We established 2 mouse models of diabetes: leptin receptor-deficient db/db diabetic mice or STZ-induced diabetic mice. All animal procedures were carried out in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Shenyang Medical College (SYYXY2021032301). Ten-weeks-old male db/db mice on a C57BLKS/J background were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China) and housed under specific pathogen-free conditions in the Laboratory Animal Center of Shenyang Medical College (Shenyang, Liaoning, China), control mice were littermate heterozygous (db/+). After a 2-week acclimatization period, db/db mice or db/m mice were divided into three groups: a control group (control, n = 10), a db/db diabetic group (db/db, n = 10) and db/db mice treated with MR409 group (db/db + MR409, n = 10). All group mice were fed a regular mouse diet. The mice on db/db + MR409 group received subcutaneous injection of MR-409 at 15 μg/mouse/day, with MR409 dissolved in a 10% of 1, 2-propanediol solution. This dose was selected based on our prior studies demonstrating its efficacy in treating diabetic vascular complications and chronic ischemic infarction without adverse effects in mice (Liu et al., 2021; Ren et al., 2023; Liu et al., 2025). The mice in control group and db/db group were subcutaneously injected with an equivalent volume of vehicle solution.

To establish STZ-induced type II diabete or DN model, 10-week-old C57BL/6J mice were fed a high-fat diet (45% kcal fat, 20% kcal protein and 35% kcal carbohydrates) for 2 weeks. Mice then received intraperitoneal injection of STZ at 50 mg/kg/day dissolved in citrate buffer for five consecutive days while maintaining the high fat diet. Control mice received equivalent volume of citrate solution injection and were fed a regular mouse diet (17% kcal fat). Fasting blood glucose levels were monitored every 3 days post-STZ injection until levels exceeded 250 mg/dL, confirming the induction of diabetes. Diabetic mice were randomized into two groups: the STZ diabetic group and STZ + MR409 group. MR409 administration followed the same protocol as described for db/db mice. Throughout the experiment, both STZ and STZ + MR409 groups remained on the high-fat diet, while the control group continued the regular mouse diet.

Body weight was measured weekly, while fasting blood glucose was assessed biweekly using an automatic blood glucose monitoring system (Roche Accu-CHEK Active, Mannheim, Germany). Urine samples were obtained by gently compressing the mouse bladder over a metal plate to stimulate urination. Urine albumin was quantified using the Bio-Rad protein assay (Beyotime Biotech, Shanghai, China), and urine levels of creatinine were determined with a creatinine assay kit (Nanjing Jiangchen Bioengineering Institute Co, Nanjing, China), respectively, in accordance with the manufacturer’s protocols. The urine albumin excretion was expressed as albumin-to-creatinine ratio.

At the end of the study, mice were subjected to an overnight fasting period and subsequently anesthetized with a combination of 100 mg/kg ketamine and 20 mg/kg xylazine. Following anesthesia, the thoracic cavity was promptly opened, and blood samples were harvested through left ventricular puncture. These blood samples were used to measure plasma levels of total cholesterol (TC) and total triglycine (TG). The kidneys were excised, weighted, and the renal index (mg/g) was calculated as the ratio of kidney weight (mg) to body weight (g), Then renal tissue samples were snap frozen with liquid nitrogen and stored in −80 °C freezer.

Serum levels of total TC and TG were quantified using the GPO-PAP enzymatic method and COD-PAP method, respectively. The serum GH concentration was measured using a mouse GH ELISA Kit (CUSABIO, Wuhan, China), according to the manufacturer’s instructions. In brief, samples were incubated in microtitration wells pre-coated with an anti-mouse GH antibody. A standard curve for the assay was generated using reference samples supplied by the manufacturer. The optical density measurements were obtained using a microplate reader.

Kidney tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 3 μm. Following deparaffinization and hydration, the sections were stained with hematoxylin-eosin (H&E) for general histological evaluation, periodic acid-Schiff (PAS) to assess glomerulosclerosis and glycogen deposition, and Masson’s trichrome and Sirius red stains to evaluate collagen deposition and interstitial fibrosis, respectively. Quantitative assessment of the fibrotic areas was performed by measuring the positive stained areas in Masson’s trichrome and Sirius red stained sections using the ImageJ software (NIH, United States). Similarly, glycogen deposition was quantified by analyzing the positively stained areas in PAS-stained sections with the same software. All procedures were conducted in accordance with established protocols and standardized methodologies.

Renal ROS was determined by dihydroethidium (DHE) fluorescence oxidative staining, as previously described with modifications (Ren et al., 2023). Briefly, paraffin-embedded tissue sections were deparaffinized and rehydrated using standard protocols. The sections were then incubated with a DHE fluorescent probe (Sigma Aldrich; 37,291), prepared by dissolving in dimethyl sulfoxide (DMSO), at a concentration of 10 µM. Incubation was carried out at 37 °C in a light-protected environment for 30 min to allow for the oxidation of DHE by intracellular ROS. After incubation, the samples were rinsed with PBS, and stained sections were stored at 4 °C, with fluorescence quantification was completed within 30 min. Fluorescence signals were visualized and captured using an inverted fluorescence microscope equipped with appropriate excitation (488 nm) and emission (610 nm) filters. Image analysis was performed using ImageJ software to quantify fluorescence intensity per unit square area, which correlates with ROS levels (Xia et al., 2024).

The GSH content in the kidney tissues was examined using the Micro-reduced GSH Assay Kit (BC1175, Solarbio). As the manufacturer’s construction, and the content of each sample was identified with a microplate reader at the wavelength at 412 nm. The GSH levels were normalized by protein amount in kidney homogenates.

To evaluate GHRHR expression in kidney tissues, immunofluorescence staining was performed. Paraffin-embedded tissue sections were deparaffinized and rehydrated using standard protocols. Antigen retrieval was conducted by incubating the sections in sodium citrate buffer (pH 6.0) at 95 °C for 20 min. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The sections were then incubated overnight at 4 °C with a primary antibody against GHRHR (Abcam, Cambridge, MA, United States; 1:200 dilution). After washing with PBS, the sections were incubated with an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, United States) for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, United States). Fluorescence signals were visualized and analyzed using a confocal laser scanning microscope (Leica or Zeiss, Germany).

To assess ferroptosis markers, immunohistochemical staining for malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) was performed. Deparaffinized and rehydrated tissue sections underwent antigen retrieval using sodium citrate buffer (pH 6.0) at 95 °C for 20 min. Endogenous peroxidase activity was quenched by incubating the sections with 3% hydrogen peroxide (H₂O₂) for 10 min at room temperature. The sections were then incubated overnight at 4 °C with primary antibodies against MDA (ab243066, Abcam, Cambridge, MA, United States; 1:200 dilution) and 4-HNE (ab48506, Abcam, Cambridge, MA, United States; 1:200 dilution), followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories, United States) for 10 min at room temperature in the dark. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB), followed by counterstaining with hematoxylin. Stained sections were analyzed, and images were captured using a light microscope (Nikon or Olympus, Japan).

Total protein was extracted from kidney tissues using RIPA lysis buffer supplemented with protease inhibitors. Protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, United States). Equal amounts of protein (20 μg) were separated by appropriate concentration of SDS-PAGE gel and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes via electroblotting. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature to prevent nonspecific binding. Following blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-p22phox (sc-271262), anti-Fibronectin (sc-271098), and anti-TGFβ1 (sc-146) from Santa Cruz Biotechnology; anti-phospho-endothelial nitric oxide synthase (eNOS at Ser1177, AF3247) from Affinity Biosciences; anti-peroxisome proliferator-activated receptors γ (PPARγ, #2435) from Cell Signaling Technology; anti-gp91phox (ab80508), anti-GHRHR (ab76263) and anti-Klotho (ab181373) from Abcam; and anti-β-actin (AC004) from AB clonal. After incubation, the membranes were washed three times with TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, United States) and quantified using ImageJ software.

Total RNA was isolated from mouse kidney tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. RNA integrity and quality were assessed using an Agilent 2,100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States) and verified by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was subsequently enriched from the total RNA using Oligo (dT) magnetic beads, while prokaryotic rRNA was depleted using the Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, United States). The enriched mRNA was fragmented into short segments using a fragmentation buffer and reverse-transcribed into first-strand cDNA using random primers. Second-strand cDNA synthesis was performed using DNA polymerase I, RNase H, dNTPs, and reaction buffer. The resulting cDNA fragments were purified using the QiaQuick PCR extraction kit (Qiagen, Venlo, Netherlands), followed by end repair, poly(A) tailing, and ligation to Illumina sequencing adapters. The adapter-ligated cDNA libraries were size-selected by agarose gel electrophoresis, amplified by PCR, and subjected to high-throughput sequencing on the Illumina HiSeq2500 platform (Illumina, San Diego, CA, United States) at Gene Denovo Biotechnology Co. (Guangzhou, China). Sequencing data were processed to quantify gene abundance, followed by pathway enrichment analysis and Gene Ontology (GO) functional enrichment analysis of differentially expressed genes. These analyses were conducted using established bioinformatics pipelines and software tools.

Statistics analysis was carried out with SPSS 24.0 and GraphPad Prism 8. The results from at least three independent experiments were expressed as mean ± SEM. Comparisons between different groups were performed using one-way ANOVA followed by Fisher’s LSD for multiple comparisons between groups. A p value <0.05 was considered statistically significant.

To evaluate the effects of MR409 on renal function, renal parameters and blood glucose level were measured in db/db diabetic mice. Blood glucose level was significantly elevated in db/db mice; however, treatment with MR409 did not alter blood glucose level (Figure 1A). Compared to control mice, db/db mice exhibited significantly higher serum levels of total TC, TG, and the urine albumin-to-creatinine ratio. These metabolic and renal abnormalities were mitigated following MR409 treatment (Figures 1B–D). The kidney index, calculated by the ratio of kidney weight to body weight, was markedly reduced in db/db mice compared to control mice, but was normalized by MR409 administration (Figure 1E).

Furthermore, renal histopathology was evaluated using H&E and PAS staining in both db/db and STZ-induced diabetic mice. H&E staining revealed a well-organized architecture of glomeruli, renal tubules, and mesangium with intact basement membranes in normal control mice. In contrast, db/db and STZ diabetic mice displayed glomerular dilatation, increased mesangial matrix deposition, cystic wall cell proliferation, and partial renal tubular atrophy. These pathological changes were ameliorated by MR409 treatment (Figures 2A,D). PAS staining indicated a significant increase in the percentage of glomerular sclerosis in db/db and STZ mice, which was reduced by MR409 (Figures 2B,C,E,F).

To investigate the effects of MR409 on renal fibrosis in both db/db and STZ-induced diabetic mice. Masson’s trichrome staining and Sirius red staining were used to assess the extent of collagen deposition. Both methods revealed a significant increase in the positively stained areas for collagen fibers in db/db and STZ diabetic mice (Figures 3A–H). However, these changes were attenuated following treatment with MR409 (Figures 3A–H). Given the established role of the TGFβ1 signaling pathway in the pathogenesis of renal fibrosis (Liu et al., 2019), the expression levels of TGFβ1 and its downstream effector, Fibronectin, were determined in the kidneys of db/db mice by Western blot. The results indicated a marked upregulation of renal TGFβ1 and Fibronectin, which was significantly downregulated by MR409 treatment (Figures 3I,J). Collectively, these findings demonstrate that MR409 improves renal function and structural damage in diabetic mice.

As MR409 is a potent GHRHR agonist analog, its effects on the expression of GHRHR and GH levels were evaluated. Immunostaining revealed a significant reduction in the relative fluorescence intensity of GHRHR in the kidneys of db/db mice, which was partially restored following MR409 treatment (Figures 4A,B). Consistent with this finding, Western blot analysis demonstrated a marked downregulation of renal GHRHR expression in db/db mice, which was upregulated by MR409 administration (Figure 4C). Notably, GH levels remained unchanged across control, db/db mice, and MR409-treated db/db mice (Figure 4D). These findings indicate that MR409 enhances renal GHRHR expression in db/db mice without influencing circulating GH levels.

Renal ROS generation was assessed using DHE staining, this staining demonstrated significantly increased fluorescence intensity in the kidney of db/db mice compared to control, and an effect attenuated by MR409 treatment (Figures 5A,B). Given NADPH oxidase is a main source of renal ROS production, we evaluated the expressions of its membrane-bound subunits gp91phox and p22phox. Western blot analysis showed significant upregulation of both subunits of gp91phox and p22phox in the kidneys of db/db mice, which was attenuated by MR409 treatment (Figures 5C,D). These data demonstrate that MR409 attenuates renal oxidative stress in db/db mice.

To elucidate the mechanism underlying MR409-mediated renoprotection in db/db mice, we conducted a broad-spectrum gene screening in kidney tissues using RNA-sequencing analysis. KEGG pathway enrichment analysis revealed the top 20 enriched pathways, with the ferroptosis-related pathway being significantly enriched in MR409-treated db/db mice compared to untreated db/db mice (Figure 6A).

Ferroptosis, a distinct form of regulated cell death characterized by iron overload and accumulation of lipid peroxides, has been implicated in the pathogenesis of DN (Li et al., 2024). We measured the iron content and observed a significant increase in the kidney of db/db mice, which was downregulated by MR409 treatment (Figure 6B). Western blot analysis demonstrated downregulation of ferroptosis suppressors glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) (Figs. C–E), alongside upregulation of the ferroptosis promoter transferrin receptor (TFR) in the kidney of db/db mice (Figs C &F). These alterations were partially reversed by MR409 treatment (Figures 6C–F). Given the established role of nuclear factor erythroid 2-related factor (NRF2) signaling pathway in regulating ferroptosis (Dodson et al., 2019). We assessed NRF2 levels and observed a significant reduction in NRF2 levels in DN mice, which was rescued by MR409 treatment (Figure 6G). Furthermore, MR409 restored depleted renal GSH content in db/db mice (Figure 6H). Critically, MDA and 4-HNE represent two important biomarkers of lipid peroxides and ferroptosis. The expressions of MDA and 4-HNE were significantly increased in the kidney of db/db mice, and these changes were significantly reduced by MR409 treatment (Figures 7A–D). Collectively, these findings demonstrate that MR409 attenuates renal injury and oxidative stress in db/db mice, likely through the inhibition of ferroptosis.

Klotho, an anti-aging protein downregulated in DN, exerts protective effects in DN pathogenesis (Wu et al., 2022). Recent evidence indicates that Klotho may mitigate DN by inhibiting oxidative stress and ferroptosis (Qian et al., 2018). Consistent with these reports, we observed a significant reduction in Klotho expression in the kidneys of db/db mice, which was upregulated by MR409 treatment (Figure 8A). Furthermore, p-eNOS was elevated in the kidneys of db/db mice and further enhanced by MR-409 (Figure 8B). Given that Klotho is a transcriptional target of PPARγ, a ligand-activated nuclear transcription factor (Zhang et al., 2008). We assessed PPARγ expression and found that MR409 upregulated the expression of PPARγ in the kidneys of db/db mice (Figure 8C). These results indicates that PPARγ/Klotho axis, potentially contributing to MR409-mediated inhibition of oxidative stress and ferroptosis.