1. Nat. Rev. Nephrol. doi:10.1038/nrneph.2017.101
Figure 1 Segmental expression and biochemical characteristics of uromodulin
Figure 1 | Segmental expression and biochemical characteristics of uromodulin. a | Uromodulin is produced by the cells that line the thick ascending limb (TAL). This tubular segment is involved in the reabsorption of NaCl, which is mediated by the apical Na+-K+-2Cl− cotransporter NKCC2 and the basolateral exporters Na+/K+-ATPase and the ClC–Kb/Barttin complex. The apical recycling of K+ via the potassium channel ROMK generates a lumen-positive transepithelial voltage, which is the driving force for paracellular reabsorption of monovalent and divalent cations. Uromodulin is cleaved at the apical membrane by the serine protease hepsin and released in the urine where it forms macromolecular polymers. b | The predicted structure of uromodulin contains a leader peptide (grey); four EGF-like domains (orange) of which EGF-II and EGF-III are predicted to be Ca2+ binding; a cysteine-rich D8C domain (red); a bipartite C-terminal Zona Pellucida domain (ZP_N and ZP_C, blue) connected by a rigid interdomain linker; and a glycosylphosphatidylinositol-anchoring site at position 614 (green). The seven N-glycosylation sites are indicated by small triangles; the high-mannose chain on residue Asn275 is shown in red. c | Schematic representation of N-linked glucomoieties carried by human urinary uromodulin. Most N-glycosylation sites carry glycans of the di-antennary, tri-antennary or mainly the tetra-antennary type that can be sialylated or sulfated at their terminal residues (various possibilities for nonreducing terminal units are indicated28,31). Fucosylation is often present at the reducing end. The GalNAcβ1,4(Neu5Acα2,3)Galβ1,4GlcNAc sequence, known as the Sda antigen is highlighted30. The Asn275 retains a high-mannose configuration (mainly Man6GlcNac2)28. Putative cleavage sites of various glycosidases are shown and a disulfide bridge is indicated between two cysteine residues. d | Biochemical properties of human urinary uromodulin as assessed by shifts in electrophoretic mobility. Reduction of disulfide bridges by dithiothreitol (DTT) leads to a narrower uromodulin band with slower migration (at ∼100 kDa). The shift in electrophoretic mobility is compatible with the high number of disulfide bridges in the protein. Removal of all N-linked glycans, of sialylated residues and of N-linked high-mannose moieties by peptide-N-glycosidase F (PNGase F), neuraminidase and endoglycosidase H (Endo H), respectively, reveals the relative contribution of these glycans to human urinary uromodulin. The shift from 100 kDa to ∼70 kDa after PNGase F treatment confirms that ∼30% of the molecular weight of uromodulin is constituted by N-linked glycans. Membranes were blotted with polyclonal sheep anti-uromodulin antibodies. e | Immunofluorescence staining showing a dense network of uromodulin filaments (green) covering a monolayer of primary TAL cells obtained from microdissected mouse kidney. These primary cells are polarized and retain endogenous expression of uromodulin186. Nuclei are counterstained with DAPI. f | Negative stain transmission electron microscope image of uromodulin polymers secreted by primary mouse TAL cells. The helical arrangement is suggested by regular protrusions at an interval of ∼100Å. Image courtesy of Gregor Weiss, ETH Zurich. g | In situ hybridization (RNAscope®) for Umod mRNAand Slc12a1 mRNA(inset) in mouse kidney cortex. Several tubule profiles show abundant co-expression of Umod mRNA and Slc12a1 mRNA, which encodes NKCC2 in the TAL (inset). Nuclei are counterstained with DAPI. h | Immunofluorescence staining for uromodulin in the mouse kidney, showing an enhanced signal at the apical membrane of TAL cells (inset). Nuclei are counterstained with DAPI. Part a reproduced with permission from Elsevier © Devuyst, O. & Bochud, M. Kidney Int. 88, 944–646 (2015).
Part a reproduced with permission from Elsevier © Devuyst, O. & Bochud, M. Kidney Int. 88, 944–646 (2015).
Devuyst, O. et al. (2017) Uromodulin: from physiology to rare and complex kidney disorders
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