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.     

High incidence of glomerulonephritis associated with inclusion body hepatitis in broiler chickens: routine histopathology and histomorphometric studies

During the routine histologic evaluation of an outbreak of inclusion body hepatitis (IBH) in Mississippi broilers, a high incidence of renal enlargement and glomerulonephropathy was observed in the birds presenting classic hepatic pathology. Characteristic intranuclear adenoviral inclusion bodies were demonstrated in the livers of these birds, and fowl adenovirus was identified by viral isolation and by PCR. The glomerular lesions were consistent with proliferative or membranoproliferative forms of glomerulonephritis. Histomorphometric evaluations were performed to generate a more quantitative analysis of altered glomerular size and cellularity, to detect statistically significant borderline changes, and to get a clearer insight into the incidence of the glomerular alterations. Marked increases in both the average glomerular size (area) and the total glomerular cellularity were observed for the affected glomeruli relative to normal controls. The average glomerular area values for normal glomeruli in the peripheral subcapsular cortical and central cortical kidney regions were 1791 microm2 and 5302 microm2, respectively. In contrast, glomerular measurements for kidneys exhibiting glomerulonephritis by routine histopathology, had average values for the two regions of 4429 microm2 and 11,063 microm2. The average glomerular cell counts for the two regions in controls were 44 and 107 cells/ glomeruli, while averages for birds with glomerulonephritis were 85 and 193 cells/glomeruli. The proportion of IBH-associated glomeruli greater than two standard deviations above the mean glomerular size of the normal controls was 52% for the central region and 62% for the peripheral region.     

Glomerulonephritis caused by murine cytomegalovirus. Potentiation by antithymocyte serum.

Murine cytomegalovirus was found to be highly nephrotropic in mice treated with antithymocyte serum. The earliest histologic change was characteristic intranuclear inclusions in the glomerular endothelial cells. This change was followed by an eosinophilic thickening of the glomerular capillary loops. In later stages, proliferation of capsular epithelial cells and glomerular sclerosis was prominent. Tubular casts were present, and perivascular lymphoid aggregations were seen in the medulla and juxtamedullary section 56 days after inoculation. No conspicuous lesions occurred in uninfected mice treated with antithymocyte serum or in untreated infected mice. The possible mechanisms of the renal lesions are discussed.     

Glomerulopathy with IgA deposition in the dog.

In 100 dogs autopsied, glomerular IgA deposition was examined by the immunofluorescence technique and the histopathological features of glomeruli with IgA deposition were examined by light and electron microscopy. The incidence of the IgA deposition was age-related but there were no sex and breed predisposition. Deposition of IgA was observed mainly in mesangial areas in approximately a half (47%) of dogs examined. IgG, IgM and C3 often co-deposited. Histopathology of the glomeruli with IgA deposition indicated increase of mesangial cells, crescent formation, hemispherical deposits in paramesangial areas and glomerular sclerosis. Ultrastructurally electron dense substances positive for IgA deposited in mesangial and paramesangial areas. The examination to know the relation between the severity of IgA deposition and the number of mesangial cells or percent of the cells to total glomerular cells indicated that mesangial cells increased at the early stage of the disease and subsequently epithelial and endothelial cells proliferated as the increasing amount of IgA. Dogs suffering from enteritis or liver diseases showed high incidence of glomerular IgA deposition.     

Natural remission of nephrotic syndrome in a dog with immune-complex glomerular disease.

A nephrotic syndrome caused by immune-complex glomerular disease was diagnosed in a 4-year-old male Great Dane. The syndrome was characterized by proteinuria, hypoproteinemia, hypoalbuminemia, hypercholesterolemia, and subcutaneous edema. Renal biopsy revealed segmental membranous glomerular disease. The edema underwent complete remission 18 days after admission. Two months after admission, there was no clinical or laboratory evidence of glomerular disease. Periodic reevaluation of the dog during the next 2 years revealed recurrence of proteinuria, but no other clinical or laboratory abnormalities. Serial renal biopsies revealed persistence, but no appreciable increase, in the severity of the segmental membranous glomerular disease. The natural course of the nephrotic syndrome and immune-complex glomerular disease has been associated with unpredictable variability. It was concluded that the widespread use of corticosteroid or immunosuppressant therapy in dogs with immune complex glomerular disease should be withheld until the natural course of the disease has been evaluated in a significant number of patients and until the results of well-controlled clinical studies confirm or deny their therapeutic value.     

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.     

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.     

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.     

Mesangioproliferative Glomerulonephritis in Mink with Encephalitozoonosis

Renal specimens from 6 mink with encephalitozoonosis were studied by light and electron microscopy and immunohistochemistry. The glomeruli of affected kidneys had a mesangioproliferative glomerulonephritis which was characterized by an increase in mesangial cells and matrix in most glomeruli. Some glomeruli were partially or completely sclerosed. There were protein or granular casts in the cortical and medullary tubules. Interstitial nephritis, vasculitis and tubular cysts were found. Electron microscopy demonstrated extensive matrix and increased cellularity in the mesangial areas. Glomeruli showed segmentally thickened or wrinkled capillary basement membranes. Electron dense deposits were found in the glomerular basement membranes and mesangium. Peroxidase-anti-peroxidase immunohistochemistry demonstrated that IgG and IgM positive material was present as granular deposits in the glomerular basement membrane and occasionally in the mesangium.     

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.     

Proteinuria in the dog: a pathomorphological study of 51 proteinuric dogs

Renal cortical biopsies of 51 dogs with spontaneous proteinuria were examined by histology, electron microscopy and immunofluorescence. Glomerular lesions were classified in four groups: mesangioproliferative, membranoproliferative and membranous glomerulonephritis and amyloidosis. The glomerular and the tubulointerstitial lesions were graded, using a semiquantitative system. The results were used for the calculation of correlation coefficients between several parameters. A positive correlation was found between the severity of glomerular and tubulointerstitial lesions. Fibrin detection by immunofluorescence and histochemical methods appeared not to be correlated. The presence of electron dense deposits correlated only with the fluorescence for IgG and C3. Fluorescence for IgA and IgM was frequently observed in cases with or without dense deposits.     

Glomerular lesions in dogs infected with Leishmania organisms

OBJECTIVE: To histologically identify glomerular lesions in dogs infected with Leishmania organisms. ANIMALS: 41 dogs (17 sexually intact males and 14 sexually intact and 10 ovariohysterectomized females) that had positive results when tested for leishmaniosis as determined by use of serologic evaluation (indirect fluorescent antibody test, titers of 1:80 to 1:640) and direct microscopic identification of the protozoal organisms. PROCEDURE: Urine samples were collected by use of cystocentesis and examined by qualitative SDS-agarose gel electrophoresis (AGE). All dogs had non-selective (glomerular) or mixed (glomerular and tubular) proteinemia. Specimens were obtained from each dog during ultrasound-assisted renal biopsy and used for histologic examination. Each specimen was stained with H&E, periodic acid-Schiff, Goldner's trichrome, methenamine silver, and Congo Red stains. Specimens were adequate for evaluation when they contained at least 5 glomeruli/section, except for specimens stained with Congo Red in which 1 glomerulus/section was adequate. RESULTS: Examination of renal biopsy specimens revealed various glomerular lesions in all dogs and interstitial or tubular (or both) lesions in 23 of 41 (55%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Glomerular lesions that develop in dogs during infection with Leishmania organisms can be classified histologically as mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, and focal segmental glomerulonephritis. Tubulointerstitial histopathologic conditions were not observed as the primary lesion, despite being evident in 23 of 41 (55%) dogs. Use of SDS-AGE for qualitative evaluation of proteinuria and successive collection of specimens during renal biopsies following diagnosis of nonselective glomerular proteinuria provides the possibility for early identification of renal lesions.     

Spontaneous early-onset glomerulonephritis in a 8-week-old male Crj:CD1 (ICR) mouse

Glomerular lesions including membranoproliferative glomerulonephritis occur spontaneously in aged mice, but they are rare in young animals. In our laboratory, spontaneous glomerulonephritis was observed in an 8-week-old male Crj:CD1 (ICR) mouse. Macroscopically, the bilateral kidneys were discolored, but no edema or ascites was observed. Glomerular lesions were characterized by a thickening of capillary walls, a double-contoured basement membrane and mesangial expansion due to increased amounts of matrix. Ultrastructurally, mesangial interposition in the capillary wall and subendothelial deposition of basement membrane-like material were observed. No evidence of immune complex deposition or amyloid was found. On the basis of the observed clinical pathology and histopathology, a secondary form of glomerular lesion was excluded. The glomerular lesion was compatible with glomerulonephritis in a young Crj:CD1 (ICR) mouse.     

Evaluation of the effects of inhibition of angiotensin converting enzyme with enalapril in dogs with induced chronic renal insufficiency

OBJECTIVE: To determine whether the angiotensin converting enzyme inhibitor enalapril would lower systemic arterial and glomerular capillary pressure and reduce the magnitude of renal injury in a canine model of renal insufficiency. ANIMALS: 18 adult dogs that had renal mass reduced by partial nephrectomy. PROCEDURE: After surgical reduction of renal mass and baseline measurements, dogs in 2 equal groups received either placebo (group 1) or enalapril (0.5 mg/kg, PO, q 12 h; group 2) for 6 months. RESULTS: Values for systemic mean arterial blood pressure determined by indirect and direct measurement after 3 and 6 months of treatment, respectively, were significantly lower in group 2 than in group 1. During treatment, monthly urine protein-to-creatinine ratios were consistently lower in group 2 than in group 1, although values were significantly different only at 3 months. At 6 months, significant reduction in glomerular capillary pressure in group 2 was detected, compared with group 1, but glomerular filtration rate in group 2 was not compromised. Glomerular hypertrophy, assessed by measurement of planar surface area of glomeruli, was similar in both groups. Glomerular and tubulointerstitial lesions were significantly less in group 2, compared with group 1. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that inhibition of angiotensin converting enzyme was effective in modulating progressive renal injury, which was associated with reduction of glomerular and systemic hypertension and proteinuria but not glomerular hypertrophy. Inhibition of angiotensin converting enzyme may be effective for modulating progression of renal disease in dogs.     

Persistent haematuria and proteinuria due to glomerular disease in related Abyssinian cats

Eight cases of glomerular disease in young, related Abyssinian cats are described. Haematuria was the most consistent feature. Six cats developed the nephrotic syndrome. The short-term prognosis was good for cats with haematuria and fair for cats with the nephrotic syndrome as oedema resolved in three of the six cats. Light microscopic examination of renal biopsies from three cats was considered normal or revealed only mild abnormalities. In the three cases subjected to necropsy, histological abnormalities included mild mesangial hypercellularity and adhesions between the glomerular tuft and Bowman's capsule consistent with a focal proliferative glomerulopathy. Further investigation into this glomerulopathy will require ultrastructural and immunohistochemical studies to characterise the glomerular abnormality and genetic analyses to investigate its potential to be an inherited disease. Glomerular disease, potentially a familial one, should be considered in the investigation of persistent haematuria or proteinuria in Abyssinian and related cats.     

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.     

Diet modulates proteinuria in heterozygous female dogs with X-linked hereditary nephropathy

Young adult heterozygous (carrier) female dogs with X-linked hereditary nephropathy (XLHN) have glomerular proteinuria but are otherwise healthy. Because data regarding dietary influences on the magnitude of proteinuria in dogs with spontaneous glomerular disease are not available, 12 such dogs were studied in a double crossover experiment intended to determine effects of altering dietary protein intake for up to 6 weeks. Dogs were blocked by urine protein : creatinine ratio (UPC) and randomly assigned to receive 2 diets: high protein (34.6% dry matter [DM], HP) or low protein (14.1% DM, LP) fed in HP-LP-HP or LP-HP-LP sequence. Food intake was measured daily, body weight (BW) was measured twice weekly, and UPC, plasma creatinine, blood urea nitrogen, phosphorus, albumin, and protein concentrations were measured at 2-week intervals. Nutrient digestibility was measured during the third treatment period. Diet had a significant effect (P < .0001) on all measured variables except plasma phosphorus (P > .5), but unintended differences in digestibility of protein and energy (P < or = .01) prevented assignment of the diet effect exclusively to protein. Proteinuria was greater (UPC 4.7 +/- 2.2 versus 1.8 +/- 1.1, P < .0001) when the HP diet was fed, but the LP diet did not maintain starting BW or plasma albumin concentration within the normal reference range. Diet greatly affects the magnitude of proteinuria in XLHN carrier females. Dietary protein restriction can reduce proteinuria in dogs with glomerular disease, but BW and blood protein concentrations may not be maintained if the restriction is too severe.     

Immune complex-mediated glomerulonephritis associated with bacterial kidney disease in the rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) developed a post-infectious chronic membranous glomerulonephritis 15 months after they had been experimentally infected with Renibacterium salmoninarum. Histologically, peritubular and periglomerular fibrosis, hypercellular glomeruli with occluded Bowman's space, and partial or complete adhesion to Bowman's capsule were constant features. Electron microscopy revealed thickened glomerular basement membranes with spikes accompanied by finely granular electron-dense deposits at the epithelial side and dense material in the mesangial matrix. Indirect immunofluorescence indicated linear immunoglobulin deposits along the glomerular basement membrane. The presence of R. salmoninarum was demonstrated by culture and by indirect immunofluorescence. Low serum hemagglutination-inhibiting antibody titers were demonstrated.     

Effects of thromboxane synthetase inhibition on immune complex glomerulonephritis.

To determine the role of thromboxane A2 in the pathogenesis of experimentally induced immune complex glomerulonephritis, 12 concanavalin A-immunized Beagles were infused with 1 mg of concanavalin A via each renal artery and treated twice daily for 8 days with either 30 mg of CGS 12970/kg, PO, a specific thromboxane synthetase inhibitor, or placebo. The effect of treatment was assessed by measuring endogenous creatinine clearance and urine protein and eicosanoid excretion, and by evaluating changes in glomerular morphometric characteristics. On postinfusion day 8, urine protein, thromboxane B2, and 11-dehydro-thromboxane B2 excretion, glomerular epithelial crescent formation, and glomerular cell proliferation in the CGS 12970-treated dogs were significantly decreased when compared with values in the placebo-treated group. Differences were not observed in endogenous creatinine clearance, urine prostaglandin E2 and 6-keto-prostaglandin F1 alpha excretion, or glomerular polymorphonuclear leukocyte infiltration between groups in this study. These findings suggest thromboxane A2 has a role in the development of immune complex glomerulonephritis and that thromboxane synthetase inhibition may be beneficial in attenuating some of the functional and histological changes associated with immune complex glomerulonephritis.