* The Research Equipment Center, Department of Pharmacology, RI Research Center, and Second Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan; || Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana; and ¶ Department of Pharmacology, Osaka City University Graduate School of Medicine, Osaka, Japan
Address correspondence to: Dr. Akira Nishiyama, Department of Pharmacology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Phone: +81-87-898-5111 ext. 2502; Fax: +81-87-891-2126; E-mail: akira{at}kms.ac.jp
Whether temporary angiotensin II (AngII) blockade at the prediabeticstage attenuates renal injury in type 2 diabetic OLETF ratslater in life was investigated. OLETF rats were treated withan AT1 receptor antagonist (olmesartan, 0.01% in food), angiotensin-convertingenzyme inhibitor (temocapril, 0.01% in food), a combinationof the two, or hydralazine (25 mg/kg per d) at the prediabeticstage (4 to 11 wk of age) and then monitored without furthertreatment until 50 wk of age. At 11 wk of age, blood glucoselevels and urinary protein excretion (UproteinV) were similarbetween OLETF and control LETO rats. However, OLETF rats showedhigher kidney AngII contents and type IV collagen mRNA expressionthan LETO rats at this age. These decreased with olmesartan,temocapril, and a combination of these but not with hydralazine.At 50 wk of age, diabetic OLETF rats showed higher BP, UproteinV,and intrarenal AngII levels than LETO rats. Temporary AngIIblockade did not affect glucose metabolism or the developmentof hypertension in OLETF rats but significantly suppressed proteinuriaand ameliorated glomerular injury. However, no parameters wereaffected by temporary hydralazine treatment. The present studydemonstrated that intrarenal AngII and type IV collagen expressionare already augmented long before diabetes becomes apparentin OLETF rats. Furthermore, temporary AngII blockade at theprediabetic stage attenuates the progression of renal injuryin these animals. These data suggest that early AngII blockadecould be an effective strategy for preventing the developmentof type 2 diabetic renal injury later in life.
Diabetic nephropathy is a major complication in diabetes anda leading cause of end-stage renal failure, which causes disabilitiesand a high mortality rate in patients with this disease (1).The mechanisms underlying the development of diabetic nephropathyare extremely complex; however, the potential role of the renin-angiotensinsystem (RAS) has been suggested (2–13). Recent studiesindicate that in diabetes, intrarenal generation of angiotensinII (AngII) is elevated despite suppressed circulating RAS (5,6).Furthermore, AT1 receptor blockers (ARB) or angiotensin-convertingenzyme inhibitors (ACEI) have been shown to attenuate the progressionof diabetic nephropathy (3, 4, 7–13). Several clinicaltrials have shown that ARB are more effective than traditionalantihypertensive therapies in reducing renal failure progressionin patients with type 2 diabetes and that the renoprotectiveeffects of ARB are independent of their antihypertensive actions(8–11). Of interest, it has also been shown that ACEItreatment of normotensive patients with diabetes and littleor no proteinuria (early stages of diabetic nephropathy) resultsin long-term stabilization of plasma creatinine levels and urinaryprotein excretion rates (UproteinV) (12, 13). These observationssuggest that angiotensin blockade has clinical benefits forpatients who have diabetes and have no or early signals of renaldisease.
It has been shown that early administration of ACEI to youngspontaneously hypertensive rats (SHR) has long-term antihypertensiveeffects even after treatment is discontinued (14, 15). However,no similar effect could be found with administration of otherantihypertensive drugs, including calcium antagonists, -blockers,and direct vasodilators (15–17). Further studies showedthat a single application of ACE antisense to neonatal ratsattenuated the development of hypertension and endothelial dysfunctionin SHR (18). Recently, Nakaya et al. (19, 20) revealed thatadministration of ARB or ACEI during a limited time window beforepuberty results in a prolonged reduction in BP and renoprotectionin stroke-prone SHR and Dahl salt-sensitive hypertensive rats.Collectively, these data suggest that sensitivity to angiotensinblockade exists before the development of hypertension as wellas associated renal injury. However, it is still not clear whetherthe renoprotective effects of angiotensin blockade are due toits antihypertensive effect or direct mechanisms. In addition,the effects of brief periods of ARB or ACEI treatment on renalinjury in type 2 diabetes have not been examined.
This study investigates whether temporary AngII blockade atthe prediabetic stage provides long-lasting protection againstrenal injury in type 2 diabetic rats. OLETF rats, which exhibitpathologic features of renal injury similar to those of humantype 2 diabetes (21–23), were treated with an ARB, olmesartan(22, 24), an ACEI, temocapril (22), or a nonspecific vasodilator,hydralazine (24), for only 7 wk at the prediabetic stage (4to 11 wk of age) and then monitored without further treatmentuntil 50 wk of age. The present study demonstrates for the firsttime that intrarenal AngII and reactive oxygen species (ROS)levels as well as type IV collagen gene expression are alreadyaugmented in young OLETF rats, long before diabetes becomesapparent. In addition, temporary AngII blockade at the prediabeticstage suppresses the development of renal injury in these animals,independent of its effects on BP and glucose metabolism. Thesedata suggest that early AngII blockade could be an effectivestrategy for preventing the development of type 2 diabetic renalinjury later in life.
Animals
All experimental procedures were performed according to theguidelines for the care and use of animals established by KagawaMedical University. Male 4-wk-old OLETF rats and LETO rats (geneticcontrol of OLETF rats; n = 12) were supplied by Otsuka PharmaceuticalCo. Ltd. (Tokushima, Japan). After basal measurements were obtained,OLETF rats were randomly treated with one of the following combinations:tap water + standard diet (n = 14); tap water + olmesartan (0.01%in food; Sankyo Co. Ltd., Tokyo, Japan); n = 14); tap water+ temocapril (0.01% in food; Sankyo Co. Ltd.; n = 14); tap water+ olmesartan (0.01% in food) + temocapril (0.01% in food; n= 14); or standard diet + tap water containing hydralazine (25mg/kg per d; n = 14). The doses of olmesartan, temocapril, andhydralazine were determined on the basis of results from previousstudies on rats (22, 24). The medications were stopped at 11wk of age. Half of the LETO and OLETF rats in each group thenwere killed, and the remaining rats were monitored without medicationuntil they were killed at 50 wk of age.
Systolic BP (SBP) was measured in conscious rats by tail-cuffplethysmography (BP-98A; Softron Co., Tokyo, Japan), and 24-hurine samples were collected using metabolic cages. After decapitation,half of one kidney was homogenized in cold methanol and processedfor measurements of kidney AngII content (24–27). Theother half of this kidney was fixed in 10% buffered paraformaldehydefor histologic examination. The remaining kidney was snap-frozenin liquid nitrogen and stored at –80°C.
Real-Time Reverse Transcription-PCR
The mRNA expression of angiotensinogen in renal cortical tissueswas analyzed quantitatively by real-time PCR using an Mx3000PSystem with a Brilliant Single-Step QRT-PCR Master Mix Kit (Stratagene,La Jolla, CA) (26). The following rat angiotensinogen primerswere used: sense 5'-AGGCAAGAGGTGTAGCCAGT-3' and antisense 5'-AGGACCTTATGTCCGTCCAG-3'.The following rat angiotensinogen probe was used: 5'-TCTTTCTACCTTGGATCGTTGGATCCC-3'.The mRNA expression of glyceraldehyde-3-phosphate dehydrogenase,renin, p22phox, gp91phox, types I and IV collagen, TGF-, andconnective tissue growth factor (CTGF) were analyzed by real-timePCR using a LightCycler FastStart DNA Master SYBR Green I kit(28). The oligonucleotide primer sequences and PCR conditionsfor p22phox, gp91phox, and types I and IV collagen are summarizedin Table 1. TGF- and CTGF mRNA expression were measured by usingTaqMan Gene Expression Assay kits (Applied Biosystems, FosterCity, CA). All data are expressed as the relative differencesbetween OLETF and LETO rats after normalization to glyceraldehyde-3-phosphatedehydrogenase expression.
Western Blot Analysis
AT1 and AT2 receptor protein levels in the renal cortical tissueswere analyzed by Western blotting using antibodies against theAT1 and AT2 receptors (Santa Cruz Biotechnology, Santa Cruz,CA), as described previously (24, 25, 27). To check for equalloading, we reprobed membranes with an antibody against -actin(Sigma Chemical, St. Louis, MO). Data are expressed as the relativedifferences between OLETF rats and LETO rats after normalizationto -actin expression.
Histologic Examination
Kidneys were fixed with 10% formalin (pH 7.4), embedded in paraffin,sectioned into 4-µm slices, and stained with hematoxylin-eosinor periodic acid-Schiff (PAS) reagents. The severity of glomerularsclerosis in the PAS-stained sections was determined using asemiquantitative score from 0 to 4: 0, no matrix expansion;1, minor; 2, weak; 3, moderate; and 4, strong (25, 28). Thediameters of the glomeruli in each experimental group were measuredusing a visual caliper (SVS 30000; Showa Electric Laboratory,Fukuoka, Japan) (28). For all measurements, 245 to 315 randomlyselected glomeruli were examined.
Other Analytical Procedures
AngII content in the kidneys was measured by RIA as describedpreviously (24–27), and UproteinV was determined usinga protein assay kit (microTP-test; Wako Co., Osaka, Japan).Blood glucose levels were measured with a glucose analyzer (Sanwa-Kagaku,Co. Ltd., Nagoya, Japan), and insulin levels were measured witha RIA kit (Amersham Biosciences, Piscataway, NJ). The degreeof lipid peroxidation was determined using biochemical assaysof the thiobarbituric acid reactive substances (TBARS) in therenal cortical tissues, as described previously (24, 27, 28).Renal cortical tissue collagen content was determined on thebasis of hydroxyproline concentration (25, 28).
Statistical Analyses
Values are presented as means ± SEM. Statistical comparisonsof the differences were performed using one- or two-way ANOVAcombined with Newman-Keuls post hoc test. P < 0.05 was consideredstatistically significant.
BP, Body Weight, and Blood Glucose
The temporal profiles of SBP, body weight, and postprandialblood glucose are depicted in Figure 1, A through C. SBP remainedunaltered in LETO rats, whereas OLETF rats progressively developedhypertension (Figure 1A). Temporary treatment with olmesartan,temocapril, a combination of these, or hydralazine at the prediabeticstage had no effect on the development of hypertension. From15 to 50 wk of age, the body weight of untreated OLETF ratswas higher than that of LETO rats (Figure 1B). None of the temporarytreatments affected the body weight of OLETF rats (Figure 1B).OLETF rats showed higher postprandial blood glucose levels thanLETO rats from 15 to 50 wk of age (Figure 1C). As shown in Table 2,the SBP and postprandial blood glucose levels were similarbetween LETO and OLETF rats at 11 wk of age. Treatment witholmesartan, temocapril, a combination of these, or hydralazineresulted in similar reductions in SBP but did not alter thepostprandial blood glucose levels in OLETF rats. The data at50 wk of age are shown in Table 3.
Figure 1. Profiles of systolic BP (SBP; A), body weight (BW; B), postprandial blood glucose (PPBG; C), and urinary protein excretion (UproteinV; D). OLETF rats develop hypertension, obesity, diabetes, and proteinuria. In these animals, temporary treatment with olmesartan, temocapril, a combination of these, or hydralazine at the prediabetic stage (4 to 11 wk of age) does not affect the development of hypertension, obesity, or high glucose levels later in life. However, the progression of proteinuria is markedly suppressed by temporary treatment with olmesartan, temocapril, or a combination of these but not by treatment with hydralazine.
Table 2. Systolic blood pressure, postprandial blood glucose level, left kidney weight, renal cortical thiobarbituric acid reactive substances, and mRNA expression of p22phox and gp91phox in LETO and OLETF rats at 11 wk of agea
Table 3. Fasting blood glucose, plasma insulin, LKW, kidney angiotensin II levels, renal cortical TBARS, and mRNA expression of p22phox and gp91phox in LETO and OLETF rats at 50 wk of agea
UproteinV, Renal Cortical Collagen, TGF-, and CTGF
The temporal profile of UproteinV is depicted in Figure 1D.The UproteinV of OLETF rats progressively increased with age.The progression of proteinuria was markedly suppressed by temporarytreatment with olmesartan, temocapril, or a combination of thesebut not by treatment with hydralazine.
At 11 wk of age, UproteinV and the renal cortical collagen contentswere similar among all groups (Figures 1D and Figure 2A). ThemRNA expression of type I collagen in the renal cortical tissueswas also similar between LETO and OLETF rats. In OLETF rats,however, treatment with olmesartan or olmesartan plus temocaprilsignificantly decreased type I collagen gene expression (Figure 2B).Temocapril also tended to decrease type I collagen geneexpression, but the change was not statistically significant.Furthermore, hydralazine did not alter type I collagen geneexpression. However, renal cortical type IV collagen mRNA expressionwas approximately twofold higher in OLETF rats than in LETOrats at this age (Figure 2C). In addition, the augmentationof type IV collagen gene expression was prevented by treatmentwith olmesartan, temocapril, or a combination of these but notby treatment with hydralazine (Figure 2C). Renal cortical mRNAexpression of TGF- was not significantly changed in OLETF rats(Figure 2D), whereas CTGF mRNA expression was approximatelyfourfold higher in OLETF rats (Figure 2E). The augmentationof CTGF collagen gene expression was prevented by treatmentwith olmesartan, temocapril, or a combination of these but notby treatment with hydralazine (Figure 2E).
Figure 2. (A) Renal cortical collagen content, and mRNA expression of type I collagen (B), type IV collagen (C), TGF- (D), and connective tissue growth factor (CTGF; E) in OLETF rats at 11 wk of age. The renal cortical collagen contents and mRNA expression of type I collagen and TGF- are similar between OLETF and control LETO rats. However, renal cortical type IV collagen and CTGF mRNA expression is already augmented in OLETF rats at this age. The augmented type IV collagen and CTGF mRNA expression is normalized by treatment with olmesartan, temocapril, or a combination of these but not by treatment with hydralazine. *P < 0.05 versus LETO; P < 0.05, OLETF versus OLETF + olmesartan, temocapril, or olmesartan plus temocapril.
At 50 wk of age, the renal cortical collagen content in untreatedOLETF rats was significantly higher than that in LETO rats.In OLETF rats, the renal cortical collagen contents were significantlydecreased by temporary treatment with olmesartan, temocapril,or a combination of these but not by treatment with hydralazine(Figure 3A). Similar results were observed for the renal corticalmRNA expression of types I and IV collagen, TGF-, and CTGF (Figure 3Bthrough Figure 3E).
Figure 3. (A) Renal cortical tissue collagen content and mRNA expression of type I collagen (B), type IV collagen (C), TGF- (D), and CTGF (E) in OLETF rats at 50 wk of age. OLETF rats show increased levels at 50 wk of age. In OLETF rats, temporary treatment with olmesartan, temocapril, or a combination of these at the prediabetic stage (4 to 11 wk of age) reduces these levels, whereas temporary treatment with hydralazine does not. *P < 0.05 versus LETO; P < 0.05, OLETF versus OLETF + olmesartan, temocapril, or olmesartan plus temocapril.
Histologic Findings
The glomerular histologic findings with hematoxylin-eosin andPAS staining are shown in Figure 4A and Figure 4B, respectively.In 11-wk-old OLETF rats, there were no obvious glomeruli alterations(data not shown). However, mesangial expansion accompanied byan accumulation of extracellular matrix and glomerular capillarywall thickening occurred at 50 wk of age. Some glomeruli showeddeveloped diabetic glomerulopathy accompanied by diffuse mesangialmatrix expansion. However, there were no obvious findings exceptminor alterations as a result of aging in the glomeruli of LETOrats even at 50 wk of age. The semiquantitative analyses datashowed that the glomerular sclerosis index in untreated OLETFrats (50 wk of age) was significantly greater compared withLETO rats (Figure 4C). Temporary treatment with olmesartan,temocapril, or a combination of these significantly decreasedthe glomerular sclerosis index in OLETF rats, whereas this valuewas similar between the untreated and hydralazine-treated OLETFrats at 50 wk of age (Figure 4C). Figure 4D shows the glomerularsize distribution and reveals that the glomeruli tended to belarger in OLETF rats than in LETO rats. In OLETF rats, temporarytreatment with olmesartan, temocapril, a combination of these,or hydralazine did not remarkably alter the glomerular sizedistribution.
Figure 4. Photomicrographs of glomeruli with hematoxylin-eosin (HE; A) and periodic acid-Schiff (PAS; B) reagents. In diabetic OLETF rats, mesangial expansion accompanied by an accumulation of extracellular matrix and glomerular capillary wall thickening occurs at 50 wk of age. Some glomeruli show developed glomerulopathy accompanied by diffuse mesangial matrix expansion. These glomerular changes are ameliorated by temporary treatment (4 to 11 wk of age) with olmesartan, temocapril, or a combination of these but not by treatment with hydralazine. However, there are no obvious findings except minor alterations as a result of aging in the glomeruli of the control LETO rats even at 50 wk. The glomerular sclerosis index (C) and glomerular size distribution (D) determined as described in the Materials and Methods section. *P < 0.05 versus LETO; P < 0.05, OLETF versus OLETF + olmesartan, temocapril, or olmesartan plus temocapril. Original magnification, x200 in A and B.
Angiotensinogen, Renin, AngII, and AT1 and AT2 Receptors
At 11 wk of age, OLETF rats showed higher renal angiotensinogenand renin mRNA levels than LETO rats (Figure 5A and B). Treatmentwith olmesartan, temocapril, or olmesartan plus temocapril significantlydecreased angiotensinogen mRNA levels but increased renin mRNAlevels in OLETF rats. Hydralazine slightly decreased angiotensinogenexpression but increased renin expression in OLETF rats (Figure 5Aand Figure 5B). OLETF rats already showed higher kidney AngIIcontents than LETO rats at this age (Figure 5C). The kidneyAngII content decreased with daily treatments of olmesartan,temocapril, or olmesartan plus temocapril, whereas hydralazinehad no effect. The protein expression of AT1 (approximately46 kD) and AT2 (approximately 44 kD) receptors was similar amongall groups (data not shown).
Figure 5. Renal cortical mRNA expression of angiotensinogen (A) and renin (B), as well as kidney angiotensin II (AngII; C) levels in OLETF rats at 11 wk of age. Compared with control LETO rats, OLETF rats already show higher renal angiotensinogen and renin mRNA expression as well as kidney AngII levels at the prediabetic stage. Augmented intrarenal AngII levels in OLETF rats are decreased by treatment with olmesartan, temocapril, or olmesartan plus temocapril but not by treatment with hydralazine. *P < 0.05 versus LETO; P < 0.05, OLETF versus OLETF + olmesartan, temocapril, or olmesartan + temocapril.
At 50 wk of age, renal cortical angiotensinogen mRNA expressionwas similar between OLETF and LETO rats (data not shown). However,mRNA renin expression was significantly higher in OLETF ratsthan in LETO rats (by 2.1 ± 0.2-fold). Temporary treatmentwith olmesartan, temocapril, a combination of these, or hydralazinehad no effect on mRNA angiotensinogen and renin expression (datanot shown). Untreated OLETF rats had higher kidney AngII contentsthan LETO rats (Table 2). Temporary treatment with olmesartan,temocapril, a combination of these, or hydralazine had no effecton these values (Table 2). The protein levels of AT1 and AT2receptors were similar among all groups (data not shown).
TBARS Content and mRNA Expression of NAD(P)H Oxidase Components
At 11 wk of age, OLETF rats already showed higher renal corticalTBARS contents and mRNA p22phox expression compared with LETOrats (Table 2). In the OLETF rats, treatment with olmesartan,temocapril, or a combination of these decreased the renal corticalTBARS content and p22phox expression. Hydralazine also significantlydecreased renal cortical p22phox mRNA expression but not TBARScontent. However, mRNA gp91phox expression did not differ amongthe groups (Table 2).
At 50 wk of age, untreated OLETF rats showed higher renal corticalTBARS contents as well as mRNA p22phox and gp91phox expressioncompared with LETO rats (Table 3). Temporary treatment witholmesartan, temocapril, a combination of these, or hydralazinedid not alter these levels.
The RAS has been implicated in the pathophysiology of diabeticnephropathy, largely on the basis of the ability of ARB or ACEIto reduce proteinuria and the progression of renal injury inpatients (7–13) and animals (21, 22, 29) with diabetes.The present study demonstrates that renal injury is associatedwith increases in intrarenal AngII levels in type 2 diabeticOLETF rats (50 wk of age). In addition, expression of AngIIreceptors was maintained in the kidneys of diabetic OLETF rats.The key finding of the present study is that intrarenal AngIIlevels are already augmented in young OLETF rats (11 wk of age)before the manifestation of diabetes. Furthermore, the augmentationof the intrarenal AngII content was prevented by concurrentadministration of an ARB or ACEI. In addition, brief treatmentwith an ARB or ACEI during a limited time window at the prediabeticstage (4 to 11 wk of age) attenuated the development of renalinjury later in life, independent of its effects on BP and glucosemetabolism. These data suggest a potential contribution of augmentedintrarenal AngII levels at the prediabetic period to the developmentof type 2 diabetic renal injury.
Uehara et al. (21) showed that the onset of proteinuria andrenal histologic changes was delayed by daily treatments withACEI (from 6 to 22 wk of age) in OLETF rats. In the presentstudy, however, the renoprotective effects of temporary angiotensinblockade cannot be explained simply as a consequence of thedelay in the onset of renal injury. If temporary angiotensinblockade for 7 wk (4 to 11 wk of age) merely delays the onsetof renal injury, then a rightward shift of 7 wk in the timecourse of proteinuria development should be observed in thetreated animals. However, such rightward shifts in the UproteinVlevels were not observed in the ARB- or ACEI-treated animalsin the present study. It also seems likely that the antihypertensiveeffects of ARB and ACEI during the treatment period are notthe main cause of this phenomenon. The present results revealedthat temporary treatment with a nonspecific vasodilator (hydralazine)resulted in a similar reduction in BP to treatments with ARBand ACE but did not attenuate renal injury in OLETF rats. Inaddition, the antihypertensive effects of these agents werediminished soon after the cessation of treatment.
Consistent with previous observations in hypertensive rats (25,27, 30), continuous AngII blockade decreased AngII kidney contentin young OLETF rats (11 wk of age). These data suggest thatsome of the renoprotective effects of temporary angiotensinblockade are accompanied by reductions in intrarenal AngII levelsduring this treatment period. However, increases in intrarenalAngII levels in diabetic OLETF rats (50 wk of age) are not preventedby temporary ARB and ACEI treatments. Furthermore, neither AT1nor AT2 receptor expression in the renal cortical tissues wasaltered by these treatments. These data suggest that the renoprotectiveeffects of temporary angiotensin blockade are not due to thesustained suppression of intrarenal RAS activity.
AngII induces cellular changes through NAD(P)H oxidase-mediatedROS production (29, 30); ROS have also emerged as importantmediators in the pathogenesis of diabetic nephropathy (29, 31–33).Similar to the alterations in intrarenal AngII levels, p22phoxexpression and TBARS content were augmented in both prediabetic(11 wk of age) and diabetic (50 wk of age) OLETF rats. We alsoobserved that augmented renal p22phox expression and TBARS contentwere reduced by daily ARB or ACEI treatments at 11 wk of age.However, the augmentation of ROS in diabetic OLETF rats (50wk of age) was not prevented by temporary treatment with ARBor ACEI. Collectively, these results suggest the potential contributionof ROS to the development of renal injury in OLETF rats; however,the renoprotective effects of temporary angiotensin blockadeat the prediabetic stage cannot be explained by sustained suppressionof intrarenal ROS.
Although the present study provides no direct information regardingthe mechanisms that are responsible for the beneficial actionsof temporary angiotensin blockade on type 2 diabetic renal injury,several possibilities can be suggested. Okada et al. (23) showedthat AngII stimulated DNA synthesis to a greater extent in mesangialcells that were isolated from young prediabetic OLETF rats (14wk of age) than in those from older diabetic OLETF rats (50wk of age). These findings suggest that a possible criticalAngII-sensitive phase exists in the renal cells of OLETF ratsat the prediabetic stage. The authors also indicated stage-specificgene regulation in the mesangial cells of OLETF rats (23). Thus,it can be speculated that AngII-induced gene regulation in therenal cells of prediabetic OLETF rats is affected by temporarytreatment with ARB or ACEI. The present study revealed that,although the collagen contents in the renal cortical tissuesremained unchanged in prediabetic OLETF rats (11 wk of age),type IV collagen gene expression was already enhanced at thisage. In addition, gene expression of CTGF, which mediates downstreamevents of TGF- (34) and stimulates fibroblast proliferationand extracellular matrix protein synthesis (35, 36), was markedlyincreased in the kidneys of OLETF rats (11 wk of age). Furthermore,temporary AngII blockade at the prediabetic stage resulted insustained reductions in these gene expression and collagen accumulationlater in life. These data suggest that AngII-induced CTGF andcollagen gene expression at the prediabetic stage may participate,at least in part, in the onset of renal injury in OLETF rats.
In summary, the present study demonstrates that intrarenal AngIIand ROS levels as well as type IV collagen and CTGF gene expressionare already augmented long before the first signs of diabetesbecome apparent in OLETF rats. Furthermore, temporary blockadeof the RAS at the prediabetic stage suppresses the developmentof diabetic renal injury, independent of its effects on BP andglucose metabolism. These data support the hypothesis that earlyblockade of the RAS could be an effective strategy for preventingthe development of type 2 diabetic renal injury later in life.However, more careful assessments of the morphology and intrarenalRAS (by using electron microscopy, HPLC separation, and receptorbinding assays, etc.) will be required for confirmation. Inaddition, further studies on patients with type 2 diabetes willbe necessary to address this hypothesis directly. In this regard,the ROADMAP (Randomized Olmesartan and Diabetes MicroalbuminuriaPrevention) study was initiated in September 2004 to determinethe difference in time to microalbuminuria between patientswith type 2 diabetes and normoalbuminuria allocated to olmesartanor conventional antihypertensive therapy (presented at the 19thScientific Meeting of the International Society of Hypertension,February 2004). Importantly, this clinical trial will test whetherthe prevention of microalbuminuria with early olmesartan treatmentleads to renal and other cardiovascular protection and improvesdisease morbidity and mortality later in life.
Acknowledgments
This work was supported by grants-in-aid for scientific researchfrom the Ministry of Education, Culture, Sports, Science andTechnology of Japan (15790136; A.N.); and by grants from theSalt Science Research Foundation (04C2), the Nankai-IkueikaiFoundation, Mitsui Life Social Welfare Foundation, Japan ResearchFoundation for Clinical Pharmacology, and the Kao Foundationfor Arts and Sciences (to A.N.)
Part of this work was presented at the 36th Annual Meeting andScientific Exposition of the American Society of Nephrology,San Diego, CA, October 2003; and the 58th Annual Fall Conferenceand Scientific Sessions of the Council for High Blood PressureResearch in association with the Council on the Kidney in CardiovascularDisease, Chicago, IL, October 2004
We thank Drs. Hidetoshi Kakari and Toshitaka Nakagawa (KagawaMedical University) for excellent technical assistance. We arealso grateful to Sankyo Co. Ltd. for supplying olmesartan andtemocapril and to Otsuka Pharmaceutical Co. Ltd. for supplyingOLETF and LETO rats
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication August 8, 2004.
Accepted for publication November 24, 2004.
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Top
Abstract
Introduction
-blockers, and direct vasodilators (15–17). Further studies showed that a single application of ACE antisense to neonatal rats attenuated the development of hypertension and endothelial dysfunction in SHR (18). Recently, Nakaya et al. (19, 20) revealed that administration of ARB or ACEI during a limited time window before puberty results in a prolonged reduction in BP and renoprotection in stroke-prone SHR and Dahl salt-sensitive hypertensive rats. Collectively, these data suggest that sensitivity to angiotensin blockade exists before the development of hypertension as well as associated renal injury. However, it is still not clear whether the renoprotective effects of angiotensin blockade are due to its antihypertensive effect or direct mechanisms. In addition, the effects of brief periods of ARB or ACEI treatment on renal injury in type 2 diabetes have not been examined. 
