
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39251681
71755
10.1038/s41598-024-71755-y
Article
The identification of key molecules and pathways in the crosstalk of calcium oxalate-treated TCMK-1 cells and macrophage via exosomes
Sun Yushi
Li Bojun
Zhou Xiangjun
Rao Ting tinart@126.com

Cheng Fan urology1969@aliyun.com

https://ror.org/03ekhbz91 grid.412632.0 0000 0004 1758 2270 Department of Urology, Renmin Hospital of Wuhan University, No.238 Jiefang Road, Wuchang District, Wuhan, 430060 Hubei People’s Republic of China
9 9 2024
9 9 2024
2024
14 2094917 5 2024
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation.

Keywords

Exosomes
MiRNA
Macrophage
Biomarkers in kidney stones
Transcriptomics of kidney stones
Subject terms

Immunology
Biomarkers
Molecular medicine
Nephrology
Urology
Sequencing
National Natural Science Foundation of China82170775 82100806 Rao Ting Cheng Fan issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Kidney stones are a common urinary system disorder that is increasingly in prevalence due to dietary, environmental, and lifestyle changes1,2. The development of percutaneous nephrolithotomy and ureteroscopy has significantly enhanced the efficiency and safety of stone treatment and removal3,4. However, kidney stones have a high recurrence rate, with approximately 50% of patients experiencing a recurrent episode within five years5, emphasizing the importance of preventive measures and long-term patient management. Early diagnosis and lifestyle modifications are crucial to prevent calculi formation.

Exosomes are extracellular vesicles approximately 30–150 nm in diameter and surrounded by a double lipid membrane. Exosomes selectively package and deliver a range of bioactive materials such as proteins, RNA, and lipids to target cells6, influencing the behavior and function of the recipient cells. Exosomes are important mediators of intercellular communications and play a key role in a variety of biological processes, such as immune responses and disease progression7. The production and secretion of exosomes represent important mechanisms of intercellular communication8.

Among the types of kidney stones, the primary component of stone is calcium oxalate monohydrate (COM). Stone formation is a complex process, that involves several mechanisms, including crystal supersaturation, crystal-cell adhesion, crystal deposition, growth, and crystal-cell interactions, that contribute to the retention of crystals in the kidney damage9,10. These crystals subsequently induce cytotoxicity and dysfunction in renal tubular epithelial cells (TECs), which in turn increases crystal formation and adhesion11. MicroRNAs (miRNAs) are short, non-coding RNAs comprised of 20–25 nucleotides that regulate gene expression by binding to complementary sequences in the 3' untranslated region of the target mRNA, thereby inhibiting translation or promoting degradation12. Macrophages, in addition to removing stones via phagocytosis of crystal fragments, can influence kidney stone formation and damage through changes in their polarization status. M1- macrophages promote stone formation and exacerbate kidney injury by producing pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, whereas M2-macrophages aid in inflammation suppression and tissue repair by secreting anti-inflammatory cytokines13. A recent study found that exosomes from HK-2 cells stimulated by human serum albumin induced M1 polarization via the miR-199a-5p/Klotho/TLR4 axis, accelerating diabetic kidney disease progression14. Another study demonstrated that exosomal miR-19b-3p mediated crosstalk between injured TECs and macrophages, inducing M1 polarization through the NF-κB/SOCS-1 pathway15.

The aim of the present study was to investigate the role of TCMK-1 cells-secreted exosomal miRNA in mediating communication between TCMK-1 and macrophages. We performed miRNA sequencing on COM-stimulated (OX_EXO) or control (NC_EXO) TCMK-1 cell-derived exosomes. In addition, macrophages were treated with these exosomes for mRNA sequencing. The results suggest that treatment with COM significantly altered the expression profile of exosomal miRNAs and in OX_EXO-treated macrophages.

Results

Acquirement and next-generation sequencing of exosomes derived from TCMK-1 cells

To investigate the effect of COM on exosomes derived from TCMK-1, we established an untreated control (NC group) and 150 mmol/L COM-stimulated (OX group) model based on the growing status and modeling effect (Fig. 1A). We used Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering sight (DLS sight), Transmission Electron Microscope (TEM) and western blot to identify that the majority of isolated extracellular vesicles were exosomes (Fig. S1). Exosomes secreted from the NC group (NC_EXO) and the OX group (OX_EXO) were collected for sequencing, to identify the differential expression of miRNAs between the two groups. A total of 1973 miRNAs were detected, with 1952 co-expressed in NC_EXO and OX_EXO, 11 miRNAs were exclusively expressed in OX_EXO, and 10 were exclusively expressed in NC_EXO (Fig. 1B). Among the 1973 miRNAs, 22 showed no significant difference (P ≥ 0.05), resulting in a total of 1951 miRNAs included in the volcano plot. Compared with NC_EXO, 293 miRNAs were significantly up-regulated and 322 miRNAs were significantly down-regulated in OX_EXO (|Log FC|> 1, P < 0.05) (Fig. 1C). To isolate miRNAs with more significant changes, a heatmap was conducted on miRNAs with |Log FC|> 2 and P < 0.05 (Fig. 1D).Fig. 1 (A) Schematic diagram of the experimental process. Exosomes from COM treatment TCMK-1 (OX_EXO) or control TCMK-1(NC_EXO) were extracted, and the exosomal miRNAs were sequenced. (B) Venn plot showed the expression of miRNAs in NC_EXO and OX_EXO. A total of 1952 miRNAs were expressed in the two groups, 11 miRNAs were only expressed in OX_EXO, and 10 miRNAs were only expressed in NC_EXO. (C) The volcano plot shows miRNA-Seq data from NC_EXO and OX_EXO (|Log FC|> 1, P < 0.05). (D) The heat map showed the difference of miRNA expression profile in NC_EXO and OX_EXO among the four groups (|Log FC|> 2 and P < 0.05).

Enrichment analysis of genes targeted by down-regulated exosomal miRNAs

We analyzed the 30 most significantly down-regulated miRNAs using the mirPath database (v4.0, http://www.microrna.gr/miRPathv4). These miRNAs were mainly co-enriched in Glycosphingolipid-lacto and neolacto series, ECM-receptors interaction, Tyrosine metabolism, Thyroid hormone synthesis, and Morphine addiction terms (Fig. 2A). Kyoto Encyclopedia of Genes and Genomes (KEGG)16–18 enrichment analysis of the union target set of the top 30 down-regulated miRNAs revealed 12 significantly enriched pathways, including Wnt signaling pathway, cAMP signaling pathway, and circadian entrainment (Fig. 2B). To more accurately understand the pathways and gene functions associated with down-regulated miRNAs, the target genes of the top 10 down-regulated miRNAs were obtained from five miRNA target prediction databases (Targetscan, miRDB, miRTarbase, miRWalk, and ENCORI), and subsequent KEGG analyses were performed for the intersection of genes. Down-regulated miRNAs from OX-EXO were related to Autophagy, mTOR signaling pathway, PI3K-Akt signaling pathway, and FoxO signaling pathway (Fig. 2C). Additionally, Gene Ontology (GO) enrichment was conducted for the intersection-set of genes. Notable entries mainly included biological process (cell quiescence), cell component (transcription factor complex), and molecular function (volume-sensitive chloride channel activity, and kainate selective glutamate receptor activity) (Fig. 2D).Fig. 2 (A) Pathway union results of top 30 down-regulated miRNAs from MirPath. (B) KEGG plot of gene union list of top 30 down-regulated miRNAs from Mirpath. (C,D) Targets genes of top 10 down-regulated miRNAs predicted by five online databases were used for KEGG and GO analysis.

Enrichment analysis of genes targeted by up-regulated exosomal miRNAs

Following the procedures outlined in Part 2, we repeated the analysis with the 30 most significantly up-regulated miRNAs. Out of these 30, 12 miRNAs were not in the microT-CDS database. Therefore, the remaining top 18 miRNAs were prominently co-enriched in Fatty acid elongation, TGF-β signaling pathway and Wnt signaling pathway (Fig. 3A). The target genes were significantly enriched in signaling pathways relating to Adherens junction, Rap1, Focal adhesion, MAPK and Ras (Fig. 3B). The target genes of the top 10 up-regulated miRNAs were primarily enriched in Notch signaling pathway, Hippo signaling pathway and Wnt signaling pathway (Fig. 3C). GO enrichment was conducted for the intersection-set of genes. Notable entries mainly included cell component (cytoplasmic ribonucleoprotein granule) and molecular function (transcription corepressor binding, and transcription factor binding) (Fig. 3D).Fig. 3 (A) Pathway union results of top 30 up-regulated miRNAs from MirPath. (B) KEGG plot of gene union list of top 30 up-regulated miRNAs from Mirpath. (C,D) Target genes of top 10 up-regulated miRNAs predicted by five online databases were used for KEGG and GO analysis.

Identification of key genes co-targeted by up and down-regulated miRNAs

We conducted a UPSET intersection analysis of the top 10 up and down-regulated miRNA target genes in both the NC_EXO and OX_EXO, as predicted by the five aforementioned miRNA target prediction databases. This analysis aimed to identify key genes central to the exosomes secreted by TCMK-1 cells. By integrating the expression patterns of target genes in COM-stimulated macrophages and utilizing data from the KPMP database (https://atlas.kpmp.org), we identified AAK1 and NUFIP2 as key genes targeted by two distinct groups of down-regulated miRNAs: miR-6900-5p, miR-5188, miR-218-2-3p, and miR-6900-5p, miR-465a-5p, and miR-218-2-3p, respectively (Fig. 4A). Additionally, PLCB1 was identified as a key gene targeted by an up-regulated miRNA group consisting of miR-343, miR-701, and miR-1971(Fig. 4D). RT-qPCR and Western blot confirmed that the expression levels of the above miRNAs, and the mRNA and protein level of AAK1, NUFIP2, and PLCB1 in OX_EXO or NC_EXO treated macrophages (Fig. 4B,C,E,F). The changes of expression of AAK1, NUFIP2 and PLCB1 were also confirmed in HK-2 cell lines (Fig. S2).Fig. 4 (A) UPSET Plot showed top 10 down exosomal-miRNAs common possible target genes. (B,C) PCR and Western blot verified the different expression of miR-465a-5p, miR-6900-5p, miR-5188, miR-218-2-3p, AAK1, and NUFIP2. (D) UPSET Plot showed top 10 up exosomal-miRNAs common possible target genes. (E,F) RT-qPCR and Western blot verified the different expression of miR-343, miR-701, miR-1971, and PLCB1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. control.

Bioinformatic analysis and verification of genes in OX_EXO and NC_EXO-stimulated macrophages

We treated RAW264.7 cells with OX_EXO and NC_EXO, and performed the exosome uptake study (Fig. S3). The RNA-sequencing analysis of two groups of macrophages revealed 288 up-regulated genes and 70 down-regulated genes (P < 0.05, |log2FC|> 0.58) (Fig. 5A). The heatmap depicts the top 20 significantly differentially expressed genes (DEGs) (Fig. 5B). To explore the signaling pathways of DEGs modulated by OX_EXO, KEGG showed that these DEGs were primarily enriched in Ras signaling pathway and pyrimidine metabolism (Fig. 5C). GO enrichment analysis delineated the biological roles of DEGs, highlighting involvement in the negative regulation of tumor necrosis factor production, pattern recognition receptor activity, and pathway-restricted SMAD protein phosphorylation (Fig. 5D). We found that the ‘gobp regulation of immune system process’ and ‘Rodwell Aging Kidney Up’ were positively related to the OX_EXO treated macrophages (Fig. 5E). Protein–protein interaction (PPI) analysis revealed potential core connections among proteins encoded by DEGs (Fig. 5F). For instance, CD83 was potentially associated with TLR9, and both were up-regulated in the OX_EXO group. Conversely, down-regulated proteins such as SAA3 and IL-27 may be correlated with CXCL10, although CXCL10 showed no change in the macrophages treated with OX_EXO compared to NC_EXO (Fig. 5G,H). In order to ensure consistency of research in multiple species, we verified these core gene expressions in HK-2 cells (Fig. S4).Fig. 5 (A) Volcano plot of differently expressed genes of macrophages with OX_EXO or NC_EXO (P < 0.05, |log2FC|> 0.58). (B) Heatmap of significant differential genes in four groups. (C,D) KEGG and GO analysis of DEGs in macrophages. (E) GSEA showed two important pathways involved in two conditions (macrophages stimulated by OX_EXO and NC_EXO). (F) PPI network displays the connections between differential genes in macrophages. (G-H) Western blot verified the protein expressions of the core genes of PPI network and the significantly changed genes in the heatmap. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. control.

Differentially expressed miRNAs in homologous human exosomes

We performed homology analysis for the top 20 dysregulated miRNAs. Five miRNAs of interest were identified, including an up-regulation of miR-27a-5p and a down-regulation of miR-136-3p, miR-185-3p, miR-218-2-3p, and miR-380-5p. Figure 6A illustrates the complementary sequences (purple-red letters) obtained from the miRBase (http://www.mirbase.org). The paralogous and orthologous nucleotide sequences from mouse and human species are shown in Fig. 6B.Fig. 6 (A) The structures of five homologous miRNAs from mouse and human species. (B) Corresponding nucleotide sequences between two species.

Validation of miRNAs and core target genes in clinical specimens

We extracted exosomes from urine samples of 10 kidney stone patients (ST) and 10 normal controls (NC). The expression levels of the five homologous miRNAs (miR-27a-5a, miR-136-3p, miR-185-3p, miR-218-2-3p, and miR-380-5p) were consistent with those in cell experiments (Fig. 7A). The pathway union result of five hsa-miRNAs from MirPath was displayed by KEGG plot. KEGG pathway analysis found that Focal adhesions, Adherens junction, PI3K-Akt signaling pathway, Ubiquitin mediated proteolysis, AMPK signaling pathway, and TGF-β signaling pathway were significantly enriched (Fig. 7B). Subsequently, we predicted potential target sets of five miRNAs in the target prediction databases. The intersections of five databases for each miRNA are listed in Table 1. The expression matrices of potential targets were extracted from GSE73680 and visualized with a heatmap (Fig. 7C). According to the opposite expression trend between the target genes and miRNA, we identified up-regulated genes, such as CNOT3, TMEM63C, BARHL1, and CACNA1Cl, and down-regulated genes, such as APEX1, and TMEM199. The protein level of TMEM63C showed no change, and BARHL1 was not detected in either ST or NC groups (Fig. 7D,E). To further validate the function of miRNAs, we used specific mimic or inhibitors to confirm the relationship between miRNAs and target genes in OX or NC TCMK-1 cells. (Fig. S5). Immunofluorescence staining showed an increased in the expression of CNOT3 in macrophages (marked with iNOS) in the ST group (Fig. 7F).Fig. 7 (A) The expression of above five miRNAs in human urine exosomes was verified. (B) The KEGG plot shows the results of five miRNAs in the pathway-union plate. (C) The expression matrix from GSE73680 of target genes of five miRNAs. (D,E) Key proteins expressions of renal tissues of kidney stone patients and healthy controls. (F) iNOS was used as the marker of macrophages, and CNOT3 expression in macrophages was increased in kidney tissues of patients with kidney stones. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. control.

Table 1 Intersection genes from 5 databases of 5 miRNAs.

has-mir-27a	has-mir-136-3p	has-mir-185-3p	has-mir-218-2-3p	has-mir-380-5p	
APEX1	HOXB6	MECP2	OLA1		
TMEM199		SOX4	ARID5B		
FSD2		SLC10A3	POLR3E		
CLEC4E		VPS37C	PPP2CA		
		PPP2CA	ZNF268		
		TMEM63C	STAU1		
		CNOT3	HOOK1		
		IDS			
		MTHFSD			
		FOLR1			
		HMGA1			
		SEMA3F			
		BARHL1			
		CACNA1C			

Discussion

Kidney stones pose a significant risk for the development of chronic kidney diseases, imposing a substantial economic burden on society. Kidney stone formation is influenced by environment, dietary, and lifestyle factors2. The formation of Randall’s plaque is regarded as the most common and crucial mechanism for the formation of kidney stones. The initial steps of this process are crystal supersaturation and damage of renal TECs, which lead to crystal adhesion and deposition19. Exosomes are membranous vesicles released after the fusion of intracellular poly-vesicles with cell membranes. They contain bioactive molecules such as proteins, mRNAs, and microRNAs are important mediators of intercellular communication20. Recent studies have indicated that exosomes secreted by COM-stimulated epithelial cells can damage healthy cells21. Macrophages play a multifunctional role in kidney stone formation. Renal macrophages can eliminate calcium oxalate (CaOx) crystals through phagocytosis22. M1 macrophages promote kidney inflammation and cell damage, while M2 macrophages facilitate damage repair and interstitial fibrosis23. Studies have demonstrated that exosomes transporting Dll4 secreted by HK-2 cells promote M1 activation24. The exosome/miR-19b-3p/SOCS1 axis plays a key pathological role in communication between damaged TECs and macrophages15. However, research on exosomes-mediated communication between TECs and macrophages in kidney stone models primarily focus on exosomes derived from macrophages25,26, and few studies investigate the role of exosomes with epithelial origin. In the current study, we found that COM-stimulated TCMK-1-derived exosomes exhibited a different miRNA expression profile compared to untreated cells. In OX_EXO-treated RAW264.7 cells, we identified significantly altered molecules and pathways, and observed a significant promotion of M1-polarization.

MiRNAs are small non-coding RNAs that interact with one or more mRNAs to down-regulate the expression of target genes through multiple mechanisms, including translation inhibition, mRNA splicing, and de-adenylation27,28. In this study, we performed miRNA sequencing of exosomes derived from COM-treated TCMK-1 cells and sequenced RNA of macrophages treated with OX_EXO or NC_EXO. TCMK-1 cell line were selected to ensure homology, consistency, and accuracy in subsequent specific mechanism studies. Multiple enrichment analyses were performed on down-regulated miRNAs and their target genes, and multiple pathways related to kidney stone formation were highlighted. Notably, ECM-receptors interaction is an important process leading to renal interstitial fibrosis induced by kidney stone and other chronic kidney diseases29. Wnt signaling pathway has been shown to promote epithelial to mesenchymal transition and osteochondral differentiation in the hypercalciuric kidney stone-forming rat models30. Studies have demonstrated that activation of the mTOR signaling pathway decreased autophagy, and facilitated crystal-cell interaction and inflammatory responses related to kidney stone formation31.Similarly, PI3K-Akt and FoxO signaling pathways have also been found to contribute to renal injury crystal deposition32,33.Furthermore, we performed functional analyses that revealed an up-regulation of miRNAs involved in Fatty acid elongation, TGF-β signaling pathway, adherens junction, MAPK signaling pathway, and Notch signaling pathway. The kidney is an organ that requires substantial energy, that is primarily supplied by β-oxidation of long-chain fatty acids34. The negative regulation of Fatty acid elongation suggests there is a dysregulated energy balance that contributes to impaired metabolism in TECs. TGF-β is a key molecular component in kidney injury and renal fibrosis induced by crystals35. Pirfenidone, a broad-spectrum anti-fibrotic drug targeting TGF-β, has been approved for the clinical treatment of idiopathic pulmonary fibrosis, renal interstitial fibrosis, liver fibrosis and other related diseases36,37. Damage to the tubular adherens junctions results in the disruption of TEC morphology, which is a key event in the biological process of stone formation38. Moreover, high calcium-induced crystal deposition contributes to the formation of Randall’s plaque via the Nox4/MAPK signaling pathway39. Notch signaling is involved in the polarization of M1 phenotype, which exacerbates calcium crystal deposition and TECs damage40. Therefore, we found that the significantly altered miRNA profile of exosomes derived from COM-treated tubular epithelial cells are involved in multiple pathological processes relating to kidney stone formation, such as stone adhesion, crystal-cell interactions, inflammatory infiltration and renal fibrosis.

The target genes of down-regulated miRNAs, AAK1 and NUFIP2, were identified through UPSET analysis. AAK1 has been reported to inhibit the NF-κB pathway and alleviate inflammatory response in lung injury41. NF-κB pathway is associated with cell-crystal adhesion, apoptosis, and M1 polarization42. NUFIP2 is one of the core proteins of stress granules, that regulates mTOR and lysosomal damage43. As mentioned above, mTOR links autophagy and kidney stone formation. Moreover, our UPSET analysis determined that PLCB1 was up-regulated miRNA target genes. Previous studies found that overexpression of miR-7-5p/PLCB1 axis inhibited the progression of acute pancreatitis by modulating the MAPK signaling pathway44, which was shown to be involved in the formation of Randall’s plaque. It is evident that the functions of these target genes mainly enriched pathways regulating inflammation and stone formation, which aligns with the findings of our RNA-seq analysis of OX_EXO-treated macrophages.

Key genes obtained by sequencing of macrophages were closely related to immune regulation. For instance, CD83 was found up-regulated in OX_EXO-treated macrophages, and it is involved in phagocytosis and phenotypic changes of macrophages45. Studies have demonstrated that exosomal miRNA-342-5p attenuates acute kidney injury in sepsis mice by inhibiting TLR946. While expression of ZC3H12C, SAA3, ATP6V0D2, and IL-27 have been reported in the skin, nervous system, and atherosclerotic inflammation47–50, we have for the first time demonstrated a potential association between their expression and kidney stones.

Five miRNAs were selected after comparing the homology of miRNAs in human and mouse species. In OX_EXO and urine of kidney stone patients, we found that miR-27a-5p was up-regulated, and miR-136-3p, miR-185-3p, miR-218-2-3p, and miR-380-5p were down-regulated. Remarkably, the PI3K-Akt signaling pathway, AMPK signaling pathway, and FoxO signaling pathway were identified as significant pathways targeted by the five identified miRNAs. By integrating miRNA target genes with kidney genetic data of patients from GSE73680 and verifying the protein levels in urine, we identified four key genes. In kidney stone patients, APEX1 and TMEM199 were down-regulated; while CNOT3 and CACNA1C were up-regulated. Their function in other tissues will guide future studies in the kidney. In a model of unilateral ureteral obstruction induced tubulointerstitial fibrosis, APEX1was shown to regulate immune function and DNA repair functions51. TMEM199 is related to Golgi homeostasis52 and hepatic steatosis53. Recently, Golgi stress has been reported in kidney injury caused by sepsis54. CNOT3 is a subunit of the CCR4-NOT complex, which stimulates the TORC1 signaling pathway and inhibits autophagy55. In this study we confirmed that CNOT3 was increased in kidneys of nephrolithiasis patients (Fig. 7). However, its specific function requires further investigations. A decrease in CNCNA1C has been associated with the impairment of mitochondrial and lysosomal function56, and the subsequent oxidative stress injury is another mechanisms of renal damage caused by kidney stones.

In conclusion, we have uncovered the differences in exosomal miRNA profiles encapsulated from TECs stimulated by OX, compared with controls. The exosomes obtained under different conditions mediated the communication between TECs and macrophages. Bioinformatic analyses showed that macrophages’ DEGs and target genes of exosomal miRNAs were enriched in inflammation-related pathways, cell adhesion junctions, ECM interactions, and energy homeostasis. Exosomes directly influence macrophages and lead to significant changes in macrophages protein expression. The differences in pathways, molecular functions, and proteins all contribute to the formation of kidney stones and subsequent injury. In addition, we further matched the interspecies homologous miRNAs and validated their changes in the urines and tissues of clinical patients.

Conclusion

The expression profile of miRNAs derived from TCMK-1 exosomes was significantly different between COM-stimulated cells and controls, as well as in the urine of patients with kidney stones compared to healthy controls. Different exosomes exert distinct effects on communication between epithelial cells and macrophages, and exosomal target genes may influence the focal adhesion, immunity, and inflammation of macrophages, ultimately contributing to the pathogenesis of kidney stones. This study identifies potential pathways and targets involved in kidney stone formation. We will investigate on the specific underlying mechanism in the future study.

Methods

Cell culture

TCMK-1 cells were cultured at 37 °C at 5% CO2 with DMEM (Gibco, 12491015) supplemented with 10% exosome-depleted fetal bovine serum (FBS, Gibco, A2720801) and 1% penicillin–streptomycin solution. To stimulate COM conditions, 150 mmol/L of COM (Sigma Aldrich, 3757668) was introduced into the medium and cultured for 48 h. Cells in the control group were cultured in normal DMEM complete culture medium without any additional treatment. Following incubation, the supernatant was collected from the experimental (OX_EXO) and controls (NC_EXO), and exosomes were isolated through a combination of low-speed centrifugation and ultra-fast centrifugation at 4℃, following established protocols detailed previously57. Nanoparticle tracking analysis (NTA), Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) were performed as described previously57.

RAW264.7 cells were cultured under 37 °C at 5% CO2 with DMEM (Gibco, 12491015), supplemented with 10% FBS (Gibco, C0235) and 1% penicillin–streptomycin solution. The cells were cultured in a medium containing NC_EXO or OX_EXO for 48 h.

Cell transfection

Mmu-miR-27a-5p inhibitor, Mmu-miR-185-3p mimic, and Mmu-miR-136-3p mimic were supplied by RiboBio (RiboBio, Guangzhou, China). TCMK-1 cells were cultured in a 6-well plate for 24 h before transfection. The inhibitor or mimics were transfected into cells via Lipofectamine2000 (Invitrogen, 12566014) according to the introduction of manufacturer, and incubated them for 6–8 h in serum free medium. Then the serum free medium was switched to a growth medium for modeling.

Next-generation sequencing

The sequencing library was constructed using the QIAseq miRNA Library Kit, and exosomal non-coding RNA-miRNA sequencing was built by Wayenbio on the Illumina Nova sequencing platform. Analysis was performed by Wayen Biotechnologies (Shanghai, China).

Four RAW264.7 samples in the OX_EXO group and four samples in the NC_EXO group were collected for RNA-sequencing. The total RNA of each cell sample was extracted using TRIzol (Invitrogen, 15596026CN). The NEBNext Ultra RNA library prep kit (Illumina) was used to establish the sequencing library for each RNA sample. Experiments and analysis were performed on an Illumina Hiseq platform completed by Wuhan SeqHealth Tech Co., Ltd. The original read sequence was quality-controlled and compared to the reference transcriptome.

Bioinformatic analysis

The genome microarray dataset GSE73680 was downloaded from the Gene Expression Omnibus (GEO) database to identify key dysregulated genes among kidney stone patients. For bioinformatics analysis, Omicstudio tools (https://www.omicstudio.cn/tool) were employed encompassing KEGG, GO, and Gene Set Enrichment Analysis (GSEA) analysis. Additionally, the PPI analysis was constructed using the STRING database.

RT-qPCR

SeraMir Exosome RNA Purification Kit (System Biosciences, Mountain View, USA) was used for exosomal miRNA isolation. Relative miRNA expression was calculated using the 2−ΔΔCt method, and the miDETECT A Track miRNA qRT-PCR Starter Kit (C10712, RIBOBIO). The small nuclear RNA U6 was used as an internal control. The miRNA-specific Forward primer sequences are listed in Table S1, and the uni-Reverse primer was provided by miDETECT A Track miRNA qPCR Kit (C10711, RIBOBIO).

Western blot

Kidney tissues and TCMK-1 cells were lysed with RIPA buffer (G2002, Servicebio). Total protein content was calculated using BCA protein assay kit. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After the membranes were blocked with 5% skim milk for 2 h at room temperature, the blots were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the study are listed in Table S2. Image J (1.8.0) performs semi-quantitative analysis with β-actin as a control.

Urine samples and human specimens collection

All participants provided informed consent, and ethical approval was acquired from the Ethics Committee of the Renmin Hospital of Wuhan University (WDRY2021-KS047). Urine and kidney tissue samples were collected from patients of Urology Department in our hospital.

Male patients (18–60 years old) with calcium oxalate kidney stones (ST, n = 10) provided urine for this study. Patients with underlying diseases such as diabetes and hypertension, urinary tract infections, congenital urinary system dysplasia, and urinary tumours were excluded. Normal control urines (NC, n = 10) were from healthy people.

Tissue samples were collected from the non-functioning kidneys of patients with CaOx calculi (ST, n = 4), and from adjacent non-tumor kidney tissues of patients treated with radical nephrectomy (NC, n = 4).

Extraction of urinary exosomes

30 mL of fresh urine was collected from the patients and steps were taken to minimize the risk of contamination. Subsequently, the urine samples were centrifuged at 500×g for 10 min, followed by centrifugation at 1200×g for 20 min, and 10,000×g for 30 min to eliminate cell debris and large particles. The supernatant was filtered with a 0.22 μm filtration membrane to remove impurities. Then supernatant was subjected to ultracentrifugation at 100,000×g for 2 h at 4 °C58. The microspheres were re-suspended with phosphate-buffered brine and stored at − 80 °C until further analysis.

Immunofluorescence

Kidney sections were fixed with paraformaldehyde for 24 h. The samples were embedded and cut into 3-5 μm slices. The sections were incubated overnight with primary antibodies at 4 °C. Subsequently, the samples were incubated with a fluorescent secondary antibody (#4412, Cell Signaling Technology, Inc., USA) and blocked using an anti-fluorescence quencher (P0131, Beyotime Biotechnology). Fluorescence signals were then visualized under a fluorescence microscope. The antibodies used in this procedure are listed in Table S2.

Statistical analysis

Statistical analyses were carried out using GraphPad Prism 9.0 (San Diego, 274 CA, USA). All data were expressed as mean ± standard deviation. Comparisons between the two groups were analyzed by T-tests. P < 0.05 was considered statistically significant.

Supplementary Information

Supplementary Information 1.

Supplementary Information 2.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71755-y.

Acknowledgements

We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.

Author contributions

Conceptualization, Yushi Sun and Bojun Li; Formal analysis, Yushi Sun and Bojun Li; Funding acquisition, Fan Cheng; Methodology, Bojun Li; Supervision, Rao Ting and Fan Cheng; Validation, Yushi Sun, Bojun Li and Xiangjun Zhou; Visualization, Yushi Sun; Writing—original draft, Yushi Sun, Bojun Li and Fan Cheng; Writing—review & editing, Yushi Sun, Bojun Li, Xiangjun Zhou and Fan Cheng.

Funding

This work was supported by National Natural Science Foundation of China (82170775, and 82100806).

Data availability

Data is provided within the manuscript or supplementary information files. The datasets used and analyzed in this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval

The clinical specimen collection (approval number: WDRY2021-KS047) was approved by the Ethics Committee of the Renmin Hospital of Wuhan University. Confirms that all experiments were performed in accordance with relevant named guidelines and regulations. Confirms that informed consent was obtained from all participants and/or their legal guardians.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yushi Sun, Bojun Li and Xiangjun Zhou.
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References

1. Thongprayoon C Krambeck AE Rule AD Determining the true burden of kidney stone disease Nat. Rev. Nephrol. 2020 16 736 746 10.1038/s41581-020-0320-7 32753740
Thongprayoon, C., Krambeck, A. E. & Rule, A. D. Determining the true burden of kidney stone disease. Nat. Rev. Nephrol. 16, 736–746 (2020).32753740 10.1038/s41581-020-0320-7
2. Peerapen P Thongboonkerd V Kidney stone prevention Adv. Nutr. 2023 14 555 569 10.1016/j.advnut.2023.03.002 36906146
Peerapen, P. & Thongboonkerd, V. Kidney stone prevention. Adv. Nutr. 14, 555–569 (2023).36906146 10.1016/j.advnut.2023.03.002
3. Ganpule AP Vijayakumar M Malpani A Desai MR Percutaneous nephrolithotomy (PCNL) a critical review Int. J. Surg. 2016 36 660 664 10.1016/j.ijsu.2016.11.028 27856356
Ganpule, A. P., Vijayakumar, M., Malpani, A. & Desai, M. R. Percutaneous nephrolithotomy (PCNL) a critical review. Int. J. Surg. 36, 660–664 (2016).27856356 10.1016/j.ijsu.2016.11.028
4. Proietti S Knoll T Giusti G Contemporary ureteroscopic management of renal stones Int. J. Surg. 2016 36 681 687 10.1016/j.ijsu.2016.11.130 27913238
Proietti, S., Knoll, T. & Giusti, G. Contemporary ureteroscopic management of renal stones. Int. J. Surg. 36, 681–687 (2016).27913238 10.1016/j.ijsu.2016.11.130
5. Wigner P Bijak M Saluk-Bijak J Probiotics in the prevention of the calcium oxalate urolithiasis Cells 2022 11 284 10.3390/cells11020284 35053400
Wigner, P., Bijak, M. & Saluk-Bijak, J. Probiotics in the prevention of the calcium oxalate urolithiasis. Cells 11, 284 (2022).35053400 10.3390/cells11020284
6. Zhang Y Liu Y Liu H Tang WH Exosomes: Biogenesis, biologic function and clinical potential Cell Biosci. 2019 9 19 10.1186/s13578-019-0282-2 30815248
Zhang, Y., Liu, Y., Liu, H. & Tang, W. H. Exosomes: Biogenesis, biologic function and clinical potential. Cell Biosci. 9, 19 (2019).30815248 10.1186/s13578-019-0282-2
7. Gurunathan S Kang M-H Kim J-H A comprehensive review on factors influences biogenesis, functions, therapeutic and clinical implications of exosomes Int. J. Nanomed. 2021 16 1281 1312 10.2147/IJN.S291956
Gurunathan, S., Kang, M.-H. & Kim, J.-H. A comprehensive review on factors influences biogenesis, functions, therapeutic and clinical implications of exosomes. Int. J. Nanomed. 16, 1281–1312 (2021).10.2147/IJN.S291956
8. Al-Madhagi H The landscape of exosomes biogenesis to clinical applications Int. J. Nanomed. 2024 19 3657 3675 10.2147/IJN.S463296
Al-Madhagi, H. The landscape of exosomes biogenesis to clinical applications. Int. J. Nanomed. 19, 3657–3675 (2024).10.2147/IJN.S463296
9. Evan AP Worcester EM Coe FL Williams J Lingeman JE Mechanisms of human kidney stone formation Urolithiasis 2015 43 Suppl 1 19 32 10.1007/s00240-014-0701-0 25108546
Evan, A. P., Worcester, E. M., Coe, F. L., Williams, J. & Lingeman, J. E. Mechanisms of human kidney stone formation. Urolithiasis 43(Suppl 1), 19–32 (2015).25108546 10.1007/s00240-014-0701-0
10. Chung H-J The role of Randall plaques on kidney stone formation Transl. Androl. Urol. 2014 3 251 254 26816774
Chung, H.-J. The role of Randall plaques on kidney stone formation. Transl. Androl. Urol. 3, 251–254 (2014).26816774
11. Thongboonkerd V Proteomics of crystal-cell interactions: A model for kidney stone research Cells 2019 8 1076 10.3390/cells8091076 31547429
Thongboonkerd, V. Proteomics of crystal-cell interactions: A model for kidney stone research. Cells 8, 1076 (2019).31547429 10.3390/cells8091076
12. Quinn JJ Chang HY Unique features of long non-coding RNA biogenesis and function Nat. Rev. Genet. 2016 17 47 62 10.1038/nrg.2015.10 26666209
Quinn, J. J. & Chang, H. Y. Unique features of long non-coding RNA biogenesis and function. Nat. Rev. Genet. 17, 47–62 (2016).26666209 10.1038/nrg.2015.10
13. Taguchi K M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development Sci. Rep. 2016 6 35167 10.1038/srep35167 27731368
Taguchi, K. et al. M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development. Sci. Rep. 6, 35167 (2016).27731368 10.1038/srep35167
14. Jia Y Extracellular vesicles from albumin-induced tubular epithelial cells promote the M1 macrophage phenotype by targeting klotho Mol. Ther. 2019 27 1452 1466 10.1016/j.ymthe.2019.05.019 31208912
Jia, Y. et al. Extracellular vesicles from albumin-induced tubular epithelial cells promote the M1 macrophage phenotype by targeting klotho. Mol. Ther. 27, 1452–1466 (2019).31208912 10.1016/j.ymthe.2019.05.019
15. Lv L-L Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury Cell Death Differ. 2020 27 210 226 10.1038/s41418-019-0349-y 31097789
Lv, L.-L. et al. Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury. Cell Death Differ. 27, 210–226 (2020).31097789 10.1038/s41418-019-0349-y
16. Kanehisa M Goto S KEGG: Kyoto encyclopedia of genes and genomes Nucleic Acids Res. 2000 28 27 30 10.1093/nar/28.1.27 10592173
Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).10592173 10.1093/nar/28.1.27
17. Kanehisa M Toward understanding the origin and evolution of cellular organisms Protein Sci. 2019 28 1947 1951 10.1002/pro.3715 31441146
Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951 (2019).31441146 10.1002/pro.3715
18. Kanehisa M Furumichi M Sato Y Kawashima M Ishiguro-Watanabe M KEGG for taxonomy-based analysis of pathways and genomes Nucleic Acids Res. 2023 51 D587 D592 10.1093/nar/gkac963 36300620
Kanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. & Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 51, D587–D592 (2023).36300620 10.1093/nar/gkac963
19. Alexander RT Fuster DG Dimke H Mechanisms underlying calcium nephrolithiasis Annu. Rev. Physiol. 2022 84 559 583 10.1146/annurev-physiol-052521-121822 34699268
Alexander, R. T., Fuster, D. G. & Dimke, H. Mechanisms underlying calcium nephrolithiasis. Annu. Rev. Physiol. 84, 559–583 (2022).34699268 10.1146/annurev-physiol-052521-121822
20. Arya SB Collie SP Parent CA The ins-and-outs of exosome biogenesis, secretion, and internalization Trends Cell Biol. 2024 34 90 108 10.1016/j.tcb.2023.06.006 37507251
Arya, S. B., Collie, S. P. & Parent, C. A. The ins-and-outs of exosome biogenesis, secretion, and internalization. Trends Cell Biol. 34, 90–108 (2024).37507251 10.1016/j.tcb.2023.06.006
21. Su X Ambra1 in exosomes secreted by HK-2 cells damaged by supersaturated oxalate induce mitophagy and autophagy-ferroptosis in normal HK-2 cells to participate in the occurrence of kidney stones Biochim. Biophys. Acta 2024 1871 119604 10.1016/j.bbamcr.2023.119604
Su, X. et al. Ambra1 in exosomes secreted by HK-2 cells damaged by supersaturated oxalate induce mitophagy and autophagy-ferroptosis in normal HK-2 cells to participate in the occurrence of kidney stones. Biochim. Biophys. Acta 1871, 119604 (2024).10.1016/j.bbamcr.2023.119604
22. He J Renal macrophages monitor and remove particles from urine to prevent tubule obstruction Immunity 2024 57 106 123 10.1016/j.immuni.2023.12.003 38159573
He, J. et al. Renal macrophages monitor and remove particles from urine to prevent tubule obstruction. Immunity 57, 106–123 (2024).38159573 10.1016/j.immuni.2023.12.003
23. Taguchi K Okada A Unno R Hamamoto S Yasui T Macrophage function in calcium oxalate kidney stone formation: A systematic review of literature Front. Immunol. 2021 12 673690 10.3389/fimmu.2021.673690 34108970
Taguchi, K., Okada, A., Unno, R., Hamamoto, S. & Yasui, T. Macrophage function in calcium oxalate kidney stone formation: A systematic review of literature. Front. Immunol. 12, 673690 (2021).34108970 10.3389/fimmu.2021.673690
24. Liu J-L Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy Mol. Ther. 2023 31 1451 1467 10.1016/j.ymthe.2023.03.027 37016580
Liu, J.-L. et al. Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy. Mol. Ther. 31, 1451–1467 (2023).37016580 10.1016/j.ymthe.2023.03.027
25. Singhto N Kanlaya R Nilnumkhum A Thongboonkerd V Roles of macrophage exosomes in immune response to calcium oxalate monohydrate crystals Front. Immunol. 2018 9 316 10.3389/fimmu.2018.00316 29535716
Singhto, N., Kanlaya, R., Nilnumkhum, A. & Thongboonkerd, V. Roles of macrophage exosomes in immune response to calcium oxalate monohydrate crystals. Front. Immunol. 9, 316 (2018).29535716 10.3389/fimmu.2018.00316
26. Shyong Y-J Chang K-C Lin F-H Calcium phosphate particles stimulate exosome secretion from phagocytes for the enhancement of drug delivery Colloids Surf. B 2018 171 391 397 10.1016/j.colsurfb.2018.07.037
Shyong, Y.-J., Chang, K.-C. & Lin, F.-H. Calcium phosphate particles stimulate exosome secretion from phagocytes for the enhancement of drug delivery. Colloids Surf. B 171, 391–397 (2018).10.1016/j.colsurfb.2018.07.037
27. Krützfeldt J Strategies to use microRNAs as therapeutic targets Best Pract. Res. Clin. Endocrinol. Metab. 2016 30 551 561 10.1016/j.beem.2016.07.004 27923450
Krützfeldt, J. Strategies to use microRNAs as therapeutic targets. Best Pract. Res. Clin. Endocrinol. Metab. 30, 551–561 (2016).27923450 10.1016/j.beem.2016.07.004
28. Djuranovic S Nahvi A Green R miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay Science 2012 336 237 240 10.1126/science.1215691 22499947
Djuranovic, S., Nahvi, A. & Green, R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336, 237–240 (2012).22499947 10.1126/science.1215691
29. Cohen C WNT-dependent interaction between inflammatory fibroblasts and FOLR2+ macrophages promotes fibrosis in chronic kidney disease Nat. Commun. 2024 15 743 10.1038/s41467-024-44886-z 38272907
Cohen, C. et al. WNT-dependent interaction between inflammatory fibroblasts and FOLR2+ macrophages promotes fibrosis in chronic kidney disease. Nat. Commun. 15, 743 (2024).38272907 10.1038/s41467-024-44886-z
30. He D The Wnt11 signaling pathway in potential cellular EMT and osteochondral differentiation progression in nephrolithiasis formation Int. J. Mol. Sci. 2015 16 16313 16329 10.3390/ijms160716313 26193266
He, D. et al. The Wnt11 signaling pathway in potential cellular EMT and osteochondral differentiation progression in nephrolithiasis formation. Int. J. Mol. Sci. 16, 16313–16329 (2015).26193266 10.3390/ijms160716313
31. Unno R Deregulated MTOR (mechanistic target of rapamycin kinase) is responsible for autophagy defects exacerbating kidney stone development Autophagy 2020 16 709 723 10.1080/15548627.2019.1635382 31257986
Unno, R. et al. Deregulated MTOR (mechanistic target of rapamycin kinase) is responsible for autophagy defects exacerbating kidney stone development. Autophagy 16, 709–723 (2020).31257986 10.1080/15548627.2019.1635382
32. Yuan H The protective role of corilagin on renal calcium oxalate crystal-induced oxidative stress, inflammatory response, and apoptosis via PPAR-γ and PI3K/Akt pathway in rats Biotechnol. Appl. Biochem. 2021 68 1323 1331 33080078
Yuan, H. et al. The protective role of corilagin on renal calcium oxalate crystal-induced oxidative stress, inflammatory response, and apoptosis via PPAR-γ and PI3K/Akt pathway in rats. Biotechnol. Appl. Biochem. 68, 1323–1331 (2021).33080078
33. Rao P Promotion of β-catenin/forkhead box protein O signaling mediates epithelial repair in kidney injury Am. J. Pathol. 2021 191 993 1009 10.1016/j.ajpath.2021.03.005 33753026
Rao, P. et al. Promotion of β-catenin/forkhead box protein O signaling mediates epithelial repair in kidney injury. Am. J. Pathol. 191, 993–1009 (2021).33753026 10.1016/j.ajpath.2021.03.005
34. Huang L Loss of nephric augmenter of liver regeneration facilitates acute kidney injury via ACSL4-mediated ferroptosis J. Cell Mol. Med. 2024 28 e18076 10.1111/jcmm.18076 38088220
Huang, L. et al. Loss of nephric augmenter of liver regeneration facilitates acute kidney injury via ACSL4-mediated ferroptosis. J. Cell Mol. Med. 28, e18076 (2024).38088220 10.1111/jcmm.18076
35. Li Y LncRNA-ATB participates in the regulation of calcium oxalate crystal-induced renal injury by sponging the miR-200 family Mol. Med. 2021 27 143 10.1186/s10020-021-00403-2 34736391
Li, Y. et al. LncRNA-ATB participates in the regulation of calcium oxalate crystal-induced renal injury by sponging the miR-200 family. Mol. Med. 27, 143 (2021).34736391 10.1186/s10020-021-00403-2
36. Li R Novel drug delivery systems and disease models for pulmonary fibrosis J. Control Release 2022 348 95 114 10.1016/j.jconrel.2022.05.039 35636615
Li, R. et al. Novel drug delivery systems and disease models for pulmonary fibrosis. J. Control Release 348, 95–114 (2022).35636615 10.1016/j.jconrel.2022.05.039
37. Tampe D Zeisberg M Potential approaches to reverse or repair renal fibrosis Nat. Rev. Nephrol. 2014 10 226 237 10.1038/nrneph.2014.14 24514753
Tampe, D. & Zeisberg, M. Potential approaches to reverse or repair renal fibrosis. Nat. Rev. Nephrol. 10, 226–237 (2014).24514753 10.1038/nrneph.2014.14
38. Guo M Syndecan-1 shedding destroys epithelial adherens junctions through STAT3 after renal ischemia/reperfusion injury iScience 2023 26 108211 10.1016/j.isci.2023.108211 37942007
Guo, M. et al. Syndecan-1 shedding destroys epithelial adherens junctions through STAT3 after renal ischemia/reperfusion injury. iScience 26, 108211 (2023).37942007 10.1016/j.isci.2023.108211
39. Xun Y Role of Nox4 in high calcium-induced renal oxidative stress damage and crystal deposition Antioxid. Redox Signal. 2022 36 15 38 10.1089/ars.2020.8159 34435888
Xun, Y. et al. Role of Nox4 in high calcium-induced renal oxidative stress damage and crystal deposition. Antioxid. Redox Signal. 36, 15–38 (2022).34435888 10.1089/ars.2020.8159
40. Song B-F Overexpression of sirtuin 1 attenuates calcium oxalate-induced kidney injury by promoting macrophage polarization Int. Immunopharmacol. 2023 121 110398 10.1016/j.intimp.2023.110398 37301123
Song, B.-F. et al. Overexpression of sirtuin 1 attenuates calcium oxalate-induced kidney injury by promoting macrophage polarization. Int. Immunopharmacol. 121, 110398 (2023).37301123 10.1016/j.intimp.2023.110398
41. Lian J Extracellular vesicle-transmitted miR-671-5p alleviates lung inflammation and injury by regulating the AAK1/NF-κB axis Mol. Ther. 2023 31 1365 1382 10.1016/j.ymthe.2023.01.025 36733250
Lian, J. et al. Extracellular vesicle-transmitted miR-671-5p alleviates lung inflammation and injury by regulating the AAK1/NF-κB axis. Mol. Ther. 31, 1365–1382 (2023).36733250 10.1016/j.ymthe.2023.01.025
42. Song Q FKBP5 deficiency attenuates calcium oxalate kidney stone formation by suppressing cell-crystal adhesion, apoptosis and macrophage M1 polarization via inhibition of NF-κB signaling Cell Mol. Life Sci. 2023 80 301 10.1007/s00018-023-04958-7 37740796
Song, Q. et al. FKBP5 deficiency attenuates calcium oxalate kidney stone formation by suppressing cell-crystal adhesion, apoptosis and macrophage M1 polarization via inhibition of NF-κB signaling. Cell Mol. Life Sci. 80, 301 (2023).37740796 10.1007/s00018-023-04958-7
43. Jia J Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage Autophagy 2023 19 1893 1895 10.1080/15548627.2022.2148900 36394332
Jia, J. et al. Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage. Autophagy 19, 1893–1895 (2023).36394332 10.1080/15548627.2022.2148900
44. Song T-J Effect of SNHG11/miR-7–5p/PLCB1 axis on acute pancreatitis through inhibiting p38MAPK pathway Cells 2022 12 65 10.3390/cells12010065 36611865
Song, T.-J. et al. Effect of SNHG11/miR-7–5p/PLCB1 axis on acute pancreatitis through inhibiting p38MAPK pathway. Cells 12, 65 (2022).36611865 10.3390/cells12010065
45. Peckert-Maier K CD83 expressed by macrophages is an important immune checkpoint molecule for the resolution of inflammation Front. Immunol. 2023 14 1085742 10.3389/fimmu.2023.1085742 36875129
Peckert-Maier, K. et al. CD83 expressed by macrophages is an important immune checkpoint molecule for the resolution of inflammation. Front. Immunol. 14, 1085742 (2023).36875129 10.3389/fimmu.2023.1085742
46. Liu W Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9 Biol. Proced. Online 2023 25 10 10.1186/s12575-023-00198-y 37085762
Liu, W. et al. Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9. Biol. Proced. Online 25, 10 (2023).37085762 10.1186/s12575-023-00198-y
47. Liu B The RNase MCPIP3 promotes skin inflammation by orchestrating myeloid cytokine response Nat. Commun. 2021 12 4105 10.1038/s41467-021-24352-w 34215755
Liu, B. et al. The RNase MCPIP3 promotes skin inflammation by orchestrating myeloid cytokine response. Nat. Commun. 12, 4105 (2021).34215755 10.1038/s41467-021-24352-w
48. Zhu M Jin T Wu D Zhang S Wang A Transcriptomics analysis revealed key genes associated with macrophage autophagolysosome in male ApoE-/- mice aortic atherosclerosis J. Inflamm. Res. 2023 16 5125 5144 10.2147/JIR.S426155 37965353
Zhu, M., Jin, T., Wu, D., Zhang, S. & Wang, A. Transcriptomics analysis revealed key genes associated with macrophage autophagolysosome in male ApoE-/- mice aortic atherosclerosis. J. Inflamm. Res. 16, 5125–5144 (2023).37965353 10.2147/JIR.S426155
49. Verstraelen P Serum amyloid A3 fuels a feed-forward inflammatory response to the bacterial amyloid curli in the enteric nervous system Cell Mol. Gastroenterol. Hepatol. 2024 18 89 104 10.1016/j.jcmgh.2024.03.013 38556049
Verstraelen, P. et al. Serum amyloid A3 fuels a feed-forward inflammatory response to the bacterial amyloid curli in the enteric nervous system. Cell Mol. Gastroenterol. Hepatol. 18, 89–104 (2024).38556049 10.1016/j.jcmgh.2024.03.013
50. Zhang M IL-27 disturbs lipid metabolism and restrains mitochondrial activity to inhibit γδ T17 cell-mediated skin inflammation Cell Death Dis. 2024 15 491 10.1038/s41419-024-06887-0 38982043
Zhang, M. et al. IL-27 disturbs lipid metabolism and restrains mitochondrial activity to inhibit γδ T17 cell-mediated skin inflammation. Cell Death Dis. 15, 491 (2024).38982043 10.1038/s41419-024-06887-0
51. Maruyama K The antioxidant and DNA-repair enzyme apurinic/apyrimidinic endonuclease 1 limits the development of tubulointerstitial fibrosis partly by modulating the immune system Sci. Rep. 2019 9 7823 10.1038/s41598-019-44241-z 31127150
Maruyama, K. et al. The antioxidant and DNA-repair enzyme apurinic/apyrimidinic endonuclease 1 limits the development of tubulointerstitial fibrosis partly by modulating the immune system. Sci. Rep. 9, 7823 (2019).31127150 10.1038/s41598-019-44241-z
52. Jansen JC TMEM199 deficiency is a disorder of golgi homeostasis characterized by elevated aminotransferases, alkaline phosphatase, and cholesterol and abnormal glycosylation Am. J. Hum. Genet. 2016 98 322 330 10.1016/j.ajhg.2015.12.011 26833330
Jansen, J. C. et al. TMEM199 deficiency is a disorder of golgi homeostasis characterized by elevated aminotransferases, alkaline phosphatase, and cholesterol and abnormal glycosylation. Am. J. Hum. Genet. 98, 322–330 (2016).26833330 10.1016/j.ajhg.2015.12.011
53. Larsen LE Defective lipid droplet-lysosome interaction causes fatty liver disease as evidenced by human mutations in TMEM199 and CCDC115 Cell Mol. Gastroenterol. Hepatol. 2022 13 583 597 10.1016/j.jcmgh.2021.09.013 34626841
Larsen, L. E. et al. Defective lipid droplet-lysosome interaction causes fatty liver disease as evidenced by human mutations in TMEM199 and CCDC115. Cell Mol. Gastroenterol. Hepatol. 13, 583–597 (2022).34626841 10.1016/j.jcmgh.2021.09.013
54. Dusabimana T GOLPH3 promotes endotoxemia-induced liver and kidney injury through Golgi stress-mediated apoptosis and inflammatory response Cell Death Dis. 2023 14 458 10.1038/s41419-023-05975-x 37479687
Dusabimana, T. et al. GOLPH3 promotes endotoxemia-induced liver and kidney injury through Golgi stress-mediated apoptosis and inflammatory response. Cell Death Dis. 14, 458 (2023).37479687 10.1038/s41419-023-05975-x
55. Johnson DL Kumar R Kakhniashvili D Pfeffer LM Laribee RN Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy J. Biol. Chem. 2024 60 107582 10.1016/j.jbc.2024.107582
Johnson, D. L., Kumar, R., Kakhniashvili, D., Pfeffer, L. M. & Laribee, R. N. Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy. J. Biol. Chem. 60, 107582 (2024).10.1016/j.jbc.2024.107582
56. Kessi M Disruption of mitochondrial and lysosomal functions by human CACNA1C variants expressed in HEK 293 and CHO cells Front. Mol. Neurosci. 2023 16 1209760 10.3389/fnmol.2023.1209760 37448958
Kessi, M. et al. Disruption of mitochondrial and lysosomal functions by human CACNA1C variants expressed in HEK 293 and CHO cells. Front. Mol. Neurosci. 16, 1209760 (2023).37448958 10.3389/fnmol.2023.1209760
57. Zhao S Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys Theranostics 2021 11 8660 8673 10.7150/thno.62820 34522205
Zhao, S. et al. Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys. Theranostics 11, 8660–8673 (2021).34522205 10.7150/thno.62820
58. Zhu S Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias Int. Immunopharmacol. 2024 132 111828 10.1016/j.intimp.2024.111828 38552294
Zhu, S. et al. Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias. Int. Immunopharmacol. 132, 111828 (2024).38552294 10.1016/j.intimp.2024.111828
