5.1 STZ-induced hyperglycaemic rat model
Animal experimentation was conducted at the Research and Development Unit, Academic Service Division, National Laboratory Animal Center, Mahidol University (NLAC-MU). Animal experimentation was performed following to the Thai Animals for Scientific Purposes Act, B.E. 2558 and the Guidelines for the use of animals of the National Research Council of Thailand. Eight week-old male (n = 20) Wistar rats were obtained from NLAC-MU. All of the rats were housed in a temperature-, humidity- and illumination-controlled room and fed ad libitum with standard diet and reverse-osmosis water. Consequent to the acclimatisation period, all rats were fasted for 6 h before being intraperitoneally injected with a single dose of 45 mg/kg streptozotocin (Sigma-Aldrich, USA) in fresh 0.1 M citrate buffer, pH 4.0 to induce hyperglycaemia [37]. Fasting blood glucose was examined in all of the rats, and a blood sugar level of ≥200 mg/dL was considered diabetic stage. Clinical manifestations were carefully observed daily by trained personnel for two weeks. Then all rats were humanely euthanised using an overdose of carbon dioxide inhalation. However, in case of moribund rats or rats that lost ≥20% of their weight before two weeks were found, an early endpoint of those rats was done as mentioned above. Their kidneys were collected, divided into two and then fixed in 10% neutral buffer formalin and 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer (SPB) for histopathologic and electron microscopic studies, respectively.
5.2 Histopathological studies
To demonstrate the presence of alpha 2u-globulin nephropathy in the STZ-induced diabetic rats and other histopathological changes in the liver and pancreas, histopathological studies were performed. Fixed kidneys, liver and pancreas underwent standard tissue processing and were cut into 5 μm thick sections. These sections were then stained with haematoxylin and eosin (H&E) and examined under a light microscope, focusing on (i) intracytoplasmic hyaline droplet deposition, tubular cast formation, tubular degeneration, tubular regeneration and Bowman’s space distension with proteinaceous fluid deposition for kidney, (ii) pyknotic nuclei, lymphocyte infiltration and centrilobular microvesicular steatosis for liver and (iii) Islet of Langerhans degeneration, vacuolar degeneration in the acinar gland and interstitial cell necrosis and inflammation of the pancreas. The lesions were semi-quantitatively graded using H-score (distribution [~0-100%/section] × severity score [0–3: 0 = absent, 1 = mild, 2 = moderate and 3 = severe]) as shown in our previous studies [39-43].
5.3 Immunohistochemical and immunofluorescence studies
To determine the pathogenesis of alpha 2u-globulin nephropathy induced by STZ injection, as relevant to water absorption and glomerular filtrate function via AQP, immunohistochemical (IHC) and immunofluorescence (IF) studies were conducted using EnVision FLEX/HRP kit (DAKO, Denmark) and VectaFluor Duet immunofluorescence double labelling kit, DyLight 488 Anti-rabbit (green)/DyLight 594 Anti-mouse (red) (VECTOR, USA), respectively. The sections were deparaffinised in xylene, hydrated in a series of graded ethanol and heat-retrieved to enhance the antigenicity in citrate buffer, pH 6.0. Polyclonal rabbit anti-AQP-1, -2, -4 and -5 (Millipore, USA) antibodies were incubated on the tissues. Appropriate secondary antibodies matching their conjugate and visualisation system from the kit were applied to the sections. The nuclei were counterstained by either haematoxylin or VECTASHIELD Antifade mounting medium with DAPI (VECTOR, USA) for IHC and IF, respectively. Immunolocalisation was measured using the H-score as mentioned above. In addition, the area of expression as a percentage was determined using an image analysis programme (ImageJ, version 1.51J8, NIH). Briefly, five images of labelled areas were captured and transformed to binary images. Immunolocalisation was defined by the threshold mode and determined as an area fraction (%).
5.4 Electron microscopic studies
To demonstrate the fine morphological structure of alpha 2u-globulin nephropathy in STZ-induced diabetes, electron microscopic studies were performed. The kidneys were again fixed with 1% osmium tetroxide in 0.1 M SPB, dehydrated in a series of graded ethanol, infiltrated and embedded in LR white resin (EMS, USA), polymerised in a 65°C oven for 24–48 h, cut into 100 nm thickness and finally stained with uranyl acetate and lead citrate. Ultrastructural changes in relation to alpha 2u-globulin nephropathy were examined under a transmission electron microscope (TEM) (Hitachi; model HT7700, Japan).
5.5 Immunogold labelling technique
To clarify the immunolocalisation of HDHD-3 (energetic maintenance protein) and NDUFS-1 (apoptotic protein) in the renal mitochondria, immunogold labelling technique were used. After the sections were blocked with 50 mM glycine and 5% bovine serum albumin (BSA) (EMS, USA), they were incubated with the described primary antibodies for 1 h at room temperature. Immunoglobulin (Ig) G conjugated with 10 nm gold particles (EMS, USA) was then applied to the sections for 1 h. Silver enhancement was performed using the Aurion R-Gent SE-EM kit (EMS, USA). Finally, the sections were stained with lead citrate and uranyl acetate and examined under TEM, focusing on the amount of gold labelling/mitochondria. Fifty mitochondria/group were assessed.
5.6 Statistical analysis
GraphPad PRISM, version 6.05, was used for statistical analysis. Either independent t-tests or analysis of variance was performed to characterise the difference between the groups and was expressed as the mean ± SEM. The 95% confidence interval p < 0.05 was considered statistically significant.