; Konrzewski nd Dimond, 1994; Hmmond et l. 1994), rising the question of whether their functionl cpcity is similrly mtched to chnges in energy input or energy ssimiltion t high energy demnds. While there hs een correltion etween smll intestine mss nd whole-orgn functionl cpcity for nutrient uptke under

2 282 K.. HMMOND ND D. N. JNES some circumstnces, it is not cler tht there is direct reltionship etween mss nd functionl cpcity for other orgns or orgn systems. The kidney is one of the most energeticlly demnding orgns in mmmlin ody (Kres, 195; Mrtin nd Fuhrmn, 1955). On mss-specific sis, the rte of oxygen consumption of the kidney in mice t room temperture is etween 1.5 nd 2 times tht of other orgns (Mrtin nd Fuhrmn, 1955). Increses in kidney mss coinciding with incresed SusMR re prticulrly drmtic. Konrzewski nd Dimond (1994) found tht, when mient temperture ws grdully decresed from 23 C to 15 C, mice incresed their food intke, incresed the mss of the digestive system nd incresed kidney wet mss y 7 %. There my e greter energy or functionl tx on the kidney in nimls living t such cold tempertures ssocited with the removl of metolic wste products tht result from higher levels of metolic output. The kidney hs two primry functions: wste excretion nd wter regultion. ecuse the mice in the Konrzewski nd Dimond (1994) study were not wterstressed, it is resonle to suggest tht wste excretion requirements increse with elevted SusMR s the functionl cpcity of the kidney to remove these wste products from the ody increses. One of the wste compounds excreted y the kidney is ure, wste product of protein metolism. In most studies of temperture-relted (i.e. energy-demnd-relted) orgn hypertrophy, the diets used hve hd similr protein contents for nimls t ech experimentl temperture (Toloz et l. 1991; Hmmond nd Dimond, 1992; Konrzewski nd Dimond, 1994). Thus, n elevted SusMR cuses n incresed food intke to meet energy needs, which cuses concomitntly incresed protein intke. Incresed protein intke is known to cuse renl hypertrophy nd n increse in glomerulr filtrtion rte (GFR; Klhr, 1989), so we hypothesized tht the incresed kidney mss ssocited with incresed SusMRs my e result of the incresed protein intke rte itself rther thn y-product of the high rte of energy ssimiltion resulting from the metolic processes (i.e. thermogenesis or milk production) ssocited with those high energy demnds. To test this hypothesis, we cclimted mice to two different tempertures nd fed them three diets with different protein levels to determine the comprtive effects of elevted SusMR nd protein intke on kidney size nd function. We chose two estimtes of kidney function or cpcity. First, we used GFR ecuse it is n indiction of the rte t which the kidney is filtering plsm nd, s stted ove, increses with kidney hypertrophy. s n estimte of the kidneys ccommodtion to incresed protein intke, we used lood ure nitrogen level (UN). The level of ure in the lood, under stedy-stte conditions, should remin reltively constnt. If UN rises, it is n indiction tht the reltive mount of circulting nitrogen or ure hs chnged, nd chnges in UN my e correlted with chnges in kidney mss. n increse in ure production, however, my not hve negtive metolic effects if ure or nitrogen filtrtion rte hs Tle 1. Dietry constituents of test diets 7% 15% 46% protein protein protein Crohydrte (sucrose) (%) Ft (cottonseed oil) (%) Protein (purified high nitrogen csein) (%) Fier (lphcel, non nutritive ulk) (%) Slt mixture (%) rewer s yest (%) Clorific content (kj g 1 ) lso incresed to remove the excess nitrogen from the lood. Therefore, the finl estimte of kidney function we mde ws nitrogen filtrtion rte, which is the product of GFR nd UN. Mterils nd methods Thirty mle nd thirty femle virgin Swiss-Wester lortory mice Mus musculus L. (Chrles Rivers Lortories, Wilmington, M, US), 7 9 dys old, were divided into three groups on the sis of dietry protein level (7 %, 15 % or 46 % csein y mss; Tle 1). Within ech protein tretment, mice were further divided into two cclimtion-temperture tretments (23 C or 5 C; herefter referred to s wrm nd cold cclimtion, respectively). There were totl of six tretment groups. Ech group contined five mle nd five femle mice. The different protein diets were isocloric to ech other with crohydrte (sucrose) used to lnce protein (ICN iochemicls). ll mice were initilly mintined on their respective diets nd housed t 23 C. fter 1 week, hlf the mice were moved to cold room t 5 C for 2 weeks nd mintined on the sme diets. ody mss, food intke rte I (g dy 1 ) nd fecl output rte O (g dy 1 ) were mesured on ech of the lst 3 dys of the 2 week period, nd digestiility ws clculted s follows: digestiility = [(I O)/I] 1. (1) t the end of the 2 week temperture-cclimtion period, glomerulr filtrtion rte ws mesured in ech mouse using single-injection technique (Stcy nd Thorurn, 1966) s follows. n initil 1 µl lood smple ws tken from leg vein, fter which pproximtely 5 Mq of 51 Cr-lelled EDT in.3 ml of sterile Ringer s solution ws injected intrperitonelly. Susequent lood smples (5 2 µl) were tken from leg veins 1, 2, 4, 6, 8, 1 nd 12 min fter the injection of isotope. lood ws centrifuged t 12 g for 5 min, the hemtocrit mesured, nd the rdioctivity of known volume of plsm (2 1 µl) counted in eckmn LS 65 scintilltion counter. Liner regressions of log e (cts min ml 1 ) versus time were clculted, nd the slope (h 1 ) nd y-intercept (q ml 1 ) were used to clculte GFR (ml h 1 ): GFR = (R )/log 1 e, (2)

3 Incresed protein intke nd kidney size nd function 283 where R is the mount (q) of the rdioctive isotope injected. fter completion of GFR mesurements, mice were killed y intrperitonel injection of n overdose of sodium pentoritol (1 mg kg 1 ), nd 2 µl of lood ws collected from the infroritl sinus for mesurement of lood ure nitrogen level (UN). The stomch, smll intestine, lrge intestine, cecum, spleen, liver, kidneys, hert nd lungs were removed. The smll intestine ws gently flushed with Ringer s solution using gvge needle, nd the stomch, cecum nd lrge intestine were cut open nd lso flushed with Ringer s solution. Orgns were riefly lotted on pper towel, weighed to the nerest.1 mg, dried to constnt mss t 5 C, nd reweighed. lood ws centrifuged t 12 g for 5 min, nd UN ws mesured in triplicte using colorimetric ssy (Sigm Kit no. 535-). Finlly, GFR ws converted to dily rte multiplied y UN to clculte the dily nitrogen filtrtion rte of the kidney. Sttistics Our dt consist of two independent vriles (cclimtion temperture nd dietry protein level) nd mny dependent vriles (food intke rte, digestive efficiency, ody mss, kidney, gut nd other orgn msses, lood ure nitrogen level nd glomerulr filtrtion rte). s originlly designed, we used three-fctor nlysis of vrince (NOV) (two levels of sex, two levels of cclimtion temperture nd three levels of dietry protein level) to test for significnt effects of cclimtion temperture nd protein content nd their interction. This NOV reveled tht there were significnt differences etween mles nd femles in mny vriles so, for further nlysis, we seprted the sexes. In ddition, within ech sex, there ws 35 5 % rnge in individul ody msses. Therefore, we used ody mss s covrite for ech sex. In most cses, ody mss ws not significnt covrite; when ody mss ws significnt covrite for prticulr vrile, it did not cuse qulittive difference in the results. Therefore, the dt presented here re for 2 3 two-wy NOV for ech sex. Unless stted otherwise, the F nd P vlues cited throughout the text re from these two-wy NOVs. Tretment nd error degrees of freedom re used s suscripts for ech F vlue (denomintor degrees of freedom re etween 22 nd 29 ecuse some mesurements were missing for some individul mice). For ech sex, we routinely tested nine priori pirwise comprisons (ll orthogonl) in ddition to the min effects of cclimtion temperture nd dietry protein level. Those nine comprisons were the following: within ech cclimtion temperture, etween the three dietry protein levels (or six in ll), nd etween the two cclimtion tempertures for ech dietry protein level (three in ll). We tested these priori differences using rithmetic mens (SS Institute, 1985). Individul priori pirwise mens comprisons re mde y reporting the d hoc t-sttistics corresponding to the two-tiled P vlues. In these comprisons, we used the root men squre (corrected for the smple sizes of the two mens in question) s the denomintor for the totl NOV model so the comprison is mde in the context of the NOV model itself (SS Institute, 1991). Vlues re presented s mens ± S.E.M. Results There were sttisticlly significnt differences in ody mss etween mles nd femles cross ll tretment groups (mles 36.3±.5 g, femles 31.4±.5g; F 1,48 =47.83, P=.1). The three-fctor NOV showed significnt differences etween mles nd femles for ll dt except digestiility, so dt for the two sexes will e considered seprtely. For oth mles nd femles, ody mss did not differ etween experimentl groups, so ll dt re presented without correction for ody mss. Food intke rte nd digestiility For mles nd femles, food intke rte (g dy 1 ) in the coldtretment group ws 48 % nd 12 % higher, respectively, thn in the wrm-cclimtion group (F 1,24 =189 for mles nd F 1,24 =22.8 for femles, P<.1; Tle 2), ut did not differ etween protein levels. Despite the fct tht food intke rte ws higher t colder tempertures, there ws no difference in digestiility etween different cclimtion tempertures or etween different protein groups (76.6±.46 %). Protein intke rte incresed in the cold-cclimted nimls tht te more food. ecuse the protein level of the diets vried etween 7 nd 46 %, nimls in the high-protein groups ingested more protein, despite the fct tht ll groups within n cclimtion-temperture tretment hd similr food intke rtes (Tle 2). Internl orgn size Vitl orgns Kidney wet mss incresed significntly with cclimtion to oth cold temperture nd incresed dietry protein level (Fig. 1,). The cclimtion-temperture-induced increse in kidney mss ws lrger for femles (37 %; F 1,29 =13, P<.1) thn for mles (22 %; F 1,29 =42.3, P=.1). Kidney wet mss incresed y % with cclimtion to high dietry protein levels (F 2,29 =27.7 for mles nd F 2,29 =25.4 for femles, P<.2). In femles, there ws sttisticlly significnt interction etween cclimtion temperture nd dietry protein level (F 2,29 =1.9, P=.4) for kidney wet mss ecuse ll of the increse in size due to incresed dietry protein level nd intke rte occurred only in the cold-cclimted group. For mles, dietry protein level ffected the size of oth cold- nd wrm-cclimted mice similrly. Kidney dry mss incresed in response to oth cclimtion temperture nd dietry protein level in mnner tht mirrored the chnges in kidney wet mss, ut the chnges were slightly smller thn in kidney wet mss (Fig. 1C,D). Chnges in kidney dry mss due to cclimtion to cold temperture were 2 nd 34 % in mles nd femles, respectively, nd chnges due to incresed dietry protein were 26 nd 28 % in mles nd femles, respectively.

4 284 K.. HMMOND ND D. N. JNES Tle 2. Men dily food nd protein intke rtes of mle nd femle mice cclimted to either 5 C or 23 C nd fed diet with one of three different protein levels Femles Mles 5 C 23 C 5 C 23 C 7 % 15 % 46 % 7 % 15 % 46 % 7 % 15 % 46 % 7 % 15 % 46 % Food intke rte (g dy 1 ) (.39) (.36) (.58) (.27) (.3) (.27) (.35) (.51) (.35) (.44) (.14) (.62) Protein intke rte (g dy 1 ).55, 1.21, 3.7 c, , 1.28, 3.83 c,.31,.59, 1.96 C, (.27) (.54) (.27) (.18) (.45) (.12) (.24) (.77) (.16) (.3) (.21) (.12) Within sex nd temperture, letters tht re different indicte sttisticlly significnt differences etween mens. Mice cclimted to 5 C hve lowercse letters nd mice cclimted to 23 C hve uppercse letters. sterisks indicte sttisticlly significnt differences etween cold- nd wrm-cclimted nimls within dietry protein level. Vlues re mens with S.E.M. given in prentheses (N=5). Femle kidney wet mss (g) c Mle kidney wet mss (g) Femle kidney dry mss (g).2.1 C c Mle kidney dry mss (g).2.1 D Protein level (% in diet) Protein level (% in diet) Fig. 1. Kidney wet (,) nd dry (C,D) mss for mle (,D) nd femle (,C) mice cclimted to either 23 C (filled columns) or 5 C (open columns) s function of dietry protein level. Within ech cclimtion temperture, lowercse letters re used for the 5 C cclimtion group, uppercse letters for the 23 C cclimtion group. Different letters indicte significnt differences s function of dietry protein content. Within ech pir of columns (sme dietry protein content), sterisks denote mens tht differ significntly from ech other s function of temperture. Vlues re mens + S.E.M. (N=5). In oth mles nd femles, liver wet nd dry mss responded to oth high-protein diets nd to low cclimtion temperture (Tle 3), ut the pttern of the response ws different etween the sexes. In generl, cclimtion to high-protein diets incresed liver size in cold-cclimted femles only (not in wrm-cclimted femles), ut in oth cold- nd wrmcclimted mles. cclimtion to cold tempertures cused n increse of 1 39 % in femle liver wet mss (F 1,29 =37, P<.1) nd of % in liver dry mss (F 1,29 =45.6, P<.1). The significnt increse in liver mss for cold-cclimted femles ws driven y 17 nd 2 % increses in liver dry nd wet mss, respectively, in nimls eting the highest protein level diet (dry mss, F 2,29 =6.6, P=.5; wet mss, F 2,29 =4.8, P=.18). For mles, cclimtion to cold tempertures cused significnt increse in liver wet mss over tht in wrm-

5 Incresed protein intke nd kidney size nd function 285 Tle 3. Men ody nd orgn msses for femle nd mle mice cclimted to either 5 C or 23 C Femles Mles 5 C 23 C 5 C 23 C Mesured vrile 7 % 15 % 46 % 7 % 15 % 46 % 7 % 15 % 46 % 7 % 15 % 46 % ody mss (g) (.79) (.22) (.49) (2.23) (.96) (1.24) (1.58) (.96) (.61) (.81) (1.92) (1.1) Smll intestine wet mss (g) (.45) (.44) (.4) (.95) (.76) (.47) (.82) (.71) (.84) (.85) (.56) (.14) Smll intestine dry mss (g) (.23) (.6) (.11) (.17) (.17) (.9) (.22) (.11) (.19) (.12) (.29) (.21) Stomch wet mss (g) , , (.13) (.6) (.14) (.2) (.7) (.1) (.12) (.2) (.9) (.8) (.1) (.8) Stomch dry mss (g) (.5) (.2) (.3) (.5) (.2) (.3) (.2) (.5) (.4) (.3) (.4) (.2) Cecum wet mss (g) (.11) (.3) (.2) (.8) (.8) (.5) (.11) (.11) (.14) (.9) (.16) (.7) Cecum dry mss (g) (.2) (.1) (.2) (.3) (.3) (.1) (.3) (.2) (.2) (.3) (.4) (.3) Lrge intestine wet mss (g) (.14) (.13) (.14) (.22) (.8) (.12) (.1) (.16) (.14) (.13) (.12) (.9) Lrge intestine dry mss (g) (.4) (.4) (.4) (.7) (.4) (.5) (.1) (.8) (.4) (.5) (.9) (.4) Liver wet mss (g) , , 1.738,, , (.31) (.55) (.86) (.88) (.55) (.31) (.13) (.86) (.87) (.17) (.27) (.11) Liver dry mss (g).515,.474,.581, , ,.537 (.12) (.15) (.21) (.24) (.17) (.8) (.32) (.23) (.15) (.36) (.1) (.25) Hert wet mss (g) (.8) (.7) (.11) (.9) (.8) (.17) (.2) (.15) (.8) (.6) (.17) (.16) Hert dry mss (g) (.2) (.2) (.2) (.2) (.2) (.4) (.5) (.4) (.2) (.1) (.3) (.5) Lung wet mss (g) (.7) (.14) (.7) (.14) (.5) (.1) (.15) (.8) (.12) (.9) (.8) (.3) Lung dry mss (g) (.3) (.3) (.2) (.6) (.1) (.2) (.6) (.4) (.3) (.3) (.3) (.1) Within sex nd temperture, letters tht re different indicte sttisticlly significnt differences etween mens. Mice cclimted to 5 C hve lowercse letters nd mice cclimted to 23 C hve uppercse letters. sterisks indicte sttisticlly significnt differences etween cold- nd wrm-cclimted nimls within dietry protein level. Vlues re mens with S.E.M. given in prentheses (N=5). cclimted mles; however, mens seprtion tests did not show tht the incresed liver mss ws significnt in the coldcclimted mice t ny one level of protein intke rte (wet mss, F 1,29 =7.3, P=.13; dry mss, F 1,29 =3.9, P=.6). Liver size (wet nd dry mss) of oth cold- nd wrm-cclimted mles incresed with dietry protein level (wet mss, F 2,29 =4.4, P=.24; dry mss, F 2,29 =5.9, P=.9). Hert mss remined lrgely the sme in the fce of incresed dietry protein intke rte, ut for oth mles nd femles it did increse with cclimtion to cold tempertures (Tle 3). For femles, the cold-cclimtion increses in hert mss were % nd % for wet nd dry mss, respectively (wet mss, F 1,29 =26.8, P<.1; dry mss, F 1,29 =25.3, P<.1). For mles, the cold-cclimtion increses in hert mss were 1 34 % nd % for wet nd dry mss, respectively (wet mss, F 1,29 =14.9, P=.8; dry mss, F 1,29 =15, P=.7). Finlly, lung mss lso remined the sme in the fce of incresed dietry protein intke rte, ut lung wet mss incresed y 1 26 % for oth mles nd femles cclimted to cold tempertures (mles, F 1,29 =19.5, P=.2; femles, F 1,29 =17.4, P=.3). Digestive trct orgn size Tle 3 contins the dt for most orgn msses including the digestive trct. There is generl tendency towrds n increse in orgn mss with decresed cclimtion temperture nd no chnge in orgn mss with dietry protein level. The

6 286 K.. HMMOND ND D. N. JNES exception for chnges with dietry protein level is for stomch mss (discussed elow). In femles, ll comprtments of the digestive trct (stomch, smll intestine, cecum nd lrge intestine) showed tendency towrds incresed mss with cold cclimtion. This increse ws not significnt, however, t every dietry protein level in ny orgn. Stomch mss incresed y 16 % in cold-cclimted femles, ut this ws significnt only t dietry protein level of 46 % (F 1,29 =9.2, P=.6). Femle smll intestine, cecum nd lrge intestine wet mss incresed on verge y % with cold cclimtion. These differences were sttisticlly significnt for the 15 % nd 46 % ut not for the 7 % dietry protein level groups (smll intestine, F 1,29 =37, P<.1; cecum, F 1,29 =8.7, P=.7; lrge intestine, F 1,29 =18.1, P=.3). Smll intestine dry mss ws on verge 2 % greter in femles cclimted to cold tempertures (F 1,29 =27.4, P<.1). This ws true for ll levels of dietry protein level. For the other prts of the femle digestive trct (stomch, cecum nd lrge intestine), there ws no significnt increse in dry mss with cclimtion to cold tempertures. Mles responded to cold-temperture cclimtion similrly to femles, ut with less drmtic chnges. There ws no significnt difference in mle stomch wet mss in response to cold cclimtion temperture (F 1,29 =3.7, P=.7). s for femles, smll intestine wet mss incresed y n verge of 16 % in cold-cclimted nimls (F 1,29 =15, P=.7), ut this difference ws significnt only t dietry protein level of 15 %. Cecum nd lrge intestine wet msses lso incresed with cold cclimtion y % (cecum, F 1,29 =5.9, P=.2; lrge intestine, F 1,29 =5.9, P=.1), ut this difference ws significnt only t dietry protein level of 46 %. Cold cclimtion did not cuse significnt difference in dry mss in ny component of the mle digestive trct. The only component of the digestive trct tht ws ffected y dietry protein level ws the stomch. For oth mles nd femles, stomch wet mss incresed y % in coldcclimted nimls exposed to the highest (46 %) dietry protein level (femles, F 2.29 =6.2, P=.7; mles, F 2,29 =6.3, P=.6). Stomch dry mss did not chnge in response to different levels of dietry protein for either sex. Femle GFR (ml h 1 ) Mle GFR (ml h 1 ) Fig. 2. Glomerulr filtrtion rte (GFR) (,), lood ure nitrogen content (UN) (C,D) nd nitrogen filtrtion rte (NFR) (GFR UN) (E,F) for mle nd femle mice cclimted to either 23 C (filled columns) or 5 C (open columns) s function of dietry protein level. Letters indicte significnt differences s function of protein level s in Fig. 1. There were no significnt differences etween cclimtion tempertures. Vlues re mens + S.E.M. (N=5). Femle UN (mmol ml 1 plsm) Femle NFR (g dy 1 ) C E Protein level (% in diet) Mle UN (mmol ml 1 plsm) Mle NFR (g dy 1 ) D F Protein level (% in diet)

7 Incresed protein intke nd kidney size nd function 287 Estimtes of kidney function ll plots of glomerulr filtrtion rte (GFR) were highly liner (regression coefficients, r 2, vlues rnging etween 98.5 nd 99.6 %). The only significnt differences etween the lines were for the vlues of the slope, which is used in the estimte of GFR. For femles, GFR did not differ mong experimentl groups (25.8±2.1 ml h 1, Fig. 2). lood ure nitrogen level (UN) ws 43 nd 74 % higher in the high-protein group thn in the medium- nd low-protein groups (F 2,23 =17.1, P<.1, Fig. 2C), ut did not differ etween cclimtion-temperture groups. Nitrogen filtrtion rte (g dy 1 ) ws 14 % higher in mice fed the highest protein diet thn in the other two groups (F 2,22 =3.58, P<.4; Fig. 2E). Mle GFR ws pproximtely 69 % higher in the highprotein group thn in the other two protein groups (F 2,22 =1.1, P=.8; Fig. 2), ut did not differ etween cclimtiontemperture groups. Mle UN ws 22 nd 5 % higher in the high-protein group thn in the medium- nd low-protein groups (F 2,23 =11.3, P=.4; Fig. 2D), ut did not differ etween cclimtion-temperture groups (P=.72). s for femles, nitrogen filtrtion rte ws 111 % higher in the mice fed the highest protein diet thn in the other two protein groups (F 2,21 =17.13, P<.1; Fig. 2F). Discussion The primry gol of this study ws to determine whether elevted protein intkes hve different effects on kidney size nd function from the high energy intkes nd ssimiltion directly resulting from elevted SusMR. lthough oth SusMR nd protein intke rte hve een shown to ffect kidney mss nd function (Toloz et l. 1991; Hmmond nd Dimond, 1992; Konrzewski nd Dimond, 1994; Hmmond et l. 1994; Klhr, 1989), the two fctors hve not een investigted independently. The food intke rte we oserved in cold-cclimted mice ws pproximtely % greter thn tht of wrmcclimted mice nd did not vry mong groups fed diets differing in protein level. Thus, the incresed food intke rte of the cold-cclimted mice corresponded to their incresed SusMR, nd mice were eting to meet energy requirements rther thn trying to regulte protein intke rte. s result of the constnt food intke rte cross the protein tretment groups, protein intke rte ws drmticlly higher in the group fed high-protein diet (Tle 2). When food intke rte incresed in cold-cclimted nimls, protein intke rte lso incresed. The specific questions ddressed in this study re: (1) does n incresed protein intke rte resulting from incresed energy chllenges nd higher food intke rtes hve n effect on kidney size nd function, nd (2) does the effect on kidney size occur independently of increses in size of the digestive trct nd other vitl orgns? Effects of protein intke on kidney size nd function oth cold cclimtion temperture nd high-protein intke Kidney wet mss (g) Protein intke (g dy 1 ) Fig. 3. Reltionship etween kidney wet mss, M k (g), nd protein intke rte, I p (g dy 1 ), in mle (filled circles) nd femle (open circles) white mice. The liner regression for mles (roken line) is M k= i p (r 2 =.66, P<.1). The liner regression for femles (solid line) is M k= i p (r 2 =.65, P<.1). rtes increse kidney size. Dietry protein level lso hs strong effect on UN nd totl dily nitrogen filtrtion rte. Dietry protein level hs smller effect on GFR, lthough this effect is significnt only in mle mice (nd see elow). Clerly, the strongest effects on oth size nd function occur with comintion of high protein intke nd cold cclimtion tempertures when nimls re forced to et the lrgest mounts of protein nd susequently to cope with the nitrogenous wste products resulting from tht protein lod. The oservtion tht incresed protein intke rte cuses kidney hypertrophy nd incresed function (GFR) hs een mde previously in other rodents (e.g. Murry et l. 1993; Trinh-Trng-Tn et l. 1993; Klhr, 1989), ut not under circumstnces where energy demnd ws incresed fr ove norml levels. Kidney size (mss) increses linerly with protein intke rte such tht protein intke rte explins etween 65 nd 66 % of the vrition in kidney mss (Fig. 3). In ddition, the totl dily nitrogen filtrtion rte increses with dietry protein (nitrogen) intke (Fig. 4). Fig. 4 shows tht the kidneys filter pproximtely 55 % of the nitrogen tht the mice et per dy. The intercept on Fig. 4 is not expected to e zero for either mles or femles, ecuse nitrogen filtrtion with no protein intke rte is not negligile; however, ecuse the intercept is not zero, the ctul dily nitrogen filtrtion rte is much higher thn 55 %. Regrdless of the ctul percentge of dietry protein tht is filtered, it is cler from Figs 3 nd 4 tht, even with incresed UNs, the kidneys hve ccommodted to the incresing nitrogen lod plced on the system y the incresed protein intke rte. n dditionl fctor to consider in the interprettion of these dt is tht the 7 % protein diet is close to eing proteindeficient. Generlly, rodents cn e mintined on % protein (Ntionl Reserch Council, 1995), so the mice on the low-protein diet were not protein-deficient. nother fctor of concern is tht, ecuse fecl nitrogen losses re the sme

8 288 K.. HMMOND ND D. N. JNES Nitrogen filtrtion rte (g dy 1 ) Nitrogen intke (g dy 1 ) Fig. 4. Reltionship etween nitrogen intke rte, I n (g dy 1 ), nd nitrogen filtrtion rte, NFR, for mle (filled circles) nd femle (open circles) white mice. The liner regression for mles (roken line) is NFR= I n (r 2 =.5, P=.1). The liner regression for femles (solid line) is NFR= I n (r 2 =.33, P=.15). regrdless of the percentge of protein in the diet, the lowprotein diets my hve lower pprent protein digestiility thn do the higher-protein diets (Roins, 1993). Thus, the mice fed the 7 % protein diets my hve hd lower reltive nitrogen sorption rte thn the mice on the 15 % protein diet; the slope in Fig. 4, showing 55 % filtrtion of dietry nitrogen intke, my e rtificilly low ecuse the pprent protein digestiility of tht diet is overestimted. The regressions in Fig. 4 ssumes 1 % protein digestiility. more relistic ssumption for protein digestiility on 15 % protein diet of 95 % (Nutritionl Reserch Council, 1995; Roins, 1993) would increse the estimte of dietry nitrogen filtered from 55 to 58 %. If we ssume n even lower protein digestiility of 9 % for the mice on the low-protein diet, the estimte for dietry nitrogen filtered increses to 6 %. The difference etween the response of mles nd femles to high dietry protein level nd, to lesser extent, to cold cclimtion is reflected y mny of the dt. oth solute vlues of kidney mss nd function nd the ptterns of chnge differ etween the sexes. For instnce, regrdless of their reltive orgn msses, kidney mss of femles t 23 C did not increse with incresing protein level, wheres mle kidney mss t oth cclimtion tempertures incresed with incresing protein level (Fig. 1). Overll, however, the generl trends of kidney mss nd function mesured y nitrogen filtrtion rte t high levels of dietry protein re the sme etween the two sexes. Mesurements of GFR were somewht prolemtic for severl resons. First, femles nd mles differed in their response to protein level: only mles showed significnt differences in GFR mong the protein levels. Yng nd irkhhn (1993) hve reported tht fter skeletl trum femle rts displyed less nitrogen loss nd proly lower ody turnover of nitrogen thn did mle rts. From these results, they suggest tht the use of protein nd susequent nitrogen turnover is different etween the sexes. If it is true tht there re different sex-specific responses, then perhps the differences we oserved in GFR etween mles nd femles re to e expected. However, we cnnot verify tht hypothesis. Second, only t the highest protein level did mle mice exhiit significntly elevted GFR (Fig. 2). Pitts (1944) found tht three different protein levels cused stepwise increses in GFR in dogs (61, 74 nd 86 ml min 1 for three diets with incresing protein levels). However, these results re from single niml, nd the ctul protein levels of the diets were not reported. Third, lthough our GFR vlues were within the 95 % confidence limits of the vlue predicted y the llometric eqution of Yokot et l. (1985), they were considerly higher thn those mesured in previous studies (cf. men vlues of 29.3±1.67 ml h 1, N=58, for ll the dt in the present study nd 12 ml h 1 in Hines nd Frmer, 1991). Our men GFR vlues might e expected to e higher thn those of other studies ecuse the mice fed the high-protein diet nd cclimted to cold tempertures hd n extremely high protein intke rte, ut even the GFRs of the mice fed the low-protein diet re reltively high. ecuse we do not know whether our overll high GFR vlues re n rtifct of our mesurement technique nd ecuse of the vriility in mesured GFR cused y different mesurement techniques (ll of which provide only estimtes of the true GFR), these results should e interpreted cutiously. Independence of chnges in kidney size nd function Finlly, we turn our ttention to our second question: re chnges in kidney mss nd function result of incresed whole-ody sustinle metolic output or of incresed protein intke rte nd nitrogen wste removl? Recll tht our mesured increse in protein intke rte does not result from n increse in food intke rte (Tle 2). We did not find protein-induced increse in the mss of ny digestive orgn except the stomch or in ny other vitl orgn mesured except the liver nd kidney. In the liver, the sme pttern of chnges etween mles nd femles rises s in the kidney (s discussed ove). Our dt show tht the size of the liver increses in mnner similr to tht of the kidney. ecuse the production nd excretion of ure re linked, it should not e unexpected tht the msses of these two orgns should chnge simultneously with chnges in dietry protein lod. However, ecuse the liver does not generlly chnge in size with lrgescle chnges in food intke rte (Hmmond et l. 1994; Konrzewski nd Dimond, 1994), we did not expect this result. It is cler tht incresed protein intke rte ffects kidney, liver nd stomch mss differently from other orgns, ut it is not initilly cler whether these chnges re ctully independent of the ntomicl nd physiologicl chnges tht occur in other orgns, especilly the smll intestine. In order to determine whether these chnges re independent, we compred dt from two specific experimentl groups. Within protein tretment group, food intke rte incresed with cold cclimtion (Tle 2). Therefore, we still could not

9 Incresed protein intke nd kidney size nd function 289 Tle 4. Comprisons of mesurements of kidney size nd function etween nimls with dissimilr food intke rte nd energy demnds, ut lmost identicl protein intkes Femles Mles 23 C 5 C 23 C 5 C Mesured vrile 15 % 7 % P 15 % 7 % P Food intke rte (g dy 1 ) 3.77±.3 8.3± ± ±.35.1 Protein intke (g dy 1 ).566± ±.27 NS.592± ±.24 NS Kidney wet mss (g).335± ± ± ±.128 NS Kidney dry mss (g).944±.36.19± ± ±.8 NS UN (mmol l 1 plsm) 6.26± ±.75 NS 8.42± ±.75 NS GFR (ml h 1 ) 23.3± ±3.4 NS 29.8± ±4.1 NS Nitrogen filtrtion rte.12±.45.16±.51 NS.173± ±.29 NS (g dy 1 ) NS, not significnt; UN, lood ure nitrogen content; GFR, glomerulr filtrtion rte. Vlues re mens ± S.E.M. (N=5). rule out the possiility tht it ws the incresed protein intke rte tht cused the incresed kidney mss rther thn just incresed whole-ody metolic wste excretion resulting from incresed metolic ctivity ssocited with thermogenesis. To seprte the effects of cclimtion temperture nd protein level, we compred kidney wet nd dry mss, GFR, UN nd nitrogen filtrtion rtes etween two groups: 23 C/15 % protein nd 5 C/7 % protein. These two groups hd dissimilr metolic demnds (one cold-cclimted nd one wrm-cclimted) nd food intke rtes ut lmost identicl protein intke rtes (Tle 4), enling us to exmine the effect of food intke rte (i.e. SusMR) without chnging protein intke rte. Sttisticl comprisons etween the two groups for ech vrile were mde within the context of the whole experimentl design y using the error men squre of the whole model in the denomintor of t-test (see Sttistics section ove). This comprison ws mde for mles nd femles seprtely. In mles, there were no differences in GFR, UN, nitrogen filtrtion rte or kidney mss etween the two groups, indicting tht the chnges in kidney mss nd UN were due to the incresed protein intke rte nd not to generlized metolic response to incresed ssimiltion of energy or energy chllenge per se. In femles, there were lso no differences in GFR, UN or nitrogen filtrtion rte, ut kidney mss ws greter in the cold-cclimtion group (wet mss incresed y 18 %, P<.5; dry mss incresed y 17 %, P<.1), indicting tht some of the increses in kidney mss were due to incresed metolic energy ssimiltion nd energy chllenges. On the sis of the results discussed ove, it seems prole tht, within the context of our experimentl design, incresed kidney function s estimted y UN, GFR nd nitrogen filtrtion rte is due lrgely to incresed protein intke rte. For the kidney s whole, it is cler tht some of the short-term dpttion is due strictly to protein intke rte. Oviously, nitrogen filtrtion rte is only one of mny mesurements tht re necessry to determine the functionl output nd cpcity of the kidney. Therefore, mesuring other orgn-level vriles, such s energy utiliztion (e.g. N + /K + - TPse ctivity), my provide dditionl insights. ecuse we otined different results for mles nd femles, however, we still cnnot seprte the effects of incresed metolic output nd incresed protein intke rte on kidney size. In ddition, there do pper to e sex-specific differences in dpttion to incresed protein levels nd incresed metolic demnd tht re more complex nd dynmic thn our experimentl design could test. For instnce, t medium to high levels of dietry protein, mles hve lrger kidney msses nd higher GFRs, UNs nd nitrogen filtrtion rtes thn femles of similr size nd with similr protein intke rtes. This implies tht vriles such s hormonl lnce nd other sex-specific whole-ody vriles my hve n impct on kidney (nd other orgn) function nd cpcity. Overll, we suggest tht incresed food intke rte comes with costs to the niml of the mintennce of energy-utilizing pthwys nd of other metolic output pthwys such s ure metolism nd metolic wste excretion. This finding is importnt for understnding the costs ssocited with the mintennce of high levels of SusMR. Ingestion of more food in response to high levels of energy demnd does id in meeting metolic costs. It is pprent tht incresed food intke rte is esily ccommodted y the digestive system in generl (Hmmond nd Dimond, 1997), ut there re lso other costs ssocited with incresed nutrient intke (here protein) tht my e incurred in other orgn systems such s the kidney. These costs re often, ut not lwys, reflected in lrger orgn size, ut my lso e reflected in loss of the functionl reserve cpcity of other orgns. The significnce of this hidden loss in functionl reserve cpcity for nimls in chllenging situtions deserves more ttention. We thnk ronwyn Mclln for her ssistnce with the glomerulr filtrtion method. We would like to thnk Cthy utchko for her tireless help in this project nd Vughn Shoemker for his vlule insight nd discussion of the mnuscript. This project ws supported y NIH wrd HD to K..H. nd Jred Dimond.

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