The Association Between RhEPO and FN Expression In
Glomerular Mesangial Cells
Shuqin ZHOU, 1，2 Ping WEN, 2 Li FANG, 2 Lei JIANG, 2 Mingxia XIONG, 2 Feifei
ZHANG 2 and Junwei YANG 2＊
1
2
Shanghai Tenth People’s Hospital of Tongji University
JiangSu Provincial Center for Diabetes, Research Center of Nephrology, 2st Affiliated
Hospital with Nanjing Medical University, Nanjing 210003, China
Corresponding author: YANG Jun-wei, E-mail: shuqin4344@126.com
[Abstract] Objective: Erythropoietin (rhEPO) is increasingly being used in the
treatment of anemia caused by miscellaneous reasons. The aim of our research is to
investigate the effects of recombinant human erythropoietin (rhEPO) on cellular growth
and fibronectin (FN) expression in glomerular mesangial cells. Methods: Western blot
was used to detect the expression of FN induced by rhEPO (1, 10, 100 and 1000U/ml). In
vivo studies, male CD-1 mice were administered rhEPO subcutaneously at a single dose
of 1000U/Kg. The cellular hypertrophy was quantified by counting cell number and
calculating the ratio of cell protein to cell number. Results: (1) Compared with the
control group, the results of mesangial cells’ growth stimulated by rhEPO were not
significantly different. (2) RhEPO lead to hypertrophy of mesangial cells. (3) rhEPO
induced FN expression of mesangial cell in a dose-dependent way. Compared to control,
100U/ml rhEPO enhanced the
expression of FN significantly. (4)
Indirect
immunofluorescence showed that rhEPO induced large amount deposition of FN in the
intercellular space of mesangial cells. (5) In vivo studies, there were markedly increase of
FN expression in mice received the injection of rhEPO. Conclusion: RhEPO could
up-regulated the expression of FN and induced glomerular mesangial cells hypertrophy.
These results suggested rhEPO could induce dysfunction of renal glomerulus through its
influence on the function of mesangial cells.
[Key words] rhEPO; Glomerular mesangial cell; Hypertrophy; Fibronectin
Introduction
RhEPO is used not only to correct anemia caused by renal dysfunction, but also is
increasingly widely applied to the treatment of onco-anemia [1-3]. Along with promoting
the use of rhEPO, its effects on structure and function of patients’ kidney have also
received special attention [4-5]. Beside the hematopoietic function, whether other
biological effects of rhEPO could change the structure and function of kidney is very
significant to patients under the treatment of it. Therefore, our research was to investigate
the effects of rhEPO on cellular growth and FN expression in glomerular mesangial cells
in vivo and vitro, so as to discuss the biological effects which may caused by rhEPO to
renal units.
Materials and Methods
1. Reagent
Recombinant human erythropoietin- (rhEPO-, benefits Biot, 10000 IU / branches
Zhunzi S20010001) was purchased from Sansei Pharmaceuticals (Shenyang, China),
anti-FN and anti-actin antibodies were purchased from Santa Cruz (USA). DMEM / F12
medium and fetal bovine serum were purchased from Gibco (USA) and Perbio Science
Company (New Zealand). BCA protein kit and used in the experiment were purchased
from Sigma Chemicals Company (USA).
2. Cell culture
The mesangial cells were cultured in DMEM / F12 medium (37 C, 5 CO2) which was
consist of 15 fetal calf serum and penicillin / streptomycin (100 g / L). The cells were
grown in 6-well culture plates (Nunc Inc.) with 0.2106 / hole density. We replaced the
prior medium with serum-free DMEM / F12 medium cultured for 16h to synchronize the
cells, after 80% cells had fused. We also add different concentrations of rhEPO to the
medium according to test requirements at the same time. All experiments are located with
control group.
3. Animal experiments and Western blot analysis
Male CD-1 mice (body weight 2225g) were randomly divided into A, B groups. A
group (n = 4) received a single intraperitoneal injection of rhEPO (1000 U / Kg body
weight / time), B group was only injected with isotonic saline as the control group (n = 4).
All mice were sacrificed 24 hours later. The excisional kidney of those mice has been
established in our laboratory based on the mechanical sieve method [6]. The methods of
western blot analysis have been described previously [7].
4. Statistical analysis
All data were analyzed by the SigmaStat statistical software (Jandel Scientific, San Rafael
CA) and S SigmaPlot (SPSS Inc. Chicago, IL). P<0.05 was used to define statistically
significant differences.
Results
1. RhEPO regulate the expression of cell protein.
The results of mesangial cells’ growth shown in Figure 1a, compared with the control
group were not significantly different. From figure 1b, we could observe the results of
protein expression effected by RhEPO. The protein concentration of mesangial cell
stimulated by rhEPO (1U/ml, for 48h) showed no significant difference (0.2530.121 vs
0.2470.058 g/103 cells). But compared with the control group, 10U/ml rhEPO
stimulate mesangial cells to increase the protein concentration (0.3780.094 g/103 cells,
P <0.05), 100U/ml of rhEPO can further increase the concentration of cell protein
(1.2690.382 g/103 cells, P <0.01), that was significant different.
2. RhEPO stimulate mesangial cell to up-regulate the FN expression.
As shown in Figure 2a, as a contrast to normal cultured mesangial cells express only trace
amounts of FN protein. Compared with this, 1 and 10U /ml of rhEPO did not significantly
increase the FN in mesangial cell protein; when rhEPO increased to the concentration of
100U/ml, the expression of FN in mesangial cells was significantly increased compared
with the control group. To further increase the concentration of rhEPO 1000 U/ml, it still
able to increase mesangial cell expression of FN protein (as shown in Figure 2b).
Meanwhile, by the application of indirect immunofluorescence method, we observed that
the distribution of FN generated by mesangial cells. We found that only trace, scattered
distribution of FN from normal cultured mesangial cells (Figure 3a). But rhEPO (100
U/ml) had effect on the mesangial cells for 24 hours, the cell gap and a large number of
reticular cell bodies can be seen, cords, and even plate-like distribution of FN deposition
(Figure 3b).
3. The mice after injection of rhEPO can increase expression of glomerular FN
In order to further evaluate the biological role of RhEPO on the glomeruli, we used a
single intraperitoneal injection, so as to observe the FN expression that the rhEPO effect
on the CD-1 mice’s renal tissue. As the Figure 4 showed, that control mice’s kidney
tissue can not be detected in almost the expression of FN. But the CD-1 mice glomerular
FN protein expression was significantly increased compared with the control group, 24
hours after a single intraperitoneal injection.
Discussion
RhEPO, which is a 34kD single-chain acid glycoprotein secreted by the renal interstitial
cells, is a key cell growth factor in regulation of the development and maturation of red
blood cell [8]. It could promote an increase in red blood cell count in blood so as to
improve the capacity of carrying Oxygen of blood by stimulating the generation and
release of bone marrow. RhEPO also have other biological functions reported in literature
such as protecting nerve tissue structure [9], playing a role in angiogenesis in tumor
stroma [10, 11], stimulating epithelial cell proliferation and antagonizing apoptosis [12-13]
and so on. Animal experiments confirmed that rhEPO can reduce the renal tissue injury of
experimental animals caused by ischemia – reperfusion and cisplatin [14-16]. It was
reported that various cells including mesangial cells have rhEPO receptors. Therefore,
rhEPO can have biological effects on them through its specific receptor [17].
Glomerular is an important part of kidney to complete the glomerular filtration function.
Mesangial cells have a very important role in the maintenance of glomerular structure and
function. The cells not only can regulate glomerular filtration by systolic and diastolic
function, but also are directly involved in the development of glomerulosclerosis by
mesangial cell proliferation, hypertrophy, and synthesis of extracellular matrix (such as
FN, etc.) , which is an important factor of progressive renal failure. Therefore, any factor
of biological functions of mesangial cells is able to change renal function.
Erythropoietin (rhEPO) is increasingly being used in the treatment of anemia caused by a
miscellaneous reasons [1, 18, 19], especially in the treatment of renal anemia, and obtain
a significant effect [20, 21]. From the application of -rhEPO in vivo, we found that
rhEPO can not lead to increase in the number of mesangial cells, but it can significantly
increase the protein concentration in mesangial cells, and up-regulated the FN expression,
which showed dose-dependent. We also observed that FN deposit at mesangial cells gap
by immunofluorescence. Animal studies also found that, rhEPO can increase the
glomerular expression of FN, which is consistent with the vitro experiments. Therefore,
our findings are that rhEPO can act directly on mesangial cells, leading to hypertrophy
and increase of its extracellular matrix formation and accumulation.
Fibronectin (FN) is a variety of important biological role of extracellular matrix
components, and its expression, synthesis, and accumulation affected by many factors
[22]. When the cell/tissue was suffering loss, stress response and/or inflammation, kidney
tissue/cell expression of FN significantly increased to the formation of local accumulation.
Therefore, FN could become an important marker in the research of kidney disease [23].
The increased expression of organization / cell FN has a close relationship with tissue
fibrosis. We found that rhEPO could stimulate the mesangial cells to produce excessive
extracellular matrix in our research of FN expression in vivo and cell culture. Since
mesangial cell hypertrophy and extracellular matrix accumulation is very significant to
the glomerular lesions, rhEPO may be involved in/mediated glomerular disease processes.
This may have important practical significance about the clinical application of rhEPO.
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Figure 1.
rhEPO induced growth and hypertrophy of mesangial cells
(The concentrations of rhEPO were shown in the figure,
P
P<0.01)
a) changes of the quantity of mesangial cells
b) changes of the protein concentration of mesangial cells
Figure 2. Different concentrations of rhEPO upregulated the FN of mesangial cells
a) Western blot showed that rhEPO induced FN expression of mesangial cell.
b) Semi-quantitative of the western blot
Figure 3. Indirect immunofluorescence showed that rhEPO induced FN
in mesangial cells.
a) control of the mesangial cells
b) mesangial cells treated with rhEPO
Figure 4 The increase of FN expression in mice received the injection of rhEPO.
a)
Western blot showed the FN expression of the glomeruli.
b)
Semi-quantitative of the western blot