Background
Acute kidney injury (AKI) is a serious disease with a high mortality rate. Rhabdomyolysis is a common clinical disorder with a broad spectrum of traumatic and non-traumatic etiologies, and approximately 10 to 50% of patients suffering from rhabdomyolysis develop some degree of AKI [
1,
2]. Renal tubular damage is a pathological characteristic of AKI. Currently, animal models of glycerol-induced AKI are widely used [
3]. Glycerol injection into the muscle causes the release of myoglobin and other muscle contents into the circulation, ultimately resulting in AKI. Recent studies have demonstrated that the pathogenesis of glycerol-induced AKI involves myoglobin toxicity [
4‐
6], reactive oxygen species (ROS) [
7‐
9], inflammation [
10], apoptosis [
11,
12] and redox-active iron [
7]. Although the pathogenesis of glycerol-induced AKI is complex, timely prophylactic and/or early therapeutic interventions can promote recovery [
8,
12,
13]. Anisodamine, derived from
Scopolia tangutica Maxim, is used for the treatment of gastrointestinal smooth muscle spasm, infective toxic shock, myocardial infarction and acute lung injury in China [
14‐
17]. Anisodamine and atropine are non-specific cholinergic antagonists with the usual spectrum of pharmacological effects typical of this drug class. However, anisodamine appears to be less potent and less toxic than atropine, which is widely used in clinical and basic research [
18].
Anisodamine has been shown to be effective in improving the microcirculation of the hydronephrotic kidney in the rat [
19]. No published report has examined the efficacy of delayed therapeutic intervention when renal dysfunction is already well established. In our previous study (data not published), anisodamine was effective in the treatment of AKI. However, the mechanisms by which anisodamine promotes recovery from renal dysfunction in the rat AKI model remain unclear, although they may involve the inhibition of apoptosis and the suppression of inflammatory cytokine production.
In this study, we used the rat glycerol-induced acute renal injury model to clarify the mechanisms underlying the therapeutic effectiveness of anisodamine. We investigated the effects of the delayed administration of anisodamine on renal function and pathology by examining biomarkers of AKI. Our findings suggest that anisodamine improves renal function by affecting leukocyte infiltration and inflammation, oxidative stress and apoptosis.
Materials and methods
Animal groups, randomisation and tissue collection
Male Sprague-Dawley rats at 8 weeks of age (190–210 g) were purchased from Hebei Medical University and housed in metabolic cages under standard conditions, with food and water available ad libitum, in a room with a 12/12-h light/dark cycle (lights on from 08:00 to 20:00 h) and controlled temperature (21 ± 1 °C). All procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics and Use Committee of Hebei Science and Technical Bureau in the People’s Republic of China.
The block randomisation scheme will be generated by a computer-generated random assignment sequence prepared in advance. First, the rats were labeled with codes of Arabic numerals in same cage (same genetic background). In each cage, there will be labeled numerically with these codes, then the labeled codes were inputted into computer. An independent statistician who is not directly participant in the conduct of the trial will generate the randomisation sequence with computer.
The rats were fasted (food and water) for 24 h before glycerol injection, and then divided randomly into nine groups (see Table
1) according to trial design with block randomization. Group 1 (
n = 5) was not given any treatment. Groups 2–5 (
n = 45) were given intramuscular injections of 50% glycerol (10 mL/kg) in their hind limbs. Groups 1 and 2 received sterile water, while group 3 received anisodamine (Raceanisodamine Hydrochloride Injection, Hangzhou Minsheng Pharmaceutical Group Co., Ltd.) by intraperitoneal injection (1 mg/kg) 20 min before the initial glycerol injection. Groups 4 and 5 each received atropine (atropine sulfate injection, Hangzhou Minsheng Pharmaceutical Group Co., Ltd.) by intraperitoneal injection (0.05 mg/kg and 2 mg/kg) 20 min before the initial glycerol injection. Groups 6–9 (
n = 37) were given intramuscular injections of 50% glycerol (15 mL/kg) in their hind limbs. Group 6 received sterile water, while group 7 was given anisodamine by intraperitoneal injection (1 mg/kg) 20 min before the initial glycerol injection. Groups 8 and 9 each received atropine by intraperitoneal injection (0.05 mg/kg and 2 mg/kg) 20 min before the initial glycerol injection. Rats were placed in metabolic cages for 24-h urine collections. The animals were euthanized with 10% chloral hydrate (4.5 ml/kg). Blood and urine were collected at different time points for estimation of serum creatinine, blood urea nitrogen and creatine kinase. The kidneys were harvested (
n ≥ 3 at each time point) for further analysis. Part of each kidney was fixed in 4% paraformaldehyde solution. The remaining tissue was frozen immediately in liquid nitrogen and stored at − 80 °C.
Glycerol (10 mL/kg) | | √ | √ | √ | √ | | | | |
Glycerol (10 mL/kg) | | | | | | √ | √ | √ | √ |
Sterile water | √ | √ | | | | √ | | | |
Anisodamine (1 mg/kg) | | | √ | | | | √ | | |
Atropine (0.05 mg/kg) | | | | √ | | | | √ | |
Atropine (2 mg/kg) | | | | | √ | | | | √ |
Sample size calculation and inclusion/exclusion criteria
This study is designed primarily to explore the mechanism of protective effect of anisodamine on glycerol-induced acute kidney injury in rats. We will aim to collect experimental data as many rats as possible according to common animal experiment design groups (6–9 rats/ each group). Eighty-seven rats are divided nine groups, which would be able to give 95% confidence. The data from this animal experiment will be used to refine sample size calculations for future randomized controlled trial.
Rat are eligible for inclusion if they are as follows:
Exclusion criteria
Rats are excluded if they have one or more of the following:
Assessment of renal function
Renal function was monitored by measuring serum creatinine (Cat. no. C011–2), blood urea nitrogen (Cat. no. C103–2) and creatine kinase (Cat. no. A032) using assay kits (Nanjing Jiancheng Bioengineering Institute, Jiang Su, China) according to the manufacturer’s instructions.
Kidney histology
Kidney tissues were fixed in 4% paraformaldehyde and routinely processed for paraffin embedding. Sections were stained with hematoxylin and eosin for histological assessment, and images were obtained with an Olympus DP70 digital camera (Olympus Optical Co, Ltd., Tokyo, Japan) and analyzed with Image-Pro Plus 6.0 Software (Media Cybernetics, Inc., Bethesda, MD, USA). The changes were limited to the tubulointerstitial areas, and were graded as follows (described previously in [
20,
21]): (I) areas of tubular epithelial cell swelling, vacuolar degeneration, necrosis and desquamation involving < 25% of cortical tubules; (II) similar changes involving > 25% but < 50% of cortical tubules; (III) similar changes involving > 50% but < 75% of cortical tubules; (IV) similar changes involving > 75% of cortical tubules.
Immunohistochemistry and evaluation of immunostaining
All incubations were carried out at room temperature, unless otherwise stated. Immunohistochemistry was conducted for KIM-1 (ab78494, Abcam), caspase-3 (#9662, Cell Signaling Technology), cleaved caspase 3 (#9664, Cell signaling Technology), RIP3 (ab62344, Abcam) and CD45 (bs-4819R, Bioss) in longitudinal sections of the kidney at the different time points. Briefly, the sections were deparaffinized and re-hydrated in water, and then immersed in citrate buffer (pH 6.0; 95 °C for 15 min) for antigen retrieval. Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide for 10 min. Thereafter, the sections were blocked with 5% goat serum or 1% BSA in TBS. Slides were incubated overnight at 4 °C with primary antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan, Beijing, China) for 30 min at room temperature. The sections were developed with 3,3′ diaminobenzidine solution. Negative control slides were treated in the same manner, but incubated with an isotype-matched non-specific immunoglobulin.
Digital images of the sections were captured and evaluated in a blind manner. All sections were evaluated for the percentage of positive cells and labeling intensity. The percentages of positive cells were assigned scores as follows: 1, < 5%; 2, 5–25%; 3, 21–50%; 4, 50–75%. The intensity was scored as follows: 0, negative staining; 1, weak staining; 2, intermediate staining; 3, strong staining. The score was calculated by multiplying the percentage of positive cells (1–4) by the staining intensity (0–3), to obtain a value of 0–12. Cells were counted under a 40× objective.
Determination of MDA levels and SOD&IL-6 activity
Kidney tissue was gently homogenized in homogenization buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and centrifuged at 5000 rpm for 10 min at 4 °C. The protein concentration in the supernatant was determined using the bicinchoninic acid assay (Nanjing Jiancheng Bioengineering Institute). The supernatant was then used for the determination of MDA levels (A003, Nanjing Jiancheng Bioengineering Institute), SOD activity (A001, Nanjing Jiancheng Bioengineering Institute) and IL-6 activity (PI328, Beyotime) using kits according to the supplier’s instructions.
Western blotting
Protein expression levels were determined by western blot analysis as previously described [
20]. In brief, the PVDF membrane was blocked with 5% w/v dried non-fat milk in Tris buffer with 0.1% Tween-20 for 1 h, and then incubated with primary antibody to KIM-1, caspase-3, RIP3, IL-6 (bs-4539R, Bioss), RIP-3 or β-actin (1:1000) (CoWin, Beijing, China) at 4 °C overnight, followed by incubation with an HRP-conjugated goat anti-rabbit secondary antibody (1:10,000). Immunoreactive bands were detected using enhanced chemiluminescence (ECL) substrate (Transgen, Beijing, China) and the Vilber Fusion FX7 system.
Statistical analysis
The results were expressed as mean ± SEM (n = 4–6). A single comparison between two groups was performed with an unpaired, two-tailed Student’s t-test or one-way analysis of variance (ANOVA). Multiple comparisons among three or more groups were performed with an ANOVA post hoc test. P < 0.05 was regarded as significant.
Discussion
In the present study, we demonstrated that anisodamine ameliorates renal damage possibly by regulating tubular leukocyte infiltration, by inhibiting apoptosis or necroptosis, and by decreasing the levels of oxidative stress. Our findings are consistent with previous studies in which oxidative stress injury, renal tubular apoptosis and systemic or local inflammation have been implicated in glycerol-induced renal dysfunction [
10‐
12,
28]. Furthermore, anisodamine treatment was more effective than atropine treatment for glycerol-induced AKI, although they are both anti-muscarinic drugs exhibiting the usual spectrum of pharmacological effects of this drug class.
In most studies, AKI is induced by intramuscular injection of 50% glycerol (10 mL/kg) in the hind limb. This dose of glycerol is non-lethal in most rats. In comparison, 50% glycerol at 15 mL/kg can result in death. However, the cause of death was not examined in this study. The rats only showed periods of oliguria and mania-like symptoms. Treatment with anisodamine reduced the mortality rate, perhaps by improving intrarenal blood flow or by protecting against myocyte injury [
1]; however, the mechanisms remain unclear.
Atropine, a centrally-acting muscarinic cholinergic receptor antagonist, may have different functions at different doses. In a study of neurons in the subfornical organ, atropine had an antagonistic action on muscarinic responses at low concentrations (0.01–1 μM), while it suppressed GABAergic synaptic transmission at high concentrations (10 μM to 1 mM) [
29]. Therefore, we investigated the effect of a therapeutic dose of atropine on glycerol-induced kidney dysfunction. The high-dose group received 2 mg/kg of atropine, whereas the low-dose group received 0.05 mg/kg of atropine. We found that both doses of atropine had a modest effect on mortality rate in rats with glycerol-induced AKI, with no significant difference between the high-dose and low-dose groups.
Anisodamine also exhibits antioxidant activity, and its therapeutic effectiveness has been demonstrated in cardiac arrest and myocardial dysfunction [
30,
31]. Previous studies have suggested that ROS-induced oxidative stress is an important mechanism in the initiation and maintenance of glycerol-induced AKI [
30‐
32]. To assess the effect of anisodamine on redox status, we measured MDA levels and SOD activity in renal tissues. The increased SOD activity and decreased MDA levels suggest that anisodamine protects against early AKI by reducing ROS-induced oxidative stress and by enhancing endogenous antioxidant defense capacity.
As oxidative stress is directly involved in the pathogenesis of early AKI, it can also result in mitochondrial-related apoptosis and exacerbate renal dysfunction [
32,
33]. Therefore, we measured levels of a key apoptotic protein, caspase-3/cleaved caspase-3, to assess apoptotic cell death. Although the protective effect of anisodamine against myocardial cell apoptosis has been demonstrated in pigs, its effect on apoptosis in glycerol-induced AKI remained unknown. Cells in the distal portion of the proximal tubule undergo both apoptosis and necrosis in AKI [
34,
35]. Here, we found that both RIP3 and caspase-3 are localized to the membranes of damaged proximal tubular epithelial cells undergoing desquamation and necrosis, although the relationship between apoptosis and necroptosis in glycerol-induced AKI in these experiments remains unclear. After treatment with anisodamine, renal function and pathological changes were significantly improved, suggesting that necroptosis mediated by RIP3 participates in the loss of renal cells and may be an important cause of AKI.
KIM-1, a biomarker of kidney injury that is localized to damaged epithelial cells in the renal proximal tubule, was continuously expressed during the processes of kidney injury and recovery after AKI [
36]. We observed that the expression of KIM-1 quickly increased (at 3 h) after the glycerol injection (data not shown), and was highly increased and sustained from 24 to 72 h. Although anisodamine and atropine both significantly decreased the expression of KIM-1 at the 24 h time point, anisodamine was much more effective in decreasing KIM-1 levels than atropine.
A pro-inflammatory response and leukocyte infiltration are typical pathophysiological characteristics of rhabdomyolysis-induced AKI, which may impair cellular functions and lead to tubular epithelial cell swelling, apoptosis, desquamation and repair [
37‐
40]. We observed that IL-6 and CD45 levels were reduced by both anisodamine and atropine, with no significant difference between these drugs. We also found that anisodamine decreased leukocyte infiltration and protected against renal dysfunction, consistent with previous studies showing that macrophage infiltration is linked to renal dysfunction in AKI [
39,
41].
In summary, we demonstrated that anisodamine promotes renal recovery in the rat model of glycerol-induced AKI. Anisodamine inhibited delayed apoptosis, and decreased inflammation and oxidative stress in renal tubular epithelial cells. Although the mechanisms underlying the nephroprotective action of anisodamine remain unclear, our findings suggest that the drug may have therapeutic potential for rhabdomyolysis-induced and other forms of AKI.
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