The characteristics of bone turnover in the second decade in relation to age and puberty development in healthy Japanese male and female subjects--Japanese Population-based Osteoporosis Study.

BACKGROUND: There are few studies that clarify the characteristics of bone turnover in children and adolescents. Furthermore, little has been published on changes in urinary CrossLaps(TM) (CTx) in Japanese subjects. AIM: To investigate biochemical markers of bone turnover in subjects, in relation to age and puberty development. SUBJECTS AND METHODS: We measured serum bone specific alkaline phosphatase (B-Alp) and CTx in 1207 Japanese subjects aged 9-18 years. As an indicator of puberty development, the age that pubic hair appeared in males and menstruation started in females was obtained from questionnaires. RESULTS: B-Alp and CTx/Cr (creatinine) had high values before and just after the indicators and was lower thereafter, reaching a plateau in both genders. There was no significant difference in these values in males 5-6 years, or 7 years and more after the appearance of pubic hair. B-Alp and CTx/Cr values 7 years and more after menarche were significantly lower than those 5-6 years after menarche, however the differences were relatively small. CONCLUSIONS: Subjects in the second decade can be divided into three groups: 'before the appearance of pubic hair for males and menarche for females', 'up to and including 3-4 years after them' and '5-6 years and more after them'.

Age-specific values and cutoff levels for the diagnosis of osteoporosis in quantitative ultrasound measurements at the calcaneus with SAHARA in healthy Japanese women: Japanese population-based osteoporosis (JPOS) study.

To establish the reference values of the quantitative ultrasound (QUS) indices in healthy Japanese women and to propose a diagnostic criterion for osteoporosis by means of the QUS indices, 659 healthy women aged 20-79 years recruited from a larger cohort study (JPOS study), were examined for bone mass measurements by QUS at the calcaneus (SAHARA, Hologic Inc., USA) and by dual-energy X-ray absorptiometry at the spine, hip, and distal forearm. We presented 10-year age-specific mean values and T-scores of the QUS indices. The pattern of decrease in the T-score appeared to be linear in the QUS indices and total hip BMD but not in BMD at the spine. The T-score of the QUS of indices of the subjects in their 70s were significantly higher than that of BMD at the spine. The prevalence rates of osteoporosis in the subjects aged 50 and older diagnosed by QUS (8.7% for SOS, 10.7% for BUA) were similar to that diagnosed by total hip BMD (11.5%) and significantly lower than that by the spine BMD (36.1%) when the WHO criteria were applied. We performed receiver-operating characteristic analysis to set a cutoff level of the QUS indices for the diagnosis of osteoporosis to accurately identify the subjects diagnosed by either the spine or total hip BMD. The highest likelihood ratios for SOS and BUA were obtained at the cutoff levels of 1,517.7 m/sec (T-score: -1.58) with the sensitivity of 0.65 and the specificity of 0.65 and 59.5 dB/MHz (T-score: -1.52) with 0.66 and 0.69, respectively. The diagnostic accuracy of QUS indices for osteoporosis was not superior to that of age. However, the QUS indices showed a significant contribution to forming the diagnosis of osteoporosis independently of age and body size in multivariate diagnostic models developed by the logistic regression analysis. Therefore, the cutoff values presented in this study may be used as a tentative criterion until the cutoff levels for the QUS indices are set according to the fracture risk.

Clinical effect of intravenous calcitriol administration on secondary hyperparathyroidism. A double-blind study among 4 doses.

BACKGROUND/AIMS: Although the PTH-suppressive effect of intravenous calcitriol has already been demonstrated by various studies, the precise dose-response to calcitriol has not been fully determined for uremic secondary hyperparathyroidism (2HPT). In order to investigate in detail the dose-response of intravenous calcitriol and the adequate initial dose against 2HPT, a randomized prospective double-blind study was conducted. METHOD: One-hundred and sixty-two patients with 2HPT undergoing hemodialysis three times per week were randomly assigned to four calcitriol (Ro21-5535) treatment groups, 0 (placebo), 1, 1.5 or 2 microg. Calcitriol or placebo was given intravenously after each dialysis for 12 weeks under double-blind conditions. RESULTS: Calcitriol dose-dependently reduced both intact-PTH and high-sensitivity assay mid-terminal (HS)-PTH levels. The rate of per-week change in intact-PTH was 0.0% in the placebo group, -7.8% in the 1-microg group, -18.9% in the 1.5-microg group and -24.1% in the 2-microg group. Calcitriol dose-dependently increased the rate of increase in serum Ca adjusted by albumin level. The per-week increases in adjusted serum Ca were -0.01, 0.08, 0.23 and 0.35 mg/dl in the placebo, 1-, 1.5- and 2-microg groups, respectively. Although the degree of PTH suppression was correlated with the adjusted serum Ca increase, by-patients investigation revealed that the number of patients with suppression of PTH despite of no or slight elevation of adjusted serum Ca level was largest in the 1-microg group among the three calcitriol groups. CONCLUSION: Intravenous calcitriol was found to have a clear dose-dependent effect on PTH reduction in patients with 2HPT, and the appropriate initial dose of this agent was determined to be 1 microg per dialysis session.

Standalone cutting balloon angioplasty for the treatment of stent-related restenosis: acute results and 3- to 6-month angiographic recurrent restenosis rates.

Despite excellent acute reperfusion results, 20%-30% of patients who undergo coronary stent implantation will develop angiographic restenosis and may require same additional treatments. Cutting Balloon angioplasty (CBA) causes less histological damage outside of the incised area than a regular balloon. However, regular plain old balloon angioplasty is sometimes required before CBA, as is adjunctive stenting and adjunctive angioplasty. These adjunctive strategies may negate the advantages of CBA. There is little data available on CBA as a standalone therapy for stent-related restenosis (SRS). The aim of this study was to evaluate the acute and 3- to 6-month angiographic recurrent restenosis rates following standalone CBA in a patient population treated for SRS and in whom optimal acute results were obtained. In this study, 40 patients with SRS (54 lesions) underwent standalone CBA with optimal acute results. For all lesions, coronary angiography was conducted before and after a standalone CBA procedure for SRS and systematically during 3-6 months to assess recurrent angiographic restenosis rates in the study population. In the study lesions, SRS was either diffuse disease (> 15 mm; 52%) or focal type (48%). Cutting Balloon diameter was 3.20 +/- 0.44 mm and maximal inflation pressure 8.7 +/- 1.2 atm. Ratio of Cutting Balloon diameter to restenotic stent diameter was 0.996 +/- 0.487. Multiple inflations (6 +/- 3 times) were performed. Number of used Cutting Balloon was 1.02 +/- 0.14. Complications were as follows; one non-Q-wave MI (1.9%); 0 death (0%), and 17 repeat target lesion revascularizations (TLRs; 32%). Follow-up coronary angiography (CAG) was not attained for one patient. The angiographic recurrent restenosis rate was 34%, with a higher rate observed when the SRS was diffuse type, 50% vs. 16% for focal-type SRS (P < 0.01). The recurrent restenosis rate for smaller vessels (vessel diameter < or = 3.0 mm) was the same as for larger ones. At follow-up CAG, diffuse-type recurrent restenosis (56%) presented nearly as frequently as that presenting in the original SRS lesions (52%). But four diffuse-type SRS (29%) changed into focal-type recurrent stenosis. In this study, standalone CBA for SRS with optimal acute results was associated with an angiographic restenosis rate of 34%. Diffuse-type disease had a higher recurrent restenosis rate. When CBA achieves acute optimal results, adjunctive stenting or adjunctive PTCA are not always necessary, particularly when the SRS is focal. As a result of CBA, some diffuse-type SRS may change into focal-type recurrent stenosis by the time of the next intervention.

Efficient and ligand-dependent regulated erythropoietin production by naked dna injection and in vivo electroporation.

The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiencies-disorders. However, the ability to regulate transgene expression will become paramount for safety and efficacy in gene transfer therapy. We have described the construction of an efficient and ligand-dependently regulated erythropoietin (EPO) production system using naked plasmid and in vivo electroporation. Two plasmids, one encoding the chimeric GeneSwitch protein and the other encoding an inducible transgene for human EPO, were developed. Modulation of the level of secretion of EPO into the serum was achieved by intraperitoneal administration of mifepristone (MFP). Rats were divided into 4 groups: one group received EPO plasmid with MFP for 30 days, a second group received with EPO plasmid MFP for 9 days, a third group received EPO plasmid without MFP, and a fourth group received control plasmid. A pair of electrodes was inserted into the muscle of the right thigh and 100 micrograms of each plasmid was injected. In vivo electrporation (8 times at 100 V for 50 milliseconds) was performed. The presence of vector-derived EPO mRNA was detected by reverse transcriptase-polymerase chain reaction only in the EPO and MFP(+) group. The hematocrit levels increased continuously from the preinjection level of 42.7% to 53.8% on day 28 in the EPO and MFP(+) group. The serum EPO levels increased only in the EPO and MFP(+) group. There was no significant change in hematocrit levels and EPO levels in the EPO and MFP(-) group. These results demonstrate that EPO gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient and drug-dependent regulated delivery of EPO.

Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Developmental expression of CLC-K1 in the postnatal rat kidney.

CLC-K1, a kidney-specific chloride channel, has been demonstrated to be involved in the urine concentration mechanism. Here, we investigated the developmental expression of CLC-K1 in the rat kidney. Using immunohistochemistry, we showed that CLC-K1 was not present in the thin ascending limb of Henle's loop during the early prenatal stages but was significantly expressed during the adult stage. CLC-K1 started to appear at day 5 and its expression increased during further development. In developing rats this increase coincided with the increase in the urine-concentrating capacity as the animals matured. We also investigated the expressions of other channels and transporters, including NKCC2, AQP-1, and AQP-2. NKCC2 was strongly expressed throughout the inner medulla in neonatal rat kidneys but was entirely undetectable at the adult stage. The decline in its expression took the form of a gradual recession from the inner medulla together with reciprocal increases in the expression of CLC-K1. AQP-1 was weakly expressed in the inner medulla during early development and showed a rapid increase in expression at a later stage. The collecting duct cells significantly expressed AQP-2 even at birth and maintained its expression throughout the development. These results suggest that CLC-K1 expression is one of the major determinants of the urine-concentrating capacity of the developing rat kidney.

Bone mineral density of the spine, hip and distal forearm in representative samples of the Japanese female population: Japanese Population-Based Osteoporosis (JPOS) Study.

Low bone mineral density (BMD) is one of the most important elements for the diagnosis of osteoporosis and screening people with higher risk of fractures. To establish the criterion value of BMD for the diagnosis of osteoporosis and to estimate the prevalence rate of osteoporosis in Japanese women, we performed a Japanese population-based osteoporosis (JPOS) study. The subjects were 4550 women aged 15 through 79 years randomly selected from seven municipalities throughout Japan. The sample size was determined to ensure that the observed mean BMD would remain within 2.5% from the real value with a probability of 0.95 in each of the 5-year age groups. The study comprised bone mass measurements by dual-energy X-ray absorptiometry at the spine (L2-4), hip and distal forearm, body size measurements and detailed interviews on medical and gynecologic history. After excluding those subjects with apparent or suggested abnormalities affecting bone mass from 3985 women (87.6%) who completed the study, 3465 women remained and served as the subjects. We present 5-year age-specific mean values of BMD and cut-off values for the diagnosis of osteoporosis according to World Health Organization (WHO) and the Japanese Society of Bone and Mineral Research (JSBMR) criteria. The cut-off levels at the spine and the distal radius proposed in this study were similar to those proposed by the JSBMR but the cut-off level at the femoral neck in this study was 4.7% higher than that of the JSBMR. The prevalence rates of osteoporosis according to WHO criteria in the present subjects aged 50 through 79 years were calculated as 38.0% at the spine, 11.6% at the femoral neck and 56.8% at the distal one-third site of the radius, and those in the Japanese female population of the same age were estimated to be 35.1%, 9.4% and 51.2%, respectively. A fivefold difference was observed among the prevalence rates at different skeletal sites, which suggests that the different definitions of osteoporosis should be established for the different skeletal sites. The prevalence rate diagnosed at the femoral neck seemed to be lower in the present study than those reported for Caucasians. This might account for a lower incidence rate of hip fracture in Japanese women.

Isolation of a novel zinc finger repressor that regulates the kidney-specific CLC-K1 promoter.

CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, are transcriptionally regulated on a tissue-specific basis. We have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, a kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated as KKLF (kidney-enriched Krüppel-like factor) and the myc-associated zinc finger protein (MAZ) were cloned. KKLF was found to be abundantly expressed in the liver, kidney, heart, and skeletal muscle. In the kidney, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments where CLC-K1 and CLC-K2 were not expressed. Gel mobility shift assay revealed that recombinant KKLF and MAZ proteins exhibited sequence-specific binding to the CLC-K1 GA element and that the consensus sequence for the KKLF binding site was GGGGNGGNG. In transient transfection, MAZ had a strong activating effect on the CLC-K1-luciferase reporter gene transcription. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue and nephron segment-specific expression of CLC-K1 and CLC-K2 channel genes through their GA cis element.

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells.

BACKGROUND: The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo. METHODS: The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis. RESULTS: The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H2O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis. CONCLUSION: The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.

Analysis of differentially expressed genes in human hepatocellular carcinoma using suppression subtractive hybridization.

The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analysed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with 2 different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. We identified 7 known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein alpha, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and 2 previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnostic markers, or determining novel therapeutic targets.

Glomerulonephritis after methicillin-resistant Staphylococcus aureus infection resulting in end-stage renal failure.

A 58-year-old man developed proteinuria and renal dysfunction following pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin was administered, and prednisolone pulse therapy and plasmapheresis were performed. Subsequently, serum creatinine was decreased. Eight months later, creatinine and CRP were again elevated, and MRSA was detected. Vancomycin was again administered and plasmapheresis was performed. However, renal function was not improved and continuous hemodialysis was initiated. This case indicates that complete eradication of MRSA is necessary to treat MRSA-associated glomerulonephritis, and if this is not attained, a permanent loss of renal function occurs.

Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant.

CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.

TT virus is shown in the liver by in situ hybridization with a PCR-generated probe from the serum TTV-DNA.

BACKGROUND AND AIM: It has been a conflicting issue whether TT virus (TTV), a newly isolated DNA virus from a patient with liver injury of unknown cause, is a causative agent of acute and/or chronic hepatitis. TT Virus DNA titers were shown to be 10-100-fold greater in liver tissue than in serum, whereas the majority of TTV-positive cases had no biochemical or histological evidence of significant liver damage. We therefore attempted in situ hybridization to investigate whether TTV is hepatotropic. METHODS: Because of the marked divergence in TTV genome types, a template for TTV-DNA (coding region for N22 clone) was amplified and labeled with digoxigenin-dUTP by using hemi-nested PCR from the serum, then DNA probes were applied to the liver sections of the same case. After hybridization, the probes were visualized immunohistochemically. Besides TTV-DNA-negative cases, competitive inhibition experiments with unlabeled probes were performed to confirm the specificity. RESULTS: There were no positive signals in the negative controls, and the intensity of positive signals was markedly diminished in the competitive inhibition experiments. No cross-hybridization with different genotype probes also confirms the specificity. Under the optimal conditions, the positive signals were located in the cytoplasm of the hepatocytes in eight of nine TTV-DNA-positive cases. The signals were not seen in non-parenchymal cells of the liver. CONCLUSION: TT Virus is proved to be hepatotropic by in situ hybridization.

Sequences in the NS5A protein of hepatitis C virus and the serum alanine aminotransferase response to interferon therapy in Japanese patients.

BACKGROUND AND AIMS: Chronic hepatitis C is a slowly progressive disease and eventually causes hepatocellular carcinoma in many patients. Although interferon (IFN) therapy has been used for viral eradication, its success rate is only about 30%. In patients in whom it has failed (non-responders), there are several patterns of serum alanine aminotransferase (ALT) values, and detection of serum HCV-RNA during and after IFN therapy and improved long term prognosis were reported in patients whose serum ALT values were normalised by IFN therapy even if HCV viraemia persisted. The present study sought to clarify the virological characteristics contributing to these differences. METHODS: Complete or partial length dominant sequences of hepatitis C virus genotype 1b (HCV-1b) were determined by direct sequencing. Firstly, the complete sequences of HCV-1b genomes were determined in six non-responders; three showed normalisation of serum ALT values during IFN-alpha therapy and the other three did not. Subsequently, the amino acid residues that were different in the two groups were further analysed retrospectively in another 82 patients. RESULTS: Comparison of the sequences suggested an association between amino acids 2154-2172 of HCV-1b and serum ALT normalisation. A retrospective analysis of 82 patients revealed that the number of amino acid substitutions in this region was the only statistically significant variable associated with ALT normalisation (odds ratio 31.0; 95% confidence interval 5.0-286) in multivariate analyses. CONCLUSIONS: A HCV genomic region that correlates with the ALT response to IFN therapy appears to be present in virologically IFN ineffective patients.

Overexpression of protein kinase G using adenovirus inhibits cyclin E transcription and mesangial cell cycle.

The cGMP-cGMP-dependent protein kinase (protein kinase G) system plays an important role in the pathogenesis of mesangial proliferative glomerulonephritis. However, the molecular mechanisms of the inhibitory effects of the cGMP-protein kinase G system in the cell cycle progression of mesangial cells are not well known. To determine the inhibitory pathway of cGMP-protein kinase G in cultured mesangial cells, we investigated the effects of cGMP- and adenovirus-mediated overexpression of protein kinase G on the promoter activities of cyclin E, cyclin D1, and cyclin A. 8-Bromo-cGMP (8-BrcGMP) and overexpression of protein kinase G reduced [(3)H]thymidine uptake, reduced the numbers of cells in S and G(2)/M phases, and decreased the phosphorylation of retinoblastoma (Rb) protein. 8-BrcGMP (10(-3) M), protein kinase G adenovirus (Ad-cGKIbeta; 10(10) plaque-forming units/ml), atrial natriuretic peptide (ANP), and C-type natriuretic peptide (CNP) inhibited the promoter activity of cyclin E to 49, 57, 77, and 78%, respectively. On the other hand, the promoter activities of cyclin D1 and cyclin A were not changed significantly. In Western blot analysis, 8-BrcGMP, Ad-cGKIbeta, ANP, and CNP also inhibited cyclin E protein expression dose and time dependently. The p44/p42 mitogen-activated protein kinase (MAPK) kinase 1-p44/p42 MAPK had no effect on cyclin E promoter activities, and the cGMP-protein kinase G pathway did not change MAPK activity. In conclusion, our findings suggest that the reduction of the cyclin E promoter activity that downregulates G(1)/S transition plays a dominant role in the cGMP- and protein kinase G-induced inhibition of mesangial cell proliferation.

Glucocorticoids stimulate p21(CIP1) in mesangial cells and in anti-GBM glomerulonephritis.

BACKGROUND: Glucocorticoids are widely used for the treatment of glomerulonephritis, but the mechanism of cell cycle inhibition by glucocorticoids is poorly understood at a molecular level. METHODS: The effects of dexamethasone on cell cycle progression were examined in rat mesangial cells. To investigate the mechanisms of cell cycle inhibition by dexamethasone, we transfected the -2.3 kb p21(CIP1) promoter-CAT construct to mesangial cells using an electroporation METHOD: We also examined whether glucocorticoids stimulate the expression of p21(CIP1) and inhibit cell proliferation in glomeruli of anti-glomerular basement membrane (GBM) glomerulonephritis in rats. RESULTS: Dexamethasone inhibited 3H-thymidine uptake and the percentages of S and G2/M phases in rat mesangial cells. Dexamethasone stimulated CAT activity of the p21(CIP1) promoter 4.5-fold. Deletion analysis of the p21(CIP1) promoter revealed that the glucocorticoid-responsive region (GRE) is present between -1.4 and -1.1 kb upstream of the transcription initiation site. Dexamethasone inducibility of p21(CIP1) promoter activity requires the presence of the C/EBP alpha DNA binding site in the GRE of the p21(CIP1) promoter and C/EBP alpha protein. Intravenous injection of anti-GBM antibody caused mesangial proliferation, crescent formation, and proteinuria in rats. Ten days of administration of prednisolone (1 mg/kg/day) reduced proteinuria and inhibited mesangial cell proliferation and crescent formation. The glomerular-sieving method revealed that prednisolone increased p21(CIP1) expression in glomeruli. CONCLUSION: These data suggest that the cell cycle arrest of mesangial cells is mediated by a functional link between the glucocorticoid receptor and the transcriptional control of p21(CIP1) not only in vitro but also in vivo. Our observations provide new insights into the molecular mechanisms of glucocorticoid action in glomerulonephritis.

Two sporadic cases of Liddle's syndrome caused by De novo ENaC mutations.

Liddle's syndrome is a rare form of hereditary hypertension caused by mutations of the epithelial sodium (Na(+)) channel (ENaC). Analysis of the diseased pedigrees indicates an autosomal dominant inheritance, and the identified mutations are heterozygotes of gain-of-function mutations. However, sporadic cases of Liddle's syndrome have been reported in the literature, including one recently reported case caused by a de novo mutation of ENaC. We identified two patients with Liddle's syndrome who did not have family histories of hypertension. Sequence analysis showed a mutation in each case (P616L in betaENaC and W576X in gammaENaC), both confirmed to be de novo mutations. These data indicate that Liddle's syndrome should be considered even in patients without a family history of hypertension.

Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype 1b infection.

To explore the relationship between responses to interferon (IFN) and the mutation patterns in the IFN sensitivity-determining region (ISDR; amino acid positions 2209-2248) in the NS5A gene of hepatitis C virus genotype 1b, a cohort of 334 patients was analyzed. The number of mutations in the ISDR was higher in patients with sustained response (SR) than in patients with transient or no response (P<.001). Patients with viruses mutated at positions 2209 (P=.02), 2216 (P=.01), or 2227 (P=.02) more frequently experienced SR than did those without these mutations. Mutation occurred most frequently at position 2218, where the presence of cysteine was significantly associated with SR. Thus, the mutation pattern in the ISDR affects the virologic response to IFN and reflects different influences on the function of the NS5A protein. ISDR sequence analysis would allow the prediction of clinical IFN efficacy in individual patients.

Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis in cardiomyocytes.

Cardiomyocytes are terminally differentiated cells characterized as withdrawal cell-cycle machinery, but nonetheless they are known to express cell-cycle regulators. Because many proteins related to the cell cycle induce apoptosis in proliferating cells, we examined the involvement of these proteins in the apoptosis pathway in cardiomyocytes. Primary rat cardiomyocytes were exposed to a severe hypoxic condition to induce apoptosis. The apoptosis rate of cardiomyocytes increased to approximately 40% under 24 hours of hypoxia as evaluated by the TUNEL method. The cyclin A protein level assessed by immunoblot analysis accumulated in a time-dependent manner in cardiomyocytes, but there was no increase in nonmyocytes. Hypoxia increased the activity of cyclin A-associated kinase but not the activity of cyclin E-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus, suggesting that cyclin A and its associated kinase play significant roles in the apoptosis of cardiomyocytes. To investigate the cyclin A-mediated apoptosis, we infected cultured cells with cyclin A adenovirus. Apoptosis was induced in 63+/-12% of the infected cardiomyocytes in contrast to only 12+/-3% of the LacZ-infected control cells. In addition, the cells in the hypoxic condition showed an increase in caspase-3 activity and a subsequent decrease in p21(cip1/waf1) protein, which is partly cleaved by caspase-3. These findings confirm that cyclin A-associated kinase mediates hypoxia-induced apoptosis in cardiomyocytes, and they also suggest that additional elements of the cell-cycle-dependent machinery participate in this mechanism.