Methodology for Measuring Steady‐State Glomerular Filtration Rate (GFR) Using Continuous Intravenous Infusion of FITC‐Inulin in Conscious Mice

Abstract

Studies were performed to determine the extent of renal disease in type II diabetic mice. GFR was measured in conscious chronically instrumented male 18 wk old diabetic (db/db; 53±1g; n=7) and control (db/m; 18±0.5g; n=6) mice. Under isofluorane anesthesia and aseptic conditions, a canula (RenaPulse) was implanted into the right jugular vein, advanced 5mm, sealed, routed subcutaneously to the scapular region, exteriorized, passed through a tether (PE320), and sutured into place using 6‐0 silk. Upon waking, mice were placed into metabolic cages and the tether attached to a counter‐balanced arm and stainless steel swivel (Instech; 375/D/22QM). Mice were infused continuously with 0.9% saline (48hr) and FITC‐inulin (24hr; Sigma, F3272; 15mg/ml) at 4.5 μl/min. Blood (300 μl) was collected from the submandibular plexus using a lancet (GoldenRod) into microtainer tubes (BD; 20U heparin) and spun in 3 microHCT tubes. Plasma was diluted (1:4) in 10mM HEPES (pH 7.4) and assayed for FITC‐inulin fluorescence (excitation 490, emission 520 nm). GFR was calculated based on the renal clearance of inulin: inulin infusion rate/plasma [inulin]. Fed blood glucose averaged 496±46 and 100±9mg/dl (p<0.05) in diabetic and control mice, respectively. HCT was not different between the groups (0.40±0.02 vs 0.44±0.05). GFR averaged 0.65±0.04 and 1.00±0.13ml/min/gKW in diabetic and control mice, respectively (p<0.05). GFR expressed/g BW was significantly reduced in the diabetic compared to control mice (5.8±0.5 vs 12.0±1.9). Measurement of GFR in the conscious mouse removes the influence of anesthesia on this parameter. In addition, GFR measured in anesthetized diabetic mice requires higher rates of fluid replacement compared to control. In summary, the db/db mouse exhibits a 35% reduction in GFR and represents an ideal animal model of human type II diabetic renal disease.