Abstract
Diabetic nephropathy (DN) is a major complication of diabetes, largely driven by chronic hyperglycaemia and oxidative stress. This study investigated the dose-dependent effects of protocatechuic acid (PCA) on DN, focusing on its potential to attenuate ferroptosis and apoptosis in a streptozotocin-induced type 1 diabetes rat model. Thirty-two Wistar-Albino rats were divided into four groups (n = 8): Control, Diabetes, Diabetes + PCA 50 mg/kg, and Diabetes + PCA 100 mg/kg. After 12 weeks, kidney tissues were evaluated histologically (Perls’ Prussian blue, Periodic Acid–Schiff (PAS)), immunohistochemically (Bax, Bcl-2, Caspase-3, Caspase-9, Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), glutathione peroxidase 4 (GPx4), transferrin receptor-1 (TfR-1), and by TUNEL assay. Diabetic rats exhibited iron accumulation, tubular dilatation, intracellular vacuolization, sclerotic glomeruli, and mesangial expansion (p < 0.05), while tubular atrophy, hyaline deposition, and mononuclear infiltration were unchanged (p > 0.05). Apoptotic and ferroptotic markers (Bax, Caspase-3, Caspase-9, ACSL4, TfR-1) increased, and Bcl-2 and GPx4 decreased (p < 0.001). PCA treatment, particularly at the 100 mg/kg dose, significantly attenuated the expression of apoptotic and ferroptotic markers and reduced the number of TUNEL-positive cells (p < 0.001). These findings suggest that PCA modulates cell death by upregulating Bcl-2 and GPx4 while downregulating Bax, Caspase-3, Caspase-9, ACSL4, and TfR-1 in renal tissues. While PCA effectively suppressed these molecular markers of injury, it did not lead to significant improvements in systemic renal function parameters such as serum creatinine and blood urea nitrogen. Thus, PCA may serve as a potential agent for mitigating cellular damage in diabetic kidney injury by targeting specific cell death pathways.
Introduction
Diabetes mellitus (DM) is a metabolic disorder marked by high blood glucose levels, requiring close monitoring. By 2045, approximately 783.2 million people are expected to be affected. 1 DM is categorized into type 1 (T1DM), an autoimmune disease-causing pancreatic beta-cell destruction and insufficient insulin production, 2 and type 2 (T2DM), characterized by insulin resistance and impaired insulin secretion. Hyperglycaemia significantly increases the risk of complications such as cardiovascular disease, stroke, neuropathy, nephropathy, and retinopathy. Diabetic nephropathy (DN) is a common, severe complication that often progresses to chronic renal failure. 3 Diabetic individuals are up to 10 times more likely to develop end-stage renal failure, and approximately 40% of diabetic patients develop DN. 4 Hyperglycaemia induces microvascular injury, especially in the renal glomeruli, contributing to DN pathogenesis. 5 Reactive oxygen species (ROS) overproduction plays a critical role in glomerular injury and podocyte apoptosis, leading to a decrease in podocyte number, which is associated with proteinuria and glomerular damage. 6 Additionally, hyperglycaemia is involved in renal tubular cell death through ROS and apoptosis gene dysregulation.7,8 Ferroptosis is a regulated form of cell death distinct from apoptosis, necrosis, and autophagy, driven by iron-dependent lipid peroxidation and ROS accumulation. 9 Ferroptosis exhibits distinct biochemical and morphological features, such as shrinkage of mitochondria, increased mitochondrial membrane density, excessive accumulation of lipid peroxides and ROS, and decreased antioxidant capacity. Iron accumulation increases lipid peroxidation. The inability to effectively clear the increased lipid peroxides leads to cell death by ferroptosis. 10 Hyperglycaemia disrupts iron homeostasis and promotes ROS production, making renal tubular cells especially vulnerable to oxidative damage. 11 Key regulators of ferroptosis include iron, transferrin receptor-1 (TfR-1), Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), and glutathione peroxidase 4 (GPx4).12–14
The primary strategies for treating DN focus on managing hyperglycaemia, hypertension, and haemodynamic parameters. 15 Recent interest has grown in phytotherapeutic agents with both hypoglycemic and antioxidant properties. The WHO estimates nearly 21,000 plant species are used for medicinal purposes, rich in compounds such as alkaloids, phenolics, flavonoids, and terpenoids. 16 Protocatechuic acid (PCA) is a naturally occurring phenolic acid that is widely distributed in nature. Chemically, PCA is known as 3,4-dihydroxybenzoic acid. Phenolic compounds are classified as secondary metabolites and are derived from phenylalanine through the shikimic acid pathway. PCA shares structural similarity with several well-known antioxidant compounds, including gallic acid, caffeic acid, vanillic acid, and syringic acid. 17 PCA is widely distributed and found in many edible plants and plant-derived products, including bran, rice, onions, fruits such as plums, gooseberries, grapes, almonds, as well as in olive oil and white wine. 18 In experimental studies, it has been suggested that administration of 2% and 4% PCA to diabetic mice lowers blood glucose levels by exerting insulin-like effects via PPARγ activation, thereby reducing advanced glycation end-product (AGE) formation and potentially preventing glycation-related diabetic complications. 19 PCA treatment also reduced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels in cardiac and renal tissues. Consequently, PCA is proposed to exert protective effects against diabetic complications through its antilipidemic, antioxidative, and anti-inflammatory properties. 20 Furthermore, it has been demonstrated that PCA reduces ROS formation in a dose-dependent manner by inhibiting the Fenton reaction and enhancing ROS scavenging activity. 21 In addition, it has been determined that PCA suppresses ferroptosis in the liver via the NRF2 signaling pathway, thereby providing protection against lipotoxicity and steatosis. 22 This study aims to investigate the protective role of PCA in inhibiting cell death via apoptotic and ferroptotic pathways in DN. It is hypothesized that PCA, as a phytotherapeutic agent, will protect renal tissues by attenuating the processes of apoptosis and ferroptosis in streptozotocin-induced diabetic rats.
Diabetic nephropathy (DN) is a serious complication of diabetes, primarily driven by chronic hyperglycaemia and oxidative stress. Both apoptosis and ferroptosis have been implicated in the pathogenesis of DN. While protocatechuic acid (PCA) has been shown to suppress ferroptosis in other tissues like the liver, its specific role in modulating these pathways within the diabetic kidney remains to be fully elucidated
This study investigates the dose-dependent effects of PCA on cell death pathways in a streptozotocin-induced type 1 diabetes rat model. We demonstrate that PCA significantly attenuates specific markers of histological renal injury, reduces iron accumulation, and modulates key apoptotic and ferroptotic pathways in renal tissues. Specifically, PCA treatment was associated with the downregulation of pro-apoptotic and pro-ferroptotic markers (Bax, Caspase-3, Caspase-9, ACSL4, and TfR-1) and the upregulation of protective markers (Bcl-2 and glutathione peroxidase 4 (GPx4)).
These findings highlight PCA as a potential candidate for mitigating cellular damage in diabetic kidneys by targeting apoptosis and ferroptosis. This study opens new avenues for research into natural compounds that modulate regulated cell death pathways, independently of systemic glucose or functional parameter changes. Future research may focus on the early-stage intervention potential of PCA to prevent the progression of diabetic renal injury before significant functional impairment occurs.
Material and methods
Chemicals
Streptozotocin (STZ) and PCA were both purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Bcl-2, active/cleaved caspase-3, caspase-9, TfR-1, and GPx4 were obtained from Cloud-Clone Corp. (Houston, TX, USA). Antibodies against ACSL4 and Bax, as well as the TUNEL assay kit, were procured from Elabscience (Houston, TX, USA).
Induced type-1 diabetes model in rats
DM was induced in rats with a single intraperitoneal injection of 50 mg/kg STZ in 0.1 M citrate buffer (pH 4.5). To prevent hypoglycemia, rats were given a glucose solution for 24 h post-injection. After a 72-h fast, blood samples were collected, and rats with fasting blood glucose >250 mg/dL were considered diabetic.
Animals and experimental design
Ethical approval for the study was obtained from the Ondokuz Mayıs University Animal Experiments Local Ethics Committee (OMU HADYEK), dated 26.05.2022 (Approval No. 2022/16). A total of 32 adult Wistar-Albino rats (8–10 weeks old, 250–300 g) were used, housed under standard conditions (22 ± 2°C, 12-h light/dark cycle), with ad libitum access to water and food. The animals underwent a 2-week acclimatization period before the experiment. The rats were randomly divided into four groups (n = 8/group). Group I served as healthy controls. DN was induced in Groups II–IV via STZ injection. Group II was the untreated diabetic control, while Groups III and IV received oral PCA at doses of 50 and 100 mg/kg, respectively. Four animals died during the experiment. The total duration was 84 days, during which body weights were monitored, and PCA was administered daily to Groups III and IV. At the end, fasting blood glucose levels were measured before euthanasia. Blood samples were collected via cardiac puncture under xylazine-ketamine anesthesia for serum analysis, and euthanasia was performed by decapitation. The blood samples were then processed to separate the serum.
Histopathological examination
During necropsy, kidney tissues were fixed in 10% buffered formaldehyde for 48 h, processed through a graded alcohol, cleared with xylene, and embedded in paraffin. Sections of 4–5 µm thickness were cut from the paraffin blocks, mounted on slides, and stained with Perl’s Prussian Blue and Periodic Acid–Schiff (PAS) Staining. The stained sections were then examined under a light microscope (Nikon Eclipse E600).
Evaluation of PAS staining
The glomerular sclerotic index (GSI) was assessed using the semi-quantitative scoring system (score 0–4). At 400× magnification, the thickness of the glomerular basement membrane (μm), the width of Bowman’s space (μm), the thickness of the visceral and parietal layers of Bowman’s capsule (μm), and the thickness of the proximal tubular basement membranes (μm) were measured using ImageJ software. Tubular and interstitial damage scores were evaluated separately at 200× magnification on the same PAS-stained sections.
Immunohistochemical staining and analyses
The streptavidin-biotin peroxidase complex method was performed according to the manufacturer’s instructions (Epredia™ Lab Vision™). Sections were heated in citrate buffer (pH 6) at 600 watts for 20 min to unmask epitopes, then incubated in 0.5% H2O2 for 10 min to block peroxidase activity. The primary antibody solution (1/100 dilution) was applied and incubated overnight at +4°C. The next day, sections were incubated with a biotin-conjugated secondary antibody for 45 min, followed by streptavidin for another 45 min. Sections were washed with PBS, and the chromogenic reaction was developed using 3-amino-9-ethylcarbazole. After counterstaining with Mayer’s hematoxylin, sections were mounted and examined under a light microscope. Immunostaining for Bcl-2, Bax, Caspase-3, Caspase-9, TfR-1, GPx4, and ACSL4 in the kidney cortex and medulla was evaluated separately. Ten images were captured at 200× magnification and analyzed using the Bs200Pro Image Analysis System. Immunopositivity percentages were calculated based on staining intensity and area.
TUNEL staining and analyses
The TUNEL Apoptosis Assay Kit (HRP-DAB) was used to detect apoptotic cell death. Sections were deparaffinized, washed with PBS, and incubated in 1× Proteinase K solution at 37°C for 20 min. After blocking with 3% H2O2 for 10 min, the sections were incubated with TdT enzyme and labeling solution for 1 h at 37°C, followed by Streptavidin-HRP incubation for 1 h. Sections were examined with DAB staining, counterstained with Mayer’s hematoxylin, mounted, and evaluated under a light microscope. Apoptotic activity was assessed by counting 1000 cells in 20 areas (10 from the cortex and 10 from the medulla) at 200× magnification.
Statistical analysis
Data were analyzed using IBM SPSS Stastistics version 22.0. Results were expressed as mean ± standard deviation. The normality of data distribution was assessed using the Shapiro-Wilk test. For multiple comparisons between groups, One-Way ANOVA followed by Tukey’s post-hoc test was performed for normally distributed data. In cases where data did not meet normality assumptions, the Kruskal-Wallis test followed by Dunn’s post-hoc test was applied. p-Values <0.05 were considered statistically significant.
Results
Postprandial blood glucose levels
Postprandial blood glucose levels were significantly lower in the control group compared to the diabetes, diabetes + 50 mg/kg PCA, and diabetes + 100 mg/kg PCA groups (p = 0.001). Although mean blood glucose levels in both PCA-treated groups (50 and 100 mg/kg) were numerically lower than those in the untreated diabetes group, this reduction did not reach statistical significance (p > 0.05). No significant difference was observed between the two PCA-treated doses (p > 0.05; Figure 1(a)).
(a) Postprandial blood glucose levels (mg/dL) across experimental groups. Different letters (a, b) above the bars indicate statistical significance (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05). (b) Perls’ Prussian blue staining of the cortex and medulla in different experimental groups. Iron deposits are visualized in blue (arrowhead). (c) Periodic Acid Schiff staining of the cortex and medulla in different experimental groups. Black arrows indicate degenerated tubules showing loss of brush border and epithelial shedding.
Biochemicals findings
Serum biochemical analysis revealed that fasting glucose (GLUC) and creatinine (CREJ) levels were significantly lower in the control group compared to all other experimental groups (p < 0.05) (Figure 2(a)). Although the highest CREJ and GLUC levels were numerically recorded in the untreated diabetes group, PCA administration did not result in a statistically significant reduction in these parameters compared to the diabetes group (p > 0.05; Figure 2(b)). Regarding blood urea nitrogen (BUN) levels, no statistically significant differences were observed between any of the experimental groups (p > 0.05; Figure 2(c)).
(a) Comparison of CREJ analysis results between experimental groups. Different letters (a, b) above the bars indicate statistical significance (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05). (b) Comparison of GLUC analysis results between experimental groups. Different letters (a, b) above the bars indicate statistical significance (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05). (c) Comparison of BUN analysis results between experimental groups. Different letters (a, b) above the bars indicate statistical significance (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05).
Histopathological findings
Perl’s Prussian Blue staining
In the histopathological examination, no iron accumulation was found in the tubules of the cortex and medulla in the control and diabetes + 100 mg/kg PCA groups. Intense iron accumulation was observed in the diabetes group, while the diabetes + 50 mg/kg PCA group showed less iron accumulation than the diabetes group. No iron accumulation was detected in the glomeruli of any group (Figure 1(b)).
PAS staining
Histopathological analysis demonstrated that tubular dilatation, intracellular vacuolization, the number of sclerotic glomeruli, and mesangial matrix expansion differed significantly among the groups (p < 0.05). These alterations were most prominent in the untreated diabetes group. In contrast, parameters such as tubular atrophy, hyaline deposition, and mononuclear cell infiltration showed no statistically significant differences (Table 1 and Figure 1(c); p > 0.05). Morphometric measurements indicated that the diabetes group had the highest mean thicknesses for both tubular and glomerular basement membranes, while Bowman’s space was widest in the diabetes + 50 mg/kg PCA group and narrowest in the diabetes group. Although numerical variations were observed across all morphometric parameters, including the visceral and parietal layers of Bowman’s capsule, none reached statistical significance (p > 0.05).
TD, TA, ICV, HA, MCI, SGC, and MM increase (med (min:max)) in the experimental groups.
TD: tubular dilatation; TA: tubular atrophy; ICV: intracellular vacuolization; HA: hyaline accumulation; MCI: mononuclear cell infiltration; SGC: sclerotic glomerulus count; MM: mesangial matrix; PCA: protocatechuic acid.
Letters in the same column with different letters indicate statistical difference.
Immunohistochemical findings
Bax immunohistochemical staining
The positive staining rate for Bax in the kidney cortex and medulla was highest in the diabetes group and lowest in the control group. The staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were lower than in the diabetes group (p < 0.001; Figure 3(a) and (e)).
(a) Immunohistochemical localization of Bax in the cortex and medulla. (b) Immunohistochemical staining for Bcl-2 in the cortex and medulla across different groups. (c) Immunohistochemical staining for Caspase-3 in the cortex and medulla across different groups. (d) Immunohistochemical staining for Caspase-9 in the cortex and medulla across different groups. Positively immunolabelled cells are visualized in red. Scale bar = 50 μm. (e)–(h) Percentage of positive staining in the cortex and medulla for Bax, Bcl-2, Caspase-3, and Caspase-9, respectively. Different letters (a, b, c, d) above the bars indicate statistically significant differences between experimental groups (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05).
Bcl-2 immunohistochemical staining
A study was conducted in which kidney sections from the cortex and medulla, immunohistochemically stained with Bcl-2 antibody, were analyzed. The positive staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were higher than those in the diabetes group (p < 0.001; Figure 3(b) and (f)).
Caspase-3 immunohistochemical staining
Caspase-3 immunohistochemical staining analysis of kidney sections revealed the highest positive staining rate in the diabetes group. The staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were lower than in the diabetes group (p < 0.05) (Figure 3(c) and (g)).
Caspase-9 immunohistochemical staining
In kidney sections stained with caspase-9 antibodies, the control group showed the lowest positive staining, while the diabetes group had the highest (p < 0.001). The positive staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were lower than in the diabetes group (Figure 3(d) and (h)).
TfR-1 immunohistochemical staining
In kidney sections stained with TfR-1 antibody, the diabetes group showed the highest immunopositive staining, and the control group had the lowest. The staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were lower than in the diabetes group (p < 0.05; Figure 4(a) and (e)).
(a) Immunohistochemical localization of TfR-1 in the cortex and medulla. (b) Immunohistochemical detection of ACSL4 in the cortex and medulla across experimental groups. (c) Immunohistochemical detection of GPx4 in the cortex and medulla across experimental groups. Positively stained cells are visualized in red. (d) TUNEL assay showing apoptotic cells in the cortex and medulla across experimental groups. Positively stained nuclei are visualized in brown. Scale bar = 50 μm. (e)–(h) Percentage of positively stained cells in the cortex and medulla for TfR-1, ACSL4, GPx4, and TUNEL, respectively. Different letters (a, b, c, d) above the bars indicate statistically significant differences between experimental groups (p < 0.05). Groups sharing at least one common letter do not differ significantly from each other (p > 0.05).
ACSL4 immunohistochemical staining
Analysis of ACSL4 immunohistochemical staining in kidney sections showed the highest immunopositive staining in the diabetes group, with the control group having the lowest. The staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were lower than in the diabetes group (p < 0.05; Figure 4(b) and (f)).
GPx4 immunohistochemical staining
Immunohistochemical staining with the GPx4 antibody showed the highest immunopositive staining in the control group, while the diabetes group had the lowest. The staining rates in the diabetes + 50 mg/kg PCA and diabetes + 100 mg/kg PCA groups were higher than in the diabetes group (p < 0.001; Figure 4(c) and (g)).
TUNEL staining
TUNEL staining revealed the highest positive staining in the diabetes group and the lowest in the control group. The diabetes + 100 mg/kg PCA group showed fewer positively stained cells compared to the diabetes group (p < 0.05; Figure 4(d) and (h)).
Discussion
It is estimated that around 80% of end-stage renal failure worldwide is caused by diabetes or hypertension. 23 DN treatments mainly focus on blocking the renin-angiotensin system (RAS) and controlling blood glucose, using ACE inhibitors, angiotensin receptor blockers, and newer agents like SGLT2 inhibitors and GLP-1 agonists. 24 Phytotherapeutic agents are also gaining interest due to their synergistic effects and fewer side effects. 25 Hyperglycaemia, caused by insulin insufficiency, is a key symptom of T1DM. Studies show that PCA, alone or combined with oral hypoglycemic drugs, can reduce blood glucose in rats. 26 Our findings showed that while 50 and 100 mg/kg PCA doses led to numerically lower blood glucose levels compared to the untreated diabetes group, these differences did not reach statistical significance (p > 0.05). This suggests that the observed protective effects of PCA on renal tissues in our study may be driven by direct cytoprotective mechanisms rather than a primary systemic hypoglycemic effect.
Measuring serum BUN and creatinine levels is a standard method for evaluating renal function and assessing various renal diseases. 27 Increased BUN and creatinine levels are commonly observed in various kidney diseases or damage. One study reported elevated creatinine and BUN levels in diabetic rats compared to control animals, 28 while another concluded that there was no significant difference in these levels between the control and diabetes groups. 29 Other studies have shown a significant increase in BUN levels in animals with STZ-induced diabetes, while the increase in creatinine levels was less pronounced. 30 In our study, although BUN and creatinine levels were numerically higher in the diabetes group, PCA administration at both 50 and 100 mg/kg doses did not result in a statistically significant reduction in these parameters (p > 0.05). This lack of functional improvement in the serum suggests that PCA may not fully reverse the decline in glomerular filtration rate within a 12-week timeframe, likely due to the persistence of high blood glucose levels and established microvascular damage. The lack of statistically significant improvement in serum BUN and creatinine levels, despite profound histopathological and molecular recovery, highlights a common chronological disconnect in chronic diabetic complications. While a 12-week experimental duration is highly sufficient to demonstrate the modulation of acute cellular stress, lipid peroxidation, and early-stage executioner proteins (such as caspases and GPx4), it is often insufficient to functionally reverse established microvascular damage and macro-structural glomerular filtration barriers under the pressure of unmitigated systemic hyperglycemia. Serum functional markers typically exhibit late-stage alterations, shifting significantly only after structural nephron loss crosses a critical functional threshold. Therefore, the absence of immediate systemic biochemical recovery does not preclude the potent, localized cytoprotective effects of PCA. Instead, it suggests that PCA operates primarily as a targeted tissue-stabilizing agent that interrupts the structural degenerative cascade (e.g., mesangial matrix expansion and tubular shedding), effectively delaying the progression of cellular injury before severe, irreversible functional collapse takes place. This pathofunctional perspective is strongly substantiated by our immunohistochemical findings, which clearly demonstrate that localized cellular protection can be successfully achieved even before being manifested in peripheral blood parameters.
Both T1DM and T2DM can cause end-stage renal disease, requiring costly treatments like dialysis and transplantation, and contributing to mortality from DN. 31 DN pathogenesis involves various mechanisms, including genetic predisposition, hyperglycaemia-induced polyol pathway and RAS activation, ROS production, protein kinase activation, AGE accumulation, and glomerular hyperfiltration. 32 Rat models exhibit histological changes similar to humans, such as lipofuscin and glycogen accumulation, tubular degeneration, thickening of basement membranes, interstitial inflammation, glomerulosclerosis, and mesangial matrix expansion. 33 Although increases in kidney weight and tubular length are typically observed within 3 months of diabetes induction in rats, simultaneous morphological changes in both glomeruli and tubules are less commonly reported. While no marked changes in glomeruli were detected 6 weeks post-diabetes 34 and thickened glomerular basement membranes after 8 months. 35 In this study, thickening of tubular and glomerular basement membranes and Bowman’s capsule layers was observed in the diabetic group, without statistically significant differences. Bowman’s space width remained unchanged. Based on these findings, the kidneys were classified as stage 2 nephropathy, likely due to the 12-week study duration. Tubular degeneration and mesangial expansion were more prominent in the diabetes group. These findings suggest that PCA may attenuate mesangial matrix expansion by downregulating extracellular matrix protein expression, while exerting protective effects on tubular epithelium through its antioxidant activity.
Apoptosis is programmed cell death, characterized by distinct morphological features and energy-dependent biochemical mechanisms. 36 The two main apoptotic pathways are the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway. The intrinsic apoptosis pathway induces apoptosis by directly activating caspase-3 or cleaving Bid, leading to mitochondrial dysfunction and subsequent release of cytochrome c, as well as the activation of caspase-9 and caspase-3. The BCL-2 protein family plays a crucial role in activating the intrinsic pathway. The BCL-2 family includes pro-apoptotic proteins (Bax, Bak, Bad, Bim) that trigger apoptosis and anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w) that inhibit apoptosis. 37 However, certain conditions can limit the reliability of TUNEL staining. Increased DNA repair in cells may lead to false TUNEL positivity, whereas necrosis and autolysis can contribute to false-positive results. Therefore, the specificity, sensitivity, and evaluation of the TUNEL technique remain controversial. 38 Interestingly, recent studies have suggested that the TUNEL staining method can identify cells undergoing ferroptosis.39–41 It has been demonstrated that lipid peroxidation causes membrane rupture in mice with ischaemia and reperfusion-induced renal damage. Consequently, markers such as ACSL4, GPx4, 4-HNE, and MDA can be used in conjunction with TUNEL staining to more accurately detect ferroptotic stress. 42 Furthermore, TUNEL-positive cells have been observed in $Gpx4$-knockout-mediated ferroptosis, highlighting its potential as a marker for this process. 43 Similarly, in mice with doxorubicin-induced cardiotoxicity, cells undergoing ferroptosis as a result of GPx4 suppression were found to be TUNEL positive. 44
In rats and mice with streptozotocin-induced DN, TUNEL-positive cells were exclusively observed in renal tubules. 45 Additionally, TUNEL-positive cells were detected in the renal glomerulus and tubules of diabetic rats. 46 ROS caused by hyperglycaemia damages glomeruli and podocytes in the kidney, triggering apoptosis. 5 A study found that high glucose concentrations in murine tubular epithelial cell lines decreased Bcl-2 expression and induced apoptosis by increasing Bax expression. 47 ROS produced resulting from high glucose in the kidneys of diabetic rats increased the expression of caspase-3, -8, and -9, which led to cell apoptosis. 48
In this study, the highest number of TUNEL-positive cells in both the cortex and medulla was observed in the diabetes group. In contrast, among the treated groups, the lowest number of TUNEL-positive cells was recorded in the diabetes + 100 mg/kg PCA group. Immunohistochemical analysis further supported these findings: Bcl-2 immunopositivity was lowest in the diabetes group, while Bax, caspase-3, and caspase-9 levels were significantly elevated. Conversely, the diabetes + 100 mg/kg PCA group showed the most significant improvement among the treatment groups, exhibiting higher Bcl-2 and lower Bax, caspase-3, and caspase-9 immunopositivity compared to the untreated diabetes group. These findings indicate that PCA enhanced the expression of the anti-apoptotic protein Bcl-2 while reducing the expression of pro-apoptotic proteins (Bax, caspase-3, and caspase-9). Consequently, PCA administration decreased the number of TUNEL-positive cells and mitigated apoptosis in renal tissues.
Recent studies highlight ferroptosis, an iron-dependent cell death process. 49 Iron along with TFR-1, ACSL4, and GPx4 are primary regulators of ferroptosis.12–14 TFR-1 facilitates Fe3+-transport into cells. An overaccumulation of Fe3+, coupled with decreased outflow, leads to a surplus of free Fe2+, which reacts with H2O2 to generate ROS via the Fenton reaction. ROS-induced peroxidation of PUFAs (Polyunsaturated Fatty Acids) catalyzed by ACSL4. Esterified PUFAs are transferred to membrane phospholipids by acyltransferase 3 (LPCAT3) and oxidized by LOXs (Lipoxygenases) to form toxic lipid peroxides, which cause membrane damage and initiate ferroptosis. 50 GPx4 prevents membrane damage by neutralizing lipid peroxides into non-toxic alcohols, thereby shielding cells from ROS-induced oxidative stress. 51 Excessive lipid peroxidation depletes GPx4 and causes ferroptosis. In our study, we observed significantly higher TFR-1 and ACSL4 immunopositivity in the diabetes group compared to the control, while GPx4 expression was significantly lower. PCA treatment attenuated the expression of pro-ferroptotic markers (TFR-1 and ACSL4) and restored GPx4 levels in a dose-dependent manner. These findings indicate that PCA administration mitigates ferroptotic stress in renal tissues,
Despite the significant molecular and histopathological findings, our study has some limitations that warrant acknowledgment. First, while the 12-week experimental duration was highly optimal for capturing the active phase of regulated cell death pathways, it represents a chronological limitation in terms of capturing long-term translational shifts in systemic renal function markers such as serum BUN and creatinine. Future longitudinal studies extending beyond this timeframe are necessary to determine whether localized cellular preservation eventually translates into peripheral biochemical recovery. Furthermore, evaluating more sensitive translational indices, such as the urinary albumin-to-creatinine ratio (ACR) or creatinine clearance rates, would provide a more comprehensive overview of early-stage glomerular filtration dynamics. Second, while our immunohistochemical analysis provided strong, spatial evidence of protein localization and intensity, further validation via Western Blotting or quantitative mRNA expression studies could offer a more precise, absolute quantification of the apoptotic and ferroptotic signaling pathways. Additionally, histological evaluations using Perls’ Prussian blue and PAS staining were primarily qualitative or semi-quantitative; although these are standard gold methodologies for assessing structural tissue alterations, they lack the pixel-level precision of automated digital morphometric analysis, which might have masked very subtle inter-group differences. Lastly, while the STZ-induced rat model effectively mimics the early phase of DN, additional investigations utilizing alternative models are required to thoroughly confirm the broader translational and clinical potential of PCA. In conclusion, our findings suggest that PCA administration in diabetic rats effectively mitigates renal cellular damage by concurrently modulating both apoptotic and ferroptotic signaling pathways. This cytoprotective action is substantiated by a significant reduction in TUNEL-positive cells and the favorable downstream regulation of Bax, Caspase-3, and Caspase-9 expression. Furthermore, PCA treatment successfully attenuates the expression of pro-ferroptotic orchestrators, including TFR-1 and ACSL4, while restoring the primary antioxidant defense enzyme GPx4, thereby highlighting its targeted tissue-stabilizing value within the diabetic renal microenvironment. Although these profound molecular and histopathological improvements were not concurrently reflected in conventional systemic renal function markers (BUN and creatinine) within this specific study window, PCA demonstrates compelling promise as a direct, glucose-independent therapeutic candidate for targeting regulated cell death cascades in DN. Future longitudinal research is warranted to fully elucidate the deeper molecular mechanisms of PCA and to evaluate whether this localized structural preservation ultimately translates into long-term systemic functional recovery in chronic diabetic kidney disease.
Footnotes
Acknowledgements
The authors acknowledge the use of artificial intelligence (ChatGPT, OpenAI) for assistance in proofreading the English language. The final responsibility for the content rests solely with the authors. This manuscript represents part of a thesis submitted by the first author to the Department of Veterinary Pathology, Lisanustu Egitim Enstitusu, Ondokuz Mayis University, in fulfillment of the requirements for a PhD degree.
Author note
The article has not been presented elsewhere or reviewed by any other journal. All the authors are aware of and approve the manuscript as submitted to this journal.
Ethical considerations
Ethical approval for the study was obtained from the Ondokuz Mayıs University Animal Experiments Local Ethics Committee (OMU HADYEK), dated May 26, 2022 (Approval No. 2022/16).
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Ondokuz Mayıs University with the project number PYO.VET.1904.22.016.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.