Abstract
Background/Aim: A variety of actions implicated in diabetic nephropathy (DN) are attributed to inflammatory cytokines and apoptosis of tubular epithelial cells. Building on our previous research demonstrating the role of different phosphodiesterase inhibitors (PDEIs) in improving renal microcirculation, glucose lowering, and antioxidant effects in rats with DN, this study aims to further explore the anti-inflammatory and anti-apoptotic properties of PDEIs by measuring their effects on renal expression of pro-inflammatory cytokines in streptozocin (STZ)-induced diabetic nephropathic rats.
Materials and Methods: Out of 50 adult male Sprague-Dawley rats, diabetes was induced in 40 rats by a single injection of STZ (45 mg/kg) dissolved in citrate buffer. Ten days after induction of diabetes, rats were divided into five groups (10/group): normal control, diabetic control, and 3 diabetic groups treated with pentoxifylline, sildenafil, and milrinone via drinking water for 15 successive days. Serum and kidney tissue samples were collected to evaluate the effect of treatment with PDEIs on diabetes-induced histopathological changes and expression levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), apoptotic marker Bcl-2 Associated X-protein (Bax) and anti-apoptotic marker B cell lymphoma-2 (Bcl-2) in rat’s kidneys.
Results: A significant increase in pro-inflammatory cytokines (TNF-α and IL-6), and apoptosis marker (Bax), with a concomitant decrease in anti-apoptotic protein (Bcl-2) were observed in diabetic rats. Treatment with PDEIs resulted in a significant decrease in renal expression of Bax, TNF-α, and IL-6, with an increase Bcl-2 expression, with slight, though not statistically significant, differences among the PDEI-treated groups.
Conclusion: The tested PDEIs, pentoxifylline, sildenafil, and milrinone, exhibit significant anti-inflammatory and anti-apoptotic effects, highlighting their potential in slowing the progression of diabetic nephropathy.
Introduction
Approximately 20-40% of diabetic patients have elevated urinary albumin levels and/or a low glomerular filtration rate (GFR), key characteristics of diabetic kidney disease (DKD), also known as diabetic nephropathy (DN). DN has become the main cause of chronic dialysis among all kidney diseases (1); it is recognized as an inflammatory disease, with various pathogenic mechanisms that involve inflammatory cytokines. Hence, modulation of inflammatory processes represents a key tool for the prevention and treatment of DN (2). One of the major features of DN is apoptosis of renal tubular epithelial cells (3). Previous studies have shown that hyperglycemia induces cell shrinkage, chromatin condensation and DNA fragmentation, characteristics of apoptosis, especially in the proximal convoluted tubular epithelial cells (4, 5).
Phosphodiesterases (PDEs) are a superfamily family of enzymes that catalyze hydrolysis of cyclic nucleotides, including the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), into 5′-AMP and 5′-GMP respectively. Thus, the inhibition of PDEs preserves intracellular levels of cAMP and cGMP, thereby enhancing their ability to activate downstream signaling cascades involved in various physiological processes, including inflammation, apoptosis, and vascular function (6). There are 11 gene-related families of PDEs (PDE-1 to PDE-11), of which PDE-4, PDE-7 and PDE-8 are specific for degradation of cAMP, while PDE-5, PDE-6 and PDE-9 are specific for cGMP degradation and others hydrolyze both cAMP and cGMP (7). Based on the specific PDE enzyme they target, there are 11 types of PDE inhibitors, among them the most widely used are four: Phosphodiesterase type 5 inhibitors (PDE-5 inhibitors), include sildenafil, tadalafil, vardenafil, and avanafil. Phosphodiesterase type 4 inhibitors (PDE-4 inhibitors), include roflumilast, apremilast, and crisaborole. Phosphodiesterase type 3 inhibitors (PDE-3 inhibitors), include cilostazol, dipyridamole, milrinone and amrinone. Non-selective PDE inhibitors (block multiple types of PDE enzymes) include theophylline, pentoxifylline, aminophylline, and methylxanthine (8).
A previous study by Kumar et al. in 2000 documented that PDEs play a role in inflammatory responses in animals and inhibition of these enzymes can therefore elicit anti-inflammatory potential (9). Precisely, the anti-inflammatory effect of PDEIs has been known since the beginning of 1970s (10). Complementary to our previous publication (11) that highlighted the role of PDEIs in reducing the progression of DN via improving renal microcirculation, lowering blood glucose, and suppressing oxidative stress, the current study aimed to explore the additional mechanisms explaining the renoprotective effect of different PDEIs against STZ-induced DN by investigating the renal expressions of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), the pro-apoptotic marker Bcl-2 Associated X-protein (Bax), the anti-apoptotic marker B cell lymphoma-2 (Bcl-2) in diabetic rats.
Materials and Methods
After approval by the Ethical Review Committee of Damietta Faculty of Medicine, Al-Azhar University, Damietta, Egypt (IRB 00012399, July 13, 2024), and in accordance with the guidelines and recommendations of the Institutional Animal Care and Use Committee (IACUC), animal experiments were performed.
Streptozocin and enzymes were obtained from Sigma Aldrich Company, Burlington, MA, USA, Pentoxifylline from Hiral Labs Ltd, Uttarakhand, India, Sildenafil from Bayer Pharma AG, Leverkusen, NRW, Germany, and Milrinone from Eli Lilly and Company, Indianapolis, IN, USA.
Experimental animals. Healthy adult male Sprague Dawleyrats (n=50, aged 9-12 weeks, weighting 150-200 g) were used in this study. The animals were housed in clean polypropylene cages (5 rats/cage) and maintained at standard conditions of temperature (22±2°C), relative humidity (30%-50% RH) and light (12 h light/12 h dark cycle). They had free access to water and standard rat chow throughout the experiment. Experimental procedures were performed in the Animal House, Faculty of Medicine, Damietta, Al-Azhar University, Egypt.
Induction of diabetes and animal grouping. After two-week adaptation period, experimental diabetes was induced in 40 rats after 16 h fast by a single intraperitoneal (i.p.) injection of STZ (45 mg/kg bodyweight) dissolved in citrate buffer (0.1 M, pH 4.5) (12). Blood samples were obtained from rat lateral tail veins 36 h after STZ injection, and the fasting blood glucose (FBG) levels were measured by a glucose strip test in a glucometer (Easy Gluco Blood Glucose Monitoring system, Infopia, Republic of Korea). Rats with FBG ≥200 mg/dl from at least 3 samplings were considered as diabetic. After induction of diabetes, rats were divided into five groups as follows: Group 1: Normal Control, 10 non-diabetic rats received citrate buffer and normal drinking water. Group 2: Diabetic Control, 10 diabetic rats received normal drinking water. Group 3: Pentoxifylline treated, 10 diabetic rats treated with pentoxifylline (30 mg/kg/day) (13). Group 4: Sildenafil treated, 10 diabetic rats, treated with sildenafil (1 mg/kg/day) (14). Group 5: Milrinone treated, 10 diabetic rats, treated with milrinone (3.17 mg/kg/day) (15). Drugs were given daily via drinking water (depending upon water consumption) as detailed in our previous publication (11) for 15 successive days (Figure 1).
Graphical study design. STZ: Streptozocin; PDEIs: phosphodiesterase inhibitors.
Kidney sampling for measurement of renal expression of TNF-α and IL6. At the end of the experimental period, rats were sacrificed under anesthesia, dissected and the kidneys were excised. The kidney sample was prepared according to the method of Ashour et al. (16), where 0.5 g of renal tissue from each animal were homogenized in 5 ml 0.9% NaCl, the obtained homogenates were centrifuged at 3000 rpm for 10 min. The homogenate supernatants were frozen at −30 °C and subsequently used to determine the renal expression of pro-inflammatory cytokines (IL-6 and TNF-α) using rat IL6 ELISA kit (Catalog no. ZKP1598; Suzhou Zeke Biotechnology Co., Ltd., Jiangsu, PR China) and rat TNF-α ELISA kit (Catalog no. ZKP1500; Suzhou Zeke Biotechnology Co., Ltd.), respectively.
Detection of Bax and Bcl-2 genes expression in rat kidneys. After homogenizing kidney tissues, RN easy Plus Mini kit was used for RNA extraction. Then the extracted RNA was transcribed into cDNA using a Script TM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was applied using an Applied Biosystems 7500 Instrument (Applied Biosystems, Foster, CA, USA) to estimate the relative expression of pro-apoptotic marker (Bax) and anti-apoptotic marker (Bcl-2) genes using (Power SYBR® Green). B-actin was applied as a housekeeping gene.
Histopathological and immunohistochemical (IHC) investigation. For detection of histopathological changes, kidney tissues were fixed in neutral buffered formalin, then sent to the Department of Pathology, Faculty of Medicine, Al-Azhar University for blocking, sectioning, and staining with Hematoxylin and Eosin (H & E) according to the method of Banchroft et al. (17). Immunohistochemical detection of Bax and Bcl-2 proteins in rat kidney tissues was performed by first fixing the samples in 10% neutral buffered formalin, followed by paraffin embedding and sectioning at 3-5 μm thickness. The tissue sections were deparaffinized using xylene, rehydrated through a graded ethanol series, and subjected to antigen retrieval in citric acid buffer (pH 6.0) using microwave heating. Endogenous peroxidase activity was blocked with 2% hydrogen peroxide in methanol, and non-specific binding was minimized using normal serum or bovine serum albumin (BSA). Sections were incubated for one hour at room temperature with primary antibodies specific for Bax (rabbit polyclonal, Santa Cruz N20) and Bcl-2 (mouse monoclonal, Dako ab-124), followed by biotinylated secondary antibodies and a streptavidin-biotin-peroxidase complex. Visualization was achieved using 3,3′-diaminobenzidine (DAB) as the chromogen, and the slides were counterstained with hematoxylin and mounted using DPX. Appropriate positive and negative controls were included to ensure the specificity and reliability of the staining (18).
Statistical analysis. The results were analyzed for statistical significance by Statistical Packages for Social Science (SPSS) software version 21.0 (IBM Corp., Armonk, NY, USA). Quantitative data were expressed as means±SD of 10 animals in each group. One-way analysis of variance (ANOVA) was used when comparing between more than two means and post hoc test (Tukey’s test) was used for multiple comparisons between different variables. Differences between groups were considered significant at p≤0.05.
Results
Effect of PDEIs on renal expression of pro-inflammatory cytokines in diabetic rats. As shown in Table I, rats in the diabetic control group had significantly higher concentrations of IL6 and TNF-α in their kidneys compared to normal rats (p<0.05). Treatment with the studied PDEIs ameliorated such effects, with no significant differences between treated groups.
Effect of phosphodiesterase inhibitors (PDEIs) on renal expression of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL- 6) in diabetic rats.
Effect of PDEIs on renal expression of apoptotic/anti-apoptotic markers in diabetic rats. Table II shows the effect of treatment with PDEIs on Bax and Bcl-2 genes expression levels in kidney tissue of diabetic rats. The levels of Bax mRNA expression were significantly increased (p<0.05), while the Bcl-2 level was significantly decreased (p<0.05) in tissues from diabetic rats compared to the non-diabetic controls. Treatment with pentoxifylline, sildenafil or milrinone revealed anti-apoptotic effects via increasing the Bcl-2 levels and restoring the Bax levels towards the control levels. There were no statistically significant differences among the three drugs. These changes suggest that PDEIs may mitigate renal apoptosis by modulating the intrinsic apoptotic pathway.
Effects of phosphodiesterase inhibitors on renal relative mRNA expression of Bcl-2 associated X-protein (Bax) and B-cell lymphoma 2 (Bcl-2) in diabetic rats.
Morphological changes of kidney tissues. Histological examination revealed distinct renal changes across experimental groups. In non-diabetic control rats, kidney architecture remained intact, with normal glomeruli, tubules, and vasculature (Figure 2A). In contrast, diabetic rats showed marked pathological alterations, such as extensive interstitial fibrosis, glomerular congestion, and acute tubular degeneration (Figure 2B). Treatment with pentoxifylline significantly ameliorated these changes, restoring renal structure to near-normal levels (Figure 2C). However, sildenafil-treated diabetic rats displayed persistent vascular congestion (Figure 2D), while milrinone treatment was associated with focal necrosis and notable infiltration of inflammatory cells (Figure 2E).
Morphological alterations in kidney tissues after induction of DN and treatment with PDEIs by Hematoxylin and Eosin staining. Representative microscopic images are shown for each group: Non-diabetic control rats show normal renal parenchyma (normal glomerulus, tubules, and blood vessels) (A); diabetic rats exhibit histopathological changes (severe interstitial fibrosis, marked glomerular congestion and diffuse sever tubular degeneration) (B); diabetic rats treated with pentoxifylline demonstrate normal histological structure (C); those treated with sildenafil display congested blood vessels (D); diabetic rats treated with milrinone show focal necrosis and inflammatory cell infiltration. Red arrows indicate cell infiltration and black arrows show the areas of focal necrosis (E). Magnification 400×.
Discussion
Diabetic nephropathy, the leading cause of end-stage renal failure, affects approximately 30-40% of people with type 2 diabetes mellitus and 15-25% of people with type 1 diabetes mellitus (2). In a previous study (11), we identified some of the underlying mechanisms by which PDEIs reduce the progression of DN, and we conducted this study to explore other mechanisms of this improving effect.
The present study demonstrated that rats with STZ-induced DN exhibited higher renal expressions of the pro-inflammatory cytokines, TNF-α and IL-6, and the apoptotic marker Bax, as well as decreased expression of the anti-apoptotic marker Bcl-2 (detected by immunohistochemical technique) compared to the normal control rats. The pro-inflammatory cytokines (TNF-α and IL-6) are key mediators in the pathogenesis of DN, contributing to glomerular and tubular injury, fibrosis, and progressive renal dysfunction (2).
The above results are consistent with a study by Javier et al. (19), which demonstrated an increase in mRNA levels of IL-6 and TNF-α in the renal cortex of diabetic nephropathic rats compared to the non-diabetic rats. In that study, the levels of IL-6 and TNF-α increased steadily as DN progressed. Sha et al. (20) also reported such increases in renal IL-6 and TNF-α levels in diabetic rats compared to normal rats. Another study conducted by Pradeep and Srinivasan (21) showed an increase in the protein levels of the pro-apoptotic regulator Bax and a decrease in the levels of the anti-apoptotic marker Bcl-2 in kidneys of STZ-induced diabetic rats. Sha et al. (20) also elucidated that apoptosis of tubular epithelial cells may contribute to the development of DN in diabetic rats.
In the present study, treatment of diabetic rats with pentoxifylline, a non-selective PDE inhibitor, the PDE-5 inhibitor sildenafil, or the PDE-3 inhibitor milrinone significantly decreased the renal expression of pro-inflammatory cytokines (TNF-α and IL-6) without significant differences between the three groups, reflecting almost equal anti-inflammatory activities of these three different subfamilies of PDE inhibitors. These anti-inflammatory activities were demonstrated by Torphy et al. (22) for non-selective PDE inhibitors and by Sekut et al. (23) for PDE-4 inhibitors in acute and chronic models of inflammation. Nunes et al. also demonstrated that sildenafil (a PDE-5 inhibitor) exerts a potent anti-inflammatory effect by significantly reducing the levels of TNF-α, IFN-γ, IL-1β, IL-2, IL-6, and cycloxygenase-2 (COX-2) (24).
In addition to the anti-inflammatory effect of PDEI treatment, we observed a significant decrease in Bax gene expression levels along with significant increase in Bcl-2 gene expression in renal tissues from the PDEIs-treated rats compared to the diabetic control. This finding demonstrated the high efficiency of PDEIs in reducing apoptosis. Similar anti-apoptotic effects were demonstrated in a study by Puzzo et al. (25), specifically on PDE-5 inhibitors, where sildenafil partially restored the anti-apoptotic molecule Bcl-2 and normalized the Bax/Bcl-2 ratio in aged rats by modulating the cAMP/cGMP signaling pathways. Park et al. (26) also reported that PDE-5 inhibitors preserve mitochondrial function and prevent neuronal apoptosis by activating the cGMP/PKG/CREB pathway and increasing neurotrophic factors such as BDNF and NGF.
Many other studies have reported that PDE-4 inhibitors resist neuronal apoptosis via increasing the Bcl-2/Bax ratio, which improves cognitive function and reduces depressive behaviors in Alzheimer’s model rats (27, 28). In another study by Parkkonen et al. (29), PDE-4 inhibitors were found to delay spontaneous apoptosis of eosinophil and neutrophil and increase their survival in vitro.
As regard the non-selective PDE inhibitors, our results are in line with those of Wang et al. (30) who observed that long term pentoxifylline treatment reverses endothelial cell apoptosis in a dose-dependent manner in patients with DKD. Hamouda et al. (31) also reported high efficacy of pentoxifylline in treating and preventing non-alcoholic steatohepatitis (NASH) and attributed this to different mechanisms including downregulation of apoptotic mediators that induce apoptosis and necroptosis pathways.
The histopathological evaluation of renal tissues provides critical insights into the progression of DN and the therapeutic efficacy of PDEIs. The findings revealed marked distortion of kidney tissues with significantly increased apoptosis due to diabetes. Treatment with pentoxifylline appeared to preserve normal renal histology, with minimal or no signs of fibrosis or tubular damage, well-maintained glomerular and vascular structures. This suggests a strong renoprotective effect, likely due to pentoxifylline’s anti-inflammatory and anti-fibrotic properties, possibly mediated through TNF-α inhibition and improved microcirculation. Although sildenafil-treated rats showed some improvement, congested blood vessels were still evident. This may indicate partial amelioration of vascular dysfunction, incomplete protection against DN-induced vascular changes. Sildenafil’s vasodilatory effects via the NO-cGMP pathway may help improve perfusion, but its impact on structural preservation appears limited compared to pentoxifylline. Unfortunately, focal necrosis and inflammatory cell infiltration persisted in diabetic rats treated with milrinone despite its inotropic and vasodilatory properties, indicating persistent or exacerbated renal injury or possible inflammatory or cytotoxic effects at the administered dose or duration.
The findings of the present study highlight the multifactorial protective role of PDEIs in STZ-induced diabetic nephropathy. As illustrated in Figure 3, PDEIs exert their renoprotective effects through several key mechanisms, including hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic actions, along with improvement of renal microcirculation. These combined effects contribute to the preservation of renal structure and function, underscoring the therapeutic potential of PDEIs in managing diabetic kidney disease.
Scheme summarizing how treatment with PDEIs reduces the progression of STZ-induced diabetic nephropathy. PDEIs: Phosphodiesterase inhibitors; STZ: streptozocin.
Despite the promising findings, this study has several limitations that should be acknowledged. First, the treatment duration was relatively short (15 days), which may not fully capture the long-term effects or safety profiles of the tested PDEIs. Future studies should extend the treatment period to evaluate sustained efficacy and potential adverse outcomes. Second, drug administration via drinking water may introduce variability in dosing due to differences in individual water intake, potentially affecting the consistency of results. Overall, future research should aim to address these limitations by incorporating longer treatment durations, standardized drug delivery methods, functional renal assessments, and molecular analyses to better understand the therapeutic potential and mechanisms of PDEIs in diabetic nephropathy.
Conclusion
This study highlights the anti-inflammatory and anti-apoptotic properties of PDEIs, which significantly contribute to the improvement of diabetes-induced renal histological changes. Combined with their previously reported hypoglycemic and antioxidant properties, as well as their efficacy in enhancing renal microcirculation, these findings underscore the multifaceted therapeutic potential of PDEIs in attenuating the progression of diabetic nephropathy.
Acknowledgements
The Authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research at Taif University, Kingdom of Saudi Arabia, for funding this work.
Footnotes
Authors’ Contributions
OMM, AE, BHE, and MGMH contributed to the conceptualization and design of the study, generated, curated, and analyzed the data. WMSA, ASAE, MAMA, and AMH interpreted the results, wrote the original draft and the final manuscript. UBE, NMG, ME, AIS and MMK edited and revised the manuscript. All Authors revised and approved the final version submitted for publication.
Conflicts of Interest
There are no conflicts of interest as declared by the Authors.
Funding
This work was funded by Deanship of Graduate Studies and Scientific Research at Taif University, Kingdom of Saudi Arabia.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received June 8, 2025.
- Revision received September 30, 2025.
- Accepted October 16, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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