Naohito Ishii

Blunted Pressure Natriuresis in Ovariectomized Dahl-Iwai Salt-Sensitive Rats

Our objective was to determine whether increased salt sensitivity after menopause precedes the development of overt hypertension. We investigated the effect of ovariectomy on pressure natriuresis in Dahl-Iwai salt-sensitive (DS) and salt-resistant (DR) rats by in vivo perfusion studies. Differences in the neural and hormonal backgrounds of the kidney were minimized by renal denervation and by holding plasma vasopressin, aldosterone, corticosterone, and norepinephrine levels constant by intravenous infusion. The pressure-natriuresis relationship was blunted in DS rats compared with DR rats (slope, 0.30 versus 0.63 mumol.min-1.g kidney wt-1.mm Hg-1, P < .01). The impaired pressure-natriuresis response of DS rats was further blunted by ovariectomy (from 0.30 to 0.14 mumol.min-1.g kidney wt-1.mm Hg-1, P < .05), and that of DR rats was not. The ovariectomized DS rats developed hypertension earlier than sham-operated DS rats by salt loading. These results show that ovariectomy enhances genetic salt sensitivity by blunting the pressure-natriuresis relationship, which precedes the development of overt hypertension in female DS rats.

Elucidating mechanisms underlying altered renal autoregulation in diabetes

Previous studies have reported that high-salt intake paradoxically activates tubuloglomerular feedback (TGF) in type 1 diabetes. Using Zucker lean (ZL) and diabetic fatty (ZDF) rats on normal and high-salt diets, renal hemodynamics and the renin-angiotensin system (RAS) were characterized. On normal salt diet, glomerular filtration rate (GFR) was higher in ZDF than ZL rats. Autoregulation of GFR was less efficient and lithium clearance was lower in ZDF rats than ZL rats. Salt load reduced GFR in ZDF rats with restoration of lithium clearance and partial improvement in autoregulatory index (AI). The administration of 8-cyclopentyl-1,3-dipropylxanthine, a selective adenosine-1 receptor antagonist to ZDF rats on a high-salt diet abolished the improvement of AI in GFR. However, this effect was seen by neither (Cx40)GAP27 nor (Cx37,43)GAP27, which inhibits connexin (Cx) 40 or Cx37. Renal ANG II was higher in ZDF than ZL rats on normal salt diet, but the difference was eliminated by a salt load. The present data provide the first demonstration for a salt paradox in type 2 diabetes and implicate that in addition to Cx alterations, an enhanced proximal reabsorption attenuates TGF, underlying glomerular hyperfiltration and RAS activation. These data suggest that a high-salt diet standardizes distal delivery in diabetes, suppressing the RAS, and improving GFR autoregulation and hyperfiltration through adenosine.

Calcitriol supplementation improves endothelium-dependent vasodilation in rat hypertensive renal injury.

Background/Aims: Vitamin D increases renal expression of klotho in normotensive rats. Klotho reduces oxidative stress. Methods: In this study, we aimed to determine if vitamin D would suppress oxidative stress using 4 groups of hypertensive rats: uninephrectomized, stroke-prone, spontaneously hypertensive rats fed a high-salt (6%) diet (controls; C); those treated with irbesartan (I); those treated with calcitriol (V); and those treated with both irbesartan and calcitriol (I+V). Results: Systolic blood pressure was higher in the C group than in the I and I+V groups. Albuminuria was attenuated in groups I, V, and I+V. Renal angiotensin II (AngII) concentration was lower in groups I and I+V than in group C, and plasma AngII levels of groups I and V were higher and lower than those in group C, respectively. Compared with group C, renal klotho expression, 8-epi-prostaglandin F2α excretion, and acetylcholine-induced decrease in blood pressure improved in the V and I+V groups. Conclusions: The data indicate that irbesartan effectively decreases blood pressure and renal AngII levels, and improves albuminuria. Our findings indicate that vitamin D enhances klotho expression, suppressing oxidative stress and albuminuria without substantial changes in renal AngII levels. These results suggest that the amelioration of endothelium function by vitamin D involves free klotho.

Angiotensin-converting enzyme inhibition curbs tyrosine nitration of mitochondrial proteins in the renal cortex during the early stage of diabetes mellitus in rats

Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O2− (superoxide anion) and NOx (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O2− and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NOx production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

Comparison of the Antioxidant Activity of Albumin from Various Animal Species

Abstract We measured the antioxidant activity of human, rat, bovine, rabbit, and guinea pig albumins against the superoxide, hydroxyl, and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals. The albumins of different animal species did not differ in antioxidant activity against superoxide. Human and rat albumins exhibited antioxidant activity against hydroxyl radicals, but bovine, rabbit, and guinea pig albumins showed weaker antioxidant activity than human and rat albumins. Human, rat, rabbit, and guinea pig albumins, but not bovine albumin, exhibited strong antioxidant activity against DPPH radicals. Human and rat albumins with strong antioxidant activity against hydroxyl radicals contained methionine-123 in domain 1, but bovine, rabbit, and guinea pig albumins did not. Rat, rabbit, and guinea pig albumins with strong antioxidant activity against DPPH radicals had methionine-264 in domain 2. Human albumin did not have methionine-264, but methionine-298 and methionine-329 in domain 2. Bovine albumin, with the weakest antioxidant activity against DPPH radicals, contained no methionine residues in domain 2. These results suggest that methionine residues in domain 1 or 2 influence the antioxidant activity of albumin.

DOES COMBINED THERAPY OF Ca-CHANNEL BLOCKER AND ANGIOTENSIN CONVERTING ENZYME INHIBITOR EXCEED MONOTHERAPY IN RENAL PROTECTION AGAINST HYPERTENSIVE INJURY IN RATS ?

Either calcium channel blocker (CCB) or angiotensin converting enzyme inhibitor (ACEi) is used as an antihypertensive agent, and we are recommended to use them in combination to refractory hypertension with evidence dependent on clinical observations. We examined the renal protective effect of the combined therapy with calcium channel blocker (amlodipine) and angiotensin converting enzyme inhibitor (enalapril) against hypertensive renal injury in 5/6 nephrectomized spontaneously hypertensive rats (SHRs) with salt loading, comparing with monotherapy of each drug. Forty males SHRs with 5/6 nephrectomy and salt loading were divided to five groups: group 1 as control (n = 8), group 2 received 0.2 mg/kg/day of amlodipine (n = 8), group 3 received 0.2 mg/kg/day of enalapril (n = 8), group 4 (n = 8) and group 5 (n = 8) that were treated with 0.1 mg/kg/day and 0.2 mg/kg/day of each drug in combination respectively. Either amlodipine or enalapril had remarkable effects on reducing the increases in blood pressure and urinary protein excretion. In histopathological examination, it also suppressed renal injury significantly. Additional significant effect of combined therapy was not observed in blood pressure and urinary protein. There were not remarkable, additional effects of the combination of CCB and ACEi on protecting the remnant kidney in 5/6 nephrectomized SHRs fed a high-salt diet, possibly because sodium retention was not alleviated by the combination.

Klotho suppresses the renin-angiotensin system in adriamycin nephropathy

Backgrounds Klotho protein interacts with the transforming growth factor β (TGF-β) receptor and Wnt, which contribute to the progression of renal disease, inhibiting their signals. Renal and circulating klotho levels are diminished in chronic kidney disease. Methods Experiments were performed to assess whether supplementation of klotho protein could have protective effects on the kidney. Rats were injected with adriamycin (5 mg/kg) and divided into three groups: those treated with vehicle, those treated with klotho protein and those treated with klotho plus 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD). Rats without adriamycin treatment were used as a control. Results Adriamycin reduced the serum klotho concentration and renal expression of klotho and E-cadherin. Adriamycin also increased the renal expression of Wnt, TGF-β, and angiotensinogen, as well as the renal abundance of β-catenin and angiotensin II. Klotho supplementation suppressed adriamycin-induced elevations of β-catenin and angiotensin II with sustained Wnt expression. Combined treatment with klotho and TDZD reversed the klotho-induced improvements in the renal abundance of β-catenin and angiotensin II as well as the expression of TGF-β and angiotensinogen without affecting E-cadherin. Conclusions Our data indicate that Wnt is involved in the pathogenesis of adriamycin nephropathy. Furthermore, klotho supplementation inhibited Wnt signaling, ameliorating renal angiotensin II. Finally, klotho protein appears to suppress epithelial-mesenchymal transition by inhibiting TGF-β and Wnt signaling.

Aminophylline increases respiratory muscle activity during hypercapnia in humans

BACKGROUND Theophylline is an old drug traditionally used as a bronchodilator, although it was recently shown to possess anti-inflammatory properties, enhance the actions of corticosteroid actions, and stimulate the respiratory neuronal network. Theophylline has been recognized as an important drug for not only asthma but also corticosteroid-insensitive chronic obstructive pulmonary disease (COPD). To clarify the role of theophylline in hypercapnic ventilatory responses in humans, we analyzed the effects of aminophylline administered at the usual clinical therapeutic doses on ventilation and augmentation of respiratory muscle contractility in room air and under 3 conditions of hypercapnia. STUDY DESIGN We performed electromyography (EMG) of the parasternal intercostal muscle (PARA) and transversus abdominis muscle (TA) in 7 healthy subjects and recorded both ventilatory parameters and EMG data in room air and under 3 conditions of hypercapnia before (control) and during aminophylline administration. RESULTS Before aminophylline administration (control), hypercapnic stimulation elicited ventilatory augmentation in a hypercapnia intensity-dependent manner. Ventilatory parameters (tidal volume, frequency of respiration, and minute ventilation) showed significant increases from lower PaCO2 levels during aminophylline administration when compared with the corresponding values before aminophylline administration. EMG activity of both PARA and TA increased significantly at each level of hypercapnia, and those augmentations were shown from lower PaCO2 levels during aminophylline administration. CONCLUSION Aminophylline administered at the usual clinical therapeutic dose increases ventilation and EMG activity of both inspiratory and expiratory muscles during hypercapnia in healthy humans.

Appearance of Apoptotic Cells and Granular Cells in the Silkworm Midgut Lumen During Larval-Pupal Ecdysis

Abstract To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.

Altered Renal Microvascular Function in Early Diabetes

The early stage of type 1 diabetes (T1D) is characterized by glomerular hyperfiltration that arises as the result of preglomerular (primarily afferent arteriolar) vasodilation. Although hyperglycemia is the trigger for this process, the mechanism linking hyperglycemia to reduced afferent arteriolar tone remains an area of active debate. It is well established that diabetic hyperglycemia provokes a condition of oxidative stress in many organs including the kidney. In this chapter, we consider the possible role of oxidative stress in producing a defect in afferent arteriolar electromechanical coupling that involves K+ channel activation, membrane hyperpolarization, and a consequent decrease in Ca2+ influx through voltage-gated channels. As voltage-dependent Ca2+ influx is a primary determinant of afferent (but not efferent) arteriolar tone and vasoconstrictor responsiveness, this scenario offers a potential mechanism whereby hyperglycemia results in the preglomerular vasodilation that underlies diabetic hyperfiltration.