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ORIGINAL ARTICLE
Kidney urinary biomarkers in patients with
branched-chain amino acid and cobalamin
metabolism defects
Felix Köpfer
1
| Sven F. Garbade
1
| Kristina Klingbeil
1
|
Brigitte Schmidt-Mader
1
| Jens H. Westhoff
1
| Jürgen G. Okun
1
|
Markus Zorn
2
| Georg F. Hoffmann
1
| Verena Peters
1
| Marina Morath
1
1
Dietmar-Hopp-Metabolic Center, Centre
for Pediatric and Adolescent Medicine,
University Hospital, Heidelberg, Germany
2
Department of Internal Medicine I
(Endocrinology) and Clinical Chemistry,
University Hospital, Heidelberg, Germany
Correspondence
Marina Morath, Centre for Pediatric and
Adolescent Medicine, Division of Child
Neurology and Metabolic Medicine,
University Hospital of Heidelberg, Im
Neuenheimer Feld 430, 69120 Heidelberg,
Germany.
Email: marina.morath@med.uni-
heidelberg.de
Funding information
Deutsche Forschungsgemeinschaft,
Grant/Award Number: SFB 1118
Communicating Editor: D. Sean Froese
Abstract
There is a clinical need for early detection of chronic kidney disease (CKD) in
patients with organic acidurias. We measured kidney markers in a longitudinal
study over 5 years in 40 patients with methylmalonic aciduria (Mut
0
), propionic
aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies.
Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney
injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-
2-microglobulin(B2MG)inurine,aswellascystatinC(CysC)inserum
were quantified. In Mut
0
patients, mean concentrations of B2MG, KIM-1, and
DKK-3 were elevated compared with healthy controls, all markers indicative of
proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were
elevated, indicating signs of proximal tubule and glomerulus damage and inflam-
mation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and con-
sidered as markers for proximal and distal tubule damage and inflammation.
B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut
0
/
CblAandwitheGFR(CysC)andKIM-1inPApatients,respectively.Noneofthe
markers were elevated in CblA patients. Significant deterioration of kidney func-
tion, as determined by steady increase in CysC concentrations was noted in seven
patients within the observation period. None of the investigated biomarker profiles
showed a clear increase or added value for early detection. In conclusion, we iden-
tified disease-specific biomarker profiles for inflammation, tubular, and proximal
damage in the urine of Mut
0
, PA, and CblC patients. Whether these biomarkers
can be used for early detection of CKD requires further investigation, as significant
kidney function deterioration was observed in only a few patients.
KEYWORDS
biomarker, chronic kidney disease, cobalamin deficiency, organic aciduria
Verena Peters and Marina Morath shared senior authorship.
Received: 4 May 2023 Revised: 2 August 2023 Accepted: 18 August 2023
DOI: 10.1002/jimd.12672
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.
J Inherit Metab Dis. 2023;1–11. wileyonlinelibrary.com/journal/jimd 1
1|INTRODUCTION
Methylmalonic acidemias (MMA) and propionic acidemia
(PA) are caused by a disturbance in the catabolism of
branched-chain amino acids. MMAs are due to a complete
(Mut
0
subtype) or partial (Mut
subtype) deficiency of the
mitochondrial methylmalonyl-CoA mutase (EC 5.4.99.2;
OMIM #251000) or due to defects in the uptake, transport,
or synthesis of its cofactor 50-deoxyadenosylcobalamin
(cblA, cblB or cblD variant 2 type; OMIM #607481,
#251110, #251100, #277410). Deficiency of the mitochon-
drial propionyl-CoA carboxylase (EC 6.4.1.3; OMIM
#606054) results in PA. CblC deficiency is the most com-
mon inborn error of intracellular vitamin B
12
metabolism
(Figure 1; OMIM #277400). In patients with Mut
0
and PA,
and to lesser extend also with CblA and CblC,
2-methylcitric acid (MCA) accumulates in tissues and
body fluids.
1,2
Higher MCA levels are seen in patients with
severe phenotype and significant long-term complications,
and MCA levels are generally higher in patients with PA
than in Mut
0
.
3
MCA increases during metabolic decom-
pensation
4
and plasma MCA correlates with disease bur-
den.
5
The clinical presentation is very heterogenous but
the majority of patients suffers from severe multi-organ
involvement in the first years of life.
6–8
Chronic renal fail-
ure (CKD) is the most important organ system long-term
complication in MMA, Mut
0
, and CblB patients,
9
while
CKD is a rare complication in PA, CblC, and CblA in
childhood. In an adult American cohort of PA patients,
CKD has been manifested in half of the patients,
10
in
CblC, kidney involvement occurs preferentially in the late-
infantile or adult-onset form.
11
A cross-sectional data from
the European Registry revealed that CKD was less fre-
quent in CblA
12
occurring primarily in adult patients.
13
In
clinical practice, glomerular filtration rate (GFR) estima-
tion and albuminuria are widely used for CKD diagnosis
and prognosis. GFR is often approximated by measuring
serum CysC concentrations,
14
and urinary B2MG excre-
tion is considered as a marker of tubular damage. How-
ever, these traditional biomarkers only increase when
kidney damage is already advanced.
15
Early identification
of CKD is important to predict and prevent or slow CKD
progression.
16
There is an urgent clinical need of addi-
tional diagnostic non-invasive tools that allow us to deter-
mine kidney disease.
3
Several new biomarkers for early
detection of deterioration of kidney function have been
identified and studied in several patient cohorts with kid-
ney involvement.
17,18
We, therefore, investigated three novel markers of
tubular damage, i.e. neutrophil-gelatinase-associated
lipocalin (NGAL), kidney injury molecule-1 (KIM-1),
dickkopf 3 (DKK-3),
19–22
albumin as a well-established
marker of glomerular injury and beta-2-microglobulin
(B2MG), as a well-established marker of proximal tubule
FIGURE 1 Metabolic overview. Metabolic pathways affected by methylmalonic acidemia (Mut
0
), propionic acidemia (PA), and
cobalamin A (CblA) and C (CblC) deficiency. Adapted from Morath et al.
60
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damage. In addition, we investigated calprotectin (CLP)
as an immunological marker released during a variety of
inflammatory processes.
23
Furthermore, MCA was
included as a marker for severe phenotype and disease
burden. These urine markers were then related to serum
CysC and eGFR (CysC). This approach should provide
information on whether the new markers offer an advan-
tage in early diagnostics of CKD.
2|METHODS
2.1 |Study design and population
A longitudinal study was conducted at Heidelberg
University Children's Hospital, Germany, from 2017 to
2022. Urine and serum samples from patients with
methylmalonic aciduria (Mut
0
;n=11), Propionic acid-
uria (PA; n=15), Cobalamin A (CblA; n=5), and
Cobalamin C (CblC; n=9) deficiencies were collected
during routine metabolic follow-up every 6–12 months
resulting in 128 urine samples and 106 serum samples
from 40 patients. Samples were collected in well-state
and never during metabolic crisis. Diagnosis for all
patients was genetically confirmed.
The control group (n=49) consisted of apparently
healthy children and adolescents aged 0–18 years. Urine
from healthy children and urine and serum samples from
patients were collected, centrifuged and the supernatant
was stored frozen (20C). This study was approved by
the ethic committee of the University Heidelberg (S-
436/2016).
2.2 |Measurement of urinary and serum
markers
Calprotectin was measured using an enzyme-linked
immunosorbent assay (ELISA) kit (Biovendor R&D;
Czech Republic; Catalog No. RD191217100R), KIM-1
was measured with the KIM-1 (human) ELISA kit
(Enzo Life Sciences GmbH, Lörrach, Germany; Catalog
No. ADI-900-226-0001) and NGAL with the NGAL
Rapid ELISA kit (BioPorto Diagnostik, Gentofte,
Denmark; Catalog No. 036CE). DKK-3 was measured
using an enzyme-linked immunosorbent assay (ELISA)
kit (Dia Ren UG; Homburg). 2-methylcitric acid (MCA)
was measured by organic acid analysis carried out by gas
chromatography-mass spectrometry.
24
Urinary B2MG and
albumin were measured immunoturbidimetrically as
described by Martin et al. (2011). All urinary markers were
related to creatinine. Serum CysC was measured immuno-
turbidimetrically as described.
25
Reference values were
defined by the Central Laboratory of University Hospital
Heidelberg.
2.3 |Determination of cystatin C-based
equation (2012)
The calculation of cystatin C-based equation [eGFR
(CysC)] was performed according to Schwartz et al.
(2012),
26
Grubb et al. (2010),
27
and Inker et al. (2012).
28
2.4 |Statistical analysis
All results are presented as mean ± SD for continuous var-
iables, however median for ordinal scaled data. For show-
ing differences in biomarker levels between groups one-
way ANOVA with Tukey's test was performed using
GraphPad Prism version 9.4.1 for macOS, GraphPad Soft-
ware, San Diego, California USA. Age-dependent effects of
urinary biomarker concentrations were analyzed by multi-
ple regression with biomarker as dependent variable, and
age in years and disease as independent variables.
3|RESULTS
3.1 |Urinary biomarker concentrations
in the patient cohort
Urine concentrations of albumin, B2MG, NGAL, CLP,
KIM-1, and DKK-3 were determined in 49 urine samples
from healthy children. 128 urine samples from 40 patients
were collected over a period of 5 years and analyzed.
Multiple regression analysis of CLP, KIM-1, and DKK-3
concentrations revealed no age-dependent effects in dis-
ease groups and respective controls. Borderline, opposing
effects were observed for NGAL in Mut
0
and CblC
patients, probably caused by the small sample size
(Figure S1). For 22 patients, multiple urine samples
(up to 8 samples per patient) were available. The data
were analyzed both in the entire collective (as shown in
Figure 2) and compared to the averaging of the values
per patient (Table S1). The biomarker concentrations
were both normalized to creatinine (crea), the non-
normalized data can also be found in Figure S2.
Compared to healthy subjects, mean albumin was
increased by twofold in Mut
0
and PA and B2MG up to
fourfold in those patients (Figure 2A). None of the bio-
markers were elevated in CblA patients. Mean urinary
excretion for NGAL was sevenfold higher in CblC
patients, mean CLP was sevenfold higher in PA and CblC
patients, mean KIM-1 was twofold higher in Mut
0
and
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PA patients and mean DKK-3 up to 30-fold higher in
Mut
0
patients (Figure 2B,Table1). Comparing the patient
groups with each other, mean urinary NGAL excretion
was two-, three- and eightfold higher in CblC compared
with samples from Mut
0
, PA, and CblA patients, respec-
tively (Table 1). Three of the CblC patients, including an
18-year-old patient with late-onset presentation of hemo-
lytic uremic syndrome history at 2 and 5 years of age, had
elevated CysC levels since study enrollment. The NGAL
and CLP concentrations in these three patients were in the
same range as for the patients with normal CysC concen-
trations (119 ± 53 vs. 312 ± 203 NGAL pg/mg creatinine
and 68 ± 73 vs. 72 ± 65 CLP ng/mg creatinine; both
p=ns). CLP excretion was approximately 3-, and 20-fold
higher in PA and CblC compared with Mut
0
and CblA
patients. KIM-1 excretion was approximately twofold
higher in Mut
0
and PA patients compared with CblC
patients (Figure 2B,Table1). The mean value for DKK-3
in Mut
0
patients was highly variable, due to individual
patients with generally high and patients with low DKK-3
concentrations. Two or more urine samples from the same
patient were available from six patients with Mut
0
.Halfof
the patients had elevated mean DKK-3 concentrations
(1193 ± 1061 mg/mg crea, n=7; 35 014 ± 22 474 mg/mg
crea, n=2; 7655 ± 8946 mg/mg crea, n=3), and the
mean eGFR (CysC) was 26, 26, and 8 mL/min/1.73m
2
in
the patients, respectively. In the other three patients, mean
DKK-3 levels were within the normal range (6.3 ± 1.7 mg/
mg crea, n=4; and 7.3 ± 6.1 mg/mg crea, n=5; 2.7
± 1.2 mg/mg crea, n=3), and mean eGFR (CysC) was
64, 49 and 26 mL/min/1.73m
2
, respectively. None of the
Mut
0
patients showed a clear increase in DKK-3 concen-
tration over time. Neither DKK-3, KIM-1, and B2MG con-
centrations correlated with urinary albumin, only DKK-3
correlated with B2MG in those patients (Table S3).
The highest mean urinary MCA concentrations were
found in PA patients, which were almost fourfold higher
compared to Mut
0
patients. MCA concentration did not
correlate with biomarker concentrations (Figure S3). Com-
bining the distribution of conventional and new kidney
biomarkers revealed marker-specific patterns (Table 2).
3.2 |Serum CysC levels as a marker of
kidney function
The patients’serum samples were divided into two
groups: in patients with eGFR(CysC) above and below
(A) (B)
FIGURE 2 Distribution of urinary markers in patients with methylmalonic and propionic aciduria and cobalamin A and C deficiency.
Urinary markers were quantified and related to creatinine in patients with methylmalonic aciduria (Mut
0
;n=11), propionic aciduria (PA;
n=15), cobalamin A (CblA; n=5), and cobalamin C (CblC; n=9) deficiency and compared to healthy controls (ctrl; n=49). The boxplot
presents the data of 128 urine samples from 40 patients, given the median and the interquartile range. In case of multiple individual
samples, mean concentration was used for calculation. (A) Distribution of the conventional markers albumin and ß-2-microglobulin (B2MG)
and of (B) novel markers neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1) and
dickkopf 3 (DKK-3). *p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001 (one-way ANOVA with Tukey's test).
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TABLE 1 Description of patients and samples.
Mut
0
PA CblA CblC
Healthy
controls
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Cohort Number of patients 11 15 5 9 49
Age (years) 15.8 ± 10.7 13.3 ± 9.2 21.8 ± 13.3 14.5 ± 7.8 8.5 ± 3.8
Female 7 10 0 6 24
Male 4 5 5 3 25
Adults (≥20 years) 4 4 2 3 0
Adolescence (10–19 years) 3 4 2 4 19
Children (0–9 years) 4 7 1 2 30
Patient multiple visits 7 9 3 3 0
Patients with HUS 0 0 0 1 0
Patients with normal CysC
f
11256 49
Patients with elevated CysC
f
10 3 0 3 0
Urinary markers Albumin/crea (mg/mg) 2.3 ± 1.1
a
2.2 ± 1.7
a
1.2 ± 0.7 1.4 ± 1.3 1.2 ± 1.1
B2MG/crea (pg/mg) 368.2 ± 27 1.0
a
256.8 ± 244.6
a
114.8 ± 74.9 285.9 ± 352.0
a
83.8 ± 45.3
bce
NGAL/crea (pg/mg) 94.5 ± 52.9
e
69.1 ± 40.1
e
31.2 ± 14.0
e
247.9 ± 189.2
abcd
34.1 ± 27.2
e
CLP/crea (ng/mg) 22.2 ± 19.7
ce
60.6 ± 52.6
abd
2.7 ± 2.6
ce
67.0 ± 64.4
abd
9.0 ± 8.7
ce
KIM-1/crea (pg/mg) 416.4 ± 310.8
a
391.1 ± 187.1
a
223.8 ± 147.6 218.5 ± 169.2 239.7 ± 147.6
bc
DKK-3/crea (pg/mg) 1396 ± 2317
ac
75.3 ± 71.2
b
126.1 ± 215.0 84.5 ± 113.6 51.0 ± 58.5
b
MCA/crea (nnmol/mol) 171.3 ± 93.5 644.0 ± 397.7 48.0 ± 46.8 31.9 ± 19.2 n.d.
MMA (mmol/mol creatinine) 8395 ± 2355 1.4 ± 0.5 716.6 ± 384.3 102.4 ± 84.4 n.d.
creatinine (mg/dl) 60.8 ± 36.2
ad
64.1 ± 44.0
ad
166.1 ± 59.9
bce
91.1 ± 50.7
d
115.9 ± 50.1
bc
Blood markers Plasma MMA (μmol/l) 2020 ± 1643 n.d. 48.4 ± 22.9 n.d. n.d.
Samples with normal CysC
(n)
6 38 13 15 n.d.
Samples with elevated CysC
(n)
32 18 0 6 n.d.
Serum creatinine (mg/dl)
f
1.8 ± 1.6
ce
0.5 ± 0.3
b
0.6 ± 0.2 0.4 ± 0.1
b
0.1–1.2
Serum CysC (mg/l)
f
2.5 ± 1.5
cde
1.0 ± 0.5
b
0.9 ± 0.2
b
1.0 ± 0.3
b
n.d.
eGFR (CysC) Samples with eGFR (CysC)
below 60 (mL/min/1.73 m
2
)
26 15 0 2 n.d.
Samples with eGFR (CysC)
above 60 (mL/min/1.73 m
2
)
7 32 10 18 n.d.
eGFR (Crea) Samples with eGFR (Crea)
below 60 (mL/min/1.73 m
2
)
14 1 0 0 n.d.
Samples with eGFR (Crea)
above 60 (mL/min/1.73 m
2
)
3 13 9 6 n.d.
Abbreviations: B2MG, ß-2-microglobulin; CLP, calprotectin; CysC, cystatin C, all concentrations were related to creatinine (crea); DKK-3, dickkopf-3; HUS,
hemolytic uremic syndrome; KIM-1, kidney injury molecule-1; MCA, 2-methylcitric acid; MMA, methylmalonic acid; n.d. =not determined; NGAL, neutrophil
gelatinase-associated lipocalin.
a
p< 0.05 versus healthy controls.
b
p< 0.05 versus Mut
0
.
c
p< 0.05 versus PA.
d
p< 0.05 versus CblA.
e
p< 0.05 versus CblC (one-way ANOVA with Tukey's test).
f
Age-dependent, normal ranges for serum CysC are provided in Table S5.
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60 mL/min/1.73 m
2
, indicating mild to severe loss of
kidney function. In the patient cohort with eGFR(CysC)
below 60 mL/min/1.73 m
2
, albumin, B2MG, NGAL, and
DKK-3 were elevated compared to the control group.
Mean NGAL and CLP concentrations were also higher
compared to controls for patients with eGFR(CysC) above
60 mL/min/1.73 m
2
(Figure 3). Correlation of biomarkers
with eGFR(CysC) revealed a significant result for B2MG
in PA patients and for KIM-1 in CblA patients (Figure 4).
3.3 |Urinary biomarkers in progression
of CKD
During the observation period, only 2 of 11 Mut
0
,1of
15 PA, and 2 of 9 CblC patients developed for the first-
time kidney function deterioration based on CysC eleva-
tion above the age-related normal range (Table S4). In
those five patients, biomarker concentrations fluctuated
and showed no clear increase over time (Figure S4). Two
further patients, one Mut
0
and one PA, with increased
CysC concentrations from the beginning of the study,
developed significant disease progression during the
observation time, measured by steadily increasing CysC
concentration (from 2.3 to 3.8 over 3.11 years and from
1.8 to 2.7 over 4.5 years, respectively). Based on the small
sample size, no statement can be made on prediction of
kidney disease development or even progression in these
patient cohorts.
4|DISCUSSION
Early diagnosis and treatment of chronic kidney disease
(CKD) in patients with organic acidurias and treatment are
two important measures to prevent further deterioration of
renal function and to delay adverse consequences. CKD is
TABLE 2 Disease-specific pattern of the conventional and new
urinary markers when compared to a healthy control group.
Mut
0
PA CblA CblC
Albumin +
B2MG ++ +
NGAL +
CLP ++
KIM-1 +
DKK-3 +
Note: Urinary markers were quantified and related to creatinine in patients
with methylmalonic aciduria (Mut
0
;n=11), propionic aciduria (PA;
n=15), cobalamin A (CblA; n=5) and cobalamin C (CblC; n=9)
deficiency and compared to healthy controls (ctrl; n=49). Differences
compared to healthy controls (p< 0.05) were indicated by +.
(A) (B)
FIGURE 3 Distribution of urinary markers in patients with eGFR(CysC) above and below 60 mL/min/1.73 m
2
and healthy controls
(ctrl). (A) Mean urinary concentration of urinary albumin and B2MG were significantly higher in patients with eGFR below 60 mL/
min/1.73 m
2
. (B) Mean urinary NGAL and DKK-3 concentrations were elevated in patients with eGFR below 60 mL/min/1.73 m
2
*NGAL
and CLP concentrations were elevated in patients with eGFR above 60 mL/min/1.73 m
2
;**p< 0.01; ***p< 0.001; ****p< 0.0001 (Oneway
ANOVA with Tukey's test). Data presented as box-plot.
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defined as a gradual loss of kidney function due to struc-
tural or functional abnormalities with or without a
decreased glomerular filtration rate.
29
Measured glomerular
filtration rate (mGFR) is considered the gold standard for
assessing kidney function, but is rarely used due to its inva-
sive nature, high cost and time.
30
An overestimation of
mGFR could be demonstrated for creatinine-based calcula-
tionsinadolescentandadultMMApatients.
9
Serum CysC
allows the calculation of estimated GFR (eGFR) and has
been considered to be more accurate than serum creatinine
determinations.
26,31–34
Serum creatinine is subject to individual variation
and is an unreliable marker for estimating renal function
in pediatrics due to dependence on muscle mass and
age
35,36
Especially in CblC deficiency, borderline reduced
serum creatinine concentrations have been observed.
37
Normalization of biomarker concentrations to urinary
creatinine levels is a common practice, but it should be
noted that in CKD and in disorders of creatinine metabo-
lism, such as in CblC deficiency, may lead to lower uri-
nary creatinine levels. For this purpose, we also
performed the analysis without creatinine normalization,
which revealed similar but less significant results. We
further provided the calculation of biomarker concentra-
tions by averaging all samples and averaging values per
patient in case of multiple samples per patient. Averaging
of all samples carries the risk of skewing the data toward
the findings of a smaller subset of patients who happen
to have more frequent sample collection. Both types of
calculation revealed a comparable disease-specific bio-
marker profile, especially for NGAL, CLP, KIM-1, and
DKK-3.
In addition, the two urinary markers, albumin, and
B2MG, were used to detect glomerular and tubular renal
damage. Despite its prognostic significance, tubular dis-
ease cannot be reliably detected with standard clinical
measures of glomerular health (eGFR and UACR) and is
invisible to the clinician except in the rare instances
when biopsies are obtained.
38
The pathomechanisms of
CKD development in the studied disease groups are only
partly understood, but seem to be based on different
mechanisms. Patients with branched-chain organic acid-
urias, that is, Mut
0
, CblA, and PA, develop chronic-
tubulo-interstitial nephritis (cTIN), demonstrated in
human
13,39
and murine
40
kidney biopsies. A severe dis-
turbance of mitochondrial network especially in renal
proximal tubule cells has been suggested as a causative
pathomechanism of kidney damage.
41,42
In CblC defi-
ciency, kidney involvement occurs preferentially in the
late-infantile or adult-onset form, in most cases as acute
or chronic thrombotic microangiopathy (TMA), some-
times resulting in hemolytic uremic syndrome or even
chronic renal failure.
43
The underlying pathophysiologi-
cal mechanism remains fairly unknown, hyperhomocys-
teinemia and elevated MMA appear not to be the
responsible nephrotoxins.
44
(A) (B)
FIGURE 4 Correlation of urinary markers with eGFR(CysC). (A) Urinary concentration of B2MG and albumin and (B) urinary
concentrations of NGAL, CLP, KIM-1, and DKK-3 were correlated with eGFR(CysC). In PA patients, B2MG correlated with eGFR(CysC)
decline. No clear correlation could be demonstrated for the other investigated markers.
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KIM-1 is expressed at low levels in both normal kid-
ney and other organs, but its expression is upregulated in
proximal tubule cells in kidney after post-ischemia/
reperfusion injury. It has been used as a sensitive indica-
tor for early detection of renal tubular injury in patients
with acute kidney injury.
20
Urinary KIM-1 is closely
related to tissue KIM-1 levels and correlates with tissue
damage.
20,45
In CKD, the role of KIM-1 is controversially
discussed. Studies in patients with diabetes and hyperten-
sion found no correlation of urinary KIM-1 with lowering
eGFR,
46,47
whereas in the systolic blood pressure inter-
vention study, urinary KIM-1 was associated with an
increased risk for CKD progression.
48,49
After acute kid-
ney injury urinary KIM-1 has been associated with a sig-
nificantly increased risk for CKD development
50
Mean
urinary KIM-1 concentrations were higher in Mut
0
patients but in no other patient cohort (Figure 2). A novel
and promising urinary biomarker, particularly for the
detection of ongoing tubular injury and early stages of
CKD, is the glycoprotein DKK-3, a stress-induced, renal
tubular epithelial cell-derived, pro-fibrotic molecule.
51
It
promotes tubulointerstitial fibrosis, a histopathological
feature of Mut
0
and CblA.
13,39,40
Urinary DKK-3 is able to predict acute kidney
injury
21,52
and identifies patients at high risk for short-
term progression of CKD, regardless of the cause of kidney
disease, baseline kidney function, and albuminuria
51
and
indicates a short-term risk of severe kidney function
impairment in children withchronickidneydisease.
53
DKK-3 was significantly elevated in some, but not all, Mut
0
patients and correlated with B2MG but not with albumin
excretion (Table S3). In six Mut
0
patients who already had
impaired renal function (eGFR 8–64 mL/min/m
2
)since
study inclusion, there tended to be a relationship between
the severity of renal function impairment and the increase
of urinary DKK-3 concentration. However, in the two Mut
0
patients who showed an increase in CysC for the first time
during the study period, no increase in urinary DKK-3 con-
centrations was observed (Table S4).
This needs to be verified in a larger cohort. The par-
tially large scatter of urinary DKK-3 values in Mut
0
patients is possibly due to a binding of plasma compo-
nents preventing the crossing of the glomerular barrier.
51
Mean NGAL concentrations were only significantly
increased in the CblC group. In Mut
0
and PA patients,
NGAL concentrations were heterogenous with mean con-
centrations higher than in respective controls, albeit not
significantly. NGAL is considered as a marker for distal
tubule, is reabsorbed by the proximal tubules and released
from damaged distal tubules in the setting of acute tubular
injury. It can be detected within hours of tubular injury,
even in the absence of functional acute kidney injury.
54
Although several studies support the use of NGAL as an
early marker for acute kidney injury, its utility as a marker
for CKD is uncertain and disease-dependent.
20
In some
conditions, plasma NGAL correlates better than urinary
NGAL with CKD progression.
55–58
Apreviousstudy
showed a strong correlation of plasma NGAL with creati-
nine and eGFR (CysC) in Mut
0
MMA subjects and
Mut
/;TgINS-Alb-Mut
mouse model.
40
The high concentra-
tions of urinary NGAL and CLP in CblC patients might
indicate distal tubular damage and inflammation processes
in these patients but this needs further investigations. For
NGAL, a correlation could be shown with hyperhomocys-
teinemia in kidney-transplanted adult patients as well as
in association with vascular damage.
59
This is another
indicator that the biomarker profiles of the investigated
disease groups are influenced by the underlying patho-
physiology. Three of the CblC patients had elevated CysC
concentrations; two of them had an early-onset and one
had a late-onset presentation with two episodes of HUS
and were all treated with parental hydroxocobalamin,
betaine, and folinic acid after diagnosis.
Mean urinary CLP levels, a mediator protein of the
innate immune system, were especially elevated in PA
and CblC and to a lesser extend in Mut
0
patients. Urinary
CLP values were six times higher in structural acute kid-
ney injury than in functional acute kidney injury.
23
In
CblC patients, Lemoine et al demonstrated IgM deposits
of the glomerular basement membrane, in addition to
arteriolar and glomerular lesions of thrombotic microan-
giopathy, indicating immunological involvement in CblC
patients.
11
However, Seibert et al found no difference of
CLP excretion between primary inflammatory and
non-inflammatory etiologies of CKD, although the differ-
entiation between “completely inflammatory and non-
inflammatory”renal disease is difficult.
58
It remains
unknown why CLP is less elevated in Mut
0
patients com-
pared to PA patients, immunological tubulointerstitial
involvement was detected in both diseases.
13,34
In the patient cohort studied, only five patients expe-
rienced an increase in age-appropriate CysC via the
upper cut-off during the observation period. No increase
in urine concentration with the onset of CysC-based
renal impairment could be identified for any of the bio-
markers investigated, including the conventional
markers albumin and B2MG (Table S4), whereas elevated
CyS and eGFR(CysC) correlated with increased urinary
excretion of albumin, B2MG, and DKK-3. Due to the very
small number of patients with an early onset of CKD
(5 patients) and progression of CKD (2 patients) during
the observation period, no statement can be made on the
suitability of the investigated novel biomarkers as early
indicators of CKD or as a marker for disease progression
on the basis of these data. Future investigations of serum
biomarker concentrations, for example, serum NGAL
8KÖPFER ET AL.
15732665, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jimd.12672 by Ruprecht Karls Universitaet, Wiley Online Library on [09/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
concentrations,
40
in comparison to urinary biomarker
concentrations in the described patient's cohort, would
be of interest and may lead to a better understanding of
the detected biomarker profiles.
5|CONCLUSION
In conclusion, a disease-specific biomarker profile in
patients with branched-chain amino acid and cobalamin
metabolism defects with a risk of CKD was found, con-
sisting of markers for proximal, distal tubule damage
and/or an inflammatory component. More samples are
needed to evaluate the diagnostic value of investigated
kidney biomarkers for early detection of CKD and disease
progression in patients with Mut
0
, PA, CblA, and CblC.
AUTHOR CONTRIBUTIONS
Conceptualization: Verena Peters, Jens H Westhoff,
Marina Morath; Methodology: Felix Köpfer, Kristina
Klingbeil, Brigitte Schmidt-Mader, Markus Zorn, Jürgen
G Okun; validation and formal analysis: Sven F Garbade,
Felix Köpfer; writing—original draft preparation: Felix
Köpfer, Marina Morath, Verena Peters; writing—review
and editing, Jürgen G Okun, Markus Zorn, Jens H
Westhoff, Georg F Hoffmann; supervision: Marina Mor-
ath, Verena Peters. All authors have read and agreed to
the published version of the manuscript.
ACKNOWLEDGMENTS
We thank all colleagues for sample collecting, especially
Friederike Hörster, Dorothea Haas, Ulrike Mütze, Tunç
Tuncel, Christian Staufner, Alboren Shtylla, Tergita Preci,
Hannelore Bartl, Emilia Vogel and Matthias Zielonka. Open
Access funding enabled and organized by Projekt DEAL.
FUNDING INFORMATION
This study was funded by grants from the Deutsche For-
schungsgemeinschaft (DFG, German Research Founda-
tion)—Project number 236360313—SFB 1118.
CONFLICT OF INTEREST STATEMENT
All authors declare that they have no conflict of interest.
DATA AVAILABILITY STATEMENT
The original data generated from experiments are avail-
able in the supplementary information. Individuals' data
are not publicly available due to the data protection law.
ETHICS STATEMENT
The clinical study was approved by the Institutional
Ethics Committee of the University of Heidelberg
(S-436/2016).
PATIENT CONSENT STATEMENT
All procedures followed were in accordance with the eth-
ical standards of the responsible committee on human
experimentation (institutional and national) and with the
Helsinki Declaration of 1975, as revised in 2013.
Informed consent was obtained from all patients for
being included in the study.
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SUPPORTING INFORMATION
Additional supporting information can be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Köpfer F, Garbade SF,
Klingbeil K, et al. Kidney urinary biomarkers in
patients with branched-chain amino acid and
cobalamin metabolism defects. J Inherit Metab Dis.
2023;1‐11. doi:10.1002/jimd.12672
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