Materials and Methods
Ex Vivo Glomeruli Assays
Glomeruli were isolated from rat kidney cortex using a differential sieving method. Freshly purified glomeruli (10 000 per well) were incubated in 2 ml of HAM-F10 medium (Invitrogen, Grand Island, NY, USA) supplemented with 1 μmol/l NECA (non-selective P1 receptors agonist), 50 nmol/l MRS1754 (A2BAR antagonist), 30 nmol/l CPA (A1AR agonist), 30 nmol/l DPCPX (A1AR antagonist), all from Tocris Cookson (Ellisville, MO, USA) at standard conditions (37 °C and 5% CO2) for 6 h. Also, supplementation with D-glucose 25 mM was carried out for 24 h. Five millimoles of D-glucose and supplementation with D-mannitol were included as controls. Following incubation, the glomeruli were collected by centrifugation and supernatants were stored at −70 °C. Total protein extracts were obtained from glomeruli resuspended in 150 μl RIPA buffer containing protease inhibitors 1 mM PMSF, 2 μM aprotinin, 1 μg/μl leupeptin and pepstatin (Roche, Indianapolis, IN, USA).
Short Interfering RNA
The generation of an adenoviral vector that codes a short interfering RNA targeting the sequence 5′-AACAGUAAAGACCGUGCCACC-3′ of rat A2BAR mRNA (nucleotides 540–560) or the scrambled sequence 5′-ACGUGAGACACCGAACCUAAC-3′ were constructed using pSilencer adeno 1.0-CMV System (Ambion, Austin, TX, USA). Efficient inhibition of A2BAR expression was obtained following 48 h from adenoviral infection of glomeruli in HAM-F10 medium (see Supplementary Figure 2).
Total proteins extracts (50 μg) fractionated by polyacrylamide gel (10%) electrophoresis were transferred to nitrocellulose membranes and probed with anti-VEGF antibody (1:1000) (ab46154 from Abcam, Cambridge, MA, USA). Membranes were washed in Tris buffer saline 0.1% Tween, and incubated 1 h in TBST/0.1% BSA containing HRP-conjugated goat anti-rabbit IgG antibody. Immunodetections were revealed by enhanced chemiluminescence and quantitated by densitometry. Following the stripping procedure, the membranes were probed with a monoclonal anti-β actin antibody (1:5000) (Sigma–Aldrich, St Louis, MO, USA) and revealed, as described above.
Enzyme-linked Immunosorbent Assay
The amounts of VEGF secreted by glomeruli were measured by a quantitative solid-phase Enzyme-Linked Immunosorbent Assay enzyme immunoassay designed to recognize rat VEGF164 (R&D Systems, Minneapolis, MN, USA). The sensitivity of the assay was 8.4 pg/ml.
Adenine Nucleotides and Adenosine Quantifications
Total glomeruli freshly purified from individual rat were incubated in 1 ml of tyrode's buffer at 37 °C and 5% CO2 for 1 h. Incubation medium and glomeruli were separated by centrifugation at 2000 g for 5 min. The adenine nucleotides and adenosine contents in supernatants were quantified using derivatization with chloroacetaldehyde and HPLC with fluorometric detection. For determination of the urinary nucleoside levels the urines of rats were recollected in metabolic cages for 24 h. Samples were processed according to previously described and derivatized for HPLC analysis.
Animals and Treatments
All animal studies were approved by the institutional animal care and use committee at Universidad Austral de Chile according to the NIH Guide for the Care and Use of Laboratory Animals. Diabetes was induced in male rats (Sprague-Dawley) weighing 250 g by single intravenous injection of streptozotocin 65 mg/kg (Merck, Darmstadt, Germany), dissolved in citrate buffer, pH 4.5. The diabetic groups included animals showing blood glucose levels ≥25 mmol/l. Following 4 weeks of diabetes induction, the rats were treated with the antagonist of A2BAR MRS1754 (0.2 or 1.0 mg/kg) for 2 weeks administered via intra peritoneal, every 2 days. Control rats received an equivalent volume of vehicle. Urines of rat were recollected in metabolic cage for 24 h and protein contents were quantified by the pyrogallol red molybdate method (Proti U/LCR, Wiener Lab, Rosario, Argentina).
Rat kidney tissues were fixed in formalin, paraffin embedded and 5 μm sections were mounted on sylanized slides. Immunodetection was performed as described previously using the primary anti-A2BAR, anti-A1AR, anti-VEGF and anti-nephrin polyclonal antibodies (ab40002, ab46154 and ab58968 from Abcam), and the monoclonal anti-α-smooth muscle actin (αSMA) (sc130617 from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunosignals were revealed using the LSAB+System–HRP system (Dako, Carpinteria, CA, USA). The podocytes number was estimated by counting Wilms tumor protein (WT)-1-stained nuclei in 30 glomeruli representatives of each group. Glomerular injury characterized by mesangial expansion was graded on the basis of the extent of glomerular staining using Periodic-acid Schiff (PAS) on a scale of 1–3 as follows: 1, normal; 2, mild; 3, moderate to severe.
Values are means±s.e.m., where n indicates number of animals. Statistical analyses were carried out on raw data using the Peritz F multiple means comparison test. Student's t-test was applied for unpaired data.
Lab Invest. 2013;93(1):135-144. © 2013 Nature Publishing Group