Mesangial cell immune injury. Synthesis, origin, and role of eicosanoids.

The synthesis, cell origin, and physiologic role of eicosanoids were investigated in a model of mesangial cell immune injury induced by a monoclonal antibody against the rat thymocyte antigen Thy 1.1 also expressed in rat mesangial cells. A single intravenous injection of the antibody resulted in enhanced glomerular synthesis of thromboxane (Tx)B2, leukotriene (LT)B4, and 12-hydroxyeicosatetraenoic acid (HETE), whereas that of PGE2 and PGF2 alpha was either unaltered or impaired. The enhanced eicosanoid synthesis was associated with decrements in glomerular filtration rate (GFR) and renal blood flow (RBF). Complement activation mediated both the increments in TxB2, LTB4, and 12-HETE and the decrements in GFR and RBF. The decrements in GFR were abolished by the TxA2 receptor antagonist SQ-29,548. Although both neutrophiles and Ia (+) leukocytes infiltrated glomeruli, glomerular LTB4 originated mainly from the latter. Platelets entirely accounted for the enhanced 12-HETE synthesis in isolated glomeruli and to a lesser extent for that of LTB4 and TxB2. Glomerular PGE2 and PGF2 alpha originated from mesangial cells as their impaired synthesis coincided with extensive mesangial cell lysis. The observations indicate that in mesangial cell immune injury vasoactive and proinflammatory eicosanoids originate from recruited or activated Ia (+) leukocytes and platelets and may exert paracrine effects on mesangial cells.

Mesangiolysis: an update.

Mesangiolysis occurs in many renal diseases, both human and experimental. At least three types of mesangiolysis may be recognized, which differ in their mode of origin and in morphologic features. The first type is severe mesangiolysis with formation of glomerular cysts and subsequent cellular proliferation resembling glomerulonephritis. In the second type, mesangiolysis is associated with extensive widening of the subendothelial space and is thought to follow endothelial injury. The third type is mesangiolysis with lamellated mesangial nodules which is believed to result from relatively mild but persistent or recurrent localized mesangial, and perhaps also endothelial damage, with lysis of mesangial anchor points.

A morphological study of radiation nephropathy in the pig.

Both kidneys of mature pigs received a single dose of 9.8 Gy 60Co gamma rays. Pigs were killed between 2 and 24 weeks after irradiation and the kidneys examined histologically. Glomerular and tubular changes were observed within 2 weeks of irradiation. Neutrophils and other leukocytes were seen within glomerular capillary loops; mesangial matrix and cell number increased. A progressive increase in thickening of the basement membrane and a decrease in capillary lumina were then noted. Basement membrane duplication occurred within 12 weeks. By 24 weeks these lesions had increased in severity, sclerotic endstage glomeruli, predominantly subcapsular or juxtamedullary, being evident. Tubular lesions initially consisted of focal areas of tubular atrophy in the juxtamedullary region. By 6 weeks subcapsular foci of tubular degeneration, regeneration, and necrosis were found; these appeared to resolve 12 weeks after irradiation. At later times the severity of the tubular lesions varied between pigs, with some exhibiting interstitial fibrosis involving a complete band of subcapsular tissue, while others showed relatively mild changes. There was no apparent change in the vasculature. These findings indicate that (a) there is no one target or dose-limiting cell, and (b) the vasculature does not play a primary role in the development of radiation nephropathy.

Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms.

Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P

Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis.

To determine the ability of radiation to modulate mesangial cell expression of various molecules involved in promoting extracellular matrix (ECM) accumulation [fibronectin, plasminogen activator-inhibitor 1 (Pai1), and tissue inhibitor of metalloproteinase-2 (Timp2)] and degradation (Tgfb, plasminogen activators u-PA or t-PA, matrix metalloproteinases Mmp2 and Mmp9), primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 48 h. Irradiation of quiescent mesangial cells resulted in significant (P < 0.05) time- and dose-dependent increases in Fn and Pai1 mRNA and/or immunoreactive protein. Despite an increase in Tgfb1 mRNA, there was little evidence for an increase in total Tgfb protein. Indeed, active levels remained unaltered after irradiation. Irradiation led to differential changes in MMP expression; active Mmp2 levels increased, while Mmp9 levels appeared unaltered. In addition, secretion of plasminogen activators into the medium was unchanged after irradiation, while secretion of Timp2 increased. We conclude that irradiating mesangial cells leads to altered production of various molecules involved in accumulation and degradation of extracellular matrix.

Origin of the rat mesangial phagocyte and its expression of the leukocyte common antigen.

We have previously characterized a subpopulation of cells in the rat glomerular mesangium that are phagocytic, display Ia antigens, and stimulate lymphocytes. We now report that total-body irradiation induces a progressive loss of the Ia+ mesangial cells, which is complete by 3 days. Experiments utilizing kidney shielding and in vitro exposure to irradiation have established that the effect is not mediated via radiation sensitivity of the mesangial phagocyte. Transplantation of syngeneic bone marrow cells results in a restoration of Ia+ mesangial cells in 3 weeks, establishing their extrarenal origin. Employing monoclonal antibodies to rat leukocyte common (LC) antigen, we have quantified a larger population of mesangial phagocytes; the Ia+ cells are a subset of the glomerular LC+ cells. In tissue culture, anti-LC antibody labels 100% of the phagocytic cells obtained from perfused glomeruli. Ultrastructural analysis, utilizing immunoperoxidase techniques, has localized the LC+ cell to the glomerular mesangium.

Glomerular macrophage modulation affects mesangial expansion in the rat after renal ablation.

Recent studies have provided evidence for the involvement of macrophages (m phi) in various types of human and experimental glomerular disease. The aim of the present study was to examine the effect of m phi depletion on glomerular injury after 3/4 renal ablation in the rat. This "remnant kidney" model is a widely used experimental model of focal and segmental glomerulosclerosis. Sustained glomerular m phi depletion was induced in remnant kidney rats by a regimen of sublethal triple systemic X-irradiation with shielding of the kidney remnants. Groups of 8 X-irradiated and 8 non-irradiated rats were studied at 5, 9, and 13 weeks after renal ablation. X-irradiated rats showed severe peripheral blood leukopenia at 5 and 9 weeks which had normalized at 13 weeks. The number of remnant glomerular m phi (immunohistochemistry with the monoclonal antibody ED1) in X-irradiated rats at 5 weeks was significantly lower when compared to non-irradiated remnant kidney rats. A rebound effect occurred at 9 and 13 weeks with increased m phi in remnant glomeruli of X-irradiated rats. Light microscopic examination disclosed significantly lower semiquantitative scores for mesangial cellularity and mesangial matrix expansion in remnant glomeruli of X-irradiated rats at 5 weeks when compared to non-irradiated remnant kidney rats. Mesangial matrix expansion had increased in X-irradiated rats at 9 and 13 weeks after ablation coincident with elevated glomerular m phi at these intervals. Multiple linear regression analysis indicated a highly significant contribution of m phi to the best fitting regression model predicting mesangial matrix expansion (multiple r2 = 0.81). In conclusion, these data provide evidence for a contributory role of m phi in the evolution of glomerular injury in the rat after renal ablation.

Heparin increases hillock formation in mesangial cell cultures.

Mesangial cells in culture develop hillocks, which are composed of aggregates of cells, necrotic cellular debris, and extracellular matrix material. The significance and mechanism of their formation are unknown. To determine whether a proliferative component is involved in hillock formation, cells were treated with heparin or irradiated to inhibit proliferation. Heparin caused a 50% inhibition of mesangial cell growth and stimulated hillock formation three-fold to fourfold. Irradiated cells developed hillocks to the same extent as did nonirradiated cells, and the addition of heparin also increased hillock formation threefold to fourfold. Dextran sulfate and chondroitin B sulfate had no effect on mesangial cell hillock formation. Mesangial cells cultured in the presence of 50 micrograms/mL of heparin were less tightly adhered than nontreated cells, as assessed by a trypsin adhesion assay (control cells, 12% detached; heparin-treated cells, 72% detached). Thus, it appears that heparin, a glycosaminoglycan with potent antimitogenic activity, stimulates mesangial cell hillock formation, possibly by decreasing cell adhesion.

Apoptosis in mesangial cells induced by ionizing radiation and cytotoxic drugs.

Mesangial proliferation contributes to the pathogenesis of many forms of glomerulonephritis. To evaluate the role of apoptosis on the pharmacologic effects of cytotoxic drugs and ionizing radiation, we studied their effects on cultured rat mesangial cells (MC), whose apoptotic response to these drugs is unknown. Mesangial cells were cultured with or without stimuli to induce apoptosis and were harvested at 24 and 48 hours. MC morphology was examined by light microscopy, in situ end labeling technique using terminal deoxy-transferase (TUNEL) and by electrophoresis of extracted total cellular DNA. MCs exposed to cytotoxic drugs or irradiation demonstrated statistically significant increases in apoptotic cells identified by light microscopy. DNA fragmentation of apoptotic cells was also visualized as characteristic staining by the TUNEL method and statistically significant increases in apoptotic cell number in cells exposed to cytotoxic drugs and irradiation were noted compared to control cultures. In general, the number of TUNEL positive cells was greater than that of morphologically apoptotic cells. DNA extracted from these cells also showed the characteristic ladder pattern of internucleosomal chromatin cleavage of 180 bp fragments on agarose gel electrophoresis. To further analyze whether MC apoptosis induced by these drugs alters the cell cycle, 3H-thymidine incorporation rates were measured in both the cell culture monolayer and in those cells shed into the supernatant when cultured with or without cyclophosphamide (N = 5). 3H-thymidine incorporation corrected for total cellular DNA showed a similar pattern in both control and cyclophosphamide treated groups, suggesting that cyclophosphamide did not alter the mesangial cell cycle. Considering that the dosage of the cytotoxic drugs utilized in these experiments in nearly the therapeutic plasma level in humans, these results suggest that cytotoxic drugs used to treat glomerular disease can induce apoptotic mesangial cell death and may operate in part via this mechanism.