The parathyroid hormone-related protein system and diabetic nephropathy outcome in streptozotocin-induced diabetes

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1 & 26 Interntionl Society of Nephrology originl rticle The prthyroid hormone-relted protein system nd dietic nephropthy outcome in streptozotocin-induced dietes A Izquierdo 1,PLópez-Lun 1, A Orteg 2, M Romero 1, MA Gutiérrez-Trrés 1, I Arris 3, MJR Álvrez 3, P Esrit 2 nd RJ Bosch 1 1 Lortory of Renl Physiology nd Experimentl Nephrology, Deprtment of Physiology, University of Alclá, Alclá de Henres, Spin; 2 Bone nd Minerl Metolism Lortory, Fundción Jiménez Díz (Cpio Group), Mdrid, Spin nd 3 Deprtment of Biochemistry, Hospitl Principe de Asturis, Alclá de Henres, Spin The pthophysiology of the dietic kidney (e.g., hypertrophy, incresed urinry lumin excretion (UAE)) is still ill-defined. Prthyroid hormone-relted protein () is overexpressed in severl nephropthies, ut its role remins uncler. We evluted the effect of high glucose on nd the PTH1 receptor () protein (y Western lot nd immunohistochemistry) in the kidney of mice with streptozotocin-induced dietes, nd in severl mouse renl cells in vitro. Dietic mice showed significntly incresed renl expression of nd proteins within 2 8 weeks from the onset of dietes. These nimls exhiited n intense immunostining for oth proteins in the renl tuules nd glomeruli. Using trnsgenic mice overexpressing trgeted to the renl proximl tuule, we found significnt increse in the renl hypertrophy index nd in UAE in these dietic mice reltive to their control littermtes. Moreover, logistic regression nlysis showed significnt ssocition etween oth nd protein levels nd UAE in ll dietic mice throughout the study. High-glucose (25 mm) medium ws found to increse nd in tuuloepithelil cells, mesngil cells, nd podocytes in vitro. Moreover, this increse in (ut not tht of ) ws inhiited y the AT 1 receptor ntgonist losrtn. Collectively, these results indicte tht the renl / system is upregulted in streptozotozin-induced dietes in mice, nd ppers to dversely ffect the outcome of dietic renl disese. Our findings lso suggest tht ngiotensin II might hve role in the upregultion in this condition. Kidney Interntionl (26) 69, doi:1.138/sj.ki.5195; pulished online 3 My 26 KEYWORDS: dietic nephropthy; dietes mellitus; prthyroid hormonerelted protein; PTH1 receptor; proteinuri Correspondence: RJ Bosch, Deprtmento de Fisiologí, Fcultd de Medicin, Cmpus Universitrio, Universidd de Alclá, Alclá de Henres, Spin. E-mil: ricrdoj.osch@uh.es Received 28 June 24; revised 14 Septemer 25; ccepted 31 Octoer 25; pulished online 3 My 26 In dietes mellitus, nephropthy the most common single cuse of end-stge renl disese in developed countries occurs relted to poor glycemic lnce. 1 Hypertrophy of mesngil nd proximl tuulr cells is n erly hllmrk of dietes renl involvement. 2 4 In fct, ecuse the tuulointerstitium comprises the ulk of the kidney, proximl tuulr hypertrophy is quntittively responsile for the lrgest component of renl enlrgement in this condition. 2,3 Recent functionl studies in the streptozotocin (STZ)-dietic model hve shown tht, in erly dietes, kidney growth cuses primry increse in proximl tuule resorption, which is responsile for dietic renl hyperfiltrtion. 5,6 This hyperfiltrtion stte my contriute to proteinuri, structurl renl lesions, nd the progression to chronic renl filure. 2,4 As mtter of fct, severl oservtions indicte tht the development of dietic glomerulosclerosis nd tuulointerstitil firosis is lwys preceded y renl hypertrophy. 2 Interctions mong severl cytokines nd hemodynmic gents re thought to e responsile for the renl chnges occurring in dietes. 2,7,8 However, the true moleculr mechnisms of dietic nephropthy remin to e fully chrcterized. In the dult kidney, oth prthyroid hormone-relted protein () nd the PTH1 receptor () re undnt throughout the renl prenchym, including the intrrenl vsculture In the kidney, ppers to modulte renl plsm flow nd glomerulr filtrtion rte, nd induces prolifertive effects on oth glomerulr mesngil nd tuuloepithelil cells Renl is overexpressed in severl experimentl nephropthies, including rt model of tuulointerstitil scrring fter protein overlod, ssocited with the development of proteinuri. 12,19 Although upregultion ppers to e common event ssocited with renl injury nd repir, its pthophysiologicl significnce remins to e clrified. 12 Moreover, no study hs yet explored the puttive involvement of in the pthophysiology of the dietic kidney. In the present study, we initilly ssessed the expression nd locliztion of nd in the kidney of Kidney Interntionl (26) 69,

2 o r i g i n l r t i c l e A Izquierdo et l.: nd dietic nephropthy outcome STZ-induced dietic mice. We then explored the functionl consequences of chronic overexpression in the dietic kidney using trnsgenic mouse strins overexpressing in the renl proximl tuule. 2 Furthermore, the effect of high-glucose medium on the expression of nd ws nlyzed in three mouse kidney cell lines: tuuloepithelil MCT cells, mesngil cells, nd conditionlly immortlized podocytes. RESULTS Renl functionl prmeters in dietic mice Dietic mice exhiited hyperglycemi nd developed renl hypertrophy nd incresed urinry lumin excretion (UAE), consistent with previous findings using similr time schedule in this model 4,8,21,22 (Figure 1 nd ). In these mice, totl plsm protein levels showed tendency to decrese (Figure 1c). Expression nd locliztion of nd in the dietic mouse kidney A single nd corresponding to n pprent moleculr weight of 18 kd ws detected in the mouse kidney y ntiserum C6, consistent with our recent findings 2,23,24 (Figure 2, left). A similr nd ws oserved in W256 tumor extrcts using this C-terminl ntiserum, in greement with previous results using n N-terminl ntiody. 25 This moleculr weight is similr to tht reported for the predominnt form isolted from other humn nd niml tumors overexpressing this protein, nd it is likely to correspond to the single full-length isoform descried in the mouse. 9 Moreover, significnt increse in this 18-kD nd ws oserved in the mouse kidney 2 weeks fter dietes induction (Figure 3). Using immunolotting mg/g c g/dl ,,, Time of dietes (weeks), mg/dy d mg/dl Time of dietes (weeks) Time of dietes (weeks) 2 1,, Time of dietes (weeks) Figure 1 Renl functionl prmeters in experimentl dietes. Index of renl hypertrophy (), UAE (), totl plsm proteins (c), nd plsm cretinine (d) were evluted t different time periods fter dietes development in -overexpressing mice (&) nd their norml littermtes ( ). Po.5, dietic vs nondietic (t time ) mice overexpressing ; Po.5, dietic vs nondietic norml littermtes; Po.5, dietic -overexpressing mice vs dietic littermtes., with ntiserum C6, n increse in single protein nd with similr pprent moleculr weight ws lso found in rt model of cute renl filure. 24 In the mouse kidney, ws detected with the ntiody A-VII s mjor 9-kD nd, consistent with our findings in the rt kidney 23,24 (Figure 2, right). This pprent moleculr weight is consistent with tht reported for the glycosylted receptor isolted from vrious tissues. 29 This ntiody lso reveled minor nd corresponding to 66 kd in mouse kidney extrcts (Figure 2, right), which might e the ntive nonglycosylted form of. 29 As preliminry experiments showed tht this minor nd chnged in ccordnce with the mjor nd, only the results of densitometric nlysis of the ltter re shown elow. Two weeks fter dietes induction, significnt increse in the protein expression ws found in the dietic mouse kidney (Figure 4). Renl levels of nd the protein peked within 4 6 weeks of dietes, decresing (18 kd) Norml WT Norml WT Norml (9 kd) Figure 2 Protein expression of nd in the kidney of -overexpressing mice nd their norml littermtes. Representtive utordiogrms corresponding to Western lot nlysis of oth (using ntiserum C6) (left) nd (using ntiody A-VII) (right) in the kidney of norml nd overexpressing trnsgenic mice () in sl conditions. A minor 66-kD nd ws detected with the ltter ntiody. Wlker 256 (W256) tumor protein extrcts (WT) were used s positive control for. Antiserum C6 ws replced y preimmune serum, s negtive control for (middle pnel). N Time of dietes (weeks) AU Time of dietes (weeks) 2 4 Time of dietes (weeks) Norml 6 8 Figure 3 Renl protein expression in experimentl dietes. Totl protein ws isolted from the mouse kidney t different time periods fter the development of dietes. Autordiogrms nd densitometric nlysis corresponding to chnges in protein y Western lot (using ntiserum C6) in overexpressing mice (&) nd their norml littermtes ( ) re shown. protein levels were corrected to those of ctin. AU: ritrry units. Po.5; Po.1, dietic vs the corresponding nondietic controls. Renl protein levels in -overexpressing mice were significntly (Po.5) higher thn in their norml (nondietic) littermtes nd unchnged y dietes for the time period studied Kidney Interntionl (26) 69,

3 A Izquierdo et l.: nd dietic nephropthy outcome o r i g i n l r t i c l e therefter, ut remined over control vlues throughout the study. Moreover, levels were two-fold higher in the renl cortex thn in renl medull in dietic mice (Po.5; n ¼ 6), ut were similr in oth cortex nd medull in nondietic mice (not shown). In the kidney of nondietic nimls, immunostining (using ntiserum C6) ws present in the tuulr epithelium, wheres stining ws minly oserved in the solterl memrne of tuulr cells (Figure 5 nd d). No positivity for either or ws detected within the glomerulus in these mice. By contrst, 6 weeks fter dietes induction, the mouse kidney showing norml morphology displyed n intense stining in most glomeruli for oth (1771/2 glomeruli, n ¼ 5) nd (1571/2 glomeruli, n ¼ 5) (Figure 5c nd e). In ddition, proximl nd distl tuules (identified y morphologicl criteri) of dietic mice showed remrkle nucler immunolocliztion of not oserved in the nondietic nimls (Figure 5 nd c). Moreover, y using doule stining, n intense immunofluorescence ws found in the tuulr cell nucleus in the mouse dietic, ut not nondietic, kidney with either ntiserum used, C6 (not shown) or C13 (Figure 6). No nucler stining ws oserved for in these mice (Figure 6). Outcome of dietic nephropthy in -overexpressing mice To explore the functionl consequences of upregultion in the dietic mouse kidney, we used trnsgenic mice overexpressing in the renl proximl tuule. 2 In these nimls, the high renl protein levels were unchnged y dietes (Figure 3). However, dietesrelted increse in renl protein occurred in these mice, lthough it ws lower thn tht in their dietic littermtes (Figure 3). Dietes in -overexpressing mice ws lso ssocited with positive stining for oth nd in the glomerulus, nd for in the tuulr cell nucleus (not shown). After dietes induction, -overexpressing mice progressively developed significnt increse in the renl hypertrophy index nd UAE, nd hd lower totl plsm protein vlues, compred to their norml littermtes (Figure 1 c). No chnges in plsm cretinine occurred in dietic mice throughout the study (Figure 1d). Using logistic regression nlysis, renl protein levels were highly ssocited with UAE vlues in ll dietic mice included in the study (Po.1; n ¼ 49). Similrly, significnt ssocition (Po.1) ws found etween the renl levels nd UAE in these mice (Tle 1). The effect of high glucose on nd the protein in mouse kidney cells We next exmined the effect of high glucose on the expression of nd in severl renl cell lines in vitro. Similr to findings in whole mouse kidney extrcts, single nd corresponding to 18 kd ws detected y ntiserum C6 in the three cell lines studied (Figure 7, left). In ddition, single 66-kD nd ws oserved with the ntiody A-IV in these cell lines (Figure 7, right). Differences in the specificity etween this ntiody nd ntiody A-VII might ccount for their reltive efficiency in detecting either the 66-kD or the 9-kD nd s oserved herein in vitro nd in vivo, respectively. 2,23,24 c N Time of dietes (weeks) Time of dietes (weeks) AU 3,, Norml d e Time of dietes (weeks) Figure 4 Renl protein expression in experimentl dietes. Totl protein ws isolted from the mouse kidney t different time periods fter the development of dietes. Autordiogrms nd densitometric nlysis corresponding to chnges in the protein y Western lot (using ntiody A-VII) in -overexpressing mice (&) nd their norml littermtes ( ) re shown. The protein levels were corrected to those of ctin. AU: ritrry units. Po.5; Po.1, dietic vs the corresponding nondietic controls; Po.5, dietic -overexpressing mice vs dietic littermtes. Figure 5 Immunostining for nd in the kidney of dietic mice t 6 weeks of dietes. Kidney sections of representtive nondietic (, d) nd dietic (c, e) nimls, showing immunolocliztion of (y using ntiserum C6) (, c) or (y using ntiody A-VII) (d, e) in the glomerulus of dietic mice re shown. () Negtive control without primry ntiody. Originl mgnifiction 25. Kidney Interntionl (26) 69,

4 o r i g i n l r t i c l e A Izquierdo et l.: nd dietic nephropthy outcome c d e f g h i Tle 1 Assocition etween proteinuri nd the / system, nd other iologicl prmeters in dietic mice Vrile OR CI (95%). Univrite nlysis Diuresis Cretinine Weight Cholesterol Glucose Multivrite logistic regression nlysis Diuresis Constnt.6 CI, confidence intervl;, prthyroid hormone 1 receptor;, prthyroid hormone-relted protein; OR, odds rtio. j k l P MMC MCT WT P MMC MCT (18 kd) (66 kd) Figure 6, ut not, loclizes into the nucleus of the renl tuules in the mouse kidney t 6 weeks of dietes. Kidney sections of representtive nondietic (, g) nd dietic (d, j) mice showing immunostining for either (using ntiserum C13) (, d) or (using ntiody A-IV) (g, j) re shown. Immunolocliztion of oth nd ws ssessed y doule stining, for the nucleus (with propidium iodide; red) (, e, h, k), nd fluorescein isothiocynte-leled IgG (green) for either (, d) or (g, j) detection. The overlid imges in red nd green yielding n ornge tone confirmed the presence of intense fluorescence in the nucleus of tuulr cells in the mouse dietic kidney (f), ut not in the nondietic mice (c). Similr results were otined with ntiserum C6 (dt not shown). By using this mneuver, no nucler stining ws oserved for in either nondietic (g, i) or dietic mice (j, l). High glucose induced n upregultion of nd protein t 24 h in MCT cells (Figure 8) nd in oth glomerulr cell lines (Figures 9 nd 1). Losrtn olished this protein overexpression only in MCT cells nd podocytes (Figures 8 1), wheres it filed to ffect the protein upregultion y high glucose in ll cells studied (Figures 8 1). DISCUSSION Renl nd ngiotensin-converting enzyme genes re upregulted in severl experimentl nephropthies. 12,16,18,19,23,24,3 Recently, we found tht AT 1 receptor lockde in rts with cute renl filure ws ssocited with improved tuulr dmge nd renl function relted to lck of upregultion. 23,24 In renl tuuloepithelil cells, Figure 7 Western lot nlysis of nd in mouse renl cell lines. Representtive utordiogrms corresponding to Western lot nlysis of oth (using ntiserum C6) (left) nd (using ntiody A-IV) (right) in mouse kidney cell lines in sl conditions: podocytes (P), mesngil cells (MMC), nd corticl tuule cells (MCT). Wlker 256 (W256) tumor protein extrcts (WT) were used s positive control for. 25 2, AU Mnnitol + Losrtn AU Mnnitol + Losrtn + Figure 8 Induction of nd the protein expression y high glucose in MCT cells. Cells were cultured with or without high-glucose (25 mm) medium (or the sme concentrtion of mnnitol), in the presence or sence of 1 mm losrtn, for 24 h. Autordiogrms nd densitometric nlysis corresponding to chnges in () nd the () protein levels y Western lot (using ntiserum C6 nd ntiody A-IV, respectively) re shown. nd the protein levels were corrected to those of ctin (n ¼ 4). HG: high glucose; AU: ritrry units. Po.5, compred to norml glucose medium; Po.5, compred to HG medium þ losrtn Kidney Interntionl (26) 69,

5 A Izquierdo et l.: nd dietic nephropthy outcome o r i g i n l r t i c l e AU Mnnitol + Losrtn + ngiotensin II (Ang II) interction with AT 1 receptors ctivtes mitogen-ctivted protein kinse nd the trnscription fctor camp-responsive element inding protein, leding to incresed expression. 24 Thus, ppers to exert reciprocl control on, or recpitulte, some Ang II effects in the dmged kidney. We presently used the STZ model, which is considered s the work horse for experimentl studies in dietic nephropthy, 31,32 condition ssocited with ngiotensinconverting enzyme nd mitogen-ctivted protein kinse ctivtion. 2,6 Consistent with previous findings in this model, 4,8,21,22 dietic mice developed renl hypertrophy AU Mnnitol + Losrtn + Figure 9 Induction of nd the expression y high glucose in mouse mesngil cells. Cells were cultured with or without high-glucose (25 mm) medium (or the sme concentrtion of mnnitol), in the presence or sence of 1 mm losrtn, for 24 h. Autordiogrms nd densitometric nlysis corresponding to chnges in () nd the () protein levels y Western lot (using ntiserum C6 nd ntiody A-IV, respectively) re shown. nd the protein levels were corrected to those of ctin (n ¼ 4). HG: high glucose; AU: ritrry units. Po.5, compred to norml-glucose medium. AU 3 2 1, Mnnitol + Losrtn + AU Mnnitol + Losrtn + Figure 1 Induction of nd the expression y high glucose (25 mm) in mouse podocyte cells. Cells were cultured with or without high-glucose (25 mm) medium (or the sme concentrtion of mnnitol), in the presence or sence of 1 mm losrtn, for 24 h. Autordiogrms nd densitometric nlysis corresponding to chnges in () nd the () protein levels y Western lot (using ntiserum C6 nd ntiody A-IV, respectively) re shown. nd the protein levels were corrected to those of ctin (n ¼ 4). HG: high glucose; AU: ritrry units. Po.5, compred to norml-glucose medium; Po.5, compred to HG medium þ losrtn. nd n increse in UAE throughout this study. We found tht oth nd the protein were upregulted in the dietic mouse kidney. However, this increse in ws prtly lunted in -overexpressing mice, suggesting tht homologous downregultion of this receptor, s oserved in these mice fter cute renl filure, 2 my counterct in prt the upregultion in the dietic kidney. An intense immunostining for oth nd within the glomerulr cpillry tuft ws found in the mouse kidney ut only in dietic mice. The presence of hs een previously disclosed in the glomerulus in frozen nd prffin-emedded renl sections in rts nd rits. 1,16,19 Glomerulr mrna hs lso een found y in situ hyridiztion in frozen sections from norml rt glomeruli. 33 Thus, the immunohistochemistry method used might require high protein expression of nd s tht found in dietic nimls to e detected. We found tht dietic mice, in contrst to nondietic nimls, hd remrkle nucler stining for in the renl tuules. The mechnism of internliztion into the nucleus of tuulr cells is currently unknown. The present findings, however, mke it unlikely tht such mechnism involves, ut rther the nucler locliztion signl domin in the region of the molecule. 34,35 The ntiser used in our immunolocliztion studies, recognizing two different C- nd N-terminl epitopes, seem to e similrly efficient in detecting fulllength (contining nucler locliztion signl domin) in the nucleus. Consistent with the / system upregultion in experimentl dietic nephropthy, high glucose ws found to stimulte this system in glomerulr nd tuuloepithelil cells in vitro. Collectively, our present findings indicte tht hyperglycemi should e dded to the incresing list of renl pthophysiologicl conditions ssocited with n ltered / system. These results lso suggest tht could ct in n utocrine/prcrine s well s n intrcrine fshion in the dietic mouse kidney. In the present study, Ang II ctivtion, which is known to occur in the dietic kidney, 2,7 might hve triggered the renl / system, s oserved in cute renl filure. 24 In fct, losrtn inhiited the high-glucose induction of in oth tuuloepithelil MCT nd mouse glomerulr cells. However, our findings lso suggest tht n AT 1 - independent mechnism might e responsile for the upregultion induced y high glucose in these cells. In this regrd, Ang II infusion into norml rts incresed nd the immunostining in the renl prenchym, ut this vsoctive gent filed to ffect the protein in rt mesngil nd MCT cells in vitro. 23 Thus, definite conclusions out the puttive role of Ang II or other unidentified hyperglycemi-relted fctors in the expression in the mouse dietic kidney cnnot e mde t present. Our findings demonstrte tht dietic -overexpressing mice, in comprison to their control littermtes, hve incresed renl hypertrophy. Although the mechnisms Kidney Interntionl (26) 69,

6 o r i g i n l r t i c l e A Izquierdo et l.: nd dietic nephropthy outcome of dietic renl hypertrophy re still ill-defined, Ang II nd trnsforming growth fctor- hve importnt roles in this condition. 2 Trnsforming growth fctor- is upregulted y high glucose nd cn trnsform mitogenic stimulus into hypertrophy in tuuloepithelil cells Of note, is mitogen for proximl tuulr cells, nd hs shown to ct s downstrem effector of trnsforming growth fctor- t lest in some cell systems. 17,4 42 As recently suggested y Clemens et l., 9 lso ppers to e n importnt regultory fctor of glomerulr hemodynmics, nd might prticipte in the pthophysiology of glomerulr hyperfiltrtion. Collectively, these dt strongly suggest tht could e involved in the mechnisms of renl hypertrophy in dietes. We lso found tht dietic -overexpressing mice hve higher UAE nd lower totl plsm protein levels thn their dietic control littermtes. Furthermore, there ws six-fold increse in the risk of developing proteinuri in those mice with the higher nd levels, ccording to the logistic regression nlysis. is potent vsorelxnt nd cn interct with vsoctive fctors ffecting glomerulr hemodynmics nd permeility. 9,1,13,14 These effects seem to occur prtly through interction with nd susequent ctivtion of the nitric oxide/cyclic gunosine monophosphte pthwy. 1 In ddition, hs proinflmmtory/profirogenic fetures in some cells Interestingly, n increse in nitric oxide nd vrious proinflmmtory cytokines pper to ply criticl roles in the dietic kidney. 1,46,47 It is thus tempting to speculte tht ny of these mechnisms would contriute to the reltionship etween the / system nd proteinuri in this dietic model. In ny cse, our dt indicte tht chnges in the renl / system hve predictive vlue on the erly development of proteinuri in mice. Although the STZ model hs limittions for ssessing long-term histomorphologicl chnges in the dietic kidney, 31,32 our findings might hve pthophysiologicl implictions, s the mount of proteinuri is relile predictor of dietic nephropthy. 48,49 In conclusion, the present results demonstrte tht the renl / system is upregulted during STZinduced dietes in mice, nd this upregultion ppers to ffect dversely the outcome of dietic renl disese. Our findings lso suggest tht Ang II might hve role in upregultion in this setting. MATERIALS AND METHODS Experimentl procedure We used two types of mice, norml CD-1 mice nd overexpressing mice, generously supplied y Professor AF Stewrt nd Dr A Grcí-Ocñ (University of Pittsurg School of Medicine, Pittsurg, PA, USA). 2 The renl specificity of the trnsgene ws conferred y the g-glutmyl trnspeptidse-l promoter, minly expressed in the renl proximl tuule. -overexpressing mice were generted y reeding two types of trnsgenic mice: one contining g-glutmyl trnspeptidse-l promoter frgment upstrem of tetrcycline trnsctivtor fusion protein which functions s strong trnscription ctivtor; nd the other with complementry DNA plced under the control of tetrcycline opertor construct. Trnsgene-ering founders were continuously outred to norml CD-1 mice to generte hemizygotes. Genotyping of these mice ws performed y til DNA PCR, s descried. 2 In ll of the experiments descried elow, dult mice (4 8 months old) were used, nd the results otined with overexpressing trnsgenic mice were compred with those using their corresponding norml littermtes. 2,5 All studies were performed with the pprovl of nd in ccordnce with guidelines estlished y Institutionl Animl Cre nd Use Committees t the University of Alclá nd Fundción Jiménez Díz. Mice were housed in temperture-controlled room (21721C) on 14/1 h light/drk cycle under pthogen-free conditions nd with free ccess to food nd wter. Dietes ws induced y three consecutive dily intrperitonel injections of STZ (Sigm, St Louis, MO, USA), 65 mg/kg ody weight in citrte uffer, ph 4.5 (vehicle). This is previously reported model of erly dietic nephropthy chrcterized y renl hypertrophy nd incresed UAE during the first month of dietes. 21,22 After the lst STZ injection, induction of dietes ws confirmed y mesurement of lood glucose levels. Forty-nine dietic nimls (25 -overexpressing mice nd 24 norml littermtes), with lood glucose 43 mg/dl, were included in the study. Animls were killed under ether nesthesi 2, 4, 6, nd 8 weeks following the development of dietes (n ¼ 6 7, t ech time period). Another group (n ¼ 6 1) of weight-mtched overexpressing mice or their corresponding norml littermtes received the sme volume of vehicle nd were used s nondietic controls. Animls were individully housed in metolic cges with free ccess to tp wter, nd 24-h urine ws collected for protein mesurement y the sulfoslicylic cid method, s previously reported. 51 Blood ws tken y crdic puncture under ether nesthesi, nd plsm glucose ws determined y the glucose oxidse technique. 52 One kidney of ech niml ws removed, weighed, frozen in liquid nitrogen, nd stored t 81C for susequent totl protein extrction. In some cses, the kidneys were dissected, the cpsule removed, nd the cortex seprted from the medull ws frozen s descried. The remining kidney of ech niml ws fixed in 4% uffered p-formldehyde for morphologicl nd immunohistochemistry studies. The degree of renl hypertrophy ws expressed s n index, the rtio of kidney weight to totl ody weight. Histology nd immunohistochemistry Fixed renl tissue sections were dehydrted y grded ethnols nd xylene, nd then emedded in prffin. Prffin-emedded tissue sections (4 mm) were stined with hemtoxylin. immunostining ws performed using rit polyclonl nti- ntiserum C6, recognizing the highly conserved C-terminl epitope in the intct molecule, 17,53 s descried. 23,24 For the stining, we tested two well-chrcterized ffinity-purified polyclonl ntiodies to this receptor: one recognizing its extrcellulr domin (A-VII; Covnce, Berkeley, CA, USA), which hs een extensively used in previous studies; 18 2,23 nd ntiody A-IV (Covnce), directed ginst the region in the molecule, contriuting to receptor ctivtion. 2,54 We found tht oth ntiodies hd similr sensitivity to detect this receptor in the mouse kidney, y immunohistochemistry. Some kidney smples were incuted without primry ntiody or with nonimmune rit serum s negtive controls Kidney Interntionl (26) 69,

7 A Izquierdo et l.: nd dietic nephropthy outcome o r i g i n l r t i c l e The numer of glomeruli showing cpillry stining for oth nd per 2 glomeruli without selection ws counted for ech mouse. 18 Histologicl evlutions were performed y two independent oservers in linded fshion. The finl score ws the men of the two evlutions. To ssess puttive chnges in the locliztion pttern of nd in the dietic mouse kidney, renl tissue smples from dietic nd nondietic mice were permeilized with.1% Triton X-1 in phosphte-uffered sline for 3 min t 371C. After locking with 1.5% norml got serum, the smples were incuted overnight t 41C with the following primry ntiodies: ntiserum C6 nd ntiserum C13 recognizing n N-terminl epitope in the intct molecule nd lso the (1 36) frgment, 53 ech t 1:1 dilution; nd the ntiody A-IV, t 1:25 dilution. Then, fluorescein isothiocynte-conjugted got nti-rit IgG (Sigm), t 1:2 dilution, ws dded for 3 min. After wshing, smples were counterstined with propidium iodide to detect cell nuclei, nd mounted with Mowiol (Cliochem, Sn Diego, CA, USA). Immunofluorescence nlysis ws performed with Leic DM-IRB confocl microscope. 24 Cell cultures We used three well-estlished mouse kidney cell lines corticl tuule MCT cells, mesngil cells, nd podocytes to evlute the effect of high glucose on the / system in vitro. MCT cells were grown in RPMI 164 with 1% fetl ovine serum nd ntiiotics in 5% CO 2 t 371C, s descried. 23 Mouse mesngil cells (ATCC CRL-1927) were cultured in Dulecco s modified Egle s medium nd Hm s F12 medium (3:1, v/v) with 5% fetl ovine serum. 55 Conditionlly immortlized mouse podocytes ( generous gift from Dr Hermn Pvenstädt, University Hospitl of Freiurg, Freiurg, Germny) were cultured s reported. 56,57 Podocytes were cultured on type I collgen (Sigm) nd grown in RPMI 164 medium with 5% fetl ovine serum nd ntiiotics, supplemented with 1 U/ml recominnt interferon-g to enhnce T-ntigen expression, t 331C (permissive conditions). To induce differentition, podocytes were mintined on type I collgen t 371C without interferon-g (nonpermissive conditions). Cells etween pssges 1 nd 16 were used in ll experiments. MCT nd mesngil cells were serum-depleted, nd podocytes were switched to 1% fetl ovine serum-contining medium for 24 h efore experiments. The cells were then treted with 25 mm glucose (or mnnitol) for 24 h, 58,59 in the presence or sence of the AT 1 receptor locker losrtn (MSD, Mdrid, Spin). 23,24 Western lot nlysis of nd Proteins were isolted from either mouse kidney homogentes or cultured cells, using stndrd procedure (TriRegent; Moleculr Reserch Center, Cincinnti, OH, USA). Fifteen microgrms of protein from totl kidney (or kidney cortex) or cultured renl cells, estimted y the Brdford s method, 6 were seprted on 1 15% sodium dodecyl sulfte-polycrylmide gels nd trnsferred to polyvinylidene difluoride memrnes (Bio-Rd, Hercules, CA, USA). The memrnes were locked using 1 mm Tris, 15 mm NCl, ph 7.5, nd.1% Tween 2 (TTBS) with 5% ovine serum lumin () or 5% deftted milk (). They were then incuted overnight t 41C with ntiserum C6, t 1/2 dilution, s reported, 2,23,24 nd the nti- ntiodies A-VII or A-IV, t 1/2 nd 1/1 dilution, respectively, in TTBS. In the kidney, A-VII ntiody detects minly 9-kD nd, 23,24 wheres single 66-kD nd ws reveled with the A-IV ntiody, 2 in greement with the mnufcturer s informtion (wesite: www. crpinc.com). Wlker 256 (W256) tumor protein extrcts were used s positive control for. 25 An nti-ctin ntiody (Sigm), t 1:5 dilution, ws used s loding control. Following incution with iotinylted nti-rit IgG (or peroxidseconjugted nti-rit IgG), nds were detected with 3,3 -diminoenzidine (DAKO, Glostrup, Denmrk) or ECL chemiluminescence (Amershm, Buckinghmshire, UK). Blots were nlyzed y densitometric scnning. Densitometric vlues were normlized ginst those of ctin. Sttisticl nlysis All results re expressed s men7s.e.m. Sttisticl significnce (Po.5) ws ssessed y Kruskl Wllis test or Mnn Whitney test, when pproprite. To ssess the ssocition etween nd the protein levels (y Western nlysis) nd UAE, quntittive vlues corresponding to ll dietic nimls included throughout the study (n ¼ 49) were converted to qulittive prmeters y grouping the upper over the medin nd the lower under the medin vlues. Then, univrite nd multivrite logistic regression nlysis ws performed. The SPSS 9. sttisticl pckge (SPSS Inc., Chicgo, IL, USA) ws used. ACKNOWLEDGMENTS We thnk Yolnd Fernández for proofreding the mnuscript. A Izquierdo is fellow of the Spnish Ministerio de Educción y Cultur. A Orteg is fellow of Conchit Rágo Foundtion. This work ws supported y grnts from MEC (SAF C3-1 nd -2), CAM (8.6/38.1/2-1 nd -2), the Spnish Society of Nephrology, nd Instituto de Slud Crlos III (C3/8). REFERENCES 1. Ritz E, Rychlick I, Loctelli F, Hlimi S. End-stge renl filure in type 2 dietes: medicl ctstrophe of worldwide dimensions. Am J Kidney Dis 1999; 34: Wolf G, Ziydeh FN. Moleculr mechnisms of dietic renl hypertrophy. 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