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The appearance of alpha-smooth-muscle-actin (alpha-smA)-positive cells during hepatic fibrosis was studied immunohistochemically in rat and human livers. In the normal rat liver, alpha-smA was observed only in vascular smooth muscle cells. With the progression of fibrosis induced by CCl4 injection, alpha-smA-positive cells appeared in the perisinusoidal space and the fibrous septa, and ultimately surrounded regenerative nodules. An increase of desmin-positive cells was recognized in the fibrotic areas and the perisinusoidal area. In the human liver, alpha-smA-positive cells appeared in the fibrotic area, whereas no desmin-positive cells were observed, except in vascular walls of the central vein and the portal tract, alpha-smA is a good marker for the detection of myofibroblast-like cells, and the appearance of alpha-smA in liver mesenchymal cells seems closely related to the process of hepatic fibrosis in both rat and man.

Background: Health-related quality of life (HQOL) of predialysis patients with chronic renal failure (CRF) has received less attention than that of dialysis patients. We investigated changes in SF-36 over 1 year and examined associations between clinical parameters and SF-36 in predialysis CRF patients. Methods: Subjects were 471 predialysis CRF patients. SF-36 and clinical parameters were measured every 8 weeks for 48 weeks. Of the 471 subjects, 294 underwent one or more follow-ups. We analyzed the pooled dataset of the 294 CRF patients and 2002 subjects from Japanese general population using analysis of covariance. Results: After adjustment for age and sex, the 1-year declines in SF-36 domains were significantly greater in the predialysis patients than in the general population. For a 10% decline in hematocrit from the baseline survey value, the decline in vitality of SF-36 was 4.5 points (p = 0.003), while for a 10% increase in serum creatinine from the baseline survey value, respective declines in physical functioning, role-physical and mental health were 1.2 (p = 0.004), 1.9 (p = 0.035), and 1.0 points (p = 0.008). Conclusion: Among these predialysis CRF patients, the decline in HQOL was faster than that in the general population, and was associated with an increase in serum creatinine and decline in hematocrit.

Muscle cramps are one of the most common complications of hemodialysis (HD), and often are a source of great pain in spite of various clinical measures. The traditional herbal medicine, Shao-Yao-Gan-Cao-Tang (Japanese name: Shakuyaku-kanzo-to), consists of equal amounts of paeony and licorice roots, and has been used in Japan and China for muscle pain or skeletal muscle tremors. To determine whether this medicine is able to prevent frequent and unendurable muscle cramps in patients undergoing HD, Shakuyaku-kanzo-to at 6 g per day was prospectively administered for 4 weeks to five patients on HD who were suffering from frequent muscle cramps. The frequency and severity of cramping before and after the treatment were carefully observed and compared. Skeletal muscle cramps completely disappeared in two of the treated patients after the start of oral administration of Shakuyaku-kanzo-to. Moreover, the frequency of cramping was significantly decreased in two of the remaining three patients after persistent administration. The severity of muscle cramps was also decreased by this treatment in the responsive patients. No serious side effects were detected during the treatment period. The inhibitory effect of Shakuyaku-kanzo-to on muscle contraction was also experimentally examined by using phrenic nerve-diaphragm preparations from male Wistar rats. Differences between the twitch responses were determined when the diaphragms and the nerves were stimulated in the presence and absence of the extract of Shakuyaku-kanzo-to. The results demonstrated that extracts of paeony and licorice roots inhibit contraction of skeletal muscles in rats. Taken together, we suggest that administration of Shakuyaku-kanzo-to is a safe, effective treatment for preventing muscle cramps in patients undergoing HD.

Cytotoxic T cells (CTLs) are believed to play an important role in the pathogenesis of chronic hepatitis C based on histological findings of the liver and in vivo experiments. Fas-ligand-Fas and perforin dependent pathways are two major killing systems when CTLs induce their target-cell death. Thus, the present study attempts to determine whether these pathways are activated, and if they are, how they are related in chronic hepatitis C. To investigate the expression of Fas-ligand and perforin, we performed double immunofluorescent staining of liver biopsy specimens from patients with chronic hepatitis C. Fas-ligand and perforin expression was observed in mononuclear cells, and the partial coexistence of the two proteins was observed. Cells expressing both proteins were positive for CD45RO(+) T cells (active T cells), whereas cells expressing perforin were negative for CD68 (macrophages). In the cases which had sustained negative HCV-RNA over 6 months after interferon treatment, Fas-ligand was not expressed, although perforin was slightly detectable. To quantitatively assess the balance of these pathways, hepatic mRNAs of Fas-ligand and perforin were measured by quantitative RT-PCR. The ratio of Fas-ligand-mRNA/perforin-mRNA was significantly correlated with serum alanine aminotransferase (ALT) levels (r=0.913). These results suggest that both pathways are activated in chronic hepatitis C and that Fas-ligand-Fas pathway may be predominant in active inflammation.

Background. The intrarenal localization of metallothionein (MT) expression has not been studied in detail. Therefore, we investigated the effects of acute exposure to cadmium on the renal expression of MT. Methods. MT mRNA expression was estimated quantitatively at 2, 6, 24, and 72 h after the injection of cadmium (1 mg/kg) to the intraperitoneum of male Sprague-Dawley rats. The site of cadmium-induced MT mRNA expression was determined, by the reverse transcription polymerase chain reaction (RT-PCR), using microdissected tubules. Immunoblotting and immunohistochemistry were also performed, to determine the localization of MT protein. Results. The mRNAs of MT-I and MT-II were significantly upregulated at 2 h and peaked at 24 and 6 h, respectively. RT-PCR revealed the expression of cadmium-induced MT mRNA mainly in the proximal tubules. Immunoblotting detected a clear 14-kDa band corresponding to MT protein, and immunohistochemistry showed abundant MT protein in the renal cortices of cadmium-treated rats. Conclusions. Our findings suggest that the proximal tubules are responsible for the cadmium-induced renal expression of MT mRNA and protein.

To date, there have been no adequate studies examining the kinetics of anticoagulant binding to the continuous hemofilter membranes used in continuous blood purification therapy. In this study, we compared the binding kinetics of conventional heparin (CH), argatroban (ARG), and nafamostat mesilate (NM) with various types of continuous hemofilter membranes [polyacrylonitrite (PAN), polymethylmethacrylate (PMMA), polysulfone (PS), and polyamide (PA)]. In-circuit circulation was conducted according to a recirculation batch method. Each drug concentration in the test solution was monitored up to 24 h after the in-circuit circulation, and amounts of the bound drug were calculated. There was little binding of CH to any of the membranes examined at 24 h after in-circuit circulation (383–1222 units/m2), except PA (2717 units/m2). ARG bound largely to PS (61.9 mg/m2) and slightly to PMMA (6.9 mg/m2). NM bound PAN (55.3 mg/m2) > PS (38.0 mg/m2) > PMMA (7.7 mg/m2). Binding of NM to PA was not detected. Our study suggested that the degree of anticoagulant binding depended on characteristics of the continuous hemofilter membranes. Accordingly, the combination of the type of membranes and the dose of anticoagulants should be carefully chosen in continuous blood purification therapy.

Background: CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3–/–) by targeted gene disruption. Results: Together with developmental retardation and higher mortality, the Clcn3–/– mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3–/– mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3–/– mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3–/– mice. Conclusions: These results indicated that the neurodegeneration observed in the Clcn3–/– mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.

Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney. Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and TGF-β is considered to play an important role in this fibrotic process. Smad7 has been recently identified as an antagonist of TGF-β signaling. To investigate whether this novel molecule can be exploited for therapy of renal fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant adenovirus vector combined with in vivo electroporation (EP), on UUO-induced renal fibrosis in rats. Methods A model of UUO was made in SD rats by ligating their left ureters. The next day, the rats were divided into four groups and adenovirus was injected into the extended pelvic space (two groups received AdCMV-LacZ and two groups received AdCMV-Smad7). Then, EP was performed in one group of AdCMV-LacZ-injected rats and one group of AdCMV-Smad7-injected rats. The renal tissues were obtained 3, 5, 10, and 14 days after the UUO operation. We detected the efficiency of transgene by immunoblots of renal cortical and medullary tissues and immunohistochemical studies for Smad7 and FLAG (the FLAG gene was introduced in the AdCMV-Smad7 as a marker). The renal fibrosis was monitored by histological scoring of Masson stainings. Results In immunoblotting, both Smad7 and FLAG were clearly detected in the renal medullary tissue of the rats given AdCMV-Smad7 with EP. In contrast, immunoblots of renal cortical tissue did not demonstrate positive bands. In immunohistological study, Smad7 was stained in the renal medulla in the rats given AdCMV-Smad7 with EP. In the rats given AdCMV-Smad7 without EP, only a weak signal was detected in renal medullary tissue. The rats given AdCMV-Smad7 with EP demonstrated significantly more suppression of renal fibrosis than rats treated with AdCMV-LacZ. The rats treated with AdCMV-Smad7 without EP did not demonstrate significant suppression of renal fibrosis. Conclusion These data indicate that gene transfer of Smad7 prevents UUO-induced renal fibrosis, suggesting that Smad7 may be applicable for the treatment of renal fibrosis. In vivo electroporation of adenovirus may be a powerful tool for gene delivery in renal tissue.

Orthostatic hypotension is one of the major factors interfering with everyday activities in hemodialysis patients, but there has been no effective agent for treating it. In order to clarify the clinical effects of L-threo-3,4-dihydroxyphenylserine (L-DOPS) on orthostatic hypotension of hemodialysis patients, we conducted a randomized, double-blind comparative trial. 149 regular hemodialysis patients with orthostatic hypotension were randomly allocated to three groups and L-DOPS at doses of 400 mg, 200 mg or placebo was orally administrated to each group 30 min before starting every hemodialysis for 4 weeks. Changes of blood pressure (BP) in orthostatic hypotension immediately after completion of hemodialysis and symptoms related to orthostatic hypotension were compared between the three groups. In the 400-mg group, systolic and diastolic BP after standing increased significantly and the drop of mean BP after standing was also reduced compared with pretreatment levels. No such changes were observed in the placebo group. Fatiguability, malaise/weakness, dizziness and light-headed feeling, the interdialytic symptoms commonly observed in hemodialysis patients who developed orthostatic hypotension, were improved to a significant extent in the L-DOPS group compared with the placebo group. In particular, the improvement was more remarkable for the L-DOPS 400-mg group than the placebo group in patients with diabetic nephropathy, lower systolic BP after standing, and the long duration type of orthostatic hypotension. The incidence of adverse events was comparable between the three groups, and all recovered after discontinuation of L-DOPS or concomitantly administered drugs, or without any treatment. These findings indicate that L-DOPS taken before hemodialysis prevents orthostatic hypotension in patients undergoing hemodialysis, and is also effective for the interdialytic symptoms related to orthostatic hypotension.

Background: Orthostatic hypotension (OH) is a serious complication observed in hemodialysis (HD) patients after HD as well as during the interdialytic period. L-Threo-3,4-dihydroxyphenylserine (L-DOPS) is a nonphysiological neutral amino acid that is directly converted to the neurotransmitter norepinephrine by aromatic L-amino acid decarboxylase. Methods: A placebo-controlled double-blind study for 4 consecutive weeks and a long-term study (24–52 weeks) were conducted to evaluate the efficacy of L-DOPS for OH after HD. The drug was administered orally 30 min before the start of each HD period in both studies. Doses of 400 mg of L-DOPS or placebo were given to HD patients with OH (45 and 41 patients, respectively) in the double-blind study, and doses of 200 or 400 mg of L-DOPS were given to 74 HD patients in the long-term study. Results: In the double-blind study, L-DOPS significantly ameliorated subjective symptoms related to OH, including dizziness/light-headed feeling, and malaise, throughout the interdialytic period. For 19 patients with delayed-type OH, hypotension with the lowest blood pressure recorded 10 min after standing, the decrease in blood pressure was suppressed significantly after L-DOPS treatment (10 patients) as compared with the placebo-treated group (9 patients). In the long-term study, the efficacy of L-DOPS was not attenuated, and the marked fluctuations in the plasma L-DOPS and norepinephrine levels were not noted after long-term use, without increases in incidence or severity of adverse reactions. Conclusions: These results indicate that L-DOPS is effective for improving OH-related interdialytic subjective symptoms in HD patients after short-term as well as after long-term administration.

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 µg. 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-µg group, –18.9% in the 1.5-µg group and –24.1% in the 2-µg 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-µg 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-µg 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 µg per dialysis session.

Background. Polymorphism of the collagen type Iα1 gene (COLIA1) has been shown to be involved in bone density in the general population. However, the significance of this polymorphism has not been examined in patients with chronic renal failure. Methods. We first determined the COLIA1 Bal I restriction fragment length polymorphism (RFLP) at the Sp 1 binding site, and, next, we measured biochemical bone markers and the bone mineral density (BMD) of the lumbar spine (L2–L4) and radius (distal one-third) in 181 hemodialyzed patients. Results. Distributions of the Bal I RFLP were: SS, 62%; Ss, 32%; and ss, 6%. The Z scores for the BMD and for the serum levels of Ca, P, alkaline phosphatase, intact-parathyroid hormone (PTH), osteocalcin, and tartrate-resistant acid phosphatase were not significantly different among the three genotype groups. The slope of the regression line in the age-BMD relationship, a measure of the rate of BMD decrease, was similar among the three genotypes in men, but was significantly (P < 0.05) larger in the ss group than in the SS and Ss groups in women. Conclusions. The COLIA1 polymorphism may be associated with the bone loss seen in female patients on hemodialysis.

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 μg 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. © 2001 by the National Kidney Foundation, Inc.

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.

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 Kruppel-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-K1GA 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.

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 α, 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 diagnosic markers, or determining novel therapeutic targets. © 2001 Cancer Research Campaign http://www.bjcancer.com

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.

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.

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.

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.

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.

Spontaneous degeneration of rapid atrial fibrillation (AF) to ventricular fibrillation has been documented in patients with hypertrophic cardiomyopathy (HCM) and Wolff-Parkinson-White (WPW) syndrome. However, the importance of rap

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.

目的: ダイアライザー接続カプラーによる透析液のエンドトキシン (ET) 汚染に対して, ET汚染対策カプラーが用いられている. そこで各種汚染対策カプラーによる透析液のET汚染防止効果を同一条件下で基礎および臨床的に評価した. 方法: 検討に用いたカプラーは, A: 1年以上使用した従来型. B: 新しいオーリングに交換した従来型. C: 抗菌オーリングに交換した従来型. D: オーリング部分の消毒可能な型. E: オーリングなしのシリコン型. F: オーリングなしでダイアライザー接触部全てが消毒可能な型を用いた. 基礎実験ではカプラーと血液回路を用い閉鎖回路を作成しET汚染後の洗浄効果を観察した. 長期臨床使用では, カプラー長期臨床使用による透析液へのET汚染, カプラー内ET汚染, ダイアライザー接続時カプラーET汚染とその初期値からのET濃度の時間経過を検討した. 結果: 洗浄実験では, A, B, Cは循環開始直後からET濃度は上昇し, D, E, FではET濃度の上昇はなかった. 長期臨床使用でのダイアライザー接続直後のET濃度は, Aで30週間高値を示し, B, Cは18週目より上昇した. D, E, Fでは30週間低値を維持した. 脱着時のカプラー内ET濃度は, Fで他に比べて低値を示した. ダイアライザー出口のET濃度はA, B, Cで灌流直後に高値を示し, 時間経過とともにET濃度は低下し, 他のカプラーでは終始低値だった. ET濃度の時間経過はカプラーA, B, Cで他のD, E, Fに比べET濃度低下に時間を要した. 結語: ダイアライザーと接触するオーリングがなく, カプラー内部を全て洗浄・消毒できる構造がET汚染対策カプラーとして有効であった.

エストロゲン受容体 (ER) のPvuII, XbaI制限酵素断片長多型が透析患者の骨病変に影響を及ぼしているかを検討した. 患者末梢血から抽出したゲノムDNAを鋳型としてpolymerase chain reaction (PCR) を行い, ERのイントロン1とエクソン1を含む領域を増幅した. 得られたPCR産物を制限酵素PvuII, XbaIと反応して遺伝子多型を決定した. また骨生化学的指標として血清Ca, P, Al-P, tartrate-resistant Ac-P, intact-PTH, osteocalcinを測定するとともにdual energy X-ray absorptiometry (DEXA) 法にて腰椎正面 (L2-L4) および橈骨 (遠位端1/3) 骨塩量測定を行った. PvuII遺伝子多型の分布は, 男性 (n=126) がPP 23.0%, Pp 51.6%, pp 25.4%であり, 女性 (n=81) がPP 19.8%, Pp 38.3%, pp 42.0%であった. XbaI遺伝子多型の分布は, 男性 (n=120) がXX 5.8%, Xx 37.5%, xx 56.7%であり, 女性 (n=78) がXX 9.0%, Xx 38.5%, xx 52.6%であった. 遺伝子多型別に比較した骨生化学的指標は, 男女のどの3群間においても有意差は認められなかった. 腰椎および橈骨骨塩量のZ scoreもすべて3群間で有意差はなかった. 腰椎骨塩量のZ scoreと年齢との相関では, 女性のPvuII遺伝子多型でPP群の回帰直線勾配がPp群, pp群より有意に高値であり (p

The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1−/− mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1−/− mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/− and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1−/− mice. These results indicate that the polyuria observed in Clcnk1−/− mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1−/− mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1−/− mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1−/− mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1−/− mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1−/− mice. We concluded that NDI in Clcnk1−/− mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems.

We treated a female patient suffering from immunoglobulin A (IgA) nephropathy and congenital deficiency of the ninth component of the complement system (C9). She was admitted with hematuria and proteinuria, and the C9 deficiency was diagnosed based on the low hemolytic activity of 50 % of the hemolytic unit of the complements (CH50) and the normal C3 level in the plasma. Renal biopsy revealed mild mesangial proliferation, and immunofluorescence examination revealed mild mesangial deposits of IgA and C3 with the same distribution. We discuss the pathogenesis of IgA nephropathy and the role of the complements in its progression.

A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.

Catheter ablation of idiopathic left ventricular outflow tract tachycardia (LVOT-VT) is rare because a safe ablation technique at this site has not been described, and serious complications may occur. This study compared the QRS morphology of LVOT-VT with that of idiopathic right ventricular outflow tract tachycardia. A comparison was made between the electrocardiographic characteristics of LVOT-VT originating from the supravalvular region of a coronary cusp (Supra-Ao group) with those of LVOT-VT originating from the infravalvular endocardial region of a coronary cusp of the aortic valve within the LV (Infra-Ao group). After precise mapping of the right ventricle, left ventricle, pulmonary artery, coronary cusps, and proximal portion of the anterior interventricular vein, there were 17 patients in whom VT was thought to be located at the LVOT by both activation and pace mapping. They were divided between a Supra-Ao group (n = 8), and an Infra-Ao group (n = 9). Analysis of the 12-lead electrocardiogram (ECG) revealed an S wave in lead I in all 17 patients. A precordial R wave transition was also observed at V1 or V2 in 16 patients (94%). In 7 of 8 patients (88%) with Supra-Ao LVOT-VT, no S wave was observed in either V5 or V6. In contrast, an Rs pattern was observed in both V5 and V6, or in V6 only, in 100% of the patients with Infra-Ao LVOT-VT. A LVOT-VT should be suspected when the ECG shows an S wave in lead I and an R/S ratio greater than 1 in lead V1 or V2, versus a coronary cusp location if there is no S wave in either lead V5 or V6.

As a part of the defense mechanism of the host to viral infection, interferons induce the transcription of several genes. These interferon-inducible genes contribute to the eradication of the viruses. Whereas some studies suggested the participation of a dsRNA-dependent protein kinase in the host reaction to hepatitis C virus infection, the involvement of other interferon-inducible genes has not been evaluated. Furthermore, there has been no analysis on the expression profile of multiple interferon-inducible genes. The aim of this study was to clarify the hepatic mRNA expression profile of interferon-inducible genes with a special concern to chronic hepatitis C. A total of 76 liver biopsy samples (28 with chronic hepatitis C, 10 with chronic hepatitis B, 9 with alcoholic liver disease, 14 with autoimmune hepatitis, 10 with primary biliary cirrhosis, and 5 of normal liver) were enrolled. The expression of the following genes was quantified by competitive reverse transcription-polymerase chain reaction and was compared according to the etiology; dsRNA-dependent protein kinase (PKR), 2′,5′-oligoadenylate synthetase (2,5-AS), latent cellular endoribonuclease (RNase L), RNase L inhibitor, and MxA. As a result, PKR mRNA was significantly overexpressed in the liver of chronic hepatitis C compared with those of other etiologies (P= .0178), and it correlated significantly with serum alanine transaminase values (r = .51, P = .0054). Also, the expression of the RNase L inhibitor showed a significant reduction in chronic hepatitis C (P = .0184). The expressions of 2,5-AS, RNase L, and MxA were not different significantly irrespective to the etiology. In conclusion, hepatic overexpression of PKR and reduced expression of RNase L inhibitor seem to contribute to the anti-HCV mechanism characteristically. (Hepatology 2000;32:1089-1095.)

CLC-K1 and CLC-K2 are two kidney-specific members of the CLC chloride channel family (1, 35). Both are present in the plasma membranes of tubular cells in the kidney (36, 37), and it has been speculated that both serve as routes for transepithelial chloride transport. Mutations of CLCNKB (the human homologue of rat CLC-K2) were recently found in patients with Bartter's syndrome (30), and the CLC-K1 gene knockout in mice results in nephrogenic diabetes insipidus (21), confirming the important role of these channels in chloride transport in the kidneys. Although the two clones are highly homologous (80% amino acid identity in the rat sequence and 90% in the human sequence), their intrarenal localizations are completely different (39). Accordingly, the analysis of transcriptional regulation of these two genes is expected to elucidate mechanisms of kidney-specific and nephron segement-specific gene expression. To this end, we previously isolated the promoters of the rat CLC-K1 (34) and CLC-K2 (26) genes. Surprisingly, proximal 5′-flanking regions that include the transcriptional start sites are highly homologous and characterized by a GA element, GGGGAGGGGAGGGGGAGGG (26). Reporter gene assays and gel retardation assays (26, 34) revealed that this GA element is indispensable for the basal promoter activities of both genes, suggesting that one or more proteins binding to this element may be involved in the kidney-specific expression of the CLC-K1 and CLC-K2 genes. In the present study, we isolated two cDNAs that bind to the GA element, i.e., MAZ, the previously isolated myc-associated zinc finger protein, and KKLF, a novel kidney-enriched Kruppel-like factor. MAZ and KKLF have opposite effects on the CLC-K1 promoter activity, suggesting that the kidney-specific expression of CLC-K genes may be regulated by a series of zinc finger proteins through the GA element. The spatial pattern of KKLF expression overlapped with negative expressions of CLC-K1 and CLC-K2 in the kidneys, supporting the idea that KKLF may contribute to the strict nephron segment-specific expression of the CLC-K genes in vivo. Furthermore, we also found that KKLF repressed the promoter activity of the α2(I) collagen gene. Given the localization of KKLF in interstitial fibroblasts in cardiac and skeletal muscle and in potentially fibrogenic cells such as the mesangial cells in the kidneys or stellate cells in the liver, it is reasonable to assume that KKLF may be involved in the fibrogenesis in these organs. In a unilateral ureteral obstruction (UUO) model of mouse kidney, a well-characterized model of progressive tubulointerstitial fibrosis, a rapid decrease of KKLF and subsequent increase of α2(I) collagen expression were observed, suggesting that KKLF is involved in type I collagen synthesis and tissue fibrosis.

Adrenomedullin (AM) is a novel vasorelaxant peptide, isolated from human pheochromocytoma. Although AM may be involved in the regulation of the cardiovascular system, a number of other mechanisms are also involved. The present study was undertaken to confirm the presence of AM in human maternal circulation and in placental function during pregnancy. Immunoreactive (ir) AM concentrations in maternal plasma were 3.4+/-0.7fmol/ml (mean+/-s.e. m.) in the first trimester, 3.3+/-1.1fmol/ml in the second trimester, 7.3+/-2.8fmol/ml in the third trimester, 4.1+/-1.9fmol/ml in early puerperium and 3.0+/-0.4fmol/ml in non-pregnant periods; the concentration in the third trimester was significantly greater than those in other periods. Plasma concentrations of estradiol (E(2)), progesterone, human placental lactogen (hPL) and human chorionic gonadotropin (hCG) were also measured, using RIA kits. Significant correlations have been demonstrated between the concentrations of irAM and those of E(2), progesterone and hPL. We therefore examined the expression of AM within the placental tissues using immunohistochemistry and northern blot analysis in order to demonstrate a correlation between the presence of AM in the placenta and maternal plasma. Using immunohistochemistry, we detected AM in the amnion at term and the expression of AM mRNA in human placental tissues using cloned human (h) AM complementary DNA as a probe. This study demonstrates the immunoreactivity of human hAM in maternal plasma during pregnancy, and suggests that hAM in maternal plasma is generated partly from placental tissue.

Activated STAT1 suppresses proliferation of cultured rat mesangial cells. Background JAK-STAT signaling has been shown to promote development and proliferation in lymphopoietic and hematopoietic lineages. We investigated the effect of activated STAT1 on mesangial cell proliferation. Methods Rat mesangial cells of primary culture (rMCs) were used in the following experiments: ( 1 ) Whole cell lysates were immunoblotted against JAK1 and JAK2. ( 2 ) Whole cell lysates and nuclear proteins were extracted from rMCs with or without treatment with interferon-γ, and immunoblotting was performed against both STAT1 and tyrosine (701)-phosphorylated STAT1. ( 3 ) rMCs and rMCs electroporated with either wild-type STAT1, mutated STAT1, or antibody against STAT1 were incubated with interferon-γ for 20 hours, followed by a further incubation with [ 3 H]-thymidine for four hours. Results JAK1, JAK2, and STAT1 were detected in whole cell lysates, suggesting that JAK-STAT signaling could be activated by interferon-γ (INF-γ). Using an antibody specific for tyrosine-phosphorylated STAT1, we detected signal in the INF-γ–treated nuclear extracts, which showed translocation of phosphorylated STAT1 to the nucleus. [ 3 H]-thymidine incorporation in the presence of INF-γ was significantly lower than that of control in a dose-dependent manner. The introduction of wild-type STAT1 enhanced the effect of interferon-γ and decreased [ 3 H]-thymidine incorporation, whereas tyrosine-mutated (Y701F) STAT1 and SH2 domain (R602T)-mutated STAT1 reversed INF-γ–induced suppression of [ 3 H]-thymidine incorporation. Electroinjected antibody against STAT1 increased [ 3 H]-thymidine incorporation upon stimulation with INF-γ. Conclusion STAT1 activated by interferon-γ suppresses mesangial cell proliferation.

Abstract —Endothelin (ET)-1, an endothelium-derived vasoconstrictor and mitogen, acts as an antiapoptotic factor against serum deprivation–induced apoptosis of endothelial cells and fibroblasts but enhances apoptosis of some cancer cells. In the present study, we examined whether nitric oxide (NO) and ET-1 modulate apoptosis of rat vascular smooth muscle cells (VSMCs) via the mitogen-activated protein (MAP) kinase pathway. Both serum deprivation and NO donors (FK409 and SNAP) caused apoptosis of VSMCs, as demonstrated by TdT-mediated dUTP-biotin nick end-labeling, appearance of fragmented DNA, and induction of caspase-3 activity. ET-1 dose-dependently antagonized apoptosis induced by serum deprivation and NO donors. A selective ET A receptor antagonist (BQ123) and a nonselective ET A/B receptor antagonist (TAK044), but not a selective ET B receptor antagonist (BQ788), inhibited the antiapoptotic effect of ET-1, indicating that the antiapoptotic effect of ET-1 is mediated via the ET A receptor. ET-1 activated MAP kinase, whose effect was inhibited by FK409. Transfection with an unphosphorylated wild-type MAP kinase kinase-1 (MAPKK-1) or its constitutively activated mutant protected VSMCs against apoptosis induced by serum deprivation and NO donors. Inhibition of MAP kinase activity with PD98059, a specific inhibitor of MAPKK-1, or by transfection of a dominant-negative MAPKK-1 mutant antagonized the antiapoptotic effect of ET-1, suggesting the involvement of MAP kinase in the antiapoptotic effect. The potent inhibitory effect of ET-1 on apoptosis of VSMCs induced by serum deprivation and NO suggests that the counterbalance between the 2 endothelium-derived factors contributes to the process of vascular remodeling by determining VSMC survival and death, respectively, via a common MAP kinase pathway.

BACKGROUND Gap junctions contain intercellular channels through which contacting cells communicate directly. The expression of connexin 32, a major gap junction protein in the liver, during the progression of chronic liver diseases has not yet been clarified. METHODS Immunohistochemical staining was performed using anti-connexin 32 antibody on 6 specimens of normal human liver, 7 of chronic viral hepatitis, and 7 of liver cirrhosis. RESULTS The number of gap junction plaques in chronic viral hepatitis and liver cirrhosis was significantly smaller than that in normal liver (10350+/-2180 and 7550+/-3040 vs 22560+/-3700 spots/mm2, p

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione- S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd3+) and an intracellular Ca2+ antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [3H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca2+-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.

Amino acid mutations in the interferon sensitivity determining region (ISDR) are closely associated with the response to interferon in patients with hepatitis C virus genotype 1b (HCV-1b) infection. In this study, 36 patients chronically infected with HCV-1b, with no history of interferon therapy with respect to ISDR changes in HCV were studied. Two serum samples were obtained from each patient, with an average interval of 3.5 years, and predominant nucleotide and amino acid sequences of the ISDR at initial and subsequent time points were compared for each patient. Three of 12 patients with the wild type ISDR (no amino acid mutation in the ISDR compared to the consensus sequence) changed to the intermediate type (1 to 3 mutations) at later time points, whereas the other 9 still had the wild type. Similarly, 2 of 18 patients with the intermediate type changed to the wild type, whereas the other 16 patients continued to have the intermediate type. One of 6 patients with the mutant type (4 or more mutations) changed to the intermediate type, and the other 5 continued to have the mutant type. Although ISDR nucleotide changes/site/year were not significantly different among the 3 groups of patients, percentages of non-synonymous nucleotide changes were greater in the mutant type (63%) than the wild (9%) or the intermediate type (20%) (P < 0.05). These results show that mutations in the ISDR do not occur frequently, suggesting that interferon sensitivity does not change greatly during the natural course of the disease in each patient.

Mechanisms determining the chronicity or the pattern of clinical course of hepatitis C virus (HCV) infections have not been clarified. Recently, CD81 was reported to bind the E2 protein of HCV and was suggested to function as a cellular receptor for HCV. Accordingly, the hypothesis was examined that CD81 polymorphism, if it exists, might correlate with certain clinical courses of HCV infection. CD81 cDNA sequences were determined from peripheral blood mononuclear cells (PBMCs). Twenty-four Japanese subjects were enrolled initially as follows: patients with chronic hepatitis C without cirrhosis (n = 3), patients with cirrhosis (n = 3), patients with cirrhosis complicated by hepatocellular carcinoma (HCC) (n = 3), patients with persistent HCV viremia without ALT elevation (n = 3), those with positive anti-HCV antibodies without evidence of HCV viremia (n = 3), and healthy volunteers (n = 9). In all PBMCs samples analyzed, no polymorphism was found in the CD81 cDNA sequence. The sequence was different, however, from the one reported previously at three nucleotide positions: a transversion to thymine instead of cytosine at nt 1130, a deletion at nt 1206, and a guanine insertion at nt 71. Subsequently, CD81 cDNA sequences from PBMCs and HCC tissue were compared among the other 6 patients with chronic hepatitis C bearing HCC. A comparative study of the CD81 sequences from HCC and PBMCs revealed that various nucleotide mutations existed only in the HCC samples in 3 out of 6 patients. Several mutations in the 3' non-coding region of CD81 cDNA were observed exclusively in HCC tissue suggesting its possible role in hepatocarcinogenesis. Because of the absence of polymorphisms, however, CD81 is unlikely to affect the progression of chronic hepatitis C in terms of chronicity, hepatitis activity, or disease stage.

The importance of the cell cycle in proliferating cells is well known, but little is known about the role of cell cycle regulatory proteins in cardiac myocytes, which are fully differentiated cells. The present study determined, in vitro, the effect of angiotensin II (Ang II) treatment of neonatal rat cardiac myocytes on protein levels of cyclins and retinoblastoma gene product (pRb) phosphorylation. The role of Gi cyclin/cdk in Ang II-induced cardiac myocyte hypertrophy by overexpressing cdk inhibitor p21 Cipl/Waf1 or p16 INK4a was also examined using recombinant adenoviral vectors encoding these genes. Western blot analysis revealed that Ang II stimulated cyclin Dl, D2, D3 and A protein levels in cardiac myocytes. Moreover, Ang II phosphorylated pRb on serine 780, which is known to occur in mitotic cells during cell cycle progression. Cultured cardiac myocytes treated with Ang 11 and infected with either control or recombinant adenovirus indicated that expression of p21 and p16 inhibited Ang II-induced cardiac myocyte hypertrophy, [ 3 H]leucine incorporation into total cellular proteins, and skeletal α-actin (SK-A) and atrial natriuretic peptide (ANP) mRNA accumulation. Control virus had no effects on these parameters. These results suggest that Gi cyclins play an important role in cardiac myocyte hypertrophy stimulated by Ang 11.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a specific heart muscle disease of unknown etiology characterized by fatty and fibrofatty replacement of the right ventricular myocardium. It often manifests life-threatened ventricular arrhythmias. Previous studies have hypothesized that myocyte apoptosis contributes to the myocyte cell loss and fatty change in ARVC and may be induced by recurrent ventricular tachycardia (VT). We examined whether these progressive pathological changes result from apoptotic cell death in both autopsied and biopsied right ventricular myocardium from 35 patients with ARVC by using in situ terminal deoxynucleotidyl transferase assay (TUNEL) and agarose gel electrophoresis. We also studied the biopsied myocardium from 30 patients with idiopathic sustained VT whose origin was the outflow tract of the right ventricle. TUNEL-positive cells indicating DNA fragments were observed in some cardiomyocytes and fibroblasts in ARVC, but the numbers of TUNEL-positive myocytes were very low in idiopathic VT. DNA laddering was confirmed in two autopsied cases in ARVC, but not in a non-cardiac case who died. These results suggest that at least some cardiomyocytes and fibroblasts are subjected to apoptosis in ARVC, leading to the loss of myocardium with characteristic pathological changes and subsequently progressive cardiomyopathy. Furthermore, the apoptotic process may not result from myocardial ischemia due to repetitive VT.

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.

Roles of E2F1 in mesangial cell proliferation in vitro. Background The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. Methods To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. Results E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. Conclusions In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins.

ClC-4 gene was isolated as a putative Cl−channel. Due to a lack of functional expression of ClC-4, its physiological role remains unknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletal muscles and stably transfected it to Chinese hamster ovary cells. Whole cell patch-clamp studies showed that the hClC-4sk channel was activated by external acidic pH and inhibited by DIDS. It passed a strong outward Cl−current with a permeability sequence of I−> Cl−> F−. The hClC-4sk has consensus sites for phosphorylation by protein kinase A (PKA); however, stimulation of PKA had no effect on the currents. hClC-4sk mRNA was expressed in excitable tissues, such as heart, brain, and skeletal muscle. These functional characteristics of hClC-4sk provide a clue to its physiological role in excitable cells.