Corticosteroids alter kidney development and increase glomerular filtration rate in larval zebrafish (Danio rerio).

Pharmaceutical drugs and other chemicals can impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants. Corticosteroids are administered to pregnant women at risk of preterm delivery in order to reduce neonatal morbidity and mortality. In addition, high-dose corticosteroid exposure of very preterm infants regularly serves to maintain blood pressure and to prevent and treat bronchopulmonary dysplasia, a form of chronic lung disease in prematurely born infants. Despite clinical benefits, there is increasing evidence of corticosteroid-mediated short- and long-term detrimental developmental effects, especially in the kidney. Here, we performed a detailed morphological and functional analysis of corticosteroid-mediated effects on pronephros development in larval zebrafish. About 24-h postfertilization (hpf) transgenic Tg(wt1b: EGFP) zebrafish larvae were exposed to a set of natural and synthetic corticosteroids (hydrocortisone, dexamethasone, 6α-methylprednisolone, betamethasone, prednisolone, fludrocortisone, 11-deoxycorticosterone) with varying glucocorticoid and mineralocorticoid potency for 24 h at different concentrations. A semiautomated, multiparametric in vivo workflow enabled simultaneous assessment of kidney morphology, renal FITC-inulin clearance, and heart rate within the same larva. All corticosteroids exerted significant morphological and functional effects on pronephros development, including a significant hypertrophy of the pronephric glomeruli as well as dose-dependent increases in FITC-inulin clearance as a marker of glomerular filtration rate. In conclusion, the present study demonstrates a significant impact of corticosteroid exposure on kidney development and function in larval zebrafish. Hence, these studies underline that corticosteroid exposure of the fetus and the preterm neonate should be carefully considered due to potential short- and long-term harm to the kidney.

Fat embolism syndrome in Duchenne muscular dystrophy: Report on a novel case and systematic literature review.

We report a non-ambulatory 13-year-old boy with Duchenne muscular dystrophy who experienced severe acute respiratory distress syndrome and cerebral fat embolism following elective soft tissue surgery. Post-surgery radiological examination revealed bilateral femoral fractures and marked osteopenia that were believed to have caused disseminated pulmonary and cerebral fat embolism. The patient had never been on glucocorticoid treatment. Five months post-surgery, he remained in a state of minimal consciousness. A literature review was performed and eleven publications included, providing case reports of a total number of 23 patients with Duchenne muscular dystrophy with fat embolism syndrome. The most common causes were falls from the wheelchair that predominantly resulted in femoral fractures. Median age at the event was around 14 years. Seven patients succumbed to complications of fat embolism. No event was described in the context of surgery. We want to raise awareness that spontaneous unnoticed fractures may occur especially in adolescents with DMD from traumatic injury of large bones and also during elective surgery with a high risk of causing fat embolism with severe sequelae.

Kidney urinary biomarkers in patients with branched-chain amino acid and cobalamin metabolism defects.

There is a clinical need for early detection of chronic kidney disease (CKD) in patients with organic acidurias. We measured kidney markers in a longitudinal study over 5 years in 40 patients with methylmalonic aciduria (Mut0 ), propionic aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies. Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-2-microglobulin (B2MG) in urine, as well as cystatin C (CysC) in serum were quantified. In Mut0 patients, mean concentrations of B2MG, KIM-1, and DKK-3 were elevated compared with healthy controls, all markers indicative of proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were elevated, indicating signs of proximal tubule and glomerulus damage and inflammation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and considered as markers for proximal and distal tubule damage and inflammation. B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut0 /CblA and with eGFR(CysC) and KIM-1 in PA patients, respectively. None of the markers were elevated in CblA patients. Significant deterioration of kidney function, as determined by steady increase in CysC concentrations was noted in seven patients within the observation period. None of the investigated biomarker profiles showed a clear increase or added value for early detection. In conclusion, we identified disease-specific biomarker profiles for inflammation, tubular, and proximal damage in the urine of Mut0 , PA, and CblC patients. Whether these biomarkers can be used for early detection of CKD requires further investigation, as significant kidney function deterioration was observed in only a few patients.

Rescue of respiratory failure in pulmonary alveolar proteinosis due to pathogenic MARS1 variants.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a heterogeneous condition with more than 100 different underlying disorders that need to be differentiated to target therapeutic options, which are generally limited. METHODS: The clinical course of two brothers with pathogenic variants in the methionyl-tRNA synthetase (MARS)1 gene was compared to previously published patients. Functional studies in patient-derived fibroblasts were performed and therapeutic options evaluated. RESULTS: The younger brother was diagnosed with PAP at the age of 1 year. Exome sequencing revealed the homozygous MARS1 variant p.(Arg598Cys), leading to interstitial lung and liver disease (ILLD). At 2 years of age, following surgery hypoglycemia was detected, the pulmonary condition deteriorated, and the patient developed multiorgan failure. Six therapeutic whole lung lavages (WLL) were necessary to improve respiratory insufficiency. Methionine supplementation was started and a high protein diet ensured, leading to complete respiratory recovery. The older brother, homozygous for the same MARS1 variant, had a long-known distinct eating preference of methionine-rich food and showed a less severe clinical phenotype. Decreased aminoacylation activity confirmed the pathogenicity of p.(Arg598Cys) in vitro. In agreement with our review of currently published ILLD patients, the presence of hepatopathy, developmental delay, muscular hypotonia, and anemia support the multisystemic character of the disease. CONCLUSIONS: Catabolic events can provoke a severe deterioration of the pulmonary situation in ILLD with a need for repetitive WLL. Although the precise role of oral methionine supplementation and high protein intake are unknown, we observed an apparent treatment benefit, which needs to be evaluated systematically in controlled trials.

Factors influencing time-dependent decannulation after pediatric tracheostomy according to the Kaplan-Meier method.

PURPOSE: The aim of this study was to determine whether there were differences in decannulation rates and time to decannulation in children depending on the indication for tracheostomy, age, and maturity at birth. STUDY DESIGN: Retrospective chart review and prospective interview by questionnaire. METHODS: The medical records of 106 pediatric patients (age 0-18 years) tracheostomized between January 1 1999 and January 1 2019 were reviewed. Patients were divided into three different groups depending on the indication for tracheostomy: unsafe airway (37.7%), long-term respiratory dependence (50.9%), or bronchopulmonary toilet for aspirations (11.3%). RESULTS: 40 patients were successfully decannulated. The time-dependent decannulation rate after 2 and 5 years was 28.3% and 40.5% for patients with an unsafe airway, 42.4% and 66.8% for patients with long-term respiratory dependence, and 41.7% and 70.8% for patients needing bronchopulmonary toilet, respectively. After 2 and 5 years, patients aged 0-12 months at the time of tracheostomy were decannulated in 13.1% and 50.2% of cases, 1-5-year-olds in 35.3% and 48.2% of cases, 6-10-year-olds in 70% and 70% of cases, and 11-18-year-olds in 66.6% and 66.6% of cases, respectively. However, in a multivariate analysis, prematurity was found to be the only significant unfavorable variable (p = 0.013). Maturely born patients had an odds ratio of 3.87 (95% CI 1.32-11.33) for successful decannulation. This effect was present only in the first 5 years of life. CONCLUSION: Factors indicating problems with decannulation are an unsafe airway, a young age at the time of tracheostomy, and prematurity at birth.

A Smart Imaging Workflow for Organ-Specific Screening in a Cystic Kidney Zebrafish Disease Model.

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift172 in Tg(wt1b:EGFP) embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.

Urinary acute kidney injury biomarkers in very low-birth-weight infants on indomethacin for patent ductus arteriosus.

BACKGROUND: Serum creatinine (SCr)- or urine output-based definitions of acute kidney injury (AKI) have important limitations in neonates. This study evaluates the diagnostic value of urinary biomarkers in very low-birth-weight (VLBW) infants receiving indomethacin for closure of a patent ductus arteriosus (PDA). METHODS: Prospective cohort study in 14 indomethacin-treated VLBW infants and 18 VLBW infants without indomethacin as controls. Urinary biomarkers were measured before, during, and after indomethacin administration. RESULTS: Indomethacin therapy was associated with significantly higher SCr concentrations at 36, 84, and 120 h compared to controls. At 36 h, three indomethacin-treated patients met the criteria for neonatal modified Kidney Disease: Improving Global Outcomes (KDIGO) AKI. The product of urinary tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 ([TIMP-2]•[IGFBP7]) was significantly elevated in the AKI subgroup at 12 h (P < 0.05), hence 24 h earlier than the increase in SCr. Urinary neutrophil gelatinase-associated lipocalin (NGAL) and calprotectin were significantly increased in the indomethacin group at 12 h (P < 0.05), irrespective of fulfillment of the AKI criteria. Urinary kidney injury molecule-1 (KIM-1) was not significantly altered. CONCLUSION: While urinary [TIMP-2]•[IGFBP7] proves valuable for the early diagnosis of neonatal modified KDIGO-defined AKI, elevated urinary NGAL and calprotectin concentrations in indomethacin-treated VLBW infants not fulfilling the AKI criteria may indicate subclinical kidney injury.

Endothelial progenitor cells accelerate endothelial regeneration in an in vitro model of Shigatoxin-2a-induced injury via soluble growth factors.

Endothelial injury with consecutive microangiopathy and endothelial dysfunction plays a central role in the pathogenesis of the postenteropathic hemolytic uremic syndrome (D + HUS). To identify new treatment strategies, we examined the regenerative potential of endothelial progenitor cells (EPCs) in an in vitro model of Shiga toxin (Stx) 2a-induced glomerular endothelial injury present in D + HUS and the mechanisms of EPC-triggered endothelial regeneration. We simulated the proinflammatory milieu present in D + HUS by priming human renal glomerular endothelial cells (HRGECs) with tumor necrosis factor-α before stimulation with Stx2a. This measure led to a time- and concentration-dependent decrease of HRGEC viability of human renal glomerular endothelial cells as detected by a colorimetric assay. Coincubation with EPCs (104-105 cells/ml) under dynamic flow conditions led to a significant improvement of cell viability in comparison to untreated monolayers (0.45 ± 0.06 vs. 0.16 ± 0.04, P = 0.003). A comparable regenerative effect of EPCs was observed in a coculture model using cell culture inserts (0.41 ± 0.05 vs. 0.16 ± 0.04, P = 0.003) associated with increased concentrations of vascular endothelial growth factor, insulin-like growth factor I, fibroblast growth factor-2, and hepatocyte growth factor in the supernatant. Treatment of Stx2a-injured monolayers with a combination of these growth factors imitated this effect. EPCs did not show distinct sings of migration and angiogenic tube formation in functional assays. These data demonstrate that EPCs significantly improve endothelial viability after Stx2a-induced injury in vitro and that this effect is associated with the release of growth factors by EPCs.

Association of angiotensin II type 1 receptor antibodies with graft histology, function and survival in paediatric renal transplant recipients.

Background: We analysed in a carefully phenotyped cohort of paediatric patients the association of serum angiotensin II type 1 receptor antibodies (AT1R-Ab) with specific histological lesions and with graft function and survival in conjunction with overall and complement-binding donor-specific human leucocyte antigen donor-specific antibodies (HLA-DSA). Methods: Sera of 62 patients at the time of renal graft biopsy for clinical indication >1 year post-transplant were assessed for AT1R-Ab by enzyme-linked immunosorbent assay (ELISA) and for DSA and C1q-fixing DSA by single-antigen bead technology. Results: Serum AT1R-Ab concentration was significantly higher in antibody-mediated rejection (ABMR) than in T-cell-mediated rejection or control. By receiver operating characteristic (ROC) curve analysis, the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR and those without was 9.5 U/mL. A total of 6 of 28 patients (21.4%) with ABMR were only positive for AT1R-Ab. Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher vascular micro-inflammation score than DSA-negative patients. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Patients with AT1R-Ab and HLA-DSA double positivity tended to have a more rapid decline of estimated glomerular filtration rate (eGFR) than patients who were only positive for AT1R-Ab or HLA-DSA. In a multivariate Cox regression model of non-invasive factors, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with accelerated graft function decline. Conclusions: Serum AT1R-Ab positivity in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is an independent non-invasive risk factor for adverse graft outcome.

Urinary calprotectin, kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin for the prediction of adverse outcome in pediatric acute kidney injury.

Early identification of patients with acute kidney injury (AKI) being at high risk for adverse outcome can influence medical treatment. This study compares urinary calprotectin, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) for their performance in predicting mortality and need for renal replacement therapy (RRT) in pediatric AKI patients. Urinary biomarker concentrations were assessed prospectively in 141 subjects aged 0-18 years including 55 patients with established AKI according to pediatric Risk, Injury, Failure, Loss, and End-stage kidney disease (pRIFLE) criteria, 27 patients without AKI, and 59 healthy children. Within the AKI group, receiver operating characteristic (ROC) curve analysis revealed moderate to poor performance of calprotectin and KIM-1 in the prediction of 30-day mortality (calprotectin area under the curve (AUC) 0.55; KIM-1 AUC 0.55) and 3-month mortality (calprotectin AUC 0.61; KIM-1 AUC 0.60) and fair performance in the prediction of RRT requirement (calprotectin AUC 0.72; KIM-1 AUC 0.71). Urinary NGAL showed good performance in predicting 30-day (AUC 0.79) and 3-month (AUC 0.81) mortality and moderate performance in predicting RRT (AUC 0.61). CONCLUSIONS: Whereas urinary calprotectin and KIM-1 can be useful for the prediction of RRT, urinary NGAL has a good diagnostic performance in predicting mortality in pediatric patients with AKI of heterogeneous etiology. What is known: • There is increasing evidence that urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are valuable for the prediction of adverse outcome in adult acute kidney injury (AKI), whereas data on pediatric AKI is scarce. What is new: • Urinary calprotectin and KIM-1 do not predict mortality in our heterogeneous pediatric AKI cohort, but they show moderate performance in the prediction of dialysis. • Urinary NGAL is a good predictor of mortality performing better than pRIFLE stage, eGFR, or creatinine, but it shows moderate performance in the prediction of dialysis.

Urinary biomarkers for the differentiation of prerenal and intrinsic pediatric acute kidney injury.

BACKGROUND: Urinary calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) have recently been identified as promising biomarkers for the differentiation of prerenal and intrinsic acute kidney injury (AKI) in adults. In the study reported here we examined the diagnostic accuracy of calprotectin, NGAL, and kidney injury molecule 1 (KIM-1) in pediatric patients. METHODS: Urinary calprotectin, NGAL, and KIM-1 concentrations were assessed in a study population of 139 pediatric subjects including 39 patients with intrinsic AKI, 14 with prerenal AKI, and 86 non-AKI subjects. RESULTS: Median urinary calprotectin and NGAL concentrations were higher in patients with intrinsic AKI than in those with prerenal AKI (calprotectin by 22-fold, NGAL by 9-fold). Receiver operating characteristic (ROC) curve analyses for the differentiation of intrinsic and prerenal AKI resulted in an area under the curve (AUC) of 0.90 [95 % confidence interval (CI) 0.81-0.98] for calprotectin and 0.73 (95 % CI 0.58-0.87) for NGAL. Median urinary KIM-1 concentrations were not significantly different between patients with prerenal AKI and those with intrinsic disease (P = 0.98; AUC 0.50, 95 % CI, 0.35-0.65). The AUC for the fractional excretion of sodium (FENa) and proteinuria was 0.78 (95 % CI 0.63-0.92) and 0.77 (CI 0.65-0.90), respectively. CONCLUSIONS: Urinary calprotectin outperforms NGAL, KIM-1, FENa, and proteinuria as a biomarker for the differentiation of prerenal and intrinsic AKI in pediatric patients with a high diagnostic accuracy.

Association of C1q-fixing DSA with late graft failure in pediatric renal transplant recipients.

BACKGROUND: We investigated the prognostic value of overall and complement-binding donor-specific HLA antibodies (DSA) in pediatric patients undergoing clinically indicated graft biopsies and their association with graft outcome and specific histological lesions. METHODS: Sera of 62 patients at time of indication biopsy ≥1 year posttransplant were assessed for DSA and C1q-fixing DSA by single-antigen bead (SAB) technology. RESULTS: Twenty-six patients (42 %) were DSA-positive at time of indication biopsy and nine (15 %) were C1q-positive. At 4 years postbiopsy, patients with C1q-positivity had a low graft survival (11 %) compared to DSA-positive, C1q-negative patients (82 %, p = 0.001) and to DSA-negative patients (88 %, p < 0.001). The majority (89 %) of C1q-positive patients were diagnosed with active chronic antibody-mediated rejection (ABMR). C1q DSA-positivity [adjusted hazard ratio (HR) 6.35], presence of transplant glomerulopathy (HR 9.54), and estimated glomerular filtration rate (eGFR) at the time of indication biopsy (HR 0.91) were risk factors for subsequent graft loss. CONCLUSIONS: The presence of C1q-positive DSA in the context of an indication biopsy identifies a subgroup of pediatric renal transplant recipients with a markedly increased risk of subsequent graft loss. Because a fraction of DSA-positive patients escape rejection or graft dysfunction, the C1q assay increases the specificity of a positive DSA result regarding unfavorable transplant outcome.

Urinary Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) • Insulin-Like Growth Factor-Binding Protein 7 (IGFBP7) Predicts Adverse Outcome in Pediatric Acute Kidney Injury.

BACKGROUND: The G1 cell cycle inhibitors tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) have been identified as promising biomarkers for the prediction of adverse outcomes including renal replacement therapy (RRT) and mortality in critically ill adult patients who develop acute kidney injury (AKI). However, the prognostic value of urinary TIMP-2 and IGFBP7 in neonatal and pediatric AKI for adverse outcome has not been investigated yet. METHODS: The product of the urinary concentration of TIMP-2 and IGFBP7 ([TIMP-2]•[IGFBP7]) was assessed by a commercially available immunoassay (NephroCheck™) in a prospective cohort study in 133 subjects aged 0-18 years including 46 patients with established AKI according to pRIFLE criteria, 27 patients without AKI (non-AKI group I) and 60 apparently healthy neonates and children (non-AKI group II). AKI etiologies were: dehydration/hypovolemia (n = 7), hemodynamic instability (n = 7), perinatal asphyxia (n = 9), septic shock (n = 7), typical hemolytic-uremic syndrome (HUS; n = 5), interstitial nephritis (n = 5), vasculitis (n = 4), nephrotoxic injury (n = 1) and renal vein thrombosis (n = 1). RESULTS: When AKI patients were classified into pRIFLE criteria, 6/46 (13%) patients fulfilled the criteria for the category "Risk", 13/46 (28%) for "Injury", 26/46 (57%) for "Failure" and 1/46 (2%) for "Loss". Patients in the "Failure" stage had a median 3.7-fold higher urinary [TIMP-2]•[IGFBP7] compared to non-AKI subjects (P<0.001). When analyzed for AKI etiology, highest [TIMP-2]•[IGFBP7] values were found in patients with septic shock (P<0.001 vs. non-AKI I+II). Receiver operating characteristic (ROC) curve analyses in the AKI group revealed good performance of [TIMP-2]•[IGFBP7] in predicting 30-day (area under the curve (AUC) 0.79; 95% CI, 0.61-0.97) and 3-month mortality (AUC 0.84; 95% CI, 0.67-0.99) and moderate performance in predicting RRT (AUC 0.67; 95% CI, 0.50-0.84). CONCLUSIONS: This study shows that urinary [TIMP-2]•[IGFBP7] has a good diagnostic performance in predicting adverse outcomes in neonatal and pediatric AKI of heterogeneous etiology.

Telomere shortening reduces regenerative capacity after acute kidney injury.

Telomeres of most somatic cells progressively shorten, compromising the regenerative capacity of human tissues during aging and chronic diseases and after acute injury. Whether telomere shortening reduces renal regeneration after acute injury is unknown. Here, renal ischemia-reperfusion injury led to greater impairment of renal function and increased acute and chronic histopathologic damage in fourth-generation telomerase-deficient mice compared with both wild-type and first-generation telomerase-deficient mice. Critically short telomeres, increased expression of the cell-cycle inhibitor p21, and more apoptotic renal cells accompanied the pronounced damage in fourth-generation telomerase-deficient mice. These mice also demonstrated significantly reduced proliferative capacity in tubular, glomerular, and interstitial cells. These data suggest that critical telomere shortening in the kidney leads to increased senescence and apoptosis, thereby limiting regenerative capacity in response to injury.

Hypertension induces somatic cellular senescence in rats and humans by induction of cell cycle inhibitor p16INK4a.

There is increasing evidence for a role of somatic cellular senescence in physiological aging but also in injury and disease. Cell cycle inhibitor p16(INK4a) is the key mediator for stress and aberrant signaling induced senescence. Here we report that elevated blood pressure markedly induced p16(INK4a) expression in rat kidneys and hearts, as well as in human kidneys. In kidneys from deoxycorticosterone acetate-salt-treated rats, p16(INK4a) induction was found in tubular, glomerular, interstitial, and vascular cells and correlated with the typical histopathologic features of hypertensive target organ damage. p16(INK4a) expression also correlated with phospho-p38, a positive upstream regulator of p16(INK4a) expression. In left ventricles, increased p16(INK4a) expression was found in myocardium and cardiac arteries. Antihypertensive medication consistent of hydrochlorothiazide, hydralazine, and reserpine ameliorated the histopathologic changes and attenuated p16(INK4a) expression in kidneys of deoxycorticosterone acetate-salt-treated rats. Nonantihypertensive administration of spironolactone also reduced kidney damage and p16(INK4a) expression. p16(INK4a) induction was further observed in kidneys from hypertensive transgenic rats heterozygous for the mouse Ren-2 gene and was prevented by the angiotensin II type 1 receptor blocker losartan. In human kidney biopsies showing hypertensive nephrosclerosis, increased p16(INK4a) expression was found compared with age-matched normotensive control subjects. Thus, hypertension induces cellular senescence via p16(INK4a), possibly through p38, thereby contributing to hypertensive target organ damage. This detrimental effect can be overcome by different therapeutic drug strategies.

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling.

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.

Differential functional interaction of two Vesl/Homer protein isoforms with ryanodine receptor type 1: a novel mechanism for control of intracellular calcium signaling.

Vesl/Homer proteins physically link proteins that mediate cellular signaling [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851] and thereby influence cellular function [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962]. A previous study reported that Vesl-1L/Homer-1c (V-1L) controls the gain of the intracellular calcium activated calcium channel ryanodine receptor type 1 (RyR1) channel [J. Biol Chem. 277 (2002) 44722]. Here, we show that the function of RyR1 is differentially regulated by two isoforms of Vesl-1/Homer-1, V-1L and Vesl-1S/Homer-1a (V-1S). V-1L increases the activity of RyR1 while important regulatory functions and pharmacological characteristics are preserved. V-1S alone had no effect on RyR1, even though, like V-1L, it is directly bound to the channel. However, V-1S dose-dependently decreased the effects of V-1L on RyR1, providing a novel mechanism for the regulation of intracellular calcium channel activity and calcium homeostasis by changing expression levels of Vesl/Homer proteins.

PDGF-BB induces expression of LTBP-1 but not TGF-beta1 in a rat cirrhotic fat storing cell line.

TGF-beta, a profibrogenic cytokine is predominantly secreted as a latent molecule complexed with one of the latent TGF-beta binding proteins (LTBP). Due to the proposed functions of LTBP-1 and -3 in regulating TGF-beta-bioavailability and -activity, we investigated the effects of PDGF-BB and TGF-beta1 on their expression levels in Cirrhotic fat storing cells (CFSC). CFSC basally express LTBP-1 and -3 and TGF-beta1. LTBP-1 colocalizes with LAP and the cells secrete some active TGF-beta1. Promoter studies showed no strong induction of the LTBP-1 promoters after stimulation, although mRNA and protein levels were increased by PDGF-BB treatment without affecting TGF-beta1 expression. Vice versa, TGF-beta1 treatment did not alter LTBP-1 expression while an autocrine induction was found. Our data indicate that LTBP-1 but not TGF-beta1 is induced by PDGF-BB and that TGF-beta1 autoinduction does not affect the expression of LTBP-beta1. This divergent regulation may represent an important mechanism for modulation of TGF-beta bioavailability.