The ability to take up substances from the surrounding environment not only provides cells with vital nutrients, but also enables the selective transport of substances from one compartment to another. Megalin and cubilin are two structurally different endocytic receptors that interact to serve such functions. Evidence has accumulated in recent years to indicate that these receptors have important functions in both normal physiology and pathology.

Megalin and cubilin: multifunctional endocytic receptors

Daily injections of any one of several foreign serum proteins produced in rabbits functional and morphological alterations similar to those seen in acute, subacute, and chronic human glomerulonephritis. The critical factor determining whether a rabbit would develop renal disease and the type of disease developed was the amount of antibody the rabbit formed. Those responding with much antibody were likely to develop an acute, self-limited glomerulonephritis and to be subsequently immune to further renal damage. Those responding with antibody barely sufficient to neutralize the antigen injected developed subacute and chronic glomerulonephritis. In the circulation of the rabbits with chronic glomerulonephritis, there was a daily recurring antigen-antibody reaction in the region of near antigen excess to near antibody excess which presumably led to the disease. Antigen apparently in the form of antigen-antibody complexes was deposited along the renal capillary basement membranes coincident with the development of subacute and chronic glomerulonephritis. Once developed, the morphologic stigmata of chronic glomerulonephritis persisted even after injections of antigen were stopped. However, in milder instances the renal function recovered in part after stopping antigen. This experimental model has several implications: first, the renal injury is precipitated by antigens with no known affinity for, or immunologic relationship to, kidney; second, antigen antibody complexes localize in the kidney, apparently on the basis of non-immunologic factors, and may be an etiologic agent of renal injury; third, severe hypersensitivity disorders can be related specifically to relatively poor as well as to good antibody responses; and finally, the pathogenesis suggested here offers an alternative to that of nephrotoxic serum nephritis for the experimental approach to the study of human glomerulonephritis.

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Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis.

Adjuvants--a balance between toxicity and adjuvanticity.

Publisher Summary No experimental model has provided greater insight into the mechanism of immune complex disease than the experimental serum sickness. The morphological, immunohistological, and serological features of the laboratory models have provided a basis for understanding the pathogenic mechanisms responsible for human glomerulonephritis, vasculitis, and a variety of systemic connective tissue diseases. The subject of experimental acute and chronic immune complex disease produced by nonliving antigens has received extensive review in this series. This chapter summarizes the data presented in these two reviews and discusses in detail more recent studies. Experiments to study acute immune complex disease (serum sickness) have been performed in rabbits almost exclusively. Experimental chronic immune complex disease has proved to be a most useful model in understanding human glomerulonephritis. When injected daily with heterologous serum protein antigens, rabbits with strong antibody responses develop chronic membranous glomerulonephritis in about 5 weeks.

Immune Complex Disease in Experimental Animals and Man

The nephritogenic antigen of Heymann's nephritis (HN) was previously purified from tubular brush-border fractions of rat kidney and found to be a 330,000- mol-wt glycoprotein (gp330). This study was conducted to determine whether gp330 is also present in the rat glomerulus, and, if so, to establish where in the glomerulus it is located. Rabbit polyclonal and mouse monoclonal antibodies were raised against purified gp330, which specifically immunoprecipitated gp330 from solubilized brush-border fractions and specifically stained microvilli and coated invaginations (located at the base of the microvilli) of proximal tubule cells. Accordingly, they were used to localize gp330 by immunoprecipitation and immunocytochemistry in glomeruli of normal Lewis rats. 

For immunoprecipitation, purified glomerular fractions were prepared from [(35)S]-methionine-labeled kidneys, extracted with Triton X-100, and the extract was used for immunoprecipitation with affinity-purified rabbit polyclonal, or mouse monoclonal, anti-gp330 IgG. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography indicated that a band corresponding in mobility to gp330 was specifically precipitated. 

When unfixed cryostat sections were incubated for indirect immunofluorescence with monoclonal or affinity-purified polyclonal IgG, a fine granular fluorescent staining was seen throughout the glomerulus. When aldehyde-fixed cryostat sections were incubated for indirect immunoperoxidase, reaction product was detected only in the epithelial cells and was not seen in the GBM, endothelium, or mesangium. Within the epithelium it was localized to the endoplasmic reticulum, occasional Golgi elements, multivesicular bodies, and coated pits at the cell surface. The reactive coated pits were distributed all along the cell membrane, including the sides and base of the foot processes. Reaction product was detected in the latter location only in sections that had been digested with neuraminidase before antibody incubation. When rats were given rabbit anti-gp330 IgG by intravenous injection and their kidneys stained for direct immunoperoxidase 3 d later, rabbit IgG was seen to be deposited beneath the slit diaphragms and in the coated pits at the base of the foot processes. 

The immunocytochemical and immunoprecipitation data indicate, in confirmation of the results of others, that the nephritogenic HN antigen is present in renal glomeruli as well as in proximal tubular brush borders. The immunocytochemical results further demonstrate that gp330 is an epithelial, rather than a glomerular basement membrane, antigen. It appears to be synthesized by glomerular epithelial cells and subsequently becomes concentrated in coated pits. As both the endogenous antigen (gp330) and exogenously administered anti-gp330 antibody were localized to coated pits, it seems likely that coated pits located at the base of the foot processes are the sites where the HN antigen (gp330) and circulating antibodies directed against gp330 meet and where immune complexes are formed.

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Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats

Summary1) A severe nephrotic syndrome was noted in 20 rats given Freund's adjuvants and a supernate of rat kidney suspension by intraperitoneal injection. When kidney tissue was replaced by liver, only 3 of 21 animals developed a much milder renal disease. Lung or muscle suspensions failed to induce proteinuria in 10 rats. 2) In large doses (0.5 ml) Freund's adjuvants alone produced a mild nephrotic disease in 3 of 10 rats. Even though the smaller amounts of the adjuvants usually used (0.25 ml) never produced proteinuria, slight histological alterations of the glomerular structure were frequently noted. 3) In contradistinction to other tissue suspensions addition of rat kidney supernate enhanced the effect of adjuvants markedly and regularly. 4) The small amounts of rat kidney proteins used, support the interpretation that an isoand autosensitization mechanism is at play.

Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions.

Coronary blood flow and myocardial oxygen and carbohydrate metabolism were studied in normal intact dogs subjected to different Degreess of hypoxia Total left ventricular oxygen consumption was maintained during administration of 10 per cent and 5 per cent oxygen owing to increase in coronary flow and myocardial oxygen extraction Ten per cent oxygen administration had little effect on carbohydrate uptake by the heart Five per cent oxygen resulted in decreased coefficients of extraction for glucose, lactate and pyruvate, but total utilization was maintained Complete anoxia resulted in negative arteriovenous differences for lactate and markedly decreased or negative values for pyruvate

Effects of Hypoxia on the Myocardial Metabolism of Intact Dogs

The nephrotic syndrome produced in rats by administration of the aminonucleoside of Puromycin® appears after a latent period which can be shortened or lengthened by increasing or reducing the daily dose. Young rats which survive the initial course lose their proteinuria, but some relapse after 3 to 4 months. Upon re-administration of the nucleoside a chronic disease can be induced which causes death in 3 to 8 months from renal failure. Older rats given a single intravenous injection develop a chronic disease with a natural history the details of which are still not known.\n It is unlikely that the pathogenesis is based on an antigen-antibody reaction, since ACTH, cortisone and inhibition of antibody formation by x-radiation fail to prevent the disease.\n The nucleoside fails to produce a similar disease in dogs, rabbits, guinea pigs, and mice. Both 3-amino-3-deoxy-D-ribose, and and 6-dimethyl aminopurine, which in combination form the nucleoside, are inactive in producing renal disease individually. It is possible that dogs, rabbits, guinea pigs and mice are able to break down this compound while rats cannot.

Aminonucleoside nephrosis in rats.

The induction of spinal anesthesia resulted in hypotension and decreased coronary blood flow and myocardial oxygen consumption. At the same time the myocardial extraction coefficient of oxygen was not increased, indicating myocardial oxygenation to be adequate for the lower work load. This does not imply that local ischemia might not occur in the presence of coronary artery sclerosis. The coronary arteriovenous differences of glucose, lactate and pyruvate were not changed during the period of hypotension.

Effect of hypotension due to spinal anesthesia on coronary blood flow and myocardial metabolism in man.

Summary 1.The importance of Freund9s adjuvants for the production of experimental autoimmune nephrosis was studied. The frequency of disease production depended on the amounts as well as on the frequency with which they were used. Incomplete adjuvants or replacement of Mycobacterium tuberculosis by Mycobacterium butyricum , did not produce renal disease. The virulence of the organism or the method by which Mycobacterium tuberculosis was killed did not affect disease production. 2.Production of a less severe, sterile peritonitis by replacement of Freund9s adjuvants by talcum suspensions containing rat kidney antigen, failed to produce renal disease. 3.Splenectomy and partial removal of the omentum before treatment did not affect incidence of ensuing renal disease. 4.When rat kidney cortex suspension was used as antigenic material, the disease incidence was 81%. With material obtained from renal medulla it was 25%. 5.ACTH and cortisone prevented the development of renal disease. Nitrogen mustard decreased the incidence from 87% to 30%. 6.Guinea pigs did not tolerate the i.p. injections and died after 2 to 4 injections. Six dogs received up to 10 injections. The degree of resulting peritonitis was less severe and was only localized around injection sites. No renal disease was noted and precipitins to dog kidney antigen could not be detected in their sera. The severity of peritonitis was more marked in 16 treated rabbits. Bands of precipitation to rabbit kidney suspensions were detected in their sera, but no evidence of renal disease was obtained.

Donald B. Hackel

Papers

Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions.

Effects of Hypoxia on the Myocardial Metabolism of Intact Dogs

Aminonucleoside nephrosis in rats.

Effect of hypotension due to spinal anesthesia on coronary blood flow and myocardial metabolism in man.

Experimental auto-immune nephrosis in rats: III.