scholarly journals Adaptive Response of HK-2 Cells to High Levels of COM Crystals Highlights CaSR as an Enhancer of Crystal Adhesion

2020 ◽  
Author(s):  
Siyu Chen ◽  
Junsheng Bao ◽  
Jianzhong Lu ◽  
Zhongyun Ning ◽  
Zhongjin Yue ◽  
...  
Keyword(s):  
Annexin V ◽  
Human Kidney ◽  
Calcium Oxalate Monohydrate ◽  
Outer Leaflet ◽  
Calcium Sensing ◽  
Outward Transport ◽  
Proteomics Approach ◽  
Specific Antagonist ◽  
Crystal Adhesion ◽  
Related Proteins

Abstract Background: Calcium oxalate monohydrate (COM) is an aetiologic factor for urolithiasis. However, how Human Kidney-2 (HK-2) cells respond to a high COM has not yet been completely elucidated.Materials and methods: A gel-based proteomics approach was applied to investigate COM-induced cellular proteomic changes. The COM-induced upregulation of calcium-sensing receptor (CaSR) in HK-2 cells was studied. Surface phospholipids (PS), which play a role in urolithiasis formation by mediating adhesion of HK-2 cells, were labelled in the inner or outer leaflet of the plasma membrane of HK-2 cells with fluorescent nitrobenzoxadiazole (NBD) to form NBD-PS to detect transmembrane movements of PS. After labelling, HK-2 cells were exposed to COM in the presence of the CaSR-specific agonist gadolinium chloride (GdCl3) or the CaSR-specific antagonist NPS2390. Inward and outward transmembrane movements of PS were tracked with a fluorescence quenching assay. Surface-expressed PS was detected by an annexin V binding assay. Changes in aminophospholipid translocase (APLT), oxidative stress (OS), levels of apoptosis-related proteins in HK-2 cells and crystal adhesion were also assessed.Results: COM increased CaSR and surface-expressed PS levels, decreased APLT activity, impaired inward transport of PS, and enhanced outward transport of PS. However, pretreatment with GdCl3 further effectively inhibited the inward movement of PS and APLT activity and increased surface-expressed PS levels compared with COM treatment alone. In contrast, NPS2390 promoted the inward movement of PS and APLT activity and decreased surface-expressed PS levels compared with COM treatment alone. COM increased OS, apoptosis of HK-2 cells and crystal adhesion onto cells, and this increase was further enhanced by GdCl3 pretreatment but attenuated by NPS2390 treatment.Conclusions: These results strongly suggest that COM-induced CaSR generation may affect crystal adhesion by regulating PS externalization, apoptosis and OS in HK-2 cells.

scholarly journals Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.

1996 ◽  
Vol 183 (3) ◽  
pp. 1037-1044 ◽  
Author(s):  
M Hedlund ◽  
M Svensson ◽  
A Nilsson ◽  
R D Duan ◽  
C Svanborg
Keyword(s):  
Escherichia Coli ◽  
Signaling Pathway ◽  
Kinase Inhibitors ◽  
Human Kidney ◽  
Cytokine Response ◽  
Protein Kinase Inhibitors ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Outer Leaflet

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

A comparative study of the adsorption of amino acids on to calcium minerals found in renal calculi

2001 ◽  
Vol 101 (2) ◽  
pp. 159-168 ◽  
Author(s):  
David E. FLEMING ◽  
Wilhelm VAN BRONSWIJK ◽  
Rosemary Lyons RYALL
Keyword(s):  
Amino Acids ◽  
Calcium Oxalate ◽  
Binding Affinity ◽  
Stone Formation ◽  
Human Kidney ◽  
Renal Calculi ◽  
Calcium Oxalate Monohydrate ◽  
Carboxyglutamic Acid ◽  
Calcium Minerals ◽  
Ph Range

To assess the binding of individual amino acids to the principal calcium minerals found in human kidney stones, the adsorption of 20 amino acids on to calcium oxalate monohydrate, CaHPO4.2H2O, Ca3(PO4)2 and Ca5(PO4)3OH crystals was determined over the physiological urinary pH range (pH 5–8) in aqueous solutions. All amino acids adsorbed most strongly at pH 5, and this decreased in all cases as the pH was increased. The amino acids which adsorbed most strongly were aspartic acid, glutamic acid and γ-carboxyglutamic acid, with the last displaying the strongest affinity. All amino acids bound more avidly to calcium oxalate monohydrate than to any of the phosphate minerals. Adsorption on to CaHPO4.2H2O was generally higher than for Ca3(PO4)2 and Ca5(PO4)3OH, for which all amino acids, with the exception of γ-carboxyglutamic acid, had only a weak affinity. The binding affinity of these acids is thought to be due to their zwitterions being able to adopt conformations in which two carboxyl groups, and possibly the amino group, can interact with the mineral surface without further rotation. The strong binding affinity of di-and tri-carboxylic acids for calcium stone minerals indicates that proteins rich in these amino acids are more likely to play a functional role in stone pathogenesis than those possessing only a few such residues. These findings, as well as the preferential adsorption of the amino acids for calcium oxalate monohydrate rather than calcium phosphate minerals, have ramifications for research aimed at discovering the true role of proteins in stone formation and for potential application in the design of synthetic peptides for use in stone therapy.

scholarly journals Curcumin Enhanced Busulfan-Induced Apoptosis through Downregulating the Expression of Survivin in Leukemia Stem-Like KG1a Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Guangyang Weng ◽  
Yingjian Zeng ◽  
Jingya Huang ◽  
Jiaxin Fan ◽  
Kunyuan Guo
Keyword(s):  
Specific Inhibitor ◽  
Annexin V ◽  
Chemotherapeutic Agents ◽  
Combination Group ◽  
Hematopoietic Stem ◽  
Leukemia Relapse ◽  
Conditioning Regimens ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Related Proteins

Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CUR- and CUR+BUS-treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin.

Regulation of renal epithelial cell affinity for calcium oxalate monohydrate crystals

2000 ◽  
Vol 278 (1) ◽  
pp. F130-F137 ◽  
Author(s):  
John C. Lieske ◽  
Erick Huang ◽  
F. Gary Toback
Keyword(s):  
Calcium Oxalate ◽  
Mdck Cells ◽  
Stone Formation ◽  
Calcium Oxalate Monohydrate ◽  
Intracellular Camp ◽  
Renal Epithelial Cell ◽  
Protective Function ◽  
Crystal Binding ◽  
Cell Affinity ◽  
Crystal Adhesion

The binding and internalization of calcium oxalate monohydrate (COM) crystals by tubular epithelial cells may be a critical step leading to kidney stone formation. Exposure of MDCK cells to arachidonic acid (AA) for 3 days, but not oleic or linoleic acid, decreased COM crystal adhesion by 55%. Exogenous prostaglandin PGE1 or PGE2 decreased crystal binding 96% within 8 h, as did other agents that raise intracellular cAMP. Actinomycin D, cycloheximide, or tunicamycin each blocked the action of PGE2, suggesting that gene transcription, protein synthesis, and N-glycosylation were required. Blockade of crystal binding by AA was not prevented by the cyclooxygenase inhibitor flurbiprofen, and was mimicked by the nonmetabolizable AA analog eicosatetryanoic acid (ETYA), suggesting that generation of PGE from AA is not the pathway by which AA exerts its effect. These studies provide new evidence that binding of COM crystals to renal cells is regulated by physiological signals that could modify exposure of cell surface molecules to which the crystals bind. Intrarenal AA, PGs, and/or other agents that raise the intracellular concentration of cAMP may serve a protective function by preventing crystal adhesion along the nephron, thereby defending the kidney against crystal retention and stone formation.

A novel p64-related Cl−channel: subcellular distribution and nephron segment-specific expression

1999 ◽  
Vol 276 (3) ◽  
pp. F398-F408 ◽  
Author(s):  
John C. Edwards
Keyword(s):  
Chloride Channels ◽  
Sequence Similarity ◽  
Fluid Phase ◽  
Human Kidney ◽  
Specific Expression ◽  
Proximal Tubule Cells ◽  
Sds Page ◽  
Nephron Segment ◽  
Related Proteins

Several closely related proteins that have been implicated as chloride channels of intracellular membranes have recently been described. We report here the molecular cloning and characterization of a new member of this family from human cells. On the basis of sequence similarity, we conclude that this new protein represents the human version of a previously described protein from rat brain named p64H1. The human version of p64H1 (huH1) is a 28.7-kDa protein that shows an apparent molecular mass of 31 kDa by SDS-PAGE. A single 4.5-kb message is detected on Northern blots and is present in all tissues probed. The protein is expressed in an intracellular vesicular pattern in Panc-1 cells that is distinct from the endoplasmic reticulum, fluid-phase endocytic, and transferrin-recycling compartments, but which does colocalize with caveolin. In human kidney, huH1 is highly expressed in a diffuse pattern in the apical domain of proximal tubule cells. huH1 is expressed less abundantly in a vesicular pattern in glomeruli and distal nephron.

scholarly journals Repair of Tea Polysaccharide Promotes the Endocytosis of Nanocalcium Oxalate Monohydrate by Damaged HK-2 Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Chuang-Ye Li ◽  
Li Liu ◽  
Yao-Wang Zhao ◽  
Qian-Long Peng ◽  
Xin-Yuan Sun ◽  
...  
Keyword(s):  
Human Kidney ◽  
Calcium Oxalate Monohydrate ◽  
Protective Mechanism ◽  
Renal Epithelial Cells ◽  
Molecular Weights ◽  
Lactate Dehydrogenase Release ◽  
Before And After ◽  
Proximal Tubular ◽  
Repair Ability ◽  
Tea Polysaccharides

Endocytosis is a protective mechanism of renal epithelial cells to eliminate retained crystals. This research investigated the endocytosis of 100 nm calcium oxalate monohydrate crystals in human kidney proximal tubular epithelial (HK-2) cells before and after repair by four kinds of tea polysaccharides with molecular weights (MWs) of 10.88 (TPS0), 8.16 (TPS1), 4.82 (TPS2), and 2.31 kDa (TPS3), respectively. When HK-2 cells were repaired by TPSs after oxalic acid injury, the cell viability, wound healing ability, mitochondrial membrane potential, percentage of cells with endocytosed crystals, and dissolution rate of the endocytosed crystals increased; the cell morphology recovered; and the reactive oxygen level and lactate dehydrogenase release decreased. Most of the endocytosed crystals were found in the lysosomes. The repair effects of the four TPSs were ranked in the following order: TPS2>TPS1>TPS3>TPS0. TPS2 with moderate MW presented the optimal repair ability and strongest ability to promote endocytosis.

scholarly journals Autophagy Protects Advanced Glycation End Product-Induced Apoptosis and Expression of MMP-3 and MMP-13 in Rat Chondrocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Wenzhou Huang ◽  
Peng Ao ◽  
Jian Li ◽  
Tianlong Wu ◽  
Libiao Xu ◽  
...  
Keyword(s):  
Western Blotting ◽  
Annexin V ◽  
Protective Role ◽  
Autophagic Flux ◽  
Advanced Glycation ◽  
Age Related ◽  
Glycation End Products ◽  
Induced Apoptosis ◽  
Related Proteins

Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis (OA). One feature of age-related changes in OA is advanced glycation end products (AGEs) accumulation in articular cartilage. Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins. However, the relationship between autophagy and AGE-associated OA is unknown. The aim of this study is to determine whether autophagy participates in the pathology of AGE-treated chondrocytes and to investigate the exact role of autophagy in AGE-induced cell apoptosis and expression of matrix metalloproteinase- (MMP-) 3 and MMP-13. AGEs induced notable apoptosis that was detected by Annexin V/PI double-staining, and the upregulation of MMP-3 and MMP-13 was confirmed by Western blotting. Autophagy-related proteins were also determined by Western blotting, and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux. As a result, autophagy significantly increased in chondrocytes and peaked at 6 h. Furthermore, rapamycin (RA) attenuated AGE-induced apoptosis and expression of MMP-3 and MMP-13 by autophagy activation. In contrast, pretreatment with autophagy inhibitor 3-methyladenine (3-MA) enhanced the abovementioned effects of AGEs. We therefore demonstrated that autophagy is linked with AGE-related pathology in rat chondrocytes and plays a protective role in AGE-induced apoptosis and expression of MMP-3 and MMP-13.

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