Cyclosporin A reduces expression of adhesion molecules in the kidney of rats with chronic serum sickness.

Treatment with cyclosporin A (CsA) improves proteinuria and reduces renal cellular infiltration in chronic serum sickness (CSS). We examined if these effects were associated with a reduced renal expression of CD54 and its ligands, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and MHC class II molecules. We studied two groups of rats in which CSS was induced by daily injections of ovalbumin (OVA): a group treated with CsA (OVA.CsA group, n = 11) and a group that received no treatment (OVA.CSS group, n = 11). An additional group of five rats (control group) received only phosphate buffer. Immunostaining techniques were used to follow CSS and to study the expression of CD54, CD18, CD11b/c, IFN-gamma, TNF-alpha and MHC class molecules. Proteinuria (mg/24 h) was reduced from 248.2 +/- 73.1 (OVA.CCS group) to 14.5 +/- 13.1 with CsA treatment (P < 0.0001). The renal expression of CD54 and its ligands (CD18 and CD11b/c) was reduced by 50% to 75%. Correspondingly, there was a 60% to 85% reduction in the number of infiltrating leucocytes. The number of cells expressing TNF-alpha, IFN-gamma and MHC II molecules was also reduced. CsA reduces expression of CD54 and its ligands. This effect is associated with a reduction of cellular infiltration, IFN-gamma, TNF-alpha-producing cells and with MHC II expression in the kidney. These findings suggest that expression of adhesion molecules plays a critical role in CSS and underline the importance of cellular immunity in this experimental model.

Differential expression of collagen type IV alpha-chains in the tubulointerstitial compartment in experimental chronic serum sickness nephritis.

The expression of collagen type IV chains in the renal tubulointerstitium was investigated during the development of chronic serum sickness (CSS) in rats, a model for immune complex-mediated renal disease. Immunohistochemical studies showed increased expression of alpha4(IV) collagen early during disease development, followed by an increase in alpha1(IV) through alpha3(IV) collagen subchain expression, especially in the tubular basement membrane. Dot-blot and in situ hybridization analysis showed a transient increase in steady-state mRNA levels for all collagen IV subchains during the development of CSS, which was most abundant for alpha1(IV), alpha2(IV), and alpha4(IV). Statistical correlations were found between the mRNA levels of alpha1(IV) and alpha2(IV) collagen and between alpha3(IV) and alpha4(IV), in line with the results of others which showed that these chains are co-distributed as heterotrimer collagen type IV molecules. However, additional correlations were found between the mRNA levels coding for alpha1(IV) and alpha3(IV) collagen, and between alpha1(IV) and alpha4(IV) mRNAs in the course of CSS. These abnormal correlations support the hypothesis that changes occur in the co-expression of the collagen IV subchains during the development of CSS. In addition, a strong correlation was found between the presence in the tubulointerstitium of alpha1(IV) and alpha2(IV) collagen chains, on the one hand, and the tubulointerstitial influx of R73+ and ED1+ cells, on the other, suggesting the involvement of inflammatory cells in the observed alterations in matrix production. Changes in the relative abundance of collagen IV chains in disease states may perturb the collagen IV network in the tubulointerstitial compartment and thereby play a role in the development of renal failure.

Differential expression of collagen IV isoforms in experimental glomerulosclerosis.

Expansion of the glomerular mesangial matrix (MM), thickening of the glomerular basement membrane (GBM), and eventually the development of glomerulosclerosis are often seen in immunologically mediated kidney diseases. In addition to quantitative changes in the extracellular matrix (ECM), qualitative changes in ECM molecules may contribute to alterations in the composition of the glomerular matrix. The expression of collagen IV, alpha 1-5(IV) mRNA, and polypeptides was therefore investigated during the development of chronic graft-versus-host disease (GvHD) in mice, a model for lupus nephritis, and in chronic serum sickness (CSS) in rats, a model for membranous nephropathy. Immunohistochemical studies showed increased mesangial expression of alpha 1 and alpha 2 early in the disease, but only late in the GBM. In contrast, alpha 3 and alpha 4 increased in the GBM during disease, but not in the MM. The mRNA levels for all collagen IV chains were increased in isolated glomeruli before morphological alterations were detectable. The mRNA increase was earlier and more profound for alpha 3, alpha 4 and alpha 5 than for alpha 1 and alpha 2. Expression of alpha 3(IV) was greatest in GvHD, whereas expression of alpha 4 was greatest in CSS. As determined by in situ hybridization (ISH), alpha 1 mRNA was observed dispersed in the glomerulus, but alpha 3, alpha 4, and alpha 5 mRNAs were mainly located in cells at the periphery of the glomerular tuft. The changes in the relative abundance of collagen IV mRNA in disease states may perturb the collagen IV network, altering glomerular structure and function, and may thereby play a central role in the development of glomerulonephritis and glomerulosclerosis.

Glomerular sequence of events concerning monocyte/macrophage accumulation and ICAM-1 expression during experimental serum sickness nephritis in the rat.

Serum sickness nephritis was induced in Fischer rats by preimmunization and repeated immunization with chicken egg albumin. This experimental model is characterized by marked accumulation of monocytes/macrophages (MO) and deposition of immune complexes (IC) in glomeruli during the inflammatory stage and, thereafter, the advancement to glomerulosclerosis. The correlations between glomerular tissue damage, MO participation, intercellular adhesion molecule-1 (ICAM-1) expression and IC deposition were analyzed during the long-term disease process. The grade of ICAM-1 expression was well correlated with MO accumulation and IC deposition, and its distribution was observed on the glomerular endothelial layer, mesangium, and along the parietal epithelial layer of the Bowman's capsule. It is suggested that glomerular MO accumulation is largely affected by the ICAM-1 expression on glomeruli and, underneath such adhesion molecules, MO may play a role in subendothelial or mesangial migration, mesangial cell activation, inducing sclerosis and monocytic-epithelial crescent formation.

Dietary fish oil supplementation exacerbates serum sickness nephritis in mice.

The effects of fish oil (FO) on immune complex nephritis induced by bovine serum albumin (BSA) were studied in female B10.Br mice. The mice were fed an experimental fat-free diet composed of either 10% FO, safflower oil (SO), or beef tallow (BT) as a lipid source throughout the study. Proteinuria was observed in 84% of the FO group (n = 19), 53% of the SO group (n = 19) and in 48% of the BT group (n = 19; p = 0.0217 vs. FO). The FO group showed a tendency toward more severe renal histologic changes than the SO and BT groups. The levels of anti-BSA antibody and circulating BSA-anti-BSA immune complexes were significantly higher in the FO group than in the SO and in the BT groups. Avidity of the anti-BSA antibodies showed a lower tendendy in the FO group. Prostaglandin E2 and thromboxane B2 productions by the renal cortex were much lower in the FO than in the other two groups. The ratios thromboxane B2/prostaglandin E2 were higher in the FO than in the BT group. These results suggest that FO oil supplementation leads to the deterioration of BSA-induced immune complex nephritis in mice due to the altered immune responses in association with suppressed prostanoid production.

Monocyte chemoattractant protein-1 in chronic proliferative immune complex nephritis.

In rats with chronic serum sickness, proliferative immune complex glomerulonephritis progresses in three discrete stages, designated mild, moderate, and severe. One distinguishing immunopathologic feature, the progressive increase in the number of glomerular macrophages, is closely correlated with decreasing kidney function. We hypothesized that monocyte chemoattractant protein-1, a beta-subfamily chemokine with potent monocyte-specific chemotactic activity, might contribute to this macrophage accumulation. Immunohistochemical methods were used to identify monocyte chemoattractant protein-1 in kidney tissue sections. Total RNA was extracted from the kidneys of rats at each stage of chronic serum sickness, and age-matched controls, and Northern blot analysis was performed with a rat monocyte chemoattractant protein-1 cDNA probe. Tissue staining localized monocyte chemoattractant protein-1 to the glomerular capillary wall and mesangium in chronic serum sickness. Minimal quantities of monocyte chemoattractant protein-1 mRNA were detected in the kidneys of normal control rats, with marked increases in mRNA as chronic serum sickness nephritis progressed to the moderate stage. There was then an apparent decrease in monocyte chemoattractant protein-1 mRNA in the severe stage. The degree of protein staining and mRNA levels paralleled each other. We conclude that monocyte chemoattractant protein-1 is a potentially important chemotactic agent in chronic serum sickness nephritis.

Cloning of the mouse fibronectin V-region and variation of its splicing pattern in experimental immune complex glomerulonephritis.

Increased mRNA and protein expression of extracellular matrix (ECM) components, including fibronectin, occurs during the development of glomerulonephritis and glomerulosclerosis in immunologically mediated kidney diseases. However, in addition to these quantitative changes in ECM expression, qualitative changes in these molecules may contribute to malformations in the composition of the glomerular matrix. These qualitative changes may include alterations in the splicing pattern of the V-region of fibronectin, since this region plays a role in its accumulation. The splicing patterns of this region have been studied in chronic graft-versus-host disease (GvHD) in mice, a model of lupus nephritis, and in chronic serum sickness (CSS) in rats, a model of immune complex nephritis. Cloning of the mouse fibronectin V-region from kidney tissue revealed 96.1 per cent homology with the corresponding domain in rat fibronectin. PCR (polymerase chain reaction) analysis of RNA from isolated glomeruli revealed three isoforms of this region in both mouse and rat fibronectin, namely inclusion or exclusion of the whole region, or exclusion of only the CS1 domain. In both models, increased exclusion of the V-region was observed early in the disease. However, in GvHD the splicing pattern returned to normal, whereas in CSS the shift persisted during the course of the experiment. Differentiated expression of fibronectin isoforms may exert an important effect on the structure and biological function of the glomerulus and may thus play a role in the development of glomerulonephritis and glomerulosclerosis.

Specific accumulation of exogenous fibronectin in experimental glomerulosclerosis.

The prognosis of patients showing glomerulosclerosis as a complication of an immunologically mediated kidney disease is poor. To improve the diagnosis and treatment of these patients, it is important to understand the processes involved in the development of glomerulosclerosis. In this study, we investigated the molecular composition of experimental end-stage glomerular sclerotic lesions and their pathogenesis in chronic graft-versus-host disease (GvHD) in the mouse and chronic serum sickness in the rat. Accumulation studies were performed to determine the degree of specific trapping of constituents from the circulation. Two different models were investigated to determine whether differences in disease initiation resulted in different compositions of the glomerulosclerotic lesions. In both models, glomerulosclerosis was preceded by expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM). The end-stage sclerotic lesions consisted mainly of fibronectin, which appeared to displace the other extracellular matrix (ECM) components peripherally in the mesangial matrix and GBM. The abundance of fibronectin in the lesions was not reflected in the mRNA levels for this component. Indeed, antibodies directed against the cellular form of fibronectin did not stain positive in the end-stage lesions. These findings, together with accumulation studies, suggest that specific accumulation rather than de novo synthesis of fibronectin plays a major role in the development of experimental glomerulosclerosis, which appears to be independent of the pathway of induction.

Anionic sites in glomerular basement membrane of rats with serum sickness nephritis: quick-freezing and deep-etching study.

Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.

Influence of subepithelial deposits on permeability of the glomerular capillary wall in serum sickness nephritis in the rat.

Serum sickness nephritis was induced in male Fisher rats by immunization with egg albumin (EA). Correlations of subepithelial deposits (SD) with size and charge barriers of the glomerular filter were investigated using native (NF) and cationized (CF) ferritin as tracer probes. In proteinuric animals large numbers of NF molecules perfused from the abdominal aorta were observed to cross the glomerular basement membrane (GBM) and enter SD. The concentration of NF molecules was higher in GBM segments with SD than in GBM segments without SD, and the concentration of these molecules was higher within SD than in the intervening GBM. In contrast, CF clusters were fewer in number in the lamina rara externa (LRE) of GBM segments with SD than in the GBM segments without SD. CF particles could not be observed within SD, even in the areas of podocyte detachment. It is suggested that permeability in GBM segments with SD increases and that the development of proteinuria in this model can be attributed to alterations in both charge- and size-selective barriers to glomerular filtration.

[Immunological injury from serum sickness on aortic connective tissue components].

Changes of aortic proteoglycan, collagen and elastic fibers following immunological injury were studied quantitatively and qualitatively under electron microscope after ruthenium red labelling. The results showed that repeated intravenous injections of bovine serum albumin in rabbits activated the synthesis of collagen, elastic fibers and proteoglycan in the aortic wall. The increase of connective tissue components was coincident with the morphological changes of smooth muscle cell.

The effects of chronic serum sickness on albumin distribution and glucose utilization in rat brain.

The level of cerebrospinal fluid (CSF) protein is elevated in diseases and disease models that are associated with circulating immune complexes such as serum sickness. Circulatory immune complexes are known to deposit in the basal lamina of fenestrated capillaries and may, as a result, affect both capillary bed and parenchymal function. Since the brain has both fenestrated and unfenestrated capillaries and immune complexes deposit to a varying extent in the fenestrated capillaries in chronic serum sickness, cerebral capillary permeability to protein may be altered in some brain areas and lead to the elevation of CSF proteins. In addition various other cerebrovascular and metabolic functions may also be affected by this condition. In this study either radio-iodinated serum albumin (RISA) or 2-[14C]deoxyglucose (14C-2DG) was intravenously injected into control Wistar rats and Wistar rats with chronic serum sickness; subsequently the tissue levels of radioactivity were measured by quantitative autoradiography in 4 brain areas with fenestrated capillaries and 11 brain areas with unfenestrated capillaries. The 2-min distribution of RISA, which demarcates the volume of circulating plasma in perfused microvessels and is generally proportional to local plasma flow, was the same in control and experimental rats. The passage of RISA from blood into brain over 30 min was negligible in both groups; thus cerebral capillary permeability to albumin was not detectably increased in any of these 15 brain areas by chronic serum sickness. The rate of local cerebral glucose utilization, an indicator of local metabolic and neural activity, was calculated from the 14C-2DG data and was virtually identical in control and experimental rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of hyperfiltration on serum sickness glomerulonephritis in rats.

The effects of hyperfiltration induced due to unilateral nephrectomy on immunologically induced glomerular injuries were studied. Glomerulonephritis was induced in rats by sensitizing them with egg albumin as an antigen. Unilateral nephrectomy did not affect the removal rate of the antigen from the glomeruli in the rats, but accelerated the rate of the glomerular injuries after cessation of the immunologically induced glomerular inflammation. The histopathological features were characterized by sclero-adhesive lesions with aneurysmal dilatation and hyalinosis of the glomerular capillaries. The parietal epithelial cells extended from the Bowman's capsule with matrices to cover the denuded basement membrane and formed adhesions. The neighboring capillaries collapsed, and the sclero-adhesive lesions progressed. These findings indicate that hyperfiltration at the capillary level did not accelerate the recovery from glomerulonephritis, but induced glomerular sclerosis with adhesions and deteriorated the trivial glomerular injuries to produce similar focal segmental lesions.

Glomerular eicosanoid production in acute serum sickness nephritis.

To determine if the induction of immune-mediated glomerular injury influences the formation of glomerular cyclooxygenase products, we measured thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) production by isolated glomeruli of rabbits induced with acute serum sickness nephritis by the administration of bovine serum ablumin (BSA). Animals were randomly assigned to one of three experimental groups: animals injected with BSA (BSA group; n = 11); animals injected with normal saline (control group; n = 11); and animals injected with BSA which were treated with the thromboxane synthetase inhibitor, OKY-046 (BSA + OKY-046; n = 6). Animals in the BSA and BSA + OKY groups developed severe proteinuria and glomerular histologic lesions of nephritis. No differences in proteinuria, serum creatinine and severity of histologic nephritis were observed between the two groups. Examination of glomerular eicosanoid production at the end of the experiment showed a marked reduction of glomerular PGE2 and 6-keto-PGF1 alpha production with a smaller reduction of glomerular TXB2 production in the BSA group. In the BSA + OKY-046 group, the production of TXB2 was significantly less than that in the BSA group; despite this, no effect on proteinuria could be discerned.

Antinephritic effect of prostaglandin E1 on serum sickness nephritis in rats (5). Effect of PGE1 on disposal of heat-aggregated bovine serum albumin in the glomerulus.

In the present study, we investigated whether prostaglandin E1 (PGE1) could accelerate the disposal of heat-aggregated BSA (a-BSA) in the glomerulus by mesangial cells and/or resident mesangial cells. ICR mice were injected i.v. with 90 mg/100 g B.W. of a-BSA 3 times at 4-hr intervals. Kidneys were isolated at various times after the first injection of a-BSA. The location of a-BSA in the glomerulus was then detected by immunohistochemical staining and immunofluorescence. A-BSA was detected in the mesangium and along capillary walls by both techniques. The amount of glomerular a-BSA increased with time, attaining a peak about 12 to 14 hr after the first administration of a-BSA and then disappeared by 36 hr after. The mice injected with a-BSA 3 times received 150, 200, 300 and 400 micrograms/mouse of PGE1, s.c., and 200 and 400 micrograms/mouse of PGE2 or PGF2 alpha, s.c., at 12 hr; and their kidneys were isolated at 16 hr. The mice with 300 and 400 micrograms/mouse of PGE1 had 34.3% and 37.6% less a-BSA than the control mice, respectively. Additionally, the mice with 400 micrograms/mouse of PGE2 had 63.2% less a-BSA than the control mice. However, PGF2 alpha failed to reduce glomerular a-BSA to a level less than that of the control. In conclusion, we confirmed that PGE1 accelerates breakup of macromolecules by mesangial cells and/or resident mesangial cells in the glomerulus.

Localization of cationic proteins derived from platelets and polymorphonuclear neutrophils and local loss of anionic sites in glomeruli of rabbits with experimentally-induced acute serum sickness.

The localization of cationic proteins (CP) derived from platelets and from polymorphonuclear neutrophils (PMN) in glomeruli of 42 rabbits injected i.v. with a large amount of bovine serum albumin, was investigated in sequential biopsies by immunofluorescence, using goat-anti-platelet CP and anti-PMN CP sera. Platelet CP deposits became detectable within 7 to 8 days after the i.v. injection of bovine serum albumin, before or coincident with the onset of proteinuria. The intensity and the extent of linear and segmental deposits of platelet CP along the glomerular capillary walls reached a peak at day 9 to 10, when proteinuria was maximal. The anti PMN-CP serum stained the cytoplasm of the few PMN present in glomeruli and only occasionally at day 11 and 12 identified focal deposits of PMN-CP along the glomerular capillary walls. The kinetic study of glomerular immune deposits showed that the first appearance of immune deposits in antigen excess was preceded by, or was concomitant, with the detection of platelet-CP in glomeruli. In the later stages of serum sickness, the immune deposits showed a progressive increase in rabbit IgG and C3. The glomerular polyanions were studied by light microscopy, using the colloidal iron technique, and by electron microscopy using polyethyleneimine as a cationic probe. The glomerular deposits of platelet-CP were associated with a reduction of colloidal iron staining, which was maximal 9 to 11 days after the i.v. injection of bovine serum albumin. At day 15, colloidal iron staining was almost completely restored. At day 9 in rabbits with acute serum sickness the anionic sites of glomerular basement membrane evidenced by polyethyleneimine, were segmentally decreased, mainly in the lamina rara interna. In rabbit studied at day 15 the anionic sites were decreased only at the base of the subepithelial electron dense deposits (humps). These results suggest that in rabbits with experimentally-induced acute serum sickness, during the early stages of glomerular immune deposit formation endogenous CP, released mainly from platelets, bind to glomerular capillary walls and possibly contribute to the neutralization of glomerular polyanions.

Absence of sodium and water retention in rats with severe proteinuria.

Studies with two models of immunologically mediated glomerular disease in the rat, chronic serum sickness and Heymann nephritis, show that fluid retention can be dissociated from other signs of the nephrotic syndrome (excessive proteinuria, hypoalbuminemia, hypercholesterolemia). Clinical evidence of fluid retention (increased body weight, decreased hematocrit, ascites) was only detected in severe chronic serum sickness and coincided with an abrupt drop in urinary sodium concentration and sodium excretion. Severe proteinuria was not associated with sodium and water retention in moderate chronic serum sickness and in Heymann nephritis. These observations support the hypothesis that, in conditions of severe proteinuria, an intrarenal defect in sodium excretion rather than a systemic factor, leads to fluid retention.