Cuproptosis was first characterized in 2022 by Peter Tsvetkov and colleagues, who showed that excess copper directly binds and aggregates lipoylated proteins of the tricarboxylic acid cycle, particularly dihydrolipoamide S-acetyltransferase, or DLAT. This aggregation produces proteotoxic stress, destabilizes iron-sulfur cluster proteins, and ultimately kills the cell. Unlike apoptosis, necrosis, pyroptosis, autophagy, or ferroptosis, cuproptosis involves the direct copper-mediated modification of mitochondrial metabolic enzymes rather than membrane rupture or caspase activation. Copper is indispensable for several enzymes involved in mitochondrial oxidative metabolism, oxidative stress defense, and biosynthetic regulation, but under physiological conditions intracellular concentrations are kept below cytotoxic thresholds by the exporters ATP7A and ATP7B. Ischemia-reperfusion injury disrupts this equilibrium, and copper accumulation has been observed in various models of acute kidney injury, raising the possibility that cuproptosis might represent an uncharacterized pathway in the disease.

To test this hypothesis, the team led by Lirong Lin, Shahong He, Ke Li, and Jurong Yang integrated single-cell transcriptomics from the public dataset GSE180420, a human renal biopsy cohort of 22 patients diagnosed with acute kidney injury according to KDIGO criteria, murine ischemia-reperfusion models, and hypoxia-reoxygenation cellular models. Using the Scanpy package, they scored each tubular epithelial cell against gene sets representing distinct regulated cell death modalities and assigned each cell to the pathway with the highest score. The cuproptosis gene set included FDX1, LIAS, LIPT1, DLAT, PDHA1, PDHB, DBT, GCSH, SLC31A1, ATP7A, and ATP7B. The analysis of roughly 110,000 cells revealed that cuproptosis was the most prominent regulated cell death modality, accounting for approximately 33 percent of the cells assigned to any regulated cell death pathway. Notably, cuproptotic death was specifically enriched in proximal convoluted and proximal straight tubule epithelial cells, the metabolically active segments most dependent on mitochondrial tricarboxylic acid cycling, and the proportion of cuproptosis increased significantly after ischemic injury compared with sham-operated controls.

The human biopsy data corroborated these findings across multiple etiologies of acute kidney injury, including nephrotoxic exposures, toxins, and infections, although the cohort included only one case of isolated ischemic injury. Immunohistochemical staining localized CTR1 and FDX1 predominantly to the plasma membrane and perinuclear regions of tubular epithelial cells. Quantitative morphometry revealed dramatic upregulation in injured tissues: CTR1-positive tubules rose from 2.05 percent in controls to 24.96 percent in patients with acute kidney injury, while FDX1-positive tubules increased from 2.53 percent to 33.44 percent. Both markers correlated strongly with established injury biomarkers. CTR1 expression correlated positively with serum creatinine, blood urea nitrogen, and urinary N-acetyl-β-D-glucosaminidase, and inversely with estimated glomerular filtration rate, suggesting that cuproptosis markers could serve as diagnostic and risk-stratification tools.

In mice subjected to 28 minutes of bilateral renal ischemia followed by 48 hours of reperfusion, histological examination revealed characteristic pathology including tubular epithelial vacuolization, brush border loss, cast formation, and interstitial inflammation. Renal function deteriorated markedly, with serum creatinine rising from 31.3 to 206.5 micromoles per liter and blood urea nitrogen increasing from 4.8 to 16.0 millimoles per liter. Inductively coupled plasma mass spectrometry demonstrated a five-fold increase in kidney copper content, from 0.9 to 4.8 micrograms per gram, coinciding with marked upregulation of CTR1, FDX1, DLAT, and HSP70 as confirmed by western blot analysis. In vitro, the combination of copper chloride and the copper ionophore elesclomol induced marked cell death in NRK-52E tubular epithelial cells, increasing lactate dehydrogenase release 4.3-fold, reducing cell viability to 38.2 percent, and raising intracellular copper levels 2.1-fold. The hypoxia-reoxygenation challenge recapitulated these findings, including a 2.2-fold copper accumulation and a 68 percent decrease in the copper exporter ATP7B, suggesting impaired copper clearance as a key event in ischemic tubular injury.

Schisandrin B, a dibenzocyclooctadiene lignan with known antioxidant, anti-inflammatory, and mitochondrial protective properties, was then tested as a potential intervention. When added at 25 micromolars at the start of reoxygenation and maintained for 24 hours, Schisandrin B significantly ameliorated hypoxia-reoxygenation-induced injury, reducing the injury markers NGAL and KIM-1 by 71 and 68 percent respectively and decreasing lactate dehydrogenase release by 54 percent. Intracellular copper levels were normalized, accompanied by downregulation of CTR1, FDX1, DLAT, and HSP70, indicating potent inhibition of cuproptosis. In vivo, Schisandrin B administered at 50 milligrams per kilogram intraperitoneally 2 hours before ischemia significantly improved renal function, reduced tissue copper content, and downregulated cuproptosis-related proteins, with histological injury scores falling from 2.75 to 1.25.

RNA sequencing of hypoxia-reoxygenation-challenged cells treated with Schisandrin B identified 1,581 differentially expressed genes, including 513 upregulated and 1,068 downregulated genes following treatment. KEGG enrichment analysis highlighted glutathione metabolism, p53 signaling, cellular senescence, and the PI3K-Akt pathway as the most significantly altered pathways, consistent with antioxidant activity and promotion of cell survival. Schisandrin B reduced malondialdehyde levels, a marker of lipid peroxidation, by 61 percent in vitro and 48 percent in vivo, while increasing glutathione levels by 2.3-fold and 1.9-fold respectively. However, the authors caution that because cuproptosis is largely independent of reactive oxygen species, these changes may reflect reduced copper accumulation and cellular stress following CTR1 modulation rather than a classical antioxidant mechanism.

Molecular docking using AutoDock Vina explored the interaction between Schisandrin B and CTR1, the principal copper importer encoded by SLC31A1. The top-ranked pose placed Schisandrin B in the predicted copper-binding pocket of CTR1 with a minimum binding energy of −5.1 kilocalories per mole, indicating favorable binding affinity. In this pose, Schisandrin B formed hydrophobic interactions with Val14 and π-π stacking interactions with Phe18, suggesting stable ligand-receptor engagement within the copper-coordinating region. To validate the functional dependency, the team used siRNA-mediated knockdown of CTR1, achieving a 53 percent reduction in protein levels. In these CTR1-deficient cells, Schisandrin B failed to produce a statistically significant further reduction in any measured parameter compared with CTR1 knockdown alone, suggesting that the protective effect requires a CTR1-competent state and that Schisandrin B provides no additional protection when CTR1-mediated copper entry is already reduced.

Compared with the classical copper chelator tetrathiomolybdate, Schisandrin B exhibited protective effects that were at least comparable under the tested conditions. Both treatments significantly reduced lactate dehydrogenase release, KIM-1 and NGAL mRNA levels, and intracellular copper content in hypoxia-reoxygenation-challenged cells. Schisandrin B produced numerically greater reductions in KIM-1 and NGAL than tetrathiomolybdate, although these between-treatment differences were not statistically significant. Molecular profiling showed greater reductions in FDX1, CTR1, DLAT, and HSP70 with Schisandrin B than with tetrathiomolybdate. The authors note that tetrathiomolybdate may disrupt physiological copper functions through non-selective chelation, whereas Schisandrin B appears to modulate copper uptake through CTR1, representing a mechanistically distinct strategy.

The study has limitations that warrant cautious interpretation. The clinical biopsy cohort was small and drawn from a single center, and the single-cell gene-set scoring approach cannot fully distinguish baseline metabolic gene expression from active cell death program activation, since proximal tubular epithelial cells constitutively express high levels of tricarboxylic acid cycle and lipoylation genes. Only ATP7B was assessed, whereas ATP7A, a major copper efflux transporter in most epithelia, was not examined. The absence of further improvement with Schisandrin B in CTR1-knockdown cells does not establish direct physical binding between the compound and the transporter or show that CTR1 is its exclusive target. Future studies using CTR1 overexpression, rescue experiments, CETSA, DARTS, or co-crystallization approaches are needed to establish direct target engagement. Nevertheless, by identifying cuproptosis as a druggable form of regulated cell death in acute kidney injury and characterizing Schisandrin B as a CTR1-dependent therapeutic candidate, the findings provide a preclinical rationale for repurposing this natural product in acute ischemic kidney disease, offering a strategy that preserves physiological copper function while limiting pathological copper accumulation.

Subject of Research: Copper-triggered cell death and Schisandrin B protection in ischemic acute kidney injury

Article Title: Schisandrin B attenuates ischemic acute kidney injury in a CTR1-dependent manner by inhibiting cuproptosis

Article References: Lin, L., He, S., Yang, X., Zheng, L., Tan, W., Li, K., & Yang, J. (2026). Schisandrin B attenuates ischemic acute kidney injury in a CTR1-dependent manner by inhibiting cuproptosis. Frontiers in Cell and Developmental Biology, 14, Article 1993380. https://doi.org/10.3389/fcell.2026.1993380

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1993380

Keywords: acute kidney injury, cuproptosis, Schisandrin B, CTR1, copper, cell death, nephrology, Schisandrin, attenuates, ischemic, acute, kidney