Renal Cell Line Research Articles

Fractionation and Characterization of Triterpenoids from Vaccinium vitis-idaea L. Cuticular Waxes and Their Potential as Anticancer Agents.

Fruit and leaf cuticular waxes are valuable source materials for the isolation of triterpenoids that can be applied as natural antioxidants and anticancer agents. The present study aimed at the semi-preparative fractionation of triterpenoids from cuticular wax extracts of Vaccinium vitis-idaea L. (lingonberry) leaves and fruits and the evaluation of their cytotoxic potential. Qualitative and quantitative characterization of obtained extracts and triterpenoid fractions was performed using HPLC-PDA method, followed by complementary analysis by GC-MS. For each fraction, cytotoxic activities towards the human colon adenocarcinoma cell line (HT-29), malignant melanoma cell line (IGR39), clear renal carcinoma cell line (CaKi-1), and normal endothelial cells (EC) were determined using MTT assay. Furthermore, the effect of the most promising samples on cancer spheroid growth and viability was examined. This study allowed us to confirm that particular triterpenoid mixtures from lingonberry waxes may possess stronger cytotoxic activities than crude unpurified extracts. Fractions containing triterpenoid acids plus fernenol, complexes of oleanolic:ursolic acids, and erythrodiol:uvaol were found to be the most potent therapeutic candidates in the management of cancer diseases. The specificity of cuticular wax extracts of lingonberry leaves and fruits, leading to different purity and anticancer potential of obtained counterpart fractions, was also enclosed. These findings contribute to the profitable utilization of lingonberry cuticular waxes and provide considerable insights into the anticancer effects of particular triterpenoids and pharmacological interactions.

The E3 ligase RBCK1 reduces the sensitivity of ccRCC to sunitinib through the ANKRD35-MITD1-ANXA1 axis.

Despite the promise of targeted tyrosine kinase inhibitors (TKIs), such as sunitinib, in the extension of survival time in patients with clear cell renal cell carcinoma (ccRCC) progression or metastasis, the patients eventually succumb to inevitable drug resistance. Protein degradation executed by the ubiquitin-dependent proteasome system played an important role in determining the sensitivity of ccRCC to sunitinib. Here, we applied the bioinformatic analysis to identify that E3 ligase RBCK1 was elevated in the sunitinib-resistant renal cancer cell lines or patient specimens. The subsequent in vitro or in vivo studies demonstrated that RBCK1 contributed to decreasing the sensitivity of ccRCC to sunitinib. Then, we showed that inhibition of RBCK1 inactivated the AKT and MAPK signaling pathways, which might be one of the main reasons why RBCK1 induces sunitinib resistance in ccRCC cells. Mechanistically, our results indicated that RBCK1 promotes the degradation of ANKRD35 and that ANKRD35 destabilizes MITD1 by binding with SUMO2 in ccRCC cells. In addition, we showed that the RBCK1-ANKRD35-MITD1-ANXA1 axis regulates the phosphorylation of AKT and ERK and contributes to the dysregulation of sunitinib in ccRCC cells. Therefore, we identified a novel mechanism for regulating the sensitivity of sunitinib in ccRCC. Therefore, we elucidated a novel mechanism by which RBCK1 regulates sunitinib sensitivity in ccRCC.

Cytotoxicity of Carvotacetones from Sphaeranthus africanus Against Cancer Cells and Their Potential to Induce Apoptosis.

Three carvotacetones (1 - 3: ) isolated from Sphaeranthus africanus were screened in 60 cancer cell lines at the National Cancer Institute (NCI) within the Developmental Therapeutics Program (DTP). At the concentration of 10-5 M, compound 1: (3,5-diangeloyloxy-7-hydroxycarvotacetone) turned out to be the most active compound against ACHN and UO-31 renal cancer cell lines with growth percent values of - 100% (all cells dead). Compound 2: (3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone) showed strong effects in SK-MEL-5 melanoma and ACHN renal cancer cells with inhibition values of 93% and 97%, respectively. Compound 3: (3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxy-carvotacetone) exhibited a quite strong effect on renal cancer cells with a growth inhibitory effect of 96% against ACHN and UO-31 cells. When treated with five different concentrations of 1: (1 × 10-8, 1 × 10-7, 1 × 10-6, 1 × 10-5, and 1 × 10-4 M), HOP-92 cells were found to be most sensitive with GI50, TGI, and LC50 values of 0.17, 0.40, and 0.96 µM, respectively. When using the ApoTox-Glo triplex assay to evaluate the apoptosis inducing effects of seven carvotacetones isolated from S. africanus in CCRF-CEM cells, compounds 1: - 6: increased caspase-3/7 activity with 1, 2: , and 4: (3-angeloyloxy-5,7-dihydroxycarvotacetone) exhibiting the highest activitiy, indicating induction of caspase-dependent apoptosis.

Synthesis, spectroscopic characterization, antibacterial activity and antiproliferative profile of a new silver(I) complex of 5-fluorocytosine

In this paper, we report the synthesis, structural characterization, and explore the potential antibacterial and antiproliferative activities of a new silver(I) complex of the nucleotide analogue 5-fluorocytosine, Ag-5FC. Elemental and thermogravimetric (TGA) analyses led to the minimal formula AgC4H4FN3O·H2O. Infrared (FT-IR) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopic analyses indicate that the ligand coordinates to the metal ion by its nitrogen and oxygen atoms. High resolution mass spectrometric (HRMS) data confirmed the proposed composition of the complex as well as the presence of recombination species in solution. Molecular modeling reinforces the proposed N,O-coordination of 5FC to silver as indicated by IR data with a possible structure of the complex in a dimeric form, of coordination formula [Ag(C4H4FN3O)(OH2)]2. The complex showed a modest antimicrobial activity (MIC = 4.3 mmol⋅L−1) against Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus cereus) strains. Despite the low solubility, the Ag-5FC complex totally inhibited the proliferation of colon (HT-29), multidrug resistant ovarian (NCI/ADR-RES), prostate (PC-3), and renal (786–0) tumor cell lines in a concentration range of 6.2 to 9.3 µmol L−1. Further studies are necessary to confirm efficiency and safety for continuing the pre-clinical evaluation of the Ag-5FC complex.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

Single-cell RNA-seq integrated with multi-omics reveals SERPINE2 as a target for metastasis in advanced renal cell carcinoma

Tumor growth, metastasis and therapeutic response are believed to be regulated by the tumor and its microenvironment (TME) in advanced renal cell carcinoma (RCC). However, the mechanisms underlying genomic, transcriptomic and epigenetic alternations in RCC progression have not been completely defined. In this study, single-cell RNA-sequencing (scRNA-seq) data were obtained from eight tissue samples of RCC patients, including two matched pairs of primary and metastatic sites (lymph nodes), along with Hi-C, transposable accessible chromatin by high-throughput (ATAC-seq) and RNA-sequencing (RNA-seq) between RCC (Caki-1) and human renal tubular epithelial cell line (HK-2). The identified target was verified in clinical tissue samples (microarray of 407 RCC patients, TMA-30 and TMA-2020), whose function was further validated by in vitro and in vivo experiments through knockdown or overexpression. We profiled transcriptomes of 30514 malignant cells, and 14762 non-malignant cells. Comprehensive multi-omics analysis revealed that malignant cells and TME played a key role in RCC. The expression programs of stromal cells and immune cells were consistent among the samples, whereas malignant cells expressed distinct programs associated with hypoxia, cell cycle, epithelial differentiation, and two different metastasis patterns. Comparison of the hierarchical structure showed that SERPINE2 was related to these NNMF expression programs, and at the same time targeted the switched compartment. SERPINE2 was highly expressed in RCC tissues and lowly expressed in para-tumor tissues or HK-2 cell line. SERPINE2 knockdown markedly suppressed RCC cell growth and invasion, while SERPINE2 overexpression dramatically promoted RCC cell metastasis both in vitro and in vivo. In addition, SERPINE2 could activate the epithelial-mesenchymal transition pathway. The above findings demonstrated that the role of distinct expression patterns of malignant cells and TME played a distinct role in RCC progression. SERPINE2 was identified as a potential therapeutic target for inhibiting metastasis in advanced RCC.

Upregulation of CENPM is associated with poor clinical outcome and suppression of immune profile in clear cell renal cell carcinoma

BackgroundThe response of advanced clear cell renal cell carcinoma (ccRCC) to immunotherapy is still not durable, suggesting that the immune landscape of ccRCC still needs to be refined, especially as some molecules that have synergistic effects with immune checkpoint genes need to be explored.MethodsThe expression levels of CENPM and its relationship with clinicopathological features were explored using the ccRCC dataset from TCGA and GEO databases. Quantitative polymerase chain reaction (qPCR) analysis was performed to validate the expression of CENPM in renal cancer cell lines. Kaplan-Meier analysis, COX regression analysis and Nomogram construction were used to systematically evaluate the prognostic potential of CENPM in ccRCC. Besides, single gene correlation analysis, protein–protein interaction (PPI) network, genetic ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) were used to predict the biological behaviour of CENPM and the possible signalling pathways involved. Finally, a comprehensive analysis of the crosstalk between CENPM and immune features in the tumor microenvironment was performed based on the ssGSEA algorithm, the tumor immune dysfunction and exclusion (TIDE) algorithm, the TIMER2.0 database and the TISIDB database.ResultsCENPM was significantly upregulated in ccRCC tissues and renal cancer cell lines and was closely associated with poor clinicopathological features and prognosis. Pathway enrichment analysis revealed that CENPM may be involved in the regulation of the cell cycle in ccRCC and may have some crosstalk with the immune microenvironment in tumors. The ssGSEA algorithm, CIBERSOPT algorithm suggests that CENPM is associated with suppressor immune cells in ccRCC such as regulatory T cells. The ssGSEA algorithm, CIBERSOPT algorithm suggests that CENPM is associated with suppressor immune cells in ccRCC such as regulatory T cells. Furthermore, the TISIDB database provides evidence that not only CENPM is positively associated with immune checkpoint genes such as CTLA4, PDCD1, LAG3, TIGIT, but also chemokines and receptors (such as CCL5, CXCL13, CXCR3, CXCR5) may be responsible for the malignant phenotype of CENPM in ccRCC. Meanwhile, predictions based on the TIDE algorithm support that patients with high CENPM expression have a worse response to immunotherapy.ConclusionsThe upregulation of CENPM in ccRCC predicts a poor clinical outcome, and this malignant phenotype may be associated with its exacerbation of the immunosuppressive state in the tumor microenvironment.

Pleiotropic Devitalization of Renal Cancer Cells by Non-Invasive Physical Plasma: Characterization of Molecular and Cellular Efficacy.

Renal cell carcinoma (RCC) is the third most common urological tumor and has an extremely poor prognosis after metastasis has occurred. Therapeutic options are highly restricted, primarily due to resistance to classical chemotherapeutics. The development of new, innovative therapeutic procedures is thus of great urgency. In the present study, the influence of non-invasive physical plasma (NIPP) on malignant and non-malignant renal cells is characterized. The biological efficacy of NIPP has been demonstrated in malignant renal cell lines (786-O, Caki-1) and non-malignant primary human renal epithelial cells (HREpC). The cell responses that were experimentally examined were cell growth (cell number determination, calculation of growth rate and doubling time), cell motility (scratch assay, invasiveness assay), membrane integrity (uptake of fluorescent dye, ATP release), and induction of apoptosis (TUNEL assay, caspase-3/7 assay, comet assay). A single NIPP treatment of the malignant cells significantly inhibited cell proliferation, invasiveness, and metastasis. This treatment has been attributed to the disruption of membrane functionality and the induction of apoptotic mechanisms. Comparison of NIPP sensitivity of malignant 786-O and Caki-1 cells with non-malignant HREpC cells showed significant differences. Our results suggest that renal cancer cells are significantly more sensitive to NIPP than non-malignant renal cells. Treatment with NIPP could represent a promising innovative option for the therapy of RCC and might supplement established treatment procedures. Of high clinical relevance would be the chemo-sensitizing properties of NIPP, which could potentially allow a combination of NIPP treatment with low-dose chemotherapy.

LIM and Cysteine-Rich Domains 1 Promotes Transforming Growth Factor β1–Induced Epithelial–Mesenchymal Transition in Human Kidney 2 Cells

Renal fibrosis is the major pathologic manifestation of chronic kidney disease (CKD). LIM and cysteine-rich domains 1 (LMCD1) is upregulated in the kidney tissue from patients with CKD and the transforming growth factor β1 (TGF-β1)–treated human renal tubular epithelial cell line human kidney 2 (HK-2) (Gene Expression Omnibus: GSE66494 and GSE23338). Previously, we have demonstrated that the knockdown of LMCD1 ameliorated renal fibrosis in mice by blocking the activation of the extracellular signal-regulated kinase pathway. In this study, we sought to further investigate whether LMCD1 affects TGF-β1–induced epithelial–mesenchymal transition (EMT) of kidney tubular epithelial cells and its potential role in the TGF-β1/Smad signaling pathway. First, we confirmed that LMCD1 expression was increased in the fibrotic kidneys of patients with CKD compared with that in normal kidneys and that LMCD1 was predominantly localized in the renal tubules. LMCD1 and mesenchymal markers were upregulated in obstructed kidney tissues of mice at 21 days after unilateral ureteral obstruction surgery compared with the tissues in sham mice. Next, we demonstrated that TGF-β1 significantly increased LMCD1 expression through Smad-mediated transcription in HK-2 cells in vitro. In turn, LMCD1 acted as a transcriptional coactivator of E2F transcription factor 1 to promote the transcription of TGF-β1. Moreover, TGF-β1 increased the interaction between LMCD1 and Smad ubiquitination regulatory factor 2 (Smurf2) and accelerated Smurf2-mediated LMCD1 degradation via the ubiquitination system. The knockdown of LMCD1 inhibited TGF-β1–induced EMT in both HK-2 cells and unilateral ureteral obstruction mice. Our results indicate a positive feedback loop between TGF-β1 and LMCD1 for EMT induction in HK-2 cells and that Smurf2 acts as a negative regulator in this process by accelerating LMCD1 degradation.

Assessment of the Cytotoxicity, Mutagenicity, and Genotoxicity of Two Traditional Chinese Herbs: Aristolochia baetica and Magnolia officinalis.

Herbal remedies used in traditional medicine often contain several compounds combined in order to potentiate their own intrinsic properties. However, herbs can sometimes cause serious health troubles. In Belgium, patients who developed severe aristolochic acid nephropathy ingested slimming pills containing root extracts of an Aristolochia species, as well as the bark of Magnolia officinalis. The goal of the study was to evaluate, on a human renal cell line, Aristolochia and Magnolia extracts for their cytotoxicity by a resazurin cell viability assay, and their genotoxicity by immunodetection and quantification of the phosphorylated histone γ-H2AX. The present study also sought to assess the mutagenicity of these extracts, employing an OECD recognized test, the Ames test, using four Salmonella typhimurium strains with and without a microsomial fraction. Based on our results, it has been demonstrated that the Aristolochia-Magnolia combination (aqueous extracts) was more genotoxic to human kidney cells, and that this combination (aqueous and methanolic extracts) was more cytotoxic to human kidney cells after 24 and 48 h. Interestingly, it has also been shown that the Aristolochia-Magnolia combination (aqueous extracts) was mutagenic with a TA98 Salmonella typhimurium strain in the presence of a microsomial liver S9 fraction. This mutagenic effect appears to be dose-dependent.

The Curcuminoid EF24 in Combination with TRAIL Reduces Human Renal Cancer Cell Migration by Decreasing MMP-2/MMP-9 Activity through a Reduction in H2O2.

Cancer cells present high levels of oxidative stress, and although an increase in reactive oxygen species (ROS), such as H2O2, can lead to apoptosis, it can also induce cell invasion and metastasis. As the increase in ROS can lead to an increase in the expression of MMP-2 and MMP-9, thus causing the degradation of the extracellular matrix, an increase in the ROS H2O2 might have an impact on MMP-2/MMP-9 activity. The natural compound curcumin has shown some anticancer effects, although its bioavailability hinders its therapeutic potential. However, curcumin and its analogues were shown to resensitize kidney cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. This study shows that the curcuminoid EF24 in combination with TRAIL increases peroxidase activity in the renal adenocarcinoma cell line ACHN, reducing the level of intracellular H2O2 and MMP-2/MMP-9 activity, a mechanism that is also observed after treatment with curcumin and TRAIL.

METTL3 alleviates D-gal-induced renal tubular epithelial cellular senescence via promoting miR-181a maturation

Methyltransferase-like protein 3 (METTL3) mediated N6-Methyladenosine (m6A) modification has been implicated in many physiological and pathological processes. However, its function and mechanism in kidney aging are not entirely clear. Here, we investigated changes in m6A levels of aging kidneys and the role of METTL3 in senescent renal tubular epithelial cells and its potential mechanisms. First, we used the naturally aged mouse model and the D-galactose (D-gal)-induced aged mouse model. Dot blot and m6A RNA methylation quantification showed significantly decreased m6A levels in both models. In addition, we observed that METTL3 was down-regulated in D-gal-induced senescent human renal tubular epithelial cell line (HK-2). METTL3 reduction was associated with senescence-related phenotypes of HK-2 cells. We also found that miR-181a-5p attenuated HK-2 senescence by targeting the NF-κB pathway. Moreover, METTL3 was able to promote the maturation of miR-181a-5p and then inhibited the expression of NF-κB and IL-1α. Taken together, we demonstrate that the METTL3/miR-181a-5p/NF-κB axis counteracts HK-2 senescence. Our results suggest that METTL3 may be a novel biomarker and a potential therapy target for kidney aging.

Effects of Dichloromethane and N-Butanol Fractions of Nigella sativa on ACHN and GP-293 Cell Line Morphology, Viability, and Apoptosis.

Renal cell carcinoma (RCC) is among the top death-causing cancers. Medicinal herbs can also have beneficial effects on RCC treatment. In this project, we aimed to study the antitumor effect of dichloromethane and N-butanol fractions of hydroalcoholic extract of Nigella sativa (N. sativa) on the morphology, viability, and apoptosis of ACHN (human renal adenocarcinoma) and GP-293 (normal renal epithelial) cell lines. In this experimental study, N-butanol and dichloromethane fractions of N. sativa were obtained, and ACHN and GP293 cell lines were treated with various concentrations of dichloromethane (0-100 μg/mL) and N-butanol (0-12.5 μg/mL) fractions for 24, 48, and 72 hours. Then, morphological changes, viability, and apoptosis were investigated. Our results indicated that dichloromethane and N-butanol fractions cause morphological changes and significant decreases in the percentage of live cells in the ACHN cell line, in a dose- and time-dependent manner. In the GP-293 cell line, however, a lower toxicity was observed in comparison with that found for ACHN. The results of flow cytometry showed an apoptotic effect of dichloromethane and N-butanol fractions on the ACHN cell line but a higher rate of apoptosis induction for the total extract compared to the two fractions in the renal cancer cell line compared to the normal cell line. Our findings demonstrated that these two fractions of N. sativa induce inhibitory effects on the ACHN cell line morphology and viability. These effects were lower than those induced by the total extract. In addition, the two fractions caused more marked effects in the renal cancer cell line compared with the GP-293 cell line.

Evaluation of Apoptosis, Cell Proliferation and Cell Cycle Progression by Inactivation of the NEAT1 Long Noncoding RNA in a Renal Carcinoma Cell Line Using CRISPR/Cas9.

Long noncoding RNAs (lncRNAs) play an important role in cellular mechanisms including transcription, translation, and apoptosis. NEAT1 is one of the essential types of lncRNAs in humans that can bind to active genes and modify their transcription. NEAT1 upregulation in various forms of cancer such as kidney cancer has been reported. Kidney cancer accounts for approximately 3% of all cancers worldwide and occurs almost twice as often in men as in women. This study has been performed to knockout the NEAT1 gene using the CRISPR/Cas9 technique in the Renal Cell Carcinoma ACHN cell line and to evaluate its effects on cancer progression and apoptosis. Two specific (single guide RNA (sgRNA) sequences for the NEAT1 gene were designed by CHOPCHOP software. These sequences were then cloned into plasmid pSpcas9, and recombinant vectors PX459-sgRNA1 and PX459-sgRNA2 were generated. ACHN cells were transfected using recombinant vectors carrying sgRNA1 and sgRNA2. The expression level of apoptosis-related genes was assessed by real-time PCR. Annexin, MTT and cell scratch tests were performed to evaluate the survival, proliferation, and migration of the knocked out cells, respectively. The results have shown successful knockout of the NEAT1 gene in the cells of the treatment group. Expressions of P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed significant increases in expression compared to the cells of the control group (P <0.01). Additionally, decreased expression of BCL2 and survivin genes was observed in knockout cells compared to the control group (p <0.05). In addition, in the cells of the treatment group compared to control cells, a significant decrease in cell viability, ability to migrate and cell growth and proliferation was observed. Inactivation of the NEAT1 gene in ACHN cell line using CRISPR/Cas9 technology elevated apoptosis and reduced cell survival and proliferation which makes it a novel target for kidney cancer therapeutics.

Deciphering key genes involved in cisplatin resistance in kidney renal clear cell carcinoma through a combined in silico and in vitro approach.

The low survival rate of Kidney renal clear cell carcinoma (KIRC) patients is largely attributed to cisplatin resistance. Rather than focusing solely on individual proteins, exploring protein-protein interactions could offer greater insight into drug resistance. To this end, a series of in silico and in vitro experiments were conducted to identify hub genes in the intricate network of cisplatin resistance-related genes in KIRC chemotherapy. The genes involved in cisplatin resistance across KIRC were retrieved from the National Center for Biotechnology Information (NCBI) database using search terms as "Kidney renal clear cell carcinoma" and "Cisplatin resistance". The genes retrieved were analyzed for hub gene identification using the STRING database and Cytoscape tool. Expression and promoter methylation profiling of the hub genes was done using UALCAN, GEPIA, OncoDB, and HPA databases. Mutational, survival, functional enrichment, immune cell infiltration, and drug prediction analyses of the hub genes were performed using the cBioPortal, GEPIA, GSEA, TIMER, and DrugBank databases. Lastly, expression and methylation levels of the hub genes were validated on two cisplatin-resistant RCC cell lines (786-O and A-498) and a normal renal tubular epithelial cell line (HK-2) using two high throughput techniques, including targeted bisulfite sequencing (bisulfite-seq) and RT-qPCR. A total of 124 genes were identified as being associated with cisplatin resistance in KIRC. Out of these genes, MCL1, IGF1R, CCND1, and PTEN were identified as hub genes and were found to have significant (p < 0.05) variations in their mRNA and protein expressions and effects on the overall survival (OS) of the KIRC patients. Moreover, an aberrant promoter methylation pattern was found to be associated with the dysregulation of the hub genes. In addition to this, hub genes were also linked with different cisplatin resistance-causing pathways. Thus, hub genes can be targeted with Alvocidib, Estradiol, Tretinoin, Capsaicin, Dronabinol, Metribolone, Calcitriol, Acetaminophen, Acitretin, Cyclosporine, Azacitidine, Genistein, and Resveratrol drugs. As the pathogenesis of KIRC is complex, targeting hub genes and associated pathways involved in cisplatin resistance could bring a milestone change in the drug discovery and management of drug resistance, which might uplift overall survival among KIRC patients.

Amelioration of Cyclosporine A-Induced Acute Nephrotoxicity by Cordyceps cicadae Mycelia via Mg+2 Reabsorption and the Inhibition of GRP78-IRE1-CHOP Pathway: In Vivo and In Vitro.

Fruiting bodies of Cordyceps cicadae (CC) have been reported to have a therapeutic effect in chronic kidney disease. Due to the rare and expensive resources from natural habitats, artificially cultivated mycelia using submerged liquid cultivation of CC (CCM) have been recently developed as an alternative to scarce sources of CC. However, little is known regarding potential protective effects of CCM against cyclosporine A (CsA)-induced acute nephrotoxicity in vivo and in vitro. In this study, male Sprague-Dawley rats were divided into six groups: control, CCM (40 mg and 400 mg/kg, orally), CsA (10 mg/kg, oral gavage), and CsA + CCM (40 mg and 400 mg/kg, orally). At the end of the study on day 8, all rats were sacrificed, and the blood and kidneys retrieved. CsA-induced acute nephrotoxicity was evident by increased levels of blood urea nitrogen (BUN). Levels of the endoplasmic reticulum (ER) resident chaperone glucose regulated protein 78 (GRP 78) were increased significantly in rats with acute nephrotoxicity. BUN and GRP 78 were significantly ameliorated in synchronous oral groups of CCM (40 or 400 mg/kg) plus CsA. Examination of hematoxylin and eosin stained kidney tissues revealed that the combined treatment of CCM slightly improved vacuolization in renal tubules upon CsA-induced damage. CsA-induced down-regulation of protein expression of magnesium ion channel proteins and transient receptor potential melastatin 6 and 7 were abolished by the combined treatment of CCM. CCM has the potential to protect the kidney against CsA-induced nephrotoxicity by reducing magnesium ion wasting, tubular cell damage, and ER stress demonstrated further by human renal proximal tubular epithelial cell line HK-2. Our results contribute to the in-depth understanding of the role of polysaccharides and nucleobases as the main secondary metabolites of CCM in the defense system of renal functions in CsA-induced acute nephrotoxicity.

LncRNA MIAT Modulates LPS-Induced Acute Kidney Injury via BECN1-Dependent Autophagy by Interacting with PTBP1.

Autophagy plays critical adaptive and nonadaptive roles in the pathogenesis of Sepsis-associated acute kidney injury (Sepsis-AKI). However, it remains unknown whether myocardial infarction associated transcript (MIAT) is involved in the process of autophagy in Sepsis-AKI. This study aimed to explore the exact association between MIAT1 and Beclin 1 (BECN1)-mediated autophagy in Sepsis-AKI in vitro. HK-2 (human renal tubular epithelial cell line) cells were stimulated by lipopolysaccharide (LPS) to construct a septic kidney injury cell model in vitro. The relative expression changes of genes or proteins in clinical samples and cells were examined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell survival was detected by cell counting kit-8 (CCK-8) and flow cytometry analysis. The production of inflammatory mediators was determined using Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assays. The interlinked relationship between polypyrimidine tract-binding protein 1 (PTBP1) and MIAT or BECN1 was validated by RNA immunoprecipitation (RIP) and RNA pull-down detections. The expression of MIAT was up-regulated in Sepsis-AKI patients and LPS-stimulated HK-2 cells. Down-regulation of MIAT strikingly lightened LPS-induced cell apoptosis and inflammation, but enhanced cell viability. Evidenced by mechanistic experiments, MIAT silencing was confirmed to activate BECN1-mediated cell autophagy by interacting with PTBP1. Furthermore, the elimination of BECN1 remarkably reversed the antiapoptotic and anti-inflammatory roles mediated by MIAT silencing. In summary, the experimental data reinforced that MIAT downregulation attenuated LPS-stimulated renal cell inflammatory injury by promoting BECN1-mediated autophagy activation through binding to PTBP1, providing some new insights into the function and mechanism of MIAT in Sepsis-associated acute kidney injury (Sepsis-AKI).

The Anti-Tumor Effect of Zinc on Renal Cell Carcinoma by Enhancing Autophagy

Renal cell carcinoma remains one of the most common malignant tumorsand is associated with significant challenges in drug therapy. Zinc has been reported to be of pharmacological importance and immense potential in the inhibition of renal cell carcinoma cells. In order to investigate the inhibitory effect of zinc on the human renal cell carcinoma cell line A498, its anti-proliferative activity was investigated using the CCK8 assay. The oxidative stress status and mitochondrial injury were detected by DCFH-DA and JC-1 staining, respectively. The expression of autophagy-related proteins was determined by western blot analysis, which aimed at exploring the antioxidant mechanisms of zinc. Over 40% yield of ROS and more than four times the number of autophagosomes were detected in the experimental group treated by zinc compared to the control group. Furthermore, zinc led to up-regulation of the Beclin 1-related signaling pathway in the late stage of autophagy degradation. Zinc demonstrated a good inhibitory effect on A498 cells with the value of IC50similar to the values of natural products. One of the mechanisms of zinc is enhancing the Beclin 1-dependent autophagy. Taken together, with its low cost and natural properties, zinc could become a promising molecule with satisfied anti-tumor effect and low cost in the treatment of renal cell carcinoma.

A new thinking: deciphering the aberrance and clinical implication of copper-death signatures in clear cell renal cell carcinoma

RationaleRecent research has indicated that cuprotosis, or copper induced cell death, is a novel type of cell death that could be utilized as a new weapon for cancer management. However, the characteristics and implications of such signatures in cancers, especially in clear cell renal cell cancer (ccRCC), remain elusive.MethodsExpression, methylation, mutation, clinical information, copy number variation, functional implication, and drug sensitivity data at the pan-cancer level were collected from The Cancer Genome Atlas. An unsupervised clustering algorithm was applied to decipher ccRCC heterogeneity. Immune microenvironment construction, immune therapy response, metabolic pattern, and cancer progression signature between subgroups were also investigated.ResultsCuprotosis related genes were specifically downregulated in various cancer tissues compared with normal tissues and were correlated with hypermethylation and copy number variation. Cuprotosis scores were also dysregulated in tumor tissues, and we found that such a signature could positively regulate oxidative phosphorylation and Myc and negatively regulate epithelial mesenchymal translation and myogenesis pathways. CPCS1 (cuprotosis scores high) and CPCS2 (cuprotosis scores low) in ccRCC displayed distinctive clinical profiles and biological characteristics; the CPCS2 subtype had a higher clinical stage and a worse prognosis and might positively regulate cornification and epidermal cell differentiation to fuel cancer progression. CPCS2 also displayed a higher tumor mutation burden and low tumor stemness index, while it led to a low ICI therapy response and dysfunctional tumor immunity state. The genome-copy numbers of CPCS2, including arm- gain and arm- loss, were higher than those of CPCS1. The prognostic model constructed based on subgroup biomarkers exerted satisfactory performance in both the training and validation cohorts. In addition, overexpression of the copper death activator DLAT suppressed the malignant ability, including cell migration and proliferation, of renal cell lines in vitro and in vivo. Finally, activation of cuprotosis in tumors could enhance antitumor immunity through dsDNA-cGAS-STING signaling in ccRCC.ConclusionThe activation of cuprotosis might function as a promising approach among multiple cancers. The cuprotosis related signatures could reshape tumor immunity in the ccRCC microenvironment via cGAS-STING signal, thus activating tumor antigen-presenting process. Upregulation of DLAT expression in ccRCC cell lines could reactivate the copper death pattern and be treated as a suitable target for ccRCC.

Heparin prevents the cytotoxic activity of Bothrops jararacussu and Apis mellifera venoms in renal cells

Envenomation by Bothrops snakes and Apis mellifera bee may imply systemic disorders which affect well-perfused organs such as kidneys, a process that can lead to acute renal failure. Nevertheless, there is scarce information regarding a direct renal cell effect and the putative antagonism by antivenoms. Here the cytotoxic effect of B. jararacussu and A. mellifera venoms was evaluated in the renal proximal tubule cell line LLC-PK1, as well as the antagonism of this effect by heparin. B. jararacussu venom showed significant cytotoxicity as assessed by LDH release and MTT reduction, with a sharp decline of the cell number after 180 min (>90% at 50 μg/mL). A. mellifera venom produced a much faster and potent cytotoxic activity, conferring almost no viable cells after 15 min at 25 μg/mL. Phase contrast microscopy revealed that while B. jararacussu venom induced a progressive loss of cell adhesion and detachment, A. mellifera venom promoted a rapid plasma membrane disruption and nuclear condensation suggestive of necrotic cell death. Pre-incubation of both venoms with heparin for 30 min significantly reduced cytotoxicity. Our results demonstrate direct toxicity of B. jararacussu and A. mellifera venoms toward renal cells but with distinct kinetics and cell pattern, suggesting different mechanisms of action. In addition, the antagonistic, cytoprotective effect of heparin ascribes such compound as a promising drug for preventing renal failure from envenomation.