Modulation of Incipient Glomerular Lesions in Experimental Diabetic

Modulation of Incipient Glomerular Lesions in Experimental Diabetic Nephropathy by Hypotensive and Subhypotensive Dosages of an ACE Inhibitor Bruno Fabris, Riccardo Candido, Michele Carraro, Francesco Fior, Mary Artero, Cristina Zennaro, Maria Rosa Cattin, Angela Fiorotto, Monica Bortoletto, Cristina Millevoi, Moreno Bardelli, Luigi Faccini, and Renzo Carretta

A glomerular permeability defect occurs early in the course of type 1 diabetes and precedes the onset of microalbuminuria and renal morphological changes. Re- icroalbuminuria is an early marker of diabetic cently, ACE inhibitors have been shown to prevent loss nephropathy (1), and its presence is indica- of glomerular membrane permselective function, but tive of an increased risk not only of overt the mechanism of this nephroprotective effect is still nephropathy (2) but also of cardiovascular being debated. The objective of the present study was to M disease and death (3,4). The onset of microalbuminuria in evaluate the effects of hypotensive and subhypotensive dosages of the ACE inhibitor quinapril ex vivo and of its diabetes is thought to result from increased glomerular active metabolite quinaprilat in vitro on the glomerular passage of albumin and an eventual decrease in the protein reabsorptive capacity of the tubules (5). albumin permeability (Palb) defect in the early phases of experimental diabetes. For the ex vivo study, six groups Recently, we demonstrated that an albumin permeabil- of male Wistar rats were evaluated for 4 weeks. One ity (Palb) defect exists in the earliest stages of streptozo- group served as a nondiabetic control (C); the other five tocin (STZ)-induced type 1 diabetes by using the model of groups were rendered diabetic and included untreated isolated nonperfused rat glomeruli, and we also showed diabetic rats (D) and diabetic rats receiving quinapril at that this alteration precedes the appearance of glomerular the dosages of 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and .(DQ4) mg ⅐ kg؊1 ⅐ day؊1. Dosage-dependent effects hyperfiltration and hypertrophy (6) 0.625 of quinapril on systolic blood pressure and the glomer- Treatment with ACE inhibitors has been shown to lower ular filtration rate were observed. In contrast, control urinary protein excretion in experimental and human of Palb in isolated glomeruli exposed to oncotic gradi- diabetes (7–10), as well as in other forms of renal disease ents, proteinuria, and glomerular and tubular hypertro- (11–14). It is widely assumed that alterations in systemic phy was obtained with subhypotensive dosages (DQ3 and renal hemodynamics underlie the reported effect of and DQ4 groups) of the ACE inhibitor. In the in vitro ACE inhibitors in reducing proteinuria (15–17). Recently, study, quinaprilat reduced P significantly in concen- ؊ alb؊ it has been suggested that low dosages of ACE inhibitors tration ranges from 10 6 to 10 14 mol/l compared with may modify glomerular size selectivity in subjects with results in control glomeruli. The effect on Palb may have occurred by mechanisms different from kidney ACE established diabetic nephropathy (18), thereby raising the inhibitor. These study results indicated that ACE inhib- possibility of a specific intrarenal effect of ACE inhibitors. itor treatment prevents the early onset of the Palb However, it is still difficult to discriminate the role of defect in experimental diabetes. This effect seemed to hemodynamic and nonhemodynamic effects of ACE inhib- occur independently of systemic or glomerular hemody- itors in the prevention of diabetes-associated renal func- namic changes and, at least partially, from kidney ACE tional and structural modifications (19). inhibition. Diabetes 50:2619–2624, 2001 The main objective of the present study was to evaluate ex vivo the effects of treatment with hypotensive and subhypotensive dosages of the ACE inhibitor quinapril on the Palb defect found in the early phases of experimental diabetes. To this end, we used an experimental procedure that excludes the presence of perfusion pressure as a variable in the analysis of permeability function. We simultaneously explored the possibility of using subhypo- From the Department of Medicina Clinica and Neurologia, University of tensive dosages of an ACE inhibitor to modulate the other Trieste, Italy. Address correspondence and reprint requests to Bruno Fabris, MD, Univer- functional and structural alterations that characterize the sity of Trieste, Dipartimento di Medicina Clinica e Neurologia, c/o Ospedale di early phases of diabetes. Cattinara, Strada di Fiume 447, 34149 Trieste, Italy. E-mail: fabris@ fmc.univ.trieste.it. Although most of the relevant literature on the nephro- Received for publication 10 January 2001 and accepted in revised form 16 protective effect of ACE inhibitors in diabetes supports the August 2001. view that inhibition of renal angiotensin II (ANG-II) gen- ANG-II, angiotensin II; ANOVA, analysis of variance; BSA, bovine serum eration by ACE inhibitors could prevent the renal hemo- albumin; GFR, glomerular filtration rate; Palb, albumin permeability; SBP, systolic blood pressure; STZ, streptozotocin. dynamic (7) and structural changes (8) associated with

DIABETES, VOL. 50, NOVEMBER 2001 2619 ALBUMIN PERMEABILITY RESPONSE TO ACE INHIBITION

diabetes, the extent to which inhibition of kidney ACE created an oncotic gradient across the basement membrane, resulting in a glomerular volume change (⌬V ϭ [V ϪV ]/V ]), which was measured activity contributes to the nephroprotective effect of ACE final initial initial off-line by a video-based image analysis program (SigmaScan Pro; Jandel inhibitors remains to be defined. Consequently, a further Scientific Software, Erkrath, Germany). The computer program determines aim of this study was to correlate the inhibition of kidney the average radius of the glomerulus in two-dimensional space, and the ACE activity by different dosages of quinaprilat, the active volume is derived from the formula V ϭ 4/3␲r3. The magnitude of ⌬V was related to the albumin reflection coefficient, ␴ , by the following equation: metabolite of quinapril, with P in vitro in isolated alb alb ␴ ϭ ⌬ ⌬ ␴ ( alb)experimental ( V)experimental/( V)control; the alb of the control glomeruli glomeruli from diabetic rats. ␴ was assumed to be equal to 1. Palb was defined as (1- alb) and described the ␴ movement of albumin subsequent to water flux. When alb is zero, albumin moves across the membrane with the same velocity as water and Palb is 1.0; RESEARCH DESIGN AND METHODS ␴ conversely, when alb is 1.0, albumin cannot cross the membrane with water Study protocol and Palb is zero. These assumptions are valid only when the response to an Ex vivo study. Six groups of adult male Wistar rats (n ϭ 10 in each group) impermeable solute, such as highϪmolecular weight neutral dextran, is not with initial weights of 250–270 g were studied for 4 weeks. One group served altered by the experimental manipulations (6). as a nondiabetic control (C); the other five groups were rendered diabetic by Metabolic and renal parameters. Serum glucose and creatinine were intravenous injection of STZ (55 mg/kg; Sigma, St Louis, MO) in citrate buffer. determined by an autoanalyzer technique (Hitachi 717, Tokyo). Proteinuria Control animals were injected with citrate buffer only. Only animals with was assessed by measurement of 24-h total urinary protein excretion using the blood glucose levels Ͼ15 mmol/l 2 days after the induction of diabetes were Coomassie dyeϪbinding technique (23). included as diabetic animals in the study. Assessment of the GFR was performed in our laboratory, as previously Rats were housed in groups of three per cage in an air-conditioned, described (6), by a single injection technique measuring the clearance of light-controlled environment and fed a normal diet containing ϳ24% protein iopamidol (a iodinated contrast media) per 100 g body wt in a single plasma by weight (Harlan Nossan Correzzana, Milan, Italy). All animals had unre- sample. (X-ray fluorescence was used to analyze the iodine concentration in stricted access to food and water. The diabetic animals were randomly the plasma [Renalyzer; Provalid Ab, Lund, Sweden].) Iopamidol was injected allocated to one of five groups. The first diabetic group (D) received no through the tail vein and the blood sample was drawn at 43 min. This method specific therapy. The other groups were treated with quinapril (Parke-Davis, was validated in diabetic animals by comparing it with a constant infusion Ann Arbor, MI) for 4 weeks at dosages of 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and method using 51Cr-EDTA for measurement of GFR (6). Ϫ Ϫ 0.625 (DQ4) mg ⅐ kg 1 ⅐ day 1 in drinking water. The diabetic rats received Assessment of renal morphology. Two midcoronal sections 3–5 ␮m thick daily evening injections of 8 IU of ultralente insulin (Ultralente MC; Novo from each kidney were stained with hematoxylin and eosin and examined by Nordisk, Copenhagen, Denmark) to maintain blood glucose at ϳ22–28 mmol/l light microscopy. Each section was placed in a microscope and recorded by and improve long-term survival. a color video camera; the image was then transferred via computer to a At 2-week intervals, the systolic blood pressure (SBP) was measured in video-based image analysis program (MCID; Imaging Research, St. Catharines, conscious, restrained, preheated rats by tail-cuff plethysmography (20). The Ontario, Canada). The areas of 100 consecutive glomerular tufts sectioned at rats were then placed in individual metabolic cages for 24 h, and their urine the vascular pole and 100 proximal renal tubules were measured by two was collected for measurement of volume and total urinary protein. The day independent observers. Interobserver variability between measurements for after the metabolic study, a venous blood sample from rats under ether all sections was Ͻ5%. The reported values of the glomerular and proximal anesthesia was also drawn and centrifuged; the serum was frozen for later tubular areas represent the mean of the measurements obtained by both analysis of glucose, creatinine, and ACE activity. examiners. At the end of the study, the glomerular filtration rate (GFR) was evaluated Fluorometric assay of ACE. Serum and kidney ACE activities were esti- by the determination of iopamidol (Iopamiro; Schering AG, Berlin, West mated by a modification of the method of Cushman and Cheung (24). On the Germany) clearance. After the rats were decapitated, their abdomens were day of assay for ACE activity, the frozen kidneys were weighed, placed in

opened and the kidneys were rapidly excised and weighed. Palb was deter- fresh test tubes, and homogenized on ice in potassium phosphate buffer (pH mined in glomeruli isolated from the renal cortex of one of the two kidneys. 8; 1:5 wt/vol) containing 0.3% Triton X-100 using an Ultra Turrax T-25 (13,500 Then 25% of the contralateral kidney was fixed in neutral formalin, processed rpm) for 10 s. After centrifugation (15 min at 15,000 rpm at 4°C), ACE was through graded ethanol solutions, cleared in xylol, and embedded in paraffin assayed in the supernatant. Plasma or tissue homogenates were diluted with for evaluation of glomerular and proximal tubular areas. The remaining 75% of 0.25 ml of the incubation mixture, composed of 0.1 mol/l phosphate buffer (pH the kidney was frozen in liquid nitrogen and stored at Ϫ80°C for ACE activity 8), 300 mmol/l NaCl, and 5 mmol/l of Hip-His-Leu as substrate. For experi- measurement. ments measuring the inhibitory potency of quinaprilat, diluted tissue prepa- In vitro study. Adult male Wistar rats were killed 4 weeks after induction of rations were mixed with 0.05 ml of serial dilution of the ACE inhibitor (from Ϫ6 Ϫ15 diabetes. In a first group of five animals, Palb was evaluated with or without 20 10 to 10 mol/l). The enzymatic reactions were stopped after 15 min by the Ϫ Ϫ Ϫ Ϫ min of preincubation with different concentrations (10 6,10 9,10 12,10 13, addition of 1.5 ml of 280 mmol/l NaOH. The liberated His-Leu was converted Ϫ Ϫ 10 14, and 10 15 mol/l) of quinaprilat (Parke-Davis), the major active metab- into a fluorescent product by adding, with rapid mixing, 0.1 ml of 1% olite of quinapril. The second series of animals was used for evaluating the orthophthaldialdehyde in methanol. After 20 min, the reaction was terminated effect of the same concentrations of quinaprilat on kidney ACE activity. by adding 0.2 ml of 3N HCl. All tubes were centrifuged for 5 min at 2,000 rpm, Concomitantly, the in vitro inhibitory potency of quinaprilat on kidney ACE and fluorescence was measured after 30 min at an excitation wavelength of Ϫ Ϫ activity was tested in a concentration range from 10 6 to 10 15 mol/l, 360 nm and an emission wavelength of 490 nm. All measurements were done Ϫ considering that the quinaprilat IC 50 has been estimated to be 4.5 ϫ 10 11 in duplicate, and all ACE samples were run in parallel with millimolar EDTA, mol/l (21). an ACE inhibitor, to correct for nonspecific fluorescence. The procedures followed in this study were in accordance with institu- Statistical analysis. Data are reported as means Ϯ SE. Comparisons among tional guidelines for experimental animal research. different rat groups over the study period were performed by analysis of

Isolation of glomeruli and measurement of Palb. Glomeruli were isolated variance (ANOVA) and a subsequent Scheffe’s test. Parameters within one by standard sieving techniques in medium containing the following (in group at different time points were compared by ANOVA for repeated mmol/l): sodium chloride 115, potassium chloride 5, sodium acetate 10, measures. Linear regression analysis was used for testing two variable dibasic sodium phosphate 1.2, sodium bicarbonate 25, magnesium sulfate 1.2, relationships. Statview 512 software for the Apple Macintosh computer was calcium chloride 1.0, and glucose 5.5. The pH was titrated to 7.4. The medium used. A value of P Ͻ 0.05 was required for signiﬁcance. also contained 50 g/l bovine serum albumin (BSA) as an oncotic agent. The isolated glomeruli, which were free from capsules and arterioles, were then washed in 1 ml fresh medium; an aliquot of 0.1 ml was then incubated in 0.9 RESULTS ml of medium for 10 min at 37°C. Using an in vitro study design, parallel incubations were run with the addition in the medium of quinaprilat at Weight and blood glucose control. Data for body different concentrations (10Ϫ6 to 10Ϫ15 mol/l). The incubated glomeruli with or weight, blood glucose control, and urine volume are without the test agent were then transferred to a glass coverslip coated with shown in Table 1. During the experimental period, diabetic poly-L-lysine as an adherent and covered with fresh medium. animals gained less weight than did control rats. Blood The rationale and methodology for the determination of Palb has been previously described in detail (22). In brief, each of 10–16 glomeruli per glucose and urine volumes were greatly increased in the animal were videotaped through an inverted microscope before and after a diabetic rats, as expected (P Ͻ 0.01). Antihypertensive medium exchange to one containing 10 g/l BSA. The medium exchange therapy did not inﬂuence body weight at any of the

2620 DIABETES, VOL. 50, NOVEMBER 2001 B. FABRIS AND ASSOCIATES

TABLE 1 Metabolic parameters Parameters C D DQ1 DQ2 DQ3 DQ4 Body weight (g) Week 0 257 Ϯ 5 256 Ϯ 6 254 Ϯ 3 257 Ϯ 6 260 Ϯ 6 252 Ϯ 3 Week 4 329 Ϯ 8 271 Ϯ 7* 300 Ϯ 7* 303 Ϯ 10† 289 Ϯ 7* 302 Ϯ 12† Blood glucose (mmol/l) Week 0 7.3 Ϯ 0.2 7.7 Ϯ 0.6 7.2 Ϯ 0.3 7.4 Ϯ 0.2 7.1 Ϯ 0.3 7.5 Ϯ 0.3 Week 4 7.4 Ϯ 0.2 27 Ϯ 1.7* 22.1 Ϯ 1.9* 27.2 Ϯ 1.9* 25.9 Ϯ 1.6* 26.5 Ϯ 1.5* Urine volume (ml/day) Week 0 8.5 Ϯ 2.2 8.7 Ϯ 0.7 4.4 Ϯ 0.6 9.7 Ϯ 2 7.5 Ϯ 1.1 10.8 Ϯ 1.7 Week 4 7.5 Ϯ 0.5 70.1 Ϯ 7.4* 72.6 Ϯ 11.6* 60.6 Ϯ 12† 71.1 Ϯ 11.6* 62.46 Ϯ 11† Data are means Ϯ SE. C, control rats; D, diabetic rats; DQ1-DQ4, diabetic rats treated with quinapril at 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and 0.625 (DQ4) mg ⅐ kgϪ1 ⅐ dayϪ1.*P Ͻ 0.01, †P Ͻ 0.05 vs. C group. dosages used. Comparable values of serum glucose and the DQ1 and DQ2 groups, after 4 weeks of treatment with urine volumes were maintained in all the diabetic groups 5 and 2.5 mg ⅐ kgϪ1 ⅐ dayϪ1, respectively, the increase in throughout the study period. GFR was completely prevented. In contrast, no effect on SBP. The SBPs for the six groups are shown in Fig. 1. this parameter was seen after an equivalent period of There was no significant difference in blood pressure treatment with 1.25 and 0.625 mg ⅐ kgϪ1 ⅐ dayϪ1 of quinapril between diabetic and nondiabetic rats. Quinapril de- (DQ3 and DQ4 groups, respectively) (Table 2). creased the SBP in a dosage-dependent manner in the four The 24-h total urinary protein excretion was signifi- treated groups, but only the higher dosages of quinapril cantly increased in the untreated diabetic animals com- (groups DQ1 and DQ2) resulted in a statistically significant pared with the control and treated diabetic animals. The reduction in blood pressure (P Ͻ 0.01). increase in proteinuria was attenuated by quinapril treat- Glomerular Palb. The Palb was increased in diabetic ment, independent of the dosages used (Table 2). No animals (Fig. 2). Treatment with the ACE inhibitor relation was found between proteinuria and GFR. quinapril markedly reduced Palb, independent of the dos- Serum creatinine rose similarly in all the diabetic ani- ϭ ages used. Palb was closely related to proteinuria (r mals (Table 2). 0.463, P ϭ 0.002), whereas no correlation was found In contrast to somatic growth, diabetes was associated between Palb and GFR. A positive but not statistically with renal hypertrophy. The ratio of two-kidney weight to significant correlation between Palb and blood pressure body weight was significantly higher in the diabetic rats was found. than in the nondiabetic control rats (P Ͻ 0.05) (Table 2). Incubation of isolated glomeruli from diabetic rats with The renal morphometric measurements showed an in- quinaprilat in concentrations ranging from 10Ϫ6 to 10Ϫ15 crease in glomerular and proximal tubular areas in the mol/l for 10 min at 37°C significantly reduced Palb as diabetic compared with the nondiabetic rats (Fig. 4). compared with results in control glomeruli (Fig. 3). Quinapril treatment at subhypotensive dosages also pre- Renal function and structure. The induction of diabetes vented the hypertrophic response of glomerular and prox- was associated with a significant increase in the GFR. In imal tubular cells (Fig. 4). Plasma and kidney ACE activity. No differences were

FIG. 1. Sequential values for SBP for the six animals groups through- FIG. 2. Palb determined in isolated glomeruli exposed to oncotic out the experimental period. C, control rats; D, diabetic rats; gradients during the course of the experiment. C, control rats; D, DQ1؊DQ4, diabetic rats treated with quinapril at 5 (DQ1), 2.5 (DQ2), diabetic rats; DQ1؊DQ4, diabetic rats treated with quinapril at 5 DQ3), and 0.625 (DQ4) mg ⅐ kg؊1 ⅐ day؊1. Data are means ؎ SE. (DQ1), 2.5 (DQ2), 1.25 (DQ3), and 0.625 (DQ4) mg ⅐ kg؊1 ⅐ day؊1. Data) 1.25 .P < 0.01 vs. C, D, DQ3, and DQ4 groups. are means ؎ SE. *P < 0.01 vs. control, DQ1, DQ2, DQ3, and DQ4 groups*

DIABETES, VOL. 50, NOVEMBER 2001 2621 ALBUMIN PERMEABILITY RESPONSE TO ACE INHIBITION

DISCUSSION The principal finding of the present study was that treatment with the ACE inhibitor quinapril prevented the Palb defect when administered at subhypotensive dosages; a secondary finding was that significant improvement in the permeability defect occurred in vitro by incubating isolated glomeruli from diabetic rats with different dosages of quinaprilat. A direct effect of ACE inhibitors in improving glomerular size selective properties has been previously found in rat models of genetically determined, age-dependent glomerulosclerosis (25) and in humans with diabetic nephropathy (26) and other nondiabetic proteinuric renal diseases (27). In contrast, glomerular size selective dysfunction and proteinuria have not been shown to be ameliorated by ACE inhibition in type 2 diabetes, probably because the structural changes in type 2 diabetes are often advanced and diffuse, involving thickening of the basement membrane, broadening of the foot processes, and diffuse sclerotic changes (28).

FIG. 3. In vitro effect of quinaprilat on glomerular Palb.Palb of isolated In our study, the administration of subhypotensive dos- glomeruli from untreated diabetic rats after incubation: D, in control ages of quinapril to diabetic rats prevented the Palb defect medium; D10؊6, D10؊9, D10؊12, D10؊13, D10؊14, and D10؊15 in medium -containing decreasing concentrations of quinaprilat. Data are means ؎ and proteinuria without affecting the glomerular hyperfil SE. *P < 0.01 vs. D and D10؊15 groups. tration response to hyperglycemia. The use of isolated glomeruli, in contrast to studies of dextran-sieving profiles (25–28), permitted us to assess the capillary Palb without observed in plasma ACE activity between diabetic and the influence of pressure and flow variables. The preven- nondiabetic control animals (Table 2). The chronic admin- tion of the Palb defect by low dosages of quinapril was istration of quinapril induced a significant dosage-depen- associated with a significant inhibition of kidney ACE dent decrease in serum and kidney ACE activities (Table activity. Collectively, these data are consistent with a 2). nonhemodynamic effect of ACE inhibitors on glomerular The results of kidney ACE inhibition in vitro by quinapri- filtration barrier permeability properties. Considering that lat are shown in Fig. 5. Inhibition of kidney ACE activity ANG-II has been implicated as a stimulus for matrix was observed at a concentration of 10Ϫ12 mol/l and was production from mesangial cells (29) and for remodeling 100% at 10Ϫ10 mol/l. As illustrated in Fig. 6, in vitro of the glomerular basement membrane in diabetes (30), inhibition of kidney ACE activity and reduction of Palb by the favorable effect of ACE inhibition on Palb could be the different concentrations of quinaprilat in the range of 10Ϫ6 consequence of long-term inhibition of ANG-II generation to 10Ϫ15 mol/l were compared. The threshold dosages for in the kidney. On the other hand, some of the favorable Palb and kidney ACE inhibition effects were different. The effects of ACE inhibition on Palb may be explained by reduction of Palb paralleled the inhibition of kidney ACE actions other than those on tissue in the renin angiotensin activity only for the highest concentrations of quinaprilat system. Comparing the dosage-related effects of quinapri- Ϫ6 Ϫ9 Ϫ12 Ϫ13 (10 and 10 mol/l). At concentrations of 10 ,10 , lat on kidney ACE activity with the effects on Palb,itwas Ϫ14 and 10 mol/l, only the effect on Palb was still evident, evident that quinaprilat reduced Palb at dosages below the whereas no effect was detectable on kidney ACE activity. threshold for ACE inhibition. Although we cannot exclude

TABLE 2 Renal parameters and plasma and kidney ACE activity Parameters C D DQ1 DQ2 DQ3 DQ4 Serum creatinine (␮mol/l) Week 0 41.5 Ϯ 1.7 38.9 Ϯ 1.7 41.5 Ϯ 0.8 42.9 Ϯ 1.7 39.8 Ϯ 0.9 39.8 Ϯ 0.9 Week 4 47.7 Ϯ 2.7 67.2 Ϯ 0.9 59.2 Ϯ 3.5 70.5 Ϯ 3.5* 63.7 Ϯ 5.3 64.5 Ϯ 5.3 Proteinuria (mg/24 h) Week 0 12 Ϯ 514Ϯ 319Ϯ 418Ϯ 815Ϯ 412Ϯ 4 Week 4 14 Ϯ 235Ϯ 7†‡ 19 Ϯ 322Ϯ 314Ϯ 319Ϯ 4 GFR (ml ⅐ minϪ1 ⅐ 100 g body wt) week 4 1.14 Ϯ 0.05 1.55 Ϯ 0.11§ 1.13 Ϯ 0.05 1.18 Ϯ 0.08 1.3 Ϯ 0.07§ 1.56 Ϯ 0.26§ Plasma ACE (nmol His-Leu ⅐ ml–1 ⅐ min–1) Week 0 85.5 Ϯ 4.8 79.3 Ϯ 3.8 83 Ϯ 5.1 86.4 Ϯ 5.4 78.9 Ϯ 4.4 85.5 Ϯ 4.7 Week 4 81.6 Ϯ 3.9 84.5 Ϯ 4.2 25.6 Ϯ 2.5࿣ 34.9 Ϯ 4.1࿣ 47.9 Ϯ 3.1࿣ 56.6 Ϯ 3࿣ Renal ACE (% inhibition) (week 4) 0 0 75.3 Ϯ 4.2 65.9 Ϯ 4.1 55.2 Ϯ 4.5 40.3 Ϯ 3.1 Ratio two-kidney weight:body weight (week 4) 7.1 Ϯ 0.3 9.1 Ϯ 0.4* 8.7 Ϯ 0.2 8.5 Ϯ 0.6 8.6 Ϯ 0.2 8.2 Ϯ 0.4 Data are means Ϯ SE. C, control rats; D, diabetic rats; DQ1-DQ4, diabetic rats treated with quinapril at 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and 0.625 (DQ4) mg ⅐ kgϪ1 ⅐ dayϪ1.*P Ͻ 0.05 vs. C group; †P Ͻ 0.01 vs. C and DQ3 groups; ‡P Ͻ 0.05 vs. DQ1, DQ2, and DQ4 groups; §P Ͻ 0.01 vs. C, DQ1, and DQ2 groups; ࿣P Ͻ 0.01 vs. C and D groups.

2622 DIABETES, VOL. 50, NOVEMBER 2001 B. FABRIS AND ASSOCIATES

FIG. 5. In vitro inhibition of converting enzyme in rat kidney by quinaprilat.

although the effects on GFR and blood pressure clearly depended on the dosage, the prevention of increased urinary protein excretion and morphometric changes of glomerular and tubular structure was also evident with subhypotensive dosages of ACE inhibitor. This would indicate that a reduction in systemic blood pressure or in the glomerular filtration rate is not a prerequisite for the antiproteinuric and antirenal growth effect of ACE inhibitors in the early phases of diabetic nephropathy. The discrepancy observed with the subhypotensive dosages of quinapril between reduction of proteinuria and the ab- sence of significant effects on glomerular hyperfiltration is consistent with the notion that the major contributor to increased glomerular filtration of albumin is not hyperfiltration but an increase in macromolecular permeability at the glomerular filtration barrier (5,34). Accordingly, in our FIG. 4. Glomerular (A) and tubular (B) areas among groups 4 weeks ,after induction of diabetes. C, control rats; D, diabetic rats; DQ1؊DQ4 diabetic rats treated with quinapril at 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), .and 0.625 (DQ4) mg ⅐ kg؊1 ⅐ day؊1. Data are means ؎ SE. *P < 0.01 vs C, DQ1, DQ2, DQ3, and DQ4 groups. the possibility that the dissociation between the effects of quinaprilat on inhibition of ACE activity and the amelioration of permeability of the glomerular barrier is a consequence of the different tissue preparation used in the in vitro experiments, the favorable effect of quinaprilat on the glomerular barrier may have involved mechanisms different from the inhibition of “classic” ACE. ACE inhibitors may block kinases other than ACE, including en- zymes that are related to or are isomers of ACE (31) or aminopeptidase P, or those that may be related to endopeptidase 24.15 (32) or matrix metalloproteinases (33). The inhibition of non-ACE kinases by quinaprilat may be an alternative or synergistic mechanism by which ACE inhibitors prevent or reduce the glomerular permeability defect. In the present study, we recapitulated many previous FIG. 6. Comparison of the in vitro effect of increasing concentrations -experiments that showed that hyperfiltration, proteinuria, of quinaprilat (10؊6,10؊9,10؊12,10؊13,10؊14, and 10؊15) on glomeru lar Palb and kidney ACE activity in diabetic (D) rats. Reduction of Palb and renal hypertrophy were prevented by ACE inhibitor and inhibition of kidney ACE activity are expressed as percentages of treatment (7,8,15). It is interesting to note, however, that the values in the untreated animals.

DIABETES, VOL. 50, NOVEMBER 2001 2623 ALBUMIN PERMEABILITY RESPONSE TO ACE INHIBITION study, a positive correlation was found between protein- analysis of glomerular function in two rat models of glomerular sclerosis. uria and P , whereas proteinuria and the GFR were not J Clin Invest 82:322–330, 1988 alb 13. Maschio G, Alberti D, Janin G, Locatelli F, Mann JF, Motolese M, Ponticelli correlated. C, Ritz E, Zucchelli P: Effect of the angiotensin-converting-enzyme inhib- The capacity of ACE inhibitors to prevent renal growth itor benazepril on the progression of chronic renal insufficiency: the is well known; however, there has been no previous Angiotensin-Converting-Enzyme Inhibition in Progressive Renal Insuffi- evidence of a beneficial effect of subhypotensive dosages ciency Study Group. N Engl J Med 334:939–945, 1996 of ACE inhibitors on the diabetes-related renal hypertro- 14. The GISEN Group (Gruppo Italiano di Studi Epidemiologici in Nefrologia): phic response. The reduction of proteinuria by ACE inhib- Randomised placebo-controlled trial of effect of ramipril on decline in glomerular filtration rate and risk of terminal renal failure in proteinuric, itors may attenuate the growth response of tubular cells non-diabetic nephropathy. Lancet 349:1857–1863, 1997 (35). However, the direct evidence in this study for kidney- 15. Zatz R, Dunn BR, Meyer TW, Anderson S, Rennke HG, Brenner BM: converting-enzyme inhibition after chronic treatment with Prevention of diabetic glomerulopathy by pharmacological amelioration of quinapril supports the notion that a specific effect of renal glomerular capillary hypertension. J Clin Invest 77:1925–1930, 1986 ACE inhibition may be important for preventing renal 16. Myers BD, Meyer TW: Angiotensin-converting enzyme inhibitors in the prevention of experimental diabetic glomerulopathy. Am J Kidney Dis structural modifications in the course of diabetes. 13:20–24, 1989 In conclusion, our data document the capacity of ACE 17. Hostetter TH, Rosenberg ME: Hemodynamic effects of glomerular perms- inhibitors to prevent the Palb defect existing very early in electivity. Am J Nephrol 10 (Suppl. 1):24–27, 1990 the course of incipient diabetic nephropathy in STZ- 18. Remuzzi A, Ruggenenti P, Mosconi L, Pata V, Voberti G, Remuzzi G: Effect administered rats. This effect seems to have been indepen- of low-dose enalapril on glomerular size-selectivity in human diabetic nephropathy. J Nephrol 6:36–43, 1993 dent of systemic and glomerular hemodynamic changes. 19. Wolf G, Ziyadeh FN: The role of angiotensin II in diabetic nephropathy: Our results also indicate a positive effect of subhypoten- emphasis on nonhemodynamic mechanisms. Am J Kidney Dis 29:155–163, sive dosages of quinapril on renal hypertrophy. Finally, we 1997 have provided evidence that inhibition of kidney ACE 20. Bunag RD: Validation in awake rats of tail-cuff method for measuring activity may not be the only determinant of the improve- systolic pressure. J Appl Physiol 34:279–282, 1973 21. Johnston CI, Fabris B, Yamada H, Mendelsohn FAO, Cubela R, Sivell D, ment in Palb, thus giving new insights into the mechanisms Jackson B: Comparative studies of tissue inhibition by angiotensin con- responsible for the nephroprotective effect of ACE inhib- verting enzyme inhibitors. J Hypertens 7 (Suppl. 5):S11–S16, 1989 itors in the course of diabetic nephropathy. However, the 22. Savin VJ, Sharma R, Lovell HB, Welling DJ: Measurement of albumin confirmation of this concept would ultimately require reflection coefficient with isolated rat glomeruli. J Am Soc Nephrol similar designed studies with ANG-II receptor antagonists. 3:1260–1269, 1992 23. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye ACKNOWLEDGMENTS binding. 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