Synthesis and characterization of PSCZ
As shown in Scheme 1, the native enzyme could be embedded in a porous nanomaterial ZIF-8, in which Zn(OAc)2, 2-HMeIM, SOD, and CAT were incubated for 5 min at room temperature. The morphology of composite [email protected]@ZIF-8 and PSCZ was characterized by SEM (Figure 1A). The SEM image showed that [email protected]@ZIF-8 and PSCZ both possessed uniform rhombic dodecahedral morphology similar to blank ZIF-8. As shown in Figure 1B, the PXRD pattern of PSZ, PCZ, and PSCZ both exhibited peaks matching well with the simulated ZIF-8 suggesting that ZIF-8 frameworks were formed in the biomimetic mineralization, and the structural integrity of ZIF-8 could be retained in the presence of SOD and CAT. FT-IR spectra of PSZ, PCZ, and PSCZ characteristic with MPEG2000-COOH peaks at 2865–2875 cm−1 that were respectively attributable to -COOH and -OCH3, indicating the successful conjunction of MPEG2000. We further investigated the antioxidant activities of PSCZ, PSZ, and PCZ. Among biologically relevant ROS, H2O2 is of greatest importance because of its membrane permeability, longer half-life than O2− and ·OH, and consequently highest intracellular concentration. As shown in Figure 1E and 1F, we investigated the cascading enzyme scavenging ability of PSCZ, a remarkable difference in the activity was observed. PSCZ showed enhanced antioxidant activities in both SOD- and CAT-like activities.
Assessment of the ability of PSCZ to scavenge ROS in vitro
Given the promising properties of PSCZ particles as stable, biocompatible antioxidant agents, we next assessed their ROS scavenging activity in vitro relative to that of free CAT and SOD. Given that AKI pathogenesis is associated with oxidative damage to the renal tubules, we utilized the HEK293 cell line model to assess the ability of PSCZ to protect against ROS-mediated injury . We additionally utilized an xCELLigence (RTCA) instrument to monitor the survival and growth of these cells in a quantitative, real-time, dynamic manner. This assay approach is superior to traditional endpoint-based viability assays that assess membrane permeability , enabling the assessment of cell survival in a label-free and automated manner throughout a given experimental period . RTCA approach was used to measure the survival and growth of cells over a 24 h period following treatment with H2O2 and these different nanoenzymes preparations. PSCZ showed superior antioxidant capacity at 5 μg/mL than PSZ and PCZ even at a higher (20 μg/mL) concentration (Figure 2A). We then treated HEK293 cells with H2O2 to induce oxidative stress to evaluate the ability of PSCZ to protect against oxidative damage (Figure 2B). While the viability of H2O2-treated cells was ~50%, PSCZ treatment was associated with a significant increase in these cells’ survival due to the observed reductions in intracellular ROS production and the consequent preservation of mitochondrial functionality (Figure 2B).
We also utilized the DCFH-DA fluorescent probe to assess intracellular ROS levels to further confirm whether PSCZ could suppress H2O2-induced ROS generation in HEK293 cells. While a significant increase in DCFH-DA fluorescence was evident in H2O2-treated cells relative to untreated controls (Figure 2D), this fluorescence intensity was significantly reduced in cells that had additionally been treated with PSCZ. This finding was further confirmed via flow cytometry (Figure 2C), revealing that PSCZ could reliably suppress H2O2-mediated free radical generation.
Mitochondrial oxidative phosphorylation is the primary mechanism whereby most cells produce ATP . Given that renal PTECs lack glycolytic activity and are under high metabolic energy demands, these cells exist under conditions of ischemia and hypoxia . This increases the susceptibility of these renal tubular epithelial cells to acute renal injury. Mitochondria are also central regulators of intracellular apoptotic signaling, with the loss of mitochondrial membrane potential (Δψm) being an early apoptotic indicator . Therefore, we assessed the impact of PSCZ on HEK293 cell Δψm using a JC-1 staining approach, with a higher red/green JC-1 ratio indicative of lower levels of mitochondrial dysfunction. We observed a significant decrease in the Δψm of these cells following H2O2 treatment (Figure S1), whereas PSCZ treatment yielded a JC-1 fluorescence ratio comparable to that in control cells, suggesting that PSCZ can preserve mitochondrial membrane integrity.
Excess ROS generation can initiate apoptotic signaling cascades within cells [20, 21]. Given the acute sensitivity of renal tubule cells to oxidative damage, we next assessed the ability of PSCZ to facilitate in vitro ROS scavenging activity in our HEK293 cell model system. When the death of these cells was assessed via flow cytometry following Annexin V/PI staining, we found that H2O2 treatment (200 μm) increased the frequency of apoptotic HEK293 cells to 36.43%, relative to control cells (3.28%) (Figure 3). In contrast, cells treated with H2O2 and PSCZ exhibited significantly lower rates of apoptotic death (6.43%). These findings thus confirmed that PSCZ could protect against oxidative damage in HEK293 cells in vitro.
PSCZ effectively facilitates in vivo ROS scavenging
Given the promising in vitro results detailed above, we next explored the in vivo utility of our MOF antioxidant platform using a murine cisplatin-induced AKI (CP-AKI) model system . Cisplatin (CDDP) is a widely used chemotherapeutic drug that accumulates within renal proximal tubule epithelial cells (PTECs), resulting in localized renal inflammation, damage, apoptotic death, and AKI that can manifest in the form of CDDP-associated nephrotoxicity . The pathogenesis of CP-AKI has been linked to ROS production and consequent acute tubular apoptosis . Thus, this CP-AKI model system was selected for further analyses of the relative safety and in vivo antioxidant activity of PSCZ.
CP-AKI mice were established as in prior reports by intraperitoneally injecting these animals with CDDP (20 mg/kg) and then measuring serum BUN and CRE levels after 24 h to gauge renal function , given that these compounds accumulate in the blood in the context of renal damage. Mice in corresponding treatment groups were intravenously treated with PSCZ 1 h before CDDP administration (Figure 4A). CRE and BUN levels were significantly higher in CP-AKI mice than healthy controls (Figure 4B-C), consistent with the successful induction of CDDP-mediated kidney damage in these animals. Saline infusions are commonly administered to patients before and during CDDP treatment , as such infusions exhibit poorly understood renoprotective activity against CDDP-induced nephrotoxicity. However, CP-AKI will manifest in roughly 30% of treated patients . Our mouse model system observed comparable findings, as only slight reductions in CRE and BUN levels were observed in CP-AKI mice treated with PBS, with these levels being significantly higher than those in control animals. In contrast, the treatment of CP-AKI mice with PSCZ resulted in significant reductions in CRE and BUN levels to concentrations comparable to those in healthy control mice. These data underscored the therapeutic utility of PSCZ as a safe and effective tool for treating CP-AKI in mice.
We additionally isolated renal tissue sections from these treated mice and stained them with H&E stain to assess treatment-related changes in tissue pathology. Renal casts can arise due to denatured protein precipitation in kidney tubules and are often considered to be an indicator of kidney disease. Similarly, inflammatory cell infiltration and the formation of vacuoles within renal tubules can be visualized to detect and gauge the severity of the renal inflammatory injury. While many casts were evident in the renal tissue samples from CP-AKI model mice (Figure 4D), relatively few were detectable in AKI model animals that had been administered PSCZ, consistent with the ability of these nanoparticles to preserve the integrity of renal tissues better.
To further assess the ability of PSCZ to suppress renal ROS generation, we next used the fluorescent ROS probe DCFH-DA to stain kidney tissue sections from CP-AKI mice in our different treatment groups and then imaged these tissues via laser scanning confocal microscopy. This approach revealed that PSCZ administration significantly inhibited renal ROS accumulation in our AKI model mice (Figure S2).
Tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) are key inflammatory cytokines and drivers of apoptotic cell death in the context of cisplatin-induced renal injury [27, 28]. Excess ROS production by cells under inflammatory conditions can induce additional proinflammatory cytokine production, thereby promoting further immune cell infiltration and renal damage. IL-1β can further amplify these inflammatory processes through feedback mechanisms . Renal tissue samples from treated mice were collected and analyzed to reveal the underlying mechanism of PSCZ. Immunofluorescence results showed that IL-1β and TNF-α levels were comparable in the PSCZ-treated and control groups, suggesting these nanoparticles did not induce direct inflammation in mice at the utilized concentrations and can reduce inflammation-induced kidney damage (Figure 5A-5D). Notably, in AKI model mice, PSCZ administration suppressed TNF-α and IL-1β expression relative to untreated model controls. We additionally assessed SOD and CAT levels in renal homogenates from these mice (Figure 5E and 5F), to confirm that these antioxidant enzymes were successfully delivered to damaged kidney tissue . Excessive ROS levels can result in SOD and CAT depletion, as was evident in CP-AKI and PBS-treated mice. In contrast, PSCZ resulted in renal SOD and CAT levels similar to those in healthy control animals. It was confirmed that PSCZ was successfully delivered to the injured renal tissue and alleviated renal inflammatory injury.
To further test the therapeutic utility of PSCZ, we next measured malondialdehyde (MDA) levels and blood biochemical parameters to evaluate renal excretory function. MDA analysis results revealed that both CP-AKI mice and PBS-treated AKI mice exhibited elevated MDA levels consistent with renal failure, while PSCZ administration lowered these levels consistent with the alleviation of AKI-related renal damage (Figure 5G). Analyses of blood biochemical parameters from these mice indicated that PSCZ treatment was not associated with obvious renal toxicity (Figure 5H-5L), and hematological parameters in PSCZ-treated animals were comparable to those in healthy controls. These findings suggested that our PSCZ particles were highly biocompatible and useful as a tool for AKI treatment. However, it is essential to note that we only tested for acute toxicity associated with this therapeutic platform, and further research will be necessary to establish whether PSCZ is retained in the kidney beyond the 24 h time point, whether it is eliminated via renal metabolic processing, and whether it induces any long-term toxicity in vivo.
RNAs-seq analysis confirmed the successful construction of the AKI model and PSCZ showed a superior protective effect in the AKI mouse model
In the current study, we set four different groups: control group without any treatment (brief as the control group), cisplatin-induced AKI group (brief as CP-AKI group), AKI group pretreated with PSCZ (brief as the PSCZ+AKIgroup), and AKI group pretreated with PBS (brief as the PBS+CP-AKI group). More detailed information is shown in the M&M section.
An average of 63.34 (range from 58.51 to 66.33) million raw reads were obtained for all samples. After removing low-quality reads, an average of 60.52 (range from 56.05 to 63.16) million clean reads were retained for further analysis. The raw data were uploaded to National Omics Data Encyclopedia (NODE) database (https://www.biosino.org/node) with the following accession number OEP000285.
To further elucidate the underlining therapeutic mechanisms, CP-AKI was chosen as the representative disease model for further transcriptomic analysis. DESeq2 identified a total of 601, 157, and 88 DEGs for the CP-AKI group, CP-AKI+PSCZ group, and CP-AKI+PBS group compared with the control group, respectively (Figure 6D). An unguided principal component analysis (PCA) of the data revealed different transcriptomic profiles between PSCZ and PBS-treated AKI mice kidneys (Figure 6C). The Venn diagram in Figure 6B showed many differentially expressed genes were produced in the CP-AKI group compared with the control group, and the differentially expressed genes were decreased in the PSCZ treatment group.
We also investigated the impact of PSCZ on gene expression related to growth arrest and DNA damage response, apoptosis, oxidation-reduction, etc. (Figure 6G). In brief, the PSCZ treatment could reverse the impact of cisplatin-induced AKI in the mouse model.
DEGs in the CP-AKI group were enriched in the p53 signaling pathway, FoxO signaling pathway, apoptosis-related pathways, etc (Figure 6E and F, Figure S3). This is consistent with previous research. Cisplatin can interfere with DNA replication and DNA repair mechanisms, cause DNA damage, and induce apoptosis in cells. Cisplatin can also increase cellular ROS and induce endoplasmic reticulum (ER) stress, contributing to cisplatin toxicity. Cell death and ER stress are characteristic of cisplatin-induced AKI.[26, 31] P53 signaling is an early-stage response to cisplatin toxicity in renal cells. Cisplatin caused renal cells to initiated the p53 dependent DNA repair pathway. .If ROS continues to be overproduced in renal cells, the intrinsic apoptotic pathway will be activated. In the early stage, when cisplatin interferes with DNA replication and DNA repair mechanism, nano inhibits the excessive production of ROS caused by cisplatin, promotes the initiation of DNA repair pathway, and alleviates renal cell damage(Figure 6A, Figure S4).
In the current study, the kidney proximal nephron tubule segment injury marker Havcr1 (also known as Kim1) slightly changed (log2FC = 0.42, adj. P = 0.437) CP-AKI group compared with the control group. This is probably because the kidneys were harvested in the very early stage of AKI. This is consistent with a previous study that cisplatin-induced AKI mainly caused proximal nephron tubule segment injury in the very early stage. Bax, Bak1 are important signals in the apoptosis cascade and normally act on the mitochondrial membrane to promote permeabilization and release of cytochrome C and ROS. In CP-AKI group, Bax and Bak1 significantly increased (log2FC = 1.16, adj. P = 0.024; log2FC = 0.99, adj. P = 0.048, respectively). This means that ROS level increased by cisplatin. Aco1 and Cyp2e1, which are important oxidation-reduction enzymes, significantly decreased (log2FC = -0.74, adj. P = 0.007; log2FC = -1.03, adj. P = 0.026, respectively). This indicated that the normal function of oxidation-reduction was compromised. Gadd45a and Gadd45b, which are growth arrest and DNA damage response genes, also increased (log2FC = 1.13, adj. P = 0.045; log2FC = 1.29, adj. P = 0.024, respectively). This indicated that kidney cells responded to cisplatin-induced DNA damage by cell cycle arrest and initiated the DNA repair system, in which case, the p53 signaling pathway. Tnfrsf12a and Tnfrsf10b (also known as Dr5), which belong to tumor necrosis factor receptor superfamily and transduces apoptosis signals, significantly increased in CP-AKI group (log2FC = 1.50, adj. P =0.003; log2FC = 1.45, adj. P =0.006, respectively). Caspase8 also significantly increased in CP-AKI group (log2FC = 0.74, adj. P =0.044). This indicated that cisplatin-induced intrinsic apoptosis through Caspase8 by upregulating Tnfrsf10b in the CP-AKI group. The results indicated that the apoptosis process was initiated in the CP-AKI group even after 24 h being treated with cisplatin. Bcl10 contains a caspase recruitment domain (CARD) and has been shown to induce apoptosis and activate NF-κB. Bcl10 slightly increased in CP-AKI group (log2FC = 0.70, adj. P =0.159).
However, this process is reversible if cell oxidative stress can be alleviated. In PBS and PSCZ pretreated group, Bax and Bak1 tended to return to normal level (Bax: log2FC = 0.89, adj. P = 0.260; log2FC = 0.52, adj. P = 0.774, respectively; Bak1: log2FC = 0.39, adj. P = 0.836; log2FC = 0.11, adj. P = 1.000, respectively), Tnfrsf12a and Tnfrsf10b also tended to return to normal level (Tnfrsf12a: log2FC = 0.54, adj. P = 0.500; log2FC = 0.13, adj. P = 1.000, respectively; Tnfrsf10b: log2FC = 0.99, adj. P = 0.177; log2FC = 0.42, adj. P = 0.860) compared with CP-AKI group. This indicated that renal cell oxidative stress was significantly ameliorated, and cell apoptosis was repressed. Gadd45a and Gadd45b also tended to return to normal level in PBS and PSCZ pretreated AKI mouse models (Gadd45a: log2FC = 0.35, adj. P = 0.897; log2FC = -0.04, adj. P = 1.000, respectively; Gadd45b: log2FC = 0.38, adj. P = 0.862; log2FC = 0.33, adj. P = 0.957, respectively). This indicated cell cycle arrest was also alleviated in both groups. Tadagavadi et al. also reported that PBS have protective effects in AKI models. This is consistent with the findings in current study. Moreover, in current study, PSCZ showed superior protective effect than PBS.