ABSTRACT: Unbiased transcriptome profiling and functional genomics approaches identified glucocorticoid-induced transcript 1 (GLCCI1) as being a transcript highly specific for the glomerulus, but its role in glomerular development and disease is unknown. Here, we report that mouse glomeruli express far greater amounts of Glcci1 protein compared with the rest of the kidney. RT-PCR and Western blotting demonstrated that mouse glomerular Glcci1 is approximately 60 kD and localizes to the cytoplasm of podocytes in mature glomeruli. In the fetal kidney, intense Glcci1 expression occurs at the capillary-loop stage of glomerular development. Using gene knockdown in zebrafish with morpholinos, morphants lacking Glcci1 function had collapsed glomeruli with foot-process effacement. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. Taken together, these data suggest that Glcci1 promotes the normal development and maintenance of podocyte structure and function.
Project description:LAT3 is a Na+-independent neutral l-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton.
Project description:Empiric steroid therapy is the first-line therapy for pediatric nephrotic syndrome, but treatment response is variable. There are few predictors of steroid-responsiveness, although evidence for genetic factors does exist. Single nucleotide polymorphisms (SNPs) have been recently identified in the promoter region of glucocorticoid-induced transcript 1 gene (GLCCI1) which affect steroid-responsiveness in asthmatic patients. Independently, GLCCI1 was identified as a podocyte protein, the loss of which disrupts the function of the glomerular filtration barrier. We therefore examined whether SNPs associated with the steroid-responsive expression of GLCCI1 might predict steroid-responsiveness in nephrotic syndrome.A cohort of 211 pediatric patients with nephrotic syndrome and 102 controls were genotyped; among the cases, 117 were initial steroid responders, while 94 did not respond to oral steroids. No statistically significant differences were noted among the groups, although there was a trend based on the comparison of the small subgroups of steroid-responsive and non-responsive patients with biopsy-proven minimal change disease.While larger cohorts are needed to ascertain the possibility of a small effect of GLCCI1 SNPs on the steroid-responsiveness of nephrotic syndrome, the GLCCI1 SNPs associated with steroid-responsiveness in asthmatic patients are unlikely to have a clinically actionable impact in pediatric nephrotic syndrome.
Project description:OBJECTIVE:The specification and differentiation of hematopoietic stem cells into red blood cells requires precise coordination by multiple transcription factors. Most genes important for erythroid maturation are regulated by the Gata family of DNA-binding proteins. Previously, we identified three novel genes kelch-repeat containing protein (krcp), kiaa0650, and testhymin/glucocorticoid inducible transcript 1 (glcci1) to be expressed in erythroid cells in a Gata-independent manner, and we sought to further understand how these transcripts are regulated during zebrafish hematopoiesis. MATERIALS AND METHODS:We employed a loss-of-function approach, using combinations of antisense morpholinos to hematopoietic transcription factors and assayed for changes in gene expression in zebrafish embryos. RESULTS:Upon examination of embryos deficient for Gata1, Gata2, Biklf, and/or Scl, we found distinct gene combinations were required for expression of the novel genes. While krcp expression was dependent upon Gata1 and Biklf, kiaa0650 expression was greatly reduced and glcci1 was maintained in Gata1/Gata2/Biklf-deficient embryos. As with the gata1 gene, kiaa0650 and krcp required Scl for blood expression. Although reduced, glcci1 was expressed in posterior blood precursors in the absence of Scl and Gata2. CONCLUSIONS:This work identifies glcci1 as having Scl-independent expression in the posterior hematopoietic mesoderm, suggesting that its posterior expression is activated by factors upstream or parallel to Scl and Gata2. Additionally, these studies establish that blood gene expression programs are regulated by transcription factors acting in combination during erythroid maturation.
Project description:Polycystic kidney disease (PKD) is one of the leading causes of end-stage renal disease in humans and is characterized by progressive cyst formation, renal enlargement, and abnormal tubular development. Currently, there is no cure for PKD. Although a number of PKD genes have been identified, their precise role in cystogenesis remains unclear. In the jcpk mouse model of PKD, mutations in the bicaudal C gene (Bicc1) are responsible for the cystic phenotype; however, the function of Bicc1 is unknown. In this study, we establish an alternative, nonmammalian zebrafish model to study the role of Bicc1 in PKD pathogenesis. Antisense morpholinos were used to evaluate loss of Bicc1 function in zebrafish. The resulting morphants were examined histologically for kidney cysts and structural abnormalities. Immunostaining and fluorescent dye injection were used to evaluate pronephric cilia and kidney morphogenesis. Knockdown of zebrafish Bicc1 expression resulted in the formation of kidney cysts; however, defects in kidney structure or pronephric cilia were not observed. Importantly, expression of mouse Bicc1 rescues the cystic phenotype of the morphants. These results demonstrate that the function of Bicc1 in the kidney is evolutionarily conserved, thus supporting the use of zebrafish as an alternative in vivo model to study the role of mammalian Bicc1 in renal cyst formation.
Project description:Glucocorticoids are the primary anti-inflammatory therapy for asthma, but their effects are characterized by some interindividual variability that might have a genetic basis.We aimed to determine the relationship between pulmonary function change and the variant of the glucocorticoid-induced transcript 1 (GLCCI1) gene in patients with asthma receiving long-term ICS treatment, the association of GLCCI1 genotypes and the level of GLCCI1 expression and cytokines production.A total of 418 patients with asthma, including 25 individuals from 11 families with a history of asthma, were enrolled. The effects of single-nucleotide polymorphisms (SNPs) in GLCCI1 on changes in lung function in response to inhaled glucocorticoids were assessed. The expression levels of GLCCI1 mRNA and cytokines were also measured.The SNP rs37973 in GLCCI1 was independently associated with changes in forced expiratory volume at one second (FEV1) and FEV1%pred. Individuals homozygous for the wild-type allele who had a percent FEV1 change greater than 5% were more common than individuals homozygous for the rare allele. When patients were stratified according to genotype, GLCCI1 expression was enhanced upon administration of low-dose dexamethasone among patients with the rs37973 A allele; however, GG homozygotes required high-dose dexamethasone to achieve enhanced GLCCI1 expression. Furthermore, the levels of some cytokines were significantly reduced after glucocorticoid treatment in individuals with the AA and AG genotypes.The genetic variant rs37973 in GLCCI1 is associated with poorer clinical therapeutic response to inhaled glucocorticoids in a Chinese asthma population.
Project description:Asthma is a serious public health problem worldwide, without effective therapeutic methods. Our previous study indicated that glucocorticoid-induced transcript 1 gene (GLCCI1) knockout reduces the sensitivity to glucocorticoid in asthmatic mouse. Here, we explored the role and action mechanism of GLCCI1 in asthma development. In ovalbumin-sensitized mice, airway resistance and tissue damage increased, the production of inflammatory cytokines were up-regulated, GLCCI1 expression was reduced and autophagy was activated. Increasing of GLCCI1 inhibited human and mouse airway epithelial cell (AEC) autophagy, while decreasing of GLCCI1 promoted autophagy. Furthermore, we found that GLCCI1 bound with WD repeat domain 45B (WDR45B) and inhibited its expression. Increasing of WDR45B partly reversed the inhibition of GLCCI1 to autophagy-related proteins expression and autophagosome formation in vitro. Increasing of WDR45B in vivo reversed the improvement of GLCCI1 on airway remodelling in asthma and the inhibition to autophagy level in lung tissues. Overall, our data showed that GLCCI1 improved airway remodelling in ovalbumin-sensitized mice through inhibiting autophagy via combination with WDR45B and inhibiting its expression. Our results proved a new idea for asthma treatment.
Project description:Prolyl 4-hydroxylases (P4Hs) catalyze the hydroxylation of collagens and hypoxia-inducible factor (HIF)-? subunits. We studied the zebrafish homologue of the recently characterized human transmembrane P4H (P4H-TM) that can hydroxylate HIF-?, but not collagens, in vitro and influence HIF-? levels in cellulo. The zebrafish P4H-TM mRNA had its highest expression in the eye and brain and lower levels in other tissues, including the kidney. Morpholino knockdown of P4H-TM in embryos resulted in a reduction in the size of the eye and head and morphological alterations in the head from 2 days postfertilization onward. In addition, pericardial edema, regarded as a sign of kidney dysfunction, developed from 3 days postfertilization onward. The phenotype was dependent on the P4H-TM catalytic activity because similar results were obtained with morpholinos targeting either translation initiation or catalytic residues of the enzyme. Structural and functional analyses of the morphant pronephric kidneys revealed fragmented glomerular basement membranes (BMs), disorganized podocyte foot processes, and severely compromised pronephric kidney function leading to proteinuria. The opacity of the eye lens was increased due to the presence of extra nuclei and deposits, and the structure of the lens capsule BM was altered. Our data suggest that P4H-TM catalytic activity is required for the proper development of the glomerular and lens capsule BMs. Many HIF target genes were induced in the P4H-TM-deficient morphants, but the observed phenotype is not likely to be mediated at least solely via the HIF pathway, and thus P4H-TM probably has additional, as yet unknown, substrates.
Project description:The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease.
Project description:Background:Previous studies have demonstrated that glucocorticoid-induced transcript 1 gene (GLCCI1) rs37973 mutant genotype is associated with poor inhaled corticosteroid (ICS) response in asthmatics. As human airway relaxation is regulated by circulation epinephrine, which can be enhanced by corticosteroid. It is unknown whether or not GLCCI1 rs37973 is associated with circulation epinephrine and cortisol concentrations in asthma. The aim of this study is to evaluate these relationships. Methods:A total of 182 asthmatics and 180 healthy controls were recruited for the study. 30 mild-to-moderate asthmatics received fluticasone propionate (125 ?g, bid) treatment for 12 weeks. GLCCI1 rs37973 genotyping was performed with the iPlex MassARRAY genotyping platform. The plasma concentrations of cortisol and epinephrine of each participant were detected by enzyme linked immunosorbent assay (ELISA) kits. Results:GLCCI1 rs37973 homozygotes mutant genotype GG had a higher plasma epinephrine concentration (median concentration 27.032 pg/ml, nGG = 36; median concentration 23.149 pg/ml, nAA+AG = 146; P = 0.015) and cortisol concentration (median concentration 1.141 ng/ml, nGG = 36; median concentration 0.921 ng/ml, nAA+AG = 146; P = 0.013). Both epinephrine concentration and cortisol concentration in plasma were positively correlated with FEV1 (r = 0.889 and r = 0.821, respectively. nasthma = 182). For asthmatics treated with ICS, rs37973 was associated with change in plasma epinephrine and cortisol concentration in a recessive model (AA + AG vs GG), with GG had less improvement in epinephrine concentration [?EPIAA+AG = 6.843 (9.26) pg/ml, nAA+AG = 26; ?EPIGG = -1.666 (6.52) pg/ml, nGG = 4; P = 0.018] and cortisol concentration [?CORAA+AG = 0.3040 (0.21) ng/ml, nAA+AG = 26; ?CORGG = -0.066 (0.24) ng/ml, nGG = 4; P = 0.009]. Conclusions:Our study suggested that the poor ICS response in GLCCI1 rs37973 mutant genotype might be related to the less increased amplitudes of plasma epinephrine and cortisol in asthmatic patients. Trial registration:ChiCTR-RCC-13003634 www.chictr.org.cn. Active since September 27, 2013.