ORIGINAL ARTICLE

Urinary Smad1 is a new biomarker for diagnosis and evaluatingthe severity of diabetic nephropathy

Qiao Li • Lie Feng • Jiaying Li • Qianqian Chen

Received: 22 April 2013 / Accepted: 2 August 2013

� Springer Science+Business Media New York 2013

Abstract The aim of this study was to analyze urinary

Smad1 level in patients with type 2 diabetes, explore the

possibility of Smad1 being a biomarker for early diagnosis

and evaluation of severity of diabetic nephropathy, and

explore the impact factors affecting urinary Smad1 con-

centration. In this study, 132 subjects with type 2 diabetes

and 50 healthy volunteers were enrolled. Subjects were

grouped according to urine albumin to creatinine ratio

(ACR) into: normal albumin in urine (NAU), low albumin

in urine (LAU), high albumin in urine (HAU), and very high

albumin in urine (VHAU). Among those, LAU, HAU, and

VHAU were regarded as the diabetic nephropathy group

(DN group), NAU was regarded as nondiabetic nephropathy

(non-DN group), and the healthy volunteers were the con-

trols. Enzyme-linked immunosorbent assay was used to

detect the urinary Smad1 concentration, urinary Smad1 to

creatinine ratio (SCR) was used as the standard reference.

Compared with non-DN group, SCR of DN group was

higher (P \ 0.05), while there was no difference between

the non-DN group and controls (P [ 0.05). There was no

significant difference for SCR between LAU and NAU

groups (P [ 0.05). The SCR was higher in VHAU group

than those in HAU and LAU groups, and higher in HAU

than that in LAU group (P \ 0.05). Pearson correlation

analysis showed that SCR measures were positively

correlated to ACR, duration and diabetic retinopathy of the

disease (r = 0.285, 0.230, 0.202; P = 0.001, 0.008, 0.019,

respectively). Multiple linear regression analysis showed

that ACR and duration were independent impact factors for

SCR (P \ 0.05). This is the first known study examining

the correlation of Smad1 and DN in clinical practice. It

suggested that the urinary Smad1 may be a potential diag-

nostic parameter for DN and may be used to evaluate the

severity of DN. However, it cannot predict those in patients

with the earliest DN and low urine albumin concentration.

Furthermore, ACR and duration may be independent impact

factors for urinary Smad1.

Keywords Urinary Smad1 � Diabetic nephropathy �Biomarker

Introduction

Diabetic nephropathy (DN) is one of the major microvas-

cular complications of diabetes. It is also a major cause of

death in patients with diabetes. It is estimated that patients

with diabetes worldwide will increase from 1.71 hundred

million in 2000 to 4.39 hundred million by 2030, among

whom, 30–40 % of patients with type 2 diabetes (T2DM)

will develop DN [1]. The incidence of DN is high, the

prognosis is dire, and the cost for diagnosis and treatment is

high. It has become an endemic public health issue. Early

diagnosis and intervention, therefore, are urgent. The cur-

rent parameters used for early diagnosis of DN include

microalbuminuria, urinary neutrophil gelatinase-associated

lipocalin [2], Cys-C, tHcy, and urine IgM and IgG measures

[3, 4] as well as kidney biopsy and other procedures. In the

past, microalbuminuria had been the first choice for early

diagnosis of DN. Its sensitivity, specificity, and accuracy in

Q. Li � L. Feng (&) � J. Li � Q. Chen

Department of Endocrinology, The First Affiliated Hospital,

Jinan University, Huangpu Avenue West 613#,

Guangzhou 510632, China

e-mail: liqiao19851226@126.com

Q. Li

Department of Endocrinology, The First Affiliated Hospital,

Traditional Chinese Medicine University of Guangzhou, Airport

Road 16#, Baiyun District, Guangzhou 510405, China

123

Endocrine

DOI 10.1007/s12020-013-0033-9

the early diagnosis of DN have been challenged in recent

studies [5–7]. Other molecules appeared at relative late

stages of DN, and their mechanisms remained unclear.

Although, kidney biopsy is the gold standard for a definite

diagnosis of nephropathy, it is not practical for most

patients with diabetes. The parameters listed above have

flaws for early diagnosis of DN and prediction of diabetic

glomerulosclerosis; therefore, a better, noninvasive bio-

marker for early diagnosis of DN is needed.

A growing number of studies of DN address the effects of

Smads on DN. Smad1 was recognized initially from the

TGF-b/Smads signaling pathway. TGF-b1 is a well-known

fibrosis-inducing factor, highly expressed in glomerular and

epithelium of tubular. Smad protein is an important down-

stream transcription factor of TGF-b, mediating intracellular

signaling transduction of TGF-b/Smads [8–10]. Early kid-

ney changes in DN are matrix accumulation at glomerular

mesangial membrane, which were caused primarily by

extracellular matrix (ECM) accumulation. Collagen IV is the

major component of ECM. Smad1 can up-regulate the

transcription activity of collagen IV [11]. It was suggested

that the BMP4/Smad1 signaling pathway was activated

during DN development, and thus collagen IV was synthe-

sized more with this activation and the matrix accumulation

increased accordingly [12]. It was also believed that AGEs

activated Smad signaling transduction pathway through

TGF-b1 dependent and independent pathways [13], and

further promoted ECM accumulation and consequently lead

to glomerulosclerosis and renal interstitial fibrosis.

In 2004, Abe et al. [11] first suggested that Smad1 can be a

marker for kidney function disorder. Since then, many

investigators studied the effect of glomerular Smad1 and

urinary Smad1 on DN with animal models. It was revealed

that the expression of glomerular Smad1 and urinary Smad1

excretion are positively correlated to the mesangial matrix

accumulation and the degree of glomerulosclerosis, but not

with the urine albumin level. Urinary Smad1 can be used as a

new noninvasive parameter for early diagnosis of DN and for

prediction of the development of kidney morphology in the

late stage [14–17]. These previous studies, however, were

limited to animal experiments and lack of clinical data.

Herein, we initiated this clinical trial on urinary Smad1 and

DN. In this study, by observing the urinary Smad1 excretion

in patients with diabetes, we analyzed the changes in urinary

Smad1 and further explored the relationship of urinary

Smad1 with the onset and development of DN.

Materials and methods

A total of 132 subjects with definite type 2 diabetes, were

enrolled from those treated at the Department of Endocri-

nology at the First Affiliated Hospital of Jinan University

between May 2011 and May 2012. Sixty-four were men

(48 %) and 68 were women (52 %); age ranged from 31 to

80 years, with a mean age of 590.05 ± 11.96 years. Dura-

tion of disease was 5–20 years with an average of

8.47 ± 3.91 years. Another 50 healthy volunteers were

enrolled from the check-up center at the same hospital,

including 6 men (52 %) and 24 women (48 %), ranging in

age from 31 to 80 years, with an average age of

56.60 ± 6.40 years. The WHO 1999 diabetes diagnosis

criteria was used to diagnose diabetes [18], and diabetic

retinopathy (DR) was diagnosed by professional ophthal-

mologist with fundus fluorescence angiography and non-

mydriatic fundus camera check according to the Interna-

tional Clinical Grading criteria [19]. The 2009 NFK and

FDA seminar-defined urine albumin grading system [20]

was used to diagnose diabetic nephropathy (normal: ACR

\10 mg/g; low: ACR 10–29 mg/g; high: ACR 30–300 mg/g;

and very high: ACR[300 mg/g). Diagnostic criteria of early

DN in this study: those diagnosed with T2DM, those having a

duration over 5 years, those with retinal pathological changes,

those with increased number of proteinuria after being ruled

out for other causes, among three times of ACR tests within

3–6 months period, there were at least twice[10–29 mg/g. If

the diagnosis is still not definite, renal biopsy was performed

for final diagnosis. In this study, low albuminuria (ACR:

10–29 mg/g) will be used for diagnosing early diabetic

nephropathy. This is different from the previous diagnostic

criteria (ACR: 30–300 mg/g). Excluded from the study were

patients younger than 30 years and over 80 years; patients

with type 1 diabetes; complications with infection, such as

DKA and others; patients with primary and secondary kidney

disease complications; patients with severe heart, liver, or

brain diseases; patients with severe mental diseases or

inability to comply with the protocol; and the pregnant or

breastfeeding women. This study was approved by the Ethics

Research Committee of the First Affiliated Hospital of Jinan

University.

Methods

Grouping

According to 2009 NFK and FDA seminar-defined urine

ACR grading, patients with type 2 diabetes were grouped

into normal albumin in urine (NAU), low albumin in urine

(LAU), high albumin in urine (HAU), and very high albumin

in urine (VHAU) groups. The LAU, HAU, and VHAU

groups comprised the patients with DN group; patients with

NAU comprised the non-DN group; and the healthy volun-

teers were the controls. This is an observational study, not a

prospective, randomized, double-blind, or controlled study.

Most of the diabetic patients were on ARB or ACEI drugs.

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123

One week before being enrolled into the study, all the sub-

jects were asked to discontinue their ARB or ACEIs.

Parameters

Parameters included urinary Smad1, urine albumin protein,

urine creatinine, C, FPG, serum lipids, liver function test,

BUN, Cr, urine acid, retinal fundus examination, and

ultrasonography of bilateral kidneys.

Test of urinary Smad1 (human signal transduction

molecule 1)

The first urine in the morning was collected from the

subjects in sterile tubes, centrifuged at room temperature at

3,000 r/min for 20 min, and the supernatant collected and

stored at -80 �C. The specimens then were thawed rapidly

before use and centrifuged to remove urine acid or phos-

phate components. Enzyme-linked immunoadsorbent assay

(ELISA) was used to measure urinary Smad1 concentration

quantitatively. All ELISA procedures were performed in a

96-well plate (R&D Inc, USA). Standards, testing samples,

and blank controls were set up on the enzyme-labeled

plate.

Human Smad1 standard solution (Santa Cruz) was

diluted with standard sample into 1, 2, 4, 8, and 12 pg/mL.

Forty microliter sample dilution was added into the testing

samples pre-embedded human monoclonal antibody for

Smad1 (Santa Cruz) and urine sample 20 lL was added (no

urine sample or enzyme-labeled solution was added to the

blank). The final dilution of urine sample was three times.

Samples were incubated at 37 �C for 60 min and rinsed

five times with concentrated solution (DW by 30 times

dilution), and patted dry. Next, HRP-labeled goat anti-

human antibody (EarthOx, USA) 50 lL was added to each

well, incubated at 37 �C for 60 min, rinsed five times, and

patted dry. After complete rinsing, TMB substrate solution

was added and the mixture was incubated at 37 �C for

30 min, in dark. The enzyme reaction was then terminated

by adding 50 lL of 2 N H2SO4. Within 15 min after

adding the terminal solution, reading was adjusted to zero

according to the blank well, and the microplate reader

(Bio-Rad Inc., USA, model 680) with wavelength of

450 nm was used. Measure the OD value for each well in

order. Each Smad1 determination was carried out in

duplicate. The calibration curve was obtained and was used

to calculate the corresponding Smad1 concentration

(Fig. 1). Assays for Smad1 demonstrated near linearity

with the squared correlation coefficient R2 = 0.989.

The inter- and intra-assay CV were 9 and 11 %, respec-

tively. To account for the influence of urinary dilution,

urinary Smad1 was normalized to the urinary creatinine

concentration.

Statistical analysis

All data were analyzed using SPSS13.0. If the SCR results

were not normally distributed, the data were log-trans-

formed into normal distribution. ANOVA was used to

compare the mean of multiple variables, and rate com-

parison of multiple variables was analyzed using v2 test.

The impact factor screening was performed with multiple

linear regressions, using ROC curve to evaluate the spec-

ificity and sensitivity of the diagnosis tests. Data were

presented as mean ± SD �x� sð Þ. P \ 0.05 is considered

statistically significant.

Results

Patients with type 2 diabetes were comparable with the

control subject for age, sex, body mass index (BMI), SBP,

BUN, and Cr at baseline (P [ 0.05). Compared with the

healthy control subjects, patients with type 2 diabetes had

higher FPG, HbA1c, ACR, and Cys-c values (P \ 0.05)

(Table 1).

Compared with the non-DN group, the DN group had

higher SCR (P \ 0.05). There was no difference between

the non-DN group and control subjects (P [ 0.05)

(Table 2).

The ROC value for the subjects was 0.712, suggesting

that SCR is valuable for the diagnosis of DN. The cutoff of

C0.75 has high sensitivity (70.7 %) and specificity

(60.0 %).

The SCR values were different among all groups, sta-

tistically significant at P \ 0.05. There was no difference

for SCR between the LAU and NAU groups (P [ 0.05),

and SCR was higher in the VHAU group than those in the

121086420

Smad1 concentration (ng/mg)

2.000

1.500

1.000

0.500

0.000

OD

Fig. 1 Urinary concentration of Smad1. Standard curve of ELISA.

The intra-assay and inter-assay coefficient variations for this assay

were 9 and 11 %, respectively

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123

HAU and LAU groups, higher in the HAU group than that

in the LAU group (statistically significant at P \ 0.05)

(Table 3).

Pearson’s correlation analysis among FPG, HbA1c,

ACR, Cys-c, and duration was performed. It showed that

SCR is linearly correlated to ACR (r = 0.285; P = 0.001),

duration (r = 0.230; P = 0.008), and DR (r = 0.202;

P = 0.019). With FPG, HbA1c, ACR, Cys-c, and duration

as independent variables and SCR as the dependent vari-

able, multiple linear regression analysis was performed.

Results showed that both ACR and duration are indepen-

dent impact factors (P \ 0.05) (Table 4).

Discussion

This is an early initiated clinical study on urinary Smad1

and DN. By using ELISA, we tested the urinary Smad1

level in the enrolled subjects, analyzed the changes of

urinary Smad1 concentrations in all groups, and thus

explored the relationship between Smad1 and the onset and

development of DN.

The origin of urinary Smad1 remains controversial.

Some investigators believed that urinary Smad1 is derived

from mesangial cells [17], others believed that urinary

Smad1 is secreted by endothelium [21] or kidney

Table 1 Clinical characteristics in DN, non-DN, and control groups

Characteristics DN Non-DN Control P

n 82 50 50

Age (years) 59.76 ± 11.40 57.88 ± 12.87 58.43 ± 10.70 0.214

Gender (men/women) 40/42 24/26 26/24 0.911

BMI (kg/m2) 23.46 ± 4.10 23.35 ± 3.07 23.79 ± 3.17 0.810

Diabetes duration (years) 9.44 ± 4.31a 6.88 ± 2.45 0.000

FPG (mmol/L) 8.89 ± 3.20a,b 7.42 ± 2.33a 50.05 ± 0.64 0.000

HbA1c (%) 9.58 ± 2.72a 9.01 ± 2.77a 5.52 ± 0.24 0.000

SBP (mm/Hg) 118.72 ± 8.92 118.96 ± 9.60 117.06 ± 7.98 0.475

BUN (mmol/L) 5.29 ± 2.31 4.98 ± 1.36 4.56 ± 1.06 0.082

Cr (lmol/L) 63.23 ± 24.77 65.30 ± 16.03 70.80 ± 13.16 0.105

Cys-c (mg/L) 1.11 ± 0.35a,b 0.99 ± 0.26a 0.82 ± 0.13 0.000

ACR (mg/g) 117.02 ± 310.43a 5.50 ± 1.63 10.30 ± 8.21 0.003

Data expressed as mean ± SD

FPG fasting plasma glucose, BMI body mass index, SBP systolic blood pressure, BUN blood urea nitrogen, Cr creatinine, Cys-C cystatin C, ACR

albumin–creatinine ratioa P \ 0.05 versus controlb P \ 0.05 versus non-DN

Table 2 Comparison of the contents of SCR in control, non-DN, and

DN groups

Groups SCR (ng/mg)

Control (n = 50) 0.66 ± 0.22

Non-DN (n = 50) 0.70 ± 0.25

DN (n = 82) 0.90 ± 0.31a

Data expressed as mean ± SDa P \ 0.05 versus control

Table 3 Comparison of the contents of SCR in patients with diabetes

in each group

Group SCR (ng/mg)

NAU (n = 50) 0.70 ± 0.25

LAU (n = 33) 0.76 ± 0.24

HAU (n = 43) 0.97 ± 0.31a,b

VHAU (n = 6) 1.17 ± 0.42a,b,c

Data expressed as mean ± SD

HAU high-grade albuminuria group, VHAU very high-grade albu-

minuria groupa P \ 0.05 versus NAUb P \ 0.05 versus LAUc P \ 0.05 versus HAU

Table 4 Pearson correlation analysis and multiple stepwise regres-

sion analysis of variables associated with urinary Smad1 in T2DM

group (n = 132)

Variable Correlation

coefficient

(r)

P value Regression

coefficient

(b)

P value

ACR 0.285 0.001 0.279 0.001

Diabetes duration 0.230 0.008 0.244 0.003

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podocytes [22]. Still others believed that urinary Smad1

derives from the blood but is excreted in urine, but this

theory is rejected because it is undetectable in blood using

ELISA.

In previous study, urinary Smad1 was detected with

Western blotting in mice, who had glomerular matrix

accumulation. The concentration of urinary Smad1, how-

ever, is very low and cannot be quantitated using the

Western blotting technique. ELISA can be used to detect

antigen. Due to its high catalytic efficacy, it indirectly

amplified the results of immunology reaction tests, and thus

enhanced the sensitivity of the test. It was found, however,

that the urinary Smad1 concentration in mice was lower

than that detectable using ELISA [14]. Matsubara et al.

[14] used a modified ELISA test, adding urine condensing

apparatus (Amicon centrifuge ultra-filter) to detect urinary

Smad1 in diabetic mice. Then, someone found urinary

Smad1 in human patients with diabetes in the same way

[23]. In this study, we used the standard ELISA test and

found that urinary Smad1 was detectable both in patients

with type 2 diabetes and in healthy volunteers, although the

concentrations were low. This may be attributable to the

differences among species or to the high sensitivity of the

human kit used, which made urinary Smad1 detectable

even using standard ELISA.

From the previous studies, it is believed that there was

no expression of Smad1 in normal glomeruli of adult mice

[24], but in AGEs stimulating glomeruli [11]. Our study

showed that urinary Smad1 was detectable both in patients

with type 2 diabetes and in healthy populations. These

inconsistent results may be due to that, in the previous

studies, the DN animal models were STZ-induced, which

represented type 1 diabetes, whereas in our study, we tested

that in type 2 diabetes. Further study is warranted if genetic

factors and species differences have some effect on the

development of DN and Smad1 expression. Although,

Smad1 was detected in the urine of all population, the

concentration was higher in the DN group than those in the

non-DN group and control subjects. Moreover, in this

study, it is found that the ROC curve was very valuable for

diagnosis of DN (e.g., usefulness for diagnosis: low

ROC B 0.7; medium ROC C 0.7 to B0.9; and high

ROC C 0.9). This suggested that urinary Smad1 can be

used as a diagnosis marker for DN. In this study, however,

ROC was derived from ACR, not the result of kidney

biopsy, the gold standard, and thus there may be some bias.

Currently, the staging method for type 2 DN was pro-

posed by Mogensen [25]; in 1989, the staging method used

was for type 1 DN. However, recent studies [26, 27] reveal

that due to the epithelia structure disorder, when urine

albumin is below 30 mg/g, GFR starts to drop, and the

risk of kidney and cardiovascular injuries consequently

increases. Therefore, according to the discussion at 2009

NFK and FDA seminar [20], the ‘‘low albumin creatinine

ratio (ACR 10–29 mg/d or 10–29 mg/g)’’ definition was

added up to the original concept. To draw attention to ‘‘low

albumin urine,’’ and to improve the sensitivity of albumin

urine for early diagnosis of DN, in this study, we took ACR

10–29 mg/g ‘‘low albumin urine’’ as early DN, which

differs from Mogensen staging. In the previous animal

experiment, the pathology study revealed that the expres-

sion of glomerular Smad1 and the excretion of urinary

Smad1 are positively correlated to the degree of early

glomerular mesangial matrix accumulation in DN, but not

with albumin level in urine. Therefore, it was believed that

urinary Smad1 can be used for early diagnosis of DN, and

it is a better marker than albumin urine [14, 15]. In this

study, it is found that for the patients with low ACR

(10–29 mg/g) at the early stage of DN, urinary Smad1 is

not different from that of the albumin urine in healthy

patients (no difference in urinary Smad1 in LAU and

NAU). This suggested that urinary Smad1 cannot be used

to predict early DN, and albumin urine seems more sen-

sitive for the early diagnosis of DN. These inconsistent

results may be because urinary Smad1 tends to reflect the

morphology changes of kidney DN, whereas albumin urine

mainly reflects the changes of kidney function in DN. In

addition, changes in morphology and function may not be

consistent [14]. This may be due to the bias of the small

sample size. It is worth note that, in this study, the con-

clusion that urinary Smad1 cannot be used to predict early

DN based on the fact that LAU (ACR: 10–29 mg/g) was

used as the criterion for the early diagnosis of DN. If

Mogensen staging was used, the conclusion would differ

(data to be published). This result suggested that the re-

grading of albumin urine at the 2009 NFK and FDA

seminar may not be a fit for new staging criteria of DN

from another aspect. Furthermore, in one study [17],

1.74 ng/mL was used as the median of urinary Smad1 in

the DN animal model, [1.74 ng/mL is regarded as high,

and \1.74 ng/mL as low. In the early stages of DN, when

the changes of kidney structure were not significant, the

expression of glomerular Smad1 in mice with high con-

centrations of urinary Smad1 is higher than that in mice

with low concentrations. By using the median, DN can be

detected even earlier. In this study, by using ROC curve, it

was found that urinary Smad1 C0.75 (actual concentration:

C5.64 ng/mg) is the cutoff for the diagnosis of DN because

both sensitivity(70.7 %) and the specificity (60.0 %) are

high. Therefore, 5.64 ng/mg can be used as the best cutoff

for urinary Smad1 in the diagnosis of DN.

The previous animal pathology study revealed that uri-

nary Smad1 concentration at the early stage of DN can be

used to predict the severe degree of glomerulosclerosis at

the late stage. In this study, urinary Smad1 in the VHAU

group was higher than that in the HAU and LAU groups,

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123

and that in the HAU group was higher than that in the LAU

group. With the development of DN, urinary Smad1 con-

centration increases with albumin urine, suggesting that

urinary Smad1 concentration can be used to evaluate the

severity of DN. In this study, we neither did follow-up on

subjects nor performed kidney biopsy. If urinary Smad1

can be used to predict the severity in degree of glomeru-

losclerosis, further study is warranted.

In this study, we found that urinary Smad1 can be used as a

parameter for diagnosis of DN. It is very important for the

prevention and treatment of DN to further understand the

impact factors of urinary Smad1. In this study, Pearson

correlation revealed that ACR and duration may be the

impact factors of urinary Smad1. Multiple linear regression

analysis results further suggested that ACR and duration may

be independent impact factors for urinary Smad1 after other

confounding factors have been excluded. It is well known

that ACR indicates kidney injury, whereas urinary Smad1 is

correlated to glomerular mesangial matrix accumulation.

With worsening kidney injury, matrix accumulation

increases, and ACR and the excretion of urinary Smad1

increases. The previous animal model study suggested that

urinary Smad1 is not related to albumin urine [17]. The

inconsistent results may be due to a small sample size in this

study, as well as missing data, and thus resulting in bias.

Furthermore, species differences and misinterpretation of

creatinine for albumin urine in animal experiment play some

role as well. In this study, we also realized that urinary

Smad1 increases with the duration of diabetes, and thus

increases the kidney injury. In this study, it showed a close

correlation between the DR and urinary Smad1. With the

progress of the DR, the levels of urinary Smad1 increases.

The DR and DN are part of the microvascular complications

of type 2 diabetes. This further explains why urinary Smad1

can be used to assess the severity of the microvascular lesions

such as DN and DR. It is worth to note that FPG and HbA1c

were not correlated with urinary Smad1 in our study.

Some previous studies revealed that captopril signifi-

cantly reduces microalbuminuria induced by exercise in

normotensive diabetics without affecting systemic blood

pressure [28]. Furthermore, Picotamide, a TXB synthetase

and receptor inhibitor, may decrease exercise-induced

albuminuria in diabetic patients through reducing circu-

lating TXB levels and inhibiting TXB action, which in turn

may lower glomerular capillary hydraulic pressure [29].

Recent DN animal model studies revealed that ARB drugs

can affect the excretion of urinary Smad1 [16, 17]. In the

early stage of diabetic nephropathy, angiotensin can regu-

late the development of mesangial matrix accumulation.

ARBs can block its effect, and thus decrease urinary

Smad1 concentration, delay the development of DN, and

improve kidney function. This protective effect of ARB on

the kidney is dose dependent and not correlated to the

decrease of blood pressure [30]. Numerous large clinical

trials also confirmed that the protective effect of ARB on

the kidney is not only attributed to its blood pressure-

lowering effect [31]. Compared with placebo and other

blood pressure-lowering drugs, ARBs improve hemody-

namic, decrease oxidative stress, and decrease pathology

injury. In this study, all subjects discontinued their ARB

and ACEIs 1 week before participating the study. Thus,

urinary Smad1 was not interfered by the ARB or ACEIs.

This is the first known clinical study suggesting that

urinary Smad1 is a new, promising biomarker for the

diagnosis and evaluation of the severity of DN. In the near

future, we will explore the relationship of urinary Smad1,

kidney pathology changes of DN, and RAS, to investigate

the cutoff of urinary Smad1 for the diagnosis of DN within

the reference range of the healthy population, to block the

signaling pathway of Smad1 and control blood glucose

effectively, as well as to suggest new strategies for the

treatment of DN. This is an early clinical study exploring

the relationship of urinary Smad1 and DN, and will serve

as a reference for future studies. However, it is a cross-

sectional study, and its testing method is different from

those used in previous studies. So, it may present bias in

the outcomes of this study. The value of urinary Smad1 in

the diagnosis of DN warrants additional study. Currently,

the follow-up study of patients are going on, and we hope it

will bring you new surprises in the near future.

Conflict of interest The authors declared no conflict of interest.

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