This study was designed to clarify the role of collagen metabolism in the pathogenesis of diabetic nephropathy. Urinary hydroxyproline (hyp), hydroxylysine (hyl) and its glycosides, indexes of collagen degradation, were measured in 46 diabetics and 10 normal subjects. The diabetics were divided into 3 groups; without complications (G-I, n=11), with retinopathy (G-II, n=17) and with nephropathy (G-III, n=18). Urinary hyp excretions was normal in diabetics, but total hyl and its glycosides were greater in G-II and G-III than in normal subjects. The ratio of glucosylgalactosyl hyl (glc-gal-hyl) to galactosyl hyl (gal-hyl) was also higher in G-II and G-III than in normal subjects. Excretion of hyp, hyl and its glycosides were high in patients with unsatisfactory control of diabetes mellitus. Ratio of glycosylated hyl (glc-gal-hyl+gal-hyl) to total hyl decreased and the ratio of glc-gal-hyl to gal-hyl increased in patients improved by insulin injection. While, in patients poorly controlled by insulin injection, the ratio of glycosy lated hyl to total hyl increased and the ratio of gic-gal-hyl to gal-hyl decreased. Patients with elevated excretion of glycosylated hyl and low ratio of hyp to glycosylated hyl developed to diabetic microangiopathy within two years. Increased excretion of hyl and its glycosides indicates that degradation of type IV collagen in GBM accerelates in patients with diabetic microangiopathy, which may develop to nephropathy. It is presumed that measurement of either urinary excretion of hyp, hyl and its glycosides or the ratio of glc-gal-hyl to gal-hyl is useful to estimate the possibility to developing to diabetic microangiopathy and outlook for its prognosis.

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The very high mortality in acute renal failure (ARF) with hepatic disorders and hepato-renal syndrome indicate that there is a close relation between the kidney and liver. When rats which had recovered from glycerol-induced ARF (recovery rats) were rechal lenged with glycerol, the development of ARF was significantly suppressed and renal and hepatic blood flows were well maintained. This indicated that hepatic clearance of substances which induced ARF or which were accumulated by ARF might have been high in recovery rats. The hepatic drug clearance depends on the hepatic blood flow and the hepatic drug metabolizing activity. In order to study the drug metabolism of the liver in recovery rats, we measured cytochrome P-450-dependent monooxygenase in the microsomes from liver in both the control and recovery rats. As a result, although there was no significant change in the total cytochrome P-450 content of the liver microsomes after intramuscular injection of glycerol in both groups, aminopyrine N-demethylase activity, NADPH-cytochrome P-450 reductase activity and cytochrome b5, content increased and these activities before glycerol injection were significantly high in recovery rats compared to control rats. These results indicated that hepatic microsomal cytochrome P-450-dependent drug metabolizing activity is significantly high in recovery rats compared to control rats before glycerol injection and therefore in recovery rats the hepatic clearance of toxic substances which deteriorate ARF is increasing.

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Plasma proteinase inhibitory activities of α₂-macroglobulin (α₂-MG) and inhibitory activities of plasma against lymphocyte blastogenesis were studied to examine the roles of α₂-MG in regulating cell immunity in idiopathic nephrotic syndrome (INS) and chronic glomerulonephritis found by chance proteinuria and/or hematuria (CPH). Proteinase in hibitory activities of α₂-MG were measured using thermolysin as a proteinase and Briliant Blue Hide Powder as a substrate. Plasma thermolysin inhibitory activities in patients with INS at relapses were significantly higher than those in INS at remission and those in CPH. Values of thermolysin inhibitory activity of plasma α₂-MG (ng/min/μl) /plasma α₂-MG concentration (ng/μl) in INS at relapses were significantly lower than those in INS at remission and those in CPH. Plasma α₂-MG concentrations and plasma inhibitory activities against lymphocyte blastogenesis showed a significant correlation in INS (r=0.39, p<0.01). Plasma thermolysin inhibitory activity and plasma inhibitory activity against lymphocyte blastogenesis also showed a significant correlation (r=0.31, p<0.05) in INS including both at relapses and remission. Of interest was the difference of the correlation coefficients in INS at relapses (-0.35) and at remission (r=0.15), although the values did not reach statistical significance. There was a significant inverse correlation (r=-0.37, p<0.01) between the values of [thermolysin inhibitory activity of plasma α₂-MG (ng/min/μl)/plasma α₂-MG concentration (ng/μl)] and plasma inhibitory activities on lymphocyte blastogenesis in INS. These results suggest a role of α₂-MG, possibly a certain type of proteinase-α₂-MG complex, in regulating cell immunity in INS through the inhibitory activity against lymphocyte blastogenesis.

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It has been recently found that glycolipids and glycoproteins having sialic acids inhibit the activation of alternative complement pathway (ACP) in their own membranes. When the author measured IgG and IgM antibody titers in sera of 75 children suffering from renal glomerular diseases by enzyme-linked immunosorbent assay (ELISA) with glycolipid antigens, such as fucosyl GM₁ (FucGM₁), asialo GM₁(GA1) and globoside selective and significant increase in the titer of anti-FucGM₁ (IgG) antibody was found in many ofthem. Also, the C3 breakdown index (=C3d/C3c) as an indicator of ACP activation in sera of the patients (0.72±0.52), was significantly differed from those of normal control subjects (0.27±0.06). Especially sera of children with mesangial proliferative glomer ulonephritis and IgA nephropathy exhibited higher indexes than those of control (p<0.01). It was noteworthy that the significant positive correlation between the C3 breakdown index and the titer of anti-FucGM₁ (IgG) antibody was seen not only in sera of the children with mesangial proliferative glomerulonephritis and IgA nephropathy, but also those of children with primary glomerular diseases and glomerulonephritis with systemic diseases. In addition, the localization of glycolipid antigens was determined in renal sections of 10 children by indirect immunofluorescence method with anti-FucGM₁ antiserum. Strong positive staining was observed in glomeruli of 1 child and weak staining was in those of 3 children. However, macro-and micro-scopically normal renal tissue, which were obtained from 3 cases at autopsy, were negative no anti-FucGM₁ antibody. These results suggest that sialoglycolipids play some role in the occurrence and aggravation of glomerular diseases in children. Further studies are required to determine possible involvement of other glycolipids in the disease.

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Continuous ambulatory peritoneal dialysis (CAPD) is now accepted widely as effective dialytic method for end stage renal disease. Abnormality of calcium metabolism is the etiology of renal osteodystrophy (ROD), however metabolic changes are yet obscure in patients on CAPD. In this work, we studied factors affecting transperitoneal calcium flux in forty seven patients. All patients (forty two men and five women) aged 25-67 years (average 47 years) were treated by CAPD for 0.2-5.4 years (average 2.2 years). They were medicated calcium carbonate as a phosphate binder and 1 a-hydroxyvitamin D3(0.25-0.75μg/day). Their serum total calcium level was 9.62±1.28 mg/dl (mean±SD) and albumin level was 3.94±0.44g/dl (mean±SD). Transperitoneal calcium flux was a significant correlation between ultrafiltration volume (UFV) [P<0.001] and serum total calcium concentration level [P<0.001]. Total calcium concentration of effluent was 135.7±15.1mg/bag (mean±SD) and net calcium influx was +14.8 mg/bag. Ionized calcium concentration in effluent showed a highly significant correlation to total calcium concentration [P<0.001]. A calcium ionized ratio (%:ionized calcium concentration/total calcium concentration×100) was constant (approx. 73%) regardless of dialysate dextrose concentration or ultrafiltration volume. There was no significant correlation between this ratio and dialysate PH. Furthermore, dialysate non-ionized calcium concentration showed no significant correlation to dialysate albumin concentration. It was suggested that diffusible calcium fraction except ionized calcium concerned with transperitoneal flux. In other words, complexed calcium fraction (diffusible, but not ionized calcium) was an important fraction in regard to transperitoneal calcium flux.

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This study examined the relationship between C3B, C3 antigens and C3 hemolytic activity both in normal subjects and in patients with hypocomplementemic glomerulonephritis. Specific antiserum against only the B determinant on human native C3 was elaborated by extensive absorption of an anti-C3 serum with preparations containing C3a, C3c and C3d. The serum levels of C3B and C3 antigens were determined by single radial immunodiffusion using anti-C3B and anti-C3. C3 hemolytic activity was assayed by the method of Nelson et al. To determine whether C3B antigen amounts were related to C3 hemolytic activity, purified human C3 in solution was treated with three different intermediate cells. Treatment of C3 with EAC142 cells caused a decrease in the amount of C3B antigen by more than 90%, whereas EA and EAC14 cells did not. The extent of this reduction was comparable to that of a decrease in C3 hemolytic activity. Furthermore, disappearance of C3B antigen from purified C3 incubated with EAC142 was confirmed by immunoelectrophoretic analysis. Treatment of fresh human serum with complement activators also caused a decrease in the amount of C3B antigen and C3 hemolytic activity, and a signi ficant correlation was recognized between these two parameters. However, no correlation was recognized between C3 antigen and C3 hemolytic activity. To clarify this relationship obtained from in vitro study, protein determinations and hemolytic titration of C3 in patient's sera were performed. Although both C3B and C3 antigens correlated with C3 hemolytic activity in the whole of normal and patient's sera, a substantially different picture was found in sera from patients with marked hypocomplementemic state. In these patients the slope value of the relationship between C3B antigen and C3 hemolytic activity was nearly 1.0, whereas that of between C3 antigen and C3 hemolytic activity was 1.5 times higher. These results indicate that C3B antigen amounts might reflect the extent of C3 hemolytic activity more directly than C3 antigen amounts. The determination of serum C3B antigen is a simple method, and may contribute to exclude high value which is given falsely by measuring C3 breakdown products.

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The ultrastructural alterations of glomerular anionic sites were studied in biopsy specimens from 34 patients with IgA nephropathy using polyethyleneimine (PEI). Prominent common findings in the glomeruli of the patients were few PEI particles in electron dense deposits in the mesangial and subepithelial area and marked reduction in glomerular anionic sites covered with deposits. The anionic sites of the glomerular basement membrane (GBM) and epithelial cell surface coat (ESC) appeared unaltered in the patients with hematuria and/or mild pro teinuria. But in patients with proteinuria in the nephrotic range, focally discrete loss of anionic sites in the lamina rara externa (LRE) was seen and the number of anionic sites of the ESC were decreased with retraction of the foot processes. The anionic sites of the lamina rara interna showed much less change in these patients . Subepithelial deposits were often seen concomitantly with focal loss of anionic sites in the LRE at the site of the deposits, but subendothelial deposits had little influence on the anionic sites of the neighboring GBM. The number of anionic sites of GBM that showed focal thinning with small GBM projections was appreciably decreased, but the number of those in split GBM was not decreased. These results suggest that loss of the negative charge of the glomerular capillary wall associated with subepithelial immune deposition or morphological changes of the GBM contributes to the progression of proteinuria in IgA nephropathy.

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We evaluated the effect of FR-900452 (FR), a specific antagonist of platelet-activating factor (PAF), on the nephrotic rats. The nephrotic rats were induced by the intraperitoneal injection of Aminonucleoside (50 mg/kg), in male Wistar rats (body weight: 150-200g). Subsequently to these nephrotic rats, FR dissolved in polyetylene glycole 400 was administrated intraperitoneally at a dose of 50 mg or 100mg/kg body wt/day, twice a day, for 12 days. Nephrotic rats were housed in metabolic cages that allowed collection of the final 24-hour urine sample. Protein excretion, and the contents of arachidonic acid metabolites (6-keto-Prostaglandin F_1α and Thromboxane B₂) in urine were measured. Urinary protein excretion was significantly (p<0.05) decreased in FR treated nephrotic rats, compared with FR untreated nephrotic rats. Furthermore, urinary Thromboxane B₂ excretion was likewise significantly decreased in FR treated nephrotic rats, but urinary 6-keto-Prostaglandin F1.. excretion was unaffected by FR administration. The data suggest that synthesis of PAF causes proteinuria and that proteinuria induced PAF may depend on excessive synthesis of Thromboxane A₂ partially in the Aminonucleoside nephrotic rats, so that a specific antagonist of PAF may be effective for the nephrotic syndrome.

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Production of beta 2 microglobulin (B2M) was studied in vivo and in vitro in mainten ance dialysis patients. Changes in serum B2M concentration was examined before and after hemodialysis with three different types of dialyzer membrane. Synthesis of B2M was also studied on cultured mononuclear cells. Plasma B2M concentration was significantly increased at the end of dialysis using cuprophane or PMMA; B2 membrane dialyzer, while a significant reduction was noticed in EVAL membrane dialyzer. These changes in plasma B2M after dialysis seems to depend on the removal capacity of dialyzer and not to depend on complement activation. B2M synthesis of cultured mononuclear cells was significantly decreased in maintenace hemodialysis patients compared to normal volunteers, however, there was no change in CAPD patients. Ex vivo recirculation of heparinized whole uremic blood enhanced B2M synthesis on mononuclear cells, which did not relate to types of sterilization method. Those changes may partly be respohsible for abnormal metabolism of B2M in chronic dialysis patients.

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Lipid metabolism of tissues and lipoproteins were examined in daunomycin induced nephrotic rats.1) Phospholipids, triglycerides and cholesterol content in VLDL, IDL+LDL and HDL were higher in daunomycin induced nephrotic rats than control rats.2) Incorporation of 3H-leucine into liver, kidney, heart, serum and VLDL+LDL were studied using whole animal. There were no difference in the incorporation into liver, kidney and heart protein between daunomycin induced nephrotic rats and control rats. But, incorporation of 3H-leucine into serum and VLDL+LDL were increased in daunomycin induced nephrotic rats.3) The incorporation of 3H-leucine into protein by liver slices was higher in daunomycin induced nephrotic rats than control rats. There was no difference in the incorporation of 3H-leucine into protein by kidney slices in two groups.4) The rate of increase of triglycerides in serum after Triton WR-1339 injection was greater in daunomycin induced nephrotic rats than control rats. These results show hyperlipidemia in daunomycin induced nephrotic rats are caused by increased secretion of lipoproteins. It is considered that the accumulation of cholesterol esters in IDL+LDL, the increase of lipoprotein secretion and lysosomal cholesterol esterase activity in liver and kidney resulted in the increased cholesterol esters in liver and kidney.

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To clarify the pathobiochemical changes in urinary tubular enzymes and the low molecular weight protein with progression of diabetic nephropathy, urinary excretions of N-acetyl-β-D-glucosaminidase (uNAG), angiotensin I-converting enzyme (uACE), γ-glutamyl transpeptidase (uγGTP) and β2-microglobulin (uβ₂MG) were measured in 9 normal subjects and 46 diabetic patients (22 without proteinuria (group A), 14 with intermittent proteinuria (group B), 10 with persistent proteinuria (group C)). U NAG excretion was slightly high in group A and significantly high in groups B and C, and increased with the severity of renal function, diabetic control or retinopathy. But there was little relation between uNAG excretion and blood pressure. The I-form of uNAG in group B and the B-form of uNAG in group C were high in diabetic patients. DACE and uβ₂MG excretions were higher in group C than groups A and B, but uγGTP excretion was normal in all groups of diabetic patients. These results suggest that proximal tubular cells have already been impaired before the occurrence of proteinuria, and that the enzyme activity related to the synthesis of NAG may be suppressed in diabetic patients. It is concluded that the measurements of urinary excretions of tubular enzymes and the low molecular weight protein are useful for the evaluation of tubular damage in early stage of diabetes mellitus.

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The author was able to induce a disease closely resemling human lobular glomerulonephritis in the right kidneys by means of 3 or 4 intravenous injections, at intervals of one month or 3 weeks, of anti-rabbit renal glomerular basement membrane hen antibody or antiserum into rabbits in the beginning of the course of clamping of the left renal arteries for 20 minutes. The mode of development of the characteristic glomerulitis with lobular feature in the same individual animals was ascertained on the basis of the findings of biopsy materials surgically resected repeatedly as well as autopsy materials. It was clarified that the glomerular changes begin with transformation of glomerular loops into reticular structure (disorganization) due to histolysis of loop walls and fixed cell proliferation, and are followed by crowding of proliferated cells accompanied by formation of basement membrane-like structures along them toward axis of the loop and recanalization at its periphery (recovery of glomerular functional structure), recurrence of cell prolifera tion at the recanalized blood spaces, crowding of the proliferated cells toward axis, and peripheral recanalization, and repetition of such processes, thus forming centrolobular, ultimately hyaline nodules. Some problems concerning the pathogenesis of lobular glomerulonephritis (glomerulitis) were also discussed.

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Present investigation was performed to assess the influence of a high calcium (Ca) intake on the development of hypertension in subtotally nephrectomized rats. The rats in which approximately 5/6 of renal mass was ablated were fed on either a control diet (0.8% Ca) or a high Ca diet (4.0% Ca) for 15 weeks, and changes in the following parameters were determined: systolic blood pressure, electrolyte concentrations in blood and urine, serum level of parathyroid hormone (PTH), and urinary excretion of cyclic-AMP (cAMP). At the end of the experiment, pressor response to intravenous adminis tration of angiotensin II (Ang II) in doses of 1-64ng/100g of body weight was also studied. A high Ca intake attenuated the development of hypertension of the subtotally nephrectomized rats (at 15th week of dietary manipulation, 188±2.0 mmHg and 165±5.3 mmHg for control diet group and high Ca diet group, respectively; Mean±SEM, n=5, p<.01). This phenomenon was accompanied by higher concentration of plasma ionized Ca in rats fed on the high Ca diet (1.33±.01mmol/l) as compared with that in rats on the control diet (1.28±.01 mmol/l, p<.01). Serum level of PTH (234±12.0 pmol/l) and urinary excretion of cAMP (52±6.2 nmol/day) in the former were significantly lower than those in the latter (268±11.3 pmol/l and 84±9.7 nmol/day, respectively, p<.05). There was no significant difference in the pressor responses to Ang II among the two groups. Present study demonstrates that dietary Ca supplementation has an antihypertensive effect in subtotally nephrectomized rats. The results suggest that the mechanism of the action involved may be related to “membrane stabilizing effect” of Ca or a change in the serum level of PTH.

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To elucidate the mechanism of blood pressure change with low calory regimen, essential hypertensive subjects with at least 20% obesity (age 51±2 yrs, n=29) were hospitalized and a 2, 000 kcal a day diet for 5 days (control period) followed by an 800 kcal a day diet for 21 days were prescribed. Salt intake was maintained at 8 to 10g a day throughout the observation. Blood pressure, plasma norepinephrine (PNE) and 24-hour urinary excretion of norepinephrine (UNE) were measured in both diet periods. During the 800 kcal/day period, blood pressure normalized in 17 subjects (R group) and 12 remained hypertensive (PR group). Blood pressure, amount of obesity, PNE and UNE during the control period did not differ in the two groups. Whereas PNE and UNE decreased significantly during the low calory period in the R group, it did not change in the PR group. These data suggest that an 800 kcal/day diet is an effective nonphamacological measure for obese hypertensive subjects and that decrease in sympathetic tone is playing an important role for the blood pressure change during the low calory regimen.

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The effects of CaCO₃ on renal osteodystrophy (ROD) were investigated in non-diabetic patients with chronic renal failure in the predialytic stage (Ccr<30 ml/min, non-HD group, N=7) and undergoing maintenance hemodialysis (HD group, N=6) not treated with phosphate binder and vitamin D metabolites. In the non-HD group, the levels of serum total calcium and ionized calcium (Ca⁺⁺) were significantly increased and the levels of serum phosphate (Pi), calcium×phosphate product (Ca×Pi), C-terminal parathyroid hormone (C-PTH) and N-terminal parathyroid hormone (N-PTH) were meaningfully decreased on the 4th week after CaCO₃ 3.0 g/day administration. One of the non-HD group, however, had a deterioration of renal function. In the HD group, there was no significant change on the 4th week. CaCO₃ was effective as phosphate binder except some of the HD group. Although we think that the therapy of ROD is necessary at the time of Ccr 30 ml/min in respect of calcium metabolism, aggravation of renal function due to hypercalcemia or elevation of Ca×Pi needs a care. It is, therefore, desirable that CaCO₃ is used from a little. In the patients of the non-HD group with the decrease of serum 1-25 dihydroxycholecalciferol concentration (1-25 (OH)₂D₃), it is suggested that (1-25 (OH)₂D₃) increase reciprocally because of the decrease of Pi for CaCO₃ administration, so that Ca rise owing to the increment of intestinal absorption of calcium and bone resorption, finally C-PTH and N-PTH diminish.

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