Tissue-specific regulation of angiotensinogen gene expression in spontaneously hypertensive rats

Angiotensinogen is expressed in many tissues besides the liver. Recent studies have suggested that abnormalities in the regulation of angiotensinogen gene expression may be involved in the development of hypertension. However, little information is available concerning the functional significance of tissue angiotensinogen. In this study, we measured plasma angiotensinogen concentration by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Although plasma angiotensinogen concentration in SHR was comparable to that in WKY at 6 weeks of age, it was increased significantly at 14 weeks of age in SHR and became higher than that in WKY. The levels of hepatic angiotensinogen mRNA were similar in SHR and WKY, and the levels of aortic, adrenal, and renal angiotensinogen mRNAs were lower in SHR than in WKY at both 6 and 14 weeks of age. Brain angiotensinogen expression in SHR was higher than in WKY at 6 weeks of age and was comparable to that in WKY at 14 weeks of age. On the other hand, cardiac and fat angiotensinogen mRNA levels were significantly increased at 14 weeks of age in SHR. These results demonstrate that the expression of tissue angiotensinogen is regulated differently in SHR and WKY and indicate that the development of hypertension is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as cardiac and adipogenic angiotensinogen mRNA in SHR.     

The influence of nephrectomy, ureteral ligation, and of estradiol on plasma renin substrate in unilaterally nephrectomized rats

The effects of three experimental conditions on the concentration of plasma renin substrate were studied with special reference to plasma renin concentration in unilaterally nephrectomized rats. After simultaneous bilateral nephrectomy the maximum increase in plasma renin substrate was 17 times higher than normal within 24 h, while in rats which were unilaterally nephrectomized 10 days previously, followed by the removal of the remaining kidney (two-step bilateral nephrectomy), the maximum increase in plasma renin substrate was markedly suppressed (6-fold of normal). The maximum increases in plasma renin substrate by estradiol treatment in normal and unilaterally nephrectomized rats were about the same, associated with similarly decreased plasma renin concentrations. The similar increase in plasma renin substrate was found after ureteral ligation in unilaterally nephrectomized rats and bilateral ligation of the ureters in normal rats. This was the case where the plasma renin concentrations changed differently after ureteral ligation. After two-step bilateral nephrectomy plus estradiol treatment the maximum increase in plasma renin substrate was found to be higher than that found after two-step bilateral nephrectomy, but was lower than that after simultaneous bilateral nephrectomy. It is suggested that under the pathological conditions that stimulate renin substrate production, the plasma rein substrate concentration is less affected by circulating renin.     

Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.     

Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats

OBJECTIVE: The conventional surgical treatment of isolated critical stenosis of the left main coronary artery (LMCA) leads to the definitive occlusion of LMCA, restores only a retrograde perfusion to a rather extensive myocardial area and consumes bypass material. Direct surgical angioplasty avoids these inconveniences. METHODS: Between June 1985 and August 1996, 49 surgical angioplasties have been performed in 47 patients. LMCA was approached posteriorly in the first 11 procedures, and an anterior approach was preferred in the last 38 because of better exposure. The onlay patch consisted of saphenous vein in 37 cases; pericardium was used in 12 cases, and only for ostial stenosis. RESULTS: No technical failure occurred in the last 28 cases. 44 procedures, (90%), succeeded, but 1 patient (2.3%) died later of a massive air embolism, and 2 patients needed conventional CABG after 3 and 5 months, respectively. The 35 survivors still benefiting from a successful LMCA angioplasty on the long term are free of ischemia after a mean follow-up of 75 months (2-136). Angiographic restudy was obtained in 30 patients (70%) at an average of 38 months and revealed an excellent result in 26 (87%). In 10 patients, a late angiographic restudy at an average of 71 months (32-119) still revealed a perfect result. CONCLUSION: Provided that well-defined contra-indications (involvement of the distal bifurcation, heavy calcification) are respected, LMCA surgical angioplasty deserves a place in the array of surgical strategies.     

Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2

Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. We demonstrated that IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increased both protein and mRNA levels of IRS-3 but decreased those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective impairment of insulin-induced PI 3-kinase activation was observed in the LDM fraction, whereas PI 3-kinase activation was conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular compartments, which may contribute to determining the insulin sensitivity in adipocytes.     

Tissue-specific regulation of cytochrome c oxidase efficiency by nucleotides

Cytochrome c oxidase from bovine heart and liver was reconstituted in liposomes in the absence or presence of nucleotides. Intraliposomal ADP, and to a smaller extent intraliposomal ATP, increased the respiratory activity of the heart but not of the liver isozyme under uncoupled but not under coupled conditions, leading to increased respiratory control ratios. In a preceding publication [Anthony, G., Reimann, A., & Kadenbach, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 1652-1656], the stimulatory effect of intraliposomal ADP could be related to interaction with the matrix domain of subunit VIa-h (heart type). The data suggest a regulatory effect of matrix nucleotides in heart and skeletal muscle mitochondria on the efficiency of energy transduction in COX.     

Renal protective effects of efonidipine in partially nephrectomized spontaneously hypertensive rats

The influence of the internal features (eyes, nose, and mouth) in the age processing of unfamiliar faces was examined. Younger and older versions of the faces of six individuals (covering three different age ranges, from infancy to maturity) were used as donor stimuli. For each individual in turn, the effects on age estimates of placing older features in the younger face version (or vice versa) were investigated. Age estimates were heavily influenced by the age of the internal facial features. Experiment 2 replicated these effects with a larger number of faces within a narrower age range (after growth is complete and before major skin changes have occurred). Taken together, these two experiments show that the internal facial features may be influential in conveying age information to the perceiver. However, the mechanisms by which features exert their influence remain difficult to determine: although age estimates might be based on local information from the features themselves, an alternative possibility is that featural changes indirectly influence age estimates by altering the global three-dimensional shape of the head.     

Ovarian sensitivity to exogenous gonadotrophins in phenobarbital treated unilaterally ovariectomized albino rats

To evaluate platelet activity in patients with non-insulin-dependent diabetes mellitus (NIDDM), we measured the mean platelet volume (MPV) and 24-hour urinary excretion of 11-dehydro-thromboxane B2 (11-dTXB2) and 6-keto-prostaglandin F1 alpha (6-kPGF1 alpha), stable metabolites of thromboxane A2 and prostacyclin, respectively. The MPV of the 103 subjects in the NIDDM group were 10.72 +/- 0.82 fl for males and 10.52 +/- 1.01 fl for females (mean +/- SD), significantly higher than those of normal controls (9.95 +/- 0.75 fl for males and 9.84 +/- 0.72 fl for females). The MPV of patients with NIDDM showed positive correlations with fasting plasma glucose level and HbA1c (r = 0.234, P < 0.05; r = 0.267, P < 0.01, respectively). The urinary excretion of 11-dTXB2 was greater in the NIDDM group (7.58 +/- 4.42 micrograms/day for males and 5.65 +/- 2.38 micrograms/day for females) than in the normal controls (4.61 +/- 2.31 and 3.83 +/- 1.60, respectively), suggesting that the synthesis of thromboxane A2 by platelets may be accelerated in vivo in patients with NIDDM. The urinary 6-kPGF1 alpha was not different between the NIDDM group and normal controls among the males, but was greater in the NIDDM group among the females. As MPV showed a positive correlation (r = 0.364, P < 0.05) with urinary excretion of 11-dTXB2, MPV may be related to platelet activity. These findings suggest that the platelets of patients with NIDDM may be in a hyperactive state.     

Tissue-specific recognition of mouse CD1 molecules

Although there is evidence that some members of the CD1 gene family may present particular types of foreign Ags, such as mycobacterial lipid Ags or synthetic hydrophobic peptides, to alphabeta T cells, most CD1 isotypes share the unusual property of being recognized by a high frequency of naturally autoreactive alphabeta T cells. In the case of mouse CD1.1 and its human counterpart CD1d, a significant fraction of the autoreactive T cells express semi-invariant TCRs. CD1.1-specific T cells have a restricted tissue distribution and very promptly secrete a large panel of potent cytokines, including IL-4 and IFN-gamma, upon primary activation through their TCR, suggesting that they might regulate some immune responses in these tissues. We show here that their autorecognition of mouse CD1.1 is highly dependent upon the cell type in which CD1.1 is expressed. For example, some of these T cells only respond to CD1.1 expressed by splenic dendritic cells, some respond preferentially to cortical thymocytes, and others respond to splenic B cells. Tissue specificity of CD1.1 recognition is also observed with various cell lines transfected with CD1.1 cDNA. These results show that different CD1.1 self Ags are expressed in different tissues and can be specifically recognized by autoreactive T cells. They suggest that CD1.1 may be naturally associated with a variety of self ligands that overlap only partially in different cell types.     

Effect of Onpi-to (TJ-8117) on TGF-beta 1 in rats with 5/6 nephrectomized chronic renal failure

We and others have already reported that Onpi-to (TJ-8117), a Kampo medicine composed mainly of Rhei Rhizoma, has a beneficial effect on an adenine-induced renal failure model and 5/6 nephrectomized renal failure. However, little is known about the detailed mechanism of this medicine when used for renal failure. The present study was designed to clarify whether or not TJ-8117 affects TGF-beta 1 production or activation in glomeruli of 5/6 nephrectomized rats. TJ-8117 (400 mg/kg/ day) and captopril (50 mg/kg/day) were administered as drinking water from the day immediately after the operation and continued throughout the experiment. All rats were sacrificed at 4 weeks and renal cortical tissue was removed to quantify the protein and activity of TGF-beta 1 and activities of metalloproteinase. TIMP expression and extracellular matrix (collagen type I, IV) in the glomeruli were analysed histologically. TJ-8117 inhibited proteinuria, and the accumulation of collagen type I and IV in glomeruli of nephrectomized rats. In addition, TJ-8117 inhibited the TGF-beta 1 positive area in the glomeruli and the elevation of mature TGF-beta 1 level in the renal cortex of nephrectomized rats. In the TJ-8117 treated group, activities of metalloproteinase 1, 2 or 9 in the renal cortex were elevated compared with the control group. Captopril failed to affect the TGF-beta 1 level. We also found that the constitutive herbs in TJ-8117, Rhei Rhizoma and Ginseng radix, inhibited the process from inactive TGF-beta 1 to mature TGF-beta 1.     

Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats

We present a minimal kinetic model for excitatory synaptic transmission to cerebellar Purkinje cells. The main components are a kinetic model for a single glutamate receptor, which is calibrated with the help of patch clamp data, and a mean field approximation for the dynamics of a population of channels, which generate an EPSC. The resulting minimal model of the parallel fiber-Purkinje cell synapse is used to estimate the dynamics of glutamate in the synaptic cleft and to clarify the role of receptor desensitization in synaptic transmission. We also apply the model to different aspects of synaptic modulation, like long-term depression and potentiation by pharmacological application of ampakines. In the framework of the minimal model these effects can be understood as the result of modified receptor kinetics.     

Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats

BACKGROUND: Tubulointerstitial fibrosis is the final common pathway to end-stage renal failure. The present study investigated the potential role of tubular epithelial cells (TEC) in progressive fibrosis in the rat remnant kidney model. METHODS: Rats underwent 5/6 nephrectomy or a sham operation (control), and groups of six animals were killed at weeks 1, 3, 5, 9, 13, 17 and 21. RESULTS: Immunohistochemistry staining and in situ hybridization at week 3 after nephrectomy demonstrated de novo expression of alpha-smooth muscle actin (alpha-SMA)--a marker of smooth muscle cells and myofibroblasts--by TEC that was invariably associated with disruption of the tubular basement membrane (TBM). This phenotypic evidence of tubular epithelial-myofibroblast transdifferentiation was supported by ultrastructural studies identifying the presence of characteristic actin microfilaments and dense bodies within TEC with a transformed morphology. In the late stage of this apparent tubular epithelial-myofibroblast transdifferentiation, TEC lost apical-basal polarity and tight junctions, became elongated, detached from the TBM, separated from neighboring cells and appeared to migrate into the peritubular interstitium through the damaged basement membrane. Indeed, focal peritubular accumulation of alpha-SMA+ myofibroblasts and local tubulointerstitial fibrosis was closely associated with alpha-SMA+ tubules, suggesting a tubular epithelial origin for some of these cells. Quantitative analysis found a significant correlation between the number of alpha-SMA+ TEC and the accumulation of interstitial alpha-SMA+ myofibroblasts and the severity of tubulointerstitial fibrosis (both P < 0.001). CONCLUSIONS: This study provides phenotypic and morphological evidence to support the hypothesis that TEC are pro-fibrogenitor cells capable of tubular epithelial-myofibroblast transdifferentiation in progressive renal fibrosis. In addition, we postulate that disruption of the TBM, which facilitates epithelial cell contact with the interstitial matrix, promotes this process of transdifferentiation.     

Sequential alterations in clinical biochemical indicators of renal function in 5/6 nephrectomized rats--basic study for renal toxicity using 5/6 nephrectomized rats

It has been established that progression of renal lesions in 5/6 nephrectomized rats, in which the total right kidney was removed and two poles of the left kidney were excised surgically, and are used as an animal model of renal failure in man, can be morphologically divided into three stages. In the present study, for establishing a renal toxicity study using this animal model on the physiological side, changes of biochemical parameters were sequentially investigated in 20 male 5/6 nephrectomized Wistar rats until 26 weeks after nephrectomy. Creatinine clearance (CLcre) and water as well as electrolyte reabsorption (FRwater and FR Na, K, Cl) were reduced at weeks 2-4, then increased slightly from weeks 6 to 10, and reduced again thereafter. On the contrary, urinary protein was elevated throughout the experimental period, while albumin fraction was increased after week 2 and low-molecular tubular protein increased after week 6 by electrophoresis. Urinary LDH also demonstrated high levels throughout the observation period, but ALP only increased after week 18. The present study thus confirmed that renal function after 5/6 nephrectomy is indeed changed in three stages with clinical biochemical parameters, especially CLcre and FR Na, K, Cl being good indicators to distinguish the three stages of glomerular and tubular dysfunction, respectively. In addition, urinary protein-fractions by electrophoresis in this animal model were examined for the first time, proving useful approach to glomerular and tubular dysfunction.     

Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites

Serine and threonine phosphorylation has been shown to down-regulate insulin signaling at multiple steps, including the receptor and downstream molecules such as insulin receptor substrate-1 (IRS-1). To further address the mechanism of this regulation at the level of IRS-1, we constructed a double serine mutant of IRS-1: S662A/S731A-IRS-1. The serines 662 and 731 mutated to alanine are surrounding tyrosines Y658 and Y727, respectively. These tyrosines are comprised in YXXM motifs, which are potential binding sites for the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. In a first series of experiments using the yeast two-hybrid system, we show that IRS-1 interacts with p85alpha, and this interaction depends on tyrosine phosphorylation, as shown with the IRS-1 mutant F18 and 3Y-IRS-1. F18-IRS-1 contains 18 potential tyrosine phosphorylation sites mutated to phenylalanine; three of them, i.e. Y608, 628, and 658, which are potential binding sites for p85alpha, have been added back in the 3Y-IRS-1 mutant. The tyrosine phosphorylation of IRS-1, which is required for the interaction with p85alpha, is thought to occur via endogenous yeast kinases that phosphorylate IRS-1 at least on these PI 3-kinase-binding sites. Next, we show that not only p85alpha but also p55PIK, another regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast. Interestingly, for both regulatory subunits their interaction with IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1. In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1. Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1. In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity.