Analysis of macrophages in urine sediments in children with IgA nephropathy.

AIM: Although infiltrating macrophages found in renal biopsy specimens have been accepted as a useful marker for evaluating the activity of IgA nephropathy (IgAN), it is difficult to perform renal biopsies repeatedly, especially in children. To establish a more convenient and noninvasive method for estimating the degree of macrophage infiltration we examined the number of macrophages in urinary sediments. PATIENTS AND METHODS: Ten ml of morning urine were collected from 30 children with IgAN, 10 with thin basement membrane disease (TBMD), 8 with idiopathic renal hemorrhage (IRH) which was defined as nonglomerular hematuria due to nutcracker phenomenon revealed on ultrasonography, and 10 healthy children as controls. Ten of the 30 children with IgAN were treated with combination therapy comprising prednisolone, warfarin and dipyridamole and urine samples were collected weekly during the period of treatment. Two microl of the urine sediment were smeared on glass slides, dried and stained with a monoclonal antibody to human macrophages (anti-CD68, PG-M1) followed by a FITC-conjugated secondary antibody. After staining with propidium iodide (PI), the cells were examined by fluorescence microscopy with cells stained with both FITC and PI being counted as macrophages. In addition, anti-CD68 staining was used to quantify macrophage infiltration in renal biopsies from the same group of IgAN patients. RESULTS: The number of urine macrophages in children with IgAN was significantly higher than in children with TBMD and IRH as well as the control group (p < 0.01), whereas that was similar among TBMD, IRH and healthy children. In IgAN, there was a significant correlation between urine macrophage number and the activity index (p < 0.01), proteinuria (p < 0.01) and urine WBC count (p < 0.01). In addition, there was also a significant correlation between urine macrophage number and glomerular (p < 0.05) as well as interstitial macrophage infiltration (p < 0.01). In children with IgAN who received combination therapy, urine macrophage number decreased significantly (p < 0.01) in the 1st week of treatment whilst the degree of proteinuria decreased significantly (p < 0.01) in the 4th week. CONCLUSION: Urinary macrophage number may represent a noninvasive and straightforward estimate of the pathological activity evident in renal biopsy specimens, and may also be a more sensitive indicator than proteinuria of the therapeutic effect of interventional treatments in childhood IgAN.

[Apoptotic cell death induced by actinomycin D arisen at different levels and cell cycle stages according to human cultured cell species].

Nine kinds of human cultured cells, including fetus cells (smooth chorion trophoblast cells, amnion epithelial cells and HE-21), adult non-carcinoma cells (HCF), and carcinoma cells (KATO-III, COLO 201, Lu-134-AH, SK-OV-3 and SKG-3a) were stimulated with Actinomycin (Act.) D for 24 h. Apoptosis induction was investigated by agarose gel electrophoresis for DNA fragmentation analysis and by flow cytometric analysis of stained cells using in situ terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling TUNEL) staining techniques for the quantification of apoptosis, and simultaneously using propidium iodide for the gain of some information about cell cycle. By agarose gel electrophoresis, DNA fragmentation of these cells except amnion epithelial and SKG-3a cells was detected, depending on concentration of Act. D. Using flow cytometric analysis, these cells were separated into four groups according to the information about cell cycle. Group 1 included amnion epithelial and SKG-3a cells, which were TUNEL negative. In group 2, all cell populations at G0/G1 and G2/M phases of HCC, KATO-III and SK-OV-3 were TUNEL staining positive. A portion of each G0/G1 or G2/M phase cell of Lu-134-AH and COLO 201 in group 3 was TUNEL stain positive. In group 4, G2/M phase cells of smooth chorion trophoblast cells and HE-21 were mostly stained and a small population of G0/G1 phase cells were also TUNEL stain positive. These results show that the stages of the cell cycle at which apoptosis was arisen by Act. D stimulation were significantly different depending on the cells types.

[Analysis of glomerular anionic charge status in renal biopsy specimens of childhood minimal change nephrotic syndrome using confocal laser scanning microscopy].

Analysis of glomerular anionic charge in human renal biopsy specimens has been restricted previously to staining of sites at the electron microscopic level, which is a product that needs skills and precludes a wide observable area. The introduction of a new tool, confocal laser scanning microscopy together with FITC conjugated poly-L-lysine as a cationic tracer, which demonstrates fixed anionic sites in thin sections from routinely formalin-fixed and paraffin-embedded renal biopsy tissue, has now enabled glomerular charge at light microscopic level. In this method, the patterns of staining in tissue showing minimal change nephrotic syndrome (MCNS) indicate that the intensity of anionic charge in 4 children with heavy proteinuria was significantly less than that in 7 children without proteinuria at remission, supporting previous observations using electron microscopy. Furthermore, staining the serial sections after methylation or saponification revealed that carboxyl components such as sialic acid may be responsible for proteinuria. We anticipate that this method may facilitate the investigation of the participation of charged components in the pathogenesis of MCNS and their role in relation to glomerular proteinuria.

[A case of poorly differentiated hepatic cell carcinoma which showed 90% decrease in size after chemotherapy with intraarterial infusion of mitomycin C and oral administration of tegafur].

The patient was a 77-year-old man, admitted complaining of abdominal fullness and appetite loss. By ultrasonography and CT an 8 x 5 cm mass was discovered in S1 of the liver. Needle biopsy specimen from the lesion revealed poorly differentiated hepatic cell carcinoma. Because of his advanced age and the size of the tumor, surgical therapy was not used. Chemotherapy with intraarterial injection of mitomycin C 2 mg once a week and 800 mg of tegafur PO daily was given for 5 weeks until bone marrow suppression developed. After recovery of hematological data, tegafur 800 mg PO daily every other week was administered for a year. In the course of these therapies, the hepatic tumor became smaller, and the ultimate decrease rate was over 90% (PR). Thus far it seems that chemotherapy with tegafur might be tried in hepatic cell carcinoma cases in which surgery is not indicated.