Complications of diabetes, including variety of macrovascular and microvascular issues, may negatively affect patient outcomes and prognoses. The most frequent microvascular complications in type 2 diabetes patients are diabetic nephropathy, which clinically causes terminal-stage renal disease (1). Prior studies have explicitly elucidated the role of oxidant stress as a key factor in the progression of diabetic complications (2,3); however, the involvement and interaction of specific molecules and pathways in the progression of these complications has remained inexplicit.
It is understood that asymmetrical dimethylarginine (ADMA) is a major endogenously derived methylated arginine which endogenously inhibits nitric oxide synthase (NOS) and participates in endothelial dysfunction (4-6). According to recent studies, ADMA could elevate the level of reactive oxygen species (ROS), thus inducing oxidant stress (7,8). In addition, patients with elevated plasma concentrations of ADMA were carrying cardiovascular risk factors such as hyperlipidemia (9), hypertension (10), diabetes (11), and end-stage renal disease (ESRD) (12). Protein arginine N-methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) are involved in ADMA regulation. Over 85% of ADMA is generated by type 1 PRMT (PRMT1) (13); DDAH in rats metabolized over 90% of the circulating ADMA (14), and type 1 DDAH (DDAH1) was responsible for more than 70% of ADMA metabolism (15). In sum, these proteins form the PRMT1-ADMA-DDAH1 metabolic axis that is related to oxidant stress and regulates the ADMA concentration in the blood and tissues; therefore, both ends of this axis are vulnerable to redox reactions (16). Furthermore, recent studies have shown impairment in the PRMT1-ADMA-DDAH1 metabolic axis in the vasculature of diabetic rats (15,17). However, the role of this metabolic axis in the progression of diabetic kidneys has remained inexplicit.
Probucol is a potent antioxidant drug which inhibits lipid peroxidation and oxidatively modifies low-density lipoprotein particles in vitro, circumnavigates endothelial impairment and reduces blood ADMA concentrations in rats (18). Few studies have demonstrated that probucol substantially decreases the ADMA level, concomitantly with a reduction in the PRMT-1 activity and elevation in the DDAH expression in endothelial cells (19). Recent studies have suggested that this has a beneficial impact on renal disease (20). However, few intervention trials have subjected this effect to evaluation of probucol on the PRMT1-ADMA-DDAH1 metabolic axis in tissues, particularly in the kidneys, kidney tissues, and reported inexplicit results.
This study aimed to research the expression of this metabolic axis in the kidneys of diabetic rats and the effect of probucol on the axis and its function. Therefore, we also investigated probucol as a supplemental therapy for renal protection, lowering oxidant stress, reducing urinary protein excretion rate, and enhancing renal function in diabetic patients.
We present the following article in accordance with the ARRIVE reporting checklist (available at http://dx.doi.org/10.21037/apm-21-417).
We made a purchase of 30 male Sprague-Dawley rats, aged 7 weeks, and weighted 160.30±20.09 g from the Laboratory Animal Center of Chengdu Medical College. Study protocols were reviewed and approved by the Animal Research Committee of Chengdu Medical College. Experiments were performed in compliance with Chinese national standard ‘Laboratory Animals and Environmental Facilities’ and the relevant laboratory animal experiment regulations of Chengdu Medical College for the care and use of animals. All rats were housed in a temperature-controlled room at 22±2 °C, humidity at 50%±5%, under a 12 h light/dark cycle, and food and tap water were freely available. Rats were housed in our laboratory environment within 7 days before the experiment. The rats were allocated to a normal control group (NC group, n=10) and an experimental group (n=20). The NC rats were fed a standard diet provided by the Laboratory Animal Center of Chengdu Medical College, whereas the experimental rats were given a fat- and sugar-rich diet (68.5% standard diet, 15% lard, 15% sucrose, 0.5% bile salt, and 1% cholesterol). At 4 weeks thereafter, the experimental group rats were injected with streptozotocin intraperitoneally (STZ; Sigma; St Louis, MO, USA) diluted in freshly prepared citrate buffer liquid (pH 4.5) at a dose of 30 mg/kg body weight after an overnight fast. The rats in NC group were injected with an equal dose of citrate buffer liquid. After 7 days, according to the measurement of the fasting plasma glucose (FPG) levels with tail- nicked blood samples. Rats with the average fasting blood glucose was >11.1 mmol/L, and the typical symptoms of diabetes were observed including polydipsia, polyphagia, polyuria and weight loss, indicated that the preparation of the diabetes model was successful. FPG levels >13 mmol/L were deemed diabetics rats. Diabetics rat modeling was established as elucidated in a prior study (21). The measurement of the glucose was performed with blood glucose test strips (Johnson & Johnson; Lifescan, CH, UK). Next, the experimental rats were randomly assigned to a diabetics group (DM group, n=7) and a diabetic under probucol treatment group (DM-P group, n=7) and rats were continued with the same enriched diet. Diabetics rats in the PM group were perfused with probucol (500 mg/kg/d) into the gastricum for 8 weeks (22), whereas rats in NC and DM groups were administered the same dosage of placebo.
All rats were subject to anesthetization with pentobarbital sodium (35 mg/kg) and euthanasia with cervical decapitation. Next, blood samplings were performed from aorta abdominalis with a heparinized syringe and centrifuged. Plasma was gathered and frozen at −80 °C. After the rapid removal of kidney and dissection into 3 pieces, we immediately immobilized one of the pieces with a conventional fixation solution and other techniques (23). The remaining 2 pieces were frozen immediately for reverse transcription polymerase chain reaction (RT-PCR) and western blotting, and immunohistochemical (IHC) analysis.
Newly diagnosed type 2 diabetes patients aged 50–65 years from the First Affiliated Hospital of the Chengdu Medical College between January 2017 and January 2018 were selected for this study. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Medical Ethics Committee of the First Affiliated Hospital of Chengdu Medical College and informed consent was taken from all the patients. All patients were cleared through oral glucose tolerance test (OGTT), had complete medical history data and underwent clinical sample collection. The history of the type 2 diabetes patients was analyzed retrospectively; 40 participants with urine albumin/creatinine ratio (ACR) >30 mg/mmol were screened and allocated to the DM group or DM-P group. We selected 20 healthy volunteers whose physical health was examined in our hospital during the same period to construct the control group. The DM group included 12 males and 8 females, and the average participant age was 56±4 years old. The DM-P group included 10 males and 10 females, and average patient age was 56±5 years old. The control group included 10 males and 10 females, and average patient age was 57±5 years old. The general characteristics of the groups were comparable (P>0.05). All diabetic patients were treated with metformin at 850 mg twice a day. The dose of metformin was adjusted according to their blood glucose levels. Random allocation of the participants was made to receive metformin alone (DM group) or metformin and probucol at 500 mg/d (DM-P group) for a period of 24 weeks.
Serum sample collection and index detection methods
We collected 4–6 mL of peripheral blood from each participant of all groups before the OGTT test; 4–6 mL of peripheral blood was collected from the control group participants during physical examination. The blood samples were centrifuged to separate the serum; thereafter, determination of superoxide dismutase SOD, catalase CAT, and malondialdehyde (MDA) content with radioimmunoprecipitation kits was performed.
Biochemical indicators of oxidant stress and renal function
Plasma NOS and nitric oxide (NO) activity were determined by resorting to nitrate reductase methods with available kits (Real-Times (Beijing) Biotechnology Co., Ltd, Beijing, China) from the commercial market. Measurement of FPG concentrations was performed via glucose oxidase method utilizing an automatic analyzer (Beckman Coulter, Brea, CA, USA). Blood urea nitrogen (BUN), creatinine (Cr), and urine ACR levels (Biosino Biotechnology and Science Inc., Beijing, China) were measured using a chemical colorimetric method as per the calculation of absorbance.
The kidney tissues were subject to finer grounding with a liquid nitrogen-cooled mortar and pestle and extraction, total RNAs were extracted according to the manufacturer’s directions (Invitrogen, Carlsbad, CA, USA), and its purity and concentration were measured with a nucleic acid protein analyzer (Amersham Biosciences, Piscataway, NJ, USA). We conducted RT-PCR according to previously described methods (24). The primers of PRMT1, DDAH1 and β-actin are shown in Table 1.
Images were obtained and quantified using Bio-Rad Laboratories Quantity One version 4.6.2 software (Bio-Rad Inc., Hercules, CA, USA).
The concentration of ADMA in kidney tissue was measured with an enzyme-linked immunosorbent assay (ELISA; R&D Systems; Minneapolis, MN, USA). This assay had a detection limit of 0.05 mmol/L; the relationship between the ADMA concentration and photometric optical densities (ODs) was linear in the range of 0.1–5.0 mmol/L. The ADMA concentration in the kidney tissue was measured in 4 separate experiments, each was run in duplicate; the results are expressions of average ± standard deviation (SD) values.
Western blotting analysis
Kidney tissues were subject to dissection into small-sized pieces and homogenization in lysate buffer (Beyotime Institute of Biotechnology; Jiangsu, China) on ice. The concentration of total protein was measured with the bicinchoninic acid (BCA) method (20). Proteins of equal concentration were subject to isolation on electrophoresis and electro-phoretical transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were plugged for 1 h and subsequently incubated with primary monoclone PRMT1 antibody (1:2,000 dilution; Cell Signaling Technology, Shanghai, China) or DDAH1 antibody (1:5,000) in Tris-buffered saline containing 4% evaporated milk at 4 °C overnight. Rinsing with phosphate buffered saline with Tween-20 (PBST) was performed and the membranes were incubated with horseradish peroxidases (HRP)-conjugated antibodies against mouse β-actin (1:5,000) and rabbit β-actin (1:2,500 dilution; 4A Biotech, Beijing, China) or HRP-conjugated anti-rabbit secondary antibody (1:1,000) for 1 hour. Images were received and quantified within a Bio-Rad VersaDoc imaging system (Amersham Biosciences; UK) with Bio-Rad Quantity One software.
The kidney sections were deparaffinized, incubated with 3% H2O2, blocking goat serum, and subsequently tinted for detection of PRMT1. The tissue sections were incubated with primary polyclonal antibody against PRMT1 (ZSGB-BIO, Beijing, China) and to diluted at 1:1,500 for 2 hours at an ambient temperature. The sections were incubated with a secondary antibody conjugated by HRP and antibody binding was detected using diaminobenzidine (DAB) solution. The average OD was determined with a spectrophotometer and analyzed with Image-Pro Plus 6.0 software (Media Cybernetics Corp., Rockville, MD, USA) for image analysis.
Results were expressed as mean ± SD values. After a normality and variance testing for homogeneity, normally distributed data was subjected to one-way analysis of variance (ANOVA), after which the Bonferroni’s post-hoc test was caried out for comparisons. Statistical analyses were performed using SPSS 23.0 statistical software (IBM, Armonk, NY, USA). A P value <0.05 was considered statistically significant.
The indexes of serum oxidant stress and renal function
The content of serum oxidant stress indexes SOD, CAT, NOS, and NO in the DM group was substantially lower in contrast to the control group, whereas MDA showed a substantially higher content in contrast to the NC group; (P<0.05; Table 2). Treatment with probucol substantially reinforced the SOD, CAT, NOS, and NO activity in comparison with the levels of DM rats (P<0.01; Table 2). Blood glucose level in the DM and PM groups were substantially more elevated than those in the NC group (P<0.01; Table 2). The same results were seen in patients with diabetes, with treatment of probucol, the level of plasma SOD and CAT activity were substantially increased (P<0.05; Table 3), whereas plasma MDA was decreased in contrast to the control group (P<0.05; Table 3). The renal function indexes BUN, Cr, and ACR levels in the PM group were substantially lower than in the DM group (P<0.05; Tables 3,4); however, the levels were higher than those in the NC group (P<0.05; Tables 3,4).
The average concentration of ADMA in the DM group was substantially increased in comparison with that in the NC group (314.70±40.85 vs. 159.99±32.08 µmol/L; P<0.01). Probucol substantially inhibited the increase in the ADMA concentration according to the observations in the DM group (218.66±25.38 vs. 314.70±40.85 µmol/L; P<0.01).
PRMT1 and DDAH1 mRNA expression
The expression of PRMT1 messenger RNA (mRNA) in the kidney tissues was higher in the DM compared with NC rats (P<0.05); and there was decreased expression in the PM group in contrast with the DM group (P<0.01). The mRNA of DDAH1 in the kidney tissues had lower expression in the DM group in comparison with the NC group (P<0.05); treatment with probucol increased the expression of DDAH1 mRNA more than that in the DM group (P<0.05; Figure 1).
PRMT1 and DDAH1 protein expression
According to the results of western blotting analysis, PRMT1 in the kidneys had a lower protein expression in the NC group in comparison with the protein expression in the DM and PM groups (P<0.01); however, PRMT1 protein expression was higher in the DM group in comparison with the PM group (P<0.05). Comparatively, DDAH1 protein expression in the kidneys was decreased more in the DM group than in the NC (P<0.01) and PM (P<0.05) groups (Figure 2). Treatment with probucol decreased the protein expression of PRMT1 and increased the protein expression of DDAH1 in diabetics rats.
The IHC staining for PRMT1 in the DM and PM groups had a higher reactivity in the glomerular and epithelial cells of the proximal tubules than that in the NC group (Figure 3). The average OD of PRMT1 was lower in the NC group compared with that in the DM and PM groups (P<0.01); however, it was higher in the DM group compared to the PM group (P<0.05).
Oxidant stress is defined as a disturbance of equilibrium between ROS production, mainly free radicals are produced and antioxidant defense mechanisms, resulting in tissue injury. Considering the physiological conditions, antioxidant defense mechanisms could defend tissues against ROS-induced damage. However, conditions that either develop more free radicals or dilute the antioxidant defense would incur cellular and tissue damage because of free radical accumulation that greatly surpassed the body’s scavenging capacity. Oxidant stress is an important change that takes place in the body under the circumstances of high blood glucose levels; further, it serves as an important pathological linkage for mediation of diabetic microvascular complications (23).
Oxidant stress activation could give rise to mass production of oxygen free radicals which participate in oxidizing reactions, damaging the cells; next, the proteins leak from the glomeruli, pass into urine, and result in proteinuria (24-26). Prior studies have confirmed that early nephropathy in diabetes was related to high intrarenal NO and NOS productivity (27,28), whereas mid-term nephropathy and ESRD were accompanied by decreased NO and NOS levels (29).
The kidneys are particularly sensitive to oxidant stress. The production of NO is promoted by ROS, and excessive NO production results in glomerular hyperperfusion and high permeability, leading to renal vasodilatation and reinforced vascular permeability in early stage (30). Meanwhile, there are a variety of antioxidants in the body, and antioxidant enzymes, such as SOD and CAT, can scavenge oxygen free radicals in the body and reduce oxidant stress injury; in the case of excessive activation of oxidant stress, antioxidant enzymes become massively consumed and their levels substantially reduce (31,32). Our analysis of the content of these antioxidant enzymes in our study showed that serum NOS, NO, SOD, and CAT content of the DM group was substantially lower than those in the control group; the higher the ACR was, the lower the serum SOD and CAT content became.
The oxidation reaction of oxygen free radicals and lipid compositions in the endothelial cells and basement membrane cells form MDA (33). In our study, we analyzed the content of these oxidation reaction products and found that the serum MDA content in the DM group was substantially higher than that in the control group; the higher the ACR, the higher the serum MDA. In contrast, the plasma BUN, Cr, and ACR levels were higher in the kidneys of diabetic rats in comparison with those in the kidneys of NC rats. Previous studies have also confirmed that decreased renal function was followed by reinforced oxidant stress in diabetic nephropathy (34,35). However, these studies could not confirm the presence of an imbalance of the PRMT1-ADMA-DDAH1 metabolic axis in reinforced oxidant stress in the kidneys and declined renal function under hyperglycemic conditions.
The chemical ADMA is an endogenous inhibitor of NOS and it has been gradually recognized as a potential risk factor in chronic kidney diseases. Prior studies have declared that oxidant stress may propel the progression of type 2 diabetes mellitus via activation of stress-signaling pathways, resulting in an increased concentration of ADMA and impaired NOS regulation (36,37). Elevated concentrations of ADMA have been observed in type 2 diabetes, hyperlipidemia, hypertension, and other diseases related with oxidant stress in animal models and humans (38). Therefore, ADMA is considered a risk factor of diabetic nephropathy. It is predominantly generated by PRMT1 and is primarily metabolized by DDAH1, which forms the PRMT1-ADMA-DDAH1 metabolic axis. Both ends of this axis are involved in redox reactions (16). This study showed that the mRNA and protein expression of PRMT1 was elevated and DDAH1 was reduced in the kidneys of diabetic rats compared to those in normal rats. Moreover, the IHC analysis showed increased expression of PRMT1 in the epithelial cells of the proximal tubules of diabetic rats in comparison with those of normal rats. Recent studies have shown the PRMT1-ADMA-DDAH1 metabolic axis in the vascular system of diabetic rats to be impaired (15,16), which is in favor of our conclusions. However, Onozato et al. (39) revealed abnormal expression of PRMT1 and DDAH1 protein in the serum and kidney tissue of type 2 diabetic rats; they also showed that ADMA concentration was related to the expression levels of PRMT1 or DDAH1 protein. Based on these results, we speculated that metabolic abnormality of the PRMT1-ADMA-DDAH1 axis in the kidney is a marker of type 2 diabetes in rats, and this could indicate a novel causative pathway of renal damage that affects the morbidity and progression of diabetic nephropathy.
The ADMA content herein was greater in the kidneys of diabetic rats in comparison with normal control rats. Western blotting analysis and RT-PCR analysis showed that the expression of PRMT1 mRNA and protein were elevated substantially in the kidneys of type 2 diabetic rats, whereas expressions of DDAH1 were reduced. The IHC results also showed that the PRMT1 levels were elevated in the kidneys of diabetic rats. We confirmed that ADMA levels are associated with the expression of PRMT1 and DDAH1, and that changes in the PRMT1-ADMA-DDAH1 axis occur in the kidneys of type 2 diabetic rats. Therefore, any factors that might affect the balance of the PRMT1-ADMA-DDAH1 axis will cause an ADMA accumulation, increase oxidant stress, increase ROS production, and ultimately lead to a vicious cycle of damage.
Probucol is a widely accepted lipid-lowering drug which is used for treating hypercholesterolemia. It is another potent antilipidemic agent that has an anti-atherosclerotic action and prevents oxidative tissue injury (40). Previous studies have demonstrated that probucol might hinder the progression of early- and advanced- stage diabetic nephropathy and reduce ROS levels by increasing plasma NO and NOS activity, and reducing the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity (41,42). Our results confirmed that probucol treatment increased the serum NOS, NO, SOD, and CAT activity. Therefore, we speculated that 8 weeks of probucol treatment could reduce oxidant stress and have a positively protective effect on STZ-induced type 2 diabetes rats’ kidneys.
Certain limitations existed herein. For all diabetics, treatment with probucol should be concomitant with metformin treatment for renal protection. Metformin is a well-known classic drug for the treatment of diabetes and has an anti-oxidant effect (43). According to our observations, when the positive effect of probucol on renal function was combined with metformin treatment, the potent antioxidant property increased the avoidance of renal impairment.
We found that probucol had a positive effect on renal function which may be on account of probucol’s role in regulating the PRMT1-ADMA-DDAH1 axis. On the basis of these results, we hypothesized that probucol may participate in metabolic regulation at both ends of the PRMT1-ADMA-DDAH1 axis. In fact, our results showed that probucol improves renal function and decreases PRMT1 levels, thereby confirming our hypothesis. Furthermore, we found that probucol had a protective effect on diabetic nephropathy which may reduce ADMA concentration and elevate NOS expression via the inhibition of PRMT1 expression and augmentation of DDAH1 expression. In sum, these changes should help to reduce oxidant stress, promote improved renal function, and delay the morbidity and occurrence of diabetic nephropathy.
Our results strongly supported the hypothesis that the PRMT1-ADMA-DDAH1 metabolic axis could fulfill a necessary role in the diabetes-related degradation of renal function. The protective effect of probucol on renal function in type 2 diabetes rats is related to its involvement in regulation of the PRMT1-ADMA-DDAH1 axis. The protective mechanism of probucol on diabetic kidney is mainly through inhibiting the generation or metabolic pathway of ADMA, regulating the expression of both ends of the metabolic axis of PRMT1-ADMA-DDAH1, inhibiting the expression of PRMT1 and ADMA, and further inhibiting oxidative stress, thus improving vascular endothelial function and protecting renal function. Probucol shows promise as a supplementary therapy to protect the kidneys in type 2 diabetes. The number of rats and human subjects studied in our trial was relatively small, and not all results of animal experiments can be readily extrapolated to humans. Therefore, further experiments are required to evaluate the role of the PRMT1-ADMA-DDAH1 axis in diabetic nephropathy.
Funding: This study was supported by the Key Discipline Construction project of Sichuan Province, Science and Technology Fund project of Chengdu Medical College (CYZ13-027), and Sichuan Collaborative Innovation Center for Elderly Care and Health of Chengdu Medical College (YLZBZ2008).
Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at http://dx.doi.org/10.21037/apm-21-417
Data Sharing Statement: Available at http://dx.doi.org/10.21037/apm-21-417
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/apm-21-417). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Medical Ethics Committee of the First Affiliated Hospital of Chengdu Medical College (NO.: 2019CYFYIRB-BA-01-01) and informed consent was taken from all the patients. Experiments were performed in compliance with Chinese national standard ‘Laboratory Animals and Environmental Facilities’ and the relevant laboratory animal experiment regulations of Chengdu Medical College for the care and use of animals.
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(English Language Editor: J. Jones)