Abstract Background Congenital Hydronephrosis (CH) is a common pediatric disorder that often leads to renal fibrosis (RF), significantly impairing kidney function. Oxidative stress (OS) plays a central role in the pathogenesis of RF. Current treatments lack effective monitoring and targeted therapies for CH, thus highlighting the need for innovative diagnostic and therapeutic approaches. This study explores a novel multifunctional nanozyme, pH-responsive PEG-SH and imidazole-modified gold nanoparticles (PMIZ-AuNPs), for both real-time ultrasound monitoring and treatment of CH-induced RF. Results We designed a pH-responsive nanozyme, consisting of PEG-SH and PMIZ-AuNPs. This nanozyme exhibits enhanced ultrasound imaging properties and dual catalytic activities, including superoxide dismutase (SOD) and catalase (CAT), under acidic conditions. In a unilateral ureteral obstruction (UUO) mouse model, PMIZ-AuNPs accumulated at injury sites, enhancing ultrasound signal intensity and improving RF. Protein sequencing and bioinformatics analysis identified C9 as a critical gene involved in RF. Further experiments showed that PMIZ-AuNPs reduced C9 expression by inhibiting OS and modulated the TGF-β signaling pathway, leading to significant attenuation of RF in both in vitro and in vivo models. Conclusion PMIZ-AuNPs demonstrate significant potential as a multifunctional tool for the diagnosis and treatment of CH-induced RF. By targeting oxidative stress and modulating C9 expression, PMIZ-AuNPs improve renal function and offer a promising strategy for the clinical management of CH. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03618-1. Keywords: Congenital hydronephrosis, Renal fibrosis, Nanozyme, Oxidative stress, Ultrasound contrast agent Graphical Abstract [32]graphic file with name 12951_2025_3618_Figa_HTML.jpg [33]Open in a new tab Framework of the Clot Cases Conference Background Congenital Hydronephrosis (CH) is a common congenital urinary system disease typically caused by urinary tract obstruction [[34]1, [35]2]. This obstruction leads to impaired urine flow, resulting in renal pelvis dilation and kidney function impairment. As the disease progresses, hydronephrosis causes renal tissue damage and fibrosis, significantly affecting patients’ quality of life and prognosis. Although current treatments, such as surgery and medication, can alleviate symptoms to some extent, they suffer from issues like delayed diagnosis, unstable efficacy, and side effects [[36]3]. Therefore, developing novel diagnostic and therapeutic methods to improve early diagnosis rates and treatment outcomes remains an urgent medical challenge [[37]4, [38]5]. Oxidative Stress (OS) plays a crucial role in the development and progression of Renal Fibrosis (RF) [[39]6, [40]7, [41]8]. Elevated levels of reactive oxygen species (ROS) lead to damage to proteins, lipids, and DNA within cells, triggering a series of pathological changes [[42]9, [43]10, [44]11]. In the pathology of hydronephrosis, OS not only directly damages renal tubular cells but also activates inflammatory responses and fibrosis signaling pathways, further exacerbating renal tissue damage and fibrosis [[45]12]. Studies indicate that antioxidant therapy can effectively alleviate RF [[46]13, [47]14, [48]15]. However, existing antioxidants face significant limitations in stability, targeting, and efficacy. Thus, identifying a novel antioxidant that can stably, efficiently, and specifically inhibit OS has become a major research focus. Nanozymes are a class of artificially synthesized nanomaterials with natural enzyme activities [[49]16]. Due to their high stability, ease of modification, and functional diversity, nanozymes exhibit broad application prospects in the biomedical field [[50]17, [51]18, [52]19]. In recent years, Multifunctional Nanozyme (MF-NZ) has achieved significant results in tumor therapy and the treatment of neurological diseases [[53]20, [54]21, [55]22]. For example, some studies show that nanozymes can serve as Ultrasound Contrast Agent (UCA), enabling early diagnosis and treatment of tumors [[56]23, [57]24]. Compared to traditional treatment methods, nanozymes not only enhance imaging quality but also have good biocompatibility and low toxicity, providing new possibilities for disease diagnosis and treatment. In this study, we apply multifunctional targeted nanozymes to the diagnosis and treatment of CH, aiming to achieve early monitoring and effective treatment of this disease. This study designs a pH-responsive PEG-SH and imidazole-modified gold nanoparticles (PMIZ-AuNPs), which possesses enhanced ultrasound imaging and dual catalytic activities. We observe the morphology and size of the nanozymes through transmission electron microscopy (TEM) and record their spectral characteristics using a spectrophotometer. Dynamic light scattering (DLS) is used to measure the zeta potential and hydrodynamic diameter (HD) of the nanozymes at different pH values. We detect the ultrasound signals within cells and use Electron Spin Resonance (ESR) spectroscopy to assess the enzyme activity of the AuNP nanozymes, characterizing their imaging properties and catalytic activity. Subsequently, we construct a left unilateral ureteral obstruction (UUO) mouse model to investigate the ultrasound signals and therapeutic effects of the AuNP nanozymes in vivo. Through protein sequencing, combined with the Gene Expression Omnibus (GEO) database of UUO-related datasets, we perform differential analysis, protein interaction, machine learning, and Receiver Operating Characteristic (ROC) analysis to identify characteristic genes of the AuNP nanozyme treatment for CH in mice and predict downstream molecular pathways. We then use mouse and human proximal kidney tubular cells (TKPTS and Human Kidney-2 (HK-2) cells) for in vitro mechanistic validation, exploring the regulation of RF and inflammation in H[2]O[2]-treated renal tubular cells by AuNP nanozymes and the characteristic genes. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Western Blot (WB) are used to detect the expression of characteristic and inflammatory genes, and immunofluorescence staining is used to investigate the expression of fibrosis marker proteins in cells. Finally, we validate the molecular mechanisms of AuNP nanozyme treatment for CH through in vivo animal experiments, assessing the degree of RF in mouse kidney tissues using Hematoxylin and Eosin (H&E) staining, Masson’s trichrome staining, Sirius red staining, and Immunohistochemistry (IHC). This study aims to develop a novel multifunctional targeted nanozyme for the real-time monitoring and effective treatment of CH. We successfully design a PMIZ-AuNPs. Under acidic conditions, PMIZ-AuNPs exhibit aggregation and enhanced ultrasound contrast properties, along with dual catalytic activities of superoxide dismutase (SOD) and Catalase (CAT). Upon injection into UUO mice, PMIZ-AuNPs accumulate at the site of renal injury, enhancing ultrasound signal intensity and improving RF associated with hydronephrosis. Protein sequencing, combined with GEO database analysis of UUO-related datasets, identifies C9 as a key gene/protein. We find that PMIZ-AuNPs exert SOD and CAT activities, reducing C9 expression by inhibiting OS and improving RF through the Transforming Growth Factor Beta (TGF-β) signaling pathway. Both in vitro cell experiments and in vivo mouse experiments confirm that PMIZ-AuNPs ameliorate OS-induced RF and inflammation, whereas C9 overexpression reverses these effects. This study provides new technological approaches for the early diagnosis and treatment of CH and opens new avenues for the application of MF-NZs in nanomedicine, holding significant scientific and clinical value. Results Preparation and characterization of PMIZ-AuNPs In the rapid development of nanoscience and nanotechnology, gold nanostructures have attracted significant interest due to their unique optical, electronic, and catalytic properties. While bulk gold is traditionally considered an inert material, AuNPs have been demonstrated to act as catalysts for various chemical reactions under specific experimental conditions. AuNPs are widely studied for their potential medical applications due to their superior optical and photothermal conversion properties, ease of synthesis, and versatile surface modification capabilities [[58]25, [59]26]. Renal-clearable nanoparticles are typically quickly filtered out through the glomerulus, leading to weak interaction with the renal tissue and negligible ultrasound signals, which poses challenges for direct imaging of kidney diseases [[60]27]. We developed PMIZ-AuNPs (Fig. [61]1A). Initially, Au was reduced from HAuCl[4] using a reduction reaction and placed in a AuNPs growth solution to form small-sized AuNPs. These nanoparticles were then subjected to high-temperature treatment to remove citrate organic ligands, enhancing their catalytic activity. Subsequently, we synthesized and functionalized PMIZ-AuNPs and used glutathione-modified gold nanoparticles (GS-AuNPs) nanozymes as a control. Fig. 1. [62]Fig. 1 [63]Open in a new tab Aggregation Characteristics and Optical Properties of PMIZ-AuNPs. Note: (A) Schematic illustration of PMIZ-AuNPs; (B) Representative TEM image of PMIZ-AuNPs with corresponding core size distribution (Scale bar: 10 nm); (C) Absorption and emission spectra of PMIZ-AuNPs, showing absorption spectrum (Abs, blue line), emission spectrum (Em, red line), and excitation spectrum (Ex, gray shaded area); (D) pH-dependent Zeta potential of PMIZ-AuNPs at different pH values; (E) HD of PMIZ-AuNPs at various pH levels; (F) Representative TEM images of PMIZ-AuNPs at different pH levels (Scale bar: 200 nm); (G) Ultrasound imaging and intensity of AuNPs at different pH values; (H-I) Intracellular ultrasound imaging of PMIZ-AuNPs at pH 5.5 and corresponding statistical ultrasound intensity; (J) Schematic diagram illustrating ultrasound aggregation enhancement of PMIZ-AuNPs in an acidic microenvironment To validate the properties of the nanozymes, we conducted a systematic characterization of the synthesized nanozymes. TEM data (Fig. [64]1B, Figure [65]S1A) revealed that both PMIZ-AuNPs and GS-AuNPs nanozymes exhibit good dispersion and uniform spherical morphology, with core sizes of 2.3 ± 0.4 nm and 2.4 ± 0.3 nm, respectively. Ultraviolet-visible spectroscopy and fluorescence spectroscopy were used to record the absorption, emission, and excitation spectra of the nanozymes. The results showed that both PMIZ-AuNPs and GS-AuNPs have similar spectral characteristics, with significant absorption peaks in the wavelength range of approximately 200–500 nm and emission peaks at around 800 nm (Fig. [66]1C, Figure [67]S1B). These properties indicate that both nanozymes are suitable for near-infrared fluorescence imaging or optical sensing applications. DLS was employed to measure the zeta potential and HD of the nanozymes at different pH levels. The results indicated that the zeta potential and HD of PMIZ-AuNPs change with pH, exhibiting charge reversal capability from negative (10.9 ± 1.0 mV, pH 7.4) to positive (17.4 ± 1.6 mV, pH 5.5) (Fig. [68]1D). PMIZ-AuNPs were negatively charged at pH levels above 6.8 and positively charged below 6.6. The HD of PMIZ-AuNPs increased from 3.9 ± 0.3 nm at pH 7.4 to 1143.18 ± 153.7 nm at pH 5.5 (Fig. [69]1E). In contrast, GS-AuNPs had a zeta potential of 24.3 ± 2.4 mV at pH 7.4 and 10.2 ± 1.8 mV at pH 5.5, with no significant change in HD at either pH level (Figure [70]S1C, Figure [71]S1D). TEM images further confirmed that PMIZ-AuNPs were monodispersed ultra-small particles at pH 7.4 but aggregated into large clusters at pH 5.5, demonstrating their aggregation in acidic microenvironments (Fig. [72]1F). These results suggest that PMIZ-AuNPs exhibit aggregation properties in acidic conditions, whereas GS-AuNPs do not. We subsequently measured the ultrasound signal of nanozymes at different pH levels. The results indicated that at pH 7.4 to 6.0, the ultrasound signal intensity of PMIZ-AuNPs was very weak, but it significantly increased at pH 5.5 to 4.0. In contrast, GS-AuNPs showed no detectable ultrasound signal across the pH range of 7.4 to 4.0 (Fig. [73]1G). Additionally, PMIZ-AuNPs at low concentrations aggregated intracellularly (Fig. [74]1H), with their ultrasound signal being 3.1 times higher than that of the cells (Fig. [75]1I). In summary, these results demonstrate that PMIZ-AuNPs exhibit aggregation properties and enhanced ultrasound performance in acidic microenvironments (Fig. [76]1J). PMIZ-AuNPs exhibit multifunctional catalytic properties Numerous nanomaterials have been reported to possess enzyme-like activities. Metals, metal oxides, sulfides, and carbon-based nanostructures have been found to exhibit oxidase-like and peroxidase-like activities, particularly in Fenton-like reactions and H[2]O[2] decomposition [[77]28, [78]29, [79]30]. Recently, AuNPs have been shown to mimic the activities of SOD and CAT. AuNPs can efficiently catalyze the decomposition of superoxide and H[2]O[2]. Notably, the decomposition of H[2]O[2] is accompanied by the formation of ·OH under low pH conditions and the formation of oxygen under high pH conditions [[80]31, [81]32]. To investigate the catalytic activity of PMIZ-AuNPs, we selected three spin-trapping agents (POBN, BMPO, and DMPO) commonly used for capturing ·OH radicals at pH 1.2 to determine if PMIZ-AuNPs induce ·OH production. ESR Spectroscopy showed (Fig. [82]2A) that in the absence of PMIZ-AuNPs (samples containing H[2]O[2] and spin trapping agents), the characteristic ESR signal for ·OH adducts was weak. In contrast, 2 min after adding PMIZ-AuNPs, a strong ESR signal for ·OH adducts appeared in all spin-trapping agent groups. When using POBN dissolved in 100 mM ethanol, a six-line ESR spectrum was observed, indicating the formation of POBN/CH(OH)CH[3] adducts containing the CH(OH)CH[3] radical, produced by the reaction of ·OH with ethanol. The ESR spectra for BMPO/OH and DMPO/OH displayed four lines with relative intensities of 1:2:2:1. The production of ·OH is highly pH-dependent. To better define the conditions for hydroxyl radical formation, we examined the PMIZ-AuNPs-assisted decomposition of H[2]O[2] across a broad pH range. The results (Fig. [83]2B-C) showed a four-line ESR spectrum with a 1:2:2:1 intensity ratio at pH 1.2 and 3.6, characteristic of DMPO/OH. The signal at pH 1.2 was significantly stronger than at pH 3.6. No DMPO/OH ESR signal was detected at pH values greater than 5.5. We further investigated the specific conditions for oxygen production during the PMIZ-AuNPs-assisted decomposition of H[2]O[2] by observing the formation of gas in sealed capillaries at different pH levels. The results (Fig. [84]2D) indicated no bubble formation at pH 1.2 and 3.6, while bubbles appeared at pH values above 5.5, with higher pH levels producing more bubbles. To confirm that the bubbles observed during H[2]O[2] decomposition were O[2], we used an ESR oximeter with the spin label CTPO to monitor the reaction products. In control samples without PMIZ-AuNPs, typical hyperfine structures and high-intensity ESR signals indicated low oxygen concentrations. However, upon adding PMIZ-AuNPs, the hyperfine splitting decreased, and the hyperfine spectrum disappeared with increasing pH, suggesting the formation of significant amounts of O[2]. These results demonstrate that at pH values below 5.5, PMIZ-AuNPs-assisted H[2]O[2] decomposition produces ·OH radicals, while at pH values above 5.5, the decomposition yields O[2]. Fig. 2. [85]Fig. 2 [86]Open in a new tab Investigation of PMIZ-AuNPs Nanozyme Activity. Note: (A) Production of ·OH detected by ESR Spectroscopy with three spin-trapping agents (POBN, BMPO, and DMPO) at pH 1.2; (B-C) Production of ·OH detected by ESR Spectroscopy using DMPO at different pH values; (D) Formation of gas by PMIZ-AuNPs in a closed capillary at different pH values; (E) Production of O[2] at different pH values detected by ESR Spectroscopy with spin-labeled CTPO; (F) Production of BMPO/OOH detected by ESR Spectroscopy with BMPO We generated superoxide in situ using the xanthine/xanthine oxidase system (Xan/XOD) and employed BMPO as a spin trap to form the BMPO/OOH spin adduct, in order to investigate the SOD-like activity of PMIZ-AuNPs. The results (Fig. [87]2E) indicate that the addition of xanthine oxidase to the PBS buffer containing xanthine, DTPA, and BMPO produced a strong BMPO/OOH ESR signal. Subsequently, the introduction of SOD significantly reduced the ESR signal intensity due to its ability to dismutate superoxide. The addition of PMIZ-AuNPs exhibited a similar effect to SOD, causing the signal to almost completely disappear, indicating that PMIZ-AuNPs possess SOD-like activity (Fig. [88]2F). These findings demonstrate that PMIZ-AuNPs exhibit both SOD and CAT-like catalytic activities. PMIZ-AuNPs improve RF in mice with hydronephrosis Hydronephrosis is one of the most common congenital abnormalities of the urinary tract, with a complex pathogenesis. The most frequent cause of pathological hydronephrosis is obstruction at the ureteropelvic junction [[89]33]. This obstruction can lead to urine retention within the kidney, increased renal pelvis pressure, tubular dilation, cellular phenotypic transformation, apoptosis, glomerular damage, and interstitial inflammation. Without timely diagnosis and intervention, these changes can progress to renal interstitial fibrosis (RIF) and renal dysplasia, ultimately resulting in renal atrophy and loss of renal function [[90]34]. We developed a UUO mouse model (Fig. [91]3A) and measured urine pH daily to investigate pH changes in the kidneys of UUO mice. Results indicated that three days post-modeling, the urine pH of UUO mice began to differ significantly from that of sham-operated mice (Figure [92]S2A). By day 7, the urine pH in UUO mice decreased to 5.67, indicating the accumulation of metabolic waste and acidic substances in the renal pelvis due to urine retention. This suggests that the microenvironment of hydronephrosis is typically mildly acidic. Seven days post-modeling, we administered 200 µL of PMIZ-AuNPs intravenously to UUO mice and performed ultrasound imaging. GS-AuNPs and saline were used as controls. The results showed that, following the injection of GS-AuNPs and saline, the ultrasound signals in both UUO and normal kidneys were weak (Figure [93]S2B). However, after the injection of PMIZ-AuNPs, the ultrasound signal in the UUO kidneys increased significantly within 10 min and continued to strengthen, while no significant change was observed in normal kidneys. This indicates that normal kidneys rapidly filtered out PMIZ-AuNPs through glomerular filtration, whereas the acidic microenvironment in UUO kidneys enhanced the tubular reabsorption of PMIZ-AuNPs, thereby significantly increasing the ultrasound signal. Ultrasound imaging combined with CDFI was performed on day 7 and day 14 post-modeling to observe kidney structure and blood flow dynamics. The results demonstrated that the imaging effect of UUO + PMIZ-AuNPs mice was significantly stronger than that of the sham, UUO, and UUO + Saline groups on day 14 and comparable to the imaging effect observed on day 7 (Figure [94]S2C, Table [95]1). On day 7, UUO mice were graded as level 2 hydronephrosis, with a 31.9% increase in the left kidney resistance index (RI) compared to sham mice. By day 14, the hydronephrosis grade in UUO and UUO + Saline mice increased to level 4, while UUO + PMIZ-AuNPs mice averaged a grade of 2.7. Compared to sham mice, UUO mice had a 78.7% increase in left kidney RI, UUO + Saline mice had a 74.2% increase, and UUO + PMIZ-AuNPs mice had a 57.4% increase. Additionally, on day 7, UUO mice exhibited decreased renal cortical thickness (RCT) and increased maximum cross-sectional area (Area^Max) compared to sham mice. On day 14, there was no significant change in UUO + Saline mice compared to UUO mice, whereas UUO + PMIZ-AuNPs mice showed increased RCT and decreased Area^Max. Fig. 3. [96]Fig. 3 [97]Open in a new tab Investigating the Therapeutic Effects of PMIZ-AuNPs on Mouse Hydronephrosis and RF. Note: (A) Experimental protocol for UUO mice; (B, D) H&E staining to assess kidney tissue damage in each group (Scale bar = 50 μm); (C, E) Masson’s trichrome staining to evaluate the fibrotic area in kidney tissues of each group (Scale bar = 50 μm); (F, G) Sirius red staining to detect the percentage of cortical interstitial collagen in each group (Scale bar = 50 μm); (H, I) IHC and WB analysis of α-SMA protein expression, a marker of myofibroblast differentiation in kidney tissues (Scale bar = 50 μm); (J) WB analysis of collagen I, collagen III, and fibronectin expression levels in kidney tissues; **p < 0.01 compared to the sham group; ***p < 0.001 compared to the sham group; #p < 0.05 compared to the UUO + Saline group; ##p < 0.01 compared to the UUO + Saline group; N = 5 Table 1. Resistive index (RI), renal cortex thickness (RCT), and renal maximum Cross-Sectional area (Area ^Max) of left kidney in each group Group RI RCT(mm) Area ^Max(cm^2) Day 7 after UUO sham 0.51 ± 0.04 3.4 ± 0.2 1.85 ± 0.04 UUO 0.67 ± 0.03* 1.3 ± 0.1* 3.65 ± 0.06* Day 14 after UUO sham 0.51 ± 0.04 3.5 ± 0.2 1.88 ± 0.06 UUO 0.91 ± 0.02*# 0.9 ± 0.2*# 4.21 ± 0.07*# UUO + Saline 0.89 ± 0.04* 1.0 ± 0.1* 4.33 ± 0.06* UUO + PMIZ-AuNPs 0.79 ± 0.03*^ 1.8 ± 0.1*^ 2.54 ± 0.05*^ [98]Open in a new tab Note: *p < 0.05 versus the Sham group and # p < 0.05 versus the UUO group of Day 7, ^p < 0.05 versus the UUO group of Day 14 After 14 days of modeling, the mice were euthanized. H&E staining results (Fig. [99]3B and D) showed significant inflammatory cell infiltration and severe tubulointerstitial damage in the UUO group compared to the sham group, with an increased tubulointerstitial damage score. In comparison to the UUO + Saline group, the UUO + PMIZ-AuNPs group exhibited improved renal tissue with a significantly lower tubulointerstitial damage score. Masson’s Trichrome staining results (Fig. [100]3C and E) demonstrated a significant increase in the area of RIF in the UUO group compared to the sham group. However, this fibrotic area was significantly reduced in the UUO + PMIZ-AuNPs group compared to the UUO + Saline group. Similarly, Sirius Red staining results (Fig. [101]3F-G) were consistent with those of Masson’s Trichrome staining. Under non-polarized light microscopy, all types of collagen appeared red, and the UUO group showed increased renal interstitial collagen compared to the sham group. In contrast, the UUO + PMIZ-AuNPs group showed a significant reduction in collagen levels compared to the UUO + Saline group, with even more pronounced effects under polarized light. IHC and WB analysis of Alpha-Smooth Muscle Actin (α-SMA), a marker of myofibroblast differentiation in renal tissue, indicated a significant increase in α-SMA protein levels in the UUO group compared to the sham group (Fig. [102]3H-I). However, the UUO + PMIZ-AuNPs group showed a significant decrease in α-SMA protein levels compared to the UUO + Saline group. Additionally, WB analysis of type I collagen (collagen I), type III collagen (collagen III), and fibronectin in the renal tissue revealed that their protein levels were significantly elevated in the UUO group compared to the sham group (Fig. [103]3J). In contrast, these protein levels were significantly reduced in the UUO + PMIZ-AuNPs group compared to the UUO + Saline group. These results indicate that PMIZ-AuNPs accumulate at the site of renal injury in mice, enhancing ultrasound intensity and improving RF associated with hydronephrosis. Multi-omics analysis reveals the significant role of C9 in PMIZ-AuNPs treatment of hydronephrosis To further investigate the specific molecular mechanisms by which PMIZ-AuNPs improve RF in mice with hydronephrosis, we employed TMT-labeled quantitative proteomics to analyze kidney samples from UUO + Saline and UUO + PMIZ-AuNPs groups. We identified 2,660 proteins and found 124 significant DEPs, with 45 upregulated and 79 downregulated (Figs. [104]4A-B). A PPI network of the DEPs was constructed (Fig. [105]4C), and the top 30 proteins with the highest number of interacting nodes were selected as candidate proteins (Fig. [106]4D). Fig. 4. [107]Fig. 4 [108]Open in a new tab Mult-Omics Analysis Identifying Characteristic Genes During PMIZ-AuNPs Treatment for Hydronephrosis. Note: (A-B) Volcano plot and heatmap of proteomics DEPs in kidney tissues of UUO + Saline group and UUO + PMIZ-AuNPs group (N = 3). Red dots represent significantly upregulated proteins, green dots represent significantly downregulated proteins, and black dots represent proteins with no differential expression. (C) PPI network of 124 DEPs. (D) Bar chart of the number of adjacent nodes of core genes in the PPI network. (E-F) Volcano plot and heatmap of DEGs in the sham group and UUO group (N = 60) based on high-throughput sequencing analysis. Red dots represent significantly upregulated genes, green dots represent significantly downregulated genes, and black dots represent genes with no differential expression. (G) The LASSO algorithm identified 46 key genes. (H) The SVM-RFE algorithm identified 40 key genes. (I) Venn diagram showing the intersection of 30 candidate proteins from the results of the two machine learning methods, identifying one characteristic gene/protein. (J-K) Expression levels of C9 in proteomics and transcriptomics. (L) WB analysis of C9 protein expression levels in kidney tissues of each group (N = 5). (M) ROC curve for C9 gene in transcriptome dataset. ** indicates p < 0.01 compared to the sham group; *** indicates p < 0.001 compared to the sham group; ## indicates p < 0.01 compared to the UUO + Saline group; N = 5 Next, we performed differential expression analysis on the UUO mouse dataset [109]GSE96101, identifying 2,730 DEGs between the sham and UUO groups, with 633 upregulated and 2,097 downregulated (Figs. [110]4E-F). Using machine learning algorithms, DEGs were further screened through Lasso regression and SVM-RFE models to identify characteristic genes of RF. In the Lasso regression, the Lambda parameter was chosen by cross-validation to find the point with the minimum error, resulting in 46 key genes (Fig. [111]4G). The SVM-RFE algorithm, using the “svmRadial” method, identified 40 key genes (Fig. [112]4H). The intersection of genes obtained from both algorithms and the top 30 candidate proteins resulted in the identification of the characteristic gene/protein C9 (Fig. [113]4I). Further validation of C9 expression and diagnostic capability showed that C9 gene expression was significantly upregulated in the UUO group compared to the sham group in the transcriptome (Fig. [114]4J). In the proteome, C9 protein expression was significantly downregulated in the UUO + PMIZ-AuNPs group compared to the UUO + Saline group (Fig. [115]4K). WB analysis confirmed these results, with C9 protein levels significantly increased in the UUO group compared to the sham group, and significantly decreased in the UUO + PMIZ-AuNPs group compared to the UUO + Saline group (Fig. [116]4L). ROC curve analysis demonstrated that the machine-learning-selected characteristic gene C9 had strong diagnostic and predictive capabilities, with an AUC value of 0.754 (Fig. [117]4M). In summary, our proteomics and transcriptomics analyses revealed that C9 is upregulated in the kidneys of UUO mice, and PMIZ-AuNPs reverse this upregulation. These findings indicate that C9 plays an important role in the PMIZ-AuNPs-mediated treatment of hydronephrosis. PMIZ-AuNPs reduce RF in hydronephrosis by inhibiting OS and downregulating C9 expression To explore the role of C9 in the treatment of hydronephrosis with PMIZ-AuNPs, we identified 154 co-expressed genes with C9 from the transcriptome, using a correlation threshold of|R| ≥ 0.5 and p < 0.001. The top genes correlated with C9 were visualized in a circos plot (Fig. [118]5A). Gene Ontology (GO) functional analysis and KEGG pathway analysis were then performed on C9 and its co-expressed genes. The GO analysis revealed that in the Biological Processes (BP) category, C9 and its co-expressed genes were mainly enriched in fibroblast proliferation, collagen fibril organization, and transforming growth factor bata production. In the Cellular Components (CC) category, they were enriched in collagen-containing extracellular matrix, fibrillar collagen trimer, and plasma membrane part. In the Molecular Functions (MF) category, they were enriched in oxidoreductase activity, cell adhesion molecule binding, and fibronectin binding. KEGG pathway analysis showed that C9 and its co-expressed genes were primarily enriched in the TGF-beta signaling pathway, ECM-receptor interaction, and focal adhesion (Fig. [119]5B). These enrichment results suggest that C9 plays a role in oxidoreductase activity, fibroblast proliferation, and collagen fibril formation, and is involved in the TGF-beta signaling pathway and ECM-receptor interaction. RF is characterized by excessive extracellular matrix (ECM) deposition leading to tissue scarring [[120]35], and TGF-β1 has long been considered a key mediator of RF, primarily inducing kidney scarring by activating its downstream Smad signaling pathway [[121]36, [122]37]. C9 is a critical protein in the complement system, an essential part of the innate immune system responsible for recognizing and clearing pathogens and damaged cells and promoting inflammatory responses. Studies have shown that complement activation in the kidney is associated with tubulointerstitial fibrosis in lupus nephritis (LN) and that C9 positively correlates with TGFβR1 and TGFβR2, indicating a link between complement activation and TGF-β signaling [[123]38], consistent with our findings. Based on these results and published literature, we propose that C9 influences RF progression by affecting kidney scarring through the TGF-β signaling pathway. Fig. 5. [124]Fig. 5 [125]Open in a new tab Exploring the Function of C9 in the Treatment of Hydronephrosis with PMIZ-AuNPs. Note: (A) Circle plot showing top-ranked genes associated with C9; (B) Bubble plot displaying GO functional analysis and KEGG pathway enrichment analysis of C9 and its co-expressed genes in biological processes (BP), cellular components (CC), and molecular functions (MF). The size of the circles represents the number of selected genes, and the color indicates the p-value of the enrichment analysis; (C-F) Measurement of renal SOD (C), CAT (D), GSH-Px (E) activity, and MDA levels (F) in each group of mice. * indicates p < 0.05 compared to the sham group; ** indicates p < 0.01 compared to the sham group; ns indicates no significant difference compared to the UUO + Saline group; # indicates p < 0.05 compared to the UUO + Saline group; ## indicates p < 0.01 compared to the UUO + Saline group; N = 5 Additionally, the literature indicates that OS can significantly increase the transcription of the C9 gene [[126]39]. OS is considered a critical factor in the development of RF, and antioxidants have been used to protect kidneys from damage induced by UUO. The extent of OS and the subsequent severity of RF may depend on the imbalance between the excessive production of ROS and the antioxidant defense systems within the kidney. OS results from increased ROS production and a reduction in endogenous antioxidant systems, including SOD, CAT, and GSH-Px, which protect cells from ROS-induced damage. SOD is a key enzyme that eliminates ROS by catalyzing the dismutation of superoxide radicals to H[2]O[2], which is then removed by CAT and GSH-Px [[127]40]. Previously, we characterized PMIZ-AuNPs as possessing dual catalytic activities of SOD and CAT. Therefore, we measured the activities of SOD, CAT, and GSH-Px in the left kidneys of mice across different groups. The results (Fig. [128]5C-E) demonstrated that compared to the sham group, SOD, CAT, and GSH-Px activities in the left kidneys of the UUO group were significantly decreased. Compared to the UUO + Saline group, the UUO + PMIZ-AuNPs group exhibited significantly increased SOD and CAT activities in the left kidney but did not show an improvement in the GSH-Px activity reduction caused by UUO. Additionally, we measured the levels of MDA, a marker of OS. The results (Fig. [129]5F) showed that compared to the sham group, MDA levels in the left kidneys of the UUO group were significantly increased, whereas, compared to the UUO + Saline group, the UUO + PMIZ-AuNPs group had significantly reduced MDA levels in the left kidney. In summary, these results suggest that PMIZ-AuNPs exert their therapeutic effects in mice with hydronephrosis by acting through SOD and CAT, inhibiting OS, reducing C9 expression, and preventing renal scarring via the TGF-β signaling pathway, thereby improving RF. TGF-β1 reverses the ameliorative effects of low C9 expression on cellular fibrosis and inflammation To investigate the relationship between OS, C9, RF, and inflammation, we conducted in vitro cell experiments. We constructed HK-2 cells with stable C9 knockdown using a lentiviral vector system and treated them with H[2]O[2]. After 24 h of exposure to TGF-β1, we measured the expression levels of C9 and TGF-β1 in each group using RT-qPCR and WB analyses. The results (Fig. [130]6A-B) showed that, compared to the control group, the H[2]O[2]-treated HK-2 cells exhibited significantly increased mRNA and protein levels of C9 and TGF-β1. In contrast, the H[2]O[2] + sh-C9 group showed a significant reduction in C9 and TGF-β1 expression levels compared to the H[2]O[2] + sh-NC group. Treatment with TGF-β1 did not affect the mRNA and protein levels of C9 and TGF-β1 mRNA but significantly increased TGF-β1 protein levels. Immunofluorescence staining results (Fig. [131]6C-D) indicated that H[2]O[2] treatment significantly increased the expression of α-SMA, collagen I, and fibronectin in HK-2 cells compared to the control group. However, the H[2]O[2] + sh-C9 group showed a significant decrease in the expression of these markers compared to the H[2]O[2] + sh-NC group. TGF-β1 treatment reversed the reduction in α-SMA, collagen I, and fibronectin expression levels. Fig. 6. [132]Fig. 6 [133]Open in a new tab Effects of C9 and TGF-β1 on Inflammation and RF in HK-2 Cells. Note: (A-B) RT-qPCR and WB analysis of C9 and TGF-β1 mRNA and protein expression levels in each group; (C-D) Immunofluorescence staining for α-SMA, collagen I, and fibronectin protein expression levels in each group (Scale bar = 25 μm); (E) RT-qPCR analysis of IL-8, MCP-1, TNF-α, and IL-6 mRNA expression levels in each group. * p < 0.05 vs. control group; ** p < 0.01 vs. control group; *** p < 0.001 vs. control group; ## p < 0.01 vs. H[2]O[2] + sh-NC group; ### p < 0.001 vs. H[2]O[2] + sh-NC group; ns indicates no significant difference vs. H[2]O[2] + sh-C9 group; ^ p < 0.05 vs. H[2]O[2] + sh-C9 group; ^^ p < 0.01 vs. H[2]O[2] + sh-C9 group. Experiments were repeated three times Additionally, RT-qPCR analysis of inflammatory cytokines in HK-2 cells (Fig. [134]6E) revealed that H[2]O[2] treatment significantly upregulated the mRNA levels of IL-8, MCP-1, TNF-α, and IL-6 compared to the control group. In the H[2]O[2] + sh-C9 group, these cytokine levels were significantly downregulated compared to the H[2]O[2] + sh-NC group. TGF-β1 treatment reversed the downregulation of IL-8, MCP-1, TNF-α, and IL-6. Consistent results were observed in TKPTS cells (Figure [135]S3). These findings indicate that low C9 expression can reduce fibrosis and inflammation in vitro, and TGF-β1 can reverse these ameliorative effects. Overexpression of C9 can reverse the improvements in fibrosis and inflammation in renal tubular cells treated with H[2]O[2] and PMIZ-AuNPs We further investigated the regulatory effects of PMIZ-AuNPs and C9 on RF and inflammation in H[2]O[2]-treated renal tubular cells through in vitro cell experiments. We constructed HK-2 cells with stable overexpression of C9 using a lentiviral vector system. After treatment with H[2]O[2] and PMIZ-AuNPs, the expression levels of C9 and TGF-β1 in each cell group were detected using RT-qPCR and WB. The results (Fig. [136]7A-B) indicated that, compared to the H[2]O[2] group, the H[2]O[2] + PMIZ-AuNPs group showed significantly downregulated expression levels of C9 and TGF-β1 mRNA and protein. Conversely, compared to the H[2]O[2] + PMIZ-AuNPs + oe-NC group, the H[2]O[2] + PMIZ-AuNPs + oe-C9 group exhibited significantly upregulated expression levels of C9 and TGF-β1 mRNA and protein. Immunofluorescence staining results (Fig. [137]7C-D) further demonstrated that, compared to the H[2]O[2] group, the H[2]O[2] + PMIZ-AuNPs group showed significantly reduced expression levels of α-SMA, collagen I, and fibronectin. In contrast, the H[2]O[2] + PMIZ-AuNPs + oe-C9 group showed significantly increased expression levels of α-SMA, collagen I, and fibronectin compared to the H[2]O[2] + PMIZ-AuNPs + oe-NC group. Fig. 7. [138]Fig. 7 [139]Open in a new tab Effects of C9 Overexpression and PMIZ-AuNPs on OS-Induced Fibrosis and Inflammation in HK-2 Cells. Note: (A-B) RT-qPCR and WB analysis of C9 and TGF-β1 mRNA and protein expression levels in each group; (C-D) Immunofluorescence staining of α-SMA, collagen I, and fibronectin protein expression levels in each group (Scale bar = 25 μm); (E) RT-qPCR analysis of IL-8, MCP-1, TNF-α, and IL-6 mRNA expression levels in each group. **p < 0.01 vs. H[2]O[2] group; ***p < 0.001 vs. H[2]O[2] group; #p < 0.05 vs. H[2]O[2] + PMIZ-AuNPs + oe-NC group; ##p < 0.01 vs. H[2]O[2] + PMIZ-AuNPs + oe-NC group; ###p < 0.001 vs. H[2]O[2] + sh-NC group; N = 5 Additionally, RT-qPCR was used to measure the expression of inflammatory factors in each group of HK-2 cells. The results (Fig. [140]7E) indicated that, compared to the H[2]O[2] group, the H[2]O[2] + PMIZ-AuNPs group exhibited significantly decreased mRNA levels of IL-8, MCP-1, TNF-α, and IL-6. However, compared to the H[2]O[2] + PMIZ-AuNPs + oe-NC group, the H[2]O[2] + PMIZ-AuNPs + oe-C9 group showed significantly increased mRNA levels of IL-8, MCP-1, TNF-α, and IL-6. Similar trends were observed in TKPTS cells (Figure [141]S4). These results suggest that PMIZ-AuNPs can ameliorate OS-induced fibrosis and inflammation in vitro, while overexpression of C9 can reverse these improvements. Overexpression of C9 reverses the therapeutic effects of PMIZ-AuNPs on mouse hydronephrosis To investigate the regulatory effects of PMIZ-AuNPs and C9 on RF and inflammation in UUO mice, we conducted in vivo experiments. Firstly, to explore the in vivo safety of PMIZ-AuNPs, we measured blood WBC, RBC, PLT counts (Figure [142]S5A) and serum biochemical parameters (Albumin, Amylase, Alkaline phosphatase, Blood Urea Nitrogen, Calcium, Creatinine, Globulin, Glucose, Potassium, Sodium, Phosphorus, Total bilirubin, Total protein) (Figure [143]S5B) in Saline and PMIZ-AuNPs groups at 14 days after injection. We found no significant differences between the two groups, indicating that PMIZ-AuNPs are safe for in vivo use. Mice were intravenously injected with AAV carrying either oe-NC or oe-C9. Two weeks post-injection, a UUO mouse model was established, followed by tail vein injections of PMIZ-AuNPs (Figure [144]S5C). Ultrasonography performed on days 7 and 14 post-UUO revealed that, compared to the UUO + PMIZ-AuNPs + oe-NC group, the UUO + PMIZ-AuNPs + oe-C9 group exhibited increased hydronephrosis grades (Fig. [145]8A). After 14 days, mice were euthanized, and the expression levels of C9 and TGF-β1 mRNA and proteins in renal tissues were analyzed using RT-qPCR and WB. Results showed significant upregulation of these markers in the UUO + PMIZ-AuNPs + oe-C9 group compared to the UUO + PMIZ-AuNPs + oe-NC group (Fig. [146]8B-C). Fig. 8. [147]Fig. 8 [148]Open in a new tab Effect of C9 Overexpression on PMIZ-AuNPs Treatment in Mice with Hydronephrosis. Note: (A) Ultrasound imaging and hydronephrosis grading of mice in each group at 7 and 14 days post-modeling; (B-C) RT-qPCR and WB analysis of C9 and TGF-β1 mRNA and protein expression levels in renal tissues of each group; (D-E) H&E staining to assess renal tissue damage in each group (Scale bar = 50 μm); (F) Masson’s trichrome staining to evaluate the fibrotic area in renal tissues of each group (Scale bar = 50 μm); (G) Sirius Red staining to determine the percentage of collagen in the renal cortex tubulointerstitium of each group (Scale bar = 50 μm); (H-I) IHC and WB analysis of α-SMA protein expression, a marker of myofibroblast differentiation, in renal tissues of mice (Scale bar = 50 μm); *p < 0.05, **p < 0.01, ***p < 0.001 compared to the UUO + PMIZ-AuNPs + oe-NC group; N = 5 H&E staining indicated increased infiltration of inflammatory cells and higher tubular interstitial injury scores in the UUO + PMIZ-AuNPs + oe-C9 group compared to the control (Fig. [149]8D-E). Masson’s Trichrome staining demonstrated a significant increase in RIF in the UUO + PMIZ-AuNPs + oe-C9 group (Fig. [150]8F). Consistently, Sirius Red staining also showed elevated collagen deposition in the renal interstitium of this group (Fig. [151]8G). IHC and WB analyses revealed a significant increase in α-SMA protein levels in the renal tissues of the UUO + PMIZ-AuNPs + oe-C9 group compared to the control (Fig. [152]8H-I, Figure [153]S6A). Furthermore, WB results indicated significant upregulation of collagen I, collagen III, and fibronectin proteins in the UUO + PMIZ-AuNPs + oe-C9 group (Figure [154]S6B). Oxidative stress arises from increased ROS generation and reduced endogenous antioxidant systems (including SOD, CAT, and GSH-Px), which protect cells from ROS-induced damage. SOD is a key enzyme that eliminates ROS by catalyzing the dismutation of superoxide radicals into H[2]O[2], which is then removed by CAT and GSH-Px [[155]40]. As previously characterized, PMIZ-AuNPs exhibit dual catalytic activities of SOD and catalase. Thus, we measured the activities of SOD, CAT, and GSH-Px in the left kidneys of mice. The results showed (Figure [156]S6C-E) that compared to the UUO + PMIZ-AuNPs + oe-NC group, the UUO + PMIZ-AuNPs + oe-C9 group showed significantly increased SOD and CAT activities in the left kidney; however, it did not improve the reduction of GSH-Px activity caused by UUO. Additionally, we measured MDA, a marker of oxidative stress, and found (Figure [157]S6F) that compared to the UUO + PMIZ-AuNPs + oe-NC group, the UUO + PMIZ-AuNPs + oe-C9 group had significantly reduced MDA levels in the left kidney. Discussion CH is a common congenital urinary system disease, often leading to RF, significantly impairing renal function [[158]1, [159]41, [160]42]. Previous studies have demonstrated that OS plays a crucial role in the development of RF [[161]43]. The inflammatory response and apoptosis induced by OS are key pathological mechanisms of RF [[162]44, [163]45]. Complement component C9, an essential part of the complement system, has recently attracted attention for its role in RF [[164]46, [165]47, [166]48]. However, current treatments for RF are limited and lack innovative methods for simultaneous diagnosis and treatment [[167]48, [168]49]. Treatment methods for congenital hydronephrosis and renal fibrosis include drug therapy, surgical intervention, and lifestyle adjustments. Congenital hydronephrosis is typically caused by urinary tract obstruction, treated with antibiotics to prevent infection, surgical procedures like pyeloplasty, and minimally invasive surgery (e.g., percutaneous nephrolithotomy) [[169]50, [170]51]. Renal fibrosis, a common pathological change in chronic kidney disease, is mainly treated with medications (e.g., ACEI/ARB, anti-inflammatory, and anti-fibrotic drugs), lifestyle changes (e.g., low-salt, low-protein diet, blood pressure, and blood sugar control), and dialysis or kidney transplantation for end-stage patients. Treatment plans need to be tailored to the patient’s specific condition, with regular follow-up and monitoring of disease progression [[171]52]. Due to their recognized anti-inflammatory and antioxidant properties, AuNPs have been emphasized as promising treatments for various inflammations, with specific advantages including: surface modifications that enable targeted delivery to kidney lesions, reducing damage to normal tissues [[172]53]; dual therapeutic and diagnostic functions for drug delivery, imaging, and photothermal therapy; good biocompatibility and low toxicity with proper modifications; and controlled drug release to reduce side effects and improve efficacy [[173]54, [174]55]. However, limitations of AuNPs include potential cell toxicity and immune responses with long-term use; possible accumulation in kidneys due to size and surface properties, affecting kidney function; challenges in large-scale production and cost; and the need for further studies to assess the long-term safety and effectiveness of AuNPs in treating kidney diseases [[175]56, [176]57]. This study aims to design a novel multifunctional targeted nanozyme, PMIZ-AuNPs, which can serve as both an UCA for real-time monitoring and a therapeutic agent for RF. In this study, we developed a responsive nanozyme, PMIZ-AuNPs, incorporating PEG-SH and imidazole groups to exhibit aggregation and enhanced ultrasound contrast under acidic conditions. TEM confirmed the appearance and size of the PMIZ-AuNPs, showing a uniform nanoscale distribution. Spectral properties recorded by spectrophotometry revealed absorption peaks at specific wavelengths, demonstrating excellent optical properties. Additionally, DLS measurements showed changes in zeta potential and HD of the nanozyme under different pH conditions, further indicating its responsiveness to acidic environments. The dual catalytic activities of SOD and CAT in PMIZ-AuNPs were verified using ESR spectroscopy, underscoring its potential as a multifunctional therapeutic tool. To verify the therapeutic effect of PMIZ-AuNPs, we established a UUO mouse model. The experimental results demonstrated that PMIZ-AuNPs accumulated at the site of kidney injury in UUO mice. Significant signal enhancement was observed through ultrasound imaging, indicating that the aggregation properties of PMIZ-AuNPs under acidic conditions significantly enhance the ultrasound contrast. Moreover, the injection of PMIZ-AuNPs notably improved hydronephrosis and RF in UUO mice. Compared to traditional treatments, PMIZ-AuNPs not only provide real-time monitoring capabilities but also significantly reduce the occurrence of RF through multi-functional catalytic actions. These findings suggest the potential of PMIZ-AuNPs in the treatment of RF. Through protein sequencing and bioinformatics analysis of the GEO database, we identified the characteristic gene/protein C9. C9 plays a critical role in the complement system, although its specific function in RF remains unclear. However, this study found that the expression of C9 significantly increased in the UUO mouse model, while PMIZ-AuNPs treatment markedly decreased C9 expression. This finding aligns with previous studies reporting the role of C9 in inflammation and fibrosis. Further cell experiments and in vivo mouse studies confirmed that PMIZ-AuNPs alleviate RF and inflammatory responses by inhibiting OS and reducing C9 expression. This study thoroughly investigates the mechanism of PMIZ-AuNPs. Experimental results indicate that PMIZ-AuNPs aggregate in acidic environments, enhancing ultrasound contrast signals. Additionally, the dual catalytic activity of SOD and CAT significantly reduces OS levels. This reduction in OS subsequently lowers C9 expression through the TGF-β signaling pathway, a crucial regulator of RF. By inhibiting fibrosis markers through this pathway, PMIZ-AuNPs offer a novel therapeutic approach for RF, demonstrating superior efficacy compared to traditional antioxidant treatments. In cellular experiments, we validated the mechanism in vitro using mouse and human proximal tubular cells (TKPTS and HK-2 cells). Results showed that PMIZ-AuNPs significantly inhibited H[2]O[2]-induced cell fibrosis and inflammation. RT-qPCR and WB analyses revealed a marked reduction in the expression of the characteristic gene C9 and inflammatory genes. Immunofluorescence staining further confirmed the decrease in fibrosis marker proteins in treated cells. In vivo mouse experiments, including H&E Staining, Masson’s Trichrome Staining, Sirius Red Staining, and IHC, demonstrated that PMIZ-AuNPs significantly alleviated RF in mouse kidney tissues. Overall, both in vitro and in vivo results consistently indicated that PMIZ-AuNPs effectively ameliorate CH and RF through their multifunctional activities. This study successfully designed and validated a novel multifunctional targeted nanozyme, PMIZ-AuNPs. PMIZ-AuNPs is a pH-responsive nanozyme, primarily composed of PEG-SH, imidazole, and AuNPs. Due to their recognized anti-inflammatory and antioxidant properties, AuNPs have been highlighted as promising therapeutic strategies for various inflammations, including rheumatoid arthritis, sepsis, and asthma. Moreover, AuNPs have shown beneficial effects on the development of kidney injury observed in subAKI [[177]53]. Functionalization with PEG-SH significantly improves the long-term colloidal stability of AuNPs [[178]58]. Compared to unmodified G5 conjugates, the use of PEG-imidazole-modified G5 as a platform for conjugation was most effective [[179]59], suggesting that PEG-imidazole-modified AuNPs can enhance their bioavailability. PMIZ-AuNPs can serve as both ultrasound contrast agents for real-time monitoring and as therapeutic agents, significantly improving congenital hydronephrosis renal fibrosis through dual catalytic activities of SOD and catalase. PMIZ-AuNPs exert SOD and catalase activities, reducing C9 expression by inhibiting oxidative stress and decreasing the occurrence of renal fibrosis through the TGF-β signaling pathway, demonstrating good therapeutic effects and potential clinical application value. However, this study still has some limitations, such as a limited sample size and the need for long-term effect observation. Future research will focus on further optimizing the nanozyme design, improving its biosafety and targeting capabilities, expanding the sample size, and verifying its potential applications in other kidney diseases. Through continuous in-depth research, we look forward to PMIZ-AuNPs bringing new breakthroughs in the diagnosis and treatment of renal fibrosis. Conclusion Based on the results, we can preliminarily conclude the following: PMIZ-AuNPs exhibit SOD and CAT activity, reduce C9 expression by inhibiting OS, and subsequently improve RF in CH through the TGF-β signaling pathway (Fig. [180]9). Although this study proposes new therapeutic approaches and molecular targets for CH, several limitations exist. Firstly, the research is primarily based on cell and animal models, which, despite providing initial mechanistic insights and therapeutic validation, may not fully represent the conditions in human patients. Further collection and analysis of clinical samples are required to validate the efficacy and safety of PMIZ-AuNPs in CH patients. Clinical sample validation could include pathological analysis of tissue samples and biomarker detection in blood and urine. Secondly, although our study found that PMIZ-AuNPs reduce C9 expression by inhibiting OS and subsequently improve RF via the TGF-β signaling pathway, the specific regulatory mechanisms remain unclear. Further research is needed to elucidate the interactions between C9 and the TGF-β signaling pathway, including the precise role of C9 in TGF-β signal transduction, potential direct or indirect regulatory relationships, and related molecular mechanisms. Lastly, this study mainly focuses on the short-term therapeutic effects of PMIZ-AuNPs and lacks an assessment of their long-term efficacy and safety. Long-term animal studies and clinical trials are necessary to observe the effects and potential side effects of prolonged PMIZ-AuNPs usage, ensuring their feasibility and safety as therapeutic agents. Fig. 9. [181]Fig. 9 [182]Open in a new tab Schematic Diagram of the Molecular Mechanisms by which Multifunctional Targeted Nanoenzyme PMIZ-AuNPs Treat CH RF Methods Preparation of gold nanoparticles (AuNPs) A 0.1 mM gold precursor solution was prepared by adding 1 mL of 0.01 M HAuCl[4] solution (484385, Sigma) to 100 mL of deionized water. Under continuous stirring, 1 mL of 0.1 M NaBH[4] solution (215511, Sigma) was rapidly added to the gold precursor solution, reducing HAuCl[4] and forming Au nanoparticle seeds. Immediately afterward, 1 mL of 1% sodium citrate solution (S5770, Sigma) was added to stabilize the Au nanoparticle seeds and prevent aggregation. The 0.1 M HAuCl4 solution and 1% sodium citrate solution were then mixed in a 1:10 ratio to prepare the AuNPs growth solution. The prepared AuNPs seed solution was added to the growth solution, stirred, and reacted at room temperature for 1 min to form small-sized AuNPs. To prepare the PAH solution, polyallylamine hydrochloride (PAH, 283215, Sigma), with an average molecular weight of 17,500 g/mol, was dissolved in Milli-Q water. The pH of the PAH solution was adjusted to 9 using 0.1 M NaOH standard solution (109137, Sigma). Silicon wafers (5 × 5 mm) with a SiO[2] oxide layer were exposed to the PAH solution for 30 min to form a PAH coating. The silicon wafers were rinsed three times with Milli-Q water to remove any unbound or loosely bound PAH molecules. The AuNPs solution was then dropped onto the PAH-coated silicon wafers and allowed to dry. The silicon wafers were rinsed three times with Milli-Q water to remove any unbound or loosely bound AuNPs. The Au/SiO[2] samples were heat-treated in a nitrogen atmosphere to remove the organic citrate ligands from the surface of the AuNPs. The heat treatment was conducted at an appropriate temperature of 250 °C for 2 h. After treatment, the samples were quickly removed from the furnace and cooled in air. A field-emission scanning electron microscope (FE-SEM, Zeiss) was used at an accelerating voltage of 3 kV and a working distance of 5.5 mm to analyze the morphology of the samples. SEM images were analyzed using ImageJ software to assess the morphological changes of the AuNPs post-heat treatment. Synthesis of PMIZ-AuNPs and GS-AuNPs PMIZ-AuNPs were synthesized by mixing AuNPs, PEG-SH (729108, Sigma), MSA-SH (M6182, Sigma), and deionized water, stirring the mixture at 95 °C for 18 h. After dialysis purification, 4 mL of NHS (1.0 M, 130672, Sigma), 4 mL of EDC (1.0 M, 39391, Sigma), 4 mL of MIZ-H (1.0 M, Y0001779, Sigma), and 5 mL of PB buffer (1 M, pH 6.0) were added to 50 mL of the solution. This mixture was slowly agitated at room temperature for 12 h. The resulting PMIZ-AuNPs were purified by centrifugation at 21,000 g and stored at 4 °C. For the synthesis of GS-AuNPs (control), AuNPs, GSH (Y0000517, Sigma), and deionized water were mixed, stirring the mixture at 95 °C in an oil bath for 0.5 h. After ethanol precipitation purification, the GS-AuNPs were obtained and stored at 4 °C. Nanocharacterization For TEM imaging, 5 µL of PMIZ-AuNPs and GS-AuNPs were added to a copper grid and dried in the air. High-resolution TEM images were captured using a JEOL JEM 2100 F TEM (Japan) at an accelerating voltage of 200 kV. The emission spectra were determined using an LS-55 spectrofluorometer (PerkinElmer, USA), and the absorption spectra were measured using a UV-Vis spectrophotometer (UV2600, Shimadzu, Japan). The hydrodynamic diameter (HD) of the nanoparticles was measured in PB buffer (200 mM), and the zeta potential (ζ-potential) was detected in PB buffer (20 mM). The average HD distribution and ζ-potential of the particles were measured using a Zeta-Potential & Particle Analyzer (ELSZ-2000, Osaka, Japan). Ultrasound signals of AuNPs Ultrasound Signals of AuNPs at Different pH Levels: AuNPs (100.0 mg/mL, with gold atom concentration measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)) were dissolved in PB buffer solutions (1 M) with different pH levels (e.g., 7.4, 6.9, 6.5, 6.0, 5.5, 5.0, and 4.0) in centrifuge tubes. Ultrasound images were acquired using a small animal ultrasound system (Vevo 2100, FUJIFILM VisualSonics, Inc, Toronto, ON, Canada) with a 30 MHz ultrasound array probe in B-mode. The transmission power and receiver gain were set to 100% and 30 dB, respectively. Ultrasound Signals of PMIZ-AuNPs in Cells: HK-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium (30-2002, ATCC) containing 10% (v/v) fetal bovine serum (FBS, 164210, Procell) at 37 °C, 5% CO[2], and a humidified environment. 1 × 10^7 HK-2 cells were seeded into 100 mm culture dishes and incubated under standard conditions for 24 h. The medium was then replaced with 5.0 mL of fresh medium containing PMIZ-AuNPs (3.0 mg/mL, pH 6.5). After incubation at 37 °C for 12 h, the pH of the solution was changed to 5.5. The cells were incubated for an additional 30 min, the PMIZ-AuNPs were removed, and the cells were washed three times with DPBS (pH 5.5, D5773, Sigma). Paraformaldehyde (PFA) (818715, Sigma) was then added to the culture dishes to fix the cells for 5 min, and the cell pellets were collected using a cell scraper and redistributed in 330 µL of fresh PFA. Ultrasound images of cells containing PMIZ-AuNPs were then acquired using a small animal ultrasound system with a 30 MHz ultrasound array probe in B-mode, with the transmission power and receiver gain set to 100% and 20 dB, respectively. A similar procedure was followed to acquire ultrasound images of HK-2 cells without PMIZ-AuNPs and pure PMIZ-AuNPs (3.0 mg/mL). ESR All ESR measurements were conducted at room temperature using a Bruker EMX ESR spectrometer (Bruker). 50 µL of control or sample solution was placed in a glass capillary tube with an inner diameter of 1 mm and sealed. The capillary tube was then inserted into the ESR cavity, and spectra were recorded at designated times. The settings were as follows: for detecting spin adducts using spin traps, the magnetic field modulation was set at 1 G, the scan range was 100 G, and the microwave power was 20 mW; for ESR oximetry with the spin label 5-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (CTPO), the magnetic field modulation was 0.04 G, the scan range was 5 G, and the microwave power was 1 mW. To verify the formation of hydroxyl radicals (·OH) during H[2]O[2] degradation in the presence of AuNPs under various conditions, spin traps 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), and α-(4-Pyridyl 1-oxide)-N-tert-butylnitrone (POBN) were used. The number of ·OH was quantified by measuring the ESR signal intensity of the hydroxyl radical spin adduct (DMPO/OH), specifically using the peak height of the second ESR spectral line. H[2]O[2] was mixed with DMPO in buffers of different pH values, and the reaction was initiated by adding AuNPs. ESR spin labeling was employed to quantitatively measure oxygen content using the water-soluble spin label CTPO. Its ESR spectrum featured hyperfine structures sensitive to oxygen molecules, allowing precise monitoring of O[2] concentration. The sample mixture included CTPO, H[2]O[2], and buffer with or without AuNPs. It was deoxygenated by purging with N[2]. To verify the ability of AuNPs to scavenge superoxide anions, xanthine and xanthine oxidase (XOD) were mixed in pH 5.5 Phosphate-Buffered Saline (PBS) buffer to generate superoxide anions and capture them with BMPO, forming the spin adduct BMPO/OOH. The control sample contained 25 mM BMPO, 0.05 mM DTPA, 1 mM xanthine, and 0.2 U/mL XOD in 10 mM pH 5.5 PBS buffer. SOD or AuNPs were added to scavenge the radicals. The reaction was initiated by adding XOD, and ESR spectra were recorded at specified intervals. Animal sources and ethical statement Two-day-old SPF-grade healthy C57BL/6J mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. All mice were housed in environmentally controlled, pathogen-free barrier facilities with a 12-hour light/dark cycle, a temperature of 24 ± 1 °C, and humidity of 50 ± 10%. Food and water were provided ad libitum. All animal experiments in this study adhered to the guidelines and regulations of our Institutional Animal Care and Use Committee (IACUC) and received appropriate approval. Efforts were made to minimize the pain and discomfort of the animals and reduce the number of animals used in experiments whenever possible. The housing, handling, and experimental procedures for the animals strictly complied with internationally recognized standards of animal welfare. Appropriate care was provided for all animals, and their proper disposition was ensured at the end of the experiments. Establishment and treatment of mouse hydronephrosis model Newborn mice underwent either a complete left-sided UUO or a sham operation within 48 h after birth. Under general anesthesia with isoflurane and oxygen, a left-side incision exposed the ureter, where two 6 − 0 sutures were placed 2 mm apart on the distal ureter without cutting it, or left untreated for the sham operation. The incision was closed with a single-layer suture and sealed with adhesive. After recovering from anesthesia, the mice were returned to their mothers. The pH value of the urine was measured daily. On day 7 post-modeling, 200 µL of PMIZ-AuNPs (10 mg/mL) was administered via intravenous injection. The hydronephrosis and Color Doppler Flow Imaging (CDFI) grading were evaluated by measuring the renal pelvis volume using Vevo 700 ultrasound on days 7 and 14 post-modeling, as detailed in Table [183]S1. Mice were euthanized with a lethal dose of pentobarbital, and kidneys were collected [[184]60, [185]27]. Before inducing the mouse model, we injected mice with adeno-associated viruses (AAV) carrying either oe-NC or oe-C9 through the tail vein. The injection volume was 100 µL, with a viral titer of 1 × 10¹¹ GC/mL, using the AAV9 vector (from HanBio) as the overexpression vector. Two weeks post-injection, the overexpression efficiency was evaluated using RT-qPCR and WB analysis (Figure [186]S7). Mouse Grouping (n = 5): Sham group (sham operation), UUO group (left UUO surgery), UUO + Saline group (UUO surgery followed by intravenous injection of saline on day 7), UUO + PMIZ-AuNPs group (UUO surgery followed by intravenous injection of PMIZ-AuNPs on day 7), UUO + PMIZ-AuNPs + oe-NC group (UUO surgery following oe-NC lentivirus infection, with intravenous injection of PMIZ-AuNPs on day 7), and UUO + PMIZ-AuNPs + oe-C9 group (UUO surgery following oe-C9 lentivirus infection, with intravenous injection of PMIZ-AuNPs on day 7). In vivo safety assessment of PMIZ-AuNPs Mouse groups (n = 5): Saline group (normal healthy mice + intravenous injection of saline); PMIZ-AuNPs group (normal healthy mice + intravenous injection of 200 µL PMIZ-AuNPs (10 mg/mL)). Blood was collected via submandibular puncture into Microvette 100 K2-EDTA collection tubes (Sarstedt) for cell counting before sacrificing the mice at 14 days after modeling. For serum biochemical analysis, blood was collected without anticoagulant, allowed to clot for at least 30 min, and then centrifuged at 3000 g for 10 min to obtain serum. Histopathological analysis The extent of kidney tissue damage and fibrosis was evaluated. After euthanizing the mice, the kidneys were extracted and fixed in 4% PFA at 4 °C for 24 h. Subsequently, the kidney tissues were embedded in OCT Tissue-Tek (Sakura, USA) and sectioned continuously at a thickness of 4 μm. The kidney tissue sections were stained using H&E staining kits (G1120, Solarbio) and Masson’s trichrome staining was performed using kits (G1340, Solarbio). Additionally, modified Sirius Red staining kits (G1472, Solarbio) were applied to the tissue sections. Following the standard procedures for Masson’s trichrome staining, Image-Pro Plus (IPP) software version 6.0 (Media Cybernetics, Rockville, MD, USA) was used to analyze 10 fields per section at 200x magnification, quantifying the percentage of blue-stained collagen areas. For evaluating interstitial fibrosis using Sirius Red staining, collagen fibers were identified as red under a light microscope and as birefringent under polarized light. Using IPP software, 10 fields per section at 200x magnification were analyzed to assess the percentage of birefringent area relative to the total area. All these semi-quantitative analyses were performed in a blinded manner. IHC Paraffin-embedded kidney tissue sections from mice were prepared and deparaffinized to water, followed by dehydration in an ethanol gradient. Antigen retrieval was performed in a hot water bath with antigen retrieval solution, and the sections were then cooled with tap water. Normal goat serum blocking solution (Catalog No. C-0005, Shanghai Haoran Biological Technology Co., China) was applied to the sections at room temperature for 20 min, and the excess liquid was removed from the slides. Primary antibodies were then added to the sections: Anti-α-SMA antibody (ab124964, 1:1000, Abcam, UK), Anti-C9 antibody (mouse, AA 136–512, 5 µg/mL, Antikoerper-Online), and Anti-C9 antibody (human, AA 22–265, 5 µg/mL, Antikoerper-Online), and incubation was done overnight at 4 °C. The sections were washed three times in 0.1 M PBS, with 5 min for each wash. The sections were then incubated with a secondary antibody, goat anti-rabbit IgG (ab6721, 1:2000, Abcam, UK). An optical microscope (IX53, Thermo Fisher) was used to capture images and quantify positive cells. For each section, 10 fields at 400x magnification were randomly selected for cell counting. The percentage of SMA-positive areas in 10 fields per section at 200x magnification was measured using IPP software. Determination of antioxidant enzyme activity and malondialdehyde (MDA) levels in kidneys The activities of SOD, CAT, and Glutathione Peroxidase (GSH-Px), as well as the levels of MDA, were measured in the left kidneys on the 14th day after UUO. Each kidney sample was quickly weighed, homogenized, and centrifuged. The supernatant was then analyzed using commercial assay kits: Total SOD (T-SOD) Assay Kit (A001-1-2, Nanjing Jiancheng Bioengineering Institute, China), CAT Assay Kit (A007-1-1, Nanjing Jiancheng Bioengineering Institute, China), GSH-Px Assay Kit (A005-1-2, Nanjing Jiancheng Bioengineering Institute, China), and MDA Assay Kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute, China). Protein sample preparation and analysis Kidney samples from the UUO + Saline group and the UUO + PMIZ-AuNPs group (n = 3) were ground in liquid nitrogen, and the resulting powder was transferred into 5 cm³ centrifuge tubes. The samples were then subjected to ultrasonic treatment in an ice bath using a Scientz-IID ultrasonic cell disruptor (Scientz, Ningbo, China). The extraction buffer used contained 10 mM Dithiothreitol (DTT) (R0861, Solarbio, Beijing, China), 1% protease inhibitor mixture (P6731, Solarbio, Beijing, China), and 2 mM EDTA (E1170, Solarbio, Beijing, China) in phenol (100206, Merck, USA). This process was repeated eight times. Next, an equal volume of pH 8.0 Tris-saturated phenol (HC1380, BIOFOUNT, Beijing, China) was added, and the mixture was vortexed for 4 min. The samples were centrifuged at 5000×g for 10 min at 4 °C, and the upper phenol layer was transferred to new centrifuge tubes. To the phenol solution, 0.1 M ammonium sulfate (101217, Merck, USA) and saturated methanol (106035, Merck, USA) were added in a 1:5 volume ratio and left overnight to precipitate the proteins. Following this, the samples were centrifuged at 4 °C for 10 min, and the supernatant was discarded. The remaining pellet was washed once with cold methanol and three times with cold acetone. The washed protein pellet was then dissolved in 8 M urea (U8020, Solarbio, Beijing, China), and the protein concentration was determined using a BCA kit (P0012, Beyotime). All procedures were performed according to the manufacturer’s instructions. Proteolysis, peptide labeling, fractionation, and nano-LC-MS/MS analysis Each sample (50 µg) was subjected to proteolysis. The protein solution was mixed with DTT to a final concentration of 5 mM and incubated at 56 °C for 30 min. Subsequently, iodoacetamide was added to a final concentration of 11 mM, and the mixture was incubated at room temperature for 15 min. The urea concentration of the samples was then diluted to less than 2 M, and trypsin (25200056, Thermo Fisher Scientific, USA) was added at a ratio of 1:50 (w/w) for overnight digestion at 37 °C. An additional aliquot of trypsin was added at a ratio of 1:100 (trypsin) to continue digestion for another 4 h. Post-digestion, peptides were desalted using HyperSep™ C18 purification columns (60108-302, Thermo Fisher Scientific, USA) and vacuum-dried. The peptides were then reconstituted in 0.5 M TEAB (90114, Thermo Fisher Scientific, USA) and processed according to the TMT kit (90064CH, Thermo Fisher Scientific, USA) manufacturer’s instructions. Briefly, a unit of TMT reagent was reconstituted in acetonitrile (113212, Merck, USA) and incubated with the peptide mixture at room temperature for 2 h. The labeled peptides were desalted and vacuum-dried again using a vacuum centrifuge. Each set of labeled peptides was then equally mixed and subjected to high-pH reverse-phase peptide fractionation using the Pierce™ kit (84868, Thermo Fisher Scientific, USA). The samples were combined into 15 fractions, dried, and reconstituted in 0.1% formic acid (159002, Merck, USA). For nano-LC-MS/MS analysis, 2 µg of peptides from each sample were separated using an Easy-nLC 1200 nano-UPLC system (Thermo Fisher Scientific, USA). The samples were first loaded onto a Trap C18 column (100 μm × 20 mm, 5 μm) and then separated on an analytical C18 column (75 μm × 150 mm, 3 μm) at a flow rate of 300 nL/min. The mobile phase A was 0.1% formic acid in water, and the mobile phase B was 0.1% formic acid in acetonitrile containing 95% acetonitrile. The gradient elution program was as follows: 0 to 2 min, 2–8% B; 2 to 71 min, 8–28% B; 71 to 79 min, 28–40% B; 79 to 81 min, 40–100% B; and 81 to 90 min, 100% B. The peptides were analyzed using a Q-Exactive HFX mass spectrometer (Thermo Fisher Scientific, USA) in positive ion mode. The ESI voltage was set to 2.1 kV, and the mass spectrometer was operated with a full MS scan range of 350–1200 m/z, a resolution of 60,000 at m/z 200, an AGC target of 3e^6, and a maximum IT of 30 ms for MS1. The MS2 spectra were acquired at a resolution of 15,000 at m/z 200, with an AGC target of 1e^6, a maximum IT of 25 ms, an MS2 activation type of HCD, an isolation window of 20 Th, and a normalized collision energy of 32. Database search and data processing The Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) data were processed using MaxQuant software (v.1.5.2.8) for peptide identification and protein quantification. Tandem mass spectrometry searches were conducted using the UniProt 14.1 (2009) database for Gossypium hirsutum and a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, allowing for up to two missed cleavages. The initial search was set at 20 ppm, with the main search at 5 ppm and a fragment ion mass tolerance of 0.02 Da. The search criteria included a peptide false discovery rate (FDR) ≤ 0.01, a protein FDR ≤ 0.01, and peptide score distribution. Differentially expressed proteins (DEPs) between the UUO + Saline and UUO + PMIZ-AuNPs groups were identified using the R software package “Limma” with a threshold of|log[2]FC| >2 and p-value < 0.05. Construction of protein-protein interaction (PPI) networks The DEPs were analyzed for PPI using the STRING database ([187]http://www.string-db.org/), with the species limited to mice and a confidence score threshold set at 0.4. The R software was then used to calculate the number of adjacent nodes for each protein, and the top 30 proteins with the highest number of adjacent nodes were selected to create a bar chart. GEO data chip source and analysis The UUO mouse dataset [188]GSE96101, which includes kidney tissue data from UUO and sham mice (n = 60), was obtained from the NCBI database ([189]https://www.ncbi.nlm.nih.gov/). Differentially expressed genes (DEGs) between sham and UUO samples were identified using the “Limma” package in R, with|log[2]FC|>0 and p-value < 0.05 as the threshold. Heatmaps of gene expression differences were generated using the “heatmap” package in R, and volcano plots of the DEGs were created using the “ggplot2” package. Machine learning for screening hydronephrosis signature genes Based on the GEO dataset [190]GSE96101, the R package “glmnet” was utilized to perform the Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression analysis. Binomial logistic regression was employed to select signature genes from DEGs, with the optimal penalty parameter λ determined by the minimum binomial deviation. For the Support Vector Machine-Recursive Feature Elimination (SVM-RFE) algorithm, the R package “e1071” was used to identify the best variables, and the “kernlab” and “caret” packages were used to select the minimum cross-validation error. The intersection of genes identified by these two algorithms and the 30 candidate proteins was used to screen for diagnostic biomarkers of hydronephrosis. ROC curve analysis The dataset was analyzed using the “pROC” package in R to plot Receiver Operating Characteristic (ROC) curves. The Area Under the Curve (AUC) was calculated to evaluate the predictive utility of the identified biomarkers. Gene function enrichment analysis Matrix correlation calculation tools were utilized to analyze the correlation between C9 and expression matrix genes, with co-expressed genes selected using a threshold of R ≥|0.5| and p < 0.001. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on C9 and its co-expressed genes using the “clusterProfiler” package in R. The “enrichplot” package was employed to generate bubble plots for the enrichment results, specifically focusing on the GO categories of Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), as well as the KEGG enrichment results. In vitro cell culture Mouse proximal tubular cells TKPTS (CRL-3361) were purchased from ATCC and cultured in F-12 Medium (30-2006, ATCC) supplemented with 7% FBS (FBS, 30-2020, ATCC) and 0.06% insulin (10 mg/mL, 19278, Sigma). HK-2 (CRL-2190) were also obtained from ATCC and cultured in Keratinocyte Serum-Free Medium (K-SFM kit, 17005-042, ATCC) containing 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL epidermal growth factor (EGF). All cells were maintained at 37 °C in a humidified atmosphere of 5% CO[2] and 95% air [[191]61]. Cell treatment and grouping Lentiviral particles containing sh-NC, sh-C9, oe-NC, and oe-C9 were packaged into HEK-293T cells (CRL-11268, ATCC). The pLenti6/V5-DEST™ Gateway™ vector (V49610, ThermoFisher) was used as the overexpression vector, and the pLenti6/BLOCK-iT™-DEST vector (K494300, ThermoFisher) was used as the knockdown vector. After 48 h, Genechem (Shanghai, China) collected and concentrated the viral supernatant. TKPTS and HK-2 cells were cultured to approximately 50% confluence and then infected with the lentivirus at a multiplicity of infection (MOI) of 5. After 48 h of infection, 10 µg/mL puromycin (HY-K1057, MCE) was used for selection, maintaining the selection for at least one week to establish stable cell lines. The shRNA target sequences are listed in Table [192]S2. The efficiency of infection was evaluated by RT-qPCR and WB after 48 h (Figure [193]S8). After C9 knockdown, C9 expression was significantly reduced in both TKPTS and HK-2 cells, with sh-C9-1 and sh-C9-4 showing the best knockdown efficiency for subsequent experiments. For cell treatment (n = 5), 20 µM H[2]O[2] was added to induce OS for 0.5 h (Figure [194]S9 confirms the establishment of the OS model), and 3.0 mg/mL PMIZ-AuNPs were added, or cells were exposed to TGF-β1 (10 ng/mL) for 24 h. The cell groups were as follows: control group (untreated cells), H[2]O[2] group (cells treated with H[2]O[2]), H[2]O[2] + sh-NC group (H[2]O[2]-treated cells infected with sh-NC lentivirus), H[2]O[2] + sh-C9 group (H[2]O[2]-treated cells infected with sh-C9 lentivirus), H[2]O[2] + sh-C9 + TGF-β1 group (sh-C9 lentivirus-infected cells treated with H[2]O[2] and exposed to TGF-β1), H[2]O[2] + PMIZ-AuNPs group (cells treated with H[2]O[2] and PMIZ-AuNPs), H[2]O[2] + PMIZ-AuNPs + oe-NC group (cells treated with H[2]O[2] and PMIZ-AuNPs infected with oe-NC lentivirus), and H[2]O[2] + PMIZ-AuNPs + oe-C9 group (cells treated with H[2]O[2] and PMIZ-AuNPs infected with oe-C9 lentivirus). Cell immunofluorescence assay Cells were washed twice with PBS to remove any residual medium and then fixed with 4% PFA for 30 min. Following fixation, the cells were incubated in 0.3% Triton X-100 for 15 min. They were then blocked with 5% bovine serum albumin (BSA) in PBS at 37 °C for 1 h to prevent non-specific antibody binding. Subsequently, the cells were incubated overnight at 4 °C with specific primary antibodies. After three washes with PBS, the cells were incubated with secondary antibodies in the dark at 37 °C for 1 h, followed by DAPI staining. The cells were observed and imaged using a confocal laser scanning microscope. Protein concentration based on fluorescence intensity was quantified using ImageJ software (Rawak Software, Germany). All antibodies used in this study are listed in Table [195]S3. WB Proteins from mouse kidney tissue and renal epithelial cells were extracted using the Tissue Protein Extraction Reagent Kit (EX2410, Solarbio) and the Cell Protein Extraction Reagent Kit (EX2170, Solarbio). Protein concentrations were determined using the BCA Protein Assay Kit (BCA1-1KT, Sigma). Equal amounts of protein (20 µg per lane) were separated by 10–12% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA for 2 h and then washed with PBS. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies (details in Table [196]S3). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:2000, Abcam, UK) at room temperature for 2 h. The signals were detected using an enhanced chemiluminescence system (iBright FL1500, Thermo Fisher). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab9485, 1:2500, Abcam, UK) was used as the internal control. The experiments were repeated three times. RT-qPCR Total RNA was extracted from cells using the RNA Extraction Kit (12183020, Thermo Fisher) following the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed into cDNA using the First Strand cDNA Synthesis Kit (K1622, Fermentas). The synthesized cDNA was then subjected to RT-qPCR using the BeyoFast™ SYBR Green One-Step qRT-PCR Kit (D7268S, Beyotime) and the ABI PRISM 7500 RT-PCR system (Applied Biosystems, Thermo Fisher). Each sample was run in triplicate. Relative mRNA expression levels were analyzed using the 2^-ΔΔCt method, with GAPDH serving as the internal control. The ΔΔCt calculation was performed as follows: ΔΔCt = (Ct_target, experimental - Ct_GAPDH, experimental) - (Ct_target, control - Ct_GAPDH, control). The RT-qPCR reactions were conducted using the StepOnePlus system (Applied Biosystems, California, USA) with the following thermal cycling conditions: 95 °C for 15 min for initial denaturation, followed by 40 cycles of 95 °C for 10 s and 60 °C for 60 s. Primer sequences used in this study are listed in Table [197]S4. All reagents and consumables were purchased from Wuhan Servicebio Technology Co., Ltd. Statistical analysis All data were analyzed using SPSS version 22.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism version 7.0. Continuous data were expressed as mean ± standard deviation (Mean ± SD). Comparisons between two groups were conducted using an unpaired t-test, while comparisons among multiple groups were performed using a one-way analysis of variance (ANOVA). Homogeneity of variances was assessed using Levene’s test. If variances were homogeneous, Dunnett’s t-test and LSD-t-test were used for pairwise comparisons. If variances were not homogeneous, Dunnett’s T3 test was employed. A p-value of less than 0.05 was considered statistically significant. Electronic supplementary material Below is the link to the electronic supplementary material. [198]12951_2025_3618_MOESM1_ESM.jpg^ (457.2KB, jpg) Supplementary Material 1: Figure S1: Characterization of GS-AuNPs. [199]12951_2025_3618_MOESM2_ESM.jpg^ (964.7KB, jpg) Supplementary Material 2: Figure S2 Ultrasound Imaging of Hydronephrosis in Mice. [200]12951_2025_3618_MOESM3_ESM.jpg^ (1.1MB, jpg) Supplementary Material 3: Figure S3: The Impact of C9 and TGF-β1 on Inflammation and RF in TKPTS Cells. [201]12951_2025_3618_MOESM4_ESM.jpg^ (964.7KB, jpg) Supplementary Material 4: Figure S4: Effects of C9 Overexpression and PMIZ-AuNPs on OS-Induced Fibrosis and Inflammation in TKPTS Cells. [202]12951_2025_3618_MOESM5_ESM.jpg^ (1,004.9KB, jpg) Supplementary Material 5: Figure S5: In vivo safety assessment and experimental flowchart of PMIZ-AuNPs [203]12951_2025_3618_MOESM6_ESM.jpg^ (644.9KB, jpg) Supplementary Material 6: Figure S6: Fibrosis Markers in Mouse Kidney Tissue. [204]12951_2025_3618_MOESM7_ESM.jpg^ (114.3KB, jpg) Supplementary Material 7: Figure S7: Detection of C9 Overexpression Efficiency in Mice. [205]12951_2025_3618_MOESM8_ESM.jpg^ (542.2KB, jpg) Supplementary Material 8: Figure S8: Detection of C9 Knockdown and Overexpression Efficiency in TKPTS and HK-2 Cells. [206]12951_2025_3618_MOESM9_ESM.jpg^ (205.9KB, jpg) Supplementary Material 9: Figure S9: Validation of the OS Model in Cells. [207]Supplementary Material 10^ (21.6KB, docx) [208]Supplementary Material 11^ (315.7KB, zip) [209]Supplementary Material 12^ (236.1KB, zip) [210]Supplementary Material 13^ (530.9KB, zip) [211]Supplementary Material 14^ (392.5KB, zip) [212]Supplementary Material 15^ (801.5KB, zip) [213]Supplementary Material 16^ (830.7KB, zip) [214]Supplementary Material 17^ (552.8KB, zip) [215]Supplementary Material 18^ (1.5MB, zip) [216]Supplementary Material 19^ (375.4KB, zip) [217]Supplementary Material 20^ (1.2MB, zip) [218]Supplementary Material 21^ (1.1MB, zip) Acknowledgements