Acridine orange (AO), a pH-sensitive fluorescent indicator, was used to study the characteristics of SO₄/HCO₃ transport in basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex. The BLMV preparation containing a low buffer concentration and preloaded with 25 mM HCO₃ was mixed with buffer containing AO and SO4 in the absence of an initial pH gradient. SO₄ influx tended to drive HCO₃ efflux, causing intravesicular accumulation of AO and fluorescence quenching. There was no AO quenching in the absence of HCO₃ with or without an external SO₄ gradient, 100μM 4, 4'-dibenzamido-2, 2'-disulfonic stilbene (DBDS) inhibited the fluorescence quenching completely. 25 and 100 mM external Cl did not cause AO quenching. There was no effect of pH (6.5-8.0) on SO₄/HCO₃ transport. The Kd for SO4 was 8.2 mM. A positive inwardly directed diffusion potential (K_in = 5 mM, K_out = 100 mM with valinomycin) did not exert any effect on the SO₄/HCO₃ transport, indicating that the transport process is insensitive to voltage. The K, for DBDS inhibition of S₄/HCO₃ transport was 2.3μM.

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The effects of arginine vasopressin (AVP^*2) and its V₂ receptor agonist, 1-deamino-8-D-AVP (dDAVP), on the intracellular calcium ion concentration ([Ca²⁺]_i) in isolated collecting tubular cells of mouse kidney were examined using fluorescent indicator fura-2 and a superfusion system. Both AVP and dDAVP evoked a rapid, transient increase followed by a sustained elevation of [Ca²⁺]_i in CCT, OMCT, and IMCT in a dose-dependent manner. In CCT, the increments in [Ca²⁺]_i by dDAVP were lower than those induced by AVP at all concentrations (10^-10-10^-6 M) of the agonists tested, while in OMCT and IMCT, the increments were comparable. The initial peak of the rise in [Ca²⁺]_i induced by AVP and dDAVP in these collecting tubule segments was partially attenuated by about 40% and the second sustained elevation was largely abolished in the absence of Ca²⁺ in the superfusate. Further, the increments [Ca²⁺]_i induced by AVP were not affected by the addition of nicardipine to the superfusate. The increases in [Ca2+]_i evoked by AVP and dDAVP were not mimicked by CAMP or forskolin. Moreover, they were not affected by α-adrenergic stimulation with epinephrine, in the presence and absence of prazosin, conditions which inhibit AVP-dependent cAMP production. These results indicate that AVP increases [Ca²⁺]_i in CCT, OMCT, and IMCT, probably through V₂ receptors, but via a mechanism which is independent of adenylate cyclase activation. In addition, the rise in [Ca²⁺]_i is due to both Ca²⁺ release from the intracellular stores and increased Ca²⁺ influx through Ca²⁺ channels insensitive to nicardipine.

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G-proteins in rat glomeruli were examined using bacterial toxin-catalyzed ADP-ribosylation and specific immunoblots. ADP-ribosylation catalyzed by cholera and pertussis toxin demonstrated the existence of Gs and Gi proteins in the glomerular membranes. Immunoblots further revealed two types of Gsα(45 and 52 kDa), Giα1 and/or Giα2 (40-41 kDa), Giα3 (40 kDa) and G β(35-36 kDa) but not Goain the membranes of the glomeruli. The predominant subspecies of Gsa was a 52 kDa protein. However, detectable amounts of G-proteins did not exist in cytosolic extracts of the glomeruli. We conclude that several subspecies of Gs and Gi proteins are present in rat glomerular membranes.

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A study was undertaken on the specificity of circulating IgA antibodies in patients with IgA nephropathy detected by immunofluorescence using avidin-biotin complexes. Renal biopsy specimens and serum samples were obtained from 33 patients with IgA nephropathy, 14 other glomerular diseases and 3 normal renal tissues. These renal specimens were treated with citrate buffer (pH 3.2), and then incubated with serum samples obtained from the same and other patients with IgA nephropathy, other glomerular diseases or healthy adults at 37°C for 30 min. The specimens were incubated with biotin conjugated gout F(ab')2 anti-human IgA antiserum at 37°C for 30 min, and then with fluorescein-labeled avidin at 37°C for 30 min. It was found that IgA antibodies in the sera from patients with IgA nephropathy specifically combined with the autologous glomerular mesangial areas, but only 25.7% of them combined with allogeneic renal tissues of IgA nephropathy patients. Conbrmatory findings were obtained using an automatic image analyzer. However, these IgA antibodies did not combine with the renal tissues from patients with other glomerular diseases or normal renal tissues. In parallel studies, in order to distinguish IgA nephropathy from other glomerular diseases before renal biopsy, the renal specimens from patients with IgA nephropathy were also incubated with serum samples obtained from 42 patients with proteinuria and/or hematuria before renal biopsy. It was demonstrated that the incidence of IgA binding in IgA nephropathy was significantly higher than that in other glomerular diseases prior to renal biopsy. IgA antibodies in the sera were concluded to have a specific reactivity to the mesangial areas of patients with IgA nephropathy and these IgA antibodies to show some heterogeneity among patients with this disease. It was inferred that the present technique might be useful for distinguishing IgA nephropathy from other glomerular diseases before renal biopsy.

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Serum IgG antibodies to polyclonal IgA, IgA₁ and IgA₂ were evaluated by enzyme-linked immunosorbent assay in 50 patients with IgA nephropathy and 30 healthy controls to elucidate the relationship between IgA and IgG in IgA nephropathy. Anti-IgA antibody was considered positive if the titer exceeded the mean value in normal controls by <2 SD. In patients with IgA nephropathy, 18 cases (36%) demonstrated anti-IgA antibody, 19 cases (38%) anti-IgA₁ antibody and 7 cases (14%) anti-IgA₂ antibody. Western blots confirmed the existence of anti-IgA antibody in these patients. There were no significant differences in serum IgA concentration, serum creatinine concentration, degree of hematuria, amount of urinary protein, and rate of glomerular IgG deposition between the "positive" group and "negative" group . Although the mechanism of production and the role of this antibody remain unknown, it may represent one of the diverse immune abnormalities of IgA nephropathy and may be involved in the pathogenesis of IgA nephropathy.

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The present study was undertaken to determine the anti-neutrophil cytoplasmic antibody (ANCA) levels in 96 patients with various collagen diseases associated with renal vasculitis and vasculitic syndrome in Japan. The results indicated that cytoplasmic(C)-ANCA is an autoantibody highly specific to Wegener's granulomatosis (WG) and that it is also active in renal injury. The relationships between ANCA and focal segmental necrotizing GN, i.e., renal vasculitis as proposed by Balow, were investigated. Perinuclear(P)-ANCA was detected with high sensitivity and specificity in renal vasculitis without WG, and the severity of necrotizing and crescentic nephritis in WG was correlated especially well with the C-ANCA titer. Detection of ANCA is considered clinically useful for the etiological differentiation of renal vasculitis, suggesting the possibility that C-ANCA may be involved in the onset of vasculitis of the glomerular capillary vessels in WG. The presence of C-ANCA and cytokines (IL-1β and TNF-α) is important in the pathogenesis of vasculitis and GN in WG.

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To investigate the ultrastructure of the glomerular basement membrane (GBM), rat GBM was treated with sodium dodecyl sulfate and 2-mercaptoethanol and observed under an electron microscope employing ultrathin sectioning and rotary shadowing methods. Further, thick sections of the treated GBM were examined by high voltage transmission electron microscopy. A fine three-dimensional meshwork structure was clearly observed through the entire thickness of the GBM treated with sodium dodecyl sulfate and 2-mercaptoethanol by conventional transmission electron microscopy and high voltage transmission electron microscopy. The diameter of the fibrils forming the meshwork structure was about 3 nm and the dimensions of the pores present in the meshwork were 3 to 4 nm. The rotary shadowing technique revealed fine fibrils disentangled from the GBM that were bound together, and corresponded morphologically type IV collagen molecules. The present findings suggested that the GBM has a fine three-dimensional meshwork structure through its entire thickness which is composed mainly of type IV collagen and may function as a size barrier in the renal glomerulus.

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Alterations of the anionic charge and/or sites of the glomerular basement membrane (GBM) in the heterologous phase of passive Heymann nephritis (PHN) were studied. Rats with PHN induced by a single injection of anti-Fx1A IgG were examined at days 1, 2, 3 and 4. The left kidney was perfused with ruthenium red (RR) solution as a cationic probe. The RR particles (= anionic sites) in the GBM were counted and expressed as the number of RR particles per unit length of GBM. For quantitative determination of the total anionic charge of the GBM, the GBM-bound ruthenium (= anionic charge) was measured with an atomic absorption spectrophotometer (AAS). Abnormal proteinuria corresponding to a decrease in anionic charge was detected at days 3 and 4. The anionic sites in the lamina rara externa (LRE) adjacent to immune complex (IC) deposits were found to have diminished earlier from day 1onwards. This diminution was largely confined to areas adjacent to the IC deposits and was significantly correlated with the amount of urinary albumin excretion. Proteinuria in the heterologous phase of PHN would thus appear to be causally related to a decrease in the number of anionic sites in the LRE adjacent to IC deposits.

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The nephrotic syndrome is often accompanied by hyperlipidemia associated with an increased risk of accelerated atherosclerosis. The present study was undertaken to evaluate the effects of pravastatin, a novel competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the serum lipids and apolipoproteins in patients with this syndrome and marked hyperlipidemia. Eleven adult patients received 10 mg of pravastatin twice daily for 4 to 8 weeks. The total serum cholesterol decreased from 426±44 to 309± 18 mg/dl (-27.4%, mean ± SE.; p <0.01) following administration of pravastatin. The serum triglyceride decreased from 332 ± 122 to 229 ± 50 mg/dl (-30.9%), although this change was not significant. Despite the fact that the HDL cholesterol level was barely changed (51 ± 7 to 51 ± 6 mg/dl), the LDL cholesterol fell from 313 ± 30 to 211 ± 16 mg/dl (-32.5%; p<0.005), and the LDL to HDL cholesterol ratio fell from 7.57 ± 1.59 to 4.94 ± 0.88 (-34.8%; p <0.05). These changes caused the atherogenic index to decline from 9.6 ± 2.4 to 6.1 ± 1.2 (-36.5%; p <0.05). No significant alterations couldbe found among apolipoproteins A-1, A-2, B, C-2, C-3, and E. During the present study period, pravastatin was well tolerated and did not affect the serum protein, albumin, serum urea nitrogen, creatinine levels, or urine protein excretion. Also, there were no serious adverse effects. Pravastatin appears to be effective for treating patients with hyperlipidemia of the nephrotic syndrome.

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In order to compare the protective effects of angiotensin converting enzyme inhibitors (ACEI) and calcium channel blockers (CCB) on the renal function in experimental nephritis, nephrotoxic serum nephritis was induced in male spontaneously hypertensive rats (SHR). The above drugs were then chronically administered to different groups, as follows: the ACEI-treated group (n=7) received captopril (150 mg/kg/day), and the CCB-treated group (n=6) was given both nifedipine (40 mg/kg/day) and nisoldipine (20 mg/kg/day). The control group (n= 8) received a placebo. Although the control group developed marked hypertension and proteinuria, the rats treated with either ACEI or CCB demonstrated a significant and equivalent decrease in mean arterial pressure and urinary protein excretion. At 15 weeks after the injection of nephrotoxic serum, all rats were anesthetized with Inactin, and the glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured. In the control group, GFR and RPF were markedly attenuated. However, both were preserved at much higher levels in the ACEI-treated group, and GFR was also maintained to a similar degree in the CCB-treated group. Histological studies were carried out after the clearance studies. As a result, it was found that the ACEI treatment significantly limited the development of glomerulosclerosis, whereas CCB modestly ameliorated the glomerular structural lesions. Moreover, ACEI significantly reduced the serum cholesterol, while CCB did not exert such an effect. These results suggest that both ACEI and CCB have a therapeutic effect in experimental glomerulonephritis models which are accompanied by hypertension. The protective effect may be more prominent in ACEI in comparison with CCB, and it is possible that lipid metabolism might be involved in the difference between the two agents.

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The present study was conducted on 8 patients with advanced diabetic nephropathy who showed a significant reduction of proteinuria through ACE inhibition. Camostat mesilate, one of the most potent protease inhibitors developed for oral use, was administered to these patients at a daily dose of 600 mg starting after 4 weeks of ACE inhibitor administration. Laboratory data were obtained 1) just before the ACE inhibition, 2) after 4 weeks of the ACE inhibitor single treatment, and 3) after another 4 weeks of the additional treatment with camostat mesilate. The urinary protein excretion decreased from 1)10 .1 ± 1.3 to 2) 7.3± 1.1, and 3) 4.6± 0.9 g/day [mean ± SEM; significance of difference 1)-2), p<0.05; 2)-3), p <0.01], and the serum total protein values increased from 1)5.0±0.3 to 2) 5.2± 0.2, and 3) 5.4± 0.3 g/dl [1)-3), p<0.05]. The plasma levels of fibrinogen, and of E fragment and D-dimer of FDP changed from 1) 476 ± 43 to 2) 477 ± 41, and 3) 374 ± 33 mg/dl [2)-3), p<0.01], from 1) 125 ± 19 to 2) 147 ± 27, and 3) 104 ± 30 ng/ml [2)-3), p<0.05], and from 1) 261 ± 60 to 2) 272 ± 86, and 3)185 ± 56 ng/ml [2)-3), p <0.05], respectively. Although the urinary excretions of E fragment and D-dimer of FDP decreased in many patients even during ACE inhibition, their urinary excretion ratio to urinary protein fell significantly only after camostat mesilate was administered. These results suggest that camostat mesilate may suppress the intravascular over-formation of fibrinogen and fibrin, and exert inhibitory effects on the hypercoagulable state induced by advanced diabetic nephropathy. This anti-coagulatory effect might be closely related to its anti-proteinuric effect in diabetic nephropathy.

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The factors related to hypotension in hemodialysis patients were evaluated. The subjects consisted of 120 hemodialysis patients (61 males and 59 females), whose ages ranged between 28 and 82 years, and who had undergone hemodialysis treatment for a period ranging between 3 and 201 months. They were divided into three groups; Group A, 53 normotensive patients; Group B, 52 patients with hypotension during hemodialysis; and Group C, 15 patients with constant hypotension. The evaluated factors were age, sex, etiology of renal failure, hemodialysis term, ultrafiltered volume, responsiveness to vasopressor drugs, Cr, Na, Ht, TP, cardiothoracic ratio, presence or absence of heart disease, and microvibration pattern. Statistical analysis was performed using multivariate analysis (Quantification II and IV methods). The results of the Quantification IV method indicated that Groups A and B were similar but Group C was quite different from the other two groups. The results of the Quantification II method indicated thatthe most important factor which characterized the difference between Group A and Group C was theresponsiveness to vasopressor drugs, and the most important factor in the difference between Group B and Group C was the microvibration pattern. Group B was characterized by abnormal microvibration patterns and Group C was characterized by a low responsiveness of the peripheral vessels to vasopressor drugs. We conclude that the hypotension during hemodialysis was caused predominantly by dysfunction of the autonomic nervous system, while the constant hypotension was caused predominantly by a deterioration of constriction of the peripheral vessels.

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We report the case of a 15-year-old Japanese female with severe mesangial proliferative IgA glomerulonephritis who showed a dramatic response to cocktail therapy for nephrotic syndrome. She had suddenly developed massive proteinuria and microscopic hematuria. The first renal biopsy at one month after onset revealed severe mesangial hypercellularity and podocytic detachment from the glomerular basement membrane (GBM). The cocktail therapy resulted in a decrease of proteinuria clinically, and a second biopsy demonstrated repair of the podocytic detachment. We suggest that the massive proteinuria in this case was due to destruction of the size barrier by detachment of podocytes from the GBM, and the repair of the podocytic covering on the GBM was accelerated by the cocktail therapy.

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