Economic evaluation of the Very Early Rehabilitation in SpEech (VERSE) intervention.

INTRODUCTION: There is limited evidence on the costs and outcomes of patients with aphasia after stroke. The aim of this study was to estimate costs in patients with aphasia after stroke according to the aphasia therapies provided. METHODS: A three-arm, prospective, randomized, parallel group, open-label, blinded endpoint assessment trial conducted in Australia and New Zealand. Usual ward-based care (Usual Care) was compared to additional usual ward-based therapy (Usual Care Plus) and a prescribed and structured aphasia therapy program in addition to Usual Care (the VERSE intervention). Information about healthcare utilization and productivity were collected to estimate costs in Australian dollars for 2017-18. Multivariable regression models with bootstrapping were used to estimate differences in costs and outcomes (clinically meaningful change in aphasia severity measured by the WAB-R-AQ). RESULTS: Overall, 202/246 (82%) participants completed follow-up at 26 weeks. Median costs per person were $23,322 (Q1 5,367, Q3 52,669, n = 63) for Usual Care, $26,923 (Q1 7,303, Q3 76,174, n = 70) for Usual Care Plus and $31,143 (Q1 7,001. Q3 62,390, n = 69) for VERSE. No differences in costs and outcomes were detected between groups. Usual Care Plus was inferior (i.e. more costly and less effective) in 64% of iterations, and in 18% was less costly and less effective compared to Usual Care. VERSE was inferior in 65% of samples and less costly and less effective in 12% compared to Usual Care. CONCLUSION: There was limited evidence that additional intensively delivered aphasia therapy within the context of usual acute care provided was worthwhile in terms of costs for the outcomes gained.

Towards an integrated blood pressure self-monitoring solution for stroke/TIA in Ireland: a mixed methods feasibility study for the TASMIN5S IRL randomised controlled trial.

BACKGROUND: Optimising blood pressure (BP) control is one of the most important modifiable risk factors in preventing subsequent stroke where the risk increases by one-third for every 10 mmHg rise in systolic BP. This study evaluated the feasibility and potential effectiveness of blood pressure self-monitoring with planned medication titration, to inform a definitive trial of the intervention, in patients with a previous stroke or transient ischaemic attack (TIA). METHODS: Patients with a history of stroke/TIA and sub-optimal BP control were invited to take part in a mixed methods feasibility study for a randomised controlled trial. Those meeting the inclusion criteria with systolic BP >130 mmHg were randomised to a self-monitoring intervention group or usual care group. The intervention involved self-monitoring BP twice a day for 3 days within a 7-day period, every month, following text message reminders. Treatment escalation, based on a pre-agreed plan by the general practitioner (GP) and patient, was initiated according to the results of these readings. Semi-structured interviews were carried out with patients and clinicians and analysed thematically. RESULTS: Of those identified, 47% (32/68) attended for assessment. Of those assessed, 15 were eligible for recruitment and were consented and randomised to the intervention or control group on a 2:1 basis. Of those randomised, 93% (14/15) completed the study and there were no adverse events. Systolic BP was lower in the intervention group at 3 months. Participants found the intervention acceptable and easy to use. GPs found it easy to incorporate into their practice activity without increasing workload. CONCLUSIONS: TASMIN5S, an integrated blood pressure self-monitoring intervention in patients with a previous stroke/TIA, is feasible and safe to deliver in primary care. A pre-agreed three-step medication titration plan was easily implemented, increased patient involvement in their care, and had no adverse effects. This feasibility study provides important information to inform a definitive trial to determine the potential effectiveness of the intervention in patients post-stroke or TIA. TRIAL REGISTRATION: ISRCTN57946500 . Registered on 12/08/2019.

Easy Identification of Optimal Coronal Slice on Brain Magnetic Resonance Imaging to Measure Hippocampal Area in Alzheimer's Disease Patients.

INTRODUCTION: Measurement of an- hippocampal area or volume is useful in clinical practice as a supportive aid for diagnosis of Alzheimer's disease. Since it is time-consuming and not simple, it is not being used very often. We present a simplified protocol for hippocampal atrophy evaluation based on a single optimal slice in Alzheimer's disease. METHODS: We defined a single optimal slice for hippocampal measurement on brain magnetic resonance imaging (MRI) at the plane where the amygdala disappears and only the hippocampus is present. We compared an absolute area and volume of the hippocampus on this optimal slice between 40 patients with Alzheimer disease and 40 age-, education- and gender-mateched elderly controls. Furthermore, we compared these results with those relative to the size of the brain or the skull: the area of the optimal slice normalized to the area of the brain at anterior commissure and the volume of the hippocampus normalized to the total intracranial volume. RESULTS: Hippocampal areas on the single optimal slice and hippocampal volumes on the left and right in the control group were significantly higher than those in the AD group. Normalized hippocampal areas and volumes on the left and right in the control group were significantly higher compared to the AD group. Absolute hippocampal areas and volumes did not significantly differ from corresponding normalized hippocampal areas as well as normalized hippocampal volumes using comparisons of areas under the receiver operating characteristic curves. CONCLUSION: The hippocampal area on the well-defined optimal slice of brain MRI can reliably substitute a complicated measurement of the hippocampal volume. Surprisingly, brain or skull normalization of these variables does not add any incremental differentiation between Alzheimer disease patients and controls or give better results.

Anthropometrical and Physical Fitness Predictors of Operational Military Test Performance in Air Force Personnel.

The objective of this study was to verify the relationships between the anthropometrical and physical fitness parameters (measured by the Physical Conditioning Assessment (PCA) of the Aeronautics Command), with the operational performance in the simulated military task performance (SMTP) performed by the infantry military of a Brazilian Air Force (BAF) unit. These evaluations were performed on two distinct days, interspersed by 48h, with PCA on the first day and the SMTP in the second. The distribution of the dependent variable was not normal (Shapiro-Wilk test, p = 0.001). Data are presented as mean and standard deviation, median and interquartile, for variables normally and non-normally distributed, respectively. The correlation between variables was determined using the Spearman's correlation coefficient. A regression model to predict performance in the SMTP, based on the anthropometrical, physiological and performance variables, was performed. The significance level was set at 5%. Based on the results, there was an association between all the PCA and SMTP variables: weight, lean body mass, trunk flexion, and estimated VO2max based on the distance covered in the 12-minute test. The following equation was generated: SMTP (s) = 350.611 - 1.556 (fat-free mass, in kg) - 0.34 (12-min running distance, in m) - 0.632 (sit-up, in repetitions). The explained variance of the SMTP was 72.3% with an estimated standard error of 3.6s. It was observed that, although the association was diagnosed in some variables, there is a need to analyze possibilities for improvement in the selection of physical fitness tests that are closer to operationality in BAF Infantry military personnel.

Efficacy of cryotherapy as first line therapy in patients with Barrett's neoplasia: a systematic review and pooled analysis.

Cryotherapy has been used as salvage therapy; however, its efficacy as first line treatment in patients with Barrett's esophagus (BE) neoplasia has not been well studied. The aim of this paper was to perform a systematic review to look at the efficacy of cryotherapy as the primary treatment of BE. An electronic database search was performed (PubMed, Embase, Cochrane, and Google Scholar) to search for studies with cryotherapy as the initial primary modality of ablation in patients with BE neoplasia. Studies that included patients with other prior forms of therapy were excluded. The primary outcomes were the pooled rates of complete eradication of intestinal metaplasia (CE-IM) and CE of neoplasia (CE-N). Secondary outcomes were recurrence rates of neoplasia and intestinal metaplasia (IM) and adverse events. The statistical software OpenMetaAnalyst was used for analysis with pooled estimates reported as proportions (%) with 95% confidence intervals (CI) with heterogeneity (I2) among studies. The search revealed 6 eligible studies with a total of 282 patients (91.5% male, average age 65.3 years) with 459 person years of follow-up. 69.35% [95% CI (52.1%-86.5%)] of patients achieved CE-IM and 97.9% (95% CI: 95.5%-100%) had CE-N. 7.3% of patients had persistent dysplasia with 4% progressing to cancer. The recurrence rate of neoplasia was 10.4 and that of IM was 19.1 per 100 patient years of follow-up. The overall rate of stricture formation was 4.9%. There are scarce data on the use of cryotherapy as the primary modality for the treatment of BE dysplasia. The published data demonstrate efficacy rates of 69% and 98% for complete eradication of metaplasia and neoplasia, respectively. These results need to be assessed in prospective, comparative trials with other forms of therapy.

Kidney enlargement and multiple liver cyst formation implicate mutations in PKD1/2 in adult sporadic polycystic kidney disease.

Distinguishing autosomal-dominant polycystic kidney disease (ADPKD) from other inherited renal cystic diseases in patients with adult polycystic kidney disease and no family history is critical for correct treatment and appropriate genetic counseling. However, for patients with no family history, there are no definitive imaging findings that provide an unequivocal ADPKD diagnosis. We analyzed 53 adult polycystic kidney disease patients with no family history. Comprehensive genetic testing was performed using capture-based next-generation sequencing for 69 genes currently known to cause hereditary renal cystic diseases including ADPKD. Through our analysis, 32 patients had PKD1 or PKD2 mutations. Additionally, 3 patients with disease-causing mutations in NPHP4, PKHD1, and OFD1 were diagnosed with an inherited renal cystic disease other than ADPKD. In patients with PKD1 or PKD2 mutations, the prevalence of polycystic liver disease, defined as more than 20 liver cysts, was significantly higher (71.9% vs 33.3%, P = .006), total kidney volume was significantly increased (median, 1580.7 mL vs 791.0 mL, P = .027) and mean arterial pressure was significantly higher (median, 98 mm Hg vs 91 mm Hg, P = .012). The genetic screening approach and clinical features described here are potentially beneficial for optimal management of adult sporadic polycystic kidney disease patients.

ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease.

Cellular senescence is a state of stable cell cycle arrest triggered by diverse stresses. Establishment of senescence occurs in conjunction with a multitude of chromatin changes, which are just beginning to be studied. These chromatin changes are hypothesized to be causative for senescence. Currently, a preferred method to study such changes is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). This is usually done by cross-linking the cells with formaldehyde and then generating chromatin fragments between 150 and 300bp by sonication. The DNA replication-independent histone chaperone HIRA plays an important role in control of chromatin in nonproliferating senescent cells. While investigating the role of HIRA in senescence, we found conventional ChIP protocols to be problematic, routinely yielding too low amounts of DNA for sequencing. To overcome these problems we adapted and optimized an alternative ChIP method that does not rely on cross-linking and sonication for chromatin fragmentation, and is able to easily isolate chromatin from senescent cells ready for immunoprecipitation. This method uses Benzonase endonuclease for solubilization of uncross-linked chromatin by digestion of DNA and RNA, in the absence of proteolytic activity. Using this protocol, we were easily able to immunoprecipitate HIRA with sufficient DNA for Illumina sequencing.

Is increased time to diagnosis and treatment in symptomatic cancer associated with poorer outcomes? Systematic review.

BACKGROUND: It is unclear whether more timely cancer diagnosis brings favourable outcomes, with much of the previous evidence, in some cancers, being equivocal. We set out to determine whether there is an association between time to diagnosis, treatment and clinical outcomes, across all cancers for symptomatic presentations. METHODS: Systematic review of the literature and narrative synthesis. RESULTS: We included 177 articles reporting 209 studies. These studies varied in study design, the time intervals assessed and the outcomes reported. Study quality was variable, with a small number of higher-quality studies. Heterogeneity precluded definitive findings. The cancers with more reports of an association between shorter times to diagnosis and more favourable outcomes were breast, colorectal, head and neck, testicular and melanoma. CONCLUSIONS: This is the first review encompassing many cancer types, and we have demonstrated those cancers in which more evidence of an association between shorter times to diagnosis and more favourable outcomes exists, and where it is lacking. We believe that it is reasonable to assume that efforts to expedite the diagnosis of symptomatic cancer are likely to have benefits for patients in terms of improved survival, earlier-stage diagnosis and improved quality of life, although these benefits vary between cancers.

Markers of circulating tumour cells in the peripheral blood of patients with melanoma correlate with disease recurrence and progression.

BACKGROUND: Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. OBJECTIVES: To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. METHODS: Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3years and 9months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0-II, n=154) and metastatic melanoma (late stages, III-IV, n=76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFß2, PAX3d and MCAM. RESULTS: In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P=0·001 and P=0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. CONCLUSIONS: Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis.

OBJECTIVES: To identify an appropriate sampling technique(s) to accurately detect the bacteria causing urinary tract infections in dogs with urolithiasis. METHODS: Twenty-one dogs with urolithiasis were included in the study. Three types of samples were taken from each dog. Urine was collected by cystocentesis, and a urinary bladder mucosal biopsy and urolith were retrieved during cystotomy. The samples were then cultured on blood agar and MacConkey's agar to identify the bacteria associated with urinary tract infections. RESULTS: Bacterial urinary tract infection was found in 16 cases (76.19 per cent). The most prevalent bacteria found to cause urinary tract infection were Escherichia coli (n=7), followed by coagulase-positive Staphylococcus species (n=4), Klebsiella pneumoniae (n=2), Pseudomonas aeruginosa (n=2) and Proteus mirabilis (n=1). In the case of a positive urine culture, the same bacteria were also cultured from the urinary bladder mucosal biopsy alone or from both the urinary bladder mucosal biopsy and urolith. However, in the case of a negative urine culture, bacteria were found to be present in the urinary bladder mucosal biopsy or urolith cultures in 23.81 per cent of dogs. The uroliths that gave positive culture results were either infection-induced uroliths composed of struvite and calcium carbonate phosphate, ammonium acid urate only or metabolic uroliths composed of calcium oxalate and calcium phosphate, or calcium phosphate only. All the uroliths that gave negative culture results were metabolic uroliths composed of calcium oxalate and/or calcium phosphate, and uric acid and calcium phosphate. CLINICAL SIGNIFICANCE: When the culture from the urine obtained by cystocentesis is negative, cultures of urinary bladder mucosal biopsy and urolith are recommended in dogs with urolithiasis in order to accurately assess the microbiological status of the urinary tract.

Adherence of cyanoacrylate which leaked from gastric varices to the left renal vein during endoscopic injection sclerotherapy: a histopathologic study.

We report a case involving leakage of cyanoacrylate (CA) to the inferior vena cava (IVC) through a gastrorenal shunt and left renal vein. A 72-year-old man with liver cirrhosis was admitted to our hospital to undergo emergency treatment for massive hemorrhage of gastric varices. Endoscopic injection sclerotherapy (EIS) using CA was performed on the varices. Radiographic fluoroscopy revealed that most of the injected CA had adhered firmly to the gastric varices, but a certain portion of the CA had flowed to the IVC through the gastrorenal shunt and left renal vein. At that point, the patient did not complain of any symptoms. However, 6 months later, he died of hepatic failure and an autopsy was performed. Histopathologic examination of the wall of the IVC and renal vein, to which CA had adhered, revealed that the CA was covered with endothelial cells of the vessel and no nearby thrombus was present. Long-term anticoagulant therapy may not be indicated in cases of leakage of CA from the gastric varices to other veins, since the leaked CA may be readily covered with endothelium without thrombus formation as in our patient. It is possible for CA to flow to the IVC and have a fatal impact. Our patient was fortunate, and for safe EIS it is important that these complications are prevented.

Isolation and characterization of kidney-specific CLC-K2 chloride channel gene promoter.

CLC-K1 and CLC-K2 are highly homologous kidney-specific chloride channels, but they are expressed in the different nephron segments. To understand the molecular mechanisms of kidney-specific and nephron-segment-specific expression of CLC-K channel genes, the rat ClC-K2 gene promoter was cloned and compared with that of CLC-K1. In the 1.5-kb pair 5'-flanking region of the CLC-K2 gene, no TATA box was identified around the transcriptional start site, and the proximal region (-32 to -68) was characterized by a GA-rich motif that had a significant sequence similarity to that of the previously isolated CLC-K1 gene promoter. In contrast, the distal portion did not have significant sequence similarity to that of CLC-K1. Reporter gene assay and gel-retardation analysis revealed that the GA-rich motif and the binding of a specific protein(s) to this element were indispensable for the basal promoter activity of the CLC-K2 gene. These results suggest that the GA-rich element may have an important role in the promoter activities of the kidney-specific CLC-K1 and -K2 genes, but that the GA-element alone is not sufficient for the strict regulation of nephron-segment-specific expression of CLC-K1 and CLC-K2 genes.

Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter.

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle's loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5'-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the approximately 2.5-kb pair 5'-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7-to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the approximately 2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5'-flanking region but was lost in the -29 construct, clearly demonstrating that the 22 bp from -51 to -30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGG-GAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.

[Niceritrol decreases serum phosphate levels in chronic hemodialysis patients].

Since phosphorus retention in hemodialysis (HD) patients is known to be an important factor in the development of secondary hyperparathyroidism and renal osteodystrophy, phosphate binders have been needed for the control of serum phosphate levels (P). However, the calcium-containing phosphate binders that have been used widely can cause a rise in serum calcium levels and cause secondary hypoparathyroidism. We have recently experienced decreases in P after the administration of niceritrol (NT), a prodrug of nicotinic acid, for the treatment of low HDL-cholesteremia (HDL-C) in HD patients. The aim of the present study was to assess the mechanism of the P-lowering effect of NT in comparison with nicomol (NC), another prodrug of nicotinic acid. NT (750 mg/day) or NC (600 mg/day) was given orally to 10 or 14HD patients respectively. Blood samples were collected before the first dialysis of each week for the determination of serum urea nitrogen (UN), Cr, Ca, P, total cholesterol (TC), triglyceride (TG) and HDL-C. Serum nicotinic acid concentration (NAC) by gas chromatograph mass-spectrometry method was determined before, 4 weeks and 8 weeks after the administration of these drugs. After NT administration, P was decreased from 6.2 +/- 0.4 mg/dl to 5.1 +/- 0.4 mg/dl (1st week, p < 0.001, Mean +/- SE) and 4.5 +/- 0.3 mg/dl (2nd weeks, p < 0.001) with no change in UN, Cr or Ca levels; these significant decreases in P lasted for 8 weeks. NAC increased significantly after NT administration from 25.5 +/- 1.3 ng/ml to 549.8 +/- 102.2 ng/ml (4 weeks, p < 0.01) and 431.7 +/- 51.4 ng/ml (8 weeks, p < 0.01). HDL-C also increased (33.6 +/- 4.0 mg/dl vs 42.7 +/- 4.6 mg/dl, p < 0.05), but TC and TG did not change. In contrast, no significant changes were observed in P, NAC and HDL-C after NC administration. These discrepancies could be ascribed to the differences in serum NAC levels. These data suggest that NT could be useful for the control of P in HD patients. However further studies are needed to confirm the mechanism of the P-lowering effect of NT.

Urinary excretion of aquaporin-2 water channel protein in human and rat.

Previous studies by the authors demonstrated that the response of urinary aquaporin-2 (AQP2) excretion to dDAVP (deamino-8-D-arginine vasopressin) infusion is an index of vasopressin action on the kidney (N Engl J Med 332: 1540-1545, 1995). In the study presented here, the characteristics of urinary excretion of AQP2 were examined further. An RIA suitable for AQP2 in the urine was established. Relatively high concentrations of detergent and bovine serum albumin in the RIA buffer allowed analysis of urine samples with a wide range of concentrations and increased the sensitivity of the assay. AQP2 in the urine existed as a high molecular weight form of approximately 190 kD by HPLC analysis. The mean urinary AQP2 concentration corrected for creatinine in spot urine samples of healthy subjects who voided in the morning was 1081 +/- 699 fmol/mg creatinine (mean +/- SD, n = 208). The amount of daily excretion of AQP2 in the urine was the same in men and women. Urinary AQP2 content was not affected by age of the subjects and showed a positive correlation with urine osmolality. Finally, the fraction of AQP2 excreted in the urine compared with whole kidney content was determined in the rat. Approximately 3% of AQP2 in the kidney was excreted daily, and this fraction did not change when rats were dehydrated for 3 d. These data demonstrate the necessity of establishing well-designed protocols to use urinary AQP2 as a marker of AVP action.

Cloning of rat and mouse aquaporin-2 gene promoters and identification of a negative cis-regulatory element.

The promoters of rat and mouse aquaporin-2 (AQP-2) genes were cloned and compared with that of human genes. Nucleotide identity up to -593 bp was 62%, and consensus sequences such as TATA box and adenosine 3',5'-cyclic monophosphate responsive element were conserved. Deoxyribonuclease I footprint assay revealed a footprinted region at -210 to -184 bp in rat AQP-2 gene promoter produced by nuclear extract from nonexpressing (liver) tissue. The sequence of this region included a GATA motif but otherwise showed no homology with any other previously known cis-elements. Electromobility shift assay and ultraviolet cross-linking analysis confirmed that specific binding proteins to this element were present in kidney, spleen, and liver and that these proteins were distinct from GATA factors. Both deletion and mutation of this cis-element abolished the protein DNA binding and increased promoter activity in in vitro reporter gene assay using rat cultured hepatocyte Ac2F cells, suggesting the negative regulatory role of this cis-element. These results indicate that tissue-specific expression of AQP-2 gene may in part be regulated by this novel negative acting cis-element.

Transcriptional regulation of aquaporin-2 water channel gene by cAMP.

Aquaporin-2 (AQP-2) water channel is a key molecule for urinary concentration whose expression is augmented by dehydration in vivo. To elucidate the regulatory mechanism of this phenomenon in vitro, mouse collecting duct cell lines were established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T antigen gene and then screened for the AQP-2 expression, using ribonuclease protection assay. In one cell line designated C4, the endogenous AQP-2 mRNA level measured by ribonuclease protection assay increased fourfold after treatment with chlorophenylthio-cAMP (cpt-cAMP) (400 microM). In contrast, phorbol 12-myristate 13-acetate did not affect the AQP-2 mRNA level. To identify the molecular mechanism(s) of cAMP-induced upregulation of AQP-2 mRNA in C4 cells, luciferase assay was performed using various 5'-flanking regions of the human AQP-2 gene. Luciferase activity in C4 cells transfected with constructs containing approximately 2.8-kbp or 224-bp 5'-flanking region showed a 3.5-fold increase by cpt-cAMP treatment, indicating that the 224-bp 5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms. This region contains cAMP-responsive element (CRE), and the deletion of the core sequence of CRE (GACGTCA) or introduction of mutation into CRE (GTGGTCA) completely abolished the responsiveness to cpt-cAMP, confirming the key role of CRE in the cAMP-induced transcriptional activation of the AQP-2 gene. Electrophoretic mobility shift assay revealed the existence of proteins binding to CRE in C4 cells and in rat kidney. The binding of CRE proteins to CRE was increased in the nuclear extract from cpt-cAMP-treated C4 cells and dehydrated rat kidney compared with those from controls. These results demonstrated that the CRE in the AQP-2 gene promoter is a key cis-element for cAMP-mediated transcriptional regulation of this gene and may be important for in vivo regulation of AQP-2 expression in a dehydrated state.

Regulation of aquaporin-2 gene transcription by GATA-3. off.

To evaluate the functional role of GATA motifs in the 5'-flanking region of a kidney-specific AQP-2 water channel gene, we sought to isolate a GATA factor(s) expressed in collecting ducts and determined the role on the AQP-2 promoter. Two cDNAs encoding GATA factors were isolated from rat kidney, whose sequences were highly homologous with human GATA-2 and -3. Reverse-transcription PCR using dissected nephron segments revealed that rat GATA-3 but not GATA-2 was expressed in collecting ducts, thus indicating that GATA-3 could interact with GATA motifs in the AQP-2 promoter. Transactivation experiments utilizing the rat GATA-3 expression vector indicated that rat GATA-3 increased the AQP-2 promoter activity about fourfold. These results indicated that GATA motifs in the 5'-flanking region of the hAQP-2 gene were functional cis-elements and that GATA-3 in collecting ducts may be one of the important regulators of AQP-2 expression in vivo.