Urinary proteins from normal children and patients with various kinds of nephropathies were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel without concentration, and were visualized with Western blotting. The following results were obtained 1. Urinary proteins from normal children were composed of mainly albumin along with small amounts of 33-112 kD proteins. 2. Electrophoretic patterns of urines from patients with various kinds of nephropathies were divided into 5 types (type I-type V). (a) In general, patients with moderate proteinuria (1 g/m²/day≥proteinuria 3.5 g/m²/day) often showed type IV pattern. (b) Patients with acute glomerulonephritis showed type IV even if they excreted trace amounts of proteins, (c) Type I or type II were often found in minimal change nephrosis or membranous nephropathy, whereas type V was frequently found in tubular diseases. 3. Mean values of glomerulo-tubular protein ratio elevated with the increase of urinary protein excretion, The mean values of selective protein index decreased with the increase of urinary protein excretion except for urinary protein over 3.5 g/m²/day, Mean values of glomerulo-tubular protein ratio were high in nephrotic syndrome due to Henoch-Schönlein purpura nephritis or membranoproliferative glomerulonephritis. On the other hand, those were low in hemolytic uremic syndrome or pyelonephritis, Mean values of selective protein index were high in minimal change nephrosis, membranous nephropathy with nephrotic syndrome or pyelonephritis, but low in Henoch-Schonlein purpura nephritis with nephrotic syndrome. 4. High molecular weight bands (aggregated and whole IgG) reacted with anti-IgG serum were prominent in crescentic glomerulonephritis, while the low molecular weight bands (IgG fragments) were dominant in pyelonephritis. 5. Electrophoretic analysis of urinary proteins by Western blotting was useful to assess the renal histopathologic changes.

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Renal biopsy specimens from a total of 59 patients with IgAA nephropathy (25 patients), Henoch-Schonlein purpura nephritis (HSPN) (13 patients), mesangial proliferative (non-IgA) glomerulonephritis (13 patients), and minimal change nephrotic syndrome (S patients) were examined for immune cells (macrophages, helper T cells, suppresser T cells, D cells and polymorphonuclear leukocytes) by immunofluorescence. Non-specific esterase (NSE) staining was also used to identify the macrophages, and compared with the identification by monoclonal antibodies. Two monoclonal antibodies (Mo2 and FMC-32) showed similar results on the detection of macrophages, and NSE was less sensitive than the monoclonal antibodies. In IgA nephropathy and HSPN, macrophages were the dominant cells infiltrating the gloeruli. In patients with mesangial proliferative (non-IgA) glomerulonephritis and minimal change nephrotic syndrome, there was no significant difference in glomerular and interstitial populations of immune cells. These findings suggest that macrophages play an important role in the development of IgA nephropathy and HSPN.

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A biological aspect of glomerular mesangial cells (MC) as an arterial smooth muscle cell (ASMC) to compared with venous smooth muscle cell (VSMC) was examined using a homogenous culture of swine mesangial cells, and was compared with cultured endow thelial cells (EC) and cultured smooth muscle cells from aorta (ASMC) and vena cava (VSMC) of the young swine by electron microscopy, cytochemistry and radioimmunoassay of cyclic nucleotides. Although morphological differences could not not be obtained between the three cultured cell groups, ATPase activity as well as cAMP contents are significantly higher in ASMC and MC than in VSMC. This tendency became more distinct when it was stimulated by prostacyclin (PGI₂) and arginine vasopressine (AVP) dosedependentry. The results obtained were suggested that glomerular MC have essentially similar characters with ASMC biologically. Under the certain conditions such as chronic glomere ulonephritides or diabetic nephropathy, MC with these characteristics may play an important roles on the mesangial proliferation and sclerosis by cell proliferation and various collagen synthesis as well as on the contraction and dilatation of glomerular capillary.

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Following the report of Sainte-Marie (J. Histochem. Cytochem. 10: 250-256, 1962), it has been recognized that ethanol-fixed paraffin-embedded tissue material can be used for immunohistochemical demonstration of various intra- and extracellular proteins. In our laboratory, renal biopsy specimens have been prepared by ethanol-fixation and paraffinembedding technique for simultaneous study on light microscopy and immunohistology. These materials were satisfactorily used for routine stainings on light microscopy. In order to evaluate the reliability of immunohistology on these materials, immunoperoxidase (IF) stainings for several immunoproteins (IgG, IgA, IgM, Clq, C3, and fibrinogen) were carried out on 71 cases. The materials were treated with proteolytic enzyme (Protease type XIV Sigma) before IP stainings. The results of IF were compared with the findings of immunofluorescence (IF) study on fresh frozen sections from the same cases. Sensitivity and specificity of IF method were calculated on the basis of IF method as the standard. The antigenicity of deposits in fixed materials could be preserved for several years after the biopsies, and reproduced by proteolytic treatment. Localization of deposits was much clearer than that in IF, and could be simultaneously studied with the serial sections of light microscopy. The results of IP stainings corresponded with those of IF in 73.2- 81. 7% except for IgM and fibrinogen (60.6%, 71.0%, respectively). The sensitivity of IF was 78.7-98.1% for all immunoproteins examined.And specificity was 70.6-91.2% except for IgM and fibrinogen (51.8%, 66.7% respectively). In conclusion, ethanol-fixed paraffin-embedded materials are suitable for serial examinations by immunohistological echnique as well as by light microscopy, and are useful for retrospective studies.

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The concept of “mesangial flow” and “mesangial channel” is generally accepted, but its nature and physiological function have not been clearly elucidated . Renal changes of 3 experimental models, which were designed to disturb the mesangial flow by increased renal interstitial and mesangial hydrostatic pressure, were examined. Seventy-two male wistar rats were divided into 3 groupes and performed following operative procedures in order to increase renal interstitial pressure by lymph flow stasis and blood congestion. Right kidneys were examined light and electron microscopically 1 hour, 6 hours, 12 hours, 24 hours, 3 days, and 7 days after the operation. (Group 1) Complete thoracic duct occulusion, (Group 2) Incomplete right renal vein occulusion, (Group 3) Complete thoracic duct occulusion with incomplete right renal vein occulusion. Pathological changes caused by the operation were seen mainly in the mesangial region in all groups. Changes were severest in group 3 of which interstitial pressure was highest. In group 3, the mesangial area was reduced in size and severe mesangial cell injuries including atrophy, degeneration, and occasional necroses were seen 12- 24 hours later, and mild focal segmental mesangial proliferation was recognized 7 days later. These findings suggest that the mesangial flow, as the flow of interstitial fluid in glomerulus, plays an important role to maintain the mesangium, and disturbance of this flow may contribute to the mesangial and the glomerular injury.

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Tubular basement membrane (TBM) antigen was purified from kidneys of mice of three inbred strains, BALB/c, C3H/He, C57BL/6, and out bred ddY mice by our purification method. Then, the genetic control of immune response to syngeneic and/or allogeneic TBM antigen and induction of interstitial nephritis (IN) in inbred mice were investigated. BALB/c (H-2_d) mice were highly susceptible to IN whenever they were immunized with any one of mouse TBM antigens, whereas C57BL/6 (H-2b) mice were not, Furthermore, immune response to TBM antigen, proliferative response and antibody response to TBM antigen, was greater in BALB/c and lower in C57BL/6. C3H/He (H-2_k) were intermediate responders to TBM antigen. Studies with their F₁ hybrids suggested that induction of IN and immune response to TBM antigen are controlled by a dominant trait. Further studies with the backcrossed mice produced by mating CBF₁ (BALB/c x C57BL/6) hybrids with the resistant C57BL/6 mice showed that all mice with H-2^d/H-2_b were high responders and all mice with H-2^b/H-2^b were low responders in induction of IN and in immune response to TBM antigen. These results suggest that induction of IN and immune response to TBM antigen are strongly related, and they are controlled by one or a few dominant gene(s), whose loci are within or closely linked to H-2 complex.

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1. To evaluate the potential contribution of thromboxane A₂ (TXA₂) and prostaglandin I₂ (PGI₂) to the development of chronic glomerulonephritis, urinary excretion of TXB₂, 2, 3-dinor-TXB₂ and 6-keto-PGF₁α was measured by radio-immunoassay in patients with chronic glomerulonephritis. In patients with nephrotic syndrome, urinary excretion of TXB₂ and 2, 3-dinor-TXB₂ was highly increased, whereas that of 6-keto-PGF₁α remained normal In patients with non-nephrotic chronic glomerulonephritis, urinary excretion of TXB₂ was significantly increased, whereas that of 2, 3-dinor-TXB₂ and 6-keto-PGF₁α remained normal. In patients with chronic renal failure, urinary excretion of TXB₂, 2, 3-dinor-TXB₂ and 6-keto-PGF₁α was markedly decreased. 2. To determine if a selective TXA₂ synthetase inhibitor is an effective drug for chronic glomerulonephritis, 10 patients including 7 with nephrotic syndrome were treated only with OKY-046 for 8 weeks, Urinary excretion of protein, TXB₂, 2, 3-dinor-TXB₂ and fibrinogen/fibrin degradation products (FDP) decreased significantly with OKY-046. Urinary excretion of 6-keto-PGF₁α increased significantly, Creatinine clearance, however, did not change significantly. Serum level of albumin increased significantly, and that of total cholesterol and plasma level of fibrinogen decreased, Platelet aggregation induced by 2μg/ml collagen was significantly inhibited with OKY-046, One patient with minimal change nephrotic syndrome showed complete remission only with OKY-046. 3. These results demonstrate that TXA₂ plays an important role as an exaggerating factor in the development of chronic glomerulonephritis particularly that accomparying nephrotic syndrome, and that the selective TXA₂ synthetase inhibitor is an effective drug for the treatment of chronic glomerulonephritis especially with nephrotic syndrome.

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Three monoclonal antibodies against laminin A and B chains, which were isolated from murine EHS sarcoma were prepared and designated Lam 1, Lam 2 and Lam 3. Lam 1 recognized laminin A and B chains, whereas Lam 2 and Lam 3 reacted with only A chain By using Immunofluorescence method, three monoclonal antibodies equally stained the hu man skin basement membrane and Descemet's membrane of cornea. In the kidney, howw ever, these monoclonal antibodies showed the different distribution; Lam 1 stained the glomerular basement membrane, the mesangium, Bowman's capsule and the tubular base ment membrane in the mouse kidney. In the human kidney Lam 1 stained the mesangium very slightly and not stained the glomerular basement membrane, Both Lam 2 and Lam 3 stained the mesangium, Bowman's capsule and proximal tubular basement membrane in the mouse kidney. In the human kidney, however, Lam 2 stained the mesangium and slightly stained the glomerular basement membrane, although Lam 3 stained only the mesangium. Oxidation of the kidney sections by sodium periodate changed the staining pattern of Lam 2 and Lam 3 in the human kidney, whereas it did not change in the mouse kidney. Furthermore, Lam 1 did not change the staining pattern in the both kidneys, This fact may indicated that the antigenic site which Lam 2 and Lam recognize is hidden by some form, Using Lam 3 which stain only the mesangium in the normal human kidney, we tried to stain the biopsy sections, As the result, Lam 3 presented in the capillary wall in membranous nephropathy. It suspected that in membranous nephropathy some qualitical change on the glomerular basement membrane.

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In order to clarify the role of cell-mediated immunity in minimal change nephrotic syndrome (MCNS), we investigated lymphocyte subpopulations, using fluorescein isothiocyanate (FITC) - and phycoerythrin (PE)-labelled monoclonal antibodies to human lymphocyte antigens and two-color flow cytometry. This study included 46 patients with CNS and 100 healthy volunteers as normal controls. Patients with MCNS, who received prednisolone (PSL), had significantly reduced Leu3a/Leu2a ratios due to both the depression of Leu3a+ cells and the elevation of Leu2a+ cells . In patients with nephrotics of MCNS, who did not receive any PSL, Leu3a+Leu8- subsets (helper T cells), Leu3a+Leu8+ subsets (suppressor inducer T cells), Leu2a+Leul5+ subsets (suppressor T cells) and Leu2a+Leul5R subsets (killer T cells) were not significantly different compared to the normal controls. However, in patients with complete remission of MCNS, who received PSL, Leu3a+Leu8+ subsets were significantly lower compared to the normal controls. Patients with MCNS showed a lower level of Leu10+ cells. MCNS in nephrotics showed a significantly lower level of Leu7+Leulla- subsets compared to the normal controls. These data suggest that the defect in T cell subpopulations are seen in patients with MCNS, and the defect in T cell subpopulations may be important in pathogenesis of MCNS.

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Phosphate transport by renal brush border membrane was studied in the rat with chronic renal failure (CRF). Chronic renal failure was produced by injection of glycoprotein (GP) in Wistar-Imamichi strain rats. Ten months after injection, renal brush border membrane vesicles (BBMV) were isolated and phosphate transport studies were performed with the rapid filtration method, Nay dependent phosphate uptake by BBMV was decreased in CRF compared with that in the control. In kinetic study a decrease of Vmax was observed. On the other hand, D-glucose uptake was not different between two groups. The effect of Na+ gradient on phosphate uptake in the BBMV of CRF revealed an increase of phosphate uptake by BBMV in proportion to extravesicular Na⁺ concentrations, whereas a sigmoidal increment was showed in the control rats. Decrease of Km for Na⁺ dependent phosphate uptake was observed in parathyroidectomized rats with CRF. In vitro, guanidino compounds inhibited Na⁺ dependent uptake of phosphate by the BBMV purified from normal rats. These results indicate that the specific inhibition of phosphate transport in tubular brush border membrane in CRF was produced by decreasing the availability of phosphate carriers of the membrane.

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Autonomic nervous function was studied in 30 patients with chronic renal failure on hemodialysis (mean age : 54.3±3.8 years) by beat-to-beat variation (BBV) during deep breathing. Eight diabetic nephropathics were included. BBV prior to hemodialysis was 6.9±0.7 and 2.4±0.4 beats/min in non-diabetic and diabetic patients respectively. The latter value was lower (p<0. 001). BBV after hemodialysis did not change significantly compared with that before it. Oral administration of a daily dosis of 1500 μg methylcobalamin for 3 months elevated BBV by 150.5% and that for 6 months produced further prolongation of improvement (151.8%), whereas patients without methylcobalamin administration did not show any significant amelioration in an interval of 3 months, It was to be noticed that 9 patients with abnormal BBV prior to methylcobalamin administration revealed marked improvement (205.3±39.2%) .These observations indicate that disturbed autonomic nervous function in chronic renal failure might be ameliorated y methylcobalamin.

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In spontaneously hypertensive rats (SHR), approximately two months age, fed a highprotein diet (containing about 45% protein) and given 1% saline for drink, the rise in blood pressure was suppresed in comparison with rats fed a low-proten diet (containing about 15% protein). Systolic blood pressure in six of nine patients with essential hypertension, who stage I to II, were also decreased under the high protein diet. Under the high protein diet, the increase of urinary urea nitrogen excretion was significantly greater than these of urine volume and urinary sodium and potassium excretion. No significant change in renal clearance was observed between the low and high protein diet during the experimental course. Therefore, the high protein diet might suppress the rise of blood pressure in sodium dependent hypertension by promoting urinary sodium excretion, presumably induced by urea diuresis.

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This study was designed to evaluate the role of the action of plasma angiotensin II on the sympathetic nervous system in the regulation of blood pressure in essential hypertension (n =26). The effects of 1-sarcosine, 8-isoleucine angiotensin II ([Sar¹, Ile⁸]-AII: 600 ng/kg/min) on plasma noradrenaline level (PNA) and blood pressure (BP) were examined in supine position on normal dietary sodium (ND) and sodium deprivation (SD: NaCI 5-8 g/day and furosemide 40-80 mg/day for 3 days). The change of PNA by 60°head-up tilt was compared before and after the administration of this agent. 1. Before the administration of this agent, PNA was significantly correlated with plasma renin activity (PRA) in supine position on normal dietary sodium (r=0.52, p<0.01). The increases in PNA were significantly related to those in PRA by sodium deprivation (r=0.57, p<0.05) or head-up tilt (r=0.76, p<0.01). 2. [Sar¹, Ile⁸]-AII infusion induced a significant decrease in PNA. This change in PNA was significantly correlated with the pretreatment PRA (ND: r=-0.62, p<0.Ol, SD : r= -0.58, p<0.05) and the change in mean BP (ND: r=0.68, p<0.01, SD:r=0.67, p<0.01). 3. On sodium deprivation, the decrease in PNA by [Sar¹, Ile⁸]-AII was significantly greater than that on normal dietary sodium, and [Sar¹, Ile⁸]-AII produced a significant fall in mean BP. 4. The increments in PNA and heart rates by head-up tilt were significantly reduced by [Sar¹, Ile⁸]-AII. These observations provide that angiotensin II may regulate, in part, the activation of the sympathetic nervous system, and that it may play some significant role in the regulation of blood pressure through the sympathetic nervous system, especially in sodium depleted states, in essential hypertension.

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A 31 year-old woman progressed to end stage renal failure within 3 to 4 months in the course of rheumatoid arthritis . She received nonsteroidal anti-inflammatory drugs (NSAIDs) for three years. Renal biopsy showed extensive tubular degeneration with karyomegaly . There were no interstitial cell infiltration and no remarkable glomerular changes . Renal function did not improve despite discontinuation of NSAIDs and she required regular hemodialysis . Although the etiology of renal failure in the present case was unknown, the karyomegaly in the renal tubuli could be the action of some antimitotic agents such as drugs or viral infections.

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We mesured erythrocyte sodium ion transport systems in essential hypertensive and normotensive subjects. Furthermore, the effects of angiotensin converting enzyme inhibitor, captopril, on sodium ion transport systems were investigated. The passive Na⁺permeability of erythrocyte membrane was significantly higher and the erythrocyte Na⁺-K⁺cotransport was lower in patients with essential hypertension than in the normal subjects. However, the Na⁺- K⁺pump activity and the Na⁺-Li⁺countertransport were not significantly different between in the hypertensive subjects and in the normal subjects. After administration of captopril, the passive Na⁺permeability was reduced and the Na⁺-K⁺cotransport was increased in the erythrocytes of hypertensive subjects . Plasma renin activity and plasma aldosterone concentration were altered by captopril, but did not correlate with the changes in Na⁺transport systems. These date suggest that the abnormal Na⁺transport systems are associated with essential hypertension and the effects of capfopril on Na⁺transport systems may contribute to its antihypertesive action.

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