The glomerular mesangial cell: an expanding role for a specialized pericyte

The mesangial cell occupies a central position in the renal glomerulus. It has characteristics of a modified smooth muscle cell, but is also capable of a number of other functions. Among these are generation of prostaglandins (PGs) and mediators of inflammation; production and breakdown of basement membrane and other biomatrix material; synthesis of cytokines; and uptake of macromolecules, including immune complexes. In terms of its smooth muscle activity, the mesangial cell contracts or relaxes in response to a number of vasoactive agents. This ability allows the cells to modify glomerular filtration locally. The cellular mechanism of action of many agents influencing mesangial cells involves activation of phospholipase C for phosphatidylinositol 4,5-bisphosphate. This results in generation of inositol trisphosphate and release of intracellular calcium. Mesangial cell relaxation can be mediated by enhanced cAMP or cGMP generation. Many vasoactive substances also stimulate PG production by mesangial cells. This involves activation of both phospholipase C and A2, the latter being responsible for the release of arachidonic acid. Mesangial cells are also capable of endocytosis of macromolecules, including immune complexes. This is initiated by binding to a specific receptor, resulting in formation of PG, platelet-activating factor, and reactive oxygen species. Mesangial cells can generate interleukin 1 and platelet-derived growth factor and respond to these in an autocrine manner. Thus, the mesangial cell not only can control glomerular filtration, but may also be involved in the response to local injury, including cell proliferation and basement membrane remodeling.     

The role of CXCL16 and its processing metalloproteinases ADAM10 and ADAM17 in the proliferation and migration of human mesangial cells

In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNγ, TNFα, and IL1β). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

Enhancement of glomerular mesangial cell neutral proteinase secretion by macrophages: role of interleukin 1

We have examined the ability of rat mesangial cells to regulate neutral proteinase production in vitro. Mesangial cells constitutively produced gelatinase when cultured in serum-free medium, and enzyme production by these cells was inhibited by cycloheximide. Coculture with thioglycollate-elicited rat peritoneal macrophages resulted in enhanced gelatinase production. The increase in enzyme released correlated directly with the number of macrophages added. Conditioned medium from LPS-activated peritoneal macrophages also enhanced gelatinase production in a dose-dependent manner. Fractionation of these macrophage supernatants on Sephacryl S-200 revealed a predominant fraction of gelatinase-enhancing activity in a m.w. range between 10,000 and 20,000. These data suggested that the enhanced mesangial cell gelatinase production was mediated through the action of interleukin 1. This was confirmed by the finding that purified interleukin 1, prepared from LPS-stimulated rat peritoneal macrophages, stimulated mesangial cells to secrete gelatinase in a dose-dependent manner. These findings may be of significance in the understanding of the pro-inflammatory role of macrophages in immune-mediated glomerulonephritis.     

Signaling role of PDE isozymes in pathobiology of glomerular mesangial cells

Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazione, inhibited (>50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with cilostamide or with rolipram increasedin situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated protein kinase. The efficacy of PDE isozyme inhibitors (IC₅₀) to suppress mitogenesis or ROM generation paralleled IC₅₀ for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazione in combination with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should be investigated for potential use for “signal transduction pharmacotherapy” of glomerulonephritis.     

Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies

Cadmium represents a major environmental pollutant that may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC₅₀ cytotoxicity index was obtained with 3.55 μmol/L using neutral red assay for 24 h. After cadmium exposure (1 μmol/L, determined as nonlethal concentration), 0.38 μg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle α-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.     

Potential role for ADAM15 in pathological neovascularization in mice

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.     

Conserved charge of glomerular and mesangial cell proteoglycans: possible role of amino acid-derived sulphate.

Sulphation of proteoglycans was studied in isolated glomeruli and cultured rat mesangial cells. Both preparations produced heparan, dermatan, and chondroitin sulphates, recoverable both from the tissue layers and the conditioned media. The proportion of heparan sulphate made by mesangial cells was independent of the age of the culture, but declined in later passage. These preparations differed from several other nontransformed cell types studied to date in that the degree of proteoglycan sulphation was independent of the concentration of inorganic sulphate provided. Even when no exogenous sulphate was added, sulphation-dependent charge density of newly synthesized proteoglycans was conserved. Both isolated glomeruli and cultured mesangial cells produced proteoglycans with 35S-labelled sulphate esters when [35S]methionine was provided as the sole source of labelled sulphate. Conversion of methionine to cysteine and subsequent oxidation of organic sulphate via the sulphinyl pyruvate pathway is the only mechanism known in eukaryotic cells that can account for this observation. We conclude that facile oxidation of sulphur-containing amino acids can contribute to sulphation of glomerular proteoglycans and may serve to sustain the charge density of these multifunctional molecules when the supply of inorganic sulphate is otherwise compromised.     

The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis

Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca²⁺]_i) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP₃R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca²⁺]_i and the percentage of Cd-induced apoptosis. However, EGTA and the IP₃R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4 h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16 h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (ΔΨ_m) was depolarized significantly and cytochrome c was released after 24 h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.     

ADAM10 is required for SCF-induced mast cell migration

A Disintegrin and Metalloproteinase (ADAM)-10 plays critical roles in neuronal migration and distribution. Recently, ADAM10 deletion was shown to disrupt myelopoiesis. We found that inducible deletion of ADAM10 using Mx1-driven Cre recombinase for a period of three weeks resulted in mast cell hyperplasia in the skin, intestine and spleen. Mast cells express surface ADAM10 in vitro and in vivo, at high levels compared to other immune cells tested. ADAM10 is important for mast cell migration, since ADAM10-deficiency reduced c-Kit-mediated migration. As with some mast cell proteases, ADAM10 expression could be altered by the cytokine microenvironment, being inhibited by IL-10 or TGFβ1, but not by several other T cell-derived cytokines. Collectively these data show that the ADAM10 protease is an important factor in mast cell migration and tissue distribution, and can be manipulated by environmental cues.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

The role of chromatin structure in cell migration

Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization appears to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow passage of cells through narrow openings, is dependent not only on changes in cytoskeletal elements but also on global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure could facilitate cellular reorganizations necessary for efficient migration.     

IGF-1 induces foam cell formation in rat glomerular mesangial cells.

When rat glomerular mesangial cells (MCs) are cultured with IGF-1 they accumulate intracellular lipid and take on foam cell morphology. These changes were characterized by electron microscopy and Nile red staining. To define the mechanism responsible for IGF-1-mediated lipid uptake, MCs were evaluated for endocytosis, scavenger receptor activity, and receptor-mediated uptake by the LDL receptor. Lipid accumulation was markedly increased when MCs were cultured with IGF. The primary route of uptake was through enhanced endocytosis. Lipid-laden MCs have decreased phagocytic capacity and disrupted cytoskeletons. These data show that IGF-1 induces MC to take on a foam cell morphology and that lipid-laden MCs have impaired phagocytic function.     

Selective modulation of integrin-mediated cell migration by distinct ADAM family members

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.     

Epidermal growth factor enhances glomerular mesangial cell soluble phospholipase A2 activity

We have previously characterized a hormonally regulated soluble form of phospholipase A2 (PLA2) in the cultured renal mesangial cell which is similar and possibly identical to the major form in rat kidney. In an attempt to further characterize the mechanisms of regulation of this enzyme we have used epidermal growth factor (EGF), which does not activate polyphosphoinositide-specific phospholipase C in these cells. EGF-enhanced PLA2 activity as assayed by the ability of the soluble extracts of cells to cleave arachidonic acid from the sn-2 position of phosphatidylcholine and phosphatidylethanolamine. This represents a direct demonstration of EGF-induced PLA2 activation which is preserved in a cell-free extract. Phorbol myristate acetate (PMA), as well as 1-oleoyl-2-acetylglycerol, also enhanced PLA2 activity. By contrast, the calcium ionophore A23187 had no effect on extract PLA2 activity. The EGF- and PMA-induced enhanced activity was recovered following fractionation by Mono-Q anion exchange chromatography. The peak of activity comigrated for both agonists, suggesting that both EGF and PMA stimulated the same form of the enzyme. Down-regulation of protein kinase C by pretreatment with PMA resulted in loss of the PMA-induced, but not the EGF-induced, enhancement in PLA2 activity. 8-Bromo-cAMP had no effect upon the PLA2 activity, and did not modulate the EGF effect. Pertussis toxin induced G protein ADP-ribosylation but had no effect upon PLA2 activity, and did not alter the EGF effect. In summary, EGF results in a stable modification of PLA2 activity in glomerular mesangial cells. This enhanced activity is independent of polyphosphoinositide hydrolysis, insensitive to protein kinase C down-regulation, and is not affected by cAMP or pertussis toxin pretreatment of the cells.     

FTY720 induces cell cycle arrest and apoptosis of rat glomerular mesangial cells

The present study aimed to examine the effect of FTY720, a new immunosuppressive agent, on the proliferation and apoptosis of glomerular mesangial cells (GMC), and investigate the underlying mechanisms. Cultured rat GMC were treated by FTY720, and the cell viability, apoptosis and cell cycle progression were examined. Furthermore, cell cycle related gene expression profile was analyzed by cDNA microarray, and the protein expression of cell cycle related genes as well as Bax and Bcl-2 were examined by Western blot. The results showed that FTY720 inhibited GMC proliferation and induced apoptosis of GMC in a dose- and time-dependent manner, and induced G(1) phase cell cycle arrest in GMC in a dose-dependent manner as well. cDNA microarray analysis revealed that FTY720 regulated the expression of cell cycle-related gene. Western blot analysis showed that FTY720 induced the downregulation of cyclin D1, cyclin E, CDK2, CDK4, Bcl-2 and E2F1 and the upregulation of Kip1/p27, Cip1/p21, Bax and Rb in GMC in a dose-dependent manner. These results demonstrated that FTY720 could inhibit the proliferation of GMC through inducing cell cycle arrest and apoptosis, probably via the regulation of the expression of cell cycle-related genes and Bax/Bcl-2.     

The role of receptor/channel activity in neuronal cell migration

Confocal laser microscopy, in conjunction with carbocyanine dyes and calcium-sensitive fluorescent indicators, was used in slices and explant cultures of developing cerebellum to study cellular mechanisms underlying a motility of neuronal cell migration. The results indicate that a combination of voltage- and ligand-activated ion channels cooperatively regulates Ca²⁺ influx into the migrating cells. We suggest that molecules, present in the local cellular milieu, affect cell motility by activating specific ion channels and second messengers that influence polymerization of stiff and contractile cytoskeletal proteins. This early interaction between postmitotic neurons and surrounding cells controls the rate of their movements, sculpts their shapes, establishes their positions, and, therefore, indirectly determines their identities to prior formation of synaptic connections. © 1995 John Wiley Sons, Inc.     

The role of Exo70 in vascular smooth muscle cell migration

Background

As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70’s function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5.

Methods

Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay.

Results

The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration.

Conclusions

This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty.