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Unlabelled: Enalapril, a long-acting inhibitor of angiotensin-converting enzyme, was given for 2 years to seven children with Alport syndrome. Five patients had a classical X-linked form of the disease; two siblings had the autosomal recessive variant. Their age was between 5.15 and 13.75 years when enalapril was started. All patients had haematuria and proteinuria, creatinine clearance was > 80 ml/min per 1.73 m2 in all, and only one patient was hypertensive. The starting dose of enalapril (0.1 mg/kg body weight per day) was increased progressively according to individual clinical tolerance. The median doses were 0.13, 0.12, 0.21 and 0.29 mg/kg at 6, 12, 18 and 24 months, respectively. Median values of mean blood pressure were 95 mmHg at the start and 84 mmHg after 24 months. Median daily proteinuria decreased from 52 mg/kg to 18 mg/kg at 6 months, 21 mg/kg at 12 months, 12 mg/kg at 18 months and 30 mg/kg at 24 months. Serum creatinine increased over time from a median of 0.64 mg/dl at baseline to 0.77 mg/dl at 24 months. Concomitantly, there was a decrease in GFR from 104 to 83 ml/min per 1.73 m2 at 18 months and an increase again to 95 ml/min per 1.73 m2 at 24 months. Analysis of the individual data showed three patterns: no response (n = 2), temporary response (n = 2) and sustained response (n = 3).Conclusion: When given enalapril at the dosages mentioned, Alport patients as a group display a marked reduction in urinary protein excretion with a nadir of 23% of the baseline figure at 18 months, a decrease that cannot be accounted for by the slight decrease in glomerular filtration rate. Although these are preliminary data, it is recommended to try an angiotensin-converting enzyme inhibitor in every paediatric Alport patient with proteinuria. [ABSTRACT FROM AUTHOR]

Hypomagnesemia in patients with type 1 diabetes (T1D) as well as in obesity has been related to insulin resistance in adults, but not yet in pediatric patients. In this observational single-center study, we aimed to investigate the relation between the magnesium homeostasis, insulin resistance, and body composition in children with T1D and in children with obesity. Children with T1D (n = 148) and children with obesity and proven insulin resistance (n = 121) and healthy controls (n = 36) were included in this study. Serum and urine samples were collected to determine magnesium and creatinine. The total daily dose of insulin (for children with T1D), results from the oral glucose tolerance test (OGTT, for children with obesity), and biometric data were extracted from the electronic patient files. Furthermore, body composition was measured via bioimpedance spectroscopy. Serum magnesium levels were decreased in both children with obesity (0.87 ± 0.07 mmol/l) and children with T1D (0.86 ± 0.07 mmol/l) compared to healthy controls (0.91 ± 0.06; p = 0.005). A lower magnesium level was associated with more severe adiposity in children with obesity, while a worse glycemic control was associated with lower magnesium levels in children with T1D.   Conclusion: Children with T1D and children with obesity have decreased serum magnesium levels. An increased fat mass is associated with lower magnesium levels in childhood obesity, indicating that the adipose tissue is an important factor in magnesium homeostasis. In contrast, glycemic control was the main determining factor for serum magnesium levels in children with T1D. What is Known: • Hypomagnesaemia has been related to insulin resistance in both adults with T1D and adults with obesity. • There is an increasing prevalence of obesity and T1D in childhood, but little is known about the relationship between magnesium and insulin resistance in these children. What is New: • Both children with T1D and children with obesity have decreased serum magnesium levels. • In childhood obesity an increased fat mass is associated with lower magnesium levels, while glycaemic control is the main determining factor for serum magnesium in children with T1D., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The Flemish Collaborative Glomerulonephritis Group (FCGG) registry is a population-based kidney biopsy registry that has been including all native kidney biopsies performed in children in Flanders (Belgium), since 2017., Methods: From 2017 to 2020, 148 pediatric (< 18 years) native kidney biopsies were included. Each biopsy received a histopathological and final nephrological diagnosis, and concordance between both was assessed. Disease chronicity, summarized by the Mayo Clinic Chronicity Score, was determined on 122 biopsies with > 5 glomeruli., Results: Kidney biopsy rate was high (29.0 biopsies per million children per year), median age was 10.0 years (IQR 5.8-14.7), and boys predominated (56.1% males). A total of 140 biopsies (94.6%) showed a representative pathology result. Glomerular disease was most prevalent, with IgA nephropathy/IgA vasculitis (43 biopsies, 29.1%) and minimal change disease (MCD) (29 biopsies, 19.6%) being the overall most frequent diagnoses. In general, diagnostic concordance was high (80.7%). In Alport syndrome and focal segmental glomerulosclerosis (FSGS), concordance was lower, as the nephrological diagnosis was often determined by results of genetic analysis. Nephrotic syndrome was the most frequent indication for kidney biopsy (31.8%) and was mainly caused by MCD and FSGS. The degree of disease chronicity on kidney biopsies was generally low, although 27.3% of biopsies with a diagnosis of FSGS showed moderate-to-severe chronic damage., Conclusions: The presented epidemiological findings validate data from previous European registry studies and may inspire kidney biopsy registries worldwide to implement novel features such as clinicopathological concordance and chronicity grading. A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

An increased blood pressure is a known comorbidity of both type 1 diabetes (T1DM) and obesity in children. Increasing evidence suggests a subtle interplay between epidermal growth factor (EGF) and renin along the juxtaglomerular system, regulating the impact of blood pressure on kidney health and the cardiovascular system. In this study, we investigated the relation between urinary EGF, serum renin and blood pressure in children with obesity or T1DM. 147 non-obese children with T1DM and 126 children with obesity, were included. Blood pressure was measured and mean arterial pressure (MAP) and the pulse pressure (PP) were calculated. Serum renin and urinary EGF levels were determined with a commercial ELISA kit. Partial Spearman rank correlation coefficients and multiple linear regression models were used to study the association between renin, the urinary EGF/urinary creatinine ratio and blood pressure parameters. The urinary EGF/urinary creatinine ratio is correlated with the SBP and the MAP in boys with obesity as well as in boys with T1DM. Multiple regression analysis showed that sex and pulse pressure in male subjects were found to be independently associated with renin. Sex, the presence of diabetes, age, the glomerular filtration rate and both pulse pressure and mean arterial pressure in male subjects were independently associated with urinary EGF/urinary creatinine. In conclusion, in boys with either obesity or diabetes, pulse pressure and mean arterial pressure are negatively associated with the functional integrity of the nephron, which is reflected by a decreased expression of urinary EGF., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ledeganck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Involving pediatric nephrological input in the clinical diagnostic work-up of children with short stature, gave rise to the hypothesis that the presence of an underlying renal tubular disorder in children with short stature is possibly underestimated. This study focussed on the added value of calculated urinary fractional excretion (FE) in the early detection of tubular disorders in children with growth failure., Methods: This trial was designed as an observational study analyzing the medical files of children between 5 and 16 years who had been referred for short stature to the pediatric endocrinology outpatient clinic at the University Hospital Antwerp between 25/01/2015 and 01/03/2019. Based on the laboratory results of the simultaneously taken blood and urine sample, the fractional excretions of Sodium, Chloride, Potassium, Calcium, Phosphate, and Magnesium were calculated., Results: Of the 299 patients, 54 patients had at least one deviating fractional excretion value, requiring further investigation (control sample of blood and urine, kidney ultrasound or 24 h urine collection). Genetic screening for tubulopathies was performed in 19 patients. In 5 patients (1.7% of the total population) a tubulopathy was confirmed based on genetic analysis., Conclusion: This study explored the possibility of using fractional excretions as a screening test to obtain an earlier diagnosis of tubular disorders in children with short stature. Of the 299 patients, 5 patients were diagnosed with a genetically confirmed tubulopathy. Based on these results, we propose a flowchart for an additional work-up in all children with a deviating fractional excretion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Becue, Ceuleers, den Brinker, Somers, Ledeganck, Dotremont and Trouet.)

Background: In this study, the profile of urinary EGF excretion (uEGF/uCreat) was mapped in children presenting with prolonged proteinuria or with nephrotic syndrome refractory to or dependent of steroids. We investigated whether uEGF/uCreat could be linked to the underlying biopsy result, taking into account its response to immunosuppressive medication and to ACE inhibition, as well as genetic predisposition. Methods: Ninety-eight pediatric patients with initial presentation of nephrotic syndrome or prolonged proteinuria were included in this study, along with 49 healthy controls and 20 pediatric Alport patients. All patients had a normal kidney function and were normotensive during the course of the study, whether or not under ACE inhibition. In repeated urine samples, uEGF was measured and concentration was normalized by urine creatinine. In order to compare diagnosis on kidney biopsy, genetic predisposition and response of uEGF/uCreat to immunosuppression and to ACE inhibition, uEGF/uCreat is studied in a linear mixed effects model. Results: Patients with Minimal Change Disease (MCD) showed a significantly different profile of uEGF/uCreat in comparison to healthy children, as well as compared to patients with Focal Segmental Glomerulosclerosis (FSGS) or another glomerulopathy on kidney biopsy. The response of uEGF/uCreat to ACE inhibition was absent in minimal change disease and contrasted with an impressive beneficial effect of ACE inhibition on uEGF/uCreat in FSGS and other proteinuric glomerulopathies. Absence of a genetic predisposition was also associated with a significantly lower uEGF/uCreat. Conclusions: Despite preserved kidney function, children with a proteinuric or nephrotic glomerular disease on kidney biopsy show a significantly lower uEGF/uCreat, indicative of early tubulo-interstitial damage, which appears reversible under ACE inhibition in any underlying glomerulopathy except in minimal change disease. In view of the distinct profile of uEGF/uCreat in minimal change disease compared to other glomerulopathies, and the link between genetic predisposition and uEGF/uCreat, our study suggests that uEGF/uCreat can be a helpful tool to decide on the need for a renal biopsy in order to differentiate minimal change disease from other proteinuric glomerular diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lodeweyckx, Wouters, Ledeganck and Trouet.)

Aims: Micro-albuminuria is considered an early clinical sign of diabetes nephropathy, however, early decrease of glomerular filtration can be present years before the presence of microalbuminuria. In this study, we explored whether urinary epidermal growth factor (uEGF) might serve as an early marker of diabetes nephropathy compared to microalbuminuria in children and adolescents., Methods: Children with type 1 diabetes mellitus (n = 158) and healthy controls (n = 40) were included in this study. Serum and urine samples were collected three times with an interval of at least one month to determine creatinine (serum and urine), epidermal growth factor and albumin (urine). Demographic data and routine lab values were extracted out of the electronic patient files., Results: uEGF was significantly lower in children with T1DM compared to healthy controls (p = 0.032). A relatively lower glomerular filtration rate (eGFR) was associated with a decreased uEGF (p < 0.001). uEGF was independently associated with eGFR in a multivariate analysis., Conclusion: This study provides evidence that uEGF can serve as an early marker of diabetes nephropathy in children and adolescents., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

There is an increasing need for suitable tools to evaluate body composition in paediatrics. The Body Composition Monitor (BCM) shows promise as a method, but reference values in children are lacking. Twenty children were included and measured twice by 4 different raters to asses inter- and intra-rater reproducibility of the BCM. Reliability was assessed using the Bland-Altman method and by calculating intraclass correlation coefficients (ICCs). The intra-rater ICCs were high (≥ 0.97) for all parameters measured by BCM as were the inter-rater ICCs for all parameters (≥ 0.98) except for overhydration (0.76). Consequently, a study was set up in which BCM measurements were performed in 2058 healthy children aged 3-18.5 years. The age- and gender-specific percentile values and reference curves for body composition (BMI, waist circumference, fat mass and lean tissue mass) and fluid status (extracellular and intracellular water and total body water) relative to age were produced using the GAMLSS method for growth curves.Conclusion: A high reproducibility of BCM measurements was found for fat mass, lean tissue mass, extracellular water and total body water. Reference values for these BCM parameters were calculated in over 2000 children and adolescents aged 3 to 18 years. What is Known • The 4-compartment model is regarded as the 'gold standard' of body composition methods, but is inappropriate for regular follow-up or screening of large groups, because of associated limitations. • Body Composition Monitor® is an inexpensive field method that has the potential to be an adequate monitoring tool. What is New • Good reproducibility of BCM measurements in children provides evidence to use the device in longitudinal follow-up, multicentre and comparative studies. • Paediatric reference values relative to age and sex for the various compartments of the body are provided.

X-linked hypophosphatemia (XLH) is the most common genetic form of hypophosphatemic rickets and osteomalacia. In this disease, mutations in the PHEX gene lead to elevated levels of the hormone fibroblast growth factor 23 (FGF23), resulting in renal phosphate wasting and impaired skeletal and dental mineralization. Recently, international guidelines for the diagnosis and treatment of this condition have been published. However, more specific recommendations are needed to provide guidance at the national level, considering resource availability and health economic aspects. A national multidisciplinary group of Belgian experts convened to discuss translation of international best available evidence into locally feasible consensus recommendations. Patients with XLH may present to a wide array of primary, secondary and tertiary care physicians, among whom awareness of the disease should be raised. XLH has a very broad differential-diagnosis for which clinical features, biochemical and genetic testing in centers of expertise are recommended. Optimal care requires a multidisciplinary approach, guided by an expert in metabolic bone diseases and involving (according to the individual patient's needs) pediatric and adult medical specialties and paramedical caregivers, including but not limited to general practitioners, dentists, radiologists and orthopedic surgeons. In children with severe or refractory symptoms, FGF23 inhibition using burosumab may provide superior outcomes compared to conventional medical therapy with phosphate supplements and active vitamin D analogues. Burosumab has also demonstrated promising results in adults on certain clinical outcomes such as pseudofractures. In summary, this work outlines recommendations for clinicians and policymakers, with a vision for improving the diagnostic and therapeutic landscape for XLH patients in Belgium., Competing Interests: ML has received lecture and consultancy fees from Alexion, Amgen, Kyowa Kirin, Menarini, Sandoz, Takeda, UCB and Will-Pharma. JS has received lecture, consultancy fees, and conference support from Kyowa Kirin, Alexion, Eli-Lily, Ferring, Ipsen, Menarini, Novo Nordisk, Pfizer, Sandoz, and Siemens Healthcare. DT has received conference support from Novo Nordisk. NG, JLa, and KH have received consultancy fees from Kyowa Kirin. EB has received conference support from Novo Nordisk and Pfizer. CH has received consultancy fees and conference support from Kyowa Kirin, Novo Nordisk, and Ferring. EC has received consultancy fees from bioMérieux, Diasorin, Fujirebio, IDS, and Menarini. PH is an employee of GlaxoSmithKline but participates in his own capacity. J-FK has received consultancy fees and conference support from Heel Belgium, Sanofi, and TRB Chemedica. KW has received conference support from Alexion, Ferring, Kyowa Kirin and Novo Nordisk. CV has received conference support from Boehringer Ingelheim. GM has received consultancy fees from Alexion, Biomarin, Kyowa Kirin, and Pfizer. EL has received consultancy fees and travel support from Kyowa Kirin, Chiesi, and Recordati. JV has received conference support and consultancy fees from Alexion, Bellco, Ferring, Medtronic, and Kyowa Kirin. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laurent, De Schepper, Trouet, Godefroid, Boros, Heinrichs, Bravenboer, Velkeniers, Lammens, Harvengt, Cavalier, Kaux, Lombet, De Waele, Verroken, van Hoeck, Mortier, Levtchenko and Vande Walle.)

Background & Objective: Epidermal growth factor (EGF) stimulates cell proliferation and differentiation after binding to its receptor. Next to its role in magnesium homeostasis, EGF disturbances have been described in oncology, diabetes and autism spectrum disorders. The aim of this study was to determine EGF serum and urine values for both healthy children and adults. Next, we investigated the relation between several variables and urinary and serum EGF concentrations., Methods: Both healthy adults (n = 50) and children (n = 78) were included. Serum and urinary EGF concentrations were measured with ELISA technology., Results: Serum EGF was inversely correlated with age (r = -0.873; p<0.001) and positively correlated with serum magnesium (r = 0.597; p<0.001). The urinary EGF was also inversely correlated with age (r = -0.855; p<0.001). In adults and children older than 13 years of age, the urinary EGF significantly differed between sexes (p = 0.001). Urinary EGF was positively correlated with serum magnesium (r = 0.583; p<0.001) and creatinine clearance (r = 0.524; p<0.001) and negatively correlated with the fractional excretion of magnesium (r = 0.248; p = 0.014). In a multivariate model, age and serum magnesium remained independently related to serum EGF while age, serum EGF and serum magnesium remained independently related to urinary EGF., Conclusions: This study provides valuable insights in urinary and serum EGF patterns in healthy subjects. By systematically correcting EGF for body surface, significant correlations with age, gender and magnesium were observed., Competing Interests: The authors have declared that no competing interests exist.

Background: It was shown in animal models and adults that the epidermal growth factor (EGF) is involved in the pathophysiology of calcineurin inhibitor (CNI) induced renal magnesium loss. In children, however, the exact mechanism remains unclear, which was set as the purpose of the present study., Methods: Children with nephrotic syndrome and renal transplant children treated with CNI ( n = 50) and non-CNI treated children ( n = 46) were included in this study. Urine and serum samples were collected at three time points to determine magnesium, creatinine, and EGF. The magnesium intake was calculated from a food frequency questionnaire., Results: Serum Mg 2+ and urinary EGF/creatinine were significantly lower in the CNI treated children, with significantly more CNI-treated children developing hypomagnesaemia. In the latter patients, the fractional excretion of magnesium (FE Mg 2+ ) was significantly higher. Urinary EGF, age, renal function, and serum magnesium were independent predictors of the FE Mg 2+ . Only 29% of the children reached the recommended daily intake of magnesium. The magnesium intake did not differ between hypomagnesemic and normomagnesemic patients and was not a predictor of the FE Mg 2+ ., Conclusions: In CNI-treated children who developed hypomagnesemia, the FE Mg 2+ was increased. The urinary EGF concentration, age, and renal function are independent predictors of the FE Mg 2+ .

Introduction: Renal papillary necrosis is not commonly seen in daily practice, but can have severe consequences when it is not diagnosed in time. It is known to be associated with sickle cell hemoglobinopathies; however a wide range of etiologies are possible, and it is therefore not the first diagnosis clinicians consider in patients with sickle cell disease who present with hematuria., Methods: A literature search was performed to summarize the current knowledge about renal papillary necrosis associated with sickle cell disease. These findings are illustrated with a case of a 9-year old girl with sickle cell disease who was referred with painless gross hematuria., Results: Typical radiologic signs for renal papillary necrosis are necrotic cavities that fill with contrast, small collections of contrast peripheral to the calyces in the papillary region (ball-on-tee sign), calcification of the papillary defect, filling defects, hydronephrosis, blunted papillary tip, clefts in the renal medulla filled with contrast, hyperattenuated medullary calcifications, non-enhanced lesions surrounded by rings of excreted contrast, and clubbed calyces., Discussion: This study focuses on the pathophysiology of renal papillary necrosis associated with sickle cell disease, the possible symptoms, as well as the diagnostic steps, with a special interest in particular presentation on old (retrograde pyelography) and new (computed tomography) gold standard in radiologic imaging, and the management for this pathology., Conclusion: This study aims to remind clinicians of this "forgotten" diagnosis and what signs to look for in pediatric patients with sickle cell disease who present with hematuria. In pediatric cases radiation protection is important, therefore knowing what radiologic signs can be found on retrograde pyelography can lead to early identification of this pathology without having to proceed to computed tomography., (Copyright © 2017 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objectives: To investigate the possible advantage of administration of preemptive oral ibuprofen in children after ambulatory pediatric urologic surgery such as penile surgery (circumcision and hypospadias repair) and inguinal surgery (communicating hydrocele and orchidopexy), a study was performed on the experience of postoperative pain, nausea or vomiting, and resumption of normal activities such as normal sleep and play activity. In addition, this study has validated a method of measurement of pain and resumption of normal activities in children., Material and Methods: In a prospective, randomized, double-blind study, 66 prepubertal boys (0-12 years) underwent an ambulatory pediatric urological intervention. One hour prior to surgery, the experimental group received 10 mg/kg oral ibuprofen along with their usual premedication (<8 years midazolam 0.5 mg/kg PO or rectal; >8 years or >30 kg alprazolam 0.5 mg, 0.25-0.5 mg PO), whereas the children of the control group received only the usual premedication. Anesthesia was achieved with Sevorane (Sevoflurane) inhalation induction (Sevoflurane 8% in 50% N(2)O/50% O(2)) and a locoregional caudal block (0.5-1.0 ml/kg levobupivacaine 0.25% with a maximum of 30 ml). Immediate postoperative pain was assessed by the child, the parents and a single observer using the Faces Pain Scale and the CHEOPS behavioral scale (Children's Hospital of Eastern Ontario Pain Scale). Vomiting and nausea were also assessed. On the first and second postoperative day the same variables were evaluated by the child and the parents, as well as quality of sleep and play, and need for pain medication., Results: No difference in the experience of pain was found at any point after the operation between the experimental and control groups; moreover, the consumption of pain medication postoperatively did not differ between the two groups. There was also no difference in the incidence of vomiting and nausea in the hospital or at home (p>0.05). The method for measuring pain and normal activities was validated as the assessments of the parents, children and the investigators were concordant throughout the study., Conclusions: Children who received preoperative oral ibuprofen (10 mg/kg) did not experience less pain or less nausea and vomiting, and did not show a better pattern of sleep and play up to 2 days after ambulatory pediatric urologic surgery than children who did not receive this medication.

Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I(Cl,swell). However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of Galpha (a Rho activating heterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway.

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I(Cl,swell), the swelling-induced Cl- current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I(Cl,swell). However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I(Cl,swell) in CPAE cells. Kinase activity was dispensable for the inhibition of I(Cl,swell), since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity., (Copyright 2001 Academic Press.)

Restoration of cell volume after cell swelling in mammalian cells is achieved by the loss of solutes (K+, Cl-, and organic osmolytes) and the subsequent osmotically driven efflux of water. This process is generally known as regulatory volume decrease (RVD). One pathway for the swelling induced loss of Cl- (and also organic osmolytes) during RVD is the volume-regulated anion channel (VRAC). In this review, we discuss the physiological role and cellular control of VRAC. We will first highlight evidence that VRAC is more than a volume regulator and that it participates in other fundamental cellular processes such as cell proliferation and apoptosis. The second part concentrates on the Rho/Rho kinase/myosin phosphorylation cascade and on compartmentalization in caveolae as modulators of the signal transduction cascade that controls VRAC gating in vascular endothelial cells.

1. Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). 2. ICl,swell, the cell swelling-induced chloride current through VRACs, was studied in three caveolin-1-deficient cell lines: Caco-2, MCF-7 and T47D. 3. Electrophysiological measurements showed that ICl, swell was very small in these cells and that transient expression of caveolin-1 restored ICl,swell. The caveolin-1 effect was isoform specific: caveolin-1beta but not caveolin-1alpha upregulated VRACs. This correlated with a different subcellular distribution of caveolin-1alpha (perinuclear location) from caveolin-1beta (perinuclear and peripheral). 4. To explain the modulation of ICl, swell by caveolin-1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

The aim of the present study is to elucidate the effects of the expression of large conductance Ca2+ activated K+ channels (BK[Ca]) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK(Ca) was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK(Ca). Membrane potential, ionic currents and Ca2+ signals were investigated in non-transfected and transfected cells using a combined patch clamp and Fura-2 fluorescence technique. In non-transfected control CPAE cells, ATP evoked a Ca2+ activated Cl- current (I[Cl,Ca]). The most prominent current component during ATP stimulation in hslo expressing cells was conducted BK(Ca) which resulted in a pronounced transient hyperpolarization. This hyperpolarization, which was absent in non-transfected cells, was enhanced if I(Cl,Ca) was blocked with niflumic acid. The sustained component of the Ca2+ response during ATP stimulation was significantly larger in hslo transfected cells than in non-transfected cells. This plateau level correlated well with the corresponding effects of ATP on the membrane potential, indicating that the expression of cloned BK(Ca) exerts a positive feedback on Ca2+ signals in endothelial cells by counteracting the negative (depolarizing)effect of stimulation of Ca2+-activated Cl- channels.

Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.