Uromodulin ELISA

(Tamm-Horsfall-Glycoprotein)
EQ 6821-9601
 Protein characteristics

• Synthesised only in renal epithelial cells forming

the thick ascending limb of the loop of Henle and
the early distal convoluted tubule

• Molecular weight: 105 kDa

• 30 % of protein mass are carbohydrates

• Highest concentrated protein in urine (50 mg/d)

• But also lower secretion rate into the blood

(conc.: 200 ng/mL)
• It is thought that Uromodulin protects the
kidney from kidney stone formation and
urinary tract infections .
• Uromodulin has immunomodulatory properties
and is capable of neutrophils and monocytes
to activate.
• Uromodulin activates dendritic cells via Toll-
like Receptor -4 ( TLR4 ) and thus combines
unspecific with specific immune defense .
• Mutations in the gene of the Uromodulin
UMOD, lead to three hereditary renal
diseases:
• Familial juvenile hyperuricemic nephropathy
(FJHM),
• Medullary cystic kidney disease type 2
(MCKD2) and
• Dominant glomerulocystic kidney disease.
• These mutations lead to inflammation
( tubulo-interstitial nephritis ) and increased
connective tissue formation ( fibrosis ) in the
kidneys
• A drop in serum-measured urromodulin concentration is
associated with a significant deterioration in renal function
• The most recent demonstration that Uromodulin, as a
kidney-specific glycoprotein from the epithelium of the
thick ascending part of Henle's loop,
• circulates not only in urine, but in serum in ng / ml, is
based on a second transport route
• Transport of Uromodulin takes place in the direction of the
tubule lumen via the apical membrane.
• A second path towards the basolateral plasmamembrane,
where the protein is embedded in cytoplasmic vesicles.
• The vesicles fuse with the basal plasma membrane,
urromodulin is released into the renal interstitium and
appears in the blood
• In contrast to traditional functional markers such as
serum creatinine, cystatin C and eGFR-cystatinC, the
serum level of urromodulin decreases significantly in
the direction of the stage 1 of renal insufficiency
(CKD-1) earlier,
• thus recognizing a renal involvement rather than all
other known measuring parameters of renal function
• Low serum concentrations of uromodulin are also
predictive of later graft dysfunction
• Mutations in the UMOD gene are closely linked to the
risk that renal insufficiency will reach the final stage of
dialysis.
• It is noticeable that even healthy family members of a
UMOD Gen Missense mutation have very low blood
values ​of Uromodulin
• In 529 patients with increased cardiovascular risk, reduced
serum uromodulin levels were associated with increased
overall mortality; Whereas those with higher values ​with a
better metabolic position, lower prevalence of further
comorbidities such as arterial hypertension, diabetes mellitus
and heart failure.
• Low serum concentrations of uromodulin were seen in 3057
patients, Which were subjected to coronary angiography, also
associated with increased overall mortality.
• In addition, arterial hypertension, diabetes mellitus, cardiac
failure were associated with lower levels of uromodulin in the
blood,
• higher serum urromodulin associated with younger age,
lower body mass index, lower blood pressure, lower
triglycerides, lower fasting blood glucose, 3, NT-proBNP, LDL
and HDL cholesterol, but with higher 25- (OH) 2-vitamin D
levels.
• In patients with multiple myeloma , it may in
the renal tubules in the presence of Tamm-
Horsfall protein precipitations of light chains in
the form of protein cylinders come.
• These precipitates have a direct toxic effect
on the renal tubule cells and can lead to a
rapid loss of renal function and thus to acute
renal failure ( myeloma kidneys ).
 Normal range
Males +
Males females children
females
n 190 89 101 443
Mean 222 199 241 204
SD 95 79 104 89
75th percentile 275 251 308 254
Median 207 188 230 193
25 percentile 149 144 165 143
 Sample stability
U ro m o d u lin [n g /m L ]

- Umod is very stable, even after 4 weeks of storage at increased temperatures

450
400
350
300
250 -20
°C
200 4 °C
150 37 °C
100
50
0
5011 5015 29 43 50 61 65 66
Sample ID#
And only 5µl serum is needed! (1:100 Dilution)
 Autoantibody positive patients
(with probable kidney involvement)

Anti-
Healthy Anti-PLA2R Anti-GBM
dsDNA
controls positive positive
positive
n 190 20 20 10
Mean [ng/ml] 222 87 110 41
SD [ng/ml] 95 54 76 22
75th percentile [ng/ml] 275 110 135 52
Median [ng/ml] 207 82 107 37
25 percentile [ng/ml] 149 48 39,0 22

500
p<0.001

p<0.001
400
p<0.001
-- -- Mean
----- Median

300

200
Urom odulin [ng/m l]

100

0
Healthy blood anti-dsDNA anti-PLA2-R anti-GBM
donors n=190 n=20 n=20 n=10
 Uromodulin in SLE patients
(with and without kidney involvment)

52 sera from clinically characterized SLE patients (48f, 4m; age 15-82) were tested for sUmod by
ELISA (EUROIMMUN, Germany). 32 of these patients had an active or inactive kidney involvement,
while 20 patients did not show any kidney damage according to the clinical reports.
 Uromodulin levels in different stages
of chronic kidney disease

Stages defined by doctor:

eGFR Cystatin C according to Hoek
eGFR and other comorbidities (Diabetis,
(values >90 unreliable)
high blood pressure…)
 Uromodulin can identify
beginning of kidney disease

ROC analysing the ability of different parameters to differentiate between patients without
chronic kidney disease (CKD stage 0) and CKD stage 1; BUN = blood-urea-nitrogen;
eGFR = estimated glomerular filtration rate; AUC = area-under-the-curve.

*Data from Scherberich at al. publication in preparation

Uromodulin levels in different stages
of chronic kidney disease
 Uromodulin can identify
beginning of kidney disease
 Comparison to established
renal markers

*Data from Scherberich at al. publication in preparation

UMOD – after kidney transplantation
n=44
 Advantages of Uromodulin

- Direct marker for kidney vitality

- No estimations/calculations necessary
• eGFR is estimated based on creatinine/cystatin C levels (serum) or
creatinine clearance (urine)
• Different equations for estimating eGFR available
- Synthesized only in the kidney (in contrast to creatinine and cystatin C)
- Independent of muscle mass and protein intake (in contrast to creatinine)
- No 24h urine collection (as for creatinine clearance)
- Early diagnostic marker (early loss of renal function can be identified in the
„creatinine-blind“ range)
 Indications

Indication: Nephropathies

 Marker for kidney function and vitality

 Decreasing levels of uromodulin with decreasing kidney function:

direct correlation to estimated glomerular filtration rate (eGFR)
 Only marker that can distinguish between controls and stage 1
CKD cases
 Risk factor for chronic kidney disease (CKD)

 Marker for monitoring therapy of CKD (hypertension, diabetes,

tumors)
 Predictive marker after kidney transplantation
 Serological markers for primary
membranous nephropathy
• Membranous nephropathy (MN) is a chronic inflammatory disease
of the glomeruli which is characterised by subepithelial in situ
deposition of immune complexes at the glomerular basement
membrane .
• For this reason it is also known as membranous glomerulonephritis
(MGN)
• The deposition of complexes results in a dysfunctional permeability
of the capillary walls of the glomeruli, leading to proteinuria and
very frequently to nephrotic syndrome.
• Besides the highly increased protein secretion in the urine (>3.5
g/24 h), the latter is also characterised by a reduced concentration
of protein in the blood (hypoproteinaemia), an increased
concentration of blood lipids (hyperlipidaemia) and oedema.
 • Around 20-30 % of MN cases are of a secondary form
and arise as a result of a different underlying disease or
drug therapy
• They must be differentiated from primary MN, for which an
underlying secondary disease has not been found.
• In 2009, autoantibodies against phospholipase A2
receptors (anti-PLA2R) were found in around 70 % of
primary MN patients.
• Since then it has been possible to differentiate between
anti-PLA2R positive and idiopathic cases (without
detectable autoantibodies and underlying secondary
disease)
• This nomenclature, however, is not used in all of the
literature and many patients who are positive for anti-
PLA2R are misleadingly called idiopathic
 Causes of membranous nephropathy
(MN)
• Primary MN
• Anti-PLA2R autoantibodies
• Idiopathic (without detectable autoantibodies and underlying
secondary disease)
• Secondary MN
• Infections (e.g. hepatitis B virus, hepatitis C virus, syphilis, malaria)
• Autoimmune diseases (e.g. rheumatoid arthritis, Sjögren's
syndrome, bullous pemphigoid)
• Carcinoses
• Other diseases (e.g. sickle cell anaemia, Guillain-Barré syndrome)
• Induced by medication (e.g. non-steroidal anti-inflammatory drugs
(NSAID), gold, mercury, penicillamine)
• Since different diagnostic procedures and
therapies are applied to the two forms of
disease,
• the differentiation between primary and
secondary MN is of major clinical importance.
• While therapy in secondary MN is aimed at
the underlying disease, patients with primary
MN are mainly treated with an
immunosuppressive therapy.
• Correct and fast diagnosis can therefore help
to avoid unnecessary medication and
extensive diagnostic procedures.
 Discovery and pathogenic role in primary
membranous nephropathy
• For decades, the target antigen in patients with primary MN
was unknown.
• In 2009, the group of David J. Salant and co-workers
identified a 185-kDa glycoprotein as the major antigen of
autoantibodies which were detected in 70 % of primary MN
patients but not in patients suffering from secondary MN.
• The target protein was identified as M-type phospholipase
A2 receptor (PLA2R) by mass spectroscopy
• PLA2R is expressed on the surface of podocytes and binds
soluble phospholipase A2
• that binding of the autoantibodies to
PLA2R results in formation of in situ
immune complexes in the area of the
glomerular basement membrane
• These may trigger local complement
activation with overproduction of type IV
collagen and laminin.
• Destruction of the cytoskeleton may
cause damage to the podocytes, which
leads to an impaired filter function and the
known symptoms (nephrotic syndrome).
• retrospective study (panel n=1061) for the
determination of anti-PLA2R
autoantibodies using an enzyme linked
immunoadsorbent assay (ELISA) (Anti-
PLA2R ELISA (IgG)) yielded a sensitivity
of 96 % .
• The specificity amounted to 99.9 %
including borderline sera
 Anti-PLA2R-
Panel n
positive/borderline

Primary MN 198* 190

Sensitivity 198 96.0 %

Secondary MN 27 0

Other glomerulonephritides 230 0

Other autoimmune diseases 315 0

Healthy blood donors 291 1

Specificity 836 99.9 %

Only patients with primary MN were found to be positive for
anti-PLA2R autoantibodies in a retrospective analysis by
Beck et al. All patients suffering from secondary MN were
negative for these autoantibodies.
 Titers are crucial for disease evaluation, therapy
and risk assessment
• Anti-PLA2R autoantibodies, moreover, provide important
information about the clinical activity of the disease.
• Hofstra and colleagues showed that a weak but
statistically significant association exists between the
anti-PLA2R titer and the clinical status of the
disease as defined by proteinuria.
• They found that anti-PLA2R titers were high in patients
with acute nephrotic syndrome (high proteinuria) but
decreased or were absent in cases of spontaneous or
treatment-induced remission (low proteinuria)
• If a relapse occurred, the antibody titer increased again
 Anti-thrombospondin type-1 domain
containing protein 7A antibody (anti-
THSD7A)
• Is a supplementary serological marker to
anti-PLA2R
• Up to 10% of anti-PLA2R-seronegative
patients with primary membranous
nephropathy are anti-THSD7A-positive
• It is known since 2014
• It increases the serological detection rate
in non-invasive , serological diagnostics of
primary MN
Thank you!