2.1. Materials

Streptozotocin (STZ, Cat# S0130), Vitamin D (Cat# C9756-5GM), Metformin (Cat# PHR1084-500MG), β-Nicotinamide adenine dinucleotide 2^′-phosphate reduced (NADPH, Cat# N7505), were procured from sigma Aldrich, St. Louis, USA. BCA protein estimation kit (Cat# 23227), Bovine serum albumin (BSA, Cat# 23209) were procured from Thermo Fischer Scientific, Waltham, MA, United States. NAD(P)H: quinone oxidoreductase 1 (NQO1, Cat# PALP69Hu01), VEGF (Cat# MAA143Hu24), TGF-β (Cat# MAB949Hu24), vitamin D receptor (VDR, Cat# sc13133), Superoxide dismutase (SOD, Cat# PAB960Hu01), Nrf2 (Cat # PAL947Hu01) from cloud clone. AMP-activated protein kinase (AMPK, Cat# 2532S). β-actin (Cat# 4967), Mouse anti-rabbit IgG horseradish peroxidase (HRP) (Cat# sc-2357), Goat anti-rabbit IgG HRP (Cat# sc-1004), Goat anti-mouse IgG HRP (Cat# sc-2005) were from Santa Cruz Biotechnology, Inc, Dallas, TX, USA. High throughput SOD assay kit (Cat# 7501-500-K) and high throughput glutathione peroxidase (GPx) assay kit (Cat# 7512-100-K) from R&D systems. Ethylenediaminetetraacetic acid (EDTA, Cat# 40648), RIPA buffer 1× (100 mL) was prepared as detailed and stored at −20˚C. The composition of 1× RIPA buffer –20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% Sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₂VO₄, 1 g/mL leupeptin. Acrylamide (Cat# 22794); bis-acrylamide (Cat# 38516); coomassie blue G-250 (Cat# 64222); EDTA (Cat# 40648); glycine (Cat# 66327); polysorbate 20 (Tween 20) (Cat #23610); sodium lauryl sulfate AR (Cat# #54468); TEMED (Cat# 52145); trichloroacetic acid (Cat# 90544); tris buffer AR (#71033) were procured from SRL – Mumbai, Maharashtra, India. PVDF Membrane from Pall Corporation (Cat# BSP0161), Precision Plus Protein WesternC Blotting Standards (Cat# 1610376), StrepTactin-HRP (Cat# 1610380), Clarity Western enhanced chemiluminescent (ECL) (Cat# 1705061) were procured from Bio-Rad.

2.2. Animal Study

A total of 102 male Wistar rats, aged 7 weeks and weighing 160–180 g, were obtained from the Experimental Animal Centre, Department of Studies in Zoology, University of Mysore, Karnataka, India. The rats were housed in standard laboratory conditions, with a controlled temperature of 25°C, a 12-h light–dark cycle, and unrestricted access to a standard pellet diet (SPD) and water. All the experimental protocols were approved by Animal Ethical Committee of University of Mysore (Approval number UOM/IAEC/14/2022) and this study was conducted as per the Committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines [13]. The study was carried out in compliance with the ARRIVE guidelines.

2.3. Induction of Diabetes

Following overnight fasting, the rats were intraperitoneally (i.p.) injected with a single dose of STZ at a dose of 45 mg/kg body weight (B.W.) to induce diabetes. Rats in the control SPD group received equivalent vehicle injections. Three days after the STZ injection, fasting blood glucose (FBG) levels were measured, and rats with FBG levels > 200 mg/dL were classified as diabetic and selected for subsequent experiments.

2.4. Experimental Design for Strategy 1: Prophylactic Impact of Vitamin D on the Prevention of DN

Following the induction of diabetes, diabetic rats were randomly divided into seven equal groups, except diabetic control group (n = 7 rats per group) to assess the prophylactic effects of vitamin D on the prevention of DN. Experimental groups were as follows: Group 1: Control; Group 2: Diabetic control; Group 3: DN control; Group 4: Diabetic group treated with vitamin D (5000 IU/kg B.W.) [14, 15]; Group 5: Diabetic group treated with vitamin D (8000 IU/kg B.W.) [16]; Group 6: Diabetic group treated with vitamin D and metformin (5000 IU/kg B.W. + 125 mg/kg B.W.) Group 7: Diabetic group treated with vitamin D and metformin (8000 IU/kg B.W. + 125 mg/kg B.W.); Group 8: Diabetic group treated with metformin (125 mg/kg B.W.). A detailed description of the treatment protocols is provided in Table S1. Metformin and Vitamin D were administered orally three times a week starting from the 3^rd week after the induction. Simultaneously, in Group 2 and 3 animals were administered sodium citrate buffer as a procedural control. FBG levels and B.W. were measured every 3 weeks, to monitor the progression of diabetes. Metformin, an antihyperglycemic drug, was used as a positive control. At the end of the 12^th week, Group 3 animals were placed in metabolic cages to collect 24 h urine samples for measuring microalbuminuria. The presence of microalbuminuria (> 30 mg/24 h) confirmed the development of DN in diabetic rats. By the 12^th week, all animals were euthanized by CO₂ asphyxiation followed by cervical dislocation, as recommended by CPCSEA guidelines. Euthanization of rat was performed in a location separate from the room where the rats were housed to minimize anxiety, pain, and distress. Blood was collected via the retro-orbital puncture and along with urine samples for biochemical analysis. The serum samples were stored at −80°C for future biochemical estimations. Following blood collection, the rats were sacrificed by cervical dislocation, and their vital organs (kidney, liver, and pancreas) were collected for further analysis.

2.5. Experimental Design for Strategy 2: Nephroprotective Effects of Vitamin D in DN

In Strategy 2, the nephroprotective effects of vitamin D were evaluated in a therapeutic context, specifically after the onset of DN. DN was confirmed in the experimental model by the presence of microalbuminuria in diabetic rats by the 12^th week of the study. Following DN confirmation, the rats were randomized into six experimental groups, each groups receiving treatment protocols consistent with those used in Strategy 1. In this protocol, however, vitamin D administration began in the 13^th week and continued through until the 21^st week, at which point all animals underwent necropsy for histological and biochemical evaluation. This strategy differs from Strategy 1, where vitamin D was administered as a prophylactic intervention starting from an earlier time point before DN onset. In contrast, Strategy 2 aims to assess the therapeutic efficacy of vitamin D as a postdiagnosis intervention. By initiating treatment after DN has developed, this approach seeks to determine the potential of vitamin D to mitigate progression or reverse damage within the renal tissues in the context of established DN.

2.6. Preparation of Kidney Homogenates

Renal tissues from all rat groups were harvested, perfused with ice-cold saline, and homogenized in RIPA buffer using a probe sonicator (Vibracell-VCX500, Sonics, Sonics & Materials Inc, Newtown, Connecticut, USA). The homogenates were then centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was collected for subsequent assays of antioxidant enzymes, OS markers, and western blot analysis. Total protein levels were quantified using a commercially available kit from Thermo Scientific, with BSA used as the reference standard.

2.7. Assessment of Renal Dysfunction

The levels of serum FBG, lipid profile (total cholesterol, HDL, triglycerides), and renal function markers (urea, creatinine, uric acid, calcium, phosphorus, and BUN) were estimated using commercially available Roche kits, according to the manufacturer’s protocol on a fully automatic Cobas c501 analyzer (Roche, USA). Serum vitamin D levels were determined using a Cobas 601 immunoassay analyzer (Roche, USA). Microalbuminuria (24-h urine) were quantified from the collected urine samples using a Cobas c501 chemistry analyzer (Roche, USA).

2.8. Renal OS Markers

Renal OS was assessed by measuring the levels of nitric oxide (NO) and hydrogen peroxide (H₂O₂) in kidney homogenates. NO levels were quantified through the Griess assay by measuring nitrite accumulation, a stable NO metabolite. In this assay, nitrite reacts with the Griess reagent to form a purple azo dye, with absorbance measured at 540 nm against a sodium nitrite standard curve [17]. H₂O₂ levels were assessed using the ferrous oxidation-xylenol orange (FOX1) assay. In this protocol, 10 μL of kidney homogenate, normalized to 20 μg of protein, was mixed with 190 μL of FOX1 reagent. The mixture was thoroughly mixed and incubated at room temperature (RT) for 30 min. After incubation, the absorbance was measured at 560 nm to determine the concentration of H₂O₂.

2.9. Renal Antioxidant Parameters

SOD activity in kidney homogenate samples was measured using the R&D Systems high throughput SOD assay kit. Protein concentrations were normalized to 50 µg, 25 µL of serially diluted standards, and samples were loaded into a 96-well plate. SOD activity was determined by measuring absorbance at 450 nm every minute for 10 min at RT using a multimode plate reader (Enspire 2300, PerkinElmer, Waltham, MA, USA). Results were calculated according to the manufacturer’s protocol. GPx activity was measured in kidney homogenates samples using the R&D Systems high throughput GPx assay kit. A total of 20 µL of GPx standards and protein samples normalized to 50µg were added to designated wells. GPx activity was determined by measuring absorbance at 340 nm every 30 s over 30 min. Results were calculated according to the manufacturer’s protocol. Glutathione reductase (GR) activity in kidney homogenate was measured by monitoring the oxidation of NADPH. For the assay, 20 μL of the sample, normalized to 50 μg of protein, was added to 200 μL of assay buffer containing 1 mM glutathione disulfide (GSSG), 0.2 mM NADPH, and 0.1 M potassium phosphate buffer with 2 mM EDTA. After mixing the contents, the decrease in absorbance at 340 nm was monitored over 2 min, as the reduction of GSSG to Glutathione (GSH) consumes NADPH, resulting in a proportional decrease in absorbance, indicative of GR activity.

2.10. Western Blot

Protein expression in kidney homogenates was assessed by Western blotting. Samples were separated using 12% SDS-PAGE, with 25 μg of total protein loaded per lane. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% nonfat skimmed milk in 1× Tris-buffered saline (TBS) for 2 h at RT. After blocking, the membrane was incubated overnight at 4°C with primary antibodies targeting VDR, NQO1, VEGF A, TGF-β, AMPK, SOD superoxide dismutase, and β-actin. Following primary antibody incubation, the membrane was washed three times with 1× TBS-Tween (TBST) for 10 min each. Secondary antibody incubation was performed for 2 h at RT on the gel rocker (Compact Digital Rocker, China). After washing, the membrane was developed using an ECL reagent (Biorad, USA). Protein bands were visualized using a chemiluminescent gel documentation system (Alliance Q9, UK) and band intensities were quantified with ImageJ software. Results were expressed as fold changes by normalizing the densitometric values of the test samples to β-actin (control).

2.11. Renal Histopathological Examination

Renal tissue samples were fixed in 10% formalin and dehydrated in a graded series of ethanol. The tissues were embedded in paraffin and sectioned into 4 μm slices. These sections were stained with hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and Masson’s trichrome stain (MTS) for histopathological examination under a light microscope. The histopathological evaluations were conducted in a blinded manner to minimize any observer bias.

2.12. Renal Immunohistochemistry (IHC)

Renal tissues were sectioned into 4 μm slices, deparaffinized, and permeabilized by incubation at 65°C in 0.1 M sodium citrate for 2 h. To block endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxides for 15 min. Following this, the slides were incubated in a 100°C water bath for 10 min in 0.01M phosphate-buffered saline (PBS). Tissue sections were blocked with 2% BSA for 30 min. Primary antibodies targeting VDR, Nrf2, TGF-β, and VEGF were applied followed by overnight incubation at 4°C. After washing, the sections were incubated with HRP-conjugated secondary antibody at 37°C for 1 h. Visualization was performed using a 3,3′-diaminobenzidine (DAB) substrate for 4 min. Observations and images were captured under a light microscope. All IHC assessments were performed by who were blinded to the treatment groups to ensure unbiased results.

2.13. Statistical Analysis

Given the small sample size (n = 7 per group), normality was assessed using visual methods, including histograms and boxplots, and was further confirmed using the Shapiro–Wilk test. Values are represented as mean ± SE for normally distributed variables. Comparisons between multiple groups were performed using a one-way analysis of variance (ANOVA). When ANOVA indicated significant differences, Tukey’s post hoc test was used for pairwise comparisons to identify specific group differences.

Western blot data were analyzed using densitometry, and protein expression differences between groups were compared using one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons. For histopathological and IHC analysis, a semiquantitative scoring system was applied to assess the degree of renal damage, and comparisons between groups were performed using the chi-square test or Fisher’s exact test for categorical data.

All statistical analyses were conducted using Graph Pad Prism 9.0 (Graph Pad Software, La Jolla, CA, USA) or SPSS version 25 (IBM Corp., Armonk, NY, USA). A p − value  < 0.05 was considered statistically significant, with Bonferroni correction applied for multiple comparisons where necessary.

3.1. Impact on B.W.

During the 12-week experiment, STZ-induced diabetic rats exhibited significant weight loss compared to a control group. By the end of the 12^th week, the B.W. of rats treated with vitamin D, either alone or in combination with metformin, was maintained at a higher level compared to the DN control group (Figure 1a,b). Similarly, in the 21-week experiment, STZ-induced DN rats exhibited significant weight loss compared to normal rats. However, by the end of the 21^st week, the B.W. of rats treated with Vitamin D, either alone or in combination with metformin, showed a significant increase compared to the DN control group. This indicated that vitamin D and metformin have a beneficial effect on B.W. maintenance in the context of DN (Figure 1c, d). A detailed week by week B.W. profile for all groups is provided in the Supporting Information (Figure S1a, b).

3.2. FBG Levels

The administration of STZ resulted in a significant increase in FBG levels in DN control groups compared to the control group throughout the 12-week study. However, by the end of the 12^th week, STZ-induced diabetic rats treated with 5000 and 8000 IU of vitamin D showed a decrease (− 49.8%) in FBG levels, while the other groups exhibited a nonsignificant reduction (Figure 2a, b). Similarly, in the 21-week study, STZ-induced DN rats displayed significantly elevated FBG levels compared to the control group. By the 21^st week, rats treated with 8000 and 5000 IU of vitamin D, either alone or in combination with metformin, showed a significant decrease (− 50%) in FBG levels compared to the DN control group (Figure 2c,d). A detailed week by week FBS profile for all groups is provided in Figure S2a, b.

3.3. Assessment of Microalbuminuria

During the 12-week experiment, 24-h urine samples were collected from all experimental groups in the 3^rd week, before initiating treatment with vitamin D or the combination of vitamin D and metformin. Microalbuminuria levels key marker for DN were measured at the 12^th week to assess disease progression, By this point, rats in the DN control group had progressed to nephropathy stage, with microalbuminuria levels reaching 30 mg/L, indicating the onset of renal damage. After confirming the development of nephropathy, the treatment was stopped. However, the remaining groups treated with vitamin D, either alone or in combination with metformin, did not reach the DN stage, demonstrating the protective effects of these treatments against the progression of kidney damage (Figure 3a, b).

In the 21-week experiment, 24-h urine samples were collected from all experimental groups in the 12^th week to confirm microalbuminuria levels, which served as an indicator of kidney damage in the DN control group. Following this confirmation, treatment with vitamin D and the combination of vitamin D and metformin was initiated. By the end of the 21^st week, microalbuminuria levels were effectively stabilized in both the vitamin D and the vitamin D with metformin treatment groups, showing marked improvement compared to the DN control group. This suggests that these treatments mitigated further kidney damage and helped maintain renal function, highlighting their potential therapeutic benefit in managing DN (Figure 3c, d).

3.4. Serum Profiles

This study demonstrates significant alterations in lipid profiles in diabetic and DN control groups. Cholesterol levels were elevated in the diabetic group compared to controls but decreased in the DN control group. Vitamin D supplementation, at both 5000 and 8000 IU, resulted in a dose-dependent reduction in cholesterol levels, with the most pronounced effect observed in the 8000 IU vitamin D combined with the 250 mg metformin group. Triglyceride levels, which were elevated in the diabetic control group, were significantly reduced (− 50%) following treatment with 8000 IU vitamin D, both alone and in combination with metformin. Notably, no significant changes were observed in HDL levels across all groups. Overall, these findings suggest that vitamin D, particularly at 8000 IU and when combined with metformin, exerts beneficial effects on lipid metabolism in diabetic and DN models, potentially enhancing nephroprotective outcomes by improving the lipid profile. This may play a key role in mitigating cardiovascular risk factors associated with DN (Supporting Information; Table S2 and Table S3; Lipid profile).

In the 12^th week of the study, phosphorus levels decreased in the diabetic group compared to controls but increased in the DN control group. However, treatment with vitamin D 5000 IU combined with 250 mg metformin significantly reduced phosphorus levels compared to both control and DN control groups. By the 21^st week, phosphorus levels had decreased in all treatment groups except for the metformin-treated group, where phosphorus levels remained similar to controls. Despite these alterations in phosphorus, no significant changes were observed in calcium, urea, uric acid, creatinine, or BUN levels across any of the groups. Uric acid levels, however, significantly decreased in all treated groups, while BUN levels showed a slight increase across all groups, except for the 8000 IU vitamin D combined with 250 mg metformin group, where BUN levels remained comparable to the control, diabetic, and DN control groups. The stability of calcium, urea, and BUN levels suggests that renal function and overall metabolic status were maintained throughout the study, explaining the lack of significant differences in these parameters. Metformin’s ability to maintain phosphorus levels close to control values may be related to its influence on glucose and lipid metabolism, which could indirectly stabilize phosphate handling in the kidneys. Additionally, metformin’s role in modulating phosphate transport and inhibiting phosphate reabsorption in renal cells, explains its influence on phosphorous homeostasis without disrupting overall renal or metabolic function (Supporting Information; Table S2 and Table S3; renal function markers).

In diabetic conditions, serum vitamin D levels progressively declined compared to Controls. This reduction in vitamin D levels correlated with the exacerbation of DN. Upon administration of vitamin D supplementation, there was a noticeable increase in serum vitamin D levels compared to the control group. This supplementation not only normalizes vitamin D status but also potentially mitigates the progression of DN by modulating the associated pathophysiological mechanisms (Supporting Information; Table S2; and Table S3; serum vitamin D levels).

3.5. OS Markers

Our study demonstrates that OS, characterized by elevated H₂O₂ levels, and plays a key role in the pathogenesis of diabetes and DN. In kidney homogenates, H₂O₂ levels were significantly increased in both diabetic and DN control groups compared to controls, reflecting heightened OS. However, treatment with vitamin D (5000 and 8000 IU) combined with metformin 250 mg, as well as metformin alone, effectively reduced (− 36.84%) H₂O₂ levels, with the most notable normalization observed in the group treated with vitamin D 8000 IU and metformin. This suggests that these interventions possess antioxidant properties capable of mitigating OS in diabetic and DN conditions (Supporting Information; Table S2; Renal function markers). Similarly, NO levels were significantly elevated in both diabetic and DN control groups compared to controls, indicating dysregulated NO metabolism. While all treatment groups exhibited increased NO levels compared to controls, the combination of vitamin D 8000 IU with metformin, and metformin alone significantly reduced (−14.29%) NO levels in a dose-dependent manner, bringing them closer to normal levels. These findings indicate that the combination of vitamin D and metformin exerts a dual modulatory effect by reducing both H₂O₂ and NO levels, thereby offering protection against oxidative damage in DN (Supporting Information; Table S3; Renal function markers).

3.6. Antioxidant Defense Mechanism

The antioxidant defense mechanisms, involving key enzymes, such as GR, SOD, and GPx were significantly compromised in both diabetes and DN control groups, as evidenced by a significant reduction in their activity. However, treatment with vitamin D and metformin significantly restored and enhanced the activity of these enzymes across all treated groups, with increases of GR (+ 250%), SOD (+ 11.33%), and GPx (+ 62.83%). These findings suggest an improved cellular antioxidant response, contributing to the protective effects observed against OS and DN (Supporting data; Table S2 and Table S3 Antioxidant defense).

3.7. Modulation of Angiogenesis, Fibrosis, and OS by Vitamin D and Metformin in DN

In this western blot study investigating the prophylactic (Figure 4) and nephroprotective effects (Figure 5) of vitamin D on DN, we examined how vitamin D (5000 and 8000 IU) alone and in combination with metformin influenced key protein markers in an STZ-induced diabetic and DN rat model. In DN control rats, VEGF dimer expression was elevated, indicative of abnormal angiogenesis associated with DN. Treatment with the combination of vitamin D 5000 IU and metformin reduced VEGF levels, suggesting a therapeutic reduction in aberrant angiogenic activity. However, treatment with vitamin D at 8000 IU combined with metformin led to an increase in VEGF expression, likely due to dose-dependent feedback effect on angiogenic pathways. This differential response highlights the nuanced effect of vitamin D dosage on angiogenesis in DN. TGF-β, a maker associated with fibrosis, was elevated across all diabetic and DN control groups. However, significant reductions were observed in the group receiving 8000 IU vitamin D with metformin, indicating an antifibrotic benefit at higher vitamin D doses in combination with metformin. This finding indicates a potential therapeutic advantage of the higher dose for mitigating fibrotic progression in DN. In the prophylactic approach, VDR expression was upregulated in response to vitamin D supplementation, likely as a compensatory mechanism to diabetic rats. However, in the nephroprotective strategy, VDR levels were lower in DN control group compared to treated groups. Treatment with vitamin D and metformin improved VDR expression, suggesting that this combination might enhance receptor activity and vitamin D pathway engagement in the nephroprotective context. Antioxidant markers SOD and NQO1, were initially low in untreated diabetic rats, indicating impaired oxidative defense mechanisms. Treatment with vitamin D and metformin led to increased expression of these antioxidant markers, reflecting an enhancement in oxidative defense and potential protective effects against oxidative damage in DN. AMPK, a marker of metabolic stress, was elevated in the untreated diabetic controls, consistent with increased metabolic strain. The combination of vitamin D and metformin, especially at 8000 IU dose, was associated with reduced AMPK expression, suggesting an alleviation of metabolic stress under treatment. These findings highlight the differential effects of vitamin D in both prophylactic and nephroprotective contexts, with a notable synergy observed when combined with metformin. The results suggest that vitamin D, especially in combination with metformin, may offer protection against DN progression through modulation of key pathways involved in angiogenesis, fibrosis, OS, and metabolic regulation.

3.8. Renal Histopathological Evaluation of the Effects of Vitamin D and Metformin

Histological and staining analyses revealed distinct renal morphological changes across the experimental groups. The control group exhibited normal kidney morphology, while the diabetic control showed moderate tubular degeneration and fibrosis. The DN control group displayed severe fibrosis, inflammation, and marked nephropathic changes. Treatment with 5000 IU of vitamin D resulted in mild tubular degeneration, but overall normal glomerular structure was maintained. In contrast, 8000 IU of vitamin D led to moderate tubular changes and mild cystic dilation. The combination of vitamin D and metformin demonstrated a protective effect, with several samples maintaining normal renal morphology and others exhibiting only mild inflammation. Treatment with metformin alone resulted in moderate tubular degeneration. PAS staining revealed improved glycogen accumulation and reduced PAS-positive staining in the treated groups. The combination of vitamin D and metformin notably reduced fibrosis compared to the diabetic and DN control groups, as shown in Figure 6. Overall, the combination therapy of vitamin D and metformin, particularly at higher doses effectively mitigated renal damage and fibrosis.

Histological analyses showed significant variations in renal morphology and pathology among the groups. While the control group exhibited normal kidney architecture, both diabetic and DN control groups displayed severe inflammation, tubular degeneration, and nephritis, which are indicative of advanced nephropathy. Treatment with vitamin D, particularly when combined with metformin, demonstrated promising protective effects in mitigating renal damage and promoting regeneration. These treatments maintained or restored normal glomerular and tubular structures, and also reduced pathological changes, such as fibrosis and sclerosis, as shown in Figure 7. Supplementation with vitamin D and metformin was particularly effective in attenuating these pathological alterations, suggesting their potential therapeutic benefits in ameliorating diabetic kidney disease by preserving renal function and reducing structural damage.

3.9. Prophylactic Effects on Molecular Targets in DN

Vitamin D supplementation, both alone and in combination with metformin, demonstrated significant modulatory effects on key molecular targets associated with DN shown in Figure 8. Treatment with 5000 IU of vitamin D resulted in mild VDR expression, while 8000 IU of vitamin D led to moderate VDR expression in damaged renal tubules. The combination of vitamin D with metformin further enhanced VDR expression, suggesting a synergistic effect that could support renal defense mechanisms against DN development. In terms of VEGF expression, vitamin D 5000 IU combined with metformin showed moderate VEGF expression in regions of inflammation, whereas vitamin D 8000 IU with metformin exhibited a mild VEGF expression profile, suggesting that early intervention with this combination might temper pathological angiogenesis more effectively. Notably, TGF-β expression was significantly reduced in the groups treated with 8000 IU of vitamin D, both alone and in combination with metformin, indicating a reduction in inflammation and fibrosis compared to the DN control group. Additionally, Nrf2 expression, which was absent in the control group, was significantly upregulated in all treated groups, indicating an enhanced antioxidant response that may fortify renal tissue against OS in DN.

3.10. Therapeutics Effects on Molecular Targets in DN

The IHC reports highlight the dynamic effects of various treatments on key molecular targets implicated in DN. VDR expression, which is significantly diminished in diabetic conditions and absent in DN, was partially restored following treatment with vitamin D, particularly in combination with metformin. This restoration suggests that vitamin D and metformin may enhance receptor activity in damaged kidneys, promoting renal resilience post- DN diagnosis. Additionally, VEGF expression, which is elevated in diabetic conditions, was modulated by vitamin D and metformin treatments in a dose-dependent manner. This suggests that vitamin D and metformin may exert a dose-dependent modulation of VEGF, potentially aiding in the control of pathological angiogenesis in established DN. Furthermore, TGF-β1, a cytokine elevated in DKD and linked to fibrosis and tissue remodeling, was notably downregulated by the combined vitamin D and metformin therapy, indicating a therapeutic benefit in mitigating fibrosis and chronic inflammation. Lastly, the significant modulation of Nrf2 expression in the treated groups suggests that these interventions enhance the kidney’s antioxidant defense mechanisms and shield against oxidative damage, as shown in Figure 9.

These findings underscore that vitamin D and metformin have distinct roles in both prophylactic and therapeutic settings. By modulating key molecular markers like VDR, VEGF, TGF-β1, and Nrf2 these treatments provide potential benefits in slowing DN progression and improving renal function, whether used to prevent or treat established DN.