The glomerular mesangium: I. Kinetic studies of macromolecular uptake in normal and nephrotic rats

This study was designed to define quantitatively the function of the rat glomerular mesangium in the uptake and processing of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, AHIgG-(125)I), to relate this function to that of the general reticuloendothelial system, and to examine the effects of increased glomerular permeability to protein on the mesangial cell system.Mesangial localization of human IgG as demonstrated by immunofluorescent microscopy showed good correlation with concentrations of AHIgG-(125)I in preparations of isolated glomeruli. In normal rats the concentrations of AHIgG-(125)I in glomeruli were similar to those of lung, liver, and spleen and demonstrated a rapid decrease with increasing time intervals after aggregate administration.In rats given aminonucleoside of puromycin a marked increase in mesangial uptake of aggregates was found while studies of nephrotic lungs, liver, spleen, and blood showed no such differences. Glomerular levels of AHIgG-(125)I in aminonucleoside animals could not be correlated with the quantity of proteinuria.Nephrotic and control animals given unaggregated human IgG showed little glomerular localization by immunofluorescent microscopy; no difference in the concentration of this protein in nephrotic as compared to control glomerular isolates was found.Thus, the mesangium in normal animals functions in a manner analogous to that of the general reticuloendothelial system. In nephrotic rats the mesangial uptake of macromolecules is makedly increased, a finding not observed in other tissues.     

Platelets and neutrophils are critical to the enhanced glomerular arachidonate metabolism in acute nephrotoxic nephritis in rats.

Nephrotoxic nephritis (NTN) is characterized by a marked increase in glomerular eicosanoid synthesis, which appears to play an important role in the pathophysiology of this disease model. In this study, we investigated the biochemical and cellular basis of this metabolic change. By examining the enzymatic conversion of exogenous substrates by intact glomeruli, we found that cyclooxygenase, TX synthase, and 5-lipoxygenase activities increased 4-, 8-, and 100-fold, respectively, in acute NTN. PGH2-PGE2 isomerase and leukotriene A4 hydrolase activities did not change. The cellular basis of these changes was examined using dissociated glomerular cells in vitro and by depleting platelets in vivo. Dissociated glomerular cells from nephritic glomeruli (largely mesangial cells and leukocytes) exhibited an enhanced arachidonate metabolism similar to intact nephritic glomeruli. Depletion of neutrophils (PMNs) from these cell preparations by 90% commensurately decreased 5-lipoxygenase and cyclooxygenase activity but had little effect on TX synthase activity. The recovered PMN fraction, however, did exhibit TX synthase activity. Immunocytochemical analysis of dissociated cells using an antiplatelet antibody demonstrated the presence of platelets, both adherent to cells and noncell associated. Depletion of platelets in vivo using this antibody substantially attenuated the increase in glomerular eicosanoid synthesis that accompanied NTN. Platelet depletion also decreased the influx of PMNs into the glomerulus by 50%. These data show that PMNs and platelets colocalize to the glomerulus in acute NTN and are coordinately essential to the increase in glomerular arachidonate metabolism.     

Essential role of Gas6 for glomerular injury in nephrotoxic nephritis

Growth-arrest specific gene 6 (Gas6) is a vitamin K-dependent growth factor for mesangial and epithelial cells. To investigate whether Gas6 is essential for progressive glomerular injury, we constructed Gas6(-/-) mice and examined the role of Gas6 in accelerated nephrotoxic nephritis (NTN), a model of progressive glomerulonephritis. We found less mortality and proteinuria in Gas6(-/-) mice than in wild-type mice following injection of nephrotoxic serum. Glomerular cell proliferation, glomerular sclerosis, crescent formation, and deposition of fibrin/fibrinogen in glomeruli were also reduced in Gas6(-/-) mice. Furthermore, administering Gas6(-/-) mice recombinant wild-type Gas6, but not Gas6 lacking a previously characterized N-terminal gamma-carboxyl group, induced massive proteinuria, glomerular cell proliferation, and glomerulosclerosis, comparable to responses seen in wild-type mice. These data indicate that Gas6 induces glomerular cell proliferation in NTN and suggest that this factor contributes to glomerular injury and the progression of chronic nephritis.     

The influence of selective thrombocytopenia on nephrotoxic nephritis.

Anticoagulation with agents that interfere with fibrin formation inhibit the development of the autologous phase of nephrotoxic nephritis (NTN). Platelet participation in the nephritic process has been suggested but not proved, therefore, the influence of selective thrombocytopenia on the autologous phase in rabbits was evaluated. NTN was produced with goat antirabbit glomerular basement membrane antiserum. Thrombocytopenia was induced with goat antirabbit platelet antiserum 24 hours prior to the onset of nephritis. Platelet accumulation within the nephritic kidney was quantitated using chromium labeled platelets. Thrombocytopenia has no inhibitory effect on the development of the autologous phase of NTN in rabbits. There was no platelet accumulation within the nephritic kidney in the presence of thrombocytopenia. Pharmacologic inhibition of platelet aggregation may be of no benefit in glomerulonephritis produced by a fixed antigen-antibody reaction within the glomerular capillary wall.     

Effects of complement depletion on glomerular eicosanoid production and renal hemodynamics in rat nephrotoxic serum nephritis

The role of complement in mediating the changes in renal hemodynamics and glomerular eicosanoid synthesis after the administration of heterologous antibody against rat glomerular basement membrane (AGBM) was studied in Munich-Wistar rats. AGBM serum decreased glomerular filtration rate (GFR) and increased glomerular thomboxane B2 (TxB2) production without associated changes in glomerular prostaglandin E2 (PGE2) or PGF2 alpha production. Pretreatment of rats with cobra venom factor to deplete complement blocked the fall in GFR produced by AGBM without altering the increment in glomerular TxB2 production. In these animals, glomerular PGE2 synthesis was elevated. The results indicate that the salutary effects of complement depletion in nephrotoxic serum nephritis are not mediated by changes in the glomerular production of the vasoconstrictor TxA2. An enhanced production of PGE2 may participate in preventing the fall in GFR after AGBM administration in the complement-depleted rats.     

肾毒性血清肾炎大鼠心肌组织形态学改变的观察

目的：观察肾毒性血清肾炎（ＮＳＮ）大鼠心肌的组织形态学改变。方法：用兔抗鼠肾毒性血清静脉注射制作ＮＳＮ模型，取ＮＳＮ大鼠心肌组织作光镜、电镜和免疫组化ＩｇＧ染色检查。结果：心肌细胞肌膜和闰盘正常，肌浆网及线粒体结构清晰，肌小节内粗、细肌丝排列正常，细胞核核膜正常，核形态规则；心肌间质水肿，毛细血管扩张、充血，毛细血管管壁有兔ＩｇＧ沉积；心肌间质有散在的炎性细胞浸润。结论：ＮＳＮ可引起间质性心肌炎。     

Effect of antimacrophage serum on the proliferation of glomerular cells in nephrotoxic serum nephritis in the rat

To evaluate the effect of a rabbit anti-rat macrophage serum (AMS) on glomerular cells in vivo, glomeruli were isolated from an accelerated autologous form of nephrotoxic serum nephritis (NTSN) in rats and grown in tissue culture. The prominent feature of the glomerular outgrowth of the glomeruli in the NTSN was the presence of large numbers of type III (macrophages) cells, which were not present in cultured normal glomeruli. In addition, there were significantly greater numbers of type II (mesangial) cells in culture from the NTSN rats as compared with glomeruli from normal rats though the numbers of type I (epithelial) cells were the same. The administration of AMS prevented the outgrowth of macrophages and reduced the number of mesangial cells in the outgrowth of glomeruli from the NTSN rats. The AMS-treated rats showed profound reduction in proteinuria. Light micrographs showed only minor histologic lesion in the AMS-treated rats. These findings suggest that AMS may be applicable to the modulation of the proliferative response seen in NTSN.     

Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.

Requirements for leukocyte adhesion molecules as well as cytokines have been determined in the rat model of acute nephrotoxic nephritis. Proteinuria (at 24 h) and neutrophil accumulation in renal glomeruli (at 6 h) have been used as the endpoints. For full accumulation in glomeruli of neutrophils as well as full development of proteinuria, requirements have been demonstrated for TNF alpha, (but not IL-1), CD11b (but not CD11a), very late arising-4 (CD49d/CD29), and intercellular adhesion molecule-1 but not endothelial leukocyte adhesion molecule-1 (E-selectin). By immunohistochemical approaches, infusion of antibody to glomerular basement membrane induced glomerular upregulation of intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular adhesion molecule-1. Treatment of rats with anti-TNF alpha or soluble recombinant human TNF receptor-1 blocked this expression. Renal arterial infusion of TNF alpha induced glomerular expression of all three endothelial adhesion molecules, but infusion of IL-1 beta did not. These data suggest that, in neutrophil and complement-dependent anti-glomerular basement membrane-induced acute nephritis in rats, there are selective requirements for cytokines, beta 1 and beta 2 integrins, and endothelial adhesion molecules. These requirements contrast with those found in other vascular beds in which complement and neutrophil-induced vascular injury has been induced by deposition of immune complexes.     

Glomerular arachidonate lipoxygenation in rat nephrotoxic serum nephritis.

Arachidonate lipoxygenation to monohydroxylated eicosatetraenoic acids (HETE) was studied in rat nephrotoxic serum nephritis (NSN). A single infusion of nephrotoxic serum enhanced conversion of [3H]arachidonic acid ([3H]C20:4) to [3H]12-HETE in glomeruli isolated from nephritic rats compared with controls. The percent conversion of [3H]arachidonic acid was 1.95 +/- 0.2% in control glomeruli and 14.2 +/- 2% in nephritic glomeruli 2 d after induction of disease. No significant changes in the conversion of [3H]C20:4 to [3H]5-, 8-, and 9-HETE were noted. Extraction of glomerular HETE by alkaline hydrolysis, to evaluate possible reacylation of HETE after their production, confirmed the presence of 12-HETE and did not provide evidence of 5-HETE synthesis. Increased glomerular 12-HETE synthesis in nephritic rats was also demonstrated by high pressure liquid chromatography-UV detection and by 12-HETE radioimmunoassay. The enhanced glomerular 12-HETE synthesis commenced as early as 3-5 h after administration of nephrotoxic serum and peaked at day 2 with 10-fold enhancement of 12-HETE production. Increments of glomerular 12-HETE persisted on day 7 and returned toward control levels by day 14. Platelet depletion, induced by antiplatelet antisera, did not decrease glomerular 12-HETE synthesis in NSN, thereby eliminating platelets as the cellular origin of 12-HETE. Glomerular epithelial and mesangial cells are the most likely sources of enhanced 12-lipoxygenase activity. The enhanced arachidonate 12-lipoxygenation in glomerular immune injury could have important proinflammatory effects in the evolution of glomerulonephritis since 12-HETE has important effects on leukocyte function.     

Renal effects of synthetic rat-atrial natriuretic peptide in nephron-reduced rats with nephrotoxic nephritis

Following nephron reduction by means of five--sixths nephrectomy (Nx), nephritis was induced in rats by injection of nephrotoxic serum (NTS). A continuous intravenous infusion of synthetic rat--atrial natriuretic peptide (r-ANP) at 2.5 micrograms/min for produced remarkable diuresis and natriuresis in these rats. Furthermore, inulin clearance, para-amino-hippuric acid clearance and filtration fraction increased in spite of the reduction of mean arterial pressure during the infusion of r-ANP. It was therefore evident that diuresis and natriuresis were mediated by a direct action of r-ANP in the kidney. We observed that r-ANP was a diuretic, natriuretic and hypotensive agent but at a higher dosage in Nx rats with NTN.     

Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.     

Progression to renal failure after nephrotoxic nephritis in rats studied by renal transplantation

We studied the relation between immunopathology and progressive renal failure after nephrotoxic nephritis (NTN) in rats. Thirty days after induction of nephritis by injection of rabbit anti-rat nephrotoxic serum, pairs of kidneys from 13 nephritic rats were transplanted into separate syngeneic recipients, one of whom had been pre-immunized with rabbit immunoglobulin G (IgG) whilst the other was naive. Progression to renal failure of the transplanted nephritic kidney was studied after removal of the recipient's own kidneys; results from right and left kidney from a single donor in pre-immunized and naive recipients were compared. There were substantial differences in autologous anti-rabbit IgG titres in naive and preimmunized recipients; despite this pairs of kidneys from the same donor had almost identical courses as assessed by proteinuria, serum creatinine and graft survival. There was substantial variation in survival of kidneys from different donors. But there were very strong correlations of graft survival with proteinuria (r = 0.97, t = 4.443, P less than 0.001) and reciprocal serum creatinine (r = 0.95, t = 4.32, P less than 0.001) in donors shortly before transplantation. We conclude that autologous antibody titres did not influence the progression to renal failure after nephrotoxic nephritis. The rate of progression was already determined at the time of transplantation.     

Selective glomerular thrombosis in rats induced by combined injections of nephrotoxic antiserum and lipopolysaccharide

Experimental glomerular thrombosis was induced in rats by combined injections of nephrotoxic antiserum and lipopolysaccharide. For the development of glomerular thrombosis, administration of nephrotoxic antiserum (greater than or equal to 0.1 ml pooled material) was required as a preparatory agent and greater than or equal to 100 ng lipopolysaccharide as a provoking agent. The severity of renal lesions was not parallel with the amounts of nephrotoxic antiserum and lipopolysaccharide injected. Transient clamping of a unilateral renal artery for 10 to 20 minutes at the time of the nephrotoxic antiserum injection partially prevented the development of glomerular thrombosis in the clamped side. Intervals between the preparatory and provoking injections were found to be -4 to 72 hours for the development of renal lesions. With the preparatory injection of 0.1 to 0.3 ml nephrotoxic antiserum a thrombotic lesion developed exclusively in glomerular capillary walls greater than or equal to 2 hours after the lipopolysaccharide injection. No thrombotic lesion was observed in other tissues such as lung, liver, or intestine, but a generalized Shwartz-manlike phenomenon was observed with the preparatory injection of 0.5 ml nephrotoxic antiserum. When rats were pretreated with nephrotoxic antiserum and 3 hours thereafter transfused with 1 to 3 X 10(8) polymorphonuclear leukocytes, which had been incubated with lipopolysaccharide for 30 minutes in vitro and washed three times with buffered physiologic saline solution, a marked glomerular thrombosis was also induced. The result indicates that lipopolysaccharide plays a role in the development of thrombosis by a direct effect on leukocytes. The development of glomerular thrombosis was prevented in a leukocytopenic state when leukocyte count was less than 600/microliter, but not in thrombocytopenic rats with a platelet count 8.7 to 30 X 10(3)/microliter. Leukocyte count and plasma fibrinogen level decreased, and prothrombin time and activated partial thromboplastin time were prolonged significantly during the pathologic course. Platelet count and FDP did not change significantly. This experimental model has a basic similarity to the generalized Shwartzman reaction, but the lesions develop exclusively in glomeruli.     

Evidence against a role for superoxide ions in the injury of nephrotoxic nephritis in rats

The accelerated model of nephrotoxic serum nephritis (NTSN) was produced in 12 Sprague-Dawley rats. Six of these rats were administered superoxide dismutase (EC 1.15.1.1; SOD) subcutaneously (8 mg/kg) 8-hourly for 4 days. The first dose was given 6 h before the nephrotoxic serum (NTS). The progression of renal disease was monitored by following (i) albumin excretion, (ii) serum creatinine and creatinine clearance and (iii) renal histopathology and immunofluorescence. There was no evidence that SOD influences the course of NTSN. SOD was scarcely excreted by control rats or rats with NTSN.     

Preservation of the glomerular capillary ultrafiltration coefficient during rat nephrotoxic serum nephritis by a specific leukotriene D4 receptor antagonist.

Leukotriene D4, a potent biologically active lipoxygenase derivative of arachidonic acid in activated leukocytes, depresses the glomerular capillary ultrafiltration coefficient (Kf) and contracts mesangial cells in culture. We therefore investigated its potential role in mediating the reduction in nephron filtration rate seen after induction of experimental nephrotoxic serum (NTS)-induced glomerulonephritis in the rat. Micropuncture measurements were performed in euvolemic Munich-Wistar rats 2 h after i.v. administration of 0.8 ml of rabbit serum (group 1, n = 6), 0.8 ml of rabbit anti-rat glomerular basement membrane antibody in the absence (group 2, n = 8), or presence (group 3, n = 7) of the new highly specific LTD4 receptor antagonist SK&F 104353. Quantitation of antibody binding and neutrophil infiltration revealed no differences between groups 2 and 3. Antagonism of endogenous LTD4 actions, however, was associated with prevention of the NTS-induced fall in SNGFR because of the abrogation of the fall in Kf which characterizes this form of experimental glomerulonephritis. Antagonism of endogenous LTD4 had no effect on the NTS-induced increases in pre- and postglomerular arteriolar resistances, and did not affect nephron plasma flow rate or net transcapillary hydraulic pressure difference. The observed highly localized protective action of the LTD4 antagonist on the glomerular capillary points to a possibly major functional role for intraglomerularly released LTD4, likely originating from infiltrating leukocytes, in the pathophysiology of this form of glomerulonephritis.     

Autoimmunity to glomerular antigens in Henoch-Schoenlein nephritis.

1. Henoch-Schoenlein nephritis and IgA nephropathy share clinical and immunological features, but the pathogenesis of neither condition is established. We have recently described IgG autoantibodies to glomerular components in active IgA nephropathy and have now sought evidence for a similar autoimmune component in Henoch-Schoenlein purpura. 2. Sera from 26 patients with Henoch-Schoenlein nephritis and six patients with Henoch-Schoenlein purpura without accompanying nephritis were studied and compared with sera from 20 patients with other forms of glomerulonephritis and 40 normal subjects. E.l.i.s.a.s were developed to detect IgA and IgG binding to the ligand from whole human glomeruli previously described, laminin, DNA, cardiolipin (diphosphatidylglycerol) and a panel of dietary constituents (BSA, alpha-caesin, beta-lactoglobulin, ovalbumin and wheat gliadin). 3. Sera from 16 of the 26 patients with Henoch-Schoenlein nephritis displayed increased IgG binding to the human glomerular extract compared with the normal control group (P < 0.001), whereas IgG binding was not significantly raised in the patients with Henoch-Schoenlein purpura without evidence of renal involvement. IgA binding was not raised compared with control subjects. Serum IgA and IgG binding to other potential autoantigens or antigens present on dietary constituents was not significantly different in patients with Henoch-Schoenlein nephritis or patients with Henoch-Schoenlein purpura without nephritis compared with control subjects. 4. Western blotting of the denatured and reduced glomerular extract revealed binding of IgG, from the sera of patients with active Henoch-Schoenlein nephritis, to glomerular components of M(r) 48,000 and 58,000, similar to the M(r) of the glomerular antigens identified in IgA nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of aspirin and sulindac in rabbit nephrotoxic nephritis

Glomerular damage in nephrotoxic nephritis is mediated by circulating cells (neutrophils, platelets, and macrophages) infiltrating the glomerular tuft and responsible for the inflammatory reactions. It has been suggested that inflammatory cells may participate in glomerular inflammation through the synthesis and release of eicosanoids derived from the metabolism of arachidonic acid. On the other hand, old data and recent evidence indicate that eicosanoids derived from renal arachidonic acid metabolism play an important role in maintaining hemodynamics, especially in disease conditions. We wanted to evaluate the differential role of arachidonic acid metabolites derived from circulating or renal cells on the evolution of nephrotoxic nephritis. We used a nonselective cyclooxygenase inhibitor, aspirin, which blocks eicosanoid synthesis both in circulating cells and in renal tissue, as compared with a selective cyclooxygenase inhibitor, sulindac, which inhibits arachidonic acid metabolism in circulating cells, partially sparing renal cyclooxygenase. Aspirin, at a dosage that almost completely inhibits both circulating cell and renal arachidonate metabolites, worsens the morphologic expression of rabbit nephrotoxic nephritis and negatively influences the clinical course of the disease. Sulindac, at a dose that suppresses circulating platelet cyclooxygenase activity by 90%, but only partially affects renal prostaglandin synthesis, prevents extracapillary proliferation and reduces proteinuria without negative influence on glomerular hemodynamics.     

Dietary fish oil intake: effects on glomerular prostanoid formation, hemodynamics, and proteinuria in nephrotoxic serum nephritis

Dietary fish oil intake improves glomerular pathology and proteinuria in murine models of autoimmune disease. We evaluated glomerular prostanoid formation, glomerular hemodynamics, and proteinuria in rats with nephrotoxic serum nephritis (NSN) to test whether this beneficial effect of marine lipids also applies to other animal models of glomerular immune injury. Rats were fed diets (8 weeks) containing either cod liver oil or sunflower oil. NSN was induced with a rabbit anti-rat glomerular basement membrane antiserum. Antibody injection significantly stimulated glomerular thromboxane B2 (TxB2) formation in animals fed cod liver oil and sunflower oil at 2 hours, 24 hours, and 7 days. TxB2 production in glomeruli of sunflower oil rats, however, was five to seven times higher when compared with that in rats fed cod liver oil. The dietary regimen led to a significant decrease of glomerular TxB2 and prostaglandin E2 formation in the animals receiving cod liver oil when compared with those fed sunflower oil. Induction of NSN resulted in a significant fall of inulin clearance (Cin) and paraaminohippurate clearance at 2 hours, 24 hours, and 7 days in both groups. The decrease in Cin at 2 hours was greater in rats fed cod liver oil when compared with animals receiving sunflower oil (p less than 0.02); it was not different, however, at 24 hours and 7 days. Animals with NSN developed proteinuria. There was no difference in protein excretion between rats fed cod liver oil or those fed sunflower oil (days 2 and 7).(ABSTRACT TRUNCATED AT 250 WORDS)