[Clinical evaluation of kampo medication, mainly with wen-pi-tang, on the progression of chronic renal failure].

Previous studies have shown that Kampo (traditional Chinese) prescriptions, mainly Wen-Pi-Tang (Onpi-to, [symbol see: text]), have a useful effect in patients with chronic renal failure (CRF). We aimed to examine the long-term effect of Kampo prescriptions on serum creatinine (Cr) among patients with CRF. Patients with serum Cr levels of 2 mg/dl more were enrolled if they had at least 4 recordings of serum Cr in the previous 6 months or more, and were followed-up until the start of dialysis. Eight patients aged 24-59 years with serum Cr 4.5 mg/dl were enrolled in the study for 40 to 402 weeks (mean; 228.1 +/- 118.8 weeks). The cause of CRF was chronic glomerulonephritis in 7 patients and systemic lupus erythematosus in 1 patient. The end points of the study were the slope of the reciprocal of the serum Cr concentration plot against time using Mitch's method, and the predicted period of pre-dialysis. The predicted pre-dialysis period was defined as an increase in serum Cr by 10 mg/dl. As a result, the individual slopes were improved in 6 of 8 cases, in particular, in 4 of 5 Wen-Pi Tang-treated cases. The average slope was improved significantly (p < 0.01) in Wen-Pi-Tang-treated cases, although it showed only a tendency to improve in all 8 cases. The predicted pre-dialysis period was prolonged from 79.2 +/- 74.8 weeks to 389.5 +/- 355.4 weeks and 55.6 +/- 37.0 weeks to 262.4 +/- 145.8 weeks in all 8 cases and Wen-Pi-Tang-treated cases, respectively. The observed pre-dialysis period was 228.1 +/- 118.8 weeks, which showed that Kampo prescriptions prolonged the predicted period for 186 additional weeks. In conclusion, this study demonstrated that the Kampo prescriptions, consisting mainly of Wen-Pi-Tang, retarded the progression of CRF.

Changes in the extracellular matrix on the surface of sintered bovine bone implanted in the femur of a rabbit: an immunohistochemical study.

The interface of implanted True Bone Ceramics (TBC; sintered bovine bone; Koken, Tokyo, Japan) was examined. In the primary experiment, TBC was implanted into the bone marrow of a rabbit's femur. The extracellular matrices (types I, II, and III collagens and fibronectin) of decalcified specimens collected 1-48 weeks postoperatively were immunohistochemically examined. Undecalcified sections collected 6 weeks postoperatively were used for line analyses of calcium and phosphorus, by a scanning electron microscope-electron probe microanalysis (SEM-EPMA) method. In a secondary experiment, TBC was implanted into an osteochondral defect of a femoral condyle, harvested 1-12 weeks postoperatively, and decalcified to examine the extracellular matrices at the interface. In the bone marrow in the early phase, TBC had absorbed quantities of fibronectin. Immature bone (containing both types I and III collagens) in direct apposition to the ceramic surface had matured (containing type I collagen alone) in the TBC pores. SEM-EPMA revealed the continuity of high levels of calcium and phosphorus at the TBC-bone interface. In the secondary experiment, enchondral ossification or fibrous tissue formation was observed near the articular surface. However, in the subchondral layer, direct bone formation was observed in the TBC pores. It was concluded that TBC has excellent bioactivity for inducing maturation of new bone matrix on porous surfaces.

Proof that green tea tannin suppresses the increase in the blood methylguanidine level associated with renal failure.

The effects of a green tea tannin mixture and its individual tannin components on methylguanidine were examined in rats with renal failure. The green tea tannin mixture caused a dose-dependent decrease in methylguanidine, a substance which accumulates in the blood with the progression of renal failure. Among individual tannin components, the effect was most conspicuous with (-)-epigallocatechin 3-O-gallate and (-)-epicatechin 3-O-gallate, while other components not linked to gallic acid showed only weak effects. Thus, the effect on methylguanidine was found to vary among different types of tannin.

Inhibitory effect of green tea on injury to a cultured renal epithelial cell line, LLC-PK1.

When cells from a cultured renal epithelial cell line, LLC-PK1, were cultured under hypoxic conditions (oxygen concentration of 2% or less) before reoxygenation was applied (95% air, 5% CO2), the leakage of lactate dehydrogenase (LDH) into the medium increased. This phenomenon was inhibited in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, suggesting the involvement of free radicals. Such oxidative stress was significantly inhibited by a green tea extract, and more potently by a tannin mixture. On the other hand, under ordinary culture conditions (95%, air, 5% CO2), there was cell injury, although the LDH leakage was less than that under hypoxia/reoxygenation, and such injury was inhibited by the green tea extract and the tannin mixture.

Magnesium lithospermate B suppresses the increase of active oxygen in rats after subtotal nephrectomy.

Subtotally nephrectomized rats were found to have decreased activities of superoxide dismutase (SOD) and catalase, and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that the amount of hydroxyl radical in the residual kidney tissue was greater than that in normal rat kidney. This indicated both direct and indirect involvement of free radicals in renal failure. In contrast, rats given magnesium lithospermate B (10 mg/kg body weight) orally for 30 days after subtotal nephrectomy showed restoration of SOD and catalase activities to almost normal levels. Hydroxyl radical, which is highly reactive and for which there is no scavenger system in the body, was decreased markedly in kidney homogenates obtained from rats given magnesium lithospermate B and in an experimental system for hydroxyl radical production to which magnesium lithospermate B was directly added. The increased levels of uremic toxins in the blood were also low in rats given magnesium lithospermate B. This indicates that magnesium lithospermate B helps to inhibit the progression of renal failure by scavenging radicals.

Magnesium lithospermate B ameliorates cephaloridine-induced renal injury.

To determine whether magnesium lithospermate B ameliorates renal injury induced by cephaloridine, the effect of cephaloridine was investigated in rats given magnesium lithospermate B for 20 days preceding cephaloridine administration and in control rats given no magnesium lithospermate B. In the control rats, blood and urinary parameters and the activity of radical-eliminating enzymes in the renal tissue deviated from the normal range, indicating damage to the kidneys. In contrast, rats given magnesium lithospermate B showed decreased urine volume, increased urinary osmotic pressure, and decreased urinary levels of glucose, protein, sodium and potassium, denoting less damage to the kidney. In this group, the urinary nitrite/nitrate ratio, and the activities of superoxide dismutase and catalase in the renal tissue were increased, while the malondialdehyde levels were decreased, suggesting the involvement of radicals in the normalizing of kidney function. The increased levels of urea nitrogen in the blood of rats with induced renal failure were also lowered by administering magnesium lithospermate B.

Magnesium lithospermate B ameliorates cisplatin-induced injury in cultured renal epithelial cells.

A study was conducted to clarify whether magnesium lithospermate B ameliorates cisplatin-induced renal injury in terms of lactate dehydrogenase and malondialdehyde leakage from LLC-PK1 cells in culture. Magnesium lithospermate B was shown to suppress the cytotoxicity of cisplatin, the suppressive effect increasing with the dose of magnesium lithospermate B.

Protective effects of Wen-Pi-Tang against cultured renal epithelial cellular injury.

A study was conducted to clarify whether extracts of Wen-Pi-Tang and its component crude drugs ameliorate renal cellular injury by assaying lactate dehydrogenase and malondialdehyde leakage from LLC-PK(1) cells in culture. The cells were cultured with various concentrations of the samples under two sets of conditions: routine and hypoxia-reoxygenation. The results demonstrated that Wen-Pi-Tang, Rhei rhizoma, Glycyrrhizae radix exerted marked protective effects on the cells; Ginseng radix showed moderate activity, whereas Zingiberis rhizoma and Aconiti tuber had virtually no such effect. Two pure compounds, epicatechin 3-O-gallate and licochalcone A isolated from Rhei rhizoma and Glycyrrhizae radix, respectively, exerted the same marked effects as the parent crude drugs. In the light of these findings, we concluded that Wen-Pi-Tang and its major components protect renal epithelial cells against injury mediated by hypoxia-reoxygenation and/or prevent such injury. The primary mechanism of these effects appeared to be antilipid peroxidant activity present in the preparation.

Molecular biological analysis of the effects of ginsenoside-Rb2 on albumin mRNA in streptozotocin-induced diabetic rats.

The mechanisms of the mRNA synthesis-promoting action of ginsenoside-Rb2, were investigated at the gene level. Rot analysis suggested that the previously reported increase in RNA polymerase activity as a result of administration of ginsenoside-Rb2 might be because of its effect on a specific gene. In this regard, albumin mRNA, which is expressed specifically in the liver, was assayed by northern blot hybridization using albumin cDNA in normal rats, diabetic control rats and diabetic rats given ginsenoside-Rb2. When the level of albumin mRNA in normal rats was set at 100, the level was reduced markedly to 32 in diabetic control rats. In contrast, in diabetic rats given ginsenoside-Rb2 the level was 0.54, significantly higher (69%) than that in diabetic rats given no ginsenoside-Rb2. In addition, poly(A)+RNA was purified from total RNA and subjected to hybridization, and poly(A)+RNA bands with different charges were measured by densitometry. The results of the measurement revealed changes dependent on the charge, and this was confirmed by autoradiography. We found no significant difference in the transcription activity of albumin mRNA, however, it showed only a tendency to increase. This suggests that ginsenoside-Rb2 has some effect on post-transcriptional regulation of the stability of mRNA itself. The results of Rot analysis suggest that ginsenoside-Rb2 affects a specific gene alone.

Effectiveness of green tea tannin on rats with chronic renal failure.

The effects of green tea tannin on nephrectomized rats were examined. There were increases in blood urea nitrogen, serum creatinine, and urinary protein, and a decrease in creatinine clearance in the nephrectomized control rats, whereas better results for these parameters were obtained in rats given green tea tannin after nephrectomy, demonstrating a suppressed progression of the renal failure. When the renal parenchyma was partially resected, the remnant kidney showed a decrease in the activity of radical scavenger enzymes. Green tea tannin, however, was found to lighten the kidney under such oxidative stress. Mesangial proliferation and glomerular sclerotic lesions, which were conspicuous in the rats that were not given green tea tannin after nephrectomy, were also relieved.

Effects of a component of green tea on the proliferation of vascular smooth muscle cells.

The effects of a component of green tea on the proliferation of smooth muscle cells were measured in terms of [3H]thymidine uptake. When green tea tannin mixture was added to the medium of cultured smooth muscle cells, it suppressed the proliferation of the cells dose-dependently. Similarly to the effects of the green tea tannin mixture, (-)-epigallocatechin 3-O-gallate, its main ingredient, had an inhibitory effect on smooth muscle cell proliferation at a low concentration. (-)-Epicatechin 3-O-gallate was also an effective component. Among four types of gallate-free tannin, (-)-epigallocatechin, (-)-epicatechin, and (+)-catechin showed significant dose-dependent inhibition of smooth muscle cell proliferation. However, caffeine and theanine were found to have no such action.

Confirmation that magnesium lithospermate B has a hydroxyl radical-scavenging action.

Male LWH: Wistar rats were given creatinine (Cr) intraperitoneally at a dose of 1.00 g/kg body weight, and the urine was collected for 3 h after administration. Magnesium lithospermate B, a compound newly isolated from Salviae Miltiorrhizae Radix, was administered intraperitoneally 30 min before and 30 min after Cr administration. The excretion of urinary creatol and methylguanidine induced by Cr decreased in a dose-dependent manner. Since we have already shown that the main contributor to the Cr-->creatol oxidation step is the hydroxyl radical, magnesium lithospermate B may act as a radical scavenger.

The promoting action of magnesium lithospermate B on the kinin-prostaglandin E2 system in the kidney.

The effect of magnesium lithospermate B purified from Salviae Miltiorrhizae Radix on the kinin-prostaglandin E2 system was investigated using kidney slices and isolated kidney microsomes. Magnesium lithospermate B markedly increased the amount of thiobarbituric acid-reactive substances produced by incubation of arachidonic acid in renal slices. Enhancement of arachidonic acid-mediated oxygenation of 1,3-diphenylisobenzofuran was also observed in renal microsomes from rats treated with magnesium lithospermate B, indicating an increase of prostaglandin biosynthesis. Furthermore, magnesium lithospermate B significantly increased the synthesis of prostaglandin E2 in renal slices, but the magnesium lithospermate B-mediated response was blunted with kinin antagonist. On the basis of the above results, it is apparent that magnesium lithospermate B exerts an influence on the prostaglandin system via kinin receptors.

Effects of a Dan Shen component, magnesium lithospermate B, in nephrectomized rats.

Magnesium lithospermate B, a compound newly isolated from Dan Shen, was given orally to rats for 70 days after excision of five-sixths of their kidney volume. As a result, mesangial proliferation, tubulo-interstitial lesions and glomerular sclerotic lesions, which were conspicuous in rats that were not given magnesium lithospermate B after nephrectomy, were inhibited. Furthermore, a decrease in blood urea nitrogen, improvement of hypoproteinemia, hypoalbuminemia and hypercholesteremia, and inhibition of urinary protein excretion were observed. The levels of creatinine, methylguanidine and guanidino-succinic acid, which accumulate in the blood with the progress of renal failure, were decreased significantly in rats given magnesium lithospermate B. These results indicate that magnesium lithospermate B, a component of an Oriental medicine has potential as a new therapeutic agent for inhibiting the progression of renal dysfunction.

Effects of ginseng in nephrectomized rats.

Ginseng extract and its active component, saponin, were administered orally to nephrectomized rats for 90 d, and changes in blood and urine parameters and renal tissue lesions were assessed. Rats given saponin showed a significant decrease in the concentrations of blood urea nitrogen, creatinine and methylguanidine and a significant increase in total protein and albumin in the blood, with reduce urinary excretion of protein. There was also slight amelioration of the degree of mesangial proliferation, the severity of extratubular lesions and glomerular sclerotic lesions, and the extent of tubular interstitial lesions. However, these histological changes were inconspicuous in nephrectomized rats given ginseng extract.

Ursolic acid inhibits aflatoxin B1-induced mutagenicity in a Salmonella assay system.

An attempt was made to isolate the active component of Eriobotrya japonica, which inhibits aflatoxin B1-induced mutagenicity in the Salmonella assay system. The number of revertants per plate was significantly decreased when a MeOH extract of Eriobotrya japonica was added to the assay system using Salmonella typhimurium TA100 or TA98. Furthermore, we examined the effect of each fraction purified from the MeOH extract, and an EtOAc fraction was found to be the most effective. Ursolic acid isolated from the EtOAc fraction markedly and significantly decreased the numbers of Salmonella typhimurium TA100 revertants per plate, thus showing antimutagenic activity.

Inhibitory effects of ginseng on proliferation of cultured mouse mesangial cells.

To evaluate the pharmacological action of ginseng, its effects on the proliferative activity of mesangial cells, which are thought to play an important role in the regulation of renal function, were determined in terms of [3H]thymidine uptake. When the extract was added to the medium of mesangial cell cultures, it suppressed the proliferation of mesangial cells, and similar proliferation-inhibitory activity was found in the total saponin and ginsenoside-Rd fractions, consistent with the renal effects observed in our previous in vivo studies. The inhibition of mesangial cell proliferation by the extract can thus be explained by the action of ginsenoside-Rd.

Inhibitory effects of crude drug components on the proliferation of cultured human mesangial cells.

The effects of a component of an Oriental medicine and green tea plants utilized for the traditional beverage on the proliferation of mesangial cells were determined in terms of the 3H-thymidine uptake. When magnesium lithospermate B was added to the medium of the mesangial cell culture, it suppressed the proliferation of mesangial cells at a concentration of 50 micrograms/ml. The 3H-thymidine uptake began to display an inhibitory action from the 6.25 micrograms/ml (-)-epicatechin 3-O-gallate, (-)-epigallocatechin 3-O-gallate or green tea tannin mixture, showing a more conspicuous inhibition at 50 micrograms/ml that did the magnesium lithospermate B at the corresponding concentration. In particular, (-)-epigallocatechin 3-O-gallate, the predominant component of the green tea tannin mixture, exerted an inhibitory effect at a relatively low concentration.

Effect of magnesium lithospermate B on the renal and urinary kallikrein activities in rats with adenine-induced renal failure.

A study was conducted to examine the effect of magnesium lithospermate B on both the urinary and renal total and active kallikrein and prokallikrein in rats with adenine-induced renal failure. In rats given magnesium lithospermate B at a dose of 10 mg/kg body weight/day for 12 days, significant increases of urinary total and active kallikrein were associated with significant increases of urine volume and urinary total and active kallikrein were associated with significant increases of urine volume and urinary creatinine excretion. The renal total and active kallikrein levels were also significantly elevated by the treatment with magnesium lithospermate B. On day 24, the urinary excretion of total and active kallikrein and prokallikrein was significantly increased. Concomitantly, a significant increase in renal kallikrein (total, active and pro-) was found in the rats given magnesium lithospermate B. A significant relationship existed between the urinary creatinine and active kallikrein excretion. These results suggest that magnesium lithospermate B may stimulate the synthesis of kallikrein and/or conversion to active kallikrein, thus improving renal function.