Human Renal Cell Research Articles

Abstract 6471: Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas

Abstract Although Epithelial-Mesenchymal Transition (EMT) in cancers remains controversial, its relevance in tumor progression is a subject of investigation. The presence of cellular changes associated with EMT in bladder tumor progression have been well investigated, unlike other human carcinomas with very little data available to date. ZEB-1 which targets E-Cadherin repression is a transcriptional regulator that has been implicated in EMT, and its nuclear expression in tumor cells has been associated with aggressive malignancies in some types of cancer. Recently, it was also reported that ZEB-1 inhibits the expression of proapoptotic factor B-cell lymphoma (Bcl)-2 interacting mediator of cell death (BIM) by binding directly to the BIM promoter and repressing its transcription. We tested this hypothesis with a human cohort of 24 Cholangiocarcinoma (CC), 18 Hepatocellular (HCC) and 31 Renal Cell (RCC) human carcinomas. Formalin fixed paraffin embedded tissue sections containing benign and tumor lesions were immunostained using the Dako Autostainers Link 48 along with ZEB-1 monoclonal OT13G6 (ABCAM), Bcl-2 monoclonal 124 (Agilent, Dako), and E-Cadherin monoclonal NCH-38 (Agilent, Dako) antibodies. Nuclear, membraneous and/or cytoplasmic immunoreactivity was scored based on the intensity and percentage of positive cells in both the benign epithelium and adjacent tumor in each case. E-Cadherin was highly expressed in both the adjacent benign and malignant components with no significant variability in all three carcinomas, with ZEB-1 and Bcl-2 evaluated for the any positivity staining in both components for all cases. Results are detailed in the table below.All positive ZEB-1 cases in HCC were in high grade cases unlike the positive ZEB-1 in RCC which were in low grade cases. But more noticeably, 8/10 (80%) positive ZEB-1 were also positive for Bcl-2 in all three carcinoma types. Despite the small number of cases analyzed in this cohort a trend in the expression of ZEB-1 and its co-expression with BCL-2 may have some prognostic implications. We are currently extending this analysis to a larger number of CC, HCC, and RCC to closely examine the significance of ZEB-1, and Bcl-2 expression across differently graded lesions to further test our hypothesis. Citation Format: Joeffrey J. Chahine, Kyungmin Ko, Bhaskar VS Kallakury, Pamela L. Tuma. Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6471.

Abstract 4702: DNA methylation enhances apoptosis resistance of renal cancer cells through the control of mitochondria related gene expression

Abstract Many gene alterations have been identified in the onset of renal carcinogenesis. However, molecular alterations might be added to renal cancer cells during the progression. In order to uncover additional molecular alterations, we established highly malignant cancer cells which possess identical background to original cancer cells, and analyzed them in the present study. Since our previous works demonstrated that interaction with cancer microenvironment aids cancer cells with malignant trait through “education” and “selection”, serial orthotopic transplantation was utilized. First, parental human clear cell renal cell carcinoma cells, OS-RC-2, were subjected to serial orthotopic transplantation. Then, novel derivatives were obtained from resulting primary renal tumors. These derivatives formed primary renal tumor faster than parental cells and exhibited increased lung metastasis, suggesting that these derivatives were defined as “highly malignant cancer cells”. Next, we tried to identify the underlying molecular mechanism(s) for renal cancer progression through the characterization of highly malignant cancer cells. Gene expressions in each cell were profiled using RNA-sequencing, and these analyses revealed that expression of several genes associated with mitochondrial functions were dramatically down-regulated in highly malignant cancer cells. Since mitochondrial electron transport chain triggers apoptotic cell death, decrease of these genes may contribute to suppression of apoptosis of highly malignant cancer cells. Cell culture and in vivo experiments demonstrated that mitochondrial dysfunction is responsible for increased survival of renal cancer cells and cancer metastasis. Finally, regulatory mechanism of downregulation of these mitochondria-related genes was analyzed. Bisulfite sequencing analysis revealed that the downregulation may occur by DNA hypermethylation of CpG islands in the promoter regions in renal cancer cells.Taken together, these findings suggested that renal cancer cells acquire additional anti-apoptotic ability through the DNA methylation during the interactions with renal microenvironment, which may in turn lead to the cancer progression and metastasis. We are currently investigating whether the effect of anti-cancer reagent is enhanced in the presence of DNA methylation inhibitors. Citation Format: Kosuke Miyakuni, Jun Nishida, Shogo Ehata, Kohei Miyazono. DNA methylation enhances apoptosis resistance of renal cancer cells through the control of mitochondria related gene expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4702.

The effect and molecular mechanism of hypoxia on proliferation and apoptosis of CD133+ renal stem cells.

Congenital hydronephrosis caused by ureteropelvic junction obstruction (UPJO) eventually leads to renal interstitial fibrosis and atrophy, after a series of pathophysiological problems. Renal repair after injury depends on renal stem cells. This study aimed to determine the expression of renal stem cell marker CD133 in children of different ages and the regulatory effect of stem cell microenvironment. Renal stem cells from children of different ages were identified and screened out by flow cytometry in the study. Children with hydronephrosis were divided into neonates, infants, preschool age, school age, and adolescents groups. A hypoxic cell model prepared with CoCl2 was developed to detect the effect of hypoxia on the proliferation and apoptosis of renal stem cells. The effect and molecular mechanism of hypoxia-inducible factor 1-alpha (HIF-1α) on the proliferation and apoptosis of renal stem cells were also explored. Both hypoxia and HIF-1α significantly promoted the proliferation of renal stem cells and inhibited cell apoptosis. HIF-1α could bind to the promoter region of proliferating cell nuclear antigen (PCNA) and PROM1 (CD133) to mediate their transcription and expression. The content of CD133+ renal stem cells was the highest in the neonatal group and it decreased with the increase of age. Taken together, this study clarified the effect of age on the content of human renal stem cells and determined the regulatory mechanism of hypoxia on renal stem cells. We expect our results to provide a research basis for the treatment and clinical application of renal stem cells.

<i>In Vitro</i> and <i>In Vivo</i> Evaluation of Amphiphilic Chitosan Derivatives for Inhibition of Organic Cation Transport Function

This study explored the interaction of amphiphilic chitosan derivatives, N-benzyl-N,O-succinyl chitosan (BSCS), N-naphthyl-N,O-succinyl chitosan (NSCS) and N-octyl-N,O-succinyl chitosan (OSCS), with renal organic cation transporter 2 (OCT2). The influence of amphiphilic chitosan derivatives on renal OCT2 transport function was determined by monitoring the transport of a positively charged substrate into human renal proximal tubular epithelial cells (RPTEC/TERT1 cells), and murine kidney. Amphiphilic chitosan derivatives inhibited 3H-MPP (a substrate of OCT2) transport in the renal cells in a concentration-reliance characteristic. OSCS reduced the accumulation of the cationic drug, cisplatin, in RPTEC/TERT1 cells. This effect was more pronounced than that of other chitosan derivatives. In addition, co-administration of cisplatin and OSCS significantly reduced cisplatin accumulation compared with receiving cisplatin alone. This result was accompanied by the decrease in nephrotoxicity induced by cisplatin. In conclusion, OSCS inhibited OCT2 function and reduced cationic drug disposition in human renal proximal tubular cells and murine kidney.

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer.

We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this “bladder-specific” region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

The circadian clock gene Bmal1 facilitates cisplatin-induced renal injury and hepatization

Cisplatin is one of the most potent chemotherapy drugs to treat cancers, but its clinical application remains limited due to severe nephrotoxicity. Several approaches have been developed to minimize such side effects, notably including chronotherapy, a well-known strategy based on the circadian clock. However, the component of the circadian clock machinery that particularly responses to the cisplatin stimulation remains unknown, including its functions in cisplatin-induced renal injury. In our present study, we demonstrated that Bmal1, as a key clock gene, was induced by the cisplatin stimulation in the mouse kidney and cultured human HK-2 renal cells. Gain- and loss-of-function studies indicated that Bmal1 facilitated cisplatin-induced renal injury both in vivo and in vitro, by aggravating the cell apoptotic process. More importantly, RNA-seq analysis revealed that Bmal1 triggered the expression of hallmark genes involved in renal hepatization, a critical event accompanied by the injury. At the molecular level, Bmal1 activated the transcription of hepatization-associated genes through direct recruitment to the E-box motifs of their promoters. Our findings suggest that Bmal1, a pivotal mediator induced renal injury in response to cisplatin treatment, and the therapeutic intervention targeting Bmal1 in the kidney may be a promising strategy to minimize the toxic side-effects of cisplatin in its clinical applications.

MO065TUBULAR EPITHELIAL CELL POLYPLOIDIZATION IS REQUIRED TO SURVIVE AKI BUT PROMOTES CKD DEVELOPMENT

Abstract Background and Aims Acute kidney injury (AKI) is a global health concern. If not lethal in the acute phase, AKI is considered reversible based on the capacity of surviving tubular cells (TECs) to re-enter cell cycle. However, even mild AKI episodes carry a substantial risk of developing chronic kidney disease (CKD). The pathophysiological basis for this phenomenon remains unclear. Recently, we demonstrated that tubular epithelial cells (TECs) can undergo endoreplication-mediated hypertrophy after AKI. Endoreplications are incomplete cell cycles that lead to the formation of polyploid cells. As polyploid cells can provide increased cell function without restoring tissue integrity, we hypothesized that this mechanism is essential to survive AKI but it can be potentially maladaptive. Method To address this hypothesis, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology to monitor cell cycle phasing in combination with YAP1 overexpression or downregulation. In the in vivo models, YAP1 overexpressing mice and YAP1 knock-out mice were subjected to unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis to induce AKI. Polyploid cells have been then characterized by microarray analysis, cell sorting, super-resolution STED microscopy and transmission electron microscopy. Results In vitro, human renal tubular cells undergo polyploidization. The fraction of polyploid cells significantly decreases when YAP1 nuclear translocation is blocked, suggesting a possible involvement of YAP1 in regulating TEC polyploidization. After AKI in mice, the inhibition of YAP1 significantly reduces the number of polyploid cells and worsens kidney function resulting in a dramatic decrease of mouse survival. In contrast, YAP1 overexpression leads to an increase in the number of polyploid cells up to 20% of all TECs, further confirming the role of YAP1 in controlling TEC polyploidization. In YAP1 overexpressing mice, electron microscopy and STED analysis revealed the presence of both mononucleated and binucleated polyploid cells. Strikingly, these mice appear to be more prone to develop tubulointerstitial fibrosis acquiring a marked senescent phenotype along with significant decline in renal function thus suggesting an association between polyploidization and CKD development. Indeed, isolation of polyploid cells proved that these cells actively transcribe and secrete pro-fibrotic and senescent factors confirming their role in CKD progression. Conclusion These data suggest that: 1) polyploidization after AKI is required to preserve renal function in the acute phase of damage and it is essential for survival 2) polyploid cells are pro-fibrotic and senescent leading in the long run to the progression of AKI to CKD.

MO068A KIDNEY-ON-THE-CHIP APPROACH USING PRIMARY HUMAN TUBULAR CELLS IN A 3D CO-CULTURE SYSTEM

Abstract Background and Aims Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, although their value is limited by poor translatability and predictive value for the in vivo or even human situation. The implementation of more sophisticated in vitro assays as routine cell culture systems is often limited by complex protocols and long lasting procedures. We aimed to establish and validate a relatively easy-to-use but yet (patho-) physiologically relevant cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Method We utilized the 3-lane OrganoPlate® system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker (Mimetas), that facilitates continuous gravity-triggered bi-directional perfusion of C1. Within 48h the cells form a leak-tight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of the C2-matrix and – upon continuous perfusion – formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells were isolated and seeded into C3 (see figure). Results Establishing the whole tubule-on-the-chip as described above takes on average three days. We investigated its operational life span by monitoring the barrier integrity of the tubular structure in C1 using a fluorescence-labeled dextran (150 kDa). Over a course of 5 days the tubular integrity did not decline, suggesting that the co-culture system remains stable and functional for at least 5 days. In accordance with other studies, the primary human tubular cells constituting the 3D tubule-on-the-chip expressed higher levels of functionally relevant proteins, e..g the tight-junction protein ZO-1 and the sodium-potassium-pump Na-K-ATPase, compared to standard 2D settings without perfusion. This emphasizes, that even primary cells show a physiologically reduced phenotype in standard 2D settings, which possibly impedes the identification and representative quantification of physiologically and hence also patho-physiologically relevant mechanisms in vitro. To study the interaction of cells in the tubule-on-the-chip, we investigated the recruitment of immune cells from C3 (vessel) across C2 (interstitium) to C1 (renal tubule), which - in vivo - represents a detrimental mechanism of action in intrarenal forms of AKI. Under baseline conditions the immune cells inserted into C3 did not leave their compartment. Upon damaging the tubular cells in C1 with extracellular histones, neutrophils and monocytes left C3 (extravasation), migrated through C2 and could be found in close contact with epithelial cells of C1. This serves as a proof of principle, that the tubule-on-the-chip is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Measuring lactate dehydrogenase release for a number of known nephrotoxic agents revealed, that tubular cells forming a 3D-structure while kept under perfusion show significantly different responses to the same dose compared to standard 2D conditions, suggesting that dose-response studies using target cells out of their tissues context can be misleading when extrapolating results from in vitro to in vivo. Conclusion The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited to allow for in depth mechanistic studies in vitro.

MO046MICROBIOTA DERIVED SHORT CHAIN FATTY ACIDS, PROPIONATE AND BUTYRATE, CONTRIBUTE TO MODULATE THE INFLAMMATORY RESPONSE IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Dysbiosis, or changes in the gut microbiota composition, had been related to the developed of several pathologies, such as chronic kidney disease. Until now, multiple studies have focused on the influence of diet on outcomes of patients with CKD. These patients with advanced disease are recommended a restricted intake of vegetable fiber due to the phosphorus and potassium levels, and low proteins to avoid the generation of uremic toxins. It is known that dietary changes lead to alterations in gut microbiota, but also in microbial metabolites production, some of which could be beneficial for the host. A recent and exciting area of research has begun to explore the role of microbiota-derived metabolites in the renal physiology. Short-chain fatty acids (SCFA, acetate, propionate and butyrate) are a type metabolite produced from dietary fiber by gut microbiota that enter in the bloodstream leading to distal effects, such as modulation of the immune cells. SCFAs are essential to maintain the permeability of the intestinal epithelial barrier, the metabolic functions and have potent anti-inflammatory effects. The aim of this study was to identify the SCFAs levels during the progression of CKD and determinate the functional role of these metabolites in the renal inflammation. Method SFCAs (acetate, propionate and butyrate) levels were determined using gas chromatography-mass spectrometry in fecal samples collected from patients with different stages of CKD (n=60) and age-matched healthy control (n=20). Moreover, common bacterial families were determined by quantitative PCR. Additionally, the in-vitro effect of the three SCFAs was evaluated in the human tubular epithelial cell line HK2 using RNA-seq, specific silencing with siRNAs and histone deacetylases (HDAC) inhibitors. To evaluate the effect in immune cells, monocyte and macrophages were treated with LPS and ATP /Nigericin to induce inflammasome activation. Results The SCFAs levels were significantly lower in patients with CKD than in healthy controls, mainly propionate and butyrate. Moreover, these levels progressively decreased with the developed of the disease, showing the patients with stage 5 (CKD5) have the lowest levels that correlates with a lesser abundance of Clostridium IV family. According to the renal function, butyrate levels were positively correlated with the glomerular filtration rate and negatively with the blood urea nitrogen and creatinine levels. Surprisingly, high propionate levels correlate with the most elevated serum calcidiol concentrations. Functionally, propionate and butyrate show a similar pattern in the modulation of inflammatory genes in HK2 cells. Most regulated pathways are associated with Inflammatory response (GO:0006954: IL6, TNF, CCL2, RELB, IRAK2, NFKB1,CCL20) and immune response (GO:0006955: CSF2, CXCL3, CD40, IL7R, LIF). Additionally, both SCFAs regulates the expression of multiple epigenetic enzymes involves in the chromatin remodeling, mainly in histone acetylation. In monocytes/macrophages, propionate and butyrate inhibits the IL1B, CASP, and ASC gene transcription damaging the IL-1β secretion. We determined that the effect of SCFAs in these in-vitro models is mediated by inhibition of HDAC although also change other histone modifications (H3K9me3, H3K27me3) and through the GPR109A receptor. Conclusion Our initial results showed that patients with advanced CKD have low levels of SCFAs, and those were correlated with the renal function. Treatment of human renal and immune cells with propionate and butyrate induces profound changes in the chromatin structure, changing the whole-genome gene expression and modulating key pathways in the renal pathology. Increasing the SCFAs levels in those patients could be a potential therapeutic strategy to slow down the disease progression.

SO082LATENT TRANSFORMING GROWTH FACTOR BETA BINDING PROTEIN (LTBP4) CAN REGULATE RENAL FIBROSIS AND ALTER INFLAMMATION AND MITOCHONDRIAL DYSFUNCTION IN CHRONIC KIDNEY DISEASE (CKD)

Abstract Background and Aims: Transforming growth factor beta (TGF-beta) has been well known as a key factor for fibrosis and inflammation. LTBPs regulate TGF-beta signalling in complicated ways. Disruption and loss of LTBP4 expression is associated with abnormal accumulation of extracellular matrix (ECM) and altered TGF-beta activation. However, the role of LTBP4 in chronic kidney disease remains largely unknown. We aim to explore the mechanisms of LTBP4-related regulation in kidney disease. Method: To investigate the impact of LTBP4 on tubulointeresitial fibrosis (TIF), we generated LTBP4-overexpression human renal proximal tubule cells (HK-2), treated with exogenous TGF-beta and established a fibroblasts-HK-2 co-culture system using rat fibroblasts (NRK-49F) and HK-2 cells. Moreover, to create TIF model, we performed unilateral ureteral ligation (UUO) in Ltbp4S-/- mice and wild-type (WT) mice to check ECM deposition and phenotypic alterations. In addition, we performed RNA-Sequencing analysis to understand transcriptomic changes associate with LTBP4 overexpression. Results: Up-regulation of Ltbp4 in fibrotic kidney was noted in TIF model with UUO and in renal tissue with diabetic nephropathy. LTBP4-overexpression reduced epithelial-mesenchymal transition (EMT) with showing increased epithelial-cadherin, reduced vimentin and collagen I in the co-culture system. Moreover, higher expression of fibronectin, collagen I and alpha-smooth muscle actin (alpha-SMA) were noted in fibrotic kidneys in Ltbp4S-/- mice compared with changes in WT mice, suggesting inflammation condition could be altered by the absence of Ltbp4S. The inflammatory change in renal tissue in UUO model was studied with F4/80 stain. Increased macrophage infiltration and increased monocyte chemoattractant protein-1 (MCP-1) expression were detected in Ltbp4S-/- mice compared with that in WT mice. In addition, LTBP4-overexpression reduced mitochondrial biogenesis. Conclusion: Ltbp4 acted as a regulator of fibrosis and inflammation in fibrotic kidneys. TGF-beta- induced EMT were clearly enhanced in ltbp4 deficient environments, accompanied with reduced macrophage infiltration, which could be the major mechanism related to LTBP4/TGF-beta pathways. Furthermore, altered ATP production and usage as well as mitochondria respiration could be regulated by LTBP4 in renal failure.

Tumor-targeted oncolytic adenovirus demonstrates high cytotoxicity for human lung and renal cell carcinomas independently of the level of tumor PD-L1 expression.

3596 Background: Immuno-checkpoint (IC) inhibitors targeting PD1-PD-L1 pathway have proven highly effective at extending survival of cancer patients. However, the clinical benefits of IC inhibitors are limited to only about 20% patients who have moderate to high levels of tumor PD1 and/or PD-L1 expression. To develop therapeutics that would provide clinical benefits to a larger proportion of cancer patients, we engineered oncolytic adenovirus for targeted infection of human tumor cells via CD46 and integrins of α3β1 or α6β4 classes, overexpressed on many epithelial human cancers. Methods: Here, we analyzed the infectivity and cytotoxicity of this novel oncolytic virus in a panel of human non-small cell lung cancers (NCSLC) cell lines, primary patient derived NSCLC xenografts, and tumor surgical explants from patients with renal cell carcinoma (RCC). Results: The in vitro analysis of NSCLC cells lines (N = 17) demonstrated that over 60% of them were highly sensitive to virus infection. The genome-wide transcriptional profiling showed that only 3 out of 12 cell lines that were sensitive to oncolytic virus infection, expressed PD-L1 ( > 4.5 Log2 RPMK). Furthermore, although the pre-treatment of these cell lines with IFN-I activated PD-L1 expression, IFN-I treatment did not reduce the efficacy of tumor cell infection by the oncolytic virus. The analysis of virus infectivity on primary human tumor cells from patients with NSCLC (N = 4) and RCC (N-24) demonstrated that primary tumors were highly sensitive to oncolytic virus infection. Specifically, tumor-targeted oncolytic virus demonstrated strong cytotoxicity in 22 out of 24 analyzed primary isolated RCC cell samples. Next, we subcutaneously grafted PD-L1-negative NSCLC A549 cells to NSG mice, treated them with oncolytic virus intravenously, and the kinetics of tumor growth and animal survival was monitored. This analysis showed that after intravenous administration, oncolytic virus was able to infect tumor cells and suppress tumor growth. Whereas the median survival in mock-treated group was 26 days, all mice survived up to 100 days post oncolytic virus therapy (endpoint). Conclusions: Our study showed that tumor-targeted oncolytic adenovirus infects human tumor cell lines independently of their PD-L1 expression status and is not sensitive to IFN-I inhibition. This novel tumor-targeted oncolytic virus has the potential to provide clinical benefits to cancer patients, who do not respond or became resistant to ICI drugs.

RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells’ Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.

The HOPS complex subunit VPS39 controls ciliogenesis through autophagy.

Primary cilia are microtubule-based organelles that assemble and protrude from the surface of most mammalian cells during quiescence. The biomedical relevance of cilia is indicated by disorders ascribed to cilia dysfunction, known as ciliopathies, that display distinctive features including renal cystic disease. In this report, we demonstrate that vacuolar protein sorting 39 (VPS39), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, acts as a negative regulator of ciliogenesis in human renal cells, by controlling the localization of the intraflagellar transport 20 protein at the base of cilia through autophagy. Moreover, we show that VPS39 controls ciliogenesis through autophagy also in vivo in renal tubules of medaka fish. These observations suggest a direct involvement of the HOPS complex in the regulation of autophagy-mediated ciliogenesis and eventually in target selection. Interestingly, we show that the impact of autophagy modulation on ciliogenesis is cell-type dependent and strictly related to environmental stimuli. This report adds a further tile to the cilia-autophagy connection and suggests that VPS39 could represent a new biological target for the recovery of the cilia-related phenotypes observed in the kidneys of patients affected by ciliopathies.

Facile synthesis of hydrophilic magnetic graphene nanocomposites via dopamine self-polymerization and Michael addition for selective enrichment of N-linked glycopeptides

The development of methods to effectively capture N-glycopeptides from the complex biological samples is crucial to N-glycoproteome profiling. Herein, the hydrophilic chitosan–functionalized magnetic graphene nanocomposites (denoted as Fe3O4-GO@PDA-Chitosan) were designed and synthesized via a simple two-step modification (dopamine self-polymerization and Michael addition). The Fe3O4-GO@PDA-Chitosan nanocomposites exhibited good performances with low detection limit (0.4 fmol·μL−1), good selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a molar ration of 10:1), good repeatability (4 times), high binding capacity (75 mg·g−1). Moreover, Fe3O4-GO@PDA-Chitosan nanocomposites were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 393 N-linked glycopeptides, representing 195 different glycoproteins and 458 glycosylation sites were identified. This study provides a feasible strategy for the surface functionalized novel materials for isolation and enrichment of N-glycopeptides.

TMEM63C, a Potential Novel Target for Albuminuria Development, Is Regulated by MicroRNA-564 and Transforming Growth Factor beta in Human Renal Cells

Introduction: Transmembrane protein (TMEM) 63C is a member of the TMEM gene family and was recently linked to glomerular filtration barrier function and albuminuria. Its molecular function and expression regulation are largely unknown. Objective: In this study, we set out to characterize the regulating impact of microRNAs (miRNAs) such as miRNA-564 (miR-564) on TMEM63C expression in renal cells. Also, we examined the influence of transforming growth factor beta (TGF-ß) on TMEM63C expression and the potential impact of TMEM63C inhibition on epithelial-mesenchymal transition (EMT) in renal cells and on cell viability in human embryonic kidney 293 cells (HEK 293). Methods: Expression analyses were done using real-time PCR and Western blot. Dual luciferase assay was performed to determine the miRNA-mediated expression control. Cell viability was assessed via trypan blue exclusion staining. Results and Conclusions: MiR-564 reduced TMEM63C expression in HEK 293 and human podocytes (hPC). The treatment of renal cells with TGF-ß led to an increased expression of TMEM63C. Moreover, a reduced TMEM63C expression was associated with a changed ratio of EMT marker proteins such as α-smooth muscle actin versus E-cadherin in HEK 293 and decreased nephrin expression in hPC. In addition, cell viability was reduced upon inhibition of TMEM63C expression in HEK 293. This study demonstrates first mechanisms involved in TMEM63C expression regulation and a link to EMT in renal cells.

Retraction notice for: "Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage" [Braz J Med Biol Res (2018) 51(2): e6611

Retraction notice for: "Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage" [Braz J Med Biol Res (2018) 51(2): e6611

Oridonin Attenuates Lipopolysaccharide-Induced ROS Accumulation and Inflammation in HK-2 Cells.

Renal tubulointerstitial inflammation plays an important role in chronic kidney disease (CKD). Inflammation reduction is a good strategy to combat CKD. Oridonin, an ent-kaurane diterpenoid isolated from Rabdosia rubescens (Donglingcao), is considered as an effective natural candidate for the treatment of anti-inflammatory, antiviral, and antibacterial activities, including liver fibrosis and many tumors; however, no study has demonstrated its effect on lipopolysaccharide- (LPS-) induced renal inflammation. To investigate the anti-inflammatory effects of oridonin on human renal proximal tubular epithelial cells (HK-2 cells), the expression levels of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS) were evaluated by Western blot analysis and 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining, respectively. The level of intracellular ROS increased in a dose-dependent manner following LPS treatment, whereas oridonin inhibited this effect, suggestive of its ability to prevent ROS accumulation. As the mitogen-activated protein kinase (MAPK) family of enzymes plays an important role in physiological responses, we examined the activation of JNK by Western blotting and found that oridonin attenuated LPS-induced JNK phosphorylation. Oridonin also attenuated RAW 264.7 cell chemotaxis towards LPS-treated HK-2 cells. Taken together, oridonin protected against LPS-induced inflammation including ROS accumulation, JNK activation, NF-κB nuclear translocation in HK-2 cells, and functionally blocked macrophage chemotaxis towards LPS-treated HK-2 cells. Oridonin may exhibit therapeutic potential by the anti-inflammation effect in LPS-treated HK-2 cells.

Study on the attenuation mechanism of aristolochia manshuriensis compatibility with zingiberis rhizoma based on different methods of boiling and blending

Objective Comparison of effects of Zingiberis Rhizoma and Aristolochia manshuriensis on diuretic effect and acute renal injury in rats by combining two methods of co-decoction and mixed-decoction. The effects of in vitro observation on normal human renal tubular epithelial cells (HK-2) were observed. Methods The rats were randomly divided into five groups: blank group, positive control group, aristolochia manshuriensis group, combined decoction group and divided decoction group, 8 rats in each group. The water-loading rat model was established by intragastric administration of normal saline. The urine of rats was collected and the volumes of urine were measured for 24 hours after the corresponding drugs were given to each group. After 2 weeks of gavage of the corresponding drugs in each group, the serum BUN, SCr and urine UCr and PRO levels were measured by 7600P automatic biochemical analyzer, and renal histopathology were observed by HE staining. The HK-2 cells were cultured in vitro and divided into control group, Aristolochia manshuriensis group, mixed decoction group and sub-decoction group. After 24 hour intervention, the activity of cells was detected by CCK-8 method and the apoptosis was observed by Hoechst stain method. Results There was no significant difference in 24 hour urine output between the groups (P>0.05). Compared with Aristolochia manshuriensis group, the kidney coefficient (0.010 1 ± 0.005 8 vs. 0.013 3 ± 0.007 8), SCr (38.52 ± 0.58 μmol/L vs. 46.61 ± 0.72 μmol/L), BUN (8.55 ± 0.12 mmol/L vs. 10.21 ± 0.30 mmol/L), UCr (52.21 ± 0.89 μmol/L vs. 57.71 ± 0.67 μmol/L), PRO (29.89 ± 0.18 mg/L vs. 34.23 ± 6.05 mg/L) of combined decoction group significantly decreased (P<0.05). The survival rate of HK-2 cells (72.45% ± 3.70% vs. 55.92% ± 8.39%) in combined decoction group significantly increased (P<0.01), and the apoptosis rate (7.9% ± 2.6% vs. 31.6% ± 9.1%) significantly decreased (P<0.01). Conclusions The traditional co-decoction method of Aristolochia manshuriensis compatibility with Zingiberis Rhizoma can achieve a certain attenuation effect, and the mixed-decoction group can not achieve the attenuating effec. Key words: Caulis aristolochiae manshuriensis; Zingiberis rhizoma; Compatibility (TCD); Decoction preparing methods; Toxicity of Chinese medicine; Acute kidney injury; Rats

Associative effects of lignin-derived biochar and arbuscular mycorrhizal fungi applied to soil polluted from Pb-acid batteries effluents on barley grain safety

While disobeying environmental regulations of Pakistan, several Pb-acid batteries recycling and repairing units discharge their effluents into water canals that irrigate arable fields. Resultantly, serious ecological risks, as well as human health hazards through consumption of edible crops grown on such Pb-polluted soils have been reported. In this experiment, we observed associative effects of amending a soil polluted from Pb-acid batteries effluents (SPB) with arbuscular mycorrhizal fungi (AMF) and lignin-derived biochar (LBC) on barley grain safety to human health. The SPB was treated with AMF inoculum (a consortium of four AMF species), lignin (LN), and LBC, as sole treatments and AMF inoculum with LN and LBC. Barley parameters involving Pb distribution in grain and other parts, grain biochemistry, and nutrition were assessed. Likewise, Pb bioavailability in SPB, AMF root colonization, soil enzymes, microbial biomass carbon (MBC), and AMF produced total glomalin related soil protein (TGSP) were also scoped. Additionally, human renal cells (HEK 293) cytotoxicity test was performed by opting barley grain-related Pb concentrations. Results show that LBC + AMF significantly reduced grain Pb concentrations below the critical limit [4.67 mg kg−1 (WHO/FAO standard)], AMF colonization, MBC, soil enzymology, and TGSP, compared to control. Likewise, rest barley parameters were also improved in this treatment. Contrary to other treatments, grain produced on LBC + AMF did not result in (a) cell apoptosis, (b) cell distortion and (c) cohesion loss. Immobilization of Pb in SPB was due to the dilution effect of Pb adsorption on LBC, AMF mycelium and TGSP which resulted in a significant drop of grain Pb concentrations below the critical limit and ultimately no harm to HEK 293 cells. Our findings endorse that grain produced at LBC + AMF treatment are safer for human consumption and will not pose health risks. The LBC + AMF application can remediate SPB for safer cereal production.

Hexavalent chromium intoxication induces intrinsic and extrinsic apoptosis in human renal cells.

Hexavalent chromium [Cr(VI)], is a well-known toxic form of the heavy metal chromium in the natural environment. Clinical evidence has indicated that exposure to Cr(VI) can cause severe renal damage. The production of reactive oxygen species (ROS) due to intracellular reduction of Cr(VI) is the main mechanism underlying the induction of cellular dysfunction and apoptosis. The present study aimed to investigate in detail the apoptotic pathways induced by Cr(VI)-exposure in a human immortalized proximal tubular epithelial cell line HK-2, in order to understand the mechanism involved therein. Exposure to 10 µM potassium dichromate (K2Cr2O7), a toxic compound of Cr(VI), significantly decreased cell viability after 24 and 48 h of incubation and induced intracellular ROS generation. The expression levels of markers that activate the apoptotic pathway including cleaved caspase-3 and poly (ADP-ribose) polymerase were significantly upregulated in K2Cr2O7-exposed HK-2 cells. In addition, the induction of intrinsic and extrinsic apoptotic markers was detected in K2Cr2O7-exposed HK-2 cells. In summary, the present study described for the first time the novel apoptotic mechanism of Cr(VI)-toxicity in human renal cells which may be beneficial in designing optimal clinical treatment for renal damage caused by acute Cr(VI) toxicity.