Influence of the electric charge of the antigen and the immune complex (IC) lattice on the IC activation of human complement

In order to understand the mechanism of complement (C) activation by immune complexes (ICs), the anti-complementary effect of ICs containing cationized antigens was compared in vitro to that using ICs formed by native antigens. ICs were prepared with affinity-purified rabbit polyclonal IgG antibovine serum albumin (BSA) antibody and either native BSA (isoelectric point 4.2) or BSA rendered cationic by treatment with ethylenediamine (isoelectric point 9.4). Native and cationized antigens were characterized by isoelectric focusing. ICs containing anti-BSA IgG or F(ab')2, formed either at equivalence or in excess of native or cationized antigen, were submitted to ultracentrifugation in a sucrose gradient for mesh size determination. The anti-complementary effect of ICs was evaluated by kinetic determination of haemolytic activity of human serum on haemolysin-sensitized sheep red blood cells. In conditions of antigen excess, the ICs formed by cationized BSA were significantly more efficient in activating human complement than those formed by native antigen. This higher activity was dependent on cationized antigen complexed with complete antibody molecules, as non-complexed cationized BSA or ICs prepared with F(ab')2 fragments were inactive under the same experimental conditions. Furthermore, this difference did not depend on the mesh size of the immune complexes. Our results suggest that the balance between antigen, antibody and C may be of importance in vivo for the onset and course of infections and other pathological processes involving IC formation. ICs containing cationized antigens should be proven of value in experimental models for studies on the regulation of C activation.     

C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.     

Anti-inflammatory activity of IFN-beta in carrageenan-induced pleurisy in the mouse.

Passive injection of mice with preformed immune complexes (IC) made from cationized bovine serum albumin (BSA) and anti-native BSA antibody gave immune deposits along the glomerular capillary walls at predominantly subepithelial sites, while similar quantities of complexes made with native, anionized BSA did not deposit. Peripheral localization could be obtained also using low avidity antibody and a great excess of native BSA. Ultracentrifugation analysis showed that the size of IC in the animals given complexes containing cationized BSA was a little larger than 7 S, whereas those formed with the native or anionized BSA were around 19 S. The anti-native BSA antibody had a low avidity for cationized BSA in vitro, and thus all the IC which could deposit peripheral capillary walls were small and contained low avidity antibody. Chemical cationization of BSA alters the precipitability of the antibody and also the size and stability of the complexes formed. In an active model, injection of cationized BSA into mice preimmunized with cationized BSA caused localization of the BSA and its antibody in the peripheral capillary walls. Analysis of the circulating IC formed in this model also revealed low avidity of antibody and small-sized IC. From these results, it is clear that chemical cationization of antigen changes the characteristics of the antigen-antibody interaction, e.g. low precipitating efficiency and the formation of small-sized IC. Therefore, in addition to interaction of cationized IC with the polyanion layer of the glomerular basement membrane (GBM), the properties of antigen-antibody interaction play an important role in the deposition of IC along the peripheral capillary walls in a model of membranous glomerulonephritis.     

Antibody-Mediated Rejection of Human Renal Allografts: An Electron Microscopic Study of Peritubular Capillaries

The role of humoral rejection in acute and chronic rejection of human renal allografts other than in hyperacute rejection has not been well established, and its importance may be underestimated. Recently, a specific histological pattern of antibody-mediated rejection of renal allografts has been recognized. The antigens targeted by this mode of rejection are not well defined but are likely located on the endothelium of small vessels (arterioles and glomerular and peritubular capillaries). In both cellular and humoral rejection, the microvasculature of transplanted organs appears to be a main target of injury. This study describes the ultrastructural changes of peritubular capillaries, over a period of up to 8 months, in 14 biopsy specimens obtained from 5 renal allograft recipients diagnosed with “pure” antibody-mediated rejection. In peritubular capillaries, there is progression of injury from necrosis of endothelial cells with lifting and denudation of basement membrane to complete disappearance of capillaries. Acutely, acute tubular necrosis is a constant finding. At 2 to 3 months posttransplantation, the remaining capillaries are dilated, misshapen, and distorted, and are surrounded by a reduplicated and thickened basement membrane. These changes are associated with increased interstitial fibrosis and tubular atrophy, comparable to a sort of renal “asphyxial” death. The author concludes that in “pure” antibody-mediated rejection, the endothelium of peritubular capillaries is a main target of injury. The potential role of antibody-mediated rejection in acute and chronic rejection of renal allografts needs to be explored further.     

Plasma protein extravasation and vascular endothelial growth factor expression with endothelial nitric oxide synthase induction in gentamicin-induced acute renal failure in rats

Microvascular hyperpermeability to plasma proteins via vascular endothelial growth factor (VEGF) with endothelial nitric oxide synthase (eNOS) induction may contribute to wound healing through matrix remodeling. However, vascular hyperpermeability is not examined in acute renal failure (ARF), a unique form of wound healing. Subcutaneous injection of gentamicin (400 mg/kg per day for 2 days in divided doses every 8 h) in rats increased serum creatinine levels and induced tubular damage, which peaked at day 6, after the last gentamicin injection. Ki67-positive regenerating proximal tubules (PTs) peaked in number at day 6 and almost covered the bare tubular basement membrane (TBM) by day 10. Staining of fibrinogen and plasma fibronectin began to increase in the peritubular regions as early as day 0, steadily increased in TBM and tubular lumen until day 6 and then decreased. Hyperpermeable peritubular capillaries were identified by extravasation of perfused-fluoresceinated dextran (both 70 kDa and 250 kDa) into peritubular regions as early as day 0 and prominently into TBM and tubular lumen at day 6. Electron microscopy further suggested the intraendothelial pathway of dextran. Immunoreactive VEGF increased in the damaged and regenerating PTs. Immunoreactive VEGF receptors-1 and -2 did not change, but immunoreactive eNOS increased in the peritubular capillaries after induction of ARF. Western blotting for VEGF and eNOS supported the immunostaining findings. In addition, we assessed the effects of NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) on vascular hyperpermeability during the recovery phase of this model. Treatment with l-NAME (s.c. at a dose of 100 mg/kg/day from day 3 to day 6) decreased extravasation of perfused-250-kDa dextran and significantly inhibited the regenerative repair of PTs at day 6 when compared with vehicle-treated rats. In conclusion, plasma protein extravasation occurred, leading to matrix remodeling, such as the process of wound healing during the tubular repair in gentamicin-induced ARF. Since VEGF-induced vascular hyperpermeability may depend on NO production, VEGF/VEGF receptor system with eNOS induction might be responsible for this process.     

Pathologic diagnosis of renal transplantation

Eighty-seven renal allografts were examined at our department between 1980 and 1989. In these specimens, 24 cases involved so-called "1 hour biopsy", including a case of donor carrying IgA nephropathy. Nine cases showed glomerulonephritis, of which three cases are suspected as de novo membranous nephropathy. Acute rejection, glomerular, tubular and vascular type were seen in 11 cases, and chronic rejection in 20 cases. Four cases showed tubular injury which may be associated with cyclosporine nephrotoxicity. At diagnosis of acute cellular rejection, immunohistological analysis was performed to characterize the composition of the micronuclear cells. In all cases we examined, T lymphocyte is dominant, and CD8+ (Leu 2) cells and CD4+ (Leu 3) cells were seen in various percentages. The Leu M1(+) macrophage was also seen in glomerular capillary lumen or peritubular space. Whether the expression of HLA-DR antigens is altered in renal transplants was examined. Expression of DR antigens increased considerably on renal tubular cells and glomerular capillary endothelial, and may be a markers of acute rejection.     

Comparison of chloride concentration and osmolality in proximal tubular fluid, peritubular capillary plasma and systemic plasma in the rat.

1. Chloride concentration and osmolalities were compared in consecutively collected samples of proximal tubular fluid, peritubular capillary plasma and systemic plasma. 2. Mean chloride concentrations (m-mole/l) were 141.3+/-2.6 in tubular fluid, 114.8+/-1.7 in peritubular capillary plasma and 119.4+/-1.8 in systemic plasma. 3. Mean osmolalities (m-osmole/kg H2O) were 297+/-2.2 in tubular fluid, 293+/-2.4 in peritubular capillary plasma and 299+/-1.8 in systemic plasma. 4. These differences are discussed in relation to the anatomical and functional organization of the peritubular capillaries and renal tubules.     

Renal biopsy findings predicting outcome in scleroderma renal crisis

Scleroderma renal crisis is irreversible in some patients despite aggressive treatment. This study was designed to identify pathologic prognostic features in scleroderma renal crisis. We retrospectively reviewed the pathology and the clinical records of 17 patients who underwent kidney biopsies during scleroderma renal crisis (group A, recovered renal function [n = 7]; group B, remained in renal failure or died [n = 10]). Multiple histologic features were assessed semiquantitatively (0-3) or as percentages. C4d staining of peritubular capillaries and small vessels was assessed semiquantitatively (0-3) in patients with scleroderma (n = 11), normotensive (n = 10), and hypertensive (n = 12) nonscleroderma native kidney controls. The percentage of thrombosed vessels (25.1 +/- 21.0 versus 5.6 +/- 12.3, P = .045) and the severity of glomerular ischemic collapse (2.9 +/- 0.3 versus 1.4 +/- 0.8, P = .001) were significantly higher in group B than in group A. Also, group B patients tended to have more severe acute tubular injury and vascular fibrinoid changes. The peritubular capillary C4d score in patients with scleroderma, normotensive controls, and hypertensive controls were 1.1 +/- 0.9, 0.3 +/- 0.7, and 0.3 +/- 0.5, respectively (P = .018, scleroderma versus other controls). Small vessel C4d score was higher in scleroderma compared to normotensive but not hypertensive controls. Within scleroderma samples, a significantly higher peritubular capillary C4d score (1.6 +/- 0.7 versus 0.3 +/- 0.5, P = .024) but not small vessel score was found in group B compared to group A. This tended to be associated with peritubular capillary leukocyte margination. Vascular thrombosis, severe glomerular ischemic collapse, and peritubular capillary C4d deposits in scleroderma renal crisis kidney biopsies correlated with increased risk of failure to recover renal function.     

Tandem Scanning Confocal Microscopy (TSCM) of normal and ischemic living kidneys

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20× water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing repidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush broders, and closure of the peritubular capillary spaces. Upon relases of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.     

Induction of Interstitial Nephritis in Rats by Basement Membrane of Human Origin

A study was conducted to investigate nephritogenic tubular basement membrane antigens common to human and rat kidneys. Brown Norway (BN) rats were immunized with human renal basement membrane in complete Freund's adjuvant simultaneously with Bordetella pertussis vaccine. The immunized rats developed polyuria and increased levels of serum creatinine one week after the second immunization. Renal histology at this time revealed marked, acute tubulointerstitial nephritis with linear deposition of IgG and C, along the tubular basement membrane and Bowman's capsule, but not along the glomerular basement membrane. Rats with this tubulointerstitial nephritis rapidly developed antibodies against renal antigens from normal BN rats such as tubular basement membrane and proximal tubule brush border, however antibodies to glomerular basement membrane appeared later. Western blotting using the same rat sera detected a 145 kDa antigen from 8 M ufea solubilized human renal basement membrane and 120-kDa, 135 kDa and 145 kDa antigens from 8M urea solubinzed BN rat renal basement membrane. This suggests that renal basement membranes of human and rat origin have common antigens involved in the pathogenesis of tubulointerstitial nephritis. Acta Pathol Jpn 39: 551 557, 1989.     

Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys

To label heparan sulfate proteoglycans and other strong anions within glomerular basement membranes (GBM) during assembly, cationized ferritin (CF), with a narrow isoelectric range of 7.7 to 8.2, was intravenously injected into newborn rats. Kidneys were then fixed and processed for electron microscopy at intervals ranging from 1 to 72 h after CF injection. One hour after injection, CF bound extensively to the lamina rara interna and externa of developing GBM and mesangial matrix and to tubular basement membranes (TBM). In double basement membranes of early stage glomeruli, large amounts of CF were also seen in central areas between the endothelial and epithelial basement membranes. In maturing-stage glomeruli, CF bound throughout interior regions of GBM outpockets projecting into the epithelial side of capillary walls as well as to the laminae rarae. Because in adult rats CF binds only to the laminae rarae, the abundant anionic sites seen here in newborns between double basement membranes and within GBM outpocket interiors may be subsequently neutralized or removed during the GBM assembly process. In addition to basement membranes, CF was also located intracellularly within endocytic vesicles and lysosomes of glomerular endothelial, mesangial, and epithelial cells 1 h postinjection. CF was also present in similar structures within the tubular epithelium. In contrast to these findings, CF was gradually lost from developing GBM 5, 15, and 24 h after injection and was essentially cleared from all GBM, mesangial matrices, and TBM after 48 h. Large CF aggregates were progressively accumulated within mesangial lysosomes, however. The transient binding of CF to GBM anionic sites seen here was most likely due to its endocytic removal by developing glomerular endothelial, mesangial, and epithelial cells. Anions in the circulation probably also competed effectively with the GBM and TBM for bound CF.     

Acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pig-to-nonhuman primate renal grafts

The major cause of xenograft loss beyond hyperacute rejection is a form of injury, traditionally termed delayed xenograft rejection (DXR), whose pathogenesis is unknown. Here we analyze the immunologic and morphologic features of DXR that develops in pig kidney xenografts transplanted into nonhuman primates. Kidneys from miniature swine were transplanted into cynomolgus monkeys (n = 14) or baboons (n = 11) that received regimens aimed to induce mixed chimerism and tolerance. No kidney was rejected hyperacutely. Morphologic and immunohistochemical studies were performed on serial biopsies, and an effort was made to quantify the pathologic features seen. The early phase of DXR (Days 0-12) was characterized by focal deposition of IgM, IgG, C3, and scanty neutrophil and macrophage infiltrates. The first abnormality recognized was glomerular and peritubular capillary endothelial cell death as defined by in situ DNA nick-end labeling (TUNEL). Damaged endothelial cells underwent apoptosis and, later, frank necrosis. The progressive phase developed around Day 6 and was characterized by progressive deposition of IgM, IgG, C3, and prominent infiltration of cytotoxic T cells and macrophages, with a small number of NK cells. Thrombotic microangiopathy developed in the glomeruli and peritubular capillaries with TUNEL+ endothelial cells, platelet aggregation, and destruction of the capillary network. Only rare damaged arterial endothelial cells and tubular epithelial cells were observed, with rare endothelialitis and tubulitis. In the advanced phase of DXR, interstitial hemorrhage and infarction occurred. During the development of DXR, the number of TUNEL+ cells increased, and this correlated with progressive deposition of antibody. The degree of platelet aggregation correlated with the number of TUNEL+ damaged endothelial cells. We conclude that peritubular and glomerular capillary endothelia are the primary targets of renal DXR rather than tubular epithelial cells or arterial endothelium and that the earliest detectable change is endothelial cell death. DXR was characterized by progressive destruction of the microvasculature (glomeruli and peritubular capillaries) and formation of fibrin-platelet thrombi. Both cytotoxic cells and antibodies potentially mediate the endothelial damage in DXR; however, in this model, DXR is largely humorally mediated and is better termed "acute humoral xenograft rejection."     

Identification of I/i, Pr1-3 and Gd antigens in the human kidney: possible relevance to hyperacute graft rejection induced by cold agglutinins.

Human cold agglutinins (CA) with I/i, Pr1--3 and Gd specificities were tested for reactivity against kidney tissue by immunofluorescent techniques. I/i antigens were found on the epithelia of the Henle's loops and distal tubules. Pr1--3 antigens were demonstrated on the glomerular capillaries. Gd antigens were localized on the endothelia of the glomerular and peritubular capillaries as well as in the kidney interstitium. From these results, it is suggested that hyperacute rejection of renal transplants in recipients with high-titre CA may not only be caused by intravascular erythrocyte agglutination but also by direct cytotoxic damage of the transplant. Since CA occur frequently in potential kidney recipients, pre-operative determinations of CA in these patients should be recommended.     

Kidney structural and ultrastructural pathological changes induced by uracoan rattlesnake (Crotalus vegrandis Klauber 1941) venom

Acute renal insufficiency related to acute tubular necrosis is the most important complication caused by crotalid bite. For structural and ultrastructural studies of renal tissue, mice injected with crude venom or C. vegrandis haemorrhagic fraction, and controls were tested. Light microscopy analysis of kidneys at 24 h after injection of crude venom showed only moderate alterations such as tubular epithelia microvacuolisation. After 120 h marked glomerular and tubular capillaries congestion and interstitial oedema were observed. At 24 h after Uracoina-1 i.p. injection, intense glomerular and peritubular capillaries congestion was observed. Electron microscopic analysis of kidneys 24 h after i.p. injection of crude venom showed, capillary endothelial cell debris and pleomorphic mitochondria. Loss of interdigitations regularity, abundant dense bodies and light widening of the basal membrane were observed. Autophagic vacuoles were present as well as endothelia unfolding to the lumen and altered forms of podocytes. At 48 h, augmented endothelia without fenestrae formation with sequestration of low optical density debris inside the protrusions were noticed. At 120 h, capillary residues with loss of the endothelium were present and the basal membrane was widened. At 15 days, the number of vesicles and vacuoles in the tubules was increased and only few interdigitations were noticed. Autophagic vacuoles and mitochondrial matrix low electron density were observed. At 120 h after injection of crude venom, vascular damage with loss of capillary cell structures and collagen fibres were observed. At 24 h of haemorrhagic fraction injection, presence of autophagic vacuoles and myelinic figures were noticed.     

Structural evidence for counter-current flow in proximal tubules versus pertitubular capillaries in the rat kidney. Evaluation of the counter-current mechanism between the proximal convoluted tubules and the peritubular capillaries in the rat nephron

BACKGROUND: In spite of the very high exchange of water and solutes between the proximal tubules and the peritubular capillaries, very little is known about flow directions in these two interrelated structures. We therefore developed a morphological technique suitable for the quantitative evaluation of a counter-current system between the proximal convoluted tubules and the peritubular capillaries in rat renal cortex. METHODS: In male pentothal-anesthetized Wistar rats (body weight 200-250 g), India ink was injected into the aorta above the renal arteries, followed by instant freezing of the right kidney in isopentane at -165 degrees C, and subsequent freeze-substitution in alcohol. In microscopic slides from kidneys in which only 20-55% of the cortical peritubular capillary loops was filled with ink--representing the arterial end of the capillaries--and in which the proximal tubular segmentation could be identified in PAS-stained sections, the segments of the convoluted proximal tubules were quantitatively compared with regard to the presence of ink-stained and unstained peritubular capillaries in nephrons from the whole renal cortex. RESULTS: In the microscopic specimens of the five animals used both the loops from the first segment (P1) of the proximal convoluted tubule and those of the second segment (P2) were systematically packed closely together, the transitional segment (P1-2) being interposed between the groups. Around the loops of P1, 8%+/-2% of the capillaries was stained with India ink. In contrast, surrounding the P2 loops 67%+/-5% of the capillaries contained ink, significantly exceeding that for P1 (p<0.01). CONCLUSION: Throughout the rat renal cortex, the most proximal fraction of the peritubular capillaries surrounds the second segments of the proximal convoluted tubules, while the first tubular segments are surrounded by the more distal fraction of the peritubular capillaries. Consequently, the flows in the peritubular capillaries and in the proximal convoluted tubules in the rat renal cortex are systematically arranged as a counter-current system. This feature was previously identified only in superficial nephrons.     

Definition of glomerular antigens by monoclonal antibodies produced against a human glomerular membrane fraction.

Experimental animal models of glomerulonephritis (GN) produced by direct antibody binding to non-basement membrane glomerular capillary wall antigens do not to date have human parallels. To examine the potential for this form of humoral glomerular injury in man, we sought to define discrete human non-GBM glomerular antigenic targets using hybridoma technology. Mice were immunised intraperitoneally with 20-100 micrograms of a human glomerular membrane fraction (HGMF). Six fusions have yielded 12 stable reagents defined by positive glomerular indirect immunofluorescence (IF) and microELISA using HGMF as the screening antigen. Subclass analysis of ascitic McAbs indicated several IgG1, one IgG2b, and three IgM reagents. Distinctive IF patterns of reactivity with epithelial, endothelial or mesangial structures have been observed, with or without peritubular capillary, tubular basement membrane and vessel wall reactivity. Seven normal non-renal human organs and the kidneys of rat, rabbit and sheep have shown patterns characteristic of each individual McAb, restricted to human or with species cross reactivity. To partially characterise McAb-reactive antigens, detergent-solubilised renal cortex and collagenase-solubilised GBM (CS-GBM) extracts have been probed by immunoblot. A unique McAb 7-5Q, reactive with glomerular and tubular epithelial structures, binds major bands of approximately 107 KD and 93 KD in detergent solubilised cortex and a single band of similar size by immunoprecipitation (110 KD). 5-3A (a human-restricted linear-reacting McAb) binds bands of 20-200 KD (major band 58 KD) in CS-GBM. In conclusion, distinct species-restricted and more broadly disposed glomerular epitopes are definable in man by McAbs and are potential targets for humoral injury. Purification of these antigens will allow assay for circulating putative nephritogenic auto-antibody and potentially, McAbs may be useful in screening urine for evidence of occult structural renal disease.     

Characterization of renal injury initiated by immunization of rats with heparan sulfate.

To investigate the role of antibody to heparan sulfate (HS) in the development of glomerular injury, male Lewis rats were immunized with HS and compared with unimmunized controls. In HS-immunized rats circulating antibodies that bound to renal basement membranes, an increase in serum creatinine (0.8 mg/dl versus 0.6 in controls P less than 0.01), and a 40% decline in creatinine clearance developed. In no animal did abnormal proteinuria develop. By histologic examination there was glomerular and interstitial capillary engorgement with erythrocytes, modest infiltration by polymorphonuclear leukocytes, and no proliferation of intrinsic glomerular cells. Immunofluorescence microscopy demonstrated deposits of rat IgG along the glomerular basement membrane. Bowman's capsule, and peritubular capillaries. Electron-microscopic examination revealed capillary engorgement with erythrocytes that appeared adherent to each other and contained entrapped areas of rarefied material. These observations demonstrate that binding of antibody to HS in the glomerulus induces a mild inflammatory reaction and a reduction in glomerular filtration rate, but no abnormal proteinuria.     

Immunohistochemical localization of C3d fragment of complement and S-protein (vitronectin) in normal and diseased human kidneys: association with the C5b-9 complex and vitronectin receptor

The localization of C3d, a fragment produced by C3 activation and S-protein (vitronectin), a regulatory factor of C5b-9, was studied immunohistochemically in normal human kidney and renal biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that C3d was present along the glomerular basement membrane (GBM), tubular basement membrane (TBM) and arterioles, and that S-protein was present in the GBM, mesangium, TBM, and arterioles. Immunoelectron microscopy of isolated basement membranes showed that C3d was localized exclusively on the epithelial side of the GBM, and that S-protein was present along both the epithelial and endothelial sides. In nephritic tissues, glomerular staining of C3d, C5b-9, and S-protein was increased when compared with that in normal tissues. S-protein, frequently co-localized with C3d and C5b-9 neoantigen, was intensely positive in the immune deposits of glomerular capillaries and the mesangial area, overlapping the background staining of GBM and mesangial matrix. S-protein and its receptor were occasionally co-localized in the glomeruli. These findings indicate that C3d and S-protein are normally present in the glomeruli. Co-staining of C3d, C5b-9 neoantigen, and S-protein within the immune deposits of nephritic kidneys suggests in situ binding of S-protein to locallyformed C5b-9 complex, or merely co-distribution of S-protein with the complex, rather than trapping of large molecular SC5b-9 complex from the circulation.     

Monoclonal antibodies to rat renal antigens

We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis.     

Effect of Elevated Interstitial Pressure on the Renal Cortical Hemodynamics

The influence of renal interstitial pressure on the resistance pattern within the superficial cortical vasculature has been investigated from determinations of I) the glomerular blood flow with a modified microsphere technique and 2) the intravascular hydrostatic pressures. Interstitial pressure was monitored via a 50 μm PVC-catheter placed into the subcapsular interstitial space. Two conditions were analyzed viz. a) elevation of iiretheral pressure to 20 mm Hg and b) venous stasis to 10–15 mm Hg. Both conditions produced an increase in the interstitial pressure from 1–2 mm Hg to about 5 mm Hg as well as an increased hilar lymph flow and protein flow of about the Same size. The vascular reactions were different, however. Urethcral stasis (but not the stasis of a single nephron) produced a decreased resistance in the afferent arteriolae with a concomitant increase in the pressures in the glomerular capillaries, and the peritubular capillary network. In contrast, venous stasis produced only small changes in the parameters studied but for the obvious rise in the peritubular capillary pressure. The results suggest that factors other than the interstitial pressure are governing the afferent vascular tone; the tubular wall tension might he one of these factors.