Morphological and Biochemical Insights Into the Renoprotective Effects of Combined N-Acetylcysteine and Glycine Treatment in Experimental Diabetic Nephropathy.

Morphological and Biochemical Insights Into the Renoprotective Effects of Combined N ‐Acetylcysteine and Glycine Treatment in Experimental Diabetic Nephropathy - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Pharmacol Res Perspect . 2026 Jun 1;14(3):e70273. doi: 10.1002/prp2.70273 Morphological and Biochemical Insights Into the Renoprotective Effects of Combined N ‐Acetylcysteine and Glycine Treatment in Experimental Diabetic Nephropathy Malik Ejubović 1 , Andrej Belančić 2, ✉ , Yun Wah Lam 3 , Farideh Javid 4 , Dina Kapić 5 , Esad Ćosović 5 , Rijad Jahić 6 , Orhan Lepara 7 , Amira Jagodić Ejubović 1 , Lejla Alić 8 , Amil Topuz 8 , Zurifa Ajanović 9 , Almir Fajkić 10, ✉ Author information Article notes Copyright and License information 1 Department of Internal Medicine, Cantonal Hospital Zenica, Zenica, Bosnia and Herzegovina 2 Department of Basic and Clinical Pharmacology With Toxicology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia 3 Department of Diagnostic Sciences, School of Nursing and Health Sciences, Hong Kong Metropolitan University, Hong Kong, Hong Kong 4 Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Huddersfield, UK 5 Department of Histology and Embryology, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 6 University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina 7 Department of Human Physiology, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 8 Department of Medical Biochemistry, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 9 Department of Human Anatomy, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 10 Department of Pathophysiology, Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina * Correspondence: Andrej Belančić ( andrej.belancic@uniri.hr ; a.belancic93@gmail.com ), Almir Fajkić ( almir.fajkic@mf.unsa.ba ) ✉ Corresponding author. Revised 2026 Apr 26; Received 2025 Dec 10; Accepted 2026 May 12; Collection date 2026 Jun. © 2026 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMC Copyright notice PMCID: PMC13239810  PMID: 42220095 ABSTRACT Diabetic nephropathy (DN) is a major microvascular complication of diabetes mellitus and the leading cause of end‐stage renal disease. Oxidative stress and inflammation are central drivers of DN progression, yet no effective therapies exist to prevent or delay renal injury. This study investigated the renoprotective effects of glycine (GLY), N‐acetylcysteine (NAC), and their combination administered at early versus late stages of streptozotocin induced diabetes. Forty‐eight male Wistar rats ( n = 48) were allocated into five groups: healthy controls (Group 1, n = 6), untreated diabetic rats (Group 2, n = 6), and three treatment groups (Groups 3–5, each n = 12). Diabetes was induced by a single intraperitoneal streptozotocin injection (55 mg/kg). Group 3 received NAC (100 mg/kg), Group 4 received GLY (250 mg/kg), and Group 5 received NAC + GLY. Each treatment group was subdivided into early (6 week, n = 6) and late (12 week, n = 6) intervention subgroups. Treatments were administered orally. Renal tissue was evaluated using classic histology, geometric morphometric analysis, and biochemical assays of superoxide dismutase (SOD) and myeloperoxidase (MPO). Statistical analyzes were performed using ANOVA with appropriate post hoc tests ( p < 0.05). Untreated diabetic rats (Group 2) showed significantly decreased SOD activity, increased MPO levels, marked mesangial matrix expansion, glomerular hypercellularity, tubular epithelial degeneration, and interstitial inflammation with fibrosis. NAC (Group 3) and GLY (Group 4) each improved oxidative stress markers and partially restored glomerular and tubular morphology, with early treatment subgroups exhibiting more substantial benefit than late subgroups. The combined NAC + GLY therapy (Group 5) demonstrated the strongest renoprotective effect, preserving renal structure and biochemical parameters closest to healthy controls. To conclude, early combined administration of glycine and N ‐acetylcysteine yields superior protection against diabetes‐induced renal injury compared with individual treatments. These findings support the therapeutic potential of antioxidant‐amino acid combinations in preventing or delaying diabetic nephropathy. Keywords: diabetic nephropathy, glycine, N ‐acetylcysteine, oxidative stress, protective agents, renal morphology 1. Introduction Diabetic nephropathy (DN) remains the leading cause of chronic kidney disease (CKD) and end‐stage renal disease (ESRD) worldwide, despite significant advances in glycemic and blood pressure management. DN affects approximately 20%–40% of individuals with diabetes and is responsible for a substantial proportion of chronic kidney disease (CKD) and end‐stage renal disease (ESRD) cases globally, with its prevalence continuing to rise in parallel with the increasing incidence of diabetes mellitus [ 1 , 2 ]. Clinically, DN presents with persistent albuminuria, declining glomerular filtration rate (GFR), and progressive histopathological changes [ 3 ]. The pathomorphological sequence involves increased production and impaired turnover of glomerular basement membrane components, leading to mesangial matrix accumulation and glomerulosclerosis. Secondary changes include the development of tubular atrophy and interstitial fibrosis caused by degenerating tubular epithelia and local inflammatory responses [ 4 ]. These structural lesions precede functional decline, yet current standard‐of‐care treatments rarely prevent or reverse them, highlighting persistent gaps in our understanding of DN pathogenesis and treatment. Oxidative stress is a key molecular driver in diabetic nephropathy progression, where chronic hyperglycemia increases reactive oxygen species (ROS) production via mitochondrial dysfunction, NADPH oxidase, and myeloperoxidase (MPO) activity, overwhelming antioxidant defenses such as superoxide dismutase (SOD), glutathione, and catalase [ 5 , 6 ]. More recently it was discovered that oxidative stress is further increased by iron overload, through ROS production via the Fenton reaction, leading to lipid peroxidation and consequently ferroptosis, an iron‐dependent form of regulated cell death [ 7 ]. Ferroptosis has been implicated in glomerular and tubular injury as well as the progression of fibrosis in DN [ 8 , 9 , 10 ]. Thus, excess renal iron exacerbates oxidative stress that directly damages glomerular and tubular cells, while also triggering inflammatory and profibrotic cascades that accelerate structural injury [ 11 ]. Inflammation reinforces and amplifies these oxidative insults. Activation of NF‐κB signaling promotes the production of pro‐inflammatory cytokines, including TNF‐α, IL‐1β, and IL‐6, thereby leading to immune cell infiltration, fibroblast activation, and extracellular matrix deposition [ 12 ]. This establishes a self‐sustaining cycle between oxidative stress and inflammation that underlies the progression of DN [ 13 ]. Therapeutic approaches capable of targeting several pathogenic arms at the same time may therefore offer improved renal outcomes than strategies targeting single pathways. N ‐acetylcysteine (NAC), a glutathione precursor, replenishes antioxidant defenses, reduces mitochondrial oxidative injury, and has shown promise in experimental models of diabetic complications [ 14 , 15 ]. Interestingly, iron and glutathione are interconnected in maintaining the redox balance and the modulation of inflammatory processes. Under normal conditions, glutathione, by the action of an enzyme, glutathione peroxidase 4 (GPX4), prevents lipid peroxidation‐induced ferroptotic cell death. However, under hyperglycemic conditions and oxidative stress, glutathione is depleted and the activity of GPX4 is impaired, leaving the cells vulnerable to iron‐mediated ROS accumulation [ 9 ]. In addition, glycine, an amino acid with immunomodulatory and cytoprotective properties, attenuates inflammatory signaling and improves cellular redox status in various metabolic and inflammatory conditions [ 16 , 17 ]. Therefore, restoring the levels of glutathione with precursors such as NAC or glycine may preserve the function of GPX4, inhibit ferroptosis, and reduce inflammation. Despite encouraging findings with antioxidant and anti‐inflammatory therapies, significant uncertainties remain regarding the optimal timing and strategy of intervention. Whether early intervention, initiated early after disease onset, can prevent irreversible histological remodeling more effectively than delayed treatment, whether combination therapy offers additive or synergistic benefits beyond single agents, and how these approaches influence the interconnected cascade of oxidative stress, inflammation, and structural injury in DN all remain incompletely defined. This lack of clarity contributes to the substantial residual risk of progression to ESRD, reflecting the heterogeneity of DN, variable therapeutic responses across individuals, and gaps in mechanistic understanding [ 18 ]. Although emerging treatments targeting novel molecular pathways, anti‐inflammatory mediators, and precision medicine approaches hold promise, none has yet fully addressed the complex burden of DN or provided definitive strategies to halt its progression [ 3 , 19 ]. To address these questions, we investigated the effects of NAC, glycine, and their combination in a diabetic rat model, comparing early versus delayed treatment initiation after disease induction by assessing oxidative stress, inflammation, and renal structural alterations. 2. Materials and Methods 2.1. Study Design and Ethical Approval This study is part of a broader experimental project entitled “Effects of N ‐acetylcysteine and glycine on serum soluble tumorigenicity suppressor 2 and morphological characteristics of the heart in a streptozotocin‐induced diabetes model,” approved by the Ethics Committee of the Veterinary Faculty, University of Sarajevo (No. 02–3‐4‐AK‐11376/23) and the Ethics Committee of the Faculty of Medicine, University of Sarajevo (No. 07–03–989‐2/23). All procedures adhered to relevant national and institutional guidelines for the care and use of laboratory animals. The study was designed as a prospective, randomized, controlled experimental trial. The sample size was determined based on preliminary data and a power analysis ( α = 0.05, power = 0.8) to ensure adequate statistical power for detecting biologically relevant differences among groups. 2.2. Experimental Animals and Group Allocation A total of 48 healthy male Wistar albino rats (10 weeks old, 180–380 g) were randomly assigned to five experimental groups (Groups 1–5). Groups 3–5 were further subdivided into early (a) and late (b) treatment subgroups (detailed description in Table 1 ). Randomization was performed using a computer‐generated sequence. Prior to the experiment, the rats were acclimatized for 7 days. TABLE 1. Experimental groups, interventions, and follow‐up periods in the STZ‐induced diabetes rat model. Group Number of animals ( n ) Diabetes Therapeutic intervention Treatment start Dose and route of administration Follow‐up period Group 1 6 No None — — Up to 12 weeks Group 2 6 Yes (STZ) None — — Up to 12 weeks Group 3 Group 3a 6 Yes (STZ) NAC Day 7 after diabetes confirmation 100 mg/kg orally, syringe Up to 12 weeks Group 3b 6 Yes (STZ) NAC Week 7 100 mg/kg orally, syringe Week 7 to 12 Group 4 Group 4a 6 Yes (STZ) Glycine Day 7 after diabetes confirmation 250 mg/kg orally, syringe Up to 12 weeks Group 4b 6 Yes (STZ) Glycine Week 7 250 mg/kg orally, syringe Week 7 to 12 Group 5 Group 5a 6 Yes (STZ) NAC + Glycine Day 7 after diabetes confirmation NAC 100 mg/kg + Glycine 250 mg/kg orally, syringe Up to 12 weeks Group 5b 6 Yes (STZ) NAC + Glycine Week 7 NAC 100 mg/kg + Glycine 250 mg/kg orally, syringe Week 7 to 12 Open in a new tab Abbreviations: NAC, N ‐acetylcysteine; STZ, streptozotocin. Type 1 diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, Sigma‐Aldrich, USA) dissolved in citrate buffer (pH 4.5) at 55 mg/kg. After 72 h, diabetes was confirmed using a glucometer (Accu‐Chek, Roche, Switzerland); control animals received citrate buffer alone [ 20 ]. Animals were allocated to early and late intervention groups based on the timing of treatment initiation. In the early intervention groups, NAC, glycine, or their combination was administered on Day 7 after diabetes induction and continued for 12 weeks. In the late intervention groups, treatment was initiated at Week 7 and continued for 6 weeks. The rationale for early versus late treatment was to distinguish preventive effects (early treatment) from therapeutic effects on established diabetic nephropathy (late treatment). NAC and glycine were administered as aqueous solutions by the oral route using a syringe. NAC was given at a dose of 100 mg/kg and glycine at a dose of 250 mg/kg. The total administered volume was 1 mL per animal, and the concentration of the prepared solution was adjusted according to the individual body weight of each animal. Experimental animals were allocated into eight groups as summarized in Table 1 . 2.3. Sample Collection and Preparation On Day 85 (12 weeks and 1 day after induction), animals were anesthetized with ketamine (50 mg/kg, i.p.) and sacrificed according to institutional protocols and current ethical regulations. Blood samples were collected from the heart and aorta. After coagulation at room temperature and centrifugation, serum was aliquoted into microtubes and stored at −80°C until biochemical analyzes. Immediately after blood sampling, left kidneys were excised and weighed. Relative kidney mass was calculated as the ratio of the absolute kidney mass to the total body mass. The left kidneys were then rinsed with ice‐cold physiological saline to remove residual blood and processed for histological evaluation. 2.4. Histological Analysis of Kidney Tissue Kidney samples were fixed in formalin, dehydrated in a graded series of alcohols and xylene, and then embedded in paraffin. Sections of 5 μm thickness were cut, deparaffinized, rehydrated, and stained with hematoxylin–eosin (H&E), periodic acid–Schiff (PAS), and Masson's trichrome. 2.4.1. Semiquantitative Analysis The following parameters were scored in a semiquantitative manner (0–3) [ 21 ]: Glomerular damage (hypercellularity, GBM thickening, mesangial expansion) Tubular damage (brush border loss, necrosis, atrophy, hypertrophy, dilatation, casts, basement membrane thickening) Inflammation Fibrosis A score of 0 indicated no changes; 1, mild; 2, moderate; and 3, severe changes. All histological assessments were performed in a blinded manner by two independent observers, with respect to the group allocation. 2.4.2. Geometric Morphometric Analysis of Renal Glomeruli The analysis of shape differences in glomeruli between the examined groups was performed using the MorphoJ software, which included geometric morphometric tests—generalized Procrustes analysis, principal component analysis, and discriminant functional analysis [ 22 ]. The analysis encompassed renal glomeruli from the studied animals, including those from the upper pole, midsection, and lower pole of each kidney from all animals (a total of 165 glomeruli per group). Each image was coded for group, animal ID, and kidney region. Images were converted into TPS format using the tpsUtil software, then processed in tpsDig to assign 16 landmarks per glomerulus (8 external and 8 internal). Landmarks were digitized in an identical sequence for all images. Coordinate data ( x , y ) were analyzed in MorphoJ software using: Generalized Procrustes Analysis (GPA) for shape standardization, Principal Component Analysis (PCA) for major axes of variation, Discriminant Function Analysis (DFA) with cross‐validation to test statistical significance among groups. 2.5. Biochemical Analyzes All biochemical analyzes were performed in duplicate using standard protocols and commercially available kits, following the manufacturers' instructions. 2.5.1. Myeloperoxidase (MPO) Activity Serum MPO activity was measured colorimetrically (ELK Biotechnology, Hubei, China; Cat. No. BC100). MPO catalyzes hydrogen peroxide reduction; the product reacts with dianisidine forming a yellow complex ( λ = 460 nm). Activity was expressed as U/L using a VWR UV1600‐PC spectrophotometer (USA). 2.5.2. Superoxide Dismutase (SOD) Activity SOD activity was determined by a colorimetric assay based on xanthine oxidase–generated superoxide anions, which react with WST‐1 forming a yellow formazan dye. The reaction is inversely proportional to SOD activity. Samples were incubated for 20 min at 37°C; absorbance was read at 450 nm using a STAT FAX 2100 reader (USA). Activity was expressed as inhibition rate (%). 2.5.3. Serum IL‐33 Quantification Serum IL‐33 was measured using a quantitative sandwich ELISA. Plates precoated with anti–IL‐33 antibodies were incubated with standards and serum samples, followed by detection antibody cocktail. After washing, TMB substrate was added; color development was stopped with acid, and optical density was read at 600 nm (STAT FAX 2100, USA). The detection range was 46.9–3000 pg/mL, with a lower limit of 6 pg/mL. 2.6. Statistical Analysis Statistical analyzes were performed using SPSS Statistics version 13.0 (SPSS Inc., Chicago, Illinois, USA). The Shapiro–Wilk test was applied to assess the normality of continuous variables. Data were expressed as mean ± standard deviation (SD) for normally distributed variables, median with interquartile range (IQR) for nonnormally distributed variables, and absolute or relative frequencies for categorical variables. Comparisons between groups were conducted using Student's t ‐test for two groups or one‐way ANOVA followed by Bonferroni post hoc test for pairwise multiple comparisons after a significant overall test for three or more groups in the case of normally distributed data. Superscript symbols in the tables denote statistically significant pairwise comparisons versus the reference groups specified in the corresponding table footnotes. For nonnormally distributed variables, the Mann–Whitney U test was applied for two groups, whereas the Kruskal–Wallis test was used for three or more groups. Categorical variables were analyzed using Pearson's chi‐squared test or Fisher's exact test, as appropriate. Effect size measures were additionally reported where appropriate to complement p ‐values. A p ‐value < 0.05 was considered statistically significant. 3. Results Results are presented in the following order: histological qualitative, semiquantitative, and morphometric analyzes, biochemical parameters, and correlation analyzes. Detailed numerical data for all parameters are provided in the Supporting Information Tables. Key findings are summarized below with emphasis on statistically significant differences among groups. 3.1. General Characteristics of Experimental Animals The average values of body and kidney masses in all observed groups are presented in Table 2 . TABLE 2. Body and kidney masses at the end of the experiment in the observed groups. Body mass Absolute kidney mass Relative kidney mass Group 1 270.5 ± 1.25 1.36 ± 0.02 0.0051 ± 0.00007 Group 2 213.3 ± 16.95 1.74 ± 0.12 0.0081 ± 0.000447 Group 3a 239.9 ± 18.1 1.57 ± 0.057 0.0065 ± 0.0007 Group 3b 237.96 ± 1.25 1.51 ± 0.03 0.0063 ± 0.00013 Group 4a 223.77 ± 11.86 1.52 ± 0.47 0.0068 ± 0.0003 Group 4b 220.8 ± 3.1 1.74 ± 0.12 0.0081 ± 0.000447 Group 5a 249.97 ± 2.15 1.48 ± 0.05 0.006 ± 0.0015 Group 5b 224.3 ± 13.83 1.49 ± 0.03 0.006 ± 0.005 p 0.000 0.000 0.000 Open in a new tab Note: Results are expressed as mean ± SD. * p < 0.05 versus Group 1; # p < 0.05 versus Group 2; ‡ p < 0.05 versus Group 3a; † p < 0.05 versus Group 3b; § p < 0.05 versus Group 4a; & p < 0.05 versus Group 4b; ¶ p < 0.05 versus Group 5a. 3.2. Histological Changes in Kidney Tissue 3.2.1. Qualitative Histological Findings Histopathological evaluation revealed distinct renal structural alterations across experimental groups (Figure 1 ). Group 1: All renal compartments, including glomeruli, tubules, and interstitium, showed normal morphology without detectable pathological changes. Group 2: Marked glomerular hypercellularity, increase in the amount of mesangial matrix, tubular epithelial degeneration, interstitial inflammation, and fibrosis were evident compared with healthy controls. Group 3a and 3b: Both groups exhibited moderate improvement in glomerular and tubular morphology, with reduced inflammatory infiltration and fibrosis compared with untreated diabetic animals. Group 4a and 4b: Renal lesions were attenuated, particularly when treatment was initiated early, showing decreased tubular damage and interstitial fibrosis. Group 5a and 5b: The combined therapy, especially early intervention, resulted in the most pronounced preservation of renal structure, with morphology approaching that of healthy controls. FIGURE 1. Open in a new tab Representative histological images showing renal morphology across experimental groups. H&E—hematoxylin–eosin; PAS—periodic acid–Schiff. Full histopathological descriptions for each experimental group are provided in Supporting Information . 3.2.2. Semiquantitative Assessment of Histological Changes The average values of histological changes in the renal tissue of the observed groups are presented in Table 3 . In Group 2, tubular damage averaged 2.4 ± 0.89, significantly exceeding values in Group 3a (1.28 ± 0.49, p < 0.05), Group 4a (1.0 ± 0.12, p < 0.05), Group 5a (0.5 ± 0.84, p < 0.05), and Group 5b (1.17 ± 0.41, p < 0.05). Inflammation differed significantly only between Group 2 and Group 5a [1.6 ± 0.89 vs. 0.33 ± 0.52, p < 0.05]. TABLE 3. Histological changes in renal tissue, including signs of fibrosis, inflammation, tubular degeneration, and glomerular damage observed in the different groups. Glomerular damage Tubular damage Inflammation Fibrosis Group 1 0 0 0 0 Group 2 2 ± 1.12 2.4 ± 0.89 1.6 ± 0.89 2.4 ± 0.54 Group 3a 1.28 ± 0.75 1.28 ± 0.49 # 1.28 ± 0.75 0.85 ± 0.34 # Group 3b 1.16 ± 0.41 1.33 ± 0.52 1.33 ± 0.52 1.5 ± 0.84 Group 4a 1.33 ± 0.52 1 ± 0.12 # 0.83 ± 0.41 1 ± 0.13 # Group 4b 1.33 ± 0.52 1.33 ± 0.52 1.17 ± 0.41 1.33 ± 0.52 # Group 5a 0.67 ± 1.03 0.5 ± 0.84 # 0.33 ± 0.52 # 0.33 ± 0.52 # ,†,& Group 5b 1 ± 0.63 1.17 ± 0.41 # 1 ± 0.63 0.52 ± 033 #,†,& p 0.000 0.000 0.004 0.000 Open in a new tab Note: Results are expressed as a mean and SD. # p < 0.05 in comparation to group 2; † p < 0.05 in comparation to group 3b; and & p < 0.05 in comparation to Group 4b. For fibrosis, Group 2 reached 2.4 ± 0.54, markedly higher than in Group 3a (0.85 ± 0.34, p < 0.05), Group 4a (1.0 ± 0.13, p < 0.05), Group 4b (0.33 ± 0.52, p < 0.05), Group 5a (0.33 ± 0.52, p < 0.05), and Group 5b (0.52 ± 0.33, p < 0.05). Notably, Groups 3b and 4b exhibited significantly greater fibrosis compared with Groups 5a and 5b ( p < 0.05). 3.2.3. Geometric Morphometric Analysis of Renal Glomeruli Figure 2 illustrates the spatial distribution of all analyzed glomeruli in morphospace, plotted along the first two principal components (PC1 and PC2) that summarize the greatest sources of shape variation in the dataset. FIGURE 2. Open in a new tab Spatial distribution of analyzed glomeruli in morphospace along the first two principal components (PC1 and PC2), stratified by experimental groups. The proportion of total variance explained by each principal component is summarized in Table 4 , accounting for the majority of shape variability across groups. TABLE 4. Principal component analysis and percentage of variance explained (first 10 components shown). Eigenvalues % Variance Cumulative % PC1. 0.00474010 53.455 53.455 PC2. 0.00089743 10.121 63.576 PC3. 0.00080621 9.092 72.667 PC4. 0.00067365 7.597 80.264 PC5. 0.00049225 5.551 85.816 PC6. 0.00042136 4.752 90.567 PC7. 0.00026490 2.987 93.555 PC8. 0.00016225 1.830 95.384 PC9. 0.00012683 1.430 96.815 PC10. 0.00009433 1.064 97.878 Open in a new tab To further illustrate morphological heterogeneity, Figure 3 visualizes the positional variability of all 16 landmarks along PC1 for every analyzed glomerulus. The blue point represents the mean position of each landmark, while the blue line indicates both the direction and magnitude of positional variation. This complementary analysis highlights subtle shape differences not fully captured by spatial clustering alone. FIGURE 3. Open in a new tab Distribution of specific landmarks in morphospace along the first principal component (PC1) for all glomeruli across experimental groups. The blue point represents the mean landmark position, while the blue line indicates the magnitude and direction of positional variability. This analysis revealed subtle but consistent morphological shifts in landmark positions across treatment groups, indicating group‐specific glomerular morphological variation. Furthermore, discriminant function analysis was performed to assess whether the observed groups exhibited statistically significant differences in glomerular shape. Overall significance values for all group comparisons are presented (Table 5 ), while histograms are shown only for selected comparisons to enhance clarity and focus on the most informative results (Figure 4 ). TABLE 5. Statistical significance of discriminant function analysis differences between groups. Group Group 1 Group 2 Group 3a Group 3b Group 4a Group 4b Group 5a Group 5b Group 1 — p = 0.03 p = 0.005 p = 0.005 p < 0.034 p < 0.034 p = 0.07 p = 0.06 Group 2 — — p = 0.351 p = 0.351 p = 0.76 p = 0.76 p = 0.05 p = 0.3 Group 3a — — — p = 0.3 p = 0.06 p = 0.058 p = 0.06 p = 0.07 Group 3b — — — — p = 0.5 p > 0.05 p = 0.67 p > 0.05 Group 4a — — — — — p > 0.05 p > 0.05 p > 0.5 Group 4b — — — — — — p > 0.05 p = 0.08 Group 5a — — — — — — — p = 0.2 Group 5b — — — — — — — — Open in a new tab FIGURE 4. Open in a new tab Discriminant functional analysis of glomerular morphological differences between Group 1 and 2 (A), Group 1 and 3a (B), Group 1 and 3b (C), Group 1 and 4a (D), Group 1 and 4b (E), Group 1 and 5a (F), Group 1 and 5b (G), Group 2 and 5a (H), Group 2 and 5b (I). Each graph displays two colors representing the compared groups, with overlapping regions indicating shared morphological features. Comparison between healthy controls (Group 1) and untreated diabetic animals (Group 2) showed significant differences ( p = 0.03; Figure 4A ). Early and late glycine‐treated diabetic animals (Groups 3a and 3b) differed significantly from healthy controls ( p = 0.005 for both; Figure 4B,C ) but not from untreated diabetic rats ( p = 0.351 for both). NAC treatment alone at early and late stages (Groups 4a and 4b) produced significant differences versus healthy controls ( p < 0.034 for both; Figure 4D,E ) and no differences from untreated diabetic animals ( p = 0.76 for both). Combined glycine and NAC treatment at early stages of diabetes (Group 5a) preserved glomerular morphology, showing similarity to healthy controls ( p = 0.07; Figure 4F ) and borderline differences from untreated diabetic animals ( p = 0.05; Figure 4G ), supporting effective maintenance of glomerular structure after 12 weeks. Late‐stage combined therapy (Group 5b) showed no significant differences compared with either untreated diabetics ( p = 0.3; Figure 4H ), or healthy controls ( p = 0.06; Figure 4I ), suggesting partial preservation of glomerular morphology. 3.3. Biochemical Analyzes The average values of biochemical parameters in all observed groups are presented in Table 6 . In Group 2, SOD activity reached 73.3 ± 5.67 U/L, markedly reduced compared with Group 1 (90.11 ± 1.25, p < 0.05), Group 3a (89.18 ± 11.38, p < 0.05), Group 3b (86.53 ± 7.03, p < 0.05), Group 4b (86.83 ± 4.40, p < 0.05), Group 5a (87.7 ± 4.98, p < 0.05), and Group 5b (88.38 ± 2.71, p < 0.05). Conversely, MPO activity in Group 2 was 413.54 ± 253.77 U/L, significantly exceeding values in Group 1 (116.35 ± 22.74, p < 0.05), Group 3a (184.55 ± 41.41, p < 0.05), Group 5a (110.17 ± 38.66, p < 0.05), and Group 5b (181.25 ± 68.72, p < 0.05). TABLE 6. The average values of biochemical parameters in all observed groups. SOD MPO IL‐33 Group 1 90.11 ± 1.25 116.35 ± 22.74 267.0 ± 103.17 Group 2 73.3 ± 5.67* 413.54 ± 253.77* 179.22 ± 12.86 Group 3a 89.18 ± 11.38 # 184.55 ± 41.41 # 282.14 ± 54.42 Group 3b 86.53 ± 7.03 # 261.44 ± 100.75 150.35 ± 22.51 Group 4a 83.2 ± 5.78 244.68 ± 95.17 352.30 ± 39.22 #,† Group 4b 86.83 ± 4.40 # 210.34 ± 46.52 † 278.75 ± 27.30 Group 5a 87.7 ± 4.98 # 110.17 ± 38.66 # 450.53 ± 25.56* ,#,‡,†,§ Group 5b 88.38 ± 2.71 # 181.25 ± 68.72 # 199.13 ± 25.56 &,¶ ( F = 3.905; p = 00.000) ( F = 4.927; p = 0.000) ( F = 15.042; p = 0.000) Open in a new tab Note: Results are expressed as mean ± SD. * p < 0.05 versus Group 1; # p < 0.05 versus Group 2; ‡ p < 0.05 versus Group 3a; † p < 0.05 versus Group 3b; § p < 0.05 versus Group 4a; & p < 0.05 versus Group 4b; ¶ p < 0.05 versus Group 5a. Abbreviations: IL‐33, interleukin‐33; MPO, myeloperoxidase; SOD, superoxide dismutase. The IL‐33 concentration peaked in Group 5a (450.53 ± 25.56 pg/mL), surpassing levels in Group 1 (267.0 ± 103.17, p < 0.05), Group 2 (179.22 ± 12.86, p < 0.05), Group 3a (282.14 ± 54.42, p < 0.05), Group 3b (150.35 ± 22.51, p < 0.05), Group 4a (352.30 ± 39.22, p < 0.05), Group 4b (278.75 ± 27.30, p < 0.05), and Group 5b (199.13 ± 25.56, p < 0.05). Additional significant differences emerged between Groups 4a and 3b ( p < 0.05), 4b and 2 ( p < 0.05), 4b and 3b ( p < 0.05), and 5b and 4b ( p < 0.05). 3.4. Correlation Analysis Between Biochemical and Histological Parameters Correlation analysis between biochemical parameters and histological changes in renal tissue revealed several group‐specific associations. In Group 5a, SOD activity showed a strong negative correlation with both glomerular damage ( r = −0.980, p < 0.01) and inflammation ( r = −0.980, p < 0.01). In the same group, IL‐33 levels were inversely correlated with tubular injury ( r = −0.953, p = 0.001), glomerular damage ( r = −0.767, p < 0.05), and inflammation ( r = −0.767, p < 0.05). In Group 4b, SOD activity correlated negatively with glomerular damage ( r = −0.832, p < 0.05). No significant correlations between SOD, MPO activity, IL‐33 levels, and histological parameters were observed in the remaining groups. The complete group‐specific correlation matrices are provided for Group 2 (Table S1 ), Group 3a (Table S2 ), Group 3b (Table S3 ), Group 4a (Table S4 ), Group 4b (Table S5 ), Group 5a (Table S6 ), and Group 5b (Table S7 ). 4. Discussion This study demonstrates that the early administration of combined NAC and glycine (GlyNAC) provided more effective protection against STZ‐induced diabetic nephropathy than individual glycine or NAC treatments. We show that early GlyNAC largely preserved renal morphology, normalized oxidative (SOD) and inflammatory (MPO) indices, whereas glycine or NAC alone achieved only partial improvements. Classic qualitative and semiquantitative histological analyzes were further supported by the geometric morphometric analysis that revealed GlyNAC shifted glomerular architecture into a reproducible morphospace distinct from untreated diabetes, suggesting a treatment‐related modulation of renal structure. Untreated diabetic rats in this study developed hallmark lesions of DN, including mesangial expansion, glomerulosclerosis, tubular atrophy, and interstitial fibrosis. These histopathological features recapitulate the lesions seen in human DN, validating the STZ model as an appropriate platform for mechanistic and therapeutic investigation [ 20 ]. Crucially, such structural changes arise early in disease progression and typically precede overt functional decline, indicating that once established, they are difficult to reverse. Within this framework, the marked preservation of renal morphology by early GlyNAC suggests its potential to intercept DN at a stage when intervention is most likely to achieve durable protection. Glycine, NAC, and GlyNAC have been widely used as health supplements. While clinical evidence for these supplements remains limited, several strands of human data point toward potential renoprotective benefits. In hemodialysis (CKD) cohorts, multiple randomized trials and meta‐analyzes have shown that NAC supplementation reduces circulating markers of oxidative stress and, in some studies, modestly lowers proteinuria or blood pressure [ 23 , 24 , 25 ]. Observational metabolomic studies further associate higher plasma glycine with a reduced risk of incident CKD, suggesting a potential protective role in renal metabolism [ 26 , 27 ]. GlyNAC supplementation has been more extensively studied in aging populations: in placebo‐controlled trials, 12–24 weeks of GlyNAC restored glutathione concentrations in blood and muscle, improved mitochondrial fuel oxidation, reduced insulin resistance, lowered oxidative stress markers, and enhanced gait speed and muscle strength [ 28 ]. A small open‐label pilot in patients with Type 2 diabetes reported that GlyNAC restored red blood cell glutathione, reduced systemic oxidative stress, and improved insulin resistance (HOMA‐IR), although kidney endpoints were not assessed [ 29 ]. To date, no clinical trial has directly evaluated the effects of GlyNAC on renal function, underscoring the translational importance of preclinical studies. In experimental models, glycine and NAC exert distinct, nonredundant renoprotective actions. In STZ‐diabetic rats, glycine attenuates mesangial expansion and tubulointerstitial fibrosis while restoring renal glutathione and reducing lipid peroxidation. Mechanistic studies have shown that glycine suppresses Nox4 activity and activates Nrf2, leading to nuclear translocation and induction of glyoxalase‐1, which detoxifies methylglyoxal and reduces advanced glycation end‐product burden [ 30 , 31 ]. NAC has also been shown to improve renal function and histology in STZ‐diabetic rats. In these models, NAC increases nitric oxide bioavailability by up‐regulating eNOS and suppressing iNOS, restores glutathione, and reduces lipid peroxidation [ 32 ]. In a large‐animal model of diabetes (beagle dogs), NAC alleviated mitochondrial dysfunction through activation of the SIRT3–SOD2–GPx4 axis [ 3 ]. Taken together, these findings suggest that glycine acts as a modulator of dicarbonyl stress and NAC as a regulator of nitrosative and mitochondrial homeostasis, both converging on glutathione‐dependent antioxidant defense. The central question of this study was whether the combined action on these two pathways by simultaneous administration of glycine and NAC (GlyNAC) provides superior renoprotection compared with either substrate alone, and whether the timing of intervention influences efficacy. The timing of treatment is critical because oxidative and inflammatory cascades become self‐sustaining once chronic injury is established [ 6 , 33 ]. If supplementation is only effective when started early, this would define its translational window and influence its feasibility as a therapy in human diabetic nephropathy. Our histopathological analyzes demonstrated that injury was most effectively prevented by GlyNAC when administered immediately after STZ induction (Table 3 and Figure 1 ). Tubular degeneration, inflammation, and fibrosis were markedly reduced with early GlyNAC, whereas glycine or NAC alone produced only partial improvements. The superiority of early intervention echoes the findings of Nogueira et al., who showed that NAC reduced proteinuria, preserved creatinine clearance, and limited tubulointerstitial damage in uninephrectomised STZ rats only when treatment began at the onset of diabetes [ 32 ]. These data underscore the principle that early interruption of oxidative and inflammatory cascades is essential for structural rescue. Biochemical indices supported the histological results. We found STZ reduced SOD activity and increased MPO (Table 6 ), consistent with oxidative stress and neutrophil‐driven inflammation. Both monotherapies partially corrected these changes, but GlyNAC restored SOD and normalized MPO when given early. Late GlyNAC also improved these markers but less effectively, again emphasizing the temporal dependence of therapeutic benefit. Collectively, these findings demonstrate that GlyNAC achieves a breadth and depth of renoprotection not attainable with either substrate alone, and that early intervention is most effective. Interestingly, we observed that STZ‐induced diabetes was associated with reduced circulating IL‐33, a result that aligns with clinical observations of suppressed IL‐33 and elevated soluble ST2 in patients with Type 2 diabetes and microalbuminuria [ 34 , 35 , 36 ]. Given the context‐dependent roles of IL‐33 in kidney injury: in some settings amplifying inflammation [ 37 ], while in others promoting repair [ 38 ]. Experimental studies have shown that exogenous IL‐33 can attenuate diabetic nephropathy, improve endothelial resilience, and reduce renal injury, although excessive IL‐33 activity has also been linked to fibrosis, emphasizing a dose‐ and context‐sensitive biology [ 39 ]. Against this background, the finding that glycine, NAC, and especially early GlyNAC restored or even elevated IL‐33 beyond normal levels is intriguing. The result may reflect either redox‐dependent activation of reparative IL‐33 signaling (via ILC2/Treg pathways) or a treatment‐related immunological steady state different from both health and disease, as also suggested by our geometric morphometric findings. Either way, these results suggest that IL‐33 may not only be seen as a marker of the altered immune–redox milieu but also as a potential mediator of renal regeneration following intervention. Further studies are needed to clarify its mechanistic role [ 34 , 40 ]. Beyond conventional histology, we applied landmark‐based geometric morphometrics to quantify subtle variations in glomerular topology. This approach represents a methodological strength of this study, since it improves sensitivity in detecting treatment‐related preservation of tissue architecture and provides a useful tool for evaluating morphological alterations in experimental diabetic nephropathy. To our knowledge, this approach has not previously been applied to study diabetic nephropathy. Principal component analysis (Figure 2 ) showed broad overlap between groups, indicating that dominant axes of variation captured features common to all kidneys. However, discriminant functional analyzes revealed significant separations between selected pairs, demonstrating that finer‐scale morphologies encode disease and treatment signatures (Figure 4 ). Glycine or NAC alone did not significantly revert STZ‐induced alterations (Table 5 ), whereas early treatment with GlyNAC shifted diabetic glomeruli into a distinct pattern in morphospace, significantly different from both diabetes and control (Figure 4F,H ). Without baseline data from nondiabetic animals treated with Gly/NAC/GlyNAC, however, it remains unclear whether this reflects partial restoration or a novel treatment‐induced configuration. Nevertheless, the consistent emergence of a GlyNAC‐associated morphospace indicates that redox–metabolic repletion may influence glomerular architecture in ways not fully captured by conventional histological scoring. The observed negative correlations between SOD activity and glomerular injury, as well as IL‐33 and tubular damage, suggest a mechanistic link between oxidative stress, inflammatory signaling, and structural renal alterations in diabetic conditions. This study has several limitations. First, the use of STZ to induce diabetes primarily models the acute β‐cell destruction of Type 1 diabetes, rather than the progressive metabolic syndrome–associated pathophysiology of Type 2 diabetes in which diabetic nephropathy most often arises. Importantly, STZ is directly nephrotoxic [ 41 ], raising the possibility that part of the observed glomerular phenotype reflects chemical injury rather than hyperglycaemia per se. Although the consistent concordance between renal structural alterations, oxidative/inflammatory indices, and GlyNAC‐mediated rescue argues against nonspecific toxicity as the sole driver, additional models, such as db/db [ 42 ] or high‐fat diet–induced diabetes [ 43 ], will be important to generalize these findings. Second, the study did not include nondiabetic baseline controls for glycine, NAC, or GlyNAC treatment. This omission, intended to limit animal use, constrains interpretation of the data, especially the observed distinct pattern in morphospace associated with GlyNAC: it cannot be determined whether the shift represents partial reversion of the diabetic phenotype or the emergence of a treatment‐specific architecture. Future inclusion of baseline treatment arms will be necessary to establish whether GlyNAC induces compensatory remodeling in healthy kidneys. Third, the duration of follow‐up was limited to 12 weeks. While sufficient to demonstrate early and intermediate structural changes, this timeframe does not capture the chronic progression to glomerulosclerosis, advanced fibrosis, or end‐stage renal disease. For instance, STZ‐treated mice typically develop glomerulosclerosis and tubulointerstitial fibrosis only after 20–30 weeks [ 44 ], whereas db/db mice manifest nodular sclerosis and advanced fibrosis by 30–40 weeks [ 45 ]. Longer‐term studies are required to determine whether early GlyNAC intervention confers durable protection and whether delayed administration retains efficacy once irreversible lesions have formed. Also, the relatively small group sizes ( n = 6) limit statistical power, particularly for morphometric analyzes where inter‐individual variability can be substantial. Although the group size was determined a priori based on preliminary data and a power analysis, larger cohorts will be necessary to confirm robustness and to uncover subtler or secondary treatment effects. Finally, the analyzes relied on structural and biochemical endpoints without direct measurement of renal function, such as serum creatinine, blood urea nitrogen, or albuminuria. While classic histology and redox markers provide mechanistic insight, their translation to physiological renal performance remains inferential. Incorporating functional assays in future experiments will be critical to demonstrate whether structural rescue by GlyNAC is accompanied by restored filtration capacity and metabolic clearance. Despite these limitations, this work demonstrates that GlyNAC is a potent modulator of diabetic renal injury, restoring antioxidant defenses, suppressing inflammatory cascades, and reshaping glomerular architecture. The combined classic histological, geometric morphometric, and biochemical data suggest that dual‐precursor supplementation achieves a level of protection not attainable with either agent alone, and that the timing of intervention is critical. By integrating geometric morphometrics with conventional histopathology, this study introduces a framework capable of detecting subtle architectural responses to therapy and suggests that redox–metabolic repletion may represent a potential strategy for disease modification in diabetic nephropathy. In future, dose–response and pharmacokinetic studies of glycine and NAC in vivo will help combination regimens, and testing in combination with established or emerging therapies such as SGLT2 inhibitors, anti‐fibrotics, or anti‐inflammatory biologics may reveal additive or synergistic benefits [ 46 ]. Given that both glycine and NAC have established human safety profiles, these preclinical data provide a rationale for moving toward early‐phase clinical trials of GlyNAC in DN. 5. Conclusions This study shows that early combined N ‐acetylcysteine and glycine therapy provides greater protection against diabetic nephropathy compared with either agent alone. Early GlyNAC was associated with preservation of glomerular architecture, marked reduction in tubular injury, inflammation, and fibrosis, accompanied by improvement in antioxidant and inflammatory markers toward control levels. Geometric morphometrics showed a distinct pattern of structural preservation not observed with monotherapy or delayed treatment, supporting the potential importance of timely interruption of redox‐inflammatory cascades. Although limited by model specificity and absence of functional renal endpoints, these results suggest that metabolic–redox repletion may represent a potent disease‐modifying strategy. Further long‐term and multimodal studies are warranted to define translational potential and to evaluate GlyNAC alongside established nephroprotective therapies. Author Contributions M.E., A.F., L.A., R.J., A.M., Z.A., D.K., E.Ć., and O.L. and collected, analyzed and generated data. M.E., A.F., A.B., Y.W.L., F.J., L.A., R.J., A.M., A.J.E., Z.A., D.K., E.Ć., and O.L. wrote the manuscript. M.E., A.B., R.J., and A.F. initiated the idea and supplied resources. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. Funding The authors have nothing to report. Disclosure The authors declare that no artificial intelligence tools, including large language model based systems, were used in the conception, writing, editing, data analysis, figure preparation, or any other stage of the manuscript development. All components of the manuscript were created entirely by the authors, who take full responsibility for the accuracy and integrity of the work. Ethics Statement This study is part of a broader experimental project entitled “Effects of N ‐acetylcysteine and glycine on serum soluble tumorigenicity suppressor 2 and morphological characteristics of the heart in a streptozotocin‐induced diabetes model,” approved by the Ethics Committee of the Veterinary Faculty, University of Sarajevo (No. 02‐3‐4‐AK‐11376/23) and the Ethics Committee of the Faculty of Medicine, University of Sarajevo (No. 07‐03‐989‐2/23). All procedures adhered to relevant national and institutional guidelines for the care and use of laboratory animals. Conflicts of Interest The authors declare no conflicts of interest. Supporting information Table S1: Correlations between biochemical and histological parameters in Group 2. Table S2: Correlations between biochemical and histological parameters in Group 3a. Table S3: Correlations between biochemical and histological parameters in Group 3b. Table S4: Correlations between biochemical and histological parameters in Group 4a. Table S5: Correlations between biochemical and histological parameters in Group 4b. Table S6: Correlations between biochemical and histological parameters in Group 5a. Table S7: Correlations between biochemical and histological parameters in Group 5b. PRP2-14-e70273-s001.docx (18.7KB, docx) Acknowledgments The authors have nothing to report. Contributor Information Andrej Belančić, Email: andrej.belancic@uniri.hr, Email: a.belancic93@gmail.com. Almir Fajkić, Email: almir.fajkic@mf.unsa.ba. Data Availability Statement Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. References 1. Młynarska E., Buławska D., Czarnik W., et al., “Novel Insights Into Diabetic Kidney Disease,” International Journal of Molecular Sciences 25, no. 18 (2024): 10222, 10.3390/ijms251810222. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. American Diabetes Association Professional Practice Committee , “11. Chronic Kidney Disease and Risk Management: Standards of Care in Diabetes‐2025,” Diabetes Care 48, no. 1 (2025): S239–S251, 10.2337/dc25-S011. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 3. Martinez Leon V., Hilburg R., and Susztak K., “Mechanisms of Diabetic Kidney Disease and Established and Emerging Treatments,” Nature Reviews Endocrinology 22, no. 1 (2026): 21–35, 10.1038/s41574-025-01171-3. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 4. Hung P. H., Hsu Y. C., Chen T. H., and Lin C. L., “Recent Advances in Diabetic Kidney Diseases: From Kidney Injury to Kidney Fibrosis,” International Journal of Molecular Sciences 22, no. 21 (2021): 11857, 10.3390/ijms222111857. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 5. Chen X., Xie N., Feng L., et al., “Oxidative Stress in Diabetes Mellitus and Its Complications: From Pathophysiology to Therapeutic Strategies,” Chinese Medical Journal 138, no. 1 (2025): 15–27, 10.1097/CM9.0000000000003230. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 6. Wang N. and Zhang C., “Oxidative Stress: A Culprit in the Progression of Diabetic Kidney Disease,” Antioxidants 13, no. 4 (2024): 455, 10.3390/antiox13040455. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Kagan V. E., Mao G., Qu F., et al., “Oxidized Arachidonic and Adrenic PEs Navigate Cells to Ferroptosis,” Nature Chemical Biology 13, no. 1 (2017): 81–90, 10.1038/nchembio.2238. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 8. Li Q., Liao J., Chen W., et al., “NAC Alleviative Ferroptosis in Diabetic Nephropathy via Maintaining Mitochondrial Redox Homeostasis Through Activating SIRT3‐SOD2/Gpx4 Pathway,” Free Radical Biology and Medicine 187 (2022): 158–170, 10.1016/j.freeradbiomed.2022.05.024. [ DOI ] [ PubMed ] [ Google Scholar ] 9. Li L., Dai Y., Ke D., et al., “Ferroptosis: New Insight Into the Mechanisms of Diabetic Nephropathy and Retinopathy,” Frontiers in Endocrinology 14 (2023): 1215292, 10.3389/fendo.2023.1215292. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 10. Jiang X., Stockwell B. R., and Conrad M., “Ferroptosis: Mechanisms, Biology and Role in Disease,” Nature Reviews. Molecular Cell Biology 22, no. 4 (2021): 266–282, 10.1038/s41580-020-00324-8. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 11. Irazabal M. V. and Torres V. E., “Reactive Oxygen Species and Redox Signaling in Chronic Kidney Disease,” Cells 9, no. 6 (2020): 1342, 10.3390/cells9061342. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Hou G., Dong Y., Jiang Y., et al., “Immune Inflammation and Metabolic Interactions in the Pathogenesis of Diabetic Nephropathy,” Frontiers in Endocrinology 16 (2025): 1602594, 10.3389/fendo.2025.1602594. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 13. Ansari Z., Chaurasia A., Neha, Sharma N., Bachheti R. K., and Gupta P. C., “Exploring Inflammatory and Fibrotic Mechanisms Driving Diabetic Nephropathy Progression,” Cytokine and Growth Factor Reviews 84 (2025): 120–134, 10.1016/j.cytogfr.2025.05.007. [ DOI ] [ PubMed ] [ Google Scholar ] 14. Alnahdi A., John A., and Raza H., “N‐Acetyl Cysteine Attenuates Oxidative Stress and Glutathione‐Dependent Redox Imbalance Caused by High Glucose/High Palmitic Acid Treatment in Pancreatic Rin‐5F Cells,” PLoS One 14, no. 12 (2019): e0226696, 10.1371/journal.pone.0226696. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 15. Rosa L. R. O., Kaga A. K., Barbanera P. O., Queiroz P. M., do Carmo N. O. L., and Fernandes A. A. H., “Beneficial Effects of N‐Acetylcysteine on Hepatic Oxidative Stress in Streptozotocin‐Induced Diabetic Rats,” Canadian Journal of Physiology and Pharmacology 96, no. 4 (2018): 412–418, 10.1139/cjpp-2017-0559. [ DOI ] [ PubMed ] [ Google Scholar ] 16. Pérez‐Torres I., Zuniga‐Munoz A. M., and Guarner‐Lans V., “Beneficial Effects of the Amino Acid Glycine,” Mini‐Reviews in Medicinal Chemistry 17, no. 1 (2017): 15–32, 10.2174/1389557516666160609081602. [ DOI ] [ PubMed ] [ Google Scholar ] 17. Aguayo‐Cerón K. A., Sánchez‐Muñoz F., Gutierrez‐Rojas R. A., et al., “Glycine: The Smallest Anti‐Inflammatory Micronutrient,” International Journal of Molecular Sciences 24, no. 14 (2023): 11236, 10.3390/ijms241411236. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 18. Efiong E. E., Bazireh H., Fuchs M., et al., “Crosstalk of Hyperglycaemia and Cellular Mechanisms in the Pathogenesis of Diabetic Kidney Disease,” International Journal of Molecular Sciences 25, no. 20 (2024): 10882, 10.3390/ijms252010882. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 19. Provenzano M., Maritati F., Abenavoli C., et al., “Precision Nephrology in Patients With Diabetes and Chronic Kidney Disease,” International Journal of Molecular Sciences 23, no. 10 (2022): 5719, 10.3390/ijms23105719. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 20. Ghasemi A. and Jeddi S., “Streptozotocin as a Tool for Induction of Rat Models of Diabetes: A Practical Guide,” EXCLI Journal 22 (2023): 274–294, 10.17179/excli2022-5720. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 21. An X., Liao G., Chen Y., et al., “Intervention for Early Diabetic Nephropathy by Mesenchymal Stem Cells in a Preclinical Nonhuman Primate Model,” Stem Cell Research & Therapy 10, no. 1 (2019): 363, 10.1186/s13287-019-1401-z. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 22. Klingenberg C. P., “MorphoJ: An Integrated Software Package for Geometric Morphometrics,” Molecular Ecology Resources 11, no. 2 (2011): 353–357, 10.1111/j.1755-0998.2010.02924.x. [ DOI ] [ PubMed ] [ Google Scholar ] 23. Ye M., Lin W., Zheng J., and Lin S., “N‐Acetylcysteine for Chronic Kidney Disease: A Systematic Review and Meta‐Analysis,” American Journal of Translational Research 13, no. 4 (2021): 2472–2485. [ PMC free article ] [ PubMed ] [ Google Scholar ] 24. Chiu A. H., Wang C. J., Lin Y. L., Wang C. L., and Chiang T. I., “ N ‐Acetylcysteine Alleviates the Progression of Chronic Kidney Disease: A Three‐Year Cohort Study,” Medicina (Kaunas, Lithuania) 59, no. 11 (2023): 1983, 10.3390/medicina59111983. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 25. Shahbazian H., Shayanpour S., and Ghorbani A., “Evaluation of Administration of Oral N‐Acetylcysteine to Reduce Oxidative Stress in Chronic Hemodialysis Patients: A Double‐Blind, Randomized, Controlled Clinical Trial,” Saudi Journal of Kidney Diseases and Transplantation 27, no. 1 (2016): 88–93, 10.4103/1319-2442.174084. [ DOI ] [ PubMed ] [ Google Scholar ] 26. Hou Y., Xiao Z., Zhu Y., Li Y., Liu Q., and Wang Z., “Blood Metabolites and Chronic Kidney Disease: A Mendelian Randomization Study,” BMC Medical Genomics 17, no. 1 (2024): 147, 10.1186/s12920-024-01918-3. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 27. McMahon G. M., Hwang S. J., Clish C. B., et al., “Urinary Metabolites Along With Common and Rare Genetic Variations Are Associated With Incident Chronic Kidney Disease,” Kidney International 91, no. 6 (2017): 1426–1435, 10.1016/j.kint.2017.01.007. [ DOI ] [ PubMed ] [ Google Scholar ] 28. Kumar P., Liu C., Hsu J. W., et al., “Glycine and N‐Acetylcysteine (GlyNAC) Supplementation in Older Adults Improves Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Insulin Resistance, Endothelial Dysfunction, Genotoxicity, Muscle Strength, and Cognition: Results of a Pilot Clinical Trial,” Clinical and Translational Medicine 11, no. 3 (2021): e372, 10.1002/ctm2.372. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 29. Kumar P., Liu C., Suliburk J., et al., “Supplementing Glycine and N‐Acetylcysteine (GlyNAC) in Older Adults Improves Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Physical Function, and Aging Hallmarks: A Randomized Clinical Trial,” Journals of Gerontology. Series A, Biological Sciences and Medical Sciences 78, no. 1 (2023): 75–89, 10.1093/gerona/glac135. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 30. Wang Z., Zhang J., Wang L., et al., “Glycine Mitigates Renal Oxidative Stress by Suppressing Nox4 Expression in Rats With Streptozotocin‐Induced Diabetes,” Journal of Pharmacological Sciences 137, no. 4 (2018): 387–394, 10.1016/j.jphs.2018.08.005. [ DOI ] [ PubMed ] [ Google Scholar ] 31. Wang Z., Zhao D., Chen L., et al., “Glycine Increases Glyoxalase‐1 Function by Promoting Nuclear Factor Erythroid 2‐Related Factor 2 Translocation Into the Nucleus of Kidney Cells of Streptozotocin‐Induced Diabetic Rats,” Journal of Diabetes Investigation 10, no. 5 (2019): 1189–1198, 10.1111/jdi.13032. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 32. Nogueira G. B., Punaro G. R., Oliveira C. S., et al., “N‐Acetylcysteine Protects Against Diabetic Nephropathy Through Control of Oxidative and Nitrosative Stress by Recovery of Nitric Oxide in Rats,” Nitric Oxide 78 (2018): 22–31, 10.1016/j.niox.2018.05.003. [ DOI ] [ PubMed ] [ Google Scholar ] 33. Ma L., Liu D., Yu Y., Li Z., and Wang Q., “Immune‐Mediated Renal Injury in Diabetic Kidney Disease: From Mechanisms to Therapy,” Frontiers in Immunology 16 (2025): 1587806, 10.3389/fimmu.2025.1587806. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 34. Anand G., Vasanthakumar R., Mohan V., Babu S., and Aravindhan V., “Increased IL‐12 and Decreased IL‐33 Serum Levels Are Associated With Increased Th1 and Suppressed Th2 Cytokine Profile in Patients With Diabetic Nephropathy (CURES‐134),” International Journal of Clinical and Experimental Pathology 7, no. 11 (2014): 8008–8015. [ PMC free article ] [ PubMed ] [ Google Scholar ] 35. Tonacci A., Quattrocchi P., and Gangemi S., “IL33/ST2 Axis in Diabetic Kidney Disease: A Literature Review,” Medicina (Kaunas, Lithuania) 55, no. 2 (2019): 50, 10.3390/medicina55020050. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 36. Singh H., Khadanga S., Goel S. K., et al., “Evaluation of Interleukin‐33 & sST2 Levels in Type‐2 Diabetic Mellitus Patients With or Without Metabolic Syndrome,” Indian Journal of Medical Research 157, no. 5 (2023): 470–476, 10.4103/ijmr.IJMR_1444_19. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 37. Nagashima R. and Iyoda M., “The Roles of Kidney‐Resident ILC2 in Renal Inflammation and Fibrosis,” Frontiers in Immunology 12 (2021): 688647, 10.3389/fimmu.2021.688647. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 38. Matsuzaka Y. and Iyoda M., “The IL‐33/ST2/ILC2 Pathway in Kidney Disease: Balancing Inflammation, Fibrosis, and Repair,” American Journal of Physiology. Cell Physiology 329, no. 3 (2025): C718–C725, 10.1152/ajpcell.00405.2025. [ DOI ] [ PubMed ] [ Google Scholar ] 39. Chen W. Y., Li L. C., Wu Y. H., Yang J. L., and Tzeng H. T., “Emerging Roles of Interleukin‐33‐Responsive Kidney Group 2 Innate Lymphoid Cells in Acute Kidney Injury,” International Journal of Molecular Sciences 21, no. 4 (2020): 1544, 10.3390/ijms21041544. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 40. Gungor O., Unal H. U., Guclu A., et al., “IL‐33 and ST2 Levels in Chronic Kidney Disease: Associations With Inflammation, Vascular Abnormalities, Cardiovascular Events, and Survival,” PLoS One 12, no. 6 (2017): e0178939, 10.1371/journal.pone.0178939. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 41. Nakai K., Umehara M., Minamida A., et al., “Streptozotocin Induces Renal Proximal Tubular Injury Through p53 Signaling Activation,” Scientific Reports 13, no. 1 (2023): 8705, 10.1038/s41598-023-35850-w. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 42. Wang L., Zhou R., Li G., et al., “Multi‐Omics Characterization of Diabetic Nephropathy in the Db/Db Mouse Model of Type 2 Diabetes,” Computational and Structural Biotechnology Journal 27 (2025): 3399–3409, 10.1016/j.csbj.2025.07.037. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 43. Xie K., Ma Y., Din J., et al., “Research and Advances in Mouse Models of Diabetic Nephropathy: A Narrative Review,” BMC Nephrology 26, no. 1 (2025): 511, 10.1186/s12882-025-04432-5. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 44. Lim B. J., Yang H. C., and Fogo A. B., “Animal Models of Regression/Progression of Kidney Disease,” Drug Discovery Today: Disease Models 11 (2014): 45–51. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 45. Giralt‐López A., Molina‐Van den Bosch M., Vergara A., et al., “Revisiting Experimental Models of Diabetic Nephropathy,” International Journal of Molecular Sciences 21, no. 10 (2020): 3587, 10.3390/ijms21103587. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 46. Khan M. S. and Lea J. P., “Kidney and Cardiovascular‐Protective Benefits of Combination Drug Therapies in Chronic Kidney Disease Associated With Type 2 Diabetes,” BMC Nephrology 25, no. 1 (2024): 248, 10.1186/s12882-024-03652-5. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials Table S1: Correlations between biochemical and histological parameters in Group 2. Table S2: Correlations between biochemical and histological parameters in Group 3a. Table S3: Correlations between biochemical and histological parameters in Group 3b. Table S4: Correlations between biochemical and histological parameters in Group 4a. Table S5: Correlations between biochemical and histological parameters in Group 4b. Table S6: Correlations between biochemical and histological parameters in Group 5a. Table S7: Correlations between biochemical and histological parameters in Group 5b. PRP2-14-e70273-s001.docx (18.7KB, docx) Data Availability Statement Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. 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