Background: The use of endogenous plasma creatinine levels and creatinine clearance as a tool to evaluate renal function in mice has come under scrutiny as prior studies have reported that the Jaffé alkaline picrate method grossly overestimates true plasma creatinine in mice. As members of the NIDDK Animal Models of Diabetic Complications Consortium (AMDCC), we evaluated the performance and feasibility of an alternative high-performance liquid chromatography (HPLC)-based method for standard determination of plasma creatinine and creatinine clearance in mice. Our purpose was to develop a simple method that provides a reliable, reproducible, and sensitive assay for small volumes (<25 microL) of mouse plasma and sera.
Methods: We compared creatinine clearance measured by HPLC with the Jaffé method and HPLC creatinine clearance with inulin clearance [fluoroscein isothiocyanate (FITC) inulin in an osmotic pump implanted in mouse] in C57BL/6J mice. Different groups of mice underwent either one of two protocols. Protocol A included dietary intervention with normal, low salt plus enalapril, or high salt. Protocol B induced diabetes using streptozotocin.
Results: First, mean plasma creatinine levels were significantly lower (P < 0.0001) by HPLC (0.128 +/- 0.026 mg/dL) vs. Jaffé (0.4 +/- 0.12 mg/dL) for mice on a normal diet. Urine creatinine concentrations measured by HPLC were 10% lower than by Jaffé (P < 0.01). Second, mean creatinine clearance by HPLC for mice on a normal diet was 255 +/- 68 microL/min. Mice on low salt diet plus enalapril had reduced creatinine clearance (72.8 +/- 24.2 microL/min) while mice on high salt diet had an elevated creatinine clearance (355 +/- 105 microL/min). Third, diabetic mice (19 to 24 weeks of diabetes) exhibited hyperfiltration as creatinine clearance was 524 +/- 214 microL/min whereas nondiabetic age/gender-matched mice showed a mean creatinine clearance of 206 +/- 41 microL/min. Finally, significant correlation was demonstrated for creatinine clearance by HPLC vs. inulin clearance (R= 0.643; P < 0.001).
Conclusion: HPLC is highly accurate, much more sensitive and specific than the Jaffé method for plasma creatinine measurements in mice. Creatinine clearance in mice measured by HPLC reflects changes in renal function induced by diet and diabetes.