Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury.

OBJECTIVE: We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy. SUBJECTS: C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone. RESULTS: The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3ß-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase. CONCLUSION: Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.

Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumours.

BACKGROUND: Adoptive transfer of ex vivo expanded autologous Vγ9Vδ2 T cells may be of therapeutic benefit for cancer because of their potent direct cytotoxicity towards tumour cells, synergistic cytotoxicity when combined with aminobisphosphonates and enhancement of antibody-dependent cell-mediated cytotoxicity. METHODS: To determine the feasibility and clinical safety of therapy with ex vivo expanded, activated Vγ9Vδ2 T cells in combination with zoledronate, we enrolled 18 subjects with advanced solid tumours into a phase I clinical study. Administered indium(111)-oxine-labelled Vγ9Vδ2 T cells were tracked in a cohort of patients. RESULTS: Administered Vγ9Vδ2 T cells had an activated effector memory phenotype, expressed chemokine receptors predictive of homing to peripheral tissues and were cytotoxic in vitro against tumour targets. Adoptively transferred Vγ9Vδ2 T cells trafficked predominantly to the lungs, liver and spleen and, in some patients, to metastatic tumour sites outside these organs. No dose-limiting toxicity was observed, but most patients progressed on study therapy. However, three patients administered Vγ9Vδ2 T cells while continuing previously ineffective therapy had disease responses, suggesting an additive effect. CONCLUSION: Therapy with aminobisphosphonate-activated Vγ9Vδ2 T cells is feasible and well tolerated, but therapeutic benefits appear only likely when used in combination with other therapies.

Isolation and characterization of pokeweed mitogen-like phytomitogens from Shoriku, Phytolacca esculenta.

From saline extracts of Phytolacca esculenta (shoriku) roots, two phytomitogenes were isolated by salting out with (NH4),SO4 and chromatography on DEAE-cellulose and Sephadex G-100 columns. Both fractions were homogeneous on disc electrophoresis and on immunoelectrophoresis. One of these (Fraction E-2) was shown to be similar to pokeweed mitogen in respect to mol. wt (32,000) and amino acid composition. The other (Fraction E-3) was a protein of 18,000 mol. wt. Both fractions had similar biological activities to pokeweed mitogen in their ability to stimulate pig blood lymphocytes in vitro to incorporate tritiated thymidine, and to induce blastoid transformation. Both fractions contained an unusually large amount of cystine, i.e., 18 half-cystine residues % for Fraction E-2 and 22 residues % for Fraction E-3. Although these mitogens were resistant to deproteinizing procedures such as perchloric acid treatment and Sevag's procedure, the DNA synthesis-stimulating activity was inactivated by digestion with Pronase E and Nagarse, but resistant to trypsin, chymotrypsin, deoxyribonuclease, ribonuclease, lysozyme and neuraminidase. The activity was stable at acidic and neutral pH (4-7) but unstable at alkaline pH. The activity at pH 7.3 was stabilized by the addition of Ca2+ or Mg2+. On the addition of more than 2 mM of Ca2+, precipitation of mitogen occurred. From the above results the molecular basis of the mitogenic activity of shoriku mitogen is discussed.

In vivo biological behavior of a water-miscible fullerene: 14C labeling, absorption, distribution, excretion and acute toxicity.

BACKGROUND: Water-soluble fullerenes have recently been shown to exhibit considerable in vitro biological activity including cytotoxicity, site-selective DNA cleavage and inhibition of HIV protease. To assess the potential of these compounds as drugs, studies on the in vivo behavior of fullerenes are needed. We therefore set out to synthesize a radiolabeled, water-soluble fullerene, in order to obtain data on the oral absorption, distribution and excretion of this class of compounds. RESULTS: We synthesized a 14C-labeled water-soluble [60]fullerene using dipolar trimethylenemethane, which undergoes cycloaddition to [60]fullerene. When administered orally to rats, this compound was not efficiently absorbed and was excreted primarily in the feces. When injected intravenously, however, it was distributed rapidly to various tissues, and most of the material was retained in the body after one week. The compound was also able to penetrate the blood-brain barrier. Acute toxicity of the water-miscible fullerene was found to be quite low. CONCLUSIONS: Although the water-soluble fullerenes (and possibly their simple metabolites) are not acutely toxic, they are retained in the body for long periods, raising concerns about chronic toxic effects. The fact that fullerenes distribute rapidly to many tissues suggests that they may eventually be useful to deliver highly polar drugs through membranes to a target tissue, however, and they may even have applications in the delivery of drugs to the brain. Recent advances in fullerene synthetic chemistry may also make it possible to control fullerene absorption/excretion profiles in the future.

Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.

OBJECTIVE: To assess the in-vivo action on the renal microvasculature of the calcium antagonists nifedipine (L-type blocker), efonidipine (L/T-type blocker), and mibefradil (predominant T-type blocker). DESIGN: An intravital needle-type charge-coupled device (CCD) camera videomicroscope was introduced to visualize the renal microcirculation directly in vivo. METHODS: In anesthetized mongrel dogs, nifedipine (0.01-1 mg/kg per min), efonidipine (0.033-0.33 mg/kg per min), or mibefradil (0.01-1 mg/kg per min) was infused intravenously after the insertion of a CCD probe into the kidney. Renal microvascular responses to calcium antagonists were directly evaluated, with concomitant observation of renal clearance. RESULTS: Each calcium antagonist caused modest vasodepressor action without affecting heart rate. Nifedipine (1 mg/kg per min, n = 9) increased renal plasma flow (RPF) (14 +/- 4%, P < 0.05) and glomerular filtration rate (GFR) (19 +/- 5%, P < 0.05), and tended to increase the filtration fraction (5 +/- 2% increment, P = 0.07). Efonidipine (0.33 mg/kg per min, n = 9), however, had no effect on filtration fraction, with 14 +/- 6% increments in RPF (P < 0.05) and 14 +/- 7% increments in GFR (P = 0.08). Rather, mibefradil (1 mg/kg per min, n = 9) elicited 6 +/- 2% decreases in filtration fraction (P < 0.05), with slight increments in RPF (6 +/- 3%) and no changes in GFR. In direct in-vivo microvasculature observations, nifedipine caused predominant (22 +/- 2%) dilatation of afferent arterioles (from 15.5 +/- 0.4 to 18.9 +/- 0.4 microm, n = 5), compared with that of efferent arterioles (10 +/- 2%; from 11.0 +/- 0.4 to 12.1 +/- 0.3 microm). In contrast, efonidipine caused a similar magnitude of vasodilatation (16 +/- 4%) compared with 18 +/- 2%; n = 6), and mibefradil caused greater dilatation of efferent arterioles (20 +/- 4%, n = 7) than that of afferent arterioles (13 +/- 4%). CONCLUSIONS: There exists marked heterogeneity in action of nifedipine, efonidipine and mibefradil on the renal microvascular in canine kidneys in vivo. Furthermore, our current observations suggest an important contribution of T-type calcium channel activity to efferent arteriolar tone in vivo.

Retinoids as Ig Isotype-Switch Modulators

The role of retinoids was analyzed in directing isotype switching to IgA and IgG1 (IgE) by LPS-stimulated murine μ(+)B-cells in the presence of two Th2-type cytokines, IL-4 and IL-5. All trans retinoic acid (RA) enhanced the production of IgA at high concentrations (10-100 nM) in the presence of IL-5. Addition of IL-4 to the system modulated the IgA response in a dose-dependent manner. Namely, IL-4 inhibited the response at concentrations higher than 250 usolidusml, but showed slight enhancement at lower concentrations (130 usolidusml). IL-4 alone, which is considered to be an IgE isotype-switch inducer, strongly enhanced the IgG1 and IgE responses. Addition of IL-5 to the system showed a synergistic effect which could be attenuated by addition of low concentrations of RA (about 1 nM). Thus, the presence of switch modulators such as IL-4 and IL-5, their concentration ratios, and concentrations of retinoids are crucial factors in initiating and directing isotype switching to IgA and IgG1 (IgE).

Intramolecular 1,3-dipolar cycloaddition strategy for enantioselective synthesis of FR-900482 analogues.

[reaction: see text] Enantioselective synthesis of FR-900482 analogues is described. The key reaction of the synthesis is intramolecular 1,3-dipolar cycloaddition of a highly functionalized nitrile oxide with complete stereo- and regioselectivities to construct the eight-membered benzazocine ring.

Renal protective effect of troglitazone in Wistar fatty rats.

Although it is known that renal injury develops in obesity and diabetes mellitus, there have been no investigations examining the impact of insulin resistance per se on the development of renal injury. The present study was undertaken to examine whether insulin resistance and obesity influence urinary protein excretion (UPE) in female heminephrectomized Wistar fatty rats (WFRs). After 24 weeks of heminephrectomy in WFRs, the body weight ([BW], 465+/-18 g; n = 6), blood pressure (155+/-5 mm Hg), serum insulin to glucose ratio (1.31+/-0.39 microU/mg), and daily UPE (24+/-7 mg/d) were greater versus Wistar lean rats ([WLRs] 258+/-8 g, 134+/-1 mm Hg, 0.19+/-0.06 microU/mg, and 5+/-1 mg/d, respectively; n = 6), whereas blood glucose levels did not increase significantly. In WFRs, long-term (ie, 24 weeks) treatment with troglitazone, an insulin-sensitizing agent, improved the serum insulin to glucose ratio (0.17+/-0.09 microU/mg), reduced blood pressure (to 140+/-4 mm Hg), and decreased UPE (to 7+/-1 mg/d), although it had no effect on BW. Of note, with troglitazone treatment, the reduction in proteinuria preceded the correction of hypertension (ie, at week 12). In conclusion, our study suggests that insulin resistance per se causes proteinuria that does not appear to depend on blood pressure. Furthermore, long-term therapy with troglitazone may be a useful tool for the treatment of renal injury in the insulin-resistant condition.

Impaired nitric oxide-independent dilation of renal afferent arterioles in spontaneously hypertensive rats.

Sustained hypertension alters vasomotor regulation in various vascular beds. We studied whether nitric oxide (NO)-dependent and NO-independent vasodilator mechanisms are altered in renal microvessels in hypertension. To directly visualize the renal microcirculation, the isolated perfused hydronephrotic rat kidney model was used. After pretreatment with indomethacin (100 micromol/l), afferent arterioles were constricted by norepinephrine (NE) or by increasing renal arterial pressure (i.e., myogenic constriction; from 80 to 180 mmHg). Acetylcholine (ACH) was then added, and the renal microvascular response was assessed by computer-assisted video image analysis. A similar protocol was conducted in the presence of nitro-L-arginine methylester (L-NAME; 100 micromol/l). During NE constriction, ACH caused dose-dependent and sustained vasodilation of the afferent arteriole, similar in magnitude in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In the presence of L-NAME, ACH (0.01-1 micromol/l) elicited only transient dilation, and the degree of vasodilation was very low in SHR. During myogenic constriction, afferent arterioles from WKY and SHR kidneys responded to ACH with only transient vasodilation, which was unaffected by NO inhibition; the transient vasodilative responses elicited by ACH (0.1-1 micromol/l) were smaller in SHR than in WKY. In conclusion, ACH has both sustained and transient vasodilative effects on the afferent arteriole. Sustained vasodilation is attributed to NO generation, which is similar in WKY and SHR. In contrast, transient vasodilation, mediated by NO-independent vasodilator factors, is impaired in SHR. Deranged vasodilatory mechanisms in hypertension may disturb the renal microcirculation, which may result in renal injury.

Impact of calcium antagonists on bleeding time in patients with chronic renal failure.

Haemorrhagic diathesis develops in chronic renal failure, in which calcium antagonists are used widely as antihypertensive agents. Although calcium antagonists are reported to impair platelet function, it has not been examined whether calcium antagonists alter bleeding time. The present study was conducted to clarify whether calcium antagonists affect bleeding time in chronic renal failure. Patients with chronic renal failure without and with calcium antagonists were enrolled (n = 156), and bleeding time (Ivy's method) as well as blood parameters (BUN, creatinine, platelet counts, and haemoglobin) were compared in patients with normal and prolonged bleeding time. Among patients not taking calcium antagonists (n = 34), three cases manifested prolonged bleeding time, whereas abnormal bleeding time was observed in 31 patients out of 122. Positive correlations were observed between bleeding time and BUN in both calcium antagonist-untreated (r = 0.46) and -treated groups (r = 0.25). The odds ratio for prolongation of bleeding time in patients taking calcium antagonists was 3.52 (95% CI, 1.01-12.33). In 12 calcium antagonist-treated patients with prolonged bleeding time, the withdrawal of calcium antagonists markedly shortened bleeding time (from 11.3 +/- 0.8 to 5.4 +/- 0.8 min, P < 0.05, n = 12). In contrast, in the additional group (n = 9), the continued treatment with calcium antagonists had no effect on bleeding time (from 11.7 +/- 0.9 to 10.0 +/- 1.0 min). Despite the inhibitory effect of calcium antagonists on bleeding time, no clinically serious events associated with haemorrhagic diathesis developed. In conclusion, calcium antagonists prolong bleeding time in patients with chronic renal failure. The subclinical (laboratory) effect of calcium antagonists however is not necessarily associated with haemorrhagic events of clinical significance.

Synthesis and biological activities of cyclopropenone antibiotic penitricin and congeners.

A number of derivatives of the cyclopropenone antibiotic penitricin have been synthesized by the reaction of metalated cyclopropenone acetals with electrophiles. Studies on the antimicrobial structure-activity relationships indicated that the penitricin skeleton, hydroxymethylcyclopropenone, is indispensable for antimicrobial activity. These compounds were also found to display cytotoxic activity.

Mouse monoclonal antibodies with restricted specificity for human renal cell carcinoma and ability to modulate the tumor cell growth in vitro.

Three mouse monoclonal antibodies (MAbs), KRC-1, -2 and -3(IgG1) were produced against cultured cells of human renal cell carcinoma (RCC) cell lines. All of them had restricted specificity for subset of RCC, but did not react with other tumor cell lines, tumor and normal tissues including kidney. The effect of these MAbs on the tumor cell growth in vitro was examined both in soft agar and in plate culture systems. All of them showed modulator effect on the tumor cell growth: KRC-1 showed stimulation effect and others, KRC-2 and -3, suppression one in the both systems. The specificity was observed in the modulator effects. KRC-1 detected a 135 kd glycoprotein in immunoblotting. KRC-2 detected a high molecular weight antigen (ca. 1200 kd) in gel filtration. All the antigens showed affinity for Con A lectin. Molecular properties of the antigens appeared to be different from those of previously reported RCC-associated antigens. The three MAbs seem to be useful to study mechanism of growth regulation in transformed cells.

Spontaneous fractures of the hip in the elderly.

The authors found 41 patients with 42 fractures of the hip who had no distinct history of trauma in the total of 1,449 hip fractures treated. They were all women ranging in age from 64 to 91 years. Most of the fractures were of intracapsular type. Three extracapsular fractures, however, were found. Fractures were randomly selected with clear history of trauma, which were matched to the patients with no trauma for age, sex, and type of fracture as a control group. The grades of osteoporosis of the femoral neck, estimated with the Singh index, and of the spine, estimated with the spinal score, were noted statistically significantly higher in the patients than those in the control group. Most of spontaneous fractures of the hip in the elderly are considered similar phenomena to the compression fractures of the dorsolumbar spine.

[Treatment of complicated urinary tract infections caused by fungi].

The predisposing factors of funguria and the clinical efficacy of treatment with ketoconazole, a new oral imidazole antifungal drug were studied in 10 patients with complicated urinary tract infection due to fungi. Preceding antibiotic therapy, presence of indwelling catheter and malignant tumor were regarded as the important factors that predisposed to the fungal urinary tract infections. Patients received 200 mg of ketoconazole once a day for 5 to 14 days. Of the 9 patients in whom clinical efficacy of ketoconazole was evaluated, excellent response was obtained in 4 patients, moderate in 2 and poor in 3 patients. Of the 9 strains of fungi isolated before treatment, 6 strains or 67% were eradicated after treatment. Neither subjective nor objective side effects were observed in any of 10 patients receiving ketoconazole, and laboratory tests showed elevated BUN and serum creatinine in 1 patient. From the results obtained in this study, ketoconazole was regarded as useful in the treatment of complicated urinary tract infection due to fungi.

[A case of a scrotal hemo-lymphangioma].

A nineteen-year-old man with a painless tumor in the right scrotal area was seen at our Department. The scrotal mass had been noticed from his childhood. The tumor was 8.2 X 5.2 X 5.0 cm weighed 47.3 g. Histological examination revealed hemo-lymphangioma.

[Spontaneous peripelvic extravasation: reports of two cases].

Spontaneous peripelvic extravasation must be distinguished from spontaneous rupture of the renal pelvis in urological emergency. The literatures revealed 42 cases of peripelvic extravasation and 35 cases of rupture of the renal pelvis in Japan. Most of them were caused by urolithiasis and malignant tumors. We report 2 cases of spontaneous peripelvic extravasation caused by urolithiasis, which were successfully treated conservatively.

[A case of renal cell carcinoma suspected of multilocular cystic nephroma].

A 52-year-old, healthy looking woman was referred for further evaluation of left renal mass, She had had a history of hematuria for ten years. Admission laboratory studies were within normal limits except for slight elevation of white blood cell count, c-reactive protein, blood sedimentation rate and immunosuppressive acidic protein. Retrograde pyelography demonstrated a large mass with ringed calcification in the middle lobe of the left kidney. The multiloculated mass was also confirmed by computed tomography and ultrasonography. Selective left renal arteriography showed stretched arteries and irregularity and tortuosity of the smaller vessels. Under a presumptive diagnosis of multilocular cystic nephroma or renal cell carcinoma, left radical nephrectomy was performed. In surgical specimen, many lobules were filled with serous fluid and clotted blood. Microscopic examination revealed that the lining of the cyst wall consisted of renal cell carcinoma cell. At present, 5 months after the operation, she is well without any signs or symptoms of recurrence.

[Malignant melanoma of male urethra: a case report].

A case of male urethral melanoma is reported. A 85-year-old male with a 2-month history of progressive, severe obstructive urinary symptoms and bloody urethral discharge was referred to us after an unsuccessful management at a local doctor. Physical examination revealed an ill looking old man with no evidence of nevi or other cutaneous pigmentation looking like malignant melanoma. Neither palpable periurethral mass nor inguinal lymphadenopathy was noted. RUG showed an irregular shadow defect in bulbous urethral regions. In cystourethroscopy, a raised nodular reddish black lesion in the urethra without adjacent satellite lesions was found. Histologic examination revealed that the tumor was made up of closely spaced, anaplastic, spheroidal or polyhedal cells. Intracellular brown pigment was richly present, gave a negative reaction for iron, but stained black with Masson-Fontana's method. Further examination for evaluating metastases including bone scintigraphy, computer tomographic scan, chest X-ray film were negative. Due to his poor risk, radical operation such as cystourethrectomy might be undesirable. We performed TUR to relieve urethral obstruction, because the patient refused cystostomy. He died of wide spread metastases at 6 months after the operation. This case seems to be the second report in the Japanese literature.

[Clinical study of male sterility patients].

We report the results of a clinical study of the male sterility in our institute during the past 55 months. A total of 187 male patients consulted us with the complaint of sterility, and the semen of 173 patients was examined, sixty two cases were diagnosed as azoospermia, 34 as severe oligozoospermia, 17 as moderate oligozoospermia and 60 cases as normospermia. Based upon the results of semen examination, endocrinological examination and so on, 78 cases were diagnosed as idiopathic spermatogenetic dysfunction. Serum follicle stimulating hormone (FSH) levels in azoospermia patients were significantly higher than those of normal males statistically, and the serum FSH level was considered to correlate with the damage of spermatogenesis. We performed serial treatment using kallikrein and/or human chorionic gonadotropin, on 44 patients. Only patient, who was treated with a high dose kallikrein, succeeded in impregnating his spouse. Four of our patients with oligozoospermia had received chemotherapy for testicular cancer. Recently, with the progress of anticancer chemotherapy, curative cases have increased gradually. However, because of adverse reaction, oligozoospermia cases have also increased. Considering the quality of life, the management of the sterility may be very important.

[Treatment of suture-thread stones in the kidney by percutaneous nephrolithotripsy. Report of a case].

Percutaneous nephrolithotripsy was performed successfully to the stones in the left kidney, in which pyelolithotomy had been done three years earlier. There were foreign body stones with silk sutures used in the previous surgery as the nuclei. All the stones were removed, and a stricture in the renal pelvis was liberated endoscopically. No reports of endoscopic management to foreign body stones in a kidney are found in the Japanese literature. A few ways of the treatment are discussed.