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Background: Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP; Agtrap gene) promotes AT1R internalization along with suppression of pathological AT1R 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 C57BL/6J background., Methods and Results: Renal ATRAP transgenic (rATRAP-Tg) mice, which exhibit renal tubule-dominant ATRAP enhancement, and their wild-type littermate C57BL/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 AT1R 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., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity., (© 2014 Kajitani et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

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(-1) for 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 alpha-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.

We reported on the relationship between base blood pressure (BP), autonomic function and health-related quality of life (HRQOL) in healthy adults. The aim of the present study was to examine the relationship between the antihypertensive effects, autonomic function, and HRQOL following the treatment of hypertensive subjects with losartan/hydrochlorothiazide in hypertensives. In the 10 hypertensive patients treated with angiotensin receptor blockers for more than 1 month, combination therapy with losartan/hydrochlorothiazide was conducted for 3 months after the cessation of treatment with angiotensin receptor blockers. Either immediately before the onset of combination therapy or 3 months after the treatment, 24-h ambulatory BP and pulse wave velocity (PWV) were measured. Sympathetic nervous activity (ratio of low frequency to high frequency component: LF/HF) and parasympathetic nervous activity (high frequency component: HF) were calculated by heart rate variability. Quality of life (HRQOL) was assessed by the Medical Outcome Study Short-Forum 36-Item Health Survey (SF-36). All of the participants completed the study. Losartan/hydrochlorothiazide combination therapy reduced base BP(from 114 +/- 5 to 100 +/- 3 mmHg; p < 0.03)and 24-h LF/HF (from 1.48 +/- 0.18 to 0.94 +/- 0.20; p < 0.02). However, heart rates and PWV were not influenced by losartan/hydrochlorothiazide treatment. The HRQOL scores improved during the study (p < 0.05). These findings indicated that losartan/hydrochlorothiazide was associated with an improvement in base BP relative to daytime BP, autonomic function and HRQOL.

A 65-year-old woman with a 48-year history of Behçet's disease associated with nephrotic syndrome is described. Immunofluorescence study revealed IgA nephropathy. Following treatment with an angiotensin II type-I receptor-blocker, an anti-platelet drug, and an HMG-CoA reductase inhibitor, accompanied by dietary restrictions of protein and sodium, proteinuria was markedly decreased. This report describes our experience with a rare entity of Behçet's disease complicated by nephrotic syndrome due to IgA nephropathy. Routine urine examination and renal biopsy are needed for the detection and diagnosis of renal problems with Behçet's disease.

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.

The purpose of this study was to examine a possible difference in the 24-h blood pressure (BP) profile between hypertensives with diabetic nephropathy (DN) and those with non-diabetic glomerulopathy (non-DN). We measured 24-h ambulatory BP in 34 type 2 DN and 34 non-DN patients who were hospitalized for the educational program in our hospital. There were no significant differences in 24-h and daytime systolic BP between DN (143 vs. 136 mmHg, NS for 24-h systolic BP) and non-DN (143 vs. 138 mmHg, NS for daytime systolic BP). Although both groups disclosed blunted nocturnal decrease in BP and were classified as "non-dipper" type, DN patients had a significantly higher nighttime systolic BP than patients with non-DN (142 vs. 132 mmHg, p = 0.0217). BP and heart rate (HR) variabilities were also estimated, and patients with DN showed a reduced nighttime HR variability than those with non-DN (4.8 vs. 6.6 beats/min, p = 0.0115). DN patients had an increase in urinary protein excretion (3.0 vs. 1.4 g/day, p = 0.0095) and a decrease in serum albumin concentration (3.1 vs. 3.7 mg/dl, p < 0.0001). Furthermore, urinary protein excretion was significantly correlated with nighttime systolic BP (r = 0.480, p = 0.0031) but not with nighttime HR variability. Taken together, these results demonstrate that the circadian rhythms of BP and HR are affected by underlying diseases and suggest that an elevated nighttime BP level may contribute to the enhanced urinary protein excretion in hypertensives with DN.

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-beta 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 AT(1) receptor, MAK-V/Hunk-immunopositive distal tubules expressed the AT(1) receptor. This is the first report on the intrarenal localization of MAK-V/Hunk and its cellular function in renal tubular cells.

Evidence suggests a relationship between short-term blood pressure (BP) variability and cardiovascular target-organ damage. Although a blunted nocturnal decrease in BP and reduced heart rate variability have been shown to be associated with cardiovascular morbidity in diabetic patients, little information is available on short-term BP variability. In this study, short-term BP variability was assessed in 36 subjects with type 2 diabetes and overt nephropathy who underwent ambulatory BP monitoring, and the factors that correlated with short-term BP variability were examined. The incidence of coronary artery disease (CAD) was significantly greater in the patients with increased 24-h systolic BP variability (67% versus 11%; p < 0.0005), while that of cerebrovascular disease was not significantly affected (61% versus 50%). Multiple stepwise regression analysis revealed that serum cholesterol (cholesterol) and plasma norepinephrine (p-NE) were significant and independent contributors to nighttime systolic BP variability (partial R2 = 0.490, p < 0.001; partial R2 = 0.470, p < 0.001) and demonstrated that body mass index and p-NE were primary determinants of nighttime diastolic BP variability (partial R2 = 0.539, p < 0.0005; partial R2 = 0.304, p < 0.05). Diabetic nephropathy patients with CAD had significantly increased daytime systolic (17.8 mmHg versus 13.1 mmHg, p < 0.0005), nighttime systolic (17.4 mmHg versus 10.5 mmHg, p < 0.0001), and nighttime diastolic (10.4 mmHg versus 7.2 mmHg, p < 0.05) BP variability. Furthermore, logistic regression analysis demonstrated that nighttime systolic BP variability was an independent risk factor for CAD (odds ratio 3.13 [95% CI 1.02-9.61]; p < 0.05). The increase in nighttime BP variability is associated with a proportional sympathetic activation in diabetic nephropathy. Elevated short-term BP variability combined with relative sympathetic prevalence during the night might represent an important risk factor for cardiovascular events in the diabetic population.

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-beta (TGF-beta) modulate cytokine-stimulated NO production and NOS-II gene expression in rat glomerular mesangial cells., Methods: Cultured rat mesangial cells were incubated with interleukin-1 beta (IL-1 beta) for 24 hours. The effects of angiotensin II and TGF-beta on stimulated nitrite accumulation and NOS-II mRNA levels were determined., Results: Angiotensin II and TGF-beta significantly decreased IL-1 beta-stimulated nitrite accumulation. The angiotensin type 1 receptor antagonist CV11974 prevented angiotensin II-mediated inhibition of NO production. TGF-beta-neutralizing antibody reversed the effect of TGF-beta without affecting angiotensin II-mediated inhibition of NO production. TGF-beta 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-beta participate in the inhibitory regulation of cytokine-induced NO production. TGF-beta 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-beta induced by angiotensin II is unlikely to contribute to angiotensin II-mediated inhibition of NO production.

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.

A 56-year-old man presented with transient anemia in minimal-change nephrotic syndrome. Following nephrotic syndrome, anemia suddenly appeared without renal dysfunction. The anemia might be attributable to hemodilution because of significant correlations between the values of hemoglobin concentration and serum total protein or blood urea nitrogen during the clinical course. A low serum level and a low urinary excretion of erythropoietin were found, and when nephrotic syndrome ameliorated with steroid therapy, urinary erythropoietin excretion and anemia disappeared. This case indicated disappearance of the exponential increase of endogenous erythropoietin in acute anemia in nephrotic syndrome probably due to urinary losses and altered biosynthesis of erythropoietin. We report a case of the simultaneous improvement of both nephrotic syndrome and anemia with steroid therapy.

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.

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.

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.

1. We investigated the toxicity of cyclosporine A (CsA) with reference to the timing of its administration in rats. 2. To elucidate the time-dependent effects of CsA on renal function and survival rate, CsA (75 mg/kg per day) or vehicle was orally administered once daily at four different times (3, 9, 15 and 21 h after lights on; HALO) over a period of 21 days to male Wistar rats (n = 56) kept in rooms with a 12 h light-dark cycle. 3. On the 7th day after treatment, creatinine clearances (Ccr) of groups dosed at 3 and 9 HALO (inactive period) were not reduced in comparison with clearances of time-matched control rats, whereas Ccr significantly decreased in rats dosed at 15 and 21 HALO (active period). Cyclosporine A markedly increased urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion in all dosed groups at the 7th day after treatment, except for rats dosed at 3 HALO. In rats dosed at 3 HALO, Ccr decreased progressively; however, it did not decrease progressively in rats dosed at 9 HALO. In surviving rats treated during the inactive period, urine NAG subsequently returned to control levels. Survival rates were greater in animals dosed during inactive periods than those in groups dosed during active periods. 4. Significant differences in CsA-induced toxicity were obvious as a result of the timing of its administration. A different time course between Ccr and urine NAG excretion was observed during repeated CsA administration. Degenerative changes in proximal tubules were demonstrated after chronic administration of CsA, suggesting that severe and persistent tubular damage cannot be assessed by urinary NAG excretion.

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.

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-a mRNA expression in fat of WKY and SHR, and the increase in tumor necrosis factor-alpha mRNA level in SHR was significantly greater than that in WKY. Therefore, the increased tumor necrosis factor-alpha 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.

The renin-angiotensin system plays an important role in the hypertrophic responses in cardiac myocytes through the activation of signal transduction pathways and expression of oncogenes. In the present study, we examined mechanical stretch-induced activation of mitogen-activated protein kinases (MAP kinases) using cultured cardiac myocytes derived from neonatal angiotensinogen gene deficient mice (Agt-/-) and neonatal wild type mice (Agt+/+). Within 2 minutes of being added to cardiac myocytes, angiotensin II activated MAP kinases and the response was completely blocked by pretreatment of the cardiac myocytes with CV-11974, a selective antagonist of angiotensin II type 1 receptors. Interestingly, mechanical stretch resulted in significantly greater activation of MAP kinases in Agt-/- cardiac myocytes than in Agt+/+ cardiac myocytes. CV-11974 failed to suppress the stretch-induced activation of MAP kinases in Agt-/- cardiac myocytes while it inhibited the activation in Agt+/+ cardiac myocytes. BQ123, an endothelin type A receptor antagonist, had no effect on stretch-induced activation of MAP kinases in cardiac myocytes from either mouse strain. These results suggest that cardiac RAS is important for stretch-induced MAP kinase activation in Agt+/+ cardiac myocytes; however, angiotensin II is not indispensable for mechanical stretch-induced activation of MAP kinases in Agt-/- cardiac myocytes.

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.

Although several alpha-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 alpha 1B-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 alpha 1B-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 alpha 1B-adrenergic receptor mRNA was detected for the first time in rat Glm, PCT, PST and TAL using RT-PCR. alpha 1BAR 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 alpha 1B-adrenergic receptor are unknown.

We reported a 27-year-old man who developed nephrotic syndrome 12 months after a bone marrow transplantation from his HLA-identical sister for chronic myelocytic leukemia. Anti-nuclear antibodies had been serially investigated after the bone marrow transplantation. They were detected in his serum 5 months before the appearance of proteinuria, but he tested negative at the onset of nephrotic syndrome. Histological analysis of the renal biopsy revealed subepithelial and subendothelial immune deposits in the glomerular basement membrane with increased mesangial matrix and cells. These findings suggested immune complex glomerulonephritis due to chronic graft-versus-host disease (GVHD) after bone marrow transplantation. In murine experimental chronic GVHD, anti-nuclear antibodies, which generate immune complexes that deposit or form in the kidney have been detected.

Angiotensinogen gene-knockout (Atg -/-) mice lacking angiotensin II exhibit chronic hypotension and an increase in renal renin gene expression. The present study was designed to provide evidence for the possible involvement of neuronal type nitric oxide synthase (N-NOS) at the macula densa in the increased renin production in Atg -/- mice. The enzyme activity of N-NOS was histochemically detected by NADPH diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. N-NOS mRNA expression in the renal cortical tissue was determined using reverse transcription-PCR in a semiquantitative manner. The levels of renal renin mRNA were evaluated by Northern blot analysis. In the kidneys of wild-type (Atg +/+) mice, N-NOS activity was localized to the macula densa as reported previously. On the other hand, N-NOS-positive macula densa cells of Atg -/- mice were distributed beyond the original location of the macula densa. They often occupy the entire cross-sectional profiles of the tubules. In addition, Atg -/- mice showed a stronger signal intensity for the enzyme reaction than Atg +/+ mice. The mean total number of N-NOS-positive cells per 100 glomeruli was 6 times higher in Atg -/- mice than in Atg +/+ mice. Semiquantitative reverse transcription-PCR revealed an increase in the N-NOS mRNA level in renal cortical tissue of Atg -/- mice compared with Atg +/+ mice. Furthermore, the selective inhibition of N-NOS activity by 7-nitrondazole significantly decreased the level of renal renin mRNA in Atg -/- mice. These results suggest that increased N-NOS activity at the macula densa is involved a renal renin overproduction in Atg -/- mice.

In this study, to investigate the mechanism of hypertension-associated induction of cardiac angiotensinogen in vivo and in vitro, we studied the regulation of angiotensinogen mRNA in the hearts of genetically hypertensive rats and in the rat cardiomyocytes. Levels of cardiac angiotensinogen mRNA were significantly increased in the hypertensive rats. Steady state mRNA levels for angiotensinogen mRNA in cardiomyocytes were increased by angiotensin II and mechanical stretch. The addition of an angiotensin II type 1 receptor antagonist (CV11974) and a transcriptional inhibitor (actinomycin D) completely blocked the induction of angiotensinogen mRNA by angiotensin II in cardiomyocytes. The addition of CV11974 significantly, but not completely, inhibited the induction of angiotensinogen mRNA by mechanical stretch. Actinomycin D completely blocked the induction of angiotensinogen mRNA by stretch in cardiomyocytes. An angiotensin II type 2 receptor antagonist (PD123319) and a protein synthesis inhibitor (cycloheximide) did not affect the induction. These results indicate that the expression of cardiac angiotensinogen mRNA is activated by the development of hypertensive cardiac hypertrophy, and that angiotensin II and mechanical stretch activates the angiotensinogen gene via the angiotensin II type 1 receptor-pathway in cardiomyocytes.

Effects of slowly performed daytime hemodialysis (slow HD) using a high-flux hemodialyzer on the pharmacokinetics of vancomycin were determined in 5 critically ill patients with renal failure. Following intravenous administration of 0.5 g of vancomycin, concentrations in the serum and dialysate were monitored. Pharmacokinetic parameters were calculated after fitting individual concentration-time curves to a two-compartment model. The volume of distribution at steady state was 0.58 +/- 0.12 liters/kg. Total body clearance was 37.46 +/- 3.20 ml/min with an elimination phase half-life of 8.72 +/- 0.99 h. Slow HD clearance was 20.19 +/- 2.30 ml/min. During a 10-hour session of slow HD, the serum vancomycin concentration decreased from 44.2 +/- 3.8 to 10.0 +/- 5.0 mg/l and 30.10 +/- 5.34% of the dose was eliminated. Dialyzer clearance of this drug and urea was 18.71 +/- 1.40 and 28.77 +/- 1.77 ml/min, respectively. Slow HD may effectively eliminate vancomycin by a diffusive mechanism and this elimination should be taken into consideration for designing the dosage schedule during the treatment.

We reported a case of type II cryoglobulinemia involving glomerulopathy associated with HCV-induced liver cirrhosis. The patient was a 57-year-old woman. Her past history included chronic hepatitis at 51 years and rheumatoid arthritis at 53 years of age. At 46 years, an erythematous lesion appeared on her legs, which was diagnosed as allergic vasculitis by skin biopsy. At 50 years, proteinuria, hematuria and hypertension were recognized. The next year, the first renal biopsy was performed and showed membranoproliferative glomerulonephritis (MPGN). Recently, the edema of her legs has progressed, and the laboratory data showed proteinuria, hematuria, hypocomplementemia, rheumatoid factor positivity, and increase of monoclonal IgG kappa chain. The second renal biopsy revealed an endocapillary proliferative glomerulonephritis-like lesion with marked infiltration of monocytes and macrophages. The subendothelial deposit showed a fine fibril-like pattern. She was treated with steroids and double filtration plasmapheresis (DFPP) therapy, but the treatment was not very effective. She died of liver cirrhosis, which was probably induced by hepatitis C virus (HCV), and sepsis. Generally, the patients of type II cryoglobulinemia often showed HCV antibody positivity, pointing to HCV as an etiological factor. In this case, renal biopsy was performed twice in the same patient, and the histologic findings suggest the clinicopathological course of cryoglobulinemia.

This case was a 51-year-old woman, who had been diagnosed as having rheumatoid arthritis at some clinic and had been treated with both non-steroidal anti-inflammatory drugs and steroid 3 years before visiting our clinic. When she noticed a decrease in visual acuity and general fatigue in June 1985, she was referred to an ophthalmologist of our hospital, and found to have blood pressure of 240/150 mmHg and KW grade IV retinal findings. She was admitted in our department to examine and treat malignant hypertension. On admission, remarkable hypergammaglobulinemia (29.3%), arthralgia, arthral deformity and pericardial effusion were present thus, she was suspected to be suffering from malignant rheumatoid arthritis. Anti-nuclear antibody (64X), anti-nuclear ribonucleoprotein antibody (64X) and anti-RNase sensitive antibody of anti-extractable nuclear antigens (ENA) antibody (81920X) were positive, while anti-RNase resistant antibody of anti-ENA antibody was negative. Immunologically, her condition was consistent with mixed connective tissue disease (MCTD). Since urinary protein was positive and creatinine clearance was 46.0 ml/min, renal function was thought to be diminished. Her chest roentgenogram revealed cardiomegaly (CTR 67.5%) and an increase in pulmonary vascular shadow. An echocardiogram demonstrated the presence of pericardial effusion. Plasma renin activity was 3.3 ng/ml/h and it was suspected that an intrarenal ischemic change resulted in increased renin release from the juxta-glomerular apparatus, leading to the marked hypertension. Treatment was started with prednisolone 60 mg/day during 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)