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The aim of the present study was to evaluate the acidification of the endosome-lysosome system of renal epithelial cells after endocytosis of two human immunoglobulin lambda light chains (Bence-Jones proteins, BJP) obtained from patients with multiple myeloma. Renal epithelial cell handling of two BJP (neutral and acidic BJP) was evaluated by rhodamine fluorescence. Renal cells (MDCK) were maintained in culture and, when confluent, were incubated with rhodamine-labeled BJP for different periods of time. Photos were obtained with a fluorescence microscope (Axiolab-Zeiss). Labeling density was determined on slides with a densitometer (Shimadzu Dual-Wavelength Flying-Spot Scanner CS9000). Endocytosis of neutral and acidic BJP was correlated with acidic intracellular compartment distribution using acridine orange labeling. We compared the pattern of distribution after incubation of native neutral and acidic BJP and after complete deglycosylation of BJP by periodate oxidation. The subsequent alteration of pI converted neutral BJP to acidic BJP. There was a significant accumulation of neutral BJP in endocytic structures, reduced lysosomal acidification, and a diffuse pattern of acidification. This pattern was reversed after total deglycosylation and subsequent alteration of the pI to an acidic BJP. We conclude that the physicochemical characteristics of BJP interfere with intracellular acidification, possibly explaining the strong nephrotoxicity of neutral BJP. Lysosomal acidification is fundamental for adequate protein processing and catabolism.

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RESUMO

The aim of the present study was to evaluate the acidification of the endosome-lysosome system of renal epithelial cells after endocytosis of two human immunoglobulin lambda light chains (Bence-Jones proteins, BJP) obtained from patients with multiple myeloma. Renal epithelial cell handling of two BJP (neutral and acidic BJP) was evaluated by rhodamine fluorescence. Renal cells (MDCK) were maintained in culture and, when confluent, were incubated with rhodamine-labeled BJP for different periods of time. Photos were obtained with a fluorescence microscope (Axiolab-Zeiss). Labeling density was determined on slides with a densitometer (Shimadzu Dual-Wavelength Flying-Spot Scanner CS9000). Endocytosis of neutral and acidic BJP was correlated with acidic intracellular compartment distribution using acridine orange labeling. We compared the pattern of distribution after incubation of native neutral and acidic BJP and after complete deglycosylation of BJP by periodate oxidation. The subsequent alteration of pI converted neutral BJP to acidic BJP. There was a significant accumulation of neutral BJP in endocytic structures, reduced lysosomal acidification, and a diffuse pattern of acidification. This pattern was reversed after total deglycosylation and subsequent alteration of the pI to an acidic BJP. We conclude that the physicochemical characteristics of BJP interfere with intracellular acidification, possibly explaining the strong nephrotoxicity of neutral BJP. Lysosomal acidification is fundamental for adequate protein processing and catabolism

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Chronic nitric oxide (NO) inhibition causes hypertension and renal injury. Concomitant salt overload promotes massive albuminuria. We investigated the mechanisms whereby these treatments impair glomerular permselectivity. Adult male Munich-Wistar rats received either a standard-salt (SS; 0.5% Na) or high-salt (HS; 3.1% Na) diet and either no treatment or the NO inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). At 30 days, albuminuria was moderate, the density of fixed anionic sites at the glomerular basement membrane (GBM), estimated by cationic ferritin binding, declined by approximately 35%, and the fractional clearance of 70-kDa neutral dextran (phi) rose moderately in rats receiving L-NAME and SS. Rats given L-NAME and HS exhibited massive albuminuria, whereas phi was nearly tripled. Depletion of GBM anionic sites was also seen in these rats. The GBM was thickened in both L-NAME-treated groups. These abnormalities were largely reversed after cessation of treatments. These results indicate that chronic L-NAME treatment promotes reversible albuminuria by impairing both glomerular size and charge selectivity. These effects likely reflect functional rather than structural disruption of the glomerular wall.

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Proximal tubule handling of two human Bence Jones proteins (neutral and acidic BJP) was evaluated using protein A-gold labelling. After 30 min of acute light-chain infusion into 6 rats (alone or in combination with dinitrophenyl-aminopropyl-methylamine [DAMP]), kidney biopsies were processed for immunoelectron microscopy. Antibodies directed at monoclonal lambda light chains, mannose-6-phosphate cation-independent receptor (MPR) and DAMP were used. Labelling density (number of pA-gold particles/micrometer2), expressed as median (25-75 percentiles), differed (p < 0.05) between the two BJP, being 94.5 (32.9-212.5) vs. 19.4 (3.7-45.6) pA-gold/ micrometer2++ in endocytic vacuoles, and 297.3 (207.1-382.1) vs. 83.2 (16. 6-197.0) pA-gold/ micrometer2 in non-vacuolar electrondense endosome-lysosome structures. Labelling density for MPR was 47.7 (22. 2-84.6) vs. 4.0 (2.7-6.3) pA-gold/micrometer2. The area of MPR-labelled structures was also different, i.e.: 0.2 (0.1-0.4) vs. 0.9 (0.5-1.8) micrometer2. The endosome-lysosome pH distribution range differed significantly: 6.8 (6.4-7.0) vs. 6.3 (5.8-7.0). There was a significant accumulation of neutral BJP in endocytic structures, an acidification deficit of pre-lysosomes/lysosomes and MPR retention, suggestive of defective receptor recycling with this BJP. Interference with the physiological process of lysosomal acidification may be an important mechanism of higher nephrotoxicity in some BJP.

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The aim of the present study was to evaluate renal and liver distribution of two monoclonal immunoglobulin light chains. The chains were purified individually from the urine of patients with multiple myeloma and characterized as lambda light chains with a molecular mass of 28 kDa. They were named BJg (high amount of galactose residues exposed) and BJs (sialic acid residues exposed) on the basis of carbohydrate content. A scintigraphic study was performed on male Wistar rats weighing 250 g for 60 min after i.v. administration of 1 mg of each protein (7.4 MBq), as the intact proteins and also after carbohydrate oxidation. Images were obtained with a Siemens gamma camera with a high-resolution collimator and processed with a MicroDelta system. Hepatic and renal distribution were established and are reported as percent of injected dose. Liver uptake of BJg was significantly higher than liver uptake of BJs (94.3 vs 81.4%) (P < 0.05). This contributed to its greater removal from the intravascular compartment, and consequently lower kidney accumulation of BJg in comparison to BJs (5.7 vs 18.6%) (P < 0.05). After carbohydrate oxidation, there was a decrease in hepatic accumulation of both proteins and consequently a higher renal overload. The tissue distribution of periodate-treated BJg was similar to that of native BJs: 82.7 vs 81.4% in the liver and 17.3 vs 18.6% in the kidneys. These observations indicate the important role of sugar residues of Bence Jones proteins for their recognition by specific membrane receptors, which leads to differential tissue accumulation and possible toxicity.

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RESUMO

The aim of the present study was to evaluate renal and liver distribution of two monoclonal immunoglobulin light chains. The chains were purified individually from the urine of patients with multiple myeloma and characterized as lambda light chains with a molecular mass of 28 kDa. They were named BJg (high amount of galactose residues exposed) and BJs (sialic acid residues exposed) on the basis of carbohydrate content. A scintigraphic study was performed on male Wistar rats weighing 250 g for 60 min after iv administration of 1 mg of each protein (7.4 MBq), as the intact proteins and also after carbohydrate oxidation. Images were obtained with a Siemens gamma camera with a high-resolution collimator and processed with a MicroDelta system. Hepatic and renal distribution were established and are reported as percent of injected dose. Liver uptake of BJg was significantly higher than liver uptake of BJs (94.3 vs 81.4 per cent) P<0.05). This contributed to its greater removal from the intravascular compartment, and consequently lower kidney accumulation of BJg in comparison to BJs (5.7 vs 18.6 per cent) (P<0.05). After carbohydrate oxidation, there was a decrease in hepatic accumulation of both proteins and consequently a higher renal overload. The tissue distribution of periodate-treated BJg was similar to that of native BJs: 82.7 vs 81.4 per cent in the liver and 17.3 vs 18.6 per cent in the kidneys. These observations indicate the important role of sugar residues of Bence Jones proteins for their recognition by specific membrane receptors, which leads to diffedential tissue accumulation and possible toxicity.

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Biozzi's Selection IV-A mice, genetically selected for 25 generations for high and low antibody response to sheep red blood cells (SRBC), 2-3 months old, were made uremic by subtotal nephrectomy and characterized for antibody production against the selection antigen. T cell activity was evaluated in vitro by lymphocyte proliferation and interleukin 2 (IL 2) production in response to the super antigen staphylococcal enterotoxin B (SEB). Total and IgM antibody titers (log2) were similar in uremic and non-uremic low responder mice (total antibody: 4.0 +/- 0.6 vs 3.6 +/- 0.6; IgG: 3.0 +/- 0.7 vs 2.4 +/- 0.4), while uremic high responders presented with non-uremic animals (total antibody: 10.8 +/- 1.6 vs 13.0 +/- 0.2; IgG: 10.3 +/- 1.5 vs 11.7 +/- 0.3). T cell proliferation and IL 2 production were similar in uremic and non-uremic groups after SEB stimulation. The results indicate a genetic effect on sensitivity to humoral immune response modulation by uremic status, with deficient antibody production despite a normal T cell proliferative response to mitogen stimulation in short-duration renal failure in high responder mice.

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Biozzi's Selection IV-A mice, genetically selected for 25 generations for high and low antibody response to sheep red blood cells (SRBC), 2-3 months old, were made uremic by subtotal nephrectomy and characterized for antibody production against the selection antigen. T cell activity was evaluated in vitro by lymphocyte proliferation and interleukin 2 (IL 2) production in response to the superantigen staphylococcal enterotoxin B (SEB). Total and IgM antibody titers (log2) were similar in uremic and non-uremic low responder mice (total antobody: 4.0 +/- 0.6 vs 3.6 +/- 0.6; IgG: 3.0 +/- 0.7 vs 2.4 +/- 0,4), while uremic high responders presented a blunted humoral immune response to SRBC when compared with non-uremic animals (total antibody: 10.8 +/- 1.6 vs 13.0 +/- 0.2; IgG: 10.3 +/- 1.5 vs 11.7 +/- 0.3). T cell proliferation and IL 2 production were similar in uremic and ...

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The effect of intravenous administration of 80 mg purified human Bence Jones protein twice weekly for 5 weeks was investigated in male Wistar rats (N = 7; 2 months old). A state of immunological tolerance was demonstrated by the absence of a B-cell response (plaque-forming cells and hemagglutination titers) and by the absence of detectable antigen or antibody deposition in glomeruli, as indicated by light and electron microscopy. No rise in blood urea level was detected (33.9 +/- 4.3 vs 32.8 +/- 1.3 mg%). There was an increase in proteinuria (5.3 +/- 0.9 vs 32.8 +/- 4.0 mg/day), mainly due to Bence Jones protein excretion (0 vs 29.2 +/- 5.2 mg/day), with a slight but significant increase in albuminuria (0.2 +/- 0.1 vs 1.0 +/- 0.2 mg/day). There was a significant increase of lysosomal N-acetyl-beta-D-glucosaminidase in the urine (6.1 +/- 1.3 vs 72.7 +/- 18.8 mU/mg in creatinine). Lysosomal accumulation of Bence Jones protein in proximal tubular cells was evidenced by immunoelectronmicroscopy with protein A-gold. These results clearly showed proximal tubular dysfunction induced by chronic Bence Jones protein administration, without interference of autologous immune response as demonstrated by immunological state of tolerance.

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The effect of intravenous administration of 80 mg purified human Bence Jones protein twice weekly for 5 weeks was investigated in male Wistar rats (N = 7; 2 months old). A state of immunological tolerance was demonstrated by the absence of a B-cell response (plaque-forming cells and hemagglutination titers) and by the absence of detectable antigen or antibody deposition in glomeruli, as indicated by light and electron microscopy. No rise in blood urea level was detected (33.9 +/- 4.3 vs 32.8 +/- 1.3 mg per cent ). There was an increase in proteinuria (5.3 +/- 0.9 vs 32.8 +/- 4.0 mg/day), mainly due to Bence Jones protein excretion (0 vs 29.2 +/- 5.2 mg/day), with a slight but significant increase in albuminuria (0.2 +/- 0.1 vs 1.0 +/- 0.2 mg/day). There was a significant increase of lysosomal N-acetyl-beta-D-glucosaminidase in the urine (6.1 +/- 1.3 vs 72.7 +/- 18.8 mU/mg in creatinine). Lysosomal accumulation of Bence Jones protein in proximal tubular cells was evidenced by immunoelectronmicroscopy with protein A-gold. These results clearly showed proximal tubular dysfunction induced by chronic Bence Jones protein administration, without interference of autologous immune response as demonstrated by immunological state of tolerance

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Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence.

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We describe a child with dermatomyositis and calcified necrosis in the middle third of both ureters. Histopathological examination showed vasculitis associated with ureteral necrosis and calcification. These findings together with a similar previous report in the literature have prompted us to correlate dermatomyositis in childhood with ureteral necrosis and to anticipate specifically a lesion in the middle third of the ureter because of the relative lack of blood flow in that segment.

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1. Normal rats and passive Heymann membranous glomerulonephritic rats were chronically treated with DOCA/NaCl for 9 weeks. Normal and untreated nephritic rats were used as controls. Urinary kallikrein excretion (UKE), proteinuria and tail blood pressure (BP) were determined in awake rats. Glomerular filtration rate (GFR), plasma renin activity (PRA), and plasma potassium (PK) concentration were measured at the end of the experiment. 2. Comparison between basal and 9th-week measurements indicated that DOCA/NaCl administration significantly increased (P less than 0.05) UKE (3.96 +/- 0.30 vs 7.60 +/- 1.51 U/24 h) and BP (118 +/- 2 vs 135 +/- 6 mmHg) in normal rats, whereas in nephritic DOCA/NaCl-treated rats, UKE was unaltered (3.80 +/- 0.50 vs 3.40 +/- 0.30 U/24 h) and BP increased to higher levels (117 +/- 2 vs 152 +/- 3 mmHg) than in the normal DOCA/NaCl group (P less than 0.05). Passive Heymann nephritis alone did not affect UKE (3.56 +/- 0.40 vs 3.60 +/- 0.80 U/24 h) or BP (124 +/- 2 vs 125 +/- 2 mmHg). 3. At the end of the study, PK was decreased and PRA totally suppressed in both normal and nephritic DOCA/NaCl-treated rats. Proteinuria was more pronounced in nephritic DOCA/NaCl-treated rats (44.8 +/- 5.2 mg/day) than in control nephritic animals (15.1 +/- 2.4 mg/day) and GFR was increased equally in both DOCA/NaCl-treated groups. 4. The failure of nephritic rats to respond to DOCA/NaCl by increasing UKE was not associated with any significant derangement of renal function or structure and may have been related to the aggravation of arterial hypertension in this group.

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1. Normal rats and passive Heymann membranous glomerulonephritic rats were chronically treated with DOCA/NaCl for 9 weeks. Normal and untreated nephritic rats were used as controls. Urinary kallikrein excretion (UKE), proteinuria and tail blood pressure (BP) were determined in awake rats. Glomuerular filtration rate (GFR), plasma renin activity (PRA), and plasma potassium (PK) concentration were measured at the end of the experiment. 2. Comparasion between basal and 9th-week measurements indicated that DOCA/NaCl administration significantly increased (P < 0.05) UKE (3.96 ñ 0.30 vs 7.60 ñ 1.51 U/24 h) and 118 ñ 2 vs 135 ñ 6 mmHg) in normal rats, whereas in nephritic DOCA/NaCl-treated rats, UKE was unaltered (3.80 ñ 0.50 vs 3.40 ñ 0.30 U/24 h) and BP increased to higher levels (117 ñ 2 vs 152 ñ 3 mmHg) than in the normal DOCA/NaCl group (P < 0.05). Passive Heymann nephritis alone did not affect UKE (3.56 ñ 0.40 vs 3.60 ñ 0.80 /24 h) or BP (124 ñ 2 vs 125 ñ 2 mmØg). At the end of the study, PK was decrease and PRA totally suppressed in both normal and nephritic DOCA/NaCl - treated rats. Proteinuria was more pronounced in nephritic DOCA/NaCl - treated rats (44.8 ñ 5.2 mg/day) than in control nephritic animals (15.1 ñ 2.4 mg/day) and GFR was increased equally in both DOCA/NaCl-treated groups. 4. The failure of nephritic rats respond to DOCA/NaCl by increasing UKE was not associated with any significant derangement of renal function or structure and may have been related to the aggravation of arterial hypertension in this group

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1. Female Wistar rats received a single subnephrotoxic dose of guinea pig anti-glomerular basement membrane (GBM) IgG1, 2.5 mg, followed by infusion of preformed immune complexes (BSA, 5.0 mg/rabbit anti-BSA, 6 mg), 10 X antigen excess. Control groups received guinea-pig IgG1 anti-GBM, or preformed immune complexes alone, or isotonic saline. Systemic reactions were observed clinically during the first 24 h, and 24 h urine was collected for the measurement of proteinuria and hematuria. 2. Blood was collected before and 2 h after the above treatment for the determination of complement (50% hemolytic assay), kininogen (isolated guinea pig assay of released bradykinin-like spasmogenic activity) and activated partial thromboplastin time (APTT). Kidney and lung tissue was examined by light microscopy, immunofluorescence and electron microscopy. 3. Rats treated with guinea pig anti-GBM IgG1 followed by BSA immune complex presented a severe systemic picture, with macroscopic hematuria (9/14), several deaths (8/14), slight proteinuria (24.6 +/- 5.2 mg/day), marked complement consumption (delta = 49.4 +/- 2.4 UCH50/ml), intravascular coagulation and severe diffuse interstitial pneumonia, obliteration of glomerular capillary walls by edema of endothelial cells, without deposition of immune complexes in kidneys or lungs. The control groups showed no signs of systemic reaction (isotonic saline alone) or slight dyspnea (guinea pig anti-GBM IgG1 or immune complexes alone), without proteinuria or macroscopic hematuria, and with foci of interstitial pneumonia. 4. Complement consumption was significant in rats receiving immune complexes alone (delta = 31.1 +/- 1.3 UCH50/ml) and even higher when associated with infusion of guinea pig anti-GBM IgG1 (delta = 49.3 +/- 2.4 UCH50/ml). APTT was significantly lengthened only for the group treated with guinea pig anti-GBM IgG1 plus immune complexes (delta = 18.5 +/- 1.9 s), with no alterations in the other groups. Kininogen consumption was demonstrable for all groups except the saline control and was more extensive in rats which received immune complexes alone or preceded by guinea pig IgG1. 5. These data show that previous infusion of a subnephrotoxic dose of guinea pig IgG1 anti-GBM aggravated the pathological effects of preformed immune complexes by promoting marked complement consumption and activation of the coagulation system, rather than by enhancing tissue deposition.

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Se describe un caso de tumor glómico solitario, el cual microscópicamente podría encuadrarse en la rara variante de glomangiomioma y que muestra un aspecto clínico atípico, relación que se pretende resaltar.

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Os tumores de testiculo em crianca sao uma patologia rara. Os autores apresentam 3 casos, todos tratados cirurgicamente, atraves de orquiectomia radical por via inguinal. Nao foi utilizada a radioterapia, nem a quimioterapia associada. Apos 3 anos de acompanhamento, dos 3 casos, nao se evidenciaram metastases, ou qualquer sinal de disseminacao

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Reportamos um caso de tratamento de valvula de uretra posterior, no qual novo acesso foi tentado com exito. O acesso proposto faculta o tratamento ja no periodo perinatal e prescinde de instrumental infantil. Foi feita uma vesicostomia como primeiro procedimento e, depois, a cauterizacao da valvula feita pelo orificio da mesma, quando a crianca estava com seis meses de vida. As vantagens do metodo sao discutidas

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