Takehiko Miyaji

Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCl in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 microl of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury.

The goal of this study was to clarify the role of p21, a cyclin-dependent kinase inhibitor, in acute renal failure (ARF). This was accomplished with the examination of the renal expression of p21 in cisplatin (CDDP)-induced ARF and in rechallenge injury with CDDP. The injection of CDDP (5 mg/kg) into rats induced increases in serum creatinine and tubular damage and the number of in situ DNA nick end labeling-positive cells, which peaked at day 5, followed by recovery to control levels by day 14. The rechallenge with the same dose of CDDP 14 d after the first dose of CDDP induced significantly less injury and no significant increase in in situ DNA nick end labeling-positive cells. The first CDDP dose significantly increased p53-positive nuclei at day 1, which disappeared by day 5, and the number of p21-positive nuclei, which had two peaks on days 3 and 9. The number of proliferating cell nuclear antigen (PCNA)-positive nuclei peaked at days 3 and 12. A significant increase in the incorporation of 5-bromo 2'-deoxyuridine (BrdU) was found at day 5 and peaked at day 7. The second injection of CDDP induced significant increases in the number of p21-, p53-, and PCNA-positive nuclei within 2 d but did not affect the incorporation of BRDU: These findings suggested that (1) CDDP induced two peaks of the increase in p21; (2) the first peak occurred shortly after CDDP and was accompanied by overexpression of p53 and PCNA but not with BrdU incorporation, possibly reflecting G1 arrest and DNA repair; (3) the second peak of p21 occurred through an p53-independent pathway and may contribute to cell differentiation; and (4) the overexpression of p21 and PCNA in rechallenge injury may contribute to acquired resistance in CDDP-induced ARF via enhanced DNA repair.

BACKGROUND: Establishment of non-invasive urinary biomarkers for the prediction of acute renal failure (ARF) is important. We evaluated whether urinary oxidative stress markers reflect intrarenal oxidative stress in cisplatin (CDDP)-induced ARF, and whether these markers can be used for the prediction of future ARF. METHODS: Urinary malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured up to day 14 post-CDDP (6 mg/kg) injection in rats. MDA and 8-OHdG expressions were examined in kidneys. RESULTS: CDDP induced an increase in serum creatinine (Scr), blood urea nitrogen (BUN), and tubular damage at day 5, increased urinary MDA excretion and MDA expression in kidneys at day 1 (but returned to basal values by day 3), increased urinary excretion of 8-OHdG at day 5 till day 14 (though the number of 8-OHdG-positive tubular cells increased at day 5 and then gradually decreased). Urinary MDA levels at day 1 correlated significantly with Scr (rho = 0.721, P < 0.01) and tubular damage score (rho = 0.840, P < 0.01) at day 5. CONCLUSION: Our findings demonstrated divergent changes of urinary oxidative stress markers in CDDP-induced ARF, and suggested that urinary MDA may be a useful marker for the prediction of the development of CDDP-induced ARF.