IntroductionNephrolithiasis, commonly known as kidney stone disease, is a prevalent and recurrent urological condition with a recurrence rate of up to 50% within the first five years [1, 2]. It is characterized by inflammation and tissue injury, the driving factors in stone re-formation [3, 4]. Repeated stone formation further aggravates tissue injury, thus forming a vicious circle [5]. Surgical crushing alone does not resolve the increasing prevalence of kidney stones (KS). While some therapeutic approaches have shown promise in limiting renal injury and fibrosis in clinical trials [2, 6], the overall clinical efficacy remains limited. A promising strategy is necessary to address the renal damage and crystal aggregation inherent to KS.A critical aspect of renal stone pathogenesis and subsequent damage involves metabolic alterations, particularly in fatty acid metabolism [7, 8]. As the primary energy source for tubular epithelial cells (TECs), fatty acid oxidation (FAO) supports critical cellular functions under normal conditions. However, alterations in fatty acid metabolism during renal injury contribute to metabolic reprogramming and oxidative stress, ultimately promoting renal fibrosis [7, 9]. Recent studies have highlighted the intricate interplay between fatty acid metabolism and renal disease progression, revealing potential therapeutic targets [8, 10].Concurrently, protein arginine methylation, a post-translational modification facilitated by the family of protein arginine methyltransferases, has been recognized as an essential regulator of cellular functions in multiple organs [11, 12]. Among the PRMT family, PRMT1 stands out due to its broad substrate specificity and abundance in various cell types, including renal cells [11, 13]. PRMT1 catalyzes the mono- and asymmetric dimethylation of arginine residues, thereby influencing protein function, stability, and subcellular localization [14, 15]. Its expression patterns and functional roles in the kidney are well-documented, providing a solid foundation for further investigation [16]. However, how PRMT1 influences CaOx stone-induced kidney injury remains largely unknown. Recent studies have implicated PRMT1 in regulating fatty acid metabolism, suggesting that it might be a pivotal node in the complex interplay between metabolic reprogramming and renal injury [17,18,19]. Perturbations in PRMT1 activity affect the expression and function of enzymes involved in FAO, thereby modulating cellular energy metabolism [20]. Therefore, targeting PRMT1 holds promise as a novel therapeutic strategy, particularly in kidney stone disease, in which metabolic derangement and inflammation are prevalent.In this study, we confirmed that PRMT1 enhanced the neddylation function of ubiquitin-conjugating enzymes E2M (UBE2m) through arginine methylation at R169, increasing the neddylation level and protein stability of NEDD4. This promoted Peroxisome proliferator activated receptor γ (PPARγ) ubiquitination and degradation, leading to the inhibition of fatty acid metabolism. Our findings elucidate the underlying mechanisms and highlight potential therapeutic targets for CaOx crystal-induced kidney injury.MethodsHuman samples studySamples were obtained from eight patients with nonfunctioning kidneys due to KS undergoing nephrectomy at Renmin Hospital of Wuhan University (mean age 49.1 ± 8.0 years; five males and three females) (Table S1). The stones were identified as CaOx monohydrate or dihydrate using stone composition analysis (Fig. S1A). Eight normal tissue samples were obtained from the adjacent non-tumorous tissues of patients who had undergone radical renal resection (mean age 59.9 ± 6.1 years; five males and three females) as the normal controls (NC) (Table S1). The study protocol adhered to the Declaration of Helsinki and was approved by the review board of Renmin Hospital of Wuhan University (WDRY2021-KS047). Informed consent was obtained from all participants prior to sample collection.Single-cell RNA sequencingHuman kidney samples were prepared as single-cell suspensions according to the manufacturer’s instructions. The single-cell library was established according to the BD Rhapsody Single-Cell 3ʹ Whole Transcriptome (WTA) Library Construction Standardization Manual. After library preparation and quality control, double-end sequencing was performed on the Illumina Nova Seq6000 sequencing platform in 2 × 150 bp mode. Raw BCL files were assembled using Illumina’s bcl2fastq converter to obtain raw fastq data, and high-quality clean reads were obtained after data filtering through primary quality control. Subsequently, the BD Rhapsody analysis process was used for quality control and filtering of data, identification of cell ID and molecule ID, mapping of reads to the reference genome, quantification of gene expression and identification of cells, and, finally, generation of the original expression matrix. Sctype software was used for automatic annotation, followed by manual inspection and correction for cell type annotations. The FindMarkers and FindAllMarkers functions, with default parameters set, were used to identify differentially expressed genes between each subgroup.Animal experimentsThe Animal Care and Use Committee of Renmin Hospital of Wuhan University approved all animal experiments (WDRM 20200604), which complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Transgenic mice expressing Cre-recombinase under the cadherin 16 (Cdh16) promoter were purchased from the Jackson Laboratory (JAX 012237) [21]. The Cdh16 (also known as Ksp-cadherin) promoter was activated in the TECs of the kidneys [22, 23]. PRMT1 floxed mice (C57BL/6J background, PRMT1fl/fl) were obtained from Shanghai Model Organisms. Renal tubular epithelial-specific deletion of PRMT1 mice were generated by crossing floxed PRMT1 mice with Cdh16-Cre± mice and wild-type littermates were used as controls. Genotyping was performed using agarose gel electrophoresis. PRMT1 identification: Homozygotes = 330 bp; Heterozygotes = 330 bp and 273 bp; Wild-type allele = 273 bp (Fig. S2H). Cdh16cre identification: Cre primers amplified 400 bp bands indicating Cdh16Cre positive (Fig. S2I).PRMT1 was specifically overexpressed in TECs in mice by injecting recombinant adeno-associated virus 2 (rAAV9) (Genomeditech, Shanghai, China) into the bilateral renal parenchyma using a microliter syringe. The injection involved inserting the needle from the lower pole to the upper pole of the kidney and gradually withdrawing it after administering the rAAV9 solution. Western blotting (WB) and EGFP fluorescence were used to detect transgene expression levels and distribution after 4 weeks (Fig S2C–E). rAAV9-PRMT1 was developed by cloning the target gene PRMT1 into an AAV serotype 9 vector containing the kidney-specific promoter KSPC (GPAAV-KSP0.3k-Mouse_Prmt1-T2A-eGFP-WPRE) (Fig. S2A).All the mice (6–8 weeks) were maintained in a pathogen-free environment with ad libitum access to food and water, and a suitable temperature range of 20–25 °C and 50–65% relative humidity. Animals were randomly assigned to experimental groups using a computer-generated sequence. Outcome assessors were blinded to group allocation. Each mouse was injected intraperitoneally with glyoxylate (Gly, 100 mg/kg/d) for 10 consecutive days to establish a hyperoxaluria mouse model. Mice within the control group were subjected to intraperitoneal administration of the same volume of saline. Mice received atorvastatin at a dose of 20 mg/kg/day via oral gavage.Sample size estimation was guided by previously published studies using similar models. For in vivo studies, a minimum of five mice per group was selected, which has been shown to be sufficient to detect biologically and statistically significant differences in similar experiments. For in vitro experiments, at least three independent replicates were performed to ensure reproducibility.Mice with signs of unrelated illness, abnormal baseline renal function, or technical failure during surgery/crystal injection were excluded from analysis. For in vitro studies, data from wells with contamination, low cell viability (