Excretion of metabolites of prostacyclin and thromboxane by rats with nephrotoxic nephritis: effects of interleukin-1.

1. To obtain direct evidence of abnormal eicosanoid biosynthesis in rats injected with anti-glomerular-basement-membrane antibodies (a-GBM), products derived from thromboxane A2 (TXA2) and prostacyclin (PGI2) were measured in 24 h urine collections before and after a-GBM. 2. Administration of a-GBM (9.5 mg) caused albuminuria, decreased creatinine clearance, increased numbers of intra-glomerular neutrophils and increased excretion of TXB2, 2,3-dinor-TXB2 (products of TXA2) and 6-oxo-PGF 1 alpha and 2,3-dinor-6-oxo-PGF 1 alpha (products of PGI2) at 24 h. 3. Interleukin-1 (IL-1 beta; 5 micrograms) alone caused an increase in PGI2 metabolite excretion but had no effect on TXA2 metabolites. It had no effect on creatinine clearance but increased numbers of glomerular neutrophils by approximately 4-5 fold compared to a-GBM. 4. Pretreatment of rats with IL-1 beta before a-GBM synergistically increased albumin excretion but only additively increased eicosanoid excretion. Numbers of intra-glomerular neutrophils and creatinine clearance were unchanged compared to IL-1 beta alone. 5. The cyclo-oxygenase inhibitor, ibuprofen (10 mgkg-1 i.p., twice daily for 4 days) inhibited both serum TXB2 production and urinary prostaglandin excretion. It also caused an almost complete attenuation of albumin excretion. Creatinine clearance and glomerular neutrophils remained unchanged after a-GBM/IL-1 beta. 6. We conclude that the 50% inhibition of thromboxane production induced by ibuprofen does not modify the fall in creatinine clearance of accumulation of neutrophils in the glomerulus caused by the a-GBM. This degree of inhibition of eicosanoid production was associated with a striking decrease in proteinuria, but this may reflect a haemodynamic rather than a disease modifying action.

Nomenclatures of anatomical distortions of the spine: a comparison.

A review of the major chiropractic, osteopathic and medical nomenclatures for describing anatomical distortions of the spine is presented. A survey of the institutions demonstrates 10 separate nomenclatures currently being employed by the three professions to describe similar anatomical distortions of the spine. This wide variety of nomenclature can only be seen as creating barriers to communication both inter- and intraprofessionally. Because there are two differing conceptual principles for the definition of an anatomical distortion of the spine, an argument is presented for the adoption of two universal nomenclatures.

Extraglomerular distribution of immunoreactive Goodpasture antigen.

The distribution of Goodpasture antigen (GA) was studied in a range of human tissues using indirect immunofluorescence and immunoperoxidase techniques. Frozen sections were stained using (1) a mouse monoclonal antibody (P1) raised against the autoantigenic component of human glomerular basement membrane, (2) autoantibodies eluted from the kidneys of patients with Goodpasture's syndrome, (3) antibodies eluted from the kidneys of a sheep with Steblay nephritis, and (4) mouse monoclonal and guinea pig polyclonal antibodies to human type IV collagen. The same pattern of staining was demonstrated using the eluted antibodies and monoclonal antibody P1. The presence of GA was confirmed in the lung and choroid plexus. GA was also detected in basement membranes at a number of previously unreported sites in the eye, thyroid, pituitary, adrenal, breast, and liver. GA was absent from other sites at which type IV collagen could be demonstrated. Direct immunofluorescence studies of tissue from a patient with Goodpasture's syndrome revealed deposition of IgG in the choroid plexus and eye, as well as in the kidney and lung.

Progressive systemic sclerosis: autoimmune arteriopathy.

In a case of progressive systemic sclerosis (PSS), widespread deposition of IgM was found in the media and internal elastic lamina of small muscular arteries. IgA and C3 were also found in some vessels. Antibody eluted from the kidney bound to smooth muscle and elastica of small arteries. These findings suggest that PSS is a vasculopathy which is mediated by complement-fixing antibodies.

Experimental interstitial renal fibrosis in rats: nephritis induced by N-(3,5-dichlorophenyl)succinimide.

The nephrotoxic properties of the chemical N-(3,5-dichlorophenyl)-succinimide were investigated in rats with a view to establishing the usefulness of this chemically-induced nephritis as a model of chronic interstitial renal fibrosis. The compound was synthesized and given daily by gastric intubation as a suspension in arachis oil B.P. to male WAG-strain rats, for periods of up to 108 days. Polydipsia and polyuria resulted rapidly in all treated animals and persisted for the duration of the experiment. There was a progressive increase in the extent of proteinuria in all treated animals and, by the end of the experiment, there was an increase in the plasma levels of urea and creatinine. Short term treatment (up to 3 days) resulted in focal areas of necrosis of some proximal convoluted tubules. Treatment for 28 days resulted in patchy but severe tubular interstitial nephritis with which was associated a moderate interstitial fibrosis. By 108 days, the nephritis was more widespread and the interstitial fibrosis was severe. The activity of proline hydroxylase, a part of the intracellular sequence of collagen synthesis, showed progressive increase in the renal cortex throughout the experiment and there was an associated increase in the cortical hydroxyproline content, a measure of the amount of collagen present. Associated with this biochemical evidence of an active, chronic fibrosis, was an increased water content of the cortical tissue. The results indicate that this chemically-induced, tubular interstitial nephritis is indeed a good and reliable model of interstitial renal fibrosis.

Cyclosporin A in the prevention and treatment of experimental autoimmune glomerulonephritis in the brown Norway rat.

Experimental autoimmune glomerulonephritis (EAG) was induced in brown Norway (BN) rats by a single i.m. injection of collagenase-solubilized homologous glomerular basement membrane (GBM) in Freund's complete adjuvant. This model of anti-GBM disease is characterized by the development, over several weeks, of circulating and deposited anti-GBM antibodies, accompanied by albuminuria. We examined the effects of treatment with oral cyclosporin A (CsA) at different doses, starting at the time of immunization and during the course of the disease. Pretreatment with CsA 5 mg kg daily produced a moderate reduction in circulating anti-GBM antibody levels, reduced deposition of antibody on the GBM and decreased albuminuria. Doses of 10 and 20 mg/kg CsA produced a marked reduction in circulating antibody, absence of detectable deposited antibody and virtual absence of albuminuria. Renal function remained normal in CsA-treated and control animals. When CsA treatment was introduced at 2 or 4 weeks after immunization, there were significant effects on the subsequent autoimmune response and albuminuria at 10 and 20 mg/kg daily. These studies demonstrate that CsA in conventional doses has a therapeutic effect in this model of anti-GBM disease, and suggest a role for T lymphocytes in the pathogenesis of EAG.

Experimental autoimmune glomerulonephritis (EAG) induced by homologous and heterologous glomerular basement membrane in two substrains of Wistar-Kyoto rat.

BACKGROUND: Goodpasture's, or antiglomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis, and is caused by auto-immunity to the NC1 domain of the alpha 3 chain of type IV collagen. In order to investigate mechanisms involved in the induction and regulation of glomerulonephritis, experimental models of Goodpasture's disease have been developed in the rat which share many characteristics with the human disease. Induction of experimental autoimmune glomerulonephritis (EAG) involves immunization of susceptible strains with either heterologous or homologous GBM in FCA. However, pathological changes have tended to be mild and/or variable, except in certain protocols using Wistar-Kyoto (WKY) rats. METHODS: We studied the susceptibility of inbred WKY rats from two different suppliers to the development of EAG. These substrains of rat had different MHC haplotypes (WKY/CR, RT1-1; WKY/Olac, RT1-k), so we proposed that they might show differences in their immune response to GBM antigens. Both substrains were immunized with sheep GBM, pH 7, or rat GBM buffered to pH 3, pH 5 or pH 7. RESULTS: All immunized rats developed circulating anti-GBM antibodies detectable at 14 days and rising until 28 days, at which time there was linear deposition of IgG on the GBM. WKY/CR rats developed severe focal segmental proliferative and necrotizing glomerulonephritis, with heavy albuminuria, following immunization with rat GBM, pH 7, but only moderate disease following sheep GBM. WKY/Olac rats showed a more variable response, with moderate disease following both rat and sheep GBM. Immunization of either substrain with rat GBM, pH 5, produced a response similar to that with rat GBM, pH 7, but disease was mild following rat GBM, pH 3. CONCLUSION: EAG in the WKY rat varies in severity according to the substrain of animal and preparation of GBM used for immunization. The model with the most severe and consistent changes was that induced in the WKY/CR rat by rat GBM at pH 7. This model of EAG will be of value for investigating mechanisms of autoimmunity and inflammation in glomerulonephritis, and for attempting novel forms of immunotherapy prior to trials in man.

A single-chain Fv reactive with the Goodpasture antigen.

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

Glomerular expression of interleukin-1 receptor antagonist and interleukin-1 beta genes in antibody-mediated glomerulonephritis.

Interleukin-1 (IL-1) is a powerful proinflammatory cytokine whose function is modulated by a natural IL-1 receptor antagonist (IL-1ra). There are few data about kinetics of in vivo synthesis of IL-1ra at tissue level, except in response to bacterial endotoxin. The purpose of this study was to examine the kinetics of local expression of IL-1ra gene in relation to IL-1 beta gene in a model of anti-glomerular basement membrane antibody-mediated glomerulonephritis. Rats were killed in groups of 5 or 6 at 0, 4, 6, 24, 48, and 96 hours after induction of glomerulonephritis. Messenger RNA for IL-1ra and IL-1 beta was undetectable by Northern blot in normal glomeruli but increased markedly 4 to 6 hours after induction of nephritis. The increase in IL-1ra mRNA was more sustained than that of IL-1 beta mRNA. In situ hybridization showed that IL-1 beta mRNA increased diffusely within glomeruli, while IL-1ra mRNA was expressed more discretely. Expression of these mRNA in noninflamed tissues, spleens and lungs, was different, particularly increase in IL-1ra mRNA was more substantial than that of IL-1 beta. These observations suggest that differential expression of IL-1ra and IL-1 beta might focus inflammation in glomeruli while protecting more distant sites. They also raise the possibility of reducing glomerular injury by therapeutic measures that upregulate glomerular synthesis of IL-1ra while reducing that of IL-1 beta.

Variability of the antigenicity of the glomerular basement membrane in nail-patella syndrome.

The difficulty of diagnosing Alport-type nephritis, and the possibility of altered antigenicity of the glomerular basement membrane in Alport-type nephritis is discussed. Attention is focused on the monoclonal antibody MCA-Pl as a specific tool in studying the Goodpasture antigenetic determinant in renal biopsy specimens. The clinical details and the results of MCA-Pl binding studies for three patients with nail patella syndrome are presented. Two of these patients proved to have an abnormal antigenicity of the glomerular basement membrane as demonstrated by the absence of binding of MCA-Pl. This is the first time that such an abnormality has been demonstrated in patients other than those with Alport-type nephritis. The significance of these results is discussed.

Passive immunization against tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta protects from LPS enhancing glomerular injury in nephrotoxic nephritis in rats.

Glomerular injury caused by injection of heterologous anti-glomerular basement membrane antibodies (anti-GBM Ab) is increased in rats pretreated with small doses of bacterial lipopolysaccharide (LPS). We have investigated the involvement of tumour necrosis factor-alpha (TNF-alpha), IL-1 alpha and IL-1 beta in this phenomenon by passive immunization against these cytokines. Anti-TNF-alpha or anti-IL-1 beta antibodies given 1.5 h before the induction of nephritis significantly decreased injury in this model, whether assessed by the magnitude of albuminuria, the prevalence of glomerular capillary thrombi or the intensity of glomerular neutrophil infiltrate. Albuminuria in anti-GBM Ab alone was 11 +/- 3, LPS/anti-GBM Ab 87 +/- 22, and anti-TNF-alpha antibodies/LPS/anti-GBM Ab 21 +/- 6 mg/24 h (mean +/- s.e.) P < 0.05. Passive immunization with antibodies to IL-1 beta had a similar effect (anti-GBM Ab, 0.6 +/- 0.1, LPS/anti-GBM Ab, 92 +/- 19, anti-IL-1 beta antibodies/LPS/anti-GBM Ab 39 +/- 8 mg/24 h, P < 0.05). The prevalence of glomerular capillary thrombi was also reduced significantly by these treatments; from 22 +/- 5% to 4 +/- 1% in the case of anti-TNF-alpha antibodies and 28 +/- 5% to 13 +/- 4% with anti-IL-1 beta antibodies. Similarly, the glomerular neutrophil infiltrate was also reduced by these treatments; from 42 +/- 3 to 25 +/- 1 in the case of anti-TNF-alpha and 47 +/- 2 to 30 +/- 1 with anti-IL-1 beta antibodies. In contrast, passive immunization against IL-1 alpha had no effect on either albumin excretion (4 +/- 3, 83 +/- 22 and 77 +/- 24 mg/24 h), glomerular capillary thrombi (2 +/- 1; 19 +/- 5 and 16 +/- 3) or glomerular neutrophil infiltrate (22 +/- 3; 47 +/- 5 and 48 +/- 5 from the three groups respectively). These results demonstrate that enhanced antibody mediated injury in the kidney is modulated by TNF-alpha and IL-1 beta but not by IL-1 alpha.

Experimental autoimmune glomerulonephritis induced by homologous and isologous glomerular basement membrane in Brown-Norway rats.

In order to study disease mechanisms and potential forms of therapy in glomerulonephritis, a model of experimental autoimmune glomerulonephritis (EAG) has been developed in the rat. We have examined the response of Brown-Norway (BN) rats to a single i.m. injection of collagenase-solubilised homologous (Sprague-Dawley, SD) or isologous (BN) glomerular basement membrane (GBM), with and without complete Freund's adjuvant (CFA). There was a dose-dependent circulating anti-GBM antibody response to all preparations of rat GBM. Animals given either antigen alone at a dose of 2 mg/kg developed circulating anti-GBM antibodies, which reached peak values by 6 weeks (63 +/- 5% following SD GBM; 53 +/- 8% following BN GBM), but did not develop glomerular deposits of IgG or nephritis. Animals given 2 mg/kg SD GBM in CFA developed greater concentrations of anti-GBM antibody by 6 weeks (122 +/- 20%) together with linear deposits of IgG on glomerular and tubular basement membranes (TBM), albuminuria (mean 7 mg/24 h), and variable focal segmental necrotising glomerulonephritis with mild interstitial nephritis. The same dose of BN GBM in CFA produced similar concentrations of circulating antibody (144 +/- 26%), with linear deposits of IgG on GBM but rarely TBM, little albuminuria, and variable mild focal glomerulonephritis. Other strains injected with SD GBM in CFA showed a variable circulating anti-GBM antibody response, which was similar to that of BN rats in PVG and DA rats but lower in LEW and WAG rats. Linear deposits of IgG on the GBM were detected in a proportion of PVG and DA rats, but not in LEW or WAG rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivity of monoclonal antibody P1 with glomerular basement membrane in thin-membrane nephropathy.

A monoclonal antibody, P1, possessing antiglomerular basement membrane specificity similar to that of naturally-occurring Goodpasture antibody, was used to study renal biopsy material from patients with thin-membrane nephropathy. Immunofluorescence and immunoperoxidase techniques were employed to detect tissue localisation after sections had been incubated with P1. Staining of glomerular and tubular basement membranes in the 14 patients with thin-membrane nephropathy was similar to that in 10 subjects with various other renal diseases, whereas three patients with Alport's syndrome all gave diminished or absent staining, as has been reported previously. These observations confirm that Goodpasture antigen is present in the basement membranes in thin-membrane nephropathy and that it reacts normally with P1 antibody. They also add to the evidence that the lesions of thin-membrane nephropathy and Alport's syndrome are fundamentally different. The staining method may be used as a differential diagnostic test.

Modulation of antibody-mediated glomerular injury in vivo by bacterial lipopolysaccharide, tumor necrosis factor, and IL-1.

We have investigated the effects of LPS, human rTNF (hrTNF) and human rIL-1 beta (hrIL-1 beta) pretreatment on the intensity of antibody-mediated injury in vivo by using a passive model of anti-glomerular basement membrane (GBM) antibody-mediated nephritis in rats. The experiments show that all three pretreatments exacerbate injury in this model whether judged by albuminuria or the prevalence of glomerular capillary thrombi. The effect on albuminuria was dose dependent with all three treatments. The lowest effective dose of LPS was 0.025 microgram while those for hrTNF and hrIL-1 beta were 0.4 microgram and 0.5 microgram, respectively. All three pretreatments also increased the prevalence of glomerular capillary thrombi which were rare in rats injected with anti-GBM antibodies without pretreatment. LPS pretreatment appeared to be more effective in causing glomerular capillary thrombi than hrTNF or hrIL-1 beta and this was reflected in the correlations between albuminuria and the proportion of glomeruli with capillary thrombi. This relation was linear for all three pretreatments but the slope was appreciably greater for rats pretreated with LPS (0.37) when compared with results from rats given either hrTNF (0.22) or hrIL-1 beta (0.29). Pretreatment of nephritic rats with both cytokines increased the slope to 0.42 demonstrating a synergistic effect. The synergism of hrTNF with hrIL-1 beta was also demonstrated by the effective doses needed to induce albuminuria which was evident in rats treated with 0.05 microgram of IL-1 beta and 0.4 microgram of TNF. Neither the cytokines nor LPS caused clinical, morphologic, or biochemical evidence of renal toxicity when given alone in the dose used here but they did cause a transient increase in the number of neutrophils marginated in glomerular capillaries. Pretreatment of rats with LPS or cytokines increased the glomerular neutrophil influx after anti-GBM antibodies by roughly sixfold. Our experiments show that TNF and IL-1 can increase the severity of glomerular injury in nephritis; they may be important in modulating glomerular injury clinically.

A detailed examination of the clinical terms and concepts required for communication by electronic messages in diabetes care.

The wider electronic exchange of clinical information between heterogeneous information systems in the delivery of diabetes care demands a common structure in the form of a message standard. A European Standard electronic diabetes message is being developed in conjunction with CEN TC251. This paper describes the methodologies that the 1998 DO IT Workshop has used to identify potential areas of difficulty in the design and implementation of the preliminary message model. To facilitate implementation and to avoid ambiguity in electronic messaging it is particularly important that there is standardisation of the definitions of the clinical terms specifically used in diabetes care across systems. Comprehensive lists of such terms to describe all areas of diabetes care do not exist and there is a lack of harmonisation of definitions in many areas. Thus, to better understand the user requirements of diabetes messaging several approaches were adopted. A review of the clinical terms and concepts contained in pre-existing datasets was undertaken with detailed study of a number of specific areas of diabetes care, analysing the conceptual structure of all the clinical terms that they comprised. Consideration of several worst case clinical scenarios for messages to communicate was also made to identify deficiencies in the message structure. This activity confirmed the importance of creating a Standard for a superset or thesaurus of diabetes specific terms, with appropriate definitions, to harmonise data communication in different IT systems to facilitate messaging. A substantial number of new terms were identified in the workshop and these will form an important first step to accomplishing a first draft superset once fully analysed. It was also apparent that certain specific areas within diabetes care, but most particularly in nursing, dietetics and podiatry, need urgent work to further develop the concepts and terms. This needs to be facilitated for an appropriate group of such professionals. To achieve such a Standard, continued co-operation with CEN/ISSS was recognised to be very important.

The Goodpasture antigen in Alport's syndrome: studies with a monoclonal antibody.

A mouse monoclonal antibody (MCA-P1), which recognizes an antigenic determinant in human glomerular basement membrane against which autoantibodies are directed in Goodpasture's syndrome, was used in indirect immunofluorescence studies to investigate glomerular basement membrane structure in Alport's syndrome. We found reduced or absent binding of MCA-P1 to glomerular and distal tubular basement membranes in renal biopsy tissue from ten patients with Alport's syndrome. Antiglomerular basement membrane antibody eluted from the kidneys of a patient who had died from Goodpasture's syndrome was used to confirm these findings. In contrast, there was bright linear fluorescence of MCA-P1 on glomerular and tubular basement membranes of normal renal material and renal biopsy tissue obtained from patients with a variety of glomerulonephritides. These results suggest an abnormality or a variable quantity of the immunoreactive autoantigen in the glomerular basement membrane of patients with Alport's syndrome. Furthermore, MCA-P1 may be of value in the diagnostic interpretation of renal biopsies from patients with familial nephritis.