title: Wild-type uromodulin prevents NFkB activation in kidney cells, while mutant uromodulin, causing FJHU nephropathy, does not.
abstract: BACKGROUND
  Uromodulin (Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals. Despite 60 years of research, its physiological role remains rather elusive. Familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease Type 2 are autosomal dominant tubulointerstitial nephropathies characterized by gouty arthritis and progressive renal insufficiency, caused by uromodulin (UMOD) mutations. The aim of this study was to compare the cellular effects of mutant and wild-type UMOD.
  
  
  METHODS
  Wild-type UMOD cDNA was cloned from human kidney cDNA into pcDNA3 expression vector. A mutant UMOD construct, containing the previously reported mutation, V273, was created by in vitro mutagenesis. Transient and stable transfection studies were performed in human embryonic kidney cells and mouse distal convoluted tubular cells, respectively. Expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Oligosaccharide cleavage by glycosidases was performed to characterize different forms of UMOD. Nuclear translocation of P65 and degradation of IκBα and IRAK1 in response to interleukin (IL)-1β were used to evaluate the effects of wild-type and mutant UMOD on the IL-1R-NFκB pathway.
  
  
  RESULTS
  The mutant protein was shown to be retained in the endoplasmic reticulum and was not excreted to the cell medium, as opposed to the wild-type protein. NFκB activation in cells expressing mutant UMOD was similar to that of untransfected cells. In contrast, cells over-expressing wild-type UMOD showed markedly reduced NFκB activation.
  
  
  CONCLUSIONS
  Our findings suggest that UMOD may have a physiologic function related to its inhibitory effect on the NFκB pathway.
authors.affiliation: Department of Nephrology and Hypertension, The Chaim Sheba Medical Center, Tel-Hashomer and the Faculty of Medicine, Sackler School of Medicine, Sheba Medical Center, 52621, Tel-Aviv, Israel, Dganit.Dinour@sheba.health.gov.il.
authors.forename: Dganit
authors.initials: D
authors.lastname: Dinour
authors.forename: Liat
authors.initials: L
authors.lastname: Ganon
authors.forename: Levin-Iaina
authors.initials: LI
authors.lastname: Nomy
authors.forename: Rotem
authors.initials: R
authors.lastname: Ron
authors.forename: Eliezer J
authors.initials: EJ
authors.lastname: Holtzman
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006073:Gout / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D033461:Hyperuricemia / Q000235:genetics / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007674:Kidney Diseases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000378:metabolism*; D014162:Transfection; D058949:Uromodulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D020672:I-kappa B Proteins; C508585:IL1B protein, human; D053583:Interleukin-1beta; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C498143:RELA protein, human; D051996:Transcription Factor RelA; C548612:UMOD protein, human; D058949:Uromodulin; D000072000:NF-KappaB Inhibitor alpha; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases