Urinary Podocyte-Associated mRNA profile in Various Stages of Diabetic Nephropathy
Citation: Zheng M, Lv L-L, Ni J, Ni H-F, Li Q, et al. (
Urinary Podocyte-Associated mRNA profile in Various Stages of Diabetic Nephropathy
Min Zheng 0 1
Lin-Li Lv 0 1
Jie Ni 0 1
Hai-Feng Ni 0 1
Qing Li 0 1
Kun-Ling Ma 0 1
Bi-Cheng Liu 0 1
Jean-Claude Dussaule, INSERM, France
0 Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine , Nanjing , China
1 We thank Mr Dai Hou-Yong (Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine) for his work in collecting samples
Background: Podocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We hypothesized that the urinary mRNA profile of podocyte-associated molecules may provide important clinical insight into the different stages of diabetic nephropathy. Methods: DN patients (N = 51) and healthy controls (N = 13) were enrolled in this study. DN patients were divided into a normoalbuminuria group (UAE,30 mg/g, n = 17), a microalbuminuria group (UAE 30,300 mg/g, n = 15), and a macroalbuminuria group (UAE.300 mg/g, n = 19), according to their urinary albumin excretion (UAE). Relative mRNA abundance of synaptopodin, podocalyxin, CD2-AP, a-actin4, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined. Results: The urinary mRNA levels of all genes studied were significantly higher in the DN group compared with controls (p,0.05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and BUN. The expression of podocalyxin, CD2-AP, a-actin4, and podocin mRNA correlated with serum creatinine (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021; r = 0.357, p = 0.006, respectively). Furthermore, podocalyxin mRNA was found to negatively correlate with eGFR (r = 20.349, p = 0.01). Conclusion: The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, a-actin4, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN.
Diabetic nephropathy (DN) is now the leading cause of
endstage renal disease (ESRD) in patients beginning renal dialysis in
the United States, and this trend is extending to developing
countries as well [1,2]. The pathogenesis of DN is complex and
has not yet been fully elucidated. Recent studies have shown
that renal podocyte injury is pathogenically and prognostically
important in DN progression. The potential mechanisms of
podocyte injury include foot process effacement, hypertrophy,
detachment, apoptosis, and perhaps epithelial-to-mesenchymal
transition (EMT), and these mechanisms are believed to be
associated with the onset and progression of DN .
Accumulating evidence suggests that podocyte-associated proteins or genes
may correlate with proteinuria and renal function . These
findings bring up the interesting possibility that screening for
podocyte-related molecules might be a novel strategy in
monitoring the progression of DN.
Currently, renal pathological examination is the gold standard
for evaluating podocytopathy in DN. However, due to the invasive
nature of renal biopsy, it is impractical for physicians to closely
monitor patients using this method. As a technique, real-time PCR
has the benefits of excellent sensitivity, quantification, and
reproducibility, and it is able to measure low-abundance genes
from even one single cell . In recent years, with the
development of reliable RNA extraction methods from urinary
sediment and real-time quantitative PCR applications, the
quantification of mRNA expression in urinary sediment has
become an emerging modality for studying renal pathology. Some
preliminary studies suggest that the determination of urinary
mRNA levels might be valuable in monitoring the progression of
renal disease . However, the exact clinical relevance of
urinary mRNA levels remained to be determined. In this study,
the expression of podocyte-associated genes in urinary sediment
and their relation to disease severity were investigated in patients
The primary clinical and laboratory characteristics of the study
subjects are summarized in Table 1. The age of participants in the
#Macroalbuminuria, Microalbuminuria, and Normoalbuminuria vs. Healthy controls, p,0.05.
*Macroalbuminuria vs. Microalbuminuria, Normoalbuminuria and Healthy controls, p,0.05.
control group was lower than in the three DN groups (p,0.05),
while the differences among the DN patients were not significant.
The macroalbuminuria group had a significant increase in serum
creatinine and BUN compared with the other three groups
Urinary mRNA expression levels of DN groups and
We first compared the levels of target gene expression between
the DN groups and the healthy controls. Figure 1 summarizes the
expression of the target genes synaptopodin, CD2-AP, a-actin4,
podocin and podocalyxin in urinary sediment from all study
subjects. The gene levels were compared by the log-transformed
ratio of target gene mRNA to b-actin expression in urinary
sediment cells. We found that all five target genes show significantly
higher levels of expression in the experimental groups compared
with the control group (p = 0.007 for synaptopodin, p = 0.047 for
CD2-AP,p = 0.01 for a-actin4, p = 0.006 for podocalyxin, and
p = 0.021 for podocin, respectively, by Mann-Whitney test).
Urinary mRNA profiles and disease severity staged by
The gene expression levels in varying stages of DN (as defined
by extent of albuminuria) compared with healthy controls are
summarized in figure 2. The mRNA levels varied significantly
among the different DN groups and healthy controls. The
expression levels of all five target genes tended to increase with
increasing progression of DN.
Figure 1. Comparison of podocyte-associated mRNA expressions in urinary between DN patients and health controls. Box plots show
the minimum value, 25th, 50th (median), 75th, and the maximum values for lg-transformed ratios of mRNA copies compared with b-actin mRNA
copies for synaptopodin, CD2-AP, a-actin4, podocin, and podocalyxin. Data were compared using the Mann-Whitney U test. DN = Diabetic
Figure 2. Comparison of podocyte-associated mRNA expressions in urinary in varying stages of DN and controls. Box plots show the
the minimum value, 25th, 50th (median), 75th, and the maximum values for lg-transformed ratios of mRNA copies compared with b-actin mRNA
copies for synaptopodin, CD2-AP, a-actin4, podocin and podocalyxin. mRNA expressions among overall four groups were compared using the
Correlation between urinary mRNA expression and
No significant correlations between urinary mRNA expression
of podocyte markers and age were found (r = 0.133, p = 0.289 for
a-actin4, r = 0.018, p = 0.894 for podocin, r = 20.007, p = 0.955
for CD2-AP, r = 0.15, p = 0.279 for podocalyxin, and r = 0.12,
p = 0.351 for synaptopodin using the Spearman rank-order
correlation). The correlations between these markers and clinical
parameters of renal function, such as urinary albumin, BUN,
serum creatinine and eGFR, are summarized in figure 3. In
Figure 3. Relationships between mRNA expression of podocyte-associated molecules in urinary sediment and baseline parameters.
albuminuria (A), serum creatinine (B) and BUN (C). Data were compared using the Spearman correlation coefficient.
general, the urinary mRNA levels of all target molecules were
significantly correlated with urinary albumin (r = 0.478, p,0.001
for a-actin4, r = 0.378, p = 0.003 for podocin, r = 0.402, p = 0.001
for CD2-AP, r = 0.457, p,0.001 for podocalyxin, and r = 0.384,
p = 0.001 for synaptopodin using the Spearman rank-order
correlation). Also, a significant positive correlation was observed
between BUN and urinary a-actin4 mRNA (r = 0.353, p = 0.004),
CD2-AP mRNA (r = 0.303, p = 0.018), podocalyxin mRNA
(r = 0.474, p,0.001) and synaptopodin mRNA levels (r = 0.359,
p = 0.004). Moreover, the expression of podocalyxin, CD2-AP,
aactin4, and podocin mRNA correlated with serum creatinine
levels (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021;
r = 0.357, p = 0.006, respectively). However, eGFR levels did not
significantly relate to the mRNA levels of synaptopodin (r = 20.146,
p = 0.255), CD2-AP (r = 20.209, p = 0.106), a-actin4 (r = 20.227,
p = 0.069), or podocin(r = 20.202, p = 0.127), but did show a
significant correlation with podocalyxin expression (r = 20.349,
p = 0.01).
ROC curve analysis of mRNA levels
ROC curves were calculated to assess the diagnostic power for
each target gene in discriminating between DN patients and healthy
controls. Figure 4 demonstrates the diagnostic performance of
synaptopodin, podocalyxin, CD2-AP, a-actin4 and podocin in
terms of AUCs when DN patients and controls were compared. As
shown in Figure 4, all target genes were effectively able to
discriminate between two groups, with an AUC above 0.5. The
AUC was 0.753 (95% confidence interval, 0.623 to 0.883) for
podocalyxin mRNA levels, which demonstrated the highest
diagnostic value. The log-transformed threshold providing optimal
sensitivity and specificity for podocalyxin mRNA was 23.24. Using
the cutoff value of 23.24 derived from the data, podocalyxin
mRNA levels predicted DN with a sensitivity of 81.4% and a
specificity of 62.5% (p = 0.006).
Podocytes are highly specialized epithelial cells that cover the
outer aspect of the glomerular basement membrane (GBM), playing
a crucial role in the regulation of glomerular function. Both foot
process effacement and a decreased number of podocytes have been
documented to be associated with DN, which is a disease process
that may be characterized as a podocytopathy . An Akita model
of type 1 DN as well as a leptin receptor-deficient db/db mouse
model of type 2 DN demonstrated that diseased podocytes lose
nephrin expression, become effaced, and detach from the GBM or
undergo apoptosis, which all correlate with the appearance of
albuminuria . More recently, we demonstrated that podocytes
presented with phenotypical change in the early stages of DN and
that could be attenuated by irbesartan, an angiotensin II receptor
A reduction in podocyte number and density has also been
linked to proteinuria and progression of disease in patients with
DN. A study of type 1 DN animal model demonstrated reduced
podocyte number , and in another study, patients with renal
biopsy-confirmed type 2 diabetes were confirmed to have a
dramatic decrease in podocyte density . A recent cross-sectional
study also suggested a significant inverse correlation between
proteinuria and podocyte number as well as podocyte density per
glomerulus . Nakamura et al. demonstrated that identification
Figure 4. ROC-curve analysis of mRNA levels. Fraction of true positive results (sensitivity) and false positive results (1-specificity) for mRNA
levels of synaptopodin, CD2-AP, a-actin4, podocin, and podocalyxin as predictors of DN. The calculated area under the curve was 0.726 for
synaptopodin mRNA levels (95% confidence interval 0.589 to 0.864), 0.671 for CD2-AP mRNA levels (95% confidence interval 0.53 to 0.811), 0.725 for
a-actin4 mRNA levels (95% confidence interval 0.582 to 0.867), 0.753 for podocalyxin mRNA levels (95% confidence interval 0.623 to 0.883), and 0.706
for podocin mRNA levels (95% confidence interval 0.559 to 0.853). A value of 0.5 is no better than that expected by chance (the null hypothesis), and
a value of 1.0 reflects a perfect indicator.
of podocytes in the urine may be a useful marker of disease activity
in diabetic nephropathy . However, a drawback to this
modality is that it is time consuming to quantify urinary podocytes
and would require an experienced cytologist. Consequently, it is
important to explore alternative approaches to detect podocyte
injury in order to identify novel biomarkers for DN.
Recently, efforts have focused on urinary gene expression as a
potential approach for identifying biomarkers in patients with
renal diseases. Li et al. demonstrated that acute renal allograft
rejection could be diagnosed by quantification of perforin and
granzyme B mRNA in urinary cells, and Muthukumar et al. found
that mRNA measurements could improve the prediction of acute
rejection outcome[22,23]. Urinary gene expression has also been
explored for monitoring therapeutic response in patients with
lupus nephritis. These studies have implicated urinary mRNA
detection as a means to provide us with a promising technique for
diagnosing kidney disease and assessing disease activity,
progression, and response to therapy .
In this study, we firstly determined the expression of
podocyteassociated genes in the urine of patients with varying stages of DN.
The results suggest that urinary synaptopodin, podocalyxin,
CD2AP, a-actin4, and podocin mRNA were significantly increased in
DN patients compared with healthy controls. Increased levels of
target gene expression were observed for all five podocyte markers,
implying an increased excretion of podocytes into the urine of
patients with DN. In a previous study, specific podocyte markers
were characterized in patients with DN. Wang et al. demonstrated
that urinary nephrin, podocin, synaptopodin, Wilms tumor-1
(WT-1), and a-actin4 were increased in DN patients . In the
present study, we confirmed these findings and identified two more
podocyte markers with high gene expression levels, providing
additional candidate molecules of urinary podocyte biomarkers.
More recently, Su et al. used WT1 as a marker to evaluate
podocyte damage and showed that podocyte number and density
was decreased in patients with early stage of DN, and which
became more dramatic as proteinuria progressed . In this
study, we investigated whether the expression of urinary podocyte
mRNAs correlated with the progression of DN. Patients with DN
were divided into three experimental groups based on their level of
albuminuria. We discovered that the expression levels of all five
molecules directly correlated with extent of albuminuria.
Specifically, mRNA expression increased with the severity of
albuminuria in experimental group, and this was consistent with previous
findings describing the critical role of podocyte injury in the onset
of albuminuria. The shedding of podocytes into urine might, in
fact, be an important contributor in albuminuria. This result,
therefore, directly supports a role for podocyte loss in the
development of albuminuria . To our knowledge, this is the
first study to evaluate changes in urinary podocyte-associated
mRNA levels in different stages of DN patients staged by
albuminuria, a key indicator of progression in DN.
To further analyze the correlation between urinary mRNA
levels and renal functional parameters, we identified a number of
gene markers that significantly correlated with BUN and serum
creatinine levels, among which a-actin4, CD2-AP, podocalyxin
and synaptopodin demonstrated a positive correlation with BUN,
and podocalyxin, CD2-AP, a-actin4, and podocin correlated
positively with serum creatinine. eGFR, on the other hand,
showed an inverse relationship with the expression of podocalyxin
mRNA. It is widely accepted that podocyte injury may trigger a
sequence of events through epithelial-mesenchymal transition and
apoptosis or detachment, to ultimately contribute to
glomerulosclerosis and decline of renal function . Our current study
suggests that the detection of urinary podocyte-associated mRNAs
may provide valuable information for evaluating the progression of
In conclusion, our study demonstrates that the mRNA expression
levels of synaptopodin, podocalyxin, CD2-AP, a-actin4, and
podocin increase with DN progression. Quantification of
podocyte-associated molecules appears to reflect the severity of
albuminuria and renal damage, suggesting that these
podocytespecific genes may be used as biomarkers for DN progression.
Materials and Methods
All studies were approved by the Ethical Committee of
Southeast University. Written informed consents were obtained
from all subjects to use their urine for research purpose.
Patient selection and clinical data
We studied 51 patients with type 2 diabetic nephropathy from
Zhong Da Hospital, Southeast University. Diabetic nephropathy
was diagnosed based on NKF KDOQI guidelines 2007. The
inclusion criteria in this study were: at least 5 years from the
diagnosis of type 2 diabetes, the presence of diabetic retinopathy,
elevated albumin-creatinine ratio (ACR). The exclusion criteria
were: infection, signs or symptoms of other systemic disease, ACEI
or ARB administration in the last 2 weeks, suspected nondiabetic
kidney disease. Patients were divided into three groups based on
extent of urinary albumin excretion (UAE): normoalbuminuria
(UAE,30 mg/g, n = 17), microalbuminuria (UAE 30,300 mg/g,
n = 15), and macroalbuminuria (UAE.300 mg/g, n = 19).
Thirteen healthy controls were from medical examination centre and the
inclusion criteria were: age.40 years, non-hypertensive,
nondiabetes, the absence of clinical or laboratory evidence of kidney
disease. Clinical data including albuminuria, blood urea nitrogen
(BUN), and serum creatinine were recorded at baseline for each of
the groups. GFR was calculated by the equation proposed by Ma et
al, which was considered to be more suitable for Chinese study
Collection of urine samples and total RNA extraction
A whole-stream early-morning urine specimen was collected
from each study participant at the first day when they were
admitted to hospital. Shortly after collection, the urine was
centrifuged at 3,000*g for 30 minutes at 4uC. The urinary
supernatant was discarded, and the remaining cell pellet was
resuspended in 1.5 ml DEPC-treated PBS and was then
centrifuged at 13,000*g for 5 minutes at 4uC . The pellet was
then resuspended in 1.0 ml RNAiso Plus (TAKARA, Dalian,
China) and was stored at 280uC until use. Total RNA was
extracted according to the manufacturers protocol (TAKARA).
All tubes and tips used for RNA extraction were 0.1% DEPC
treated to inhibit the RNase and total RNA was lysised in 10
30 ml 0.1% DEPC-treated ddH2O. We confirmed the integrity of
RNA by running agarose gel, which shown to be adequate for
PCR. The RNA concentration and purity were confirmed using
the relative absorbance ratio at 260/280 on a nanodrop 2000
(Thermo, Wilmington, USA). RNA samples with a ratio higher
than 1.8 were used for RT PCR.
For reverse transcription, 2 mg total RNA was mixed with 8 ml
5X PrimeScriptTM Buffer, 2 ml PrimeScriptTM RT Enzyme MixI,
2 ul Oligo dT Primer (50 mM), 2 ml Random 6 mers (100 mM),
(TAKARA), the solution and was increased to a volume of 40 ml
with dH2O. Reverse transcription was performed at 37uC for 15
minutes, followed by an inactivation reaction at 85uC for 5
seconds. The resulting cDNA was stored at 220uC until use.
In the present study, relative abundance of synaptopodin,
podocalyxin, CD2-ap, and a-actin4, podocin mRNA were
quantified using the ABI Prism 7300 Sequence Detection System
(Applied Biosystems, California, USA). Human b-actin was used
as a reference housekeeping gene. The following oligonucleotide
primer sequences were used: synaptopodin: forward
59CTTACGGCGGTGACATCTC, reverse 59-
GGTCCTGAGCCTCGATCC; podocalyxin: forward 59-
CTTGAGACACAGACACAGAG, reverse 59- CCGTATGCCGCACTTATC;
CD2AP: forward 59- AGGCTGGTGGAGTGGAAC, reverse 59-
CAGAGAAGGTATAGGTGAAGTAGG; a-actin4: 59-
GATGGTCTTGCCTTCAATG, reverse 59-
TGTTCACGATGTCCTCTG ; podocin: forward 59- TGGCTGTGGAGGCTGAAG,
reverse 59- TGAAGGGTGTGGAGGTATCG; b-actin: forward
59- TGGCACCCAGCACAATGAA, reverse 59-
CTAAGTCATAGTCCGCCTAGAAGCA (designed and synthesized by
TAKARA). For real-time PCR, 2 ml cDNA, 10 ml SYBR Premix Ex
TaqTM, 0.4 ml forward primer (10 mM), 0.4 ml reverse primer
(10 mM), 0.4 ml ROX Reference dye (50X; all from TAKARA)
and 6.8 ml dH2O were mixed to make a 20 ml reaction volume. All
samples were run in duplicate. The PCR technique was performed
using a two-step process: 95uC for 30 s, 40 cycles at 95uC for 5 s
and 60uC for 31 s. Then, dissociation curves (DC) and melting
temperatures (Tm) were recorded. The results were analyzed using
Sequence Detection Software version 1.4 (Applied Biosystems).
The relative gene expression of each target was quantified with a
standard curve method. The pre-PCR product of each gene was
used as standard, and the standard curve was established with a
10-fold serial dilution of the product. The standard curve was
included in all PCR runs. The equation of target gene abundance/
housekeeping gene abundance was used to evaluate the level of
expression of each gene. Controls consisting of ddH2O were
negative in all runs.
SPSS 13.0 was used for data analysis. All results are presented as
mean6SD unless otherwise specified. Baseline data were
compared by a one-way analysis of variance (ANOVA) between four
groups. Since gene expression levels were highly skewed, lg
transformation was used before the analysis. We used the b-actin
normalized level as the dependent variable in a Kruskal-Wallis test
to identify the differences between the three DN groups. The
MannWhitney test was used for gene comparison between two
groups. Correlations between gene expression and clinical
parameters (urinary albumin, BUN, serum creatinine, eGFR)
were calculated using the Spearman rank-order correlation. If the
difference between two groups was statistically significant, the
receiver-operating-characteristic (ROC) curves were established
and cutoff points that yielded the highest combined sensitivity and
specificity were calculated. All p-values were two tailed, and a
value ,0.05 was considered to be statistically significant.
Conceived and designed the experiments: B-CL. Performed the
experiments: MZ JN. Analyzed the data: MZ L-LL K-LM. Wrote the paper: MZ
L-LL B-CL. Performed sample collection: MZ JN H-FN QL.
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