PRPF19 mediates the proteasomal degradation of VDR to exacerbate ferroptosis in diabetic nephropathy

PRPF19 mediates the proteasomal degradation of VDR to exacerbate ferroptosis in diabetic nephropathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PRPF19 mediates the proteasomal degradation of VDR to exacerbate ferroptosis in diabetic nephropathy Qiongyao He, Wu He, Yanlin Ren, Wenbin Wu, Hui Dong, Gang Yuan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6083539/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 May, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted 8 You are reading this latest preprint version Abstract Ferroptosis, an iron-dependent form of programmed cell death, is closely associated with tubular damage in diabetic nephropathy (DN). Glutathione peroxidase 4 (GPX4) is an important anti-oxidant enzyme, and plays a crucial role in protecting against ferroptosis. However, the regulatory mechanism of GPX4 expression levels in renal tubular epithelial cells (RTECs) remains elusive. This study reveals that ferroptosis occurs in the late-stage of DN, and the GPX4 level is significantly downregulated in DN patients, animal models and cell models. By applying database predictions, luciferase reporter assays and chromatin immunoprecipitation, we find that vitamin D receptor (VDR) transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs. VDR knockout exacerbates ferroptosis in RTECs and worsens renal function, while intraperitoneal injection of VDR agonist paricalcitol significantly improves renal injury. Proteomics analysis suggests that E3 ligase PRPF19 mediates ubiquitination degradation of VDR and is an important therapeutic target for DN. Therefore, through molecular docking, targeted fishing technology using high-performance affinity beads, and surface plasmon resonance (SPR), we screen and identify berberine (BBR) as a novel inhibitor of PRPF19, which offers renal protection by inhibiting VDR degradation and tubular ferroptosis. These findings elucidate the role of ferroptosis in DN renal tubular injury, and suggest that PRPF19 is a promising therapeutic target. Ferroptosis Diabetic nephropathy Renal tubule VDR PRPF19 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction DN is a common and serious microvascular complication of diabetes mellitus (DM), and it is the leading cause of end-stage renal disease[ 1 ]. Regarding the pathogenesis of DN, the “glomerulocentric view” has been dominant for a long time. However, accumulating evidence indicates that renal tubular injury also plays a key role in the pathogenesis of DN, and tubulopathy may precede glomerular alterations[ 2 – 4 ]. The renal proximal tubular cells are uniquely susceptible to a variety of metabolic and hemodynamic factors associated with diabetes, especially to hyperglycemia, leading to enhanced O 2 consumption and increased hypoxic tubular damage[ 5 ]. Impaired tubular uptake explains albuminuria in early DN[ 6 ], and tubular damage is a reliable predictor of renal functional deterioration[ 7 – 9 ]. Furthermore, protective effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors validate the proximal tubule cell as a target of therapy[ 10 , 11 ]. Better understanding of the pathobiology of renal tubular injury will lead to identification of novel therapeutic targets for the treatment of DN. Ferroptosis is a form of regulated cell death characterized by iron-dependent membrane lipid peroxidation[ 12 – 14 ]. Remarkably, accumulating evidence shows that ferroptosis in RTECs is an important driver for the progression of DN[ 15 – 17 ]. The glutathione (GSH)-dependent lipid hydroperoxidase GPX4 prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols[ 18 , 19 ], which is one of the key members in ferroptosis defense systems[ 20 ]. Yet, the specific regulatory mechanism of GPX4 expression level in RTECs remains largely undefined. Some studies have reported that GPX4 is reduced via ubiquitin-proteasomal degradation during ferroptosis in RTECs[ 21 – 23 ]. However, the exact role that gene expression regulation mechanisms plays in GPX4 expression level is still not fully elucidated. Coptis chinensis Franch is a common traditional Chinese medicine (TCM) and has a long application history, which is widely used for treating DM and its complications, hyperlipidemia and gastrointestinal infections[ 24 , 25 ]. The active ingredients and agents of the bitter taste of Coptis chinensis Franch are mainly protoberberine-type alkaloids such as berberine (BBR), coptisine, jatrorrhizine, palmatine, columbamine, epiberberine, magnoflorine and groenlandicine[ 26 , 27 ]. Reportedly, Coptis chinensis Franch and its pharmacological active ingredients can significantly ameliorate DN by protecting glomerular podocyte damage[ 28 – 30 ]. However, the role of its active ingredients in tubular injury still needs further exploration. Here, we found that ferroptosis occurred in the late-stage of DN through two animal models of DN (STZ-induced diabetic mice and db/db mice) and human plasma samples. We then identified VDR transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs. Tubule-specific deletion of VDR exacerbated renal morphological abnormalities and renal dysfunction. Additionally, we demonstrated that the level of VDR was predominantly controlled by the PRPF19-mediated ubiquitin-proteasome degradation system, and the natural compound BBR could bind to PRPF19 to inhibit VDR degradation, and protect renal injury. Collectively, this study proposed a new perspective on the potential of targeting PRPF19 to inhibit ferroptosis in DN therapy. 2. Methods and materials Animals The RTEC-specific VDR knockout mice were generated by crossing Pax8-Cre mice (provided by Shulaibao (Wuhan) Biotechnology Co., Ltd.) with VDR-flox mice mice (provided by Shulaibao (Wuhan) Biotechnology Co., Ltd.). The DN model was established by intraperitoneal injection of STZ (40 mg/kg, Sigma #S0130) for 7 days and high-fat diet (HFD) (60% Kcal fat, #D12492, Research Diets, New Brunswick, NJ) for 4 weeks. Nec-1 (5 mg/kg, MedChemExpress #HY-1576), Fer-1 (5 mg/kg, MedChemExpress #HY-100579), Emricasan (12.5 mg/kg, MedChemExpress #HY-10396), and 3-MA (10 mg/kg, MedChemExpress #HY-19312) were injected intraperitoneally once a week until the animals were euthanized, with saline as a control. PAR (1 ug/kg, MedChemExpress #HY-50919) were injected intraperitoneally once a week for 12 weeks, with saline as a control. Intragastric administration of BBR (300 mg/kg/d, Aladdin #B414323) or vehicle was started at 16 weeks of age and maintained for 4 weeks. The dosage of BBR (300 mg/kg/d) used in our experiment was chosen according to animal studies and clinical trials previously reported[ 31 – 33 ]. Mice were housed in a 12-h light/dark cycle with free access to a standard rodent diet and water. All animal euthanasia by anesthesia (overdose of pentobarbital, intraperitoneal injection) and all animal care procedures were performed according to the guidelines of the Committee for Animal Research of Huazhong University of Science and Technology. Cell culture, transfection, and treatments Human renal tubular cell line HK-2 (Procell Life Science&Technology Co,.Ltd #CL-0109) was cultured in DMEM/F12 media plus 10% FBS (Procell Life Science&Technology Co,.Ltd #CM-0109). HEK293T cells (Cell Health, CHCH-0004) were cultured in DMEM media containing 10% FBS (Cell Health, CHCH-0004-025). HEK293T were transfected with 5 µg of EV, pENTER-VDR or pcDNA3.1-PRPRF19 using exfect transfection reagent (Vazyme, T101). 48 h after transfection, cells were treated with or without AGEs (100 ug/ml) and MG-132 (30 nM) for additional 24 h. siVDR or siCtrl were transfected into HK-2 cells by RNAiMAX transfection reagent (Thermo Fisher Scientific, 13778030) for 72 h. HK-2 were preincubated with 10 µM BBR, 5 µM Fer-1 or basic medium for 12 h. Then cells were cultured with 5 µM erastin (MedChemExpress #HY-15763) for 12 h and collected for subsequent assay. Dual-luciferase reporter assay HEK293 cells were cultured on 12-well cell culture plates overnight. Cells were then transiently transfected with wild-type (WT) or mutant PGL3-GPX4-luc (luciferase reporter) and pLR-TK (renilla internal control) and vectors carrying full-length human gene (pCDH-puro-POU2F2, pENTER-VDR, pENTER-YY1, pcDNA3.1-MAZ, pENTER-AR, pENTER-USF2, pENTER-ATF3) using exfect transfection reagent. After 48 h, cells were washed once with PBS and lysed for measurement of luciferase activity using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega, Mannheim, Germany). All measurements were performed with a Tecan Infinite M200 PRO luminometer (Tecan, Crailsheim, Germany). For determination of specific luciferase activity, activity of the firefly luciferase was normalized to the activity of the renilla luciferase. CUT&RUN CUT&RUN was performed as described in Hyperactive pG-MNase CUT&RUN Assay Kit (Vazyme, HD101) protocol. Briefly, 0.5 million live HEK293 cells were collected and resuspended in 100 ul wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM Spermidine, supplemented with Protease Inhibitor EDTA-Free tablet (Sigma-Aldrich #5056489001)). Activated Concanavalin A was incubated with cells at room temperature for 10 min to let the cells bind to the beads. 0.5 µg VDR or H3K4me3 histone antibodies were added to each sample and incubated in the antibody buffer (wash buffer + 0.01% Digitonin and 2 mM EDTA) at 4°C for 4 h. The beads were then washed twice with digitonin buffer (wash buffer + 0.01% Digitonin), and 2.5 µl pG-MNase was added to each sample. After 10 min of incubation at room temperature, excessive pG-MNase was washed out by a two-time digitonin buffer wash. Then targeted chromatin was digested and released from cells by 2 h of incubation with the presence of 2 mM CaCl 2 at 4°C, which were collected from the supernatant, and subjected to phenol/chloroform DNA extraction and finally to qRT-PCR detection. EMSA Nuclear proteins were extracted using a commercial kit (Carlsbad, CA, USA). The extracted proteins were incubated in reaction buffer on ice, followed by the addition of biotin-labeled probes. For supershift assays, anti-VDR antibodies were added to the reaction mixture prior to probe incubation. All steps were performed using the light shift Kit (Pierce, Rockford, IL, USA). IP-MS The kidney tissue of mice was ground and digested to extract lysates. After transfection and treatments, HK-2 cells were lysed with pre-lysis buffer. The tissue and cell lysates were incubated with VDR antibody or respective IgGs antibody with protein A/G magnetic beads (MedChemExpress #HY-K0202) overnight at 4°C. After washing (1×PBS + 0.5% Tween-20, PH 7.4), the beads were boiled in loading buffer and subjected to mass spectrometry detection and immunoblotting. Preparation of BBR-immobilized beads Magnetic FG beads (Linker beads, TAS8848N1110, Tamagawa Seiki, 1 mg) were incubated with a 10 mM or 12 mM solution of the berberrubine (with DMSO as a control) in N, N-dimethylformamide and 14 mg of potassium carbonate for 16–20 h at 60°C. Unreacted residues were masked using 50% methanol, and the resulting beads were stored at 4°C. Affinity purification with BBR-immobilized beads BBR-immobilized beads (0.5 mg) were equilibrated with 100 mM KCl buffer containing 20 mM HEPES-NaOH (pH 7.9), 100 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EDTA,10% (v/v) glycerol, 0.1% NP-40, 1 mM DTT, and 0.2 mM PMSF. Cell extracts were prepared from HK-2 cells and were incubated with the beads for 4 h at 4°C. The beads were washed three times with 100 mM KCl buffer, and bound proteins were eluted with 1× loading dye solution containing 62.5 mM Tris-HCl (pH 6.8), 0.005% bromophenol blue, 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol. SPR We used SPR to determine the real-time interactions between BBR and PRPF19. The device used is BiacoreT200 (Cytiva). PRPF19 protein from AtaGenix at 1.67 mg/ml was diluted with 10mM sodium acetate, pH 4.0, and immobilized on Series S Sensor Chip CM5 (Cytiva) (7 min activation with blocking buffer) at 10µl/min to get immobilization signal of 5550 RU. Samples were prepared as dilution series with the starting concentration of 25 µM in the running buffer for BBR (0.78, 1.5625, 3.125, 6.25, 12.5, 25 µM). Running and sample buffer contained 1×PBS, 0.005% Tween 20, 1% DMSO, and pH 7.4 at 25°C. Flow and injections scheme consisted of 30 µl/min for 120 s followed by 200 s of dissociation. Reference channel was activated with EDC/NHS and deactivated with 1 M ethanolamine, pH 8.3. Sensorgram response data were analyzed by using the BIA evaluation kinetics software, and the equilibrium binding and disassociation constants were calculated. RNA-sequencing analysis The mRNA of mice kidney tissue was extracted and sent to Shanghai Gene Co., Ltd. for mRNA sequencing. Differential gene expression analysis was completed using the limma R package (version 3.44.3). The fold change (FC) in expression of each gene was log 2 transformed and further analyzed using RStudio version 1.1.442 (RStudio, Inc., USA). Urine and serum analyses Urinary albumin, creatinine and BUN were measured using urine protein test kit (Nanjing Jiancheng, China), creatinine assay kit (Nanjing Jiancheng, China) and urea assay kit (Nanjing Jiancheng, China). Blood glucose was measured using Haier Blood Glucose Meter (China). MDA, GSH, GPX4, ferritin, TfR and 1,25-(OH)2D3 content of plasma was analyzed by lipid peroxidation test kit (Beyotime, China), reduced glutathione assay kit (Nanjing Jiancheng, China), human GPX4 ELISA kit (Bioswamp, China), human ferritin ELISA kit (Raybiotech, USA), human TfR ELISA kit (Raybiotech, USA) and human 1,25-(OH)2D3 ELISA kit (Nanjing Jiancheng, China). Histologic analysis Paraffin-embedded mouse kidney sections (5 µm thickness) were prepared by a routine procedure. Sections were stained with H&E, masson and PAS. TEM was performed on glutaraldehyde-fixed, epoxy-embedded kidney samples and stained with uranyl acetate and lead citrate. TEM images were collected by RADIUS Software v2.1. Immunofluorescence The paraffin-embedded sections were deparaffinized and stained with primary antibodies overnight at 4°C. After washing in PBS, sections were visualized by secondary antibody. Nuclear counterstaining was performed using 4’,6-diamidino-2-phenylindole. Cultured cells were first fixed and blocked with serum. After that, sections or cells were incubated with antibodies. Staining was observed by a fluorescence microscope. RNA extraction and qRT-PCR RNA was extracted from samples using the Trizol reagent (Takara, Japan). Then, cDNA was synthesized using the reverse transcriptase kit (Takara, Japan). Quantitative analysis of mRNA expression was conducted with a SYBR premix EX TaqTM kit (Takara, Japan) with StepOne PCR system (Applied Biosystems, USA). The relative quantity of mRNA was expressed as 2 −△△CT . Sequences of the primers were listed in Table S2. Western blotting Freshly collected kidney or cultured cells were sonicated in ice-cold RIPA buffer (Beyotime), 20–80 µg protein from each sample was separated by SDS-PAGE. The proteins were transferred onto PVDF membranes for immune detection. The antibodies used were provided in Table S3. Statistics Statistical analyses were performed with Prism 7 (GraphPad Software, Inc., USA) or RStudio version 1.1.442 (RStudio, Inc., USA). For data with a normal distribution and homogeneity of variance, one-way ANOVA was performed for comparisons among more than two groups. Two-tailed Student’s t tests were performed to evaluate significant differences between two groups. All data were expressed as mean ± SEM. Differences for which P < 0.05 were considered significant. 3. Results 3.1 Ferroptosis is the predominant form of cell death in the late-stage of DN. To acquire a full understanding of the relative contributions of different types of cell death in DN renal tubular injury, high-fat-fed STZ-induced diabetic mice were intraperitoneally injected with different inhibitors of cell death once a week. A time series analysis of urine albumin-to-creatinine ratio (uACR) and fasting food glucose of mice at different phase of DN (Fig. 1 A) was performed. The results revealed that in the early-stage of DN, necrostatin-1 (Nec-1, inhibitor of necroptosis), rather than emricasan (inhibitor of apoptosis), 3-methyladenine (3-MA, inhibitor of autophagy) or ferrostatin-1 (Fer-1, inhibitor of ferroptosis) significantly improved fasting blood glucose and uACR in mice (Fig. 1 B and 1 C). Nec-1 had a protective effect on early renal tubular lesions and mitochondrial morphological abnormalities (Fig. S1 ), which suggested that necroptosis might be involved in the early renal tubular injury in DN. However, in the late-stage of DN, renal function and blood glucose levels were improved more significantly in the Fer-1 group (Fig. 1 B and 1 C). The results of Hematoxylin and Eosin (H&E), Masson and Periodic Acid-Schiff staining(PAS)staining showed that Fer-1 treatment markedly improved renal tubule swelling, epithelial cell exfoliation and death, tubulointerstitial fibrosis and glycogen deposition (Fig. 1 D and 1 F). In addition, decreased mitochondrial count, the rupture of mitochondrial cristae, and swelling of mitochondrial morphology were prominently alleviated in the Fer-1 group (Fig. 1 E and 1 G). This indicated that, unlike in the early-stage, ferroptosis might be the main form of cell death in the late-stage of DN. Furthermore, 80 plasma samples from late-stage DN patients or normal individuals were collected for enzyme-linked immunosorbent assay (ELISA) analysis of the markers related to ferroptosis (Fig. 1 H). Clinical demographics of these subjects were provided in Table S1 . The results showed that the expression of molecules that promoted lipid peroxidation and iron overload, such as malondialdehyde (MDA), ferritin and transferrin receptor 1 (TfR1), were increased in plasma of DN patients (Fig. 1 I). In contrast, molecules with antioxidant properties, such as GSH and GPX4, were down-regulated in DN patients (Fig. 1 I). Subsequently, in 24-week-old db/db mice, we validated the disorders of renal function and blood glucose level (Fig. S2A and S2B), and found an increase in ferroptosis markers (ACSL4, TfR1, 4-HNE, MDA, Fe 2+ ) level, significant mitochondrial cristae breakage, and decrease in GPX4 and GSH levels (Fig. 8 C- 8 H and Fig. S2C-S2H). These results suggest that ferroptosis plays an important role in prolonged renal tubular damage in DN. 3.2 VDR is a key transcriptional regulator of GPX4 We first confirmed the reduction of GPX4 in the kidney of DN mice by RNA sequencing (RNA-seq) (Fig. 2 A). Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis results also showed that the expression level of GPX4 decreased in STZ and HFD-induced DN mice, db/db mice and HK-2 cells treated with advanced glycation end-products (AGEs) (Fig. 2 B- 2 E). Importantly, the expression of GPX4 decreased by degrees with the progression of DN (Fig. 2 D), and the mRNA level of GPX4 were positively correlated with the protein level (Fig. 2 D and 2 E). Thus, we speculated that the decrease of GPX4 was related to the mechanism of transcriptional regulation. By intersecting the prediction results of 4 transcription factor databases ( http://jaspar.genereg.net , http://bioinfo.life.hust.edu.cn/AnimalTFDB#!/ , http://gtrd20-06.biouml.org , http://alggen.lsi.upc.es ), we obtained 7 candidate GPX4 transcription factors (Fig. 2 F). And by copying and selectively inserting the GPX4 promoter sequence into the PGL3 vector, we obtained the PGL3-GPX4-luc plasmid (Fig. S3A). The results of dual-luciferase reporter assay and cleavage under targets and release using nuclease (CUT&RUN) showed that VDR was the main transcription factor of GPX4 (Fig. 2 G and 2 H, the results of POU2F2 were negative, while the results of YY1 lacked specificity) and could target the AGGGGTCA base sequence (1423–1430 bp upstream from transcription start site) in the GPX4 promoter sequence (Fig. 2 I). Mutation of the AGGGTCA motif (Fig. 2 J) significantly attenuated VDR-mediated luciferase activity (Fig. 2 K), confirming this sequence as the functional VDR binding site. Furthermore, electrophoretic mobility shift assay (EMSA) directly demonstrated VDR binding to the GPX4 promoter (Fig. S3B). Notably, we measured the content of VDR active ligand-1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in the plasma of DN patients by ELISA, and found that there was a significant negative correlation between 1,25-(OH)2D3 and uACR levels (R=-0.77, P = 5.5e-09) (Fig. 2 L). These data indicate that VDR is a key transcription factor of GPX4 and may affect renal function. 3.3 VDR deletion promotes ferroptosis to exacerbate renal tubule injury in DN mice To explore the contribution of VDR to the maintenance of GPX4 within RTECs, we generated RTEC-specific VDR knockout mice (Pax8-Cre/VDR fl/fl , VDR-cKO) by crossing Pax8-Cre and VDR-flox mice. VDR-cKO mice presented no obvious kidney dysfunction physiologically (Fig. 3 B and 3 C). However, post-STZ and HFD induction, VDR-cKO mice exhibited an amplified renal hypertrophy (Fig. 3 A, 3 B, Fig. S4A and S4B). VDR deficiency significantly increased fasting blood glucose, uACR, and blood urea nitrogen (BUN) levels (Fig. 3 C). In addition, compared with VDR fl/fl mice, VDR-cKO mice showed more severe renal tubular enlargement, brush edge destruction, and epithelial cell shedding after STZ and HFD induction (Fig. 3 D). The VDR-cKO group showed a significant decrease in GPX4 levels (Fig. 3 E and 3 F). Meanwhile, there was more lipid peroxidation product 4-hydroxynonenal (4-HNE) accumulation in RTECs of VDR-cKO mice than VDR fl/fl mice treated with STZ and HFD (Fig. 3 G), accompanied by significantly reduced GSH levels (Fig. 3 H). We further performed RNA-seq in the cortex of kidney from VDR-cKO mice and VDR fl/fl mice treated with STZ and HFD. Transcriptomic analysis showed that VDR knockdown resulted in down-regulation of GPX4 (Fig. 3 I), and KEGG pathway enrichment analysis of the differentially expressed genes high-lighted cellular processes such as ferroptosis and glutathione metabolism (Fig. 3 J). Simultaneously, GO enrichment analysis was performed on differentially expressed genes (Fig. S4C), and it was found that the molecular functions emphasized glutathione peroxidase activity (Fig. S4D). These findings indicate that VDR affects RTECs death primarily through a ferroptosis-mediated mechanism. 3.4 VDR activation alleviates renal pathological damage in DN mice We also investigated whether activation of VDR would alleviate renal tubular injury in vivo. After the induction of STZ and HFD, DN mice were intraperitoneally injected with VDR agonist-PAR or vehicle once a week (Fig. 4 A). After 12 weeks of treatment, the mice showed significant improvement in kidney hypertrophy, fasting blood glucose, uACR, and BUN levels (Fig. 4 B, 4 C, Fig. S4E and S4F). Histological analysis of the lesions revealed a reduction in swelling of renal tubules in the PAR group (Fig. 4 D). GPX4 and GSH expression in RTECs was significantly increased in the PAR group (Fig. 4 E, 4 F and 4 H), while 4-HNE level was significantly reduced (Fig. 4 G). These data indicate that VDR protects RTECs by stabilizing intracellular GPX4 levels and reducing lipid peroxidation. 3.5 VDR promotes GPX4 expression to inhibit ferroptosis in HK-2 cells Similarly, to determine whether VDR played a crucial role in the process by which GPX4 regulated ferroptosis, we transfected pENTER-VDR plasmids or empty vector (EV) into HK-2 cells in high-glucose medium supplemented with AGEs. Western blotting showed a significant increase in the content of VDR and its target protein-GPX4 after plasmid transfection (Fig. 5 A). Using CCK-8 assay kit to detect cell viability, it was found that overexpression of VDR significantly increased cell viability (Fig. 5 B). In addition, increased expression of VDR could increase intracellular GSH content (Fig. 5 C) and reduce the accumulation of 4-HNE (Fig. 5 D). The lipid reactive oxygen species (ROS) accumulation was detected by BODIPY (581/ 591) C11 probe. Normal probe displays red fluorescence, and the probe bound to peroxidized lipids showed green fluorescence. The results showed that the level of lipid peroxidation was obviously downregulated after VDR overexpression in HK-2(Fig. 5 E). On the contrary, when HK-2 cells were transfected with VDR specific siRNA (siVDR) or negative control siRNA (siCtrl), compared with the siCtrl group, VDR and GPX4 decreased significantly (Fig. 5 F), cell viability (Fig. 5 G) and GSH (Fig. 5 H) content decreased, while 4-HNE (Fig. 5 I) and ROS (Fig. 5 J) accumulation increased accordingly in the siVDR group. 3.6 E3 ubiquitin ligase PRPF19 mediates ubiquitination degradation of VDR To explore the mechanism of VDR-induced RTECs ferroptosis, we investigated the expression of VDR in mice during the progress of DN. Western blotting showed a gradual decrease in the protein level of VDR (Fig. 6 A), but no significant change in the mRNA level by qRT-PCR (Fig. 6 B). This suggested that the downregulation of VDR might be related to protein post-translational modifications rather than transcriptional regulatory mechanisms. Therefore, we used Immunoprecipitation-mass spectrometry (IP-MS) to elute VDR related interacting proteins and perform mass spectrometry analysis (Fig. 6 C). The results of 4-dimensional data-independent acquisition (4D DIA) quantitative proteomics demonstrated that compared with the IgG group, 119 molecules were upregulated and 233 molecules were downregulated in the elution proteins of the VDR group (Fig. S5A). VDR-related proteins were enriched in the ferroptosis pathway and involved in regulation of gene expression (Fig. S5B, S5C and Fig. 6 D). Interestingly, they also participated in the ubiquitin-dependent protein catabolic process (Fig. 6 D). By comparing the proteins involved in the ubiquitin-dependent protein catabolic process, we identified the molecule-PRPF19 (an E3 ubiquitin ligase) with the strongest binding affinity to VDR (Fig. 6 E). To verify the involvement of the ubiquitin-proteasome pathway in VDR degradation, we utilized different inhibitors of protein degradation mechanisms. Western blotting showed that only MG-132 (ubiquitin-dependent proteasome inhibitor), rather than bafilomycin A1 (BafA1, autophagy-dependent lysosome inhibitor) or oroxylin A (mitochondrial autophagy inhibitor), restored the VDR levels in cultured HK-2 cells (Fig. 6 F). The immunofluorescence staining proved that VDR and PRPF19 were colocalized in cytoplasm, and the overlapping fluorescence intensity of VDR and PRPF19 was stronger in the AGEs group (Fig. 6 G). Thus, we preliminarily determined that PRPF19 was involved in the ubiquitin-proteasome degradation process of VDR. The results of co-immunoprecipitation (Co-IP) experiments confirmed the interactions between VDR and PRPF19, which were enhanced after treatment with AGEs (Fig. 6 H). To investigate the role of PRPF19 in VDR regulation, we transfected HK-2 cells with either Myc-PRPF19 plasmids or PRPF19-targeting sgRNA (Sg-PRPF19). Notably, PRPF19 overexpression significantly reduced VDR protein levels, whereas PRPF19 knockout increased VDR expression (Fig. 6 I and 6 J). Co-IP assays further demonstrated that PRPF19 enhances VDR ubiquitination, as overexpression of PRPF19 markedly increased VDR ubiquitination levels, whereas PRPF19 knockout reduced them (Fig. 6 K). These findings demonstrate that PRPF19 is involved in the degradation of VDR by a ubiquitination-dependent pathway. 3.7 BBR competitively binds to PRPF19 to inhibit VDR degradation To test whether the active ingredients of Coptis chinensis Franch could controll ferroptosis via PRPF19, we used molecular docking to predict the binding modes and affinities between 8 alkaloids and PRPF19. The analysis results showed that BBR displayed the lowest binding energy of − 8.3 kcal/mol (Fig. 7 A), forming hydrogen bond with PHE-483 residues of PRPF19 and favorable hydrophobic interactions with GLN-445 and ILE-442 (Fig. 7 B). Furthermore, molecular dynamics (MD) simulations showed the root mean square deviation (RMSD) of the BBR-PRPF19 system stabilizes between 60–100 ns (Fig. S6A), indicating a stable binding interaction. Root mean square fluctuation (RMSF) analysis revealed conformational flexibility in specific regions of PRPF19 upon BBR binding (Fig. S6B). Notably, residues 255–270, 340–350, 370–390, 430–440, and 470–480 exhibited higher RMSF values, suggesting increased flexibility in these regions due to BBR recognition. We next investigated whether BBR could directly bind to PRPF19. We used high-performance affinity beads, which the active BBR derivative berberrubine could covalently conjugated to, to purify drug-targeted proteins from cell extracts (Fig. 7 C). Using BBR-immobilized beads or naked beads for pull-down experiments, and performing mass spectrometry analysis on the fractions eluted from the beads. 5335 proteins were identified in the fractions (Fig. S6C), and KEGG pathway classification analysis indicated that BBR was involved in the regulation of cell growth and death (Fig. S6D). Importantly, the results of 4D DIA quantitative proteomics demonstrated that BBR-related proteins were also involved in ubiquitin-mediated proteolysis (Fig. S6E), and PRPF19 was a key E3 ubiquitin ligase (Fig. 7 D). Again, fractions eluted from BBR-immobilized beads were subjected to immunoblotting and probed with a PRPF19-specific antibody. The results showed that PRPF19 was clearly isolated as a BBR-specific binding protein (Fig. 7 E), and high concentration of BBR covalently conjugated to beads could increase the production of PRPF19 compared with low concentration (Fig. 7 E). To further establish that BBR directly bound to PRPF19, we used SPR, a technique for detecting the interactions between ligands and analytes on biosensor chips. SPR demonstrated that BBR directly bound to PRPF19, with an estimated equilibrium dissociation constant (K D ) of 3.38× 10 − 3 M (Fig. 7 F). Next, to verify whether BBR could competitively bind to PRPF19 with VDR, we conducted co-immunoprecipitation experiments. It was found that by adding additional BBR to the co-incubation system of cell extracts and beads, the content of PRPF19 protein pulled down by VDR was reduced (Fig. 7 G). In addition, we performed immunoprecipitation experiments in DN mice treated with BBR or vehicle, and found that BBR treatment significantly decreased the ubiquitination level of VDR (Fig. 7 H). These results demonstrate that BBR can competitively bind to PRPF19 with VDR to reduce VDR ubiquitination degradation, which may play a role in inhibiting renal tubular ferroptosis. 3.8 BBR inhibits ferroptosis and attenuates renal injury in vivo Next, to examine the therapeutic efficiency of BBR in DN, we treated mice with intragastric administration of BBR (300 mg/kg/d) for 4 weeks. The STZ and HFD-induced elevation of kidney volume (Fig. 8 A and Fig. S7A), elevated fasting blood glucose, uACR, BUN and serum creatinine (Scr) levels were reversed by BBR treatment (Fig. 8 B). We further found STZ and HFD-induced DN mice exhibited significant renal tubular tissue damage, interstitial fibrosis and glycogen deposition, while BBR treatment effectively blocked these pathological changes (Fig. 8 C). Moreover, DN mice induced a significant decrease in VDR, GPX4 and ferritin heavy chain 1 (FTH1, a molecular biomarker inhibiting ferroptosis) protein levels, while acyl-CoA synthetase long-chain family member 4 (ACSL4) and TfR1 (molecular biomarkers promoting ferroptosis) protein levels increased, which were effectively reversed by BBR treatment (Fig. 8 D). The mRNA level of GPX4 was significantly reduced in DN mice, but BBR treatment could salvage the decrease in GPX4 mRNA level (Fig. 8 E). After BBR gavage, the morphological changes of mitochondria in DN mice were obviously mitigated (Fig. 8 F). Furthermore, high levels of lipid peroxidation products (4-HNE and MDA) and iron content were effectively suppressed by BBR, and the low level of GSH was apparently recovered (Fig. 8 G, 8 H and Fig. S7B). 3.9 BBR diminishes ferroptosis of RTECs by a PRPF19-dependent pathway In vitro experiments, using Fer-1 as a positive control, it was notable that the BBR-treated group was resistant to erastin-induced ferroptosis, as evidenced by the increase in cell viability (Fig. 9 A), as well as reduction in lipid peroxidation and iron accumulation (Fig. 9 B, 9 C and Fig. S7C). In vitro, BBR treatment could also reverse the decrease in GPX4 mRNA and protein levels (Fig. 9 B and 9 D). In addition, by utilizing JC-1, an ideal fluorescent probe widely used for detecting mitochondrial membrane potential Δᴪ m, we discovered that the decrease in membrane potential was evidently reversed by BBR (Fig. 9 E). Our data demonstrate that BBR can inhibit ferroptosis of RTECs in vitro. To test whether BBR played a protective role by a PRPF19-dependent pathway, we employed a PRPF19-overexpression cell model. After transfection of pcDNA3.1-PRPF19 plasmids into HK-2 cells, we observed that BBR failed to further reverse the decrease in cell viability caused by AGEs stimulation (Fig. 9 F). In agreement, in PRFP19-overexpressing cells, BBR could not further salvage the AGEs-induced decrease in VDR, GPX4 and FTH1 levels (Fig. 9 G). GSH is an important antioxidant substance, while MDA and 4-HNE are by-products of lipid peroxidation. After overexpression of PRPF19, the ability of BBR to reduce lipid peroxidation significantly decreased (Fig. 9 H, 9 I and 9 K). Moreover, in PRPF19-overexpressing cells, the ability of BBR to reduce Fe 2+ and ROS levels was greatly weakened (Fig. 9 J and 9 L). These results convincingly show that BBR diminishes renal tubular ferroptosis by a PRPF19-dependent pathway. 4. Discussion In this study, we revealed the significant involvement of PRPF19/VDR/GPX4 axis in DN through the findings of in vivo, in vitro, and pharmacological investigations. The major findings of this work include: (i) ferroptosis was the predominant form of cell death in the late-stage of DN; (ii) VDR transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs; (iii) mechanistically, E3 ligase PRPF19 mediates ubiquitination degradation of VDR; and (iv) BBR was verified as a novel inhibitor of PRPF19 and effectively alleviated ferroptosis in renal tubules. Renal injury in DM is a long-term and continuous process. Multiple studies have reported that DN involves in various forms of cell death, such as apoptosis, necrosis, autophagy, and newly discovered ferroptosis[ 34 – 42 ]. However, it is still unclear which mode of cell death plays a more important role at different stages of DN. In this study, we found that in diabetic mice with long-term renal injury, the ferroptosis inhibitor Fer-1 significantly reduced urinary protein excretion, lowered blood glucose, ameliorated tubular pathological damage and renal interstitial fibrosis. Ferroptosis phenomenon was more significant in late-stage DN mice and aged db/db mice. This phenomenon may be attributed to the combined effects of elevated oxidative stress[ 43 ], impaired antioxidant defense mechanisms[ 44 ], disrupted iron homeostasis[ 45 ] and sustained chronic inflammation[ 46 ] in the advanced stages of DN. In addition, compared with normal individuals, the levels of lipid peroxidation and iron accumulation markers were increased in the plasma of late-stage DN patients. These data show that ferroptosis may be involved in the late-stage of DN renal injury. The elucidation of the ferroptosis time window provides a basis for the clinical translation of intervention strategies for DN. Non-open-loop steroid 1,25-(OH)2D3 is a hormone form of vitamin D and an endocrine hormone with multiple physiological functions[ 47 ]. VDR is a nuclear receptor for 1,25-(OH)2D3 and the activation of VDR depends on 1,25-(OH)2D3 ligands[ 48 – 56 ]. Previous studies have found that PAR and 1,25-(OH)2D3 have renal protective effects in DN[ 57 – 62 ]. In a multinational, placebo-controlled, double-blind trial, de Zeeuw D et al. stated addition of 2 µg/day PAR to renin–angiotensin–aldosterone system (RAAS) inhibition safely lowered residual albuminuria in patients with DN (P = 0.014 vs placebo) [ 60 ]. Another analysis included 14,709 participants with type 2 diabetes from the UK Biobank observed that higher serum 1,25-(OH)2D3 concentrations were significantly associated with lower risk of diabetic microvascular complications, including DN (P<0.05; aHR: 0.54, 95% CI: 0.38–0.78)[ 57 ]. Here, our research findings indicate that VDR, as an important transcription factor, binds to the GPX4 promoter to suppress tubular cell ferroptosis and renal dysfunction, and provide evidence for the explanation of the therapeutic effects of PAR and 1,25-(OH)2D3 on DN. Regarding the degradation pathway of VDR, previous studies have shown that the AF-2 domain of VDR interacts with mSUG1, a component of the 26S proteasome, which may target VDR towards proteasome-mediated degradation[ 63 ]. In addition, in some cellular environments, unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation, while 1,25-(OH)2D3-dependent heterodimerization with retinoid X receptor and subsequent nuclear localization protect VDR from these modifications[ 64 , 65 ]. This is consistent with our research results. By applying inhibitors targeting the ubiquitin-proteasome system, lysosome-autophagy system, and mitochondrial autophagy system, we found that ubiquitin-dependent degradation was an important regulatory mechanism affecting VDR levels. PRPF19 has been identified as a key E3 ubiquitin ligase of VDR to promote this process. Our work confirmed the interactions between VDR and PRPF19, which were enhanced during high-glucose induction. As a natural compound, prior research has demonstrated the therapeutic effect of BBR in diabetes and its complications[ 66 – 68 ]. However, there is limited evidence on how BBR acts on renal tubules. Since the proximal tubules require high energy and rely on aerobic metabolism, they are vulnerable to ischemic injury in diabetes due to increased consumption, impaired utilization and reduced oxygen delivery[ 69 ]. Therefore, the death of tubular cells has been considered as one of the key pathogenesis of DN. Here, our investigation provides evidence that by targeting PRPF19 to inhibit ubiquitination degradation of VDR, BBR can effectively improve renal function, alleviate lipid peroxidation, iron accumulation, and abnormal morphology of mitochondria of RTECs in vitro and in vivo. BBR exhibits extremely low oral bioavailability (< 1% in rat models) attributable to poor intestinal absorption and extensive first-pass metabolism[ 70 ]. Following administration, BBR demonstrates preferential tissue distribution to metabolic (liver) and excretory organs (kidneys), with hepatic cytochrome P450 enzymes (particularly CYP2D6 and CYP3A4) mediating its biotransformation into three primary metabolites: berberrubine (M1), thalifendine (M2), and jatrorrhizine (M4)[ 71 ]. These metabolites undergo dual elimination pathways via both hepatobiliary excretion and renal clearance[ 72 ]. To advance therapeutic translation, further comprehensive investigation is needed to quantify BBR and its bioactive metabolites in diabetic renal tissue using advanced mass spectrometry techniques, and develop novel delivery systems (including nanoparticle formulations and prodrug approaches) to overcome BBR's current bioavailability limitations. On the whole, our findings propose that ferroptosis of RTECs is one of the essential mechanisms of renal injury in diabetes, and inhibition of ferroptosis by targeting PRPF19 may be a potential therapeutic strategy to restore renal function in diabetes. Compared with physiological kidneys, the level of ubiquitination and proteasome degradation of VDR is up-regulated in renal tubules of DN. The degradation of VDR prevents it from entering the nucleus and promoting the transcription of GPX4, which leads to downregulation of GPX4 and the aggravation of lipid peroxidation, ultimately resulting in ferroptosis of RTECs. BBR reduces VDR degradation by binding to PRPF19 and inhibiting its function, thus protecting against ferroptosis of renal tubules and renal injury. Declarations Data Availability All data generated in this study are provided in the Supporting Information file. The datasets generated during the current study have been deposited in the GEO datasets under the accession number GSE295215. Acknowledgements This work was supported by National Natural Science Foundation of China (Grant NO.82274470 and NO.81974567). Author information Authors and Affiliations Institute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Qiongyao He, Wenbin Wu Division of Cardiology, Department of Internal Medicine and Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiologic Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430000, China Wu He Department of Traditional Chinese Medicine, Zhongshan Hospital of Hubei Province, Wuhan 430030, China Yanlin Ren Department of Integrated Traditional Chinese and Western Medicine, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, China Hui Dong, Dingkun Wang, Fuer Lu Department of Endocrinology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Gang Yuan, Huihui Ren College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China Xinwei Wang Contributions Q.H. and W.H. conducted in vivo and in vitro experiments, performed data analysis, and helped write the manuscript. H.D. and W.W. contributed to the experimental design and performed in vitro experiments. X.W. performed in vivo animal studies. Y.R., G.Y. and H.R. helped design experiments. F.L. and D.W. designed the experiment, interpreted the data, wrote the manuscript. All the authors approved the final version of the manuscript for publication. Corresponding authors Correspondence to Fuer Lu or Dinkun Wang Ethics declarations Competing interests The authors declare no competing interests. Ethical approvals Human serum samples were collected by Tongji Hospital affiliated to Huazhong University of Science and Technology, and the study protocol was approved by the Ethics Committee of Tongji Hospital (permit number: TJ-IRB202406035). All animal care and experimental procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Animal Research of Huazhong University of Science and Technology. References Alicic RZ, Rooney MT, Tuttle KR. 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Supplementary Files SupplementaryMaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 25 May, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Accepted 17 May, 2025 Reviews received at journal 16 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 26 Apr, 2025 Reviewers invited by journal 25 Apr, 2025 Submission checks completed at journal 25 Apr, 2025 First submitted to journal 24 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6083539","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448541316,"identity":"11f0ea35-52fc-4323-90ce-4a219f8a4f4f","order_by":0,"name":"Qiongyao He","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qiongyao","middleName":"","lastName":"He","suffix":""},{"id":448541317,"identity":"1eb86418-45c0-42f9-b231-b54ad8320334","order_by":1,"name":"Wu He","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"He","suffix":""},{"id":448541319,"identity":"5e51c68d-01d5-43a8-908f-bbe27012b46d","order_by":2,"name":"Yanlin Ren","email":"","orcid":"","institution":"Zhongshan Hospital of Hubei Province","correspondingAuthor":false,"prefix":"","firstName":"Yanlin","middleName":"","lastName":"Ren","suffix":""},{"id":448541321,"identity":"561c9292-94a5-42a3-a824-dc0a6b27e6fa","order_by":3,"name":"Wenbin Wu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Wu","suffix":""},{"id":448541324,"identity":"3e68e8c8-8d17-49bb-80a2-69e3d2388eb7","order_by":4,"name":"Hui Dong","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Dong","suffix":""},{"id":448541326,"identity":"2efabf9d-10d8-45f7-be0a-dd86c29124dc","order_by":5,"name":"Gang Yuan","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Yuan","suffix":""},{"id":448541327,"identity":"df8e3887-22ab-4be0-b465-37824295a0a0","order_by":6,"name":"Huihui Ren","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huihui","middleName":"","lastName":"Ren","suffix":""},{"id":448541328,"identity":"0f8f5d3f-34d4-4a38-8c7d-ea7b2b7eb4c8","order_by":7,"name":"Xinwei Wang","email":"","orcid":"","institution":"Hubei University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinwei","middleName":"","lastName":"Wang","suffix":""},{"id":448541329,"identity":"67e60e8a-98aa-47b5-abf5-fcae209d8bc0","order_by":8,"name":"Fuer Lu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Fuer","middleName":"","lastName":"Lu","suffix":""},{"id":448541330,"identity":"d13963c4-ef4e-4d70-8272-9348c49a0709","order_by":9,"name":"Dingkun Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYFCCNBBhw8PP30CaljQZyRkHSNNy2MagIYFIDQbH09KkC36d5zFgOMD44WMOMVrOPDsmPbPvNo85cwOz5MxtRGgxu5HeJs3bc5vHsuEAGzMvCVrO8RgcSCBaS9oxaZ4fB0jQYn/mWbI1b0Myj+SMg83E+UWyPc3wNs8fO3t+/uaDHz4SowUMGNvAZAOx6kHgDymKR8EoGAWjYMQBAKohNq8lwXWeAAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Dingkun","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-02-22 06:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6083539/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6083539/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-025-02253-5","type":"published","date":"2025-05-25T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82075184,"identity":"5d6b014d-c8a2-41f7-8702-597e0f1966e7","added_by":"auto","created_at":"2025-05-06 13:40:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119921177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFerroptosis is the predominant form of cell death in the late phase of DN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of DN mice receiving treatment with four types of inhibitors. (B and C)\u003cstrong\u003e \u003c/strong\u003eLevels of fasting food glucose and uACR in DN mice treated with Nec-1 (5 mg·kg-1·d-1), Fer-1 (5 mg·kg-1·d-1), Emricasan (12.5 mg·kg-1·d-1), 3-MA (10 mg·kg-1·d-1), or vehicle (n = 5 or 6). (D) H\u0026amp;E, Masson and PAS staining of kidney sections collected from mice in the indicated groups at week 16 (bar = 50 μm). (E)\u003cstrong\u003e \u003c/strong\u003eRepresentative transmission electron microscopy images of PTECs from mice in the indicated groups at week 16 (bar = 10 μm).\u003cstrong\u003e \u003c/strong\u003e(F)\u003cstrong\u003e \u003c/strong\u003eQuantification of tubulointerstitial fibrosis in the kidney cortex. (G)\u003cstrong\u003e \u003c/strong\u003eAbsolute counting of the number of cristae per mitochondria. (H) Schematic diagram of collecting plasma samples from DN patients and normal individuals. (I)\u003cstrong\u003e \u003c/strong\u003eDetection of ferroptosis marker molecules in the plasma of DN patients and normal individuals using ELISA (n = 40). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/44e3e58b33bbaadf710d510f.png"},{"id":82075744,"identity":"475e1491-6ebe-46a3-b8ad-8289d48d3e7b","added_by":"auto","created_at":"2025-05-06 13:48:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27592711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDR is a key transcriptional regulator of GPX4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Heatmap showing the expression of ferroptosis marker proteins in the cortex of kidney from mice (n = 3). The kidneys were collected at week 16. (B and C) Western blotting and qRT-PCR of GPX4 in db/db mice and HK-2 cells (n = 6). (D and E) Western blotting and qRT-PCR of GPX4 in the cortex of kidney at serial time points (n = 6). (F)\u003cstrong\u003e \u003c/strong\u003eVenn diagram showed the intersection results of 4 transcription factor databases, and the table showed the prediction scores of 7 candidate transcription factors. (G)\u003cstrong\u003e \u003c/strong\u003eFirefly/renilla luciferase activity ratios in HEK293 cells, transfected with PGL3-GPX4-luc, pLR-TK, pCDH-puro-POU2F2, pENTER-VDR, pENTER-YY1, pcDNA3.1-MAZ, pENTER-AR, pENTER-USF2, pENTER-ATF3 or empty vector (n = 10). (H) qRT-PCR detection based on predicted binding sites of Cut\u0026amp;Run (n = 5). (I) Schematic diagram showing the binding site of VDR on the GPX4 promoter fragment. (J)\u003cstrong\u003e \u003c/strong\u003eSchematic diagram showing complementary mutations of VDR binding site. (K) Firefly/renilla luciferase activity ratios in HEK293 cells, transfected with WT or mutant PGL3-GPX4-luc, pLR-TK and pENTER-VDR (n = 10). (L)\u003cstrong\u003e \u003c/strong\u003eLinear analysis between uACR and 1,25-(OH)2D3 level in DN patients (n = 40). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/e125f9336375e5a12e49ca12.png"},{"id":82075765,"identity":"646e3ebd-eb8c-47de-bf0b-346d6e00e55a","added_by":"auto","created_at":"2025-05-06 13:48:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80297351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDR deletion promotes ferroptosis to exacerbate renal tubule injury in DN mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of VDR knockout mice receiving STZ and HFD induction. (B) Representative photographs of kidneys from mice in the indicated groups (bar = 5 cm). (C) Fasting blood glucose, uACR, and BUN levels of mice (n = 6). (D) H\u0026amp;E staining of kidney sections collected from mice (bar = 50 μm). (E) qRT-PCR of VDR and GPX4 in the kidney tissues of mice (n = 6). (F) Western blotting of VDR and GPX4 in the kidney tissues of mice (n = 6). (G) Immunofluorescence staining of 4-HNE of PTECs from mice (n = 6; bar = 50 μm). (H) GSH levels of mice (n = 6). (I) Volcanic diagram of differentially expressed genes in VDR-cKO and VDR\u003csup\u003efl/fl\u003c/sup\u003e mice treated with STZ and HFD. (J) KEGG pathway analysis of differentially expressed genes in VDR-cKO and VDR\u003csup\u003efl/fl\u003c/sup\u003e mice. Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/b4e6def63bb7e6d6eff564ad.png"},{"id":82076790,"identity":"01cf342b-c9dc-447f-b3df-2e36c3273aa6","added_by":"auto","created_at":"2025-05-06 13:56:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31822298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDR activation alleviates renal pathological damage in DN mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of DN mice receiving PAR treatment. (B)\u003cstrong\u003e \u003c/strong\u003eRepresentative photographs of kidneys from DN mice treated with PAR or vehicle for 12 weeks (bar = 5 cm). (C) Fasting blood glucose, uACR, and BUN levels of DN mice treated with PAR or vehicle (n = 6). (D) H\u0026amp;E staining of kidney sections collected from DN mice treated with PAR or vehicle (bar = 50 μm). (E) qRT-PCR of GPX4 in the kidney tissues of DN mice treated with PAR or vehicle (n = 6). (F) Western blotting of GPX4 in the kidney tissues of DN mice treated with PAR or vehicle (n = 6). (G) Immunofluorescence staining of 4-HNE of PTECs from DN mice treated with PAR or vehicle (n = 5; bar = 20 μm). (H) GSH levels of DN mice treated with PAR or vehicle (n = 6). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/0e18a58bd53d9d589ba06abd.png"},{"id":82075130,"identity":"e33feba6-4d69-49ed-b2ae-9d94a06551de","added_by":"auto","created_at":"2025-05-06 13:40:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36165250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDR promotes GPX4 expression to inhibit ferroptosis in HK-2 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blotting of VDR and GPX4 in HK-2 cells treated with AGEs (100 ug/ml) for 24 h after pENTER-VDR or EV transfection (n = 6). (B) Cell viability of HK-2 cells treated with AGEs after pENTER-VDR or EV transfection (n = 5). (C) GSH levels in HK-2 cells treated with AGEs after pENTER-VDR or EV transfection (n = 6). (D)Immunofluorescence staining of 4-HNE in HK-2 cells treated with AGEs after pENTER-VDR or EV transfection (n = 5; bar = 50 μm). (E) Representative images of BODIPY (581/591) C11 staining in HK-2 cells treated with AGEs after pENTER-VDR or EV transfection (n = 6; bar = 20 μm). (F) Western blotting of VDR and GPX4 in HK-2 cells treated with AGEs after siVDR or siCtrl transfection (n = 6). (G) Cell viability of HK-2 cells treated with AGEs after siVDR or siCtrl transfection (n = 5). (H)\u003cstrong\u003e \u003c/strong\u003eGSH levels in HK-2 cells treated with AGEs after siVDR or siCtrl transfection (n = 6). (I)Immunofluorescence staining of 4-HNE in HK-2 cells treated with AGEs after siVDR or siCtrl transfection (n = 6; bar = 50 μm). (J) Representative images of BODIPY (581/591) C11 staining in HK-2 cells treated with AGEs after siVDR or siCtrl transfection (n = 6; bar = 20 μm). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/b7590947951e79d96c63ebb2.png"},{"id":82075148,"identity":"07e5c2c0-a73f-4946-8078-45a674d26ae6","added_by":"auto","created_at":"2025-05-06 13:40:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59570350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE3 ubiquitin ligase PRPF19 mediates ubiquitination degradation of VDR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blotting of VDR in the kidney tissues of mice at serial time points (n = 6). (B)\u003cstrong\u003e \u003c/strong\u003eqRT-PCR of VDR in the kidney tissues of mice at serial time points (n = 6). (C)\u003cstrong\u003e \u003c/strong\u003eSchematic diagram showing the process of IP-MS. (D)\u003cstrong\u003e \u003c/strong\u003eKEGG pathway and biological process analysis of VDR interacting proteins. Blue represents high enrichment significance, and blue-and-white represents low enrichment significance. * represents P value\u0026lt;0.05, * * represents P value\u0026lt;0.01, * * * represents P value\u0026lt;0.001. (E) The table shows the binding affinity of 11 candidate proteins involved in VDR ubiquitin-dependent degradation. (F)\u003cstrong\u003e \u003c/strong\u003eWestern blotting of VDR in HK-2 cells treated with AGEs and 3 inhibitors of protein degradation pathways for 24 h (n = 6). (G)Immunofluorescence staining of VDR (green) and PRPF19 (red) in HK-2 cells treated with AGEs or vehicle for 24 h (bar = 20 μm). (H) Precipitation and Western blotting showing the interaction between VDR and PRPF19 in cells treated with AGEs or vehicle after Flag-VDR, Myc-PRPF19 or EV transfection.\u003cstrong\u003e \u003c/strong\u003e(I)\u003cstrong\u003e \u003c/strong\u003eWestern blotting of PRPF19 and VDR in HK-2 cells treated with AGEs after Myc-PRPF19 or EV transfection (n = 6). (J)\u003cstrong\u003e \u003c/strong\u003eWestern blotting of PRPF19 and VDR in HK-2 cells treated with AGEs after Sg-PRPF19 or Co.sgRNA transfection (n = 6). (K) Precipitation and Western blotting showing PRPF19 promotes the ubiquitination of VDR. Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/40e794a9a2b878ef1a426f7b.png"},{"id":82075114,"identity":"ef3076b2-18d5-4c42-9d2f-85f65b423603","added_by":"auto","created_at":"2025-05-06 13:40:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28386852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBBR competitively binds to PRPF19 to inhibit VDR degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The table shows the binding modes and affinities between 8 alkaloids and PRPF19. (B) Images of the interaction between BBR and PRPF19 by molecular docking. (C) Schematic diagram showing the process of IP-MS with high-performance affinity beads. (D) The image shows E3 ubiquitin ligases involved in ubiquitin-mediated proteolysis in sequencing results, and PRPF19 is a key ligase. (E) BBR-binding proteins from HK-2-cell extracts were analyzed with western blotting using an anti-PRPF19 antibody. (F) SPR showing binding of BBR to PRPF19. KD of 3.38× 10\u003csup\u003e-3\u003c/sup\u003e M. (G) Precipitation and Western blotting showing BBR competitively bind to PRPF19 with VDR. (H) Precipitation and western blotting showing the ubiquitination of VDR in DN mice treated with BBR or vehicle.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/35e4cf00e9e2b97ca5313b1b.png"},{"id":82075161,"identity":"a183f96c-d082-4710-9060-c76248b68985","added_by":"auto","created_at":"2025-05-06 13:40:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71143183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBBR inhibits ferroptosis and attenuates renal injury in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative photographs of kidneys in DN mice treated with BBR or vehicle for 4 weeks (bar = 5 cm). (B) Fasting blood glucose, uACR, BUN and Scr levels in DN mice treated with BBR or vehicle for 4 weeks (n = 5 or 6). (C) H\u0026amp;E, Masson and PAS staining of kidney sections collected from DN mice treated with BBR or vehicle, and quantification of tubulointerstitial fibrosis in the kidney cortex (n = 6; bar = 50 μm). (D) Western blotting of VDR, GPX4, FTH1, ACSL4 and TfR1 in the kidney tissues of DN mice treated with BBR or vehicle (n = 4). (E) qRT-PCR of GPX4 in the kidney tissues of DN mice treated with BBR or vehicle (n = 6). (F) Representative transmission electron microscopy images of PTECs from DN mice treated with BBR or vehicle, and absolute counting of the number of cristae per mitochondria (n = 6; bar = 10 μm). (G) Immunofluorescence staining of 4-HNE of PTECs from DN mice treated with BBR or vehicle (n = 6; bar = 50 μm). (H) GSH, MDA and iron levels of DN mice treated with BBR or vehicle (n = 5). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/5fa8cd5f96e717fe358ac697.png"},{"id":82075125,"identity":"f55d6bb3-f2e7-4929-aaab-0574125a8946","added_by":"auto","created_at":"2025-05-06 13:40:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":34850034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBBR diminishes ferroptosis by a PRPF19-dependent pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell viability of HK-2 cells treated with series concentrations of BBR and Fer-1(n = 3). (B) Western blotting of VDR, GPX4 and FTH1 in HK-2 cells treated with erastin (5 μM), BBR (10 μM), Fer-1 (5 μM) or vehicle (n = 6). (C) GSH, MDA and iron levels in HK-2 cells treated with erastin (5 μM), BBR (10 μM), Fer-1 (5 μM) or vehicle (n = 4-6). (D) qRT-PCR of GPX4 in HK-2 cells treated with erastin (5 μM), BBR (10 μM), Fer-1 (5 μM) or vehicle (n = 3). (E)\u003cstrong\u003e \u003c/strong\u003eFluorescence staining of JC-1 in HK-2 cells treated with erastin (5 μM), BBR (10 μM), Fer-1 (5 μM) or vehicle. In cells with high mitochondrial membrane potential (ΔΨm), JC-1 forms complexes known as J-aggregates that emit an orange to red fluorescence. In cells with low ΔΨm, JC-1 remains in the monomeric form, which emits a green fluorescence (n = 6; bar=50μm). (F) Cell viability of HK-2 cells transfected with pcDNA3.1-PRPF19 or EV followed by AGEs and BBR treatment (n = 3). (G) Western blotting of VDR, GPX4 and FTH1 in HK-2 cells transfected with pcDNA3.1-PRPF19 or EV followed by AGEs and BBR treatment (n = 5). (H-J)\u003cstrong\u003e \u003c/strong\u003eGSH, MDA and iron levels in HK-2 cells transfected with pcDNA3.1-PRPF19 or EV followed by AGEs and BBR treatment (n = 6). (K) Immunofluorescence staining of 4-HNE in HK-2 cells transfected with pcDNA3.1-PRPF19 or EV followed by AGEs and BBR treatment (n = 6; bar = 50μm). (L) Representative images of BODIPY (581/591) C11 staining in HK-2 cells transfected with pcDNA3.1-PRPF19 or EV followed by AGEs and BBR treatment (n = 6; bar = 20 μm). Data are expressed as means ± SEM. Student’s t-test was employed for comparisons between two groups; one-way ANOVA followed by Tukey’s post-test for multiple comparisons was used for groups of three or more.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/0de3b922daa573e560783767.png"},{"id":82075107,"identity":"32491289-bccc-4a45-b836-dc0335435f93","added_by":"auto","created_at":"2025-05-06 13:40:36","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":11995399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic depictingBBR binds to PRPF19 and suppresses RTECs ferroptosis to alleviate DN.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/eb34a2c8257e50494a37416c.png"},{"id":82075083,"identity":"716c58ec-6189-4ca1-a789-205638e360dd","added_by":"auto","created_at":"2025-05-06 13:40:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1334518,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/cc7503ce-56ab-407c-b729-6a728e737a78.pdf"},{"id":82075133,"identity":"a1105fee-d7a7-44a4-8802-d26ec62b60c7","added_by":"auto","created_at":"2025-05-06 13:40:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21664386,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6083539/v1/330b312f9a36cf37b9148869.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PRPF19 mediates the proteasomal degradation of VDR to exacerbate ferroptosis in diabetic nephropathy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDN is a common and serious microvascular complication of diabetes mellitus (DM), and it is the leading cause of end-stage renal disease[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Regarding the pathogenesis of DN, the \u0026ldquo;glomerulocentric view\u0026rdquo; has been dominant for a long time. However, accumulating evidence indicates that renal tubular injury also plays a key role in the pathogenesis of DN, and tubulopathy may precede glomerular alterations[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The renal proximal tubular cells are uniquely susceptible to a variety of metabolic and hemodynamic factors associated with diabetes, especially to hyperglycemia, leading to enhanced O\u003csub\u003e2\u003c/sub\u003e consumption and increased hypoxic tubular damage[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Impaired tubular uptake explains albuminuria in early DN[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and tubular damage is a reliable predictor of renal functional deterioration[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, protective effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors validate the proximal tubule cell as a target of therapy[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Better understanding of the pathobiology of renal tubular injury will lead to identification of novel therapeutic targets for the treatment of DN.\u003c/p\u003e \u003cp\u003eFerroptosis is a form of regulated cell death characterized by iron-dependent membrane lipid peroxidation[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Remarkably, accumulating evidence shows that ferroptosis in RTECs is an important driver for the progression of DN[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The glutathione (GSH)-dependent lipid hydroperoxidase GPX4 prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which is one of the key members in ferroptosis defense systems[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Yet, the specific regulatory mechanism of GPX4 expression level in RTECs remains largely undefined. Some studies have reported that GPX4 is reduced via ubiquitin-proteasomal degradation during ferroptosis in RTECs[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the exact role that gene expression regulation mechanisms plays in GPX4 expression level is still not fully elucidated.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCoptis chinensis Franch\u003c/em\u003e is a common traditional Chinese medicine (TCM) and has a long application history, which is widely used for treating DM and its complications, hyperlipidemia and gastrointestinal infections[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The active ingredients and agents of the bitter taste of \u003cem\u003eCoptis chinensis Franch\u003c/em\u003e are mainly protoberberine-type alkaloids such as berberine (BBR), coptisine, jatrorrhizine, palmatine, columbamine, epiberberine, magnoflorine and groenlandicine[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Reportedly, \u003cem\u003eCoptis chinensis Franch\u003c/em\u003e and its pharmacological active ingredients can significantly ameliorate DN by protecting glomerular podocyte damage[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the role of its active ingredients in tubular injury still needs further exploration.\u003c/p\u003e \u003cp\u003eHere, we found that ferroptosis occurred in the late-stage of DN through two animal models of DN (STZ-induced diabetic mice and db/db mice) and human plasma samples. We then identified VDR transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs. Tubule-specific deletion of VDR exacerbated renal morphological abnormalities and renal dysfunction. Additionally, we demonstrated that the level of VDR was predominantly controlled by the PRPF19-mediated ubiquitin-proteasome degradation system, and the natural compound BBR could bind to PRPF19 to inhibit VDR degradation, and protect renal injury. Collectively, this study proposed a new perspective on the potential of targeting PRPF19 to inhibit ferroptosis in DN therapy.\u003c/p\u003e"},{"header":"2. Methods and materials","content":"\u003cp\u003e \u003cb\u003eAnimals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe RTEC-specific VDR knockout mice were generated by crossing Pax8-Cre mice (provided by Shulaibao (Wuhan) Biotechnology Co., Ltd.) with VDR-flox mice mice (provided by Shulaibao (Wuhan) Biotechnology Co., Ltd.). The DN model was established by intraperitoneal injection of STZ (40 mg/kg, Sigma #S0130) for 7 days and high-fat diet (HFD) (60% Kcal fat, #D12492, Research Diets, New Brunswick, NJ) for 4 weeks. Nec-1 (5 mg/kg, MedChemExpress #HY-1576), Fer-1 (5 mg/kg, MedChemExpress #HY-100579), Emricasan (12.5 mg/kg, MedChemExpress #HY-10396), and 3-MA (10 mg/kg, MedChemExpress #HY-19312) were injected intraperitoneally once a week until the animals were euthanized, with saline as a control. PAR (1 ug/kg, MedChemExpress #HY-50919) were injected intraperitoneally once a week for 12 weeks, with saline as a control. Intragastric administration of BBR (300 mg/kg/d, Aladdin #B414323) or vehicle was started at 16 weeks of age and maintained for 4 weeks. The dosage of BBR (300 mg/kg/d) used in our experiment was chosen according to animal studies and clinical trials previously reported[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Mice were housed in a 12-h light/dark cycle with free access to a standard rodent diet and water. All animal euthanasia by anesthesia (overdose of pentobarbital, intraperitoneal injection) and all animal care procedures were performed according to the guidelines of the Committee for Animal Research of Huazhong University of Science and Technology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture, transfection, and treatments\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman renal tubular cell line HK-2 (Procell Life Science\u0026amp;Technology Co,.Ltd #CL-0109) was cultured in DMEM/F12 media plus 10% FBS (Procell Life Science\u0026amp;Technology Co,.Ltd #CM-0109). HEK293T cells (Cell Health, CHCH-0004) were cultured in DMEM media containing 10% FBS (Cell Health, CHCH-0004-025). HEK293T were transfected with 5 \u0026micro;g of EV, pENTER-VDR or pcDNA3.1-PRPRF19 using exfect transfection reagent (Vazyme, T101). 48 h after transfection, cells were treated with or without AGEs (100 ug/ml) and MG-132 (30 nM) for additional 24 h. siVDR or siCtrl were transfected into HK-2 cells by RNAiMAX transfection reagent (Thermo Fisher Scientific, 13778030) for 72 h. HK-2 were preincubated with 10 \u0026micro;M BBR, 5 \u0026micro;M Fer-1 or basic medium for 12 h. Then cells were cultured with 5 \u0026micro;M erastin (MedChemExpress #HY-15763) for 12 h and collected for subsequent assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual-luciferase reporter assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHEK293 cells were cultured on 12-well cell culture plates overnight. Cells were then transiently transfected with wild-type (WT) or mutant PGL3-GPX4-luc (luciferase reporter) and pLR-TK (renilla internal control) and vectors carrying full-length human gene (pCDH-puro-POU2F2, pENTER-VDR, pENTER-YY1, pcDNA3.1-MAZ, pENTER-AR, pENTER-USF2, pENTER-ATF3) using exfect transfection reagent. After 48 h, cells were washed once with PBS and lysed for measurement of luciferase activity using the Dual-Luciferase Reporter Assay System according to the manufacturer\u0026rsquo;s instructions (Promega, Mannheim, Germany). All measurements were performed with a Tecan Infinite M200 PRO luminometer (Tecan, Crailsheim, Germany). For determination of specific luciferase activity, activity of the firefly luciferase was normalized to the activity of the renilla luciferase.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCUT\u0026amp;RUN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCUT\u0026amp;RUN was performed as described in Hyperactive pG-MNase CUT\u0026amp;RUN Assay Kit (Vazyme, HD101) protocol. Briefly, 0.5\u0026nbsp;million live HEK293 cells were collected and resuspended in 100 ul wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM Spermidine, supplemented with Protease Inhibitor EDTA-Free tablet (Sigma-Aldrich #5056489001)). Activated Concanavalin A was incubated with cells at room temperature for 10 min to let the cells bind to the beads. 0.5 \u0026micro;g VDR or H3K4me3 histone antibodies were added to each sample and incubated in the antibody buffer (wash buffer\u0026thinsp;+\u0026thinsp;0.01% Digitonin and 2 mM EDTA) at 4\u0026deg;C for 4 h. The beads were then washed twice with digitonin buffer (wash buffer\u0026thinsp;+\u0026thinsp;0.01% Digitonin), and 2.5 \u0026micro;l pG-MNase was added to each sample. After 10 min of incubation at room temperature, excessive pG-MNase was washed out by a two-time digitonin buffer wash. Then targeted chromatin was digested and released from cells by 2 h of incubation with the presence of 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e at 4\u0026deg;C, which were collected from the supernatant, and subjected to phenol/chloroform DNA extraction and finally to qRT-PCR detection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEMSA\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNuclear proteins were extracted using a commercial kit (Carlsbad, CA, USA). The extracted proteins were incubated in reaction buffer on ice, followed by the addition of biotin-labeled probes. For supershift assays, anti-VDR antibodies were added to the reaction mixture prior to probe incubation. All steps were performed using the light shift Kit (Pierce, Rockford, IL, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIP-MS\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe kidney tissue of mice was ground and digested to extract lysates. After transfection and treatments, HK-2 cells were lysed with pre-lysis buffer. The tissue and cell lysates were incubated with VDR antibody or respective IgGs antibody with protein A/G magnetic beads (MedChemExpress #HY-K0202) overnight at 4\u0026deg;C. After washing (1\u0026times;PBS\u0026thinsp;+\u0026thinsp;0.5% Tween-20, PH 7.4), the beads were boiled in loading buffer and subjected to mass spectrometry detection and immunoblotting.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of BBR-immobilized beads\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMagnetic FG beads (Linker beads, TAS8848N1110, Tamagawa Seiki, 1 mg) were incubated with a 10 mM or 12 mM solution of the berberrubine (with DMSO as a control) in N, N-dimethylformamide and 14 mg of potassium carbonate for 16\u0026ndash;20 h at 60\u0026deg;C. Unreacted residues were masked using 50% methanol, and the resulting beads were stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAffinity purification with BBR-immobilized beads\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBBR-immobilized beads (0.5 mg) were equilibrated with 100 mM KCl buffer containing 20 mM HEPES-NaOH (pH 7.9), 100 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EDTA,10% (v/v) glycerol, 0.1% NP-40, 1 mM DTT, and 0.2 mM PMSF. Cell extracts were prepared from HK-2 cells and were incubated with the beads for 4 h at 4\u0026deg;C. The beads were washed three times with 100 mM KCl buffer, and bound proteins were eluted with 1\u0026times; loading dye solution containing 62.5 mM Tris-HCl (pH 6.8), 0.005% bromophenol blue, 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSPR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used SPR to determine the real-time interactions between BBR and PRPF19. The device used is BiacoreT200 (Cytiva). PRPF19 protein from AtaGenix at 1.67 mg/ml was diluted with 10mM sodium acetate, pH 4.0, and immobilized on Series S Sensor Chip CM5 (Cytiva) (7 min activation with blocking buffer) at 10\u0026micro;l/min to get immobilization signal of 5550 RU. Samples were prepared as dilution series with the starting concentration of 25 \u0026micro;M in the running buffer for BBR (0.78, 1.5625, 3.125, 6.25, 12.5, 25 \u0026micro;M). Running and sample buffer contained 1\u0026times;PBS, 0.005% Tween 20, 1% DMSO, and pH 7.4 at 25\u0026deg;C. Flow and injections scheme consisted of 30 \u0026micro;l/min for 120 s followed by 200 s of dissociation. Reference channel was activated with EDC/NHS and deactivated with 1 M ethanolamine, pH 8.3. Sensorgram response data were analyzed by using the BIA evaluation kinetics software, and the equilibrium binding and disassociation constants were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-sequencing analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe mRNA of mice kidney tissue was extracted and sent to Shanghai Gene Co., Ltd. for mRNA sequencing. Differential gene expression analysis was completed using the limma R package (version 3.44.3). The fold change (FC) in expression of each gene was log\u003csub\u003e2\u003c/sub\u003e transformed and further analyzed using RStudio version 1.1.442 (RStudio, Inc., USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUrine and serum analyses\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUrinary albumin, creatinine and BUN were measured using urine protein test kit (Nanjing Jiancheng, China), creatinine assay kit (Nanjing Jiancheng, China) and urea assay kit (Nanjing Jiancheng, China). Blood glucose was measured using Haier Blood Glucose Meter (China). MDA, GSH, GPX4, ferritin, TfR and 1,25-(OH)2D3 content of plasma was analyzed by lipid peroxidation test kit (Beyotime, China), reduced glutathione assay kit (Nanjing Jiancheng, China), human GPX4 ELISA kit (Bioswamp, China), human ferritin ELISA kit (Raybiotech, USA), human TfR ELISA kit (Raybiotech, USA) and human 1,25-(OH)2D3 ELISA kit (Nanjing Jiancheng, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistologic analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eParaffin-embedded mouse kidney sections (5 \u0026micro;m thickness) were prepared by a routine procedure. Sections were stained with H\u0026amp;E, masson and PAS. TEM was performed on glutaraldehyde-fixed, epoxy-embedded kidney samples and stained with uranyl acetate and lead citrate. TEM images were collected by RADIUS Software v2.1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe paraffin-embedded sections were deparaffinized and stained with primary antibodies overnight at 4\u0026deg;C. After washing in PBS, sections were visualized by secondary antibody. Nuclear counterstaining was performed using 4\u0026rsquo;,6-diamidino-2-phenylindole. Cultured cells were first fixed and blocked with serum. After that, sections or cells were incubated with antibodies. Staining was observed by a fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and qRT-PCR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA was extracted from samples using the Trizol reagent (Takara, Japan). Then, cDNA was synthesized using the reverse transcriptase kit (Takara, Japan). Quantitative analysis of mRNA expression was conducted with a SYBR premix EX TaqTM kit (Takara, Japan) with StepOne PCR system (Applied Biosystems, USA). The relative quantity of mRNA was expressed as 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e. Sequences of the primers were listed in Table S2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFreshly collected kidney or cultured cells were sonicated in ice-cold RIPA buffer (Beyotime), 20\u0026ndash;80 \u0026micro;g protein from each sample was separated by SDS-PAGE. The proteins were transferred onto PVDF membranes for immune detection. The antibodies used were provided in Table S3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStatistical analyses were performed with Prism 7 (GraphPad Software, Inc., USA) or RStudio version 1.1.442 (RStudio, Inc., USA). For data with a normal distribution and homogeneity of variance, one-way ANOVA was performed for comparisons among more than two groups. Two-tailed Student\u0026rsquo;s t tests were performed to evaluate significant differences between two groups. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences for which P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Ferroptosis is the predominant form of cell death in the late-stage of DN.\u003c/h2\u003e\n\u003cp\u003eTo acquire a full understanding of the relative contributions of different types of cell death in DN renal tubular injury, high-fat-fed STZ-induced diabetic mice were intraperitoneally injected with different inhibitors of cell death once a week. A time series analysis of urine albumin-to-creatinine ratio (uACR) and fasting food glucose of mice at different phase of DN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) was performed. The results revealed that in the early-stage of DN, necrostatin-1 (Nec-1, inhibitor of necroptosis), rather than emricasan (inhibitor of apoptosis), 3-methyladenine (3-MA, inhibitor of autophagy) or ferrostatin-1 (Fer-1, inhibitor of ferroptosis) significantly improved fasting blood glucose and uACR in mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Nec-1 had a protective effect on early renal tubular lesions and mitochondrial morphological abnormalities (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), which suggested that necroptosis might be involved in the early renal tubular injury in DN. However, in the late-stage of DN, renal function and blood glucose levels were improved more significantly in the Fer-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results of Hematoxylin and Eosin (H\u0026amp;E), Masson and Periodic Acid-Schiff staining(PAS)staining showed that Fer-1 treatment markedly improved renal tubule swelling, epithelial cell exfoliation and death, tubulointerstitial fibrosis and glycogen deposition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, decreased mitochondrial count, the rupture of mitochondrial cristae, and swelling of mitochondrial morphology were prominently alleviated in the Fer-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG). This indicated that, unlike in the early-stage, ferroptosis might be the main form of cell death in the late-stage of DN.\u003c/p\u003e\n\u003cp\u003eFurthermore, 80 plasma samples from late-stage DN patients or normal individuals were collected for enzyme-linked immunosorbent assay (ELISA) analysis of the markers related to ferroptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). Clinical demographics of these subjects were provided in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. The results showed that the expression of molecules that promoted lipid peroxidation and iron overload, such as malondialdehyde (MDA), ferritin and transferrin receptor 1 (TfR1), were increased in plasma of DN patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). In contrast, molecules with antioxidant properties, such as GSH and GPX4, were down-regulated in DN patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). Subsequently, in 24-week-old db/db mice, we validated the disorders of renal function and blood glucose level (Fig. S2A and S2B), and found an increase in ferroptosis markers (ACSL4, TfR1, 4-HNE, MDA, Fe\u003csup\u003e2+\u003c/sup\u003e) level, significant mitochondrial cristae breakage, and decrease in GPX4 and GSH levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC-\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eH and Fig. S2C-S2H). These results suggest that ferroptosis plays an important role in prolonged renal tubular damage in DN.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 VDR is a key transcriptional regulator of GPX4\u003c/h2\u003e\n\u003cp\u003eWe first confirmed the reduction of GPX4 in the kidney of DN mice by RNA sequencing (RNA-seq) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis results also showed that the expression level of GPX4 decreased in STZ and HFD-induced DN mice, db/db mice and HK-2 cells treated with advanced glycation end-products (AGEs) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Importantly, the expression of GPX4 decreased by degrees with the progression of DN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD), and the mRNA level of GPX4 were positively correlated with the protein level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Thus, we speculated that the decrease of GPX4 was related to the mechanism of transcriptional regulation. By intersecting the prediction results of 4 transcription factor databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://jaspar.genereg.net\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfo.life.hust.edu.cn/AnimalTFDB#!/\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gtrd20-06.biouml.org\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://alggen.lsi.upc.es\u003c/span\u003e\u003c/span\u003e), we obtained 7 candidate GPX4 transcription factors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). And by copying and selectively inserting the GPX4 promoter sequence into the PGL3 vector, we obtained the PGL3-GPX4-luc plasmid (Fig. S3A). The results of dual-luciferase reporter assay and cleavage under targets and release using nuclease (CUT\u0026amp;RUN) showed that VDR was the main transcription factor of GPX4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH, the results of POU2F2 were negative, while the results of YY1 lacked specificity) and could target the AGGGGTCA base sequence (1423\u0026ndash;1430 bp upstream from transcription start site) in the GPX4 promoter sequence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI). Mutation of the AGGGTCA motif (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ) significantly attenuated VDR-mediated luciferase activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eK), confirming this sequence as the functional VDR binding site. Furthermore, electrophoretic mobility shift assay (EMSA) directly demonstrated VDR binding to the GPX4 promoter (Fig. S3B). Notably, we measured the content of VDR active ligand-1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in the plasma of DN patients by ELISA, and found that there was a significant negative correlation between 1,25-(OH)2D3 and uACR levels (R=-0.77, P\u0026thinsp;=\u0026thinsp;5.5e-09) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL). These data indicate that VDR is a key transcription factor of GPX4 and may affect renal function.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 VDR deletion promotes ferroptosis to exacerbate renal tubule injury in DN mice\u003c/h2\u003e\n\u003cp\u003eTo explore the contribution of VDR to the maintenance of GPX4 within RTECs, we generated RTEC-specific VDR knockout mice (Pax8-Cre/VDR\u003csup\u003efl/fl\u003c/sup\u003e, VDR-cKO) by crossing Pax8-Cre and VDR-flox mice. VDR-cKO mice presented no obvious kidney dysfunction physiologically (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, post-STZ and HFD induction, VDR-cKO mice exhibited an amplified renal hypertrophy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, Fig. S4A and S4B). VDR deficiency significantly increased fasting blood glucose, uACR, and blood urea nitrogen (BUN) levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, compared with VDR\u003csup\u003efl/fl\u003c/sup\u003e mice, VDR-cKO mice showed more severe renal tubular enlargement, brush edge destruction, and epithelial cell shedding after STZ and HFD induction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). The VDR-cKO group showed a significant decrease in GPX4 levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). Meanwhile, there was more lipid peroxidation product 4-hydroxynonenal (4-HNE) accumulation in RTECs of VDR-cKO mice than VDR\u003csup\u003efl/fl\u003c/sup\u003e mice treated with STZ and HFD (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG), accompanied by significantly reduced GSH levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). We further performed RNA-seq in the cortex of kidney from VDR-cKO mice and VDR\u003csup\u003efl/fl\u003c/sup\u003e mice treated with STZ and HFD. Transcriptomic analysis showed that VDR knockdown resulted in down-regulation of GPX4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI), and KEGG pathway enrichment analysis of the differentially expressed genes high-lighted cellular processes such as ferroptosis and glutathione metabolism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Simultaneously, GO enrichment analysis was performed on differentially expressed genes (Fig. S4C), and it was found that the molecular functions emphasized glutathione peroxidase activity (Fig. S4D). These findings indicate that VDR affects RTECs death primarily through a ferroptosis-mediated mechanism.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 VDR activation alleviates renal pathological damage in DN mice\u003c/h2\u003e\n\u003cp\u003eWe also investigated whether activation of VDR would alleviate renal tubular injury in vivo. After the induction of STZ and HFD, DN mice were intraperitoneally injected with VDR agonist-PAR or vehicle once a week (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). After 12 weeks of treatment, the mice showed significant improvement in kidney hypertrophy, fasting blood glucose, uACR, and BUN levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. S4E and S4F). Histological analysis of the lesions revealed a reduction in swelling of renal tubules in the PAR group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). GPX4 and GSH expression in RTECs was significantly increased in the PAR group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH), while 4-HNE level was significantly reduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). These data indicate that VDR protects RTECs by stabilizing intracellular GPX4 levels and reducing lipid peroxidation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 VDR promotes GPX4 expression to inhibit ferroptosis in HK-2 cells\u003c/h2\u003e\n\u003cp\u003eSimilarly, to determine whether VDR played a crucial role in the process by which GPX4 regulated ferroptosis, we transfected pENTER-VDR plasmids or empty vector (EV) into HK-2 cells in high-glucose medium supplemented with AGEs. Western blotting showed a significant increase in the content of VDR and its target protein-GPX4 after plasmid transfection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Using CCK-8 assay kit to detect cell viability, it was found that overexpression of VDR significantly increased cell viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, increased expression of VDR could increase intracellular GSH content (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC) and reduce the accumulation of 4-HNE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). The lipid reactive oxygen species (ROS) accumulation was detected by BODIPY (581/ 591) C11 probe. Normal probe displays red fluorescence, and the probe bound to peroxidized lipids showed green fluorescence. The results showed that the level of lipid peroxidation was obviously downregulated after VDR overexpression in HK-2(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). On the contrary, when HK-2 cells were transfected with VDR specific siRNA (siVDR) or negative control siRNA (siCtrl), compared with the siCtrl group, VDR and GPX4 decreased significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF), cell viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG) and GSH (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH) content decreased, while 4-HNE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI) and ROS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ) accumulation increased accordingly in the siVDR group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 E3 ubiquitin ligase PRPF19 mediates ubiquitination degradation of VDR\u003c/h2\u003e\n\u003cp\u003eTo explore the mechanism of VDR-induced RTECs ferroptosis, we investigated the expression of VDR in mice during the progress of DN. Western blotting showed a gradual decrease in the protein level of VDR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), but no significant change in the mRNA level by qRT-PCR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). This suggested that the downregulation of VDR might be related to protein post-translational modifications rather than transcriptional regulatory mechanisms. Therefore, we used Immunoprecipitation-mass spectrometry (IP-MS) to elute VDR related interacting proteins and perform mass spectrometry analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). The results of 4-dimensional data-independent acquisition (4D DIA) quantitative proteomics demonstrated that compared with the IgG group, 119 molecules were upregulated and 233 molecules were downregulated in the elution proteins of the VDR group (Fig. S5A). VDR-related proteins were enriched in the ferroptosis pathway and involved in regulation of gene expression (Fig. S5B, S5C and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Interestingly, they also participated in the ubiquitin-dependent protein catabolic process (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). By comparing the proteins involved in the ubiquitin-dependent protein catabolic process, we identified the molecule-PRPF19 (an E3 ubiquitin ligase) with the strongest binding affinity to VDR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003eTo verify the involvement of the ubiquitin-proteasome pathway in VDR degradation, we utilized different inhibitors of protein degradation mechanisms. Western blotting showed that only MG-132 (ubiquitin-dependent proteasome inhibitor), rather than bafilomycin A1 (BafA1, autophagy-dependent lysosome inhibitor) or oroxylin A (mitochondrial autophagy inhibitor), restored the VDR levels in cultured HK-2 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). The immunofluorescence staining proved that VDR and PRPF19 were colocalized in cytoplasm, and the overlapping fluorescence intensity of VDR and PRPF19 was stronger in the AGEs group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). Thus, we preliminarily determined that PRPF19 was involved in the ubiquitin-proteasome degradation process of VDR. The results of co-immunoprecipitation (Co-IP) experiments confirmed the interactions between VDR and PRPF19, which were enhanced after treatment with AGEs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). To investigate the role of PRPF19 in VDR regulation, we transfected HK-2 cells with either Myc-PRPF19 plasmids or PRPF19-targeting sgRNA (Sg-PRPF19). Notably, PRPF19 overexpression significantly reduced VDR protein levels, whereas PRPF19 knockout increased VDR expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Co-IP assays further demonstrated that PRPF19 enhances VDR ubiquitination, as overexpression of PRPF19 markedly increased VDR ubiquitination levels, whereas PRPF19 knockout reduced them (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eK). These findings demonstrate that PRPF19 is involved in the degradation of VDR by a ubiquitination-dependent pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 BBR competitively binds to PRPF19 to inhibit VDR degradation\u003c/h2\u003e\n\u003cp\u003eTo test whether the active ingredients of \u003cem\u003eCoptis chinensis Franch\u003c/em\u003e could controll ferroptosis via PRPF19, we used molecular docking to predict the binding modes and affinities between 8 alkaloids and PRPF19. The analysis results showed that BBR displayed the lowest binding energy of \u0026minus;\u0026thinsp;8.3 kcal/mol (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), forming hydrogen bond with PHE-483 residues of PRPF19 and favorable hydrophobic interactions with GLN-445 and ILE-442 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Furthermore, molecular dynamics (MD) simulations showed the root mean square deviation (RMSD) of the BBR-PRPF19 system stabilizes between 60\u0026ndash;100 ns (Fig. S6A), indicating a stable binding interaction. Root mean square fluctuation (RMSF) analysis revealed conformational flexibility in specific regions of PRPF19 upon BBR binding (Fig. S6B). Notably, residues 255\u0026ndash;270, 340\u0026ndash;350, 370\u0026ndash;390, 430\u0026ndash;440, and 470\u0026ndash;480 exhibited higher RMSF values, suggesting increased flexibility in these regions due to BBR recognition. We next investigated whether BBR could directly bind to PRPF19. We used high-performance affinity beads, which the active BBR derivative berberrubine could covalently conjugated to, to purify drug-targeted proteins from cell extracts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Using BBR-immobilized beads or naked beads for pull-down experiments, and performing mass spectrometry analysis on the fractions eluted from the beads. 5335 proteins were identified in the fractions (Fig. S6C), and KEGG pathway classification analysis indicated that BBR was involved in the regulation of cell growth and death (Fig. S6D). Importantly, the results of 4D DIA quantitative proteomics demonstrated that BBR-related proteins were also involved in ubiquitin-mediated proteolysis (Fig. S6E), and PRPF19 was a key E3 ubiquitin ligase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Again, fractions eluted from BBR-immobilized beads were subjected to immunoblotting and probed with a PRPF19-specific antibody. The results showed that PRPF19 was clearly isolated as a BBR-specific binding protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE), and high concentration of BBR covalently conjugated to beads could increase the production of PRPF19 compared with low concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). To further establish that BBR directly bound to PRPF19, we used SPR, a technique for detecting the interactions between ligands and analytes on biosensor chips. SPR demonstrated that BBR directly bound to PRPF19, with an estimated equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) of 3.38\u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). Next, to verify whether BBR could competitively bind to PRPF19 with VDR, we conducted co-immunoprecipitation experiments. It was found that by adding additional BBR to the co-incubation system of cell extracts and beads, the content of PRPF19 protein pulled down by VDR was reduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG). In addition, we performed immunoprecipitation experiments in DN mice treated with BBR or vehicle, and found that BBR treatment significantly decreased the ubiquitination level of VDR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH). These results demonstrate that BBR can competitively bind to PRPF19 with VDR to reduce VDR ubiquitination degradation, which may play a role in inhibiting renal tubular ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.8 BBR inhibits ferroptosis and attenuates renal injury in vivo\u003c/h2\u003e\n\u003cp\u003eNext, to examine the therapeutic efficiency of BBR in DN, we treated mice with intragastric administration of BBR (300 mg/kg/d) for 4 weeks. The STZ and HFD-induced elevation of kidney volume (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA and Fig. S7A), elevated fasting blood glucose, uACR, BUN and serum creatinine (Scr) levels were reversed by BBR treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). We further found STZ and HFD-induced DN mice exhibited significant renal tubular tissue damage, interstitial fibrosis and glycogen deposition, while BBR treatment effectively blocked these pathological changes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). Moreover, DN mice induced a significant decrease in VDR, GPX4 and ferritin heavy chain 1 (FTH1, a molecular biomarker inhibiting ferroptosis) protein levels, while acyl-CoA synthetase long-chain family member 4 (ACSL4) and TfR1 (molecular biomarkers promoting ferroptosis) protein levels increased, which were effectively reversed by BBR treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). The mRNA level of GPX4 was significantly reduced in DN mice, but BBR treatment could salvage the decrease in GPX4 mRNA level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE). After BBR gavage, the morphological changes of mitochondria in DN mice were obviously mitigated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eF). Furthermore, high levels of lipid peroxidation products (4-HNE and MDA) and iron content were effectively suppressed by BBR, and the low level of GSH was apparently recovered (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG, \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eH and Fig. S7B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.9 BBR diminishes ferroptosis of RTECs by a PRPF19-dependent pathway\u003c/h2\u003e\n\u003cp\u003eIn vitro experiments, using Fer-1 as a positive control, it was notable that the BBR-treated group was resistant to erastin-induced ferroptosis, as evidenced by the increase in cell viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA), as well as reduction in lipid peroxidation and iron accumulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC and Fig. S7C). In vitro, BBR treatment could also reverse the decrease in GPX4 mRNA and protein levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD). In addition, by utilizing JC-1, an ideal fluorescent probe widely used for detecting mitochondrial membrane potential \u0026Delta;ᴪ m, we discovered that the decrease in membrane potential was evidently reversed by BBR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eE). Our data demonstrate that BBR can inhibit ferroptosis of RTECs in vitro.\u003c/p\u003e\n\u003cp\u003eTo test whether BBR played a protective role by a PRPF19-dependent pathway, we employed a PRPF19-overexpression cell model. After transfection of pcDNA3.1-PRPF19 plasmids into HK-2 cells, we observed that BBR failed to further reverse the decrease in cell viability caused by AGEs stimulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eF). In agreement, in PRFP19-overexpressing cells, BBR could not further salvage the AGEs-induced decrease in VDR, GPX4 and FTH1 levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eG). GSH is an important antioxidant substance, while MDA and 4-HNE are by-products of lipid peroxidation. After overexpression of PRPF19, the ability of BBR to reduce lipid peroxidation significantly decreased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eH, \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eI and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eK). Moreover, in PRPF19-overexpressing cells, the ability of BBR to reduce Fe\u003csup\u003e2+\u003c/sup\u003e and ROS levels was greatly weakened (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eJ and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eL). These results convincingly show that BBR diminishes renal tubular ferroptosis by a PRPF19-dependent pathway.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we revealed the significant involvement of PRPF19/VDR/GPX4 axis in DN through the findings of in vivo, in vitro, and pharmacological investigations. The major findings of this work include: (i) ferroptosis was the predominant form of cell death in the late-stage of DN; (ii) VDR transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs; (iii) mechanistically, E3 ligase PRPF19 mediates ubiquitination degradation of VDR; and (iv) BBR was verified as a novel inhibitor of PRPF19 and effectively alleviated ferroptosis in renal tubules.\u003c/p\u003e \u003cp\u003eRenal injury in DM is a long-term and continuous process. Multiple studies have reported that DN involves in various forms of cell death, such as apoptosis, necrosis, autophagy, and newly discovered ferroptosis[\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, it is still unclear which mode of cell death plays a more important role at different stages of DN. In this study, we found that in diabetic mice with long-term renal injury, the ferroptosis inhibitor Fer-1 significantly reduced urinary protein excretion, lowered blood glucose, ameliorated tubular pathological damage and renal interstitial fibrosis. Ferroptosis phenomenon was more significant in late-stage DN mice and aged db/db mice. This phenomenon may be attributed to the combined effects of elevated oxidative stress[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], impaired antioxidant defense mechanisms[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], disrupted iron homeostasis[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and sustained chronic inflammation[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] in the advanced stages of DN. In addition, compared with normal individuals, the levels of lipid peroxidation and iron accumulation markers were increased in the plasma of late-stage DN patients. These data show that ferroptosis may be involved in the late-stage of DN renal injury. The elucidation of the ferroptosis time window provides a basis for the clinical translation of intervention strategies for DN.\u003c/p\u003e \u003cp\u003eNon-open-loop steroid 1,25-(OH)2D3 is a hormone form of vitamin D and an endocrine hormone with multiple physiological functions[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. VDR is a nuclear receptor for 1,25-(OH)2D3 and the activation of VDR depends on 1,25-(OH)2D3 ligands[\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52 CR53 CR54 CR55\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Previous studies have found that PAR and 1,25-(OH)2D3 have renal protective effects in DN[\u003cspan additionalcitationids=\"CR58 CR59 CR60 CR61\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In a multinational, placebo-controlled, double-blind trial, de Zeeuw D et al. stated addition of 2 \u0026micro;g/day PAR to renin\u0026ndash;angiotensin\u0026ndash;aldosterone system (RAAS) inhibition safely lowered residual albuminuria in patients with DN (P\u0026thinsp;=\u0026thinsp;0.014 vs placebo) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Another analysis included 14,709 participants with type 2 diabetes from the UK Biobank observed that higher serum 1,25-(OH)2D3 concentrations were significantly associated with lower risk of diabetic microvascular complications, including DN (P\u0026lt;0.05; aHR: 0.54, 95% CI: 0.38\u0026ndash;0.78)[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Here, our research findings indicate that VDR, as an important transcription factor, binds to the GPX4 promoter to suppress tubular cell ferroptosis and renal dysfunction, and provide evidence for the explanation of the therapeutic effects of PAR and 1,25-(OH)2D3 on DN.\u003c/p\u003e \u003cp\u003eRegarding the degradation pathway of VDR, previous studies have shown that the AF-2 domain of VDR interacts with mSUG1, a component of the 26S proteasome, which may target VDR towards proteasome-mediated degradation[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In addition, in some cellular environments, unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation, while 1,25-(OH)2D3-dependent heterodimerization with retinoid X receptor and subsequent nuclear localization protect VDR from these modifications[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. This is consistent with our research results. By applying inhibitors targeting the ubiquitin-proteasome system, lysosome-autophagy system, and mitochondrial autophagy system, we found that ubiquitin-dependent degradation was an important regulatory mechanism affecting VDR levels. PRPF19 has been identified as a key E3 ubiquitin ligase of VDR to promote this process. Our work confirmed the interactions between VDR and PRPF19, which were enhanced during high-glucose induction.\u003c/p\u003e \u003cp\u003eAs a natural compound, prior research has demonstrated the therapeutic effect of BBR in diabetes and its complications[\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, there is limited evidence on how BBR acts on renal tubules. Since the proximal tubules require high energy and rely on aerobic metabolism, they are vulnerable to ischemic injury in diabetes due to increased consumption, impaired utilization and reduced oxygen delivery[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Therefore, the death of tubular cells has been considered as one of the key pathogenesis of DN. Here, our investigation provides evidence that by targeting PRPF19 to inhibit ubiquitination degradation of VDR, BBR can effectively improve renal function, alleviate lipid peroxidation, iron accumulation, and abnormal morphology of mitochondria of RTECs in vitro and in vivo.\u003c/p\u003e \u003cp\u003eBBR exhibits extremely low oral bioavailability (\u0026lt;\u0026thinsp;1% in rat models) attributable to poor intestinal absorption and extensive first-pass metabolism[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Following administration, BBR demonstrates preferential tissue distribution to metabolic (liver) and excretory organs (kidneys), with hepatic cytochrome P450 enzymes (particularly CYP2D6 and CYP3A4) mediating its biotransformation into three primary metabolites: berberrubine (M1), thalifendine (M2), and jatrorrhizine (M4)[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. These metabolites undergo dual elimination pathways via both hepatobiliary excretion and renal clearance[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. To advance therapeutic translation, further comprehensive investigation is needed to quantify BBR and its bioactive metabolites in diabetic renal tissue using advanced mass spectrometry techniques, and develop novel delivery systems (including nanoparticle formulations and prodrug approaches) to overcome BBR's current bioavailability limitations.\u003c/p\u003e \u003cp\u003eOn the whole, our findings propose that ferroptosis of RTECs is one of the essential mechanisms of renal injury in diabetes, and inhibition of ferroptosis by targeting PRPF19 may be a potential therapeutic strategy to restore renal function in diabetes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with physiological kidneys, the level of ubiquitination and proteasome degradation of VDR is up-regulated in renal tubules of DN. The degradation of VDR prevents it from entering the nucleus and promoting the transcription of GPX4, which leads to downregulation of GPX4 and the aggravation of lipid peroxidation, ultimately resulting in ferroptosis of RTECs. BBR reduces VDR degradation by binding to PRPF19 and inhibiting its function, thus protecting against ferroptosis of renal tubules and renal injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are provided in the Supporting Information file. The datasets generated during the current study have been deposited in the GEO datasets under the accession number GSE295215.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (Grant NO.82274470 and NO.81974567).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiongyao He, Wenbin Wu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDivision of Cardiology, Department of Internal Medicine and Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiologic Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430000, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWu He\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Traditional Chinese Medicine,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Zhongshan Hospital of Hubei Province, Wuhan 430030, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanlin Ren\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Integrated Traditional Chinese and Western Medicine, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui Dong, Dingkun Wang, Fuer Lu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Endocrinology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGang Yuan, Huihui Ren\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollege of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXinwei Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.H. and W.H. conducted in vivo and in vitro experiments, performed data analysis, and helped write the manuscript. H.D. and W.W. contributed to the experimental design and performed in vitro experiments. X.W. performed in vivo animal studies. Y.R., G.Y. and H.R. helped design experiments. F.L. and D.W. designed the experiment, interpreted the data, wrote the manuscript. All the authors approved the final version of the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Fuer Lu or Dinkun Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approvals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman serum samples were collected by Tongji Hospital affiliated to Huazhong University of Science and Technology, and the study protocol was approved by the Ethics Committee of Tongji Hospital (permit number: TJ-IRB202406035). All animal care and experimental procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Animal Research of Huazhong University of Science and Technology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlicic RZ, Rooney MT, Tuttle KR. Diabetic Kidney Disease: Challenges, Progress, and Possibilities. Clin J Am Soc Nephrol. 2017;12:2032\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu C, Zhou X, Xie T, Zhou Y, Zhang Q, Jiang S, Zhang R, Liao L, Dong J. Renal tubular Bim mediates the tubule-podocyte crosstalk via NFAT2 to induce podocyte cytoskeletal dysfunction. Theranostics. 2020;10:6806\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBagby SP. Diabetic nephropathy and proximal tubule ROS: challenging our glomerulocentricity. 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J Pharm Sci. 2009;98:4391\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ferroptosis, Diabetic nephropathy, Renal tubule, VDR, PRPF19","lastPublishedDoi":"10.21203/rs.3.rs-6083539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6083539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFerroptosis, an iron-dependent form of programmed cell death, is closely associated with tubular damage in diabetic nephropathy (DN). Glutathione peroxidase 4 (GPX4) is an important anti-oxidant enzyme, and plays a crucial role in protecting against ferroptosis. However, the regulatory mechanism of GPX4 expression levels in renal tubular epithelial cells (RTECs) remains elusive. This study reveals that ferroptosis occurs in the late-stage of DN, and the GPX4 level is significantly downregulated in DN patients, animal models and cell models. By applying database predictions, luciferase reporter assays and chromatin immunoprecipitation, we find that vitamin D receptor (VDR) transcription factor promotes GPX4 expression and plays a key role in inhibiting ferroptosis of RTECs. VDR knockout exacerbates ferroptosis in RTECs and worsens renal function, while intraperitoneal injection of VDR agonist paricalcitol significantly improves renal injury. Proteomics analysis suggests that E3 ligase PRPF19 mediates ubiquitination degradation of VDR and is an important therapeutic target for DN. Therefore, through molecular docking, targeted fishing technology using high-performance affinity beads, and surface plasmon resonance (SPR), we screen and identify berberine (BBR) as a novel inhibitor of PRPF19, which offers renal protection by inhibiting VDR degradation and tubular ferroptosis. These findings elucidate the role of ferroptosis in DN renal tubular injury, and suggest that PRPF19 is a promising therapeutic target.\u003c/p\u003e","manuscriptTitle":"PRPF19 mediates the proteasomal degradation of VDR to exacerbate ferroptosis in diabetic nephropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 13:40:29","doi":"10.21203/rs.3.rs-6083539/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-05-17T15:29:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T15:40:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T01:42:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161041006129331295595983796783408635963","date":"2025-04-27T06:58:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107803571769891881875971823169814501486","date":"2025-04-27T00:24:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-25T12:43:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T09:36:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2025-04-24T09:14:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e0a2dcf6-144d-47d7-aa36-f95e7aa7d573","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T16:11:33+00:00","versionOfRecord":{"articleIdentity":"rs-6083539","link":"https://doi.org/10.1186/s12964-025-02253-5","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2025-05-25 15:57:28","publishedOnDateReadable":"May 25th, 2025"},"versionCreatedAt":"2025-05-06 13:40:29","video":"","vorDoi":"10.1186/s12964-025-02253-5","vorDoiUrl":"https://doi.org/10.1186/s12964-025-02253-5","workflowStages":[]},"version":"v1","identity":"rs-6083539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6083539","identity":"rs-6083539","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}