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Introduction: Despite several therapeutic efforts, lung cancer remains a highly lethal disease. Novel therapeutic approaches encompass immune-checkpoint inhibitors, targeted therapeutics and antibody-drug conjugates, with different results. Several studies have been aimed at identifying biomarkers able to predict benefit from these therapies and create a prediction model of response, despite this there is a lack of information to help clinicians in the choice of therapy for lung cancer patients with advanced disease. This is primarily due to the complexity of lung cancer biology, where a single or few biomarkers are not sufficient to provide enough predictive capability to explain biologic differences; other reasons include the paucity of data collected by single studies performed in heterogeneous unmatched cohorts and the methodology of analysis. In fact, classical statistical methods are unable to analyze and integrate the magnitude of information from multiple biological and clinical sources (eg, genomics, transcriptomics, and radiomics)., Methods and Objectives: APOLLO11 is an Italian multicentre, observational study involving patients with a diagnosis of advanced lung cancer (NSCLC and SCLC) treated with innovative therapies. Retrospective and prospective collection of multiomic data, such as tissue- (eg, for genomic, transcriptomic analysis) and blood-based biologic material (eg, ctDNA, PBMC), in addition to clinical and radiological data (eg, for radiomic analysis) will be collected. The overall aim of the project is to build a consortium integrating different datasets and a virtual biobank from participating Italian lung cancer centers. To face with the large amount of data provided, AI and ML techniques will be applied will be applied to manage this large dataset in an effort to build an R-Model, integrating retrospective and prospective population-based data. The ultimate goal is to create a tool able to help physicians and patients to make treatment decisions., Conclusion: APOLLO11 aims to propose a breakthrough approach in lung cancer research, replacing the old, monocentric viewpoint towards a multicomprehensive, multiomic, multicenter model. Multicenter cancer datasets incorporating common virtual biobank and new methodologic approaches including artificial intelligence, machine learning up to deep learning is the road to the future in oncology launched by this project., Competing Interests: Disclosures Arsela Prelaj certifies that all conflicts of interest reported can be considered outside the present paper: consulting or advisory role for BMS, AstraZeneca; had travel, accommodations, or other expenses paid or reimbursed by Roche, Italfarmaco; principal investigator of Spectrum Pharmaceuticals. Alessandra Laura Giulia Pedrocchi holds shares of Agade srl. Giuseppe Lo Russo has received fees for acting as a consultant from Roche, Novartis, BMS, MSD, AstraZeneca, Takeda, Amgen, Sanofi, Italfarmaco, Pfizer; has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Roche, Novartis, BMS, MSD, AstraZeneca, Takeda, Amgen, Sanofi, has received support for attending meetings and/or travel from Roche, BMS, MSD; has participated on data safety monitoring board or advisory board for Roche, Novartis, BMS, MSD, AstraZeneca, Sanofi, has acted as principal investigator in sponsored clinical trials for Roche, Novartis, BMS, MSD, AstraZeneca, GSK, Amgen, Sanofi. Rossana Berardi has received fees for acting as a consultant, for lectures and/or for participating to advisory board from BI, EISAI, GSK, Italfarmaco, Otsuka, Lilly, MSD; has received funding to Institution from AZ, BMS, Pfizer, Novartis, Roche; AMGEN. Giulia Galli declares the following conflicts of interest: Italpharma (advisory board); Roche (travel accommodation); Astra Zeneca, BMS, MSD (honoraria for lectures). Federica Bertolini has received consultant fees from MSD, Astra-Zeneca, Lilly, Eisai, Sanofi and speakers fee from BMS, MSD, Astra Zeneca. Filippo de Braud reports a patent for PCT/IB2020/055956 pending and a patent for IT201900009954 pending; and Roche, EMD Serono, NMS Nerviano Medical Science, Sanofi, MSD, Novartis, Incyte, BMS, Menarini Healthcare Research & Pharmacoepidemiology, Merck Group, Pfizer, Servier, AMGEN, Incyte. No disclosures were reported by the other authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The patient selection for optimal adjuvant therapy in gastrointestinal stromal tumors (GISTs) is provided by nomogram based on tumor size, mitotic index, tumor location, and tumor rupture. Although mutational status is not currently used to risk assessment, tumor genotype showed a prognostic influence on natural history and tumor relapse. Innovative measures, such as KIT/PDGFRA-mutant-specific variant allele frequency (VAF) levels detection from next-generation sequencing (NGS), may act as a surrogate of tumor burden and correlate with prognosis and overall survival of patients with GIST, helping the choice for adjuvant treatment., Patients and Methods: This was a multicenter, hospital-based, retrospective/prospective cohort study to investigate the prognostic role of KIT or PDGFRA-VAF of GIST in patients with radically resected localized disease. In the current manuscript, we present the results from the retrospective phase of the study., Results: Two-hundred (200) patients with GIST between 2015 and 2022 afferent to 6 Italian Oncologic Centers in the EURACAN Network were included in the study. The receiver operating characteristic (ROC) curves analysis was used to classify "low" vs. "high" VAF values, further normalized on neoplastic cellularity (nVAF). When RFS between the low and high nVAF groups were compared, patients with GIST with KIT/PDGFRA nVAF > 50% showed less favorable RFS than patients in the group of nVAF ≤ 50% (2-year RFS, 72.6% vs. 93%, respectively; P = .003). The multivariable Cox regression model confirmed these results. In the homogeneous sub-population of intermediate-risk, patients with KIT-mutated GIST, the presence of nVAF >50% was statistically associated with higher disease recurrence., Conclusion: In our study, we demonstrated that higher nVAF levels were independent predictors of GIST prognosis and survival in localized GIST patients with tumors harboring KIT or PDGFRA mutations. In the cohort of intermediate-risk patients, nVAF could be helpful to improve prognostication and the use of adjuvant imatinib., (© The Author(s) 2023. Published by Oxford University Press.)

Introduction: Axonal loss is an important feature of Multiple Sclerosis (MS), being strongly related to irreversible disability accumulation. Nonetheless, the exact mechanisms underlying axonal loss remain unclear. Cerebrospinal fluid (CSF) levels of Tau and Beta-amyloid (Abeta) currently represent diagnostic biomarkers in other neurodegenerative diseases. In MS, studies on CSF Tau and Abeta provided preliminary informations on disease prognosis, but results have not yet been replicated., Methods: We investigated whether CSF Tau and Abeta levels could predict early disability accumulation in MS patients. 100 patients underwent CSF analysis during their diagnostic work-up. Demographic, clinical, radiological features and CSF were collected at baseline. MS severity score (MSSS) and age-related MSSS (ARMSS) were calculated at last follow-up. We performed Mann-Whitney test, Spearman's coefficient, and multiple regression analysis for significant predictors of disability based on CSF Abeta and Tau levels, gender, age at diagnosis and MRI characteristics at baseline., Results: Baseline CSF Tau levels moderately correlated with MSSS (r=0.372 p=0.0001) and weakly with ARMSS (r=0.237 p=0.0176) after a mean two years follow-up. Predictors of early disability evaluated with MSSS and ARMSS were CSF Tau (Beta:0.258 p=0.009 and Beta:0.252 p=0.01) and spinal cord involvement (Beta:0.196 p=0.029 and Beta:0.240 p=0.008); as well as age at MS diagnosis (Beta:0.286 p=0.001) for MSSS, and high brain lesion load (Beta:0.207 p=0.02) for ARMSS., Conclusion: CSF Tau levels at diagnosis possibly has a predictive value along with MRI features and age at diagnosis. We hypothesize that Tau levels may express chronic axonal damage, possibly contributing to early MS disability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cerebrospinal fluid (CSF) kappa free light chain (KFLC) index has been described as a reliable marker of intrathecal IgG synthesis to diagnose multiple sclerosis (MS). Our aims were: (1) to compare the efficiency of KFLC through different interpretation approaches in diagnosing MS. (2) to evaluate the prognostic value of KFLC in radiologically and clinically isolated syndromes (RIS-CIS). We enrolled 133 MS patients and 240 with other neurological diseases (93 inflammatory including 18 RIS-CIS, 147 non-inflammatory). Albumin, lambda free light chain (LFLC) and KFLC were measured in the CSF and serum by nephelometry. We included two groups of markers: (a) corrected for blood-CSF barrier permeability: immunoglobulin G (IgG), KFLC and LFLC indexes. (b) CSF ratios (not including albumin and serum-correction): CSF KFLC/LFLC, CSF KFLC/IgG, CSF LFLC/IgG. KFLC were significantly higher in MS patients compared to those with other diseases (both inflammatory or not). KFLC index and CSF KFLC/IgG ratio showed high sensitivity (93% and 86.5%) and moderate specificity (85% and 88%) in diagnosing MS. RIS-CIS patients who converted to MS showed greater KFLC index and CSF KFLC/IgG. Despite OB are confirmed to be the gold-standard to detect intrathecal IgG synthesis, the KFLC confirmed their accuracy in MS diagnosis. A "kappa-oriented" response characterizes MS and has a prognostic impact in the RIS-CIS population.

Anorexia is one of the most widespread eating disorders that appears to contribute to malnutrition in patients with advanced kidney dysfunction. The changes of neuropeptides controlling feeding behaviors synthesized in the hypothalamus under several physiological condition could induce anorexia. While several mechanisms underlying uremic anorexia have been proposed, the changes of hypothalamic neuropeptides controlling feeding behaviors of uremic patients are poorly understood. The gene expressions of hypothalamic neuropeptides controlling feeding behaviors were evaluated after bilateral nephrectomy, which is a model of acute kidney dysfunction, by in situ hybridization histochemistry. Food consumption decreased markedly in bilateral nephrectomized rats. The mRNA levels of corticotrophin-releasing hormone, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, which suppress feeding behavior, were significantly higher in bilateral nephrectomized rats than in sham-operated rats. On the other hand, the mRNA levels of Agouti-related peptide, neuropeptide Y, melanin-concentrating hormone, and orexin, which promote feeding behavior, were significantly lower in bilateral nephrectomized rats than in sham-operated rats. In addition, the plasma level of leptin, which has an anorexic effect, increased after bilateral nephrectomy. The results suggest that hypothalamic neuropeptides controlling feeding behaviors may be involved in the development of anorexia in bilateral nephrectomized rats. This report is the first step to elucidating the physiological mechanisms of anorexia in patients with kidney dysfunction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Cerebrospinal fluid (CSF) kappa free light chains (KFLC) have been suggested as quantitative alternative to oligoclonal bands (OB) in multiple sclerosis (MS) diagnosis. Despite OB have been associated to poor disease prognosis, little is known on KFLC in predicting MS early progression. Our aim is to evaluate the prognostic value of KFLC in a cohort of Italian MS patients., Methods: 100 patients (64 females) underwent CSF analysis during their diagnostic MS work-up. We collected clinical/paraclinical features (gender, age at onset, clinical course, early MS treatments (within 1 year), gadolinium-enhancing (Gd+) lesions), calculated K index (ratio CSF-serum KFLC and albumin), and MS severity score (MSSS) at last follow up (minimum 1 year). Statistical analysis included Mann-Whitney descriptive analysis, Spearman correlation for independent samples, and linear regression for significant predictors., Results: K index resulted a significant predictor for disability over time being higher in patients who developed greater MSSS. Accordingly, K index was also significantly increased in patients undergoing early versus delayed treatment (N = 50/100, p = 0.046). A similar role in predicting MS disability was confirmed for age at onset. No other factors were retained in our regression model. Of note, K index was not associated to known MS prognostic markers such as gender, age at onset, and Gd+ lesions (N = 31/96)., Conclusion: Our study suggests KFLC as a CSF quantitative marker to predict early disability in MS (despite not being a substitute for OB)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acute loss of kidney function is a critical internal stressor. Arginine vasopressin (AVP) present in the parvocellular division of the paraventricular nucleus (PVN) plays a key role in the regulation of stress responses. However, hypothalamic AVP dynamics during acute kidney dysfunction remain unclear. In this study, we investigated the effects of bilateral nephrectomy on AVP, using a transgenic rat line that expressed the AVP-enhanced green fluorescent protein (eGFP). The eGFP fluorescent intensities in the PVN were dramatically increased after bilateral nephrectomy. The mRNA levels of eGFP, AVP, and corticotrophin-releasing hormone in the PVN were dramatically increased after bilateral nephrectomy. Bilateral nephrectomy also increased the levels of Fos-like immunoreactive cells in brainstem neurons. These results indicate that bilateral nephrectomy upregulates the AVP-eGFP synthesis. Further studies are needed to identify the neural and/or humoral factors that activate AVP synthesis and regulate neuronal circuits during acute kidney dysfunction.

The K free light chain (K) index has been suggested as a reliable marker of intrathecal synthesis,despite the 2017 McDonald criteria for multiple sclerosis (MS) suggesting to "interpret with caution positiveimmunoglobulin G (IgG) index when testing for oligoclonal bands (OB) is negative or not performed". Theaim of this study was to compare the performance of K and IgG indexes for MS diagnosis and OB detectionin a cohort of Italian patients. We enrolled 385 patients (127 MS, 258 non-MS) who had cerebrospinal fluid(CSF) analysis, including isoelectric focusing (IEF), to detect OB in the diagnostic work-up. Albumin, IgGand free light chains were measured by nephelometry and used to calculate IgG and K indexes. Althoughthe two markers were highly related (r = 0.75, r2 = 0.55, p < 0.0001), the K index showed greater sensitivity andnegative predictive value (versus the IgG index) for OB detection (97% versus 48% and 97% versus 71%) andMS diagnosis (96% versus 50% and 98% versus 78%). These results support K index (and not IgG index) as afirst-line marker for MS, followed by IEF, according to a sequential testing approach in CSF analysis., Competing Interests: Domizia Vecchio received a Merk–Serono fellowship. All the other authors have nothing to disclose and declare no conflict of interest.

Furosemide, which is used worldwide as a diuretic agent, inhibits sodium reabsorption in the Henle's loop, resulting in diuresis and natriuresis. Arginine vasopressin (AVP) is synthesized in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) of the hypothalamus. The synthesis AVP in the magnocellular neurons of SON and PVN physiologically regulated by plasma osmolality and blood volume and contributed water homeostasis by increasing water reabsorption in the collecting duct. Central AVP dynamics after peripheral administration of furosemide remain unclear. Here, we studied the effects of intraperitoneal (i.p.) administration of furosemide (20 mg/kg) on hypothalamic AVP by using transgenic rats expressing AVP-enhanced green fluorescent protein (eGFP) under the AVP promoter. The i.p. administration of furosemide did not affect plasma osmolality in the present study; however, eGFP in the SON and magnocellular divisions of the PVN (mPVN) were significantly increased after furosemide administration compared to the control. Immunohistochemical analysis revealed Fos-like immunoreactivity (IR) in eGFP-positive neurons in the SON and mPVN 90 min after i.p. administration of furosemide, and AVP heteronuclear (hn) RNA and eGFP mRNA levels were significantly increased. These furosemide-induced changes were not observed in the suprachiasmatic AVP neurons. Furthermore, furosemide induced a remarkable increase in Fos-IR in the organum vasculosum laminae terminals (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), locus coeruleus (LC), nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVLM) after i.p. administration of furosemide. In conclusion, we were able to visualize and quantitatively evaluate AVP-eGFP synthesis and neuronal activations after peripheral administration of furosemide, using the AVP-eGFP transgenic rats. The results of this study may provide new insights into the elucidation of physiological mechanisms underlying body fluid homeostasis induced by furosemide. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

We investigated the effects of bicarbonate/lactate-buffered peritoneal dialysis fluid (B/L-PDF) and lactate-buffered PDF (L-PDF) on cell viability and apoptosis, focusing on monocarboxylate transporters (MCTs). MCT-1 transports lactate into cells. Cell viability and apoptosis of human peritoneal mesothelial cells (HPMCs) were examined by water-soluble tetrazolium salt-1 and TUNEL assays, respectively. The relative number of viable HPMCs was significantly decreased by L-PDF at 48 h (8.8 ± 0.4%) compared with cells cultured in M199, but not by B/L-PDF (66.7 ± 1.1%). Apoptosis was markedly induced by L-PDF at 48 h (69.3 ± 16.2%), but not by B/L-PDF (2.6 ± 0.3%). Knockdown of MCT-1 by small interfering RNA (siRNA) attenuated the L-PDF-induced reduction of viable cells and increased apoptosis compared with control siRNA, but MCT-4 knockdown had no effect. B/L-PDF had lesser effects on cell viability and apoptosis of HPMCs compared with L-PDF. These results suggest that B/L-PDF biocompatibility occurs by avoiding the induction of apoptosis in HPMCs., (© 2016 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

Background: Continuous exposure to peritoneal dialysis fluids (PDFs) is associated with pathological responses such as persistent micro-inflammation, which leads to ultrafiltration failure. Pentraxin-3 (PTX3), a multifunctional soluble pattern recognition receptor, is produced at sites of inflammation by a wide range of cell types. This study investigates the in vivo expression of PTX3 in the peritoneal membrane of a rat continuous peritoneal dialysis (PD) model, as well as the effect of high glucose on the in vitro expression of PTX3., Methods: The expression of PTX3 was analyzed using RT-PCR, real-time PCR, immunohistochemistry and western blotting in a PD rat model receiving saline or conventional PDF containing 3.86% glucose for 8 weeks. The effects of high glucose on the expression of PTX3 were examined in cultured rat peritoneal mesothelial cells (RPMCs), mouse macrophage-like cells, and mouse fibroblasts., Results: In a rat model of PD, eight-week instillation of the conventional PDF produced increased submesothelial thickening, followed by substantially enhanced PTX3 protein levels in the submesothelial layer of peritoneal membrane. PTX3 was detected in peritoneal mesothelial cells, macrophages and fibroblasts in the thickened submesothelial area. Glucose was found to induce PTX3 protein expression in RPMCs as well as macrophage-like cells and fibroblasts., Conclusion: Continuous exposure to conventional PDF induces PTX3 expression in the peritoneal membrane of rats. High glucose may be involved in the mechanism of PDF-induced local micro-inflammation in the peritoneum.

Background: In addition to corticosteroids and inhibition of the renin-angiotensin-aldosterone system, tonsillectomy with steroid pulse therapy (TSP) may have a beneficial impact on the clinical course of IgA nephropathy (IgAN). However, there is still much uncertainty regarding the indications for therapy, treatment protocol, and therapeutic options for IgAN., Methods: In this multicenter retrospective cohort study, we enrolled 284 patients with biopsy-proven IgAN who received TSP or corticosteroid therapy or conservative therapy. The effects of TSP on clinical remission (CR) were evaluated after a median follow-up period of 4.1 years in relation to histological classifications., Results: Among the 284 participants, 161 patients received TSP. During the observation time, 141 patients (49.6%) achieved CR, with a median time to remission of 397 days. In multivariate Cox regression analyses, TSP had an impact on achieving CR in only the group with histological grade 3 defined as glomerulosclerosis, crescent formation or adhesion to Bowman's capsule in 10-30% of all biopsied glomeruli, or mild cellular infiltration in the interstitium (hazard ratio (HR) 4.29, 95% confidence interval (95%CI) 1.88-11.19, P < 0.001). TSP independently contributed to a higher incidence of CR, particularly in the patient group showing evident mesangial hypercellularity (HR 2.54, 95%CI 1.38-5.08, P = 0.002)., Conclusions: TSP may have a beneficial effect on the clinical course in IgAN patients with mild to moderate glomerular and interstitial lesions, particularly with distinct mesangial cell proliferation.

Uromodulin-associated kidney disease (UAKD) is an autosomal dominant disease caused by a mutation in the uromodulin (UMOD) gene, leading to end-stage renal disease. We herein report the case of a family with UAKD caused by a novel mutation (C135G) in the UMOD gene. A 31-year-old woman had a low estimated glomerular filtration rate (59.7 mL/min per 1.73 m(2)). Her father, grandfather and paternal aunt had received maintenance hemodialysis therapy since their 40's. This case underscores the importance of performing genetic testing in young patients even in cases involving only moderate abnormalities in the kidney function.

Introduction: The increasing demand for therapeutic monitoring in patients receiving antiplatelet therapy has been paralleled by the development of instruments and tests whose clinical usefulness is still under debate. We devised a laboratory approach to detect patients with antiplatelet resistance at risk to develop thrombotic events., Methods: One hundred and eighty patients, under aspirin and clopidogrel after angioplasty and stent implantation, were studied by PFA100(®) with collagen/epinephrine (CoEPI, cutoff 165s) cartridge and by Multiplate(®) using arachidonic acid (ASPItest, pos < 862AUC), ADP (ADPtest, pos < 417AUC), and collagen (COLtest, pos < 607AUC)., Results: Only 67 of 173 patients with ASPI < 862 displayed a prolonged CoEPI and up to 65 patients had normal CoEPI despite ASPI < 300. Patients with ASPI < 300 had significantly lower COL than patients with ASPI > 300. One hundred and thirty-eight patients displaying ADP < 417 had significantly lower COL than those with ADP > 417. Association between COL and ADP remained after ASPI stratification: in patients with suboptimal (ASPI 300-892) or maximal (ASPI < 300) response to aspirin, having ADP < 417 (clopidogrel responsive) increased COL positivity, respectively, from 9.5 to 58.8% and from 47.6 to 82.7%., Conclusion: A combination of specific tests may be useful in identifying higher-risk patients with poor compliance or drug resistance who potentially may benefit from therapy change., (© 2012 Blackwell Publishing Ltd.)

Aims: Exposure to glucose and its metabolites in peritoneal dialysis fluid (PDF) results in structural alterations of the peritoneal membrane. Icodextrin-containing PDF eliminates glucose and reduces deterioration of peritoneal membrane function, but direct effects of icodextrin molecules on peritoneal mesothelial cells have yet to be elucidated. We compared the impacts of icodextrin itself with those of glucose under PDF-free conditions on wound healing processes of injured mesothelial cell monolayers, focusing on integrin-mediated cell adhesion mechanisms., Main Methods: Regeneration processes of the peritoneal mesothelial cell monolayer were investigated employing an in vitro wound healing assay of cultured rat peritoneal mesothelial cells treated with icodextrin powder- or glucose-dissolved culture medium without PDF, as well as icodextrin- or glucose-containing PDF. The effects of icodextrin on integrin-mediated cell adhesions were examined by immunocytochemistry and Western blotting against focal adhesion kinase (FAK)., Key Findings: Cell migration over fibronectin was inhibited in conventional glucose-containing PDF, while icodextrin-containing PDF exerted no significant inhibitory effects. Culture medium containing 1.5% glucose without PDF also inhibited wound healing of mesothelial cells, while 7.5% icodextrin-dissolved culture medium without PDF had no inhibitory effects. Glucose suppressed cell motility by inhibiting tyrosine phosphorylation of FAK, formation of focal adhesions, and cell spreading, while icodextrin had no effects on any of these mesothelial cell functions., Significance: Our results demonstrate icodextrin to have no adverse effects on wound healing processes of peritoneal mesothelial cells. Preservation of integrin-mediated cell adhesion might be one of the molecular mechanisms accounting for the superior biocompatibility of icodextrin-containing PDF., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Strongyloidiasis, a chronic infection caused by the intestinal parasite Strongyloides stercoralis, is prevalent in the Nansei Islands of Japan. Here, we report our findings on a case of strongyloidiasis complicated with steroid-resistant minimal change nephrotic syndrome in a 69-year-old male resident of Fukuoka Prefecture who had lived in Yakushima, one of the Nansei Islands, until age 15. In October 2006, he developed proteinuria and edema, and was diagnosed with minimal change nephrotic syndrome on the basis of the renal biopsy findings. Following treatment with prednisolone, the level of proteinuria decreased to 0.29 g/day by day 35. However, 5 days later (day 40), the patient developed persistent watery diarrhea and vomiting, leading to dehydration and malnutrition. Pneumonia and bacterial meningitis subsequently developed (day 146); filarial (infectious-type) and rhabditiform (noninfectious-type) S. stercoralis larvae were detected for the first time in the patient's sputum, gastric juice, feces, and urine. Although treatment with ivermectin was started immediately and the parasitosis responded to the treatment, the patient died of sepsis. Consequently, although strongyloidiasis is a rare infection except in endemic regions, it is essential to consider the possibility of this disease and begin treatment early for patients who have lived in endemic areas and who complain of unexplained diarrhea during steroid-induced or other immunosuppression.

Background: Bioincompatible peritoneal dialysis fluids (PDFs) cause pathological changes in the peritoneal membrane, related to membrane dysfunction and progressive peritoneal fibrosis. We investigated the effects of Pro-His-Ser-Arg-Asn (PHSRN) peptide, one of the fibronectin cell-binding domains that activates integrins and reinforces wound healing, on peritoneal remodelling in a rat peritoneal injury model undergoing peritoneal dialysis., Methods: The peritoneal mesothelial monolayer was removed by a stripping procedure in rats receiving conventional high glucose-containing PDF supplemented with or without PHSRN or control His-Ser-Pro-Asn-Hrg (HSPNR) peptides. Effects of PHSRN on cell motility and signalling molecules were examined in cultured rat peritoneal mesothelial cells (RPMCs) and normal rat kidney fibroblasts (NRKs)., Results: The cytokeratin- and HBME-1-positive mesothelial cell monolayer was selectively removed by the procedure. By day 6, HBME-1-positive cells had regenerated to 53.3 +/- 6.5% of the peritoneal surface in the control group. Regeneration of the mesothelial layer was delayed in the PDF group (35.2 +/- 10.2%, P < 0.05), but PHSRN reversed the effects of PDF (51.7 +/- 9.6%, P < 0.05). PDF treatment increased thickening of granulomatous submesothelial tissue and numbers of ED1-, CD31- and alpha-smooth muscle actin-positive cells, but PHSRN ameliorated these effects. HSPNR had no effects on mesothelial regeneration or peritoneal wound healing. PHSRN, but not HSPNR, recovered glucose-induced inhibition of cell motility and phosphorylation of focal adhesion kinase and its downstream p130(Cas) in RPMCs and NRKs., Conclusions: These results suggest that PHSRN has beneficial effects on peritoneal regeneration by reducing the inhibitory effects of conventional PDF on integrin-mediated wound healing.

Background: Angiotensin II receptor blockers (ARBs) provide renoprotective effects in patients with mild-to-moderate chronic kidney disease (CKD). However, there have been few reports regarding whether ARBs show clinical efficacy and safety in patients with advanced CKD., Methods: Seventy-two hypertensive patients with Stages 3 - 4 CKD receiving no ARBs were enrolled in this study and observed up to 48 months. Telmisartan was added to conventional antihypertensive agents (n = 36, mean estimated glomerular filtration ratio [eGFR] 19.7 ml/min/1.73 m2) whilst the remaining control patients were not treated with ARBs (n = 36, mean eGFR 19.2 ml/min/1.73 m2). Urinary protein excretion, kidney function, and the occurrence of end-stage renal disease requiring renal replacement therapy, hyperkalemia, and death were analyzed., Results: Baseline characteristics of each group were similar. During the observation period, the blood pressures of each group decreased at similar rates. In the telmisartan group, 17 patients (47.2%) were introduced to renal replacement therapy, as compared with 31 patients (86.1%) in the control group (relative risk 0.55, 95% confidence interval 0.19 - 0.92, p < 0.05). Telmisartan significantly reduced proteinuria levels (from 3.47 +/- 3.00 to 2.41 +/- 2.46 g/g . creatinine, p < 0.05) and was associated with a reduction of 49.6% in the decline rate of eGFR. The incidence of major adverse events in both groups was similar., Conclusions: The addition of telmisartan to conventional antihypertensive therapy is associated with significant improvement in kidney outcome without increased incidence of adverse effects, even in patients with advanced CKD.

Aims: Insulin-like growth factor (IGF)-1 is a major mitogenic growth factor for mesangial cells (MCs). Statins slow the progression of chronic kidney disease by affecting inflammatory cell signaling pathways, in addition to improving lipid profile, however, no studies have investigated the effects of fluvastatin on mitogen-activated protein (MAP) kinase activity or MC proliferation in kidney cells. We investigated the effects of fluvastatin on IGF-1-induced activation of intracellular signal pathways and MC proliferation, and examined the inhibitory mechanisms of fluvastatin., Main Methods: Western blotting and cell proliferation assay were used., Key Findings: IGF-1 induced phosphorylation of extracellular-related kinase (ERK)1/2, MAP or ERK kinase (MEK)1/2, and Akt, expression of cyclin D1, and MC proliferation in cultured human MCs. Fluvastatin or PD98059, an MEK1 inhibitor, completely abolished IGF-1-induced MEK1/2 and ERK1/2 phosphorylation and MC proliferation, whereas inhibition of Akt had no effect on MC proliferation. Mevalonic acid prevented fluvastatin inhibition of IGF-1-induced MEK1/2 and ERK1/2 phosphorylation, cyclin D1 expression, and MC proliferation., Significance: Fluvastatin inhibits IGF-1-induced activation of the MAP kinase pathway and MC proliferation by mevalonic acid depletion, and might have renoprotective effects by inhibiting IGF-1-mediated MC proliferation.

Mizoribine (MZR) has shown to be effective against antineutrophil cytoplasmic antibody (ANCA)-related vasculitis; however, no reports have described the successful treatment of steroid-resistant ANCA-related vasculitis with MZR in patients with renal insufficiency requiring hemodialysis. We herein report the case of a 39-year-old man undergoing hemodialysis in whom MZR successfully lowered the myeloperoxidase (MPO)-ANCA titer accompanied by remission of interstitial pneumonia, together with the pharmacokinetics of MZR. The patient developed severe renal insufficiency and interstitial pneumonia, and was started on hemodialysis. Although prednisolone was administered followed by azathioprine, the MPO-ANCA level and interstitial pneumonia showed insufficient improvement. Azathioprine was replaced by MZR and the administered dose of MZR was determined by measuring serum concentrations of MZR at the start of the dialysis session; this was because we confirmed that MZR could only be removed via dialysis, and that the serum concentration of MZR was maintained until the next dialysis session. The maintenance dose was finally set at MZR 75 mg after each dialysis. Subsequently, the ANCA titer decreased and interstitial pneumonia resolved without any MZR-related side effects. This case demonstrates that MZR is safe and effective, even in patients with steroid-resistant ANCA-related vasculitis undergoing hemodialysis, and can be monitored by measuring serum concentrations of MZR.

Lupus anti-coagulants (LA) are a variety of anti-phospholipid antibodies characterized by their capacity to interfere with phospholipid-dependent coagulation assays. LA are increasingly recognized as important predictors of thrombosis. However, the antigen specificity of LA is still poorly characterized. Growing evidence indicates that oxidized phospholipids are among the targets of anti-phospholipid antibodies. This prompted us to investigate the role of IgG directed against different oxidized phospholipids in 164 subjects without clotting factor defects that were tested for the presence of LA using a LA-sensitive activate partial thromboplastin time (aPTT-FSL) and a screening/confirmation assay based on diluted Russell's viper venom test (dRVVT-PL). The response to aPTT-FSL was significantly (P < 0.0005) associated with high titres of IgG against oxidized phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, whereas positivity to dRVVT-PL was associated with the elevation of IgG against oxidized phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine (P < 0.0005) and phosphatidylinositol (P < 0.01). No difference in reactivity against oxidized cardiolipin was evident between the different groups. Positivity to the dRVVT-PL test was also associated significantly (P < 0.005) with the elevation of anti-cardiolipin and anti-beta(2)-glycoprotein-1 IgG. However, stepwise logistic regression demonstrated that IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylcholine were the only independent predictors of the response to dRVVT-PL assay, while IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylinositol were independent predictors of the response to aPTT-FSL test. In conclusion, autoantibodies against defined oxidized phospholipids are independent predictors of LA detection by aPTT-FSL or dRVVT-PL assays and might contribute to the variability often observed in the responses to the functional tests detecting LA.

Background/aims: Chronic hepatitis C (CHC) is often associated with auto-immune reactions. In the light of the role of alcohol in promoting CHC progression, we have investigated the possible presence of auto-reactivity against the ethanol-inducible cytochrome P4502E1 (CYP2E1) in CHC patients with and without alcohol consumption., Methods: The IgG reactivity against recombinant human CYP2E1 was evaluated by solid-phase immunoassays in 102 CHC patients with different alcohol consumption and 59 HCV-free controls., Results: Auto-antibodies against CYP2E1 were significantly (p<0.0001) increased in CHC patients as compared to controls. Anti-CYP2E1 IgG above the 97th percentile in the controls were evident in 41 (40%) CHC patients. Competition experiments revealed that CYP2E1 recognition was not due to the cross-reactivity with CYP2D6. The detection of anti-CYP2E1 IgG was unrelated to alcohol consumption and no difference in gender, age, aminotransferase levels and virus genotype was evident among the patients with or without anti-CYP2E1 auto-antibodies. However, anti-CYP2E1 auto-reactivity was significantly (p=0.025) associated with the severity of periportal/periseptal interface hepatitis. Moreover, confocal microscopy demonstrated that anti-CYP2E1 IgG associated with CHC recognized CYP2E1 exposed on the outer side of hepatocyte plasma membranes., Conclusions: HCV infection favours the breaking of self-tolerance against CYP2E1 that might contribute to hepatocyte injury.

The mechanism of peritoneal fibrosis in patients on continuous ambulatory peritoneal dialysis (CAPD) is poorly elucidated. We investigated the cellular mechanism of high-glucose-induced expression of monocyte chemoattractant protein-1 (MCP-1), which is important in recruiting monocytes into the peritoneum and progression of peritoneal fibrosis, and examined the inhibitory mechanism of glucocorticoids. Rat peritoneal mesothelial cells were cultured in high-glucose-containing medium and then analyzed for phosphorylation levels of p42/44 and p38 mitogen-activated protein (MAP) kinases (MAPK), MAPK or extracellular signal-regulated kinase kinase (MEK)1/2, c-Jun N-terminal kinase (JNK)1/2, and protein kinase C (PKC) by Western blotting. Expression of MCP-1 was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. DNA-binding activity of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay. High glucose increased MCP-1 mRNA and MCP-1 protein expression. Although glucose increased phosphorylation of MEK1/2, p42/44 MAPK, p38 MAPK, JNK1/2, and PKC, and DNA-binding activity of NF-kappaB, its effect on MCP-1 expression was suppressed only by PKC and NF-kappaB inhibitors. Mannitol caused a similar increase in PKC and NF-kappaB activation and MCP-1 synthesis. Prednisolone increased I-kappaB-alpha expression and inhibited glucose/mannitol-induced NF-kappaB DNA binding and MCP-1 expression without affecting PKC phosphorylation. The inhibitory effects of prednisolone on MCP-1 expression were reversed by mifepristone, a glucocorticoid receptor antagonist. Our results indicate that glucose induces MCP-1 mainly through hyperosmolarity by activating PKC and its downstream NF-kappaB, and that such effect was inhibited by prednisolone, suggesting the efficacy of prednisolone in preventing peritoneal fibrosis in patients on CAPD.

Background: Growth factors, extracellular matrix and its receptor integrins are upregulated in various glomerular diseases. We investigated the mechanism of collaboration between integrins and platelet-derived growth factor (PDGF) in focal adhesion kinase (FAK)- and extracellular signal-related kinase (ERK)1/2-mediated signal pathways that lead to monocyte chemoattractant protein (MCP)-1 expression in cultured rat mesangial cells (MCs)., Methods: Serum-starved MCs were plated on fibronectin- or polylysine-coated plates with or without PDGF, and examined for phosphorylation of ERK1/2, mitogen-activated protein or ERK kinase (MEK)1/2 and FAK by western blotting, and for expression of MCP-1 mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The effects of dominant-negative FAK on MCP-1 expression were examined., Results: Cell adhesion to fibronectin increased phosphorylation of FAK, MEK1/2 and ERK1/2, and induced MCP-1 mRNA and protein expression. PDGF increased phosphorylation of FAK, MEK1/2 and ERK1/2 even without cell adhesion to fibronectin, and induced MCP-1 mRNA and protein expression. PDGF with integrin activation by fibronectin synergistically increased phosphorylation of FAK, MEK1/2 and ERK1/2, and expression of MCP-1 mRNA and protein. Dominant-negative FAK attenuated fibronectin enhancement of PDGF-induced ERK1/2 phosphorylation and MCP-1 expression, indicating involvement of FAK in this signalling., Conclusions: Our results suggest the cooperative role of integrin and PDGF receptor in activation of the ERK pathway possibly via FAK in MCs. The synergistic activation of integrin and PDGF signalling may play an important role in the progression of glomerular diseases through the induction of MCP-1.

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.

The discovery of neuropeptides has resulted in an increased understanding of novel regulatory mechanisms of certain physiological phenomena. Here we identify a novel neuropeptide of 36 amino-acid residues in rat brain as an endogenous ligand for the orphan G protein-coupled receptor FM-4/TGR-1, which was identified to date as the neuromedin U (NMU) receptor, and designate this peptide 'neuromedin S (NMS)' because it is specifically expressed in the suprachiasmatic nuclei (SCN) of the hypothalamus. NMS shares a C-terminal core structure with NMU. The NMS precursor contains another novel peptide. NMS mRNA is highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression is restricted to the core of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular administration of NMS in rats activates SCN neurons and induces nonphotic type phase shifts in the circadian rhythm of locomotor activity. These findings suggest that NMS in the SCN is implicated in the regulation of circadian rhythms through autocrine and/or paracrine actions.

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

Renal concentrating ability is known to be impaired with aging. The antidiuretic hormone AVP plays an important role in renal water excretion by regulating the membrane insertion and abundance of the water channel aquaporin-2 (AQP2); this effect is primarily mediated via the V2 subtype of the AVP receptor (V2R). This study evaluated the hypothesis that decreased renal sensitivity to AVP, with subsequent altered renal AQP2 expression, contributes to the reduced urinary concentrating ability with aging. Our results show that under baseline conditions, urine osmolality is significantly lower in aged Fischer 344 and Brown-Norway F1 hybrid (F344BN) rats despite equivalent plasma AVP concentrations as in young rats. Levels of kidney V2R mRNA expression and AQP2 abundances were also significantly decreased in aged F344BN rats, as was AQP2 immunostaining in collecting duct cells. In response to moderate water restriction, urine osmolality increased by significantly lesser amounts in aged F344BN rats compared with young rats despite similar increases in plasma AVP levels. Moderate water restriction induced equivalent relative increases in renal AQP2 abundances in all age groups but resulted in significantly lower abundances in total kidney AQP2 protein in aged compared with young F344BN rats. These results therefore demonstrate a functional impairment of renal concentrating ability in aged F344BN rats that is not due to impaired secretion of AVP but rather appears to be related to impaired responsiveness of the kidney to AVP that is secondary, at least in part, to a downregulation of renal V2R expression and AQP2 abundance.

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.

To determine the role of adrenomedullin (AM) in the fluid electrolyte homeostasis and endotoxin shock, cerebral spinal fluid (CSF) and plasma were sampled from rats after respective challenges. The AM levels were measured by a highly sensitive immunoassay. The AM levels in the CSF of the rats anesthetized with ether (10.7 +/- 0.60 fmol/ml) were significantly higher than those with isoflurane 5.17 +/- 0.70 fmol/ml, P < 0.01), while the plasma level did not differ significantly. The CSF levels of the rats received 2% saline drinking increased to 3 and 4 folds at day 5 and day 7, respectively, while the plasma levels did not differ from controls at both time points. The AM levels in CSF or plasma increased to 1.5 and 3 folds at 1.5 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS, 5 mg/kg), reached 6.5 and 30 folds at 6 h, respectively, while no change was observed in the controls. The present findings suggest that AM in the CSF is regulated independently from that in the plasma, the centrally synthesized AM plays and important role in the regulation of the fluid electrolyte homeostasis. Furthermore, the circulatory AM plays an important role in the endotoxin shock., (Copyright 2004 Elsevier Inc.)

The effect of short-term selective REM sleep deprivation (RSD) on the gene expression of galanin in the rat hypothalamus was examined using in situ hybridization histochemistry. Monitoring an electroencephalogram (EEG) and electromyogram (EMG) on an on-line computer screen, as the RSD rats entered REM sleep, they were gently stroked on their backs using a brush to wake them during the RSD period. Galanin mRNA levels in the preoptic area (POA) were significantly increased by RSD for a period of 6 h. RSD had no significant effect on the mRNA levels of corticotrophin-releasing factor (CRF), arginine vasopressin (AVP), oxytocin (OXT) or orexins. These results suggest that 6-h selective RSD may not be sufficient to induce the activation of the hypothalamo-pituitary adrenal axis, and that the expression of the galanin gene in the hypothalamus reacts more readily against the loss of REM sleep in comparison to other hypothalamic neuropeptides such as arginine vasopressin, oxytocin and orexins.

Background: Integrins are major adhesion receptors that not only regulate cytoskeletal organization, but also trigger a variety of intracellular signal transduction pathways. We examined the effects of increased extracellular matrix (ECM) accumulation on monocyte chemoattractant protein-1 (MCP-1) expression, which is known to play an important role in the progression of various glomerular diseases., Methods: MCP-1 mRNA and protein expression in cultured rat mesangial cells (MC) attached to ECM proteins were examined by reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting, respectively. Phosphorylation of focal adhesion kinase (FAK) was measured by Western blotting. Effects of wild-type and dominant-negative FAK on MCP-1 expression were examined by a transient transfection assay., Results: Cell adhesion to fibronectin-induced phosphorylation of FAK and MCP-1 mRNA expression in time- and dose-dependent manners followed by increased MCP-1 protein expression. All integrin-interacting substrates (laminin and types I, III, and IV collagens) also increased levels of FAK phosphorylation and MCP-1 expression, whereas nonspecific adhesive substrates (polylysine and concanavalin A) had no significant effects. Overexpression of wild-type FAK increased phosphorylation of FAK and expression of MCP-1 mRNA and protein, whereas transfection of dominant-negative FAK abolished adhesion-induced MCP-1 expression. Adhesion-induced expression of MCP-1 mRNA was inhibited by genistein and tosyl phenylalanyl chloromethylketone (TPCK), suggesting that tyrosine kinases [e.g., FAK, and nuclear factor kappa B (NF-kappa B)] are necessary in this signaling., Conclusion: Our results indicate that integrin-mediated cell adhesion to the ECM can induce MCP-1 expression through activation of FAK, and suggest a role for altered ECM deposition in the progression of glomerular diseases by affecting gene expression.

Emotional stress inhibits vasopressin release from the pituitary but may facilitate its release from the dendrites in the hypothalamus. We examined effects of intermittently applied footshock upon the amount of vasopressin heteronuclear RNA in the hypothalamus. The footshock decreased plasma vasopressin concentration but increased its extracellular concentration within the supraoptic nucleus. The contents of the vasopressin heteronuclear RNA in the supraoptic nucleus were significantly decreased after the shock. These data suggest that intermittent footshock decreases not only vasopressin release from the axon terminals in the pituitary, but also vasopressin synthesis in the cell bodies in the hypothalamus while the stimulus facilitates vasopressin release from the dendrites in the hypothalamus. The data also suggest differential control of dendritic vasopressin release and synthesis in the hypothalamus.

The expression of the corticotropin-releasing hormone (CRH) gene and the arginine vasopressin (AVP) gene in the hypothalamus examined in bilateral nephrectomized rats by in situ hybridization histochemistry. The expression of the CRH gene was significantly increased in the parvocellular part of the paraventricular nucleus (PVN) 12 and 20 h after bilateral nephrectomy in comparison with that after sham operation. The plasma concentration of adrenocorticotropic hormone (ACTH) in nephrectomized rats was significantly higher than that in sham operated rats 20 h after surgery. In contrast, the expression of the AVP gene in both the parvocellular and magnocellular parts of the PVN and throughout the supraoptic nucleus (SON) was significantly decreased 20 h after bilateral nephrectomy in comparison with that after sham operation. These results suggest that nephrectomy-induced upregulation of the CRH gene with elevation of plasma ACTH may be due to the activation of the hypothalamo-pituitary adrenal (HPA) axis.

We have examined the effects of 3 weeks of food restriction on both the activity of neurons containing hypothalamic orexin (OX)-A and the level of OX receptor type 2 (OX2R) mRNA in the paraventricular nucleus (PVN) of rats. Double immunohistochemistry was used to examine the expression of OX-A and Fos in the lateral hypothalamic area (LHA), and in situ hybridization histochemistry was used to measure levels of OX2R mRNA in the PVN. After the period of restricted feeding, 20-30% of OX-A-containing neurons exhibited Fos-like immunoreactivity (LI). The distribution of OX-A-LI/Fos-LI cells in the food-restricted rats was similar to that observed in glucose-deprived rats after intracerebroventricular (icv) administration of 2-deoxy-D-glucose (2-DG). In addition, 3 weeks of food restriction caused a significant decrease in the expression of the OX2R gene in the parvocellular division of the PVN. These results suggest that the activation of OX-A-containing neurons induced by restricted feeding may be involved in neuroendocrine responses to food restriction.

The plasma concentration of arginine vasopression (AVP) and the expression level of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular nucleus (PVN) and the Supraoptic nucleus (SON) of Sprague-Dawley (SD). Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats on a high salt diet were examined by radioimmunoassay for AVP and in situ hybridization histochemistry for nNOS. The high salt diet containing 8.0% NaCl was given for 4 weeks. The concentrations of AVP in hypertensive Dahl S rats were significantly increased in comparison with those in SD rats and Dahl R rats on a high salt diet. The levels of nNOS mRNA and NADPH-diaphorase activity in the PVN and SON of hypertensive Dahl S rats were greater than those in Dahl R rats on a high salt diet. The antihypertensive drugs, either nicardipine or captopril were administered to the Dahl S rats for 2 weeks beginning 2 weeks after the start of the high salt diet The nNOS mRNA in the PVN and SON of Dahl S rats given a high salt diet was not upregulated by treatment with nicardipine, while the nNOS mRNA in salt loaded Dahl S rats was greater upregulated by treatment with captopril to that greater than without the antihypertensive drug. Our results suggest that the increased NO production in the PVN and SON of hypertensive Dahl S rats may be ineffective in decreasing blood pressure or inhibiting AVP secretion.

The expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) in rats with lithium (Li)-induced polyuria was examined by using in situ hybridization histochemistry. The state of the thyroid axis in these rats was also examined by in situ hybridization histochemistry for thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) mRNAs and radioimmunoassay for circulating thyroid hormones. Adult male Wistar rats consuming a diet that contained LiCl (60 mmol/kg) for 4 weeks developed remarkable polyuria. The urine in the Li-treated rats was hypotonic and had a large volume and low ionic concentration. The nNOS mRNA in the PVN and SON was significantly increased in the Li-treated rats in comparison with that in control. The increased levels of the nNOS mRNA in the PVN and SON were confirmed by NADPH-diaphorase histochemical staining. There were no differences of TRH mRNA in the PVN, TSH mRNA in the anterior pituitary and plasma concentrations of free T3 and free T4 between Li-treated rats and control rats. These results suggest that Li-induced diabetes insipidus may activate nNOS in the PVN and SON without change of the thyroid axis.

The effects of chronic salt loading (2% saline to drink for 5 and 10 days), gestation, lactation and adrenalectomy on the expression of synapsin IIa and IIb genes were examined in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. In each control, synapsin IIa and IIb genes were moderately expressed in the magnocellular division of the PVN and SON, while few transcripts of synapsin IIa and IIb were observed in the parvocellular division of the PVN. Chronic salt loading, gestation on day 21 and lactation on day 10 caused significant increases in synapsin IIa and IIb transcripts in the magnocellular division of the PVN and SON, compared to each control. Although corticotropin-releasing hormone transcripts in the parvocellular division of the PVN were significantly increased in the adrenalectomized rats, no changes in the transcripts of synapsin IIa and IIb were observed throughout the PVN. These results suggest that physiological stimuli such as osmotic challenge and lactation potently increase synapsin IIa and IIb mRNAs in the magnocellular neurons of the PVN and SON.

The effects of i.c.v. administration of orexin/hypocretin on plasma ACTH, corticosterone and c-fos mRNA in the paraventricular nucleus (PVN) of the rat were examined. Plasma ACTH levels were markedly increased at 30 min after i.c.v. administration of orexin-A. Plasma corticosterone levels were significantly increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. In situ hybridization histochemistry revealed that the induction of the c-fos mRNA in the parvocellular division of the PVN was increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. These results suggest that central orexin/hypocretin activates hypothalamo-pituitary-adrenal (HPA) axis and may be involved in stress-induced activation of the HPA axis.

We examined developmental changes of orexins/hypocretins and their receptors (OX1R and OX2R) in the rat hypothalamus from postnatal day 0 to 10 weeks, using in situ hybridization histochemistry for the prepro-orexin, OX1R and OX2R mRNAs and immunohistochemistry for orexin-A and orexin-B. The prepro-orexin mRNA was weakly detected in the lateral hypothalamic area (LHA) from days 0 to 15. Orexin-A- and -B-like immunopositive cells and fibers were not detected from days 0 to 10, but they were observed after day 15. The prepro-orexin mRNA in the LHA markedly increased between days 15 and 20. The OX1R mRNA was detected in the ventromedial hypothalamic area (VMH) at day 0. The OX2R mRNA was not detected in the paraventricular nucleus (PVN) at days 0 and 1, but weakly observed on day 5. The OX1R mRNA in the VMH and OX2R mRNA in the PVN gradually increased throughout the postnatal period. Next, we examined the effects of milk deprivation and intraperitoneal (i.p.) administration of leptin on the hypothalamic prepro-orexin mRNA in pups. Although 24-h milk deprivation did not affect the level of the prepro-orexin mRNA at days 5 and 10, i.p. administration of leptin from days 0 to 3 caused a significant increase in the prepro-orexin mRNA on days 5 and 10. These results suggest that the development of orexins may be associated with developmental changes such as increase of leptin, weaning, feeding and sleep/wakefulness states.

We examined the effects of kainic acid (KA)-induced seizure on the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in the paraventricular nucleus (PVN) of rats using in situ hybridization histochemistry. Subcutaneous administration of KA (12 mg/kg) in adult male Sprague-Dawley rats caused a progressive development of seizure behavior. An induction of the PACAP gene expression in the medial parvocellular part of the PVN (mpPVN) was observed 3, 6, 12, 24 and 48 h after subcutaneous administration of KA. From a nearly undetectable level, PACAP gene expression increased in the mpPVN and reached maximum 12 h after subcutaneous administration of KA. PACAP gene expression returned to near basal level 48 h after stimulation with KA. Using a specific monoclonal PACAP antibody, PACAP immunoreactivity (-IR) gradually increased during the following 24 h after KA administration. In controls, PACAP-IR was located exclusively in nerve fibers of the mpPVN, whereas KA administration induced PACAP-IR in cell bodies of the mpPVN, and a dense accumulation of PACAP-IR nerve fibers in the external zone of the median eminence was observed. Induction of the PACAP gene expression following KA-induced seizure was significantly reduced by pretreatment with diazepam or MK-801 (nonselective N-methly-D-aspartate receptor antagonist). These results suggest that PACAP in the hypothalamo-adenohypophysial system may have a hypophysiotropic role during KA-induced seizure., (Copyright 2000 S. Karger AG, Basel)