AA-treated Rats Research Articles

Voltage-Gated Potassium Channels in IB4-Positive Colonic Sensory Neurons Mediate Visceral Hypersensitivity in the Rat

Irritable bowel syndrome (IBS) is associated with a state of chronic visceral hypersensitivity, but the underlying molecular mechanisms of visceral hyperalgesia remain elusive. This study was designed to examine changes in the excitability and alterations of voltage-gated K+ currents in subpopulations of colonic dorsal root ganglion (DRG) neurons in a rat model of IBS-like visceral hypersensitivity. The model of IBS-like visceral hypersensitivity was induced by intracolonic infusion of 0.5% acetic acid (AA) in saline from postnatal days 8 -21. Experiments were conducted when rats became adults. DRG neurons innervating the colon were identified by 1,1'-dioleoyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate (DiI) fluorescence labeling and were immunostained for isolectin B4 (IB4) binding to classify these colonic neurons. Patch-clamp recordings were made from acutely dissociated DiI-labeled DRG neurons, and the expression of K+ channel in L6-S2 DRG was examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. (1) Neonatal AA treatment induced long-lasting visceral hypersensitivity without significant inflammation but with mast cell hyperplasia. (2) Colonic DRG neurons contained IB4-positive and negative neurons with different electrophysiological properties. IB4-positive colonic neurons have longer action potentials (APs) and larger A-type K+ currents (I(A)) than the IB4-negative neurons, and IB4 phenotypic changes of colonic neurons were not involved in the chronic visceral hypersensitivity. (3) Neonatal AA treatment decreased I(A) density and changed the electrophysiological properties of I(A) and I(K) by shifting the steady-state inactivation toward a negative direction in IB4-positive colonic neurons. The excitability of these cells increased. (4) Kv4.3 was downregulated in neonatal AA-treated rats compared with control rats, which suggests a possible mechanism regarding the changes in electrical activity of DRG neurons in these rats. A new model for chronic visceral hypersensitivity following a diluted AA stimulus in the neonatal period is described. The hypersensitivity may be associated with mast cell hyperplasia in the colon and increased excitability of IB4-positive colonic neurons as a result of suppression of I(A) density and a shift in the inactivation curves of I(A) and I(K) in a hyperpolarizing direction in these cells. This study identifies for the first time a specific molecular mechanism in subpopulations of colonic DRG neurons that underlies chronic visceral hypersensitivity.

Ischemic injury underlies the pathogenesis of aristolochic acid−induced acute kidney injury

Aristolochic acid nephropathy (AAN) is a progressive tubulointerstitial renal disease caused by aristolochic acid intake. To determine the contribution of renal ischemia to the pathogenesis of AAN, we characterized changes in the expression of angiogenic factors and vasoactive substances, and then we evaluated the expression of a marker of hypoxia in an acute AAN rat model. Rats were orally administrated either a decoction of Aristolochiae manshuriensis that contained 20 mg/kg of aristolochic acid-I or an equal volume of distilled water (control group) once daily for 4 days or 7 days. Renal histology and serum creatinine were assessed. Expression of endothelin-1 (ET-1) and hypoxia inducible factor-1 alpha (HIF-1alpha) mRNA within renal cortex were determined by semiquantitative reverse-transcription polymerase chain reaction. Levels of ET-1, nitric oxide (NO), vascular endothelial growth factor (VEGF), and HIF-1alpha in kidneys were determined by radioimmunoassay, Griess method, Western blot, and immunohistochemistry, respectively. Tubular injury scores and ET-1 mRNA expression were increased in the AA-treated rats at both days 4 and 8, whereas serum creatinine level and ET-1 protein expression was increased only at day 4. In contrast, NO production in AA-treated rats was decreased at day 8 compared with the control group. Similarly, VEGF protein expression was reduced in the AA-treated rats at both days 4 and 8. A dramatic increase in nuclear staining for HIF-1alpha was observed mainly in the tubular cells of tubulointerstitial damage area in the AA-treated rats at day 8. The observed increase in HIF-1alpha protein expression, decrease in VEGF protein expression, and imbalance of vasoactive substances after induction of acute kidney injury by AA suggests that ischemic injury contributes to the pathogenesis of AAN.

P2X receptor-mediated visceral hyperalgesia in a rat model of chronic visceral hypersensitivity

Background:Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterised by abdominal pain and bloating in association with altered bowel movements. Its pathogenesis and the underlying molecular mechanisms of visceral...

DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32P-postlabeling and mutant frequency (MF) was determined using the λ Select- cII Mutation Detection System. Three major adducts (7-[deoxyadenosin- N 6-yl]-aristolactam I, 7-[deoxyadenosin- N 6-yl]-aristolactam II and 7-[deoxyguanosin- N 2-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95–4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10 −6 in liver compared with the MFs of 78–1319 × 10 −6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.

Mutations induced by carcinogenic doses of aristolochic acid in kidney of Big Blue transgenic rats

Aristolochic acid (AA) is present in at least 65 different kinds of plants, many of which are used as herbal folk remedies. AA is considered one of the most potent plant carcinogens in humans and animals. It has been associated with the development of urothelial cancers in humans, and kidney and forestomach tumors in rats. In the present study, we used the Big Blue transgenic rat model to evaluate the mutagenicity of AA in kidney of rats and to define the mechanism of action for the tumor induction by AA. Groups of six male Big Blue transgenic rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight 5 times a week for 12 weeks, a treatment protocol that resulted in tumors in kidneys and other tissues. The animals were sacrificed 1 day after the final treatment and the kidneys were isolated for assays to determine the mutant frequencies (MFs) and types of mutations induced by AA in the transgenic cII gene. AA treatment resulted in a strong linear relationship between MF inductions and treatment dose ( R 2 = 0.998). The cII MFs were 29 ± 6 × 10 −6, 78 ± 21 × 10 −6, 242 ± 104 × 10 −6 and 1319 ± 360 × 10 −6 in the control, low, medium and high dose treatment groups, respectively ( p < 0.001 for all pair wise comparisons among the four treatment groups). These MFs correlated strongly with tumor incidences induced by the different doses of AA ( Mengs et al., 1982). Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutational spectra in the AA-treated and control rats ( p < 0.05). A:T → T:A transversion was the predominant type of mutation in the AA-treated rats whereas G:C → A:T transition was the main type of mutations in the control rats. These results suggest that AA induces kidney tumors in rats though a mutagenic mechanism of action.

Effects of Supplementation with Ascorbic Acid for a Period of 120 days on the Myosin‐V and NADPHd Positive Myenteric Neurons of the Ileum of Rats

We investigated the effect of ascorbic acid (AA) supplementation on the NADPH-diaphorase (NADPHd) and myosin-V myenteric neurons in the ileum of rats, after 4 months of treatment. Two groups were compared, i.e. controls rats (C) and AA-treated rats (CA). Myosin-V immunohistochemistry and NADPHd histochemistry were employed. We investigated the areas of 500 cell bodies of myosin-V neurons and of 500 NADPHd stained neurons from all groups. The quantitative analysis was performed using an area of 8.96 mm2 from each ileum. There was an increase of 21.9% in the myosin-V immunoreactive myenteric neurons (P > 0.05) and of 22.5% in the NADPHd in group CA when compared with C (P < 0.05). There were no significant differences when we compared the area of myosin-V stained neurons between groups C and CA. However, we verified an area reduction of 7.5% in NADPHd neurons when comparing group C to group CA (P < 0.05).

The nephrotoxicity of Aristolochia manshuriensis in rats is attributable to its aristolochic acids.

Although Aristolochia manshuriensis (AM), which was incriminated in the Japanese variety of Chinese herbs nephropathy, has been recently shown to be nephrotoxic in rats, less is known about whether this toxicity is attributable to its aristolochic acids (AA). We compared the renal effect of AM with that of Akebia quinata (AQ), which has similar components to AM but is free of AA; we also compared this effect of AM with that of pure AA, and studied its possible mechanism. In study 1, rats were divided into four groups. Each group was orally given either 0.4 g of AM, 4 g of AM, or 4 g of AQ per day, or vehicle, for 5 days. In study 2, rats were given 4 g of AM (which contained 4 mg of AA), or 4 mg of pure AA per day, or vehicle, for 5 days. Renal function, and renal histology were evaluated on day 5 in study 1 and on days 1, 3, 5, and 14 in study 2. In study 2, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) staining were also performed. In study 3, rats were treated in the same way as in study 2, but were all killed on day 5. Concentrations of AA (I + II) were measured in serum and urine in study 2, and in the kidney, lung, heart, liver, and spleen in study 3. In study 1, only the rats that received 4 g/day of AM developed tubular necrosis, azotemia, proteinuria, and glucosuria. In study 2, both AM- and AA-treated rats showed progressive tubular damage, decreased renal function, and increased urinary protein excretion. The degree of these alterations was comparable in the AM and AA groups. Moreover, the concentrations of AA (I + II), in serum, urine, and kidney were also comparable in the AM- and AA-treated rats. High levels of AA were detected in the lung and kidney. Apoptotic tubular cells increased on day 5 and had decreased by day 14 after the withdrawal of AM or AA. Meanwhile, proliferating tubular cells progressively increased from day 3 to day 14. Our study indicates that the renal toxicity of AM can be attributed to its AA component. Tubular cell apoptosis might be one of the mechanisms involved in this renal injury.

Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats.

Chinese-herb nephropathy (CHN) is a rapidly progressive renal fibrosis associated with the intake of a Chinese herb (Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acids (AA). This study attempted to reproduce the main features of human CHN (renal failure, tubular atrophy, and interstitial fibrosis) in a rat model similar to that of cyclosporin-induced nephropathy. Salt-depleted male Wistar rats received daily subcutaneous injections of either 1 mg/kg body wt AA (low-dose AA group), 10 mg/kg body wt AA (high-dose AA group), or vehicle (control group) for 35 d. On days 10 and 35, assessment of renal function, measurements of urinary excretion of glucose, protein, and leucine aminopeptidase, and histologic analyses were performed (six rats euthanized/group). High-dose AA induced glucosuria, proteinuria, and elevated serum creatinine levels and reduced leucine aminopeptidase enzymuria on days 10 and 35, whereas low-dose AA had no significant effect. Tubular necrosis associated with lymphocytic infiltrates (day 10) and tubular atrophy surrounded by interstitial fibrosis (day 35) were the histologic findings for the high-dose AA-treated rats. In both AA groups, urothelial dysplasia was also observed, as well as fibrohistiocytic sarcoma at the injection site. A short-term model of AA-induced renal fibrosis was established in salt-depleted Wistar rats. These results support the role of AA in human CHN and provide a useful model for examination of the pathophysiologic pathways of renal fibrosis.

Effects of Antioxidant Therapy on Leukocyte Myeloperoxidase andCu/Zn‐Superoxide Dismutase and Plasma Malondialdehyde Levels in ExperimentalColitis

The aim of the present study was to evaluate the effects of N-acetylcysteine (NAC) and L-carnitine (LCAR) supplementations on polymorphonuclear leukocytes myeloperoxidase (MPO) and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and plasma malondialdehyde (MDA) in acetic acid (AA)-induced ulcerative colitis model. The mean polymorphonuclear leukocyte MPO and Cu/Zn-SOD activity was significantly higher in the colitis group than in the control group. Both NAC and LCAR pretreatment markedly decreased MPO and Cu/Zn-SOD activity compared to colitis group. AA administration significantly increased the levels of plasma MDA in comparison with controls. However, NAC and LCAR administration to the AA-treated rats significantly reduced the MDA levels compared to colitis group. In conclusion NAC and LCAR could be beneficial agents in restoring the circulating proinflammatory mediators.

Allylamine Cardiovascular Toxicity: Evidence for Aberrant Vasoreactivity in Rats

Allylamine (AA, 3-aminopropene) is a specific cardiovascular toxin used experimentally to model myocardial necrosis and atherosclerosis. In these physiologic experiments, 10-day AA exposure (100 mg · kg−1· day−1by gavage) produced severe myocardial necrosis and increased heart rate but did not affect systolic blood pressure in rats. Mid-thoracic aortic ring segments were removed, and reactivity to contractile and relaxant agonists was tested. Aortic rings (∼3 mm) from AA-treated rats were contracted significantly more by high potassium (100 mM) and slightly more by norepinephrine (NE, 10 μM) than anatomically matched control aortic rings. No difference in aortic ring NE sensitivity or percentage relaxation in response to acetylcholine (1 μM) or sodium nitroprusside (100 μM) was detected between control and AA-treated rat aortic rings. Allylamine (1 μM–1 mM) induced modest, concentration-dependent contractions and tension oscillations in aortic rings from both control and AA-treated rats. Aortic rings from AA-treated rats, however, were more sensitive to AA. Vascular smooth muscle cells derived from control and AA-treated rat aortas had similar toxic sensitivity to AAin vitrousing the MTT viability assay. The mechanisms by which AA exposure increased heart ratein vivoand contractility of aortic rings are unknown. These experiments support the previously proposed concept that AA-induced acute myocardial necrosis is due to coronary vasospasm and myocardial ischemia and cell injury.

Effect of D, L-alpha-aminoadipate on the mediobasal hypothalamus and endocrine function in the rat.

Using the glutamate analog, D,L-alpha-aminoadipic acid (D,L-alpha AA), experiments were conducted to examine the nature, extent, and specificity of its toxicity in the mediobasal hypothalamus and to determine its effect on endocrine homeostasis. Neonatal rats received daily injections of D,L-alpha AA (4 g/kg BW) on postnatal days 5-10 and were killed at various post-treatment intervals. Sex-matched littermates were given equimolar amounts of NaCl and served as controls. Treated rats killed 18 days post injection weighed slightly less than controls and had reduced testicular, ovarian, and uterine weights, but the differences were not statistically significant. In D,L-alpha AA treated rats serum and pituitary levels of TSH and PRL were comparable to control values. Pituitary content of LH (male's and female's) and FSH (female's), however, was lower (P less than 0.05) in D,L-alpha AA treated rats than in controls, but serum levels were not significantly different. Distinct cytopathologic changes were evident in the arcuate nucleus and median eminence of D,L-alpha AA-treated rats killed at 2 and 6 h post injection only. By 12 h evidence of acute damage had largely disappeared. Both glial and ependymal cells underwent edematous swelling and necrosis, but neurons were largely unaffected. Evidence of reactive changes, such as gliosis, infiltration of microglia, and removal of debris, however, were not very conspicious. A random sample of mediobasal hypothalami of rats killed 18 days post injection failed to show any detectable lesion or residual effects of earlier pathology. Age at the time of exposure to the gliotoxin was found to be an important variable affecting both extent and duration of injury. The most deleterious effects were observed when the gliotoxin was administered in the form of a single injection on postnatal day 5 only. The results suggest that normal neuronal activity and endocrine homeostasis, specifically gonadotropin, may be irreversibly altered as a consequence of transient disruption of the glial compartment.