Cornus officinalis var. koreana Kitam extracts alleviate cadmium-induced renal fibrosis by targeting matrix metallopeptidase 9.

ETHNOPHARMACOLOGICAL RELEVANCE: Cornus officinalis var. koreana Kitam (Cornus officinalis) is a commonly used Chinese herbal medicine and has a good clinical efficacy in kidney and liver diseases. Recent years, a number of studies reported the significant effects of Cornus officinalis on renal fibrosis. However, it is still unclear about the underlying specific mechanism, the bioactive ingredients, and the target gene regulatory network. AIM OF THE STUDY: We investigated the impact of Cornus officinalis extract on cadmium-induced renal fibrosis, screened the bioactive ingredients of Cornus officinalis using a pharmacological sub-network analysis, and explored the regulatory effects of Cornus officinalis extracts on target gene matrix metallopeptidase 9 (MMP9). METHODS: Male C57BL/6N mice were treated with single or combinatorial agents such as saline, cadmium chloride, Cornus officinalis, Isoginkgetin and FSL-1. Isoginkgetin is a compound with anti-MMP9 activity. FSL-1 can induce MMP9 expression. Masson staining and Western blot and immunohistochemistry analyses were used for assessing renal fibrosis. In addition, wound healing model was established using BUMPT (Boston university mouse proximal tubular) cells to investigate how Cornus officinalis affected cadmium-induced cell migration. The main Cornus officinalis bioactive compounds were identified by UHPLC-MS (Ultra-high-performance liquid chromatography - mass spectrometry). The MMP9 target for Cornus officinalis active ingredients were confirmed through a pharmacological sub-network analysis. RESULTS: Aqueous extracts of Cornus officinalis protected from renal dysfunction and kidney fibrosis induced by cadmium chloride in mice. In vitro experiments validated that Cornus officinalis extracts inhibited cell migration ability especially in cadmium chloride condition. The sub-network analysis and chemical components profiling technique revealed the active compounds of Cornus officinalis. Cellular thermal shift assay verified the binding abilities of three active components Daidzein, N-Acetyl-L-tyrosine or Swertisin with matrix metalloproteinase-9. Gelatin zymography assay revealed that the activity of MMP9 was inhibited by the three active components. We further confirmed that MMP9 was involved in the process of Cornus officinalis extracts reducing renal fibrosis. Cornus officinalis attenuated the cadmium-induced renal fibrosis was correlated with decreased expression of MMP9, collagen I, α-SMA (alpha-smooth muscle actin) and vimentin. CONCLUSIONS: This study demonstrated that Cornus officinalis extracts could alleviate the cadmium chloride-induced renal fibrosis by targeting MMP9, and might provide new insights into the mechanism of treating renal fibrosis by Cornus officinalis.

Tim-3 deficiency aggravates cadmium nephrotoxicity via regulation of NF-κB signaling and mitochondrial damage.

Kidney is the target organ of serious cadmium injury. Kidney damage caused by cadmium exposure is greatly influenced by the inflammatory response and mitochondrial damage. T cell immunoglobulin domain and mucin domain 3 (Tim-3) is an essential protein that functions as a negative immunological checkpoint to regulate inflammatory responses. Mice were given cadmium treatments at various dosages (0, 1.5, 3, 4.5 mg/kg) and times (0, 3, 5, 7 days) to assess the effects of cadmium on kidney damage. We found that the optimal way to induce kidney injury in mice was to inject 4.5 mg/kg of cadmium intraperitoneally for five days. It is interesting that giving mice 4.5 mg/kg of cadmium intravenously for seven days drastically lowered their survival rate. After cadmium exposure, Tim-3 knockout mice exhibited higher blood concentrations of urea nitrogen and creatinine compared to control mice. Tim-3 impacted the expression of oxidative stress-associated genes such as UDP glucuronosyltransferase family 1 member A9 (Ugt1a9), oxidative stress-induced growth inhibitor 2 (Osgin2), and S100 calcium binding protein A8 (S100a8), according to RNA-seq and real-time RT-PCR data. Tim-3 deficiency also resulted in activated nuclear factor-kappa B (NF-κB) signaling pathway. The NF-κB inhibitor 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) significantly alleviated cell apoptosis, oxidative stress response, and renal tubule inflammation in Tim-3 knockout mice exposed to cadmium. Furthermore, cadmium caused obvious B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) translocation from cytoplasm to mitochondria, which can be inhibited by TPCA-1. In conclusion, Tim-3 prevented mitochondrial damage and NF-κB signaling activation, hence providing protection against cadmium nephrotoxicity.

Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation.

The impact of Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3) on cisplatin-induced acute kidney injury was investigated in this study. Cisplatin-induced Tim-3 expression in mice kidney tissues and proximal tubule-derived BUMPT cells in a time-dependent manner. Compared with wild-type mice, Tim-3 knockout mice have higher levels of serum creatinine and urea nitrogen, enhanced TUNEL staining signals, more severe 8-OHdG (8-hydroxy-2' -deoxyguanosine) accumulation, and increased cleavage of caspase 3. The purified soluble Tim-3 (sTim-3) protein was used to intervene in cisplatin-stimulated BUMPT cells by competitively binding to the Tim-3 ligand. sTim-3 obviously increased the cisplatin-induced cell apoptosis. Under cisplatin treatment conditions, Tim-3 knockout or sTim-3 promoted the expression of TNF-α (tumor necrosis factor-alpha) and IL-1ß (Interleukin-1 beta) and inhibited the expression of IL-10 (interleukin-10). NF-κB (nuclear factor kappa light chain enhancer of activated B cells) P65 inhibitor PDTC or TPCA1 lowed the increased levels of creatinine and BUN (blood urea nitrogen) in cisplatin-treated Tim-3 knockout mice serum and the increased cleavage of caspase 3 in sTim-3 and cisplatin-treated BUMPT cells. Moreover, sTim-3 enhanced mitochondrial oxidative stress in cisplatin-induced BUMPT cells, which can be mitigated by PDTC. These data indicate that Tim-3 may protect against renal injury by inhibiting NF-κB-mediated inflammation and oxidative stress.

Expression, purification and oligomerization of the S-adenosylmethionine transporter.

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.