Norberto Perico, Susana C. Amuchastegui, Vittoria Colosio, Giovanni Sonzogni, Tullio Bertani, and

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1 Evidence That an AngiotensinConverting Enzyme Inhibitor Has a Different Effect on Glomerular Injury According to the Different Phase of the Disease at Which the Treatment Is Started1 Norberto Perico, Susana C. Amuchastegui, Vittoria Colosio, Giovanni Sonzogni, Tullio Bertani, and Giuseppe Remuzzi2 N. Perico, S.C. Amuchastegui, V. Colosio, G. Sonzogni, I. Bertani, G. Remuzzi, Mario Negri Institute for Pharmacological Research, Bergamo, Italy N. Perico, I. Bertani, G. Remuzzi, Division of Nephrology and Dialysis, Bergamo, Italy (J. Am. Soc. Nephrol. 1994; 5: ) ABSTRACT In rats with streptozotocin-induced diabetes, the effect of an angiotensin-converting enzyme (ACE) inhibitor on the evolution of glomerular injury according to the time at which the treatment Is started with respect to the onset of the disease was studied. Three groups of animals were used, a control Group 1 and two groups of diabetic rats treated with insulin (Groups 2 and 3). The latter were monitored until urinary protein excretion reached 4 to 5 mg/24 h (on average, 23 wk after the induction ofthe diabetes). Atthis time, Group 2 continued to receive Insulin alone, whereas Group 3 was also given the ACE inhibitor moexipril for 8 more wk. Untreated diabetic rats showed a moderate increase in systolic blood pressure that was normalized by moexipril administration. Urinary protein excretion progressively increased during the 8-wk follow-up in untreated diabetics that, at the end of the study, developed moderate glomerular sclerosis. Moexipril treatment lowered urinary protein excretion to a normal range and completely prevented glomerular injury. Three other groups of rats were similarly treated, except that moexipril treatment was started later on (when proteinuria reached 1 to 2 mg/24 h, on average, 32 wk after the induction of diabetes), and were monitored for another 8 wk. Untreated and treated diabetics had comparable blood glucose levels throughout. Systolic blood pressure, significantly increased in untreated diabetic rats, was effectlvely controlled by moexipril administration. Unlike in the early phase of the disease, moexipril was unable r Received March 8, Accepted July 6, Correspondence to Dr. G. Remuzii, Mario Neg Institute for Pharmacological Research, Via Gavazzeni 1 1, Bergamo, Italy / / Journal of the American Society of Nephrology Copyright C 1994 by the American Society of Nephrology to prevent the progressive increase in urinary protein excretion as well as the glomerular sclerotic lesions that developed in untreated diabetics. These results indicates that, in a model of streptozotocin-induced diabetes, ACE inhibition affords remarkable protection against the development of diabetic nephropathy, but only when treatment is started early in the course of the disease. Key Words: Angiotensin-converting enzyme inhibitor. diabetes, proteinuria, glomerular injury P rogressive nephropathy, characterized by protelnurla and a decline In GFR, develops in more than 3% of patients with insulin-dependent (Type I) diabetes mellitus ( 1,2). There are data that most patients who develop diabetic nephropathy have higher than normal GFR early in the course of the disease, but this has been challenged by other studies (3). Early hyperfiltration, associated with glomerular capillary hypertension, is also a feature of streptozotocin (STZ) diabetes In rats that develop protelnuria and glomerular structural injury with time (4-7). In rats, angiotensin-converting enzyme (ACE) Inhibitors, in addition to effectively limiting early hyperfiltratlon, also reduced systemic blood pressure (8), a well-known aggravating factor for renal disease progression (9, 1). However, unlike ACE inhibitors, reserpine, hydralazine, and hydrochlorothiazmde, equaily effective in reducing systemic hypertension, did not reduce urinary albumin excretion and glomerular injury ( 1 1). The different effect of various antihypertensive regimens likely reflects the fact that ACE Inhibitors are unique In their properties of effectively reducing glomerular capillary hypertension, a common finding of experimental renal diseases, Including diabetes, that develop proteinuria and progressive glomerular Injury ( 12-15). The fact that ACE Inhibitors are superior to other antthypertensive molecules in retarding renal disease progression also applies to insulin-dependent diabetes in humans ( 16-19). The decrease in blood pressure induced by ACE Inhibitors in most studies did not correlate with the renoprotective effect of the various molecules used to the point that a beneficial effect could be documented, even In patients who had normal systemic blood pressure ( 16,2). Thus, in a metaregression analysis of 1 clinical trials, ACE inhibitors reduced urinary protein excretion more than other antihypertensive drugs at a Journal of the American Society of Nephrology 1 139

2 ACE Inhibition in Experimental Diabetes comparable level of systemic blood pressure (2 1 ). In a recent study in Type I diabetes with nephropathy, low-dose enalapril that did not reduce systemic blood pressure to any significant extent effectively improved glomerular permeability to macromolecules (22). Altogether, these data converge to indicate that ACE inhibitors are superior to other antthypertensives, a property that could be explained by their effect on glomerular hemodynamics or on glomerular membrane size-selective properties (23). Most studies, however, were uncontrolled, the number of patients were small, and the degree of protection was dependent on the duration of the treatment and whether or not renal disease was advanced. This study, performed in rats with experimental diabetes, was designed to address the issue of the potential differences in the renoprotective properties of ACE inhibitors according to the time at which the treatment is started with respect to the onset of the disease. METHODS Adult male Sprague-Dawley rats with initial body weights of25 to 275 g were studied. Animal care and treatment were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/69. OJL 358, Dec 1987: NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No , 1985). All animals were allowed free access to standard diet (containing 2% protein by weight and 2.5% sodium) and tap water. Animals were divided into six groups. Groups 1 and 4 received no specific therapy and are later referred to as control groups. Groups 2. 3, 5, and 6 were made diabetic by an iv tail injection ofstz (Sigma Chemical Co.. St Louis, MO). 6 mg/kg body wt. under thiopental sodium anesthesia (5 mg/kg ip). The induction of diabetes was confirmed, 2 days later. by the measurement of the tail blood glucose level with a reflectance meter (Miles Ames Div. Miles Laboratories, Inc.. Elkhart, IN). Diabetic rats received daily evening injections of insulin (Ultratard HM, Novo Nordisk, Rome, Italy) in doses individually adjusted to maintain a blood glucose level between 2 and 4 mg/dl. Blood glucose levels were monitored at least once a week in all diabetic rats and occasionally in control animals for comparison. Diabetic (DM) Groups 2 (N = 8) and 3 (N = 8) were monitored thereafter until urinary protein excretion reached values of 4 to 5 mg/24 h and were given no therapy other than insulin. At this time, DM Group 2 continued to receive insulin alone for 8 more wk, whereas Group 3 was given insulin and the ACE inhibitor Moexipril (Schwarz Pharma AG, Monheim, Germany) administered in the drinking water at the daily starting dose of 3 mg/kg for the same period. The dose of moexipril was adjusted according to the water intake in order to maintain a constant daily dose of the drug and as needed to maintain the systolic blood pressure (SBP) in the range of 1 to 135 mm Hg. Thus, the dose of moexipril ranged from 3 to 35 mg/kg during the entire treatment period. Control rats (Group 1 ) were also monitored for the same 8-wk period. The time for starting observation was, on average. 23 wk (range, 2 to 24 wk) after the STZ injection for Groups 2 and 3. In all of these animals, SBP was measured at baseline, at monthly intervals after the DM induction, at the time of randomlzation, and at monthly intervals thereafter by the tall cuff method (24). The protein excretion rate was monitored from 24-h urine samples collected in metabolic cages at baseline, every month for the first 4 months after STZ injection, and then every week until the established range of proteinuria was reached. After the beginning of the treatment, proteinuria was monitored every 4 wk. At the end of the study period, whole-kidney function was determined by solute clearance techniques. Rats were then euthanized, kidneys were removed, and tissue specimens were processed for morphologic studies. DM Groups 5 (N = 8) and 6 (N = 8) were monitored until the urinary protein excretion rate reached 1 to 2 mg/24 h, a time that corresponded. on average, to 32 wk (range. 3 1 to 33) after the STZ injection. Up to this time, DM rats received no therapy other than insulin. Then, DM Group 5 continued to receive insulin alone for an additional 8 wk, whereas Group 6 was treated with insulin and with moexipril at the daily starting dose of 3 mg/kg for the same time period in the drinking water and with the same adjustment of dosage as for the early study. Thus, the moexipril daily dose ranged from 3 to 35 mg/kg. Control rats (Group 4) were also monitored for the same time interval. SBP, urinary protein excretion, whole-kidney function measurement, and renal tissue morphologic examination were performed throughout the study. Whole-Kidney Function Studies Renal clearance studies were performed as previously described (25). Briefly, after blood glucose measurement, rats were anesthetized with thiopental sodium (6 mg/kg body wt ip) placed on a temperature-regulated table, and tracheotomized. A catheter was inserted into the left femoral artery for subsequent periodic blood sampling and continuous blood pressure monitoring with an electronic transducer (Battaglia Rangoni, Bologna. Italy). A catheter was also placed In the left femoral vein for the infusion of clearance markers. Urine was collected by bladder incannulation. After the completion of surgery. a bolus of 2 ml/kg of 5% inulin and.2% p-aminohippurate (PAH) in normal saline was infused as a priming load, followed by a sustaining infusion of the same solution at the rate of 3 ml/h per kilogram. Fifty to 6 mm after the bolus infusion, three timed clearance periods of about 3 mm each were started. Arterial blood samples were obtained before and at the end of each clearance period for the evaluation of plasma mnulin and PAH concentration. Urine collection was started 2.5 mm after the first blood sample to allow for the transit time from Bowman s space to the tip of the bladder catheter. Morphologic Studies After whole-kidney function evaluation, kidney slices were processed for light microscopy examination. Fragments of kidneys were fixed in Dubosq-Brazll fluid and embedded in paraffin. Sections 3 im in thickness were stained with Masson s trichrome, with hematoxylin and eosin, and by the periodic acid-schiff techniques. Sections including superficial andjuxtamedullary glomeruli were evaluated. At least 8 to 1 glomeruli were examined for each animal, and the extent of glomerular damage was expressed as the percentage ofglomeruli with focal or global sclerotic lesions. Tubular changes (atrophy, cast formation, and tubular dilatation) and interstitial fibrosis and inflammation were graded from to 4+ (. no changes: 1 +. changes affecting <25% of sample: 2+,changes affecting 25 to 5% of sample; 3+. changes affecting 5 1 to 75% ofsample: 4+. changes affecting >75% of 1 14 Volume 5 Number

3 Perico et al sample). Kidney tissue specimens were analyzed by the same pathologist blind to the nature of the experimental groups. 18 Moexipnl Other Analysis Inulin concentration in plasma and urine samples was measured by the thiourea-resorcinol method (26). The PM! concentration In the same samples was determined by the method of Smith et a]. (27). GFR and RPF, measured as inulin and PM! clearances, respectively, were calculated by the use of standard formulas. Protein concentration in 24-h urine samples was measured by the Coomassie blue G-dye binding method (28). Statistical Analysis All results are expressed as mean ± SD. Data were analyzed by one-way or two-way analysis of variance as appropriate. The significant level of difference between individual group means, subjected to the analysis of variance, was established by the use ofthe Tukey-Cicchetti test for multiple comparisons (29). Values for urinary protein excretion, which were not normally distributed. were subjected to log transformation before statistical analysis. Estimates of renal injury by morphologic studies were compared with the Mann-Whitney test for nonparametric data. Statistical significance was defined as P <.5. RESULTS Diabetic Animals of Groups 2 and 3 Studied From 2 to 24 Weeks of Diabetes With Baseline Proteinuria of 4 to 5 mg/24 h Total food intake was comparable in all diabetic groups for the entire study period. At the end of the observation period, the average food intake was 3 1 ± 6 and 32 ± 6 g/day for untreated diabetic and moexipril-treated diabetic rats, respectively. Diabetic an- Imals gained weight during the follow-up, although they were not at levels comparable to those of control rats. Thus, the final body weight was significantly (P <. 1) lower in untreated and treated diabetics than in controls (Table 1 ). Serial values of blood glucose levels and awake SBP are reported in Figure 1. Stable and comparably moderate hyperglycemia was maintained throughout the duration of the experiment in both 4- E a- C,) :g) ) (I, ) lao Time 5 DM. DM + Moexipril L otiti L (weeks) Time (weeks) Figure 1. Awake SBP and blood glucose concentration during the early phase ofthe study in controls and untreated and moexipril-treated diabetic (DM) rats. Values are mean ± SD. < versus untreated and moexipril-treated diabetic groups. #{176}P <.1 versus premoexiprll treatment (2 to 24 wk). diabetic groups. Untreated diabetic rats exhibited a moderate Increase in SBP that was relatively constant during the observation period. Before treatment was started in the moexipril group, SBP was comparable with that of untreated diabetics. The ACE Inhibitor TABLE 1. Body weight, kidney weight, systemic hemodynamic, and whole-kidney function parameters in controls and diabetic animals of Groups 2 and 3 studied from 2 to 24 wk of diabetes with baseline proteinuria of 4 to 5 mg/24 h Group Body (g) Wt Left Kidney wt (g) BG (mg/dl) (mm SBP Hg) GFR RPF (ml/min per 1 g) FF (%) DM (N = 8) 526 ± ± ± ± 3.67 ± ±.15 3 ± 3 DM/Moexipril 563 ± ± ± ± 3.68 ± ± ± 2 (N = 8) Control 675 ± 35C 1.6 ±.8d 16 ± 13C 127 ± 3b.67 ± ±.18 3 ± 2 (N = 8) a Values are mean ± SD. BG. blood glucose; FF. filtration fraction. b p <.1 versus DM. C p <.1 versus DM and DM/moexiprll. d p <.5 versus DM and DM/moexiprll. Journal of the American Society of Nephrology 1141

4 ACE Inhibition in Experimental Diabetes given for 8 wk reduced SBP to normal values, which were indeed comparable to those of control, agematched, nondiabetic animals. Urinary protein excretion rates during the study period are given in Figure 2. In untreated diabetics, urinary protein excretion progressively increased up to 143 ± 21 mg/24 h at the end of the study. Diabetic rats given moexipril were completely protected from the progressive increase In urinary proteins (Figure 2). Of interest, values of urinary protein excretion were lower at the end of the study in moexipril-treated animals than before the treatment was started, and the differences were statistically significant. Control rats had a slow but progressive increase in urinary protein excretion. As reported in Table 1, GFR was not significantly different among untreated diabetic and control rats at the end of the study period. Moexipril administration did not affect GFR to a significant extent. Similarly, RPF was comparable in untreated and moexipril-treated diabetic animals, which had values that did not differ to a significant extent from those of control rats (Table 1). As a consequence, the filtration fraction was comparable in the three experimental groups of rats. Kidney weight was higher in all diabetic animals as compared with controls (Table 1). At the end of the observation period, none of the control rats developed focal or segmental glomerulosclerosis (Figure 3) nor was there any evidence of interstitial or tubular damage. Three of eight diabetic rats exhibited a modest degree of glomerular sclerosis (Figure 3). Interstitial fibrosis and tubular casts were roughly proportional to the degree of glomerular involvement (interstitial fibrosis: mean score,.44; range. to 1 ; tubular casts: mean score,.63; range, to 1 ). In diabetic rats treated with moexipril, glomerular sclerosis (Figure 3) as well as tubular and Interstitial changes were completely prevented (Interstitial C E :, C ci) a- 2 lao DM. DM#{247} Moexipd Control Moexipnl if Time (weeks) Figure 2. Serial values of urinary protein excretion rate during the early phase of the study in controls and untreated and moexipril-treated diabetic (DM) rats. Values are mean ± SD. <.1 versus controls. #{176}P <.1 versus premoexipril treatment (2 to 24 wk). 4z 2 ci) C,) LI Control DM+ DM Moexipril Figure 3. Percentage of sclerotic glomeruli in individual animals of control, untreated, and moexipril-treated diabetic (DM) rats at the end of the early phase of the study. <.5 versus controls and moexipril-treated diabetic rats. FSG, focal and segmental glomerulosclerosis. fibrosis: mean score,.6; tubular casts: mean score, ). Diabetic Animals of Groups 5 and 6 Studied From 31 to 33 Weeks of Diabetes With Baseline Proteinuria of 1 to 2 mg/24 h Mean body weight at the end of observation period was comparable in both diabetic groups, although they did not reach that of control animals (Table 2). Values of blood glucose concentration and SBP are given in Figure 4. The two diabetic groups had comparable levels of blood glucose throughout the followup. Values of blood glucose at the end of the observation period averaged 358 ± 59 and 35 ± 54 mg/dl in untreated and moexipril-treated diabetic animals, respectively. SBP values, comparable in the two diabetic groups until starting moexipril treatment, were significantly elevated as compared with nondlabetic controls. In untreated diabetics, blood pressure remained elevated thereafter. Moexipril treatment was effective in controlling SBP during the subsequent 8-wk followup, and the values were comparable with those of controls by the end of the study period (Table 2). Serial values of urinary protein excretion are given in Figure 5. Nondiabetic control rats developed a modest increase in urinary protein excretion with time that was age related. Untreated diabetic rats had a progressive increase in urinary protein excretion with time. Moexipril, given when animals had proteinuria of >1 mg/24 h, was unable to prevent the progressive further increase in urinary protein excretion. Thus, at the end of the study, values of urinary proteins were only numerically lower than in untreated diabetics, but the differences were not significant. Whole-kidney func Volume 5. Number

5 Perico et a] TABLE 2. Body weight, kidney weight, systemic hemodynamic, and whole-kidney function parameters in controls and diabetic animals of Groups 5 and 6 studied from 31 to 33 wk of diabetes with baseline proteinuria of 1 to 2 mg/24 h Group Left Kidney Body Wt BG SBP GFR RPF FF wt (g) (g) (mg/dl) (mm Hg) (ml/min per 1 g) (%) DM (N = 8) DM/Moexipril 588 ± ± ± ± ± ± ± ± 3b.67 ± ± ± ± ± 1 29 ± 2 (N = 8) Control 738 ± 79C 1.83 ±.1 1C 98 ± 9d 126 ± 3b.69 ± ±.2 29 ± 1 (N -8) a Values are mean ± SD. BG. blood glucose; FE, filtration fraction. b p <.1 versus DM. C p <.5 versus DM and DM/moexiprll. d p <.1 versus DM and DM/moexipril DM S DM + Moexipril Control 14 C,) 12.c I. I Time(weeks) DM DM + Moexipril TIme (weeks) : 4 - Control Figure 5. Serial values of urinary protein excretion rate during g) the late phase of the study in controls and untreated and -, moexipril-treated diabetic (DM) rats. Values are mean ± SD. 3 P <.1 versus untreated and moexipril-treated diabetic rats. g2 ) #{149} 8 1 comparable In diabetic and nondiabetic animals. Kid- ney weight was higher In all diabetic groups as com- - I. I. I pared with controls, but no differences were found among treated and untreated diabetics (Table 2). Two control rats of eight showed a negligible degree Time (weeks) of glomerular sclerosis (mean..25% of glomeruli) Figure 4. Awake SBP and blood glucose concentration (Figure 6), whereas tubulointerstitial changes, albeit during the late phase of the study in controls and untreated modest, were observed in five rats (interstitial fibrosis: and moexiprll-treated diabetic (DM) rats. Values are mean mean,.8; range, to.5; tubular casts: mean, ± SD. P <.1 versus untreated and moexiprll-treated.3 1 ; range, to.5). SIx of eight diabetic rats left diabetic groups. #{176} P <.1 versus premoexipril treatment (31 untreated developed glomerulosclerosis (Figure 6). Six to 33 wk). of eight diabetic animals given moexipril developed a- glomerulosclerosls. which Involved 2.9% of glomeruli. The degree of tubulointerstitial damage was comparation was comparable in untreated diabetic and control ble In both groups of untreated and moexipril-treated rats (Table 2). Moexipril did not modify GFR or RPF; diabetic animals (mean score: Interstitial fibrosis, these values were comparable to those of untreated.69 versus.3 1 ; tubular casts, 1. versus.69, diabetics. Consequently, the ifitration fraction was respectively). Journal of the American Society of Nephrology 1 143

6 ACE Inhibition in Experimental Diabetes Control DM+ Moexipril Figure 6. Percentage of sclerotic glomeruli in individual animals of control, untreated, and moexipril-treated diabetic (DM) rats at the end of the late phase of the study (39 to 41 wk). P <.5 versus controls. FSG, focal and segmental glomerulosclerosis. DISCUSSION DM We have found that, in moderately hyperglycemic diabetic rats, the ACE inhibitor moexipril, started when protelnuria was 4 to 5 mg/24 h, i.e., on average, 23 wk after the induction of the disease, significantly reduced systemic arterial pressure and normalized urinary protein excretion, which remained within the normal range thereafter. By contrast, animals who started moexipril later in the course of the disease, at the time when proteinurla was higher than 1 mg/ 24 h, despite an effective control of systemic blood pressure, had only a transient effect of stabiliz- Ing urinary protein excretion, which actually progressively increased thereafter, reaching values that at the end of the observation (i.e., 39 to 4 1 wk after the Induction of the disease) were comparable to the ones of untreated diabetic animals. Groups of treated and untreated diabetics were euthanized at 28 to 32 and 39 to 4 1 wk, respectively. When euthanized earlier, moexipril-treated diabetic rats did not show any evidence of glomerular sclerotic lesions. In contrast, at the end of the 39 to 4 1 wk, untreated diabetics and diabetics on moexipril exhibited a comparable degree of glomerulosclerosls. All previous studies in STZ diabetes that addressed the issue of the prevention of diabetic glomerulopathy with ACE inhibitors or other antthypertensives have been performed by starting drug administration at the moment of the induction of diabetes and continuing thereafter until the end of the experiment. Thus, Zatz and coworkers (8) first documented that enalapril treatment, started 2 to 3 days after STZ injection, limited the late development of albuminuria and gbmerulosclerosis. In a subsequent study, the same mode of treatment, again started a few days after STZ injection, largely before diabetic nephropathy developed, afforded better long-term protection than did triple therapy with reserplne, hydralazlne. and hydrochlorothlazide ( 1 1). Studies in unlnephrectomlzed diabetic rats showed that fosinopril, started from Day 2 after STZ injection, protected against diabetic gbmerubopathy, a property that was not shared by the calcium channel blocker nifedipine ( 14). On the same line were studies in spontaneously hypertensive diabetic rats that the ACE inhibitor perindopril, started a few days after the induction of diabetes, reduced albuminuria (3). Of course, the relevance of such experimental studies to the treatment of human patients with diabetes who may present often with microalbuminurla or clinical proteinurla would be lim- Ited unless one could document that the drug is also effective when diabetic renal disease is already established. These results offer indeed the demonstration that ACE inhibitors can be used to slow renal disease progression in animals that have already developed signs of renal Injury, as documented by an increased urinary protein excretion rate. However, they also Indicate that an effective protection can be achieved only If the treatment is started reasonably early in the course of the disease. Later on, urinary protein excretion is not reduced by the treatment nor Is the number of gbomeruli with focal sclerosis lesions. One cannot exclude that If the ACE Inhibitor were given for more than 8 wk, more benefit could be observed, even in animals with elevated proteinuria. Thus, these results do not allow us to definitely conclude whether ACE Inhibitors given once the lesion is established are not Ineffective, but rather, are less effective in reducing the speed of renal disease progression. In apparent contrast with these results are data that ACE inhibitors given chronically to rats with renal mass reduction reduce the further progression ofthe disease (31). However, in the above study, treatment started when the level of urinary protein excretion was around 5 mg/day, a value absolutely comparable to the one of those animals in our study that did respond to moexipril treatment. The idea that the renoprotective effects of ACE Inhibitors is somehow dependent on the stage of diabetic nephropathy at which the treatment is started appears consistent with some human studies already available that ACE inhibitors normalize albuminuria and stabilize renal function in both normotensive and hypertensive patients (17,18,2,21). Short-term studies in small groups of patients indicate that ACE Inhibition reduces urinary protein excretlon and may prevent the progression to overt nephropathy in the early microalbuminuric phases of the disease both oftype I and Type II diabetes (2,32-34). In the more advanced phases, the effect of ACE inhibitors Is less clear. In the most recent randomized control trial of 49 patients with Type I diabetes in 1144 Volume 5 - Number 4 #{149}1994

7 Perico et a] whom urinary protein excretion was 5 mg/day, captopril significantly reduced proteinuria as compared with placebo (35). Data are not available in that article to allow us to establish whether patients with more proteinuria benefited less from ACE inhibitors. Moreover, Lebovitz et a!. (36), studying patients with clinical protemnuria of Type II diabetes, found an only transient decrease of urinary proteins with a slight slowing of GFR decline over 3 yr that, however, failed to achieve statistical significance. Thus, whether diabetic patients who benefit from ACE inhibitors are only those with less proteinuria is a question that remains to be adequately addressed. Of interest in our study is the fact that the completely different effect of ACE inhibitors on the kidney-that is achieved according to the different time at which the treatment is started-is totally independent from systemic blood pressure control. The fact that, in diabetic rats, the control of systemic hypertension per se may not be enough to prevent the development of renal injury is also supported by previous observations that antihypertensive regimens with triple therapy with reserpine, hydralazine, and hydrochlorothiazide effectively reduced systemic blood pressure but had no effect on subsequent structural injury ( 1 1). This reinforces the already known evidence that ACE inhibitors reduce proteinuria quite independently from blood pressure control. Human studies have linked the effect oface inhibitors of reducing protemnuria in diabetes to the property of this class of compounds of reducing the mean radius of functional pores perforating the glomerular barrier (22,23). Other studies in experimental animals have indicated that ACE inhibitors, besides inhibiting membrane size-selective properties, increase singlenephron GFR and whole-kidney GFR by remarkably enhancing the ultrafiltration coefficient (37). That and other findings of a consistent increase in K associated with the use of ACE inhibitors in various models of disease progression (8, 13,38) were taken to suggest that this class of compounds also enhances either the hydraulic permeability of the glomerular membrane or, alternatively, the capillary surface area available for filtration. Thus, it is possible that the beneficial effect oface inhibitors on renal disease progression is related to an ameliorating effect on gbomerular membrane functional properties. The mechanism(s) underlying the favorable effect of ACE inhibitors ofpreserving the glomerular size selectivity and hydraulic function is not clarified yet. There are data that angiotensin II infusion to rats remarkably reduces K even when native or synthetic angiotensin II is infused to mildly or nonpressor doses (39), a finding consistent with the possibility that the effect of angiotensmn II on K is not necessarily the consequence of its effect of producing systemic hypertension. Angiotensin II increases the quantity of filamentous actin in the cytoskeleton of the filtration barrier (4), a finding that is consistent with angiotensin Il-induced actin polymerization. It was speculated that increased actin polymerization reduces K by determining foot process contraction, which also can alter the size of glomerular pores (4). It is possible that the ACE inhibitor-induced increase in K, is due to its capacity to normalize actin polymerization. It is conceivable that, early in the course of the disease, epithelial cell contraction and slit diaphragm changes are more easily reversible, whereas later on, more advanced structural changes are less likely influenced by reducing angiotensin II. In conclusion, this study indicates that, in a model of STZ-induced diabetes: ( 1 ) the administration of the ACE inhibitor moexipril normalized the urinary protein excretion when treatment was started early in the course of the disease, but not at the time when proteinuria is elevated, despite a comparable and effective control of systemic blood pressure; and (2) the effect of moexipril on protemnuria was associated with protection against the development of glomerular structural changes only early but not later on in the course of the disease. ACKNOWLEDGMENTS Moexpril was provided by Schwarz Pharma AG. Monheim. Germany. Insulin was kindly provided by Dr. Gustavo Coronel from Novo Nordisk Farmaceutici SpA.. Rome. Italy. Dr. S.C. 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