Human Kidney Epithelial Cells Research Articles

Enhanced stability and dissolution of curcumin nanocrystals stabilized by octenyl succinic anhydride modified starch

Curcumin (CUR) has known to have poor oral bioavailability due to its poor aqueous solubility and stability in gastrointestinal fluid condition as well as its limited permeability which greatly limits its applications in food and medical fields. The present study reported the development of CUR nanocrystal suspension (CUR-NS) for enhanced bioavailability, stabilized with octenyl succinic anhydride modified starch (OSA-S), a naturally renewable and biocompatible biomolecule. This preparation was achieved via an antisolvent precipitation combined with ultrasonic dispersion method, following which the suspension was freeze-dried to form powder CUR nanocrystals (CUR-NCs). Physicochemical properties of the prepared CUR-NS and CUR-NCs powders were extensively characterized. The particle size of the nanocrystals was found to be around 322.8 ± 15.7 nm with a small polydispersity index (PDI) of about 0.230 ± 0.039. Rod like crystal shape was observed by the SEM. Results from powder X-ray diffraction (pXRD) and differential scanning calorimetry (DSC) indicated that the crystal form of CUR in CUR-NCs was a monoclinic polymorph Form 2. Stability results indicated that CUR-NCs possessed an excellent storage-, thermal-, and photo-stabilities. CUR-NCs had a significantly higher dissolution rate, a 68-fold increase in its solubility than that of raw CUR. The cytotoxicity assay demonstrated that CUR-NCs exhibited lower toxicity towards normal human kidney epithelial cells compared to raw CURs. Parallel artificial membrane permeability assay (PAMPA) characterization confirmed that the flux of CUR was significantly enhanced after forming nanocrystals with OSA-S, probably resulting from its increased solubility and dissolution rate. The study showed that OSA-S could effectively stabilize CUR-NCs, and CUR-NCs developed in the current work exhibited significant enhancement in the stability and dissolution rate leading to an improved bioavailability which might be helpful for further development of CUR based products.

DNA binding, potential anticancer, antioxidant and molecular docking simulations of some isonicotinohydrazide metal complexes with the impact of high energy γ-rays irradiation doses: Synthesis and structural characterization

This work focuses on the preparation and characterization of some VO(II), Co(II), Cu(II), Zn(II) and Hg(II) complexes (1–5) of N'-(2-aminobenzoyl)isonicotinohydrazide ligand (HL). The compounds were characterized using various analytical and spectral tools. The resulting powder samples of the as prepared complexes (3,5) were exposed to two gamma ray doses up to 150 and 200 kGy from highly-energetic 60Co γ-irradiation (hereafter referred to as (3F,5F) at dose value of 150 kGy and (3F*,5F*) at dose value of 200 kGy). The impact of γ-irradiation on the possible structural changes of these samples after gamma irradiation was studied. The ligand and as prepared Cu(II) (3), Hg(II) (5) and their γ-irradiated (3F,5F) and (3F*,5F*) samples were tested for free radical scavenging activity using the DPPH assay. The DNA cleavage activity using supercoiled plasmid DNA (calf thymus ct-DNA) was explored. The tested ligand (HL) and complexes (3,5) showed excellent DNA binding. Additionally, the in vitro antitumor activity of (HL) and its Cu(II) (3) and Hg(II) (5) complexes was scrutinized against the human breast carcinoma (MCF-7), human hepatocarcinoma (Hep-G2) cell lines and kidney epithelial normal cells extracted from African green monkey kidney (Vero) to investigate the potential safety towards the normal cells. Also, docking simulation was done on the active site of 4FM9 and 1H7K protein for liver cancer and breast cancer cell line, respectively and showed excellent results.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Epigallocatechin-3-gallate attenuates arsenic-induced fibrogenic changes in human kidney epithelial cells through reversal of epigenetic aberrations and antioxidant activities.

Renal fibrosis is a pathogenic intermediate stage of chronic kidney disease (CKD). Nephrotoxicants including arsenic can cause kidney fibrosis through induction of oxidative stress and epigenetic aberrations. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, is known to have antioxidant and epigenetic modulation properties. Whether EGCG, through its antioxidant and epigenetic modulating activities, can attenuate fibrogenesis is not known. Therefore, the objective of this study was to determine whether EGCG can attenuate arsenic-induced acute injury and long-term exposure associated fibrogenicity in kidney epithelial cells. To address this question, two human kidney epithelial cell lines Caki-1 and HK-2 exposed to arsenic for both acute and long-term durations were treated with EGCG. The protective effect of EGCG on arsenic-induced cytotoxicity and fibrogenicity were evaluated by measuring the cell growth, reactive oxygen species (ROS) production, genes expression, and epigenetic changes in histone marks. Results revealed that EGCG has a protective effect in arsenic-induced acute cytotoxicity in these cells. EGCG scavenges the increased levels of ROS in arsenic exposed cells. Aberrant expression of fibrogenic genes in arsenic exposed cells were restored by EGCG. Abrogation of arsenic-induced fibrogenic changes was also associated with EGCG-mediated restoration of arsenic-induced aberrant expression of epigenetic regulatory proteins and histone marks. Novel findings of this study suggest that EGCG, through its antioxidant and epigenetic modulation capacities, has protective effects against arsenic-induced cytotoxicity and fibrogenic changes in kidney epithelial cells.

Abstract B104: U18666A-induced cholesterol accumulation decreases tumor-derived exosomes load and modulates malignant transformation

Abstract Background: Tumor-derived exosomes (TDEs) play an important role in the horizontal transfer of oncogenic information from the tumor to other sites, where they regulate pre-metastatic niche formation, organotropism, migration, invasion and survival. Cellular cholesterol homeostasis emerges as an important factor for exosomal biogenesis and cellular release. In this study, we utilized U18666A to trigger cholesterol accumulation within late endosomes of MDA-MB231 cells and evaluated the ability of exosomes derived from both U18666A-treated and untreated cells to initiate malignant transformation in recipient human epithelial kidney (HEK293) cells. Methods: Exosomes were isolated and characterized from MDA-MB231 (untreated and U18666A-treated) and HEK293 cells. Cholesterol content of the cells and their associated exosomes were measured to confirm its accumulation following U18666A treatment. The effects of exosomes derived from untreated MDA-MB231 (UCE) and U18666A-treated MDA-MB231 (UTCE) cells on HEK293 cells were examined to determine their transforming potential. Results: Exosomes derived from MDA-MB231 cells induced proliferation, migration, malignant transformation and epithelial-mesenchymal transition (EMT) process in non-cancerous recipients’ cells. U18666A treatment led to accumulation of cholesterol within late endosomes and significantly reversed the EMT process in MDA-MB231 cells. It also reduced TAEs load from the cancer cells and rendered them less oncogenic which was evident from their inability to induce malignant transformation in the recipient HEK293 cells. Conclusion: Modulation of cholesterol homeostasis and disruption of cholesterol supply to aggressive cancer cells can be an attractive strategy for limiting TAEs release as well as their subsequent role in cancer progression and metastasis. Citation Format: Syed Sultan Beevi, Vinod Kumar Verma. U18666A-induced cholesterol accumulation decreases tumor-derived exosomes load and modulates malignant transformation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B104.

Enhanced fatty acid oxidation through metformin and baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-β-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-β-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage.

Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

Malignant transformation of human cells is associated with their re-programming which results in uncontrolled proliferation and in the same time biochemical activation of immunosuppressive pathways which form cancer immune evasion machinery. However, there is no conceptual understanding of whether immune evasion machinery pathways and expression of immune checkpoint proteins form a part of the process of malignant transformation or if they are triggered by T lymphocytes and natural killers (NK) attempting to attack cells which are undergoing or already underwent malignant transformation. To address this fundamental question, we performed experimental malignant transformation of BEAS-2B human bronchial epithelium cells and RC-124 non-malignant human kidney epithelial cells using bracken extracts containing carcinogenic alkaloid called ptaquiloside. This transformation led to a significant upregulation of cell proliferation velocity and in the same time led to a significant upregulation in expression of key immune checkpoint proteins – galectin-9, programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO1). Their increased expression levels were in line with upregulation of the levels and activities of HIF-1 transcription complex and transforming growth factor beta type 1 (TGF-β)-Smad3 signalling pathway. When co-cultured with T cells, transformed epithelial cells displayed much higher and more efficient immune evasion activity compared to original non-transformed cells. Therefore, this work resolved a very important scientific and clinical question and suggested that cancer immune evasion machinery is activated during malignant transformation of human cells regardless the presence of immune cells in microenvironment.

CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells

ABSTRACT Background CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. Methods We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. Results In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells’ normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. Conclusion APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.

Combination Organelle Mitochondrial Endoplasmic Reticulum Therapy (COMET) for Multidrug Resistant Breast Cancer

It is time for the story of mitochondria and intracellular communication in multidrug resistant cancer to be rewritten. Herein we characterize the extent and cellular advantages of mitochondrial network fusion in multidrug resistant (MDR) breast cancer and have designed a novel nanomedicine that disrupts mitochondrial network fusion and systematically manipulates organelle fusion and function. Combination Organelle Mitochondrial Endoplasmic reticulum Therapy (COMET) is an innovative translational nanomedicine for treating MDR triple negative breast cancer (TNBC) that has superior safety and equivalent efficacy to the current standard of care (paclitaxel). Our study has demonstrated that the increased mitochondrial networks in MDR TNBC contribute to apoptotic resistance and network fusion is mediated by mitofusin2 (MFN2) on the outer mitochondrial membrane. COMET consists of three components; Mitochondrial Network Disrupting (MiND) nanoparticles (NPs) that are loaded with an anti-MFN2 peptide, tunicamycin, and Bam7. The therapeutic rationale of COMET is to reduce the apoptotic threshold in MDR cells with MiND NPs, followed by inducing the endoplasmic reticulum mediated unfolded protein response (UPR) by stressing MDR cells with tunicamycin, and finally, directly inducing mitochondrial apoptosis with Bam7 which is a specific bcl-2 Bax activator. MiND NPs are PEGylated liposomes with the 21 amino acid (2577.98 MW) anti-MFN2 peptide compartmentalized in the aqueous core. Hypoxia (0.5% oxygen) was used to create MDR derivatives of MDA-MB-231 cells and BT-549 cells. Mitochondrial networks were quantified using 3D analysis of 60× live cell images acquired with a Keyence BZ-X710 microscope and MiND NPs effectively fragmented mitochondrial networks in drug sensitive and MDR TNBC cells. The IC50 values, combination index, and dose reduction index derived from dose response studies demonstrate that MiND NPs decrease the apoptotic threshold of both drug sensitive and MDR TNBC cells and COMET is a synergistic drug combination. Complex V (ATP synthase) extracted from bovine cardiac mitochondria was used to assess the effect of MiND NPs on OXPHOS; both MiND NPs and anti-MFN2 peptide solution significantly decrease the activity of mitochondrial complex V and decrease the capacity of OXPHOS. A BacMam viral vector based fluorescent biosensor was used to quantify the unfolded protein response (UPR) at the level of the endoplasmic reticulum and tunicamycin specifically induces the UPR in drug sensitive and MDR TNBC cells. A caspase 3 colorimetric assay demonstrated that the synergistic triple drug combination of COMET increases the ability of Bam7 to specifically induce apoptosis. Dose limiting toxicity and off target effects are a significant challenge for current chemotherapy regimens including paclitaxel. COMET has significantly lower cytotoxicity than paclitaxel in human embryonic kidney epithelial cells and has the potential to fulfill the clinical need for safer cancer therapeutics. COMET is a promising early stage translational nanomedicine for treating MDR TNBC. Manipulating intracellular communication and organelle fusion is a novel approach to treating MDR cancer. The data from this study has rewritten the story of mitochondria, organelle fusion, and intracellular communication and by targeting this intersection, COMET is an exciting new chapter in cancer therapeutics that could transform the clinical outcome of MDR TNBC.

90 Complement activation during hypoxia-reoxygenation in human kidney epithelial cells

90 Complement activation during hypoxia-reoxygenation in human kidney epithelial cells

RhMYDGF Alleviates I/R-induced Kidney Injury by Inhibiting Inflammation and Apoptosis via the Akt Pathway.

Renal ischemia/reperfusion (I/R) injury is one of the crucial factors affecting the outcome of renal transplantation. In recent years, myeloid-derived growth factor (MYDGF) has received a lot of attention for its extensive beneficial effects on cardiac repair and protection of cardiomyocytes from cell death. Therefore, we hypothesized that the recombinant human MYDGF (rhMYDGF) protein might play an essential role in safeguarding renal I/R injury. In vivo experiments were conducted using a mouse unilateral I/R model. Mice were pretreated with rhMYDGF by intraperitoneal injection to study the potential mechanism of renal protection. In vitro, we established hypoxia/reoxygenation and H 2 O 2 treatment models to pretreat cells with rhMYDGF. The expression levels of oxidative stress, inflammation, and apoptosis-related factors in tissues and cells were detected. Finally, we explored the role of the protein kinase B (Akt) pathway in the renal protective mechanism of rhMYDGF. In this study, we found that intraperitoneal injection of 1.25 μg rhMYDGF could significantly improve renal function of I/R mice, and reduce oxidative stress, inflammation, and apoptosis. For the human proximal tubular epithelial cell line and human kidney cell line, pretreatment with 0.3 μg/mL rhMYDGF for 24 h significantly downregulated oxidative stress, inflammation, and apoptosis via the phosphorylation of Akt, which could be ameliorated by LY294002. rhMYDGF protects kidney from I/R injury by attenuating oxidative stress, inflammation, and apoptosis through the activation of the Akt pathway.

OCT4 induces long-lived dedifferentiated kidney progenitors poised to redifferentiate in 3D kidney spheroids

Upscaling of kidney epithelial cells is crucial for renal regenerative medicine. Nonetheless, the adult kidney lacks a distinct stem cell hierarchy, limiting the ability to long-term propagate clonal populations of primary cells that retain renal identity. Toward this goal, we tested the paradigm of shifting the balance between differentiation and stemness in the kidney by introducing a single pluripotency factor, OCT4. Here we show that ectopic expression of OCT4 in human adult kidney epithelial cells (hKEpC) induces the cells to dedifferentiate, stably proliferate, and clonally emerge over many generations. Control hKEpC dedifferentiate, assume fibroblastic morphology, and completely lose clonogenic capacity. Analysis of gene expression and histone methylation patterns revealed that OCT4 represses the HNF1B gene module, which is critical for kidney epithelial differentiation, and concomitantly activates stemness-related pathways. OCT4-hKEpC can be long-term expanded in the dedifferentiated state that is primed for renal differentiation. Thus, when expanded OCT4-hKEpC are grown as kidney spheroids (OCT4-kSPH), they reactivate the HNF1B gene signature, redifferentiate, and efficiently generate renal structures invivo. Hence, changes occurring in the cellular state of hKEpC following OCT4 induction, long-term propagation, and 3D aggregation afford rapid scale-up technology of primary renal tissue-forming cells.

Potent saccharinate-containing palladium(II) complexes for sensitization to cancer therapy

Palladium(II) (PdII) complexes are among the most promising anticancer compounds. Both 2`-benzoylpyridine thiosemicarbazone (BpT) and saccharinate (Sac) are efficient metal chelators with potent anticancer activity. To explore a more effective new anticancer drug, we synthesized a series of Sac and BpT-containing PdII complexes coordinated with thiosemicarbazone (TSC)-derived ligands, and characterized them through nuclear magnetic resonance (NMR), Fourier transformed infrared spectroscopy (FT-IR), elemental analysis, ultraviolet-visible spectroscopy (UV–Vis) and thermogravimetric analysis (TGA). Each target complex was composed of PdII, BpT, and one or two Sac molecules. Both the in vitro and in vivo anti-growth effects of those ligands and the obtained PdII complexes were investigated in the human lung adenocarcinoma cell lines A549 and Spc-A1. The coordination of PdII with the TSC-derivatives and Sac resulted in clearly greater anticancer activity than single ligands. These compounds were demonstrated to be safe for 293 T normal human kidney epithelial cells. The introduction of Sac into the TSC-derived PdII complex significantly enhanced anti-growth effects, and induced apoptosis of human lung cancer cells in vitro and in vivo in a dose dependent manner. Moreover, the PdII complex containing two Sac molecules showed the most promising therapeutic effects, thereby confirming that Sac increases the cancer therapeutic efficacy of PdII complexes and providing a new strategy for exploring anticancer drugs for potential clinical treatment.

Identification of an unrecognized circRNA associated with development of renal fibrosis.

Backgroud: Renal fibrosis is the common characteristic of chronic kidney disease. Circular RNA plays an essential role in the occurrence and development of Renal fibrosis, but its regulative mechanism remains elusive. Methods: The animal and cell model of Renal fibrosis was established, and RNA-sequencing and real-time polymerase chain reaction (qRT-PCR) experiments were implemented. Subsequently, experiments for detecting apoptosis and proliferation of cell, were carried out, and the isobaric tags for relative and absolute quantification proteomics analyses were performed accordingly. Results: It was found that a newly discovered Circular RNA (circRNA_0002158), is highly expressed in kidneys or cells with fibrosis, implying that this Circular RNA might be associated with the occurrence and development of Renal fibrosis. Subsequently, the overexpression and knockdown of circRNA_0002158 were conducted in the human kidney epithelial cell line (HK-2) cells, and the results indicated that the circRNA_0002158 could inhibit apoptosis, and promote proliferation of cells. The kidney injury-related factors, including Fibronectin and plasminogen activator inhibitor-1 (PAI-1), were decreased in HK-2 cells with overexpression of circRNA_0002158, while the results were reversed in cells with knockdown of circRNA_0002158. Finally, to explore the regulative mechanism of circRNA_0002158, the iTRAQ proteomics analyses were implemented for the cell samples with OE of circRNA_0002158 and its control, it showed that multiple genes and functional pathways were associated with the occurrence and development of Renal fibrosis. Conclusion: CircRNA_0002158 is associated with regulating Renal fibrosis, and may contribute to ameliorating the progression of Renal fibrosis in the future.

Overexpressed c-Myc Sensitizes Cells to TH1579, a Mitotic Arrest and Oxidative DNA Damage Inducer.

Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation.

Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.

Chloride channel-3 (ClC-3) Cl-/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A, and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labeled vesicles were sorted to MVBs labeled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.

Targeting of Heparanase in the Murine Model Protects against Radiation-Induced Nephropathy

<h3>Purpose/Objective(s)</h3> Adverse effects are common in virtually all types of treatment modalities in cancer including that of radiation therapy. Several agents are clinically used to aid in the reduction of side effects of anti-cancer therapy, however these agents <i>per se</i> are typically not endowed with tumor-suppressing properties. Herein we report that the extracellular matrix (ECM)-degrading enzyme heparanase (a valid target for anti-cancer drug development) is also a promising candidate for interventions aimed at preventing development of radiation nephropathy - a significant side effect of radiation therapy in the treatment of abdominal/pelvic malignancies. Heparanase is the sole mammalian endoglucuronidase that cleaves heparan sulfate in the ECM. The enzymatic action of heparanase has not only been found to promote tumor development and progression at various anatomic sites but is upregulated in essentially all tumor types found in the abdominal/pelvic cavity, whose radiation treatment may result in the development of radiation nephropathy. <h3>Materials/Methods</h3> Eleven-week-old female C3H/HeNHsd mice were administered 10 Gy of radiation using brachytherapy to induce radiation nephropathy. Subcutaneous injections of Roneparstat, a specific heparanase inhibitor, were administered to the mice and urine samples were collected over 24h. Urinary albumin was measured using an ELISA kit. Prior to and following irradiation, heparanase and early growth response (Egr1) transcription factor mRNA levels were determined by qRT-PCR in both HK-2 human proximal tubule epithelial cells and HEK-293 human embryonic kidney cells. <h3>Results</h3> In this study, we evaluated the potential of targeting heparanase to diminish radiation therapy-induced kidney damage. We demonstrated that clinically relevant doses of ionizing radiation upregulate heparanase expression in renal cells (most likely through a transcription factor Egr 1 –dependent mechanism). Moreover, utilizing a murine model of abdominal radiation therapy, we found that the specific heparanase inhibitor, Roneparstat, abolished radiation-induced albuminurea –the hallmark of radiation nephropathy. <h3>Conclusion</h3> Given the well-documented anti-cancer effects of heparanase inhibition, our present findings attest this enzyme as a unique target in the landscape of cancer therapy due to its dual action. Indeed, targeting of heparanase exerts not only direct anti-tumor effects but also protects from kidney damage induced by radiation - the backbone of cancer therapy across a broad range of abdominal/pelvic malignancies.

Significance of chlorine-dioxide-based oral rinses in preventing SARS-CoV-2 cell entry.

ObjectiveThis work aims to determine the efficacy of preprocedural oral rinsing with chlorine dioxide solutions to minimize the risk of coronavirus disease 2019 (COVID‐19) transmission during high‐risk dental procedures.MethodsThe antiviral activity of chlorine‐dioxide‐based oral rinse (OR) solutions was tested by pre‐incubating with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pseudovirus in a dosage‐dependent manner before transducing to human embryonic kidney epithelial (HEK293T‐ACE2) cells, which stably expresses ACE‐2 receptor. Viral entry was determined by measuring luciferase activity using a luminescence microplate reader. In the cell‐to‐cell fusion assay, effector Chinese hamster ovary (CHO‐K1) cells co‐expressing spike glycoprotein of SARS‐CoV‐2 and T7 RNA polymerase were pre‐incubated with the ORs before co‐culturing with the target CHO‐K1 cells co‐expressing human ACE2 receptor and luciferase gene. The luciferase signal was quantified 24 h after mixing the cells. Surface expression of SARS‐CoV‐2 spike glycoprotein and ACE‐2 receptor was confirmed using direct fluorescent imaging and quantitative cell‐ELISA. Finally, dosage‐dependent cytotoxic effects of ORs were evaluated at two different time points.ResultsA dosage‐dependent antiviral effect of the ORs was observed against SARS‐CoV‐2 cell entry and spike glycoprotein mediated cell‐to‐cell fusion. This demonstrates that ORs can be useful as a preprocedural step to reduce viral infectivity.ConclusionsChlorine‐dioxide‐based ORs have a potential benefit for reducing SARS‐CoV‐2 entry and spread.

Endoplasmic-reticulum-stress-induced lipotoxicity in human kidney epithelial cells.

Accumulation of lipids and their intermediary metabolites under endoplasmic reticulum (ER) stress instigates metabolic failure, described as lipotoxicity, in the kidney. This study aimed to determine ER-stress-related sphingolipid and polyunsaturated fatty acid (PUFA) changes in human kidney cells. Tunicamycin (TM) was employed to induce ER stress and an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was given to minimize cytotoxicity. Cell viability was determined by MTT assay. Sphingomyelin (SM), ceramide (CER), and PUFA levels were measured by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78), cleaved caspase-3 and cyclooxygenase-1 (COX-1) levels were assessed by immunofluorescence. Cytosolic phospholipase A2 (cPLA2), total COX, and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. Decreased cell viability was observed in TM treated cells. Administration of TUDCA following TM treatment significantly increased cell viability compared to TM treatment alone. Tunicamycin-induced ER stress was confirmed by significantly increased protein levels of GRP78. A significant increase was observed in C18-C24 CERs and caspase-3 activity, while a significant decrease occurred in sphingosine-1-phosphate (S1P) and cPLA2 activity in cells treated with TM versus controls. The decrease in cPLA2 activity was accompanied by significantly increased PUFA levels in TM treated cells. TUDCA treatment in conjunction with TM significantly decreased ER stress, C18-C24 CERs, caspase 3 activity, and increased S1P levels. Results show the buildup of long chain CERs and PUFAs in kidney cells undergoing ER stress alongside increased apoptotic activity. TUDCA administration, along with TM treatment alleviated the buildup of CERs and TM-induced apoptotic activity in kidney epithelial cells.

Enterohemorrhagic Escherichia coli and a Fresh View on Shiga Toxin-Binding Glycosphingolipids of Primary Human Kidney and Colon Epithelial Cells and Their Toxin Susceptibility.

Enterohemorrhagic Escherichia coli (EHEC) are the human pathogenic subset of Shiga toxin (Stx)-producing E. coli (STEC). EHEC are responsible for severe colon infections associated with life-threatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological disturbances. Endothelial cells in various human organs are renowned targets of Stx, whereas the role of epithelial cells of colon and kidneys in the infection process has been and is still a matter of debate. This review shortly addresses the clinical impact of EHEC infections, novel aspects of vesicular package of Stx in the intestine and the blood stream as well as Stx-mediated extraintestinal complications and therapeutic options. Here follows a compilation of the Stx-binding glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) and their various lipoforms present in primary human kidney and colon epithelial cells and their distribution in lipid raft-analog membrane preparations. The last issues are the high and extremely low susceptibility of primary renal and colonic epithelial cells, respectively, suggesting a large resilience of the intestinal epithelium against the human-pathogenic Stx1a- and Stx2a-subtypes due to the low content of the high-affinity Stx-receptor Gb3Cer in colon epithelial cells. The review closes with a brief outlook on future challenges of Stx research.