see commentary on page 226 Fructokinase activity mediates dehydration-induced renal injury

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1 sic reserch & 13 Interntionl Society of Nephrology see commentry on pge 6 Fructokinse ctivity medites dehydrtion-induced renl injury Crlos A. Roncl Jimenez 1, Tkuji Ishimoto 1, Miguel A. Lnsp 1, Christopher J. Rivrd 1, Tkhiko Nkgw 1, A. Ahsn Ejz, Christin Cicerchi 1, Shinichiro In 1, MyPhuong Le 1, Mkoto Miyzki 1, Json Glser 3, Ricrdo Corre-Rotter 4, Mrvin A. González 5, Auror Argón 5, Cthrin Wesseling 6, Lur G. Sánchez-Lozd 7,9 nd Richrd J. Johnson 1,8,9 OPEN 1 Division of Renl Diseses nd Hypertension, University of Colordo, Auror, Colordo, USA; Division of Nephrology, University of Florid, Ginesville, Florid, USA; 3 L Isl Foundtion, León, Nicrgu; 4 Instituto Ncionl de Ciencis Médics y Nutrición Slvdor Zuirán, Mexico City, Mexico; 5 Reserch Centre on Helth, Work nd Environment (CISTA), Ntionl Autonomous University of Nicrgu t León, (UNAN-León), León, Nicrgu; 6 Progrm Work, Helth nd Environment in Centrl Americ, Universidd Ncionl, Heredi, Cost Ric; 7 Instituto Ncionl de Crdiologí Igncio Chávez, Mexico City, Mexico nd 8 Division of Nephrology, Estern Colordo Helth Cre System, Deprtment of Vetern Affirs, Denver, Colordo, USA The epidemic of chronic kidney disese in Nicrgu (Mesomericn nephropthy) hs een linked with recurrent dehydrtion. Here we tested whether recurrent dehydrtion my cuse renl injury y ctivtion of the polyol pthwy, resulting in the genertion of endogenous fructose in the kidney tht might susequently induce renl injury vi metolism y fructokinse. Wild-type nd fructokinsedeficient mice were sujected to recurrent het-induced dehydrtion. One group of ech genotype ws provided wter throughout the dy nd the other group ws hydrted t night, fter the dehydrtion. Both groups received the sme totl hydrtion in 4 h. Wild-type mice tht received delyed hydrtion developed renl injury, with elevted serum cretinine, incresed urinry NGAL, proximl tuulr injury, nd renl inflmmtion nd firosis. This ws ssocited with ctivtion of the polyol pthwy, with incresed renl corticl soritol nd fructose levels. Fructokinse-knockout mice with delyed hydrtion were protected from renl injury. Thus, recurrent dehydrtion cn induce renl injury vi fructokinse-dependent mechnism, likely from the genertion of endogenous fructose vi the polyol pthwy. Access to sufficient wter during the dehydrtion period cn protect mice from developing renl injury. These studies provide potentil mechnism for Mesomericn nephropthy. Kidney Interntionl (14) 86, 94 3; doi:1.138/ki.13.49; pulished online 11 Decemer 13 KEYWORDS: ldose reductse; fructokinse; fructose; hydrtion; Mesomericn nephropthy Correspondence: Crlos A. Roncl Jimenez, Division of Renl Diseses nd Hypertension, University of Colordo, Auror, Colordo 845, USA. E-mil: Crlos.Roncl@ucdenver.edu 9 The lst two uthors re co-senior uthors. Received 7 April 13; revised August 13; ccepted 3 Octoer 13; pulished online 11 Decemer 13 An epidemic of chronic kidney disese is occurring in the hot costl communities of Centrl Americ, especilly mong men working in sugrcne fields. 1 3 The cuse of the disese, which hs een clled Mesomericn nephropthy, 1 3 hs remined mystery, s it is not ssocited with the usul cuses of chronic kidney disese such s dietes or hypertension. Studies to dte hve not identified hevy metls, grochemicls, or other toxins s likely cndidtes for cusing the epidemic. However, common predisposing feture ppers to e exposure to het nd the development of recurrent dehydrtion. 1 3 Dehydrtion is commonly ssocited with prerenl dysfunction with retention of ure, ut clssiclly this is thought to reflect hemodynmic chnges nd not to e ssocited with true renl injury. If dehydrtion is severe, sujects cn develop cute kidney injury from het stroke, rhdomyolysis, nd hypotension, ut such individuls would likely hve sustntil symptoms nd not present with the insidious onset of chronic kidney disese s typiclly occurs. However, fvorite hypothesis is tht recurrent dehydrtion my led to low-grde renl injury tht over time results in chronic kidney disese. Dehydrtion from swet nd het is known to ctivte the relese of vsopressin. In turn, vsopressin hs recently een recognized to hve potentil role s meditor of chronic kidney disese. 4 6 However, lthough role for vsopressin in incresing the rte of progression in chronic kidney disese hs een shown in nimls, 4,7 its role in cute renl injury ssocited with dehydrtion remins lrgely unexplored. Dehydrtion cn lso result in n increse in serum osmolrity nd in the ctivtion of ldose reductse. 8 Activtion of ldose reductse is importnt in the renl medull, s here it genertes soritol tht cn help protect tuulr cells from the high osmolrity in the extrcellulr environment t this site, thus fcilitting urinry concentrtion. However, ctivtion of ldose reductse in the proximl tuule my not e eneficil, s the conversion of glucose to soritol my 94 Kidney Interntionl (14) 86, 94 3

2 CA Roncl Jimenez et l.: Dehydrtion nd fructokinse sic reserch result in its further degrdtion y soritol dehydrogense to fructose. Although fructose itself my not e toxic, when it is metolized y fructokinse, it results in trnsient denosine triphosphte depletion with the genertion of uric cid, oxidnts, nd inflmmtory meditors The proximl tuule is one of the mjor sites where fructokinse is expressed. 1 This rises the hypothesis tht recurrent dehydrtion my result in repeted stimultion of ldose reductse with the genertion of fructose in the proximl tuule, leding to tuulr injury nd inflmmtion. To test this hypothesis, we designed the following study. RESULTS Effect of dehydrtion on dily weight The dehydrtion protocol resulted in the mice sweting nd losing weight cutely. Figure 1 shows the chnge in men weight during the 7-h dehydrtion period. Mice tht were not provided wter during the dy (wter t night (WAN)) lost men of 15% ody weight over the 7-h dehydrtion period (men 15.4% for wild-type (WT) mice nd 14.6% for the fructokinse (ketohexokinse) A nd C knockout () mice, P ¼ nonsignificnt). In contrst, mice who hd ccess to wter during the dy (wter ll time ()) showed significntly less weight loss during the dy (7.8% nd 8.1%, respectively, for the WT nd mice, P ¼ nonsignificnt). This degree of weight loss ws oserved oth t the eginning nd t the end of the study nd there ws no evidence of dpttion over time (dt not shown). Not surprisingly, the mice tht hd wter withheld during the dy drnk more wter t night (Figure 1), nd the mice tht hd wter ll the time during the dy drnk more wter compred with the control group in 7 h (Figure 1c), such tht totl wter intke over 4 h ws identicl in the nd WAN groups (Figure 1d). The effect of the dehydrtion procedure on overll chnges in morning sl weight ws lso ssessed. At the end of 5 weeks, oth WT nd mice exposed to dehydrtion tended to mintin ut not increse their weight (Figure 1e). In contrst, control WT nd control KHK mice tended to increse their weight over the 5-week period. Dehydrtion ctivtes the ldose reductse pthwy in the renl cortex of WT mice WT nd mice tht were dehydrted without wter (WAN) showed n increse in serum osmolrity t the end of the 7 h dehydrtion period (Figure ). Urinry osmolrity ws incresed in the mice exposed to het in smples collected t oth week 1 nd week 4 (Figure nd c). Hyperosmolrity is expected to increse ldose reductse levels, ut no differences were oserved in ldose reductse protein levels y western lot of the renl cortex t the time of killing, lthough there ws tendency for higher ldose reductse levels in the WT WAN group (Figure d). Nevertheless, oth renl corticl soritol (Figure e) nd renl corticl fructose levels (Figure f) were incresed in WT mice dehydrted without wter during the dy (WAN group), consistent with n increse in ldose reductse ctivity. Soritol levels were not incresed in the mice independent of groups. In contrst, renl fructose levels were elevted in ll three groups of mice, consistent with erlier studies showing tht fructose levels re high in mice in which fructokinse is sent. 13 Effect of dehydrtion on lood pressure, renl function, nd renl histology As shown in Figure the WT mice tht were dehydrted during the dy without wter (the WAN group) constituted the group tht showed evidence for n increse in ldose reductse ctivity, s noted y n increse in renl soritol nd fructose levels. This ws lso the only group tht showed evidence of renl injury, s noted y n increse in urinry neutrophil geltinse-ssocited lipoclin (Figure 3), serum cretinine (Figure 3), nd evidence of proximl tuulr injury y renl histology (Figure 4), nd with loss of proximl tuulr rush order sed on stining for ngiotensin-converting enzyme using computer imge nlysis (Figure 5). Injury ws present in oth proximl tuules in the renl cortex nd in the outer stripe. The injury ws not due to rhdomyolysis, s serum cretine phosphokinse levels t the time of killing were norml in ll mice regrdless of group (cretine phosphokinse levels o1 ng/ml). Consistent with the known effects of fructose to stimulte the chemokine monocyte chemotctic protein-1 (MCP-1) in proximl tuulr cells, 1 we lso found oth n increse in renl corticl MCP-1 levels (Figure 6) nd n increse in infiltrting mcrophges in the WAN group compred with the other groups of WT mice (Figure 6) s we cn see in the F4/8 stin (Figure 7). An increse in interstitil firosis (collgen III stining) ws lso oserved in the WT WAN group (Figures 8 nd 9). Importntly, mice were protected from renl injury. Despite the WAN group hving the sme degree of weight loss nd increse in serum osmolrity, the mice did not show n increse in serum cretinine, tuulr injury, inflmmtion, or firosis y histology, or incresed expression of the chemokine MCP-1 (Figures 6 9). Blood pressure ws lso mesured in oth WT nd KHK- KO mice t the end of dehydrtion period t week 4 (Figure 1). Blood pressure ws incresed in the WT WAN mice compred with ll other groups. mice tht were dehydrted without wter (WAN) lso showed higher lood pressure, ut it ws significntly less thn tht of the WAN WT mice (Figure 1). Uric cid levels Previous studies hve found tht uric cid generted during fructose metolism my e prtilly responsile for the stimultion of MCP-1. 1 Consistent with this hypothesis, n increse in kidney corticl uric cid ws oserved in the WT WAN mice (Figure 11). However, renl corticl uric cid levels were lso high in the WAN mice despite no stimultion of MCP-1 (Figure 11). We lso noted Kidney Interntionl (14) 86,

3 sic reserch CA Roncl Jimenez et l.: Dehydrtion nd fructokinse % Weight loss fter dehydrtion WAN WAN Wter intke overnight (ml) WAN CON WAN CON c Wter intke during dehydrtion (ml) 3 1 WAN CON WAN CON d 4 h wter intke h dehydrtion Overnight WAN CON WAN CON e Body weight (g) W WAN WAN Control Control -5 W WAN WAN -5 W -5 W -5 W Control Control -5 W Figure 1 Effects of dehydrtion. () Percent weight loss fter 7 h of dehydrtion. () Wter intke overnight. (c) Wter intke during dehydrtion. (d) The 4 h wter intke (light shde, t night; drk shde, during dehydrtion). (e) Body weight t seline nd fter 5 weeks. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5; Po.1; Po.1; Po.1. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. no difference in serum uric cid levels mong ll groups (Figure 11). These results suggest tht uric cid my not e the primry mechnism y which KHK medites renl injury. DISCUSSION The purpose of this study ws to explore the potentil mechnisms y which chronic or recurrent dehydrtion could cuse kidney injury. The hypothesis ws tht dehydrtion-ssocited hyperosmolrity my ctivte the ldose reductse pthwy in the renl cortex nd thus led to the genertion of fructose during the reclmtion of glucose y the proximl tuule. In turn, the metolism of fructose y fructokinse in the proximl tuule would led to the genertion of oxidnts nd chemokines (MCP-1) tht would result in locl tuulr injury nd inflmmtion. To test this hypothesis, we developed model of recurrent dehydrtion y plcing mice in heted chmers for totl of 3.5 h per dy, for 5 dys per week, for totl of 5 weeks. In the sence of eing provided wter, the verge mouse lost B15% of their ody weight, only to fully hydrte itself during the night in preprtion for the next dy. A second group ws dehydrted ut llowed ccess to wter oth during the dy nd during the night. These mice lost B8% of their ody weight during the dy. Importntly, this group lso hydrted themselves during the evening, such tht oth groups recovered their norml weight nd drnk the sme totl mount of wter in the 4 h period. The first mjor finding ws tht the mice tht were severely dehydrted during the dy nd hd delyed rehydrtion ctivted the ldose reductse pthwy in their kidneys, s noted y n increse in renl soritol nd fructose 96 Kidney Interntionl (14) 86, 94 3

4 CA Roncl Jimenez et l.: Dehydrtion nd fructokinse sic reserch Serum osmolrity (mosm) WAN CON WAN CON Urine osmolrity 1 week (mosm) WAN CON WAN CON c Urine osmolrity 4 weeks (mosm) WAN CON WAN CON d Aldose reductse per β-ctin densitometry ritrry vlues WAN CON WAN CON e Cortex soritol (nmol per mg protein) f Cortex fructose (nmol per mg protein) 6 4 WAN CON WAN CON WAN CON WAN CON Figure Dehydrtion ctivtes the polyol pthwy in the renl cortex. () Serum osmolrity t the end of the 7 h dehydrtion period. Urinry osmolrity fter week 1 () nd week 4 (c). (d) Renl corticl ldose reductse protein level (densitometry of western lot). (e) Renl cortex soritol levels. (f) Renl cortex fructose levels. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5; Po.1; Po.1; Po.1. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. Urine (ng NGAL per mg Cr) WAN CON WAN CON WAN CON Serum cretinine - μg per ml WAN CON Figure 3 Dehydrtion cuses renl dysfunction. () Urinry neutrophil geltinse-ssocited lipoclin (NGAL)/cretinine (Cr) rtio. () Serum cretinine (mesured y high-performnce liquid chromtogrphy (HPLC)). Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. Kidney Interntionl (14) 86,

5 sic reserch CA Roncl Jimenez et l.: Dehydrtion nd fructokinse WT- WT-WAN c WT-Control d - e -WAN f Control Figure 4 Renl histology. Proximl tuulr injury (loss of rush order with tuulr diltion; rrows) is oserved in wild-type mice tht were dehydrted without wter (WAN) (). Compring with the groups WT- (); WT-Control (c); - (d); -WAN (e); nd Control (f). Scle r ¼ 5 mm., fructokinse-knockout; WAN, wter t night;, wter ll time; WT, wild type. levels, nd this ws ssocited with the development of renl injury, s noted y n increse in urinry neutrophil geltinse-ssocited lipoclin, n increse in serum cretinine, proximl tuulr injury y iopsy, n increse in renl MCP-1 nd mcrophge infiltrtion, nd erly renl firosis. A key finding ws tht the mice tht were exposed to the sme het ut who hydrted during the dy were protected. The oservtion tht only the group tht ws not well hydrted during the dy developed renl injury, despite oth groups receiving the sme mount of totl hydrtion in 4-h period, emphsizes the importnce of dequte hydrtion during het exposure to prevent renl dmge. The second mjor finding ws tht mice lcking fructokinse were protected from renl injury despite similr degrees of dehydrtion. In prticulr, these mice did not show n increse in serum cretinine or the presence of renl injury, inflmmtion, or firosis. These studies strongly suggest tht it is the metolism of endogenous fructose tht is driving the renl injury, nd this lso implictes n importnt role for fructokinse. These dt re consistent with recent studies tht suggest tht high dietry intke of fructose my lso cuse tuulointerstitil disese in the mouse nd rt.14,15 An interesting spect ws tht renl soritol ws not elevted in the WAN mice despite similr increse in serum osmolrity. This suggests tht the ldose reductse pthwy ws inhiited in the setting in which fructokinse is locked. The mechnism is not evident, ut could potentilly 98 relte to negtive feedck pthwy from fructose itself tht ccumultes in mice with fructokinse deficiency. Further studies for exploring this pthwy re in order. We lso noted n increse in renl corticl uric cid in oth WT nd fructokinse-ko mice tht were dehydrted without ccess to wter during the dy. We hd postulted tht the increse in renl uric cid my e limited to the WT mice s they would hve n intct fructokinse system tht would e expected to generte uric cid during fructose metolism. However, incresed proximl tuulr uptke of uric cid lso occurs during dehydrtion, nd this likely ccounts for the lck of difference etween the groups. It does suggest, however, tht the renl injury oserved my not e due to the uric cid, s the fructokinse-knockout mice were protected. We previously reported tht lowering uric cid ws not completely effective t locking oxidnts in isolted proximl tuules exposed to fructose.1 These findings could e relevnt to the etiology of the epidemic of kidney disese in Centrl Americ, which lso ppers cliniclly nd histologiclly to e type of primry tuulointerstitil injury, lthough with some glol glomerulosclerosis.16 To dte, the only common risk fctor seems to e exposure to het nd recurrent dehydrtion. Mny of the workers lso hydrte themselves with fructose-rich juices or everges tht might compound the prolem. Thus, we would like to suggest tht repeted renl injury my e occurring from the effects of endogenously generted fructose Kidney Interntionl (14) 86, 94 3

6 CA Roncl Jimenez et l.: Dehydrtion nd fructokinse sic reserch d WT- - WT-WAN e -WAN ACE stin- 4 c WT-control f -control g ACE % positive re h Serum cretine kinse-ng/ml NS. WAN CON WAN CON WAN CON WAN CON Figure 5 Proximl tuulr rush order stining using ngiotensin-converting enzyme (ACE) ntiody. Proximl tuulr rush order is decresed in the renl cortex (lck rrows) of wild-type mice with dehydrtion-ssocited kidney injury () compred with other groups (, c f). Percentge of positively stined re is shown in (g). Cretine kinse levels re norml nd similr mong groups (h). Scle r ¼ 5 mm., fructokinse knockout; WAN, wter t night;, wter ll time; WT, wild type. Kidney cortex MCP-1 (pg per μg prot) F4/8 cells (% positive) 3 1. WAN CON WAN CON WAN CON WAN CON Figure 6 Inflmmtion occurs with chronic dehydrtion. () Renl corticl MCP-1 protein levels; () infiltrting mcrophges F4/8 stin. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5; Po.1. CON, control;, fructokinse knockout; MCP-1, monocyte chemotctic protein-1; WAN, wter t night;, wter ll time. Kidney Interntionl (14) 86,

7 sic reserch CA Roncl Jimenez et l.: Dehydrtion nd fructokinse WT- WT-WAN WT-Control - -WAN Control Figure 7 Mcrophge (F4/8) infiltrtion with chronic dehydrtion. Wild-type mice with wter provided only t night show incresed mcrophge infiltrtion (rrows) () compred with other groups WT- (); WT-Control (c); - (d); -WAN (e); nd Control (f). Scle r ¼ 5 mm., fructokinse knockout; WAN, wter t night;, wter ll time; WT, wild type. WT- WT-WAN WT-Control - -WAN control Figure 8 Interstitil collgen III deposition with chronic dehydrtion. Wild-type mice with wter provided only t night show incresed interstitil firosis (), compred with other groups. WT- (); WT-Control (c);, (d); WAN (e); nd Control (f). Scle r=5 mm., fructokinse knockout; WAN, wter t night;, wter ll time; WT, wild type. nd tht this might e potentited y fructose provided in the drinks. We recognize tht other mechnisms re lso likely importnt nd could e cting synergisticlly with the consequences of ctivtion of the ldose reductse pthwy. Dehydrtion nd volume depletion, for exmple, re known to ctivte vriety of meditors tht re cple of inducing renl injury, including the renin ngiotensin ldosterone system, the sympthetic nervous system, nd vsopressin. Dehydrtion my led to incresed resorption of toxins into the kidney, such s hevy metls nd grochemicls. Nevertheless, the importnce of this study is tht it identifies new potentil meditor of chronic kidney disese tht is ctivted y het nd dehydrtion. This could e importnt s glol wrming leds to the emergence of new diseses. Besides Centrl Americ, there is concern tht kidney diseses re incresing in other hot griculturl communities, such s in Sri Lnk, Indi, Mexico, Ecudor, nd Egypt. 3 Kidney Interntionl (14) 86, 94 3

8 CA Roncl Jimenez et l.: Dehydrtion nd fructokinse sic reserch Clerly, more studies re needed. However, we would like to suggest tht the ctivtion of fructokinse could represent one new plyer in driving cute nd chronic kidney disese. Collgen III (% positive re) WAN CON WAN CON Figure 9 Interstitil firosis (type III collgen stining) occurs with chronic dehydrtion. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.1. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. Systolic BP (mm Hg) WAN CON WAN CON Figure 1 Systolic lood pressure (BP) t 4 weeks. Systolic lood pressure ws incresed in the WT WAN mice compred with ll other groups. mice tht were dehydrted without wter (WAN) lso showed higher BP, ut it ws significntly less thn tht in WAN WT mice. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5; Po.1. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. MATERIALS AND METHODS Animls mice were provided y Dr Dvid Bonthron (Leeds, UK) nd red in our vivrium for this study. The experimentl protocols were pproved y the University of Colordo Animl Cre nd Use Committee. Seven-week-old mle nd WT mice from Jckson Lortory (Br Hror, ME) of the sme C57BL/6 ckground were used. Mice were kept under temperture- nd humidity-controlled specific pthogen-free conditions nd mintined on 14-h drk/1-h light cycle. Mice were llowed d liitum ccess to norml lortory chow (Hrln Tekld; 9X, Mdison, WI) nd given wter d liitum until the strt of the study. This chow is strch-sed diet nd does not contin ny fructose. Mice were housed three per cge nd ll were treted simultneously under equivlent conditions. Study design The dehydrtion protocol consists of plcing mice in heted environment (Isotemp-Fisher Scientific incutor, Duuque, IA) t C for 3 min ech hour for totl of 7 h, 5 dys week, for totl durtion of 5 weeks. This procedure results in the nimls sweting (primrily in the foot pds) nd losing B15% of ody weight per dy in the sence of hydrtion (see Results). Mice were divided into three groups. Group I received dily dehydrtion ut with wter provided during the periods etween the het exposure (). Group were dehydrted ut provided wter only for 1 h ech night (WAN). Group 3 consisted of mice tht were never dehydrted nd wter ws provided d liitum (Control). Ech group consisted of six nd six WT mice for totl of 36 mice. Stndrd mouse chow (Hrln Tekld, 9X) ws provided oth dy nd night (except during the periods of het exposure). Blood testing Mice were killed t the end of the dehydrtion period (tht is, fter the 7 h dehydrtion protocol t 5 weeks) y nesthesi nd crdic exsnguintion. Serum uric cid ws mesured y modified cronte-phosphotungstte method. 17 Serum fructose levels were mesured using n ssy kit (EnzyChrom Fructose ssy kit, BioAssy System, Hywrd, CA). Serum cretinine concentrtion ws nlyzed with the high-performnce liquid chromtogrphy tndem mss spectrometry method. 18 Serum cretine kinse ws mesured using the enzyme-linked immunosorent ssy (ELISA) kit for Cretine Kinse, Muscle (CKM; USCN Life Science, Houston TX). Serum osmolrity ws mesured using The 5. 3 Uric cid (μg per mg protein) Serum uric cid (mg per dl) WAN CON WAN CON WAN CON WAN CON 1 Figure 11 Uric cid levels fter 7 h of dehydrtion. () Renl corticl uric cid levels. () Serum uric cid levels. Dt represent nlysis of vrince (ANOVA) (Bonferroni post test) Po.5; Po.1. CON, control;, fructokinse knockout; WAN, wter t night;, wter ll time. Kidney Interntionl (14) 86,

9 sic reserch CA Roncl Jimenez et l.: Dehydrtion nd fructokinse Advnce Micro Osmometer (Model 33, Advnced Instruments, Norwood, MA). Urine testing Urine ws collected t 1 nd 4 weeks t the end of the dehydrtion period y stimulting excretion y holding the mice over collection plte, nd were nlyzed for urine osmolrity (Advnce Micro Osmometer), urinry neutrophil geltinse-ssocited lipoclin (mouse neutrophil geltinse-ssocited lipoclin ELISA Kit, Immunology Consultnts Lortory, Portlnd, OR), nd urinry cretinine (y high-performnce liquid chromtogrphy tndem mss spectrometry method). 17 Renl histology One kidney ws removed nd tissue sections were fixed in 1% formlin or methyl Crnoy s nd emedded in prffin. Sections of 3 mm thickness were stined with periodic cid Schiff regent. Mcrophge infiltrtion ws ssessed y immunostining with monoclonl ntimouse monocyte mcrophge mrker F4/8 diluted 1:5 (Serotec, Oxford, UK). The numer of positive cells for F4/8 ws counted using n Aperio scnner (Aperio Technologies, Vist, CA). The softwre llows color recognition nd positive cells were identified s % positive color sturtion t mgnifiction in linded mnner using t lest 15 fields for ech iopsy smple. Proximl tuulr rush order loss ws ssessed y immunostining for ngiotensin-converting enzyme (ntimurine ngiotensin-converting enzyme ntiody; R&D, Minnepolis, MN) nd digitl imges were quntified t mgnifiction using Imge scope softwre (Aperio Technologies, Vist, CA). Renl firosis ws determined y stining for type III collgen with got nti-type III collgen ntiody diluted 1:1 (Southern Biotech, Birminghm, AL) nd digitl imges t mgnifiction were nlyzed using Imge scopesoftwre.thepercentgeofpositiverewsdeterminedsthe 3,3-diminoenzidine-positive pixel vlues per exmined interest re in ech section. Renl tissue nlysis The second kidney ws removed, the renl cortex ws seprted from the medull, nd the corticl tissues were lysed with lysis uffer nd used for tissue nd western lot nlyses. Western lot for ldose reductse ws performed using rit polyclonl mouse ntiody to ldose reductse (provided y Dr Mrk Petrsh t the University of Colordo Denver, Denver, CO), nd corrected for - ctin. Renl MCP-1 ws mesured on corticl lystes y ELISA (BD Biosciences Phrmingen, Sn Diego, CA) nd corrected for totl protein (Pierce, Rockford, IL). Renl fructose content ws mesured with the EnzyChrom fructose ssy kit (BioAssy Systems, Houston, TX) nd renl soritol ws mesured using the Biovision (Mountin View, CA) enzymtic ssy sed on the conversion of soritol to fructose with the genertion of NADH (color) y recominnt soritol dehydrogense. Renl corticl uric cid ws mesured y modified cronte-phosphotungstte method. 18 Blood pressure Systolic lood pressure ws mesured in conscious mice using til-cuff sphygmomnometer (Visitech BP; Apex, NC,) t week 4 t the end of the dehydrtion period. For ech mouse, five mesurements were recorded nd the men ws otined. Preconditioning ws not necessry s the mice remined docile throughout the procedure. Sttisticl nlysis Sttisticl nlysis ws performed using one-wy nlysis of vrince followed y t-tests with the Bonferroni correction. All vlues presented re expressed s men±s.d. Significnce ws defined s Pp.5. DISCLOSURE RJJ nd memers of his lortory (TI, MAL, CJR, nd CARJ) hve ptent pplictions relted to locking fructose metolism s mens to prevent cute nd chronic kidney disese. RJJ lso hs funding from Amwy nd is on the Scientific Bord of Amwy. RJJ lso hs grnts from Questcor, the NIH, nd the Stte of Colordo. All the other uthors declred no competing interests. ACKNOWLEDGMENTS We thnk Dnone Reserch for providing the funding for this investigtor-initited tril. This study ws supported y generous funds from Dnone Reserch, Pliseu, Frnce. REFERENCES 1. Torres C, Argon A, Gonzlez M et l. Decresed kidney function of unknown cuse in Nicrgu: community-sed survey. Am J Kidney Dis 1; 55: O Donnell JK, Toey M, Weiner DE et l. Prevlence of nd risk fctors for chronic kidney disese in rurl Nicrgu. Nephrol Dil Trnsplnt 11; 6: Perz S, Wesseling C, Argon A et l. Decresed kidney function mong griculturl workers in El Slvdor. Am J Kidney Dis 1; 59: Bouy N, Fernndes S. Mild dehydrtion, vsopressin nd the kidney: niml nd humn studies. Eur J Clin Nutr 3; 57(Suppl ): S39 S Bolignno D, Zoccli C. Vsopressin eyond wter: implictions for renl diseses. Curr Opin Nephrol Hypertens 1; 19: Bnkir L, Bouy N, Ritz E. Vsopressin: novel trget for the prevention nd retrdtion of kidney disese? Nt Rev Nephrol 13; 9: Bouy N, Bchmnn S, Bichet D et l. 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To view copy of this license, visit cretivecommons.org/licenses/y-nc-nd/3./ 3 Kidney Interntionl (14) 86, 94 3