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.     

Mitochondrial damage as an early event of monensin-induced cell injury in cultured fibroblasts L929

The present study was designed to identify, submicroscopically, the primary organelle or target structure for monensin in cultured murine fibroblasts L929. In addition, the effect of the drug on cell size and surface membranes of the cells were analysed; cellular proliferation, collagen secretion, and necrosis and apoptosis were re-evaluated. At the lowest concentration of monensin the foremost ultrastructural alteration occurred in the mitochondria, characterized by increased matrix density with disorganized and less distinct crystae. Incubation with monensin at higher concentrations resulted in severe mitochondrial damage and marked dilatation of the Golgi apparatus and rough endoplasmic reticulum cisternae. Fibroblasts exposed to higher concentrations of monensin were enlarged with decreased number of filopodia and hollows in the surface membrane. Moreover, monensin inhibited the cell proliferation, increased immunohistochemical positiveness for collagen type I in a dose-dependent manner, and, at high concentrations, caused cell necrosis whereas apoptosis was not induced. Taken together, these results show that monensin induces early mitochondrial damage, possibly causing an energy deficit that led to inhibition of fibroblasts proliferation and accumulation of collagen causing dilatation of Golgi apparatus and rough endoplasmic reticulum. Moreover, the mitochondrial damage would also explain the monensin-induced necrosis.     

Abnormalities of extracellular matrices and transforming growth factor beta1 localization in the kidney of the hereditary nephrotic mice (ICGN strain).

ICR-derived strain with glomerulonephritis (ICGN) is a strain of mice with hereditary nephrotic syndrome with an unidentified cause. Based on histopathological and biochemical data, ICGN mice are considered to be a good experimental model for human idiopathic nephrotic syndrome. In the present study, we histochemically investigated the changes in localization of extracellular matrix (ECM) components and transforming growth factor beta1 (TGF-beta1). Strong immunohistochemical staining of basal membrane ECM components (collagen IV and laminin) and interstitial ECM components (type III collagen and fibronectin) were demonstrated in glomeruli and tubulointerstitum of ICGN mice as compared with those of sex and age-matched ICR mice, used as normal healthy controls. Marked type I collagen and tenascin deposition, which were not detected in the glomeruli of ICR mice, were seen in the glomeruli of ICGN mice. A remarkable increase in active-TGF-beta1 was also detected only in glomeruli of ICGN mice, but not in those of ICR mice. Furthermore, strikingly increased alpha-smooth muscle actin, a marker of activated glomerular mesangial cells, was demonstrated in the glomeruli, mainly in the mesangial cells, of ICGN mice. These findings indicated that ECM components are increased in the glomerulus and tubulointerstitum of ICGN mice, and that active-TGF-beta1 induces such increases in ECM components. The present findings may contribute to elucidation of the pathogenic mechanisms of hereditary nephrotic syndrome in ICGN mice and, in future, human idiopathic nephrotic syndrome.     

Cloning and expression of type III collagen in normal and injured tendons of horses

OBJECTIVE: To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. ANIMALS: 14 healthy adult horses. PROCEDURE: The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis. RESULTS: Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease. CONCLUSIONS AND CLINICAL RELEVANCE: Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated.     

Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis

OBJECTIVE: To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS: 8 horses. PROCEDURES: Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS: Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE: ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.     

Diverse functions of perlecan in central nervous system cells in vitro

Therapeutic treatment targeting one cell type is considered ineffective in remedying any injury to the central nervous system (CNS). Perlecan, a multi‐functional, heparan sulfate proteoglycan, shows diverse effects on distinct cell types, suggesting that it is one of the candidates that can augment the regenerative mechanisms in the injured CNS. Therefore, we examined the functions of perlecan in CNS cells in vitro by using perlecan purified from bovine kidney. Perlecan‐coated cell culture plates, unlike their type I/III collagen‐coated counterparts, did not inhibit the adhesion of neural stem/progenitor cells (NS/PCs) and neurons. The coated perlecan and the perlecan added to the culture medium suppressed astrocyte proliferation; however, perlecan added to the medium promoted NS/PC proliferation. Neurons were promoted to extend their neurites on the perlecan‐coated substrate, and perlecan added to the medium also showed a similar effect. NS/PC proliferation and neurite extension is a major regenerative reaction in CNS injury, whereas excess proliferation of astrocytes cause hypertrophy of glial scars, which repels neurons. Our in vitro study suggests that perlecan is an attractive candidate to promote regenerative mechanisms and to suppress reactions that hamper regenerative processes in cases of CNS injury.     

Ovarian Fibrothecoma in an Arabian Mare: A Rare Case

The occurrence of ovarian tumors is uncommon in domestic animals. Reports are documented more often in the bitch, mare, and cow. In the mare, granulosa-thecal cell tumor is the most common ovarian neoplasms, and other tumors occur less often. Fibrothecoma is a rare gonadal-stromal tumor of the ovary. There is only one reported case in the veterinary literature. This tumor consists of theca cells and fibrous tissues. The present study describes a rare case of fibrothecoma in a 10-year-old Arabian mare that was presented due to infertility problems. The abnormal left ovary was detected via rectal palpation and ultrasonography. Postoperation study showed a solid tumoral mass that completely replaced the normal ovarian tissues. The tumor was large, firm in consistency, with smooth surface. On the cut surface, the mass was white-gray to yellow with cystic areas containing green gelatinous materials. Microscopic examination revealed the structure was composed of fibroblastic cell producing collagen fibers and theca cells containing lipids. Based on histopathologic features, diagnosis of fibrothecoma was confirmed postoperatively.     

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.     

Functional role of type VI collagen during early feather development of the chicken embryo in vitro

The regulatory function of type VI collagen during early feather development in embryonic chickens was investigated at the cellular and organ levels. Immunohistochemical studies of embryonic chicken skin showed that type VI collagen was distributed in spatial‐specific and temporal‐specific manners related to early feather development. To clarify the role of type VI collagen, we studied the feather development in intact, reconstituted and reconstituted gel skin cultures. When ethyl‐3,4‐dihydroxybenzoate (EDHB) was added to the medium of intact skin as an inhibitor of type VI collagen synthesis, the feather buds did not elongate and the number of neural cell adhesion molecule (NCAM)‐positive cells was reduced. However, the magnitudes of both suppressive effects of EDHB were reduced by the addition of liquid type VI collagen. Similar improvement was also observed in the reconstituted skin with liquid type VI collagen and in the reconstituted gel skin with solid type VI collagen at a low concentration. Moreover, type VI collagen promoted feather bud development in the absence of EDHB. However, a high concentration of solid type VI collagen in the reconstituted gel skin arrested the feather bud elongation, and antitype VI collagen antibodies caused feather buds to become longer and smaller in the reconstituted skin. At the cellular level, type VI collagen affected the proliferation, migration and NCAM expression of mesenchymal cells. These results suggest that type VI collagen regulates early feather development by controlling mesenchymal cell behavior.     

Proportion of types I and III collagen in longissimus collagen from bulls and steers

The proportion of types I and III intramuscular collagen in longissimus muscles of Simmental bulls (n = 8) and steers (n = 8) 17 mo of age was studied. Longissimus samples taken 7 d after slaughter were evaluated for total collagen, types I and III collagen, heat-soluble collagen, sensory panel traits and Warner-Bratzler shear force. Intramuscular collagen (IMC) was isolated and digested with cyanogen bromide, and peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Percentage of type III IMC was calculated from the total of types I and III collagen as determined from the peak area of densitometric scans of the cyanogen bromide peptides alpha 1(I)CB8 and alpha 1(III)CB8. Longissimus muscles from steers had lower (P less than .05) Warner-Bratzler shear values, less (P less than .05) sensory panel-detectable connective tissue and more (P less than .05) tender panel ratings for muscle fiber tenderness and overall tenderness. Muscles from steers had more (P less than .05) heat-soluble collagen than those from bulls, but no differences (P greater than .05) were found for total collagen and percentage of type III collagen. Some intramuscular-collagen characteristics may have contributed to the less tender muscle of bulls. However, the proportion of types I and III collagen did not account entirely for the tenderness difference between steer and bull muscles. Because there were differences in collagen solubility in muscles from steers and bulls, other collagen characteristics such as crosslinking or fiber size may have been more important than collagen type.     

Glomerular Ultrastructural Findings Similar to Hereditary Nephritis in 4 English Cocker Spaniels

Renal disease affecting 3 male and 1 female English Cocker Spaniels was studied. Clinical features of the disease included proteinuria and progressive deterioration of renal function. Dogs were 11 to 27 months old when euthanized because of severe chronic renal failure. Grossly, the renal cortices were thin. Light microscopic evaluation revealed diffuse glomerular disease characterized by mesangial thickening, glomerular fibrosis, periglomerular fibrosis, and glomerular obsolescence. Based on these clinical and pathologic features, familial nephropathy of English Cocker Spaniels was suspected despite the fact that the individual dogs were not closely related. On transmission electron microscopy, a distinctive ultrastructural lesion was observed in the glomerular basement membranes (GBM) of all dogs. The GBM exhibited extensive thickening, multilaminar splitting, and fragmentation. Electron dense deposits, suggestive of immunocomplex glomerular disease, were notably absent. A similar ultrastructural GBM lesion is found in human beings and Samoyeds with hereditary nephritis, diseases caused by mutations in the type IV collagen genes. Familial nephropathy in English Cocker Spaniels may be a form of hereditary nephritis caused by a mutation in one of the collagen IV genes.     

Glomerular lipidosis accompanied by renal tubular oxalosis in wild and laboratory-reared Japanese rock ptarmigans (Lagopus mutus japonicus)

Glomerular lipidosis is a disease characterized by lipid accumulation in mesangial cells but that has not been fully investigated in avian species. We examined four wild and two laboratory-reared Japanese rock ptarmigans (Lagopus mutus japonicus)--an endangered avian species--presenting vacuolar deposits in the glomeruli. All cases had vacuolar deposits in the glomeruli. In the wild cases, fewer than 30% of all glomeruli were affected, compared with more than 90% in the laboratory-reared cases. In the wild cases, most deposits were mild and restricted to the mesangial areas of glomeruli. In the laboratory-reared cases, nearly all of the deposits covered entire glomeruli. Electron microscopy of mild deposits revealed vacuoles in the cytoplasm of mesangial cells. These vacuoles were positive for Sudan III, Sudan black B, oil red O, Nile blue, periodic acid-Schiff, Schultz test, and digitonin stain and were negative for performaric acid-Schiff stains. Based on these results, we diagnosed the glomerular lesion as glomerular lipidosis caused by uptake of low-density lipoprotein in mesangial cells. Except for one wild case, all cases exhibited renal tubular oxalosis. The severity of tubular oxalosis tended to be related to the severity of glomerular lipidosis: In cases of mild glomerular lipidosis, tubular oxalosis was also mild or absent. We therefore diagnosed the primary lesion as glomerular lipidosis accompanied by tubular oxalosis. The four wild cases came from different zones and therefore had no opportunities to interbreed and no common relatives. We believe these data support the hypothesis that glomerular lipidosis is a disease of the general population ofJapanese rock ptarmigans. This is the first report of glomerular lipidosis accompanied by renal tubular oxalosis in an avian species.     

Amyloid-producing odontogenic tumor in a Shih-Tzu dog

A 9-month-old male Shih-Tzu dog had a right mandibular tumor composed of strands, or nest-like proliferation of epithelial cells with abundant fibrous stroma characterized by spheroid to large nodular deposition of amyloid with Congo-red stain. Globule calcification was also seen throughout the tumor tissue and the spheroid depositions often had a concentrically laminated structure (Liesegang rings). The case was diagnosed as amyloid-producing odontogenic tumor in a dog.     

Experimental studies on the therapy of epiglottis hypoplasia in horses--transendoscopic injection of collagen and polytetrafluoroethylene

Epiglottic augmentation by transendoscopic injection of an implant material was performed on ten clinically healthy horses. In six cases bovine collagen (Zyplast) was used, in the remaining four horses the injection was done with polytetrafluorethylene (PTFE-Paste). The results of the surgery were observed endoscopically and by contrast radiography. Using the radiographs, epiglottic length and thickness were measured. For necropsy and histologic assessment the horses, three and two animals of the two groups, were euthanized at three and 12 weeks after surgery. The data obtained from the measurements at necropsy were correlated with those from the radiographs. The minimal invasive technique was very easy to perform with the bovine collagen while PTFE was less suitable for the transendoscopic injection. The data concerning the epiglottic length as well as its thickness measured on the radiographs correlated well to those obtained from the measurements at necropsy. After injection of collagen as well as PTFE an organisation of the implant was noticed histologically, which increased in accordance with the increasing time after injection. As far as the collagen implant is concerned, there was fibrous granulation tissue accompanied by an inflammatory reaction consisting mainly of lymphocytes and histiocytes. In contrast the injection of PTFE led to the development of a foreign body granuloma. In addition, some cases showed ulcers. Both implant materials led to an increase in epiglottic thickness, so that one can expect an improvement in its stability which could be of benefit to the therapy of epiglottic hypoplasia. But this increase in thickness had its maximum level directly after injection and declined over the period observed. Still, especially in the case of the collagen used here, the development of a fibrous granulation tissue and the immigration of fibroblasts and fibrocytes into the implant indicates that there is a stabilisation to some extent. Bovine collagen was proven to be highly suitable for transendoscopic injection. Although there is report about good clinical results using PTFE via laryngotomy, this material seems to be unsuitable for the transendoscopic technique due to the circumstances that lead to a higher number of complications.     

Molecular and ultrastructural studies of the fibrotic lesions of bovine focal proliferative fibrogranulomatous panniculitis (Lechiguana)

This work aimed to investigate some aspects related to the pathogenicity of Lechiguana, a bovine fibroproliferative lesion characterized by rapid collagen accumulation. Light and transmission electron microscopy and in situ hybridization studies were performed in order to elucidate the fibrogenic activity of this lesion. The characterization of fibroblastic plasticity in the lesion was done by immunohistochemical study for α-smooth-muscle cell actin. The ovoid-shaped cells presented positive reaction for α-smooth-muscle cell actin in their cytoplasm and, at the electron-microscopic level demonstrated basal lamina-like material adjacent to the external surface and collagen fibrils that corresponded to a cell population phenotypically similar to the myofibroblast. We also investigated α1 collagen type I mRNA at different times of evolution of Lechiguana lesions, using isotopic and non-isotopic in situ hybridization. The results strongly suggest the involvement of a myofibroblast-like cell population that expresses mRNA for type I collagen and is probably associated with the increase of collagen deposition.     

Immunohistochemical and scanning electron microscopic comparison of the collagen network constructions between pig, goat and chicken livers

The distribution and three-dimensional architecture of collagen fibers were compared between pig, goat and chicken livers. Immunohistochemical staining revealed that collagen type I was identified in the interlobular connective tissue region and intralobular areas in pigs and goats. Type III collagen was also identified in the interlobular connective tissue region and intralobular sinusoidal walls. In the chicken liver, only the circumference region of the vessels was immunostained with collagen type I and III antibodies and the interlobular connective tissue wall could not be distinguished clearly. In the intralobular region, collagen type I antibody immunoreacted around the hepatic cells but collagen type III antibody immunoreacted weakly. In the NaOH macerated specimen, well-developed collagen bundles formed the prominent interlobular walls in pigs. In contrast, the wall in the goat liver comprised a thin layer of the bundles. In the chicken liver, there were no notable collagen septa between lobules. The intralobular collagen construction was quite different between the animals, indicating a fragile collagen fibril networks in pigs, a robust framework in goats and dense fabric-like septa in chickens. These results indicate that the distinct collagen frameworks may contribute to the histological strength of the livers in each of the animal species.     

Two different types of malignant fibrous histiocytomas from pet dogs

We describe 2 cases of malignant fibrous histiocytomas (MFHs) that spontaneously developed in young pet dogs. To classify these tumors, we applied a panel of antibodies (vimentin, desmin, α-SMA, and ED1) and Azan staining for collagen. The MFHs were most consistent with osteoclast-like giant and inflammatory cell types. The first case had positive staining for ED1 and vimentin, and given the osteoclast-like giant cells, calcification sites accompanying peripheral giant cell infiltrates. The latter case, the inflammatory cell type, exhibited a storiform-pleomorphic variant of neoplastic cells, including an ossifying matrix. MFHs are among the most highly aggressive tumors occurring in soft tissue sarcomas in elderly dogs; however, MFHs have been poorly studied from a diagnostic point of view. Herein, we describe the histologic and immunohistologic features of MFHs in detail, thus classifying the subtypes of these tumors.     

A novel vitamin K1 2,3-epoxide reductase (VKOR) inhibitor, 3-acetyl-5-methyltetronic acid, reduces experimental glomerulonephritis

Excessive proliferation of mesangial cells is observed in various types of glomerular disease including glomerulonephritis (GN), which is progressive in nature and eventually results in end-stage renal disease (ESRD). Vitamin K(1) 2,3-epoxide reductase (VKOR) and the vitamin K-dependent growth arrest-specific gene 6/Axl pathway play a key role in mesangial cell proliferation in GN. In the present study, we indicate the potential of a VKOR inhibitor, 3-acetyl-5-methyltetronic acid (AMT), to prevent the proliferation of glomerular mesangial cells and suppress the progression of GN. AMT was administered intravenously to rats once daily for 12 days and a mouse anti-Thy1 monoclonal antibody was injected intravenously after the AMT treatment on Day 6. Creatinine clearance (CCr) significantly increased and the albumin-to-creatinine ratio (ACR) significantly decreased in the AMT-treated group of the Thy-1 GN rats. In addition, glomerular and tubular damage was improved histopathologically in the AMT-treated group. AMT did not affect blood coagulation due to its unique pharmacokinetic properties. The concentration of AMT reached the IC(50) for VKOR in kidney, but not in liver. A novel VKOR inhibitor, AMT, reduced renal mesangial cell proliferation and could be a supportive treatment for GN.