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.

Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus.

The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K(m) of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.

Intravenous low-dose erythromycin administration for infants with feeding intolerance.

BACKGROUND: This study was undertaken to determine the efficacy of low-dose intravenous erythromycin (EM) administration in infants with feeding intolerance. METHODS: The subjects were 26 infants who would not accept enteral feeding within 5 days after birth. Fourteen infants (gestational age: 30.6+/-5.4 weeks and birthweight: 1466+/-825 g) were given EM intravenously at a dose of 1 mg/kg, three times daily (EM group). Doses were increased to 2 mg/kg in five infants who showed a poor response. Twelve infants (gestational age: 30.5+/-5.0 weeks and birthweight: 1317+/-672 g) were observed without EM administration (non-EM group). Blood concentrations of EM at 2 h after administration were measured on 8 (+/-2) days after the start of EM administration in the EM group. RESULTS: Digestive perturbations and intestinal gasless and/or atonic shadows on X-ray findings markedly improved in the EM group soon after the treatment. Comparing the EM group and non-EM group, the postnatal ages at the start of successful enteral feeding were 9.1+/-3.2 days and 14.0+/-4.1 days, respectively (P<0.01). The postnatal ages at feeding of 100 mL/kg per day were 15.2+/-4.0 days and 23.4+/-6.2 days, respectively (P<0.01). The blood EM concentrations of 1 mg/kg and 2 mg/kg were 0.29+/-0.28 microg/mL and 0.57+/-0.20 microg/mL, respectively (P<0.05). No adverse effect on cardiac status or in blood examinations was observed in any infant in the EM group. CONCLUSION: These results suggest that intravenous low-dose EM administration is a useful and safe treatment of feeding intolerance in infants including extremely low-birthweight infants.

[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.

Plasma levels of granulocyte elastase-alpha1-proteinase inhibitor complex in children with hemolytic uremic syndrome caused by verotoxin-producing Escherichia coli.

BACKGROUND: Activated neutrophils play an important role in the pathogenesis of renal injury in humans and in experimental models of hemolytic uremic syndrome (HUS). To evaluate the clinical significance of the circulating granulocyte elastase-alpha1-proteinase inhibitor complex (GEPIC), which is a marker of neutrophil activation, we investigated the plasma concentrations of GEPIC in children with hemolytic uremic syndrome (HUS) associated with verotoxin-producing Escherichia coli (VTEC), VTEC gastroenteritis without HUS and in normal controls. METHODS: Of 22 children (1-19 years of age; mean age 5.5 years) with VTEC infection, nine were diagnosed with HUS. Plasma GEPIC, soluble thrombomodulin (sTM) and thrombin-antithrombin-III complex (TAT) levels were measured by ELISA. RESULTS: The number of polymorphonuclear leukocytes and the levels of plasma GEPIC in patients with HUS were significantly higher than those in non-HUS (9850+/-5091 vs. 5278+/-3327 /microL, P<0.05; 432.1+/-211.7 vs. 188.3+/-117.0 ng/mL, P<0.01) or control subjects (9850+/-5091 vs. 4728+/-1977 /microL, P<0.05; 432.1+/-211.7 vs. 105.9+/-51.1 ng/mL, P<0.001). Furthermore, plasma GEPIC levels showed a positive correlation with sTM (r = 0.522; P<0.01), a marker of endothelial cell injury, and TAT (r = 0.594; P<0.01), a marker of thrombin activity. CONCLUSIONS: These results suggest that an increase in circulating GEPIC levels in patients with VTEC-associated HUS may be related to endothelial injury, which may possibly lead to a more severe episode of this disease.

Elevated tissue factor circulating levels in children with hemolytic uremic syndrome caused by verotoxin-producing E. coli.

BACKGROUND: Microvascular thrombosis in the kidney plays an important role in the pathogenesis of hemolytic uremic syndrome (HUS). Tissue factor (TF), present on the vascular surface of endothelial cells, binds factor VIIa. The complex initiates the coagulating cascade by activating factors X and IX. PATIENTS AND METHODS: In cases of HUS associated with verotoxin-producing E. coli (VTEC) infection, VTEC gastroenteritis without HUS and normal controls, we measured plasma concentrations of TF and tissue factor pathway inhibitor (TFPI) to evaluate their clinical significance. In children with non-HUS chronic renal failure (CRF), the TF levels were also measured as another control group. RESULTS: In the acute phase of HUS, plasma levels of TF and TFPI were significantly elevated, then returned to normal range in the recovery phase. The TF levels were closely correlated with the thrombin antithrombin-III complex, a marker of thrombin activity in circulating blood, and with creatinine clearance (Ccr). Furthermore, a positive correlation was noted between plasma TF levels and plasma soluble thrombomodulin (sTM) levels, which is a marker of endothelial cell injury. The influence of decreased excretion from damaged kidneys should be considered since a definite lot correlation was observed between plasma TF levels and Ccr in children with non-HUS CRF. CONCLUSION: From these findings, we concluded that elevated TF circulating levels may also play an important role in blood-clotting activation observed in VTEC-HUS patients, and may also be a useful marker for renal damage.

Immunogold labelling of cytokines in glomeruli in children with various renal diseases.

Precise localization of cytokines such as transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and IL-6 was observed in glomeruli using immunogold electron microscopy in 21 children with various types of renal diseases. The distribution pattern of these cytokines, as well as immunoglobulins, C3c and fibrinogen (Fg), was essentially confined to the electron-dense deposits (EDDs) regardless of their location. Frequency of positive labelling of each cytokine was different among various types of renal disorder, that is, TGF-beta was found mainly in lupus nephritis (LN), membranous nephropathy and IgA nephropathy, TNF-alpha in LN, and IL-1 in Henoch-Schönlein purpura nephritis. IL-6 was detected only in 1 case of LN. TNF-alpha was also found in the cytoplasm of glomerular epithelial cells. Furthermore, in order to evaluate the relation of cytokines to mesangial expansion, extracellular matrix components such as type IV collagen, laminin and fibronectin were stained. The result was that there was no significant correlation between the signal intensity or distribution pattern of cytokines and that of extracellular matrix components. These findings indicate that these cytokines could be associated with the formation of EDDs together with immunoglobulins, C3c and Fg. The involvement of each cytokine in renal pathophysiology might also depend upon the type of renal disease. They also raise the possibility that the glomerular epithelial cells might produce or absorb TNF-alpha. However, these results did not show significant correlation between cytokine involvement and mesangial expansion.

Percutaneous renal biopsy in children: survey of pediatric nephrologists in Japan.

Between 1996 and 1997, 2,045 percutaneous renal biopsies were performed on native kidneys in 2,013 patients in pediatric nephrology units in Japan. Of these, 50.8% were performed by automated needle biopsy gun under ultrasound guidance, and the standard biopsy needle, Tru-cut needle or Vim-Silverman needle, under fluoroscopic guidance was used in 12.4% and 12.3% of the biopsies, respectively. Adequate renal tissue for histological diagnosis was obtained in 98.7% of cases, and the success rates for the techniques were not significantly different. The overall complication rate was 5.8%; gross hematuria occurred in 2.7% and large perirenal hematoma in 0.9% of cases. These complication rates were higher when a standard needle under fluoroscopic guidance was used compared with an automated needle under ultrasound guidance. We conclude that pediatric nephrologists in Japan perform percutaneous renal biopsies safely, partly due to technical improvements, such as the automated needle or ultrasound guidance.

Neonatal erythema infectiosum.

A report is presented of a patient with neonatal erythema infectiosum who developed petechiae, transient thrombocytopenia and transient cardiac failure due to transplacental transmission of human parvovirus B19 (HPV B19) infection. It is suggested that the thrombocytopenia was caused by platelet-associated IgG produced by the patient, and that the cardiac failure may have been caused by direct entry of HPV B19 into the cardiac tissue.

Detection of activated platelets in urine by double immunofluorescence in children with IgA nephropathy.

The relationship between the activation of platelets (PLT) in urine and renal histological findings in children with IgA nephropathy (IgAN), thin basement membrane disease (TBMD), and minimal-change nephrotic syndrome (MCNS) was examined. The ratio of activated PLT to total PLT (activated and nonactivated PLT) was examined by double immunofluorescence using rhodamine-conjugated P selectin antibody (activated PLT) and fluorescein isothiocyanate conjugated PLT membrane glycoprotein antibody (GPIIb/IIIa, total PLT); the effect of urine on activation on PLT was also investigated. The number of activated PLT and the ratio of activated PLT to total PLT in urinary sediments were significantly higher in children with IgAN with diffuse mesangial proliferation than in those with TBMD or MCNS. PLT were activated by addition of urine in 13 out of 27 children with IgAN, and the activity was higher in the urine of those with active glomerular or interstitial lesions, while the urine of children with TBMD or MCNS had no effect. The presence of activated PLT and the effect of urine on PLT activation may be associated with the active glomerular or interstitial lesions in IgAN.

[Ristocetin induced platelet aggregation in children with nephrotic syndrome].

In 22 children with steroid responsive nephrotic syndrome (SRNS), we examined platelets aggregability to ristocetin, and the data obtained wre compared with negative charge on platelet membrane based on the binding of cationic dye alcian blue 8GX (AB) or plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (Rcof) activity. At the initial or relapse stage of SRNS, the enhanced platelet aggregation to ristocetin was observed, and correlated with the decreased alcian blue binding to platelets. Ristocetin-induced vWF:Ag binding to platelets by using 125I-vWF was significantly increased. In addition, ristocetin-induced platelets aggregation (RIPA) using washed nephrotic platelets still enhanced as found in the patient's platelets rich plasma (PRP), even when it was resuspended into normal plasma. These results suggest that the decrease of platelet surface negative charge play an important role of heighten RIPA found in children with SRNS.

Enhanced ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib in patients with steroid-responsive nephrotic syndrome.

To elucidate the mechanism of enhanced ristocetin-induced platelet aggregation (RIPA) in steroid-responsive nephrotic syndrome (SRNS), plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RCof) were examined in 6 patients and the amount of ristocetin-induced vWF binding to platelets was determined. At the initial or relapse stage, the plasma vWF:Ag level was 415 +/- 137% and the RCof level was 364 +/- 117%. The ratio of RCof/vWF:Ag was 0.90 +/- 0.15 and no abnormalities of vWF:Ag multimers were observed, indicating that neither functional nor structural abnormalities were present in patient's plasma. The amount of ristocetin-induced normal vWF binding to nephrotic washed platelets, when ristocetin was used at concentrations of 0.5, 0.75, and 1.0 mg/ml, was 152-163% above the binding to normal platelets. In addition, nephrotic washed platelets resuspended in either normal or nephrotic plasma aggregated at a low concentration of ristocetin (0.75 mg/ml) which did not induce aggregation of normal platelets. In accordance with these observations, the decrease of Alcian blue 8GX binding to platelets, reflecting diminished surface negative charge, was also observed. These results appear to indicate that the plasma vWF level and the altered surface-negative charge in platelets both contribute to heightened vWF binding to GPIb, thus lowering the ristocetin concentration required for RIPA in SRNS.

Clinical evaluation of a pasteurized factor XIII concentrate administration in Henoch-Schönlein purpura. Japanese Pediatric Group.

Arthral, abdominal and renal symptoms in Henoch-Schönlein purpura (HSP) were scored. Coagulation factor XIII (F XIII) activity was determined in fifty-six children with HSP and the correlation with the severity score of the clinical symptoms was investigated. As a result, it was found that the decrease in F XIII level was correlated with the severity score of clinical symptoms, particularly abdominal symptoms. Based on the results, a controlled study was performed in 24 cases with moderate symptoms divided into a group treated with F XIII concentrate and a non-treated group to investigate clear-cut efficacy as a next study. In three days after the administration the symptoms were improved remarkably in accordance with the increase of F XIII level compared with non-treated group and scoring of clinical symptoms was confirmed to be useful for assessing the application of the F XIII concentrate to HSP.

[Immunoelectron microscopic observation of membranous nephropathy in children using protein A-gold complex method].

The ultrastructural localization of immunoglobulins (IgG, IgA, IgM), complement component (C3c), or fibrinogen-related antigen (FRA) was investigated on 5 biopsy samples from 5 children with idiopathic MN or lupus nephritis using protein A-gold (PAG) complex method. The immunoreactivity of IgG was essentially confined to the mesangial and subepithelial electron-dense deposit (EDD) in all of 5 children, and more intense staining of IgG was observed in 3 children with stage II of MN than those with stage I and stage III of MN. Double immunocytochemical staining showed the same distribution of both IgG and IgM or both IgG and FRA in subepithelial EDD in 2 of 5 children. These findings suggest that IgG deposits are associated with the formation of subepithelial EDD in MN, and raise the possibility that IgM and FRA deposits may result from entrapment and/or immunological reaction.

Comparison of the vascular and glomerular changes in aortic-ligature and DOCA-salt hypertension.

1. Animals and histology from two previous investigations were used to test the hypothesis that a similar elevation in blood pressure may result in a different sequence of pathological changes in different experimental models of hypertension, DOCA-salt and aortic-ligature hypertension. 2. To asses differences in morphological parameters, individual animals in the two groups were paired (n = 12) for the same level of blood pressure at sacrifice. 3. Vascular damage was significantly less in the DOCA-salt group (P less than 0.01). Glomerular lesions however were more severe in the DOCA-salt group. In rats with aortic-ligature hypertension significantly fewer glomeruli had fibrinoid and/or crescents than in DOCA-salt rats (P less than 0.001). There were fewer glomeruli with epithelial cell droplets (ECD) and fewer ECD per glomerulus in aortic-ligature when compared with DOCA-salt hypertension (P less than 0.001, P less than 0.001, respectively). 4. This study highlights the significance of factors other than blood pressure per se in producing vessel and glomerular lesions in experimental hypertension.

Antithrombin III in children with various renal diseases.

Levels of antithrombin III (AT-III) activity and antigen in plasma and urine in children with renal diseases, and their correlation with the light microscopic findings of kidney tissue and the fluorescence of glomeruli, were investigated. AT-III activity in plasma was reduced slightly during the acute stage of acute glomerulonephritis and moderately in the relapse stage of nephrotic syndrome, whereas a small increase of AT-III antigen level in urine was noted in the acute stage of glomerulonephritis and considerably more was observed during the relapse stage of nephrotic syndrome. During the acute stage of glomerulonephritis or in some primary persistent glomerulonephritis (IgA nephritis, non-IgA nephritis), Henoch-Schönlein purpura nephritis and nephrotic syndrome, localization of small amounts of AT-III was noted on the capillary walls of glomeruli. These findings were in parallel with the proliferative changes of glomeruli. However, the AT-III localization did not change in parallel with the light microscopic findings or degree of the fluorescence of the fibrinogen/fibrin-related antigen. It was thought that the existence of AT-III antigen on the capillary walls of the glomeruli might be associated with the inhibition of excessive fibrin formation by AT-III.

Fragments of urinary fibrin/fibrinogen degradation products and cross-linked fibrin degradation products in various renal diseases.

In children with several kinds of glomerular disease, fragments of fibrin/fibrinogen degradation products (FDP) and cross-linked fibrin degradation products (XLFDP) in the urine were investigated by autoradiography using western blotting method. Results were compared with selectivity of proteins observed in cases of proteinuria, or with histological findings. Patients with nephrotic syndrome exhibited slightly increased amount of urinary FDP, consisted mainly of X and Y fragments. On the other hand, in cases of proliferative glomerulonephritis, such as acute glomerulonephritis, purpura nephritis, Ig A nephropathy, systemic lupus erythematosus, or hemolytic uremic syndrome, increased FDP, including XLFDP, was detected in the urine. In these cases, FDP was consisted mainly of fragments X, Y, and D-dimer, and could not be correlated with the degree of mesangial proliferation or with urinary protein selectivity. It was concluded that the increased urinary FDP and XLFDP were derived not only from filtration of plasma fibrinogen or FDP, but also from fibrinolysis of intraglomerular fibrin deposits.

Intraglomerular deposition of fibrin/fibrinogen-related antigen in children with various renal diseases.

The localization of intraglomerular deposits of fibrin (Fb)/fibrinogen (Fg)-related antigen (FRA) in children with various glomerular diseases was determined by an immunohistopathologic method using an anti-Fg antibody capable of detecting FRA, an anti-D-dimer antibody capable of detecting crosslinked Fb (XLFb) and its derivatives (XLFbDP), and by a method using the effect of monochloroacetic acid (MCA) treatment on kidney sections. In proliferative glomerulonephritis (PGN), XLFbs were detected within the capillaries and extension beyond the mesangium was seen in severe PGN. The FRA within the mesangium of minimal or mild PGN was composed of the non-XLFb substance. The FRA within Bowman's space of most PGN had disappeared after MCA treatment, suggesting a non-XLFb substance. The presence of FRA within electron-dense deposits (EDD) suggested that FRA deposits are associated with immune-complex deposits in the glomeruli.

Urinary cross-linked fibrin degradation products in glomerular disease with crescents.

The concentration of cross-linked fibrin degradation products (XLFDP) in urine were determined by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) in patients with several types of glomerulonephritis with crescents (CrGN). In patients with "active" cellular crescents, increased levels of XLFDP correlated with the percentage of glomeruli containing crescents. Dysmorphic erythrocytes, suggestive of glomerular bleeding, were observed in all of the patients with CrGN, and the urinary red cell counts (URCC) also correlated with percentage of glomeruli containing crescents. The absence of correlation between urinary XLFDP and URCC or urinary protein suggested that lysis of fibrin within crescents may contribute to the urinary excretion of XLFDP in CrGN. The measurement of urinary XLFDP in CrGN is likely to be of value in assessing the activity of glomerular lesions but not renal function.