Effect of compression fracture on trabecular bone score at lumbar spine.

Bone mineral density (BMD) may be increased due to vertebral compression fractures (VCF). Our study showed trabecular bone scores (TBS) was less affected than BMD by fractured vertebrae. The TBS of most compression fractures, including old and recent VCF with mild or moderate deformity and old VCF with severe deformity, could still be used in predicting fracture risk. INTRODUCTION: Trabecular bone score (TBS), a noninvasive tool estimating bone microarchitecture, provides complementary information to lumbar spine bone mineral density (BMD). Lumbar spine BMD might be increased due to both degenerative disease and vertebral compression fractures (VCF). Lumbar spine TBS has been confirmed not influenced by osteoarthrosis, but the effects of VCF are still not been well evaluated. This study aimed to investigate whether lumbar spine TBS was affected by fractured vertebrae. METHODS: We studied postmenopausal women and men above 50 years old who underwent DXA between January 1, 2017, and May 31, 2019. By calculating the difference of BMD and TBS between L1 and the mean of L2-3, the study compared the difference of values between the control group and fracture group to determine the effects of fractured vertebrae on BMD and TBS. RESULTS: A total of 377 participants were enrolled with 202 in the control group (157 females; age: 68.06 ± 6.47 years) and 175 in the fracture group (147 females; age: 71.71 ± 9.44 years). The mean BMD of the L1 vertebrae in the fracture group was significantly higher than that in the control group (p < 0.0001). There was no significant difference between the mean differences of TBS between L1 and the means of L2-3 vertebrae in the control group and the most compression fractures, including old and recent VCF with mild or moderate deformity and old VCF with severe deformity. CONCLUSION: Lumbar spine TBS, unlike BMD, is less affected by fractured vertebrae. The TBS of most compression fractures, including old and recent VCF with mild or moderate deformity and old VCF with severe deformity, could still be used in predicting fracture risk.

Effect of benign prostatic hyperplasia on the development of spine, hip, and wrist fractures.

Benign prostatic hyperplasia is one of the most common diseases in the elderly male population. The urinary tract symptoms may increase the risk of falls and fractures. The results indicated that patients with benign prostatic hyperplasia could increase the risk of vertebral compression fractures in both the thoracic and lumbar spine and also hip fractures, but did not increase the risk of wrist fracture. INTRODUCTION: The relationship between benign prostatic hyperplasia and the development of fall-related fractures, especially vertebral compression fractures, has been seldom mentioned in the literature. This study aimed to evaluate the risk of developing vertebral compression fracture, hip fracture, and wrist fracture in patients with benign prostatic hyperplasia. METHODS: This study obtained claims data retrospectively from the National Health Insurance Research Database of Taiwan and identified 48,114 patients who were diagnosed as having benign prostatic hyperplasia. Subjects of the control cohort were individually matched at a ratio of 4:1 with those in the benign prostatic hyperplasia cohort according to age and the index day. Comorbidities were classified as those existing before the index day and included a previous fracture history, osteoporosis, myocardial infarction, congestive heart failure, diabetes mellitus, hypertension, cerebrovascular accident, etc. The end of the follow-up period of the analyses was the day when the patient developed new vertebral compression fractures, hip fractures, or wrist fractures, terminated enrollment from the National Health Insurance, or died or until the end of 2012. The study used the Cox proportion hazard model to determine the hazard ratio for developing new hip fractures. RESULTS: Patients with benign prostatic hyperplasia were significantly more likely than those in the control cohort to develop new vertebral compression fractures in the thoracic spine (0.43% vs. 0.40%, adjusted hazard ratio 3.03, confidence interval 2.12-4.31) and lumbar spine (1.26% vs. 1.23%, adjusted hazard ratio 4.12, confidence interval 3.39-5.01), and hip fracture (1.47% vs. 2.09%, adjusted hazard ratio 1.22, confidence interval 1.10-1.36), but does not increase the risk of wrist fracture (0.61% vs. 0.67%, adjusted hazard ratio 1.07, confidence interval 0.85-1.34). CONCLUSIONS: Patients with benign prostatic hyperplasia exhibited an increased risk of developing vertebral compression fractures in both the thoracic and lumbar spine and also hip fractures, but did not increase the risk of wrist fracture. However, more research is needed to confirm this trend in the clinical setting.

Benefits and pitfalls of iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) imaging in clinical application of the cervical spine MR.

AIM: To evaluate efficacy of T2-weighted (T2W) iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL)-fast spin echo (FSE) imaging of the cervical spine. MATERIALS AND METHODS: The cervical spine of 100 symptomatic patients was imaged using routine magnetic resonance imaging (MRI) versus IDEAL-FSE imaging. The signal-to-noise ratios (SNRs), contrast-to-noise ratios (CNRs), and image quality were analysed. To compare the diagnostic efficiency of degenerative spondylopathy, evaluations of spondylolisthesis, retrolisthesis, disc herniation, myelopathy, disc degeneration, and bone marrow oedema were also performed. RESULTS: IDEAL-FSE showed significantly higher SNRs and CNRs (all p<0.001) than fat-suppressed (FS) T2W-FSE. Sixteen of 100 patients had cervical spine instrumentation; in those patients, IDEAL-FSE provided significantly better uniformity of fat suppression (p<0.001) and fewer metallic artefacts (p<0.001). For patients without instrumentation, FS T2W-FSE showed significantly better overall image quality (p<0.001) and homogeneity of the cerebrospinal fluid (CSF; p<0.001) with fewer motion artefacts (p<0.001). IDEAL-FSE, however, provided significantly better uniformity of fat suppression (p<0.001). There were no significant differences in the diagnoses of spondylolisthesis, retrolisthesis, disc herniation, or myelopathy between IDEAL and FS T2W images. The only significant differences between the IDEAL and FS T2W images were noted when diagnosing degenerative disc disease at the C2-3 and C5-6 disc levels (p=0.019, p=0.002, respectively) and bone marrow oedema at C3 vertebral body (p=0.029). CONCLUSION: T2W IDEAL-FSE imaging should only be considered as an additional sequence to conventional FS T2W images in patients with poor fat suppression or severe metallic artefacts.

High-concentration iodinated contrast media for T1-weighted direct magnetic resonance arthrography.

AIM: To quantify in vitro the T1-weighted (T1W) expression of iodinated contrast media (CM), and to compare the in vivo performances of iodinated CM and gadolinium-based CM for T1W direct magnetic resonance imaging (MRI) arthrography. MATERIALS AND METHODS: An in vitro study on a 1.5 T MRI system was performed using Gd-DOTA, a mixture of iopromide and Gd-DOTA, and iopromide alone. The fat-suppressed (FS) T1W signal intensities were measured and analysed. In an in vivo study, 15 normal rabbits were used to compare the expression of iopromide (370 mg iodine/ml), and the mixture of iopromide and diluted Gd-DOTA. In nine of the 15 rabbits, extra-articular administrations of CM were performed to mimic the situation of CM leak. The rabbits were scanned on a 1.5 T MRI system, and the FS T1W sequence and an axial iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) T1W sequence were acquired. Signal intensities were measured and signal-to-noise ratios (SNR) were analysed. RESULTS: In the in vitro study, a higher SNR was noted in a higher concentration of iopromide, and the highest SNR of iopromide was 45.9% of that of Gd-DOTA. In the in vivo study, the iopromide and the mixture were well identified in all rabbits. The SNRs of the intra-articular and extra-articular iopromide and the mixture were significantly higher than the SNR of the muscles in the FS T1W images (all, p<0.01) and the IDEAL images (all, p<0.01). CONCLUSIONS: A high-concentration iodinated CM can provide good imaging quality for T1W direct MRI arthrography, and may be an alternative option in certain clinical situations.

A Point-based Mortality Prediction System for Older Adults with Diabetes.

The mortality prediction models for the general diabetic population have been well established, but the corresponding elderly-specific model is still lacking. This study aims to develop a mortality prediction model for the elderly with diabetes. The data used for model establishment were derived from the nationwide adult health screening program in Taiwan in 2007-2010, from which we applied a 10-fold cross-validation method for model construction and internal validation. The external validation was tested on the MJ health screening database collected in 2004-2007. Multivariable Cox proportional hazards models were used to predict five-year mortality for diabetic patients ≥65 years. A total of 220,832 older subjects with diabetes were selected for model construction, of whom 23,241 (10.5%) died by the end of follow-up (December 31, 2011). The significant predictors retained in the final model included age, gender, smoking status, body mass index (BMI), fasting glucose, systolic and diastolic blood pressure, leukocyte count, liver and renal function, total cholesterol, hemoglobin, albumin, and uric acid. The Harrell's C in the development, internal-, and external-validation datasets were 0.737, 0.746, and 0.685, respectively. We established an easy-to-use point-based model that could accurately predict five-year mortality risk in older adults with diabetes.

Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

Circadian rhythm of urinary endothelin-1 excretion in mild hypertensive patients.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide with diverse physiologic actions and has been considered to be involved in the pathogenesis of hypertension. We sought to investigate the role of renal synthesis of ET-1 in the regulation of daily sodium homeostasis and the possible contribution of renal synthesized ET-1 in the pathogenesis of essential hypertension (EHT). Urinary ET-1-like immunoreactivity (ET-1-L1) was measured by a radioimmunoassay after extraction in 23 EHT patients without detectable target organ damage, and in 11 normotensive controls. All study subjects received a controlled diet during an 8-day study period. Urinary and blood samples were collected by four sampling periods/day from the 4th to 6th days, and on the 7th day, study subjects were given an intravenous infusion of 1250 mL normal saline over 2 h. In the basal state, the urinary sodium and ET-1-L1 excretions showed diurnal patterns in both the normal and hypertensive groups, and urinary ET-1-L1 excretion rate correlated well with urinary sodium excretion rate. There were no differences found in plasma ET-1 levels, urinary ET-1-L1, and sodium excretion rates between the control and hypertensive groups. After saline infusion, ten hypertensive patients showed nonexaggerated natriuresis, and the 24-h urinary ET-1-L1 excretion (47.0+/-4.0 pmol/day), collected during the day of saline infusion, was significantly lower than that of the control group (86.3+/-10.0 pmol/day) or the exaggeratedly natriuretic hypertensive patients (91.7+/-8.4 pmol/ day). Our results suggest that renal ET-1 may be responsible for the renal handling of sodium homeostasis, and alteration of renal ET-1 synthesis may be a contributory factor in the pathogenesis of essential hypertension and salt sensitivity.

Neuroprotective effects of Hu-Yi-Neng, a diet supplement, on SH-SY5Y human neuroblastoma cells.

OBJECTIVES: Oxidative stress is considered the potential risk to the development of dementia. Some medicines, vitamins, and diet supplements have been suggested to have possible benefits via the antioxidative effects to slow the decline of cognitive function in demented and non-demented individuals. However, few studies were conducted to examine their functions, especially in composite diet supplements. Hu-Yi-Neng is a composite diet supplement, including ginkgo biloba, extract of pine bark, phosphatidyl serine, docosahexaenoic acid, and folic acid, used extensively in Taiwan. Therefore, our aim is to investigate the potential protective effects of Hu-Yi-Neng on human neuron cells. MATERALS AND METHODS: H2O2-induced neuronal toxicity was characterized in SH-SY5Y human neuroblastoma cells by the decrease of cell viability using PrestoBlue™ assay and by the increase of intracellular reactive oxygen species (ROS) level using DCFH-DA (2', 7'-dichlorodihydrofluorescin diacetate) assays. HO-1 mRNA expression was detected by real-time PCR. Akt and Erk 1/2 proteins were detected by western blotting. RESULTS: Pretreatment with Hu-Yi-Neng significantly reversed the decrease in cell viability induced by H2O2 in SH-SY5Y cells. Furthermore, Hu-Yi-Neng dose-dependently suppressed the elevation of intracellular reactive oxygen species (ROS) level. Hu-Yi-Neng protected SH-SY5Y cells from oxidative stress may via the increase in mRNA expression of heme oxygenase-1 (HO-1), an antioxidant enzyme. In addition, Hu-Yi-Neng inhibited H2O2-induced phosphorylation of Akt kinase but further increased the phosphorylation of Erk 1/2. CONCLUSION: Our results suggest that Hu-Yi-Neng has protective effect against oxidative stress-induced neuron cell loss and it could be an ideal composite diet supplement for preventing neurodegenerative diseases.

Metabolic effects of mulberry leaves: exploring potential benefits in type 2 diabetes and hyperuricemia.

The leaves of Morus alba L. have a long history in Traditional Chinese Medicine and also became valued by the ethnopharmacology of many other cultures. The worldwide known antidiabetic use of the drug has been suggested to arise from a complex combination effect of various constituents. Moreover, the drug is also a potential antihyperuricemic agent. Considering that type 2 diabetes and hyperuricemia are vice-versa in each other's important risk factors, the use of mulberry originated phytotherapeutics might provide an excellent option for the prevention and/or treatment of both conditions. Here we report a series of relevant in vitro and in vivo studies on the bioactivity of an extract of mulberry leaves and its fractions obtained by a stepwise gradient on silica gel. In vivo antihyperglycemic and antihyperuricemic activity, plasma antioxidant status, as well as in vitro glucose consumption by adipocytes in the presence or absence of insulin, xanthine oxidase inhibition, free radical scavenging activity, and inhibition of lipid peroxidation were tested. Known bioactive constituents of M. alba (chlorogenic acid, rutin, isoquercitrin, and loliolide) were identified and quantified from the HPLC-DAD fingerprint chromatograms. Iminosugar contents were investigated by MS/MS, 1-deoxynojirimycin was quantified, and amounts of 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin and fagomine were additionally estimated.

Widespread white matter alterations in patients with end-stage renal disease: a voxelwise diffusion tensor imaging study.

BACKGROUND AND PURPOSE: ESRD results in excessive accumulation of urea and toxic metabolites. Hemodialysis is usually performed to maintain health in patients with ESRD; however, it may cause silent white matter alterations in the earlier stages. Hence, this study aimed to perform voxelwise diffusion tensor analysis for global detection of subtle white matter alterations in patients with ESRD. MATERIALS AND METHODS: Twenty-eight patients with ESRD and 25 age-matched control subjects were enrolled in this study. Each subject underwent CASI assessment and DTI. After spatial normalization of DTI images, voxelwise statistical analyses were performed to compare DTI parameters between the 2 groups. RESULTS: In patients with ESRD, AD, RD, and MD values were significantly increased, whereas the FA value was significantly decreased, mostly in the corpus callosum, bilateral sagittal stratum, and pons. Multiple regression analysis further revealed that both RD and MD were positively correlated with the duration of hemodialysis in the pons; however, no significant correlation was observed with FA. Negative correlations of RD and MD and a positive correlation of FA with the CASI score were observed in the corona radiata. CONCLUSIONS: We concluded that voxelwise DTI analysis is helpful in the detection of white matter alterations caused by hemodialysis.

MRI of multifocal kaposiform haemangioendothelioma without Kasabach-Merritt phenomenon.

Kaposiform haemangioendothelioma is a rare soft-tissue tumour of infants and children, and presents as a moderately aggressive malignancy. We present the MRI findings of a histologically proven case of Kaposiform haemangioendothelioma without Kasabach-Merritt phenomenon or typical skin changes. Our case also reveals that the multiple foci of the cutaneous tumour have different MRI morphologies. These findings have not been reported in the literature to date.

Distinction between pyogenic brain abscess and necrotic brain tumour using 3-tesla MR spectroscopy, diffusion and perfusion imaging.

The purpose of this study is to compare the effectiveness of relative cerebral blood volume, apparent diffusion coefficient and spectroscopic imaging in differentiating between cerebral abscesses and necrotic tumours. In the prospective study, a 3-tesla MR unit was used to perform proton MR spectroscopy, diffusion and perfusion imaging in 20 patients with cerebral abscesses and 26 patients who had solitary brain tumours (14 high-grade gliomas and 12 metastases). We found the mean apparent diffusion coefficient value at the central cavities of the cerebral abscesses to be significantly lower than in necrotic tumours. The mean relative cerebral blood volume values of the necrotic tumour wall were statistically significantly higher than the mean relative cerebral blood volume values of the cerebral abscess wall by the Student's t-test. The proton spectra obtained revealed amino acids only in the cerebral abscesses. Although the conventional MRI characteristics of cerebral abscesses and necrotic tumours may sometimes be similar, diffusion, perfusion-weighted and spectroscopic MRI enables distinction between the two.

Huge renal lipoma with prominent hypervascular non-adipose elements.

A primary renal lipoma is a very rare neoplasm of the kidney and only 20 cases have been reported in the literature. We report a case of a huge retroperitoneal mass that presented as a hypervascular tumour with a prominent fat component, mimicking an angiomyolipoma or a liposarcoma in imaging studies. This finding presented a diagnostic challenge in terms of selecting medical versus surgical intervention. Surgical excision of the lesions was performed and the pathological evaluation revealed a renal lipoma with extrarenal growth.

Transforming growth factor-beta 1 stimulates angiotensinogen gene expression in kidney proximal tubular cells.

The present study investigated whether transforming growth factor-beta 1 (TGF-beta1) exerts an autocrine positive effect on angiotensinogen (ANG) gene expression in rat kidney proximal tubular cells, and delineates its underlying mechanism(s) of action. Rat immortalized renal proximal tubular cells (IRPTCs) and freshly isolated mouse renal proximal tubules were incubated in the absence or presence of active human TGF-beta1. IRPTCs were also stably transfected with rat TGF-beta1 or p53 tumor suppressor protein (p53) cDNA in sense (S) and antisense (AS) orientations. ANG mRNA and p53 protein expression were assessed by reverse transcription-polymerase chain reaction and Western blotting, respectively. Reactive oxygen species (ROS) generation was quantified by lucigenin assay. Active TGF-beta1 evoked ROS generation and stimulated ANG mRNA and p53 protein expression, whereas a superoxide scavenger and inhibitors of nicotinamide adenine dinucleotide oxidase and p38 mitogen-activated protein kinase (p38 MAPK) abolished the TGF-beta1 effect. Stable transfer of p53 cDNA (S) enhanced and p53 cDNA (AS) abolished the stimulatory effect of TGF-beta1 on ANG mRNA expression in IRPTCs. Our results demonstrate that TGF-beta1 stimulates ANG gene expression and its action is mediated, at least in part, via ROS generation, p38 MAPK activation, and p53 expression, suggesting that angiotensin II and TGF-beta1 may form a positive feedback loop to enhance their respective gene expression, leading to renal injury.

Upregulation of osteopontin gene expression in diabetic rat proximal tubular cells revealed by microarray profiling.

Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5-70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-beta1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-beta1) cDNA or antisense TGF-beta1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin-angiotensin system activation, TGF-beta1 expression, and PKC-beta1 signaling.

RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.

Angiotensinogen (ANG) is the sole substrate of the renin-angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury.

Cytotoxic constituents of the fruits of Cananga odorata.

A new guaipyridine sesquiterpene alkaloid, cananodine (1), and two new eudesmane sesquiterpenes, cryptomeridiol 11-alpha-L-rhamnoside (2) and gamma-eudesmol 11-alpha-L-rhamnoside (3), along with gamma-eudesmol (4), were isolated from the fruits of Cananga odorata. The structures of compounds 1-3 were established on the basis of NMR and MS methods. In addition, compounds 1-4 and four previously reported alkaloids, cleistopholine (5), N-trans-feruloyltyramine (6), (+)-ushinsunine-beta-N-oxide (7), and lyscamine (8), were evaluated for cytotoxicity against two human hepatocarcinoma cell lines.

Increased atrial natriuretic peptide mRNA expression in the kidney of diabetic rats.

To investigate whether renal synthesis of atrial natriuretic peptide (ANP) is influenced in diabetes, we measured renal ANP mRNA levels, urine volume, urinary ANP and sodium excretion rates in streptozotocin (STZ)-induced diabetic rats. By using reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis, we found that renal cortical and outer medullary ANP mRNA levels in untreated diabetic rats were markedly increased as early as the second day after the onset of hyperglycemia and remained elevated for the entire 42-day study period. Plasma ANP concentrations in untreated diabetic rats were increased on the 42nd day, whereas plasma renin activity were suppressed. The urine volume, urinary ANP and sodium excretion rates in untreated diabetic rats were also significantly elevated on the second day and remained elevated for the entire 42-day study period. Urinary ANP excretion rates were well correlated with urine volume, and urinary sodium excretion rate in normal rats and diabetic rats on days 2, 4, 7, 14 and 42. Our results indicate that renal ANP mRNA expression is enhanced in diabetic rats, and that renal-synthesized ANP as one of regulators to handle water and sodium balance in diabetic rats is worthy of further investigation.

Increased renal atrial natriuretic peptide synthesis in rats with deoxycorticosterone acetate-salt treatment.

To investigate the role of renal synthesis of atrial natriuretic peptide (ANP) as a contributor to the water-sodium homeostasis, we studied the effects of electrolyte-water imbalance on renal ANP mRNA levels, plasma ANP concentrations, and urinary ANP excretion rates by using reverse transcription-polymerase chain reaction (PCR) and radioimmunoassay. Male Wistar rates divided into the following three groups: 1) the control group, 2) deoxycorticosterone acetate (DOCA)-salt-treated group, and 3) low-salt-treated group. The urinary sodium excretion rate and urine volume in the DOCA-salt rats were significantly elevated at 2 days and for the 10-day study. The urinary ANP excretion rate in DOCA-salt rats was significantly increased at 2 days after treatment and was well correlated to the urinary sodium excretion rate (r = 0.76, P < 0.01). Plasma ANP levels in the DOCA-salt rats were elevated on the day of death. In contrast, plasma renin activities were markedly suppressed in DOCA-salt rats and increased in low-salt rats. By immunohistochemical study, immunoreactive ANP materials were mainly localized in the proximal and distal cortical tubules of the kidney. With the PCR cloning and sequencing technique, ANP cDNA was cloned from the rat kidney, and the sequences were identical to that of ANP identified in the atria. By semiquantitative PCR technique, the expression of ANP mRNA in the ventricle and renal cortex tissues was significantly enhanced in the DOCA-salt rats. Our results confirm that the rat kidney is a site of ANP synthesis and indicate that renal ANP synthesis is enhanced in a volume-expansion state. We propose that renal synthesized natriuretic peptide participates in the intrarenal regulation of water-electrolyte homeostasis and may contribute to renal adaptation during the mineralocorticoid escape phenomenon.

Decreased GTPase activity of K-ras mutants deriving from human functional adrenocortical tumours.

Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K-ras gene mutation. In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61). To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K-Ras oncogene and the alterations of its biocharacteristics, mutant K-Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV. Mutant K-Ras genes were expressed at high levels in Escherichia coli and the resultant K-Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. The purified K-Ras protein from E. coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase-activating protein for Ras. The results showed that the wild-type cellular K-Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K-Ras gene, which mutated at codon 60. With regards to the codon 15, 16, 18 and 31 mutant K-Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K-Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K-Ras protein. These results indicated that mutations at these hot spots of K-Ras gene were indeed activated K-Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove.