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Abstract

Diabetic nephropathy is a progressive kidney disease resulting from long-term hyperglycaemia in diabetic patients, and the underlying mechanism is complex and lacks effective treatments. Various active ingredients in Chinese herbs have been shown to alleviate renal injury and improve DN in recent years. Phillyrin, a natural medicinal active compound extracted from the Oleaceae family, has various pharmacological effects, including antioxidative, antiapoptotic and antiobesity effects. However, the role of phillyrin and its underlying mechanism in DN have not yet been explored. To investigate the effects of phillyrin on DN and its potential mechanisms of action, we performed experiments using streptozotocin (STZ)-induced DN mice as models. Phillyrin significantly reduced the levels of fasting blood glucose (FBG) and glycosylated haemoglobin A1c (HbA1c), downregulated the levels of serum blood urea nitrogen (BUN), serum creatinine (Scr), serum and urine β2-microglobulins (β2-MG) and improved the pathological changes of the kidney in a DN mouse model. Phillyrin also increased the level of antioxidants and attenuated oxidative damage in DN model mice. In addition, phillyrin inhibited Glycogen synthase kinase-3β (GSK-3β) activity by activating the PI3K/Akt signalling pathway, increased the Bcl-2/Bax ratio, reduced the release of cytochrome c from the mitochondria to the cytoplasm, subsequently inhibited the activation of caspase-3 and ultimately suppressed renal cell apoptosis. These findings suggested that phillyrin could be a new promising therapeutic strategy for DN, and this protective effect might be related to suppressing oxidative stress and apoptosis via the PI3K/Akt/GSK-3β pathway.

Keywords

Phillyrin diabetic nephropathy PI3K/Akt pathway GSK-3β oxidative stress apoptosis

Introduction

Diabetes is becoming a worldwide chronic metabolic disease, and its incidence increases annually. Diabetic nephropathy, one of the most serious clinical complications of diabetes, is the major cause of end-stage renal disease.¹ The main symptoms of DN are kidney damage and blood glucose disorders. The metabolic and microvascular abnormalities caused by sustained hyperglycaemia can lead to glomerular mesangial matrix thickening, renal interstitial hyperplasia, tubular basement membrane injury, glomerulosclerosis and tubulointerstitial fibrosis and result in chronic renal failure, which ultimately endangers the survival of DN patients.² The pathogenesis of DN is complex and has not been fully elucidated. Recent experimental and clinical evidence has shown that the pathogenesis of DN involves many principal factors, such as mitochondrial dysfunction,³ oxidative stress,⁴ inflammation⁵ and apoptosis.⁶

The Ser/Thr protein kinase (Akt) signalling pathway regulates a series of cellular functions and is known to delay the progression of diseases, such as coronary heart disease,⁷ Alzheimer’s disease⁸ and liver injury,⁹ by inhibiting apoptosis. Increasing evidence suggests that many signalling pathways in DN have been implicated in Akt phosphorylation, and activation of Akt is required for DN occurrence and development.^10–12 Mitochondria are the main site of oxidative cellular respiration and have been regarded as the central regulatory link in many metabolic pathways.¹³ In a high glucose (HG) environment, mitochondrial dysfunction is a vital process in the oxidative burst and activation of apoptosis.¹⁴ Recent studies have shown that Akt activation is related to mitochondrial function, oxidative stress and apoptosis.^15,16 Glycogen synthase kinase-3β, a protein downstream of Akt, is widely involved in the regulation of mitochondrial functions.¹⁷ Akt is activated by phosphorylation, and activated Akt (p-Akt) can inversely regulate GSK-3β.¹² In DN, Akt phosphorylation is inhibited, activation GSK-3β.¹⁸ Then, activated GSK-3β regulates the proportion of Bax/Bcl-2, subsequently affecting the permeability of mitochondria, stimulating mitochondrial permeability transition pore opening, promoting the release of cytochrome C (Cyto C) from the mitochondria and finally participating in the regulation of cell apoptosis.^19,20 In addition, the Akt/GSK-3β pathway participates in the regulation of oxidative stress by inhibiting NADPH oxidase activity and then affecting the levels of SOD and MDA.²¹ Therefore, inhibiting the activation of GSK-3β could maintain mitochondrial function and restrain cell apoptosis, indicating that GSK-3β may be a therapeutic target to improve DN progression.

Phillyrin, a major active component of Forsythia suspensa (Thunb.), has various biological and pharmacological activities, including antiapoptotic,²² antioxidative,²³ antiviral,^24,25 lipid reducing,²⁶ antiobesity²⁷ and anti-inflammatory activities.²⁸ However, no studies have addressed the effects and the underlying mechanisms of phillyrin in preventing and improving DN. According to reports, phillyrin can scavenge reactive oxygen species (ROS) and inhibit apoptosis,^22,24 indicating that phillyrin could prevent diabetes-induced kidney damage. Therefore, the objective of this study was to investigate the potential protective effects of phillyrin on oxidative stress and apoptosis and to explore whether the underlying mechanisms are associated with the Akt/GSK-3β pathway in a DN mouse model.

Materials and methods

Materials and reagents

Phillyrin, citric acid, sodium citrate, streptozotocin (STZ) and metformin (Met) (>99% purity) were purchased from Solarbio Science & Technology Co. (Beijing, China). A glucometer and blood glucose test strips were purchased from Sanuo Biosensor Co., Ltd. (Guangxi, China).

Anti-p-Akt antibody (#4060), anti-Akt antibody (#9272), anti-Bcl-2 antibody (#15071), anti-Bax antibody (#89477), anti-pro-caspase-3 antibody (#9665), anti-cleaved caspase-3 antibody (#9664), anti-cytochrome C antibody (#11940), anti-COX 4 antibody (#4850), anti-GAPDH antibody (#5147) were purchased from Cell Signalling Technology (Cell Signalling Technology, Danvers, MA, USA). Anti-GSK-3β antibody (sc-81462) and anti-p-GSK-3β antibody (sc-373800) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Animals

Male Kunming mice (8 weeks, 20 ± 2 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China SCXK (XIAN) 2016-0002). All of the mice were maintained in a specific pathogen-free animal room under controlled conditions with a temperature of 22 ± 2°C, 50 ± 10% humidity and a 12-h light/12-h dark cycle. Food and water were provided ad libitum. The animals were allowed to acclimate to the environment for 5 days before the experiment. All of the animal experimental procedures in this study were performed in accordance with the administration committee of experimental animals of Yichun University (Approval No: IACUC-2020001)

Modelling and administration

After 1 week of acclimation, followed by fasting for 12 h, DN mouse models were induced by single intraperitoneal injection of 100 mg/kg STZ,^29–32 while the normal control mice were injected with the same amount of citrate buffer. Blood samples were obtained from the tails of the mice, and fasting blood glucose (FBG) levels were measured with a glucometer (Sinocare Inc., Hunan, China). Mice with FBG levels above 11.1 mmol/L were considered to have diabetes.

After 8 weeks for model establishment,^33,34 six normal control mice were randomly selected as the control group (Con group, n = 6), and the diabetic mice were randomly divided into 4 groups, including diabetic nephropathy group (DN group, n = 6), DN + 5 mg/kg/d phillyrin group (DN + L group, n = 6), DN + 15 mg/kg/d phillyrin group (DN + H group, n = 6) and DN + 100 mg/kg/d metformin group (DN + Met group, n = 6).

The treatment of phillyrin (5 or 15 mg/kg)^25,28,35 for DN + L or DN + H group mice and metformin (100 mg/kg) were implemented for DN + Met group mice, respectively. The mice in the Con group and DN group were treated with an equal volume of normal saline at the same time. All drugs were administered intraperitoneal injection once per day for 4 weeks. During the 4 weeks, FBG levels were tested once a week using tail vein blood samples, and the mouse body weights were recorded every day.

On the last day of the 4th week, the mice were sacrificed, and blood samples were collected for serum biochemical assays. The kidneys were removed and weighed immediately. One part of the kidneys was fixed in 10% formalin for histopathological examination, and the remaining parts were freshly snap frozen and preserved at −80°C, collected for biochemical analysis.

Serum biochemical assay

In this study, physiological functions were evaluated with serum levels of renal enzymes (blood urea nitrogen (BUN), serum creatinine (Scr), serum and urine β2-microglobulins (β2-MG)). These parameters were all assayed using commercially available enzymatic assay kits (Leadman Group Co., Ltd., Beijing, China).

Histopathological examinations

The kidneys were individually excised and immediately immersed in 4% formaldehyde solution for 24 h. Sections of 3 μm thickness were cut and stained with haematoxylin and eosin; the slides were observed, and photos were taken using an optical microscope (Leica DM5000B, Leica DFC500, Germany). The pathologist was blinded to the identity of all slides for analysis.

Measurement of renal oxidative stress

The kidneys were homogenized with phosphate buffered saline using a homogenizer on ice and then centrifuged for 10 min at 12,000 r/min. The supernatant was collected to evaluate superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels using assay kits (Beyotime, Jiangsu, China).

Western blot analysis

Lysate preparation and Western blot analysis were performed as previously reported.³⁶ The renal tissues were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) on ice to prepare the total protein fractions. Mitochondrial Proteins extracts were prepared using a Cell Mitochondria Isolation Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer’ s instructions.

Lysates were resolved by SDS-PAGE and transferred to PVDF membrane. The membranes were blocked with 5% skim milk at 1 h, then incubated overnight at 4°C with the primary antibody. After the incubation, membrane was washed 3 times in TBST and incubated with secondary antibody for 1 h at room temperature. The protein bands analysed with a chemiluminescence imaging system by using enhanced chemiluminescence Kit (Beyotime, Shanghai, China) and relative protein levels were quantified by Image J programme.

Statistical analysis

SPSS v22.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The values are expressed as the means ± S.D. Statistical comparisons were conducted using Student’s t-test and one-way analysis of variance, followed by Tukey’s test. The levels of significance were set at p < 0.05, p < 0.01 or p < 0.001.

Results

Successful establishment of the animal model

The FBG and renal function of the mice was investigated to verify whether the DN mouse model was successfully established. The level of FBG in the DN model mice (16.73 ± 4.99 mm) was markedly higher than that in the Con group (4.41 ± 0.75 mm) (p < 0.05). Compared with the Con group, the mice in the DN model also developed common signs of diabetes, including polydipsia, polyuria and noticeable hypoactivity and debility. In our study, DN model mice had diabetes-induced kidney dysfunction with a significant increase in BUN, Scr, β2-MG and the relative kidney index. Histological examination by light microscopy revealed damage to the kidneys of DN model mice.

Phillyrin intervention decreased the levels of FBG and HbA1c in DN mice

FBG and HbA1c are significant indicators used to assess the severity of diabetes.³⁷ The level of HbA1c is closely related to blood glucose levels, and control of HbA1c levels contributes to an improved outcome in diabetic nephropathy patients. As shown in Figure 1(A), the FBG level in the DN group was significantly increased compared with that in the Con group (p < 0.05), while phillyrin treatment at two different dosages decreased the FBG level in DN mice. Compared to the DN group, the increase in HbA1c was effectively attenuated by phillyrin at 15 mg/kg/d (Figure 1(B)). Treatment with Met, which was used as a positive control, also significantly reduced the levels of FBG and HbA1c in DN mice.

Figure 1.

Effects of phillyrin treatment on glycaemic indexes in diabetic mice. After different treatments, the level of FBG (A) and the level of serum glycated haemoglobin A1c (HbA1c) (B) were measured. Metformin (Met) was used as a positive control. Data are presented as the mean ± SD. *p < 0.05 vs. Con. **p < 0.01 vs. Con. ***p < 0.001 vs. Con; ^#p < 0.05 vs. DN. ^##p < 0.01 vs. DN. ^###p < 0.001 vs. DN. n = 6.

Phillyrin attenuated histopathological kidney injury in DN mice

After treatment with phillyrin for 4 consecutive weeks, the kidney weight and pathological changes were analysed. The relative kidney index (kidney weight to body weight ratio) was adopted as an indicator of renal function. As shown in Figure 2(A), compared to the Con group, the mice in the DN group showed a significant increase in the relative kidney index, while phillyrin reversed the upregulated relative kidney index. In addition, an overall view of the distribution of renal morphology and ultrastructure at the light microscopy level is shown in Figure 2(B). Obvious abnormalities were not evident in the Con group, while severe renal injury was found in the DN group, characterized by glomerular basement membrane thickening, tubular atrophy and obvious deformation and abscission of renal tubular epithelial cells. Phillyrin treatment effectively alleviated these changes induced by diabetes. These results indicated that phillyrin could ameliorate kidney injury in DN model mice.

Figure 2.

Effects of phillyrin treatment on histopathological kidney injury in diabetic mice. After different treatments, the relative kidney weight index was determined (A). The effects of different treatments on the histological changes in diabetic mouse kidney tissues by HE staining were determined (×400 magnification) and characterized by (1) glomerular basement membrane thickening, (2) tubular atrophy and (3) obvious deformation and abscission of renal tubular epithelial cells (B). Data are presented as the mean ± SD. **p < 0.01 vs. Con; ^#p < 0.05 vs. DN. ^##p < 0.01 vs. DN. n = 6.

Phillyrin reduced the renal dysfunction of DN mice

To investigate whether renal function was improved by phillyrin, biochemical parameters in serum were measured. Increases in BUN, Scr and β2-MG are considered markers of renal function injury.³⁸ The levels of BUN, Scr and β2-MG were markedly upregulated in the DN group but prominently decreased after phillyrin treatment (Figure 3). These results suggested that phillyrin could prevent functional injury to the kidney in DN mice.

Figure 3.

Effects of phillyrin treatment on kidney function in diabetic mice. After different treatments, the levels of BUN (A), Scr (B) and β2-MG (C) in serum were measured. Data are presented as the mean ± SD. *p < 0.05 vs. Con. **p < 0.01 vs. Con. ***p < 0.001 vs. Con; ^#p < 0.05 vs. DN. ^##p < 0.01 vs. DN. ^###p < 0.001 vs. DN. n = 6.

Phillyrin inhibited oxidative stress in renal tissues of DN mice

Evidence suggests that oxidative stress is involved in the development and progression of DN.³⁹ Oxidative stress and antioxidative ability were assessed by measuring the levels of MDA and SOD in renal tissues. As shown in Figure 4, compared to the Con group, the DN group had significantly decreased SOD activity but increased MDA levels, indicating oxidative stress in DN mouse kidneys. Treatment with phillyrin remarkably restored SOD activity and MDA levels. These results showed that phillyrin could inhibit oxidative stress in DN mouse kidneys.

Figure 4.

Effects of phillyrin treatment on oxidative stress in diabetic mice. After different treatments, the levels of MDA (A) and SOD (B) in kidney tissue were measured. Data are presented as the mean ± SD. *p < 0.05 vs. Con. **p < 0.01 vs. Con. ***p < 0.001 vs. Con; ^#p < 0.05 vs. DN. ^##p < 0.01 vs DN. n = 6.

Phillyrin activates the PI3K/Akt/GSK-3β signalling pathway in renal tissues of DN mice

Recent studies have shown that the development of DN and the apoptosis of renal cells are related to the PI3K/Akt signalling pathway.¹¹ As shown in Figure 5, compared to the Con group, the western blotting analyses revealed that the ratio of phospho-Akt/Akt was downregulated in the DN group. However, treatment with phillyrin markedly alleviated these changes in renal tissues of DN model mice. GSK-3β is considered one of the pivotal downstream proteins of the PI3K/Akt pathway, and renal cell apoptosis is associated with activating GSK-3β, accompanied by changes in the downstream proteins Bax and Bcl-2 in the kidney induced by DN.⁴⁰ As shown in Figure 5, compared with the Con group, DN significantly reduced the phosphorylation of GSK-3β in renal tissues, while phillyrin treatment visibly inhibited GSK-3β activity. In addition, DN conditions decreased Bcl-2 and increased Bax, which led to the release of Cyto C from the mitochondria into the cytoplasm and subsequently activated the caspase pathway to induce apoptosis.⁴¹ Compared with the DN group, phillyrin increased the levels of Bcl-2 and Cyto C in mitochondria and decreased the levels of Bax and Cyto C in the cytoplasm (C-Cyto C) and cleaved caspase-3. These findings indicated that phillyrin could attenuate DN-induced renal cell apoptosis by modulating the activity of the PI3K/Akt/GSK-3β pathway.

Figure 5.

Effects of phillyrin treatment on the PI3K/Akt/GSK-3β signalling pathway in renal tissues of DN mice. After different treatments, the protein levels of Akt, p-Akt, GSK-3β and p-GSK-3β in renal tissues of DN mice were analysed by western blot. The Cyto C in the mitochondrial and cytosolic protein extracts was detected, and the levels of Bcl-2 and Bax were analysed. The levels of the apoptosis-related protein cleaved caspase-3 were detected. GAPDH and COX 4 were used as internal references. Data are presented as the mean ± SD. *p < 0.05 vs. Con. **p < 0.01 vs. Con. ***p < 0.001 vs. Con; ^#p < 0.05 vs. DN. ^##p < 0.01 vs. DN. ^###p < 0.001 vs. DN. n = 6.

Discussion

Diabetes is one of the most common metabolic diseases characterized by hyperglycaemia in the world. Due to insulin secretion defects and/or insulin action disorder, long-term hyperglycaemia and metabolic disorders lead to systemic tissue dysfunction, especially in the kidneys, eyes, heart and blood vessels.⁴² DN is one of the most important complications of diabetes, affecting quality of life and endangering patients’ lives.⁴³ In recent years, the treatment strategies for DN have usually been to strictly control blood glucose and blood pressure levels by drug treatment, diet therapy and organ transplants. However, these treatment strategies are not only expensive but also have poor therapeutic effects to stop the development of kidney disease.^41,44 Therefore, preventing the occurrence of DN in a timely manner and effectively delaying the progression of DN is of great significance. Phillyrin has various pharmacological effects, including antioxidative and antiapoptotic activities, and can improve the outcome of some diseases, such as obesity.²⁷ Thus, we used STZ-induced DN mice as models to investigate the protective effects of phillyrin in delaying the progression of DN. Metformin, as the positive control in this study, is one of the first drugs for type 2 diabetes due to its effective hypoglycemic effect.⁴⁵

The cytotoxicity of STZ on pancreatic islet β-cells results in a sharp decrease in insulin secretion, significantly increasing the levels of FBG and HbA1c (Figure 1), and is applied to establish a DN mouse model.⁴⁶ Hyperglycaemia can lead to renal dysfunction, including the elevation of BUN, Scr and β2-MG levels in serum, as well as the pathomorphological changes of mesangial dilatation and renal tubular injury, which will eventually develop into DN (Figure 2).⁴⁰ Phillyrin(5 or 15 mg/kg) significantly reduced the levels of FBG and HbA1c, reversed the levels of serum BUN, Scr and β2-MG and improved the pathological changes of the kidney in DN mice (Figure 3). Our results showed that the protective effects of high dose of phillyrin (DN+H group) against diabetic nephropathy are comparable to metformin. Thus, phillyrin showed a therapeutic effect in improving kidney damage caused by hyperglycaemia.

Under physiological conditions, there is a balance between the endogenous antioxidant defence system and the production of ROS, including the action of SOD and MDA. SOD is an important intracellular antioxidant enzyme that scavenges reactive oxygen radicals and eliminates harmful metabolites to protect against kidney damage. MDA, a common index of peroxidation, is the end product of lipid oxidation, which can damage the stability of the mitochondrial membrane and aggravate the degree of injury to mitochondria.⁴⁷ Cyto C is the terminal protein of the mitochondrial oxidative respiratory chain, and an increased level of C-Cyto C indicates mitochondrial damage. Mitochondria are key players in producing physiological sources of ROS, and free radicals can also attack mitochondria. Maintaining the integrity of mitochondria is necessary for this oxidative balance to maintain normal renal cell function. Once the balance of oxidative/antioxidant stress is destroyed by the long-term hyperglycaemic environment, a series of cell signalling pathways can be activated, leading to severe injury and dysfunction in the kidney.^48,49 The present study showed that the downregulated level of SOD and the upregulated level of MDA (Figure 4) caused by sustained hyperglycaemia led to disruption of redox homoeostasis and damaged mitochondrial membrane lipids, resulting in the release of Cyto C from the mitochondria into the cytosol (Figure 5). In contrast, these changes were observably alleviated by phillyrin treatment, suggesting that the antioxidant stress properties of phillyrin can mitigate diabetes-induced kidney damage.

The phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) pathway is a classical signalling pathway that promotes cell growth and survival in response to extracellular signals. The phosphorylation of PI3K activates its downstream factor Akt, and then phosphorylated Akt (p-Akt) mediates a variety of physiological cellular functions, such as cell differentiation, proliferation, migration and apoptosis.⁵⁰ The PI3K/Akt signalling pathway is a crucial component involved in the pathogenesis of DN, and Akt activation can enhance the antioxidant and antiapoptotic capacity of diabetic animals.^51,52 Previous studies have confirmed that the PI3K/Akt pathway is suppressed in DN animal kidneys and that downregulation of PI3K/Akt activation contributes to renal proximal tubular cell apoptosis.^53,54 Phosphorylated Akt is a negative regulator of GSK-3β through the phosphorylation of GSK-3β at Ser9. Active GSK-3β is associated with the intrinsic apoptotic pathway by decreasing the level of Bcl-2, increasing the level of Bax, stimulating mitochondrial permeability transition pore opening and promoting the release of Cyto C from the mitochondria. Cytoplasmic Cyto C triggers cellular apoptosis by activating the apoptosis initiator caspase-9, and then caspase-9 cleaves and hydrolyses downstream caspase family members, such as caspase-3.⁵⁵ In this study, the treatment of DN model mice with phillyrin significantly upregulated the activity of the PI3K/Akt pathway, inhibited the activation of GSK-3β and reduced the levels of Bax, Cyto C and cleaved caspase-3 (Figure 5). Phillyrin counteracts the DN-induced inhibition of the PI3K/Akt/GSK-3β pathway and subsequently maintains mitochondrial function, alleviates oxidative stress and ultimately suppresses renal cell apoptosis.

Based on evidence from these experiments, persistent high blood glucose levels could induce mitochondrial dysfunction, oxidative stress and apoptosis in renal cells and ultimately cause severe renal injury. Phillyrin significantly reduced the levels of blood glucose and HbA1c in the model group and improved renal pathological changes. Phillyrin activated the PI3K/Akt pathway and subsequently blocked GSK-3β signal transduction to alleviate DN-induced cellular injury, including mitochondrial disorders and ROS accumulation. Then, the upregulation of antioxidant enzymes and suppression of mitochondrial dysfunction induced by phillyrin could reduce the activities of the caspase family and inhibit the apoptosis pathway, serving as a beneficial intervention for diabetes-induced renal injury. However, because the pathogenesis of DN is complex and may involve multiple factors, the effects and mechanisms of phillyrin on DN will be explored in our follow-up research. In summary, according to these findings, phillyrin may have the ability to ameliorate DN through the PI3K/Akt/GSK-3β signalling pathway and has promise as a prospective therapeutic option for DN in clinical practice.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Grant No. 81660593) and the Projects in Education Department of Jiangxi Province (Grant No. GJJ180863).

ORCID iD

Jie Zhou

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Phillyrin ameliorates diabetic nephropathy through the PI3K/Akt/GSK-3β signalling pathway in streptozotocin-induced diabetic mice

Abstract

Keywords

Introduction

Materials and methods

Materials and reagents

Animals

Modelling and administration

Serum biochemical assay

Histopathological examinations

Measurement of renal oxidative stress

Western blot analysis

Statistical analysis

Results

Successful establishment of the animal model

Phillyrin intervention decreased the levels of FBG and HbA1c in DN mice

Phillyrin attenuated histopathological kidney injury in DN mice

Phillyrin reduced the renal dysfunction of DN mice

Phillyrin inhibited oxidative stress in renal tissues of DN mice

Phillyrin activates the PI3K/Akt/GSK-3β signalling pathway in renal tissues of DN mice

Discussion

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References