Dunn Stephen R, Qi Zhonghua, Bottinger Erwin P, Breyer Matthew D, Sharma Kumar
Dorrance Hamilton Research Laboratories, Division of Nephrology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Kidney Int. 2004 May;65(5):1959-67. doi: 10.1111/j.1523-1755.2004.00600.x.
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.
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.
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).
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.
由于先前的研究报告称，Jaffé碱性苦味酸盐法严重高估了小鼠体内真实的血浆肌酐水平，因此使用内源性血浆肌酐水平和肌酐清除率作为评估小鼠肾功能的工具受到了质疑。作为美国国立糖尿病、消化和肾脏疾病研究所糖尿病并发症动物模型联盟（AMDCC）的成员，我们评估了一种基于高效液相色谱（HPLC）的替代方法在小鼠血浆肌酐和肌酐清除率标准测定中的性能和可行性。我们的目的是开发一种简单的方法，为小体积（<25微升）的小鼠血浆和血清提供可靠、可重复且灵敏的检测。
我们将通过HPLC测定的肌酐清除率与Jaffé法进行了比较，并将HPLC肌酐清除率与菊粉清除率[通过植入小鼠体内的渗透泵注入异硫氰酸荧光素（FITC）菊粉]在C57BL/6J小鼠中进行了比较。不同组的小鼠接受了两种方案中的一种。方案A包括正常饮食、低盐加依那普利或高盐饮食干预。方案B使用链脲佐菌素诱导糖尿病。
首先，对于正常饮食的小鼠，HPLC测定的平均血浆肌酐水平（0.128±0.026毫克/分升）显著低于Jaffé法（0.4±0.12毫克/分升）（P<0.0001）。通过HPLC测定的尿肌酐浓度比Jaffé法低10%（P<0.01）。其次，正常饮食小鼠通过HPLC测定的平均肌酐清除率为25
