Uromodulin

and Chronic Kidney Disease

M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
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.
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.





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.



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.

N-glycan moieties are further trimmed in
the Golgi apparatus.

Later secreted, and
glycosylphosphatidylinositol (GPI)
anchored to the apical tubular cell
membrane.
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.

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.



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.

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.



Normal urinary protein excreation per Day
is 80-150 mg.

1. UMOD: 20 70 mg / L

2. Albumin:- 5 mg / L

3. - 1 microglobulin: - 5 mg / L

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.


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.


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.


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.

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.

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.
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.
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.
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.
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.


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.
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.


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.
A Unifying Hypothesis
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.
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.
The data of Prajczer et al. and Kttgen 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.


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.
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.
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 .
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.

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 .

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.
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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