464

ORIGINAL
Molecular composition of adiponectin in urine is a usefulbiomarker for detecting early stage of diabetic kidney disease
Tomoharu Kawano1, Motohiro Aiba1, Masashi Ishizu2, Hiroyasu Mori2, Munehide Matsuhisa2, Seiichi Hashida3,
Tracey Colpitts4, and Toshihiro Watanabe4
Human Life Science, Tokushima Bunri University, Tokushima, Japan, 2Diabetes Therapeutics and Research Center, Institute of Advanced
Medical Sciences, Tokushima University, Tokushima, Japan, 3Department of Diabetes and Molecular Genetics, Ehime University Graduate
School of Medicine, Ehime, Japan, 4R&D division, Sysmex R&D Center Americas, Inc., Mundelein, IL, America
1

Abstract : We previously developed two immune complex transfer enzyme immunoassays (ICT-EIA) to measure
total adiponectin (T-AN) and high molecular weight adiponectin (H-AN) in urine and have verified their usefulness as biomarkers for diabetic kidney disease. In this study, we developed T-AN and H-AN assays using the sandwich EIA (Sand-EIA). The reactivities of Sand-EIAs were compared with ICT-EIAs by measuring size exclusion
chromatography (SEC) fractions of urine and adiponectin standard. As a result, ICT-EIAs showed higher macromolecular specificity. We then analyzed the molecular profile of adiponectin in the urine of 5 patients with different eGFR stages by measuring SEC fractions of urine. The results showed that smaller adiponectin correlated
relatively well with eGFR stage. Finally, because SEC is time-consuming, we investigated that the ratio of T-ANs
by Sand-EIA and ICT-EIA could be a good indicator of the monomer adiponectin. The ratio was evaluated using
77 urine samples from patients with diabetes and showed a significant decrease at an earlier stage comparedwith other biomarkers. In conclusion, we demonstrated a new index to estimate monomer adiponectin in urine
by using Sand-EIA and ICT-EIA, and urinary monomer adiponectin can be a good early indicator of deteriorationof renal function in diabetic patients. J. Med. Invest. 70 : 464-470, August, 2023
Keywords : Immune complex transfer enzyme immunoassay, Ultrasensitive immunoassay, Urinary adiponectin, Kidney disease, Biomarker

INTRODUCTION
Chronic kidney disease (CKD) is a disease that is initiallyasymptomatic, but leads to a gradual decline in kidney function, causing cardiovascular disease (CVD) such as myocardial
infarction, stroke, and heart failure (1) and eventually requires
dialysis or a kidney transplant (2). Diabetes mellitus is one of the
major contributing causes, and when CKD develops as a complication of diabetes mellitus, it is called diabetic nephropathy
or diabetic kidney disease (3). Therefore, it is important to findthe high-risk group of worsening renal function among diabetic
patients for therapeutic intervention, and biomarkers that enable
early diagnosis are considered useful. Past studies have shownthat adiponectin in urine may be useful as a biomarker for CKD(4-6). In particular, unlike the Glomerular filtration rate (GFR)
and urinary albumin excretion rate (UAER), which have beenused as biomarkers, adiponectin can predict the onset and progression of CKD (7, 8). It is expected to be used as an early CKD
biomarker which can detect the change of the renal function
from earlier stages compared with the conventional biomarkers.
We have developed two assays to measure trace amountsof adiponectin in urine using an ultra-sensitive immunoassay
technique called immune complex transfer enzyme immunoassay (ICT-EIA). One is a total adiponectin (T-AN) assay thatmeasures all of the high molecular weight (HMW), medium
molecular weight (MMW), low molecular weight (LMW), andmonomers in urine. The other is the HMW adiponectin (H-AN)
Received for publication November 21, 2022 ; accepted July 18, 2023.
Address correspondence and reprint requests to Toshihiro Watanabe,
R&D division, Sysmex R&D Center Americas, Inc., Mundelein, IL,
America. E-mail : Watanabe.Toshihiro@sysmex.co.jp

assay, which specifically measures only HMW. Previously, using
these two assays, we have shown that T-AN and H-AN levels arehigher with advancing eGFR stage and may be elevated earlier
than the conventional biomarker, UAER (9, 10). Furthermore,
we showed that baseline H-AN levels may be associated with adecline in eGFR after 2 years, which suggested that H-AN may
predict CKD progression (11).
In previous studies, several urine samples were size-fractionated by SEC and measured for T-AN and H-AN using ICT-EIAs,
and it was found that the molecular weight of adiponectin in
urine changed with eGFR stage. Higher molecular weight adiponectin tended to be discharged into urine as CKD progressed(9, 10). The aim of this study was to analyze the molecularweight composition of adiponectin in the urine of patients withdifferent eGFR stages, and to examine the relationship withCKD progression and the potential as a new biomarker.
In addition, the ICT-EIA method is a technique to improve
the signal-to-noise ratio by decreasing the background signalof immunoassays, which results in higher sensitivity. However,it has been found that the immune complexes in the assay, once
formed, dissociate during the reaction process (12). The effect of
this phenomenon on T-AN and H-AN measured levels has notbeen investigated. In this study, we tested not only the previously developed ICT-EIAs, but also the new conventional sandwich
EIAs, which were used to measure urine samples in order toevaluate the measured levels with and without dissociation.

MATERIALS AND METHODS
Urinary adiponectin assays

Buffers, antibodies and antigen used in the present study
were described previously and the assay reagents were preparedusing the same protocol (10). The sandwich EIA method for

The Journal of Medical Investigation Vol. 70 2023

The Journal of Medical Investigation Vol. 70 August 2023
T-AN and H-AN measurement on HI-1000 was as follows : 30 μL
of sample was mixed with 80 μL of capture and detection antibody conjugates and incubated for 1.0 min. 30 μL of streptavidin
coated magnetic particles was added and incubated for 14 min.
After washing, the bound alkaline phosphatase activity was
assayed by chemiluminescence with HISCL substrate reagent(Sysmex, Hyogo, Japan) for 5.0 min at 42 ℃. The ICT-EIA method for T-AN and H-AN measurement on HI-1000 was the sameas described previously (10).

Subjects and samples
Seventy-seven outpatients with type 1 or type 2 diabetes inTokushima University Hospital were enrolled in this study.
Patient backgrounds are shown in Table 1. Blood samples weredrawn from an antecubital vein of the subjects. Early morning
urine samples were collected after overnight fasting. The urine
samples (10 mL) were mixed with 0.1 mL of 10% BSA and NaN3
and dialyzed overnight at 4 ℃. Dialyzed urine samples were kept
frozen at -30 ℃ until analysis.

Analysis of urine samples using size exclusion chromatography (SEC)
Two randomly chosen patient urine samples (1.0 mL) out of
the 77 outpatients were subjected to size exclusion chromatography (SEC) using a HiLoad 16/60 Superdex 200 prep grade (1.6x 60cm) column on an AKTA Explorer 10S FPLC chromatography system (GE Healthcare, Tokyo, Japan). An aliquot of eachfraction was tested for T-AN and H-AN by both sandwich EIA
and ICT-EIA methods. Urine from subjects in the 25th and 75th

percentiles of each eGFR stage (G1, G2, G3a, G3b, G4), 10 samples in total, were selected and separated using the same chromatography method and each fraction was tested for T-AN by
the sandwich EIA method. HMW, MMW, LMW and monomer
adiponectin concentrations were calculated based on concentrations of fraction No. 50-52, 54-56, 62-64 and 85-89, respectively
using recombinant human adiponectin (Oriental yeast, Tokyo,
Japan) as calibrator.

Statistical analysis
For biomarker and index correlation analyses, Spearman’s
correlation coefficients were calculated. Due to the small sample
size, the p-values may not be accurate, but they are provided forreference.

Ethical considerations
The study protocol was approved by the ethical committee ofTokushima Bunri University (No. H29-17) and by the ethicalcommittee of Tokushima University Hospital (No. 2894-1). Alldiabetic participants gave written informed consent. All methods were performed in accordance with the relevant guidelines
and regulations.

Data Availability
The datasets generated and / or analyzed during the current
study are available from the corresponding author on reasonable
request.

Table 1. Patient background
Outpatients (n = 77)
Gender (Male / Female)

38 / 39Age

62 (52, 69)

BMI (kg / m )2

465

24.5 (21.6, 28,3)

HbA1c

7.24 (0.82)

Type of diabetes (T1DM / T2DM)

31 / 46

eGFR stage (G1 / G2 / G3a / G3b / G4)

9 / 43 / 14 / 8 / 3

ALB stage (1 / 2 / 3)

57 / 13 / 7

Microvascular complications
Neuropahthy

42 (54.5%)

Retinopathy (NDR / SDR / PPDR / PDR / Unknown)

38 / 17 / 9 / 7 / 6 (49.4% / 22.1 % / 11.7% / 9.1 % / 7.8%)

Medications
Insulin

51 (66.2%)

GLP-1 receptor agonist

15 (19.5%)

Sulfonylurea

5 (6.5%)Glinide1 (1.3%)

DPP-4 inhibitor

22 (28.6%)

Biguanide

23 (29.9%)

Thiazolidine

2 (2.6%)α-glucosidase inhibitor

8 (10.4%)

Sodium glucose transporter 2 inhibitor

11 (14.3%)

Antihypertensive

38 (49.4%)

Statins

35 (45.5%)

NDR : No visible diabetic retinopathy, SDR : Simple diabetic retinopathy, PPDR : Pre-proliferative diabetic
retinopathy, PDR : Proliferative diabetic retinopathy

T. Kawano, et al. Urinary adiponectin as a biomarker for DKD

466
RESULTS

Different reactivity of sandwich EIA and ICT-EIA methods
Adiponectin standard material and urine samples collectedfrom diabetic patients were size-fractionated by SEC, and eachfraction was tested for T-AN and H-AN by sandwich EIA andICT-EIA methods as shown in Fig 1.
The adiponectin standard consisted of MMW and HMW components. Both components were detected by the T-AN assays,
with the ICT-EIA method generating slightly higher signals
compared to the sandwich EIA method. The H-AN assays
demonstrated more specificity for the HMW component withlittle to no detection of the MMW component. For these assays,
the sandwich EIA method generated more signal than the ICTEIA method.
In the measurement of the urine sample using the two T-AN
assay methods, similar levels of HMW, MMW, and LMW wereobserved. However, the monomeric adiponectin was detected ingreater quantities by the sandwich EIA method.
Regarding the analysis of the urine sample using the two
H-AN assay methods, the SEC profile and signals generated
by each assay were similar to that of the adiponectin standard
material. The sandwich EIA method generated more signal thanthe ICT-EIA method of the H-AN assays, opposite to the T-AN
assays.

Analysis of adiponectin monomer and multimers in the urine fromdiabetic patientsSeventy-seven outpatients were classified based on eGFR
stage, and urine samples from patients in the 75th percentile ofeGFR in each group (G1, G2, G3a, G3b, G4) were size-fractionated by SEC, and each fraction was assayed by the T-AN sandwich
EIA method, which best detects all adiponectin forms (monomer
and multimers), and the results are shown in Fig 2. Relatively
high levels of monomeric and LMW adiponectin are detected inurine samples from diabetic patients with stages G1-G3a, whereas in G3b-G4, the presence of MMW and HMW adiponectin
were found to be increased.
The same analysis was then performed for subjects at the 25th
percentile of each eGFR stage, and the amounts of monomeric,
LMW, MMW, and HMW adiponectin, as well as their percentage of total adiponectin were calculated. Correlation between the
adiponectin biomarkers / index values and eGFR are shown in
Table 2. The correlation coefficients (r) were relatively high between eGFR and the LMW measure, the LMW / total adiponectin ratio, and the monomer / total adiponectin ratio, indicatingthat the variation of low-molecular-weight adiponectin may be
a strong indicator reflecting the progression of kidney disease.
The relationship between eGFR stage and the biomarkers
including adiponectin indexes is shown in Fig 3. Albumin, theconventional biomarker, T-AN, H-AN, and LMW adiponectintended to be elevated in eGFR stages G3b and G4. LMW adiponectin had a higher correlation coefficient with eGFR than theother biomarkers. On the other hand, the LMW / total adiponectin ratio and monomer / total adiponectin ratio, unlike other biomarkers, showed increasing and decreasing trends even in theearly stages of eGFR, indicating that they may be biomarkers
that fluctuate from earlier stages. These results suggested thatthey may be variable biomarkers from earlier stages.

Figures○ Sand● ICT
LMW

T-AN (pg/mL)

140
120
100

Adiponectin standard

Monomer

1200

MMW

80

HMW

60
40

40

1800
1600
1400
1200
1000
800
600
400
200
0

60

80

800
600
400
0

100

MMW

40

40

2500

HMW

H-AN (pg/mL)

H-AN (pg/mL)

MMW

1000

200

20
0

HMW

1400

T-AN (pg/mL)

Urine

160

60

80

60

80

100

80

100

HMW

2000
1500

MMW

1000
500

100

Fraction No.
Fig 1. Specificities of T-AN and H-AN assays by sandwich EIA and ICT-EIA.

0

40

60

Fraction No.

T-AN : Total
H-AN :ofHigh
molecular
weightadiponectin,
LMW : Low
weight, MMW : Middle molecular weight,
Figadiponectin,
1. Specificities
T-AN
and H-ANassaysby sandwich
EIAmolecularand ICT-EIA.
HMW : High molecular weight, Sand ; Sandwich EIA, ICT ; ICT-EIA. Urine samples from patients with diabetes and adiponectin standard
were separated by SEC and fractions were measured by the assays.

The Journal of Medical Investigation Vol. 70 August 2023

467

Fig 2. HMW, MMW, LMW and monomer of adiponectin in urine from patients with G1-G4 stages of diabetic kidney disease. Sixty SEC

2. HMW,
MMW,forLMW
andmonomer
adiponectin in urine from patients with G1-G4 stages offractionFig
samples
were measured
one casefromeach eGFRof
stage.
T-AN : Total adiponectin, H-AN : High molecular weight adiponectin, LMW : Low molecular weight, MMW : Middle molecular weight,
diabetic kidney disease.
HMW : High molecular weight. Urine samples from patients with diabetes were separated by SEC and fractions were measured by sandwich
T-AN assay.

Table 2. Correlation between eGFR and urinary biomarker / index
values.

Estimation of adiponectin indexes

Although the LMW / total adiponectin ratio and monomer / total
adiponectin ratio were shown to be potentially useful biomarkers
that reflect changes in diabetic kidney disease at an early
ALB
-0.58
0.082 N.S.
stage of eGFR, sample analysis by SEC is time-consuming andlabor-intensive, making it unsuitable for practical use. TheresT-AN-0.54
0.11 N.S.
fore, we investigated the possibility of estimating the values of
iT-AN
-0.54
0.11 N.S.
LMW / total adiponectin ratio and monomer / total adiponectin
ratio from the results of T-AN and H-AN measurements by
sH-AN
-0.47
0.17 N.S.
sandwich EIA and ICT-EIA methods, which are measurements
iH-AN
-0.47
0.17 N.S.
directly obtained from urine samples.
T-AN and H-AN levels were measured by sandwich EIA andsMonomer-0.09
0.80 N.S.
ICT-EIA methods and indexes using the measured values were
calculated. The correlations between those indexes and the
sLMW
-0.67
0.033 p < 0.05
values of target parameters, including LMW / total adiponecsMMW-0.58
0.082 N.S.
tin ratio and monomer / total adiponectin ratio, are shown in
sHMW
-0.45
0.187 N.S.
Table 3. It was difficult to estimate the value of LMW / total
adiponectin ratio using any of the indexes as correlations weresTOTAL-0.58
0.082 N.S.
poor. On the other hand, T-AN and H-AN levels measured by
the sandwich EIA and ICT-EIA methods correlated well withsMonomer / sTOTAL
0.65
0.043 p < 0.05
the monomer / total adiponectin ratio. Furthermore, the ratio of
3. Trend of urinary
dependingon eGFRbystages.
sLMWFig/ sTOTAL-0.67 biomarker/index
0.033 p <values
0.05
T-AN as measured
two assays (sandwich EIA method / ICTEIA method) was newly identified as a potential index whichsMMW / sTOTAL-0.39
0.26 N.S.
correlates with monomer / total adiponectin ratio and reflects thesHMW / sTOTAL-0.09
0.80 N.S.
progression of diabetic kidney disease.
Biomarker / Indexrps : Measured by sandwich EIA, i : Measured by ICT-EIA. ALB : Albumin, T-AN : Total adiponectin, H-AN : High molecular weightadiponectin, LMW : Low molecular weight, MMW : Middle molecular weight, HMW : High molecular weight. Spearman’s correlation coefficients were calculated. Due to the small sample size, thep-values may not be accurate, but they are provided for reference.

Early biomarker for diabetic kidney disease
Urine samples collected from a total of 77 diabetic patients(G1 : 9, G2 : 43, G3a : 14, G3b : 8, G4 : 3) were tested using bothsandwich EIAs and ICT-EIAs for T-AN and H-AN for a total of
4 measures. Adiponectin assay results and indexes were plottedfor each eGFR stage (Fig 4) along with the levels of albumin, aconventional biomarker.
For T-AN, neither the sandwich EIA values nor the ICT-EIA
values were significantly elevated compared to the G1 stage. The

Fig 2. HMW, MMW, LMW and monomer of adiponectin in urine from patients with G1-G4 stages ofdiabetic kidney disease.
T. Kawano, et al. Urinary adiponectin as a biomarker for DKD
468

Fig 3. Trend of urinary biomarker / index values depending on eGFR stages.

s : Measured
by sandwich
EIA, i : Measured
by ICT-EIA. ALB: Albumin,
T-AN : Total
adiponectin,
H-AN : High molecular weight adiponectin,
Fig 3. Trendof urinary
biomarker/index
valuesdependingon eGFR
stages.
LMW : Low molecular weight, Mono : Monomer.

Table 3. Correlation between urinary biomarker / index values, LMWs/TOTALs andMonomers / TOTALs
Taget parameter

Biomarker / Index

sLMW / sTOTAL

sMonomer / sTOTAL

r

p

sT-AN

0.25

0.49 N.S.

iT-AN

0.26

0.47 N.S.

sT-AN / iT-AN-0.18

0.63 N.S.

sH-AN

0.16

0.65 N.S.

iH-AN

0.16

0.65 N.S.

sH-AN / iH-AN

0.067

0.85 N.S.

sT-AN / sH-AN

0.055

0.88 N.S.

sT-AN / iH-AN

0.018

0.96 N.S.

iT-AN / sH-AN

0.14

0.70 N.S.

iT-AN / iH-AN

0.055

0.88 N.S.

sT-AN

-0.93

0.00010 p < 0.001

iT-AN

-0.89

0.00050 p < 0.001

sT-AN / iT-AN

0.94< 0.0001 p < 0.0001

sH-AN

-0.90

0.00030 p < 0.001

iH-AN

-0.90

0.00030 p < 0.001

sH-AN / iH-AN

0.36

0.31 N.S.

sT-AN / sH-AN

0.81

0.0049 p < 0.01

sT-AN / iH-AN

0.79

0.0061 p < 0.01

iT-AN / sH-AN

0.73

0.016 p < 0.05

iT-AN / iH-AN

0.75

0.013 p < 0.05

s : Measured by sandwich EIA, i : Measured by ICT-EIA. ALB : Albumin, T-AN : Total
adiponectin, H-AN : High molecular weight adiponectin. Spearman’s correlation coefficients were calculated. Due to the small sample size, the p-values may not be accurate,
but they are provided for reference.

The Journal of Medical Investigation Vol. 70 August 2023

469

urinary biomarker/ indexvalues and eGFR stages.
Fig 4. Levels
Fig 4.ofLevelsof urinary
biomarker/index
values and eGFR stages.

s : Measured by sandwich EIA, i : Measured by ICT-EIA. ALB : Albumin, T-AN : Total adiponectin, H-AN : High molecular weight adiponectin.
* : p < 0.05 vs. G1 stage by Steel-Dwass test.

H-AN levels as measured by sandwich EIA and ICT-EIA methods as well as albumin levels showed a significant upward trend
in the G3b stage compared to the G1 stage.
Against these trends, the ratio of T-AN as measured by sandwich EIA / ICT-EIA methods (sT-AN / iT-AN), which was newly
shown to be a potential indicator of diabetic kidney disease progression, showed a significant decrease at stage G3a comparedto the G1 stage.

DISCUSSION
We previously reported that T-AN and H-AN assays by an
ICT-EIA method may be useful as biomarkers for diabetic kidneydisease (9-11). In this study, we also developed sandwich EIAs
for T-AN and H-AN and compared their reactivity with that ofthe ICT-EIAs. For each of the T-AN and H-AN assays, the same
antibody pair was used in both the sandwich EIA and the ICTEIA assay, only the assay method was different.
For the T-AN assays, the ICT-EIA method proportionallydetected higher molecular weight adiponectin species comparedto the sandwich EIA method. This is observed more easily in theanalysis of the urine sample. The adiponectin standard looked
similar between sandwich-EIA and ICT-EIA methods.
For the H-AN assays, the ICT-EIA method exclusively detected higher molecular weight adiponectin species compared to thesandwich EIA method, which also detected some MMW species.
This is observed in the analysis of both the urine sample and theadiponectin standard.
This difference in reactivity is likely due to the difference inassay method. In the ICT-EIA method, it has been found thatafter the first wash, dissociation of the antibody / adiponectin
complex occurs due to the absence of excess antibody reagent in
the reaction buffer (12). In this process of dissociation, it is possible that lower molecular weight forms are more prone to dissociate compared to higher molecular weight forms of adiponectin,

resulting in the observed reactivity profile.
Next, we examined the abundance of different forms of adiponectin in the urine of diabetic patients using the T-AN sandwich EIA, which is capable of detecting all adiponectin forms,
ranging from monomeric to HMW forms, at the highest efficiency (Fig 2). In a previous report, we performed a similar analysis
using the ICT-EIA (9, 10) and observed more HMW adiponectinin patients with advanced eGFR stages. In the present study, we
similarly found that urine from patients with advanced eGFR
stage contained more high-molecular-weight adiponectin. For
more detailed analysis, urine samples (10 samples in total) fromsubjects selected at the 25th and 75th percentile of each eGFR
stage (G1, G2, G3a, G3b, and G4) were size-fractionated by
SEC before testing of fractions by T-AN and H-AN assays. The
amounts of monomeric, LMW, MMW, and HMW adiponectin
were measured and the ratio of monomeric, LMW, MMW, and
HMW to total adiponectin was calculated. Correlation of thecalculated adiponectin indexes (ratios) between eGFR stage
and the LMW measure, the LMW / total adiponectin ratio andthe monomer / total adiponectin ratio showed relatively strong
correlation (Table 2). Among them, the LMW / total adiponectin
ratio and the monomer / total adiponectin ratio demonstrated alinear correlation with eGFR stage over the entire dataset (Fig3). Urine collected from patients with relatively early eGFR
stages contained mainly monomer and LMW adiponectin, andas diabetic kidney disease progressed, MMW and HMW adiponectin became more abundant, suggesting that the relatively
low-molecular-weight adiponectin in urine may reflect changes
caused during earlier stages of kidney disease.
However, the calculation of LMW / total adiponectin ratio andmonomer / total adiponectin ratio requires size fractionation by
SEC, which is time-consuming and labor-intensive, making it
unsuitable as a clinical test. Therefore, we investigated a method
to estimate these ratios. Results of the T-AN and H-AN assays bysandwich EIA and ICT-EIA methods were used to calculate adiponectin indexes. The correlation between the target parameters

470

T. Kawano, et al. Urinary adiponectin as a biomarker for DKD(LMW / total adiponectin and monomer / total adiponectin) andthe adiponectin indexes representing results measured fromdifferent assay methods were examined (Table 3). As a result,it was difficult to estimate the LMW / total adiponectin ratiousing any of the assays and indexes, but several indexes showed
relatively strong correlation with the monomer / total adiponectin
ratio, among which the ratio of T-AN by sandwich EIA / ICT-EIA
may be useful as a new index of diabetic kidney disease.
Finally, we examined the usefulness of the ratio of T-AN as
measured by sandwich EIA / ICT-EIA methods as a new indicator of diabetic kidney disease. All other biomarkers showedsignificant changes starting at stage G3b compared to the G1stage, while the ratio of T-AN by sandwich EIA and ICT-EIA(sT-AN / iT-AN) methods showed significant differences at stage
G3a.
In the present study, we found that adiponectin in the urine ofpatients with diabetic kidney disease increased from monomer
and LMW to high molecular weight adiponectin (MMW and
HMW) as the disease progressed. Also, the LMW / total adiponectin ratio and monomer / total adiponectin ratio were foundto strongly correlate with eGFR stage in a linear fashion acrossall stages of disease. The monomer / total adiponectin ratio canbe estimated by calculating the ratio of T-AN as measured bysandwich EIA and ICT-EIA as sT-AN / iT-AN, indicating that
this index may be useful as a biomarker for early diabetic kidneydisease.
The limitations of this study are the lack of comparison withhealthy individuals, limited clinical data, cross-sectional studies
and the lack of accurate diagnosis of diabetic nephropathy that
may be done by biopsies. At this time, the mechanism of adiponectin accumulation in urine is not fully understood. Further
studies are needed to validate the biological significance of urinary adiponectin variability and whether it brings new significance compared to the eGFR currently used in clinical practice.
In the future, the usefulness of urinary adiponectin levels as abiomarker for DKD should be tested in a larger prospective study
currently underway.

ACKNOWLEDGEMENTS
The authors thank the following laboratory researcher : YumikoHisabae.

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