All chemicals were reagent grade, and all solvents were HPLC grade. Aristolochic acid sodium (AANa, containing 41% AAI and 56% AAII), p-aminobenzoic acid potassium salt (≥99%), and p-aminohippuric acid sodium salt were obtained from Sigma. Inulin was obtained from Fluka.

The general physiology of rabbits is similar to that of humans [¹⁹]. In addition, histopathological features of the tissues of AAN of rabbits are similar to those of humans. Therefore, male New Zealand white rabbits (2.0~3.0 kg) were used for this study. Before the experiment, rabbits were starved for at least 12 h, but water was given ad libitum. Two groups of rabbits were iv-administrated with 20 mg/kg of PAH (n = 10) and inulin (n = 10) before and on the 7th day after a single intravenous dose of 0.5 mg/kg AANa administration. The data collected before AANa administration is shown as control groups. Blood samples were collected at 5, 10, 20, 30, 45, 60, and 90 min and 2, 3, 4, and 6 h after dosing. After blood sampling, rabbits were sacrificed to obtain kidney specimens. In addition, 3 more rabbits without AANa administration were used as control group for histological examination. All animal experiments conformed to institutional guidelines.

The HPLC instrument consisted of a pump (Shimadzu LD-10AD), UV-VIS detector (Shimadzu-10A), autoinjector (Shimadzu SIL-9A), and integrator (Shimadzu C-R7A). A Cosmosil 5C-18AR (5 μm, 4.6 × 250 mm) column was used to analyze PAH and inulin in plasma samples. The flow rate was set to 1.2 mL/min, and the UV wavelength was set to 290 nm. The mobile phase for PAH consisted of 4% acetonitrile and 0.3% tetramethylammonium chloride in a 0.01 M phosphate butter solution (pH 4.0). The mobile phase for inulin was composed of 2.5% acetonitrile and 0.3% tetramethylammonium chloride in a 0.01 M phosphate butter solution (pH 2.9).

One hundred microliters of 1 μg/mL p-aminobenzoic acid (PABA) used as the internal standard with a 4.2% perchloric acid solution was added to 200 μL of a plasma sample and then mixed and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to another tube and then supplemented with 200 μL dichloromethane. The tubes were vortexed and centrifuged at 13,000 rpm for 5 min again, and the supernatant was transferred to another tube which contained 200 mg ammonium sulfate and 200 μL ethyl acetate. The tubes were vortexed and centrifuged at 13,000 rpm for 5 min once again, and then the supernatant was transferred to another tube. After desiccation under nitrogen gas, the sample was reconstituted with 200 μL of the mobile phase, and 50 μL was used for HPLC. The concentration range of the standard curve was 0.1~30 μg/mL. This analytical method was validated according to the “Guidance of Bioanalytical Method Validation.” [²⁰] The respective retention times of PAH and PABA were 4.4 and 10.3 min, respectively, with no significant endogenous peak being coeluted in the corresponding chromatograms. The linear regression equation of the standard curve was Y = 0.2615X + 0.0064 with a correlation coefficient of 0.9999. The limit of quantitation of PAH was 0.1 μg/mL.

One hundred microliters of 1 μg/mL PABA as the internal standard with 7% perchloric acid solution was added to 200 μL of a plasma sample and then mixed and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to another tube and then supplemented with 200 μL dichloromethane. After vortexing and centrifuging at 13,000 rpm for 5 min, this supernatant was transferred to another tube and left in boiling water for 30 min to hydrolyze inulin to fructose and allow the fructose to be converted to 5-(hydroxymethyl)-2-furaldehyde (HMF). Then the samples were cooled in cold water for 5 min and transferred to another tube containing 200 mg ammonium sulfate and 200 μL ethyl acetate. The tubes were vortexed and centrifuged at 13,000 rpm for 5 min again. The supernatant was transferred to another tube. After desiccation under nitrogen gas, the sample was reconstituted with 200 μL of the mobile phase, and 50 μL was used for HPLC. The concentration range of the standard curve was 0.1~30 μg/mL. This analytical method was validated according to the “Guidance of Bioanalytical Method Validation.” [²⁰] The respective retention times of inulin and PABA were 8.6 and 11.7 min with no significant endogenous peak being coeluted in the corresponding chromatograms. The linear regression equation of the standard curve was Y = 0.1282X − 0.1386 with a correlation coefficient of 0.9995. The limit of quantitation of inulin was 5 μg/mL.

The plasma concentration-time data of PAH and inulin of the rabbits were entered into the computer program, PKCALC [²¹], to obtain the initial estimated parameters, and these were individually weighted with another computer program, WinNonlin [²²]. All of the pharmacokinetic parameters, including the distribution half-life (α-t_1/2), elimination half-life (β-t_1/2), clearance (CL), volume of the distribution at steady state (Vss), volume of the distribution (V), volume of the distribution in the central compartment (Vc), elimination rate constant of the central compartment (k₁₀), and the transfer rate constant between the central and peripheral compartments (k₁₂, k₂₁), were directly obtained from the WinNonlin output. The area under the curve (AUC) was determined using the trapezoidal rule with the area from blood sampling time to infinity estimated as the last drug concentration divided by the estimated terminal elimination rate constant, k. This was calculated by dividing 0.693 by β-t_1/2.

RPF was calculated using the PAH extraction ratio (E_PAH) which was obtained from Gouyon and Guignard [²³] values of 92.9% ± 1.9% observed in 29 normoxemic rabbits and of 90.0% ± 2.7% observed in 21 hypoxemic rabbits were used. The other renal function parameters were calculated with the following equations: filtration fraction = GFR/RPF, GFR = CL_inulin, RPF = CL_PAH/E_PAH, and net secretion of PAH = CL_inulin − CL_PAH [²⁴, ²⁵].

All data are expressed as the mean ± SE. Statistical significance was determined by two-way analysis of variance (ANOVA) for PK parameters.

Blocks of renal tissues were fixed in 10% buffered formalin for a routine histological examination. Because of minimal changes in the glomeruli, the severity of renal changes was graded by the degree of tubulointerstitial changes. Periodic acid-Schiff- (PAS-) stained sections were evaluated at a magnification of 100×, and findings for the cortex were semiquantitatively scored. The tubulointerstitial histological score was determined as described by Sato et al. [¹⁸]. The scores used to evaluate changes in the epithelial tubules were defined as follows: 0, no degeneration of the tubular epithelium; 1, one group or a single degenerated tubule; 2, several clusters of degenerated tubules; 3, moderate degeneration of the tubular epithelium; 4, more severe degeneration of the tubular epithelium; 5, very severe degeneration of the tubular epithelium, with massive necrosis and atrophy present. Mononuclear cell infiltration into the interstitium was evaluated by the following four-point scale: 0, absent; 1, a few scattered cells; 2, groups of mononuclear cells; 3, dense and widespread infiltration. The existence of hyaline cylinders in distal tubules was evaluated as follows: 0, absent and 1, cylinders present. Interstitial fibrosis was defined as follows: 0, absent; 1, mildly diffuse fibrosis; 2, moderate fibrosis; 3, severe fibrosis. The histological score of each rabbit was expressed as the sum of these 4 scores. Nonparametric variables were analyzed by the Mann-Whitney U-test. Statistically significant differences between groups were defined at P < .05.

The plasma concentration-time curves of PAH after a single iv-administrated dose of 0.5 mg/kg AANa are shown in Figure 1. The PK data before and after AANa administration are given in Table 1. It was observed that after AA administration, CL (P = .010) and k₁₀  (P < .001) were reduced by half. α-t_1/2, β-t_1/2, and AUC significantly increased (P < .05). RPF was calculated using E_PAH which was obtained from the work by Gouyon and Guignard [²³]. In the control group, the E_PAH was 92.9% and RPF was 22.36 ± 4.01 mL/kg/min. Thus, in the group administrated AANa, the extraction ratio was 90.0% and the RPF was 10.11 ± 2.72 mL/kg/min, which significantly differed from control rabbits (P < .05).

Table 2 shows the PK data of inulin before and on the 7th day after 0.5 mg/kg of AANa iv administration. The plasma concentration-time curves are shown in Figure 2. The CL and k  (P < .001) significantly decreased, and the AUC significantly increased. The GFR significantly decreased from 5.95 ± 1.79 to 0.89 ± 0.18 mL/kg/min in the AAN group.

On the 7th day after a single iv administration of 0.5 mg/kg AANa, rabbits were sacrificed to obtain renal specimens. Moderate proximal tubular damage with 0.5 mg/kg AANa treatment was seen (Figure 3). The histological score in the control included degeneration of tubular epithelium (1.55 ± 0.38), cell infiltration (0.49 ± 0.39), and hyaline cylinders (0.05 ± 0.05), but interstitial fibrosis was absent. However, after AANa treatment on day 7, the degeneration of the tubular epithelium (2.66 ± 0.09, P < .01), cell infiltration (1.11 ± 0.10), hyaline cylinders (0.78 ± 0.06, P < .05), interstitial fibrosis (0.49 ± 0.11, P < .05) were observed. The sums of the histological scores on day 7 were 2.09 ± 0.71 in the control and 5.03 ± 0.23 (P < .01) in AANa-treated rabbits (Figure 4). Moreover, compare with the previous study [¹⁷], there was no significant difference in histological results of 0.5 mg/kg AANa-treated rabbits. Despite these tubular changes, there was no remarkable glomerular change.