EXPERIMENTAL NEPHRITIS IN RATS INDUCED BY INJECTION OF ANTI-KIDNEY SERUM : I. PREPARATION AND IMMUNOLOGICAL STUDIES OF NEPHROTOXIN

Nephritis can be induced in rats by the injection of anti-kidney sera obtained from rabbits immunized with suspensions of perfused rat kidney. Anti-kidney sera, thus prepared, contain a number of antibodies capable, on injection into rats, of inducing a severe anaphylactoid reaction with general vascular manifestations that involve the kidney as well as other organs. These sera also contain a nephrotoxic agent that affects the kidney primarily. The nephrotoxic effect is characterized clinically by severe persistent albuminuria with casts, and transient anasarca during the acute disease, but no significant hematuria occurs. When a severe anaphylactoid reaction is superimposed on the nephrotoxic injury, hematuria is an outstanding feature. Nephrotoxin is demonstrable in vivo and is not related quantitatively to the precipitins in the anti-kidney serum against kidney extract. It is most readily obtained by immunization with kidney suspensions, but may occasionally appear after injections of other organ preparations; it does not result from immunization with erythrocytes or serum. Nephrotoxin is present in the globulin fraction of anti-kidney serum. The nephrotoxic action of anti-kidney serum is easily removed by absorption with kidney cells or fat-free kidney tissue. Similar preparations of liver likewise remove it, but less readily. Neither kidney, liver, or brain lipids affect it, nor does absorption with red blood cells or serum. Nephrotoxin appears to be an antibody that is relatively organ specific in its affinities. It differs from the more common antibodies involved in reverse anaphylaxis in one respect, at least: The animal rapidly becomes desensitized against the latter and fails to react, whereas desensitization to nephrotoxin is difficult to secure

EXPERIMENTAL NEPHRITIS IN RATS INDUCED BY INJECTION OF ANTI-KIDNEY SERUM : III. PATHOLOGICAL STUDIES OF THE ACUTE AND CHRONIC DISEASE

Administration of the relatively organ specific antibody, so called nephrotoxin, present in anti-kidney serum, is followed by a diffuse glomerulonephritis. This is characterized early by swelling of the intercapillary substance of the glomerular tuft and by tubular degeneration. Fibrin thrombi are only present in the glomerular capillaries when the injection of anti-kidney serum results in a severe anaphylactoid reaction, and are due to factors other than nephrotoxin. The urinary abnormalities which develop in all rats after a suitable injection of nephrotoxin usually continue until the animal dies or is sacrificed. Microscopic renal lesions of the early phase merge into scarring of the glomeruli and tubules. Histological study of those animals which die from 3 to 11 months after treatment reveals a chronic progressive glomerulonephritis with generalized vascular lesions

: III. PATHOLOGICAL STUDIES OF THE ACUTE AND CHRONIC DISEASE

Page 501, Table I, 3rd column, 3rd line of Experiment II, for 12/19/21 read 12/19/13

THE LS-ANTIGEN OF VACCINIA : II. ISOLATION OF A SINGLE SUBSTANCE CONTAINING BOTH L- AND S-ACTIVITY

Virus-free filtrate, obtained from suspensions of vaccine virus-infected dermal pulp of rabbits and rich in the soluble substances of vaccinia, was shown to contain four distinct components in electrophoresis experiments. Electrophoretic and serological observations served as a guide in developing a method for separating these components from one another. This method depended upon changes in the solubilities of the components with alterations of pH. Three of the four components appeared to be serologically inert when tested with anti-vaccinia sera. All of the L- and S-activity was found to be associated with a single component which was electrically homogeneous at several values of pH and which was homogeneous in the ultracentrifuge. This single substance, designated as LS-antigen, precipitates in equal titers with optimal amounts of L- and of S-antibody and is completely removed from solution by absorption with either antibody. The LS-antigen of vaccinia appears to be a protein molecule with two antigenically distinct parts, L and S. Heating modifies the L-portion in such a manner that the substance no longer precipitates with L-antibody; this degraded antigen still combines with L-antibody, as is shown by inhibition tests, and still precipitates with S-antibody. Similarly, treatment with heat and dilute alkali modifies the S-portion of LS-antigen so that it combines but does not precipitate with S-antibody; and at the same time all recognizable immunological properties of the L-portion are destroyed

ELECTROPHORETIC STUDIES ON ELEMENTARY BODIES OF VACCINIA

Electrophoretic studies were made on vaccine virus, collodion particles, and glass particles suspended in 0.01 molar buffer solutions at pH 7.9, in which the moving boundary method was used. In some experiments, uncoated particles were used; in others, particles were coated with proteins and then resuspended in the buffer solution after a washing; in still others, an excess of protein which had been used to coat the particles was included in the buffer medium. Streaming boundaries were obtained with all dilute suspensions of particles in solutions containing no soluble protein instead of the flat ones usually observed with the Tiselius moving boundary technique. This boundary artifact was suppressed by maintaining a density gradient of sufficient magnitude in association with the moving boundary to counteract the tendency of endosmotic flow. This was done partially by increasing the concentration of the particles in the suspensions, and almost completely by retaining an excess of soluble-coating substance in the solutions containing the particles. The mobility of elementary bodies of vaccinia corresponds to that found for the heat-stable (S) antigen. This value was not altered by drying, heating, ether extraction, or simple washing, but was materially increased by treatment with the surface active detergent (duponol) which presumably altered the nature of the surface of the virus particles. Collodion particles coated with the heat-stable antigen of vaccinia had the same mobility as elementary bodies under comparable conditions. Glass particles coated with normal rabbit serum moved at the rate of albumin, the fastest serum component in the buffer solutions used. However, both collodion particles and vaccine virus moved at a somewhat slower rate when they were similarly coated and measured in the presence of an excess of serum in the solutions. This was probably due to adsorption of a small amount of one of the slower components (globulin) of rabbit serum on the surface of the particles. Simple washing after treatment seemed to remove the coating of serum proteins, at least in part, from both collodion particles and elementary bodies of vaccinia

EXPERIMENTAL NEPHRITIS IN RATS INDUCED BY INJECTION OF ANTI-KIDNEY SERUM : II. CLINICAL AND FUNCTIONAL STUDIES

The glomerulonephritis induced in rats by nephrotoxin was characterized clinically during its initial phase by severe albuminuria, cylindruria, and anasarca, but not by hematuria. Rapidly fatal nephritis was produced by injecting relatively large amounts of anti-kidney serum at frequent intervals. In such cases the blood urea mounted rapidly; the urea clearance fell; and death occurred within about 2 weeks. A milder nephritis of the chronic type was induced by giving smaller quantities of anti-kidney serum in either single or divided doses. In these instances there was no immediate alteration of the urea clearance. Lipemia and plasma protein deficit appeared with the development of anasarca. The majority of rats which survived the initial stage of this experimental nephritis continued to show marked albuminuria with casts until they died or were sacrificed months later. Some of these animals showed retardation of growth and a progressive fall of the urea clearance. Terminally there developed marked retention of urea, plasma protein deficit, anemia, and hypertension

THE EFFECT OF DIETARY PROTEIN ON THE COURSE OF NEPHROTOXIC NEPHRITIS IN RATS

Acute nephritis of medium severity, affecting both glomeruli and tubules, was produced in rats by injections of anti-rat-kidney serum, given on 3 consecutive days. The course of the nephritis was markedly influenced by the type of diet which was fed. Rats tended to recover promptly from the induced nephritis when a low protein-high carbohydrate diet was given. On the other hand, in nephritic rats maintained on a medium protein diet the nephritis almost invariably became chronic and half the animals died of renal insufficiency during the 10½ months of observation. Finally none of the rats which received a high protein-low carbohydrate diet recovered from the acute renal injury; all developed chronic progressive nephritis and the majority died of renal failure after some months

STUDIES ON SCRUB TYPHUS (TSUTSUGAMUSHI DISEASE) : III. HETEROGENICITY OF STRAINS OF R. TSUTSUGAMUSHI AS DEMONSTRATED BY CROSS-VACCINATION STUDIES

Antigenic differences among strains of R. tsutsugamushi are sufficiently great that vaccines prepared from certain strains fail to induce resistance in mice to infection with other strains. Although the results of cross-vaccination tests indicate varying degrees of relationship between a number of the strains, there is no correlation between source of the rickettsia and antigenic pattern of the agent

EXPERIMENTAL NEPHRITIS IN RATS INDUCED BY INJECTION OF ANTIKIDNEY SERUM : V. CHRONIC NEPHRITIS OF INSIDIOUS DEVELOPMENT FOLLOWING APPARENT RECOVERY FROM ACUTE NEPHROTOXIC NEPHRITIS

1. Three different inbred lines of rats were found to vary in their response to antikidney serum: rats of the Whelan strain were most susceptible to nephrotoxin and most prone to develop chronic glomerulonephritis immediately following the acute injury induced by this agent; animals of the Evans strain were almost as vulnerable to the acute effects of nephrotoxin; Wistar rats were the least affected. 2. Both Evans and Wistar rats usually recovered quickly from the acute injury, and between the 2nd and 5th months after injection they excreted normal or only slightly abnormal urines. During this period of absence of clinical signs of disease, histopathological examination of their kidneys revealed only minor scarring in the glomerular tufts. 3. Most of these apparently recovered rats subsequently developed a slowly progressing chronic glomerulonephritis irrespective of whether they were fed a basal or high protein diet. 4. Histopathologically similar renal lesions were present in all three strains of rats with active chronic nephritis regardless of whether the chronic disease followed immediately the acute nephrotoxic injury or was separated from it by an interval of months. These lesions were somewhat more severe, however, in Whelan rats. 5. Some intraglomerular scarring was present in the kidneys of all rats which survived acute nephrotoxic nephritis. It was especially prominent in those animals that remained clinically cured for as long as a year. 6. The permanent clinical recovery of certain animals, which were found to have moderate glomerular fibrosis on postmortem examination, suggests that factors other than this residual scarring contributed to the development of the recurrent nephritis observed in most of the Evans and Wistar rats. These unknown factors may produce varying degrees of renal functional trauma affecting both glomeruli and tubules

ELEMENTARY BODIES OF VACCINIA FROM INFECTED CHORIO-ALLANTOIC MEMBRANES OF DEVELOPING CHICK EMBRYOS

By a method of differential centrifugation and tryptic digestion suspensions of elementary bodies have been prepared from chorioallantoic membranes of chick embryos infected with vaccine virus. The infective titer of the final suspension of elementary bodies was usually the same as that of the original tissue emulsion. Elementary bodies from infected chick membranes were agglutinated as well by antivaccinal serum obtained from different mammalian species as were bodies prepared from inoculated rabbit skin. Seitz filtrates of infected chick material contained soluble precipitable substances of vaccinia; these filtrates and filtrates from infected rabbit skin, respectively, reacted equally well with rabbit serum which contained either L or S antibodies