The animals were weighed and subsequently anaesthetised using Isoflurane for induction of at a concentration of 4% in Oxygen. An intraperitoneal injection of Inactin (Thiobutabarbital sodium salt hydrate, Sigma Aldrich) (100g/ml) was subsequently administered at a dose of 1ml/kg. If the animal showed signs of arousal, then it was additionally maintained on isoflurane 0.5-1 % in 1L/min Oxygen. A heated surgical table was utilised to maintain body temperature of the animal at 37°C.
The femoral vessels were then exposed in animals weighing more than 280 grams (the common iliac vessels were utilised for smaller animals). Fine bore polyethylene tubing (Smiths Medical) (0.96mm) was inserted into the vein under microscopic vision and secured in place using 3/0 silk sutures. A bolus of 0.4mls 1% FITC Inulin was administered via a 1ml syringe with a 23 gauge needle, which fitted exactly into the polyethylene tubing emerging from the vessel. An intravenous infusion of 0.2% FITC Inulin was subsequently commenced at a rate of 3mls/hr using a programmable syringe pump (World Precision Instruments – NE300). A similar sized cannula was then inserted into the accompanying artery and a sample of 0.4 mls blood is removed from the animal.
This was centrifuged at 5000rpm for 10 minutes and the plasma transferred to a 200àl eppendorf for storage up to 72 hours at 4°C. The arterial line was flushed with heparin /saline (1%/0.9%) with a v/v ratio of 1:5 so as to maintain patency. Laboratory biochemical analysis was then performed on the stored plasma sample for multiple parameters as indicated in the text below (Companion Animal Diagnostics, University of Glasgow).
A midline laparotomy was then made and the ureters of each kidney identified and ligated distally. A fine microcatheter (0.61mm) was inserted into each ureter and secured using 3/0 silk sutures. Urine was collected from each kidney into a separate tube at 30 minute
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intervals and a blood sample (80àl) was taken simultaneously. The 30 minute interval blood samples were spun at 11,000 rpm for 8 minutes and the plasma aliquoted into a fresh eppendorf taking care not to remove any of the interphase. After every blood sample, the arterial line was flushed with heparin/saline.
Equilibrium is reached in the plasma, when the amount of FITC inulin infused is equal to filtration by the kidneys. Several initial experiments indicated that equilibrium is established between 60-120 minutes after the 0.4ml whole blood sample is obtained for laboratory analysis (data not shown). A 0.4ml volume of whole blood is needed (to give approximately 200àl plasma for laboratory biochemical analysis) which results in temporary elevated plasma inulin levels, disturbing equilibration phase. 30 minute sampling is maintained for up to 3 hours in order to ensure a steady state of plasma inulin from which to calculate individual kidney GFR.
Subsequently, the kidney pedicle of the left kidney was dissected in order to clearly display the renal artery and vein and a vascular occluding clamp was applied for exactly 10 minutes. This produced a recoverable injurious state (primarily Acute Tubular Necrosis – ATN) lasting approximately 30-40 minutes (information based on several preliminary tests). Urine samples were then collected at 15 minute intervals post clamp release in order to quantitatively characterize the animal’s response to ischaemia-reperfusion (IR) injury.
A maximum of two hours of urine collection post clamping was performed.
The procedure was terminated at approximately 6 hours from initiation of anaesthesia by Schedule 1 killing. Tissues were immediately placed in 10% formalin for future histological analysis, RNA Later® for genetic expression analysis and liquid nitrogen for immunohistochemistry.
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Figure 2.1 Depiction of surgical setup
Rat Model Clearance study depicting the surgical setup in characterizing the renal phenotype of the AS/AGU mutant. Note cannulation of individual ureters to asses function of both kidneys. Also note cannulation of left femoral or iliac vessels to allow infusion of inulin and repeated arterial blood sampling.
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Figure 2.2 Images of surgical technique
Dissection and cannulation of the left femoral vessels and ureters. The groin is dissected and the femoral vessels are exposed beneath the inguinal ligament (a). The vein is carefully dissected from the artery and a clamp is placed proximally. A venotomy is made and the primed cannula containing inulin and is ready to be introduced (b). The venous cannula is introduced and the artery is subsequently dissected. Similarly, a clamp is placed proximally and an arteriotomy is made to allow the passage of a cannula for repeat blood sampling (c & d). The ureters of both kidneys are then cannulated. Here, the left kidney is visible with a cannula inserted into the ureter (e). The procedure continues with minimal physiological disturbance and the urine is collected in separate eppendorfs (f)
a b
c d
e f
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