Takayoshi Yamauchi

Effects of chronic inhibition of ACE and AT1 receptors on glomerular injury in Dahl salt-sensitive rats

To elucidate the contribution of the renin-angiontensin system (RAS) to glomerular injury in salt-sensitive hypertension, we investigated the chronic effects of the angiotensin I-converting enzyme inhibitor cilazapril and the angiotensin II type 1-receptor antagonist (AT1a) TCV-116 in Dahl-Iwai rats. Dahl salt-sensitive (S) rats receiving 8% salt diet for 6 wk were simultaneously treated with cilazapril ( n = 6), TCV-116 ( n = 6), or saline ( n = 14). The 8% salt diet markedly increased systolic blood pressure (SBP), urinary protein, and N-acetyl-β-glucosaminidase (NAG) excretion compared with 0.3% salt-treated S ( n = 6) or salt-resistant ( n = 6) rats. Although neither cilazapril nor TCV-116 reduced the elevated SBP, TCV-116 significantly lowered urinary protein and NAG excretion. Histologically, 8% salt treatment in S rats induced progressive sclerotic and proliferative glomerular changes, which were ameliorated by both drugs. TCV-116 increased the glomerular diameter. Immunofluorescence demonstrated the increased level of type III collagen in the mesangium of 8% salt-treated S rats, which was completely reversed by TCV-116. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that 8% salt treatment significantly increased the levels of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B-chain and that TCV-116 significantly reduced the levels of PCNA and transforming growth factor-β1 (TGF-β1). Thus, although the chronic RAS-inhibition in salt-sensitive hypertension exerted a histologically renoprotective effect by both ways without lowering blood pressure, the RAS inhibition due to AT1a had more beneficial advantages of reducing proteinuria and attenuating the levels of glomerular TGF-β1 and extracellular matrix.To elucidate the contribution of the renin-angiontensin system (RAS) to glomerular injury in salt-sensitive hypertension, we investigated the chronic effects of the angiotensin I-converting enzyme inhibitor cilazapril and the angiotensin II type 1-receptor antagonist (AT1a) TCV-116 in Dahl-Iwai rats. Dahl salt-sensitive (S) rats receiving 8% salt diet for 6 wk were simultaneously treated with cilazapril (n = 6), TCV-116 (n = 6), or saline (n = 14). The 8% salt diet markedly increased systolic blood pressure (SBP), urinary protein, and N-acetyl-beta-glucosaminidase (NAG) excretion compared with 0.3% salt-treated S (n = 6) or salt-resistant (n = 6) rats. Although neither cilazapril nor TCV-116 reduced the elevated SBP, TCV-116 significantly lowered urinary protein and NAG excretion. Histologically, 8% salt treatment in S rats induced progressive sclerotic and proliferative glomerular changes, which were ameliorated by both drugs. TCV-116 increased the glomerular diameter. Immunofluorescence demonstrated the increased level of type III collagen in the mesangium of 8% salt-treated S rats, which was completely reversed by TCV-116. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that 8% salt treatment significantly increased the levels of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor B-chain and that TCV-116 significantly reduced the levels of PCNA and transforming growth factor-beta1 (TGF-beta1). Thus, although the chronic RAS-inhibition in salt-sensitive hypertension exerted a histologically renoprotective effect by both ways without lowering blood pressure, the RAS inhibition due to AT1a had more beneficial advantages of reducing proteinuria and attenuating the levels of glomerular TGF-beta1 and extracellular matrix.

The role of nitric oxide and the renin-angiotensin system in salt-restricted Dahl rats

To elucidate the role of nitric oxide (NO) and renin-angiotensin system (RAS) in the development of salt-sensitive hypertension, we investigated the pressor responses and renal histologic changes after long-term inhibition of endogenous NO synthesis in Dahl-Iwai salt-sensitive (DS) and salt-resistant (DR) rats under salt-re-stricted conditions that exaggerate RAS activation. Male DS and DR rats (6 weeks old) were fed with a low-salt (0.3%) diet for 5 weeks. NG-nitro-L-arginine (L-NA; dissolved in 60 mg/L deionized water), an arginine analogue acting as a NO-inhibitor, was also administered for 5 weeks. L-NA administration induced a gradual increase in systolic blood pressure (SBP) in both strains, and the pressor response in DS rats was apparently more enhanced relative to that in DR rats. Urinary nitrate plus nitrite (u-NOx) excretion was decreased by L-NA, with a significant negative correlation between SBP and u-NOx excretion in DS rats but not in DR rats. Plasma renin activity and urinary aldosterone level were significantly increased in L-NA-treated DS rats on week 5. Marked histologic changes with glomerular sclerosis and increased proteinuria and urinary N-acetyl-beta-glucosaminidase excretion were found in L-NA-treated DS rats but not DR rats. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that angiotensin II type 1 receptor (AT1R) mRNA level was significantly lower in DS rats than in DR rats at week 2, and that L-NA administration significantly reduced glomerular AT1R level of DS rats at week 5, possibly because of downregulation. Our results showed that, even under sodium restriction, the pressor response and renal injury induced by chronic NO inhibition were markedly more enhanced in DS rats than in DR rats, which indicates that depletion of NO participates in both the development of hypertension and glomerular injury in DS rats through a potential activation of RAS irrespective of sodium loading. These data suggest that endogenous NO is an essential determinant of salt-sensitive hypertension in DS rats.

Quantitative analysis of growth-related factors in human pituitary adenomas: Lowered insulin-like growth factor-I and its receptor mRNA in growth hormone-producing adenomas

To elucidate the contribution of growth factors to the development, growth and behavior of human pituitary adenomas, the authors used competitive reverse transcription-polymerase chain reactions to quantify expression of mRNAs for growth factors extracted from pituitary adenomas. As previously diagnosed by endocrinologic evaluation, the pituitary adenomas in this study consisted of six prolactin-producing, six growth hormone (GH)-producing, four follicle-stimulating hormone producing and six nonfunctioning adenomas. The mRNAs examined included those for platelet-derived growth factor (PDGF) B-chain, transforming growth factor (TGF)-beta1, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF)-I and -II; proliferating cell nuclear antigen (PCNA) as an indicator of cell proliferation; and pituitary-specific transcription factor-1 (Pit-1) which is a nuclear transcription factor expressed in the anterior pituitary. All factors except the last were expressed in all adenomas, and expression of PDGF B-chain, TGF-beta1, EGF, bFGF and IGF-II did not differ between the four adenoma varieties. Pit-1 was expressed only in GH- and prolactin-producing adenomas. PCNA expression also showed no differences. However, IGF-I mRNA in GH-producing adenomas was significantly lower than in prolactin-producing and nonfunctioning adenomas despite high serum IGF-I levels (1121+/-253 ng/ml). The analysis on IGF-I receptor mRNA was significantly lowered in GH-producing adenoma compared with the other types of adenoma. These findings suggest that the attenuation of negative feedback through the pituitary GH-IGF-I axis may be involved in development of GH-producing adenoma.

In vitro Macro-and Microautoradiographic Localization of V1 and V2 Receptors in the Rat Kidney Using OPC-21268 and OPC-31260

To elucidate the precise localization of vasopressin (VP) V1 and V2 receptors in the kidney, we utilized in vitro macroautoradiography (macro-ARG) and microautoradiography (micro-ARG) of these receptors in Wistar rat kidneys. This was done by using OPC-21268 and OPC-31260, two newly developed selective V1 (OPC-21268) and V2 (OPC-31260) receptor antagonists. For macro-ARG, 10-microm kidney sections were incubated with Tris-HCl buffer containing [3H]-VP with or without unlabeled ligand (VP, OPC-21268, or OPC-31260) at 20 degrees C for 40 min. These sections were then loaded into X-ray cassettes with Hyperfilm-[3H] and exposed in the dark for 2 months. The autoradiograms were quantitatively analyzed by using the research analysis system RAS 1,000; the V1 and V2 receptors were quantitated by subtracting the nonspecific binding (incubated with OPC-21268 and OPC-31260, respectively) from the total binding. To assess a more precise localization of the V1 and V2 receptors, we also investigated the micro-ARG of the renal V1 and V2 receptors by dipping the kidney section slides used for macro-ARG into a photographic emulsion and observing the receptors under light microscopy. [3H]-VP binding to the rat kidney was completely displaced by unlabeled excess VP, but not by unlabeled angiotensin II, indicating that [3H]-VP binding was specific for VP receptors. Computerized quantification showed that V2 receptors, visualized by OPC-31260, were the predominant type of VP receptor in the kidney. Conversely, V1 receptors, visualized by OPC-21268, were fewer in number. V1 receptors were partly localized to the glomerulus, cortical vessels, interstitial cells, and the medullary vessels. The V2 receptors localized to the collecting ducts and medullary tubules. Our findings indicated that renal V1 and V2 receptors can be detected by in vitro macro- and micro-ARG by using OPC-21268 and OPC-31260.

Differential effect of chronic inhibition of calcium channel and angiotensin II type 1-receptor on aldosterone synthesis in spontaneously hypertensive rats.

We have investigated the in vivo effect of chronic blockade of Ca(2+)-channels and angiotensin II type 1 (AT(1))-receptors on aldosterone (Aldo)-synthesis in the adrenal glands of spontaneously hypertensive rats (SHR). Male SHR were administered Ca(2+)-antagonist, amlodipine (10 mg/kg per day) or AT(1)-receptor-antagonist, TCV-116 (1 mg/kg per day) from 7 until 11 weeks of age. Systolic blood pressure (SBP) and heart rate (HR) were significantly higher in SHR than Wistar-Kyoto (WKY) rats. Both treatments resulted in equivalent and significant reduction in SBP in SHR. Aldo-secretion in SHR, which was significantly higher than in WKY rats, was profoundly suppressed by TCV-116 compared with amlodipine. Both treatments resulted in thickening of the zona glomerulosa, which immunohistochemically contains Aldo, at the end of therapy. Competitive reverse transcription-polymerase chain reaction (RT-PCR) showed that CYP11A (P450scc) mRNA regulating the first step of Aldo-synthesis was significantly reduced from week 9 of age by amlodipine, and that CYP11B2 (P450aldo) mRNA regulating the last step of Aldo-synthesis was potently suppressed from 9 weeks of age by TCV-116. Our results indicate that chronic treatment with different antihypertensive agents directly modulates adrenocortical aldosterone synthesis in SHR in vivo via different mechanisms.

Effect of renin-angiotensin inhibition on glomerular injuries in DOCA-salt hypertensive rats

To determine whether growth factors in the glomerulus are induced in the renin suppressed hypertensive model, we examined the mRNA expressions of platelet-derived growth factor (PDGF) B-chain, transforming growth factor (TGF)-beta 1 and angiotensin II type 1 (AT1) receptors in the glomeruli of deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats (DOCA-treated rats). We also examined the effects of treatment with cilazapril, an angiotensin I-converting enzyme inhibitor (ACEI), and L-158,809, an AT1 receptor antagonist, on these expressions in DOCA-treated rats. We administered oral 10 mg/kg of cilazapril (CILAZA group) and 1 mg/kg of L-158,809 (L158 group) to DOCA-treated rats daily. Systolic blood pressure in the two groups was not decreased compared with that in DOCA-treated rats given saline. The mRNA expressions were examined using reverse transcriptase polymerase chain reaction (RT-PCR) methods. The mRNA expressions of these genes were higher in DOCA-treated rats than in age-matched control rats. After treatment with these agents for 4 weeks, the mRNA expressions of growth factors were suppressed in both the CILAZA and L158 groups. Mesangial expansion and cell proliferation observed in DOCA-treated rats were suppressed in both the CILAZA and L158 groups. Decreases in the size of the glomerulus were observed only in the CILAZA group. These findings suggested that suppression of growth factors and glomerular proliferative changes of these agents are mediated by blocking tissue renin-angiotensin system (RAS) in the renin-suppressed model.

Angiotensin II induces in vivo c-fos expression from rat renal cortex and medulla

In order to verify whether angiotensin II (Ang II) induced in vivo protooncogene, c-fos, expression in the rat renal cortex and medulla, we administered various concentrations of Ang II to Wistar rats and measured the c-fos expression from the renal cortex and medulla using the method of Northern hybridization. c-fos expression induced by 1 microgram (1.6 x 10(-6) M) of atrial natriuretic peptide (ANP) was also examined. The result was that the peak expression of c-fos mRNA was observed at approximately 10 min after Ang II administration in both rat renal cortex and medulla. This expression was reduced to the control level at 30 min. The measurement of the concentration of injected-Ang II and c-fos mRNA expression revealed that the peak expression of c-fos mRNA in the renal cortex and medulla was detected at the concentration of 1.0 x 10(-8) M and 1.0 x 10(-9) M Ang II, respectively. Nevertheless, ANP had no significant effect on the increase in c-fos mRNA expression. These data revealed that Ang II transiently increases the in vivo c-fos expression in both rat renal cortex and medulla but ANP does not. This protooncogene expression may induce vascular and mesangial proliferation in the kidney.

Chronic Treatment with Angiotensin II Type 1 Receptor Antagonist Suppresses Glomerular Activator Protein–1 Activity in Salt–Sensitive Hypertensive Rats

We examined the effects of angiotensin–converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor antagonist (AT1a) on the action of protooncogene c–fos in salt–sensitive hypertensive rats. Seven–week old Dahl salt–sensitive rats fed a high (8%)–salt diet were treated with ACEI, cilazapril (10 mg/kg) or AT1a, TCV–116 (1mg/ kg) every day for 6 weeks. The control animals were fed a low (0.3%)–salt diet. Systolic blood pressure gradually increased in high–salt–loaded rats and was higher than low–salt–treated rats throughout the study. However, both medications had no significant antihypertensive effect. After 6 weeks of therapy, glomerular mRNA and nuclear protein were extracted from the resected kidneys. Competitive reverse transcription–polymerase chain reaction showed a high level of glomerular c–fos mRNA in high–salt–loaded rats and that ACEI or AT1a treatment did not significantly change its level. Electrophoretic mobility shift assay demonstrated that treatment with AT1a significantly decreased the activator protein–1 (AP–1) binding activity in the glomerular nuclear extract compared to ACEI. Our findings suggest that, compared with ACEI treatment, long–term treatment with AT1a may contribute to attenuation of the glomerular injury in salt–sensitive hypertension by inhibiting AP–1 transcription activity independent of its antihypertensive effect.

Testosterone modulates serum leptin concentrations in a male patient with hypothalamic hypogonadism

Serial measurements of body mass index (BMI), serum concentrations of testosterone (T), estradiol (E) and leptin (L) were performed before and after gonadotropin (Gn) therapy in an 18-year-old male subject (BMI 25.4 kg/m2) with idiopathic hypothalamic hypogonadism (IHH). We also measured the BMI and serum concentrations of L in 99 age-matched healthy subjects. Serum L correlated significantly with BMI in control subjects (r=0.84, p<0.0001). Baseline serum concentrations of L in our case were markedly high and both T and E were very low, but Gn therapy resulted in a gradual decrease in L and improvement in T and E, finally reaching the control levels of BMI-matched subjects. Our results demonstrate that T is a powerful negative modulator of serum L independent of BMI in conditions associated with low T levels, such as IHH.

Renal c-fos expression induced by angiotensin II is enhanced in spontaneously hypertensive rats.

We compared the effect of a bolus injection of angiotensin II (Ang II) on the expression of protooncogene c-fos in the renal cortex and medulla of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Intravenous infusion of 5 ng/kg body weight of Ang II resulted in an immediate rise in systolic blood pressure (SBP) in both SHR and WKY rats. The percent rise in SBP was similar in both strains. Pretreatment with Ang II type 1 (AT1)-receptor antagonist, L-158,809 (1 mg/kg) abolished the pressor response in both strains. Competitive reverse transcription-polymerase chain reaction (RT-PCR) showed that administration of Ang II increased the expression of c-fos mRNA within 10 min in both the renal cortex and medulla of SHR significantly higher than WKY rats. Moreover, the enhanced c-fos mRNA expression due to Ang II was significantly suppressed by the pretreatment of L-158,809 in both strains. These findings indicate that c-fos expression in the kidney is mediated by AT1-receptors and that the renal c-fos response to exogenous Ang II was significantly augmented in SHR compared with WKY rats, suggesting that this hyperresponsiveness on renal AT1-action may partly contribute to the progression of renal injury in SHR.