Prostaglandin E2 (PGE2) is an important lipid mediator modulating various aspects of kidney function. PGE2 exerts its effects via four PGE2 receptors, EP1-EP4. The EP3 receptor is expressed in the thick ascending limb (TAL) and the collecting duct, where it is proposed to inhibit cAMP generation and NaCl and water reabsorption. However, EP3 is also expressed in endothelial cells of arteries and arterioles, which also play a role in kidney function. Therefore, to assess the tubular role of EP3 in adult mice we generated a mouse model based on the Pax8Cre system with doxycycline-dependent deletion of EP3 along the renal tubule and assessed their renal phenotype in respect to water handling. RNAscope confirmed that EP3 was highly expressed in cortical and medullary TAL and collecting ducts, but it was non-detected in proximal tubule and thin limbs. Two weeks after treatment with doxycycline, EP3 mRNA expression was reduced by >80% in whole kidney (RT-q-PCR) and non-detectable (RNAscope) in tubules of knockout mice compared to control mice. There were no compensatory changes in other EP receptors. Under basal conditions, there were no significant differences in food and water intake, bodyweight, urinary output or plasma and urine biochemistries in both male or female control and knockout mice. There were no differences between genotypes in their renal handling of water during an acute water load, or in their response to the vasopressin V2 receptor agonist dDAVP. Rats drinking 1% NaCl for several days in combination with an EP3 antagonist have increased urine output (Hao et al., 2016). However, relative to controls, we could not detect significant differences in urine volume or osmolality in tubular EP3 knockout mice during 1% NaCl intake. In conclusion, EP3 in the renal tubule is not important for renal water handling or compensatory mechanisms exist. This new model provides a novel tool for examination of the role of EP3 in other aspects of renal function or kidney disease independently of potential developmental abnormalities or systemic effects.
GLC. Results from the gasâ liquid chromatography analysis. On the left are in bold averages of mol% for three biological replicates and on the right their respective standard deviations. Raw data is provided for each replicate in separate sheets. (XLSX 48 kb)
Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.
SILAC transVA-OA-CLA. Data from the SILAC investigation of the cellular response to transVA, OA and CLA. In sheet 1, “full data”, unfiltered data from this second SILAC triplex is displayed. In sheet 2, “signif. 1.3 fold”, proteins significantly regulated ≥ 1.3 fold in ≥ one comparison are displayed. Proteins are reported with Uniprot accession number, Name, Fold regulation in individual comparisons, the geometric standard deviation (Geo.SD.), and selected GO-terms: GO:0006950 response to stress (x) and GO:0002526 acute inflammatory response (y). Black bold: significant regulatory data (p 1.3, black italic: significant regulatory data (p 0.01). (XLSX 925 kb)
Abstract Background and Aims Nuclear Factor of Activated T-Cells 5 (NFAT5), also called TonEBP/OREBP, helps protecting kidney cells from the stress of extracellular hypertonic challenge. In response to hypertonicity, NFAT5 rapidly translocates into the nucleus and enhances the transcription of osmoprotective genes. Mice lacking NFAT5 have embryonic lethality, with those surviving having severe renal atrophy and hydronephrosis. Overexpression of a dominant-negative NFAT5 in all epithelial cells of the thick ascending limb, distal tubule and collecting duct (CD) leads to an impairment in the urine concentration, with reduced expression of AQP2 and urea transporters UT-A1 and UT-A2. Here, we generated a novel mouse model to assess the role of NFAT5 in the water transporting principal cells of the CD. Method The mouse model was generated by breeding floxed NFAT5 mice with mice specifically expressing Cre recombinase in AQP2 expressing cells (NFAT5f/f-AQP2cre+/-). AQP2cre+/- mice served as controls. For renal function analysis, mice were kept in metabolic cages for 5 days and water intake, urinary volume, osmolality, and serum and urine electrolytes were evaluated. Mice were euthanized and the kidneys were used for western blot analysis. Results NFAT5f/f-AQP2cre+/- mice show a significant increase of 24 hour urine output compared to the control (KO: 6.267 ± 0.9367 µl/h/g BW vs WT: 0.9593 ± 0.0796 µl/h/g BW), a decrease in the urine osmolality (KO: 505.6 ± 107.6 mOsm/Kg H2O vs WT: 6205 ± 752.7 mOsm/Kg H2O) and urea concentration. After 3h and half intraperitoneal injection of dDAVP (1ug/kg) the osmolality was still significantly low in NFAT5f/f-AQP2cre+/- mice (KO: 743.7 ± 81.77 vs WT: 3342 ± 80.14 mOsm/Kg H2O), as well as after 8h water restriction (KO: 732.8 ± 24.90 vs WT: 3784 ± 450.9 mOsm/Kg H2O). Immunoblotting demonstrated a significant decrease in AQP2, AQP3 and AQP4 abundance in cortical kidney fractions and inner medulla. There were no differences in vasopressin V2 receptor (V2R) and AVP-regulated transporters NKCC2, ROMK and alpha-ENaC. Conclusion We demonstrate that NFAT5 ablation from AQP2-expressing cells results in a urinary concentration defect. NFAT5f/f-AQP2cre+/- mice have a hypoosmotic polyuria, without major effects on the AVP-V2R axis, suggesting Nephrogenic Diabetes Insipidus.
Studies of the interaction between Bikunin proteins, tumor necrosis factor-stimulated gene-6 protein (TSG-6), and glycosaminoglycans have revealed a unique catalytic activity where TSG-6/heavy chain 2 transfer heavy chain subunits between glycosaminoglycan chains. The activity is mediated by TSG-6/heavy chain 2 and involves a transient SDS stable interaction between TSG-6 and the heavy chain to be transferred. The focus of this study was to characterize the molecular structure of this cross-link to gain further insight into the catalytic mechanism. The result showed that the C-terminal Asp residue of the heavy chains forms an ester bond to Ser(28) beta-carbon of TSG-6 suggesting that this residue plays a role during catalysis.
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