Effects of Low-Dose and Early versus Late Perindopril Treatment on the Progression of Severe Diabetic Nephropathy in (mREN-2)27 Rats
Sally A. Mifsud*,
Sandford L. Skinner*,
Mark E. Cooper,
Darren J. Kelly and
Jennifer L. Wilkinson-Berka*
*Department of Physiology, University of Melbourne, Parkville, Australia; Department of Medicine, Austin and Repatriation Medical Centre, West Heidelberg, Australia; and Department of Medicine, St. Vincent’s Hospital, Fitzroy, Australia.
Correspondence to Dr. Jennifer L. Wilkinson-Berka, Department of Physiology, University of Melbourne, Parkville, Victoria, Australia, 3010. Phone: 61-3-8344-5849; Fax: 61-3-8344-5818; E-mail: j.berka{at}physiology.unimelb.edu.au
ABSTRACT. It was previously reported that transgenic (mRen-2)27rats with streptozotocin-induced diabetes mellitus progressivelydevelop advanced nephropathy in 12 wk. These lesions are largelyprevented when the angiotensin-converting enzyme inhibitor perindoprilis administered from the time of induction of diabetes mellitus.This study aimed to determine the lowest dose of early perindopriltreatment required for substantial improvement of renal functionand structure and to investigate whether late intervention preventsor reverses the progression of established renal lesions. At6 wk of age, female heterozygous Ren-2 rats were randomizedto receive either streptozotocin (diabetic) or citrate buffer(control). Rats were gavaged, beginning early after the inductionof diabetes mellitus or the administration of control vehicle,with 0, 0.02, 0.2, or 2 mg/kg per d perindopril for 12 wk. Aseparate group of diabetic Ren-2 rats received late treatmentwith 2 mg/kg per d perindopril throughout week 8 to week 12,when rats were hypertensive and albuminuric and exhibited increasedkidney weight and glomerulosclerotic index (GSI). Among diabeticrats, early 0.02 mg/kg per d perindopril treatment reduced systolicBP, GSI, and renal collagen staining but had no effect on albuminuriaor kidney hypertrophy. Early 0.2 or 2 mg/kg per d perindopriltreatment further reduced systolic BP, GSI, and renal collagenstaining and decreased albuminuria and kidney hypertrophy. Lateintervention was as antihypertensive and antialbuminuric asearly 0.2 or 2 mg/kg per d perindopril treatment but did notprevent a moderate increase in GSI. In conclusion, early treatmentwith 0.2 mg/kg per d perindopril was the lowest dosage to largelyprevent severe diabetic nephropathy in transgenic Ren-2 rats.Late-onset perindopril treatment of diabetic rats with establishednephropathy was as efficacious as early treatment with respectto various renal parameters, such as albuminuria, but was associatedwith moderate progression of glomerulosclerosis.
In the past two decades, angiotensin-converting enzyme (ACE)inhibitors have been an effective therapy for the ameliorationof both human and experimental diabetic nephropathy (1–5).Hypertensive, transgenic, (mRen-2)27 rats with streptozotocin(STZ)-induced diabetes mellitus provide an excellent model forthe evaluation of such therapies because, unlike other rodentmodels of insulin-dependent diabetes mellitus, these rats progressivelydevelop severe glomerulosclerosis and tubulointerstitial injury,with many similarities to the human condition (3,6). The diabeticrenal lesions in Ren-2 rats are associated with upregulationof the juxtaglomerular and proximal tubule renin-angiotensinsystems (RAS), indicating a role for the tissue RAS in the pathogenesisof diabetic renal lesions (3,6).
We previously examined the effects of the ACE inhibitor perindopril,the angiotensin type 1 receptor blocker valsartan, and a combinationof the two agents on diabetic renal lesions in transgenic Ren-2rats (7). Although all regimens largely prevented diabetic nephropathy,we noted that high doses of perindopril (6 mg/kg per d, administeredin drinking water) could be reduced (to 0.5 mg/kg per d) whenthe drug was administered by gavage and combined with low dosesof valsartan (7). Furthermore, most studies of experimentalrenal disease demonstrated renoprotective effects when ACE inhibitorswere administered at the time of or shortly after the nephrotoxicinsult (1,3,8–10). Fewer studies examined the effectsof ACE inhibition initiated in the presence of established renallesions (11–16).
The first objective of this study was to determine the lowestdose of perindopril that would prevent severe diabetic kidneydisease in Ren-2 rats. The second objective was to examine thepossibility that late intervention would prevent or retard thedevelopment of diabetic renal lesions, because, in the clinicalsetting, commencement of ACE inhibitor therapy often beginsafter the onset of diabetic nephropathy. Late-onset perindopriltreatment was administered to diabetic Ren-2 rats that werehypertensive and albuminuric and exhibited kidney hypertrophyand mild glomerulosclerosis.
Animals
Six-week-old, female, heterozygous (mRen-2)27 rats, weighing130 ± 5 g, were randomized to receive either 55 mg/kgSTZ (Sigma Chemical Co., St. Louis, MO) diluted in 0.1 M citratebuffer (pH 4.5) or citrate buffer alone (nondiabetic animals),by tail vein injection, after an overnight fast. For the early-treatmentand dose-response study, rats were rerandomized to receive theACE inhibitor perindopril (Servier Laboratories, Paris, France)at doses of 0, 0.02, 0.2, or 2 mg/kg per d, by gavage, from2 d after diabetes mellitus induction or vehicle administration.Rats were routinely studied for 12 wk. For assessment of theeffects of late perindopril treatment, separate groups of nondiabeticand diabetic Ren-2 rats were administered perindopril (2 mg/kgper d), by gavage, from week 8 to week 12. The control animalsfor the late-intervention study were untreated nondiabetic anddiabetic Ren-2 rats assessed at 8 wk, for determination of theextent of renal damage at that time.
All rats were housed in a stable environment (maintained at20 ± 2°C, with a 12-h light/dark cycle) and allowedfree access to tap water and standard rat chow (GR2; Clark-King& Co., NSW, Australia). Each week, rats were weighed andblood glucose levels (nondiabetic, 4 to 8 mM; diabetic, 18 to27 mM) were estimated by using an Accutrend Alpha glucometer(Boehringer Mannheim, Victoria, Australia). Diabetic animalsreceived daily injections of insulin (Ultratard, 4 to 8 U, administeredintraperitoneally; Novo Nordisk, Bagsvaerd, Denmark), to promoteweight gain and prevent ketonuria. Every 2 wk, systolic BP (SBP)measurements in prewarmed conscious rats were made by usingtail-cuff plethysmography (17). Arterial pressure changes detectedwith a PE-300 pneumatic pulse transducer (Narco Biosystems Inc.,TX) were recorded by using the Chart program (version 3.5) ona Maclab/2E system (AD Instruments, Victoria, Australia). SBPwas recorded at the same time each day (2:00 to 5:00 p.m.),to minimize circadian influences (5 to 6 h after treatment administration),from an average of at least three consecutive measurements,to reduce variability (17). All experimental procedures adheredto the guidelines of the National Health and Medical ResearchCouncil of Australia Code for the Care and Use of Animals forScientific Purposes and were approved by the Bioethics Committeeof the University of Melbourne.
Renal Function
Plasma creatinine levels were determined before euthanasia byusing an autoanalyzer (ASTRA; Beckman, Palo Alto, CA) (18).For estimation of the albumin excretion rate, Ren-2 rats wereindividually housed in metabolic cages for 24 h, at 4, 8, and12 wk after STZ or vehicle administration. During the 24-h period,rats continued to have free access to tap water and standardlaboratory chow. Albumin concentrations were determined in aliquotsof urine by using a double-antibody RIA, as described previously(3).
Collection of Kidneys for Renin Assays and Histologic Assessments
At 8 or 12 wk after STZ or vehicle administration, Ren-2 ratswere anesthetized with pentobarbital sodium (Nembutal, 50 mg/kgbody wt, administered intraperitoneally; Boehringer Ingelheim,NSW, Australia) and perfused, via the abdominal aorta, with0.1 M phosphate-buffered saline (approximately 150 ml, pH 7.4,180 to 220 mmHg) for 1 to 2 min, for removal of circulatingblood. For renin assays, the left kidney was clamped at therenal artery, rapidly removed, weighed, placed in phosphate-bufferedsaline (1 ml), and snap-frozen in liquid nitrogen. For histologicanalyses, the right kidney was then perfusion-fixed for 5 minwith 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4;BDH Laboratory Supplies, Poole, England), sliced transversely,and postfixed overnight. After routine processing through gradedalcohols and Histolene (Australia Biostain, Traralgon, Victoria,Australia), the kidneys were embedded in paraffin and sectionedat 3 µm.
Kidney Renin Assays
Before assay, the kidney samples were thawed, homogenized, andrefrozen twice. Kidney samples were analyzed for renin contentsby using an enzyme kinetic method, with hog renin (NationalStandards Laboratory, London, UK) as the reference standardand 24-h nephrectomized rat plasma as the angiotensinogen substrate(3). All duplicate standards and samples were incubated for30 min at 37°C with adequate concentrations of angiotensinogen(1 µM) and angiotensinase inhibitors. The renin amountspresent in the samples were estimated by extrapolation froma standard curve constructed with serial dilutions of angiotensinI, referenced against the amount of angiotensin I generatedby 2 x 10-6 Goldblatt U of the hog renin standard (NationalStandards Laboratory).
Kidney Histopathologic Analyses
At least six randomly selected sections from each kidney werestained with hematoxylin and eosin (for examination of cellstructure), periodic acid-Schiff reagent (for identificationof basement membrane changes and glycogen deposition), or Masson’smodified trichrome stain (for demonstration of collagen matrix)(3). Changes in kidney structure were then analyzed in a double-blindedmanner. In sections stained with periodic acid-Schiff reagent,a glomerulosclerotic index (GSI) was assessed. Briefly, 150to 200 randomly chosen glomeruli from each rat kidney were gradedfor sclerosis. Glomeruli were carefully graded in a sequentialmanner, to avoid grading the same glomerulus twice. Glomerulosclerosiswas defined as glomerular basement membrane thickening, mesangialhypertrophy, and capillary occlusion. The degree of sclerosisin each glomerulus was subjectively graded on a scale of 0 to4, as follows: grade 0, normal; grade 1, sclerotic area of upto 25% (minimal); grade 2, sclerotic area of 25 to 50% (moderate);grade 3, sclerotic area of 50 to 75%; grade 4, sclerotic areaof 75 to 100% (severe). The GSI was then calculated using theformula
(1)
where Fi is the percentage of glomeruli in the rat with a givenscore of i.
Kidney Collagen Staining
In sections stained with Masson’s trichrome stain, collagenstaining was separately quantitated in the kidney cortex andmedulla. Six slides per group were analyzed in a blinded manner.The proportional area of blue stain in each section was recordedby using Analytical Imaging software (version 4.0; AIS, Ontario,Canada) and the average from four fields was calculated, toyield a relative value of collagen staining per field (19).
Statistical Analyses
All results are presented as means ± SEM. Comparisonsof normally distributed variables between nondiabetic and diabeticgroups were analyzed by ANOVA, followed by Fisher’s posthoc comparisons. Kidney renin contents were analyzed by usingtwo-sample t tests for unequal variance. When the data consistedof repeated measures at successive time points, an ANOVA forrepeated measures was used to identify significant between-groupdifferences. P < 0.05 was considered statistically significant.
SBP
Baseline (t = 0) SBP values recorded for 6-wk-old Ren-2 ratsbefore treatment were similar in all groups (151 ± 3mmHg) (Figure 1). During the study, SBP values were not significantlydifferent between untreated nondiabetic (Figure 1A) and diabetic(Figure 1B) Ren-2 rats. Early treatment with 0.02 mg/kg perd perindopril decreased SBP, compared with values for untreatednondiabetic or diabetic rats. SBP was further reduced with early0.2 or 2 mg/kg per d perindopril treatment. Late interventionreduced SBP to a similar extent, compared with early treatmentwith 0.2 or 2 mg/kg per d perindopril (Figure 1).
Figure 1. Systolic BP (SBP) in nondiabetic (A) and streptozotocin (STZ) diabetic (B) female, heterozygous, transgenic (mRen-2)27 rats during the 12-wk study period. The dosage of perindopril was 0 (untreated) (), 0.02 mg/kg per d (), 0.2 mg/kg per d (), or 2 mg/kg per d (). Late treatment with 2 mg/kg per d perindopril (x) was 8 to 12 wk after diabetes mellitus induction or control vehicle administration. Values are expressed as mean ± SEM. *P < 0.001, compared with the untreated nondiabetic group. #P < 0.001, compared with the untreated diabetic group. P < 0.001, compared with nondiabetic rats treated with 0.02 mg/kg per d perindopril. P < 0.001, compared with diabetic rats treated with 0.02 mg/kg per d perindopril.
Body and Kidney Weights
After 12 wk, diabetes mellitus was associated with reduced weightgain, compared with untreated nondiabetic rats at 8 and 12 wk(Table 1). Only early treatment with 2 mg/kg per d perindoprilattenuated the decrease in body weights among diabetic rats.Kidney weights were increased to similar extents for 8- and12-wk untreated diabetic rats, compared with the respectiveuntreated nondiabetic animals (Table 1). For diabetic rats,kidney weights were reduced with early 0.2 mg/kg per d perindopriltreatment and were further improved with early or late 2 mg/kgper d perindopril treatment.
Table 1. Physical and biochemical parameters 8 and 12 wk postinduction of streptozotocin diabetes or control vehicle in female heterozygous transgenic (mRen-2)27 ratsa
Kidney Renin Contents
Diabetes mellitus was associated with an increase in kidneyrenin contents (Table 1). For both nondiabetic and diabeticRen-2 rats, early treatment with 0.02 mg/kg per d perindoprildid not alter kidney renin contents. Early treatment with 0.2or 2 mg/kg per d perindopril and late intervention increasedkidney renin contents in both nondiabetic and diabetic rats;the increase was less marked in diabetic animals.
Renal Function
Plasma creatinine levels were increased with diabetes mellitusat 12 wk and were reduced to nondiabetic control values withearly or late 2 mg/kg per d perindopril treatment (Table 1).The albuminuria results are depicted in Figure 2. At 8 and 12wk of diabetes mellitus, albuminuria was increased, comparedwith age-matched, nondiabetic, control animals. For diabeticrats, albuminuria was reduced with early 0.02 mg/kg per d perindopriltreatment and was further reduced with higher doses of perindopril.Late intervention reduced albuminuria in diabetic Ren-2 ratsto a similar extent, compared with early treatment with 0.2or 2 mg/kg per d perindopril.
Figure 2. Twenty-four-hour urinary albumin output by nondiabetic (A) and STZ diabetic (B) female, heterozygous, transgenic (mRen-2)27 rats. The dosage of perindopril was 0 (untreated) ), 0.02 mg/kg per d (), 0.2 mg/kg per d (), or 2 mg/kg per d (). Late treatment with 2 mg/kg per d perindopril (x) was 8 to 12 wk after diabetes mellitus induction or control vehicle administration. Values are expressed as mean ± SEM. *P < 0.01, compared with untreated diabetic rats at 4 wk. **P < 0.05, compared with all nondiabetic groups at 8 wk. #P < 0.05, compared with all nondiabetic groups at 12 wk. ##P < 0.01, compared with all nondiabetic groups at 12 wk. P < 0.01, compared with untreated diabetic rats at 12 wk.
Glomerulosclerosis
In the kidney cortex of nondiabetic Ren-2 rats, most glomeruliappeared normal at 8 and 12 wk and there was no evidence ofinterstitial fibrosis or tubular degeneration (Figure 3A). Incontrast, after 8 wk of diabetes mellitus, glomerular basementmembrane thickening was evident and the GSI was increased (Figures 3B and 4A).By 12 wk of diabetes mellitus, the GSI was furtherincreased and many severely sclerotic glomeruli were apparent(Figures 3C and 4A). In addition, the cortical interstitiumappeared to be expanded, and numerous tubules were vacuolatedand contained glycogen deposits (Figure 3C). In diabetic ratstreated with early 0.02 mg/kg per d perindopril administration,glomerular basement membrane thickening and the GSI were decreased,compared with untreated diabetic rats (Figures 3D and 4A). Indiabetic rats treated with early 0.2 or 2 mg/kg per d perindopriladministration, the GSI was further reduced (Figures 3E and 4A).In diabetic rats treated with late-intervention perindopril,the GSI was similar to that for rats treated with early 0.2or 2 mg/kg per d perindopril administration but was moderatelyincreased, compared with 8-wk, untreated, diabetic, Ren-2 rats(Figures 3F and 4A).
Figure 3. Histopathologic features of the kidney cortex from nondiabetic and STZ diabetic heterozygous, transgenic, (mRen-2)27 rats. Sections were stained with periodic acid-Schiff reagent. G, glomerulus. Magnification, x300. (A) Untreated nondiabetic rat at 12 wk. (B) Untreated diabetic rat at 8 wk. (C) Untreated diabetic rat at 12 wk, with damaged glomeruli, vacuolated cortical tubules (asterisk), and expanded cortical interstitium (arrow). (D) Diabetic Ren-2 rat treated with early 0.02 mg/kg per d perindopril. (E) Diabetic Ren-2 rat treated with early 0.2 mg/kg per d perindopril. (F) Diabetic Ren-2 rat treated with late intervention.
Figure 4. Glomerulosclerotic index (A) and the proportional area of collagen staining in the kidney cortex (B) and medulla (C) in nondiabetic () and STZ diabetic () heterozygous, transgenic, (mRen-2)27 rats (n > 6/group). P, dose of perindopril (0, 0.02, 0.2, or 2 mg/kg per d). Values are expressed as mean ± SEM. *P < 0.05, compared with the respective nondiabetic group. **P < 0.01, compared with the respective nondiabetic group. #P < 0.05, compared with untreated diabetic rats at 12 wk. ##P < 0.01, compared with untreated diabetic rats at 12 wk. P < 0.01, compared with untreated diabetic rats at 8 wk. P < 0.05, compared with untreated nondiabetic rats at 12 wk. P < 0.01, compared with untreated nondiabetic rats at 12 wk.
Kidney Collagen Staining and Medullary Pathologic Analyses
Among untreated Ren-2 rats, cortical collagen staining was similarin nondiabetic and diabetic rats at 8 wk but was increased innondiabetic rats and further increased in diabetic animals by12 wk (Figure 4B). All doses of perindopril and late interventionreduced cortical collagen staining to similar extents in bothnondiabetic and diabetic rats (Figure 4B). The kidney medullaof untreated nondiabetic rats appeared normal at 12 wk (Figures 4C and 5A).In contrast, in four of six kidneys from untreateddiabetic Ren-2 rats at 12 wk, severe pathologic features wereobserved in the medulla, consisting of dilated tubules, inflammatorycells, and large areas of collagen deposition (Figures 4C and 5B).One of the six kidneys from untreated diabetic rats exhibitednecrosis of the inner medulla. The lowest dose of perindopril(0.02 mg/kg per d) reduced medullary collagen staining in diabeticrats, compared with untreated diabetic animals (Figure 4C).Higher-dose, early perindopril treatment further reduced medullarycollagen staining in diabetic rats; however, some tubules remaineddilated (Figures 4C and 5C). Late intervention prevented anincrease in medullary collagen staining in diabetic Ren-2 rats,compared with untreated diabetic Ren-2 rats, at 8 wk (Figures 4C and 5D).
Figure 5. Histopathologic features of the kidney medulla from nondiabetic and STZ diabetic heterozygous, transgenic, (mRen-2)27 rats. Sections were stained with Masson’s trichrome stain. Magnification, x150. (A) Untreated nondiabetic rat at 12 wk. (B) Untreated diabetic rat at 12 wk, with marked tubular dilation (asterisk), inflammation, and collagen deposition. (C) Diabetic Ren-2 rat treated with early 0.2 mg/kg per d perindopril. (D) Diabetic Ren-2 rat treated with late intervention.
This study demonstrates that 0.2 mg/kg per d is the lowest doseof perindopril required to prevent severe cortical and medullaryrenal pathologic changes and to normalize albuminuria and BPin diabetic Ren-2 rats. A tenfold lower dose of perindoprilmoderately improved SBP and glomerulosclerosis and normalizedcortical collagen staining but did not reduce albuminuria andkidney hypertrophy. With respect to the efficacy of late intervention,treatment with 2 mg/kg per d perindopril beginning at a timewhen diabetic Ren-2 rats exhibited albuminuria, kidney hypertrophy,and mild glomerulosclerosis was equally antihypertensive andantialbuminuric, compared with 0.2 or 2 mg/kg per d perindopriladministration from the time of diabetes mellitus induction.However, late intervention did not prevent the progression ofglomerulosclerosis in diabetic Ren-2 rats.
ACE inhibitors such as perindopril represent first-line therapyfor diabetic patients, reducing BP and albuminuria and preservingrenal function (20–24). The few studies that have examinedthe effects of perindopril in experimental diabetic nephropathyhave demonstrated improvements in the albuminuria and mild renalpathologic features that develop in diabetic Sprague-Dawleyrats and spontaneously hypertensive rats (25,26). To our knowledge,this study is the first to determine the lowest dose of perindoprilrequired to preserve renal function and structure in a modelof advanced diabetic renal disease. The dose of 0.2 mg/kg perd is th of the dose that we previously reported conferred renoprotection in diabetic Ren-2 rats (3). In contrastto the previous study, in which perindopril was administeredin drinking water (3), in this study rats received perindoprilby daily gavage, to achieve accurate dosing and mimic the clinicalsetting of drug administration. The 0.2 mg/kg per d dose ofperindopril in rats is equivalent to 2.3 mg for a 75-kg humanpatient, on a body weight basis, which is close to the minimaldaily recommended dose of perindopril for hypertensive humansubjects (27–30).
Some of the beneficial effects of ACE inhibition on hypertensiveorgan pathologic changes have been observed at nonhypotensivedoses, indicating a role for the tissue-based RAS in the developmentof organ damage (31–33). In transgenic Ren-2 rats, whichprogressively develop hypertension-related cardiac and renalinjuries, nonhypotensive doses of ramipril or the angiotensintype 1 receptor antagonist telmisartan reduced organ damage,including glomerulosclerosis (32,33). In this study, the lowestdose of perindopril (0.02 mg/kg per d) was initially nonhypotensive;however, a moderate reduction in BP was observed by week 4 oftreatment and was then sustained throughout the experimentalperiod. A similar finding was reported for rabbits, with a doseof 0.01 mg/kg per d perindopril (34). Although we observed thatthe lowest dose of perindopril reduced the progression of corticaland medullary collagen deposition, glomerulosclerosis and albuminuriawere prevented only with higher and more antihypertensive dosesof perindopril. These results suggest a role for the hypertensionof Ren-2 rats in the pathogenesis of advanced diabetic renaldisease. Hypertension has been demonstrated to be an importantfactor in the development of albuminuria and glomerulosclerosisin both nondiabetic and diabetic models of nephropathy (35,36).At variance with these findings are reports by us and by othersof an association between the tissue-based RAS and experimentaldiabetic nephropathy (3,6,19,37–39). In diabetic Ren-2rats, kidney renin contents are increased and upregulation ofjuxtaglomerular and proximal tubular renin is associated withglomerulosclerosis, tubulointerstitial injury, and overexpressionof prosclerotic cytokines (3,6). In addition, some antihypertensiveregimens, such as combined endothelin type A and type B receptorantagonism, do not confer renoprotection in this diabetic model(6). Overall, these findings suggest that upregulation of thesite-specific tissue RAS contributes to the pathogenesis ofdiabetic nephropathy in Ren-2 rats.
There is considerable evidence that ACE inhibition initiatedshortly after the induction of renal disease prevents or substantiallyreduces the development of nephropathy (3,8,9,11,14,16,40).Less clear is the effect of late ACE inhibition on the progressionand reversal of established nephropathy; however, in general,early treatment seems to be more renoprotective (1,8–11,14,16).The few studies that examined early versus late ACE interventionin experimental diabetes mellitus demonstrated that early butnot late treatment normalized glomerulosclerosis and albuminuriain rat models of mild diabetic nephropathy (12,41). The noveltyof the study presented here is the use of diabetic transgenicRen-2 rats, which develop severe nephropathy (3). Consistentwith previous studies, we observed that ACE inhibition in animalswith established renal lesions normalized hypertension and didnot retard the progression of glomerulosclerosis; however, albuminuriawas improved. Whether even later ACE inhibition would retardmore advanced glomerulosclerosis or tubulointerstitial diseasewas not addressed in this study; however, it was previouslyreported that RAS blockade has both beneficial (15) and nonbeneficialeffects (14,42) on severe glomerulosclerosis.
In both animal and human studies, there is evidence of a causalrelationship between albuminuria and the development of glomerulosclerosis(43,44). Less well defined is whether albuminuria induces tubulardisease early in the pathogenesis of diabetic nephropathy. Inthis study, Ren-2 rats at 8 wk of diabetes mellitus exhibitedincreased albuminuria and a moderate increase in glomerulosclerosis,with no evidence of tubulointerstitial damage. The advancedrenal disease observed at 12 wk of diabetes mellitus consistedof an additional increase in albuminuria and glomerulosclerosisand the appearance of marked renal collagen deposition, tubularvacuolation, and inflammation. These findings are consistentwith enhanced protein trafficking and reabsorption occurringlate in the course of diabetic nephropathy and eventually leadingto chronic nephropathy (45). Furthermore, our findings supporta recent study of type 1 diabetic patients, for whom mesangialexpansion was determined to be the central variable in the transitionto microalbuminuria or early diabetic nephropathy (46). Expansionof the renal interstitium was not detected in this study, indicatingthat tubulointerstitial pathologic changes may occur as a consequenceof glomerular injury.
In conclusion, this study provides evidence that, in diabeticRen-2 rats with advanced renal disease, 0.2 mg/kg per d is thelowest dose of perindopril required to attenuate diabetic nephropathy.Administration of perindopril to animals with established lesionsdoes confer renoprotection but does not prevent the progressionof glomerulosclerosis after mild lesions have been established.
Acknowledgments
This project was supported by grants from the National Healthand Medical Research Council of Australia. Dr. Kelly is a JuvenileDiabetes Foundation International Research Fellow. We thankServier Laboratories (Paris, France) for providing perindopriland Prof. Detlev Ganten and Ursula Ganten for donating the transgenicrats required to establish our existing colony. We acknowledgeBronwyn Rees for providing technical assistance and BelindaDavis for conducting the albuminuria assays.
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Received for publication June 27, 2001.
Accepted for publication September 17, 2001.
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Abstract
Introduction
th of the dose that we previously reported conferred renoprotection in diabetic Ren-2 rats (3). In contrast to the previous study, in which perindopril was administered in drinking water (3), in this study rats received perindopril by daily gavage, to achieve accurate dosing and mimic the clinical setting of drug administration. The 0.2 mg/kg per d dose of perindopril in rats is equivalent to 2.3 mg for a 75-kg human patient, on a body weight basis, which is close to the minimal daily recommended dose of perindopril for hypertensive human subjects (27–30). 
), 0.02 mg/kg per d (
), 0.2 mg/kg per d (
), or 2 mg/kg per d (
). Late treatment with 2 mg/kg per d perindopril (x) was 8 to 12 wk after diabetes mellitus induction or control vehicle administration. Values are expressed as mean ± SEM. *P < 0.001, compared with the untreated nondiabetic group. #P < 0.001, compared with the untreated diabetic group. 
) heterozygous, transgenic, (mRen-2)27 rats (n > 6/group). P, dose of perindopril (0, 0.02, 0.2, or 2 mg/kg per d). Values are expressed as mean ± SEM. *P < 0.05, compared with the respective nondiabetic group. **P < 0.01, compared with the respective nondiabetic group. #P < 0.05, compared with untreated diabetic rats at 12 wk. ##P < 0.01, compared with untreated diabetic rats at 12 wk. 
P < 0.05, compared with untreated nondiabetic rats at 12 wk. 