Engineering Bioactive Liposomal Nanoparticles for Kidney-Targeted ECFC Backpacks.

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Background: The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Methods: Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Results: Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Conclusions: Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake.

We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.

Histone acetylation plays an important role in regulating gene expressions by modulating chromatin structure. Histone deacetylase (HDAC) inhibitors have been reported to have an antifibrogenic effect in some organs, such as the liver, skin, and lung, but the underlying mechanisms remain to be clarified. In the kidney, bone morphologic protein 7 (BMP-7) and hepatocyte growth factor are reported to antagonize TGF-beta1-induced tubular epithelial-to-mesenchymal transition (EMT), but nothing is known concerning the effect of HDAC inhibitors on EMT. It was shown that trichostatin A (TSA), an HDAC inhibitor, prevented TGF-beta1-induced EMT in cultured human renal proximal tubular epithelial cells. Treatment with TGF-beta1 induced morphologic changes such as EMT in human renal proximal tubular epithelial cells. However, co-treatment with TSA completely prevented TGF-beta1-induced morphologic changes and significantly prevented TGF-beta1-induced downregulation of E-cadherin and upregulation of collagen type I. Treatment with TSA did not alter TGF-beta1-induced phosphorylation of Smad2 and Smad3 but induced several inhibitory factors of TGF-beta1 signals, such as inhibitors of DNA binding/differentiation 2 (Id2) and BMP-7. Chromatin immunoprecipitation assay confirmed that histone acetylation was involved in the downregulation of E-cadherin and upregulation of Id2 and BMP-7. These results suggest that TSA and other HDAC inhibitors could be new therapeutic agents for tubular EMT.

Cytolethality of hemolytic Escherichia coli to primary human renal proximal tubular cell cultures obtained from different donors

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22phox protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22phox protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22phox protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22phox protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22phox protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

Background: High blood glucose is characteristic of diabetic nephropathy (DN). Both lectin-like ox-LDL receptor-1 (LOX-1) and renal tubular epithelial cells apoptosis reportedly are important for the pathogenesis and progression of DN. In this study, we explored the regulatory effects of high glucose on the expression of LOX-1 and its impact on oxLDL-induced apoptosis in human renal proximal tubular epithelial cells (HRPTEpCs). Methods: Primary HRPTEpCs were treated with high glucose with or without concurrent treatment with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 or lentiviral knockdown of LOX-1. HRPTEpCs cultured in normal glucose concentration (5.5 mmol/l) was used as a control. Results and Conclusion: High glucose concentration dependency increased the expression of LOX-1, which led to increased ox-LDL binding in HRPTEpCs. In addition, high glucose upregulated the LOX-1 gene promoter activity but not its mRNA stability in HRPTEpCs; the effect was abolished by PD169316. Furthermore, high glucose markedly enhanced oxLDL-induced apoptosis in HRPTEpCs, which was largely abolished by knockdown of LOX-1. This study demonstrates that high glucose induces the expression of LOX-1 at the gene promoter/transcription level mainly by a p38 MAPK-dependent mechanism, which enhances oxLDL-induced apoptosis in renal tubular epithelial cells. It adds new insights into the molecular mechanisms underlying DN.

Expression of TGF-β–induced matrix protein βig-h3 is up-regulated in the diabetic rat kidney and human proximal tubular epithelial cells treated with high glucose

Modified Zhenwu Tang delays chronic renal failure progression by modulating oxidative stress and hypoxic responses in renal proximal tubular epithelial cells

Arachidonic acid can induced several injury effects on renal tubular cells. The present study was aimed to investigate the effects of arachidonic acid on cellular apoptosis in human proximal tubular epithelial cells (HK‐2) and its signaling mechanisms. Treatment with various doses of arachidonic acid resulted in dose‐dependent decreases of cell viability and increases of reactive oxygen species (ROS). Arachidonic acid induced the phosphorylation of p38 MAPK, extracellular signal‐regulated kinase (ERK1/2), and c‐Jun N‐terminal kinase (JNK), the nuclear translocation of NF‐κB, and IκB‐degradation. Cell death detection assay revealed arachidonic acid induced apoptotic cell death of HK‐2 cells. Arachidonic acid‐induced apoptosis was significantly attenuated by inhibition of the NF‐κB and mitogen‐activated protein kinase (MAPK) signaling pathways. Moreover, the antioxidant N‐acetyl‐L‐cysteine (NAC), significantly inhibited arachidonic acid‐induced apoptosis and NF‐κB and MAPK activation. Taken together, these results indicate that arachidonic acid induced apoptosis mainly by stimulating ROS production and through NF‐κB and MAPK signaling pathways in human renal proximal tubular cells.

IL-6 augments angiotensinogen in primary cultured renal proximal tubular cells

Tubulointerstitial fibrosis is a relatively common and sinister complication of cyclosporine A (CsA) therapy that limits its clinical use. CsA may have direct effects on renal tubular epithelial cells by promoting epithelial-mesenchymal transition (EMT). EMT plays an important role in embryonic development and tumourigenesis and has been described in organ remodelling during fibrogenesis. In this study, we investigated the effects of CsA on a human renal cell line as a model system to test the hypothesis that CsA can induce renal EMT. Human renal proximal tubular cells were treated with CsA (0.42-42 microm) for periods up to 72 h. Viability was assessed by the Alamar Blue assay. Morphological changes were assessed by phase contrast microscopy. The effects on epithelial adherens molecule, beta-catenin and stress fibre protein, F-actin were analysed by indirect immunofluorescence. Reverse transcription--polymerse chain reaction was performed to measure the mRNA levels of extracellular matrix components. Expression of transforming growth factor-beta was measured by western blotting. Expression and activity of matrix metalloproteinases were measured by gelatin zymography. CsA induced striking morphological changes in epithelial cells, including changes in cellular morphology, F-actin stress fibre formation, delocalization of the adherens junction protein beta-catenin and increased levels of collagen IV and fibronectin. In addition, CsA-induced EMT was associated with increased TGF-beta1 protein levels and EMT was markedly attenuated in the presence of anti-TGF-beta1 antibody. CsA-induced EMT was also associated with increased expression of connective tissue growth factor (CTGF) suggesting that this molecule may serve as downstream mediator of TGF-beta1 pro-fibrotic activity in this setting. In aggregate, these data suggest that CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-beta1 and CTGF as mediators of this response. The further delineation of the molecular components of this pro-fibrogenic response may suggest novel strategies through which to prevent CsA-induced tubulo-interstitial fibrosis in vivo.

The signal transduction pathways modulating bFGF effects in renal tubular epithelial cells (RTEc) are not completely understood. Since the cAMP and the mitogen-activated protein kinase (MAPK) pathways can modulate the growth of RTEc, we studied whether two cAMP elevating agents, isoproterenol and 8-bromo-cAMP, would modulate basic fibroblast growth factor (bFGF) induction of MAPK activity (ERK-2) and cell proliferation in human renal proximal tubular epithelial cells (RPTEc) and Madin-Darby canine kidney cells (MDCK clone EI1). Isoproterenol, but not bFGF, stimulated cAMP production in RPTEc and MDCKEI1 cells. bFGF, isoproterenol, and 8-bromo-cAMP alone increased ERK-2 activity in both cell types. However, isoproterenol and 8-bromo-cAMP partially inhibited the bFGF induction of ERK-2 activity, but only isoproterenol inhibited the proliferation of both cell types. PD098059 (25 microM), an inhibitor of MAPK kinase (MEK 1/2), blocked the bFGF mitogenic effects, but did not affect the 8-bromo-cAMP-induced mitogenic effects in MDCKEI1 cells. These findings suggest that activation of ERK-2 is required but not sufficient for mitogenesis in RTEc. We conclude that isoproterenol inhibits the growth-promoting effects of bFGF in RTEc via MEK-dependent and -independent pathways.

Objective To investigate the effect of calcium oxalate crystals on the protein expression profiles in human proximal renal tubular epithelial cells, screen differentially expressed proteins and explore the relationship between kidney stones and renal injury. Methods The clinical kidney stone samples were analyzed and chose to culture with human proximal tubular epithelial cells HK-2. The cell total protein was extracted and qualified by SDS-PAGE. The protein concentration was determined by BSA method, and the difference of protein expression profiles between the experimental group and the control group were screened by TMT-labeled quantitative proteomics analysis. Cluster analysis, GO enrichment analysis and differential gene-related interaction network analysis were performed by bioinformatics method. Results The calcium oxalate crystal showed obvious cytotoxicity to HK-2 cells, and the cell growth slowed down and caused cell shape change significantly. The TMT-labeled quantitative proteomic analysis showed that after treatment with calcium oxalate crystals, a total of 1 141 proteins were differentially expressed in HK-2 cells, of which 699 were up-regulated and 442 were down-regulated. Bioinformatics analysis indicated that these differential proteins played important roles in extracellular complexes, organelles and various cytological processes, differential protein co-expression network analysis demonstrated that proteins involved in cytoskeletal maintenance (such as ACTB, CFL1) and several specific functions such as secretion and capping (MYH9, MYH14) were most closely related. Conclusion Calcium oxalate crystal has a significant effect on protein expression profiling in human renal epithelial cells, and these differentially expressed protein molecules may be involved in the formation of kidney stones and kidney injury. Key words: Calcium oxalate crystal; Kidney stones; Protein expression profile; Renal epithelial cells; Cluster analysis