Both type I and type II diabetic patients are at risk to develop nephropathy. Diabetic nephropathy is characterized by changes in both glomerular and tubular structure and function. Most studies have focused on alterations in the glomerulus, including abnormalities in glomerular permeability and capillary pressure, glomerular hyperplasia or hypertrophy and increase in mesangial volume.

Thickening of the basement membrane is a widely accepted characteristic of diabetic small blood vessel disease. Marked thickening of basement membranes is observed in capillaries and glomeruli in kidneys of diabetic patients and this change is believed to cause premature degeneration of the kidneys. Although glomerular basement membranes (GBMs) are thicker, they are more porous. As such, many experiments have focused on studying alterations in specific elements of diabetic GBM in order to understand the pathogenesis of diabetic kidney disease. The metabolism of proteins that are components of capillary GBMs has been shown to be altered in diabetic patients. Loss of heparan sulfate in GBMs is an early event in diabetic nephropathy and this leads to an impaired selective permeability of GBMs and consequently to the onset of proteinuria.

Progressive expansion of the mesangial matrix is one of the most characteristic histological feature of diabetic nephropathy. There are multiple components of the extracellular matrix (ECM) and neither the exact composition nor the factors responsible for its increase in thickness in diabetic nephropathy are known. It has been shown that not only synthesis of ECM components is increased but also degradation of the matrix is decreased.

Angiotensin-converting enzyme (ACE) inhibitors are a group of agents effective in reduction of blood pressure. The importance of renal circulation as a potential site of action for ACE inhibitors has been established. Several studies have shown that the control of hypertension not only reduces proteinuria but also delays progression of diabetic nephropathy. Among anti-hypertensive drugs, the ACE inhibitors have been found to be therapeutically more efficacious in protecting kidneys than other types of anti-hypertensive drugs. Recent long-term studies in diabetic patients with nephropathy showed that ACE inhibition can reduce proteinuria independently of their effects on blood pressure and improves the filtration properties of the GBM. On the other hand, other studies reported that ACE inhibitors prevent the increase of GBM thickness, glomerular volume, and total mesangial volume.

Furthermore, suggested that the nephroprotective effects of ACE inhibitors are partially due to the modulation of effects on the metabolism of GBM proteins. It has also been demonstrated that ACE inhibitors protect against damage of GBMs by preventing loss of glomerular heparan sulfate. Recent clinical trials have shown that ACE inhibitors delay loss of renal function effectively in diabetic and non-diabetic patients with nephropathy, but the mechanisms by which ACE inhibitors prevent loss of GBM function remains uncertain. Therefore, the purpose of the present study was to examine whether the ACE inhibitor perindopril, which has been shown to be more effective in protection of kidneys than other ACE inhibitors, prevents glomerular structural and functional alterations and to study whether the mechanism of the renoprotective effects is based on prevention of increased synthesis or reduced degradation of type IV collagen and laminin.

In the present study, 24 Wistar albino rats, weighing 150–200 g, were used. At the beginning of the study, blood glucose levels and body weights of all animals were measured. Animals were divided into three groups each containing eight animals. The first group was the control group. The other two groups were given 65 mg/kg streptozotocin (STZ; Sigma, St. Louis, MO, USA) dissolved in serum as a single dose intraperitoneally. No further treatment was given to the second group of rats. The ACE inhibitor perindopril (Servier, Istanbul, Turkey), was given by gavage in a dose of 0.66 mg/kg/day diluted in distilled water to the third group for 6 weeks. Blood glucose levels of all groups were measured weekly in blood samples from the tail vein by using reagent strips (Glucostix; Bayer, Istanbul, Turkey) with a glucometer (glucometer II model 5550; Ames, Indianapolis, IN, USA). All animals had free access to rat chow and drinking water. Animals were sacrificed at the end of the experimental period and tissue samples from kidney cortex were prepared for light and electron microscopic evaluation.

Renal tissue samples were fixed in formol saline and embedded in paraffin. Periodic acid Schiff (PAS)-stained preparations were used for histological examination. For the calculation of glomerular size, 10 PAS-stained sections were used per animal (n=8) and 30 glomeruli from each PAS-stained section were selected randomly. Measurements were performed using a micrometer ocular system (Beck, Kassel, Germany). Each experimental group contained eight rats. The mean of the measurements was calculated for each rat. Afterwards, means were calculated for each animal group.

Sections (thickness, 5 ?m) were collected onto slides coated with poly -lysine and were left overnight to dry at 37°C. Sections were then dewaxed and rehydrated. After a washing step in phosphate-buffered saline, sections were immersed in a solution of 3% H₂O₂ for 10 min. The sections were then preincubated with non-immune goat serum for 10 min. For the localization of laminin and collagen, type IV sections were incubated overnight with rabbit anti-laminin antibodies (Sigma) in a dilution of 1:25 and rabbit anti-type IV collagen antibodies (Biogenex Laboratories, San Ramon CA, USA) in a dilution of 1:250 at 4°C. Specific staining was performed with biotinylated secondary antibodies (goat anti-rabbit-IgG), the streptavidin peroxidase complex, and 3-amino-9-ethyl carbazole as chromogen. Detection procedures were carried out as described by the manufacturer (Biogenex). Sections were counterstained with hematoxylin.

The specificity of immunolabelling was assessed by omission of the primary antibody, incubation with non-immune serum as the first layer, and incubation with an irrelevant antibody (against insulin) in the same dilutions. All these control experiments gave negative results. Positive control experiments were carried out with known positive tissue and were included in each experiment. Immunostaining of kidney tissues was assessed independently by two blinded investigators.

Immunohistochemical staining was scored in a semiquantitative manner to determine differences between the control group and experimental groups in the distribution patterns of intensity of immunolabelling of basement membranes and mesangial matrix. The intensity of the staining was recorded as weak (+), moderate (++), strong (+++) and very strong (++++). This analysis was performed in at least 10 glomeruli per kidney section, in two sections from each animal and in three animals per group at ×100 magnification.