Uromodulin

and Chronic Kidney Disease

M.Prasad NaiduMSc 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 Henle’s 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 20–70 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. α- 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 leadingto 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 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.

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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