Two-color flow cytometry was carried out to determine the correlation between cell mediated immunity and the development of the nephrotic stage in patients with mem-branous nephropathy (MN) and minimal change nephrotic syndrome (MCNS). In this study, lymphocyte subpopulations were measured by two-color flow cytometry using various monoclonal antibodies of the Leu series Thirty patients with MN and 25 patients with MCNS were examined. Clinically, those patients were divided into four stages as follows: (1) untreated nephrotic stage, (2) prednisolone (PSL) treated nephrotic stage, (3) persistent proteinuria stage (incomplete remission, ICR), and (4) complete remission (CR). Pathologically, the patients with MN also divided into four stages I '-lv, according to Churg's classification. The values of the Leu 3a/Leu 2a ratio in patients in the untreated nephrotic stage of MN and MCNS were significantly higher than those in the remission stage in both diseases (P<0.01, P<0.05, respectively). Two-color flow cytometry showed that the reduction of Leu2a possitive cells was mainly due to a decrease of Leu 2a+15+ substs (suppresser T cells) in the untreated nephrotic stage and relative increase of Leu 3a⁺8⁺ subsets (sup-pressor inducer T cells). There was no significant difference in these findings among the histopathological stages in patients with MN. Patients with MN and MCNS showed a significant elevation of Leu 2a⁺DR⁺ cells after the treatment of PSL. The abnormalities of suppressor T cells and suppressor inducer T cells in the peri-pheral blood appear to be correlated with clinical activities of the nephrotic syndrome due to MN or MCNS, but not to be related to the pathogenesis of either disease. It is postulated that PSL might stimulate Leu 2a positive cells and Leu 3a positive cells, and then increase the number of Leu 2a⁺15⁺ cells in the peripheral blood of patients with MN and MCNS.

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In order to examine the ability of complement to solubilize the immune complexes (IC) in vivo, we made acute or chronic serum sickness nephritis in 10 rabbits or 12 rats with injection of BSA as antigen (Ag). Animals were devided into two groups. The 500μ or 50 μ/week of cobra venom factor (CVF) for rabbits or rats was administered intraperitoneally in one group of animals to reduce the serum complement levels and complementmediated solubilization of immune complex. On the other hand, the 2m1 or 0.2 ml/week of turpentine oil (TO) was injected intramuscularly in another group of animals to elevate the serum complement levels. The renal biopsy was performed with weekly intervals in each animals, and the specimens were stained for antigen (BSA), antibody (IgG) and C3 by immunofluorescence techniques. The result in this experiments showed the significant difference between TO-, and CVF-treated animals in the degree of disappearence of IC in the kidney lesions. The undetectable amount of IC (especially BSA) was remained in the renal tissue of TO-treated animals. This fact indicated that complement might act to solubilize the IC deposited in the tissue in vivo also.

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In order to investigate the early renal damage in diabetes mellitus, 89 diabetics without proteinuria by dipsticks and 67 normal control subjects were examined by means of SDS-PAGE. The relationships between electrophoretic patterns of urinary protein and duration of diabetes, age of patients, metabolic controls and stages of retinopathy were examined. 1) The percentage of higher molecular weight (MW) proteins (67, 000<MW) was larger in diabetics than that in controls. Especially the percentage of proteins with MW between 67, 000 and 94, 000, which include transferrin was 13.9±6.9% in diabetics, significantly higher than that in controls (10.3±5.1%) (P<0.01). On the contrary, the percentage of low MW proteins (MW<67, 000) was relatively small in diabetics. 2) The excretion of higher MW proteins increased until 16 years of diabetic duration, however that decreased after 16 years. Especially in the group with duration longer than 20 years, excretion of low MW proteins increased. 3) Electrophoretic patterns of urinary proteins in patients with good metabolic control were similar to those in normal controls. 4) Excretion of higher MW proteins increased in patients with retinopathic complication suggesting the progression to microangiopathy. From the above results, we concluded that increased excretion of higher MW proteins in diabetics may be the results of GBM damages in protein selectivity. In patients with longer history of diabetes, predominant excretion of urinary low MW proteins may be the result of tubular dysfunction due to macroangiopathy.

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To examine the subclinical renal damage in collagen disease, we analized the excre-tion pattern of microproteinuria. We studied 58 collagen disease patients including 25 RA (rheumatoid arthritis) patients, 15 SLE (systemic lupus erythematosus), 5 PSS (progressive systemic sclerosis), 4 MCTD (mixed connective tissue disease), and 9 others. Urinary protein was not detected by urine dipsticks in all patients. Urinary proteins, which were concentrated to 5 mg/ml, were subjected to linear gradient (4-30%) SDS-PAGE and then transferred to nitrocellulose membrane by electrophoretic blotting method. The membrane was stained with Auro Dye and the blotted proteins were identified by enzyme immunoassay using specific antibodies. The percentages of albumin of whole urinary proteins were 27.2±13.7% in RA and 25.8±12.6% in PSS, which were significantly lower than that of controls (42.1±15.3%). However no significant difference in the percentage of urinary albumin was noted between SLE and controls. The percentages of low molecular weight (MW) proteins (proteins having smaller MW than albumin) were higher in RA and PSS. Especially the bands with MW of 25, 200 were prominent and these percentages were 11.3±6.1% in RA and 14.6±9.1% in PSS, which were significantly higher than controls (5.1±3.5%). These bands with MW of 25, 200 were de-monstrated to be free light chains of immunoglobulins by western blotting method. From the above observations, protein excretion patterns in RA or PSS patients were so-called tubular proteinuria, and especially free light chain excretion was increased. We proposed that tubular dysfunction and abnormal production of free light chain might exist frequently in collagen diseases, especially RA and PSS.

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The purpose of this study is to examine the immunochemical changes of the glomerular basement membrane (GBM) and the mesangium, in pretreated paraffin-embedded sections with trypsin by utilizing monoclonal antibodies to type III(anti-III), type IV (anti-IV) and type V (anti-V) collagens. We observed 6 normal kidneys and 44 kidneys with various renal diseases. In normal human kidney the staining with anti-N demonstrated GBM, mesangium, Bowman's BM, tubular BM and capillary BM. Anti-V was also seen in the interstitium. On the other hand, anti-III stained only interstitium. Thickened GBM in membranopro-liferative glomeruloneph ritis (MPGN) and diabetic nephropathy, and irregular GBM in Membranous Nephropathy and Alport's syndrome were also evident in anti-IV stain, while widened mesangial area was seen in anti-V rather than anti-IV stain. In severely pro-liferative GN, anti-III as well as anti-IV and anti-V was detected in the mesangium in spite of existence of neither adhesion nor Bowman's gap. In MPGN type II, anti-III was observed along the GBM. In obsolescent glomeruli, anti-IV was not always detected although anti-V was constantly seen. On the other hand, anti-III was markedly positive in the crescents and obsolescent glomeruli. These results suggest that it is possible for mesangial, endothelial and epithelial cell to produce several types of collagens and type III collagen is closely related to the process of the glomerular obsolescence.

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In previous papers, we have reported that methylguanidine (MG), a known uremic toxin, was synthesized from creatinine (Cr) by active oxygen generated not only by chemicall reagents but also by isolated rat hepatocytes. In this paper, we studied whether or not active oxygen generated by stimulated human neutrophils produces MG from Cr. MG was measured after incubating 2×10⁶ human neutrophils for 2 h in 1 ml of Hanks' balanced salt solution (pH 7.4) containing 100 mg/dl Cr at 37°C after the addition of phorbol myristate acetate (PMA). MG was measured by high pressure liquid chromato-graphy followed by reaction with 9, 10-phenanthrenequinone. MG was synthesized by the stimulated neutrophils and not by the unstimulated ones. MG synthesis reached a plateau (1.11±0.03 nmol/120 min/2×10⁶ cells) at a concentration of 0.125 pM PMA and reached a maximum value (1.95±0.03 nmol/120 min/2×10⁶ cells) at a concentration of 100 mg/dl Cr. MG synthesis increased depending on the concentration of neutrophils between 1 and 8×10⁶/ml and increased depending on the duration of in-cubation up to 4 h. MG synthesis was strongly inhibited by superoxide dismutase, by the scavengers of hypochloride (taurine and methionine) and by sodium azide. Catalase and the scavenger of the hydroxyl radical (dimethyl sulfoxide) inhibited MG synthesis less effectively. The effects of the scavengers of active oxygen suggest the participation of active oxygen in MG synthesis from Cr in this system. Among the active oxygen species, superoxide anion and hypochloride play an important role in this system. And less im-portant role for hydrogen peroxide and hydroxyl radical are also suggested. Moreover, the participation of singlet oxygen cannot be ruled out.

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A comparative study was designed to evaluate the contribution of calmodulin to renin release from isolated glomeruli of spontaneously hypertensive rats (SHR, Okamoto and Aoki). Male 7-week-old SHR and age-matched control Wistar-Kyoto rat (WKY) were used in the present study. Glomeruli were isolated by the method of Beierwaltes et al. Isolated glomeruli were placed within the superfusion chamber and perfused with Krebs-Ringer solution at a constant flow of 0.3 ml/minute at 37... Renin release was increased by calmodulin inhibitor, W-7 in both SHR and WKY. SHR showed higher maximal levels of renin release by W-7 compared to WKY. This result indicates that calmodulin plays an inhibitory role in renin release from juxtaglomerular cells. Calmodulin-mediated suppression mechanism in renin release is augmented in SHR.

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A compound containing cyclostatine ES-6864 (N-[(2R) -3-morpholinocarbonyl-2-(1-naph-thylmethyl)propionyl]-(4-thiazolyl)-L-alanyl-cyclostatine-(2-morpholinoethyl)amide) was found to be a competitive inhibitor of human renin with an inhibitory constant (Ki) value of 7.3×10^-9 M. The compound was also potent against monkey renin but was less effec-tive against renins from pig, goat, dog, rabbit and rat. ES-6864 did not inhibit cathepsin D, pepsin, urinary kallikrein, angiotensin converting enzyme, trypsin and chymotrypsin at a concentration of 10⁵ M. ES-6864 also inhibited the tissue renin-like activity from dog tissues with IC₅₀ values of 10^-7-10^-8 M in vitro. Oral administration of ES-6864 at 30 mg/kg to conscious, sodium-depleted marmosets produced a significant blood pressure reduction and a significant inhibition of plasma renin activity, which persisted for 6 hours. Plasma concentration of ES-6864 reached a maximum of 1.2 μg/ml at 1 hour after an oral administration. Oral administration of ES-6864 to hog renin-infused rats produced dose-related decreases in blood pressure. The results demonstrate that ES-6864 is an orally active renin inhibitor with high potency and specificity for human renin, Thus, ES-6864 is a candidate compound for development of renin inhibitors that can be used clinically.

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The purpose of this study was to assess the effects of chronic insulin infusion on blood pressure and urinary sodium excretion in Wistar rats. Fifteen Male Wistar rats weighing about 220 g were used. The rats were housed in metabolic cage and measured urine volume. Osmotic minipumps filled with insulin (0.57 U/day, Insulin group, n=9) or saline (0.014 cc/day, Control group, n=6) were implanted subcutaneously under ether anaesthesia, and blood pressure, urine volume, urinary sodium excretion (U_NaV), plasma renin activity (PRA), plasma norepinephrine concentration (PNE) were measured for 4 weeks. In insulin group, there were no significant changes on plasma glucose levels, but systolic blood pressure rose significantly from 119 mmHg to 140 mmHg after 4 weeks. In this group, urine volume, U_NaV, and PRA were significantly lower than those of control group and PNE was tended higher but not significant (P<0.1). Exogenous NE was given intravenously to assess the endogenous NE activity. Blood pressure elevation caused by exogenous NE in insulin group was suppressed significantly than that of control group. On the basis of these findings, we conclude that insulin can cause high blood pressure due to sodium retension and activation of endogenous NE.

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A case of nephrotic syndrome found by pulmonary infarction associated with renal vein thrombosis was reported. The renal biopsy showed the presence of membranous glomerulonephritis. The patient had the increase of serum level of the coagulation factor III, V, fibrinogen and FDP, and the level of urine FDP. These laboratory data suggested that the hyper coagulation state of his blood caused by nephrotic state, induced his mul-tiple thromboembolism. We surveyed 47 case report abstracts of nephrotic syndrome patients having thromboembolism on the Japanese Journal of Nephrology published from 1974 to 1986. The histopathology of their renal biopsies showed the high incidence of membranous glomerulonephritis, followed by minimal change nephrotic syndrome and focal glomerular sclerosis. Renal vein thrombosis was the most common thromboembolism in the reports, followed by thrombosis of cerebral vessels, peripheral vessels and pulmonary artery. Some patients died of pulmonary infarction or myocardial infarction. These data showed that we must keep in mind of the possibility of thromboembolism in the treatment of nephrotic syndrome.

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Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hemolytic disorder characterized by a membrane abnormality of red cells, and characterized by two major clinical features of gross hemoglobinuria and diffuse venous thrombosis. In Japan, the present report records the first case of acute renal failure complicating PNH with treated by hemodialysis and was almost completely reversible. Case : A 41 year-old woman was admitted for high fever (39.8°C), dyspnea and clinical signs of a respiratory infection. She was started on Cefotax 1, 000 mg 3 times daily. She subsequently developed acute renal failure and which treated by hemodialysis and was almost completely reversible. Following treatment of her renal failure, respiratory infection and anemia, she initialy made good progress and was discharged.

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