Normal Human Kidney Research Articles

Synthesis of M-Ag3PO4, (M = Se, Ag, Ta) Nanoparticles and Their Antibacterial and Cytotoxicity Study.

Silver Phosphate, Ag3PO4, being a highly capable clinical molecule, an ultrasonic method was employed to synthesize the M-Ag3PO4, (M = Se, Ag, Ta) nanoparticles which were evaluated for antibacterial and cytotoxicity activities post-characterization. Escherichia coli and Staphylococcus aureus were used for antibacterial testing and the effects of sonication on bacterial growth with sub-MIC values of M-Ag3PO4 nanoparticles were examined. The effect of M-Ag3PO4 nanoparticles on human colorectal carcinoma cells (HCT-116) and human cervical carcinoma cells (HeLa cells) was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay and DAPI (4′,6-diamidino-2-phenylindole) staining. Additionally, we analyzed the effect of nanoparticles on normal and non-cancerous human embryonic kidney cells (HEK-293). Ag-Ag3PO4 exhibited enhanced antibacterial activity followed by Ta-Ag3PO4, Ag3PO4, and Se-Ag3PO4 nanoparticles against E. coli. Whereas the order of antibacterial activity against Staphylococcus aureus was Ag3PO4 > Ag-Ag3PO4 > Ta-Ag3PO4 > Se-Ag3PO4, respectively. Percentage inhibition of E. coli was 98.27, 74.38, 100, and 94.2%, while percentage inhibition of S. aureus was 25.53, 80.28, 99.36, and 20.22% after treatment with Ag3PO4, Se-Ag3PO4, Ag-Ag3PO4, and Ta-Ag3PO4, respectively. The MTT assay shows a significant decline in the cell viability after treating with M-Ag3PO4 nanoparticles. The IC50 values for Ag3PO4, Se-Ag3PO4, Ag-Ag3PO4, and Ta-Ag3PO4 on HCT-116 were 39.44, 28.33, 60.24, 58.34 µg/mL; whereas for HeLa cells, they were 65.25, 61.27, 75.52, 72.82 µg/mL, respectively. M-Ag3PO4 nanoparticles did not inhibit HEK-293 cells. Apoptotic assay revealed that the numbers of DAPI stained cells were significantly lower in the M-Ag3PO4-treated cells versus control.

Dapagliflozin induces apoptosis by downregulating cFILPL and increasing cFILPS instability in Caki-1 cells.

Dapagliflozin is a sodium/glucose cotransporter 2 inhibitor used recently to treat patients with type 2 diabetes. A recent study has demonstrated that dapagliflozin induces apoptosis in human renal and breast tumor cells. However, to the best of our knowledge, the molecular mechanism underlying dapagliflozin-mediated apoptosis in Caki-1 human renal carcinoma cells has not been elucidated. The present study demonstrated that the dapagliflozin treatment dose-dependently increased cell death in Caki-1 cells. Dapagliflozin treatment also induced apoptosis as confirmed by FITC-conjugated Annexin V/PI staining. Additionally, treatment with dapagliflozin reduced the expression levels of anti-apoptotic proteins, cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein (cFLIP)L and cFLIPS in Caki-1 cells. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone inhibited dapagliflozin-induced apoptosis, implying that dapagliflozin–induced apoptosis is regulated by a caspase-dependent pathway. By contrast, N-acetylcysteine had no effect on dapagliflozin–induced apoptosis and downregulation of cFLIPL and cFLIPS expression. Furthermore, overexpression of cFLIPL, but not cFLIPS, partially inhibited apoptosis induced by dapagliflozin. cFLIPL and cFLIPS mRNA levels remained constant in Caki-1 cells after treatment with 0, 20, 40, 60, 80 and 100 µM dapagliflozin. Notably, it was confirmed that cFLIPS protein levels were reduced due to the increased cFLIPS instability in dapagliflozin-treated Caki-1 cells. The present study also demonstrated that dapagliflozin had no effect on HK-2 normal human kidney cells. Taken together, the present study revealed that dapagliflozin induced apoptosis via the downregulation of cFLIPL and an increase in cFLIPS instability, suggesting that dapagliflozin may be a feasible drug candidate for the treatment of human renal cancer.

The effects of amitriptyline on human embryonic kidney cells growth and proliferation in vitro

Many studies have shown that antidepressants, including amitriptyline, can cause side effects on normal human cells, although the exact effects of amitriptyline on normal human cells are unclear. Accordingly, the present study investigated the cytotoxic effects of amitriptyline on the human embryonic kidney cell line. In this experimental-laboratory study, cells were divided into control group (no drug-treated) and amitriptyline treatment groups (treated with 39.06, 12.78, 156.25, 312.5, 625, 1250 and 2500 μg/ml of amitriptyline). MTT method was used to assay the cytotoxic effects of amitriptyline at 24 and 48 hours. The data were analyzed by one-way analysis of variance. Treatment with 39.06 and 78.12 μg/ml of amitriptyline for 24 hours had no significant effect on the viability of Hek-293 cells, but treatment with 156.25 μg/ml and higher concentrations of amitriptyline significantly reduced the viability of Hek-293 cells. Treatment of cells for 48 hours showed a significant reduction in cell viability at all concentrations. Amitriptyline has cytotoxic effects on normal human embryonic kidney cells and these effects depend on the dose and duration of treatment. The findings of this study can be of significant importance in terms of the side effects of amitriptyline consumption on normal cells of the human body.

Scattered Tubular Cells Markers in Macula Densa of Normal Human Adult Kidney.

Background: The scattered tubular cells (STCs) are a population of resident progenitor tubular cells with expansion, self-renewal and epithelial differentiation abilities. Although these cells are localized within the proximal (PTs) and distal (DTs) tubules in a normal adult kidney, their presence has never been demonstrated in human macula densa (MD). The purpose of the present study is to describe the presence of STCs in MD using specific markers such as prominin-1 (CD133), cytokeratin 7 (KRT7) and vimentin (VIM). Methods: We analyzed two sets of three consecutive serial sections for each sample. The first sections of each set were immunostained for nNOS to identify MD, the second sections were immune-stained for CD133 (specific STCs marker) while the third sections were analyzed for KRT7 (another STCs specific marker) and VIM (that stains the basal pole of the STCs) in the first and second sets, respectively, in order to study the co-expression of KRT7 and VIM with the CD133 marker. Results: CD133 was localized in some MD cells and in the adjacent DT cells. Moreover, CD133 was detected in the parietal epithelial cells of Bowman’s capsule and in some proximal tubules (PT). KRT7-positive cells were identified in MD and adjacent DT cells, while KRT7 positivity was mostly confined in both DT and collecting ducts (CD) in the other areas of the renal parenchyma. CD133 and KRT7 were co-expressed in some MD and adjacent DT cells. Some of the latter cells were positive both for CD133 and VIM. CD133 was always localized in the apical part of the cells, whereas the VIM expression was evident only in the cellular basal pole. Although some cells of MD expressed VIM or CD133, none of them co-expressed VIM and CD133. Conclusions: The presence of STCs was demonstrated in human adult MD, suggesting that this structure has expansion, self-renewal and epithelial differentiation abilities, similar to all other parts of renal tubules.

Anticancer and antimicrobial activities of Imine containing compounds

A series of imine derivatives (3a-m) including thio-semicarbazone, semicarbazone, thiazole and oxazole functional moieties were examined for anti-cancer activity in-vitro by MTT assay using glioblastoma cell line (U87). Among all compound 3m was most potent compound with IC50 value of 8.86 ± 0.15 µM. Cytotoxicity of compounds were evaluated using normal human embryonic kidney cell line i.e HEK-293. Moreover, antibacterial activity of synthesized derivatives was also evaluated by disc diffusion method.

Synthesis and in vitro anticancer evaluation of 8b-hydroxy-1-(6-substitutedbenzo[d]thiazol-2-yl)-3-(3-substitutedphenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(3aH)-ones

A series of 1,8b-dihydroindeno[1,2-c]pyrazol-4(3aH)-ones has been efficiently synthesized on reaction of 2-(3-substitutedbenzoyl)-(1H)-indene-1,3(2H)-diones 1 and 2-hydrazinylbenzo[d]thiazole/2-hydrazinyl-6-substitutedbenzo[d]thiazoles 2 in presence of dry ethanol under reflux conditions in good yields. The structures of all the synthesized derivatives were elucidated by using different physical and spectral techniques (FTIR, 1H NMR, 13C NMR, 2D NMR and HRMS). The synthesized hydroxypyrazolones were assayed for their anticancer activity against two human cancer cell lines (i) lung carcinoma (A549) and (ii) breast carcinoma (MCF7) using MTT assay. Among all the tested compounds, 3d and 3k with IC50 values in the range of 30.68 ± 0.57 µM to 34.87 ± 0.76 µM exhibited moderate inhibitory ability against both the tested cancer cell lines in comparison to the standard drug Doxorubicin (0.95 ± 0.10 µM for A549 and 1.09 ± 0.11 µM for MCF7). The moderately active compounds of the series i.e.3d and 3k were further tested against normal human embryonic kidney cell line (HEK 293T) to examine their toxicity against normal cell lines. Selectivity Index (S.I.) of the most active compounds 3d and 3k was calculated for both the cancer cell lines which revealed that these derivatives i.e. 3d (S.I = 2.82 for A549 and 2.90 for MCF7) and 3k (S.I = 2.78 for A549 and 2.95 for MCF7) are safer anticancer agents.

Organoantimony (III) Derivative Induces Necroptosis in Human Breast Cancer MDA-MB-231 Cells.

This study aimed to investigate the anticancer effect and the underlying mechanisms of organoantimony (III) fluoride on MDA-MB-231 human breast cancer cells. Five cancer and one normal cell line were treated with an organoantimony (III) compound 6-cyclohexyl-12- fluoro-5,6,7,12-tetrahydrodibenzo[c,f][1,5]azastibocine (denoted as C4). The cell viability was detected by MTT assay. Induction of cell death was determined by Hoechst 33342/PI staining and Annexin-V/PI staining. The effect of C4 on the necroptotic relative protein was determined by Western blot analysis. Among the five cancer cell lines, C4 decreased the viability of MDA-MB-231, MCF-7 and A2780/cisR, and showed less toxicity on normal human embryonic kidney cells. In breast cancer cell line MDA-MB-231, the C4 treatment induced necrotic cell death as well as LDH release in a time- and dose-dependent manner. Moreover, C4 could increase the expression of phosphorylated RIPK3 and MLKL proteins. Overall, the C4 treatment resulted in the reduction of mitochondrial transmembrane potential and accumulation of ROS in MDA-MB-231 cells. C4-induced necroptosis could be ascribed to glutathione depletion and ROS elevation in MDA-MB-231 cells. Our findings illustrate C4 to be a potential necroptosis inducer for breast cancer treatment.

Essential role of Wtip in mouse development and maintenance of the glomerular filtration barrier.

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using β-galactosidase-neomycin (β-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using β-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier.

Spatially Resolved Transcriptomes of Mammalian Kidneys Illustrate the Molecular Complexity and Interactions of Functional Nephron Segments.

Available transcriptomes of the mammalian kidney provide limited information on the spatial interplay between different functional nephron structures due to the required dissociation of tissue with traditional transcriptome-based methodologies. A deeper understanding of the complexity of functional nephron structures requires a non-dissociative transcriptomics approach, such as spatial transcriptomics sequencing (ST-seq). We hypothesize that the application of ST-seq in normal mammalian kidneys will give transcriptomic insights within and across species of physiology at the functional structure level and cellular communication at the cell level. Here, we applied ST-seq in six mice and four human kidneys that were histologically absent of any overt pathology. We defined the location of specific nephron structures in the captured ST-seq datasets using three lines of evidence: pathologist's annotation, marker gene expression, and integration with public single-cell and/or single-nucleus RNA-sequencing datasets. We compared the mouse and human cortical kidney regions. In the human ST-seq datasets, we further investigated the cellular communication within glomeruli and regions of proximal tubules–peritubular capillaries by screening for co-expression of ligand–receptor gene pairs. Gene expression signatures of distinct nephron structures and microvascular regions were spatially resolved within the mouse and human ST-seq datasets. We identified 7,370 differentially expressed genes (padj < 0.05) distinguishing species, suggesting changes in energy production and metabolism in mouse cortical regions relative to human kidneys. Hundreds of potential ligand–receptor interactions were identified within glomeruli and regions of proximal tubules–peritubular capillaries, including known and novel interactions relevant to kidney physiology. Our application of ST-seq to normal human and murine kidneys confirms current knowledge and localization of transcripts within the kidney. Furthermore, the generated ST-seq datasets provide a valuable resource for the kidney community that can be used to inform future research into this complex organ.

Abstract 2594: Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor

Abstract DNA-damaging poisons and replicative stress commonly result in increased double-strand breaks (DSBs) in tumor cells. Non-homologous end joining (NHEJ), driven by the enzymatic function of DNA-PK, is one of the critical pathways employed to repair such DSBs. Inhibition of DNA-PK kinase activity impairs NHEJ, potentiates the activity of DNA-damaging agents, and inhibits survival of tumor cells incapable of repairing DSBs via other means, including homologous recombination (HR). We have developed an orally bioavailable, highly potent, and selective pharmacological inhibitor of DNA-PK activity, ZL-2201, for the treatment of cancer. ZL-2201 demonstrates enzymatic inhibition of DNA-PK in the single-digit nM range, with nearly 500-fold or greater selectivity against other phosphatidylinositol 3-kinase-related kinases (PIKKs). In addition, ZL-2201 showed high selectivity for DNA-PK kinase activity in a kinase panel screening study and a clean off-target profile with no appreciable inhibition against a panel of diverse, safety-relevant targets. We demonstrate that ZL-2201 effectively inhibits DNA-PK autophosphorylation and its downstream target phospho-RPA in multiple cell lines in a concentration- and time-dependent manner. DNA-PK has been shown previously to have synthetically lethal interactions with ATM. Therefore, we profiled the impact of ATM expression on the antiproliferative effects of ZL-2201. Cancer cell lines possessing defects in ATM displayed greater sensitivity to ZL-2201 compared to ATM-WT cancer cell lines. These observations were recapitulated in two ATM knockout isogenic cell lines. Moreover, ZL-2201 displayed minimal effects in a DNA-PK deficient model or in normal human and canine colon and kidney cell lines. Independent of ATM status, ZL-2201 showed strong synergy in vitro with topoisomerase II inhibitors. Following oral administration of ZL-2201 in vivo, ZL-2201 demonstrated dose-dependent and exposure-dependent antitumor activity against multiple models, with the greatest activity against ATM deficient human cancer xenografts. Antitumor activity correlated with duration of ZL-2201 exposure and level of target engagement. In addition, ZL-2201 significantly enhanced the activity of DNA-damaging agents in vivo, even in human cancer xenograft models with limited monotherapy antitumor activity for ZL-2201. The projected human PK properties, based on the ADME properties across species and PK properties in preclinical species of ZL-2201, suggest that ZL-2201 is suitable for oral administration. In conclusion, ZL-2201 demonstrates potent and selective inhibition of DNA-PK enzymatic activity, strong synergy with DNA damaging agents in vitro and in vivo, antitumor activity against multiple human xenograft models, and a favorable projected human PK profile. ZL-2201 is expected to enter clinical development in 2022. Citation Format: Shruti Lal, Bee-Chun Sun, Yuping Chen, Tom Huang, Neil Bhola, Vivian Morton, Kevin Chen, Shanghua Xia, Haoyu Zhang, Qiuping Ye, Petter Veiby, David I. Bellovin, Yuhua Ji. Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2594.

Abstract 3112: Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models

Abstract Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3112.

Kidney epithelial cells are active mechano-biological fluid pumps

The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.

Synthesis of carbon dots from taurine as bioimaging agent and nanohybrid with ceria for antioxidant and antibacterial applications

Here we have synthesized water soluble and biocompatible carbon dots (CDs) from taurine via thermal decomposition method. The CDs showed nearly spherical shape with diameter less than 10 nm. The CDs exhibited excitation dependent fluorescence emission and could be used for mammalian cell imaging. The CDs showed excellent DPPH and hydrogen peroxide radical scavenging activity in cell free system. Besides, the CDs also displayed significant intracellular radical scavenging activity in human normal kidney epithelial (NKE) cells. Furthermore, nanohybrids consisting of both CDs and nanoceria (CeO2) were prepared and tested for their biomedical applications. The nanohybrids showed significant antioxidant activities in both cell free and intracellular conditions. The CDs and nanohybrids possessed very little toxicity upto the concentration of 100 μg/mL when treated for 24 hours in human NKE cells. The CDs as well as nanohybrids further displayed significant bacterial growth inhibition against both gram-positive and gram-negative bacteria under dark as well as light illumination condition via the bacterial membrane damage. However, under the light illumination, the bacterial growth inhibition of CDs and nanohybrids was further enhanced due to the generation of reactive oxygen radicals and subsequent DNA degradation. A higher dose-dependent intracellular antioxidant and antibacterial activities of the nanohybrid is attributed to the synergistic effect of nanoceria and CDs. All these results clearly reflected that our synthesized CDs and their nanohybrids can be used for several biomedical applications.

Identification of histone deacetylase 10 (HDAC10) inhibitors that modulate autophagy in transformed cells

Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.

Role of ACSL4 in the chemical-induced cell death in human proximal tubule epithelial HK-2 cells.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) activates polyunsaturated fatty acids (PUFAs) to produce PUFA-derived acyl-CoAs, which are utilised for the synthesis of various biological components, including phospholipids (PLs). Although the roles of ACSL4 in non-apoptotic programmed cell death ferroptosis are well-characterised, its role in the other types of cell death is not fully understood. In the present study, we investigated the effects of ACSL4 knockdown on the levels of acyl-CoA, PL, and ferroptosis in the human normal kidney proximal tubule epithelial (HK-2) cells. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses revealed that the knockdown of ACSL4 markedly reduced the levels of PUFA-derived acyl-CoA, but not those of other acyl-CoAs. In contrast with acyl-CoA levels, the docosahexaenoic acid (DHA)-containing PL levels were preferentially decreased in the ACSL4-knockdown cells compared with the control cells. Cell death induced by the ferroptosis inducers RSL3 and FIN56 was significantly suppressed by treatment with ferrostatin-1 or ACSL4 knockdown, and, unexpectedly, upon treating with a necroptosis inhibitor. In contrast, ACSL4 knockdown failed to suppress the other oxidative stress-induced cell deaths initiated by cadmium chloride and sodium arsenite. In conclusion, ACSL4 is involved in the biosynthesis of DHA-containing PLs in HK-2 cells and is specifically involved in the cell death induced by ferroptosis inducers.

Protein stabilized seabuckthorn fruit oil Nanoemulsion：Preparation, characterization and performance research

Protein-stabilized nanoemulsion was successfully prepared by ultrasound-assisted method under optimal ultrasound conditions, so as to improve the defects of sea buckthorn fruit oil (SBFO) and increase its bioavailability. This paper investigated the performance of nanoemulsion obtained by sodium caseinate (SC) and whey glycoprotein (WGP) at different concentrations, including polymer dispersity index (PDI), particle size, zeta potential (ZP). The SBFO nanoemulsion prepared under the optimal concentration of protein has good microstructure and rheological properties. Under different temperature (20–80 °C), pH (3–9), salinity (50–200 mM) and storage time (4 weeks), the performance of SBFO nanoemulsion has been measured to prove that SBFO nanoemulsion can maintain stable state in most cases. The safety of the SBFO nanoemulsion was evaluated by cytotoxicity (MTT) method and the results showed non-toxic to normal human embryonic kidney cells (HEK 293T).

IMPORTANCE OF APOPTOSIS MARKERS (MDM2, BCL-2 AND Bax) IN CONVENTIONAL RENAL CELL CARCINOMA

The goal of the current study was to analyze the expression of Bcl-2, MDM2 and Bax in benign and malignant renal tissue samples and assess their possible association with different clinical parameters. Prognostic significance of the markers in recurrence-free and cancer-specific survivals has also been evaluated.Activity of MDM2, Bcl-2 and Bax was evaluated in: 24 normal human kidney tissues resected from the patients of different ages (range: 21-80 years), and in 52 conventional RCC samples. Intensity of the markers’ expression was compared between the groups and correlation was analyzed with different clinical parameters.Activity of anti-apoptotic MDM2 and Bcl-2 was significantly elevated while activity of pro-apoptotic Bax was decreased in RCC as compared with normal kidney tissues. Bax expression was positively correlated with patient age. Significant association has been detected between the evaluated markers and cancer clinical parameters like: tumor stage, grade, lymph node and distant metastases. The markers’ activity was associates with the tumor morphological features, in particular: presence of tumor necrosis and microvascular invasion. Disease recurrence and 5-year patient survival were associated with the markers’ activity. Cox regression analyses have shown that tumor size, pathological stage and grade are the risk factors for disease recurrence and patient death.Expression of MDM2 and Bcl-2 is significantly up-regulated, while Bax is down-regulated in RCC as compared with normal kidney tissue. Intensity of the markers’ activities is associated with the tumor pathological and clinical parameters (stage, grade, lymph node and distant metastases, tumor recurrence and patient survival). Further studies with more patients and longer follow-up will uncover the clinical importance of the evaluated markers in RCC.

Single-cell chromatin accessibility landscape in kidney identifies additional cell-of-origin in heterogenous papillary renal cell carcinoma

Papillary renal cell carcinoma (pRCC) is the most heterogenous renal cell carcinoma. Patient survival varies and no effective therapies for advanced pRCC exist. Histological and molecular characterization studies have highlighted the heterogeneity of pRCC tumours. Recent studies identified the proximal tubule (PT) cell as a cell-of-origin for pRCC. However, it remains elusive whether other pRCC subtypes have different cell-of-origin. Here, by obtaining genome-wide chromatin accessibility profiles of normal human kidney cells using single-cell transposase-accessible chromatin-sequencing and comparing the profiles with pRCC samples, we discover that besides PT cells, pRCC can also originate from kidney collecting duct principal cells. We show pRCCs with different cell-of-origin exhibit different molecular characteristics and clinical behaviors. Further, metabolic reprogramming appears to mediate the progression of pRCC to the advanced state. Here, our results suggest that determining cell-of-origin and monitoring origin-dependent metabolism could potentially be useful for early diagnosis and treatment of pRCC.

Synthesis of Gold Nanoparticles Using Centella Asiatica Extract and its In Vitro Cytotoxic Activity Against Human Breast Adenocarcinoma MCF-7 and Normal Vero Cell Line

In the past few decades, the gold nanoparticles (AuNPs) have attained much attention among all metal nanoparticles and it is greatly contributed for cancer photo-diagnostics, photo-thermal therapy, bio-labelling, drug delivery etc. In the present investigation, AuNPs were green synthesized using Centella asiatica leaf aqueous extract. The physicochemical properties of synthesized AuNPs were characterized using UV–visible and Transmission Electron Microscopy (TEM) analysis. Further, in vitro anticancer efficacy of biosynthesized AuNPs were assessed against human breast adenocarcinoma (MCF-7) and normal African green monkey kidney Vero cell line. Bio approach synthesized gold nanoparticles were highly stable, monodisperse particles in the size ranging