Takaaki Oteki

Association of ecNOS gene polymorphisms with end stage renal diseases

Nitric oxide (NO) is a very potent regulator of intrarenal hemodynamics and is thought to be an important factor in the deterioration of renal function. Several polymorphisms of the endothelial NO synthase (eNOS) gene have been reported. For instance, tandem 27-bp repeats in intron 4 of the eNOS gene are polymorphic, i.e. eNOS4a allele has 4 and eNOS4b has 5 tandem repeats, and the association between eNOS4a and myocardial infarction has been reported. In addition, a missense Glu298Asp mutation in exon 7 of the eNOS gene is reported to be a risk factor for hypertension or myocardial infarction. In this study, we investigated the frequencies of these 2 polymorphisms of eNOS gene in patients with end-stage renal diseases (ESRD), and compared them with those of healthy subjects.Genomic DNA was obtained from regularly hemodialyzed patients and healthy volunteers. The allele frequencies of eNOS4a and eNOS4b in intron 4 were analyzed by PCR and the missense Glu298Asp mutation in exon 7 were determined by PCR FMLP analysis.The allele frequency of eNOS4a (eNOS4a/b and eNOS4a/a) in non-diabetic group is significantly higher than that in healthy controls (27.3% vs. 19.0%, p = 0.01) though there is no significant difference between diabetic group and healthy controls. On the other hand, the frequencies of missense Glu298Asp mutation in both non-diabetic and diabetic groups are significantly higher than that in healthy controls (22.5% in non-diabetic, 20.8% in diabetic and 7.4% in control group, p = 0.002: non-diabetic vs. control, p = 0.01: diabetic vs. control).This study clarified that the polymorphisms in intron 4 and exon 7 of eNOS gene are the genetic risk factors for ESRD. The polymorphisms in intron may change the transcriptional activity and those in exon may alter the 3 dimensional structure of the enzyme, and may affect the progression of renal diseases via decreased NO synthesis. Further study is required to clarify the detailed mechanisms.

Tubulointerstitial nephritis and uveitis syndrome associated with hyperthyroidism

We report a 17-year-old male patient with tubulointerstitial nephritis and uveitis (TINU) associated with hyperthyroidism. He presented with a 2-month history of fatigue, loss of appetite, low-grade fever, and a 12-kg weight loss when he was admitted to our hospital. He had iritis, which was complicated by fibrin in the anterior chamber, diagnosed by slit-lamp examination. On laboratory examinations, deteriorated renal function (blood urea nitrogen level was 25.9 mg/dl and creatinine level was 2.82 mg/dl) and elevated urinary levels of N-acetyl-β-D-glucosaminidase (33.1 U/l) and β2-microglobulin (78 600 µg/l) were observed. Serum thyroid-stimulating hormone (TSH) was undetectable, at less than 0.01 µIU/ml, and free triiodothyronine and free thyroxine were elevated, up to 5.23 pg/ml and 2.85 ng/dl, respectively. The titers of antithyroglobulin and antithyroid microsomal and TSH-receptor antibodies were not elevated. Abdominal and thyroidal ultrasonography showed evident bilateral enlargement of the kidneys and diffuse enlargement of the thyroid gland. Iodine-123 scintigraphy showed low uptake in the thyroid gland. The biopsied renal specimen showed mild edema and severe diffuse infiltration of mononuclear cells and few eosinophils in the interstitium, without any glomerular or vascular abnormalities. Based on the clinical features and pathological findings, a diagnosis of TINU syndrome with associated hyperthyroidism was made. Treatment was started with 30 mg/day of prednisolone. The iritis disappeared, and the patients clinical status improved remarkably. This case suggests the possibility of thyroid dysfunction in some patients with TINU syndrome, and we believe thyroid function should be measured in all TINU patients. Moreover, histopathological diagnosis of the thyroid glands before treatment is necessary for TINU patients with thyroid dysfunction.

Nutcracker syndrome associated with severe anemia and mild proteinuria

A 70-year-old man was referred to our hospital with the chief complaint of gross hematuria. Urinalysis revealed gross hematuria (3+, RBC 100/HPF or more) and mild proteinuria (3+, 1.8 g/day) with no urinary casts. Computed tomography of the abdomen showed compression of the left renal vein between the superior mesenteric artery and the aorta. Ultrasonography showed an increased flow velocity at the stenotic portion of the left renal vein. An aortography and selective left renal arteriography showed that there was no evidence of tumor vessels or arterial abnormalities in the arterial phase. However, the venous phase revealed a stenosis of the left renal vein just lateral to the aorta as well as a reflux of contrast material toward the left gonadal vein which was dilated. In addition, cystoscopy revealed left ureteral bleeding. Based on these findings, we made the diagnosis of gross hematuria caused by nutcracker syndrome (NCS). We concluded that the main cause of the anemia and proteinuria in our patient was leakage of blood and this is confirmed by the relationship of red blood cells to protein in the urine because we proved whole blood and plasma protein loss in the urine by calculation. Fourteen months after discharge, both the gross hematuria and proteinuria spontaneously disappeared. This case strongly suggested that the first therapy for hematuria and proteinuria with NCS should be observation.

Evaluation of the Effect of Dietary Lycopene, the Main Carotenoid in Tomato (Lycopersicon esculentum), on the In Vivo Renal Reducing Ability by a Radiofrequency Electron Paramagnetic Resonance Method

Although it has been reported that dietary lycopene, the main carotenoid in tomato, improved drug-induced nephropathy, there are no reports on the effect of orally administered lycopene on the in vivo renal reducing (i.e., antioxidant) ability. The radiofrequency electron paramagnetic resonance (EPR) method is a unique technique by which the in vivo reducing ability of an experimental animal can be studied. In this study, the in vivo changes in the renal reducing ability of rats orally administered lycopene were investigated using a 700 MHz EPR spectrometer equipped with a surface-coil-type resonator. Rats were fed either a control diet or a diet containing lycopene. After 2 weeks, in vivo EPR measurements were conducted. The renal reducing ability of lycopene-treated rats was significantly greater than that of the control. This is the first verification of in vivo antioxidant enhancement via dietary lycopene administration.

In Vivo Imaging of Renal Redox Status during Azelnidipine Treatment

The effect of the calcium channel blocker azelnidipine on the redox status of a murine hypertension model was analyzed and imaged using in vivo low frequency electron paramagnetic resonance (EPR). A murine two kidney–one clip (2K1C) hypertension model was produced by a clipping of the right renal artery. The resulting hypertensive mice were treated with low-dose azelnidipine (1 mg/kg/d), with high-dose azelnidipine (3 mg/kg/d) or without azelnidipine (HT group). An EPR system equipped with a loop-gap resonator and an imaging system was employed. Redox status was evaluated as organ reducing activity measured by means of the decay rate (half-lives) of the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl- PROXYL). Four weeks after clipping the mice demonstrated hypertension as expected. After the additional 2 weeks of azelnidipine treatments, the Carbamoyl-PROXYL half-lives of the Low and High azelnidipine groups measured in the upper abdominal area were significantly shorter than those of the HT group, suggesting improvements in the reducing activity. The blood pressures of the three groups showed no significant differences at this time, and there was no correlation between the renal reducing activity and either blood pressure or serum creatinine values. EPR imaging studies revealed that the improvement in abdominal reducing activity was mainly recognized in the kidney but not in the liver. These results indicate that azelnidipine ameliorates renal redox status through an improvement in reducing activity independent of blood pressure control.

Estimation of the in vivo decay rate of EPR signals for a nitroxide radical in rat brains by a region-selected intensity determination method

The region-selected intensity determination (RSID) method was proposed to obtain the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region by a stationary magnetic field gradient. To select the region, the subtraction field that was derived from the distance between the center and the projection of the selected region to the direction of the field gradient was applied to the main field. The directions of the stationary magnetic field gradient at a constant strength were systematically changed in a three-dimensional space after each acquisition of the spectrum. All spectra under the field gradient were accumulated and the resultant spectrum was deconvoluted by a spectrum without the field gradient. The center height of the deconvoluted spectrum indicates the signal intensity of the selected region. To verify this method, a phantom or in vivo study was conducted on a 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator. In the temporal EPR measurements of phantoms including a nitroxide radical aqueous solution with and without ascorbic acid, the selected regions were alternatively changed at the position of the two phantoms. The signal intensity derived from the one phantom showed an exponential decay, and for the other phantom, no temporal changes. The spatial resolution of this method was estimated to be 2.7 mm by using a pinpoint phantom that included diphenylpicrylhydrazyl powder. In the in vivo temporal EPR measurements, the selected regions were alternatively changed at the cerebral cortex and the striatum of rats that had received a blood-brain barrier-permeative nitroxide radical. The decay rate of the signal intensity at each region obtained by this method was consistent with those previously reported.

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis.

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.

Nitric oxide protection against adriamycin-induced tubulointerstitial injury.

It is well known that oxidative stress is related to the pathogenesis of adriamycin (ADR) nephropathy. However, it is unclear how nitric oxide (NO) is associated with the pathophysiological process after ADR administration. The NO level in a kidney homogenate was assayed by electron paramagnetic resonance (EPR) spectrometry using a direct in vivo NO trapping technique after ADR administration. N-(3-(aminomethyl)benzyl)acetamidine (1400W) was used as a specific, inducible nitric oxide synthase (iNOS) inhibitor. The levels of NO after ADR administration gradually increased for 6 h and then decreased until 24 h after ADR administration. The fractional excretion of Na (FENa) in the urine was elevated in the ADR group on day 1. Pre-treatment of the animals with 1400W attenuated the increase in NO levels despite further elevation of FENa. These findings suggest that iNOS-derived NO does not produce a harmful effect but rather protects the ADR-treated kidney against sodium excretion.

Electron paramagnetic resonance imaging of nitric oxide organ distribution in lipopolysuccaride treated mice

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)2, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)2 complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)2 complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)2 spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)2 distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.