Glomerular lesions in unilateral nephrectomized and diabetic (UN-D) mice.

Experimental diabetes was induced in both control and unilaterally nephrectomized male mice by injecting streptozotocin (SZ) (50 mg/kg x 5 days) one week after nephrectomy. The time course changes in the glomerular lesions were examined for up to 12 weeks after completion of the SZ-injection (12WAI). In unilateral nephrectomized and diabetic mice, mild segmental expansion of the mesangial area developed at 4WAI, and it progressed to prominent segmental glomerulosclerosis at 12WAI. In the electron microscopic examination at 12WAI, marked expansion of the mesangial area, segmental thickening of the glomerular basement membrane, fusion of the foot processes of podocytes and a prominent increase in the number of microvilli of capillary endothelial cells were observed. On the other hand, mild to moderate expansion of the glomerular mesangial area was only sporadically found in unnephrectomized diabetic mice at 12WAI. Interestingly, Bowman's capsules of diabetic mice were generally lined with flattened epithelia but those of non-diabetic mice with cuboidal or low columnar epithelia.     

Glomerular lesions associated with proteinuria in clinically healthy dogs.

Spontaneous proteinuria in otherwise clinically normal adult Beagles 4-6 years old was studied for 2 years. Eighteen dogs, representing a population of 218 Beagles, were placed into three groups: group I, nonproteinuric; group II, intermittently proteinuric; group III, persistently proteinuric. The groups were alike on the basis of laboratory tests, except urinary protein loss. Proteinuria was persistent in most affected dogs but not progressive during the 2 years. The loss of proteins with high molecular weight, including alpha-, beta-, and gamma-globulins, suggested the proteinuria was of glomerular origin. There were glomerular lesions but no other significant change in the kidneys and urogenital system. Lesions were generalized and characterized by prominent, local or diffuse mesangial proliferation and by thickening, wrinkling, and splitting of the glomerular basement membrane. The subendothelial space was often widened and contained electron-dense deposits. Similar electron-dense deposits, as well as lipid and mineral, were in the mesangium. Alterations in visceral epithelial cells and endothelium were prominent. Periglomerular sclerosis was present but tended not to correlate with the severity of mesangial change in any given renal corpuscle. The severity of both mesangial and periglomerular changes increased with increasing proteinuria. Immunofluoescence studies demonstrated granular discontinuous localization of IgG and betaIC-globulins in the glomerular capillaries and mesangium. Similar localization was seen but to a lesser extent in nonproteinuric dogs. The glomerular lesions seen in these clinically healthy, proteinuric dogs are similar to those described in various canind diseases associated with terminal renal failure.     

Early sequential ultrastructural renal alterations induced by 2-bromoethylamine hydrobromide in the Swiss ICR mouse.

Thirty-two male Swiss ICR mice were injected intraperitoneally with 300 mg 2-bromoethylamine hydrobromide/kg body weight, anesthetized, and perfused with glutaraldehyde-paraformaldehyde solution at 5, 15, 30, 60, 90, 120, 150, and 180 minutes after treatment. Eight control mice were injected intraperitoneally with sterile diluent, and one was perfused at each of the same time periods as the treated mice. Proximal tubule epithelial alterations progressed over time from increased secondary lysosome and myeloid body formation to cellular and mitochondrial swelling and eventually cell necrosis. The glomerular, peritubular, and vasa recta capillaries had endothelial cell swelling and desquamation and platelet aggregation. Bromoethylamine nephrotoxicosis in the male Swiss ICR mouse is an ischemic necrosis of the proximal tubules and papilla initiated by endothelial cell damage and makes an excellent model of chemically induced damage to endothelial cells and tubular necrosis.     

Diagnostic relevance of qualitative proteinuria evaluated by use of sodium dodecyl sulfate-agarose gel electrophoresis and comparison with renal histologic findings in dogs

OBJECTIVE: To evaluate results of SDS-agarose gel electrophoresis (AGE) of urinary proteins for use in defining glomerular and tubulointerstitial derangements, investigate patterns of high-molecular-weight (HMW) proteins for differentiating among glomerular disorders, and assess low-molecular-weight (LMW) proteins as markers of severity of tubulointerstitial disease in dogs. ANIMALS: 49 dogs with increased serum creatinine concentrations or abnormal renal protein loss. PROCEDURE: Urinary proteins were examined by use of SDS-AGE and differentiated on the basis of molecular weight. The HMW proteins (> or = 69 kd) were considered indicative of glomerular origin, whereas LMW proteins (< 69 kd) were of tubular origin. Renal specimens were examined by use of light microscopy. Glomerular and tubulointerstitial lesions were differentiated by use of the classification for the World Health Organization and semiquantitative grading, respectively. RESULTS: Sensitivity of SDS-AGE was 100% for detection of glomerular lesions and 92.6% for tubulointerstitial lesions; specificity was 40% and 62.5%, respectively. Although HMW urinary proteins were not significantly associated with the type of glomerular lesion, LMW urinary proteins were significantly associated with the grade of tubulointerstitial damage. Detection of 12- or 15-kd proteins or both was highly indicative of a severe tubulointerstitial lesion. CONCLUSIONS AND CLINICAL RELEVANCE: SDS-AGE of urinary proteins in dogs represents a noninvasive test with high sensitivity for identifying glomerular and tubulointerstitial damage, but low specificity limits its validity as a stand-alone test to differentiate between glomerular and tubulointerstitial lesions. The test is particularly useful for identifying dogs with advanced tubulointerstitial disease but cannot be used to characterize glomerular disorders.     

Brain viral persistence and myelin damage in nude mice.

Multiple foci of secondary demyelination were observed in the cerebellum of nude mice given two inoculations of avirulent Semliki Forest virus compared to single lesions seen in similarly treated Swiss A2G mice. The increased number of demyelinative plaques were attributed to brain viral persistence for 35 days in the nude mice with correspondingly low serum antibody titres. No neurological signs were observed in any of the mice.     

Spontaneous and experimental aflatoxicosis in goats.

Naturally occurring aflatoxicosis is described in goats fed a concentrate mixture containing polkudu meal (defatted residue from grated coconut after juice extraction). Toxigenic strains of Aspergillus flavus were present in the concentrate and aflatoxins were recovered from the liver and urine of affected animals. Hepatic lesions in poisoned goats consisted of bile duct hyperplasia and periportal fibrosis; renal lesions included necrosis of tubular epithelial cells and proteinaceous exudation in the glomerular space. Similar lesions were produced experimentally in goats with aflatoxin and the coconut cultures of A flavus. High doses of aflatoxin B1 produced, in addition, hepatic centrilobular congestion, haemorrhage and periportal fatty change.     

The sequence of events in the development of bilateral renal cortical necrosis accompanying the generalized Shwartzman reaction.

The generalized Shwartzman reaction, or Shwartzman-like conditions, were induced in a variety of experimental mammalian species by systemic injections of disintegrated cells of Gram negative bacteria, live Salmonella cholerae-suis or Liquoid. A comparative study of the renal lesions showed that the initial step in the development of bilateral cortical necrosis is stagnation and disintegration of red cells in glomerular capillaries. The glomerular “microthrombi” consist mainly of erythrocytic debris, which frequently has staining properties akin to those of fibrin; even wide-spread glomerular “thrombosis” is not accompanied by obvious destruction of renal parenchyma. A second step is necrotic mural lesions in afferent arteries, with ensuing thrombosis. These vascular lesions lead to the formation of individual infarcts which fuse to form total bilateral cortical necrosis in fulminant cases of the generalized Shwartzman reaction.     

Immunohistochemical analysis of molecular events in tubulo-interstitial fibrosis in a mouse model of diffuse mesangial sclerosis (ICGN strain)

Diffuse mesangial sclerosis (DMS) is one of the hereditary glomerular diseases and histologically characterized by severe glomerulosclerosis and subsequent tubulo-interstitial fibrosis (TIF). In DMS patients, renal dysfunction correlates well with TIF, rather than with glomerular lesions. Thus, molecular mechanisms whereby TIF in DMS progresses should be addressed. Previously, we found that nephrotic ICGN mice manifest DMS-like lesions and develop renal dysfunction in accordance with onset of TIF. In the present study, we investigated fibrogenic events involved in the progression of TIF after DMS manifestation, using the DMS mouse model. Immunohistochemistry revealed that expression of transforming growth factor-beta (TGF-beta) was rare in the interstitial cells of the nephrotic mice at the early-stage of DMS, while the TGF-beta expression became evident in the late-stage DMS mice. Platelet-derived growth factor (PDGF) was mildly expressed in the distal tubules of the early-stage DMS mice, whereas the PDGF expression markedly increased at the late-stage of DMS. As a result, alpha-actin-positive myofibroblastic cells were found dominant in the interstitial spaces of the late-stage DMS mice. Finally, TIF became severe in accordance with the overexpressions of these molecules. Our results suggest that in our murine model: 1) persistent proteinuria leads to over-expression of TGF-beta and PDGF in non-glomerular areas; 2) these cytokines provoke interstitial myofibroblast accumulation; and 3) the myofibroblasts produce fibrotic matrix proteins in the interstitial spaces. This process may possibly contribute to the development of TIF in DMS patients.     

Urinary gamma-glutamyl transferase and the degree of renal dysfunction in 75 bitches with pyometra

In 75 bitches with pyometra single urine samples were examined for gamma-glutamyl transferase (gamma-GT), protein, glucose, specific gravity, bacteria, red blood cells and white blood cells. Serum samples were examined for urea, creatinine, inorganic phosphate and gamma-GT. Biochemical findings were compared with the degree of illness (clinical signs). Twenty one bitches had no signs of renal disease. Seventeen showed only glomerular damage indicated by proteinuria without signs of proximal tubular damage. Thirty seven bitches had increased urinary gamma-GT levels, indicating proximal tubular lesions, which were associated with proteinuria in 35 and renal failure in 16 of them, and worse clinical findings. In all bitches with pyometra serum levels of gamma-GT were comparable to values in control bitches. Glomerular dysfunction seemed to precede proximal tubular lesions, so that gamma-GT-uria in bitches with pyometra was not an early but rather a late indication of a more profound degree of renal dysfunction, that is, proximal tubular renal damage developed after glomerular dysfunction and preceding renal failure.     

Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay was used to detect serum antibodies to mouse thymic virus, a herpesvirus that causes thymic lesions and immunosuppression. Antibodies were detected in mice that had received single or multiple injections of the virus and were also found in mice housed in contact with the experimentally infected animals. By contrast, mice not exposed to mouse thymic virus or those inoculated with an uninfected thymus preparation remained seronegative. A serological survey of eight mouse colonies revealed one positive colony, confirmed by virus isolation. These results show that the test is sufficiently sensitive and specific to be used for routine screening of mice.     

Comparison of the lesions of Aleutian disease in mink and hypergammaglobulinemia in ferrets.

Gross and microscopic lesions of Aleutian disease (AD) in mink and hypergammaglobulinemia in ferrets were compared. Both conditions were characterized by widespread proliferation of plasma cells, but proliferation was more prominent in mink infected with AD. Arteritis did not occur in hypergammaglobulinemic ferrets. Minimal or no glomerular alterations occurred in infected ferrets, but were severe in mink infected with AD. Bile duct proliferation was more prominent in diseased mink. Tissue alterations suggested that AD in Aleutian genotype mink is more rapidly progressive than is AD in ferrets, causing overt clinical disease and death. In contrast, hypergammaglobulinemia in ferrets appeared to progress more slowly, with little clinical evidence of disease. This is probably the result of a paucity of glomerular lesions in ferrets. Possible mechanisms to explain the differences in the development of lesions are discussed.     

T cell mediated and humoral immunity in a mouse Chlamydia psittaci systemic infection

Immunity against Chlamydia psittaci, an obligate intracellular parasite, was studied in a mouse model of systemic infection. Sera (0.1 ml) and splenic cells (2 X 10(8)) from immunised mice were given intravenously to susceptible mice 16 hours before intravenous challenge with 1 X 10(5) plaque forming units (pfu) of virulent strain AB7. Transfer of immune cells primed with virulent strain AB7 or vaccinal strain 1B, lowered splenic and hepatic colonisation by approximately 5.5 log pfu. Treatment of immune cells with antithymocyte serum plus complement, before transfer, abrogated the protection. Transfer of sera raised against the virulent strain AB7, or the attenuated vaccinal strain 1B, lowered hepatic colonisation by approximately 1.5 log pfu. Sera containing antigenus antibodies, raised against heat-killed chlamydiae from strain AB7 or the non-virulent intestinal strain iB1, were not protective. Cellular immunity is mainly responsible for the observed protection, although humoral immunity may play some role.     

Glomerulopathy in dogs with congenital portosystemic shunts

Kidney specimens from 12 dogs with congenital portosystemic shunts were examined histologically. Glomerulopathy of variable severity was present in the kidney sections of all 12. Marked irregular thickening of the glomerular capillary wall was the most prominent pathological change, the renal interstitium being largely unaffected. The severity of lesions was not correlated with the age of dogs at the time of necropsy. An immunoperoxidase technique failed to demonstrate significant IgA or IgG deposition in affected glomeruli. Proteinuria was generally mild or absent despite significant glomerular lesions, except in dogs with concurrent urinary tract infection.     

Spontaneous Glomerular and Tubulointerstitial Lesions in Common Marmosets (Callithrix jacchus)

Spontaneous progressive nephropathy dominated by glomerular lesions in common marmosets has been reported. However, the histopathologic characteristics, including the relationship between glomerular and tubulointerstitial lesions, have not been described in detail. In the present study, the authors examined the histopathologic characteristics of the background renal lesions in common marmosets (3 males and 9 females, 3 to 8 years old). The severity of glomerular lesions was graded into 3 classes: grade I, no alteration; grade II, hilar/focal increase of mesangial matrix; grade III, global/diffuse increase of mesangial matrix. Tubulointerstitial lesions (tubular regeneration and hyperplasia and interstitial inflammation and fibrosis) were scored according to the area of each lesion. The renal lesions were characterized by enlargement of glomeruli, expanded mesangial area with increase of periodic acid-Schiff reaction-positive matrix, tubular regeneration and hyperplasia, and interstitial inflammation and fibrosis. Glomerular lesions progressed with increasing mesangial matrix and aging. Additionally, the tubulointerstitial lesions became exacerbated with progressing glomerular lesions. Tubular hyperplasia was divided into 4 types according to the structure of the cell layer (simple or stratified-like), the area of increased lining cells (partial or entire), cytoplasmic staining (eosinophilic or basophilic), brush border and thickness of basement membrane, and the activity of cell proliferation. In conclusion, the background renal lesions in common marmosets were characterized by glomerular lesions with increase of mesangial matrix, which progressed with aging, and secondary tubulointerstitial lesions, including tubular hyperplasia. Those lesions were thus diagnosed as progressive glomerulonephropathy in common marmosets.     

Toxicity of microcystin LR, a cyclic heptapeptide hepatotoxin from Microcystis aeruginosa, to rats and mice

Rats (Sprague-Dawley) and mice (Balb/c) were given microcystin LR intraperitoneally and were killed at intervals up to 24 hr (rats) or 90 min (mice) and necropsied. The lowest consistently lethal dose was 160 micrograms/kg in rats and 100 micrograms/kg in mice. Rats that were clinically unaffected had no lesion. All clinically affected rats in all dose groups died (from 20 to 32 hr after toxin) and had similar hepatic lesions. Livers were enlarged and dark red beginning 40 to 60 min after toxin. Mild disassociation and rounding of centrilobular hepatocytes developed within 20 min. By 60 min after toxin, degeneration and necrosis of hepatocytes involved most of the lobules except for small periportal zones. Weights of livers and kidneys were significantly increased. Eosinophilic fibrillar material filled renal glomerular capillaries as early as 9 hr after toxin. At 18 to 24 hr there was moderate vacuolation of proximal tubular epithelium with mild tubular dilatation. Beginning at 1 hr, intact hepatocytes and hepatic debris were present in pulmonary vessels. Analysis of serum revealed an increase in alanine aminotransferase 40 min after toxin; at 6 to 12 hr there were significant increases in alkaline phosphatase, total bilirubin, blood urea nitrogen, and creatinine. Mice survived only 60 to 90 min after toxin. Hepatic lesions were similar to those in rats, but renal and pulmonary lesions were not seen.     

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.     

Electron Microscopy of Bactericidal Effects Produced by the Alternative Complement Pathway of Channel Catfish

Abstract

The activation of the alternative complement pathway (ACP) was investigated by incubating serum of channel catfish Ictalurus punctatus with three gram-negative bacteria. Bactericidal activity and ultrastructural damage were not apparent when cells were incubated with serum that had been treated to eliminate complement activity. Proteins of the ACP were responsible for damaging susceptible Escherichia coli and Pseudomonas fuorescens, whereas there was no effect on Aeromonas salmonicida, which was apparently protected by an A-layer. Scanning electron micrographs revealed damaged cells, and transmission electron micrographs showed circular lesions oriented perpendicularly to the cell membrane. These lesions resemble those resulting from ACP activation in sera from frogs, sharks, trout, lizards and mammals. The existence of membrane attack complexes (MACs) in channel catfish, which are similar to MACs seen in other animals, emphasizes the importance of the ACP in host defense against bacterial infections.     

Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

Interleukin (IL)-10 exerts potent anti-inflammatory effects by suppression of both T-help (Th) 1 and Th2 cells. Previous studies have reported that IL-10 can ameliorate various inflammatory disorders. The present study was performed to examine whether IL-10 plasmid DNA could suppress development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice, as an initial step towards the development of an appliance for use in dogs with AD. Intradermal injection of IL-10 plasmid DNA markedly inhibited the development of AD-like skin lesions, as evidenced by a marked decrease in skin symptoms and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Moreover, relative mRNA expression levels of IL-4 and interferon-γ in the skin lesions of mice injected with IL-10 plasmid DNA were also decreased compared with those of control mice. Of note, higher serum IL-10 levels in mice injected with IL-10 plasmid DNA were maintained compared with those in control mice. Taken together, the results indicate that IL-10 plasmid DNA can suppress the development of AD-like skin lesions by suppressing both Th1 and Th2 cell responses. Beneficial effects of IL-10 plasmid DNA may be expected in dogs with AD.     

Mechanism of protection induced in mice against Erysipelothrix rhusiopathiae infection by treatment with porcine antiserum to the culture filtrate of an attenuated strain

The mechanism of protection induced in mice against challenge with a virulent strain of Erysipelothrix rhusiopathiae by porcine antiserum to the culture filtrate (CF) of an attenuated strain was investigated. Death and bacterial growth in the spleens of mice challenged with the virulent strain were completely prevented by treatment with the antiserum. The protective effect of the serum was markedly decreased in mice in which polymorphonuclear leucocytes (PMN) were depleted by cyclophosphamide (CY) treatment but not in mice in which macrophages were blocked selectively by carrageenan (CG). The phagocytic rate of PMN and the number of bacteria ingested by PMN were significantly higher in mice treated with the antiserum than in mice treated with normal serum. These results indicate that anti-CF serum exerts its protective effect by opsonic activity and that opsonized E. rhusiopathiae are eliminated mainly by PMN.