Validation of High-Throughput Methods for Measuring Blood Urea Nitrogen and Urinary Albumin Concentrations in Mice

Comparative Medicine Vol 56, No 6 Copyright 2006 December 2006 by the American Association for Laboratory Animal Science Pages 482-486 Validation of High-throughput Methods for Measuring Blood Urea Nitrogen and Urinary Albumin Concentrations in Mice Susan Grindle,1 Cheryl Garganta,2 Susan Sheehan,1 Joe Gile,1 Andree Lapierre,1 Harry Whitmore,1 Beverly Paigen,1 and Keith DiPetrillo1,†,* Chronic kidney disease is a substantial medical and economic burden. Animal models, including mice, are a crucial component of kidney disease research; however, recent studies disprove the ability of autoanalyzer methods to accurately quantify plasma creatinine levels, an established marker of kidney disease, in mice. Therefore, we validated autoanalyzer methods for measuring blood urea nitrogen (BUN) and urinary albumin concentrations, 2 common markers of kidney disease, in samples from mice. We used high-performance liquid chromatography to validate BUN concentrations measured using an autoanalyzer, and we utilized mouse albumin standards to determine the accuracy of the autoanalyzer over a wide range of albumin concentrations. We observed a significant, linear correlation between BUN concentrations measured by autoanalyzer and high-performance liquid chromatography. We also found a linear relationship between known and measured albumin concentrations, although the autoanalyzer method underestimated the known amount of albumin by 3.5- to 4-fold. We confirmed that plasma and urine constituents do not interfere with the autoanalyzer methods for measuring BUN and urinary albumin concentrations. In addition, we verified BUN and albuminuria as useful markers to detect kidney disease in aged mice and mice with 5/6-nephrectomy. We conclude that autoanalyzer methods are suitable for high-throughput analysis of BUN and albumin concentrations in mice. The autoanalyzer accurately quantifies BUN concentrations in mouse plasma samples and is useful for measuring urinary albumin concentrations when used with mouse albumin standards. Abbreviations: B6, C57BL6/6J; BUN, blood urea nitrogen; CKD, chronic kidney disease; D2, DBA2/J; RDRP, Renal Disease Research Plan Chronic kidney disease (CKD) is an important medical prob- renal function in mice will provide a key tool for forward genetic lem in the United States, affecting roughly 25 million people at strategies to identify genes underlying CKD, an important goal an estimated cost of $22 billion.6 In light of the substantial health set forth in the RDRP. risks associated with CKD, the National Institutes of Diabetes and High-throughput phenotyping of renal function and damage Digestive and Kidney Diseases, in conjunction with the Council is facilitated by clinical chemistry autoanalyzers, which can rap- of American Kidney Societies, devised the Renal Disease Research idly quantify the 3 principal clinical markers of kidney function Plan (RDRP) as a roadmap to improve the prevention, diagnosis, or damage: 1) glomerular filtration rate estimated from plasma and treatment of CKD. Several of the recommendations outlined and urine creatinine concentrations;4 2) blood urea nitrogen in the RDRP stress the need for better animal models of kidney (BUN) concentration;1 and 3) the ratio of albumin concentration disease, such as mutant mouse strains and novel mouse mod- to creatinine concentration in the urine.4 Because glomerular fil- els to study the progression of kidney disease. The RDRP also tration rate cannot be estimated reliably or measured easily in recommends new tools for identifying genes underlying kidney small animal models, such as mice, plasma creatinine levels have disease in current animal models. These mouse models represent been used as an alternative marker of kidney function for animal an important component of research into the onset and progres- studies. However, recent evidence confirms that chromagens sion of CKD because the chromosomal regions underlying kid- present in mouse plasma interfere with the Jaffe method com- ney disease in rodents often predict the location of genetic factors monly used in autoanalyzers to measure creatinine concentra- influencing kidney disease in humans.3 Common strategies for tions.2 This problem hinders high-throughput analysis of plasma identifying genes underlying disease in mice include mutagenesis creatinine concentrations in mouse studies. Although BUN is and quantitative trait locus analysis, but these strategies require not a sophisticated marker of renal function, BUN concentration large-scale phenotyping protocols dependent on high-throughput is an easy way to investigate renal function in mice and is ame- methods. Validation of high-throughput methods for measuring nable to high-throughput analysis required of primary pheno- typic screens in genetic studies. Therefore, we sought to validate Received: 10 Mar 2006. Revision requested: 4 Aug 2006. Accepted: 20 Aug 2006. the commonly used autoanalyzer methods for measuring BUN 1The Jackson Laboratory, Bar Harbor, Maine; 2Department of Genetics, Yale University and urinary albumin concentrations in mice as high-throughput School of Medicine, Biochemical Disease Detection Laboratory, New Haven, Connecticut. methods for assessing kidney function and damage, respectively, †Present address: Novartis Institutes for BioMedical Research, East Hanover, New Jersey. to facilitate forward genetic strategies to identify genes underly- *Corresponding author. Email: [email protected] ing CKD. 482 Validation of BUN and urinary albumin measurements in mice Materials and Methods Column eluent was derivatized with ninhydrin and the absor- Animals. We collected urine and blood samples from 7- to 10- bance monitored at 570 nm. Urea eluted at 4.9 min, and the inter- wk-old, male DBA2/J (D2), A/J, and C57BL/6J (B6) mice and nal standard eluted at 6 min. Between samples, the column was from separate groups of 10- to 14-mo-old and 20- to 24-mo-old washed at 70 pC for 2 min with 1% lithium hydroxide, pH 13. The D2 mice. All mice were obtained from The Jackson Laboratory. concentration of urea was determined by comparison of the ratio Blood (approximately 150 Nl) was collected by retro-orbital bleed- of peak areas (urea:internal standard) with a standard curve of 5 ing through heparin-coated capillary tubes into microcentrifuge to 80 mg urea/dl (3 to 27 mg BUN/dl) in 70% (v/v) horse serum tubes containing 5 Nl of 200 NM EDTA, and plasma was isolated (previously dialyzed against phosphate buffered saline). The coef- by centrifuging blood samples at 20,200 × g for 10 min. Spot urine ficient of variation of this method in the Yale Biochemical Disease samples were collected from individual mice into microcentrifuge Detection Laboratory over the last 5 y has been less than 5%. tubes over the course of several mornings. Measuring urinary albumin concentrations. To validate the auto- Additional groups of A/J and D2 mice underwent 5/6-nephrec- analyzer method for quantifying mouse albumin concentrations, tomy as a model of kidney disease. Mice were anesthetized with we purchased mouse albumin standards from Kamiya Biomedi- 250 mg/kg tribromoethanol injected intraperitoneally. Fur on the cal Company (Seattle, WA) and Sigma (St Louis, MO). Series of left and right sides was shaved from the last rib to the iliac crest, mouse albumin standards were measured on the autoanalyzer. and skin was disinfected with povidone iodine and 70% ethanol. Albumin was detected with a goat anti-human albumin antibody To remove the left kidney, a 5- to 8-mm incision through the skin to form antigen–antibody complexes, which increase the turbidity was made parallel to the last rib midway between the last rib and of the sample. The autoanalyzer measured the change in absor- iliac crest. A 4- to 7-mm incision then was made in the abdominal bance at 380 nm, which is proportional to the concentration of wall, and the kidney was elevated through the incision to expose albumin in the sample. the renal artery and vein. A single ligature was placed around the Measuring urinary creatinine concentrations. Urinary creatinine renal artery, renal vein, and ureter, and the kidney was excised. concentrations were measured on an autoanalyzer (Synchron CX5 The sutured vasculature was returned to the abdominal cavity, Clinical Chemistry Analyzer, Beckman Coulter, Brea, CA; reagent the abdominal incision was sutured with absorbable suture, and kit 442760) using the Jaffe method, where creatinine combines the skin incision was closed with wound clips. The mouse was re- with picric acid to form a creatinine-picrate complex that changes positioned, and the right kidney was elevated from the abdomen the absorbance of the solution. Dunn and colleagues have recent- through the same procedure as for the left kidney. Epinephrine ly demonstrated that this method accurately measures urinary 2 was applied topically to the right kidney, and 2/3 of the kidney creatinine concentrations in mice. was removed from the poles and dorsal portions. The kidney was Statistics. Prism software (GraphPad Software, San Diego, CA) returned to the abdomen and the abdominal and skin incisions was used for all statistical analyses, including correlation of BUN were closed as described for the left kidney. Mice were checked concentrations between HPLC and autoanalyzer methods, as well daily to ensure proper healing and normal behavior and were as linear regression of albumin standards. Mean values were ana- administered postoperative analgesia according to animal wel- lyzed by Student t tests

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