Mesangial cell migration precedes proliferation in Habu snake venom-induced glomerular injury.

BACKGROUND: Mesangial cells migrate in response to platelet released products in vitro (Am J Pathol 1991;138:859). Cell migration, in addition to proliferation might play a role in cell remodeling during the course of proliferative glomerular disease. EXPERIMENTAL DESIGN: In this study, we examined mesangial cell migration in vivo in a platelet-dependent model of proliferative glomerulonephritis induced by Habu snake venom. Mesangial cell migration was assessed by phenotypic identification and temporal location of mesangial cells within glomerular lesions in serial time studies from 8 to 48 hours after Habu snake venom. Autoradiography of [3H]thymidine incorporation into cells was employed to identify and temporally separate cell division and proliferation from cell motility and other related events. RESULTS: Early (8-hour) lesions consisted of microaneurysms devoid of mesangial cells. By 24 hours, glomeruli showed mesangial cells at the margins of lesions adjacent to intact glomerular tufts, followed by the presence of clusters of cells at 30 and 36 hours. By 48 hours, most lesions were filled with proliferating mesangial cells. Cells containing [3H]thymidine were rarely observed until 30 hours, at which point they were found in advanced lesions. Marginating cells did not contain [3H]thymidine, suggesting that the location of these cells was not related to cell division but rather to migration. Platelet depletion eliminated platelets from lesions and substantially retarded mesangial cell migration into glomerular lesions indicating mesangial cell migration is, in part, dependent on platelets or their secretory products. CONCLUSIONS: These studies show that mesangial cells can migrate in vivo and suggest that cell migration is an important early step in cell redistribution and remodeling during glomerular injury in this model of proliferative glomerulonephritis.

Glomerular mesangial cell migration. Response to platelet secretory products.

Glomerular mesangial cells migrate in response to platelet-derived growth factor (PDGF), but to date these cells have not been examined for migratory behavior in response to other platelet secretory products. Because migration might provide an additional mode of cell redistribution and local mesangial hypercellularity in certain forms of glomerular disease, we examined, in vitro, the potential of isolated rat mesangial cells to migrate toward gradients of platelet releasate and selected platelet secretory proteins. Chemotaxis assays were performed in two compartment blind well chambers, each compartment separated by a porous membrane. Releasate of activated platelets was added in incremental concentrations (25, 50, and 100 micrograms/ml) to lower compartments, and mesangial cells were placed in upper compartments. The chambers were then incubated at 37 degrees C for 4 hours. Mesangial cell migration through the membranes was quantitated by scanning electron microscopy. Mesangial cells migrated toward platelet releasate in a linear dose-response, achieving cell numbers of approximately 40 times those of controls. Examination of specific platelet alpha granule secretory proteins disclosed a potent mesangial cell migratory response to platelet-released fibronectin (Fn), but not to transforming growth factor-alpha (TGF-alpha), -beta (TGF-beta), epidermal growth factor (EGF), or platelet factor 4 (PF4). Secretory levels of platelet Fn (1 to 25 micrograms/ml) induced a maximum migratory response of approximately 60-fold over controls. Mesangial cell migration in response to both platelet Fn and platelet releasate was abrogated by blocking the integrin receptor for Fn with RGDS tetrapeptide. Thus, platelet Fn appears to be a prominent component of platelet releasate responsible for mesangial cell migration.

Glomerular mesangial cell migration in response to platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic protein for a variety of cell types. Glomerular mesangial cells also respond to PDGF in terms of proliferation, but, to date, have not been examined for migratory behavior in response to a specific growth factor. Here, we examine the ability of isolated rat mesangial cells to migrate toward gradients of purified PDGF. Chemotaxis assays were performed in two-compartment blind well chambers, each compartment separated by a 14-microns porous filter membrane. Human PDGF was added to 200 microliters of RPMI 1640 medium in the lower compartment beneath the filters to make incremental concentrations from 2.5 to 50 units/ml. Control compartments received diluent without PDGF. Mesangial cells in RPMI 1640 medium were added to the upper compartments and the chambers were incubated for 8 hours at 37 degrees C. After fixation, the number of cells on the underside of the filter were counted by scanning electron microscopy. A linear dose response of mesangial cell migration toward increasing concentrations of PDGF was observed, achieving cell numbers of 9-fold over controls at 50 units/ml. Migratory cells were verified as mesangial cells by fluorescence expression of actin, myosin, and desmin and absence of expression of leukocyte common antigen and Ia antigen. Addition of equimolar concentrations of PDGF on both sides of the filter or addition of anti-PDGF antibody to the lower chamber containing PDGF negated the chemotactic response. These studies indicate that mesangial cells migrate in response to PDGF. This mechanism may, in part, play a role in some forms of mesangial proliferative glomerular disease.

Hepatocyte cell surface polarity as demonstrated by lectin binding.

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.