Evaluation of the nephrotoxicity and safety of low-dose aristolochic acid, extending to the use of Xixin ( Asurum ), by determination of methylglyoxal and D -lactate

Background: Most Aristolochiaceae plants are prohibited due to aristolochic acid 25 nephropathy (AAN), except Xixin ( Asarum spp. ). Xixin contains trace amounts of 26 aristolochic acid (AA) and is widely used in Traditional Chinese Medicine. 27 Methylglyoxal and D -lactate are regarded as biomarkers for nephrotoxicity. Thus, this 28 study aimed to evaluate tubulointerstitial injury and interstitial renal fibrosis by 29 determining urinary methylglyoxal and D -lactate after withdrawal of low-dose AA in 30 a chronic mouse model. 31 Methods: C3H/He mice in the AA group ( n = 24/group) were given ad libitum access 32 to distilled water containing 3 µg/mL AA (0.5 mg/kg/day) for 56 days and drinking 33 water from days 57 to 84. The severity of tubulointerstitial injury and fibrosis were 34 evaluated using the tubulointerstitial histological score (TIHS) and Masson’s 35 trichrome staining. Urinary and serum methylglyoxal were determined by high- 36 performance liquid chromatography (HPLC); urinary D -lactate were determined by 37 column-switching HPLC. Results: After AA withdrawal, serum methylglyoxal in the AA group increased from 39 day 56 (429.4 ± 48.3 μg/L) to 84 (600.2 ± 99.9 μg/L), and peaked on day 70 (878.3 ± 40 171.8 μg/L; p < 0.05); TIHS and fibrosis exhibited similar patterns. Urinary 41 methylglyoxal was high on day 56 (3.522 ± 1.061 μg), declined by day 70 (1.583 ± 42 0.437 μg) and increased by day 84 (2.390 ± 0.130 μg). Moreover, urinary D -lactate 43 was elevated on day 56 (82.10±18.80 μg) and higher from day 70 (201.10 ± 90.82 μg) 44 to 84 (193.28 ± 61.32 μg). 45 Conclusions: Methylglyoxal is induced after AA-induced tubulointerstitial injury, 46 thus methylglyoxal excretion and metabolism may be a detoxification and repair 47 strategy. A low cumulative AA dose is the key factor that limits tubulointerstitial 48 injury and repair. Thus, AA-containing herbs, especially Xixin, should be used at low 49 doses for short durations (less than one month).


Tubulointerstitial histological score 141
The tubulointerstitial histological score (TIHS) was used to evaluate the severity 142 of tubulointerstitial damage in the PAS-stained sections. Ten non-overlapping fields of 143 view were scored for each mouse. The TIHS assesses three major items: the severity 144 of mononuclear cell infiltration into the interstitium (0, absent; 1, few scattered cells; 145 2, groups of mononuclear cells; and 3, dense widespread infiltrate); the severity of 146 degeneration in the tubular epithelium (0, no degeneration of the tubular epithelium; 147 1, one group or a single degenerated tubule; 2, several clusters of degenerated tubules; 148 3, moderate degeneration of the tubular epithelium; 4, more severe degeneration of 149 the tubular epithelium; and 5, extremely severe degeneration of the tubular 150 epithelium, with massive necrosis and atrophy); and the severity of interstitial fibrosis 9 (0, absent; 1, mild diffuse fibrosis; 2, moderate fibrosis; and 3, severe fibrosis). The 152 THIS of each mouse was expressed as the sum of the three scores [14,15,20,21,23]. 153

Semi-quantitative analysis of fibrosis 154
Interstitial renal fibrosis was assessed in the Masson trichrome and Picro Sirius 155 Red-stained sections by determining the percentage area positive for aniline blue and 156 red in ten non-overlapping fields of view for each mouse using ImageJ (National 157 Institutes of Health, MD, USA), respectively [20]. 158

Determination of methylglyoxal 159
Urinary and serum methylglyoxal were determined by high-performance liquid The intensity of the bands were measured using Image J (National Institute of Health, 218 Bethesda, Maryland, USA) and the levels of GLO1 were normalized to β-actin and 219 expressed relative to the N group. 220

Urinary D,L-lactate and D-lactate contents 280
The AA group had significantly higher urinary D,L-lactate contents than the N 281  (Fig. 5g). There was no significant change 284 in the urinary D,L-lactate content of the N group from day 56 to 84. However, the 285 urinary D/L-lactate content of the AA group significantly increased between day 56 286 and day 70 (p < 0.05) and remained high at day 84 (Fig. 5g). 287 The urinary D-lactate contents of the AA group were significantly higher than (AA: 193.28 ± 61.32 μg vs. N: 7.15 ± 3.28 μg; p < 0.001) (Fig. 5h). The urinary D-291 lactate amount of the N group did not significantly change between day 56 and 84. 292 However, the urinary D-lactate amount of the AA group significantly increased 293 between day 56 to 70 (p < 0.05) and remained high at day 84 (Fig. 5h). 294

Expression of GLO1 295
The relative levels of GLO1 in the kidney of the AA group on day 56 (180.9 ± 296 12.8%), day 70 (150.8 ± 14.9%), and day 84 (167.2 ± 13.3%) were significantly 297 higher than the N group (100.0 ± 9.0%; p < 0.05) (Fig. 6b). week for more than 17 months [29], a much longer duration than the current study (56 318 days). Interstitial fibrosis was also induced in rats by subcutaneous administration of 18 AA (10 mg/kg/day) for five weeks; however, the changes after withdrawal of AA 320 were not observed [30,31]. In the current study, slight elevation of biochemical 321 parameters were observed but rare fibrosis by day 28, compared to those on day 56 322 and 70. Moreover, while another acute mouse model used a much higher dose (10 323 mg/kg/day) than the current study (0.5 mg/kg/day), tubular necrosis-but rarely 324 interstitial renal fibrosis-were observed after intravenous administration of AA for 325 five days [14,16]. Consequently, administration of a low cumulative dose (i.e., a low 326 dose for a short period) of Xixin and monitoring NAG and renal function are essential 327 to avoid exacerbation of tubulointerstitial injury. 328 Methylglyoxal and D-lactate are regarded as biomarkers for nephropathy and 329 diabetes, thus most previous studies focused on how methylglyoxal is induced 330 endogenously, how methylglyoxal reacts with proteins and nucleic acids to produce 331 AGEs [27], and testing drugs that may lower methylglyoxal contents. Therefore, 332 urinary excretion of methylglyoxal and D-lactate are considered to reflect excessive 333 production of methylglyoxal due to renal injury and inflammation. Based on the 334 biochemical parameters and renal biopsies in this study, cell infiltration and renal 335 injury such as tubule degeneration and moderate fibrosis truly existed in the injured 336 kidney after administration of AA for 56 days. In a previous study, inflammation 337 markers, such as F4/80 and tumor necrosis factor-α (TNF-α), were induced in the 338 19 damaged kidney after long-term administration of AA [21]. On the other hand, 339 methylglyoxal is produced after injury or inflammation and is also a pro-340 inflammatory factor. Methylglyoxal promotes inflammation via upregulating the 341 nuclear factor-kappa B (NF-κB) signaling pathway [28]. Furthermore, AGEs, 342 methylglyoxal-derivatized adducts, react with receptors for advanced glycation end 343 products (RAGE) to further evoke inflammation [32]. 344 Another essential finding of this study was that the level of serum methylglyoxal 345 peaked on day 70, while the levels of MG excreted in urine were lower at this time 346 point than at day 70.These findings indirectly reflects accumulation of methylglyoxal 347 in the injured kidney. In agreement with these observations, severe renal injury and 348 interstitial fibrosis were detected at day 70. As previously described, accumulation of 349 methylglyoxal is harmful and aggravates fibrosis in a variety of pathologies [33][34][35].