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The use of human glomerular epithelial cells (HGEC) in research has been severely restricted by several obstacles, which have been circumvented by the generation of T-SV40 immortalized human visceral glomerular epithelial cells (Delarue et al, 1991). In this work, we compared the primary and immortalized HGEC for expression of integrin and some nonintegrin surface receptors. We also studied the adhesion of both types of HGEC to glomerular basement membrane (GBM), type IV collagen (tIV), and its major noncollagenous NC1 domain. The integrins mediating adhesion of HGEC to tIV were also examined. Expression of integrin and some nonintegrin cell surface receptors was analyzed by flow cytometry. Adhesion to GBM, tIV, and its major noncollagenous NC1 domain was studied by direct solid phase cell adhesion assays. Identification of integrins mediating adhesion of HGEC to tIV was achieved by inhibition of cell adhesion using monoclonal antibodies to integrin subunits. The primary and immortalized HGEC share phenotypic characteristics, and alpha3beta1 appeared to be the major integrin present on both HGEC types. The kinetics of binding to GBM, tIV, and its noncollagenous NCI domain were similar in both the primary and immortalized HGEC, although the latter displayed a somewhat weaker binding. Both the primary and immortalized HGEC displayed significantly better adhesion to NC1-alpha3 compared with NC1-alpha1, alpha3beta1 appears to be the major integrin mediating the adhesion of HGEC to tIV. Our studies suggest that alpha3beta1 is the major integrin present on HGEC. This has been confirmed by flow cytometric analysis. In addition, we demonstrated a functional role for this integrin in mediating attachment of HGEC to tIV. Our data also demonstrate a preference in binding of HGEC to alpha3 chains of NC1 compared with alpha1 chains of NC1. These findings were seen in both the primary and immortalized HGEC. The T-SV40 immortalized HGEC can therefore serve as a very useful tool to study glomerular visceral cell biology.

MHC class II-encoded molecules HLA-DR, -DP and -DQ play a pivotal role in the human immune response. Their constitutive expression is restricted to a number of immunocompetent cells referred to as antigen-presenting cells. However, gamma-interferon (gamma-IFN) has been shown to induce MHC class II molecule expression in several epithelia. Using flow cytometric analysis, we show here that normal and SV40-transformed human podocytes in culture constitutively expressed gamma-IFN receptors. We also show that MHC class I molecules are constitutively expressed in these cells and that HLA-DR, -DP and -DQ expression, which is not found in unstimulated cells, can be induced by gamma-IFN stimulation. This induction was a time-dependent event, a lag phase of 24-48 h being necessary for MHC class II molecules to become detectable at the cell surface by flow cytometric analysis. Induction of MHC class II molecules in human podocytes also showed a concentration dependence, a plateau being reached at a concentration of 500 IU of gamma-IFN/ml of culture medium. This effect was blunted by coincubation of the cells with an antihuman gamma-IFN receptor monoclonal antibody. HLA-DR expression was associated with specific mRNA accumulation, as detected by Northern blot analysis. By indirect immunofluorescence, the intercellular adhesion molecule 1 was also induced by gamma-IFN stimulation. Induction of DR, DP and DQ in human podocytes may be involved in the pathogenesis of immune glomerulonephritis in man.

Extracapillary glomerulonephritis are associated with fibrin deposition in the urinary space of the glomerulus. Such deposits were correlated with the severity of the disease and with a poor renal outcome. Fibrin formation involves an activation of the coagulation cascade either through the intrinsic pathway, Hageman factor being activated by the altered glomerular basement membrane, either by the extrinsic pathway, infiltrating monocytes and glomerular cells exhibiting a procoagulant activity i.e. thromboplastin or tissue factor. Treatments with heparin or warfarin were shown to decrease the severity of experimental glomerular diseases. A similar beneficial effect was obtained with a monocyte-depleting serum and more recently with a treatment by a tissue type plasminogen activator. Glomerular cells also produce a fibrinolytic activity which could be too low or uneffective on extracapillary fibrin deposits if they contain high amounts of plasminogen activator inhibitors. Thrombin has procoagulant activity, antifibrinolytic activity and has cellular chemotactic and proliferative effects. It could play a major role in the pathogenesis of crescent formation.

To investigate the mechanisms which may influence fibrin deposition in the remnant kidney, glomerular morphology and the haemostatic properties of isolated glomeruli were assessed in two groups of rats, 30 days after surgical removal of three-quarters of the total renal parenchyma, and compared to glomeruli in sham-operated controls. One group was given the thromboxane synthetase inhibitor OKY 046 and the other was not. Fibrin deposition occurred in about 20% of glomeruli, of which 4% exhibited segmental necrotizing lesions. Concomitantly, glomerular procoagulant activity dropped to 40% of the control level and glomerular fibrinolytic activity rose to 120-130% of this level. Inhibition of thromboxane synthesis did not affect fibrin deposition, glomerular haemostasis or the development of renal insufficiency. In an additional group of unilaterally nephrectomized rats, procoagulant activity also markedly decreased in the remaining kidney. These results indicate that in the rat remnant kidney, alterations in glomerular haemostatic properties tend to have antithrombotic effects which seem to constitute an adaptive reaction by an autacoid system to glomerular fibrin deposition.

Isolated rat renal glomeruli contain an adenylate cyclase system and guanylate cyclase system. Adenylate cyclase was strikingly activated by purified parathyroid hormone, epinephrine, prostaglandin I2 and histamine. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role of this hormone in regulation of glomerular filtration rate. Guanylate cyclase was strikingly activated by CA2+, nitrate derivatives such as sodium nitroprusside. Its role remained still unknown.

The glomerular fibrinolytic activity (GFA) was measured during the development of glomerulonephritis induced by the intravenous injection of horse or rabbit antiglomerular basement membrane antibodies in rats either untreated or preimmunized with horse or rabbit Ig. A variety of nephritides was produced ranging from proteinuria with no or mild changes in glomerular architecture to severe diffuse proliferative glomerulonephritis with close to 100% crescent formation. GFA was measured on preparations of isolated glomeruli using a radioassay based on lysis of 125I fibrin adsorbed on a solid phase. In some experiments a fibrin plaque technique was also used. Both methods clearly demonstrated a marked increase in GFA with a good correlation between the two sets of results when the glomerular architecture is preserved. The increase in GFA is related both to the severity of the nephritis estimated by the percentage of glomeruli showing crescent formation and to the extent of the fibrin deposits. The results therefore indicate that the persistence of fibrin in the glomeruli and particularly in crescents is not due to a loss of GFA.

Angiotensin II (Ang II) controls renal plasma flow, which depends on perfusion pressure and on renal vascular resistances; the glomerular filtration rate of single nephrons (SNGFR) also depends on these two parameters. The glomerulus can synthesize all components of the renin-angiotensin system. Renin is produced by mesangial cells and cells of afferent arterioles, angiotensinogen by mesangial cells, and angiotensin I-converting enzyme (ACE) by endothelial and mesangial cells. Ang II binds to afferent and efferent arterioles and to mesangial cells, which subsequently contract and enhance their production of vasodilator prostaglandins. Ang II participates in the regulation of the SNGFR by acting on arteriolar resistance, on the glomerular capillary surface area, and finally on the ultrafiltration coefficient. The synthesis of renin is initiated when the cAMP concentration increases under the influence of hormones (parathyroid hormone, catecholamines), of mediators (e.g., prostanoids), or of changes in composition of tubular fluid (in sodium, chlorine, or calcium) detected by the macula densa. Moreover, renin synthesis is stimulated by decreases in intracellular calcium concentration. The renin-angiotensin system plays an important role in the regulation of glomerular filtration. A decrease in renal plasma flow is followed by an increase in glomerular efferent arteriole resistance, so that perfusion pressure and glomerular filtration remain constant. Furthermore, if the decrease in renal plasma flow is more marked, vasodilator prostaglandins, whose synthesis is stimulated by Ang II, dilate afferent arterioles, which sustains glomerular filtration. Prostaglandins are also apt to bind to the juxtaglomerular apparatus and to increase the intracellular cAMP level.(ABSTRACT TRUNCATED AT 250 WORDS)

PGE2 production in the presence of mercuric chloride was measured by specific radioimmunoassay in isolated whole glomeruli and in cultured glomerular cells. Under basal conditions, mercuric chloride at 10 micrograms.ml-1 caused a twofold stimulation of PGE2 in isolated whole glomeruli, and at 1 to 10 micrograms.ml-1 it caused a more marked increase of PGE2 (eight times the control value) in mesangial or epithelial glomerular cells. An excess of arachidonic acid, however, blunted the stimulatory effects of mercury. Furthermore, the effect of mercuric chloride on PGE2 production was only observed with intact preparations but not with cell homogenates. Mercury inhibited cyclooxygenase activity in purified glomerular microsomes. It also inhibited phospholipase activity in purified glomerular cell membranes, whereas it stimulated phospholipase activity in intact cells. These results demonstrate that mercury modifies PGE2 synthesis via multiple mechanisms. Stimulation occurring at low doses of mercury can be attributed to the indirect activation of phospholipase. We suggest that mercuric chloride suppresses the inhibitory effect of lipomodulin on this enzyme.

The PGE2, PGF2 alpha and 6-keto-PGF1 alpha contents of the incubation medium of glomeruli isolated from rat kidney were measured at different times with or without addition of arachidonic acid. These prostaglandins accumulated progressively with time and reached equilibrium after 60--120 min incubation. Synthesis of the 3 prostaglandins was inhibited when indomethacin was added whereas it was markedly enhanced, mainly for PGE2, at increasing doses of arachidonic acid. Plateaus were reached above 5 micrograms/ml and concentrations corresponding to 50% of the maximum values were 2 micrograms/ml for PGE2 and PGF2 alpha, and 0.8 microgram/ml for 6-keto-PGF1 alpha. There were strictly linear relationships between PGE2 or PGF2 alpha productions and the concentration of glomerular protein. PGE2 and PGF2 alpha synthesis with or without arachidonic acid were maximum at 30--37 degrees C. PGE2 glomerular content was almost undetectable initially and increased with time. These data demonstrate that PGE2, PGF2 alpha and PGI2, in order of decreasing abundance, are synthesized by the glomerular cells and suggest that PGE2 and PGI2-sensitive glomerular adenylate cyclase activities and PGE2-sensitive renin synthesis may be stimulated by prostaglandins formed in the glomeruli themselves.

The effects of thrombin perfusion on glomerular fibrinolytic activity (GFA) have been assessed quantitatively in isolated rat glomeruli. Gfa was augmented by increasing total thrombin perfused to a maximal dose of 120 units per 100 gm. Fifty per cent maximal GFA occurred at a dose of 38 to 42 units of thrombin per 100 gm. independent of duration or rate of perfusion. After termination of infusion, GFA diminished and thrombi were no longer observed. Simultaneous administration of thrombin and epsilon-aminocaproic acid was associated with sustained high GFA and persistence of thrombi, presumably due to inhibition of the action of plasmin on fibrin, but it had no effect on tissue activators of plasminogen.

In 9 patients aged 16 to 71 years with acute renal failure, histological examination disclosed polyarteritis nodosa. Symptoms of rapidly progressive glomerulonephritis (GN) were present in 7 cases. Hypertension was a constant feature. Fever and muscular articular and cutaneous signs occured at times simulating Henoch-Schönlein purpura. Necrotizing angiitis was a constant finding, with perivascular granuloma in 7 cases. Lesions affecting smaller arteries were associated with diffuse or segmental extracapillary GN, and lesions of larger arteries with ischaemic appearance of the glomeruli. Diagnosis was obtained during life by percutaneous renal biopsy in 5 patients and muscular or cutaneous biopsy in 2 other patients, whereas the evidence was only obtained on post mortem examination in the 2 remaining patients. In the 6 renal biopsies examined by immunofluorescence, fibrinogen was always present in glomeruli and/or arteries. In one patient there was a linear deposit of IgG along glomerular basement membrane (GBM) and there were circulating antibodies directed against GBM. Arteries of different sizes may be damaged in various organs, as showed by arteriography and autopsy. Steroid therapy and immunosuppressive drugs had no effect on renal symptoms. 8 out of 9 patients died rapidly, in 6 the death was due to extrarenal localization of the disease, even though uraemia was controlled by haemodialysis. The last patient died 2 years after onset of the disease, while being on maintenance haemodialysis treatment.

Normal pregnancy in women and rats is characterized by an increased glomerular filtration rate. Because prostaglandin glomerular synthesis has been reported to be increased in 2 other circumstances with glomerular hyperfiltration (streptozotocin-induced diabetes and high protein dietary intake in Heymann nephrilis), we studied prostaglandin biosynthesis in glomeruli obtained from pregnant Wistar rats in comparison with non pregnant rats during the estrus cycle. Comparatively to the 3 first phases of the cycle, and wether or not arachidonic acid was present, PGE2 and PGF2 alpha production rates were found significantly higher in diestrus 2 (2-3 fold increase for both) at 15 days of pregnancy (2 fold increase for PGE2, 5-6 fold increase for PGF2 alpha) and at 21 days of pregnancy (5-6 fold increase for both). On the other hand synthesis of TXB2 was not increased during pregnancy nor during diestrus 2. Plasma renin activity (PRA) was increased during pregnancy. It is concluded that in presence of increased PRA, the increased synthesis of PGE2 may be the hormonal factor explaining, at least in part, the hemodynamic mechanism of glomerular hyperfiltration which has been previously described by micropuncture techniques and characterized by increased renal plasma flow, increased glomerular filtration pressure and decreased ultrafiltration coefficient.