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J Am Soc Nephrol 13:684-692, 2002
© 2002 American Society of Nephrology

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{dagger}, Darren J. Kelly{ddagger} and Jennifer L. Wilkinson-Berka*

*Department of Physiology, University of Melbourne, Parkville, Australia; {dagger}Department of Medicine, Austin and Repatriation Medical Centre, West Heidelberg, Australia; and {ddagger}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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. It was previously reported that transgenic (mRen-2)27 rats with streptozotocin-induced diabetes mellitus progressively develop advanced nephropathy in 12 wk. These lesions are largely prevented when the angiotensin-converting enzyme inhibitor perindopril is administered from the time of induction of diabetes mellitus. This study aimed to determine the lowest dose of early perindopril treatment required for substantial improvement of renal function and structure and to investigate whether late intervention prevents or reverses the progression of established renal lesions. At 6 wk of age, female heterozygous Ren-2 rats were randomized to receive either streptozotocin (diabetic) or citrate buffer (control). Rats were gavaged, beginning early after the induction of diabetes mellitus or the administration of control vehicle, with 0, 0.02, 0.2, or 2 mg/kg per d perindopril for 12 wk. A separate group of diabetic Ren-2 rats received late treatment with 2 mg/kg per d perindopril throughout week 8 to week 12, when rats were hypertensive and albuminuric and exhibited increased kidney weight and glomerulosclerotic index (GSI). Among diabetic rats, early 0.02 mg/kg per d perindopril treatment reduced systolic BP, GSI, and renal collagen staining but had no effect on albuminuria or kidney hypertrophy. Early 0.2 or 2 mg/kg per d perindopril treatment further reduced systolic BP, GSI, and renal collagen staining and decreased albuminuria and kidney hypertrophy. Late intervention was as antihypertensive and antialbuminuric as early 0.2 or 2 mg/kg per d perindopril treatment but did not prevent a moderate increase in GSI. In conclusion, early treatment with 0.2 mg/kg per d perindopril was the lowest dosage to largely prevent severe diabetic nephropathy in transgenic Ren-2 rats. Late-onset perindopril treatment of diabetic rats with established nephropathy was as efficacious as early treatment with respect to various renal parameters, such as albuminuria, but was associated with moderate progression of glomerulosclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the past two decades, angiotensin-converting enzyme (ACE) inhibitors have been an effective therapy for the amelioration of both human and experimental diabetic nephropathy (1–5). Hypertensive, transgenic, (mRen-2)27 rats with streptozotocin (STZ)-induced diabetes mellitus provide an excellent model for the evaluation of such therapies because, unlike other rodent models of insulin-dependent diabetes mellitus, these rats progressively develop severe glomerulosclerosis and tubulointerstitial injury, with many similarities to the human condition (3,6). The diabetic renal lesions in Ren-2 rats are associated with upregulation of the juxtaglomerular and proximal tubule renin-angiotensin systems (RAS), indicating a role for the tissue RAS in the pathogenesis of diabetic renal lesions (3,6).

We previously examined the effects of the ACE inhibitor perindopril, the angiotensin type 1 receptor blocker valsartan, and a combination of the two agents on diabetic renal lesions in transgenic Ren-2 rats (7). Although all regimens largely prevented diabetic nephropathy, we noted that high doses of perindopril (6 mg/kg per d, administered in drinking water) could be reduced (to 0.5 mg/kg per d) when the drug was administered by gavage and combined with low doses of valsartan (7). Furthermore, most studies of experimental renal disease demonstrated renoprotective effects when ACE inhibitors were administered at the time of or shortly after the nephrotoxic insult (1,3,8–10). Fewer studies examined the effects of ACE inhibition initiated in the presence of established renal lesions (11–16).

The first objective of this study was to determine the lowest dose of perindopril that would prevent severe diabetic kidney disease in Ren-2 rats. The second objective was to examine the possibility that late intervention would prevent or retard the development of diabetic renal lesions, because, in the clinical setting, commencement of ACE inhibitor therapy often begins after the onset of diabetic nephropathy. Late-onset perindopril treatment was administered to diabetic Ren-2 rats that were hypertensive and albuminuric and exhibited kidney hypertrophy and mild glomerulosclerosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Six-week-old, female, heterozygous (mRen-2)27 rats, weighing 130 ± 5 g, were randomized to receive either 55 mg/kg STZ (Sigma Chemical Co., St. Louis, MO) diluted in 0.1 M citrate buffer (pH 4.5) or citrate buffer alone (nondiabetic animals), by tail vein injection, after an overnight fast. For the early-treatment and dose-response study, rats were rerandomized to receive the ACE inhibitor perindopril (Servier Laboratories, Paris, France) at doses of 0, 0.02, 0.2, or 2 mg/kg per d, by gavage, from 2 d after diabetes mellitus induction or vehicle administration. Rats were routinely studied for 12 wk. For assessment of the effects of late perindopril treatment, separate groups of nondiabetic and diabetic Ren-2 rats were administered perindopril (2 mg/kg per d), by gavage, from week 8 to week 12. The control animals for the late-intervention study were untreated nondiabetic and diabetic Ren-2 rats assessed at 8 wk, for determination of the extent of renal damage at that time.

All rats were housed in a stable environment (maintained at 20 ± 2°C, with a 12-h light/dark cycle) and allowed free access to tap water and standard rat chow (GR2; Clark-King & Co., NSW, Australia). Each week, rats were weighed and blood glucose levels (nondiabetic, 4 to 8 mM; diabetic, 18 to 27 mM) were estimated by using an Accutrend Alpha glucometer (Boehringer Mannheim, Victoria, Australia). Diabetic animals received daily injections of insulin (Ultratard, 4 to 8 U, administered intraperitoneally; Novo Nordisk, Bagsvaerd, Denmark), to promote weight gain and prevent ketonuria. Every 2 wk, systolic BP (SBP) measurements in prewarmed conscious rats were made by using tail-cuff plethysmography (17). Arterial pressure changes detected with a PE-300 pneumatic pulse transducer (Narco Biosystems Inc., TX) were recorded by using the Chart program (version 3.5) on a Maclab/2E system (AD Instruments, Victoria, Australia). SBP was 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 adhered to the guidelines of the National Health and Medical Research Council of Australia Code for the Care and Use of Animals for Scientific Purposes and were approved by the Bioethics Committee of the University of Melbourne.

Renal Function
Plasma creatinine levels were determined before euthanasia by using an autoanalyzer (ASTRA; Beckman, Palo Alto, CA) (18). For estimation of the albumin excretion rate, Ren-2 rats were individually housed in metabolic cages for 24 h, at 4, 8, and 12 wk after STZ or vehicle administration. During the 24-h period, rats continued to have free access to tap water and standard laboratory chow. Albumin concentrations were determined in aliquots of 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 rats were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg body wt, administered intraperitoneally; Boehringer Ingelheim, NSW, Australia) and perfused, via the abdominal aorta, with 0.1 M phosphate-buffered saline (approximately 150 ml, pH 7.4, 180 to 220 mmHg) for 1 to 2 min, for removal of circulating blood. For renin assays, the left kidney was clamped at the renal artery, rapidly removed, weighed, placed in phosphate-buffered saline (1 ml), and snap-frozen in liquid nitrogen. For histologic analyses, the right kidney was then perfusion-fixed for 5 min with 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 graded alcohols and Histolene (Australia Biostain, Traralgon, Victoria, Australia), the kidneys were embedded in paraffin and sectioned at 3 µm.

Kidney Renin Assays
Before assay, the kidney samples were thawed, homogenized, and refrozen twice. Kidney samples were analyzed for renin contents by using an enzyme kinetic method, with hog renin (National Standards Laboratory, London, UK) as the reference standard and 24-h nephrectomized rat plasma as the angiotensinogen substrate (3). All duplicate standards and samples were incubated for 30 min at 37°C with adequate concentrations of angiotensinogen (1 µM) and angiotensinase inhibitors. The renin amounts present in the samples were estimated by extrapolation from a standard curve constructed with serial dilutions of angiotensin I, referenced against the amount of angiotensin I generated by 2 x 10-6 Goldblatt U of the hog renin standard (National Standards Laboratory).

Kidney Histopathologic Analyses
At least six randomly selected sections from each kidney were stained with hematoxylin and eosin (for examination of cell structure), periodic acid-Schiff reagent (for identification of basement membrane changes and glycogen deposition), or Masson’s modified trichrome stain (for demonstration of collagen matrix) (3). Changes in kidney structure were then analyzed in a double-blinded manner. In sections stained with periodic acid-Schiff reagent, a glomerulosclerotic index (GSI) was assessed. Briefly, 150 to 200 randomly chosen glomeruli from each rat kidney were graded for sclerosis. Glomeruli were carefully graded in a sequential manner, to avoid grading the same glomerulus twice. Glomerulosclerosis was defined as glomerular basement membrane thickening, mesangial hypertrophy, and capillary occlusion. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4, as follows: grade 0, normal; grade 1, sclerotic area of up to 25% (minimal); grade 2, sclerotic area of 25 to 50% (moderate); grade 3, sclerotic area of 50 to 75%; grade 4, sclerotic area of 75 to 100% (severe). The GSI was then calculated using the formula

Go


(1)

where Fi is the percentage of glomeruli in the rat with a given score of i.

Kidney Collagen Staining
In sections stained with Masson’s trichrome stain, collagen staining was separately quantitated in the kidney cortex and medulla. Six slides per group were analyzed in a blinded manner. The proportional area of blue stain in each section was recorded by using Analytical Imaging software (version 4.0; AIS, Ontario, Canada) and the average from four fields was calculated, to yield a relative value of collagen staining per field (19).

Statistical Analyses
All results are presented as means ± SEM. Comparisons of normally distributed variables between nondiabetic and diabetic groups were analyzed by ANOVA, followed by Fisher’s post hoc comparisons. Kidney renin contents were analyzed by using two-sample t tests for unequal variance. When the data consisted of repeated measures at successive time points, an ANOVA for repeated measures was used to identify significant between-group differences. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SBP
Baseline (t = 0) SBP values recorded for 6-wk-old Ren-2 rats before treatment were similar in all groups (151 ± 3 mmHg) (Figure 1). During the study, SBP values were not significantly different between untreated nondiabetic (Figure 1A) and diabetic (Figure 1B) Ren-2 rats. Early treatment with 0.02 mg/kg per d perindopril decreased SBP, compared with values for untreated nondiabetic or diabetic rats. SBP was further reduced with early 0.2 or 2 mg/kg per d perindopril treatment. Late intervention reduced SBP to a similar extent, compared with early treatment with 0.2 or 2 mg/kg per d perindopril (Figure 1).



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  		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) ({circ}), 0.02 mg/kg per d ({diamond}), 0.2 mg/kg per d ({triangleup}), or 2 mg/kg per d ({square}). 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. {dagger}P < 0.001, compared with nondiabetic rats treated with 0.02 mg/kg per d perindopril. {ddagger}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 weight gain, compared with untreated nondiabetic rats at 8 and 12 wk (Table 1). Only early treatment with 2 mg/kg per d perindopril attenuated the decrease in body weights among diabetic rats. Kidney weights were increased to similar extents for 8- and 12-wk untreated diabetic rats, compared with the respective untreated nondiabetic animals (Table 1). For diabetic rats, kidney weights were reduced with early 0.2 mg/kg per d perindopril treatment and were further improved with early or late 2 mg/kg per d perindopril treatment.


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  		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 kidney renin contents (Table 1). For both nondiabetic and diabetic Ren-2 rats, early treatment with 0.02 mg/kg per d perindopril did not alter kidney renin contents. Early treatment with 0.2 or 2 mg/kg per d perindopril and late intervention increased kidney 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 mellitus at 12 wk and were reduced to nondiabetic control values with early or late 2 mg/kg per d perindopril treatment (Table 1). The albuminuria results are depicted in Figure 2. At 8 and 12 wk of diabetes mellitus, albuminuria was increased, compared with age-matched, nondiabetic, control animals. For diabetic rats, albuminuria was reduced with early 0.02 mg/kg per d perindopril treatment and was further reduced with higher doses of perindopril. Late intervention reduced albuminuria in diabetic Ren-2 rats to a similar extent, compared with early treatment with 0.2 or 2 mg/kg per d perindopril.



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  		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) {circ}), 0.02 mg/kg per d ({diamond}), 0.2 mg/kg per d ({triangleup}), or 2 mg/kg per d ({square}). 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. {dagger}P < 0.01, compared with untreated diabetic rats at 12 wk.

 
Glomerulosclerosis
In the kidney cortex of nondiabetic Ren-2 rats, most glomeruli appeared normal at 8 and 12 wk and there was no evidence of interstitial fibrosis or tubular degeneration (Figure 3A). In contrast, after 8 wk of diabetes mellitus, glomerular basement membrane thickening was evident and the GSI was increased (Figures 3B and 4A). By 12 wk of diabetes mellitus, the GSI was further increased and many severely sclerotic glomeruli were apparent (Figures 3C and 4A). In addition, the cortical interstitium appeared to be expanded, and numerous tubules were vacuolated and contained glycogen deposits (Figure 3C). In diabetic rats treated 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). In diabetic rats treated with early 0.2 or 2 mg/kg per d perindopril administration, 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 0.2 or 2 mg/kg per d perindopril administration but was moderately increased, compared with 8-wk, untreated, diabetic, Ren-2 rats (Figures 3F and 4A).



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  		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.

 


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  		Figure 4. Glomerulosclerotic index (A) and the proportional area of collagen staining in the kidney cortex (B) and medulla (C) in nondiabetic ({square}) and STZ diabetic ({blacksquare}) 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. {dagger}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 similar in nondiabetic and diabetic rats at 8 wk but was increased in nondiabetic rats and further increased in diabetic animals by 12 wk (Figure 4B). All doses of perindopril and late intervention reduced cortical collagen staining to similar extents in both nondiabetic and diabetic rats (Figure 4B). The kidney medulla of untreated nondiabetic rats appeared normal at 12 wk (Figures 4C and 5A). In contrast, in four of six kidneys from untreated diabetic Ren-2 rats at 12 wk, severe pathologic features were observed in the medulla, consisting of dilated tubules, inflammatory cells, and large areas of collagen deposition (Figures 4C and 5B). One of the six kidneys from untreated diabetic rats exhibited necrosis of the inner medulla. The lowest dose of perindopril (0.02 mg/kg per d) reduced medullary collagen staining in diabetic rats, compared with untreated diabetic animals (Figure 4C). Higher-dose, early perindopril treatment further reduced medullary collagen staining in diabetic rats; however, some tubules remained dilated (Figures 4C and 5C). Late intervention prevented an increase in medullary collagen staining in diabetic Ren-2 rats, compared with untreated diabetic Ren-2 rats, at 8 wk (Figures 4C and 5D).



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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that 0.2 mg/kg per d is the lowest dose of perindopril required to prevent severe cortical and medullary renal pathologic changes and to normalize albuminuria and BP in diabetic Ren-2 rats. A tenfold lower dose of perindopril moderately improved SBP and glomerulosclerosis and normalized cortical collagen staining but did not reduce albuminuria and kidney hypertrophy. With respect to the efficacy of late intervention, treatment with 2 mg/kg per d perindopril beginning at a time when diabetic Ren-2 rats exhibited albuminuria, kidney hypertrophy, and mild glomerulosclerosis was equally antihypertensive and antialbuminuric, compared with 0.2 or 2 mg/kg per d perindopril administration from the time of diabetes mellitus induction. However, late intervention did not prevent the progression of glomerulosclerosis in diabetic Ren-2 rats.

ACE inhibitors such as perindopril represent first-line therapy for diabetic patients, reducing BP and albuminuria and preserving renal function (20–24). The few studies that have examined the effects of perindopril in experimental diabetic nephropathy have demonstrated improvements in the albuminuria and mild renal pathologic features that develop in diabetic Sprague-Dawley rats and spontaneously hypertensive rats (25,26). To our knowledge, this study is the first to determine the lowest dose of perindopril required to preserve renal function and structure in a model of advanced diabetic renal disease. The dose of 0.2 mg/kg per d is 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).

Some of the beneficial effects of ACE inhibition on hypertensive organ pathologic changes have been observed at nonhypotensive doses, indicating a role for the tissue-based RAS in the development of organ damage (31–33). In transgenic Ren-2 rats, which progressively develop hypertension-related cardiac and renal injuries, nonhypotensive doses of ramipril or the angiotensin type 1 receptor antagonist telmisartan reduced organ damage, including glomerulosclerosis (32,33). In this study, the lowest dose of perindopril (0.02 mg/kg per d) was initially nonhypotensive; however, a moderate reduction in BP was observed by week 4 of treatment and was then sustained throughout the experimental period. A similar finding was reported for rabbits, with a dose of 0.01 mg/kg per d perindopril (34). Although we observed that the lowest dose of perindopril reduced the progression of cortical and medullary collagen deposition, glomerulosclerosis and albuminuria were prevented only with higher and more antihypertensive doses of perindopril. These results suggest a role for the hypertension of Ren-2 rats in the pathogenesis of advanced diabetic renal disease. Hypertension has been demonstrated to be an important factor in the development of albuminuria and glomerulosclerosis in both nondiabetic and diabetic models of nephropathy (35,36). At variance with these findings are reports by us and by others of an association between the tissue-based RAS and experimental diabetic nephropathy (3,6,19,37–39). In diabetic Ren-2 rats, kidney renin contents are increased and upregulation of juxtaglomerular and proximal tubular renin is associated with glomerulosclerosis, tubulointerstitial injury, and overexpression of prosclerotic cytokines (3,6). In addition, some antihypertensive regimens, such as combined endothelin type A and type B receptor antagonism, do not confer renoprotection in this diabetic model (6). Overall, these findings suggest that upregulation of the site-specific tissue RAS contributes to the pathogenesis of diabetic nephropathy in Ren-2 rats.

There is considerable evidence that ACE inhibition initiated shortly after the induction of renal disease prevents or substantially reduces the development of nephropathy (3,8,9,11,14,16,40). Less clear is the effect of late ACE inhibition on the progression and 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 intervention in experimental diabetes mellitus demonstrated that early but not late treatment normalized glomerulosclerosis and albuminuria in rat models of mild diabetic nephropathy (12,41). The novelty of the study presented here is the use of diabetic transgenic Ren-2 rats, which develop severe nephropathy (3). Consistent with previous studies, we observed that ACE inhibition in animals with established renal lesions normalized hypertension and did not retard the progression of glomerulosclerosis; however, albuminuria was improved. Whether even later ACE inhibition would retard more advanced glomerulosclerosis or tubulointerstitial disease was not addressed in this study; however, it was previously reported that RAS blockade has both beneficial (15) and nonbeneficial effects (14,42) on severe glomerulosclerosis.

In both animal and human studies, there is evidence of a causal relationship between albuminuria and the development of glomerulosclerosis (43,44). Less well defined is whether albuminuria induces tubular disease early in the pathogenesis of diabetic nephropathy. In this study, Ren-2 rats at 8 wk of diabetes mellitus exhibited increased albuminuria and a moderate increase in glomerulosclerosis, with no evidence of tubulointerstitial damage. The advanced renal disease observed at 12 wk of diabetes mellitus consisted of an additional increase in albuminuria and glomerulosclerosis and the appearance of marked renal collagen deposition, tubular vacuolation, and inflammation. These findings are consistent with enhanced protein trafficking and reabsorption occurring late in the course of diabetic nephropathy and eventually leading to chronic nephropathy (45). Furthermore, our findings support a recent study of type 1 diabetic patients, for whom mesangial expansion was determined to be the central variable in the transition to microalbuminuria or early diabetic nephropathy (46). Expansion of the renal interstitium was not detected in this study, indicating that tubulointerstitial pathologic changes may occur as a consequence of glomerular injury.

In conclusion, this study provides evidence that, in diabetic Ren-2 rats with advanced renal disease, 0.2 mg/kg per d is the lowest dose of perindopril required to attenuate diabetic nephropathy. Administration of perindopril to animals with established lesions does confer renoprotection but does not prevent the progression of glomerulosclerosis after mild lesions have been established.


   Acknowledgments
 
This project was supported by grants from the National Health and Medical Research Council of Australia. Dr. Kelly is a Juvenile Diabetes Foundation International Research Fellow. We thank Servier Laboratories (Paris, France) for providing perindopril and Prof. Detlev Ganten and Ursula Ganten for donating the transgenic rats required to establish our existing colony. We acknowledge Bronwyn Rees for providing technical assistance and Belinda Davis for conducting the albuminuria assays.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication June 27, 2001. Accepted for publication September 17, 2001.




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