UROMODULIN

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• Uromodulin and Chronic Kidney
  Disease
• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Discovery of UROMODULIN by Igor Tamm and Frank
Lappin Horsfall

• In 1950, Tamm and Horsfall isolated a substance
  from human urine that acted as a potent in vitro
  inhibitor of virus-mediated hemagglutination.
• They determined its inhibitory and
  physicochemical properties and concluded that the
  substance was similar in structure to
  mucoproteins.

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Uromodulin(UMOD) or Tamm-Horsfall protein(THP)

• Uromodulin (UMOD) is a 85-90 Kd glycoprotein.
• It consists of 640 amino acids including 48
  cysteine residues that are completely engaged in
  disulfide bond formation.

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• UMOD is a protein expressed solely in the
  mammalian kidney, namely in TALH
• (thick ascending limb of Henles loop) and early
  DCT(distal convoluted tubules) epithelial cells
  in humans.

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• During biosynthesis, the UMOD precursor is
  cotranslationally translocated into the ER.
• There the signal peptide is cleaved, and the
  protein is glycosylated on 7 of its 8 potential
  N-glycosylation sites, disulfide bridges are
  formed and glypiation on its C-terminus (probably
  on S614) occurs.

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• N-glycan moieties are further trimmed in the
  Golgi apparatus.
• Later secreted, and glycosylphosphatidylinositol
  (GPI) anchored to the apical tubular cell
  membrane.

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• Here, UMOD forms organized structures,probably
  ensuring water impermeability and countercurrent
  gradient.
• Besides this, it might contribute to various
  biological processes such as receptor-mediated
  endocytosis, mechanosensation of urinary
  flow,cell cycle regulation and planar cell
  polarity.

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• A specific, but as yet unidentified, protease(s)
  cleaves off and releases UMOD into urine, where
  it can be found in the highest concentrations
  compared to other urinary proteins.
• It modulates aggregation and growth of
  supersaturated salts and their crystals,
  respectively.

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• In urine, UMOD might contribute to the colloid
  osmotic pressure, retard passage of positively
  charged electrolytes and prevent a number of
  bacteria strains from attaching to tubular and
  bladder epithelia, therefore helping to prevent
  urinary tract infections.

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• Healthy individuals excrete about 2070 mg of
  uromodulin per day,(average 50 mg / day) making
  it the most abundant protein in the urine.
• In the urine, the protein precipitates and is
  the main constituent of hyaline urinary casts.

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• Normal urinary protein excreation per Day is
  80-150 mg.
• UMOD 20 70 mg / L
• Albumin- 5 mg / L
• a- 1 microglobulin - 5 mg / L

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Uromodulin Storage Diseases

• These are autosomal dominant diseases clinically
  present with hyperuricemia and gout with a low
  renal fractional excretion of uric acid, and
  progressive renal failure leading
• to ESRD in adulthood.
• more than 50 UMOD mutations have been identified.

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• They are mainly localized in exons 3 and 4, of
  UMOD gene located at cytogenetic band 16p12.3
  according to major gene databases.
• Most of them are missense mutations or small
  inframe deletions.

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• Many of them cause an amino acid change at
  cysteine sites. Cysteine residues form disulfide
  bonds and determine correct protein folding.
• Therefore, it is assumed that UMOD mutations
  causing uromodulin storage disease lead to
  defective protein folding.

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• Misfolded immature uromodulin is retained in the
  ER and not expressed at or released by the apical
  cell membrane.
• Accumulation of misfolded proteins in the ER
  causes ER stress and the unfolded protein
  response with increased synthesis of chaperones
  and foldases and activation of ER-associated
  degradation in order to eliminate the misfolded
  proteins.

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• When the capacity of the cell to remove these
  molecules is working to full capacity, the
  unfolded protein response may trigger apoptosis
  and autophagy or alternatively lead to cell
  activation via MAP kinases and NF- B.
• It is highly likely that these pathways
  eventually result in TAL cell damage and loss
  with progressive renal failure.

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• Jennings et al.reported normal basolateral
  secretion of mutated uromodulin and increased
  serum levels in some patients.
• Higher basolateral secretion of uromodulin may
  cause an inflammatory response and
  tubulointerstitial damage.

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Hyperuricemia

• Hyperuricemia is a consequence of volume
  depletion.
• Scolari et al. hypothesize that due to the lack
  of uromodulin on the luminal surface of the
  TAL,water reabsorption is increased.

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• This would lead to a reduction of sodium and
  chloride reabsorption by the TAL,which is
  compensated by an increase in proximal tubular
  uptake, a process that is coupled to urate
  reabsorption.
• They also showed that a reduction in
  urine-concentrating capability was associated
  with higher uric acid serum levels in these
  patients.

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Uromodulin and CKD

• The pathogenetic link between high uromodulin
  excretion and CKD is at present unknown.
• So far, low uromodulin levels have mostly been
  considered a consequence of TAL damage and
  correlate with reduced renal function in various
  nephropathies.

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• Recently Prajczer et al. presented new data that
  may shed some light on the role of uromodulin in
  CKD.
• They measured uromodulin in serum and urine of 14
  healthy individuals and 77 CKD patients.

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• In agreement with others,they found that the
  lower the GFR the lower the urinary uromodulin
  excretion.
• Low urinary uromodulin was also associated with
  tubular atrophy and interstitial infiltration as
  detected in renal biopsies.

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• In serum, however,there was a trend towards
  higher uromodulin levels in individuals with low
  GFR.
• Furthermore, high serum uromodulin was associated
  with higher serum levels of the proinflammatory
  cytokines TNF- , IL-1 , IL-6, IL-8, and of
  vascular endothelial growth factor VEGF, but not
  hepatocyte growth factor HGF.

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• The authors speculate that uromodulin entering
  the renal interstitium, either via basolateral
  secretion by TAL cells or via backleakage urine,
  may react with cells of the immune system and
  stimulate an inflammatory response, which then
  promotes further tubulointerstitial damage.

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A Unifying Hypothesis
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• Uromodulin is, by its carbohydrate structures, a
  very sticky multipurpose molecule.
• It binds and neutralizes all sorts of objects
  that might appear in urine such as crystals,
  bacteria, various proteins and exosomes.

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• Once uromodulin finds its way into the renal
  interstitium, either by cellular secretion or
  urinary back-leak, this stickiness becomes
  dangerous.
• Uromodulin will bind to cells of the immune
  system such as neutrophils, monocytes, dendritic
  cells and lymphocytes and thereby stimulate in an
  unspecific way an already ongoing immune reaction
  that may lead to tubulointerstital damage and
  progressive renal failure.

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• The data of Prajczer et al. and Köttgen et
  al.suggest that high urine or serum levels of
  uromodulin are potentially dangerous.
• Therefore, downregulation of synthesis and
  secretion of uromodulin might be a therapeutic
  option for slowing CKD progression.

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Biochemical Analysis

• Isolation of UMOD is usually performed by
  classical salt-out precipitation of urine , with
  several modifications in postprecipitation steps.
• The resulting preparation can be further purified
  by gel filtration .
• As a quicker, as well as low-cost, alternative to
  precipitation, a method employing diatomaceous
  earth as a filter material was developed and
  evaluated for clinical purposes.

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• Qualitative and semiquantitative analysis of UMOD
  in complex samples such as urine, cultured cell
  lysates and tissue homogenates is usually
  performed by SDSPAGE followed by detection with
  Coomassie brilliant blue staining, or by Western
  blotting and immunodetection.
• These methods are easy to set up, inexpensive,
  easily scalable and informative.

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• UMOD antibodies are commercially available
  therefore,they can be routinely used to examine
  urinary UMOD excretion as a 1st step in the
  diagnostic process for UMOD-associated kidney
  diseases (UAKD).
• Qualitative and quantitative UMOD analyses by
  mass spectrometry offer promising but
  technically more demanding approaches .

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• Quantitative analysis of UMOD has been mostly
  performed by enzyme-linked immunosorbent assay
  (ELISA).
• Both indirect as well as direct ELISA setups have
  been reported for urinary and serum measurements.
  

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• Besides ELISA, radioimmunoassay , radial
  immunodiffusion and electroimmunoassay have also
  been used in the past.
• An analytical method based on high-performance
  liquid chromatography with native fluorescence
  detection offers an alternative to immunoassay.
• Finally, SDS-PAGE and densitometry quantitation
  of bands after staining has been used as a fully
  quantitative approach in several studies .

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• In situ detection of UMOD in kidney tissue
  specimens and cultured cells is performed using
  standard immunohistochemical, immunofluorescence
  and immunoelectron microscopy methods.
• Quantitative in situ analysis of UMOD may be
  performed by immunogold-electron microscopy with
  particle counting.

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• Quantitative assessment of the UMOD display on
  the surface of cultured cells is performed mainly
  by fluorescence-activated cell sorter analysis
  ,but ELISA can be also used for this purpose .

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