Tumor necrosis factor production by glomerular macrophages in anti-glomerular basement membrane glomerulonephritis in rabbits

The production of tumor necrosis factor (TNF) by nephritic glomeruli and glomerular macrophages was studied in antiglomerular basement membrane antibody induced glomerulonephritis (anti-GBM GN) in rabbits. Autologous phase injury was associated with glomerular macrophage infiltration and augmented TNF production by isolated nephritic glomeruli (day 8, 1.15 +/- 0.10 ng/10(3) glomeruli/24 hours; normal, 0.01 +/- 0.01 ng/10(3) glomeruli/24 hours; p less than 0.05). In contrast, during the heterologous phase, in which macrophages were not prominent, injury was not associated with augmented glomerular TNF production. Glomerular TNF bioactivity had a molecular weight and isoelectric point consistent with rabbit TNF and was inhibitable by an anti-TNF antibody. TNF was also identified in nephritic glomerular supernatants by Western blotting. Macrophages isolated from glomeruli of rabbits developing autologous phase anti-GBM GN produced significantly more TNF (0.14 +/- 0.02 ng/10(3) macrophages/24 hours) than blood monocytes (0.03 +/- 0.02 ng/10(3) monocytes/24 hours, p less than 0.05) from the same rabbits. Macrophage depletion of rabbits with autologous phase anti-GBM GN significantly reduced proteinuria, prevented glomerular macrophage accumulation, and blocked augmentation of glomerular TNF production. These studies demonstrate the association of glomerular TNF production with the development of glomerular macrophage infiltration and injury in anti-GBM GN and suggest that infiltrating glomerular macrophages are the major source of glomerular TNF.     

Role of glomerular epithelial cell injury in the pathogenesis of glomerular scarring in the rat remnant kidney model.

We investigated the roles of glomerular epithelial cell (GEC) pathology and dysfunction in the pathogenesis of glomerular scarring and attempted to separate them from direct hypertensive injury in the 5/6 nephrectomy (RK) model of glomerular injury. Male WKY rats weighing 200 g were studied 6 weeks after RK, when approximately one-half had developed systemic hypertension (systolic blood pressure > or = 150 mm Hg) (HT), and one-half were normotensive (NT). The incidence of glomerular necrosis and scarring was greatest in the HT rats (P = 0.0259), and vascular necrosis was only seen in 4 of 11 HT rats. The RK group had increased glomerular diameters (HT, 174 mu mean; NT, 171 mu; sham, 142 mu; P = 0.0014 by analysis of variance). There was foot process effacement in the HT and NT groups (HT, 104 filtration slits/100 mu glomerular basement membrane; NT, 112 mu; sham, 143 mu; P < 0.005 by analysis of variance), but GEC separation from the glomerular basement membrane was not significant in either HT or NT rats. GEC function was determined from protamine-heparin aggregate disappearance curves, and the curves, representing GEC endocytosis, were not different in either HT or NT groups compared with the sham-operated groups. These findings suggest that GEC function is preserved in RK, and the changes in glomerular size and GEC morphology are nonlethal and adaptive. The morphological appearance of the acute glomerular and vascular lesions and their presence only in HT animals is consistent with a hypertensive pathogenesis. The glomerular sclerosis seen in both HT and NT may result from either resolution of acute lesions with scarring and/or adaptive changes in glomerular structure and cellular functions other than the GEC clearance function we studied.     

The mechanism of action of corticosteroids on glomerular injury in acute serum sickness in rabbits.

Macrophages have recently been identified as the predominant mediators of the glomerular injury in acute serum sickness (AcSS) in rabbits. Corticosteroids have been shown to prevent this lesion, but the mechanism of this effect is unknown. As corticosteroids are potent anti-macrophage agents, the effect of prednisolone treatment (2 mg/kg/day) on glomerular macrophage accumulation and injury was assessed in rabbits developing AcSS. Eleven untreated animals all developed a proliferative endocapillary glomerulonephritis (mean 71.7 +/- 1.9 sem cells per glomerular cross section, c/gcs) with glomerular macrophage accumulation (46.3 +/- 5.7 macrophages per glomerulus, macs/glom) and proteinuria (555 +/- 379 mg/24 h). Eight animals were treated with prednisolone commencing not more than 48 h prior to immune elimination (IE). Glomerular injury was markedly attenuated with significantly less cellular proliferation (49.1 +/- 2.1 c/gcs, P less than .005), fewer macrophages within glomeruli (10.5 +/- 7.7 macs/glom, P less than .005) and minimal proteinuria (19.3 +/- 5.5 mg/24 h, P less than 0.01). Treatment did not alter the amount of circulating BSA-anti-BSA immune complex; its time of IE (11.1 +/- 0.4 days treated, 11.4 +/- 0.4 days untreated) its renal deposition (2.36 +/- 0.64 micrograms BSA/g renal cortex treated, 2.66 +/- 0.52 mg BSA/g renal cortex untreated) or its glomerular localization. These results indicate that prednisolone treatment can effectively reduce the glomerular injury of AcSS. This effect is not dependent on any alteration of immune complex formation or deposition, but involves reduction of macrophage accumulation at the inflammatory site.     

Glomerular injury and proteinuria in rats after intrarenal injection of cobra venom factor. Evidence for the role of neutrophil-derived oxygen free radicals.

The purpose of these studies was to determine how intravascular complement activation could lead to glomerular injury. Cobra venom factor (CVF) infused into the renal artery of rats resulted in increased excretion of protein in urine, which was maximal over the first 24 hours (51.2 +/- 6.0 mg/24 hours in CVF versus 14.1 +/- 0.9 mg/24 hours in saline-treated animals; P less than 0.001). Depletion of circulating neutrophils with anti-neutrophil serum significantly reduced the CVF-induced proteinuria in the first 24 hours (neutrophil depleted rats 22.7 +/- 2.8 mg/24 hours versus 63.4 +/- 9.9 mg/24 hours in neutrophil intact rats; P less than 0.005). Morphologic abnormalities (which were quantitated morphometrically) included accumulation of neutrophils in glomerular capillary loops, blebbing of endothelial cells, and epithelial cell foot process fusion. The increased protein excretion was reduced by 70% by simultaneous administration of catalase (23 +/- 4.3 mg/24 hours in CVF plus catalase versus 52.1 +/- 10 mg/24 hours in CVF alone; P less than 0.05). Catalase reduced glomerular endothelial cell blebbing and epithelial cell foot process fusion but not neutrophil accumulation in glomeruli as assessed by morphometry. In similar experiments superoxide dismutase, dimethyl sulfoxide, and deferoxamine did not prevent CVF-induced proteinuria. These studies, therefore, suggest that intravascular activation of complement in the rat causes glomerular injury and proteinuria which is dependent on neutrophils and upon the generation of hydrogen peroxide and/or its metabolites.     

Changes in glomerular heparan sulfate in puromycin aminonucleoside nephrosis.

Changes in glomerular anionic sites in puromycin aminonucleoside nephrosis (PAN) in the rat are controversial. The authors examined glomerular anionic sites in PAN by in vivo staining with polyethyleneimine (PEI). They also quantitated and characterized glomerular heparan sulfate (HS), which is known to be a major glomerular polyanion in PAN, using in vivo incorporation of 35S-sulfate. PAN rats had a mean protein excretion of 96 +/- 23 mg per 24 hours. Staining of anionic sites with PEI showed 15.3 +/- 2.8 sites per 1000-nm length of glomerular basement membrane in controls, 13.7 +/- 1.9 sites in PAN rats (P greater than 0.05), and 50% of rats with early PAN had absent staining. Total 35S-sulfate incorporation was similar in both the controls and established PAN rats (2900 +/- 150 dpm/mg dry wt of glomeruli versus 3005 +/- 260, P greater than 0.05) but decreased in early PAN rats (2025 +/- 148). The percentage of 35S-sulfate incorporated into chondroitin sulfate was similar in all three groups of animals. HS uronic acid was also similar (1.8 +/- 0.2 g/mg dry wt of glomeruli versus 1.7 +/- 0.3, P greater than 0.05) but decreased in early PAN (1.1 +/- 0.2). The distribution of 35S-sulfate activity within the HS subfractions was examined by ion-exchange chromatography and showed a shift in percent present from 1.0 M to 1.25 M fraction in established and early PAN animals (control 1.0 M 37% +/- 3.2% versus PAN 19% +/- 3.4%, P less than 0.01, and 1.25 M 36% +/- 2.9% versus 53% +/- 2.9%, P less than 0.01). These results demonstrate that glomerular heparan sulfate is unchanged in established PAN but decreased in early PAN. SO4 incorporation is unchanged in established PAN and diminished in early PAN. Thus, early in PAN HS synthesis is impaired, but in established PAN the HS is normal, and changes in glomerular HS cannot explain the increased permeability.     

Induction of glomerulosclerosis by dietary lipids. A functional and morphologic study in the rat

Clinical and experimental data indicate that glomerular function and morphology may be influenced by plasma lipids. In familial lecithin-cholesterol-acyltransferase (LCAT) deficiency and in Fabry's disease, lipids accumulate in glomeruli and are assumed to induce sclerosis. The present study was undertaken to examine if dietary lipids could exert effects on the glomeruli of normal, unilaterally nephrectomized rats, and of rats with two-kidney, one clip (2-K,1C) hypertension. In rats with two kidneys on a diet rich in fat and cholesterol, cholesterol concentrations in very low density lipoproteins increased. In these rats the number of glomeruli with sclerotic foci was significantly higher than in rats on a low fat, cholesterol free diet. After 6 months on the diet the percentage of glomeruli with sclerosis (SC) was 13.2 +/- 4.1 (N = 9) in rats with a cholesterol diet and 1.8 +/- 0.6 (N = 11) in control rats (p less than 0.05). The fat and cholesterol diet exacerbated glomerular lesions in the remnant kidney model of uninephrectomized rats. The sclerosis in rats with only one kidney was 38.2 +/- 9.5 (N = 6) on a cholesterol diet compared with 8.7 +/- 3.0 (N = 6) in control rats after 6 months (p less than 0.05). After 3 to 4 months on a fat rich diet cholesterylester was increased in isolated glomeruli. The composition of the dietary lipids influenced the development of glomerular lesions. A linseed oil diet that is rich in unsaturated fatty acids, especially linolenic acid, did not cause major plasma lipid abnormalities and was accompanied by a low sclerosis (1.2 +/- 0.3; N = 9) for rats with two kidneys. In rats with chronic 2-K, 1C hypertension the percentage of glomeruli with partially sclerosed tufts in the unclipped kidney was significantly higher on a fat and cholesterol diet (F) than on a control diet (N) (SC: diet F 31.0 +/- 4.0, N = 13; diet N 12.2 +/- 2.6, N = 12; P less than 0.05). In the clipped kidney, protected against the arterial hypertension, only an increased number of glomeruli with mesangial expansion was noted in rats with the cholesterol diet. Glomerular hemodynamic factors seem to play an important pathogenetic role in the induction of glomerular sclerosis by a lipid rich diet. The fact that dietary lipids can aggravate glomerular lesions in states of arterial hypertension and nephron loss may have implications for the progression of renal disease in humans.     

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis

The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.     

Quantitation of the intrarenal uptake of immunoglobulin aggregates by macrophages in diffuse proliferative glomerulonephritis.

The intrarenal processing of circulating immune-complex-like-material is traditionally attributed to resident glomerular mesangial cells. To assess the contribution of infiltrating mononuclear cells to macromolecule processing by diseased glomeruli we have utilized an isolated perfused kidney system (IPK) to quantify the specific glomerular uptake of heat-aggregated immunoglobulin (AIgG) (as micrograms of aggregated IgG per 10(4) glomeruli (microgram 10(4)glom)). Mononuclear cell infiltrated glomeruli from animals with diffuse proliferative glomerulonephritis had a significantly augmented uptake of AIgG (48.9 +/- 3.8 micrograms/10(4)glom; normal 11.8 +/- 0.6 micrograms/10(4)glom; P less than 0.01). Specific blockade of mononuclear cell function by perfusion with anti-macrophage-serum (AMS) prevented increased AIgG uptake (12.8 +/- 1.2 micrograms/10(4)gloms; P less than 0.01), but had no effect on the AIgG uptake of normal kidneys (13.1 +/- 1.2 micrograms/10(4)glom). Thus, in diffuse proliferative glomerulonephritis the observed increase in the glomerular clearance of AIgG was due to phagocytosis by mononuclear cells. This study suggests that infiltrating, bone-marrow derived mononuclear cells may significantly contribute to the glomerular handling of circulating immune complexes by nephritic glomeruli.     

Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells : role of cyclooxygenase-1 and -2

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG) E(2) in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A(2) was associated with a marked stimulation of PGE(2) production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE(2) production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury.     

Role of the terminal complement pathway in accelerated autologous anti-glomerular basement membrane nephritis.

The terminal complement pathway (C5b to C9) has been demonstrated to have an important role in the mediation of glomerular immune injury in various models of experimental glomerulonephritis. In the present studies, the role of the terminal complement pathway in the accelerated autologous phase of anti-glomerular basement membrane (GBM) nephritis in the rabbit was investigated. Normocomplementemic rabbits and rabbits deficient in C6 (C6D) who are therefore unable to form the terminal complement pathway were immunized with sheep immunoglobulin G (IgG) before being injected with a subnephrotoxic dose of the gamma 2 fraction of sheep anti-rabbit GBM. C6D animals had a delay in the onset of the glomerular injury, as manifested by proteinuria. At 72 hours, controls had a greater degree of proteinuria (15.2 +/- 8.8 mg protein/mg creatinine vs. 2.6 +/- 2.1, P = 0.197), but at 120 hours there were no differences in proteinuria between C6D and control animals (11.1 +/- 3.6 mg protein/mg creatinine vs. 12.2 +/- 6.2, P = 0.89). Light microscopy demonstrated more severe glomerular injury in C6D animals with marked cellular proliferation and large areas of glomerular necrosis. Interestingly, C6D animals had significantly higher levels of sheep IgG remaining in their glomeruli at 120 hours (0.95 +/- 0.12 micrograms sheep IgG/1 x 10(4) glomeruli, N = 11, vs. 0.57 +/- 0.07, N = 11, P = 0.014) and 72 hours (1.22 +/- 0.25 micrograms, N = 3, vs. 0.60 +/- 0.15, N = 3, P = 0.104) compared with 24 hours when there was no difference (1.25 +/- 0.22 micrograms, N = 7, vs. 1.08 +/- 0.14, N = 7, P = 0.53). C6D rabbits had a greater rise in serum creatinine at 120 hours (2.3 +/- 0.5 mg/dl vs. 1.3 +/- 6.4, P = 0.132). We conclude that in C6D animals, the persistence of glomerular immune deposits is responsible for more severe renal injury and renal failure.     

Glomerular tissue factor expression in crescentic glomerulonephritis. Correlations between antigen, activity, and mRNA.

Correlations between glomerular expression of tissue factor (TF) activity and antigen and cellular localization of TF mRNA was studied in crescentic glomerulonephritis (GN) in rabbits. Glomerular TF activity increased 8.7-fold 24 hours after initiation of GN (234 +/- 49 mU/10(3) glomeruli; normal, 27 +/- 10 mU/10(3) glomeruli; P = 0.003) in association with a 2.1-fold increase in TF antigen (154 +/- 34 ng/10(3) glomeruli; normal, 72 +/- 10 ng/10(3) glomeruli; P = 0.055), early macrophage infiltration, and no significant increase in TF mRNA. At the peak glomerular macrophage infiltration (day 4), TF activity remained augmented (230 +/- 63 mU/10(3) glomeruli) and TF mRNA, colocalized within macrophages, was significantly increased compared with normal (267 +/- 42%; P = 0.001). TF antigen was not increased in glomeruli (114 +/- 17 ng/10(3) glomeruli), although significant urinary excretion of TF antigen was detectable (478 +/- 121 ng/24 hours; normal, < 1 ng/24 hours; P = 0.032). At this time, the M(r) of glomerular TF (49 to 61 kd) was increased compared with TF in normal glomeruli (49 to 58 kd) as a result of increased glycosylation. At day 7, TF activity and antigen within glomeruli had decreased, although urinary excretion of TF antigen and glomerular TF mRNA remained elevated. These studies suggest that early up-regulation of TF activity is largely a result of functional up-regulation of constitutive TF in intrinsic glomerular cells. In more advanced disease, infiltrating macrophages are the major site of TF synthesis. The increased M(r) of glomerular TF, as a result of synthesis of more highly glycosylated protein by macrophages and the shedding of TF into the urine, suggests that substantial turnover of glomerular TF occurs at this stage.     

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage-dependent glomerulonephritis.

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(s)-15-methyl PGE1 (M-PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage-dependent glomerular injury. M-PGE1 ameliorated proteinuria (day 4; 61 +/- 13 mg/24 h, n = 9; vehicle treated, 164 +/- 17 mg/24 h, n = 11; P less than 0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44 +/- 9/glomerulus; vehicle treated, 119 +/- 15/glomerulus; P less than 0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8 +/- 5%; vehicle treated 25 +/- 4% P less than 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M-PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M-PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6 +/- 1.5/glomerulus; vehicle treated, 13.2 +/- 1.3/glomerulus) or alter Ia antigen expression (4.1 +/- 0.2 Ia + cells/glomerulus; vehicle treated, 3.9 +/- 0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage-mediated inflammation.     

Glomerular volume in type 2 (noninsulin-dependent) diabetes estimated by a direct and unbiased stereologic method

Glomerular volume was estimated in 20 type 2 diabetic patients (age 64 +/- 6 years, duration of diabetes 6 +/- 5 years) compared with 14 sex- and age-matched controls, as well as in a group of 11 very long-term type 1 diabetic patients (age 61 +/- 12 years, duration of diabetes 44 +/- 11 years). One whole autopsy kidney was obtained prospectively, and a known fraction (approximately equal to 1/140) was sampled systematically and embedded in plastic (JB-4 glycolmetacrylate), thereby essentially eliminating shrinkage. Sections 15-microns thick were stained with periodic acid-Schiff. Mean glomerular volume was estimated on a random sample of glomeruli using the disector method. Frequency of glomerular occlusion and mean volume of open glomeruli was estimated. Mean glomerular volume was not different between type 2 diabetic patients and controls (5.3 +/- 1.7 M mu3/1.73 m2 versus 5.3 +/- 1.9 M mu3/1.73 m2) nor was total glomerular volume or kidney weight. Frequency of glomerular occlusion was 4.8 +/- 5.7% in controls, 8.9 +/- 7.8% (p = 0.10) in type 2 patients, and 16.8% +/- 20.7 (p less than 0.05) in type 1 patients. In type 2 patients there was a correlation between frequency of glomerular occlusion and mean volume of open glomeruli (r = 0.44, p = 0.05), and the same tendency was seen in type 1 patients (r = 0.49, p = 0.12). By the present method the absolute level of glomerular volume was increased by at least a factor of two compared with previous studies. This illustrates the problems arising from shrinkage of tissue in paraffin and stresses the importance of using an unbiased stereological method. The lack of increase in total glomerular volume is in accordance with clinical findings of lack of glomerular hyperfiltration in type 2 patients, findings in contrast to those in type 1 diabetes. It is suggested that hyperfiltration per se is not the cause of glomerulopathy.     

Glomerular nitrite synthesis in in situ immune complex glomerulonephritis in the rat.

Nitrite (NO2-) is the major end product of nitric oxide (NO) production in cell culture. The authors have examined nitrite production by glomeruli in in situ immune complex glomerulonephritis in the rat. Glomerulonephritis was induced by unilateral renal perfusion of cationized human gamma G immunoglobulin (IgG) in preimmunized rats. NO2- was measured in culture supernatants of isolated glomeruli after 48 hours. NO2- was produced by nephritic glomeruli with a maximum 4 days after induction of glomerulonephritis (24.4 +/- 11.4 pmol/glomerulus/48 hours). Production was increased by lipopolysaccharide (LPS; 1 micrograms/ml) (54 +/- 4.9 pmol/glomerulus; P less than 0.001). NO2- production was inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine demonstrating synthesis through NO. Dexamethasone (10(-7) mol/l [molar]) reduced LPS-stimulated production by peritoneal macrophages and nephritic glomeruli (P less than 0.01). Macrophages isolated from nephritic glomeruli produced NO2- (4.9 +/- 0.6 nmols/10(5) cells). The production of NO by nephritic glomeruli has implications for mechanisms of glomerular injury and glomerular hemodynamics. The effect of dexamethasone may explain in part the ameliorative effect of steroids in glomerulonephritis.     

Enhanced glomerular procoagulant activity and fibrin deposition in rats with mercuric chloride-induced autoimmune nephritis

The mechanism involved in glomerular fibrin deposition was investigated during mercuric chloride (HgCl2)-induced autoimmune glomerulonephritis in the Brown Norway rat. To ascertain whether the local hemostatic system was activated secondarily to the immunological conflict, the ability of glomerular lysates to induce coagulation in vitro was assessed in treated and control rats. Glomerular procoagulant activity (PCA) of HgCl2-injected rats was measured on day 12 (latent phase of the disease), day 20 (acme), and days 32 and 42 (recovery phase) after the first mercury injection. PCA rose 3-fold (p less than 0.02) at day 20 and then almost returned to control values. Proteinuria, PCA, and the incidence of glomerular fibrin deposits peaked concomitantly at day 20. Glomerular PCA was characterized as thromboplastin. The number of Ia positive cells detected by monoclonal OX-6 antibody was not different from the control number at any phase of the disease; the number of macrophages per glomerular section detected by electron microscopy at day 20 in HgCl2-injected rats was 1.80 +/- 0.60, versus 0.30 +/- 0.11 in the controls. No correlation was found between glomerular PCA and either the number of monocytes/macrophages or of Ia-positive cells present in the glomeruli. Since glomerular PCA was maximal at the onset of fibrin formation in the glomeruli and then decreased toward its basal level, and since the fibrin disappeared, it is concluded that increased production of thromboplastin by glomeruli, with activation of the extrinsic coagulation pathway, may contribute to intraglomerular fibrin deposition in HgCl2-induced glomerulonephritis.     

Glomerular epithelial cells synthesize endothelin peptides.

Glomerular mesangial and endothelial cells have been reported to synthesize and secrete endothelin-1 (ET-1). Whether glomerular epithelial cells (GEC) have the ability to synthesize ET-peptides is not known. Employing immunocytochemistry we report that the GEC in vitro constitutively express ET-1 and ET-3. ET-1 synthesis by the GEC was further confirmed by detection of a specific 2.3kb mRNA that hybridizes with rat prepro ET-1 genomic DNA on Northern blot analysis. ET-1 is secreted into the medium in a time-dependent manner as measured by radioimmunoassay and radiobinding assay. Synthesis of endothelin peptides by the GEC may have important implications in the pathogenesis of glomerular diseases where GEC injury figures prominently.     

Markers of complement-dependent and complement-independent glomerular visceral epithelial cell injury in vivo. Expression of antiadhesive proteins and cytoskeletal changes.

BACKGROUND: Visceral glomerular epithelial cells (GEC) are an important component of the glomerular filtration barrier to proteins. While ultrastructural GEC changes have frequently been observed in proteinuric states, no suitable light microscopic markers of GEC injury have yet been identified. EXPERIMENTAL DESIGN: We have analyzed in vivo the GEC expression of proteins known to be involved in cell shape changes. SPARC (osteonectin, BM-40) and tenascin (cytotactin, J1, hexabrachion) belong to a group of anti-adhesive glycoproteins, that modulate cell-matrix interactions. We also studied cytoskeletal intermediate filament proteins, including desmin and vimentin. The GEC expression of SPARC, tenascin, desmin, and vimentin was analyzed in various types of GEC injury in the rat, including complement-mediated injury (passive Heymann nephritis, autologous immune complex nephritis, conA anti-conA nephritis), complement-independent injury (nephrotoxic nephritis), toxic injury (aminonucleoside nephrosis) and hypertensive injury (5/6 nephrectomy, angiotensin-II infusion). A complement-mediated model of mesangial cell injury (anti-Thy 1.1 mesangial proliferative nephritis) served as a control. RESULTS: SPARC mRNA and protein were constitutively expressed in normal rat glomeruli. Immunostaining and immunoelectron microscopy primarily localized SPARC to the cytoplasm of GEC. Markedly increased glomerular SPARC synthesis and GEC immunostaining was observed in all instances of complement-mediated GEC injury but in none of the other conditions. In contrast, glomerular immunostaining for tenascin, that also stained in a GEC pattern, either remained unchanged or increased to a minor degree (complement-mediated models). GEC immunostaining for desmin in normal rats was low and variable, and increased significantly in any form of GEC injury but not in anti-Thy 1.1 nephritis. No concomitant increase of GEC immunostaining for vimentin was detectable, which could have been due to the constitutively high expression of vimentin in GEC. CONCLUSIONS: SPARC and desmin, but not tenascin or vimentin, are suitable light microscopic markers of GEC injury. The combined staining for these proteins may be useful in differentiating the mechanisms of GEC injury.     

Glomerular tumor necrosis factor and interleukin 1 during acute aminonucleoside nephrosis. An immunohistochemical study

Acute aminonucleoside nephrosis progresses to glomerulosclerosis. The mechanisms for this phenomenon are not entirely known. Our objectives were to identify macrophage (m phi)-derived peptide growth factors (i.e., tumor necrosis factor and interleukin 1), using immunohistochemical means, in glomeruli of rats with acute aminonucleoside nephrosis. Recently, a role for glomerular m phi s has been suggested as one of the possible mechanisms responsible for this transition from acute glomerular injury to glomerulosclerosis. Since peptide growth factors are elaborated by m phi s and produce alterations in mesangial cell proliferation and protein biosynthesis, we investigated whether these cytokines were present in glomeruli during aminonucleoside nephrosis, which has been typically regarded as a nonimmune toxic glomerulopathy. Fourteen days after puromycin aminonucleoside (PA) delivery, nephrotic control rats (PA/control) and nephrotic animals that had been maintained on an essential fatty acid-deficient (EFAD) diet (PA/EFAD) for 8 weeks before PA, manifested cytoplasmic tumor necrosis factor and interleukin 1 within cells located in the glomerular mesangium as detected by immunohistochemical means. Despite equivalent levels of albuminuria and fasting total cholesterol during peak nephrosis, the PA/EFAD rats had significant reductions in the number of tumor necrosis factor-positive glomerular cells (1.8 +/- 0.1 versus 8.5 +/- 0.4, p less than .001) and interleukin 1-positive glomerular cells (1.5 +/- 0.1 versus 7.2 +/- 0.5, p less than .001) in comparison with the PA/control group. These data correlated with a reduction in the number of ED-1-positive cells (i.e. glomerular m phi s) in glomeruli of PA/EFAD animals as compared with PA/control rats (2.2 +/- 0.3 versus 10.9 +/- 1.4, p less than .001), suggesting that m phi-derived peptide growth factors may be important determinants in initiating a pathobiologic sequence culminating in glomerulosclerosis in this model.     

The glomerular changes in children with reflux nephropathy

While heavy proteinuria and focal segmental glomerulosclerosis (FSGS) are well-recognized features of progressive reflux nephropathy in adults, little is known of their early evolution. We have studied the glomerular changes in renal biopsy specimens obtained from 24 patients aged 5.2-18.8 years, in whom urinary protein excretion was measured as early morning urine protein creatinine ratios, using the Coomassie blue dye-binding method. Segmental sclerotic lesions were found in eight biopsies and traced through serial sections to a hilar origin in every instance. There was a strong positive correlation between the extent of glomerular involvement and the amount of proteinuria (P less than 0.0001). Parahilar hyaline deposits were observed in 16 biopsies, including five of the eight showing FSGS. All unsclerosed glomeruli were enlarged, and the hilar arterioles showed both enlargement and thickening, their walls frequently containing subendothelial hyaline deposits. Since in most patients renal function was comparatively well preserved, despite extensive loss of renal substances, we believe that these glomerular and vascular changes represent the stages in the evolution of hyperfiltration. Microproteinuria is the earliest clinical manifestation of FSGS, and should be sought routinely in all patients with reflux nephropathy.     

Heparin alters epidermal growth factor metabolism in cultured rat glomerular epithelial cells.

Extracellular matrix plays an important role in regulating cell growth. The authors have previously shown that heparin and heparan sulfate inhibit glomerular visceral epithelial cell (GEC) growth and that epidermal growth factor (EGF) can partially reverse the effect of heparin. The authors studied EGF processing by GEC in an attempt to clarify the mechanism by which heparin inhibits GEC growth. Control and heparin-treated GEC rapidly internalized 125I-EGF (within 15 minutes). In heparin-treated cells, 125I-EGF reappeared on the cell surface during the course of a 1-hour incubation and the percent internalized dropped significantly to 59.0% +/- 8.6%, suggesting recycling of 125I-EGF-occupied receptors. After incubation with 125I-EGF, heparin-treated cells also released significantly more cpm of 125I into EGF-free medium (2526 +/- 68 cpm-H; 903 +/- 32-C). Analysis of the released 125I by gel filtration chromatography showed more totally degraded 125I-EGF in media from heparin-treated cells (30.8% +/- 1.6% in heparin-treated versus 17.8% +/- 3.2 in control; P less than 0.05). Analysis of EGF-induced dimerization of receptors showed no effect of heparin on this ratio. These studies suggest that heparin decreases GEC response to EGF by accelerating its uptake and degradation. Matrix alterations in disease states may thus play a role in altering cell responsiveness to growth factors.