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ATRAP [ANG II type 1 receptor (AT1R)-associated protein] is a molecule which directly interacts with AT1R and inhibits AT1R signaling. The aim of this study was to examine the effects of continuous ANG II infusion on the intrarenal expression and distribution of ATRAP and to determine the role of AT1R signaling in mediating these effects. C57BL/6 male mice were subjected to vehicle or ANG II infusions at doses of 200, 1,000, or 2,500 ng·kg−1·min−1for 14 days. ANG II infusion caused significant suppression of ATRAP expression in the kidney but did not affect ATRAP expression in the testis or liver. Although only the highest ANG II dose (2,500 ng·kg−1·min−1) provoked renal pathological responses, such as an increase in the mRNA expression of angiotensinogen and the α-subunit of the epithelial sodium channel, ANG II-induced decreases in ATRAP were observed even at the lowest dose (200 ng·kg−1·min−1), particularly in the outer medulla of the kidney, based on immunohistochemical staining and Western blot analysis. The decrease in renal ATRAP expression by ANG II infusion was prevented by treatment with the AT1R-specific blocker olmesartan. In addition, the ANG II-mediated decrease in renal ATRAP expression through AT1R signaling occurred without an ANG II-induced decrease in plasma membrane AT1R expression in the kidney. On the other hand, a transgenic model increase in renal ATRAP expression beyond baseline was accompanied by a constitutive reduction of renal plasma membrane AT1R expression and by the promotion of renal AT1R internalization as well as the decreased induction of angiotensinogen gene expression in response to ANG II. These results suggest that the plasma membrane AT1R level in the kidney is modulated by intrarenal ATRAP expression under physiological and pathophysiological conditions in vivo.

We cloned a novel molecule interacting with angiotensin II type 1 receptor, which we named ATRAP (for angiotensin II type 1 receptor–associated protein). Previous in vitro studies showed that ATRAP significantly promotes constitutive internalization of the angiotensin II type 1 receptor and further attenuates angiotensin II–mediated hypertrophic responses in cardiomyocytes. The present study was designed to investigate the putative functional role of ATRAP in cardiac hypertrophy by angiotensin II infusion in vivo. We first examined the effect of angiotensin II infusion on endogenous ATRAP expression in the heart of C57BL/6J wild-type mice. The angiotensin II treatment promoted cardiac hypertrophy, concomitant with a significant decrease in cardiac ATRAP expression, but without significant change in cardiac angiotensin II type 1 receptor expression. We hypothesized that a downregulation of the cardiac ATRAP to angiotensin II type 1 receptor ratio is involved in the pathogenesis of cardiac hypertrophy. To examine this hypothesis, we next generated transgenic mice expressing ATRAP specifically in cardiomyocytes under control of the α-myosin heavy chain promoter. In cardiac-specific ATRAP transgenic mice, the development of cardiac hypertrophy, activation of p38 mitogen-activated protein kinase, and expression of hypertrophy-related genes in the context of angiotensin II treatment were completely suppressed, in spite of there being no significant difference in blood pressure on radiotelemetry between the transgenic mice and littermate control mice. These results demonstrate that cardiomyocyte-specific overexpression of ATRAP in vivo abolishes the cardiac hypertrophy provoked by chronic angiotensin II infusion, thereby suggesting ATRAP to be a novel therapeutic target in cardiac hypertrophy.

MAK-V/Hunk is an SNF1-related serine/threonine kinase which was previously shown to be highly expressed in the mammary gland and central nervous system. In this study, we found MAK-V/Hunk is abundantly and specifically expressed in the thick ascending limbs and distal convoluted tubules (DCT) of the kidney from the embryonic stage to the adult stage. We demonstrated that dietary salt depletion significantly enhances renal MAK-V/Hunk mRNA levels compared with a normal-salt diet. To analyze the possible renal cellular function of this kinase, we employed mouse distal convoluted tubule (mDCT) cells. The results of reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that MAK-V/Hunk is expressed endogenously in mDCT cells. Overexpression of MAK-V/Hunk by adenoviral gene transfer significantly inhibited the ANG II-induced stimulation of c- fos gene transcription and suppressed the ANG II-mediated increases in transforming growth factor-β production into the medium. This phenomenon was accompanied by inhibition of ANG II-induced activation of BrdU incorporation. On the other hand, the MAK-V/Hunk knockdown by siRNA activated the ANG II-induced c- fos gene expression. In the consecutive sections stained for MAK-V/Hunk and AT1receptor, MAK-V/Hunk-immunopositive distal tubules expressed the AT1receptor. This is the first report on the intrarenal localization of MAK-V/Hunk and its cellular function in renal tubular cells.

Background Angiotensin II type 1 receptor ( AT 1R)–associated protein ( ATRAP; Agtrap gene) promotes AT 1R internalization along with suppression of pathological AT 1R activation. In this study, we examined whether enhancement of ATRAP in the renal distal tubules affects sodium handling and blood pressure regulation in response to high salt ( HS ) loading, using ATRAP transgenic mice on a salt‐sensitive C57 BL /6J background. Methods and Results Renal ATRAP transgenic ( rATRAP ‐Tg) mice, which exhibit renal tubule–dominant ATRAP enhancement, and their wild‐type littermate C57 BL /6J mice on a normal salt diet (0.3% NaCl) at baseline were subjected to dietary HS loading (4% NaCl) for 7 days. In rATRAP ‐Tg mice, the dietary HS loading–mediated blood pressure elevation was suppressed compared with wild‐type mice, despite similar baseline blood pressure. Although renal angiotensin II level was comparable in rATRAP ‐Tg and wild‐type mice with and without HS loading, urinary sodium excretion in response to HS loading was significantly enhanced in the rATRAP ‐Tg mice. In addition, functional transport activity of the amiloride‐sensitive epithelial Na + channel was significantly decreased under saline volume–expanded conditions in rATRAP ‐Tg mice compared with wild‐type mice, without any evident change in epithelial Na + channel protein expression. Plasma membrane AT 1R expression in the kidney of rATRAP ‐Tg mice was decreased compared with wild‐type mice. Conclusions These results demonstrated that distal tubule–dominant enhancement of ATRAP inhibits pathological renal sodium reabsorption and blood pressure elevation in response to HS loading. The findings suggest that ATRAP ‐mediated modulation of sodium handling in renal distal tubules could be a target of interest in salt‐sensitive blood pressure regulation.

The renin–angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro , which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo .

We report a case of a 23-year-old Japanese woman who had severe hyperparathyroidism associated with chronic renal failure before the start of dialysis treatment. Her chief complaints were swelling and pain in both shoulders. Laboratory examination revealed renal failure (BUN 134 mg/dl, serum Cr 7.3 mg/dl), severe normocytic normochromic anemia (hemoglobin 4.3 g/dl), hypercalcemia (11.8 mg/dl), and hyperphosphatemia (9.7 mg/dl). Serum PTH levels were extremely increased (intact PTH >1,000 pg/ml: normal range 10-50 pg/ml). X-ray examination of the skull and shoulders showed a salt and pepper appearance, and cauliflower-like deformity of the distal end of both clavicles, respectively. Accelerated ectopic calcification was observed in the costal cartilages, internal carotid arteries, and splenic arteries. Ultrasonographic examination revealed enlargement of the four parathyroid glands. Thallium-technetium subtraction scintigraphy of the parathyroid glands showed increased uptake into the upper two. Renal needle biopsy revealed severe impairment of the interstitium and tubules with much milder changes in glomeruli. The etiology of the renal failure could not be identified. Hemodialysis, total parathyroidectomy and auto-transplantation into the forearm were immediately performed. The pathological diagnosis was chief cell hyperplasia of the parathyroid glands. Based on the presence of chronic renal failure, remarkable hyperphosphatemia with mild hypercalcemia, an unusually high level of serum PTH, and accelerated ectopic calcification, the patient was diagnosed to have severe secondary hyperparathyroidism caused by chronic renal failure with major impairment of the renal interstitium and tubules.

Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor gene-knockout mice. Angiotensin type-1a (AT1a) receptor gene-knockout ( AT1a -/- ) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a -/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a -/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a -/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a -/- mice (205.5 ± 26.1 ng/ml/hr) than in wild-type mice (8.0 ± 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin gene expression were 12-times and 2.6-times higher in AT1a -/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a -/- mice compared with angiotensinogen-knockout mice. When AT1a -/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a -/- mice.

The present study investigates whether neuronal type nitric oxide synthase (N-NOS) in the macula densa participates in the regulation of renal renin expression during altered dietary salt intake in angiotensinogen gene-knockout (Atg-/-) mice. Wild-type (Atg+/+) and Atg+/+ mice were fed a low-salt (0.04% NaCl), normal-salt (0.3% NaCl), or high-salt (4% NaCl) diet for 2 wk. Histochemical staining for NADPH diaphorase (NADPHd) and renin were analyzed morphometrically. Levels of N-NOS and renin mRNA in renal cortical tissues were determined by reverse transcription-PCR and Northern blot analysis, respectively. In animals fed a normal-salt diet, the renal expressions of N-NOS and renin were markedly increased in Atg-/- mice compared with Atg+/+ mice. When mutant mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. These changes were paralleled by decreases in renal renin-immunoreactive areas and the levels of renin mRNA. On the other hand, salt restriction did not produce further significant increases in the renal N-NOS and renin expressions in mutant mice, whereas a parallel inverse relationship was observed between these enzyme expressions and the levels of salt intake in wild-type mice. These results suggest that the N-NOS expression in the macula densa is inversely regulated by salt intake and that the enzyme activity is functionally linked to renal renin production. Salt-modulated renal N-NOS and renin expressions are independent on angiotensin formation in Atg-/- mice.

Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice. Angiotensinogen gene-knockout ( Atg −/− ) mice lacking angiotensin II exhibit chronic hypotension. The present study was designed to investigate pathophysiology of Atg −/− mice from the renal functional view. Wild-type ( Atg +/+ ) and Atg −/− mice at 10 weeks of age were housed in metabolic cages for 24-hour urine collection. When provided free access to water, Atg −/− mice showed an increased urine output and a decreased urine osmolality compared with Atg +/+ mice. Urinary excretion and plasma levels of vasopressin were significantly higher in mutant mice than in wild-type mice. On the other hand, urinary excretion of aldosterone in mutant mice was suppressed to the levels under the detection limit of the assay system. The mean plasma aldosterone level of Atg −/− mice was suppressed to 30% of that of Atg +/+ mice. Plasma levels of creatinine, endogenous creatinine clearance, and urinary electrolyte excretion were not different between these mice. In Atg +/+ mice, urine osmolality was markedly increased from 1929 ± 21 to 3314 ± 402 mOsm/kg during water deprivation, whereas this parameter in Atg −/− mice did not change significantly (from 1413 ± 121 to 1590 ± 92 mOsm/kg). Urinary vasopressin excretion increased during water deprivation from 0.24 ± 0.04 and 0.70 ± 0.08 to 0.42 ± 0.06 and 2.31 ± 0.35 ng/mg creatinine in wild-type and mutant mice, respectively. Histologic study revealed interstitial inflammation, and atrophic changes in the tubules and papilla in Atg −/− mice. In conclusion, a genetic deficiency of angiotensinogen produced an impaired urine concentrating ability and tubulointerstitial lesions, indicating the critical role of angiotensinogen in developing normal tubular function and construction.

Abstract —Two subtypes of angiotensin II (Ang II) receptors, type 1 (AT 1 -R) and type 2 (AT 2 -R), have been identified in the heart. However, little is known about the regulation of cardiac AT 1 -R and AT 2 -R by Ang II in vivo. Thus, we examined cardiac AT 1 -R and AT 2 -R in angiotensinogen-deficient ( Atg-/- ) mice that are hypotensive and lack circulating Ang II. Cardiac Ang II receptors (Ang II-R) were assessed by radioligand binding with 125 I-[Sar 1 ,Ile 8 ]–Ang II in plasma membrane fractions. AT 1 -R and AT 2 -R were distinguished using their specific antagonists CV-11974 and PD123319 , respectively. Total densities of Ang II-R and AT 1 -R density were significantly greater in the Atg-/- mice than Atg+/+ mice (31.1±2.8 versus18.8±2.1, 28.7±3.0 versus16.9±2.3 fmol/mg protein, P 2 -R showed a slight but not significant increase in Atg-/- mice relative to Atg+/+ control animals. K d values were not different between the two groups. In contrast to binding experiments, levels of Ang II type 1a receptor (AT 1a -R) and AT 2 -R mRNA did not differ between Atg-/- and Atg+/+ mice. These results suggest that lack of Ang II may upregulate AT 1 -R through translational and/or posttranslational mechanisms in Atg-/- mice.

The protein product of the retinoblastoma susceptibility gene, RB, is a nuclear phosphoprotein that modulates transcription of genes involved in growth control via interactions with transcription factors. Renin is a rate-limiting enzyme of the renin-angiotensin system that regulates blood pressure and water-electrolyte balance. Renin gene expression is regulated in a tissue-specific and developmentally linked manner. Similarly, the expression of RB is controlled in a differentiation-linked manner. Thus, to investigate whether RB is involved in the regulation of renin gene expression, we examined the effects of RB on transcriptional activity of the mouse renin (Ren-1C) promoter. The Ren-1C promoter contains two transcriptionally important elements; the RU-1 (-224 to -138) and RP-2 (-75 to -47) elements. RB activated the Ren-1C promoter in human embryonic kidney cells. The promoter element responsible for RB-mediated transcriptional regulation was the RP-2 element. The results of DNA-protein binding experiments showed that RB increased nuclear binding activity to the RP-2 element, and site-directed mutation which disrupted binding of nuclear factors to the RP-2 element markedly reduced RB-mediated activation of Ren-1C promoter in human embryonic kidney cells. These results indicate that the RP-2 element plays an important role in RB-mediated transcriptional regulation of Ren-1C promoter activity in human embryonic kidney cells, thereby suggesting an interesting mechanism by which RB may modulate the renin-angiotensin system.

頸部リンパ節結核によるrHuEPO抵抗性を示した53歳女性の血液透析患者の1例を報告する.本例は1986年慢性腎盂腎炎による慢性腎不全にて血液透析導入となった. 必要と判断された場合には鉄剤の投与を行いながら, 腎性貧血に対し1990年よりrHuEPOを週4500単位より投与開始した. 1991年からは週6000単位に増量したが, Htは上昇せず24%以上に維持されることはなかった. 1992年3月に頸部リンパ節結核を発症したため, isoniazid 0.4g/日およびrifampicin 0.45g/日の投与を開始した. 服薬は不規則であり臨床症状は改善しなかった. 1993年1月streptomycin 2g/週, 5週間の投与を開始した. これにより症状が改善すると同時にrHuEPOに対する反応性が良好となり, rHuEPo週4500-9000単位の投与量にてHt28-30%に維持されるようになった. isoniazidおよびrifampicinの投与はそれぞれ1993年5月, 11月まで行った. 現在のところ結核再発の兆候は認められていない. 本例は頸部リンパ節結核発症以前にすでにrHuEPO抵抗性を示していた. その原因として結核菌の持続感染の存在が考慮された. 結核は, 血液透析患者においてrHuEPO抵抗性を生じうると考えられた.

Nephrotic syndrome due to renovascular hypertension is uncommon. We herein report a case of nephrotic syndrome associated with unilateral atherosclerotic renal artery stenosis. A 76-year-old woman who had been taking antihypertensive medication for more than 15 years was referred to our hospital for treatment of uncontrolled hypertension and massive proteinuria in the nephrotic range. An abdominal bruit was heard, and laboratory findings showed high plasma renin activity and hypokalemia. Renal computed tomography angiography showed severe stenosis of the ostium of the right renal artery and an atrophic right kidney. The left renal artery was normal and the left kidney was compensatorily enlarged. After admission, we started treatment with an angiotensin II receptor blocker and subsequently performed percutaneous transluminal renal angioplasty with renal artery stent placement. As a result, her blood pressure became well controlled and the massive proteinuria disappeared. In addition, her stenotic-side renal atrophy was resolved, concomitant with an improvement in her renal function. The contralateral renal hypertrophy was also resolved.

今回, 我々は, 肝硬変に伴う意識障害に対して, 血液濾過透析が著効した, 維持血液透析下の慢性腎不全の1例を経験したので報告する. 患者は, 53歳, 女性で, 以前よりC型肝炎の進行による肝硬変の治療を受け, また, 腎機能低下の進行のため維持血液透析を受けていた. 平成4年9月28日, 意識障害のため入院した. 高アンモニア血症による肝性昏睡の進行が原因と診断し, 高アンモニア対策と血液透析を施行したが, 意識障害の軽減は見られずさらに悪化したため, 高性能膜を用いた血液濾過透析を行ったところ, 血中アンモニア濃度の低下と意識障害の改善が認められた. 慢性腎不全において, 肝硬変による肝性昏睡を合併した場合には, 血液濾過透析が有効と考えられた.

Net sodium balances in humans are maintained through various ion transporters expressed along the entire nephron. Among these ion transporters, epithelial sodium channels (ENaC) located along the aldosterone-sensitive distal nephron (ASDN) play a pivotal role in the homeostasis of sodium balance. This is supported by analyses of inherited hypertensive disorders, showing that genes encoding ENaC and other modulatory proteins cause hereditary hypertension, such as Liddle syndrome. Among various modulating proteins, E3 ubiquitin ligase, Nedd4L, binds the PY motif of ENaC COOH terminals and catalyzes ubiquitination of the NH 2 terminus of the protein for subsequent degradation. Both evolutionarily conserved and evolutionarily new C2 domains of human Nedd4L, a cryptic splice variant resulting in a disrupted isoform product formed by a frame-shift mutation, were reported previously. We focused on one of the isoforms, isoform I, generated by SNP (rs4149601), and studied its expression and interactions with other isoforms by molecular biological, immunohistochemical, and electrophysiological methods. We found that isoform I may interact with other human isoforms in a dominant-negative fashion. Such interactions might abnormally increase sodium reabsorption. Taken together, our analyses suggest that the human Nedd4L gene, especially the evolutionarily new isoform I, is a candidate gene for hypertension.

We studied the localization of angiotensinogen mRNA in rat nephron segments and the differences in angiotensinogen mRNA levels between male Sprague-Dawley rats at 6 and 12 wk of age using reverse transcription and polymerase chain reaction (RT-PCR). Each nephron segment of the rat kidney was microdissected. Total RNA was prepared and used in the following RT-PCR assay. The PCR products were size-fractionated by agarose gel electrophoresis, visualized with ethidium bromide staining, and identified by Southern blot analysis. The relative amounts of products were determined by densitometry. Strong bands corresponding to angiotensinogen mRNA were detected from proximal convoluted and straight tubules, and weaker bands were found in glomeruli. The signals in all tissues in 12-wk-old rats were weaker than those in 6-wk-old rats. Since local angiotensinogen is the unique substrate of the tissue renin-angiotensin system and exerts an autocrine-paracrine influence on renal function, the changes in tubular angiotensinogen may be related to physiological and morphological changes in the rat kidney during development.

Abstract The angiotensinogen (AGT) gene M235T variant is associated with essential hypertension and elevated plasma AGT concentrations, although the underlying mechanisms are unknown. Recent studies have suggested that AGCE 1 (human AG T gene c ore promoter e lement 1) located in the 5′ upstream core promoter region (position −25 to −1) of the human AGT gene has an important part in the expression of AGT mRNA by binding with transcription factor AGCF 1 (human AG T gene c ore promoter element binding f actor 1), and a mutation at −20 from adenine to cytosine (A-20C) increases the level of expression of this transcript. We therefore examined subjects with this mutation to study the association with increased plasma AGT concentrations and with essential hypertension. One hundred eighty-eight subjects receiving no antihypertensive medication were examined with regard to the correlation between A-20C and plasma AGT concentrations, and 234 subjects were studied with respect to the association between A-20C and essential hypertension. A-20C was determined by polymerase chain reaction–restriction fragment length polymorphism analysis with Eco OR 109I. Multiple regression analysis showed a weak but significant correlation between A-20C and plasma AGT concentrations ( P =.047) and essential hypertension ( P =.049). The results suggest that A-20C may underlie the increase in plasma AGT concentrations and be involved in the development of essential hypertension.

Abstract There is now convincing evidence that various tissues express their own tissue renin-angiotensin system, which may be regulated independently of the systemic renin-angiotensin system. However, little information is available on the regulation of the tissue renin-angiotensin system. We investigated the regulation of tissue angiotensinogen gene expression with respect to the development of hypertension. We measured basal and lipopolysaccharide-stimulated plasma angiotensinogen concentrations by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at 4 and 13 weeks of age. Basal plasma angiotensinogen concentration in SHR was comparable to that in WKY at 4 weeks of age and was significantly higher than that in WKY at 13 weeks of age. Lipopolysaccharide induced a significant increase in plasma angiotensinogen concentration in both WKY and SHR at 4 and 13 weeks of age. At 4 weeks of age, the basal levels of angiotensinogen mRNA in the liver, fat, adrenal, and aorta were higher in WKY than in SHR. At 13 weeks of age, the basal levels of angiotensinogen mRNA in the fat, adrenal, aorta, spleen, and kidney were higher in WKY than in SHR, while that in the liver did not differ significantly between the two strains. At 4 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, fat, adrenal, and aorta in both WKY and SHR. At 13 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, aorta, and adrenal; decreased those in the spleen; and had no effect in the kidney in both WKY and SHR. Interestingly, lipopolysaccharide increased the angiotensinogen mRNA level in fat only in SHR, with no effect in WKY, at 13 weeks of age. Lipopolysaccharide stimulated tumor necrosis factor-α mRNA expression in fat of WKY and SHR, and the increase in tumor necrosis factor-α mRNA level in SHR was significantly greater than that in WKY. Therefore, the increased tumor necrosis factor-α mRNA expression may be involved in the increased lipopolysaccharide-induced expression of angiotensinogen gene in fat of SHR at 13 weeks of age. These data suggest that the transcriptional and probably posttranscriptional regulation of angiotensinogen mRNA differs between SHR and WKY, that the regulation of angiotensinogen gene expression is tissue-specific, and that the altered expression of the angiotensinogen gene may be involved in the development of hypertension.

A close relationship between obesity and hypertension has been recognized, and plasma angiotensinogen concentrations (p-AGT) have been reported to correlate with blood pressure (BP). However, little is known about AGT in obese patients with hypertension. To define the role of AGT in obese hypertension, we measured p-AGT in obese patients. The subjects were 42 obese patients diagnosed on the basis of a body mass index (BMI) of more than 25 kg/m2, and 21 sex- and age-matched nonobese patients, whose BMI was less than 25 kg/m2. The hypertensive patients had not previously received antihypertensive drugs. P-AGT (P < .05) and mean BP (P < .0001) was increased in the obese patients as compared with the nonobese patients. Positive correlations were observed between BMI and p-AGT, mean BP and p-AGT, and BMI and mean BP (all P < .05). However, after adjustment for blood pressure, p-AGT was not different between groups, and after adjustment a positive correlation remained only between BMI and mean BP. These results suggested the possible involvement of increased p-AGT in hypertension in obese patients, although this may be a secondary change to hypertension or obesity.

Molecular evidence, using DNA fingerprint analyses, of extensive genetic heterogeneity between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and even within some of the WKY colonies has been reported. Thus we investigated the genetic relations between Dahl S and R rats newly inbred by Dr. Iwai. Genomic DNA was isolated from the liver of four Dahl S and four Dahl R rats, digested with the restriction enzyme HinfI or AluI, and separated in 1.2% agarose gel by electrophoresis. Then, DNA fingerprinting was performed by Southern blot analysis using the human myoglobin 33.6 minisatellite probe. Bands were detected in an alkaline phosphatase reaction system. Within the same strains, there was no heterogeneity of these fingerprinting patterns. The S and R rats shared 82% of the bands in the HinfI-digested DNA and 93% of those in the AluI-digested DNA. These shared values were much greater than the reported value (54%) between SHR and WKY from Charles River Laboratories. These newly inbred Dahl S and R rats may be appropriate, although still limited, experimental animals for investigating the pathophysiology of salt-sensitive hypertension.

Summary: The antihypertensive effects and pharmacokinetic properties of cilazapril, a long-acting angiotensin-converting enzyme (ACE) inhibitor, were investigated in hypertensive patients with normal renal function (NRF; n = 5) and those with impaired renal function (IRF; n = 7). A 1.25-mg dose of cilazapril was administered orally once a day for 5 or 8 days. Measurement of blood pressure and sampling of blood specimens were done on the first and last days of treatment. Cilazapril induced significant falls in systolic and diastolic blood pressures as early as 1 h after administration. The antihypertensive effects were still present at 24 h postdose. Serum ACE activity was markedly suppressed over 24 h, with the enzyme inhibition greater in the IRF group. Plasma levels of the active diacid in the IRF group were higher than those in the NRF group, with significant differences in the peak levels and areas under the curve (AUC). A significant inverse correlation was found between the creatinine clearance and the AUC for the diacid. Cilazapril was well tolerated by all the patients, and no adverse reactions were observed. These results suggest that cilazapril has a long-lasting action and that it is a useful antihypertensive agent for controlling blood pressure in patients with either NRF or IRF.

Distribution of α1B-adrenergic receptor mRNA expression along rat nephron segments. Although several α-adrenergic receptor genes are expressed in the rat kidney, little information is available on their expression in the renal nephron segments. We investigated the distribution of α1B-adrenergic receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of six- to eight-week-old male Sprague-Dawley rats were microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. The PCR products were size-fractionated with electrophoresis, visualized with ethidium bromide staining and confirmed by Southern blot analysis. Because the PCR primers spanned an intron, the amplification product of the predicted size was considered to be from α1B-adrenergic receptor cDNA and not from genomic DNA. The PCR products were detected in glomerulus (Glm), proximal convoluted and straight tubules (PCT, PST) and cortical and medullary thick ascending limbs of Henle (CTAL, MTAL). No signals were detected in cortical or medullary collecting ducts (CCD, MCD). Large signals were detected in the PCT, and PST, while small signals were found in the Glm, CTAL and MTAL. The α1B-adrenergic receptor mRNA was detected for the first time in rat Glm, PCT, PST and TAL using RT-PCR. α1BAR mRNA seems to be expressed in the specific sites along the nephron and may play significant roles in renal functions, although the specific physiological effects of the renal α1B-adrenergic receptor are unknown.

Angiotensin II inhibits interleukin-1β–induced nitric oxide production in cultured rat mesangial cells. Background Macrophage-type nitric oxide synthase (NOS-II) is expressed in glomerular mesangial cells in response to inflammatory cytokines. Nitric oxide (NO) has antithrombotic and cytostatic activities in glomerular diseases. Recent studies have suggested that several vasoactive substances and growth factors modulate NO production in a tissue-specific manner. The aim of this study was to examine whether angiotensin II and transforming growth factor-β (TGF-β) modulate cytokine-stimulated NO production and NOS-II gene expression in rat glomerular mesangial cells. Methods Cultured rat mesangial cells were incubated with interleukin-1β (IL-1β) for 24hours. The effects of angiotensin II and TGF-β on stimulated nitrite accumulation and NOS-II mRNA levels were determined. Results Angiotensin II and TGF-β significantly decreased IL-1β–stimulated nitrite accumulation. The angiotensin type 1 receptor antagonist CV11974 prevented angiotensin II-mediated inhibition of NO production. TGF-β–neutralizing antibody reversed the effect of TGF-β without affecting angiotensin II-mediated inhibition of NO production. TGF-β markedly decreased steady-state levels of NOS-II mRNA and the half-life of the message, whereas angiotensin II did not alter these parameters. Conclusions These results suggest that in mesangial cells, angiotensin II and TGF-β participate in the inhibitory regulation of cytokine-induced NO production. TGF-β inhibits NO production by decreasing NOS-II mRNA levels, whereas angiotensin II may regulate NO production at the levels after NOS-II gene expression. An autocrine action of TGF-β induced by angiotensin II is unlikely to contribute to angiotensin II-mediated inhibition of NO production.

Possible role of c-Jun in transcription of the mouse renin gene. Background Renin is a rate-limiting enzyme for activity of the circulating renin-angiotensin system (RAS) and expression of the renin gene is regulated by a variety of stimuli. In this study, we examined a possible role of c-Jun in the transcription of renin gene. Methods The renin promoter, chloramphenicol acetyltransferase (CAT), fusion genes with or without c-Jun expression vector (pSV-c-Jun) were transfected into human embryonic kidney (HEK) cells, and the effects of c-Jun were examined by deletion and mutation analyses of CAT assay and by in vitro transcription-primer extension assay. We also examined the effects of c-Jun on DNA-binding activity to the renin promoter by electrophoretic mobility shift assay (EMSA). Furthermore, we examined the effects of c-Jun on transcription of the renin gene in enriched juxtaglomerular (JG) cells by cotransfection with pSV-c-Jun and by treatment with antisense c- jun oligodeoxynucleotides. Results Promoter activity of the renin gene was increased by c-Jun overexpression in HEK cells, and the proximal promoter region from -47 to +16 was sufficient for transcriptional activation by c-Jun. Although mutation of activator protein-1 (AP-1) element-like sequences in the proximal promoter did not affect c-Jun-mediated stimulation, mutation of the core promoter including the TATA box inhibited c-Jun-mediated transcription. The results of EMSA showed that c-Jun overexpression produced a binding of nuclear factor, which was HEK cell-specific and distinct from TATA box-binding protein and AP-1 family transcription factor, to the renin core promoter region (RC element) from -36 to -20. The overexpression of c-Jun activated the renin promoter in renin-expressing JG cells, and antisense c- jun decreased the activity of renin promoter and expression of renin mRNA in JG cells. Conclusions These results indicate that the RC element plays a role in c-Jun-mediated transcriptional regulation of the renin gene in HEK cells, and suggest that c-Jun participates in the regulation of renin gene expression in JG cells of the kidney.