Experimental immune complex glomerulonephritis and the nephrotic syndrome in cats immunised with cationised bovine serum albumin

Membranous nephropathy was induced in four cats by repeated intravenous injections of 120 mg cationic bovine serum albumin (BSA, pI 9.5). All four cats developed diffuse granular deposits of IgG and C3 along the glomerular capillary walls as early as five weeks which persisted until the end of the experiment at 17 weeks. Ultrastructural studies revealed many subepithelial electron dense deposits. Two cats developed severe proteinuria and the nephrotic syndrome characterised by hypoalbuminaemia and oedema. An additional four cats received repeated injections of unmodified native BSA (pI 4.5) and remained basically normal. This is the first report of membranous nephropathy and the nephrotic syndrome in an experimental animal model which, unlike other animal models, is subject to the spontaneously occurring disease.     

Effects of a Specific Thromboxane Synthetase Inhibitor on Development of Experimental Dirofilaria immitis Immune Complex Glomerulonephritis in the Dog

Twelve Beagle dogs were immunized with aqueous-soluble Dirofilaria immitis antigens, and subsequent to at least fivefold increases in serum antibody titer, 6 mg of homologous antigen was infused into the left renal artery. Six dogs were treated once daily starting the day of infusion with 0.75 mg/kg of 1-benzylimidazole (1-BIM) in saline. Six control dogs were given saline only. Light, immunofluorescent, and transmission electron microscopic examinations of renal tissue from control dogs, 10 days after antigen infusion, showed a mesangioproliferative glomerulonephritis in the left kidney with polymorphonuclear leukocyte (PMNL) infiltration and fibrin deposition. Immunoglobulin (Ig) G, M, C3, and Dirofilaria antigen deposits were observed in a segmental granular pattern. Mesangial, subendothelial, and intramembranous electron dense deposits were observed, and anti-Dirofilaria antibodies were demonstrated in kidney eluates from each dog. Administration of 1-BIM had no significant effect on IgG, IgM, C3, or antigen deposits, electron dense deposits, or concentration of antibody in kidney eluates. However, 1-BIM-treated dogs had less glomerular cell proliferation, periodic acid-Schiff (PAS) positive glomerular staining, PMNL infiltration, and fibrin deposition. These data suggest that thromboxane is an important mediator in the development of immune complex glomerulonephritis, and that in certain circumstances, inhibition of thromboxane synthesis may be an effective therapy for immune complex glomerulonephritis in the dog.     

Development of canine nephrotic syndrome model

To develop an experimental animal model for immune-mediated glomerulonephritis and nephrotic syndrome, nine healthy dogs were sensitized by intravenous injection with 1 microg of endotoxin and 5 mg of native bovine serum albumin. After 1 week, 120 mg of cationized bovine serum albumin was injected intravenously 5 times a week. Among nine dogs, five dogs were confirmed as having developed glomerulonephritis and nephrotic syndrome by increase of urine protein-to-creatinine ratio (>1.0), hypoalbuminemia (<1.5 g/dl), hypercholesterolemia (>240 mg/dl), and edema. This model should be useful for studying immune-mediated glomerulonephritis and nephrotic syndrome.     

Renal lesions in MRL mice

Female MRL-Mp-lpr/lpr mice spontaneously develop autoimmune disease at three to five months of age and die most commonly from immune complex glomerulonephritis. Kidneys of two-month-old females appeared nearly normal by electron microscopy, and glomerular deposits of IgG an complement component 3 (C3) barely were detectable. In five-month-old females, immunofluorescence revealed numerous deposits of IgG and C3; glomerular mesangial cells were hypertrophic and hyperplastic and contained electron-dense material. There were subepithelial and subendothelial deposits of electron-dense material with swelling of epithelial cell cytoplasm. This disease has many features similar to the immune complex glomerulonephritis observed in New Zealand Black and White hybrid mice and in man.     

Idiopathic immune complex glomerulonephritis in dogs with multisystem involvement

Renal specimens obtained by biopsy and/or at necropsy from 4 dogs with nephrotic syndrome were studied using light, immunofluorescence, and electron microscopies. The glomerulonephritis observed in these dogs was considered an idiopathic immune complex glomerulonephritis associated with multisystem involvement because causes of glomerulonephritis in these dogs could not be established. Immunoglobulin A was observed in granular deposits in the mesangial and subendothelial regions of the glomeruli. The relationship of the clinical and pathologic features of this disease in dogs to various renal syndromes in human beings are described.     

Membranous glomerulonephritis in dogs infected with Dirofilaria immitis.

Membranous glomerulonephritis was diagnosed in five dogs with patent Dirofilaria immitis infections. Electron-dense deposits were present on the epithelial side of the glomerular basement membrane. An immunofluorescent study demonstrated immunoglobulins in the capillary wall and mesangium of the glomeruli. The glomerular lesions were considered to represent an immune complex form of glomerulonephritis induced by the D. immitis infection.     

Pathological changes in kidneys of dogs with natural Leishmania infection.

A study was made of the nephropathy in canine leishmaniasis produced in ten adult dogs naturally infected with Leishmania infantum. Renal function analyses were performed (uraemia, creatinaemia, plasma proteins, biochemistry and urinary sediment), the humoral immune response (fluorescent antibodies and levels of serum IgG, IgM and IgA) was assessed and histopathological studies were carried out. Correlation of the results showed acute renal insufficiency which was reversible in two animals (endotheliomesangial glomerulonephritis) and irreversible in four cases corresponding to glomerulonephritis in its Type I and Type II proliferative forms; extensive increase in the glomerular basal membrane, proliferation of mesangial cells and growth of the mesangial matrix were observed, as was a widespread incidence of immune complex deposits. Two animals showed chronic renal insufficiency. Lack of renal changes (minimal-changes glomerulonephritis) in two dogs was accounted for in one animal by an almost complete absence of symptoms and in the other by chronic viscerocutaneous symptoms; neither showed more than a slight immunoglobulin response.     

The use of concentrated bovine serum albumin in canines.

In humans it has been estimated that for each 2.5 g L^–1 decrease in serum albumin, risk of death increases by 24–56%. Clinical impression suggests this may be similar in veterinary patients. Species‐specific albumin (plasma) is often unavailable and concentrated solutions are not. Our experience using 25% human serum albumin (HSA) in critically ill dogs suggests a positive effect (results submitted), however it is expensive. Bovine serum albumin (BSA) may be a more cost effective and readily available alternative. The purpose of this study was to assess the immediate and long‐term safety of an intravenous dose (500 mg kg^–1) of bovine albumin administered to healthy dogs. Ten mature dogs (eight males, two females, 28 ± 6 kg) were to receive BSA (250 mg mL^–1) twice (BSA1 and BSA2) with 14 days between treatments. Temperature, blood pressure, and pulse and respiration rate were continuously monitored to identify a reaction to BSA. All dogs received BSA1. One dog immediately developed mild urticaria and pruritus, otherwise the infusion was well tolerated. No immediate reaction was noted in the other nine dogs. Two dogs received BSA2. One dog developed a mild immediate reaction similar to that occurring with BSA1, and one dog (the dog immediately reacting to BSA1) developed a severe anaphylactic reaction. Due to these reactions, no other dogs received BSA2. During a two‐week observation of the remaining eight dogs given BSA1, five developed a mild or severe generalized type‐III hypersensitivity reaction. The dog experiencing a mild reaction during BSA2 administration also developed a generalized type‐III hypersensitivity reaction. Delayed reactions occurred 15 ± 2.7 days after BSA exposure. Three dogs did not develop a reaction. All reacting dogs recovered fully. The severity of reactions, and the number of dogs affected, suggests prior (natural) exposure and immunological sensitization to bovine albumin. Bovine serum albumin is not suitable for therapeutic use in dogs.     

Elimination of homologous and heterologous (bovine) serum albumin from the blood circulation in the dog

The elimination of intravenously administered I¹³¹-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively.The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

Evaluation of adverse effects of long-term orally administered carprofen in dogs

OBJECTIVE: To evaluate the adverse effects of carprofen in dogs after oral administration for 2 months. DESIGN: Prospective, randomized, blinded, placebo-controlled clinical trial. ANIMALS: 22 dogs with osteoarthritis in the hip or elbow joint. PROCEDURE: 13 dogs received orally administered carprofen daily for 2 months, and 9 dogs received a placebo for 2 months. Dogs were weighed, and serum and urine samples were collected before initiation of treatment and 4 and 8 weeks after initiation of treatment. Serum concentrations of total protein, albumin, urea, and creatinine and serum activities of alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were measured. Urinary ALP-to-creatinine, gamma-glutamyltransferase (GGT)-to-creatinine, and protein-to-creatinine ratios were calculated. Dogs were observed by owners for adverse effects. RESULTS: Serum protein and albumin concentrations were lower in treated dogs than in those that received placebo at 4 weeks, but not at 8 weeks. No changes were observed in serum urea or creatinine concentrations; ALP or ALT activity; or urinary ALP-to-creatinine, GGT-to-creatinine, or protein-to-creatinine ratios. Dogs' weights did not change. Severity of vomiting, diarrhea, and skin reactions did not differ between groups, but appetite was better in dogs receiving carprofen than in dogs in the placebo group. CONCLUSIONS AND CLINICAL RELEVANCE: It is possible that the transient decreases in serum protein and albumin concentrations in dogs that received carprofen were caused by altered mucosal permeability of the gastrointestinal tract because no indications of renal or hepatic toxicity were observed. Carprofen appeared to be well tolerated by dogs after 2 months of administration.     

Effect on systemic antibody concentrations of topical application of choleratoxin to skin of sheep

OBJECTIVE: To examine the ability of a vaccine formulation combining choleratoxin with an experimental antigen to induce a systemic antibody response when applied topically on unbroken skin of sheep. DESIGN: Seven treatment groups of five adult sheep received systemic or topical priming followed 4 weeks later by systemic or topical boosting with choleratoxin and/or bovine serum albumin. Topical vaccines were administered to clipped skin on the ventral abdomen for 2 h. Booster immunisations were repeated 8 weeks after initial boosting. Serum antibody titres to choleratoxin and bovine serum albumin were determined by ELISA. RESULTS: An antibody response to choleratoxin was observed in serum, but no antibody response to bovine serum albumin was detected. CONCLUSION: Transdermal delivery may be feasible for livestock vaccines, however, further work is necessary to develop formulations that induce protective immunity by this route.     

Electron microscopic structure of mesangioproliferative glomerulonephritis (MesPGN) in animals

In a murine lupus model, the kidneys of 42 MRL mice of different age groups (substrains MRL/Mp-lpr/lp and -+/+) were studied by light and transmission electron microscopy. The results demonstrate the ultrastructural feature of the mesangial-proliferative glomerulonephritis which is one of the observed forms of glomerulonephritis. The most important alterations are a severe proliferation of mesangial cells (with an increase in rough endoplasmic reticulum and mitochondria) and electron dense deposits in different sites of the glomerular basement membrane (subepithelial, subendothelial, intramembranous). These deposits are proposed to be immune complexes. Osmiophilic intracytoplasmic inclusions of the mesangial cells are indications of an altered renin production. Alterations of the glomerular epithelial cells are characterized by fusions of the epithelial pedicles, an increase of microvilli and intracytoplasmic concentrations of electron dense material which are also proposed to be immune complex deposits. The morphological feature of the mesangial-proliferative glomerulonephritis is completed by an activation and focal edema of endothelial cells. The described alterations are discussed and compared with findings in other species.     

Treatment of Membranopr oliferative Glomerulonephritis and Nephrotic Syndrome in a Dog with a Thromboxane Synthetase Inhibitor

A 2-year-old spayed female Whippet with membranoproliferative glomerulonephritis and nephrotic syndrome was treated with a specific thromboxane synthetase inhibitor (3-methyl-2[3-pyridyl]-1-indoleoctanoic acid), resulting in decreased proteinuria and resolution of ascites and edema. Glomerular histology, however, appeared unaffected by treatment. Discontinuation of treatment for 10 weeks resulted in increased proteinuria and decreased serum albumin concentrations that were again attenuated when treatment was reinitiated. Thromboxane synthetase inhibitors have been used successfully to treat experimentally induced glomerulonephritis in several species and this treatment appears to hold promise for naturally occurring glomerulonephritis in dogs.     

Investigation of renal protein loss in dogs with acute experimentally induced Ehrlichia canis infection.

Urinary protein-to-creatinine ratios and serum albumin concentrations were measured in 8 adult male dogs experimentally inoculated with Ehrlichia canis. Urinary protein concentration increased significantly, but transiently, during the acute phase of infection. Urinary protein-to-creatinine ratios were highest (mean, 8.6) during the third and fourth weeks after infection, and decreased to less than 0.5 by 6 weeks after infection. Correspondingly, albumin concentration decreased significantly during the acute phase. Serum albumin concentrations were lowest (mean, 2.1 g/dl) the fourth week after infection and increased to greater than 3.0 g/dl by 11 weeks after infection. There was an inverse linear correlation between urinary protein-to-creatinine ratio and serum albumin concentration. The magnitude of proteinuria and its inverse relationship with serum albumin concentration suggested that hypoalbuminemia associated with acute E canis infection may be attributable primarily to increased renal loss of protein, rather than decreased hepatic synthesis as previously suggested. Another dog was subsequently inoculated with E canis from 1 of the experimentally infected dogs and a renal biopsy was performed during peak proteinuria (urinary protein-to-creatinine ratio = 22 and serum albumin = 1.1 g/dl). Immunofluorescent staining revealed mild to moderate deposits of anti-canine IgM, and to a lesser extent, anti-canine IgG and complement factor C3 in the glomerular tufts and mesangium. Ultrastructural evaluation revealed distortion and fusion of podocyte foot processes and increased microvilli on podocytes. These morphologic changes were consistent with transient glomerular leakage of protein of a magnitude that would significantly contribute to hypoalbuminemia during acute E canis infection. An underlying immunologic mechanism was suggested by positive glomerular immunofluorescence and previously described histologic findings.     

A Comparison Between the Active Cutaneous Arthus Reaction and Immune-mediated Enteroluminal Neutrophil Emigration in Pigs

The induction of neutrophil emigration into the intestinal lumen in bovine serum albumin immune and nonimmune pigs by mucosal exposure to bovine serum albumin was studied using a ligated intestinal loop technique. In order to compare the response in the skin to that in the intestine, test materials were inoculated intracutaneously as well as enterally. Several histochemical procedures were applied to the intestinal mucosa and skin for evaluation of responses.

In immune animals, mucosal exposure to bovine serum albumin evoked the emigration of neutrophils into the intestinal mucosa and lumen. The neutrophil emigration tended to occur focally. Denudation of a few epithelial cells occurred at emigration sites. Hemorrhage, thrombosis, and edema, quite obvious after intracutaneous inoculation were not apparent after enteroluminal inoculation of bovine serum albumin into immune animals.

Enteroluminal inoculation of bovine serum albumin or bovine serum albumin plus anti-bovine serum albumin into nonimmune animals did not elicit neutrophil emigration or any other pathological lesion in the intestine, whereas intracutaneous inoculation of bovine serum albumin plus anti-bovine serum albumin into the same animals elicited an Arthus reaction.

     

Amiodarone-induced keratopathy in healthy dogs

Amiodarone has a broad spectrum as an antiarrhythmic agent and is indicated for patients with atrial and ventricular arrhythmias. Amiodarone-induced corneal deposits are the most common reversible side effect (70-100%) in humans. Additional ocular effects in humans include deposits in the lens, retina and optic nerve. This study was conducted to determine ocular effects of chronic oral amiodarone in healthy dogs. Six chronically amiodarone-treated dogs and four controls were used for this study. Ophthalmic examination was performed using biomicroscopy and indirect ophthalmoscopy at the end of 4th, 7th and 11th weeks when dogs received amiodarone. Corneal microdeposits were observed at the end of the 7th week in one eye and at end of the 11th week in the other eye of one dog. Immediately following euthanasia, corneas and optic nerves were harvested for light and electron microscopic analysis. Light microscopic analysis showed corneal deposits in the basal epithelial cells of the cornea of the clinically affected dog. In addition, a significant increase in basal cell turnover as indicated by mitotic index was observed in the affected dog compared to both nondeposit amiodarone and control groups. All remaining animals were normal. One out of six dogs treated with amiodarone demonstrated corneal deposits (16%). This prevalence is low compared to humans. Explanations for this may include species variations particularly in volume of lacrimal secretion, or the need for longer administration. In addition, sunlight is believed to exacerbate the corneal deposits in humans and all dogs in this study were housed indoors.     

Effects of dexamethasone on development of immunoglobulin G subclass responses following vaccination of horses

OBJECTIVE: To determine the effects of dexamethasone on development of IgG subclass responses following vaccination of healthy horses. ANIMALS: 11 mature Thoroughbreds. PROCEDURE: Horses received 2 IM injections at 2-week intervals of a vaccine containing inactivated infectious bovine rhinotracheitis, bovine viral diarrhea, and parainfluenza-3 viral antigens and were then randomly assigned to 2 groups. Six horses received dexamethasone (0.2 mg/kg of body weight, IM) twice weekly for 8 weeks starting the day of the first vaccination. Five control horses received an equivalent volume of saline (0.9% NaCl) solution. Antigen-specific serum IgG subclass titers were determined weekly after vaccination by use of an ELISA. RESULTS: Vaccination resulted in similar antigen-specific serum IgG(T) titers in dexamethasone-treated and control horses. In contrast, although control horses developed IgGa and IgGb responses after vaccination, corticosteroid administration completely inhibited these responses in treated horses. CONCLUSIONS AND CLINICAL RELEVANCE: Cortico steroids can have profound effects on primary immune responses in horses and can significantly affect IgG responses to inactivated vaccines. Corticosteroid treatment regimens commonly used to treat diseases in horses may result induction of a nonprotective IgG subclass response, leaving treated horses susceptible to disease. Additionally, mechanisms regulating IgGa and IgGb responses appear to differ from those regulating IgG(T) responses. Further defining these mechanisms is a critical step in designing effective vaccines, and corticosteroid-induced immunomodulation may be a valuable tool for studying immune responses in horses.     

Evaluation of serum obtained from atopic dogs with dermatitis attributable to Malassezia pachydermatis for passive transfer of immediate hypersensitivity to that organism

OBJECTIVE: To determine the functionality of canine anti-Malassezia IgE via the passive transfer of immediate hypersensitivity localized to the skin (ie, cutaneous anaphylaxis) from atopic dogs with dermatitis attributable to overgrowth of Malassezia pachydermatis (Malassezia dermatitis [MD]) to healthy recipient dogs by use of the Prausnitz-Küstner (P-K) technique. ANIMALS: 7 clinically normal dogs, 32 atopic dogs with MD, serum from 11 atopic dogs with MD, and 3 healthy dogs without prior sensitization to M pachydermatis. PROCEDURE: Serum from atopic dogs with MD was used for P-K tests in 3 clinically normal recipient dogs. Serial dilutions of untreated, heat-inactivated, IgE-absorbed, and bovine serum albumin (BSA)-absorbed (control) aliquots of serum were injected ID in triplicate for dermal sensitization. Twenty-four, 48, and 72 hours later, a crude extract of M pachydermatis was injected ID into the sites used for sensitization injections, and immediate hypersensitivity reactions were graded on a 4-point scale. RESULTS: Untreated serum caused P-K reactivity beginning 24 hours after passive sensitization and persisting through 72 hours (titers, 1:32 to 1:64). Heat inactivation and IgE-absorption of serum eliminated P-K reactivity, whereas treatment of serum with BSA did not. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of P-K test results supports the passive transfer of cutaneous anaphylaxis by anti-Malassezia IgE and indicates it is functional in type-1 hypersensitivity reactions of atopic dogs with MD. Reduction or blockade of anti-Malassezia IgE in atopic dogs with MD may provide better clinical control of the disease.     

Serum antibodies against human albumin in critically ill and healthy dogs

OBJECTIVE: To characterize the magnitude and duration of the antibody response against human albumin (HA) in critically ill and healthy dogs. DESIGN: Cohort and cross-sectional study. ANIMALS: Fourteen critically ill dogs that received 25% HA as part of their treatment protocol, 2 healthy dogs with no known previous exposure to HA that received 2 infusions of 25% HA (positive control dogs), and 47 healthy dogs and 21 critically ill dogs with no known exposure to HA (negative control dogs). PROCEDURES: An ELISA to detect IgG against HA was developed. Serum samples were obtained from the critically ill dogs prior to infusion of HA, at the time of hospital discharge, and 4 to 6 weeks and 6 months after HA administration. Serum samples were obtained at 2- to 4-week intervals from both positive control dogs for 101 weeks. A single serum sample was obtained from each of the negative control dogs. RESULTS: All 14 critically ill dogs developed serum IgG against HA. Peak antibody response was detected 4 to 6 weeks after HA administration. In both positive control dogs, IgG against HA was detected 10 days after HA administration and continued past 97 weeks. The peak antibody response was detected at 3 weeks in 1 dog and at 9 weeks in the other. Five of the 68 (7%) negative control dogs had a positive antibody response. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that dogs developed a pronounced IgG response following exposure to HA and that some dogs with no history of HA administration were positive for anti-HA IgG.     

Effect of vaccination on serum concentrations of total and antigen-specific immunoglobulin E in dogs

OBJECTIVE: To determine the effect of vaccination on serum concentrations of total and antigen-specific IgE in dogs. ANIMALS: 20 female Beagles. PROCEDURE: Groups of 5 dogs each were vaccinated repeatedly between 8 weeks and 4 years of age with a multivalent and rabies vaccine, a multivalent vaccine only, or a rabies vaccine only. A fourth group of 5 dogs served as unvaccinated controls. Serum concentrations of total immunoglobulins and antigen-specific IgE were determined following vaccination. RESULTS:-The multivalent vaccine had little effect on serum total IgE concentrations. The concentration of IgE increased slightly following vaccination for rabies at 16 weeks and 1 year of age and increased greatly after vaccination at 2 and 3 years of age in most dogs, with a distinct variation between individual dogs. Vaccination had no effect on serum concentrations of IgA, IgG, and IgM as measured at 2 and 3 years of age. The rabies vaccine contained aluminum adjuvant in contrast to the multivalent vaccine. An increase of IgE that was reactive with vaccine antigens, including bovine serum albumin and bovine fibronectin, was detected in some of the dogs vaccinated for rabies. There was no significant correlation between serum concentrations of total IgE and antigen-specific IgE following vaccination. Serum total IgE concentration rapidly returned to preimmunization concentrations in most dogs, but high concentrations of antigen-specific IgE persisted. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination of dogs for rabies increases serum concentrations of total IgE and induces IgE specific for vaccine antigens, including tissue culture residues. Vaccination history should be considered in the interpretation of serum total IgE concentrations.