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Introduction
The MDR1 (multidrug resistance gene encoding for
P-gp) gene product P-glycoprotein (ABCB1) is a membrane
protein, which functions as an ATP-dependent exporter of
xenobiotics from cells. P-glycoprotein is expressed in
normal tissues with excretory function such as the intestine,
liver and kidneys, in capillary endothelial cells of brain,
placenta, and testis and in peripheral blood
cells[1]. In kidneys, P-glycoprotein is expressed in the brush border membrane
of proximal tubular cells[1,2]. It mediates active secretion of
its substrates into urine. Renal P-glycoprotein is likely to
function as a protective mechanism against toxic substances
in the glomerular filtrate. Both clinical and experimental
studies have reported the renoprotective effects of removing
uremic toxins by peritoneal dialysis and oral charcoal adsorbent
in delaying the progression of chronic renal
disease[3_7]. Thus, individuals with a low renal P-glycoprotein expression would
potentially be exposed to higher concentrations of toxic
agents and should be more susceptible to their damaging
effects.
Multiple mutations were found in the human
MDR1 gene[8,9]. We selected two single nucleotide polymorphisms (SNP) that
had been previously reported to be associated with the
expression or activity of MDR1. People with mutation C3435T
were associated with a lower P-glycoprotein expression in
the kidneys, compared with subjects homozygous for the
wild-type allele[10,11]. Another SNP G1199A has also been
reported to increase the intracellular accumulation of
rhodamine-123 in vitro[12].
As uremic toxins have been suggested to promote the
progression of chronic renal failure by damaging tubular
cells, based on these observations, we hypothesized that
genetically predisposed subjects carrying T mutation allele
at C3435T or the A mutation allele in G1199A might be at high
risk for developing end-stage renal disease (ESRD).
Therefore, we decided to examine whether
MDR1 is a susceptible gene for renal disease in patients.
Materials and methods
Subjects We studied 244 ESRD patients from the
Division of Nephrology (Ruijin Hospital, Shanghai Jiaotong
University, Shanghai, China), and 284 healthy controls.
Clinical information and biochemical parameters were retrieved
retrospectively from hospital records. The subject
characteristics are presented in Table 1. According to the "Practice
of Internal Medicine"[13], patients with serum creatinine >442
µmol/L were allocated to the ESRD group. Two hundred and
eighty-four healthy patients were randomly selected and used
for comparison with the ESRD patients. The healthy
patients were determined by their medical history, physical
examination, routine blood tests, and
electrocardiography, and had no history of hypertension, diabetes, renal failure,
vascular disease, stroke and cardiomyopathy. This research
was approved by the Ethics Committee of Ruijin Hospital.
Informed consent was obtained from the patients and
controls participating in the study, and the hospital ethical
committee approved the study.
DNA isolation and genotyping analysis Genomic DNA
was obtained from peripheral blood by proteinase K
digestion and phenol-chloroform extraction and ethanol
precipitation. Genotyping of the C3435T polymorphism was
carried out by polymerase chain reaction-restriction
fragment length polymorphism assay according to the method
of Hoffmeyer et al with minor
modifications[9]. Allele specific-polymerase chain reaction (AS-PCR) was used to
deter-mine the genotype of G1199A. The AS-PCR consisted of 2
rounds of PCR. In the first round, 1 µL genomic DNA sample
was added to 25 µL of reaction volume composed of PCR
buffer, 1.5 mmol/L MgCl2, 0.5 unit of Taq polymerase,
0.2 mmol/L dNTP, 6.25 pmol of forward primer, and 6.25 pmol
of reverse primer; 35 cycles were carried out in a GeneAmp
PCR system 2700 (Biocompare, CA, USA). Each cycle
consisted of 30 s at 94 °C for denaturation, 30 s at 54 °C for
annealing, and 30 s at 72 °C for elongation. The second
round was carried out in 2 reaction tubes. Each tube
contained 25 µL of reaction volume composed of PCR buffer,
1.5 mmol/L of MgCl2, 0.5 unit of
Taq polymorphism, 0.2
mmol/L dNTP, and 6.25 pmol of allele-specific primer as the
reverse primer, and the same forward primer as that used in
the first round of PCR. First-round products (20 times
diluted) 1 µL was used as template for the second round.
The second round consisted of 20 cycles performed in the
same GeneAmp PCR system (30 s at 94 °C for denaturing; 30
s at 64 °C for annealing, and 30 s at 72 °C for elongation).
Products (from each second-round reaction tube) in the
volume of 10 µL were then analyzed directly on 1.5% agarose
gel with 0.5 mg/mL of ethidium bromide. The sequence of
the oligonucleotide primers employed in the AS-PCR assays
are depicted in Table 2.
Statistical analyses We used the
Hardy-Weinberg equilibrium for frequency deviation. The comparison of the
allele and genotype frequencies between the different groups
was evaluated by Chi-square test. ANOVA tests were used
to compare genotype groups in terms of clinical and
laboratory characteristics. The SPSS software package version
11.0 (SPSS Inc, Chicago, IL, USA) was used to perform these
statistical analyses with P<0.05 as the minimal level of
statistical significance.
Results
The different genotypic and allele frequency
distributions for MDR1 C3435T in ESRD patients and controls are
shown in Table 3. The genotype distribution was consistent
with the Hardy-Weinberg equilibrium. No significant
difference was observed in genotype frequencies between the
ESRD patients and the control through the Chi-square test
(P=0.573). However, as shown in Figure 1, the level of serum
creatinine was significantly different between carriers with
genotype CC and TT in ESRD patients, although the
difference between the 3 genotypes, 3435CC, 3435CT, and 3435TT
did not reach statistical significance. The value of serum
creatinine for genotype 3435CC, 3435CT, and 3435TT were
753.8±276.0 µmol/L, and 849.6±342.2 µmol/L, 987.0±512.0
µmol/L, respectively. Of the 284 Chinese healthy subjects
and 244 ESRD patients, no variant allele 1199G>A was found.
All subjects were homozygous for 1199GG.
Discussion
To date, 48 SNP have been reported in the MDR1
gene[14]. The single nucleotide polymorphisms 1236C>T,
2677G>T/A, and 3435C>T are the most common variants in the coding
region of ABCB1[10]. 1236C>T and 3435C>T are
synonymous SNP, while the nonsynonymous 2677G>T/A causes
an amino acid substitution (899Ala>Ser/Thr). These 3 SNP
are in strong linkage disequilibrium, accounting for 2
abundant haplotypes (ABCB1*1: 1236C-2677G-3435C; and
ABCB1*13: 1236T-2677T-3435T)[14,16]. So we selected the
polymorphism 3435C>T to represent the other 2 SNP,
1236C>T and 2677G>T/A. It was reported that individuals
homozygous for 3435TT showed significantly lower P-gp
expression in the intestines, liver and kidneys, with increased plasma
levels of the P-gp substrate[10,16,17]. Similar to its protective
role at many biological barriers, P-glycoprotein as a plasma
membrane efflux pump may be involved in the clearance of
toxic compounds via the brush border of the tubular lumen
and is critical in the processes of re-absorption and secretion.
So we postulated that polymorphism C3435T should be
associated with the severity of ESRD, or the frequency of
mutation allele 3435C>T should be higher in ESRD patients
compared with the controls.
In our results, we did not find any difference for the
frequencies of the C3435T genotype and allele between the
ESRD patients and the controls. The frequencies of C3435T
in our study were consistent with other reports, as is shown
in Table 3. However, the level of serum creatinine is higher in
homozygote 3435TT than heterozygote 3435CT and
homozygote 3435CC in ESRD patients. The difference between
3435TT and 3435CC reached statistical significance.
According to the clinical characteristics in our subjects, the
most common causes for ESRD were diabetes mellitus (34%),
hypertension (25%), chronic glomerulonephritis (16%) and
other (25%).The low expression of P-glycoprotein was not
the etiological factor for the kidney disease, but it can
contribute to the progression of ESRD and affect the severity.
The cause of ESRD and interindividual differences in
susceptibility remain elusive. Many studies have recently
focused on this aspect. Kim et al found that SNP and
haplotypes of the SLC12A3 [solute carrier family12 member
(sodium/chloride) 3] gene, especially Arg913Gln, are
significantly associated with ESRD caused by diabetic
nephropathy in the Korean
population[18]. It was reported that the
polymorphism of promoter -511, exon -5+3953 in
IL-β and a variable number of tandem repeats in the interleukin-1
receptor antagonist gene affects the risk of development of
ESRD[19]. Meanwhile, Lamnissou et
al[20] reported that patients with autosomal dominant polycystic kidney disease, who carried
allele A in the nitric oxide synthase (NOS3-4) gene, progressed
to ESRD more quickly. The result from Koupepidou
et al[21] support the hypothesis that Caucasian patients with
essential hypertension, homozygous for 677TT or doubly
heterozygous for 677CT/1298AC genotypes in the
methylenetetra-hydrofolate reductase gene, are predisposed to develop
hypertensive nephrosclerosis and chronic renal failure (CRF).
Since more than 1 single gene was associated with the
progression of ESRD, a study with the multiple linear regression
method is needed in the future.
The substitution of G to A in the position 1199 of
MDR1 results in a serine-to-asparagine substitution at amino acid
400 in a cytoplasmic domain of P-gp. Alteration in the efflux
transport of P-gp owing to the G1199A transition has been
observed in a recombinant expression system. Mean
intracellular R123 fluorescence for
MDR1wt and
MDR1G1199A cells were 3.91±0.11 and 18.56±0.46
(P<0.001), respectively, an approximate 4.75-fold higher accumulation of R123 in
MDR1G1199A cells[12], which meant that G1199A mutation
resulted in the decrease of the transport function of P-gp.
Accordingly, the decrease efflux function of P-gp made the
toxins more easily accumulated in the body, so the individual
with variant 1199G>A was more likely to suffer from ESRD. It
is expected that the frequency of variant allele will be higher
in ESRD patients than in healthy controls. However, no
variant allele was found in our study, neither in healthy
controls nor in ESRD patients. Comparatively, the frequency of
genotype 1199GG, 1199GA, and 1199AA is 88.9%, 11.1%,
and 0 in 461 German volunteers,
respectively[22]. Hoffmeyer et
al reported that the frequency of 1199GA was 12.9% and
1199AA was zero in Caucasians[9], so there is no evidence to
associate 1199G>A with the progression or severity of ESRD.
As the human body is a complex organism, data obtained
from in vitro experiments sometimes cannot be applied to
the human body directly.
In conclusion, we found that SNP of the
MDR1 gene, especially C3435T, were significantly associated with the
severity of ESRD in the Chinese population. Chinese people
do not carry the 1199G>A variant allele. More study is needed
to clarify the cause and interindividual differences in the
susceptibility for the risk of ESRD.
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