Protective Effect of Aminoguanidine upon Capillary and Submesothelial Anionic Sites

Microvascular Research 61, 166 –178 (2001) doi:10.1006/mvre.2000.2293, available online at http://www.idealibrary.com on

Protective Effect of Aminoguanidine upon Capillary and Submesothelial Anionic Sites Avshalom Shostak, Valery Wajsbrot, and Lazaro Gotloib 1 Department of Nephrology & Hypertension, and the Research Center for Experimental Nephrology, “Ha’Emek” Medical Center, Afula, Israel Received June 27, 2000; published online January 24, 2001

This study evaluates albuminuria and peritoneal permeability to albumin in control and diabetic rats, as well as in diabetic animals treated with subcutaneously injected aminoguanidine hydrochloride (Ag) (5 mg/100 g/day), during a follow-up period of 6 months. Aminoguanidine effectively prevented albuminuria and albumin extravasation in the mesenteric interstitial tissue (control, 0.43 ⴞ 0.11 ␮g EB/100 g of dry tissue, Ag, 0.60 ⴞ 0.44; untreated diabetic animals, 1.22 ⴞ 0.73; control and Ag group vs untreated diabetic rats, P < 0.001). Albumin D/P ratio of the aminoguanidine-exposed rats (0.017 ⴞ 0.011) was higher than that of controls (0.008 ⴞ 0.002), but significantly lower (P < 0.001) than values observed in the untreated group of animals (0.046 ⴞ 0.003). Administration of aminoguanidine preserved both submesothelial and subendothelial electronegative charges. For capillary basement membrane (BM), control at zero time, 32 ⴞ 4 AS/␮m BM; control at 6 months, 33.4; aminoguanidine-treated rats, 35 ⴞ 2. For submesothelial BM, control at zero time, 33 ⴞ 3; control at 6 months, 32 ⴞ 3; aminoguanidine-treated rats, 35 ⴞ 3. Splitting and thickening of both basement membranes were not prevented by the therapeutic intervention. We conclude that the shielding effect of aminoguanidine upon the permselectivity capabilities of the endothelial and mesothelial 1

To whom correspondence should be addressed at Department of Nephrology & Hypertension, “Ha’Emek” Medical Center, Afula 18101, Israel. Fax: (972-6) 6525436.

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monolayers appears to be connected, basically to the preservation of anionic fixed charges. © 2001 Academic Press Key Words: diabetes; anionic fixed charges; capillary permeability; peritoneal permeability; aminoguanidine.

INTRODUCTION Research done in the past 25 years confirmed, as suggested by Lendrum in 1967 (Lendrum, 1967), that the abnormally increased capillary permeability to albumin plays a key role in the development of the microvascular complications derived from long-standing diabetes. This phenomenon has been observed in the capillary bed of skeletal muscle, kidney, eye, brain, skin, myocardium, and mesentery (Williamson et al., 1987; Alpert et al., 1972; Viberti, 1983; Pinter et al., 1991; Shostak and Gotloib, 1996). A strong body of literature supports the contention that this deviation of the normal permselectivity of the microvascular wall derives from a markedly decreased proteoglycan content of subendothelial capillary basement membranes (Olgemoller et al., 1992), leading also to an substantially reduced density of the microvascular fixed anionic charges. This manifestation has already been observed in the capillary basement membrane of retina (Bollineni et al., 1997), anterior and posterior uvea, sciatic nerve, aorta (Williamson et al., 1987), large arteries 0026-2862/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Aminoguanidine Preserves Anionic Charges

(Raz et al., 1988), myocardium (Fisher, 1986), kidney glomeruli (Rorhbach et al., 1982), and mesentery of experimental animals within 3 to 6 months after the onset of experimentally induced diabetes (Shostak and Gotloib, 1996). The pathophysiological mechanisms involved in this process have not yet been completely understood. Some lines of research suggested that the already mentioned reduced heparan-sulfate-proteoglycans content of the basement membrane appears to result from the nonenzymatic reaction of glucose in high concentrations with amino groups. The outcome of this reaction results in the formation of stable, longlived, cross-linking substances defined as advanced glycosylation end products (AGE) (Brownlee et al., 1988; Vlassara et al., 1992). Aminoguanidine, a nucleophilic hydrazine and small molecular size compound (MW around 74.1 Da), prevents the formation of AGE by irreversibly reacting with the Amadori products (Nyengaard et al., 1997; Brownlee et al., 1986). Studies done in experimental diabetic animals or nondiabetic rats injected with AGE-modified albumin have shown that administration of aminoguanidine prevented the increase of capillary permeability to albumin in several microvascular beds: ocular tissues, sciatic nerve, lung, aorta, glomerular capillaries, vena cava, brain, skin, heart, and intestine (Vlassara et al., 1992; Nyengaard et al., 1997; Soulis-Liparota et al., 1991; Tilton et al., 1993; Vlassara et al., 1994). The present study was designed to investigate whether administration of aminoguanidine as the only treatment could prevent, at least in part, the previously observed reduced density distribution of anionic fixed charges of mesenteric capillaries, and the consequent increased albumin loss in the mesenteric interstitial tissue, as well as in the peritoneal dialysate of streptozotocin diabetic rats (Shostak and Gotloib, 1996).

RESEARCH DESIGN AND METHODS Animals and Induction of Diabetes and Administration of Aminoguanidine Animals were bred, housed, and treated according to the UK legislation relevant to the maintenance and

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use of laboratory animals for scientific research (Ray and Scott, 1972). Furthermore, the experimental design of this study was done in conformity to the regulations required by the local ethics committee for animal experimentation. A total number of 133 male albino rats of the Sprague-Dawley strain were included in this study. Groups of 4 to 5 animals were housed in polycarbonate cages (1800-cm 2 surface area and 20-cm height; Tecniplast, Bugugiatte, Italy), at temperatures ranging between 19° and 22°C, exposed to a 12-h light-dark cycle, and maintained on standard Purina chow for rodents (Maabarot; Kibbutz Maabarot, Israel) and water ad libitum. For 24-h urinary collections, rats were located in the same room and under the same conditions, in polycarbonate metabolic cages (Tecniplast). Animals were divided in the following experimental groups: (a) control at zero time; (b) age-matched intact rats for a follow-up period of up to 6 months (control 3 and 6 months); (c) untreated diabetic rats; and (d) aminoguanidine-treated diabetic animals. Rats to be made diabetics were injected, via the tail vein, with a freshly prepared solution of streptozotocin (75 mg/ kg) in 0.1 M citrate buffer, at pH 4.5. Animals from the control group were sham-injected with citrate buffer alone. Rats were considered to be diabetic on the basis of nonfasting blood sugar levels of at least 22.20 mm/L, estimated 48 h after administration of streptozotocin. Blood glucose levels of diabetic animals, estimated at least once a month, remained higher than 22.20 mmol/L through the whole experimental study (mean, 39.53 ⫾ 6.16 mmol/L; range, 25.53– 48.52 mmol/L; 95% confidence interval, 37.23– 40.83 mmol/ L). Weekly estimation of glycosuria, done in diabetic rats by means of reagent strips (Ames, Bayer, Puteaux, France), ranged between 58 and more than 109 mmol/L. Ketonuria was observed during the whole period of follow-up. Glycated hemoglobin (GHb) was determined in the blood of control and diabetic animals by affinity microchromatography (Klenk et al., 1982), using the glycotek column method (Helena, Beaumont, TX). Controls had GHb blood levels within the upper limit of 7% (Gabbay et al., 1977), whereas for diabetic untreated rats values were 16.64 ⫾ 0.81 and 16.38 ⫾ 0.57% at 3 and 6 months, respectively. Animals treated with aminoguanidine showed levels of

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15.76 ⫾ 1.25 and 16.32 ⫾ 0.98% at the same time intervals. The mean body weight for controls was 162.81 ⫾ 26.61 g at zero time and 469.86 ⫾ 31.89 g at the end of the 6-month observation period. The corresponding values for untreated diabetic rats were 195.97 ⫾ 31.44 and 322.26 ⫾ 67.07 g; whereas for aminoguanidinetreated animals the recorded figures were 205.72 ⫾ 42.26 and 363.70 ⫾ 53.82 g. When the growth curve of the control animals was placed side by side with those observed in both groups of diabetic rats, it became evident that both experimental groups exposed significant growth retardation. These observations are in agreement with those reported in previously published studies (Mauer et al., 1989). Aminoguanidine hydrochloride (Sigma, Rejovot, Israel) was subcutaneously injected on a daily basis to the corresponding group of diabetic animals. Dosage was settled at 0.4 ml/100 g (5 mg of aminoguanidine/ 100 g body weight) of a solution containing 1 g of the drug, prepared in 80 ml of PBS (phosphate-buffered solution). Fresh solution was prepared once a week and sterilized by filtration through 0.22-␮m bacteriological filters (Migada, Kiryat Shmona, Israel).

Peritoneal Dialysis This procedure was carried out at the end of the 6-month observation period in 6 rats of the control group, as well as in 8 additional aminoguanidinetreated and 6 untreated diabetic animals at completion of the same time interval. The procedure was performed under neuroleptanalgesia, done by intramuscular injection (hind limbs) of a combination of fentanyl and droperidol (Janssen, Beerse, Belgium) in normal saline, in a ratio of 1/50 (0.004 mg of fentanyl and 0.2 mg of droperidol for 100 g of body weight). Animals were placed on a pad, electrically warmed at 37°C. Then, a thin-wall silicone sterile needle (1.10 mm outer diameter, 0.85 mm internal diameter), attached to a 100-mm infusion set (Labomed, Italy), with a total volume capacity of 0.2 ml (dead space), was gently introduced into the abdominal cavity, and a first exchange volume (10 ml/100 g body weight) of a commercially available lactated solution for peritoneal dialysis, at pH 5.2, with a glucose concentration of 1.5

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Shostak, Wajsbrot, and Gotloib

g/100 ml (83 mmol/L; Dialine, Teva Medical, Ashdod, Israel) was infused and left to dwell for 60 min. After drainage by gravity, a second exchange of the same solution, having a similar volume, was instilled over 60 to 80 s and also left to dwell for 60 min, counted since the completion of the infusion period. Samples of the dialytic fluid were taken at zero time (before the infusion), and after 60 min of dwell, when the abdominal cavity was drained by gravity. All infused fluids were warmed at 36 to 37°C. Recovered volumes ranged between 92 and 103% of the injected fluid (mean, 97.6 ⫾ 2.17%; coefficient of variation, 2.22%). Samples of the effluent were used for estimating total proteins with the Bradford assay (Bollag and Edelstein, 1991) and electrophoresis, done with the agarose gel procedure used for urinary proteins (Titan gel-urine protein procedure, Helena, Beaumont, TX). Plates were read in a Cliniscan 2-scanning densitometer (Helena). Albumin concentration in the effluent was calculated according to data obtained from total protein content and the corresponding value of albumin observed in the electrophoretic plate. After 60 min of dwell time, blood samples were obtained from the tail vein for estimating total proteins, using the methodology already described. Separation and quantitation of serum proteins were done by agarose gel electrophoresis (Titan gel system, Helena).

Evaluation of Albuminuria Before peritoneal dialysis, animals were housed in metabolic cages for 3 days. Twenty-four-hour urine collections were obtained during the third day. Urinary concentrations of total proteins and albumin were determined with the same techniques used for dialysate effluent. At the end of the collection period and in order to estimate the level of glycemia, samples of blood were obtained from the tail vein and processed using the glucose oxidase method (Trace Scientific PTY, Melbourne, Australia).

Leakage of Albumin to the Interstitial Compartment Albumin permeation from the blood microvessels to the mesenteric interstitial tissue was evaluated with

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Aminoguanidine Preserves Anionic Charges

Evans blue, as done in a previous study of our laboratory (Shostak and Gotloib, 1996). This azo dye, showing a molecular weight of 960.83 Da, is tightly bound to serum albumin just a few seconds after intravenous injection (Sirois et al., 1988; Patterson et al., 1992; Lange et al., 1994). This part of the study was done in three groups of rats each, at the end of the 6-month follow-up period: intact controls (n ⫽ 20), aminoguanidinetreated (n ⫽ 18), and untreated diabetic animals (n ⫽ 17). These animals were also used to estimate the growth curve. Under neuroleptanalgesia, rats were given a bolus injection of Evans blue through the exposed femoral vein (Merck, Darmstadt, Germany), at a dosage of 10 mg/kg, taken from a stock solution of Evans blue (15 mg/ml) in 0.9% normal saline. The same Evans blue stock solution was used along the whole experiment. After toracolaparotomy, the mesentery was perfused with 200 ml of phenol red-free HBSS (Biological Industries, Kibbutz Beit Ha Emek, Israel) at 37°C, at a flow of 5 ml/min, via a cannula inserted in the left ventricle. The abdominal aorta was previously ligated at the level of the renal arteries. This procedure was performed to remove the dye still present in the mesenteric vascular bed. Exsanguination was done by sectioning the inferior vena cava. Pieces of macroscopically avascular segments of mesentery were removed and immediately weighted. Then, samples were split in two pieces. One was dehydrated in an oven at 62°C for 24 h and weighted again to obtain the dry weight. The other one, after weighting, was immersed in formamide (Sigma, Rejovot, Israel) (4 ml/g of wet tissue) for 24 h, being the main purpose the extraction of the dye (Sirois et al., 1988). Care was taken to have both pieces of each sample show equivalent weight. Concentration of Evans blue in formamide was determined by an Elisa reader (Elisa Reader, SLT, Spectra A ⫺ 5082, Salzburg, Austria) at 620 nm wavelength and 96-well microplates (Nunc, Roskilde, Denmark). Readings were plotted on a standard curve of Evans blue (0.1 to 20.0 ␮g/ml) in formamide. Results showing Evans blue content of each sample were expressed as Evans blue ␮g/100 mg of dry weight tissue.

Assessment of Mesenteric Subendothelial and Submesothelial Electronegative Fixed Charges by Electron Microscopy The methodology followed for the electron microscopic part of this study was done using ruthenium red (RR) (Merck, Darmstadt, Germany). The procedure was carried out with the animals in neuroleptanalgesia. After exposure and ligation of the femoral artery, a fine polyethethylene tubing was introduced up to the thoracic portion of the aorta and fixed in place by means of a ligature. The next surgical step was dissection and exposure of the left jugular vein. Whole-body perfusion was started with Hank’s balanced salt solution (HBSS), injected through the femoral artery polyethylene tubing, whereas the left jugular vein was sectioned and used as an outlet for blood and the perfused fluids. HBSS was infused at a flow rate of 4 to 5 ml/min, during approximately 5 min, using an infusion pump (Model 201, Medix, Rejovot, Israel) to wash out the blood, and followed by 30 ml of 1% paraformaldehyde (Polaron, Watford, Hertforshire, England) in 0.1 M sodium-cacodylate (Electron Microscopy Sciences, Washington, PA) at the same flow rate. Perfusion was completed by means of a 0.2% RR solution prepared in Karnovsky’s aldehyde fixative, 1% paraformaldehyde, and 3% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (pH 7.2) with 4.4 M CaCl 2 (Kanwar and Farquhar, 1979). Before use, the dye-fixative solution was passed through a Millipore filter (pore size 0.22 ␮m) to remove large dye particles. Fluids for perfusion were kept at 37°C. After perfusion was completed, a laparotomy was performed. Two samples of macroscopically avascular mesentery were taken from different areas. Small pieces of tissue (⬇1 mm 3) were immediately excised and immersed in the same dye fixative for approximately 20 h. Then, they were washed for 2 h with sodium cacodylate buffer (25 ml at pH 7.2), containing 0.05 g of RR, and postfixed in 2% osmium tetroxide for 1 h, and, after dehydration in graded alcohols (Frutarom, Haifa, Israel), the small samples were embedded in Epon 812 (Electron Microscopy Sciences) according to standard procedures, cut with an LKB-III Ultratom (LKB, Sweden), stained with lead citrate (Electron Microscopy Sciences), and

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examined in a Phillips-300 electron microscope. Perfusion with RR was done in a total number of 15 rats: (a) 5 from the control group at zero time; (b) 5 more from the control, nondiabetic animals at the end of the 6-month follow-up period; and (c) 5 additional aminoguanidine-treated diabetic rats at the end of the observation period of 6 months. For this morphometric part of the study, 2 blocks were prepared from samples obtained from each animal, resulting in 10 blocks for each experimental group. Two to three electron micrographs were randomly taken from each grid on crosssectional areas of the mesenteric mesothelium, at a magnification of ⫻41500, making a total of 25 photographs for each experimental group. The same procedure was applied to cross-sectional areas of submesothelial continuous capillaries. Additional photographs were taken focused to record specific alterations of the submesothelial and subendothelial basement membrane, namely splitting and thickening. The length of submesothelial and subendothelial basement membranes was measured by planimetry (Table 1). RR particles showing a diameter larger than 1 ␮m (0.5 mm in electron micrographs taken at ⫻41500), decorating the anionic fixed charges, were counted along both aspects of the basement membrane. Counts were made twice by two different operators, not knowing the identity of specimens. Differences between counts were lower than 5%. Values reported in this study represent pooled numbers of both counts.

Statistical Treatment of Data Data are presented as means ⫾ SD. Differences between groups were evaluated with the one-way analysis of variance and the Tukey test for multiple comparisons. The two-tailed Fisher exact test was used to assess data dealing with categorical variables. Minimal required sample size was determined on the base of ␣ ⫽ ␤ ⫽ ⫺0.95. In cases not complying with this requirement, it was inferred that differences were devoid of statistical significance, even if the Tukey or the Fisher exact test pointed at rejecting the null hypothesis. Analysis of data was done using the GraphPad 3.0 Prism software (GraphPad Software, San Diego, CA).

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Shostak, Wajsbrot, and Gotloib

FIG. 1. At the observation period of 6 months, the urine albumin excretion of the diabetic untreated rats was several times higher than that in the control and the aminoguanidine-treated groups of animals (P ⬍ 0.001; for ␣ ⫽ ␤ ⫽ 0.05, n ⫽ ⬍4). (Control zero time) n ⫽ 10; control 6 months; n ⫽ 10; aminoguanidine 6 months; n ⫽ 8; diabetic untreated rats, n ⫽ 6). (No asterisk, not significant. ***P ⬍ 0.001.)

RESULTS Twenty-Four Hour Urinary Albumin Excretion As shown in Fig. 1, after 3 and 6 months of diabetes treated by means of aminoguanidine, albumin excretion in urine was higher than values seen in intact control animals at zero time. However, the small difference between means of values detected at the end of the 6-month observation period in control and diabetic aminoguanidine-treated rats failed to show statistical significance. This inference supports the view that administration of aminoguanidine substantially prevented the development of significant albuminuria. This contention becomes much more evident when urinary albumin losses, in control and aminoguanidine-treated rats, were compared with the corresponding values seen in diabetic untreated rats at the end of the 6-month observation period (Fig. 1). Indeed, the albumin excretion seen in the latter group of animals was 6 times higher than that detected in the aminoguanidine group.

Evans Blue Concentration in the Interstitial Compartment Albumin extravasation detected in the mesenteric interstitial tissue of diabetic rats was 2 to 3 times

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Aminoguanidine Preserves Anionic Charges

FIG. 2. After 6 months of follow-up, permeation of albumin into the mesenteric interstitial tissue was significantly higher in diabetic untreated rats, compared with values evaluated in control and aminoguanidine-treated animals (P ⬍ 0.001; for ␣ ⫽ ␤ ⫽ 0.95, n ⫽ ⬍9). (Control 6 months; n ⫽ 20; aminoguanidine 6 months; n ⫽ 18; diabetic untreated rats at 6 months, n ⫽ 17.) (No asterisk, not significant; ***P ⬍ 0.001.)

higher than values recorded in control and aminoguanidine-treated diabetic animals (Fig. 2). The narrow difference identified between the aminoguanidine and the control groups was devoid of statistical significance. Indeed, for the null hypothesis to be rejected with 95% power, it would have required around 45 rats for each group.

FIG. 3. Serum albumin levels at the end of the follow-up period of 6 months. The lower values observed in the diabetic group of animals did not reach statistical significance. (Control, n ⫽ 10; aminoguanidine-treated rats, n ⫽ 8; diabetic untreated animals, n ⫽ 6; for ␣ ⫽ ␤ ⫽ 0.05, n ⫽ ⬎10.)

Density Distribution of RR-Decorated Anionic Fixed Charges As stated before, this parameter was evaluated in both submesothelial and subendothelial basement membrane. In mesothelium, these charges were located at both aspects of the basement membrane, even though there was a preferential distribution along the

Albumin D/P Ratio Serum albumin levels observed in the three experimental groups at the end of the 6-month follow-up period were not significantly different (Fig. 3), even though the mean value seen in untreated diabetic rats was well within the frame of hypoalbuminemia (32.4 ⫾ 5.80 g/L). Notwithstanding, on the same point of the observation period, the albumin D/P ratio evaluated in the untreated diabetic rats was more than two times higher than that calculated for the aminoguanidineinjected group (P ⬍ 0.001), as shown in Fig. 4. Nevertheless, the latter had an albumin D/P ratio significantly higher than values collected from intact nondiabetic controls (P ⬍ 0.001). So far, aminoguanidine substantially restricted the increase of albumin D/P ratios, but failed to completely prevent the change.

FIG. 4. At 6 months of follow-up, the albumin D/P ratio of diabetic rats exposed to aminoguanidine was significantly higher than that in the control group (P ⬍ 0.001; for ␣ ⫽ ␤ ⫽ 0.95, n ⫽ ⬍3). However, values obtained from the diabetic untreated rats doubled those in the aminoguanidine group (P ⫽ ⬍ 0.001; for ␣ ⫽ ␤ ⫽ 0.05, n ⫽ ⬍3). (Control, n ⫽ 6; aminoguanidine, n ⫽ 8; untreated diabetic animals; n ⫽ 6.) (No asterisk, not significant. ***P ⬍ 0.001).

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FIG. 5. (A) Mesothelium of an intact control rat taken after a follow-up period of 6 months. Ruthenium red-decorated electronegative charges, the anionic sites (black arrows), are regularly distributed along the submesothelial basement membrane (black star), preferentially on its abluminal aspect. Additional negative charges can be seen in the submesothelial interstitial tissue (open star), indicating the presence of anionic glycosaminoglycans, decorated by the cationic tracer. (S, peritoneal space; V, microvilli; M, mesothelial cell; asterisk, interstitial tissue; black star, basement membrane; arrows, RR-decorated anionic fixed charges; open star, RR labeling anionic charges in the interstitial tissue.) (B) Sample of mesothelium taken from an untreated diabetic rat, after a follow-up period of 6 months. Thick straight arrow points at ruthenium red-labeled anionic sites, the density distribution of which is substantially lower than that observed in intact controls (compare with A). Curved arrow indicates an area of thickened basement membrane. Compare with the area showing normal thickness (black star). (S, peritoneal space; M, mesothelial cell; open star, pinocytotic vesicle; asterisk, submesothelial interstitial fluid.) (⫻41500)

lamina rara externa (Fig. 5A). This pattern was also observed in the capillary basement membrane (Fig. 7A). Back to the mesothelium, the density distribution of RR-decorated anionic sites counted in control rats at zero time, control at 6 months (Fig. 5A), and aminoguanidine-treated animals at the end of the same observation period (Fig. 6) was not significantly different (between 32 and 35/␮m of basement membrane) (Table 1). These values are, to a large extent, considerably higher than those of previous observations of our laboratory done on diabetic untreated rats, where the fixed electronegative charges were reduced to a density of 12 ⫾ 2/␮m of basement membrane (P ⬍ 0.001) (Shostak and Gotloib, 1996). A similar situation was unveiled when the capillary basement membrane was explored. Again, counts of RR-decorated anionic fixed charges done in samples of mesentery taken from intact rats at zero time after 6 months of follow-up (Fig. 7A), as well as from aminoguanidine-treated diabetic rats (Fig. 8), failed to show

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significant differences between means obtained from the three experimental groups (Table 1). Again, as previously reported (Shostak and Gotloib, 1996), the density distribution of RR-labeled negative charges in the mesenteric microvascular basement membrane of untreated diabetic rats of 13 ⫾ 3 ␮m was considerably lower than the value observed in the present study, in capillaries of aminoguanidine-treated diabetic rats (P ⬍ 0.001) (Fig. 7B). In both cases, quantitative estimation of the fixed negative charges was done after an observation period of 6 months.

Splitting and Increased Thickness of the Submesothelial and Capillary Subendothelial Basement Membrane Thickening of the basement membrane, characteristic of diabetic microangiopathy (Kilo et al., 1972; Giannini and Dyck, 1995), has also been observed in the mesenteric submesothelial basement membrane of di-

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FIG. 6. Mesothelium of a diabetic rat treated for 6 months with aminoguanidine. Thick arrows point at ruthenium red-decorated anionic sites, regularly distributed along the submesothelial basement membrane (black arrow). Curved arrow indicates one area of thickened basement membrane, showing a high density of anionic sites labeled by the cationic tracer. Also, negative charges can be seen in the submesothelial interstitial tissue (open arrow). (S, peritoneal space; V, microvilli; M, mesothelial cell; asterisk, submesothelial interstitial tissue.) (⫻41500)

abetic rats (Shostak and Gotloib, 1996) (Fig. 5B). The influence of aminoguanidine upon these changes is somehow controversial. Whereas some investigators reported prevention of basement membrane thickening in experimental diabetes (Ellis and Good, 1991; Yamauchi et al., 1997), other groups presented evi-

dence that aminoguanidine administration failed to put a stop to basement membrane widening (SoulisLiparota et al., 1996). As shown in Fig. 9, this is also our experience. Furthermore, layering or splitting of the microvascular basement membrane was not prevented by aminoguanidine (Fig. 10).

TABLE 1 Density Distribution of Ruthenium-Red-Decorated Sites (AS) along Both the Mesenteric Capillary Subendothelial and the Submesothelial Basement Membranes Subendothelial basement membrane Group (5 rats/group) Control, zero time Control, 6 months Diabetes ⫹ aminoguanidine, 6 months P

Submesothelial basement membrane

Length of BM (␮m)

AS/␮m BM

95% Confidence interval

Length of BM (␮m)

AS/␮m BM

95% Confidence interval

66.80 (n ⫽ 5) 62.70 (n ⫽ 5) 71.30 (n ⫽ 5)

32 ⫾ 4

29–33

33 ⫾ 3

31–36

33 ⫾ 4

31–35

32 ⫾ 3

30–35

35 ⫾ 2

33–36

76.30 (n ⫽ 9) 59.40 (n ⫽ 5) 77.25 (n ⫽ 5)

35 ⫾ 3

34–36

NS

NS

Note. The numbers given represent mean number ⫾ SD of particles of the cationic tracer per micrometer length of basement membrane (BM). Since the statistical unit (AS) exhibits a discontinuous distribution, values are presented without decimals. The density distribution of anionic sites along subendothelial and submesothelial basement membranes, observed in the three groups of experimental animals, is devoid of statistical significance (one-way ANOVA and Tukey test). Values between parentheses indicate the number of animals included in each experimental group, at each time interval.

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Shostak, Wajsbrot, and Gotloib

FIG. 7. (A) Cross section of a mesenteric capillary observed in a sample taken from a control, intact rat, after a follow-up period of 6 months. Notice the presence of regularly distributed anionic sites decorated by particles of the cationic tracer (arrows), along both aspects of the subendothelial basement membrane (black star). (L, microvascular lumen; E, endothelial cell; asterisk, subendothelial interstitial tissue.) (⫻41500) (B) Partial aspect of a blood capillary seen in a sample of mesentery obtained from diabetic untreated rats at the completion of the 6-month follow-up period. Notice the scarcity of ruthenium red-decorated anionic fixed charges (arrows) along the subendothelial basement membrane. (L, microvascular lumen; E, endothelial cell; asterisk, subendothelial interstitial tissue.) (⫻41500)

DISCUSSION The main message provided by this study is that despite severe hyperglycemia, administration of aminoguanidine prevented the loss of electronegative charges, normally present in the microvascular and submesothelial basement membranes. This fact coincided with an almost entire preservation of the permselectivity capabilities of the microvascular and mesothelial barrier, as endorsed by the absence of abnormally high albuminuria and increased albumin leakage in the mesenteric interstitial compartment, as well as by the considerably lower albumin D/P ratio seen in the aminoguanidine-treated group, compared with the corresponding values recorded in the untreated diabetic animals. Certainly, we cannot disregard the fact that other changes characterizing experimentally induced diabetes appear distinctly insensitive to the “protective effect” of aminoguanidine. Growth rate was significantly impaired and, not less important, splitting and

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thickening of subendothelial and submesothelial basement membranes were noticeable, as seen in untreated diabetic rats. It should be noted that after longer periods of follow-up (9 and 14 months), two studies evaluating glomerular basal membrane thickness in rats exposed to aminoguanidine (Ellis and Good, 1991; Yamauchi et al., 1997) showed that the treatment administered from the onset of experimental diabetes quantitatively limited the magnitude of the GBM widening. However, the change was not absolutely prevented. Conversely, electron microscopic observations made on glomerular capillaries taken from streptozotocin-treated rats after 6 months of diabetes revealed that food glucose control by means of insulin prevented basement membrane thickness within the normal values (Rasch, 1979). In addition, it has been postulated that long-term exposure to abnormally high levels of hydrogen peroxide can bring thickening of the microvascular basement membrane (Asahina et al., 1995). Certainly, the pathophysiology of the microvascular and tissue changes induced by diabetes are still pon-

Aminoguanidine Preserves Anionic Charges

FIG. 8. Blood mesenteric capillary. The sample was taken from a diabetic rat treated with aminoguanidine for 6 consecutive months. The subendothelial basement membrane shows regularly distributed ruthenium red-labeled anionic fixed charges (arrows) along both aspects of the subendothelial basement membrane (star). (R, red blood cells; L, microvascular lumen; E, endothelial cell; C, collagen interstitial fibers.) (⫻41500)

dered. Several groups postulate that, besides accumulation of AGE (Brownlee et al., 1988; Vlassara et al., 1992), certain additional mechanisms appear to be also entangled within a multifactorial combination, leading to the diabetic-induced alterations at the molecular level. Actually, AGE is thought to induce a decreased presence of heparan sulfate proteoglycans through blocking the heparan sulfate-binding protein, fibronectin (Brownlee, 1992). However, nonenzymatic glycation of albumin modifies the net charge of both circulating albumin and tissue proteins (Ghiggeri et al., 1984; Pietravalle et al., 1991). Serum albumin with a pH ranging between 4.0 and 5.8 is an anion that, at pH 7.4, holds more than 200 negative charges per molecule. Glycation of native albumin generates for-

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mation of cationic subunits which, in turn, determine a remarkable higher isoelectric point (Pietravalle et al., 1991) that makes way for increased transcapillary permeation not only of glycosylated albumin, but also of native anionic albumin (Purtell et al., 1979). The fact that positively charged albumin is freely excreted in urine (Purtell et al., 1979) supports the relevance of charge selectivity of the capillary wall. At the same time, the increased glomerular permeability for anionic albumin suggests neutralization of the microvascular anionic fixed charges by the glycosylated form of albumin (Shostak and Gotloib, 1996; Vlassara et al., 1992). These considerations have also been applied to the diabetic peritoneum (Shostak and Gotloib, 1996). However, evidence reported by other investigators points to the presence of additional pathophysiological mechanisms lying behind the decreased capillary permeability detected in diabetes: basically, oxidative stress derived from glucose autoxidation, from increased H 2O 2 formation stimulated by Amadori products, and lipoxidation (Monnier et al., 1996; Wolf et al., 1991; Baynes and Thorpe, 1999), as well as a relative or absolute increase in nitric oxide production (Tilton et al., 1993; Hasan et al., 1993). It has been shown that aminoguanidine has some capability of limiting glycoxidative reactions (Wells-Knecht et al., 1995), and is a potent inhibitor of nitric oxide synthase activity (Tilton et al., 1993; Hasan et al., 1993). So far, in view of these facts, it may be presumed that the shielding effect of aminoguanidine upon the negatively fixed submesothelial and microvascular charges, and the consequent nonappearance of increased albumin permeation into the interstitial compartment, may be connected to all the aforementioned properties of the compound. However, the proportional contribution of each one of them still remains to be established. In this sense, our observations with albumin are not far from the recently reported protective effect of aminoguanidine upon the extravasation of macromolecular FITC dextran 70 and 20 kDa seen in the cheek pouch of diabetic hamsters (Mayhan and Sharpe, 2000). Even though aminoguanidine apparently is not going to be used in clinical grounds, it still remains an important tool in probing the molecular mechanisms leading to the long-term microvascular complications of diabetes. So far, the information presented in this

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176

Shostak, Wajsbrot, and Gotloib

FIG. 9. This blood capillary was exposed to view in a sample of mesentery obtained from a diabetic rat treated by means of aminoguanidine for 6 months. Note the substantially increased thickness of the subendothelial basement membrane (thick arrow). Ruthenium red-labeled anionic fixed charges (small arrows) are regularly distributed along both aspects of the basement membrane. (L, microvascular lumen; E, endothelial cell; asterisk, subendothelial interstitial tissue.) (⫻41500)

study confirms the suitability of the pathophysiological approach on which the working hypothesis of aminoguanidine was based.

Finally, the persistent presence of basement membrane thickening and splitting, despite aminoguanidine administration as the only therapeutic interven-

FIG. 10. Section of a mesenteric capillary taken from a diabetic rat treated with aminoguanidine for 6 months. Note the splitting of the basement membrane (thick arrows), as well as the normal density distribution of ruthenium red-decorated anionic fixed charges (curved arrows). (L, microvascular lumen; E and E⬘, adjoining endothelial cells; thin long arrow, intercellular junction; asterisk, subendothelial interstitial tissue.) (⫻41500)

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Aminoguanidine Preserves Anionic Charges

tion, suggests that additional measures, like appropriate control of glycemia (Nicholls and Mandel, 1989; Lyons et al., 1991) (not performed in the present study), antioxidants (Monnier et al., 1996; Wolf et al., 1991; Baynes and Thorpe, 1999), inhibitors of nitric oxide production (Tilton et al., 1993; Hasan et al., 1993), and/or perhaps supplementary procedures still to be identified are to be used at once to effectively prevent the microvascular anatomo-functional alterations resulting from long-standing diabetes.

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