anti-GBM Nephritis Research Articles

Blockade of TGF-β action ameliorates renal dysfunction and histologic progression in anti-GBM nephritis

We tested whether the entire soluble extracellular domain of the human transforming growth factor-beta (TGF-beta) type II receptor, fused to the Fc portion of human immunoglobulin G (IgG1) (Tbeta-ExR) and expressed in skeletal muscles by adenovirus-mediated gene transfer (AdTbeta-ExR), can ameliorate renal dysfunction and histologic progression in a rat experimental anti-glomerular basement membrane (GBM) nephritis. Anti-GBM nephritis was induced in Wistar Kyoto rats by an intravenous injection of anti-rat glomerular basement membrane (GBM) sera. At day 1 (24 hours after induction), AdTbeta-ExR (1 x 109 pfu/mL) was injected into the femoral muscle in the treatment group, and an adenovirus vector-expressing bacterial beta-galactosidase (AdLacZ) was injected into the control group. Then, clinical and histologic changes were examined for 3 weeks after the induction of anti-GBM nephritis. Tbeta-ExR was detected in the serum at day 7, but the serum concentration of Tbeta-ExR had decreased below the detectable level by day 14. Although blood pressure and the degree of proteinuria were similar in both groups, the deterioration of renal function was significantly blunted in the treatment group. Crescent formation and interstitial fibrosis were also ameliorated in the treatment group. These histologic improvements were accompanied by the decreased interstitial infiltration of macrophages and the decreased alpha-smooth muscle actin (alpha-SMA)-positive cells in the glomeruli and the interstitium. This study demonstrated for the first time that the blockade of TGF-beta action by AdTbeta-ExR in the early stage of anti-GBM nephritis ameliorates the clinical and histologic progression. In addition, this study shed light on the development of a specific gene therapy for human crescentic glomerulonephritis.

Molecular characterization of the target antigens of anti-glomerular basement membrane antibody disease.

The abnormal immune response to renal antigens is a significant cause of progressive glomerulonephritis and end-stage renal disease, leading to the need for dialysis or kidney transplantation. Type IV collagen of the glomerular basement membrane (GBM), an important component of the blood filtration barrier, is the target of pathogenic antibodies in two forms of anti-GBM antibody nephritis. Type IV collagen is a family of six chains that assemble into three networks with distinct composition and tissue-specific distribution. The GBM contains an alpha3.alpha4.alpha5(IV) network essential for the maintenance of kidney ultrafiltration function: the absence of this network in patients with Alport's syndrome leads to progressive glomerulonephritis. In some Alport patients that receive a kidney transplant, anti-GBM alloantibodies develop against the non-collagenous (NC1) domains of the alpha3.alpha4.alpha5(IV) collagen network, which is present in the renal allograft but absent in the Alport kidneys, causing Alport post-transplant nephritis. In Goodpasture's (GP) syndrome, anti-GBM autoantibodies target the NC1 domain of the alpha3 (IV) chain in the GBM, causing rapidly progressing glomerulonephritis. The GP epitopes have been localized to two homologous regions of the alpha3 NC1 domain, E(A) and E(B), and several populations of autoantibodies with distinct epitope specificity were purified and characterized. The epitopes of GP autoantibodies are sequestered in the NC1 hexamer that connects two adjoining triple-helical molecules. Hydrophobic amino acids have been identified in the epitope of the immunodominant GP(A) autoantibodies, suggesting that the cryptic nature of the GP epitopes is due to interactions among NC1 domains in the NC1 hexamer. Experimental anti-GBM nephritis can be induced in animal models by passive transfer of anti-GBM antibodies or by active immunization with NC1 domains of the alpha3.alpha4.alpha5(IV) network.

Macrophage metalloelastase as a major factor for glomerular injury in anti-glomerular basement membrane nephritis.

Rat anti-glomerular basement membrane (GBM) nephritis is a model of crescentic glomerulonephritis induced by injection of anti-GBM antiserum. To elucidate the mechanism of glomerular injury, we analyzed the gene expression patterns in the kidneys of anti-GBM nephritis rats using DNA arrays, and found that macrophage metalloelastase/matrix metalloproteinase (MMP)-12 was one of the highly expressed genes in the kidneys on days 3 and 7 after the injection of anti-GBM antiserum. Enhancement of MMP-12 mRNA expression was confirmed by Northern blot analysis, and in situ hybridization revealed that MMP-12 mRNA was expressed in ED-1-positive macrophages and multinuclear giant cells in the glomeruli with crescent. Moreover, these cells were positive with anti-rat rMMP-12 Ab on the section of the kidneys of anti-GBM nephritis rats on day 7. To clarify the role of MMP-12, we conducted a neutralization experiment using anti-rat rMMP-12 Ab, which had an ability to inhibit rMMP-12 activity of degrading natural substrate such as bovine elastin or human fibronectin in vitro. Anti-rat rMMP-12 Ab or control Ig was injected in each of six rats on days 0, 2, 4, and 6 after the injection of anti-GBM antiserum. Consequently, crescent formation and macrophage infiltration in the glomeruli were significantly reduced in the rats treated with anti-rat rMMP-12 Ab, and the amount of urine protein was also decreased. These results disclosed that MMP-12 played an important role in glomerular injury in a crescentic glomerulonephritis model, and inhibition of MMP-12 may lead to a new therapeutic strategy for this disease.

Heterogeneity of antigen expression explains controversy over glomerular macrophage accumulation in mouse glomerulonephritis.

Many antibody labelling studies have suggested that there are few or no glomerular macrophages in mouse models of glomerulonephritis, despite the presence of a prominent interstitial macrophage infiltrate. These findings conflict with studies of human and rat glomerulonephritis. Therefore, we examined whether heterogeneity of macrophage antigen expression could explain this apparent discrepancy. Kidneys were collected from normal mice and mice killed at 2 and 10 days after induction of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis. Following fixation, macrophages were detected by immunoperoxidase staining in serial kidney sections using antibodies recognising CD11b, F4/80 and CD68. Induction of anti-GBM nephritis caused a progressive increase in glomerular and interstitial leukocytes. At days 2 and 10, there were more CD68+ macrophages in glomeruli than macrophages expressing CD11b or F4/80. At day 10, CD68+ macrophages accounted for almost all glomerular CD45+ total leukocytes. In contrast, CD11b+ and F4/80+ macrophages at day 10 accounted for only 65 and 13% of glomerular leukocytes, respectively. However, in the interstitium the number of macrophages expressing CD68, CD11b and F4/80 were not different. Antibody detection of mouse CD68 identifies all glomerular macrophages in mouse anti-GBM nephritis, indicating a similar infiltrate to that seen in human and rat anti-GBM nephritis. Our finding of substantial heterogeneity in glomerular macrophage antigen expression in this model suggests that previous studies of mouse glomerulonephritis may have underestimated glomerular macrophages and their role in glomerular injury.

Inflamed site-specific delivery of bone marrow-derived cells carrying IL-1Ra.

A gene delivery system using bone marrow-derived CD11b(+)CD18(+) cells and their interaction with adhesion molecules was established. After transplantation into mice, these vehicle cells may be recruited into glomeruli upon lipopolysaccharide (LPS) treatment, and the number of recruited cells corresponds to the expression of intercellular adhesion molecule-1 (ICAM-1) in glomeruli. Using this system, interleukin-1 receptor antagonist (IL-1Ra) was delivered into animal models of glomerulonephritis evoked by anti-glomerular basement membrane antibody (anti-GBM nephritis). Urinary albumin excretion and the serum creatinine level were significantly elevated after anti-GBM antibody injection in mock-treated mice, whereas they were suppressed in the IL-1Ra-treated mice. Histological analysis revealed that glomerular injuries were also suppressed in IL-1Ra-treated mice. We further confirmed that specificity for inflamed glomeruli was significantly enhanced by a combination of the Cre/loxP system with the IL-1beta promoter. These data suggested that our novel system may be used as a therapeutic intervention for glomerulonephritis.

Anti-glomerular basement membrane nephritis and bullous pemphigoid caused by distinct anti-α3(IV)NC1 and anti-BP180 antibodies in a patient with Crohn's disease

Background: Anti-glomerular basement membrane (GBM) nephritis is a rare disease induced by antibodies directed against α3(IV)NC1, the Goodpasture antigen. We report a patient with Crohn's disease who developed anti-GBM nephritis and the skin blistering disorder bullous pemphigoid, owing to distinct autoantibodies. Methods: Frozen sections of skin and kidney biopsies were incubated with antisera specific for human IgG, IgA, IgM, fibrin, and C3. Reactivity of the patient's serum with GBM antigens was studied by Western blot using bovine solubilized type IV collagen and by enzyme-linked immunosorbent assays using α1(IV), α3(IV), and α5(IV)NC1 recombinant proteins. Reactivity studies against skin antigens were done by Western blot using human keratinocyte and dermal extracts and three recombinant forms of the bullous pemphigoid antigen180 (BP180, also called BPAG2 or type XVII collagen). The patient's serum was affinity fractionated on a (IV)NC1 column, and the bound and unbound fractions were analyzed for their reactivity against GBM and skin antigens. Results: The patient had deposits of IgG along the GBM and the epidermal basement membrane zone. Circulating IgG antibodies against α3(IV)NC1 were detected. The patient's autoantibodies immunoblotted the intracellular domain but not the extracellular domain of BP180. Reactivity of the patient's IgG with BP180 was found only in the unbound fraction of the serum. Conclusion: The simultaneous development of a rare renal and skin autoimmune disorder, resulting from non-cross-reactive autoantibodies, suggests that a common triggering event could be responsible for the autoimmune injury. © 2002 by the National Kidney Foundation, Inc.

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis.

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

Plasmapheresis: Technical aspects and indications

In summary, use of plasmapheresis has changed in recent years given advances in medical technology that have allowed a wider clinical application in the critical care setting. Membrane filtration technology has provided an alternative to centrifugation that can be easily applied in intensive care units. Use of plasmapheresis has also changed in recent years reflecting the availability of evidence largely obtained from controlled prospective studies. However, the clinical efficacy of plasmapheresis for many acute renal conditions is still controversial. Plasmapheresis appears to be a useful adjunct to conventional therapy in the treatment of anti-GBM nephritis, severe dialysis-dependent forms of pauciimmune RPGN, cryoglobulinemia, and HUS-TTP. Reported data also suggest a possible benefit of plasmapheresis in patients with myeloma cast nephropathy, sepsis, and poisoning/overdose, but the case for plasmapheresis in these disorders is largely unproven and the reported evidence insufficient to recommend its use outside research settings. In contrast, data from controlled trials do not support a role for plasmapheresis in immune complex-mediated RPGN, such as lupus nephritis, and acute allograft rejection. The more widespread application of prospective, randomized, controlled clinical trials should help to better define the value of plasmapheresis for treatment of acute renal diseases.

Interactions between inducible nitric oxide synthase and heme oxygenase-1 in glomerulonephritis

In anti-glomerular basement membrane (GBM) nephritis, inducible nitric oxide synthase (iNOS) and heme oxygenase (HO-1) are co-induced. Moreover, in glomerular mesangial cells iNOS-derived nitric oxide (NO) production stimulates HO-1 while HO-1 activation reduces iNOS expression/activity. These observations prompted us to explore regulatory interactions between iNOS and HO-1 in anti-GBM nephritis. Rats with anti-GBM nephritis were pretreated with the iNOS inhibitor l-N6-(1-iminoethyl) lysine (L-NIL) or with the HO-1 inducer hemin. Glomerular HO-1 levels were assessed by Western blot analysis. iNOS activity was assessed by calculating conversion of l-arginine to l-citrulline. iNOS inhibition reduced glomerular HO-1 levels without altering the inflammatory response to anti-GBM antibody induced injury. Induction of HO-1 reduced glomerular iNOS activity. In anti-GBM nephritis iNOS up-regulates HO-1 presumably via high output NO production. Suprainduction of HO-1 attenuates iNOS activity. This negative feedback interaction points to HO-1 as a target for pharmacologic manipulation to reduce activity of prooxidant heme containing enzymes such as iNOS.

Lack of in vivo function of osteopontin in experimental anti-GBM nephritis.

Osteopontin (Opn) is a potent chemoattractant for mononuclear cells that is upregulated in various inflammatory states of the kidney. Opn is believed to contribute to mononuclear cell infiltration and renal injury. The importance of Opn was examined in vivo in rapidly progressive glomerulonephritis in Opn knockout mice. Glomerulonephritis was induced by intravenous injection of rabbit anti-mouse glomerular basement membrane antiserum in mice that had been presensitized to rabbit IgG. Immunologic responsiveness to rabbit IgG (assessed by cutaneous delayed-type hypersensitivity and antibody titers) showed no significant difference between wild-type and Opn -/- mice. Proteinuria was also similar in both groups. Glomerular crescent formation was not different in Opn +/+ and -/- groups (26 +/- 6% versus 29 +/- 7%). Tubulointerstitial infiltration was assessed qualitatively and showed no significant difference between the two genotypes. Formation of thrombi in the glomerular capillaries on a scale from 0 to 3 also showed no significant difference (1.3 +/- 0.3 for Opn +/+ and 1.4 +/- 0.3 for Opn -/- mice). Northern blot analysis of total kidney RNA showed a 5.1-fold increase of Opn expression in Opn +/+ mice compared with untreated controls and the absence of expression in Opn -/- mice, as expected. Regulated upon activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 mRNA levels were also markedly upregulated with no significant difference between the two strains, excluding compensatory overexpression of these two chemokines in Opn -/- mice. It is concluded that the known upregulation of Opn in murine anti-glomerular basement membrane nephritis does not significantly contribute to the glomerular and tubulointerstitial mononuclear cell infiltration in this model.

Immunoelectron microscopic study on type I, II and III TGF-beta receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats.

Transforming growth factor (TGF)-beta is a regulator of extracellular matrix accumulation. Both TGF-beta receptors, type I (TbetaRI) and type II (TbetaRII), may be required for signal transduction in the TGF-beta pathway. The aim of this study was to investigate the relationship between the TGF-beta pathways and glomerular basement membrane (GBM) accumulation in vivo. We examined TbetaRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats. In normal control rats TbetaRI was not detected on GEP, TbetaRII expression was very occasionally found on GEP and TbetaRIII was seen in the cytoplasm of the GEP. TbetaRI, TbetaRII, and TbetaRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TbetaRI, TbetaRII, and TbetaRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TbetaRI and TbetaRII on GEP increased in association with GBM thickening. Expression of TbetaRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TbetaRI and TbetaRII on GEP was noted, but from day 7 onwards, expression of TbetaR II on GEP dramatically decreased. Expression of TbetaRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis. The results suggest that persistent upregulation of expression of TbetaRI, TbetaRII and possibly TbetaRIII on GEP may contribute to GBM matrix accumulation in vivo.

Identification of alpha3, alpha4, and alpha5 chains of type IV collagen as alloantigens for Alport posttransplant anti-glomerular basement membrane antibodies.

Alport syndrome is a hereditary disorder of basement membranes especially affecting the kidneys, ears, and eyes. Some patients who undergo renal transplantation lose their kidneys as a result of posttransplant anti-glomerular basement membrane (anti-GBM) disease. In the present study, we analyzed serum from 21 unselected Alport patients who underwent renal transplantation. Eleven samples were from patients without posttransplant anti-GBM nephritis, and 10 were from patients with this disease. Thirteen serum samples [10 alport posttransplant nephritis serum (APTN) and three Alport posttransplant serum (APT)] revealed linear binding to the GBM by indirect immunofluorescence. By using direct ELISA and immunoblotting with GBM constituents and type IV collagen NC1 domains from bovine, human, and recombinant sources, we detected anti-GBM antibodies in all Alport patients in varying titers. Five samples showed specific reactivity to the alpha3 chain, four to the alpha5 chain, six to both alpha3 and alpha5 chains, one to the alpha3 and alpha4 chains, and two to the alpha3, alpha4, and alpha5 chains of type IV collagen. The varied spectrum of reactivities was present equally in nephritic and non-nephritic sera. Ten control samples from non-Alport transplant patients did not exhibit specific binding to the GBM. These results suggest that the absence of alpha3, alpha4, and alpha5 chains of type IV collagen in the Alport kidney leads to alloantibodies in all Alport patients who receive transplants, irrespective of whether they develop nephritis or not. Although all Alport transplant patients develop this humoral response, only a select few develop anti-GBM disease. We suggest that this difference could be attributable to a genotypic effect on the ability of some individuals to launch a cell-mediated immune response.

The possible role of adhesion molecule, &alpha;3 integrin, in the synthesis of intracrescentic extracellular matrix in accelerated anti-GBM nephritis

The possible role of adhesion molecule, &alpha;<SUB>3</SUB> integrin, in the synthesis of intracrescentic extracellular matrix in accelerated anti-GBM nephritis

Mechanism of antinephritic effect of proteinase inhibitors in experimental anti-GBM glomerulopathy.

We have previously documented amelioration of rat autologous anti-GBM nephritis with the antiproteolytic drugs epsilon-aminocaproic acid (EACA) and aprotinin, given from the day of induction or later in the course of disease. In the present study we investigated potential mechanisms of this effect by assessing interactions of the drugs with proteinase-dependent generation of superoxide anion in glomeruli, and their influence on both GBM degradation in vitro and activity of glomerular proteolytic enzymes. Release of O2- by enzymatically disrupted glomeruli, isolated from nephritic control or EACA/aprotinin-treated rats, was measured with the ferricytochrome reduction method and its activity was correlated with proteinuria and glomerular cellularity at the early phase of the disease. The hydroxyproline release assay was used to quantitate degradation of rat GBM in vitro by leukocyte proteinases stimulated by phorbol myristate acetate (PMA), in the presence or absence of EACA and aprotinin. Finally, the activities of elastase, cathepsins B and L, and plasmin, together with collagenase-like activity, were assessed fluorimetrically in homogenates of glomeruli isolated from control and antiproteolytic-drug-treated nephritic rats. EACA and aprotinin notably inhibited production of superoxide by nephritic glomeruli (by 47% and 66%, respectively), and this effect was not significantly correlated with proteinuria or glomerular hypercellularity at the early stage of disease. On the other hand, generation of O2- by glomeruli of untreated nephritic rats was notably correlated with total glomerular cell counts and numbers of macrophages infiltrating glomeruli. PMA-stimulated neutrophils and macrophages caused degradation of isolated rat GBM in vitro, markedly attenuated in the presence of EACA (P<0.0005) and, to a lesser extent, by addition of aprotinin (P<0.01). The activity of elastase was significantly reduced in glomeruli of nephritic rats treated with EACA or aprotinin (both P<0.001), while activities of remaining proteinases were not appreciably affected. The beneficial influence of proteinase inhibitors on rat anti-GBM disease may be due, at least in part, to abrogation of superoxide generation in nephritic glomeruli. EACA and aprotinin also have potential to interfere with digestion of GBM, and both these effects may be related to suppression of glomerular elastase.

Alport syndrome. An inherited disorder of renal, ocular, and cochlear basement membranes.

Alport syndrome (AS) is a genetically heterogeneous disease arising from mutations in genes coding for basement membrane type IV collagen. About 80% of AS is X-linked, due to mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen (alpha 5[IV]). A subtype of X-linked Alport syndrome (XLAS) in which diffuse leiomyomatosis is an associated feature reflects deletion mutations involving the adjacent COL4A5 and COL4A6 genes. Most other patients have autosomal recessive Alport syndrome (ARAS) due to mutations in COL4A3 or COL4A4, which encode the alpha 3(IV) and alpha 4(IV) chains, respectively. Autosomal dominant AS has been mapped to chromosome 2 in the region of COL4A3 and COL4A4. The features of AS reflect derangements of basement membrane structure and function resulting from changes in type IV collagen expression. The primary pathologic event appears to be the loss from basement membranes of a type IV collagen network composed of alpha 3, alpha 4, and alpha 5(IV) chains. While this network is not critical for normal glomerulogenesis, its absence appears to provoke the overexpression of other extracellular matrix proteins, such as the alpha 1 and alpha 2(IV) chains, in glomerular basement membranes, leading to glomerulosclerosis. The diagnosis of AS still relies heavily on histologic studies, although routine application of molecular genetic diagnosis will probably be available in the future. Absence of epidermal basement membrane expression of alpha 5(IV) is diagnostic of XLAS, so in some cases kidney biopsy may not be necessary for diagnosis. Analysis of renal expression of alpha 3(IV)-alpha 5(IV) chains may be a useful adjunct to routine renal biopsy studies, especially when ultrastructural changes in the GBM are ambiguous. There are no specific therapies for AS. Spontaneous and engineered animal models are being used to study genetic and pharmacologic therapies. Renal transplantation for AS is usually very successful. Occasional patients develop anti-GBM nephritis of the allograft, almost always resulting in graft loss.

Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.

In this study we examined if an association exists between expression of an alternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and alpha-smooth muscle actin (alpha-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and alpha-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and alpha-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and alpha-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and alpha-SMA in anti-GBM disease and suggest a functional link for these two proteins.

LONG TERM OUTCOME OF RENAL TRANSPLANTATION FOR ALPORT'S SYNDROME

581 PURPOSE: To determine the long term outcome of renal transplantation in patients with Alport's Syndrome (AS). METHODS: A single-center, retrospective review of 1531 renal transplants between 1974 and 1998 identified 38 patients with end-stage renal disease secondary to AS. A total of 48 renal transplants (41 CAD; 7 LRD) were performed in these patients (83% Caucasian; 15% African-American; 2% Hispanic). Endpoints examined included patient and graft survival, incidence of delayed graft function and rejection, death with functioning graft, impact of cyclosporine and race, and causes of graft loss. Maintenance immunosuppression consisted of prednisone and azathioprine only until 1984 when CSA was added to the regimen. Mycophenolate mofetil replaced azathioprine in 1995. Mean follow-up was 59 months (range 1 month to 213 months). (Table)TableRESULTS: Patient and graft survival in recipients with AS were not statistically different from all other recipients. There was no difference in patient survival among different AS racial groups. Incidences of delayed graft function and rejection were similar to other recipients during the same time period. A total of 27 grafts failed; 8 grafts (3 pre-CSA era, 1 retransplant, 1 venous thrombosis) were lost within 1 year. Rejection accounted for 90% of graft failures. Two patients died with functioning grafts. Only one graft failure was due to recurrent anti-GBM nephritis. CONCLUSION: Our results substantiate other reports indicating a 3-4% incidence of post-transplant anti-GBM nephritis. We conclude that over a 24 year period, with changing immunosuppressive regimens, patients with Alport's Syndrome can have excellent patient and graft survival with a low incidence of anti-GBM nephritis.

Glomerular deposition of mannose-binding lectin in human glomerulonephritis.

Mannose-binding lectin (MBL), a member of the collectin family, binds to various oligosaccharides and activates the classical pathway of complement independent from C1q. At present it is unknown whether this so-called lectin pathway of complement activation plays a role in the pathogenesis of human glomerulonephritis. Direct immunofluorescence of 84 renal biopsies using an MBL-specific monoclonal antibody and antibodies directed against IgG, IgA, IgM, C1q, C3, and terminal complement complex (TCC) was performed. Serum MBL levels of 50 patients were determined by enzyme-linked immunosorbent assay. MBL was detected in the glomeruli of patients with lupus nephropathy (15 of 16), membranous nephropathy (10/15), membranoproliferative glomerulonephritis type I (5/6) and anti-GBM nephritis (2/4). MBL deposition paralleled that of immunoglobulins, C1q, C3, and TCC but was less intense as compared to C1q. Focal segmental deposits of MBL were present in focal segmental glomerulosclerosis (4/6), IgA nephropathy (3/11), amyloidosis AL (1/4), and advanced renal fibrosis (2/2). Here MBL staining was identical to IgM and C3 and considered an unspecific entrapment of MBL in sclerotic lesions in these cases. No significant difference in MBL serum levels was observed between normal controls and patients with lupus nephritis, membranous nephropathy, membranoproliferative glomerulonephritis, focal segmental sclerosis, minimal change disease or IgA nephropathy. In patients suffering from membranous nephropathy with (n=10) or without (n=5) glomerular MBL deposits serum creatinine, C3, C4, serum protein, and proteinuria were not statistically different. MBL is present in the glomeruli of patients with glomerulonephritis involving deposition of IgG and activation of the classical pathway of complement. We propose that MBL binds to agalactosyl oligosaccharides of IgG that terminate in N-acetylglucosamine. The extent to which the lectin pathway of complement contributes to overall complement activation in the glomeruli remains unknown, but is likely to be marginal.

Angiotensin II plays a pathogenic role in immune-mediated renal injury in mice.

Several lines of evidence show the importance of angiotensin II (AII) in renal injuries, especially when hemodynamic abnormalities are involved. To elucidate the role of AII in immune-mediated renal injury, we studied anti-glomerular basement membrane (GBM) nephritis in AII type 1a receptor (AT1a)-deficient homozygous (AT1a-/-) and wild-type (AT1a+/+) mice. A transient activation of the renin-angiotensin system (RAS) was observed in both groups of mice at around day 1. A renal expression of monocyte chemoattractant protein-1 (MCP-1) was transiently induced at six hours in both groups, which was then downregulated at day 1. In the AT1a+/+ mice, after RAS activation, the glomerular expression of MCP-1 was exacerbated at days 7 and 14. Thereafter, severe proteinuria developed, and the renal expressions of transforming growth factor-beta1 (TGF-beta1) and collagen type I increased, resulting in severe glomerulosclerosis and interstitial fibrosis. In contrast, glomerular expression of MCP-1, proteinuria, and tissue damage were markedly ameliorated in the AT1a-/- mice. Because this amelioration is likely due to the lack of AT1a, we can conclude that AII action, mediated by AT1a, plays a pathogenic role in anti-GBM nephritis, in which AII may contribute to the exacerbation of glomerular MCP-1 expression. These results suggest the involvement of AII in immune-mediated renal injuries.

Experimental Goodpasture's syndrome in Wistar-Kyoto rats immunized with α3 chain of type IV collagen

Glomerulonephritis and lung hemorrhage of autoimmune Goodpasture syndrome develop due to immune reactions against epitope(s) of the non-collagenous (NC1) domain of alpha3-chain of type IV collagen [alpha3(IV) NC1]. Whether thymic mechanisms have a role in the loss of tolerance to the Goodpasture epitope has not been established. We studied the renal and pulmonary effects of immunization with different forms (monomer, dimer, or hexamer) of alpha3(IV) NC1 collagen in Wistar-Kyoto (WKY) rats, and assessed whether the intrathymic inoculation of the antigen may protect against anti-GBM disease. WKY rats were immunized with bovine alpha3(IV) monomer, dimer, or hexamer, or with alpha3(IV) NC1 synthetic peptide. Renal function, kidney and lung immunohistology, and circulating and tissue bound antibodies to type IV collagen chains were analyzed. Effects of intrathymic inoculation of antigen on subsequent disease induction were analyzed in WKY rats given alpha3(IV) NC1 dimer or GBM preparation intrathymically 48 hours before immunization. Proteinuria, linear IgG deposition in GBM, and crescentic glomerulonephritis developed in WKY rats immunized with alpha3(IV) NC1 dimer or hexamer. Lesions were dose-dependent upon injections of 10 to 100 microgram dimer. The alpha3(IV) NC1 monomer induced less severe proteinuria and no crescents. Pulmonary hemorrhage was detectable in 35% of rats immunized with 25 to 100 microgram alpha3(IV) NC1 dimer; alpha3(IV) synthetic peptide (36 carboxyl terminal) did not induce disease. Rats injected intrathymically with up to 100 microgram alpha3(IV) NC1 dimer or with GBM 48 hours before immunization were not protected against subsequent development of proteinuria and glomerulonephritis. These findings document that glomerulonephritis and lung hemorrhage can be elicited in WKY rats by immunization with alpha3(IV) NC1. Failure of the intrathymic inoculation of antigen to prevent disease suggests that immunological tolerance cannot be achieved by this intervention, in contrast to other autoimmune conditions, and may imply independent roles for cellular and humoral nephritogenic pathways in anti-GBM nephritis.