Endocytosis In Epithelial Cells Research Articles

Nonspecific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells

BackgroundAllergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. ObjectiveWe show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. MethodsWe used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. ResultsNsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. ConclusionsOur work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans.

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1β and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

A dual action small molecule enhances azoles and overcomes resistance through co-targeting Pdr5 and Vma1

Fungal infection threatens human health worldwide due to the limited arsenal of antifungals and the rapid emergence of resistance. Epidermal growth factor receptor (EGFR) is demonstrated to mediate epithelial cell endocytosis of the leading human fungal pathogen, Candida albicans. However, whether EGFR inhibitors act on fungal cells remains unknown. Here, we discovered that the specific EGFR inhibitor osimertinib mesylate (OSI) potentiates azole efficacy against diverse fungal pathogens and overcomes azole resistance. Mechanistic investigation revealed a conserved activity of OSI by promoting intracellular fluconazole accumulation via inhibiting Pdr5 and disrupting V-ATPase function via targeting Vma1 at serine 274, eventually leading to inactivation of the global regulator TOR. Evaluation of the in vivo efficacy and toxicity of OSI demonstrated its potential clinical application in impeding fluconazole resistance. Thus, the identification of OSI as a dual action antifungal with co-targeting activity proposes a potentially effective therapeutic strategy to treat life-threatening fungal infection and overcome antifungal resistance.

Cigarette smoke increases susceptibility to infection in lung epithelial cells by upregulating caveolin-dependent endocytosis.

Cigarette smoke exposure is a risk factor for many pulmonary diseases, including Chronic Obstructive Pulmonary Disease (COPD). Cigarette smokers are more prone to respiratory infections with more severe symptoms. In those with COPD, viral infections can lead to acute exacerbations resulting in lung function decline and death. Epithelial cells in the lung are the first line of defense against inhaled insults such as tobacco smoke and are the target for many respiratory pathogens. Endocytosis is an essential cell function involved in nutrient uptake, cell signaling, and sensing of the extracellular environment, yet, the effect of cigarette smoke on epithelial cell endocytosis is not known. Here, we report for the first time that cigarette smoke alters the function of several important endocytic pathways in primary human small airway epithelial cells. Cigarette smoke exposure impairs clathrin-mediated endocytosis and fluid phase macropinocytosis while increasing caveolin mediated endocytosis. We also show that influenza virus uptake is enhanced by cigarette smoke exposure. These results support the concept that cigarette smoke-induced dysregulation of endocytosis contributes to lung infection in smokers. Targeting endocytosis pathways to restore normal epithelial cell function may be a new therapeutic approach to reduce respiratory infections in current and former smokers.

Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549)

BackgroundTuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be a predisposing factor that enhances the incidence of pulmonary disorders. For example, uncontrolled macropinocytosis in alveolar epithelial cells following exposure to water-pipe smoke may predispose subjects to pulmonary infection. Here, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium Bovis BCG by macropinocytosis in the alveolar epithelial cell line A549.MethodsA549 cells were exposed to WPC (4 mg/ml) for 24, 48, 72 and 96 h. Cell viability was studied using the methyl thiazolyldipenyl-tetrazolium bromide (MTT) reduction assay and proliferation by bromodeoxyUridine (BrdU) incorporation. Cells were exposed to FITC-Dextran (1 mg/ml) (as a control) and FITC-BCG (MOI = 10) for 20 min at 37 °C before cells were collected and the uptake of BCG-FITC determined by flow cytometry. Similar experiments were performed at 4 °C as a control. The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μM) was used to assess the mechanism by which WPC enhanced BCG uptake.ResultsWPC (4 mg/ml) increased the uptake of BCG-FITC after 72 (1.3 ± 0.1 fold, p < 0.05) and 96 (1.4 ± 0.05 fold, p < 0.05) hours. No effect on BCG-FITC uptake was observed at 24 or 48 h. WPC also significantly increased the uptake of FITC-Dextran (2.9 ± 0.3 fold, p < 0.05) after 24 h. WPC significantly decreased cell viability after 24 (84 ± 2%, p < 0.05), 48 (78±, 3%, p < 0.05), 72 (64 ± 2%, p < 0.05) and 96 h (45 ± 2%, p < 0.05). Y-27632 completely attenuated the increased uptake of BCG by WPC. Cell proliferation showed a decreasing trend in a time-dependent manner with WPC exposure.ConclusionWPC exposure increased epithelial cell endocytosis activity and death as well as enhancing their capacity for macropinocytosis. Our in vitro data indicates possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacterium.

The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis.

ABSTRACTOropharyngeal candidiasis (OPC), caused predominantly by Candida albicans, is a prevalent infection in patients with advanced AIDS, defects in Th17 immunity, and head and neck cancer. A characteristic feature of OPC is fungal invasion of the oral epithelial cells. One mechanism by which C. albicans hyphae can invade oral epithelial cells is by expressing the Als3 and Ssa1 invasins that interact with the epidermal growth factor receptor (EGFR) on epithelial cells and stimulate endocytosis of the organism. However, the signaling pathways that function downstream of EGFR and mediate C. albicans endocytosis are poorly defined. Here, we report that C. albicans infection activates the aryl hydrocarbon receptor (AhR), leading to activation of Src family kinases (SFKs), which in turn phosphorylate EGFR and induce endocytosis of the fungus. Furthermore, treatment of oral epithelial cells with interferon gamma inhibits fungal endocytosis by inducing the synthesis of kynurenines, which cause prolonged activation of AhR and SFKs, thereby interfering with C. albicans-induced EGFR signaling. Treatment of both immunosuppressed and immunocompetent mice with an AhR inhibitor decreases phosphorylation of SFKs and EGFR in the oral mucosa, reduces fungal invasion, and lessens the severity of OPC. Thus, our data indicate that AhR plays a central role in governing the pathogenic interactions of C. albicans with oral epithelial cells during OPC and suggest that this receptor is a potential therapeutic target.

Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis.

Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection.

Commensal Bacterial Endocytosis in Epithelial Cells Is Dependent on Myosin Light Chain Kinase–Activated Brush Border Fanning by Interferon-γ

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.

Early Endosome Antigen 1 (EEA1) decreases in macrophages infected withParacoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis.

EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection

The fungus Candida albicans is the major cause of oropharyngeal candidiasis (OPC). A key feature of this disease is fungal invasion of oral epithelial cells, a process that can occur by active penetration and fungal-induced endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C. albicans, in part by binding to E-cadherin. However, inhibition of E-cadherin function only partially reduces C. albicans endocytosis, suggesting that there are additional epithelial cell receptors for this organism. Here, we show that the EGF receptor (EGFR) and HER2 function cooperatively to induce the endocytosis of C. albicans hyphae. EGFR and HER2 interact with C. albicans in an Als3- and Ssa1-dependent manner, and this interaction induces receptor autophosphorylation. Signaling through both EGFR and HER2 is required for maximal epithelial cell endocytosis of C. albicans in vitro. Importantly, oral infection with C. albicans stimulates the phosphorylation of EGFR and HER2 in the oral mucosa of mice, and treatment with a dual EGFR and HER2 kinase inhibitor significantly decreases this phosphorylation and reduces the severity of OPC. These results show the importance of EGFR and HER2 signaling in the pathogenesis of OPC and indicate the feasibility of treating candidal infections by targeting the host cell receptors with which the fungus interacts.

Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.

Impairment of epithelial cell endocytosis by mistargeted SP‐C I73T mutant protein

Interstitial lung disease in both children and adults has been linked to mutations in the lung‐ specific Surfactant Protein C (SP‐C) proprotein. Among these, the missense mutation, SP‐CI73T, accounts for more than 30% of all reported SP‐C mutations. We previously reported that in A549 and HEK293 cell lines, wild type SP‐C was directly targeted to lysosomal‐like vesicles, while SP‐CI73T was uncharacteristically routed to the plasma membrane, and subsequently sorted to the lysosomes via early endosomes. This mistargeting causes impaired cellular lipid uptake and degradation. To determine whether the diminution of lipid uptake is the product of a globally dysfunctional endocytic pathway, cellular uptake of dextran was examined in HEK293 cells stably expressing either the wild type or the mutant SP‐C isoforms. We found that internalization of dextran was significantly reduced in cells expressing SP‐CI73T compared to the wild type control. Thus, our data further establishes the functional impairment caused by the SP‐CI73T mutation, and supports our previously proposed cellular mechanism and paradigm for mistargeted proteins involved in the disruption of endosomal/lysosomal sorting machinery.Supported By NIH HL090732 (SM), NIH HL19737 (MFB)

Characterization of clathrin‐independent endocytosis in epithelial cells

Major histocompatibility complex class I (MHCI) and more recently, membrane transport glycoprotein, CD98, were shown to be internalized via clathrin‐independent endocytosis (CIE) in HeLa cells. Following internalization, a fraction of vesicles containing MHCI fuse with early endosomes, defined by the presence of early endosomal autoantigen 1 (EEA1) and containing the transferrin receptor, a clathrin cargo. MHCI is then routed to late endosomes for degradation or to distinct tubular carriers that return CIE cargo back to the plasma membrane. In contrast to MHCI, CD98 bypasses the merge with early endosomes and directly enters into the tubular carriers. To study CIE in different epithelial cell types, surface antibody internalization assays were used to follow the trafficking of MHCI and CD98 in A431 and ARPE‐19 cells. Like HeLa cells, MHCI enters via CIE in vesicles that fuse with early endosomes and traffic to late endosomes for degradation. However, after endocytosis, CD98 reaches late endosomes although it bypasses the EEA1‐early endosome. Recycling of MHCI and CD98 is observed in both cell lines although tubular carriers seen in HeLa cells are absent. These observations confirm that CIE exists in different cell types but that itineraries for distinct cargo may vary.

Immunolocalization of cubilin, megalin, apolipoprotein J, and apolipoprotein A-I in the uterus and oviduct.

Spermatozoa maturation and capacitation occurring in the male and female reproductive tracts, respectively, involves the remodeling of the spermatozoa plasma membrane. Apolipoprotein J (apoJ) and apolipoprotein A-I (apoA-I) have been implicated in the process of lipid exchange from the spermatozoa plasma membrane to epithelial cells lining the male reproductive tract. Evidence suggests that this process is mediated by the cooperative action of the endocytic lipoprotein receptors megalin and cubilin, which are expressed at the apical surface of absorptive epithelia in various tissues, including the efferent ducts and epididymis. Here, we investigated the possibility that these receptors and their lipid-binding ligands, apoJ and apoA-I, might function similarly in the female reproductive tract. We show that megalin and cubilin are expressed in the uterine epithelium at all stages of the estrous cycle, maximally during estrous and metestrous stages. In the oviduct, there is pronounced expression of both megalin and cubilin in the nonciliated cells of the proximal oviduct and epithelial cells of the distal oviduct, particularly during estrous and metestrous stages. In both uterine and oviduct epithelial cells, megalin and cubilin were located on the apical regions of the cells, consistent with a distribution at the cell surface and in endosomes. ApoJ and apoA-I were both detected in apical regions of uterine and oviduct epithelial cells. Secretory cells of the uterine glands were found to express apoJ and apoA-I suggesting that the glands are a site of synthesis for both proteins. In summary, our findings indicate that megalin and cubilin function within the female reproductive tract, possibly mediating uterine and oviduct epithelial cell endocytosis of apoJ/apoA-I-lipid complexes and thus playing a role in lipid efflux from the sperm plasma membrane, a major initiator of capacitation.

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

Actin microfilaments et al.—the many components, effectors and regulators of epithelial cell endocytosis

The aim of this review is to introduce the advances made over the past several years regarding the participation of actin and actin-associated proteins in clathrin-mediated endocytosis in simple cell models, and then to consider the evidence for the involvement of these effectors in apical clathrin-mediated endocytosis in epithelial cells. Basic mechanisms of clathrin-mediated endocytosis are initially addressed, followed by a detailed description of the actin cytoskeleton: its organization, function and, most importantly, the essential role played by proteins and signaling pathways responsible for the regulation of actin filament dynamics. Our focus then shifts to the GTPase, dynamin and its pivotal role as a bridge between various components of the clathrin endocytic machinery and the actin cytoskeleton. Mechanisms and effectors of dynamin-dependent endocytosis are then described, with a particular emphasis on novel proteins, which link dynamin to actin filaments. We consider additional effectors proposed to interact with actin to facilitate clathrin-mediated endocytosis in a dynamin-independent manner. The multiple roles which actin filaments are thought to play in endocytosis are addressed followed by a more detailed characterization of actin filament participation specifically in apical endocytosis. We conclude by discussing how these concepts may be integrated to improve drug internalization at the apical plasma membrane of epithelial cells.

Inhibition of Na+-H+ exchanger-3 interferes with apical receptor-mediated endocytosis via vesicle fusion.

1. Receptor-mediated endocytosis in epithelial cells is a crucial mechanism for transport of macromolecules and regulation of cell-surface protein expression. Na+-H+ exchanger type 3 (NHE3) has been shown to cycle between the apical plasma membrane and the early endosomal compartment and to interfere with endocytosis. 2. In the present study we investigated in detail the NHE3-dependent step of apical endocytosis in an epithelial cell line (opossum kidney cells). 3. Inhibition of NHE3 led to a rapid dose-dependent inhibition of apical albumin endocytosis but did not affect basolateral transferrin endocytosis. Re-exocytosis of albumin was not increased by NHE3 inhibition. 4. NHE3 dependency of albumin endocytosis was still observed at 20 degrees C or when microtubules had been disrupted. This was not the case for inhibition of vacuolar H+-ATPase. 5. NHE3 inhibition rapidly blocked internalisation of pre-bound albumin and attenuated degradation of internalised albumin without changing general protein degradation. 6. Furthermore, NHE3 inhibition reduced the rate of endocytic vesicle fusion significantly. 7. In summary, our data indicate that NHE3 is important for the early phase of the apical endocytic pathway, located between the plasma membrane and early endosomes, at least in part due to its involvement in endocytic vesicle fusion.

FIBRONECTIN AS A POTENT INHIBITOR OF CALCIUM OXALATE UROLITHIASIS

Fibronectin (230 kD.) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. Many investigators have demonstrated that fibronectin, because of its cell adhesive action, is related to biological processes such as morphogenesis, wound healing and metastasis. Recent studies have shown that a variety of molecules, including fibronectin, inhibit endocytosis of calcium oxalate crystals in vitro. We investigated other roles of fibronectin in calcium oxalate stone formation. Immunoblotting of the crystal surface binding substance obtained from pooled healthy male urine samples was used to analyze whether fibronectin was adsorbed onto the surface of calcium oxalate crystals. To clarify the relationship between fibronectin and calcium oxalate crystals, we performed 6 experiments. Experiment 1 was immunohistochemical examination of fibronectin expression in stone forming rat model kidneys, and experiment 2 examined the fibronectin content of stone forming rat kidney models with the enzyme-linked immunosorbent assay. Experiment 3 was designed to determine fibronectin content of Madin-Darby canine kidney (MDCK) cells stimulated by addition of calcium oxalate crystals and experiment 4 identified the inhibitory effect of fibronectin on calcium oxalate crystal growth by the seed crystal method. For experiment 5 we used an aggregometer system to clarify the inhibitory effect of fibronectin on calcium oxalate crystal aggregation and experiment 6 examined the inhibitory effect of fibronectin on the adhesion of calcium oxalate crystals to MDCK cells. In the crystal surface binding substance immunoreactive bands at 230 kD., which correspond to the molecular weight of fibronectin, were detected by Western blot analysis. In stone forming rat kidneys strong expression of fibronectin was found on the renal tubules to which the crystals were attached. The fibronectin content of these kidneys was significantly greater than that of kidneys without calcium oxalate crystals. The fibronectin content of MDCK cells tended to increase in proportion to the concentration of calcium oxalate crystals added to the culture medium. The growth inhibition assay showed that the inhibitory effect of fibronectin on calcium oxalate crystal growth was small in relation to the quantity of fibronectin excreted. However, fibronectin had inhibitory effects on calcium oxalate crystal aggregation and adhesion of the crystals to MDCK cells. Fibronectin secretion can be stimulated by calcium oxalate crystals, and this protein, which is excreted from the tubular cells, may inhibit calcium oxalate crystal aggregation and attachment to cells.

Regulation of renal epithelial cell endocytosis of calcium oxalate monohydrate crystals

The earliest events in the formation of kidney stones are unknown. The most common crystal in kidney stones, calcium oxalate monohydrate (COM), when added to cultures of monkey kidney epithelial cells (BSC-1 line), was internalized by 19% of the cells after 30 min. COM crystal endocytosis was enhanced by serum, ADP, and epidermal growth factor, which are potent mitogens for these cells. Endocytosis of COM crystals was inhibited by diverse molecules including Tamm-Horsfall glycoprotein (THP), the tetrapeptide arginine-glycine-aspartic acid-serine, fibronectin, transforming growth factor-beta 2, and heparin. The capacity of THP, fibronectin, or heparin to inhibit endocytosis was mediated by an interaction of these molecules with cells, not by coating the crystals. Thus renal epithelial cell endocytosis of COM crystals can be regulated by diverse molecules including THP, the most common protein found in human urine. Crystal endocytosis and subsequent cellular responses could be important pathogenic steps in nephrolithiasis.

Glomerular Epithelial Cell Endocytosis in Puromycin-Induced Glomerulopathy

Glomerulopathy and nephrotic syndrome were induced in rats by intravenous puromycin aminonucleoside. Ten days after the injection of puromycin, the animals have developed heavy proteinuria. During this phase, glomerular epithelial cell endocytosis was studied by injecting a conjugate of horseradish peroxidase and poly-L-lysine. This conjugate has been shown to be endocytosed by glomerular epithelial cells. The rats were serially sacrificed from 1 min to 24 h after this injection. Peroxidase was localised cytochemically and observed at light and electron microscopy. The early events of endocytosis in glomerulopathy (namely the binding to the plasma membrane, the membrane invagination and the formation of the early vesicles) were qualitatively similar to those in the normal. The later events (the fusion of the vesicles and their movement within the cells) were inhibited. The results show that puromycin aminonucleoside damages epithelial cell endocytotic activity and affects the later processing of the conjugate within the cells.