Alterations of collagen content in kidney of diabetic rabbits.

Abstract

Collagen functions as the major structural element of renal connective tissue. This role is primarily carried out by the fibrous interstitial collagens, types I and 111 [l]. In addition to their structural role, the interstitial collagens are also involved in the pathology of the kidney, such as the diabetic nephropathy. Diabetes mellitus is generally believed to be the major cause in the development of chronic renal failure, which is characterized by thickening of the glomerular and tubular basement membranes [2]. The aim of this study was to investigate the alterations in total collagen content and proportions of types I and 111 collagen in the kidney of rabbits made diabetic by alloxan treatment. Male albino rabbits, aged 1 .O-1.5 years and weighing 1.4 to 2.0 kg were randomized into two groups consisting of 12 rabbits in the control and 10 rabbits in the diabetic group. The animals in the diabetic group were treated once a week for four weeks with alloxan at a dose of 120 mg/kg, dissolved in 0.5 ml saline solution, prepared immediately prior to use, administrated via the ear vein. Before and after the alloxan treatments, blood and urine glucose levels of the animals in both groups were determined. Rabbits were sacrificed after one month of treatment. Following decapitation of the rabbits in both groups, kidneys were removed and defatted with acetone and ether. Dry defatted weight (DDW) was determined for each specimen. Then, tissue samples were submitted to hydrolysis by 6 N HCI. Total collagen was estimated based on the measurements of Hyp content of kidney samples, by using the convertingfactor of 7.46 [3]. As previously described, defatted kidneys were subjected to pepsin digestion (10 mg/g dry tissue weight) with three repetitions (41. After a centrifugation step, the supernatant was pooled and a crude mixture of collagen was precipitated from the 0.5 M acetic acid solution by addition of crystalline sodium chloride up to a final concentration of 0.9 M. The precipitate was redissolved in 0.1 5 M NaCI, 0.05 M Tris-HCI, pH 7.4 and collagen types sequentially precipitated at 1.7 M (Type 111) and 2.6 M NaCl (Type I). Both collagen types were further purified twice with the buffer solution and reprecipitated at the appropriate molarity. Then, they were dialyzed against distilled water,followed by lyophilization [ 5 ] . SDS-PAGE of collagens was perfomed in slab gel with a stacking gel of 3.5 % and a separation gel of 5.0 % acrylamide in 0.05 M Trisglycine buffer, pH 8.8, containing 0.1 % SDS [6]. Samples reduced with 2-mercaptoethanol were run in the presence of 3.6 M urea. The mean fasting blood giucose levels in the diabetic group, during the treatments for two and four weeks (297.9 + 6.3 mg/dl and 366.0 L 9.3 mgidl) were found to be significantly higher than those measured at the initial state of theexperiment (101.9~4.0 mg/dl; p<O.OOl). Dry weightsof the kidney samples in the group of alloxan-treated diabetic rabbits were not observed to be significantly different than those of the control group. Total collagen level in the kidneys of diabetic animals, expressed as percentage of the defatted dry weight, was found to be significantly increased (1 4.5 %; p<O.OI). Type I collagen level was also 9.9 % greater than in the control group. However, the proportion of type Ill collagen was reduced by 31.4 % when compared to control rabbits. Table 1 represents the distribution of collagen types I and 111 determined in the kidneys of alloxan-induced diabetic and control rabbits. Table 1. Distribution of collaaen in the kidnev samples