Mentha-mediated selenium nanoparticles attenuate adriamycin-induced chronic kidney disease via multi-target modulation of oxidative, inflammatory, and fibrotic pathways

Highlights

• •Mentha-mediated SeNPs protect against Adriamycin-induced chronic kidney injury via multi-target molecular modulation.
• •Green-synthesized SeNPs restore renal function and preserve kidney histoarchitecture in CKD rats.
• •SeNPs reduce oxidative stress and suppress apoptosis by inhibiting Bax and p53 signaling pathways.
• •Anti-inflammatory and anti-fibrotic actions of SeNPs involve downregulation of TNF-α, TGF-β, Collagen, and α-SMA.
• •Biosynthesized SeNPs represent a promising nanotherapeutic strategy for chronic kidney disease management.

Abstract

Background

Chronic kidney disease (CKD) is characterized by progressive renal dysfunction associated with oxidative stress, inflammation, apoptosis, podocyte injury, and fibrotic remodeling. Adriamycin (ADR)-induced nephropathy is a widely used experimental model that reproduces key features of CKD, including proteinuria, glomerular and tubular injury, and renal fibrosis. Green-synthesized selenium nanoparticles (SeNPs) have emerged as potential renoprotective agents owing to their antioxidant activity and favorable biocompatibility.

Objective

This study investigated the renoprotective efficacy of Mentha-mediated biosynthesized SeNPs against ADR-induced CKD in rats and explored the underlying molecular mechanisms.

Methods

SeNPs were biosynthesized using sodium selenite and Mentha extract as reducing and capping agent. The synthesized nanoparticles were characterized by UV–visible spectroscopy, transmission electron microscopy (TEM), zeta size and zeta potential analysis, X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). Eighteen male Sprague–Dawley rats were allocated into control, ADR-induced CKD, and ADR + SeNPs groups. At the end of the experimental period, renal function markers, oxidative stress parameters, proteinuria, histopathological alterations, alpha smooth muscle actin (α-SMA) immunoreactivity, and the mRNA expression of apoptotic, inflammatory, and fibrotic genes were assessed.

Results

Characterization confirmed the formation of predominantly spherical SeNPs with a primary particle size of approximately 45–50 nm, a crystalline trigonal selenium structure, moderate colloidal stability, and surface functional groups consistent with biological capping. ADR administration caused significant renal dysfunction, increased urinary protein-to-creatinine ratio (UPCR), decreased renal Wilms tumor 1 (WT1) protein concentration, increased malondialdehyde (MDA), reduced superoxide dismutase (SOD) activity, severe histopathological damage. ADR also increased collagen deposition and α-SMA immunoreactivity and upregulation of Bax, p53, TNF-α, TGF-β, and Collagen-1 genes. SeNPs treatment improved serum renal-function parameters, significantly reduced UPCR, increased renal WT1 concentration, restored SOD activity, decreased MDA levels, and restored renal histoarchitecture. SeNPs also reduced α-SMA immunoreactivity, attenuated fibrotic alterations, and downregulated pro-apoptotic, pro-inflammatory, and pro-fibrotic gene expressions.

Conclusion

Mentha-derived SeNPs effectively attenuated ADR-induced CKD through coordinated antioxidant, anti-inflammatory, anti-apoptotic, and anti-fibrotic actions. These findings highlight the therapeutic potential of green-synthesized SeNPs as a promising nano-based therapeutic approach for chronic renal injury.

1. Introduction

Chronic kidney disease (CKD) is a progressive condition marked by functional or structural abnormalities of the kidney that persist for >3 months and have implications for health. Functional abnormalities include a reduced glomerular filtration rate (GFR), blood electrolyte disturbances and loss of red blood cells or albumin into the urine (haematuria or albuminuria, respectively); structural abnormalities include interstitial fibrosis and glomerulosclerosis [1]. The global burden of CKD is substantial and growing. Its prevalence is estimated to be approximately 850 million cases and because of kidney failure there are about 4 million patients needing kidney replacement therapy. By 2050, CKD is projected to become the fifth leading underlying cause of death worldwide [2]. Oxidative stress and inflammation are closely interconnected processes that contribute significantly to the development and progression of CKD. Excessive production of reactive oxygen species, together with impaired antioxidant defenses, causes persistent damage to renal cells and tissues. At the same time, chronic inflammation sustains and aggravates renal injury. Together, these mutually reinforcing processes promote progressive deterioration of kidney function and contribute to CKD-related complications [3,4]

Adriamycin (doxorubicin), an anthracycline antibiotic, is one of the most effective chemotherapeutic agents in the clinic. It is used in the treatment of various solid tumors such as liver cancer, neurofibromatosis and breast cancer [[5], [6], [7]]. However, there are restrictions on its use because of its toxicity in multiple organs and tissues [8]. Adriamycin (ADR)-induced nephropathy model in mice and rats is widely utilized. When administered at specific doses, ADR induces kidney damage closely resembling the histologic and clinical manifestations of CKD. It has the ability to replicate numerous pathophysiologic features of human podocytopathy and the progression to CKD. These features include glomerulosclerosis, proteinuria, tubulointerstitial fibrosis and podocyte injury [9,10]. The quinone structure of ADR undergoes redox cycling, generating excessive reactive oxygen species (ROS) that overwhelm cellular antioxidant systems, such as glutathione and superoxide dismutase (SOD), depleting them. ROS also induce DNA damage, oxidative modification of proteins, and lipid peroxidation collectively leading to cellular dysfunction [11,12].

Selenium (Se) is a vital micronutrient that has antioxidant and anti-inflammatory properties and plays a vital role in detoxifying and enhancing the body's immunity [13]. The nanoform of selenium attracts even more attention, thanks to its high bioavailability and lower toxicity than inorganic and organic forms [[14], [15], [16]]. Selenium nanoparticles (SeNPs), in which selenium is in zero oxidation state(Se⁰) present the advantage of lower toxicity and excellent bioavailability compared to other oxidation states (Se^+IV, Se^+VI) [15,17]. SeNPs have found extensive applicability in several medicinal fields, such as combating fungal and bacterial infections, treating cancer, managing diabetes, boosting the immune system, and reducing inflammation [18].

Originally, SeNPs were synthesized by various chemical methods like other nanoparticles. However, the presence of toxic by-products and the high cost of production led to the development of novel methods of nanoparticles synthesis [19]. Biological organisms such as plants, fungi, or bacteria have the ability to convert some toxic metal ions to less toxic forms including metal precipitants or nanoparticles [[20], [21], [22]]. Many investigators focused on their use for synthesizing nanoparticles with an ecofriendly approach [23,24]. Because plant-mediated synthesis does not require specialized conditions and is relatively inexpensive, it offers several advantages over other biological methods [25].

Although several studies [[26], [27], [28]] have reported the nephroprotective potential of SeNPs synthesized using various plant extracts, Mentha-mediated SeNPs possess distinctive features that justify their specific investigation in the context of CKD. Unlike SeNPs produced from other plant sources, Mentha-derived SeNPs exhibit unique surface chemistry characterized by amide bands, carboxylate, and hydroxyl functional groups originating from Mentha-specific peptides, proteins, and polyphenolic compounds—which form a protective biocorona around the nanoparticles. This distinctive capping profile imparts not only enhanced colloidal properties but also additional bioactivity that complements the intrinsic antioxidant function of SeNPs. Furthermore, Mentha species are particularly rich in bioactive flavonoids and phenolic acids with well-documented intrinsic nephroprotective properties, having been shown to protect against oxidative renal damage, restore antioxidant enzyme activities, and suppress inflammation. Network pharmacology analyses have identified key biotargets of Mentha including TNF, CASP3, and MAPK14 are directly implicated in apoptosis, inflammation, and kidney disease progression [29] . Beyond these scientific considerations, Mentha offers practical advantages including wide cultivation, year-round availability, cost-effectiveness, and an established safety profile in traditional medicine. Collectively, the unique physicochemical characteristics, synergistic bioactivity, intrinsic nephroprotective properties, and practical availability of Mentha provide a compelling rationale for its selection as a reducing and capping agent over other medicinal plants and support the enhanced therapeutic potential of Mentha-mediated SeNPs against ADR-induced CKD.

Therefore, the present study aimed to evaluate the renoprotective efficacy of Mentha-mediated biosynthesized SeNPs against ADR-induced chronic kidney injury in rats.

2. Materials and methods

2.1. Chemicals

Sodium selenite was obtained from Sigma Aldrich, USA. All the reagents and other chemicals were of high analytical grade.

2.1.1. Biosynthesis of SeNPs using crude Mentha extract

SeNPs were biologically synthesized using sodium selenite and Mentha extract was used as reducing and capping agent. SeNPs were synthesized by carefully adding 0.0526 g of pentahydrate Na₂SeO₃ powder to 100 ml of Mentha extract to make a final concentration of 2.0 mM. The reaction mixture was adjusted to obtain the best yield at 30 °C, pH = 7.2 after salt addition with shaking at 120 rpm in the dark to prevent photooxidation for 48 h until the color change occurred from blue to red- orange. Sodium selenite solution (2 mM) without Mentha extract was also kept under identical conditions as a control. The prepared nanoparticles were immediately used after the preparation process with no evidence of agglomeration. The synthesized SeNPs were then collected by centrifugation at 12,000 rpm for 20 min. The pellet of SeNPs obtained was washed twice with double distilled water and acetone to remove unreduced salts and unused capping agents. Afterward, the washed SeNPs were dried overnight at room temperature to obtain purified SeNPs for further characterization.

2.2. Characterizations of SeNPs

2.2.1. UV- visible spectroscopy

The formation of SeNPs was measured using a spectrophotometer (200–1100 nm range). A blank containing only the solvent was used for baseline correction. The characteristic absorption peak of SeNPs was recorded using a UV–visible spectrophotometer (Unico 2100, USA).

2.2.2. Transmission electron microscopy (TEM)

The synthesized SeNPs were dispersed and deposited onto a carbon-film-supported copper grid. The samples were examined using field high-resolution transmission electron microscopy (JEOLH-7650, Hitachi High-Technologies Corp., Tokyo, Japan).

2.2.3. Zeta size and potential of synthesized SeNPs

Freeze-dried nanosuspension was characterized for size, and zeta potential by dynamic light scattering using Zetasizer Nano, Malvern, UK.

2.2.4. X-ray diffraction (XRD) analysis

XRD analysis of the prepared SeNPs was carried out at room temperature with an Empyrean diffractometer by analytical (Almelo, The Netherlands), using Cu X-ray tube operated at 30 mA and 45 kV, and using a Ni filter to eliminate Kβ radiation and a Pixcel3D detector. The diffraction patterns were collected with the step scanning mode in the 2θ range 5°–80° with step size of 0.03° and counting time of 20 s/step.

Particle size can be also estimated using Scherrer equation:

$$D=\frac{k\mathit{\lambda }}{\beta Cos\theta }$$

Where D is the average crystallite size (in nm),

k is the Scherrer constant (= 0.9),

λ is the X-ray wavelength (0.15,406 nm for a standard Cu-Kα source),

β is the Full Width at Half Maximum (FWHM) of the diffraction peak at the Bragg angle in radians.

2.2.5. Fourier transform infrared spectroscopy (FTIR)

The FTIR spectrum of the biosynthesized SeNPs was recorded over the wavenumber range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ using a recording spectrometer (type Bruker VERTEX 80v, Germany), The molecules in the sample absorb specific wavelengths of infrared radiation, which corresponds to the vibrational frequencies of bonds in the molecules. The peaks in the IR spectrum represent specific functional groups or molecular bonds within the sample.

2.3. Experimental animals

A total of 18 adult male Sprague Dawley rats weighing 220–280 g were supplied by Medical Experimental Research Center (MERC) at the Faculty of Medicine, Mansoura University, Egypt. The animals were housed under standard laboratory conditions in well-ventilated cage and were provided with a balanced diet and water ad libitum. Throughout the period of experiment, they were caged in an environment of 12- hour light/dark cycle and 25±2 °C air temperature. Rats were acclimatized for 10 days before the start of the experiment. Animal treatments in this study were performed in compliance with the guidelines for the care and use of laboratory animals and approved by Mansoura University, animal care committee (Code number: SC.PhD.23.06.11).

2.4. Experimental design

After a 10-day acclimatization period, rats were allocated to experimental groups using stratified randomization based on body weight, ensuring comparable mean body weights across all groups (n = 6 per group) as follows: I) Control group: animals were injected into the tail vein with 0.9% saline; II) ADR group: CKD was induced by tail vein injection of ADR (4 mg/kg) twice at a 14-day interval [30]; III) ADR + SeNPs group: CKD was induced as ADR group, and 5 days after injection of the first ADR dose, rats received SeNPs (0.5 mg/kg) [31] orally twice a week. At the end of the 6-week experimental period, rats were anesthetized using an intraperitoneal injection of sodium thiopental (50 mg/kg) prior to euthanasia and blood samples and tissue collection.

2.5. Collection of blood samples

The blood samples were collected in Serum Separator Tubes and allowed to clot for 30 min at room temperature, followed by centrifugation at 1000 g for 10 min. The serum was transferred to a clean Eppendorf tube and stored at – 20 °C for subsequent biochemical analysis parameters.

2.6. Kidney samples collection

The kidney was immediately dissected out and washed in sterile ice-cold saline and blotted between 3 pieces of filter paper. A small section was stored immediately in liquid nitrogen for gene expression. The second part was stored at – 20 °C for oxidative stress and antioxidant markers. The third part of the kidney was fixed in 10% neutral-buffered formalin for histopathological evaluation and immunohistochemical analysis.

2.7. Preparation of homogenate

Fifty mg of kidney tissue was homogenized in 1 ml of ice-cold 0.9% saline for 1 min using a homogenizer (HGD-10 homogenizer, version 03/10, KOREA). The homogenate was centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was used for measuring activity of SOD and malondialdehyde (MDA).

2.8. Histopathological and immunohistochemical (IHC) investigations

Formalin-fixed kidney tissues were paraffin-embedded, sectioned (5 µm), and stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), Masson’s trichrome (MT), and picrosirius red following the standard protocols. Depending on H&E stain the tubulointerstitial injury level was evaluated by grading the tubular dilatation, brush border loss, and epithelial simplification. According to the extent of cortical involvement, the degree of damage between none (normal), <25, 25–50, 50–75, and >75% were assigned as 0, 1, 2, 3, and 4 points, respectively [32]. Tubulointerstitial injury was scored semiquantitatively on PAS-stained tissues, with a 20x objective. Specifically, tubulointerstitial injury was defined as tubular dilation, tubular atrophy, sloughing of tubular epithelial cells, or thickening of the tubular basement membrane and was scored on a scale of 0 to 4, as follows: 0, no tubulointerstitial injury; 1, <25% of tubulointerstitial injured; 2, 25 to 50% of the tubulointerstitial injured; 3, 51 to 75% of the tubulointerstitial injured; 4, >75% of the tubulointerstitial injured [33]. At least 10 randomly non-overlapping cortical fields of each section were selected and evaluated in a blind manner. Finally, the mean score of each tissue was calculated as the final tubulointerstitial injury score. The collagen deposition was assessed by Masson’s trichrome and Picrosirius Red staining. Immunohistochemical detection of alpha smooth muscle actin α-SMA was performed using 3,3′-diaminobenzidine (DAB) chromogenic detection. Microscopic examination and image capture were performed using a light microscope fitted with a digital camera (OPTIKA, Italy), and images were analyzed using ImageJ software.

2.9. Assessment of albumin and renal functions

Serum albumin, creatinine and urea levels were assessed spectrophotometrically using commercially available kits from spectrum diagnostic (Cairo, Egypt).

2.10. Assessment of superoxide dismutase activity

The enzymatic activity of SOD in the kidney tissue homogenates was evaluated spectrophotometrically according to the manual method described by Nishikimi et al. [34]. This reaction depends on the ability of the enzyme to block the phenazine methosulphate-mediated reduction of nitro blue tetrazolium dye. The inhibition of NBT reduction was measured spectrophotometrically at 560 nm. The results were expressed as Units per milligram of tissue protein (U/mg protein) which assessed using Bicinchoninic acid (BCA) kit.

2.11. Quantification of malondialdehyde (MDA) levels

To evaluate the extent of lipid peroxidation, MDA levels in kidney tissue homogenates were measured by a method described previously by Ohkawa et al. (1979). Proteins were precipitated using trichloroacetic acid (TCA) then thiobarbituric acid (TBA) reacts with MDA in acidic medium at 94 °C for 30 min to yield thiobarbituric acid reactive product which is pink and measured at 532 nm.

2.12. Assessment of urinary protein-to-creatinine ratio (UPCR)

Twenty-four-hour urine samples were collected individually from each rat. Urinary protein and creatinine concentrations were measured in the same sample using commercial colorimetric kits. The (UPCR) was calculated as urinary protein concentration (mg/dL) divided by urinary creatinine concentration (mg/dL) and expressed as mg protein/mg creatinine.

2.13. ELISA assessment of wilms tumer 1(WT1)

Renal WT1 protein concentration was quantified in rat kidney tissue homogenates using a Rat Wilms tumor protein homolog (WT1) ELISA kit (Cat. No. RTEB0708). All procedures were carried out in line with the manufacturer's instructions (; Assay Genie, 25 Windsor Place, Dublin 2, Ireland, D02 VY42).

2.14. Gene expression analysis

Total RNA was isolated from the homogenized kidney tissues using TRIZOL reagent (Applied Biotechnology) following the manufacturer's standard protocol. RNA concentration and purity (A260/A280 ratio) were evaluated using a NanoDrop spectrophotometer. Subsequently, equivalent amounts of extracted RNA were reverse-transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biotechnology). Specific primers for Bax, p53, TNF-α, and Collagen, TGF-β genes and the housekeeping gene GAPDH were designed by using Primer3.

Real-time PCR was performed using a SYBR Green PCR Master Mix to quantify the mRNA expression levels of targeted apoptotic (Bax, p53), inflammatory (TNF-α), and fibrotic (Collagen I, TGF-β) genes. The primer sequences were F:5′- AGA CAG CCG CAT CTT GT −3′, R: 5′- TTC CCA TTC TCA GCC TTG AC −3′; F: 5′- ACG TCC TGG TGA AGT TGG TC −3′, R: 5′- CAG GGA AGC CTC TTT CTC CT −3′; F: 5′- CAC TCC CGT GGC TTC TAG TG −3′, R: 5′- GGA CTG GCG AGC CTT AGT TT −3′; F: 5′- GGC GAT GAA CTG GAC AAC AA −3′, R: 5′- CAA AGT AGA AAA GGG CAA CC −3′; F: 5′- CAT AGT GTG CCC TAT GAG −3′, R: 5′- CAA AGC TGT TCC GTC CCA GTA GA −3′;F:5′-TCTTCAAGGGACAAGGCTGC-3′, R:5′- CTTGATGGCAGAGAGGAGGC-3′ for GAPDH, Collagen I, TGF-β, Bax, p53 and TNF-α genes respectively. The relative fold change in gene expression for each sample was calculated utilizing the 2^-ΔΔct method, with GAPDH employed as the internal housekeeping gene for normalization.

2.15. Statistical analysis

Data are expressed as mean ± SD. One-way ANOVA was selected to compare differences among multiple independent groups, followed by Tukey's post hoc test for pairwise comparisons. Renal tubulointerstitial scores were analyzed as ordinal variables using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Statistical analysis was performed using GraphPad Prism 8, with p value < 0.05 was considered statistically significant.

3. Results

A strong absorption band was observed in the ultraviolet region of the UV–Vis spectrum of the synthesized SeNPs, together with two less intense peaks located at approximately 270 nm and 400 nm (Fig. S1). The intense absorption in the UV region corresponds to the fundamental band gap of selenium, confirming its semiconducting nature. Critically, the distinct peak at approximately 270 nm is the surface plasmon resonance (SPR), which serves as primary evidence for the successful formation of nanoparticles, indicating their small size and narrow size distribution. The additional small peak at around 400 nm is typically attributed to the presence of organic capping agents used to stabilize the colloidal suspension, though it can also signal the initial stages of particle aggregation. The peak at 400 nm, in particular, might also indicate the presence of larger particles or aggregates. Additionally, the spectrum provides evidence of the successful biological synthesis approach. Overall, this spectrum confirms the nano-scale nature of the selenium, provides insight into its size uniformity, and reveals the active surface chemistry.

Fig. S2 shows the TEM micrograph, revealing that the nanoparticles are predominantly spherical in morphology and exhibit a narrow size distribution, with diameters ranging between 45 and 50 nm (Fig. S2C). The corresponding Selected Area Electron Diffraction (SAED) pattern, shown in (Fig. S2B), consists of a series of distinct, well-arranged bright spots. This specific pattern confirms the high crystallinity and single-crystalline nature of the individual nanoparticles, as the spots can be indexed to specific lattice planes of the material's crystal structure.

The presented zeta size and zeta potential data (Fig. S3A, B) reveal critical information about the colloidal state and stability of the nanoparticle suspension. The zeta size distribution, showing two intense peaks at approximately 50 nm and 480 nm, indicates a bimodal population. The peak at 50 nm likely corresponds to the primary, individual nanoparticles, which align well with the particle size observed in TEM analysis. However, the prominent second peak at 480 nm strongly suggests a significant degree of aggregation or agglomeration within the sample, where individual nanoparticles have clustered together to form larger secondary structures. This is a common challenge in nanoparticle suspensions, often driven by high surface energy. The zeta potential results, displaying two overlapped peaks centered at −18 mV and −25 mV, provide further insight into the system's stability and complexity. The negative values confirm that the particle surfaces possess a negative charge, which is beneficial as like charges cause electrostatic repulsion between particles, hindering aggregation. However, the presence of two distinct peaks, rather than one, indicates there are two populations of particles with different surface charge densities. This can be directly correlated with the two size populations: the more negative peak (−25 mV) probably belongs to the well-dispersed 50 nm primary particles, which have a high surface charge ensuring good stability, while the less negative peak (−18 mV) likely corresponds to the larger 480 nm aggregates. The reduced zeta potential magnitude in the aggregates suggests that the aggregation process may have shielded some of the surface charges or that the larger particles have different electrostatic properties. While a zeta potential of −18 mV to −25 mV indicates a moderately stable suspension, the coexistence of these two populations and the presence of aggregates signal a metastable system that may be prone to further settling or coagulation over time, which is a crucial consideration for any practical application.

Fig. S4 reveals XRD pattern of the SeNPs. The observed XRD diffraction pattern, with its sharp peaks at specific 2θ values, provides definitive evidence for the successful biosynthesis of highly crystalline SeNPs. The presence of these distinct peaks itself confirms that the product is not amorphous but possesses a well-ordered, crystalline structure, a result facilitated by biosynthesis. Crucially, the perfect alignment of these peaks at 23.1°, 29.2°, 41.5°, 43.2°, 45.3°, and 51.8° with the Miller indices (100, 101, 110, 012, 111, and 003) acts as a fingerprint, uniquely identifying the crystal phase as pure trigonal (or hexagonal) selenium, which is the most thermodynamically stable and semiconducting form of elemental selenium. The variation in peak intensities is a direct consequence of the unique helical chain structure of this trigonal phase. Furthermore, while the peaks are sharp, they exhibit slight broadening that enables estimation of the nanoscale crystallite size via the Scherrer equation. This broadening directly implies the phytochemicals from Mentha extract during biosynthesis, yielding the desired nanocrystalline material. Using the Scherrer equation with FWHM of the (101) peak at 29.2°, the estimated average crystallite size of the SeNPs is approximately 45 nanometers. This result confirms the successful formation of nanoparticles. The small size is a direct consequence of the Mentha extract that act as an effective capping agent during the biosynthesis, which controlled crystal growth and prevented the formation of larger, bulk crystals and this finding is consistent with the previously obtained results from TEM and zeta size measurements.

The FTIR vibrational spectrum provides definitive evidence for the capping and stabilizing role of phytochemical constituents of Mentha extract on the synthesized SeNPs as depicted in Fig. S5. The appearance of the characteristic amide bands—specifically the primary amide (C O stretch) at 1651 cm⁻¹ (Amide I), the secondary amide (N—H bend) at 1559 cm⁻¹ (Amide II), and the tertiary amide (C—N stretch) at 1233 cm⁻¹ (Amide III)—collectively forms the unique fingerprint of a peptide/protein backbone. This confirms that biomolecules from Mentha extract are not merely present but are intimately associated with the nanoparticle surface. Furthermore, the additional bands offer deeper insight into the specific functional groups involved in this interaction. The symmetric stretching band of the carboxylate group (COO⁻) at 1417 cm⁻¹ suggests that acidic amino acid residuals within the plant-derived peptides or proteins are potentially coordinated to the selenium surface, acting as key anchoring points. Simultaneously, the very broad band at 3400 cm⁻¹, attributed to overlapping O—H and N—H stretching vibrations, indicates the presence of surface-adsorbed water molecules and the involvement of hydroxyl and amine containing phytochemicals such as polyphenols, flavonoids, or amino acid side chains in stabilizing the nanoparticles through hydrogen bonding.

Microscopic pictures of HE stained kidney sections (Fig. 1) showing normal glomeruli, tubules with minimal interstitial tissue in control group. In contrast kidney sections from ADR group showed a marked damage characterized by shrunken glomerular tuft with dilated Bowman's capsule (arrowhead), marked leukocyte infiltration in interstitial tissue (thick black arrow), tubular dilation (red arrow), and tubular eosinophilic cast formation (blue arrow). Conversely kidney sections from ADR + SeNPs group showing very mild tubular cast formation (thin black arrow) with few leukocyte infiltration (thick black arrow). These microscopic observations were validated by histopathological scoring. The ADR group showed a significantly higher tubulointerstitial injury score compared to the normal control (p = 0.0001). Conversely, the administration of SeNPs significantly downregulated the injury score compared to the ADR group (p = 0.0106). Also, a semiquantitative assessment of tubulointerstitial damage in PAS-stained kidney sections (Fig. S6) revealed that the ADR + SeNPs group had a significantly lower tubulointerstitial injury score than the ADR group (P = 0.0485).

Fig. 1.

Representative microscopic photomicrographs of H&E-stained kidney sections, Magnification: × 100 (scale bar = 100 μm) and × 400 (scale bar = 50 μm). Individual data are presented as dot plots with the mean ± SD of tubulointerstitial injury scores in three experimental groups (n = 6) analyzed by Kruskal–Wallis followed by Dunn's test to compare all means. Statistical significance: *** means highly significant compared to control group when P < 0.001, # means mildly significant compared to ADR group when P < 0.05 and * means mildly significant compared to control group when P < 0.05.

MT-stained kidney sections (Fig. S7) show no interstitial fibrosis in control group. However, kidney sections from ADR group show marked bluish interstitial fibrosis (thin black arrow), whereas kidney sections from ADR + SeNPs group show markedly decreased bluish interstitial fibrosis (thin black arrow). The percentage of the MT-positive fibrotic area was significantly elevated in the ADR group (p = 0.0001) compared to the normal control baseline. Conversely, the administration of SeNPs significantly suppressed the extent of fibrosis, recording a marked reduction in the fibrotic area percentage (p = 0.0006) compared to the ADR group.

Similarly, picrosirius red stained kidney sections (Fig. S8) show no interstitial fibrosis in control group but kidney sections from ADR group showing marked red stained interstitial fibrosis (thin black arrow) while sections from ADR + SeNPs group showing markedly decreased red stained interstitial fibrosis (thin black arrow).

Consistent with these microscopic observations, there was a statistically significant increase (p = 0.0003) in the proportional area of Picrosirius red-positive fibrosis in the ADR group compared to the control baseline. However, SeNPs significantly (p = 0.0008) decreased the fibrotic area percentage compared to the ADR group.

Immuno-stained kidney sections (Fig. 2) showing mild brown α-SMA-positive tubular staining in control group, while kidney sections from ADR group show strong positive brown tubular reaction for α-SMA in many tubules (thin black arrow), remarkably kidney sections from ADR + SeNPs group reveal markedly decreased positive brown reaction for α-SMA (thin black arrow) in a few tubules. Quantification of the α-SMA positive area confirmed these visual findings. The ADR group showed a massive, statistically significant elevation (p = 0.0001) in α-SMA expression compared to the control group. However, SeNPs significantly (p = 0.0007) down-regulated the α-SMA positive area percentage compared to the ADR group.

Fig. 2.

Representative microscopic photomicrographs of immunohistochemistry staining for α-SMA, Magnification: × 100 (scale bar = 100 μm) and × 400 (scale bar = 50 μm). Individual data are presented as dot plots with the mean ± SD of scores of α-SMA positive areas % by IHC in three experimental groups (n = 6) analyzed by one-way ANOVA followed by Tukey's test to compare all means. Statistical significance: *** means highly significant compared to control group when P < 0.001, ### means highly significant compared to ADR group when P < 0.001 and ** means significant compared to control group when P < 0.

The administration of ADR induced significant renal impairment, as evidenced by a marked disruption in serum biochemical profiles. Compared with control group, ADR-treated rats exhibited a significant reduction in albumin levels (p = 0.0021, Fig. 3A), coupled with a sharp elevation in serum urea (p = 0.0020, Fig. 3B) and creatinine concentrations (p = 0.0341, Fig. 3C). These changes indicate severe glomerular and tubular damage characteristic of nephrotoxicity. However, co-administration of SeNPs effectively mitigated this renal damage. The SeNPs-treated group showed a significant restoration of albumin levels (p = 0.0232) and a substantial decline in nitrogenous waste markers; urea (p = 0.0021) and creatinine (p = 0.0027), bringing them closer to the control values. The biochemical improvements were closely associated with the modulation of the redox status in kidney tissue. Compared to control group, ADR induced oxidative stress, significantly increasing MDA levels—a key indicator of lipid peroxidation (p = 0.0018, Fig. 3D) while simultaneously suppressing the activity of the antioxidant enzyme SOD (p = 0.0458 Fig. 3E). Notably, treatment with SeNPs significantly suppressed MDA production (P = 0.0244) and restore SOD activity (P = 0.0466) compared to the ADR group.

Fig. 3.

Effect of SeNPs on renal function and oxidative stress markers in ADR-induced nephrotoxicity; (A–C) Serum biochemical analysis showing albumin, urea, and creatinine levels across Control, ADR, and SeNPs groups, (D–E) Assessment of oxidative status via MDA concentration and SOD activity. Individual data are presented as dot plots with the mean ± SD (n = 6). Statistical significance: *p < 0.05 and **p < 0.01).

Fig. 4 showed that ADR administration significantly increased the UPCR compared with the control group (p = 0.0100), confirming the development of marked proteinuria and impairment of the glomerular filtration barrier. Treatment with Mentha-mediated SeNPs significantly reduced UPCR compared with the untreated ADR group (p = 0.0239), indicating attenuation of ADR-induced urinary-protein loss.

Fig. 4.

Urinary protein-to-creatinine ratio in experimental groups. The urinary protein-to-creatinine ratio (UPCR) was determined in the Control, Adriamycin-treated (ADR), and ADR+SeNPs groups and expressed as milligrams of urinary protein per milligram of urinary creatinine. Individual data are presented as dot plots with the mean ± SD. *p < 0.05.

Consistent with these functional findings, renal WT1 protein concentration was significantly reduced in the ADR group compared with the control group (p = 0.0001, Fig. S9), supporting ADR-associated podocyte injury. SeNP treatment significantly increased WT1 concentration relative to the ADR group (p = 0.017), suggesting partial preservation or restoration of the podocyte-associated WT1 protein level.

To investigate the molecular mechanisms underlying the nephroprotective effects of SeNPs, the mRNA expression levels of key pathological markers were quantified via RT-qPCR. ADR administration up regulated the pro-apoptotic markers Bax (p = 0.1227, Fig. 5A) and p53 (p = 0.0394, Fig. 5B) compared to the control group, indicating the induction of programmed cell death. Furthermore, ADR challenge triggered a robust inflammatory and fibrotic response, as evidenced by a significant increase in the fold change of Collagen-1 (p = 0.0117), TNF-α (p = 0.0217) and TGF-β (p = 0.0171) mRNA levels (Figs. 5C-E). Notably, co-treatment with SeNPs effectively attenuated these molecular alterations. SeNPs administration resulted in a dramatic downregulation of Bax (p = 0.0017), p53 (p = 0.0179), and TNF-α (p = 0.0051) expression. Additionally, a significant reduction in Collagen mRNA expression (p = 0.0038) was observed in the SeNPs-treated group, although the decline in TGF-β expression did not reach statistical significance (ns) relative to the ADR group.

Fig. 5.

Impact of SeNPs on the mRNA expression of apoptotic, fibrotic, and inflammatory markers in ADR-induced nephrotoxicity. Relative gene expression (Fold change normalized to GAPDH) of (A) Bax, (B) p53, (C) Collagen, (D) TNF-α, and (E) TGF-β. Individual data are presented as dot plots with the mean ± SD (n = 6). Statistical significance: *p < 0.05, **p < 0.01, and ns (non-significant).

4. Discussion

The formation of SeNPs can be checked via UV/vis spectral analysis, which was carried out in wavelength range 200–1100 nm showing exhibited absorption maxima at a wavelength around 388 nm which is the characteristic absorption of SeNPs as reported [35,36]

The data from the TEM analysis provides critical insights into the fundamental characteristics of the synthesized SeNPs. The TEM micrograph itself offers direct visual evidence of the particles' physical form, confirming a successful synthesis that yielded predominantly spherical nanoparticles with a highly uniform size distribution, as their diameters are consistently confined to a narrow range of 45–50 nm. This morphological control is significant for ensuring predictable physical and chemical behavior. More profoundly, the accompanying Selected Area Electron Diffraction (SAED) pattern, which displays a series of distinct and well-arranged bright spots, serves as a definitive diagnostic tool for determining the nanoparticles' internal structure [37]. Unlike the diffuse rings that would be characteristic of polycrystalline or amorphous materials, this specific pattern of sharp spots irrefutably confirms that each individual nanoparticle is a high-quality single crystal. This means the atomic lattice within each nanosphere is a continuous, unbroken, and perfectly ordered structure. The presence of this single crystallinity is a crucial finding, as it implies superior electronic and structural properties, which are essential for high-performance applications in areas such as catalysis, optoelectronics, and as functional nanofillers in composite materials.

The zeta size and zeta potential analyses for SeNPs collectively suggest that the nanoparticle formulation exhibits a heterogeneous colloidal system characterized by two distinct size populations. The primary particles (∼50 nm) appear well-dispersed, whereas the second peak (∼480 nm) indicates the formation of secondary aggregates. This bimodal distribution reflects a partial loss of colloidal uniformity that is associated with elevated surface energy in nanoparticulate systems. Furthermore, the zeta potential values ranging from –18 to –25 mV denote only moderate electrostatic stabilization. The more negative potential likely corresponds to the primary nanoparticles, while the less negative values are attributable to the aggregated fraction, implying partial surface-charge shielding during agglomeration. Taken together, these findings indicate that the system is metastable, with a measurable tendency toward further aggregation or sedimentation, which should be considered when interpreting the material’s behavior in biological or applied settings [38,39].

The XRD pattern results provide clear confirmation of the successful formation of highly crystalline SeNPs. The presence of sharp diffraction peaks at 2θ values of 23.1°, 29.2°, 41.5°, 43.2°, 45.3°, and 51.8°, which correspond precisely to the Miller indices (100), (101), (110), (012), (111), and (003), respectively, verifies that the synthesized material adopts the trigonal crystalline phase of selenium, the most thermodynamically stable form. The well-defined nature of these peaks indicates a high degree of structural order, while their slight broadening reflects the nanoscale crystallite size. This nanoscale dimension is consistent with TEM observations and zeta size measurements and underscores the effective role of phytochemicals from Mentha extract as capping agents that limited crystal growth and prevented particle agglomeration [40,41].

The FTIR spectrum provides strong evidence for the successful capping and stabilization of the SeNPs by phytochemical constituents of Mentha extract. Moreover, it illustrates a multi-faceted stabilization mechanism where the protein wraps around the SeNPs, utilizing its hydrophobic peptide backbone and hydrophilic functional groups (like carboxylate and hydroxyl) to form a protective biocorona, effectively preventing aggregation and ensuring the colloidal stability of the synthesized SeNPs in an aqueous environment [42].

While several studies have reported the nephroprotective effects of SeNPs synthesized using various plant extracts, Mentha-mediated SeNPs possess distinctive features that warrant specific consideration. Unlike SeNPs produced using other plant sources, Mentha-derived SeNPs exhibit a unique capping profile characterized by amide bands (Amide I at 1651 cm⁻¹, Amide II at 1559 cm⁻¹, and Amide III at 1233 cm⁻¹) along with carboxylate and hydroxyl functional groups, reflecting the presence of Mentha-specific peptides, proteins, and polyphenolic compounds on the nanoparticle surface. Comparative studies have demonstrated that Mentha-derived SeNPs often exhibit superior physicochemical properties, including smaller particle size and higher purity, compared to SeNPs synthesized using other plant extracts. Furthermore, the specific phytochemical composition of Mentha, particularly its rich flavonoid and phenolic acid content may confer synergistic therapeutic effects beyond the antioxidant activity of SeNPs alone. This combination of intrinsic nephroprotective activity, unique surface chemistry, and synergistic phytochemical interactions distinguishes Mentha-mediated SeNPs from previously reported SeNPs formulations and supports their enhanced therapeutic potential against CKD [[26], [27], [28]].

The present study demonstrated that Mentha-mediated biosynthesized SeNPs attenuated ADR-induced chronic renal injury, as evidenced by increased serum albumin, reduced serum urea and creatinine, decreased renal MDA levels, restoration of SOD activity, improvement of renal histoarchitecture, and suppression of apoptotic, inflammatory, and fibrotic markers. In addition to these biochemical changes, the UPCR, renal WT1 protein concentration, and histopathological findings provided complementary functional, molecular, and structural evidence of glomerular, podocyte-associated, tubular, and tubulointerstitial injury. The marked increase in UPCR together with the reduction in renal WT1 observed in the ADR group is consistent with podocyte-associated injury and impairment of the glomerular filtration barrier. Recent evidence has similarly demonstrated that podocyte dysfunction contributes directly to the progression of ADR-induced glomerular injury [43] . In parallel, renal WT1 protein concentration was markedly reduced following ADR administration and was significantly increased after SeNPs treatment. WT1 is strongly expressed in differentiated glomerular podocytes, and reduced WT1-positive podocyte number or expression has been repeatedly demonstrated in ADR nephropathy in association with proteinuria, nephrin loss, foot-process abnormalities, and progressive glomerulosclerosis [30,44,45]. Although WT1 was measured in whole-kidney homogenates by ELISA rather than by glomerular immunohistochemical counting, the combined reduction in renal WT1 and elevation of UPCR provide supportive evidence of podocyte-associated injury, whereas improvement of both parameters following SeNPs treatment suggests preservation of the podocyte phenotype and glomerular filtration-barrier integrity.

These functional and molecular findings were strongly supported by the renal histopathological observations. The PAS-detected tubular abnormalities observed in the ADR group, including tubular dilation, epithelial simplification, brush-border loss, and intratubular proteinaceous casts, may also represent an important component of subsequent tubulointerstitial fibrotic remodeling. Persistent tubular epithelial injury can promote chronic inflammation, cellular senescence, and activation of interstitial fibroblasts. This interpretation is supported by recent evidence demonstrating that tubular epithelial cell senescence can aggravate CKD through enhanced profibrotic signaling and paracrine fibroblast activation [46]. The prominent interstitial leukocytic infiltration observed in H&E-stained ADR kidneys further supports the coexistence of tubular injury and an inflammatory tubulointerstitial response, whereas the reduction in inflammatory infiltration and tubular abnormalities following SeNPs administration is consistent with attenuation of this injury process. Taken together, the concordance among increased UPCR, reduced renal WT1, H&E-detected inflammatory and tubular injury, and PAS-detected glomerular and tubular abnormalities strengthens the evidence that ADR produced substantial podocyte-associated, filtration-barrier, and tubulointerstitial damage. Conversely, the simultaneous improvement of these parameters following SeNP treatment supports a renoprotective effect extending beyond serum biochemical changes and involving preservation of both glomerular and tubulointerstitial compartments

At the molecular level, the increased expression of Bax, p53, TNF-α, collagen, and TGF-β in the ADR group further supports the contribution of oxidative stress-associated apoptosis, inflammatory activation, and extracellular-matrix deposition to ADR-induced renal damage, consistent with evidence that renal reactive oxygen species accumulation can activate TNF-α/NF-κB-related inflammatory signaling, enhance Bax-dependent mitochondrial apoptosis, and stimulate TGF-β-mediated fibrotic remodeling [47]. The restoration of SOD activity and reduction in MDA following SeNPs treatment may be associated with enhancement of endogenous antioxidant defenses, particularly the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Activation of Nrf2 promotes the transcription of antioxidant and cytoprotective genes, including HO-1, and restoration of Nrf2/Kelch-like ECH-associated protein 1 (Keap1)/HO-1 signaling has been associated with improved antioxidant status and renal function in experimental ADR-induced renal injury [48]. Plant-mediated SeNPs have also been reported to enhance HO-1 expression, restore SOD activity, reduce MDA, and improve serum urea and creatinine in experimental nephrotoxicity [27]. Similarly, the reduction in TNF-α expression observed in the present study may be consistent with attenuation of the redox-sensitive NF-κB pathway, which contributes to inflammatory cytokine production and renal injury in ADR nephropathy [49]. The downregulation of p53 and Bax further suggests attenuation of mitochondrial apoptotic signaling and may consequently limit activation of the executioner caspase-3; however, caspase-3 cleavage or activity was not directly measured. Previous evidence has linked Nrf2/HO-1 activation with reduced Bax expression and cleaved caspase-3 in renal tissue and ADR-injured podocytes [50].

These findings are also consistent with AlBasher et al., who reported that SeNPs protected against glycerol-induced acute kidney injury through antioxidant, anti-inflammatory, and anti-apoptotic effects [51], and with Al-Brakati et al., who demonstrated that lycopene-coated SeNPs improved renal function, reduced MDA, restored antioxidant enzymes, decreased TNF-α and other inflammatory mediators, and reduced pro-apoptotic markers in renal injury [52]. Although the reduction in TGF-β expression in the SeNPs-treated group did not reach statistical significance, the marked decreases in collagen expression, α-SMA immunoreactivity, and collagen deposition demonstrated by Masson’s trichrome and Picrosirius red staining indicate attenuation of downstream fibrotic remodeling. These findings are compatible with modulation of the canonical TGF-β/Smad2/3 pathway, in which phosphorylated Smad2/3 promotes myofibroblast activation, α-SMA expression, and extracellular-matrix accumulation [48]. Nevertheless, because Nrf2 nuclear translocation, HO-1 expression, NF-κB activation, caspase-3 cleavage, and Smad2/3 phosphorylation were not directly evaluated, the involvement of these pathways should be regarded as a plausible mechanistic interpretation rather than a conclusively demonstrated mechanism. Collectively, the combined improvement in UPCR, renal WT1 protein level, PAS-detected renal injury, renal-function indices, oxidative status, inflammatory and apoptotic markers, and fibrotic remodeling provides convergent functional, structural, and molecular evidence that Mentha-mediated SeNPs treatment attenuated ADR-induced chronic renal injury.

Although the present study demonstrates that SeNPs ameliorated ADR-induced renal injury, direct translation of these findings to patients with CKD requires caution. The biological performance of nanomaterials is formulation-specific, because particle size and size distribution, morphology, surface charge, coating, aggregation, physical stability, and route of administration may influence bioavailability, biodistribution, residence time, therapeutic activity, and toxicity. Therefore, the results obtained with the SeNPs preparation used in the present study should not be generalized to other SeNPs formulations without comparable physicochemical characterization and manufacturing-quality control [53]. Animal doses should also not be transferred directly to humans using a simple mg/kg body-weight conversion. Although body-surface-area scaling may provide a preliminary human-equivalent dose, selection of a first-in-human starting dose should be based on an adequately established no-observed-adverse-effect level, systemic exposure and pharmacokinetic data, absorption, distribution, metabolism and excretion characteristics, and an appropriate safety factor. Consequently, a calculated human-equivalent dose should not be interpreted as a recommended clinical dose [54]. In rats, orally administered elemental SeNPs were absorbed, distributed, metabolized, and excreted; selenium was detected in the liver and kidneys, and higher exposure was associated with a larger non-metabolized elemental-selenium pool in these organs [55].

Moreover, although Nano-Se exhibited lower subchronic toxicity than selenite and high-selenium protein in one experimental study [56], another repeated-dose study reported reduced body-weight gain, increased relative liver weight, alterations in creatinine clearance and urinary pH, and overt toxicity at high SeNPs exposure [57]. The pharmacokinetic profile, systemic exposure, organ distribution, biotransformation, clearance, and long-term accumulation of the SeNPs formulation used in the present study were not determined and should therefore be acknowledged as limitations. Whether established CKD modifies SeNPs or selenium-metabolite clearance also remains uncertain. Accordingly, formulation-specific pharmacokinetic and biodistribution studies, repeated-dose toxicity testing, and determination of the no-observed-adverse-effect level and maximum tolerated exposure are required before clinical evaluation.

5. Conclusion

Mentha-mediated biosynthesized SeNPs attenuated ADR-induced chronic renal injury in the present experimental model. SeNPs treatment improved renal function and histoarchitecture, reduced oxidative stress, inflammation, apoptosis, α-SMA expression, and collagen deposition, and modulated selected pro-inflammatory, pro-apoptotic, and profibrotic markers. These findings support the preclinical renoprotective potential of Mentha-mediated biosynthesized SeNPs. However, formulation-specific pharmacokinetic, biodistribution, dose-equivalence, and repeated-dose toxicity studies are required before their safety, efficacy, and clinical applicability in human CKD can be established. Therefore, Mentha-derived SeNPs may represent a promising experimental nanotherapeutic candidate for further investigation in CKD rather than a currently validated strategy for clinical CKD management.

Funding sources

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Clinical trial number

Not applicable.

CRediT authorship contribution statement

Ayman E. Abdelaziz: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Ahmed A. Shokeir: Writing – review & editing, Validation, Supervision, Data curation, Conceptualization. Basem I. Awad: Writing – review & editing, Supervision, Investigation, Data curation, Conceptualization. Amira Awadalla: Writing – original draft, Validation, Methodology, Formal analysis. A.M. Abdelghany: Writing – original draft, Validation, Investigation, Formal analysis, Conceptualization. Omali Y. El-Khawaga: Writing – review & editing, Validation, Supervision, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.