The glomerular mesangium. II. Studies of macromolecular uptake in nephrotoxic nephritis in rats

These studies were designed to explore the effects of nephrotoxic serum (NTS) in rats on the uptake and processing by the glomerular mesangium of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, [125I]AHIgG). Measurements of [125I]AHIgG levels in preparations of isolated glomeruli correlated well with qualitative estimates of glomerular IgG deposition seen by immunofluorescent microscopy. Rats given 2 ml NTS received 25 mg/100 g body wt [125I]AHIgG 48 h later and were sacrificed at varying time intervals thereafter. NTS-treated animals demonstrated a marked increase in glomerular uptake of [125I]AHIgG as compared with concurrent controls but a normal ability to clear [125I]AHIgG from the mesangium over 72 hr. Animals given 1.0, 0.5, and 0.25 ml NTS had neither proteinuria nor significant light microscopic changes, yet had increased uptake of [125I]AHIgG of 4.4, 3.0, and 2.1 times control values, respectively at 8 h after the infusion of aggregates. Rats given 1 ml NTS and 12.5, 6.25, and 3.125 mg [125I]AHIgG/100 g body wt had increased glomerular uptake at 8 h, but there was, within both NTS and control groups, a disproportionate decrease in uptake at lower [125I]AHIgG doses. Rats given cobra venom factor (CoF) followed by a NTS shown to be complement dependent (proteinuria reduced by prior complement depletion with CoF) had less mesangial [125I]AHIgG uptake than NTS controls but greater uptake compared with normal controls. CoF itself had no effect on mesangial or reticuloendothelial system [125I]AHIgG uptake. These studies demonstrate a striking change in glomerular mesangial activity after fixation of nephrotoxic antibodies to the glomerular basement membrane, even in the absence of proteinuria and suggest that subtle alterations of the filtration surface can influence mesangial function. The effect of NTS on the mesangium is due, in large part, to the glomerular injury and proteinuria which nephrotoxic antibodies produce.     

Mesangial Function in Ureteral Obstruction in the Rat: BLOCKADE OF THE EFFERENT LIMB

The kinetics for mesangial uptake and transport of radiolabeled aggregated human immunoglobulin (Ig)G (AHIgG(125)I) deviated markedly from normal in male Sprague-Dawley rats with ureteral obstruction. Four experimental groups, each containing 25 rats, were used: (a) bilateral ureteral ligation (BUL) with release of one ureter 24 h later; (b) unilateral ureteral ligation with release 24 h later [UUL(R)]; (c) unilateral ureteral ligation without release (unreleased) [UUL(U)]; (d) uremia-control, which consisted of rats with ligated left ureter and a severed right ureter. A similar number of sham-operated rats served as control for each group. AHIgG(125)I (45 mg/100 g body wt) was given intravenously 1 h after release of the ureteral obstruction (25 h after ureteral obstruction or sham surgery). Groups of five control and five experimental animals were sacrificed at 2, 4, 8, 16, and 24 h after injection.At all time intervals, concentrations of AHIgG(125)I in isolated glomeruli from control animals were similar to values obtained from nonobstructed kidneys of UUL(U) and UUL(R) rats: a linear decrease in concentration over a period of 24 h was observed when the logarithm of glomerular AHIgG(125)I concentration was plotted against time. Aberrations in the kinetics were apparent in obstructed kidneys but not in liver, spleen, or blood concentrations of AHIgG(125)I: (a) At 2 h in all obstructed kidneys, glomerular concentration of AHIgG(125)I was markedly reduced. (b) In BUL (released or unreleased), glomerular concentrations of AHIgG(125)I from 4 to 16 h were congruent with 10-fold those in UUL(U) or UUL(R) kidneys. (c) The significant decline in glomerular concentration between 4 and 16 h in control and nonobstructed kidneys was not observed in UUL(R), UUL(U), or BUL (released or unreleased) kidneys; in all obstructed kidneys, a plateau in glomerular concentrations of AHIgG(125)I was observed between 4 and 16 h. (d) After 16 h at a time when the blood level of AHIgG(125)I had decreased to 3% of initial values, there was progressive fall in glomerular AHIgG(125)I. Similar results were obtained in the uremia-control group in rats, which indicated that uremia per se had no measurable effect on mesangial kinetics. These studies demonstrate that ureteral occlusion induces alterations in mesangial uptake (afferent limb) and egress (efferent limb) of macromolecules. Particularly evident is the "blockade" of the efferent limb which is demonstrable at high blood levels of AHIgG(125)I. These alterations in the transit of macromolecules through the mesangium may be mediated in part by the hemodynamic changes that accompany ureteral obstruction.     

Reticuloendothelial system and glomerular deposition of heat-aggregated human IgG

The influence of functional impairment of reticuloendothelial system on glomerular deposition of heat aggregated human IgG (HAIgG) was studied. I-125 HAIgG was injected to mice treated either with dextran-containing 0.9% saline or with the saline alone. The HAIgG clearance rate from the blood decreased in the dextran-treated mice. The chronologically measured HAIgG contents in the liver, spleen and glomeruli increased in the dextran-treated animals. Immunofluorescent technique also demonstrated an increase in HAIgG and host C3 in the glomeruli. The glomerular hypercellularity was not demonstrated morphologically. Assuming that the function of the hepatic Kupffer cells principally affect the clearance rate of the foreign macromolecules from the blood, it is likely that the dextran administration impaired the function of these cells to digest the phagocytosed HAIgG, and that consequently the injected HAIgG stayed longer in the blood. Thus, increased supply of the HAIgG from the blood and/or the dextran-induced reduction of digesting function of the mesangial cell per se might have led to the accumulation of glomerular HAIgG. We suggest, therefore, that even small amount of immune complex may induce immune complex-mediated glomerular lesion, when associated with long lasting functional impairment of the Kupffer cells.     

THE GLOMERULAR MESANGIUM : III. ACUTE IMMUNE MESANGIAL INJURY: A NEW MODEL OF GLOMERULONEPHRITIS

A mechanism of immune glomerular injury is described based on the fixation of antibody (Ab) to an antigen (Ag) that has localized in the glomerular mesangium. Rabbits were given, intravenously (i.v.), aggregated human IgG (AHIgG) or albumin (AHSA) and 10 h later, when the Ag by immunofluorescent microscopy was present in the mesangium, a kidney was removed and transplanted into a normal rabbit. The recipient then received, i.v., rabbit anti-HIgG or anti-HSA. Within minutes of Ab infusion, glomeruli of the donor kidney had polymorphonuclear (PMN) infiltration that over the next few hours became marked and was associated with glomerular cell swelling. At 24 h a decrease in PMN's and early mesangial proliferation was seen. By 3 days there was marked mesangial hypercellularity and increased mesangial matrix. Within minutes after Ab administration rabbit IgG, C3, and fibrin were seen in the glomerular mesangium. There was a fall in complement titer by 1 min after Ab infusion that was due to complement consumption by the donor kidney. Complement then returned to normal levels by 48 h. Significant glomerular injury did not occur (a) in the recipient's own kidney, (b) from Ag administration and transplantation without recipient Ab administration, or (c) from transplantation and Ab administration without prior Ag administration. These studies demonstrated that Ag localized in the glomerular mesangium can react with circulating Ab and complement resulting in severe glomerular injury.     

Ameliorative effects of dietary protein restriction in chronic aminonucleoside nephrosis

Because many investigators have demonstrated the efficacy of dietary protein restriction in various experimental models of glomerular injury that progress to focal and segmental glomerulosclerosis (FSGS), we used this dietary maneuver in chronic aminonucleoside nephrosis. This model of glomerular disease, which uses a single intravenous injection of the puromycin aminonucleoside (PAN), slowly progresses over 18 weeks with mesangial cell proliferation and FSGS as the pathologic hallmarks. We performed renal functional and histopathologic studies in 21 rats with chronic aminonucleoside nephrosis. Group 1 rats (n = 10) were fed a standard rodent diet containing 23.4% protein, whereas group 2 animals (n = 11) were maintained with a 6% protein diet replete with electrolytes, minerals, and vitamins. In those animals subjected to dietary protein restriction, proteinuria was significantly reduced at 14, 28, 84, and 126 days after PAN administration. In rats maintained with the 6% protein diet at 126 days after PAN delivery, there was also a significant reduction in the percent of glomeruli with segmental areas of glomerulosclerosis or hyalinosis and mesangial cell proliferation. We conclude that the renal functional and histologic consequences of chronic aminonucleoside nephrosis can be blunted by dietary protein restriction.     

Effect of alterations in glomerular charge on deposition of cationic and anionic antibodies to fixed glomerular antigens in the rat

Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for 5 days (controls: 68 +/- 21 mg/day; protamine sulfate-treated: 65 +/- 14 mg/day; n = 25, P greater than 0.08). These results demonstrate that treatment to reduce glomerular polyanion does not significantly alter the ratio of cationic to anionic antibodies to fixed glomerular antigens that deposit in the glomerulus, or reduce proteinuria caused by deposition of antibody to a fixed subepithelial antigen.     

Modification of glomerular immune complex deposition in mice by activation of the reticuloendothelial system.

To determine the effect of activation of the reticuloendothelial system on the localization of immune complexes in the kidney, a model of passive serum sickness nephritis in the mouse was used, with activation of the reticuloendothelial system with Corynebacterium parvum. Groups of mice, control and C. parvum-treated animals, were injected with BSA-125I-anti-BSA complexes containing 3 mg 125I-anti-BSA. Blood was obtained at 5 min, at 3 h, and at 12 h, when the animals were killed. Blood concentrations of BSA-125I-anti-BSA complexes were reduced in C. parvum-treated animals compared with controls. This appeared to be mediated by two effects, increased uptake of complexes in the liver and spleen, and enhanced degradation of immune complexes as measured by TCA-soluble radioactivity. In vitro studies using cultures of peritoneal macrophages also showed enhanced uptake of immune complexes. The amount of immune complexes deposited in the glomeruli of C. parvum-treated animals was reduced as determined by quantitation of radiolabeled material bound to isolated gomeruli and by immunofluorescence techniques. The results of the study emphasize the role of the reticuloendothelial system in the modulation of immune complex localization in the kidney and suggest a potential use of stimulants of the reticuloendothelial system in the therapy of immune complex nephritis.     

Proteinuria and structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin G

Antibodies against laminin, which is a defined glycoprotein of basement membranes, were produced in sheep and affinity purified by immunoadsorption on laminin-Sepharose (S alpha L). When injected intravenously into rats, S alpha L rapidly bound in a linear pattern to the glomerular basement membrane (GBM) in the peripheral and mesangial regions of all glomeruli, and, when greater than 0.5 mg S alpha L was injected, to some tubular BM as well. 1-2 h after the injection of conjugates of horseradish peroxidase (HRP) and S alpha L, HRP reaction product was present throughout the full thickness of the GBM and mesangial matrix. [125I]S alpha L binding to the kidney in vivo increased linearly over the dose range of 40-950 micrograms of IgG and accounted for approximately 2% of the injected dose/g kidney. When 4 mg of [125I]S alpha L was injected, 1.5% or 62 micrograms/g kidney was bound. Proteinuria did not develop within 7 wk of injection in rats that received 0.5-1.6 mg of S alpha L. In contrast, all animals that received injections of 4 mg of S alpha L gradually became proteinuric within 3-6 wk. Thickening, reduplication, and flocculent subendothelial deposits were observed in the GBM of these animals. In addition, mononuclear cells adhered to the GBM and infiltrated beneath the endothelium. However, the deposition of rat C3 was infrequently observed, and rat IgG was not seen in the glomeruli of any rat that received S alpha L. 10 wk after injection, much greater amounts of S alpha L appeared within the mesangium than the peripheral GBM. These results demonstrate that the interaction of S alpha L with the GBM, possibly in concert with infiltrating mononuclear cells, gradually altered the structure and permeability characteristics of the glomerulus independent of a host anti-S alpha L humoral response.     

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus.     

The Immunohistopathology of Glomerular Antigens THE GLOMERULAR BASEMENT MEMBRANE, COLLAGEN, AND ACTOMYOSIN ANTIGENS IN NORMAL AND DISEASED KIDNEYS

The immunofluorescent localization of antisera to human glomerular basement membrane (GBM), collagen, and smooth muscle actomyosin was examined in 15 specimens of normal renal tissue and 98 specimens from patients with renal disease. The anti-GBM and anticollagen antisera normally localize to GBM, while antiactomyosin localizes to the mesangium. Diabetic nephropathy revealed a striking expansion of mesangial material reacting with antiactomyosin. In contrast, the expanded mesangium in membranoproliferative glomerulonephritis did not react with antiactomyosin, and the GBM localization of anti-GBM and anticollagen sera was similarly lost. The thickened GBM in diabetes mellitus and membranous nephropathy reacted with anti-GBM and anticollagen, but with accentuation of staining on the inner aspect of the GBM. In proliferative glomerulonephritis there was a moderate increase in the distribution of actomyosin. Glomerular sclerosis and hyalinization in all diseases studied was accompanied by a loss of immunofluorescent staining for all glomerular antigens, including collagen.     

Effect of aminonucleoside nephrosis on immune complex localization in autologous immune complex nephropathy in rats.

The effect of increased capillary permeability on glomerular immune complex localization was studied in rats immunized with proximal tubular antigen (Fx1A) to induce autologous immune complex nephropathy (AICN). AICN rats were made proteinuric by injection or unilateral renal perfusion with aminonucleoside of puromycin (PA) before developing subepithelial complex deposits. Control AICN kidneys developed diffuse granular deposits of IgG and Fx1A on the subepithelial surface of the glomerular basement membrane (GBM) at 3 wk by immunofluorescence and electron microscopy, and deposits increased in subsequent weekly biopsies. In contrast, PA-nephrotic AICN kidneys developed few or no GBM deposits and a significant increase in mesangial localization of IgG and Fx1A during the period of PA-induced proteinuria. These alterations in complex localization were documented both in rats with PA nephrosis and in unilaterally PA-nephrotic kidneys compared with contralateral controls in the same animals, thus excluding any effect of PA on the immunopathogenetic mechanism in AICN as an explanation for these findings. The absence of GBM deposits closely correlated with reduced staining for polyanionic glomerular sialoprotein in proteinuric kidneys, since PA-perfused kidneys studied 2 wk after resolution of proteinuria demonstrated return of normal staining for sialoprotein and development of subepithelial complex deposits similar to those in contralateral control kidneys. These studies demonstrate that properties of the glomerulus itself play an important role in determining the site of complex deposition in experimental AICN and suggest that electrophysical characteristics of the glomerular capillary wall may influence complex localization on the GBM.     

Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis.

Mesangial injury and cell proliferation are frequent findings in various glomerular diseases in man. Previous studies have demonstrated that basic fibroblast growth factor (bFGF) is a potent mesangial cell mitogen in vitro. To further elucidate the role of bFGF in rat mesangial cell (RMC) proliferation, we examined whether RMC synthesize bFGF in vitro and whether bFGF is involved in mesangial proliferation in vivo. Cultured RMC expressed bFGF protein (23, 21.5, and 18 kD forms) and bFGF mRNA, and released biologically active bFGF into the culture medium after antibody- and complement-mediated injury. Normal rat glomeruli in vivo contained no detectable bFGF mRNA, but bFGF protein (23 and 21.5 kD) could be demonstrated, which immunolocalized to the mesangium. Glomerular bFGF decreased markedly during the acute phase of glomerulonephritis induced by anti-Thy 1.1 antibody, compatible with mesangial bFGF release after complement-mediated mesangiolysis. During the subsequent mesangial proliferative phase, glomerular bFGF protein and mRNA increased above normal. Intrarenal infusion of heparin did not affect the bFGF immunostaining of glomeruli at this stage, indicating a predominantly intracellular localization of the bFGF. The capability of bFGF to mediate proliferation in the anti-Thy 1.1 model was further supported by experiments in which intravenous bFGF given 24 h after a subnephritogenic dose of anti-Thy 1.1 antibody led to a 4.9- to 5.1-fold increase in glomerular cell proliferation (with > 60% of the cells identified as mesangial cells by double immunolabeling). No such increase was observed in normal rats injected with bFGF. These data show that mesangial cells produce and release bFGF after injury and that bFGF is mitogenic for injured mesangial cells in vivo. Release of mesangial cell bFGF thus may be an important mechanism involved in the initiation of mesangial cell proliferation in vivo.     

Mesangial cell autoantigens in immunoglobulin A nephropathy and Henoch-Schönlein purpura.

The autoantigen(s) that we have previously described in human glomeruli, recognized in IgA nephropathy, has (have) been identified as mesangial cell in origin. Cultured mesangial cells expressed 48- and 55-kD components binding IgG isotype autoantibodies (IgG-MESCA) present in sera of patients with both IgA nephropathy and Henoch-Sch?nlein purpura (HSP). IgG-MESCA were not detected in sera of normals, or patients with other autoimmune-mediated glomerulonephritides: anti-glomerular basement membrane disease, Wegener's granulomatosis, or in IgM-mesangial proliferative disease. Binding specificity was proven by F(ab')2 studies in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and there was no significant affinity of IgA or IgM immunoglobulins. Fluorescein isothiocyanate-conjugated IgG from ELISA-positive sera localized to the mesangium and peripheral capillary loops of glomeruli, supporting the belief that the antigen is expressed in normal human renal tissue. However, only about one third of mesangial cells in culture showed affinity for IgG from ELISA-positive sera, suggesting variable expression of the antigen(s) in vitro. The only autoantigen(s) present in glomeruli, and extractable from whole normal glomeruli by the techniques employed, localized on the mesangial cell. In both IgA nephropathy and HSP, autoimmunity was intermittently present, with fluctuating levels of IgG-MESCA detectable in sera. There were positive correlations with the degree of glomerular injury assessed by erythrocyturia and proteinuria in IgA nephropathy, but significance was reached with only the degree of hematuria in HSP. These findings suggest a contributing role in the pathogenesis of the mesangial proliferative lesions and demonstrate autoimmunity common to both IgA nephropathy and HSP.     

New mechanism for glomerular injury. Myeloperoxidase-hydrogen peroxide-halide system.

Reactive oxygen species, particularly hydrogen peroxide (H2O2), participate in neutrophil-mediated glomerulonephritis. However, the mechanism of H2O2 neptrotoxicity is unknown. Myeloperoxidase (MPO), a neutrophil cationic enzyme that localizes in glomeruli, can react with H2O2 and halides to form highly reactive products. We tested the hypothesis that the MPO-H2O2-halide system may induce glomerular injury by infusing MPO followed by H2O2 in a chloride-containing solution into the renal artery of rats. Controls received MPO or H2O2 alone. MPO-H2O2-perfused rats developed significant proteinuria, endothelial cell swelling, and epithelial cell foot process effacement, whereas control kidneys were normal. In the presence of free 125I, MPO-H2O2-perfused rats incorporated large amounts of 125I, localized to the glomerular basement membrane and mesangium by autoradiography, into glomeruli. Glomerular iodination was greatly decreased or absent in controls. The MPO-H2O2-halide system causes glomerular injury and may be important in neutrophil-mediated glomerulonephritis.     

RENAL TRANSPLANTATION IN DIABETES MELLITUS IN RATS

Immunoglobulins and complement are deposited in the glomerular mesangium of rats with progressive glomerulosclerosis secondary to chemically induced diabetes mellitus. Isotransplantation of a kidney from a rat diabetic for 6 mo into a normal recipient results within 2 mo in the disappearance of IgG, IgM, and β₁C from the mesangium and arrest or reversal of the light microscopic glomerular lesions. Kidneys isotransplanted from normal donors into diabetic rats developed mesangial matrix thickening and deposition of IgG, IgM) and β₁C in the mesangium. No glomerular changes occur upon transplantation of a normal kidney into a normal rat. These findings indicate that diabetic glomerular changes in the rat are reversible and are secondary to the diabetic state rather than to the inducing agent.     

Serial micropuncture analysis of glomerular function in two rat models of glomerular sclerosis.

We have recently developed a micropuncture technique to assess repeatedly function of the same nephrons in chronic renal disease and subsequently examine the morphology of their glomeruli by serial thin-section histological analysis. Using this approach, a potential causal linkage between early functional patterns and late structural abnormalities was examined in glomeruli of two established rat models of glomerular sclerosis. The models are (a) puromycin aminonucleoside (PAN) administration in unilaterally nephrectomized Munich-Wistar rats and (b) adriamycin (ADM) treatment in nonnephrectomized Munich-Wistar rats. Single nephron GFR (SNGFR) and glomerular capillary hydraulic pressure (PGC) were measured repeatedly for 8 (PAN rats) or 31 wk (ADM rats). In all animals studied, values for PGC remained at, or slightly below, levels measured before PAN or ADM administration. SNGFR values declined progressively in all glomeruli in PAN rats. Although some glomeruli in ADM rats had an increase in SNGFR above levels observed in nonnephrectomized control rats, these hyperfiltering glomeruli did not have abnormally high PGC nor did they exhibit glomerular sclerosis at the completion of the study. Histological analysis revealed the existence of a significant inverse correlation between the degree of sclerosis and SNGFR assessed at the time of sacrifice in both PAN and ADM groups. Chronic administration of captopril, an angiotensin I converting enzyme inhibitor, in PAN rats substantially attenuated development of glomerular sclerosis without affecting PGC in earlier stages. The observations in these models indicate that glomerular hyperfiltration and hypertension are not required for the development of glomerular sclerosis in renal diseases, and angiotensin I converting enzyme inhibitor can exert its protective effect independently of its effect on glomerular capillary pressure.     

Receptor antagonist of platelet activating factor inhibits inflammatory injury induced by in situ formation of immune complexes in renal glomeruli and in the skin

Experiments were designed to test the effect of a receptor antagonist of platelet activating factor (PAF), SRI63072, on inflammatory injury induced by in situ formation of immune complexes in two vascular districts of the rat that have different structural and hemodynamic characteristics: unilateral glomerulonephritis induced by perfusion of the left kidney of preimmunized animals with cationic human IgG, and passive reversed Arthus reaction in the skin. Three days after perfusion the left kidneys of rats not treated with SRI63072 (group I) were greatly enlarged, and pale and severe exudative and proliferative lesions were present in glomeruli. Granular deposits of human IgG, rat IgG, and rat C3 were seen by immunofluorescence microscopy, and subepithelial electron-dense deposits were visualized by electron microscopy. The right kidneys were consistently normal. The animals were severely proteinuric and had increased levels of PAF in the circulation. In contrast, rats treated with SRI63072 (group II) and studied at the same interval of time developed only mild, focal glomerulonephritis in the perfused kidneys. By immunofluorescence microscopy the glomerular deposits of human IgG and rat IgG were similar in quantity and distribution to those observed in rats of group I. Despite the fact that SRI63072 did not influence the level or the activity of the rat serum complement system, the deposits of C3 were less abundant and more focal. As in animals of group I, electron-dense deposits were present at the epithelial side of the glomerular basement membrane. Proteinuria was slight, and levels of circulating PAF were not significantly increased. These effects cannot be ascribed to interference of SRI63072 with antigen "implantation," antibody binding, or local hemodynamic conditions, because the amounts of glomerular human and rat IgG were the same in treated and untreated rats; SRI63072 did not decrease the glomerular filtration rate; and SRI63072 prevented the increase in vascular permeability and the exudative lesions in passive Arthus reaction in the skin, a model less affected by hemodynamic changes than glomerulonephritis. The beneficial effect of SRI63072 indicates that PAF is an important mediator of the inflammatory process generated either in glomeruli or in the skin by in situ immune complex formation.     

Targeted enzyme therapy of experimental glomerulonephritis in rats.

We sought to determine whether systemic administration of proteases ameliorates membranous nephritis induced in rats by immunization and challenge with cationic bovine gamma globulin, and whether targeting of protease to glomerular capillaries increases efficacy. Proteases substituted with biotin were targeted via the cationic protein avidin A, which by virtue of its charge has affinity for the glomerular basement membrane. Despite identical pretreatment proteinuria, rats given untargeted protease (biotin-conjugated without avidin, or unconjugated plus avidin) had significantly less proteinuria than saline-treated controls and nephrotic rats given avidin plus biotin-conjugated (targeted) protease had even less proteinuria and reduced glomerular rat IgG and C3. Among more severely nephrotic rats, targeted protease was again more effective than untargeted protease at reducing proteinuria, and also decreased the size of electron-dense glomerular deposits, hypercholesterolemia, and creatininemia. Inactivated targeted proteases had no effect on proteinuria, hypercholesterolemia, or azotemia. Finally, active targeted protease did not affect proteinuria in the nonimmune mediated nephrosis induced by puromycin aminonucleoside. We conclude that systemic protease can specifically diminish glomerular immune deposits, proteinuria, hyperlipidemia, and creatininemia associated with experimental immune complex glomerulonephritis but not toxic nephrosis, and that targeted protease is more effective than untargeted protease.     

Localization of renal erythropoietin and the role of Ia antigen expression in hypoxic rat glomeruli

Erythropoietin (EPO) producing cells were examined by the immunofluorescent antibody technique, and the alterations in the Ia antigen expression in glomeruli by hypoxic stimuli. Antisera were obtained from rabbits immunized with pure human EPO. Positive fluorescence was localized in glomerular mesangial regions of the hypoxic rat kidney. Although hypoxia showed neither increase in the number of glomerular cells nor hypertrophy of the mesangial matrix, it induced an increase of the Ia antigen expressions in glomeruli. The peak of the increase occurred 2 hr after hypoxia and then decreased until 24 hr later. The increase of Ia antigen after 2 hr of hypoxic stimuli was 176.5 +/- 7.0% of normal controls, and ranged from 151 to 209%. In conclusion, Ia positive cells in glomeruli may have some relationship to the stimulation of EPO production.