Isolated perfused kidney experiment

The sensitized rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). At 5 min after intra-arterial heparinization (500 U/kg) following catheterization of the right carotid artery and laparotomy, the right renal artery was catheterized via the superior mesenteric artery with a stainless-steel catheter (19 G) and then, renal perfusion was begun with the 5% bovine albumin (Sigma-Aldrich Co, St Louis, MO, USA) in Krebs solution (118 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 25.5 mM NaHCO₃, and 5.6 mM glucose). Then, the inferior vena cave was catheterized just beneath the right renal vein to obtain the outflow pathway following its ligation above the right renal vein. The right kidney was excised and put in the bath in which warm saline (37 °C) was continuously perfused. The isolated kidney was perfused with the albumin-Krebs solution (50 ml) that was pumped using a Masterflex roller pump from the reservoir through a heat exchanger (37 °C) in a recirculating manner at a constant flow rate so as to obtain the baseline renal arterial blood pressure (RBP) of 78 ± 6 mmHg. The perfusate was oxygenated in the reservoir by continuous bubbling with 95% O₂ and 5% CO₂ (perfusate PO₂ = 300 mmHg). The RBP and RVP were measured using pressure transducers (TP-400T, Nihon-Kohden) attached by sidearm to the appropriate cannulas with the reference points at the kidney pelvis. Renal blood flow rate (Q) was measured with an electromagnetic flow meter (MFV 1200, Nihon-Kohden), and the flow probe was positioned in the inflow line. RVR was calculated by the following equation: RVR = (RBP − RVP)/Q. RBP, RVP, Q and RVR were continuously recorded at 40 Hz by PowerLab.

Hemodynamic parameters were observed at least for 20 min after the start of perfusion until a stable state was obtained by adjusting Q and the height of the reservoir to a RVP of 0.5 ± 0.2 mmHg and a Q of 6.1 ± 1.3 ml/min/g. After the baseline measurements, the perfused kidneys excised from the sensitized and non-sensitized rats were randomly assigned to four groups, as for the in vivo experiments (n = 6/each group). At 20 min after an injection of l-NAME (100 μM) or d-NAME (100 μM), the antigen (2 mg) was injected into the reservoir.