Research

DOI:https://doi.org/10.65613/736542 Zhen Wang1, Yuantao Zhao1 ,Qing Wang2, Fa Sun1,a,*,Kehua Jiang2,b,* 1 Guizhou Medical University, 550000, ,Guiyang,china 2 Department of Urology, Guizhou Provincial People’s Hospital,550000, ,Guiyang,china aEmail:18984159914@163.com bEmail:gzykdx3627@163.com Abstract Objective: Calcium oxalate (CaOx) stones are the most common type of kidney stones, and hyperoxaluria is a major risk factor. Although high oxalate levels are closely associated with crystal formation, the underlying molecular mechanisms remain incompletely understood. This study aims to investigate whether the IRF‑1/miR‑301/CSF‑1 signaling axis in renal tubular epithelial cells regulates macrophage polarization and thereby contributes to CaOx crystal formation. Methods: Human renal tubular epithelial cells (HK‑2) were stimulated with sodium oxalate (NaOx) and co‑cultured with macrophages to assess macrophage polarization. IRF‑1 and miR‑301 were overexpressed or inhibited using plasmid transfection and oligonucleotides, respectively. The direct targeting of CSF‑1 by miR‑301 was validated using a dual‑luciferase reporter assay. Crystal deposition was evaluated by Von Kossa staining. Expression levels of key molecules were analyzed by qRT‑PCR, Western blot, immunofluorescence, and immunohistochemistry. Results: NaOx stimulation upregulated IRF‑1 expression in HK‑2 cells. When these cells were co‑cultured with macrophages under oxalate treatment, macrophages polarized toward the M1 phenotype. Mechanistically, IRF‑1 in HK‑2 cells transcriptionally activated miR‑301 expression, and CSF‑1 was identified as a direct target of miR‑301. Inhibition of miR‑301 in HK‑2 cells or supplementation with recombinant CSF‑1 shifted macrophage polarization toward the M2 phenotype. These cellular findings were consistent with in vivo results: IRF‑1 knockout mice exhibited reduced renal crystal deposition, decreased miR‑301 expression, and increased CSF‑1 levels. Conclusion: The IRF‑1/miR‑301/CSF‑1 signaling axis plays a critical role in a hyperoxaluric environment by promoting M1 macrophage polarization and facilitating CaOx crystal formation. Targeting this pathway may provide new strategies for the prevention and treatment of CaOx kidney stones. Keywords: Sodium oxalate; IRF‑1; miR‑301; CSF‑1; Macrophage           1.Introduction Kidney stone disease is a common urinary tract disorder, often associated with renal colic, urinary tract infections, and impaired renal function. In China, its prevalence is approximately 6.4% and continues to rise. The pathogenesis of kidney stones is not fully understood, and effective preventive measures remain limited. The 10‑year recurrence rate for calcium oxalate stones, the most predominant type of kidney stones, can reach up to 50%. Several theories have been proposed, including Randall‘s plaque theory, supersaturated crystallization, renal injury theory, and inflammation/oxidative stress theory. Recent studies have indicated a strong relationship between inflammation and CaOx crystal formation. Macrophages, as key immune cells, are significantly increased in number in the kidneys during stone formation. Macrophages exhibit two polarization states: the M1 phenotype promotes inflammation, facilitating crystal adhesion and aggregation, whereas the M2 phenotype reduces inflammation and alleviates tissue injury. Macrophage polarization is modulated by various molecules, among which the interferon regulatory factor (IRF) family serves as important nuclear transcription factors. Previous research has demonstrated upregulated expression of IRF‑1 in renal tissues of hyperoxaluric mice. However, how IRF‑1 in renal tubular epithelial cells regulates macrophage polarization remains to be investigated. As a transcription factor, IRF‑1 may facilitate this process by modulating downstream molecules. MicroRNAs are important regulators of gene expression that bind to the 3′‑untranslated region (3′‑UTR) of target mRNAs to inhibit translation or promote degradation, thereby influencing numerous disease processes. Certain miRNAs have been found to play significant roles in the development and progression of nephrolithiasis. Through bioinformatic analysis, this study predicts that IRF‑1 may bind to the promoter region of miR‑301 and potentially promote its transcription. miR‑301 has been reported to regulate inflammatory responses and participate in macrophage polarization. Notably, CSF‑1 is known to promote M2 macrophage polarization, and further prediction suggests that CSF‑1 is a potential downstream target of miR‑301. Based on the above background, we hypothesize that high oxalate stimuli upregulate IRF‑1 expression in renal tubular epithelial cells, which in turn enhances miR‑301 transcription by binding to its promoter region. Increased miR‑301 inhibits CSF‑1 expression, reduces M2 macrophage polarization, disrupts the M1/M2 balance, and exacerbates inflammatory responses and crystal deposition. This study aims to investigate the role of the IRF‑1/miR‑301/CSF‑1 signaling axis in CaOx stone formation through in vitro and in vivo experiments, thereby providing new therapeutic targets and a theoretical basis for clinical prevention and treatment. Materials and Methods Cell Culture and Treatment The human renal tubular epithelial cell line (HK‑2) was purchased from Boster Biological Technology (Wuhan, China). HK‑2 cells were cultured in Dulbecco‘s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified atmosphere containing 5% CO₂. To establish an in vitro hyperoxaluria model, HK‑2 cells were treated with 800 μM sodium oxalate (NaOx, Sigma, USA) for 24 or 48 hours when cell confluence reached approximately 80%. 2.2.  Plasmid and Oligonucleotide Transfection The IRF‑1 overexpression plasmid (pcDNA3.1‑IRF‑1) and its empty vector control (pcDNA3.1) were constructed by GenePharma (Shanghai, China). The miR‑301 mimic, miR‑301 inhibitor, and their corresponding negative controls (mimic‑NC, inhibitor‑NC) were also synthesized by GenePharma. All transfections were performed using Lipofectamine 3000 reagent according to the manufacturer’s instructions. Briefly, 2.5 μg of plasmid or 50 nM of miRNA oligonucleotides were diluted in serum‑free DMEM, mixed with Lipofectamine 3000, and then added to the cells. Cells were harvested 48 hours post‑transfection for further analysis. 2.3. Co-culture System Human monocytic THP‑1 cells were differentiated into M0 macrophages by treatment with 100 ng/mL phorbol 12‑myristate 13‑acetate (PMA, Sigma, USA) for 48 hours. Differentiated macrophages were seeded into the upper chamber of a Transwell system. HK‑2 cells subjected to various treatments were cultured in the lower chamber. The two cell types were co‑cultured for 48 hours, after which macrophages in the upper chamber were collected for RNA extraction and qRT‑PCR analysis of polarization markers. 2.4. Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer‘s protocol. For miRNA analysis, cDNA was synthesized using the Synthesis SuperMix Kit (Yeasen Biotechnology). qPCR was performed on a 7500 Real‑Time PCR System (Biometra, Germany) using SYBR Green PCR Master Mix (Yeasen Biotechnology). The expression of target genes was normalized to GAPDH, and the expression of miR‑301