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Year : 2023  |  Volume : 55  |  Issue : 1  |  Page : 34--42

Nephroprotective potential of syringic acid in experimental diabetic nephropathy: Focus on oxidative stress and autophagy

Bhoomika Sherkhane1, Veera Ganesh Yerra2, Anjana Sharma3, K Anil Kumar4, Gundu Chayanika5, Arruri Vijay Kumar5, Ashutosh Kumar6,  
1 Department of Pharmacology and Toxicology, NIPER, Hyderabad, Balanagar, Telangana, India; Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Sweden
2 Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada
3 Chemical Biology Unit, Institute of Nano Science and Technology, Mohali, Punjab, India
4 College of Pharmacy and Pharmaceutical Science Florida A and M University Tallahassee, FL, USA
5 Department of Neurosurgery, University of Wisconsin-Madison, Madison, WI, USA
6 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER) Kolkata, Chunilal Bhawan, Maniktala Main Road, Kolkata; Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER) SAS Nagar, Mohali, Punjab, India

Correspondence Address:
Ashutosh Kumar
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER) SAS Nagar, Mohali Bypass, Sector 67, Sahibzada Ajit Singh Nagar, Punjab - 160062


BACKGROUND: Diabetic nephropathy (DN) is a chronic hyperglycemic manifestation of microvascular damage in the kidneys. Widespread research in this area suggests the involvement of perturbed redox homeostasis and autophagy in renal cells phrase- promote the progression of DN. MATERIALS AND METHODS: Reframed sentences-The present study investigates the pharmacological effect of Syringic acid (SYA), in streptozotocin (STZ, 55 mg/kg, i.p) induced diabetic nephropathy model and in high glucose (30 mM) challenged rat renal epithelial cells (NRK 52E) cells with a focus on oxidative stress and autophagy mechanisms. RESULTS: Both in vivo and in vitro experimental data revealed elevated oxidative stress markers along with compromised levels of nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal cellular redox-regulated transcription factor in renal cells upon glycemic stress. Elevated blood glucose also reduced the autophagy process as indicated by low expression of light chain (LC) 3-IIB in diabetic kidney and in NRK 52E cells subjected to excess glucose. SYA (25 and 50 mg/kg, p.o.) administration for 4 weeks to diabetic rats, Reframed sentence-preserved the renal function as evidenced by reduced serum creatinine levels as well as improved urine creatinine and urea levles as compared to non treated diabetic animals. At the molecular level, SYA improved renal expression of Nrf2 and autophagy-related proteins (Atg5, Atg3, and Atg7) in diabetic rats. Similarly, SYA (10 and 20 μM) co-treatment in high glucose-treated NRK 52E cells displayed increased levels of Nrf2 and autophagy induction. CONCLUSION: Results from this study signify the renoprotective effect of SYA and highlight the modulation of oxidative stress and autophagy mechanisms to mitigate diabetic kidney disease.

How to cite this article:
Sherkhane B, Yerra VG, Sharma A, Kumar K A, Chayanika G, Kumar AV, Kumar A. Nephroprotective potential of syringic acid in experimental diabetic nephropathy: Focus on oxidative stress and autophagy.Indian J Pharmacol 2023;55:34-42

How to cite this URL:
Sherkhane B, Yerra VG, Sharma A, Kumar K A, Chayanika G, Kumar AV, Kumar A. Nephroprotective potential of syringic acid in experimental diabetic nephropathy: Focus on oxidative stress and autophagy. Indian J Pharmacol [serial online] 2023 [cited 2023 Oct 3 ];55:34-42
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Diabetes and its associated complications represent a major health problem in the industrialized world, and diabetic kidney disease is one of the common and life-threatening complications of hyperglycemia.[1] Several pathways which are related to hyperglycemic injury promote the progression of diabetic nephropathy (DN), where oxidative stress due to excess glucose levels plays a key role in renal injury.[2] Oxidative stress is produced as a result of hyperglycaemia induced metabolic stress resulting in chronic ROS generation and insufficient induction of cellular antioxidant response, mediated primarily by transcriptional activation of nuclear factor (erythroid-derived 2)-like 2 Nrf2. Nrf2 is a cellular redox sensor responsible for the transcriptional expression of antioxidant enzymes via binding to the antioxidant response element (ARE) of the genome. Impaired Nrf2 activity in the DN pathogenesis and restoration of renal health by induction of Nrf2 is well documented by several preclinical studies.[3]

Several studies have revealed that in addition to oxidative stress, the hyperglycaemia induced meatbolic load in renal cells impaires several homeostatic mechanims including AMPK/SIRT1 pathway and autophagy.[4] Autophagy is a highly conserved cellular homeostatic mechanism activated under stress conditions. Initial stages of autophagy form autophagosome that sequesters the damaged proteins and older organelles in double-membrane vesicle and fuses with the lysosome to degrade the sequestered cargo.[5] Hyperglycemia-induced metabolic alterations compromise autophagy in renal cells and contribute to podocyte apoptosis and tubular interstitial fibrosis. As observed in several reports, autophagy is required for proteostasis in terminally differentiated cells such as podocytes and also, to ensure timely nutrient supply in proximal tubular cells.[6] In renal tissue, autophagy plays an essential role in maintaining proper renal function, as even under physiological conditions, the podocytes possess basal autophagic activity that ensures the effective removal of damaged proteins and cellular organelles. Recent data shows that actiavting the autophagy machinery using pharmacological agents prevent podocyte and tubular injury and also, aids in restoring renal function under diabetic condition.[7] Syringic acid (SYA) is a natural phenolic compound found in many plant species. Several reports are available on the preventive effects of SYA against various diseases, including cancer, diabetes, and inflammatory and infectious diseases which are attributable to its antioxidant and anti-inflammatory activities.[8],[9] Further, modulation of autophagy may serve as a reliable target to halt the progression of DN, and thus, there is a prerequisite for screening of pharmacological agents which enhance autophagy under hyperglycemic stress. Hence, in the current study, we have assessed the effects of SYA on the autophagy process under diabetic conditions, along with its antioxidant effect.

 Materials and Methods


The majority of the chemicals utilized in the current study were purchased from Sigma Aldrich (St. Louis, MO, USA).


Induction of diabetes and study design

Male Sprague–Dawley rats weighing around 240-260 g were kept under standard laboratory settings with room temperature 22°C ± 2°C and 12 h light/dark cycles, and all the experimental methods were authorized by Institutional Animal Ethics Committee-NIPER-Hyderabad. Animals were given a single intraperitoneal dose (55 mg/kg) of streptozotocin (STZ) to induce diabetes. Post 48 hours of induction, the plasma glucose levels were assessed, animals with plasma glucose levels >250 mg/dl were considered as diabetic and divided into 3 groups as follows, diabetic control (STZ), diabetic rats treated with SYA at 25 and 50 mg/kg (STZ+SYA-25 and STZ+SYA-50). Accordingly, age-matched normal animals were included as a control group (nondiabetic [ND]), and the normal animals were treated with SYA 50 mg/kg (ND + SYA-50) to see if SYA alone showed any physiological effects. The animal study consisted of 8-week duration, and the SYA treatment was initiated post 4 weeks of diabetes induction. SYA dosages were selected based on the available studies and SYA was administered orally every day in a single dose for 4 weeks (4th week to 8th week). Urine and serum were collected on the last day of the 8th week, and then the animals were transcardially perfused to collect the renal tissues. The animal study design was formulated based on several studies in the experimental model of DN.[10]

Biochemical analysis of plasma and urine

All serum and urinary biochemical estimations for creatinine, blood urea nitrogen (BUN), urea, and urinary albumin were performed with the standard Kits (Accurex, India) as per the manufacturer's protocol.

Assessment of renal oxidative stress markers

Quantification of lipid peroxidation

The MDA levels were estimated by thiobarbituric acid reactive substance assay performed as demonstrated in the previous report.[11]

Measurement of reduced glutathione

Glutathione (GSH) levels in renal tissue were assessed by Ellman's method.[12]

Estimation of superoxide dismutase activity

The renal superoxide dismutase (SOD) levels were measured using the commercially available kit, as per the instructions provided by the manufacturer (Sigma, USA).[13]

Estimation of catalase activity

The catalase assay was performed based on Aebi's method.[14] The renal lysates were treated with hydrogen peroxide, and the absorbance of the mixture was measured. The Catalase activity is proportional to the rate of decomposition of hydrogen peroxide.

Estimation of glutathione reductase activity

Carlberg and Mannerwick's method, which is based on the reduction of glutathione disulfide by NADPH by glutathione reductase (GR), was used to estimate GR activity.[15]

Estimation of glutathione peroxidase activity

Paglia and Valentine method with slight modification was used to check the glutathione peroxidase activity.[16]

Histopathological evaluation

Formaldehyde fixed renal samples were treated with different concentrations of alcohol, xylene, and set in paraffin. 5-μm renal sections were obtained and stained with hematoxylin-eosin and periodic acid–Schiff base (PAS) staining (PAS staining kit, Sigma, USA). The change in glomerulus volume and mesangial expansion was quantified using Image J software (NIH).[17] The following formula was used to calculate the glomerular volume.

GV = (β/k) (glomerular area [GA]) 3/2 (β =1.38 refer to the sphere; k = 1.10 refer to distribution coefficient; GA).

Immunohistochemistry and immunofluorescence studies

The renal sections were deparaffinized, rehydrated, and subjected to blocking after the antigen retrieval method. Then, probed with primary antibody Nrf2 (1:50) at 2°C–4°C. In subsequent steps, slides were incubated with a secondary antibody, stained with 3, 3'-diaminobenzidine followed by nucleus staining by hematoxylin. Slides were mounted using DPX prior to observing under the microscope. For immunofluorescence studies, the sections were incubated with an anti-LC-3B antibody (1:100) and then incubation with a secondary antibody (Fluorescein isothiocyanate tagged). DAPI was used to mount the slides and visualized using a confocal microscope (Leica, Germany).[18]

Western blotting

The protein samples were prepared from the renal tissues, and an equal quantity was loaded and separated by gel-electrophoresis and transferred onto Polyvinylidene fluoride (PVDF) blotting membrane. After blocking, the membrane was probed with antibodies of Nrf-2 (Santa Cruz Biotechnology, USA), Atg3, Atg5, Atg7, and β-Actin (1:1000) (cell signalling technology, USA) for 12 h at 2°C–6°C. After washing, the membranes were probed with appropriate secondary antibodies, then the chemiluminescence method was used to visualize protein bands (Vilber Lourmat, Germany) and quantified with NIH Image-J software (version 1.48, NIH, USA).[19]

NRK 52E cell culture

NRK 52E cell (rat renal epithelial cells) cell line was obtained from National Centre for Cell Science, Pune and maintained in Dulbecco's Modified Eagle Medium (glucose concentration 1 g/L) accompanied with 10% Fetal bovine serum, streptomycin/penicillin (1%) at 37°C with 95% air and 5% CO2. Cells were added with a glucose concentration of 30 mM, simultaneously, cells were treated with SYA (at concentrations 10 and 20 μM) for 48 h.[20]

Immunofluorescence staining

NRK 52E cells seeded on coverslips and after completion of the treatment period, they were fixed in paraformaldehyde (4%), followed by permeabilization by TritonX-100 (0.1%). After blocking with 3% BSA and probed with LC-3B antibody for overnight at 2°C–4°C. Next, the cells were incubated with appropriate fluorochrome tagged secondary antibody and mounted with flouroshield using DAPI. Fluorescence was visualized, as mentioned in section 2.5. For Lysotacker co-staining with LC-3B immunostaining, cells were incubated with Lysotacker deep red (50 nM) for 30 min prior the paraformaldehyde fixation step.[21]

Western blotting analysis

NRK 52E cells were cultured as required and incubated with glucose (30 mM) and SYA (at concentrations 10 and 20 μM) for 48 h. Following the treatment period, proteins were extracted, and the western blot analysis was performed, as mentioned in section 2.6.

Statistical analysis

Graph Pad Prism (version 5.0) was used to analyze the data, and the results were expressed as mean ± standard error of the mean. One-way analysis of variance was used to compare the mean values of different groups, followed by post hoc analysis by “Bonferroni's multiple comparison test.” Results indicating P < 0.05 were deemed statistically significant.[22]


Syringic acid treatment recovers renal function in diabetic rats

Preliminary data indicated an increased kidney-to-body weight ratio (P < 0.001) in the diabetic group and notifiable raise in serum levels of creatinine (P < 0.001) and BUN (P < 0.001) levels that positively correlated with decreased levels of creatinine in urine (P < 0.001) in diabetic control group highlighting diabetes associated renal functional abnormality [Table 1]. The SYA treatment in diabetic rats normalized the serum creatinine and BUN levels as well as increased urinary creatinine levels (P < 0.05; 25 mg/kg, p.o). Similarly, a steep upsurge in renal injury marker, urinary albumin in the diabetic group was reversed upon SYA treatment indicating the reno-protective effect of SYA in DN.{Table 1}

Syringic acid attenuates diabetes-induced renal oxidative damage in streptozotocin-induced renal injury

Biochemical analysis indicated a substantial elevation in MDA (P < 0.001) levels and a notable fall in GSH (P < 0.05) content in renal tissues of the diabetic group compared to the normal control group, indicating an increased renal lipid peroxidation as a result of excess oxidative species in tissue. SYA treatment resulted in enhanced GSH (P < 0.05; 25 mg/kg and P < 0.001; 50 mg/kg) levels as well as a significant reduction in MDA [P < 0.01; 25 mg/kg and P < 0.001; 50 mg/kg, [Figure 1]b] levels in renal tissue of hyperglycemic rats, indicating improved ROS scavenging under hyperglycemic condition.{Figure 1}

Syringic acid restricts diabetes-induced histological changes

Mayer's hematoxylin and eosin staining and PAS staining in renal sections revealed enlarged glomerulus and excess deposition of glomerular mesangial matrix, respectively (P < 0.001) in renal tissue exposed to hyperglycemia than the normal renal tissue. However, as shown in [Figure 1]a, SYA treatment reduced the mesangial matrix accumulation in diabeteic rats, therefore ameliorating the diabetes in glomerular hypertrophy.

Syringic acid enhances the expression of antioxidant enzymes in diabetic kidney

In addition to GSH, several antioxidant enzymes SOD, GR, GPx, and catalase are involved in ROS scavenging and as evidenced by the results of biochemical analysis, antioxidant enzyme activity was significantly reduced in untreated diabetic rats [Figure 1]b. Expression of the aforementioned antioxidant enzymes is regulated by Nrf2, and untreated diabetic rats displayed diminished expression of Nrf2 in renal cells [Figure 2]a. SYA treatment in diabetic rats displayed an improved expression of Nrf2 as demonstrated by western blot analysis and increased immunopositivity in immunohistochemistry staining compared to untreated diabetic rats [Figure 2]b. Consequently, SYA treatment in diabetic rats also resulted improved levels of antioxidant enzymes in the kidney.{Figure 2}

Syringic acid improves the expression of proteins related to autophagy mechanism and formation of autophagosomes in renal tissue of streptozotocin induced diabetic rats

The effect of hyperglycemia on the autophagy process in renal tissues was evaluated by immunoblot analysis of autophagy proteins. Expression of proteins implicated in the initiation of the phagophore, such as Beclin 1 and Atg5, were reduced to a greater extent in the renal cortex of diabetic rats demonstrating poor autophagy initiation. Furthermore, proteins involved in the subsequent elongation of the phagophore, such as E1-like Atg7 and E2-like Atg3, were considerably declined in the renal tissue of hyperglycemic rats [Figure 3]a. Upregulation of expression of autophagy-related proteins and enhanced immuno-localization of LC-3 in renal tissues of SYA-treated hyperglycemic implied improved formation autophagosomes [Figure 3]b. Besides the formation of autophagosomes, the efficient degradation of sequestered proteins is a crucial step in autophagy, the sequestome p62 is degraded along with autophagy cargo proteins. Increased p62 levels in renal tissues of diabetic rats are indicative of poor autophagy clearance, which was reduced by SYA treatment. Together, these results indicate the autophagy activation effect of SYA.{Figure 3}

Syringic acid treatment improves antioxidant mechanisms in hyperglycemic normal rat kidney 52E cells

Incubation of NRK 52E cells with elevated levels of glucose resulted in the downregulation of the antioxidant enzyme NQO-1 and its upstream regulator Nrf2. Co-treating the excess glucose-treated cells with SYA resulted in the upregulation of Nrf2 and NQO-1 expression [Figure 4].{Figure 4}

Syringic acid improves expression autophagy machinery proteins in normal rat kidney 52E cells treated with high glucose

In consistent with our in vivo data, immunoblot analysis of NRK 52E cell lysates revealed declined expression of autophagy protein Atg7 [Figure 4]. In addition, poor immunoco-localization of LC-3B and lysotracker in renal cells demostrated imapired autophagy in upon high glucose exposure. Accumulation of autophagy adaptor protein p62 in high glucose-treated renal cells further suggested a poor autophagy process. We observed a 2-fold increase in the expression of Atg7 and improved co-localization of LC-3 and lysotracker stain, suggesting the improved formation of autolysosome in SYA-treated NRK 52E cells. Added, with these results, we observed reduced band intensity of p62 in SYA-treated NRK 52E cells under high glucose stress, thus indicating restoration of autophagy function.


DN, the major reason behind increased renal failure cases and there is an indispensable necessity for the identification of novel targets and the development of new therapeutic entities.[4] The current work demonstrated the nephroprotective role of SYA, in STZ-induced type 1 DN model as well as in NRK 52E cells under hyperglycemic stress and investigated the mechanism of action of SYA. Chronic diabetes effect renal function, which is observed by altered renal clearance of creatinine and excessive albuminuria, which is the major marker of renal injury.[23] The hyperglycemia is also responsible for the ultrastructural changes in the glomerulus, such as excess deposition of mesangial matrix leading to glomerular hypertrophy, altered glomerular basement membrane biochemistry, and podocyte apoptosis.[24] Similarly, the STZ-induced diabetic rats displayed changes in serum and urinary creatinine and also, increased albuminuria which were reversed on SYA treatment. In addition, SYA reduced mesangial expansion and normalized the glomerular volume in renal tissues of diabetic rats, indicating overall protection from hyperglycemic injury.

At cellular levels, there are several factors that influence the progression of DN, among which hyperglycemia-induced metabolic alteration and a state to nutrient excess in renal cells emerged as prominent aspects in the pathogenesis of renal injury.[25] The hyperglycaemia-induced metabolic stress results in perturbed cellular redox that produces a state of imbalance between ROS-generating pathways and several antioxidant enzymes.[27] The antioxidant mechanism is modulated by transcription factor Nrf2, which under basal conditions binds to keap-1 and is subjected to proteasomal degradation, whereas under acute oxidative stress conditions, Nrf2 gets translocated into the nucleus, there it binds to ARE region and induces various antioxidant enzymes transcription.[27] Chronic oxidative stress in diabetes hampers Nrf2 activity and aggravates ROS signaling, and recent reports suggest that the high glucose-mediated inhibition of Nrf2 mediates oxidative renal injury in DN.[26] Similarly, a substantial reduction in the levels of Nrf2 along with the declined activity of various antioxidant enzymes such as GPx, GR, SOD, and catalase was noticed in diabetic kidney. Whereas, SYA treatment reversed these effects. Consistent with the in vivo results, SYA treatment in hyperglycemic stressed NRK 52E cells, demonstrated enhanced levels of Nrf2 and its downstream target NQO.

Hyperglycemia, in addition to impairing redox balance in renal cells, it also leads to a nutrient excess state in cells, resulting in impaired nutrient sensing pathways, among which autophagy is a crucial mechanism for nutrient homeostasis. Autophagy, is a cellular nutrient-regulating mechanism that removes damaged proteins or old organelles during stress conditions or starvation and aids in recycling the nutrient pool. Recent studies have reported the implication of autophagy in maintaining glomerular health, and loss of autophagy during chronic diabetes results in podocyte apoptosis resulting in the alteration of the glomerular filtration barrier that aggravates albuminuria worsening the renal injury.[28] Autophagy process involves phagophore initiation, via activation of Unc-51 Like Autophagy Activating Kinase 1 complex followed by activation of Class II PI3K complex, which mediates nucleation of phagophore, where the damaged protein aggregates are engulfed by phagophore. Beclin 1 is a Class II PI3K complex required for the nucleation of the phagophore, afterward, the autophagy-related proteins Atg5, Atg12, and Atg16 form a conjugate and promote the phagophore elongation. For the generation of the autophagosome, the cytosolic form of LC-3 conjugates to phosphatidylethanolamine via ubiquitination-like reactions catalyzed by the Atg7 and the Atg3, to form LC3II that serves as a marker for autophagosome formation.[29] Results from this study indicate that SYA restored the diabetes-induced autophagy impairment as per hypothesis by enhancing the expression of autophagy proteins including Beclin 1, Atg5, Atg7, and Atg3, indicating enhanced phagophore formation. Moreover, the increased expression of Atg7 and Atg3 by SYA demonstrated enhanced LC-3 lipidation. Further investigation of LC-3 expression by immunofluorescence staining displayed increased immunopositivity of LC-3 on SYA administration, while mild immunoreactivity of LC-3 in the diabetic-control group is noticed. In addition, the immuno-localization assay performed in NRK 52E cells revealed improved autophagosome formation by SYA, as demonstrated by increased colocalization of LC-3 and Lysotracker red. Improved expression of autophagy proteins, along with increased Nrf2 expression in renal lysates of diabetic rats, indicates that SYA ameliorates renal injury through activation of autophagy and antioxidant mechanisms.


The current data from in vivo and in vitro depict the nephroprotective role of SYA against hyperglycemic renal damage. These findings anticipate future molecular studies to delineate the underlying mechanism of SYA on the induction of autophagy in renal tissue.


The support from the National Institutes of Pharmaceutical Education and Research (NIPER) in Hyderabad and Kolkata is gratefully acknowledged by the authors.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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