Molecular mechanisms of glomerular injury in rat experimental membranous nephropathy (Heymann nephritis)

The molecular pathogenesis of human membranous nephropathy (MN) is unknown, despite the relatively high incidence and severity of this glomerular immune disease. Heymann nephritis (HN) in rats is considered an instructive experimental model of MN. This study summarizes current molecular aspects of two key events common to both MN and HN, i.e., formation of characteristic subepithelial immune deposits in the glomerular basement membrane (GBM), and development of glomerular capillary wall damage resulting in proteinuria. In HN, the antigenic targets of immune deposit-forming antibodies were identified in cell membranes of glomerular epithelial cells as a 515-kd glycoprotein (megalin, or gp330), which is a polyspecific receptor related to the low-density lipoprotein receptor family, and an associated 44-kd protein (receptor associated protein, RAP). One epitope was recently narrowed to 14 amino acids in RAP, and several others on megalin/gp330 are under investigation. Proteinuria requires formation of the complement C5b-9 membrane attack complex, which is presumably triggered by antibodies directed against lipid antigens that associate with immune deposit-forming megalin/gp330 immune complexes. Sublytic C5b-9 attack on glomerular epithelial cells causes upregulation of expression of the NADPH oxidoreductase enzyme complex by glomerular cells, which is translocated to their cell surfaces, similar to activated neutrophil granulocytes in the respiratory burst reaction. Subsequently, reactive oxygen species (ROS) are produced locally, which reach the GBM matrix. Here formation of lipid peroxidation (LPO) adducts is found, preferentially on monomeric and dimerized NCl domains of covalently crosslinked Type IV collagen. These structural changes within the GBM could be of functional relevance because treatment with the potent LPO-antagonist probucol reduces proteinuria by < 80%. Intact or fragmented apoprotein E-containing lipoproteins were identified as potential sources of the polyunsaturated lipids required for the production of LPO adducts. Lipoproteins accumulate within immune deposits and show signs of oxidative damage, similar to oxidized LDL within atherosclerotic lesions. Collectively, the results obtained so far in HN permit the compilation of a sequence of events, linking formation of immune deposits with proteinuria. However, despite this relatively detailed knowledge of pathogenic events in HN, the bridge to human NM remains to be built.

Mast cells and type VIII collagen in human diabetic nephropathy.

Renal injury in diabetes mellitus is associated with progressive interstitial fibrosis and extracellular matrix accumulation. However, the phenotypes of cells forming the interstitial infiltrate in diabetic nephropathy have not been precisely defined. There is increasing evidence for the association of mast cells with angiogenesis, chronic inflammatory conditions and fibrosis. We have recently shown that human mast cells can produce the non-fibrillar short chain type VIII collagen in vivo. Using immunohistochemistry, in situ hybridisation and reverse transcriptase-polymerase chain reaction, we examined the contribution of mast cells and type VIII collagen to the fibrotic changes occurring in biopsy-proven diabetic nephropathy. We observed that the number of interstitial mast cells was significantly increased in diabetic nephropathy compared with normal kidney tissue. In specimens from diabetic subjects, intense immunohistochemical staining for type VIII collagen was detected in mast cells, on periglomerular fibres and in perivascular and interstitial sites. The expression of type VIII collagen in periglomerular and interstitial sites coincided with that of alpha smooth muscle actin, a marker for myofibroblastic differentiation mRNA for type VIII collagen was detected by reverse transcriptase-polymerase chain reaction in diabetic nephropathy and in a human mast cell line. By in situ hybridisation the transcripts for type VIII collagen were localised to renal mast cells. The increased number of mast cells and the elevated type VIII collagen deposition in human diabetic nephropathy provides a potential link between the extracellular matrix accumulation and the fibrosis observed in this condition.

Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy.

Measurement of immune markers in the serum and cerebrospinal fluid of multiple sclerosis patients during clinical remission.

Magnetic resonance imaging of multiple sclerosis (MS) patients often shows active inflammatory lesions despite clinical remission. No immunological marker of disease activity has been identified in these patients. Concentrations of neopterin, interleukin-2 (IL-2), soluble interleukin-2 receptor (sIL-2R) and tumour necrosis factor-alpha (TNF-alpha) were measured in the serum and cerebrospinal fluid of 19 clinically-inactive MS patients and compared with those of 19 non-inflammatory controls. Cerebrospinal fluid (CSF) neopterin concentrations were significantly higher in the MS group than in controls (mean 9.1 mM vs 3.4 nM, P < 0.01) and 10 of 19 MS patients had levels above the control range. This finding provides evidence of ongoing T-cell-directed and interferon-gamma-mediated macrophage activation in the central nervous system. Analysis of IL-2, sIL-2R and TNF-alpha concentrations revealed no significant differences between MS patients and controls. We conclude that CSF neopterin concentration may correlate with disease activity in asymptomatic patients.

Human mast cells produce type VIII collagen in vivo.

Mast cells are assuming importance not only in their familiar role in acute allergic and parasitic diseases but also in chronic inflammatory, immunologic and fibrotic states. The processes by which human extracellular matrices are influenced by mast cells have remained obscure. We report here the production of type VIII collagen by human mast cells. Mast cells representing each of the known phenotypes were identified in a variety of tissues using histochemical techniques, and monoclonal antibodies specific for tryptase, chymase, and c-kit. Mast cells in normal and pathologic tissues expressed type VIII collagen alpha-1 chain protein and mRNA, detected by immunohistochemistry using monoclonal and polyclonal antibodies, and non-isotopic oligonucleotide in situ hybridization using digoxigenin-labelled oligonucleotide probes based on the published human alpha-1 collagen VIII sequence. Perivascular location of type VIII collagen positive mast cells was a striking finding. The secretion of type VIII collagen by mast cells in vivo may contribute to angiogenesis, tissue remodelling, and fibrosis.

Immunoassay of platelet-derived growth factor in the blood of patients with diabetes mellitus.

Platelet-derived growth factor (PDGF) is a powerful mitogen for many cell types, and is believed to play a major role in wound healing when released from platelets at sites of injury. In diabetes mellitus, it has been proposed that premature release of PDGF from platelets impairs the ability of platelets to initiate healing, and also accelerates the development of diabetic complications such as angiopathy by increasing plasma-borne PDGF. However, plasma samples from diabetic patients have not previously been assayed for PDGF using suitable techniques. A sensitive monoclonal enzyme-linked immunoassay for PDGF was applied to plasma and serum samples from 18 healthy control subjects and 60 diabetic patients. Neither plasma nor serum PDGF concentrations differed significantly between control subjects, insulin-dependent, and non-insulin-dependent diabetic patients. However, 23% of the diabetic subjects had serum PDGF levels above the control range. Limited joint mobility, which is characterised by joint contractures and collagen deposition in the skin, and is associated with microvascular disease, was used as a marker of diabetic complications. Limited joint mobility affected 43% of the diabetic subjects. Patients with moderate limited joint mobility had had diabetes significantly longer than those without limited joint mobility (means 17 years and 9 years, respectively, p = 0.008). However, limited joint mobility was not associated with elevated serum or plasma PDGF in insulin-dependent or non-insulin-dependent diabetes. We conclude that complications of diabetes are unlikely to be caused by changes in systemic levels of PDGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the ability of probucol to inhibit proteinuria support a causal role for LPO in the the alteration of the glomerular permselectivity which results in proteinuria.

The immunolocalization and urinary quantitation of 7-5Q/A, a unique human renal antigen.

In this communication 7-5Q/A, a recently described human kidney-specific acidic membrane glycoprotein, was immunolocalized by immunofluorescence in kidney and placenta, and by immunogold electron microscopy in the kidney. The antigen is located above and below the slit diaphragm on epithelial cell foot processes in the glomerulus, and also on proximal tubular brush border membranes. In the placenta it was present on epithelial membranes. 7-5Q/A and dipeptidylpeptidase IV were compared by a variety of techniques because of their similar size and possible location, and found to be distinct. A sensitive sandwich ELISA was developed using polyclonal and monoclonal antibodies to 7-5Q/A, and applied to the quantitation of this antigen in the urines and sera of patients with a variety of kidney diseases, and in the urines and sera of normal subjects. The concentration of 7-5Q/A in normal urines ranged from 0 to 2 ng/ml but levels of up to 150 ng/ml were found in urines of patients with renal disease. 7-5Q/A was not detectable in either normal or nephritic sera. Urinary quantitation of this unique antigen may be of value in the early diagnosis of renal disease.

Immunoassay of platelet-derived growth factor in the plasma of patients with scleroderma.

It has been postulated that platelet-derived growth factor (PDGF) is responsible for the abnormal fibroblast proliferation observed in scleroderma. In one previous study, plasma samples from patients with scleroderma caused increased mitogenesis in cultured fibroblasts, suggesting that the pathogenesis of scleroderma is related to increased plasma PDGF concentrations. To test this hypothesis, we used a sensitive, monoclonal antibody-based ELISA to measure PDGF in the plasma of 12 scleroderma patients. A rigorous sampling protocol prevented false elevations in plasma PDGF levels from ex vivo platelet degranulation: beta-thromboglobulin concentrations were measured in each plasma sample to monitor platelet lysis. Plasma PDGF concentrations in the scleroderma patients were not statistically different from those observed in age- and sex-matched normal controls, and patients with RA. While it is possible that changes in PDGF activity at a local level alter fibroblast function, we cannot conclude that elevated plasma concentrations of PDGF play a role in the pathogenesis of scleroderma.

Urinary neopterin quantification indicates altered cell-mediated immunity in healthy subjects under psychological stress.

In an effort to quantify changes in cell-mediated immunity (CMI) in healthy subjects under stress, we measured levels of neopterin, a well-validated marker of CMI activation, in the urine of medical students undergoing academic examinations. Neopterin/creatinine ratios measured on the first day of examinations (mean 46 mumol/mol) were significantly lower than those measured two weeks before (mean 78 mumol/mol, p = .004). Minimum neopterin production coincided with maximum subjective stress, as measured by a visual analogue scale. After examinations, neopterin/creatinine ratios rose (means 62 mumol/mol immediately after, and 65 mumol/mol two weeks after examinations), and these levels were not statistically different from those two weeks before examinations. Over this post-examination period, subjective distress was significantly lower than at either time point before examinations. We conclude that urinary neopterin/creatinine ratios may change significantly during periods of psychological stress, indicating concomitant alterations in CMI activation.

Reactive oxygen species and neutrophil respiratory burst cytochrome b558 are produced by kidney glomerular cells in passive Heymann nephritis.

Reactive oxygen species (ROS) have been implicated in the production of glomerular damage in passive Heymann nephritis (PHN), an experimental form of membranous nephropathy with neutrophil-independent proteinuria. Immunohistochemistry with monoclonal antibodies specific for cytochrome b558 (a major component of the oxidoreductase complex of the respiratory burst in stimulated neutrophilic granulocytes) showed that this enzyme is localized within visceral glomerular epithelial cells (GECs) in a dense, granular pattern in rats with PHN and proteinuria. By immunoelectron-microscopy, the cytochrome was found in membrane vesicles within the GEC and also extracellularly on the GEC membranes facing the glomerular basement membrane (GBM). By immunoblotting, cytochrome b558 was detected in highest concentration in lysates of isolated glomeruli from proteinuric rats. By contrast, only traces were found in normal glomeruli by immunohistochemistry. Depletion of complement abolished the expression of the cytochrome. Using an ultrastructural cerium-H2O2 histochemistry technique, the functional activity of the glomerular ROS-generating system was demonstrated exclusively in proteinuric PHN, where H2O2 was found in highest concentration within the GBM. These results provide evidence that in rats with PHN and proteinuria, the GECs express and externalize respiratory-burst enzymes that generate ROS in a manner similar to neutrophilic granulocytes, which could then lead to glomerular damage.

Familial cystic nephroma and pleuropulmonary blastoma.

BACKGROUND: Cystic nephroma (CN) and pleuropulmonary blastoma (PPB) are rare tumors without any previously recognized familial association. METHODS: Two cases of CN and one case of PPB in three siblings are reported. RESULTS: A 27-month-old girl and a 31-month-old boy underwent nephrectomy for CN and are free of disease in the contralateral kidney 16 and 14 years later, respectively. Their 28-month-old sister underwent pleuropneumonectomy with postoperative chemotherapy for PPB and died of recurrent disease 9 months later. CONCLUSION: To the knowledge of the authors, these cases represent the first reported familial occurrence of CN and the second of CN and PPB among siblings. The inheritance of a germline mutation predisposing to the development of these tumors is postulated.

Cognitive function and quality of life in end-stage renal failure.

The Sickness Impact Profile (SIP) and the Wechsler Memory Scale--Revised (WMS-R) were administered to a small sample of end-stage renal failure patients. The memory test successfully discriminated between patients who were rated by their nurses to be well adjusted or poorly adjusted to dialysis treatment. It is concluded that this instrument may be useful in investigating cognitive function in this patient population.

Neopterin measurement provides evidence of altered cell-mediated immunity in patients with depression, but not with schizophrenia.

Neopterin is a validated marker of the activation of cell-mediated immunity in a variety of disease states. We measured neopterin and creatinine concentrations in the plasma and urine of 22 schizophrenic and 26 depressed patients admitted acutely to hospital, and compared results with those in a large group of normal controls. Neopterin/creatinine ratios were normal in the schizophrenic patients, but significantly elevated in the plasma of depressed patients. In each diagnostic group, the use of psychotropic drugs before admission had no effect on the neopterin ratios observed. Our findings indicate altered cell-mediated immunity in depression.

Cleavage of type VIII collagen by human neutrophil elastase.

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.

Effects of steroid hormones and immunosuppressant drugs on defences against uterine microbial contamination in the rat.

This study used rats as a mammalian model to investigate the effects of steroid hormones and immunosuppressant drugs on uterine microflora. It was shown that prednisolone acetate predisposed to uterine contamination unlike oestradiol, ciclosporin and cyclophosphamide. It therefore appears that the leucocyte component of the immune system does not normally play a role in preventing bacterial colonization of the uterus and that prednisolone is producing its effect via some other mechanism, possibly an effect on cervical mucus.

Neopterin quantification in acute severe asthma.

To investigate the pathogenic role of cell-mediated immunity (CMI) in acute severe asthma, 10 patients with acute asthma requiring admission to hospital had measurements of serum and urine absolute neopterin and neopterin/creatinine ratios. Serum was collected upon entry to hospital, 24 hr later and at 2 weeks; and urine at 24 hr and at 2 weeks after admission. Comparisons were made with 12 stable asthmatic patients, healthy control subjects, and six renal transplant patients undergoing transplant rejection. In contrast to the renal transplant patients who demonstrated T-lymphocyte-macrophage activation, neopterin concentrations and neopterin/creatinine ratios in both acute and stable asthmatic patients were not elevated. We have not found functional evidence that CMI participates in the pathogenesis of acute severe asthma.

Diabetic end stage renal failure--the Wellington experience 1975-1988.

Since the late 1970s patients with diabetic nephropathy have formed an increasing proportion of new entrants to the Hospital renal dialysis and transplantation programme, reaching 28% for the three year period to December 1988. Between 1 January 1975 and 31 December 1988, 87 diabetic patients were accepted for treatment. Fifty-one per cent were European, predominantly type I diabetics. Maori (9% of the total reference population) accounted for a disproportionately high 47% due to an over-representation by type II diabetic patients (34 of 41 Maori). These findings cannot be explained by the higher prevalence in Maori of type II diabetes but appear to be due to a more prevalent and/or aggressive diabetic renal lesion in this group. On commencing treatment, nearly all patients had retinopathy and the majority had evidence of peripheral vascular disease, hypertension and neuropathy. CAPD was the initial mode of renal replacement therapy in 70% of patients. Overall patient survival was 77% at one year and 42% at three years, and survival on CAPD was 76% and 37% at one and three years, respectively. Patient survival on transplantation was 63% at one year and 58% at three years. Graft survival was 51% at one year and 46% at three years. Although the short term outlook for diabetic patients on renal replacement therapy is encouraging, longer term survival compared to non-diabetic patients is poor. Vascular disease is the major cause of death and an important factor in patient morbidity.

Acute pulmonary and renal injury after administration of heterologous anti-lung antibodies in the rat. Characterization of ultrastructural binding sites, basement membrane epitopes, and inflammatory mediation systems.

Male Sprague-Dawley rats developed acute lung and renal injury after administration of heterologous anti-lung antibody. Both organs demonstrated an increase in protein permeability after antibody binding to basement membrane (BM) antigens (lung permeability index 0.342 +/- 0.009 versus control 0.214 +/- 0.011: p less than 0.05. Urinary protein excretion 5.112 +/- 0.899 mg/hour versus control 0.402 +/- 0.008 mg/hour: p less than 0.01). The threshold value for the development of lung injury was 27.2 +/- 4.8 micrograms of antibody globulin/g of tissue (micrograms/gm). Immunoblot analysis probing with the anti-lung antibody revealed at least one common antigenic determinant (82 to 84 kilodaltons) bound within collagenase-solubilized pulmonary and glomerular BMs. Increasing doses of antibody produced hemorrhagic pneumonitis and diffuse alveolar damage. Immunofluorescence microscopy confirmed linear alveolar and glomerular BM antibody binding. Immunogold electron microscopy allowed precise identification, in intense linear patterns, of BM binding sites within lung and glomeruli. Functional lung injury was prevented by either leukocyte-depletion or complement-depletion (lung permeability index antibody-treated, complement-depleted 0.235 +/- 0.034: both p greater than 0.05 compared with controls). Injury mediation in this acute humoral model of lung damage is both complement- and leukocyte-dependent, as previously described for the renal component of heterologous anti-glomerular BM antibody-induced inflammatory disease.