TITLE: U Michigan - Glomerular Filtration Rate Determination with Minipump Inulin Clearance
AUTHORS: Jeff Hodgin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">This is the protocol for measuring Glomerular Filtration Rate (GFR) with minipump FITC-inulin clearance in mice. In brief, Alzet micro-osmotic pumps (minipumps, Model 1007D) are filled with 3% FITC-inulin solution. These pumps release FITC-inulin at a rate of 0.5 μl/hr at least seven days. Mouse is temporarily anesthetized and then two mini-pumps are implanted in mouse peritoneal cavity through a tiny midline incision. After surgery, 24-hour urine is collected with metabolic cage and 100 μl of blood from saphenous vein is obtained at the same time. The concentrations of FITC-inulin in urine and blood samples are measured by flurometer and GFR is calculated by the concentration of FITC-inulin in 24-hour-urine and the concentration of FITC-inulin in plasma.</div></div>

[STEPS]
?. Minipump preparation:
The micro-osmotic pumps are filled with approximately 100 μl of a 3 % FITC inulin solution.
?. Procedures of surgery:
2.1 Mice are anesthetized with isoflurane in gas anesthetic machine rented from Unit for Laboratory Animal Medicine (ULAM), University of Michigan.
2.2 Two minipumps are inserted into peritoneal cavity of mouse through an approximate 5 mm length of abdominal midline incision.
?. Blood and urine sample collection:
3.1 After the recovery period, mice are placed into metabolic cages and 24-hour urine is collected.
3.2 Immediate after urine sample collection, mice are restrained inside a 50-mL centrifuge tube with air-holes drilled in the tip.
3.3 The inner thigh is closely shaven and wiped with 70% ethanol, revealing the saphenous vein. A small incision is made with a scalpel, and 100-μl-blood is collected with a heparinized capillary tube (Fisher Scientific). The blood sample is centrifuged at 4000 rpm for 10 min and then take out plasma for the latter measurement.
?. Measurement of inulin in plasma and urine:
4.1 The urine samples are diluted 1:5 and 1:10 in HEPES, respectively.
4.2 The plasma samples are mixed with HEPES (pH 7.4) by 4:1.
4.2 The processed samples are loaded onto a 96-well plate (COSTAR 3595), 50μl of sample/well.
4.3 The fluorescence is measured with Fluoroscan Ascent FL (Labsystems), at excitation 485 nm, and emission 530 nm.
4.4 Standard curve: For each point on the urine standard curve, 200 μl of normal mouse urine is diluted into 300 μl HEPES. Then, 500 μl of FITC-inulin solution of varying known concentrations (dissolved in HEPES) is added to each tube. Five points are included on the urine standard curve, with inulin concentrations ranging from 0.05 mg/ml to 0.0008 mg/ml. For each point on the plasma standard curve, 160 μl of normal mouse plasma are mixed with 40μl of an inulin solution (made in HEPES) of known concentration. Four points are included on the plasma standard curve, with inulin concentrations ranging from 0.005 mg/ml to 0.0006 mg/ml. Then the standard curves are used to determine the concentration of FITC-inulin in the samples.
?. Calculation of GFR:
GFR is calculated by the concentration of inulin excreted in urine divided by the concentration of plasma inulin and expressed in ml/min/g body weight.