Nephrol Dial Transplant (2006) 21: 1514–1524

doi:10.1093/ndt/gfk089

Advance Access publication 31 January 2006

Original Article

Role of TGF-b/GLUT1 axis in susceptibility vs resistance

to diabetic glomerulopathy in the Milan rat model

Carlo Ricci1, Carla Iacobini1,2, Giovanna Oddi1,2, Lorena Amadio1,2,Stefano Menini1, Maria Pia Rastaldi3, Aurora Frasheri1, Flavia Pricci2,Francesco Pugliese1 and Giuseppe Pugliese1

1Department of Clinical Sciences, ‘La Sapienza’ University, Rome, 2Department of Cell Biology andNeurosciences, Istituto Superiore di Sanita, Rome and 3Laboratory of Renal Immunopathology,San Carlo Borromeo Hospital, Milan, Italy

Abstract

Background. GLUT1 upregulation and increased glu-cose transport activity may contribute to extracellullarmatrix (ECM) accumulation characterizing diabeticnephropathy (DN). Rats of the Milan hypertensivestrain (MHS) are resistant to both hypertensive anddiabetic renal disease, due to a haemodynamic protec-tion. On the contrary, those of the Milan normotensivestrain (MNS) develop spontaneous glomerulosclerosis,and when rendered diabetic, show typical morpholo-gical and haemodynamic changes.Methods. To assess whether susceptibility to diabeticglomerulopathy in MNS rats is associated with higherglucose transporter 1 (GLUT1) expression (andglucose transport activity) vs MHS rats, diabetic andnondiabetic MNS and MHS rats were followed for 6months and mesangial cells derived from these animalswere exposed to high glucose (HG) vs normal glucose(NG) conditions.Results. Glomerular expression of GLUT1 proteinand ECM and transforming growth factor-b (TGF-b)mRNA was significantly upregulated in diabetic vsnondiabetic MNS, but not MHS rats. Upon exposureto HG and/or TGF-b, mesangial cells from 1- and 8-month-old MNS rats showed higher glucose transportactivity and GLUT1 membrane expression than thosefrom age-matchedMHS rats. Likewise, ECM and TGF-bproduction increased more markedly in response toHG and/or TGF-b in MNS vs MHS mesangial cells.Conclusions. These data indicate that susceptibility todiabetic glomerulopathy in MNS rats is associatedwith increased GLUT1-dependent glucose transportactivity in response to hyperglycaemia and/or TGF-b,

which may amplify ECM overproduction. Conversely,the haemodynamic protection from glomerulosclerosisin MHS rats is associated with lack of upregulationof TGF-b/GLUT1 axis, thus supporting the conceptthat this axis may represent the link between haemo-dynamic and metabolic mechanisms of injury.

Keywords: diabetic nephropathy; extracellularmatrix; glucose transport; GLUT1;renal haemodynamics; TGF-b

Introduction

The injurious effects of hyperglycaemia are character-istically observed in tissues which are not dependenton insulin for glucose entry into the cell and, hence,are not capable of down-regulating glucose transportalong with elevation of extracellular sugar levels [1].Indeed, glucose uptake was shown to increase incultured mesangial cells upon exposure to high glucose(HG) containing media, via an upregulation of glucosetransporter 1 (GLUT1) expression [2]. Moreover,treatment of mesangial cells with antisense GLUT1blocked HG-induced GLUT1 and fibronectin over-expression [3]. Additionally, transfection of mesangialcells with GLUT1 cDNA mimicked the effects ofhyperglycaemia at normal glucose (NG) concentra-tions, including the upregulation of extracellullarmatrix (ECM) production [4] and the activation ofpolyol pathway and protein kinase C (PKC) a and b1[5,6]. Likewise, GLUT1 was found to be overexpressedin glomeruli from diabetic rats [7] and mice [8].Furthermore, diabetic db/db mice carrying an anti-sense-GLUT1 transgene were protected against thedevelopment of diabetic nephropathy (DN) [8], whereastransgenic overexpression of GLUT1 driven by a

Correspondence and offprint requests to: Giuseppe Pugliese, MD,PhD, Dipartimento di Scienze Cliniche (Endocrinologia), Viale delPoliclinico, 155 - 00161 Rome, Italy.Email: giuseppe.pugliese@uniroma1.it

� The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.org

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modified b-actin promoter in glomeruli of nondiabeticdb/mmice produced features of DN, as indicated by theincreased albuminuria and mesangial expansion [9].

In cultured mesangial cells, the prosclerotic cytokinetransforming growth factor-b (TGF-b), known to playa pivotal role in the pathogenesis of DN [10], wasshown to enhance GLUT1 expression and glucosetransport activity, whereas the addition of neutralizinganti-TGF-b antibody prevented the stimulatory effectof HG on GLUT1 expression [11]. Thus, it waspostulated that the overexpression of TGF-b inducedby hyperglycaemia upregulates GLUT1 that, in turn,increases glucose uptake and, hence, further enhancesthe stimulation of TGF-b production, ultimatelyleading to mesangial ECM accumulation [12,13].

A recent report showed that, in addition to HG,stretching is also capable of activating the TGF-b/GLUT1 axis, which may represent the link betweenhaemodynamic and metabolic mechanisms in glomer-ular injury associated with conditions in which systemichypertension is transmitted to the glomerular micro-circulation [14]. In fact, TGF-b, GLUT1 and ECMwere found to be upregulated in the Dahl salt-sensitive(Dahl-S) rat, characterized by glomerular hypertensionand injury, but not in the young spontaneouslyhypertensive rat (SHR), when glomerular capillarypressure is still normal and renal disease has not yetdeveloped [14]. Glomerular hypertension has beenshown to occur also in diabetes, as a consequence ofthe loss of autoregulation due to afferent arteriolevasodilation [15]. Raised intraglomerular pressure isthought to play a pivotal role in the pathogenesis ofDN, though the relative importance of haemodynamicvs metabolic factors has not been clarified yet [16].

In order to address this issue, we have utilized aunique animal model, the Milan rats, consisting of twogenetically-related rat strains derived from a commonWistar ancestor, the Milan normotensive strain (MNS)and the Milan hypertensive strain (MHS). The MNSrats develop an age-dependent focal and segmentalglomerulosclerosis, whose mechanisms are poorlyunderstood, since classical risk factors cannot beidentified [17,18]. The MHS rats develop a mild tomoderate form of arterial hypertension [19], that hasbeen attributed, at least partly, to a missense mutationof the a and b subunits of the adducin heterodimer,a protein participating in the assembly of spectrin–actincytoskeleton [20]. This mutation is associated withaltered cytoskeleton assembly, increased expression ofNaþ/Kþ ATPase and upregulation of Naþ/Kþ pumpactivity of apical and basolateral membranes ofrenal tubules, with consequent sodium and waterretention and increased blood pressure [21]. In contrastto the MNS, no glomerular disease occurs inMHS rats, despite the elevated blood pressure levels,possibly due to the marked hypertrophy of theintrarenal arteries protecting glomerular capillariestowards systemic hypertension and development ofglomerulosclerosis [17,22].

Induction of diabetes in MNS rats resulted in typicallesions superimposed onto (and distinguished from)

age-dependent glomerulosclerosis and associated withincreased glomerular filtration rate (GFR) and filtra-tion fraction. Diabetes also produced significant,though less marked changes in renal function andstructure in progenitor Wistar rats. On the contrary,diabetic MHS rats did not show the characteristicchanges in renal haemodynamics and remained freeof renal disease, even after 6 months of uncontrolleddiabetes, thus pointing to the importance of haemo-dynamic factors, which exert a permissive role towardshyperglycaemia-induced injury [23]. Conversely,in vitro, i.e. under conditions in which haemodynamicsis not operating, mesangial cells isolated from bothrat strains (before the onset of glomerulosclerosisand hypertension, respectively) responded to HG withan upregulation of TGF-b and ECM production.However, this response of ECM, but not TGF-bproduction was significantly higher in cells from MNSthan in those from MHS rats [24]. This finding andthe previous report of an age-dependent increase inECM production and proliferative response to serumin mesangial cells from MNS, but not MHS rats [25]indicate a genetically determined hyperresponsivenessto sclerosing stimuli in the MNS rats that may underlietheir susceptibility to spontaneous glomerulosclerosis.

This in vivo and in vitro study was aimed at verifyingthe hypothesis that the TGF-b/GLUT1 axis is activatedin the glomerulosclerosis-prone MNS rats but not inthe haemodynamically protected MHS rats, thusexplaining their susceptibility and resistance, respec-tively, to DN and confirming that this axis representsthe link between haemodynamic and metabolicmechanisms of injury.

Materials and methods

Design

In vivo studies. Adult (aged 12 weeks, weighing �280 g)male Milan rats of both strains (kindly provided by PrassisResearch Institute sigma tau, Settimo Milanese, Milan, Italy)were divided into the following groups: time 0 control rats,that were killed immediately after initiating the study, andnondiabetic and diabetic MNS and MHS rats, that weresacrificed 3 and 6 months after diabetes induction (n¼ 6 pergroup). Rats were made diabetic by a single injection (via thecaudal vein) of streptozotocin (Sigma Chemical Co., St Louis,MO), at a dose of 55mg/kg body weight in citrate buffer(pH 4.5) [23]. The animals were housed and cared in keepingwith the EC regulations and received water and food atlibitum; when needed, diabetic animals were given supportiveinsulin treatment (Ultratard, Novo Nordisk, Denmark) toprevent ketosis without affecting significantly glycaemia.Need for treatment was defined depending on body weightmeasurements, as follows: in case of increased body weight,once a week injection of 4 IU/kg body weight; in case ofunchanged body weight, twice a week injections of 4 IU/kgbody weight; in case of decreased body weight, three timesa week injections of 4 IU/kg body weight (or more). The meanweekly insulin dose was similar in the two diabetic groups.Metabolic control was monitored throughout the study by

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measuring blood glucose and body weight at regular intervals;blood pressure was also measured at time 0 and every3 months thereafter. At time 0 (all nondiabetic rats, n¼ 18)and 3 months (3- and 6-month nondiabetic and diabetic rats,n¼ 12) and 6 months (only 6-month nondiabetic and diabeticrats, n¼ 6) after diabetes induction, rats were placed intometabolic cages to collect urines for the assessment of urinevolume and proteinuria. Then, rats were anesthetized with i.p.ketamine (Imalgene�, 60mg/kg body weight) and xylazine(Rompum�, 7.5mg/kg body weight) and a blood sample waswithdrawn for the assessment of glycated haemoglobin (Hb)and serum creatinine. The kidneys were quickly removed,cleaned of the surrounding fat, washed in sterile salinesolution and weighed. A sagittal section of the right kidneywas immediately fixed by immersion in phosphate buffered4% paraformaldehyde solution and routinely embedded inparaffin for morphologic analysis. The remaining tissuewas immerse in OCT and then frozen in isopenthane–liquidnitrogen for measuring GLUT1 protein expression byimmunofluorescence. Renal cortex from the left kidney wasseparated from medulla and used for glomeruli isolationby standard sieving techniques, followed by total RNAextraction for the assessment of ECM and TGF-b1 geneexpression by reverse transcription polymerase chain reaction(RT-PCR).

In vitro studies. Glomerular mesangial cells were isolatedfrom 1- and 8-month-old MNS and MHS rats (i.e. beforeand after the onset of glomerulosclerosis or hypertension,respectively) and characterized as previously described [24].Cells between the third and the tenth passage were culturedfor 10–15 days (over 2–3 passages) in Dulbecco-modifiedEagle’s medium (Sigma, St Louis, MO) supplemented with17% fetal bovine serum, 2mmol/l L-glutamine and antibiotics(all obtained from Flow Laboratories, Irvine, Scotland, UK),but without insulin, at 37�C in 95% air and 5% CO2

humidified atmosphere, under NG (5.5mmol/l) vs HG(30mmol/l) conditions [24]. Then, monolayers were incubatedfor 18 h in serum-free medium containing (a) vehicle or25–200 pmol/l TGF-b (Calbiochem, San Diego, CA)±theinhibitor of protein synthesis cycloheximide (5 mmol/l,Sigma), the blocker of sodium-dependent glucose transportphloridzin (10mmol/l, Sigma) or the inhibitor of GLUT1-dependent glucose transport cytochalasin B (1 mmol/l,Sigma); and (b) a-TGF-b blocking antibody or controlchicken IgG (30 mg/ml, R&D Systems, Minneapolis, MN).ECM and TGF-b1 mRNA levels and protein releaseinto conditioned media, GLUT1 protein expression andglucose transport were measured under these experimentalconditions by RT-PCR ELISA western blot analysis and[3H]2-deoxyglucose ([3H]DOG) incorporation, respectively.All the experiment had a control for osmolarity, i.e. mediacontaining iso-osmolar mannitol concentrations(NGþ 24.5mM mannitol).

Methods

Metabolic and cardiovascular parameters. Body weightswere measured twice a week and served as a guide forsupportive insulin treatment. Urine volume was measuredat 3-month intervals. Blood glucose levels were measuredbiweekly by the use of an automated colorimetric instrument(Glucocard Memory 2�, Menarini Diagnostics, Florence,

Italy) from blood obtained by tail venepuncture. Glycated Hblevels were assessed by boronate affinity gel chromatographyusing the Glyc-Affin GHb kit (PerkinElmer, Norwalk, CT)[23]. Blood pressure was recorded by the tail cuff method.

Renal function. Total proteinuria was measured using theBradford dye-binding protein assay kit (Pierce Chemical,Rockford, IL), whereas serum creatinine was assessed by theJaffe method [23].

Renal structure. Renal morphology was evaluated semi-quantitatively in sections stained with periodic acid Schiff(PAS) [23]. At least 60 glomerular tuft profiles per sample,selected on PAS stained sections by moving from external todeep cortex in a serpentine manner (with exclusion of profilescontaining <3 mesangial tracts), were analyzed. A patholo-gist blinded to the group assignment of the specimens scoredmesangial expansion; in each experimental animal, the meanscore of each rat was derived from the individual scores of allthe glomeruli. The expansion of mesangial matrix was scored(scale 0 to 4) as previously reported [26] and glomerularsclerosis was assessed as percent of involved glomeruli [23];�150 glomerular tuft profiles per animal were evaluated.Tubulo-interstitial damage was evaluated using a semi-quantitative scale (0: absent; 1: <10%; 2: 10–30%; 3:30–70%; 4: >70%) and the extension of cortical lesions ineach animal was expressed as the mean value obtainedmeasuring a whole sagittal section [23].

ECM and TGF-�1 production. Transcripts for the ECMcomponents fibronectin, laminin B1 and collagen IV a1 chainand the pro-sclerotic cytokine TGF-b1 were measured bycompetitive RT-PCR [23]. Total RNA was extracted fromisolated glomeruli and mesangial cells by the guanidinethiocyanate–phenol–chloroform method using Trizol(Invitrogen Italia SRL, San Giuliano Milanese, Italy) andthe purity of RNA preparation was confirmed by anabsorbance 260:280 ratio >1.9, as measured in a BeckmanDU-65 spectrophotometer (Beckman Instruments, Inc.,Fullerton, CA). Then, 1 mg of total RNA was reversetranscribed using Retroscript kit (Ambion, Austin, TX).The following primers were used: fibronectin sense 50-AGCGGT GTG GTC TAC TCT GT-30—antisense 50-GAT GCACTG ATC TCG GAF CT-30; laminin B1 sense 50-TGT CAGTCA CCT GCA GGA TG-30—antisense 50-CAG GAT CCAGCA CAC GAT AG-30; collagen IV a1 chain sense 50-TCGGCT ATT CCT TCG TGA TG-30—antisense 50-TCT CGCTTC TCT CTA TGG TG-30; TGF-b1 sense 50-ATA CAGGGC TTT CGC TTC AG-30—antisense 50-GTC CAG GCTCCA AAT GTA GG-30; b-actin (for normalization) sense50-TCT AGG CAC CAA GGT GTG-30—antisense 50-TCATGA GGT AGT CCG TCA GG-30. Competitive PCR wasperformed by using increasing amounts of mutants madeby creating a deletion in the original PCR product andpreliminary experiments were performed to establish therange of mutant concentrations producing a slope of the lineclose to one and within which the equivalence point falls.After electrophoresis of PCR products, the ratio of unknowncDNA/mutant was quantified by scanning densitometryusing the ImageJ software, a public domain Java imageprocessing program inspired by NIH Image, and results wereexpressed as the ratio of each target to b-actin mRNA level.The levels of fibronectin in conditioned media from culturedmesangial cells were quantified by ELISA using a rabbit

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polyclonal antibody against rat fibronectin (Calbiochem),whereas release of total and bioactive TGF-b1 was measuredusing the Quantikine TGF-b1 Kit (R&D Systems), with andwithout prior acidification, respectively. Values were normal-ized to the DNA content of monolayers, as assessedfluorimetrically in 0.5N NaOH extracts after reaction with0.6 mM 4,6-diamidino-2-phenylindole (Sigma), as previouslydescribed [24].

GLUT1 protein expression. GLUT1 protein expressionin kidney sections was measured by immunofluorescence,as previously reported [27]. Briefly, 5 mm-thick acetone-fixedkidney sections were sequentially hydrated and incubatedfor 1 h at room temperature with a rabbit polyclonal antibodyraised against human GLUT1 and crossreacting with therat and mouse protein (Diagnostic International, Schrieseim,Germany). Then, sections were incubated for 30min at roomtemperature with a fluorescein isothiocyanate-labelled goatanti-rabbit secondary antibody (Alezafluor 488, MolecularProbe). Specificity of antibody labelling was demonstratedafter substituting proper control immunoglobulins (Zymed,Histo-Line Laboratories, Milan, Italy) for the primaryantibody. Slides were mounted with Vectashield aqueousmounting medium (Vector Laboratories, DBA Italia SRL,Milan, Italy) and observed and photographed usinga Nikon Eclipse E600 microscope (Nikon Europe B.V.,Badhoevedorp, The Netherlands). A semi-quantitativeevaluation (0¼ negative, 1¼mild positivity, 2¼ intensepositivity) of GLUT1 staining was performed by examining30 glomeruli per section. GLUT1 protein expression inmesangial cell cultures was measured by western blot analysis.Cells were grown in 100mm Petri dishes (Falcon) underthe above experimental conditions, then total or plasmamembrane extracts were obtained. To obtain total cell lysates[28], cells were scraped, pelleted by centrifugation, disruptedby incubation for 15min at 4�C in lysing buffer (25mmol/lTris-HCl, pH 7.4, 0.2% sodium dodecyl sulfate (SDS),50mmol/l NaCl, 0.5% sodium deoxycholate, 2% NonidetP-40 (NP-40), 1mmol/l phenylmethylsulfonyl fluoride (PMSF),2 mg/ml aprotinin, 2mg/ml leupeptin) under continuousagitation, and centrifuged to collect the supernatant. Toobtain plasma membrane extracts [29], cells were scrapedand homogenized in a ground glass dounce homogenizer at4�C using an ice-cold buffer consisting of PBS containing200mmol/l HEPES, 1mmol/l disodium ethylenediamine-tetraacetic acid (Na2EDTA), 30mmol/l KCl, 100mmol/lMgCl2, 2mmol/l PMSF, 10mmol/l benzamidine, 2mmol/ldithiotreitol (DTT), 25mg/ml leupeptin and 6 mg/ml apro-tinin, then centrifuged at 500 g for 15min at 4�C to pelletunbroken cells and nuclei. The supernatant was centrifugedat 40 000 g for 2 h at 4�C to collect the membrane fraction,which was washed three times in the extraction buffer andresuspended in a lysis buffer containing 0.1% SDS and NP-40Tergitolþ protease inhibitors. Extracts were assayed forprotein content by the Bradford dye-binding protein assaykit (Pierce Chemical). For western blot analysis [28,29],protein samples (10–15mg) were added with an equal volumeof sample buffer 2� (100mmol/l Tris-HCl, pH 7.4, 5% SDS,10% saccharose, 1mmol/l Na2EDTA, 0.025% bromophenolblue, 0,1mol/l DTT), separated by SDS-polyacrylamidegel electrophoresis (PAGE, 10% acrylamide, Bio-RadLaboratories, Hercules, CA) and transferred by electroblot-ting using a MINI PROTEAN IITM (Bio-Rad Laboratories)onto polyvinylidene difluoride membranes (Amersham,

Amersham, UK). The membranes were incubated overnightat 4�C under agitation with Tris-buffered saline (TBS)-Tween(TBSþ 0.5% Tween 20)þ 5% nonfat dry milk (NFDM) toblock the nonspecific reactivity, then probed for 1 h at roomtemperature under agitation with the rabbit polyclonal anti-GLUT1 antibody, diluted 1:100 in TBS-Tweenþ 3%NFDM. Subsequently, the membranes were incubatedfor 45min at room temperature with a goat anti-rabbitIgG antibody conjugated with peroxidase (Dako), diluted1:1500 in TBS-Tweenþ 3% NFDM, washed and developedwith ECL reagent (Amersham). Immunocomplexes wererevealed by autoradiography and quantified by scanningdensitometry using the ImageJ software. Results of totaland cytosolic fraction analysis were normalized to thesignal of b-actin, revealed by the use of a goat polyclonalantibody raised against the C-terminus of humanb-actin (Santa Cruz Biotechnology, Santa Cruz, CA), diluted1:1000.

Glucose transport. Confluent monolayers grown in 16mmmulti-well culture dishes (Falcon, Becton Dickinson, LincolnPark, NJ) under the above experimental conditions. [3H]DOGincorporation was assessed after 15min incubation at 37�Cin 95% air and 5% CO2 humidified atmosphere understirring conditions [2,11]. For this purpose, 1mCi of [3H]DOG(12Ci/mmol, Amersham) in PBS with Ca and Mg was addedto each well and, at the end of the incubation period, theincorporation was stopped by addition of 1ml ice-cold PBScontaining 20mmol/l glucose. Monolayers were then washedthree times with PBS, solubilized overnight at 4�C in 1ml0.1% SDS and processed for liquid scintillation counting in aTri-Carb 2100 TR liquid scintillation analyzer (PackardInstruments, Meriden, CT). Results of [3H]DOG uptakewere normalized per protein content of each well, as measuredby the Bradford method on 10ml aliquots of samples usingBSA as standard. To measure glucose transport kinetics,[3H]DOG incorporation was assessed over 5min in thepresence or absence of increasing amounts (0.1–5mM) ofunlabelled 2-deoxyglucose (Sigma) [11]. Data were analysedby Hanes plot analysis and maximal velocity (Vmax) andMichaelis-Menten constant (Km) were calculated.

Statistical analysis

Values are expressed as mean±SD; the percent change wasalso calculated. Statistical significance was evaluated by one-way ANOVA followed by the Student–Newman–Keuls testfor multiple comparisons. All statistical tests were performedon raw data.

Results

In vivo studies

Metabolic and cardiovascular parameters. Impairmentof body growth as well as increases in blood glucoseand glycated Hb levels and urine volume induced bydiabetes were similar in the two rat strains and attestedto the presence of severe metabolic derangement(Table 1). As previously reported [23], blood pressure

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was normal in the MNS and elevated in the MHSthroughout the study, and was unaffected by diabetes(data not shown).

Renal function. As previously shown [22], both pro-teinuria and serum creatinine were similar in the two ratstrains at time 0. Proteinuria increases progressivelywith time inMNS rats (�30-fold increment at 6 monthsvs time 0, P<0.001), with significantly higher levels indiabetic vs nondiabetic animals at 3, but not 6 months.Conversely, proteinuria did not change with eitherage or diabetes in the MHS rats. Serum creatininealso increased with age in MNS rats (P<0.001), withsignificantly higher values in both nondiabetic anddiabetic MNS at 6, but not 3 months, as compared withthe corresponding MHS rats (Table 2).

Renal structure. As previously described [17,22], nosign of glomerular disease was detected in the twostrains at time 0 and in MHS rats at both 3 and 6months, whereas MNS rats showed clear evidenceof glomerular disease, particularly at 6 months.In keeping with our previous morphometric data [23],mesangial expansion, but not glomerular sclerosisand tubulo-interstitial damage, was more pronounced(þ87%) in diabetic vs nondiabetic MNS rats; con-versely, no change in these parameters was detected indiabetic vs nondiabetic MHS rats (Table 2).

ECM and TGF-�1 gene expression. Transcripts for theECM components fibronectin, laminin B1 and collagenIV a1 chain and the prosclerotic cytokine TGF-b1 weresimilar in the two rat strains at time 0 (data not shown).The gene expression for these proteins increasedsignificantly with age in the MNS (by �30%,P>0.001, except for laminin), but not in the MHSrats. At 3 months of disease duration, higher mRNAlevels (fibronectin þ38%, laminin B1þ 39%, collagenIV a1 chain þ27%, and TGF-b1þ 29%, P<0.001)were detected in the diabetic vs nondiabetic MNS rats;at 6 months, ECM and TGF-b gene expression was stillincreased in the diabetic MNS vs the correspondingnondiabetic controls, with maximal increase observedfor collagen IV (þ49%) (Figure 1). No significantincrease was detected in the diabetic vs nondiabeticMHS rats, at both 3 months (data not shown) and6 months (Figure 1).

GLUT1 expression. At time 0, GLUT1 protein wasdetected in glomeruli from nondiabetic MNS rats,though at a low level of positivity, but not in those fromMHS rats; GLUT1 expression increased significantlyin nondiabetic MNS (from 0.14±0.03 to 0.34±0.12,P<0.01), but not MHS rats throughout the study.GLUT1 fluorescence was increased in diabetic vsnondiabetic MNS rats at 3 months (0.56±0.20vs 0.20±0.05, P<0.01) and 6 months (1.01±0.18 vs0.34±0.12, P<0.001) of disease duration, whereas itwas only barely detectable in MHS rats, in which it waslocalized mainly in podocytes (Figure 2).T

able

1.Effectofdiabetes

andagingonmetabolicparameters,asassessedasbodyweight(g),bloodglucose

(mmol/l),glycatedhem

oglobin

(%)andurinevolume(m

l/day),in

time0(0)MNS

andMHSrats

anddiabetic

(D3andD6)andage-matched

nondiabetic

(ND3andND6)MNSandMHSrats

at3and6monthsofdisease

duration(m

ean±

SD;n¼6anim

als

inper

group,

exceptforurinevolumeattime0,n¼18,and3months,

n¼12)

MNS

MHS

MNS

MHS

MNS

MHS

(tim

e)(tim

e)ND3

D3

ND3

D3

ND6

D6

ND6

D6

Bodyweight

351.33±

11.41

346.00±

35.01

477.50±

29.30

369.17±

18.24*

484.17±

54.63

380.67±

22.57*

548.00±

34.70

337.50±

51.93*

558.50±

23.52

319.67±

33.56*

Bloodglucose

6.45±

0.53

6.22±

0.53

6.52±

0.69

25.29±

2.98*

6.36±

0.59

25.10±

3.81*

6.40±

0.52

26.54±

2.74*

6.00±

0.63

27.86±

2.49*

GlycatedHb

5.02±

0.86

5.13±

0.29

5.05±

0.97

15.32±

0.74*

5.35±

1.04

15.30±

0.75*

5.38±

0.92

16.63±

1.16*

5.28±

1.00

15.90±

2.00*

Urinevolume

16.61±

5.21

18.17±

2.74

17.00±

4.39

163.52±

46.11*

21.60±

7.19

183.73±

79.17*

19.50±

4.55

181.33±

41.15*

21.00±

5.37

185.50±

72.86*

Significantlydifferentat*P<

0.001vs

thecorrespondingnondiabetic

rats.

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In vitro studies

ECM and TGF-�1 mRNA and protein expression. Thegene expression for fibronectin, laminin B1, collagen IVa1 chain and TGF-b1 was similar in mesangial cellsfrom 1-month-old MNS and MHS rats grown underNG conditions. It was stimulated by HG in cells fromboth strains, with increases in ECM, but not TGF-b1mRNA levels that were significantly more pronounced(� þ100% vs þ50%) in those obtained from MNSthan MHS rats (Figure 3). In mesangial cells from8-month-old MNS, but not MHS rats, gene expressionfor the ECM proteins under NG conditions was20% higher than in cells from 1-month-old animals.HG-induced increases in ECM, but not TGF-b1mRNA levels were also more pronounced in MNSthan MHS cells (Figure 3). The increases in ECM geneexpression induced by HG were prevented by co-incubation with a-TGF-b blocking antibody, but notcontrol chicken IgG (data not shown). Fibronectin andtotal TGF-b1 release in conditioned media showed thesame trend of mRNA expression (Table 3); bioactiveTGF-b1 was also higher in HG- vs NG-treated cellsof both genotypes (data not shown). Iso-osmolarmannitol did not mimic the effect of HG in theseparameters.

GLUT1 expression. At western blot analysis, differ-ences were more pronounced at membrane than totalcell level, with no change detected in cytosolic extracts(data not shown). GLUT1 protein membrane levelsbehaved as those for ECM proteins in that they werenot significantly different in cells from 1-month-oldMNS and MHS rats under NG conditions (thoughslightly higher in MNS cells) and were upregulated byHG, but not by iso-osmolar mannitol, in cells fromboth strains, with more marked increases (þ44% vsþ22%) in those obtained from MNS than MHS rats.TGF-b also increased GLUT1 expression under bothNG and HG conditions, with more pronouncedincrements in MNS vs MHS cells (Figure 4A).The increases induced by HG were prevented byco-incubation with a-TGF-b blocking antibody, butnot control chicken IgG (data not shown). Similarresults were obtained in cells from 8-month-old animals(Figure 4B).

Glucose transport. Basal glucose transport was slightlyhigher, though not significantly, in mesangial cells fromMNS vs MHS rats. Incorporation of 3H-DOGwas shown to increase in a dose-dependent fashionupon exposure to 25–200 pmol/l TGF-b. Increases weremore pronounced in mesangial cells from MNS rats(2.19� and 2.60� with 50 and 100 pmol/l, respectively)than in those from MHS rats (1.75� and 2.11� with50 and 100 pmol/l, respectively). Glucose transportstimulated by TGF-b was inhibited by cycloheximide,thus indicating that it was dependent on newprotein synthesis, as well as by cytochalasin B, butnot phloridzin, thus showing that it was GLUT1—andnot sodium—glucose co-transporter-dependent, in cellsfrom both strains (data not shown). When grown inT

able

2.Effectofdiabetes

andagingonrenalfunction,asassessedasproteinuria(m

g/24h)andserum

creatinine(mmol/l),andstructure,asassessedaskidney

wet

weight(g),

mesangial

expansion(score),glomerularsclerosis(%

),andtubulo-interstitiallesions(score),in

time0(0)MNSandMHSrats

anddiabetic

(D3andD6)andage-matched

nondiabetic

(ND3andND6)

MNSandMHSrats

at3and6monthsofdisease

duration(m

ean±

SD;n¼6anim

als

inper

group,exceptforproteinuriaattime0,n¼18,and3months,

n¼12)

MNS

MHS

MNS

MHS

MNS

MHS

(tim

e)(tim

e)ND3

D3

ND3

D3

ND6

D6

ND6

D6

Proteinuria

20.76±

5.34

22.03±

6.73

266.05±

160.96

464.08±

206.23*

22.43±

3.80z

21.98±

10.29z

578.28±

80.79

662.15±

210.69

20.40±

6.49z

20.06±

3.37z

Serum

creatinine

28.34±

1.28

28.73±

2.16

29.74±

2.12

30.29±

1.68

29.62±

1.20

30.11±

1.21

44.87±

4.72

47.62±

6.02

34.09±

6.83z

32.99±

6.02z

Kidney

wet

weight

2.30±

0.18

2.20±

0.11

2.73±

0.20

3.09±

0.30y

2.22±

0.20z

2.35±

0.22z

3.12±

0.26

4.75±

0.95*

2.68±

0.51

3.16±

0.37z

Mesangialexpansion

0.12±

0.07

0.11±

0.05

0.58±

0.15

1.25±

0.37*

0.19±

0.08§

0.25±

0.10z

1.18±

0.46

2.19±

0.59*

0.25±

0.12z

0.34±

0.12z

Glomerularsclerosis

0.02±

0.01

0.01±

0.01

10.46±

3.47

10.19±

2.06

0.29±

0.15z

0.45±

0.18z

20.22±

7.14

20.65±

8.69

0.48±

0.31z

1.36±

0.91z

Tubulo-interstitiallesions

0.02±

0.01

0.01±

0.01

0.72±

0.30

0.74±

0.27

0.02±

0.02z

0.04±

0.03z

1.97±

0.73

2.10±

1.00

0.03±

0.03z

0.07±

0.06z

Significantlydifferentat*P<

0.001or

yP<

0.01vs

thecorrespondingnondiabetic

rats

and

zP<

0.001or§P

<0.01vs

thecorrespondingMNSrats.

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HG vsNG conditions, mesangial cells from 1-month-oldanimals showed enhanced [3H]DOG uptake withincreases which were significantly more marked in thosefrom MNS vs MHS rats (þ47% vs þ27%, P<0.001).Iso-osmolar mannitol did not affect [3H]DOG uptake

by monolayers. Co-incubation with TGF-b produceda further stimulation of [3H]DOG incorporation inboth mesangial cell lines, with more pronouncedincreases vs HG-treated monolayers in MNS than inMHS mesangial cells (þ74% vs þ53%) (Figure 5A).

Fig. 2. Glomerular protein expression of GLUT1 in MNS and MHS rats. Representative kidney sections from rats of the MNS(nondiabetic, A and diabetic, C) and the MHS (nondiabetic, B and diabetic, D) at 6 months of disease duration (100� magnification).

Fig. 1. Glomerular mRNA levels of fibronectin (A), laminin B1 (B), collagen IV a1 chain (C) and TGF-b1 (D) (expressed as OD ratio tob-actin mRNA level) from diabetic (black bars) and age-matched nondiabetic (white bars) MNS and MHS rats at 6 months of diseaseduration (mean±SD; n¼ 4 per group). Significantly different at *P<0.001 vs the corresponding nondiabetic rats and or yP<0.001 vs thecorresponding MNS rats.

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HG-induced increases were prevented by co-incubationwith a-TGF-b blocking antibody, but not controlchicken IgG (data not shown). Kinetic analysisshowed that Vmax, but not Km (data not shown),increased in response to HG and/or TGF-b in cellsobtained from both strains, again to a more pro-nounced extent in MNS than in MHS cells (Figure 5C).Again, similar results were obtained in cells from8-month-old animals (Figure 5B–D).

Discussion

In the in vivo studies, diabetic MNS rats developed DNsuperimposed onto spontaneous glomerulosclerosis,whereas diabetic MHS rats remained free of renaldisease, as reported in our previous publication [23].

Glomerular expressions of GLUT1 protein and ECMand TGF-b mRNA were significantly upregulated indiabetic vs nondiabetic MNS, but not MHS rats, ascompared with the corresponding age-matched non-diabetic controls. In the in vitro studies, mesangial cellsisolated from 1- and 8-month-old MNS rats showedhigher glucose transport activity and GLUT1 mem-brane expression than those from age-matched MHSrats, when exposed to and/or TGF-b levels. Likewise,ECM (but not TGF-b) production increased moremarkedly in response to HG and/or TGF-b in MNSvs MHS mesangial cells, as previously reported [24].

In our previous publication [23], MHS rats werefound to be protected from diabetes-induced haemo-dynamic changes, at variance withMNS, which showedincreased GFR and filtration fraction, indicating anincrease of efferent/afferent resistance with consequent

Fig. 3. Mesangial cell mRNA levels of fibronectin (A), laminin B1 (B), collagen IV a1 chain (C) and TGF-b1 (D) (expressed as OD ratioto b-actin mRNA level) from cells isolated from 1- and 8-month-old MNS and MHS rats and incubated under HG (black bars) and NG(white bars) conditions for 10 days (mean±SD; n¼ 4 per group in 3–4 replicates per experimental condition). Significantly different at*P<0.001 vs the corresponding NG monolayers, yP<0.001 or zP<0.01 vs the corresponding cells from MNS rats, and §P<0.001 or||P<0.01 vs cells from the corresponding 1-month-old rats.

Table 3. Medium fibronectin (mg/mg DNA) and total TGF-b1 (ng/mg DNA) release from mesangial cells isolated from 1- and 8-month-oldMNS and MHS rats and incubated under HG and NG conditions for 10 days (mean±SD; n¼ 4 per group in 3–4 replicates perexperimental condition)

Fibronectin Total TGF-b1

NG HG NG HG

1 monthMNS 0.950±0.039§ 1.766±0.083*§ 0.768±0.039 1.280±0.115*MHS 0.921±0.052 1.435±0.076*z 0.742±0.036 1.201±0.088*8 monthsMNS 1.155±0.064y 2.335±0.242* 0.830±0.037 1.441±0.088*MHS 0.964±0.050 1.492±0.065*z 0.765±0.063 1.300±0.123*

Significantly different at *P<0.001 vs the corresponding NG monolayers, yP<0.001 or zP<0.01 vs the corresponding cells from MNSrats, and §P<0.001 vs cells from the corresponding 1-month-old rats.

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increment of glomerular capillary pressure, drivingenhanced GFR. In the present study, virtually noupregulation of the TGF-b/GLUT1 axis (and no ECMoverproduction and renal disease) occurred in thehaemodynamically-protected MHS rats, at variancewith the glomerulosclerosis-prone MNS rats, in whichglomerular GLUT1 expression increased progressively,in parallel with ECM and TGF-b gene expressionand glomerular injury. Upregulation of the

TGF-b/GLUT-1 axis under diabetic conditions didnot lead to a further increment of glomerular sclerosisnor to differences in the appearance of sclerotic lesions,but only to a more marked matrix deposition thannondiabetic animals, as reported in our previouspublication, as reported in our previous publication[23]. Conversely, HG was capable of inducing changesin TGF-b/GLUT1 axis and ECM production inmesangial cells isolated from MHS rats, i.e. under

Fig. 5. Mesangial cell glucose transport activity, as measured as 3H-DOG incorporation (CPM� 103/mg protein), and kinetics,as expressed as Vmax (nmol/mg/min), from cells isolated from 1-month-old (A and C) and 8-month-old (B and D) MNS and MHSrats and incubated under HG (black bars) and NG (white bars) conditions for 10 days, then exposed overnight to 100 pmol/l TGF-b (þ)or vehicle (�) (mean±SD; n¼ 4 per group in 3–4 replicates per experimental condition). Significantly different at *P<0.001 or yP<0.01or zP<0.05 vs the corresponding NG monolayers, or §P<0.001, ||P<0.01 or |P<0.01 vs the corresponding untreated monolayers, and�P<0.001 vs the corresponding cells from MNS rats.

Fig. 4. Mesangial cell membrane expression of GLUT1 protein (expressed as OD ratio % to b-actin) from cells isolated from 1-month-old(A) and 8-month-old (B) MNS and MHS rats and incubated under HG (black bars) and NG (white bars) conditions for 10 days, thenexposed overnight to 100 pmol/l TGF-b (þ) or vehicle (�) (mean±SD; n¼ 4 per group in 3–4 replicates per experimental condition).Significantly different at *P<0.001 or yP<0.05 vs the corresponding NG monolayers, or zP<0.001 or §P<0.01 vs the correspondinguntreated monolayers, and ||P<0.001 vs the corresponding cells from MNS rats.

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conditions in which haemodynamics is not operating,though to a significantly lesser extent than in mesangialcells isolated from MNS rats (except for TGF-bproduction).

These results confirm and extend previous observa-tions from Gnudi et al. in the hypertensive renal diseaseoccurring in the Dahl-S rat, but not in the young SHR,showing that glomerular hypertension and injurycorrelate to the activation of the TGF-b/GLUT1 axis[14]. Our data indicate that the TGF-b/GLUT1 axisplays a major role also in the pathogenesis ofDN by linking the haemodynamic and metabolicabnormalities triggered by hyperglycaemia. Hence,the permissive role of haemodynamic changes towardhyperglycaemia-induced injury would be mediated bythe upregulation of this axis induced by stretching(possibly amplified by hyperglycaemia), but not byhyperglycaemia alone. The view that this glucosetransporter is induced predominantly by haemo-dynamic stimuli is further supported by a recentreport in mice carrying the oligosyndactyly (Os) allele,which have a 50% reduction in nephron number andare susceptible to (haemodynamically-mediated) renaldisease. ragged oligosyndactyly pintail (ROP) Os/þmice backcrossed into the mouse strain (fvb) back-ground developed rapidly progressive renal disease andrenal failure at variance with classical ROP Os/þ mice,showing only glomerulosclerosis, and also had higherglomerular GLUT1 expression and glucose uptake [30].

The finding that upregulation of GLUT1-dependentglucose transport and ECM production in response toTGF-b, either stimulated by HG or added to the culturemedium, was more marked in mesangial cells fromMNS than MHS rats suggests an increased activityof the TGF-b/GLUT1 axis in the glomerulosclerosis-prone MNS rats. This conclusion is supported by theobservation that HG-induced changes in glucosetransport, GLUT1 and ECM in the MNS were moremarked than those detected not only in mesangial cellsfrom the glomerulosclerosis-resistant MHS rats, butalso in cells isolated from Sprague–Dawley or other ratstrains (unpublished observations). The observationthat GLUT1 and ECM, but not TGF-b expression,was more pronounced in MNS vs MHS mesangialcells suggests that, in the MNS, a similar HG-inducedTGF-b upregulation resulted in an increased responsevs theMHS in terms of GLUT1 and consequent glucosetransport activity, which produced a more markedECM deposition, independent of further, glucose-dependent TGF-b induction. This view is in keepingwith a recent report indicating that increased GLUT1expression, independent of elevated extracellularglucose levels, can directly enhance ECM productionthrough a PKC and AP-1 dependent pathway, withoutcausing mitogen-activated protein kinase activation,TGF-b induction or reactive oxygen species generation[6], which have all been implicated in the pathogenesisof DN.

This increased activity of the TGF-b/GLUT1 axisin the MNS rats seems to be genetically determined,since an enhanced GLUT1 response to TGF-b

upregulation was detectable in mesangial cells isolatedfrom them before the onset of spontaneous renaldisease. It underlines the susceptibility to DN in MNSrats, by amplifying the effects of hyperglycaemia andassociated TGF-b upregulation on ECM overproduc-tion, thus leading to excess matrix deposition underdiabetic conditions.

In conclusion, these results indicate that the TGF-b/GLUT1 axis plays a major role also in the pathogenesisof DN by linking the haemodynamic and metabolicabnormalities triggered by hyperglycaemia, with thepermissive role of haemodynamic changes towardhyperglycaemia-induced injury being mediated throughthe upregulation of this axis. These data also indicatethat susceptibility to diabetic glomerulopathy inMNS rats is associated with increased (genetically-determined) GLUT1-dependent glucose transportactivity in response to hyperglycaemia and/or TGF-b,which may amplify ECM overproduction occurringunder diabetic conditions.

Acknowledgements. This work was supported by grants from theMinistry of Education of Italy (40 and 60%), the Ministry ofHealth of Italy, the International Center for the Study of Diabetes,and the Diabetes, Endocrinology and Metabolism Foundation,Rome, Italy.

We are indebted to Prof. Giuseppe Bianchi (Division ofNephrology and Hypertension, San Raffaele Hospital, ‘Vitae Salute’ University, Milan, Italy) for providing us with theMilan rats.

Conflict of interest statement. None declared.

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Received for publication: 3.10.05Accepted in revised form: 3.1.06

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