Nephrotoxicity of Bence-Jones proteins: interference in renal epithelial cell acidification.

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.

Nephrotoxicity of Bence-Jones proteins: interference in renal epithelial cell acidification

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

Defective proximal tubule lysosomal acidification by Bence Jones proteins. An immunoelectron microscopy study.

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.

The renal and hepatic distribution of Bence Jones proteins depends on glycosylation: a scintigraphic study in rats.

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.

The renal and hepatic distribution of Bence Jones proteins depends on glycosylation: a scintigraphic study in rats

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.

Reduced humoral immunity in uremic mice genetically selected for high antibody response.

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.

Reduced humoral immunity in uremic mice genetically selected for high antibody response

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 ...

Nephrotoxicity of human Bence Jones protein in rats: proteinuria and enzymuria profile.

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.

Nephrotoxicity of human Bence Jones protein in rats: proteinuria and enzymuria profile

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

Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

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.