2.1 Materials
Chemicals: cis-Diamineplatinum (II) dichloride (Cat#479306) was purchased from Sigma Aldrich (MO, USA) and human AngII (Cat#51480) was purchased from Mimitopes (VIC, Australia).
Antibodies: rabbit monoclonal CD31 (Cat#182981) was purchased from abcam (VIC, Australia) and rabbit polyclonal antibodies AT1R (Cat#sc-515884) and AT2R (Cat#sc-9040 (Cat#sc-138439) were purchased from Santa Cruz Biotechnology (TX, USA).
2.2 Ethics approval and animals
Male C57BL/6 mice (n = 8) at 9 weeks of age were purchased from the Animal Resource Centre (VIC, Australia). Animals were housed 4 per cage at the Victoria University Werribee Campus Animal Facilities until 18 weeks of age. Animals were kept on a 12-hr day/night circadian rhythm cycle and maintained at a constant temperature of 21 °C. Relative humidity level was kept between 40 and 70 %. Food and water were supplied ad libitum. All experimental procedures were conducted in accordance with the National Health and Medical Research Council ‘Australia Code of Practice for the Care and Use of Animals for Scientific Purposes’ (8th edition, 2013; (8th edition, 2013, https://www.nhmrc.gov.au/about-us/publications/australian-code-care-and-use-animals-scientific-purposes), and were approved by the Victoria University Animal Ethics Committee (VUAEC: 20/003).
2.3 Acute cisplatin model and euthanasia protocol
Animals were randomly assigned into vehicle (n = 4), or cisplatin (n = 4) treated groups using a number table. On day 0 vehicle and cisplatin treated mice received an intraperitoneal (i.p.) injection of 0.9 % sterile saline. On day 1 vehicle treated mice received and i.p. of sterile saline and cisplatin treated mice received single cisplatin 12.5 mg/kg dose, dissolved in saline. Mice were euthanized 72 hours following cisplatin injection.
Animals were anaesthetised using isoflurane (4 %) and when absence of pain reflex (i.e., nail bed pinch) was observed animals were euthanized via cervical dislocation. The kidneys and blood vessels, including abdominal aorta (AA), thoracic aorta (TA), brachiocephalic artery (BC) and iliac arteries (IL), were retrieved from each mouse for immunohistochemical analysis and vasoactive studies. Under a light microscope blood vessels were cleaned of connective and adipose tissues and dissected into 2-3 mm rings in preparation for isometric tension analysis.
2.4 Drug incubation and isometric tension analysis
Arterial rings were immediately and sequentially placed into adjacent organ baths (Zultek Engineering, VIC, AUS) filled with 5 mL of Krebs solution, maintained at 37 °C and continuously bubbled with 95 % carbogen. Rings were allowed to acclimatize for 15 minutes and were then mounted between two metal organ hooks attached to force displacement transducers, stretched (AA/TA: 0.4-0.5 g; IL: 0.25-0.3 g; and BC: 0.1-0.2 g) and rested for 15 minutes. Rings were refreshed, re-stretched, and allowed to acclimatize for a further 15 minutes. To determine vasoconstriction responses, an AngII dose response was performed [10-8.0 M – 10-5.0 M, half log units].
2.5 Immunohistochemistry technique
Blood vessels and organs were fixed overnight in paraformaldehyde (4 %, pH: 7.4) in preparation for paraffin processing using a Thermo Scientific Spin Tissue Processor Microm STP 120. All processing was performed in one batch to avoid confounders. Organs were vertically embedded into paraffin blocks and cut to 5 µm sections. Ribbon sections placed into a 45 C water bath containing gelatine, mounted onto slides, and dried in a 37 C oven for 4 days. Tissues were deparaffinized using a xylene, ethanol and Tris buffer (pH: 7.4) gradient, as previously established by our laboratory (19, 20). Non-specific binding was blocked using 1% goat or donkey serum for 20 minutes prior to antibody incubation. Slides were then incubated overnight in a humidified chamber with the following antibodies: AT1R (renal tubules 1:150; blood vessels 1:10) and AT2R (renal tubules 1:250; blood vessels 1:10). For positive controls, CD31 (1:100) was used in blood vessels to determine presence of the endothelium. Negative control slides were not incubated with primary antibody. Next, slides were incubated for 60 minutes with anti-rabbit and goat secondary antibodies (ImmPRESS, Vector Labs, USA), stained with the 3,3′-diaminobenzidine chromogen for 5 minutes, and counterstained with haematoxylin for 2 minutes [19, 20]. Slides were then dehydrated and prepared for quantification.
2.6 Immunohistochemistry semi-quantification and analysis
To determine protein expression in the blood vessels and organs 8 photographs per tissue were taken at x400 magnification under an Olympus microscope using the program Leica acquisition software (Leica DFC 450F, Lecia Germany). Each image was loaded into the Micro Computer Imaging Device 6.0 program (MCID, Interfocus, UK). For blood vessels and renal vasculature, the ribbon tool was selected to trace the endothelium and the outline tool was used to trace the media and adventitia. In the kidneys, the tubules and glomeruli were independently assessed. Tubules were imaged at 40x magnification, and glomeruli and interlobar vessels were photographed at 100x magnification under immersion oil. To quantity peptide expression in glomeruli, the circle outline tool was selected and used to trace individual glomeruli and the area of the circle was kept consistent. To quantify protein expression in the renal tubules, the glomeruli were first blocked out using the circle outline tool. The outline tool was the selected to trace the remaining area. A specific range of colour intensity and hue was selected to detect the expression of the chromogen stain and buffer any non-specific binding. The colour intensity and proportional area were recorded and averaged, and these values were placed into a spreadsheet and the proportional intensity was calculated using the following equation: proportional intensity (PI) = (1/colour intensity x proportional area). The PI was averaged for each tissue. All data are expressed as arbitrary units.
2.7 Statistical analysis
GraphPad prism (Version 9.2.0, GraphPad Software Incorporated, CA, USA) was utilized to analyze isometric tension and immunohistochemistry data. The significant p-value was set at p < 0.05, and a two-way or one-way ANOVA followed by Sidak’s multiple comparisons post hoc test was performed to determine significance in isometric tension and immunohistochemistry data, respectively. All data are represented as mean ± standard error of the mean (SEM).