A novel approach to adenine-induced chronic kidney disease associated anemia in rodents

Abstract

To date, good experimental animal models of renal anemia are not available. Therefore, the purpose of this study was to establish a novel approach to induce chronic kidney disease (CKD) with severe anemia by oral administration of adenine in rodents. Adenine was administered to 6-week-old male C57BL/6 mice (25 and 50 mg/kg body weight) by oral gavage daily for 28 days. Serum creatinine and BUN as well as hematocrit, hemoglobin (Hb) and plasma erythropoietin (EPO) levels were monitored to assess renal function and anemia, respectively. Adenine at 25 mg/kg for 28 days slightly increased plasma creatinine levels, but did not induce anemia. In contrast, 50 mg/kg of adenine daily for 28 days showed severe renal dysfunction (plasma creatinine 1.9 ± 0.10 mg/dL) and anemia (hematocrit 36.5 ± 1.0% and EPO 28 ± 2.4 pg/mL) as compared with vehicle-treated mice (0.4 ± 0.02 mg/dL, 49.6 ± 1.6% and 61 ± 4.0 pg/mL, respectively). At the end of experiment, level of Hb also significantly reduced in 50 mg/kg adenine administration group. Remarkable histological changes of kidney tissues characterized by interstitial fibrosis and cystic appearance in tubules were observed in 50 mg/kg of adenine treatment group. These results have demonstrated that oral dosing with adenine at 50 mg/kg for 28 days is suitable to induce a stable anemia associated with CKD in mice.

Fig 1. High dose of adenine by oral gavage reduces body weight in mice and rats.

Changes of body weight during adenine administration and the observation period in (A) mice and (B) rats. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle-treated mice or rats, respectively.

Fig 2. Administration of adenine by oral gavage decreases renal function.

Changes in plasma creatinine levels after adenine administration and during the observation period in (A) mice and (B) rats. Levels of plasma BUN in (C) mice and (D) rats at day 70 and 66, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle-treated mice or rats (corresponding time points) respectively; ^† P < 0.05, ^†† P < 0.01 vs. vehicle-treated mice at 28 days.

Fig 3. Administration of adenine by oral gavage reduces hematocrit and Hb levels.

Changes in hematocrit levels after adenine administration and during the observation period in (A) mice and (B) rats. Levels of plasma Hb in (C) mice and (D) rats at day 70 and 66, respectively. ***P < 0.001 vs. vehicle-treated mice or rats (corresponding time points) respectively.

Fig 4. Administration of adenine by oral gavage impacts on plasma erythropoietin and ferritin levels.

Changes in plasma erythropoietin (EPO) levels after adenine administration and during the observation period in (A) mice and (B) rats. Levels of plasma ferritin in (c) rats at day 66. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle-treated mice or rats (corresponding time points) respectively; ^†† P < 0.01, ^††† P < 0.001 vs. vehicle-treated mice at 28 days.

Fig 5. Adenine treatment via oral gavage induces tubulointerstitial fibrosis.

Representative photomicrograph of azan staining of cortical sections of kidney tissues to demonstrate tubulointerstitial fibrosis in (A) mice and (B) rats after treatment with vehicle or adenine (50 mg/kg for mice and 600 mg/kg for rat) at different time points. Photomicrographs taken at 100× magnification.

Fig 6. Oral administration of adenine increases the gene expression related to fibrosis and bone regeneration.

Relative changes of mRNA expression of (A) α-Sma and (B) Fgf23 in the renal cortical tissues of rats at day 84. * P < 0.05, *** P < 0.001 vs. vehicle-treated rats.

Fig 7. Erythropoietin treatment improves renal anemia in adenine-induced renal injury model.

Plasma levels of (A) BUN, (B) hematocrit, and (C) Hb following subcutaneous injections of rhEPO (5IU) every 2 days interval for 5 weeks in mice with adenine-induced renal injury (50 mg/kg adenine for 4 weeks). *** P < 0.001 vs. vehicle-treated mice; ^§§ P < 0.01, ^§§§ P < 0.001 vs. adenine-treated (50 mg/kg) mice.