Tubular Epithelial Cells In Response Research Articles

Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-β (IFN-β). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-β, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-β or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/β (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

TGFβ instructs mTORC2 to activate PKCβII for increased TWIST1 expression in proximal tubular epithelial cell injury.

The plasticity of proximal tubular epithelial cells in response to TGFβ contributes to the expression of TWIST1 to drive renal fibrosis. The mechanism of TWIST1 expression is not known. We show that both PI3 kinase and its target mTORC2 increase TGFβ-induced TWIST1 expression. TGFβ enhances phosphorylation on Ser-660 in the protein kinase C βII (PKCβII) hydrophobic motif site. Remarkably, phosphorylation-deficient PKCβIIS660A, kinase-dead PKCβII, and PKCβII knockdown blocked TWIST1 expression by TGFβ. Inhibition of TWIST1 arrested TGFβ-induced tubular cell hypertrophy and the expression of fibronectin, collagen I (α2), and α-smooth muscle actin. By contrast, TWIST1 overexpression induced these pathologies. Interestingly, the inhibition of PKCβII reduced these phenomena, which were countered by the expression of TWIST1. These results provide the first evidence for the involvement of the mTORC2-PKCβII axis in TWIST1 expression to promote tubular cell pathology.

Sirtuin 7 mitigates renal ferroptosis, fibrosis and injury in hypertensive mice by facilitating the KLF15/Nrf2 signaling.

Hypertension is one of the leading causes of chronic kidney disease characterized with renal fibrosis. This study aimed to investigate roles and mechanisms of sirtuin 7 (SIRT7) in hypertensive renal injury. Mini-pumps were implanted to male C57BL/6 mice to deliver angiotensin (Ang) Ⅱ (1.5mg/kg/d) or saline for 2 weeks. Ang Ⅱ infusion resulted in marked increases in systolic blood pressure levels, renal ferroptosis and interstitial fibrosis in hypertensive mice, concomitantly with downregulated SIRT7 and Krüppel-like factor 15 (KLF15) levels. Notably, administration of recombinant adeno-associated virus-SIRT7 or ferroptosis inhibitor ferrostatin-1 effectively mitigated Ang Ⅱ-triggered renal ferroptosis, epithelial-mesenchymal transition (EMT), interstitial fibrosis, renal functional and structural injury in hypertensive mice by blunting the KIM-1/NOX4 signaling and enforcing the KLF15/Nrf2 and xCT/GPX4 signaling, respectively. In primary cultured mouse renal tubular epithelial cells (TECs), Ang Ⅱ pretreatment led to repressed SIRT7 expression and augmented ferroptosis as well as partial EMT, which were substantially antagonized by rhSIRT7 or ferrostatin-1 administration. Additionally, both Nrf2 inhibitor ML385 and KLF15 siRNA strikingly abolished the rhSIRT7-mediated beneficial roles in mouse renal TECs in response to Ang Ⅱ with reduced expression of Nrf2, xCT and GPX4. More importantly, ML385 administration remarkably amplified Ang Ⅱ-mediated ROS generation, lipid peroxidation and ferroptosis in renal TECs, which were significantly reversed by ferrostatin-1. In conclusion, SIRT7 alleviates renal ferroptosis, lipid peroxidation, and partial EMT under hypertensive status by facilitating the KLF15/Nrf2 signaling, thereby mitigating renal fibrosis, injury and dysfunction. Targeting SIRT7 signaling serves as a promising strategy for hypertension and hypertensive renal injury.

Inhibition of METTL3 attenuates renal injury and inflammation by alleviating TAB3 m6A modifications via IGF2BP2-dependent mechanisms.

The role of N6-methyladenosine (m6A) modifications in renal diseases is largely unknown. Here, we characterized the role of N6-adenosine-methyltransferase-like 3 (METTL3), whose expression is elevated in renal tubules in different acute kidney injury (AKI) models as well as in human biopsies and cultured tubular epithelial cells (TECs). METTL3 silencing alleviated renal inflammation and programmed cell death in TECs in response to stimulation by tumor necrosis factor-α (TNF-α), cisplatin, and lipopolysaccharide (LPS), whereas METTL3 overexpression had the opposite effects. Conditional knockout of METTL3 from mouse kidneys attenuated cisplatin- and ischemic/reperfusion (I/R)-induced renal dysfunction, injury, and inflammation. Moreover, TAB3 [TGF-β-activated kinase 1 (MAP3K7) binding protein 3] was identified as a target of METTL3 by m6A methylated RNA immunoprecipitation sequencing and RNA sequencing. The stability of TAB3 was increased through binding of IGF2BP2 (insulin-like growth factor 2 binding protein 2) to its m6A-modified stop codon regions. The proinflammatory effects of TAB3 were then explored both in vitro and in vivo. Adeno-associated virus 9 (AAV9)-mediated METTL3 silencing attenuated renal injury and inflammation in cisplatin- and LPS-induced AKI mouse models. We further identified Cpd-564 as a METTL3 inhibitor that had better protective effects against cisplatin- and ischemia/reperfusion-induced renal injury and inflammation than S-adenosyl-l-homocysteine, a previously identified METTL3 inhibitor. Collectively, METTL3 promoted m6A modifications of TAB3 and enhanced its stability via IGF2BP2-dependent mechanisms. Both genetic and pharmacological inhibition of METTL3 attenuated renal injury and inflammation, suggesting that the METTL3/TAB3 axis is a potential target for treatment of AKI.

Migration of Human Renal Tubular Epithelial Cells in Response to Physiological Electric Signals.

Restoration of proximal tubular cell integrity and function after ischemic injury involves cell migration and proliferation. Endogenous fields are present during embryonic development and wound healing. Electric field (EF)-induced effects on cell migration have been observed in many cell types. This study investigated the effect of physiological direct current EF (dc EF) on the motility of renal epithelial cells. Human renal tubular epithelial (HK-2) and human-derived renal epithelial (HEK-293) cells were exposed to dc EF at physiological magnitude. Cell images were recorded and analyzed using an image analyzer. Cell lysates were used to detect protein expression by western blot. Scratch wounds were created in monolayers of HK-2 cells, and wound areas of cells were measured in response to EF exposure. Cells migrated significantly faster in the presence of an EF and toward the cathode. Application of an EF led to activation of the Erk1/2, p38 MAPK, and Akt signaling pathways. Pharmacological inhibition of Erk1/2, p38 MAPK, and Akt impaired EF-induced migratory responses, such as motility rate and directedness. In addition, exposure of the monolayers to EF enhanced EF-induced HK-2 wound healing. Our results suggest that EFs augment the rate of single renal epithelium migration and induce cell cathodal migration through activation of Erk1/2, p38 MAPK, and Akt signaling. Moreover, exposure of the renal epithelium to EF facilitated closure of in vitro small wounds by enhancing cell migration.

MicroRNA‑93 inhibits the apoptosis and inflammatory response of tubular epithelial cells via the PTEN/AKT/mTOR pathway in acute kidney injury.

Renal tubular epithelial cell injury is the main cause of septic acute kidney injury (AKI), which is characterized by the excessive inflammatory response and apoptosis. Numerous studies have demonstrated that miRNAs are associated with inflammatory response and apoptosis in numerous diseases. The present study mainly focuses on investigating the association between microRNA (miRNA/miR) expression and inflammatory response and apoptosis in the pathogenesis of AKI. In vitro and in vivo models of AKI were simulated using Escherichia coli lipopolysaccharide (LPS)-administrated kidney epithelial cells and mice, respectively. The miRNA expression profile was examined using miRNA microarray in kidney tissues. Next, the effects of miR-93 upregulation on the apoptosis, cytokine expression and oxidative stress in the LPS-stimulated TCMK-1 were tested. The target genes of this miRNA were investigated, and the regulatory association between miR-93 and the AKT/mTOR pathway was investigated. The results demonstrated that miR-93 was the most downregulated miRNA in mice kidney. Furthermore, in LPS-induced renal tubular epithelial cells (TECs) injury model, that upregulation of miR-93 was found to attenuate the apoptosis and inflammatory response, as well as reactive oxygen species generation. Mechanistically, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was identified as a target of miR-93. Further experiments revealed that LPS-induced the decrease of phosphorylated (p)-AKT and p-mTOR protein expression in vitro are reversed by the overexpression of miR-93. The results of the present study suggested that the protective effect of miR-93 on AKI may be associated with the activation of PTEN/AKT/mTOR pathway. miR-93 may serve as a potential therapeutic target in sepsis-induced AKI.

Comprehensive study of altered proteomic landscape in proximal renal tubular epithelial cells in response to calcium oxalate monohydrate crystals

BackgroundCalcium oxalate monohydrate (COM), the major crystalline composition of most kidney stones, induces inflammatory infiltration and injures in renal tubular cells. However, the mechanism of COM-induced toxic effects in renal tubular cells remain ambiguous. The present study aimed to investigate the potential changes in proteomic landscape of proximal renal tubular cells in response to the stimulation of COM crystals.MethodsClinical kidney stone samples were collected and characterized by a stone component analyzer. Three COM-enriched samples were applied to treat human proximal tubular epithelial cells HK-2. The proteomic landscape of COM-crystal treated HK-2 cells was screened by TMT-labeled quantitative proteomics analysis. The differentially expressed proteins (DEPs) were identified by pair-wise analysis. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEPs were performed. Protein interaction networks were identified by STRING database.ResultsThe data of TMT-labeled quantitative proteomic analysis showed that a total of 1141 proteins were differentially expressed in HK-2 cells, of which 699 were up-regulated and 442 were down-regulated. Functional characterization by KEGG, along with GO enrichments, suggests that the DEPs are mainly involved in cellular components and cellular processes, including regulation of actin cytoskeleton, tight junction and focal adhesion. 3 high-degree hub nodes, CFL1, ACTN and MYH9 were identified by STRING analysis.ConclusionThese results suggested that calcium oxalate crystal has a significant effect on protein expression profile in human proximal renal tubular epithelial cells.

MiR-211 alleviates ischaemia/reperfusion-induced kidney injury by targeting TGFβR2/TGF-β/SMAD3 pathway

ABSTRACT MicroRNA-211 (miR-211) is closely related to apoptosis and plays an important role in ischemia/reperfusion (I/R) injury. Whether miR-211 is involved in the protective effects in renal I/R injury is unknown. In this study, we evaluated the role of miR-211 in human tubular epithelial cells in response to hypoxia-reoxygenation (H/R) stimulation and I/R injury in vitro and in vivo. The results revealed that miR-211 was down-regulated and TGFβR2 was up-regulated in human kidney (HK-2) cells subjected to H/R. Luciferase reporter assay showed that TGFβR2 was a direct target of miR-211. Enforced miR-211 expression decreased H/R-induced HK-2 cell apoptosis and increased cell viability, and targeting miR-211 further increased H/R-induced HK-2 cell apoptosis and decreased cell viability. However, the effect of miR-211 was reversed by targeting TGFβR2 or enforced TGFβR2 expression in miR-211 overexpressing cells or miR-211 downexpressing cells. Moreover, we confirmed that miR-211 interacted with TGFβR2, and regulating TGF-β/SMAD3 signal. In vivo in mice, miR-211 overexpression ameliorates biochemical and histological kidney injury, reduces apoptosis in mice following I/R. On the contrary, miR-211 downexpressing promoted histological kidney injury and increased apoptosis in mice following I/R. Inhibition of miR-211 or miR-211 overexpression inhibited TGF-β/SMAD3 pathways or activated TGF-β/SMAD3 signal pathways in vitro and in vivo, which are critical for cell survival. Our findings suggested that miR-211 suppress apoptosis and relieve kidney injury following H/R or I/R via targeting TGFβR2/TGF-β/SMAD3 signals. Therefore, miR-211 may be as therapeutic potential for I/R- induced kidney injury.

Analysis of Differentially Expressed Long Noncoding RNA in Renal Ischemia-Reperfusion Injury

Background: Renal ischemia-reperfusion (IR) injury is one of the major causes of acute renal failure which seriously endangers the health and life of patients. Currently, there is still lack of comprehensive knowledge of the molecular mechanism of renal IR injury, and the regulatory role of long noncoding RNA (lncRNA) in renal IR damage remains poorly understood. Aim: The aim of this study was to analyze the expression spectrum of lncRNA in renal IR damage in mice and to explore specific lncRNA that may be involved in regulating the development of human renal IR injury. Methods: RNA-Seq was used to investigate the lncRNA profile of renal IR injury in a mouse model, and conservation analysis was performed on mouse lncRNAs with differential expression (fragments per kilobase of transcript per million mapped reads ≥2) by BLASTN. The potential functions and associated pathways of the differentially expressed lncRNA were explored by bioinformatics analysis. The cell hypoxia model was used to detect the expression of the candidate lncRNA. Results: Of the 45,923 lncRNA transcripts detected in the samples, and 5,868 lncRNAs were found to be significantly differentially expressed (p < 0.05 and fold change ≥ 2) in 24-h IR kidney tissue compared to the expression in the control group. It was found that 56 differently expressed mouse lncRNA transcripts have human homology by analyzing the conserved sequences. We also found that lncRNA-NONHSAT183385.1 expression significantly increased in HK2 cells after 24 h of hypoxia and increased further 6 h after reoxygenation, and after 24 h of reoxygenation it was dramatically downregulated, indicating that NONHSAT183385.1 may be involved in the pathophysiological process of renal tubular epithelial cells in response to ischemia in human renal IR. Conclusion: Our study revealed differentially expressed lncRNAs in renal IR damage in mice and identified a set of conserved lncRNAs, which would help to explore lncRNAs that may play important regulatory roles in human renal IR injury.

The regulatory role of HIF-1 in tubular epithelial cells in response to kidney injury.

The high sensitivity to changes in oxygen tension makes kidney vulnerable to hypoxia. Both acute kidney injury and chronic kidney disease are almost always accompanied by hypoxia. Tubular epithelial cells (TECs), the dominant intrinsic cells in kidney tissue, are believed to be not only a victim in the pathological process of various kidney diseases, but also a major contributor to kidney damage. Hypoxia inducible factor-1 (HIF-1) is the main regulator of adaptive response of cells to hypoxia. Under various clinical and experimental kidney disease conditions, HIF-1 plays a pivotal role in modulating multiple cellular processes in TECs, including apoptosis, autophagy, inflammation, metabolic pattern alteration, and cell cycle arrest. A comprehensive understanding of the mechanisms by which HIF-1 regulates these cellular processes in TECs may help identify potential therapeutic targets to improve the outcome of acute kidney injury and delay the progression of chronic kidney disease.

Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals

Objective To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals. Methods HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA). Results Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P 0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50±20.51)pg/ml, P>0.05] in the ATV group was lower than that in the control group, but the difference were not statistically significant (P>0.05). The concentrations of IL-1β[(850.50±48.79)pg/ml vs. (183.50±7.78)pg/ml, P 0.05). The relative expression of NF-κB (0.295±0.035 vs. 0.100±0.014, P<0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group. While the relative expression of NF-κB (0.160±0.012 vs. 0.295±0.035, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting. Conclusions Calcium oxalate crystals can induce the inflammatory response of HK-2 cells, while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β, IL-18 and the expression of NF-κB. Key words: Inflammation; Cytokines; Atorvastatin; Calcium oxalate; Human renal tubular epithelial cells

Loss of receptor interacting protein kinases 3 and caspase-8 augments intrinsic apoptosis in tubular epithelial cell and promote kidney ischaemia-reperfusion injury.

Ischaemia-reperfusion injury (IRI) is associated with programmed cell death that promotes inflammation and organ dysfunction. Necroptosis is mediated by members of receptor interacting protein kinases (RIPK1/3). Inhibition of RIPK1/3 provides a pro-survival benefit in kidney IRI. Caspase-8 initiates apoptosis and contributes to IRI. We studied whether inhibiting both RIPK3 and caspase-8 would provide an additional benefit in kidney IRI. A clamp was applied to the left kidney pedicle for 45 min followed by right kidney nephrectomy. Kidney and serum from wild type, RIPK3-/- , and RIPK3-/- caspase-8-/- double knockout (DKO) mice were collected post-IRI for assessment of injury. Tubular epithelial cells (TEC) isolated from wild type, RIPK3-/- , and DKO mice were treated with interferons-γ and interleukin-1β to induce apoptotic death. Kidney IRI of DKO mice did not show improvement over RIPK3-/- mice. We have found that DKO triggered 'intrinsic' apoptosis in TEC in response to interleukin-1β and interferons-γ. Up-regulation of the B-cell lymphoma 2 (Bcl-2)-associated death promoter, the Bcl-2-homologous antagonist killer and Bcl-2-associated X protein and enhanced activation of caspase-3 and 9 were found in DKO TEC. TEC infected with Murine cytomegalovirus that encodes multiple cell death inhibitors resist to death. We show that the deletion of both RIPK3 and caspase-8 does not provide additive benefit in IRI or TEC death and may enhance injury by up-regulation of intrinsic apoptosis. This suggests blocking multiple death pathways may be required for the prevention of kidney IRI clinically.

Hsa-miR-500a-3P alleviates kidney injury by targeting MLKL-mediated necroptosis in renal epithelial cells.

MLKL is a central mediator for necroptosis. Its knockout significantly relieves acute kidney injury (AKI). However, its upstream regulatory mechanism in AKI has not been fully elucidated. We recently reviewed how microRNAs (miRNAs), a type of well-studied epigenetic regulator, play critical roles in AKI. Here, we evaluated miRNAs that potentially target MLKL and evaluated their function in human tubular epithelial cells in response to toxic and ischemic insults. TargetScan analysis showed that miR-194-5P, miR-338-3P, miR-500a-3P, and miR-577 had MLKL binding sites. Although all 4 miRNAs are reduced in AKI, our data show that only hsa-miR-500a-3P was significantly suppressed in cisplatin-treated human tubular epithelial (HK2) cells. We further found that hsa-miR-500a-3P alleviated cisplatin-induced HK2 cell death, which was confirmed by transmission electron microscopy and flow cytometry. In addition, overexpression of hsa-miR-500a-3P decreased kidney injury molecule-1 mRNA and protein levels. Real-time PCR, ELISA, and immunofluorescence data show that hsa-miR-500a-3P protected against inflammatory response, evidenced by decreased monocyte chemotactic protein-1 and proinflammatory cytokines TNF-α and IL-8. Further, hsa-miR-500a-3P attenuated P65 NF-κB phosphorylation and promoter activity. Mechanistically, luciferase reporter assay showed that hsa-miR-500a-3P bound the 3'UTR of MLKL, thereby suppressing phosphorylation and membrane translocation of MLKL. In agreement with these findings, we identified that overexpression of hsa-miR-500a-3P attenuated cell injury and the inflammatory response in response to sodium azide treatment in an in vitro model. Results show that circulating exosomes from patients with AKI down-regulated miR-500a-3P, which suppressed cell injury and inflammation in HK2 cells. hsa-miR-500a-3P alleviated toxic and ischemic insults that were triggered by cell necroptosis and the inflammatory response in human HK2 cells by targeting MLKL. This may serve as a novel therapeutic target for treatment of AKI.-Jiang, L., Liu, X.-Q., Ma, Q., Yang, Q., Gao, L., Li, H.-D., Wang, J.-N., Wei, B., Wen, J., Li, J., Wu, Y.-G., Meng, X.-M. hsa-miR-500a-3P alleviates kidney injury by targeting MLKL-mediated necroptosis in renal epithelial cells.

Removal of Cell-Activating Substances Using Dialyzers With Various Permeability Profiles.

Despite multiple efforts to target an improvement in clinical outcomes of patients with end-stage renal disease, several challenges must still be addressed. Dialysis patients are at a high risk for complications, as reflected by increasing mortality rates. The objective of this study is to assess the impact of the application of dialyzers with varying permeability profiles on the removal of cell-activating substances from the blood of hemodialysis (HD) patients. Dialysate samples were collected using Revaclear 400 (RC) and MCO-Ci400 (MCO-CI). Total protein and solute marker concentrations were determined for the concentrated sample. The response of tubular epithelial cells (TECs) to the dialysate samples was assessed via measurement of interleukin 6, cell viability, and morphology. Proteomic analysis of the dialysate samples was performed using liquid chromatography coupled to tandem mass spectrometry. Treatment of TECs with the MCO-CI dialysate resulted in significantly decreased cell viability compared with the RC dialysate. TECs incubated with samples from MCO-CI lost their typical brick-like shape and cell-cell connections. Proteomic analysis of dialysate samples indicated multiple pro-apoptotic and pro-inflammatory proteins, supporting the observed phenotype. Additionally, application of the MCO-CI dialyzer allowed for more efficient removal of proteins associated with advanced chronic kidney disease stages. Collectively, the use of dialyzer with a higher permeability profile enabled more efficient removal of cell-activating and toxic substances from the blood of HD patients. However, a further large-scale study is needed to address benefits and associated risks for patients.

Abstract P137: Mir-122 is Associated With Stress-response Protein, Hemeoxygenase-1, for Regulation of Extracellular Matrix Remodeling in Renal Hypertension

Oxidative stress is a major contributing factor in hypertension-induced kidney injury. Hemeoxygenase-1 (Ho-1) is stress response protein constitutively expressed by the proximal tubular epithelial cells in response to oxidative stress. MicroRNAs are single stranded RNA involved in the regulation of gene expression. MicroRNA-122 has been shown to regulate Ho-1 expression in hepatitis; however whether miR-122 regulates Ho-1 in hypertensive kidney is not known. The purpose of the study was to investigate the miRNA-122 Ho-1 regulation and determine its role in extracellular matrix remodeling in renal hypertension. In vitro experiments were done using mesangial cells, treated with/without 200 μM of Angiotensin-II (Ang-II). Ho-1 was induced by ~3.5 folds with Ang-II treatment. miR-122, Ho-1 regulator, was downregulated by &gt;15 times in Ang-II treated cells. In vivo experiments were performed on WT (C57BL6/J) mice aged 12-14 wk and 75-78 wk. The animals were treated with Ang-II (1000ng/kg/min) for 4 weeks. Ho-1 is ~6 folds less in kidney of aged mice as compared to that in the young mice. Hypertension increases miR-122 expression to a greater extent (~5 folds) in aged animals. In Ho-1 knocked down mesangial cells, the extracellular matrix component, Collagen 1A1 (Col1a1), was increased by ~2 folds. In contrast, vascular endothelial growth factor ( Vegf ) and hypoxia-inducible factor ( Hif1 α ) were downregulated in Ho-1 depleted cells. In conclusion, micro RNA, miR-122, transcriptionally regulates Ho-1 as a repressor in kidney and thus affects Ho-1 mediated regulation of the extracellular remodeling in hypertension-induced renal damage.

CXCL16 regulates cisplatin-induced acute kidney injury.

The pathogenesis of cisplatin-induced acute kidney injury (AKI) is characterized by tubular cell apoptosis and inflammation. However, the molecular mechanisms are not fully understood. We found that CXCL16 was induced in renal tubular epithelial cells in response to cisplatin-induced AKI. Therefore, we investigated whether CXCL16 played a role in cisplatin–induced tubular cell apoptosis and inflammation. Wild-type and CXCL16 knockout mice were administrated with vehicle or cisplatin at 20 mg/kg by intraperitoneal injection. CXCL16 knockout mice had lower blood urea nitrogen and less tubular damage following cisplatin-induced AKI as compared with wild-type mice. Genetic disruption of CXCL16 reduced tubular epithelial cell apoptosis and decreased caspase-3 activation. Furthermore, CXCL16 deficiency inhibited infiltration of macrophages and T cells into the kidneys following cisplatin treatment, which was associated with reduced expression of the proinflammatory cytokines in the kidneys. Taken together, our results indicate that CXCL16 plays a crucial role in the pathogenesis of cisplatin–induced AKI through regulation of apoptosis and inflammation and maybe a novel therapeutic target for cisplatin-induced AKI.

Autophagy activation reduces renal tubular injury induced by urinary proteins

Autophagy is shown to be beneficial for renal tubular injury caused by nephrotoxic drugs. To investigate whether autophagy could protect renal tubular epithelial cells (TECs) from injury induced by urinary proteins, we studied the activity and action of autophagy in TECs after urinary protein overload in vivo and in vitro. We found that autophagic vacuoles increased in TECs from patients with minimal change nephrotic syndrome (MCNS) and rat models with severe proteinuria induced by cationic BSA. In HK-2 cells, exposure to urinary proteins extracted from patients with MCNS led to a significant increase in autophagosome and autolysosome formation and decrease in SQSTM1/p62 protein level. Urinary protein addition also induced lysosomal turnover of LC3-II and perinuclear clustering of lysosomes. These changes were mediated by a reactive oxygen species (ROS)-dependent mechanism. Furthermore, pretreatment of HK-2 cells with rapamycin reduced the production of LCN2/NGAL and HAVCR1/KIM-1 and the level of apoptosis induced by urinary proteins. In contrast, blocking autophagy with chloroquine or BECN1 siRNAs exerted an opposite effect. Similar results were also observed in animal models with proteinuria after treatments with rapamycin and chloroquine. Taken together, our results indicated an increase in autophagic flux, which mounts an adaptive response in TECs after urinary protein overload.

Critical Role of CXCL16 in Hypertensive Kidney Injury and Fibrosis

Recent evidence indicates that inflammation plays a critical role in the initiation and progression of hypertensive kidney disease. However, the signaling mechanisms underlying the induction of inflammation are poorly understood. We found that chemokine (C-X-C motif) ligand 16 (CXCL16) was induced in renal tubular epithelial cells in response to angiotensin II in a nuclear factor-κB-dependent manner. To determine whether CXCL16 plays a role in angiotensin II-induced renal inflammation and fibrosis, wild-type and CXCL16 knockout mice were infused with angiotensin II at 1500 ng/kg per minute for up to 4 weeks. Wild-type and CXCL16 knockout mice had comparable blood pressure at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between wild-type and CXCL16 knockout mice. CXCL16 knockout mice were protected from angiotensin II-induced renal dysfunction, proteinuria, and fibrosis. CXCL16 deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidneys of angiotensin II-treated mice, which was associated with less expression of extracellular matrix proteins. Furthermore, CXCL16 deficiency inhibited infiltration of F4/80(+) macrophages and CD3(+) T cells in the kidneys of angiotensin II-treated mice compared with wild-type mice. Finally, CXCL16 deficiency reduced angiotensin II-induced proinflammatory cytokine expressions in the kidneys. Taken together, our results indicate that CXCL16 plays a pivotal role in the pathogenesis of angiotensin II-induced renal injury and fibrosis through regulation of macrophage and T cell infiltration and bone marrow-derived fibroblast accumulation.

Viral double-stranded RNA sensors induce antiviral, pro-inflammatory, and pro-apoptotic responses in human renal tubular epithelial cells

Viral infection in the kidney is characterized by tubular injury induced directly by the virus and/or by cytotoxic lymphocytes. Previously, we found that human tubular epithelial cells express Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene-I (RIG-I), all sensors of double-stranded RNA (dsRNA) and potent inducers of antiviral activity. Here, we demonstrate increased expression of these three dsRNA sensors in kidney transplant biopsies during cytomegalovirus or BK virus infection. In primary tubular epithelial cells, dsRNA sensor activation induced the production of pro-inflammatory TNF-α and antiviral IFN-β. Notably, dsRNA also enhanced the expression of pro-apoptotic proteins; however, dsRNA alone did not cause cell death due to the expression of anti-apoptotic proteins. The dsRNA sensitized tubular epithelial cells to apoptosis induced by an agonistic antibody against the Fas receptor (CD95), an apoptotic pathway that eliminates infected cells. These findings indicate that tubular epithelial cells require at least two signals to undergo apoptosis, which can help preserve tubular integrity even under inflammatory conditions. Thus, sensors of viral dsRNA promote antiviral, pro-inflammatory, and pro-apoptotic responses in tubular epithelial cells, which may orchestrate the control of viral infection in the kidney.