Effects of lysophosphatidylcholine on expression of monocyte chemoattractant protein-1 in glomerular endothelial cells

Glomerular endothelial cells(GEC) produce monocyte chemoattractant protein-1(MCP-1), which is considered to be an important factor for the recruitment of macrophages into the glomeruli. Recent reports have suggested an association between oxidized low-density lipoprotein(ox-LDL) and progression of glomerular disease. In this study, the effects of lysophosphatidylcholine(LysoPC), a modified phospholipid produced during LDL oxidation, on MCP-1 mRNA expression in cultured bovine GEC were examined. GEC from the 8th through 10th passages were used. LysoPC substantially increased expression of MCP-1 mRNA when compared to the control. These findings led us to examine the mechanism of LysoPC-induced MCP-1 expression in GEC. LysoPC-induced MCP-1 mRNA expression in GEC was suppressed by genistein and staurosporine. It was suggested that both the tyrosinekinase(TK) and proteinkinase C(PKC) pathways were involved in LysoPC-induced MCP-1 expression in GEC. MCP-1 mRNA induction by LysoPC was also attenuated by Vitamin E. This effect may be related to the beneficial effects of Vitamin E on experimental glomerular disease models. In conclusion, LysoPC increased MCP-1 expression in GEC. This phenomenon is believed to be mediated by both the TK and PKC signaling pathways, in contrast with other vascular endothelial cells. Vitamin E also attenuated LysoPC-induced MCP-1 expression in GEC.     

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes

BACKGROUND: Decay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF. METHODS: Function-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats. RESULTS: DAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab')2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were "planted" on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model. CONCLUSION: In cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC.     

Adhesion of rat glomerular epithelial cells to extracellular matrices: role of beta 1 integrins.

Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.     

Isolation and characterization of rat glomerular epithelial cells in vitro

Rat glomeruli were isolated by a graded sieving technique, and the nature and purity of the preparation were examined by scanning electron microscopy (SEM). A great majority of the glomeruli (86.0 +/- 6.0%) were not encapsulated, and there was very little contamination of the preparations with tubular fragments. By supplementing tissue culture medium with conditioned medium (CM) and insulin, we were able to grow single cells from dissociated rat glomeruli. From these single cells, we were able to clone and maintain in culture three distinct cell types. Also, with this specialized medium, we were able to clone these three cell types from outgrowths of whole golmeruli. One of these cell types was characterized as glomerular epithelial cells (GEC). GEC, as opposed to the other two cloned cell types obtained in this study, had cilia on their surfaces and also possessed receptors for complement (C3) in vitro. Low concentrations of the aminonucleoside of puromycin (AMNS), which have been shown to be specifically cytotoxic to GEC in vivo, were found to be cytotoxic to GEC but not to the other two glomerular cells in vitro. In addition, GEC did not contain antihemophilic factor (factor VIII), a marker for endothelium, nor were they able to phagocytose polystyrene spherules in vitro, as can mesangial cells in short-term culture.     

Antigen processing and presentation by glomerular visceral epithelium in vitro

Although macrophages are considered the prototype of antigen presenting cells (APC), recent studies have emphasized the potential role of several parenchymal and mesenchymal cells in this process. We have studied the capacity of cultured glomerular visceral epithelial cells (GEC) to act as effective APC and compared this capacity with that demonstrated by peritoneal macrophages. Affinity-purified and in vitro propagated rat GEC were exposed to hen egg lysozyme, keyhole limpet hemocyanin, and cationic ferritin. As effector cells, we used antigen-specific T cell hybridomas; the level of antigen presentation was assessed by determining the level of interleukin 2 (IL-2) present in tissue culture supernatants. Cytokine-treated GEC were capable of processing and presenting all antigens in a dose-dependent manner. Crucial for antigen presentation were intracellular processing of antigen and the presence of Ia on the cell surface. Our findings indicate that GEC can act as effective APC, and further suggest that this capacity may be relevant to cell-mediated immune injury at the level of the glomerular capillaries in vivo.     

Evaluation of Liver Function Tests to Predict Operative Risk in Liver Surgery

Despite numerous studies in the past it is not possible yet to predict postoperative liver failure and safe limits for hepatectomy. In this study the following liver function tests ICG-ER (indocyaninegreen elimination rate), GEC (galactose elimination capacity) and MEGX-F (monoethylglycinexylidid formation) are examined with regard to loss of liver tissue and prediction of operative risk. Liver function tests were assessed in 20 patients prior to liver resection and on the 10th. postoperative day. Liver and tumor volume were measured by ultrasound and pathologic specimen and the parenchymal resection rate was calculated. In patients without cirrhosis (n = 10) ICG-ER and MEGX-F remained unchanged after resection, GEC was reduced but did not correspond to the resection rate. Patients with cirrhosis (n = 10) had a significantly lower ICG-ER and GEC before resection than patients without cirrhosis. After resection these tests were unchanged. Patients with liver related complications and cirrhosis (n = 5) had lower ICG-ER and GEC than patients with cirrhosis and no complications. In the postoperative course all liver function tests in these patients were significantly lower compared to preoperative results. Comparing liver function tests ICG serves best to indicate postoperative liver failure. Liver function tests do not correspond with loss of liver tissue.     

Control of rat glomerular epithelial cell growth in vitro

The interaction of cultured rat GEC1 with several growth factors was explored in order to obtain a better understanding of in vivo factors which might stimulate GEC proliferation. GEC proliferated in response to EGF but not IGF-1, MSA or PDGF. Specific, saturable receptors for EGF were detected in saturation and competition binding studies utilizing 125I-EGF with an approximate Kd of 1.7 nM and 86,000 binding sites per cell. TGF-beta inhibited GEC growth in a time and dose dependent manner with a brief early exposure resulting in prolonged growth inhibition which was not reversible by EGF. Exposure to TGF-beta sufficient to maximally inhibit growth had no effect on EGF binding to GEC. More prolonged exposure to TGF-beta, however, did result in an increase in the apparent number of EGF receptors on GEC but no change in Kd. These studies suggest that EGF and TGF-beta released by inflammatory cells or platelets during the course of glomerular injury may play a role in modulating glomerular cell proliferation.     

Glomerular epithelial cell, polyanion neutralization is associated with enhanced prostanoid production

We have studied the effect of neutralizing the surface charge of rat glomerular epithelial cells (GEC) in culture on prostanoid production. Incubation of rat GEC with polycations poly-L-lysine (PL) resulted in a dose dependent increase of 6-keto-PGF1 alpha (up to 8 to 10-fold) and PGE2 (up to 7 to 8-fold) production. Other polycations such as protamine sulfate (PS) and lysozyme (LY) produced a similar effect. The stimulation of prostaglandin (PG) production by PL treated GEC was prevented by the addition of polyanions such as bovine serum albumin (BSA) and heparin (HP). The effect of PL on prostaglandin (PG) synthesis by GEC was suppressed by the cyclooxygenase inhibitor sulindac sulfide. Addition of exogenous arachidonic acid (C20:4) increased the basal production of PG and under these conditions the effect of PL was masked. We conclude that neutralization of the surface charge of rat GEC by polycations results in profound increase of prostanoid synthesis. The polycation caused increase in PG synthesis appears to be the result of increased availability of intracellular C20:4.     

Molecular mechanisms of experimental glomerulonephritis: an overview

Summary: Most glomerular diseases are autoimmune in nature with injury occurring as a consequence of antibody reacting with antigens on glomerular cell membranes or in glomerular matrix, or immune complex formation occurring on or near glomerular cell membrnes. Many of the structural and functional consequences of these events result from the response of resident glomerular cells to the injury, or may be induced by inflammatory effector cells derived from the circulation. In terms of glomerular cells, injury to the glomerular epithelial cell (GEC) may be induced by: (i) non-complement fixing antibodies which mimic the glomerular lesions of minimal change nephrotic syndrome/focal glomerular sclerosis; or (ii) by antibodies which fix complement leading to GEC attack by C5b-9 resulting in a lesion analogous to membranous nephropathy. C5b-9 also mediates antibody induced injury to the mesangial cell resulting in a mesangioproliferative glomerulonephritis (IgA nephropathy, systemic lupus erythematosus; SLE) as well as to the glomerular endothelial cell (thrombotic microangiopathy, haemolytic uraemic syndrome). the effects of C5b-9 may be lytic (mesangial cell, glomerular endothelial cell; GEN) or sublytic (GEC) resulting in stimulation of local oxident and protease production. Both lytic and sublytic effects are substantially modulated by cell-bound complement regulatory proteins such as CD59 and Crry. When fixed or planted antigens are present in larger quantities and accessible to the circulation, complement activation generates chemotactic factors leading to neutrophil infiltration and producing injury through the myeloperoxidase (MPO)-H₂-O₂-halide system, a mechanism substantially augmented by platelet-neutrophil interaction (post-infectious glomerulonephritis [GN], SLE). Macrophages may also be localized in glomeruli by either immune adherence mechanisms or as a consequence of cell mediated immune reactions in the glomerulus. Macrophages differ from neutrophils in producing large amounts of transforming growth factor-β (TGF-β) and procoagulants which contribute to crescent formation rapidly progressive glomerulonephritis (RPGN).     

Glomerular epithelial cells secrete a glomerular basement membrane-degrading metalloproteinase.

Cultured rat glomerular epithelial cells (GEC) were examined for their ability to release extracellular matrix-degrading proteinases with [3H]gelatin as substrate. GEC-conditioned media, under serum-free conditions, contained modest amounts of gelatinase activity (1 to 10 U/mg of protein); the activity was maximal at neutral pH, was inhibited by zinc chelators, was not inhibited by tissue inhibitor of metalloproteinase-2, and could not be further activated by trypsin or organomercurials. Gelatin substrate sodium dodecyl sulfate-polyacrylamide gels of GEC-conditioned medium revealed several zones of lysis, with molecular sizes of 150 kd (major band), and 220, 86 to 93, and 52 to 54 kd (minor bands). Northern blot analysis demonstrated that the GEC metalloproteinase(s) were distinct from the 68- to 72-kd type IV collagenase/gelatinase present in mesangial cells or the 92-kd type IV collagenase present in neutrophils. The GEC gelatinolytic activity also degraded insoluble type IV collagen in glomerular basement membrane in a dose-dependent manner. The major metalloproteinase activity responsible for the type IV collagen degradation has a molecular size of 150 kd with a type IV collagen substrate gel. Thus, GEC produce several neutral metalloproteinases, which, by virtue of their substrate specificity, may play an important role in glomerular basement membrane remodeling and in glomerular diseases characterized by alterations in basement membrane permeability.     

SRC-family kinase Fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin

Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Src-family kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was co-transfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.     

Acute renal failure secondary to leukocyte-mediated acute glomerular injury.

Acute glomerulonephritis can cause acute renal failure. Activated neutrophils and monocytes are major effectors of glomerulonephritic renal failure. Adhesion molecules, granule enzymes, reactive oxygen radicals, lipid metabolites, and cytokines of activated neutrophils and monocytes mediate glomerular capillary constriction, occlusion, and destruction. Injurious products and biologically active mediators released by activated leukocytes have profound functional effects on mesangial cells and endothelial cells, which in turn participate in the disturbance of glomerular function, for example, by altering capillary diameter and surface area. The glomerular inflammatory events result in decreased glomerular capillary ultrafiltration coefficient and glomerular filtration rate, as well as other functional perturbations.     

The interaction of glomerular mesangial cells and epithelial cells

The interaction of cells within the glomerulus plays an important role in the development and progression of glomerular disease. To investigate the interaction of glomerular mesangial cells (GMC) and epithelial cells (GEC), and mediator(s) of this interaction, we investigated the effect of Adriamycin (doxorubicin hydrochloride)-induced (ADR) rat GMC-conditioned medium (GMC-CM) on the incorporation of ³⁵S, ³H-leucine, and ³H-thymidine in normal rat GEC, as well as ³H-thymidine uptake by normal rat GMC in response to ADR-rat GEC-CM. In addition, changes in the responsiveness to interleukin-6 (IL-6) and the products of IL-6 were assessed in ADR-rat GMC. The results showed that: (1) GMC-CM of ADR-rat with heavy proteinuria stimulated GEC proliferation and the synthesis of sulfated compounds and protein, while the GEC-CM of ADR-rat from the same nephrotic period increased GMC proliferation; (2) the ADR-rat GMC had altered responsiveness to IL-6 and its products. The stimulation index results demonstrated the interaction of GMC and GEC in the ADR-induced rat model, and that this interaction related closely to the degree of proteinuria and was mediated by soluble products of the damaged glomerular cell. Received March 29, 1996; received in revised form July 1, 1997; accepted July 2, 1997     

Glomerular epithelial cell injury accelerates the progression of antibody-induced mesangial proliferative nephritis

BACKGROUND: Anti-Thy-1.1 antibody induces mesangioproliferative glomerulonephritis; however, the mesangial lesion is spontaneously recovered to the normal feature. Glomerular epithelial cells (GECs) play a crucial role in the glomerular function. Very little is known about the involvement of GECs in this disease model. This study is designed to investigate whether GEC injury prolongs the mesangial lesion. METHODS: The effects of GEC damage on mesangioproliferative nephritis were studied with combined treatment using puromycin aminonucleoside (PAN) and monoclonal antibody (MAb) against rat mesangial Thy-1.1. Urinary protein, BUN, Pcr and Ccr were measured. To clarify the underlying mechanisms, morphological study and immunohistochemistry for alpha-SMA, FGF-2 and PCNA were carried out. RESULTS: Simultaneous administration of PAN plus MAb induced progressive mesangioproliferative nephritis compared to PAN or MAb alone. Rats with combined treatment displayed renal dysfunction with massive proteinuria. Morphological studies showed that the glomeruli in combined group had features resembling those of progressive mesangial proliferative glomerulonephritis in humans. Morphologic lesions of GECs in acute nephritic phase were severer than those in other groups. Immunohistochemistry revealed that glomeruli of combined treatment exhibited persistent overexpression of alpha-SMA and FGF-2. CONCLUSIONS: Simultaneous dysfunction of GECs and mesangial cells can lead to persistent glomerular perturbations with prolonged phenotypic change of mesangial cells, resulting in end-stage renal deficiency. GEC damage during the acute nephritic phase contributes to the progression of irreversible renal disease.