Korstanje1 project protocol

Aging study: Urine albumin and creatinine in 30 inbred strains of mice   (2008)

Korstanje R
With: Tsaih SW, Schultz D, Godfrey D




DiPetrillo1_Protocol

Project protocol — Contents
 Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for measuring urine albumin and creatinine
Data
References

Workflow and sampling

Workflow

Day
Procedure performed
Ages (mo)
Data collected
1-3
urine collection
12, 18, 24
-
4-
measurement of albumin and creatinine concentrations in the mouse urine
12, 18, 24
microalbumin and creatinine concentrations in the urine

Equipment and supplies

Autoanalyzer Synchron CX5 Clinical Chemistry Analyzer (Beckman Coulter, Brea, CA)

Reagents and solutions

  • Eppendorf microcentrifuge tubes for urine collection
  • Pasteur pipettes with rubber bulbs
  • Mouse albumin standards, Kamiya Biomedical Company (Seattle, WA) and Sigma (St Louis, MO)
  • Goat anti-human albumin antibody
  • Picric acid

Acclimation to test conditions

Mice are allowed to acclimate to testing room over the weekend.

Procedure for measuring urine microalbumin and creatinine

I. Urine collection in mice.
a. Freshly voided urine samples are collected in the mornings from individual mice for three consecutive days.
b. At least 125 µL of urine is needed for testing microalbumin (MA) and creatinine.
c. Each mouse is picked-up gently by cuffing the skin in the cervical region, behind the ears, and securing the head.
d. For a male mouse, it is positioned at approximately 60° angle over microcentrifuge tube, allowing the mouse to void urine freely.
e. For a female mouse, it is positioned at approximately 45° angle over microcentrifuge tube, allowing the mouse to void urine freely. Proper positioning of the mouse over the microcentrifuge tube is critical to avoid loosing urine sample.
f. With a full bladder that is readily palpable, a mouse can be stimulated to void by gently massaging the inguinal area of the abdomen using the free index and middle fingers.

II. Measuring mouse urinary microalbumin concentrations.
a. To validate the autoanalyzer method for quantifying mouse albumin concentrations, a commercial mouse albumin standard is used.
b. The series of mouse albumin standard concentrations are first measured using the autoanalyzer.

Kamiya Mouse Standards
Known microalbumin
Measured microalbumin
0
0.00
1.5625
0.67
3.125
1.01
6.25
1.82
12.5
3.60
25
5.87


c. Microalbumin in urine samples is then detected with a goat anti-human albumin antibody to form antigen–antibody complexes increasing the turbidity of the sample.
d. The change in absorbency at 380 nm spectral wavelength is measured by the autoanalyzer, and the value obtained is proportional to the concentration of albumin in the urine sample.
e. Calculation: measured microalbumin sample (mg/dL) ÷ standard slope (0.249) = actual urine microalbumin concentration (mg/dL).

III. Measuring mouse urinary creatinine concentrations.
Urinary creatinine concentrations are measured on an autoanalyzer using the Jaffe method, where creatinine combines with picric acid to form a creatinine-picrate complex that changes the absorbency of the solution.

Data collected by investigator

Urinary microalbumin (MA) and creatinine (CREA) concentrations.

MPD calculated measurement: [urine microalbumin (mg/dL) ÷ urine creatinine (mg/dL)]*1000 = urine microalbumin-creatinine ratio (ACR in mg/g).