Purified hepatitis B virus induces human Mesangial cell proliferation and extracellular matrix expression In Vitro
Purified hepatitis B virus induces human Mesangial cell proliferation and extracellular matrix expression In Vitro
Zongli Diao 0
Jiaxiang Ding 0
Chenghong Yin 1
Liyan Wang 0
Wenhu Liu 0
0 Department of Nephrology, Beijing Friendship Hospital, Capital Medical University , 95 Yong-An Road, Beijing 100050 , China
1 Department of Infectious Disease, Beijing Friendship Hospital, Capital Medical University , 95 Yong-An Road, Beijing 100050 , China
Background: Hepatitis B virus (HBV) induces proliferation of human mesangial cells (HMCs), and extracellular matrix expression through the deposition of immune complexes in renal tissue. However, it is unclear whether HBV can directly affect HMCs. In this study, the effects of purified HBV on HMC proliferation and extracellular matrix expression in vitro was determined. Findings: HBV was purified using sucrose density gradient centrifugation. HMCs were co-cultured with purified HBV (104-106 copies/ml) for 48 h, and cell proliferation determined using 5-ethynyl-2-deoxyuridine immunofluorescence assays. After HMCs were co-cultured with 106 copies/ml purified HBV for 0, 12, 24, 36 and 48 h, expression of type IV collagen and fibronectin was measured using enzyme-linked immunosorbent assays. Three titers of purified HBV (104, 105, and 106 copies/ml) induced HMC proliferation, with the proportion of increases in cell numbers at 24.7 4.3, 31.2 9.4, and 36.8 7.5%, respectively. All these increases were significantly higher than those for the control group (13.6 4.2%) (All p < 0.05). Purified HBV (106 copies/ml) significantly increased the levels of type IV collagen and fibronectin in supernatants compared with the control group at 12 and 48 h (All p < 0.05). Conclusions: Purified HBV can directly promote HMC proliferation and expression of type IV collagen and fibronectin, and could be involved in the pathogenesis of HBV-associated glomerulonephritis.
Hepatitis B virus; Human mesangial cells; Proliferation; Type IV collagen; Fibronectin
Hepatitis B virus (HBV) infections are prevalent in China
, and infection with this virus can result in the
development of HBV-associated glomerulonephritis (HBV-GN).
HBV-GN has become a prevalent type of secondary
glomerulonephritis in China . Human mesangial cell
(HMC) proliferation and increases in the expression of
extracellular matrix proteins, including type IV collagen and
fibronectin (FN), were involved in various pathological types
of HBV-GN. The most common pathological type of
HBV-GN is HBV-associated membranous nephropathy
(HBV-MN). Proliferation of HMCs, and increases
in extracellular matrix protein levels are more common in
HBV-MN compared with the idiopathic form .
Additionally, HMC proliferation and increases in
extracellular matrix protein expression are common in
other pathological types of HBV-GN such as
The most widely accepted mechanism for increases in
HMC proliferation, and expression levels of extracellular
matrix proteins in HBV-GN is through the deposition of
HBV immune complexes . Many studies have shown
that expression of HBV DNA in glomeruli suggests direct
viral-induced pathological alterations of HMC could
contribute to the pathogenesis of HBV-GN [5,6]. The
main effect of HBV DNA in renal tissues on HMCs was
because of HBV antigens (HBAg) and HBV antibodies
(HBAb) forming immune complexes in situ [5,7]. It
remains unclear whether HBV can directly affect HMCs;
therefore, in this study we purified HBV virons and
subviral particles from the serum of HBV-infected patients, and
investigated the effects of purified HBV on HMC
Figure 1 Various titers of HBV can induce HMC proliferation after 48 h. (A) Detection of EdU (left) and Hoechst3342 (center) incorporation
in cultured HMCs, and overlay images (right). (B) HMC proliferation in control and HBV co-culture groups. **p < 0.05 compared with that in
normal MsCM. **p < 0.05 compared with that in MsCM containing 104 copies/ml HBV. Results are presented as means SEM.
purified by sucrose density gradient centrifugation as
previously described .
Isolation and purification of HBV
We isolated HBV from the HBV DNA-positive sera of
chronic HBV patients, in our hospital, containing more
than 5.0 108 copies/ml of circulating HBV. Virus was
Cell culture and cell proliferation assays
HMCs (ScienCell, San Diego, CA, USA) were cultured
in Mesangial Cell Medium (MsCM; ScienCell, Carlsbad,
USA) at 37C/5% CO2. Cells were seeded into 12-well
culture plates at a density of 1.0 104 cells/well and
incubated for 24 h in RPMI 1640 medium. We then
applied HBV at various doses (104, 105, and 106 copies/ml)
to HMC cultures. At 48 h post-seeding, cell proliferation
was determined using 5-ethynyl-2-deoxyuridine (EdU)
immunofluorescence assays according to the
manufacturers instructions (Ribobio, Guangzhou, Guangdong,
China). The culture medium was replaced with normal
MsCM containing 50 M EdU. After a 3-h incubation,
cells were fixed with formaldehyde. After washing with
phosphate-buffered saline (PBS) containing 0.5% Triton
X-100, cells were washed with PBS and counterstained
with Hoechst 33342. Cells were visualized by
fluorescence microscopy and using Image-Pro Plus 6.0 (Media
Cybernetics, Rockville, MD, USA). The level of HMC
proliferation was determined using the following formula:
HMC proliferation number of EdUpositive cells=all cells
Enzyme-linked immunosorbent assays (ELISAs)
Cells were seeded into 12-well culture plates at a density
of 1.0 104 cells/well and incubated for 24 h in normal
RPMI 1640 medium. We then applied MsCM alone, or
106 copies/ml of HBV in MsCM. At 0, 12, 24, 36, and
48 h post-seeding, the supernatants of each well were
collected, and type IV collagen and FN levels measured
by ELISA according to the manufacturers instructions
(Blue-gene, Shanghai, China).
All data were expressed as the mean the standard error
of the mean (SEM). Statistical analysis was carried out
using Tamhanes T2 test for HMC proliferation, and the
Independent-Samples T-test for type IV collagen and FN.
Differences were considered significant when the P-value
was less than 0.05. All results were repeated for three
Extracellular matrix protein levels
Using 106 copies/ml HBV significantly increased the
expression levels of type IV collagen and FN in HMCs
(p < 0.05; Figure 2).
In our study, we found that purified HBV significantly
increased HMC proliferation, and also increased
expression levels of type IV collagen and FN. The major
mechanism of increased HMC proliferation and extracellular
matrix expression in HBV-GN is suggested to be due to
HBV immune complex deposition [6,9]. However, many
studies have detected HBV DNA, as both free and
integrated forms, in mesangial cells. He et al. have reported
that the expression of HBV DNA in the nucleus and
cytoplasm of HMCs in HBV-GN patients verified the
presence of HBV DNA in HMC . Wang et al. showed
that HBV DNA as the integrated form was detected in
82% (41/50) of cases in HMCs by in situ hybridization
. These observations suggest that HBV DNA can
infect HMCs, and that it can directly induce pathological
alterations in HMCs that might be involved in HBV-GN
. However, these studies were all conducted in renal
tissues, and the effects of HBV on HMCs were suggested
to be a result of immune complex formation in situ
[7,11]. Because of confounding factors involving immune
complexes that are circulating, it was difficult to show
whether HBV can directly affect HMCs in these studies. In
our study, we purified HBV from the sera of HBV-infected
patients. The HMCs were co-cultured with purified HBV
in vitro, thereby overcoming the confounding factors
Figure 2 HMCs were co-cultured with purified HBV. Type IV collagen (A) and FN (B) levels in supernatants increased significantly compared
with controls at 12, 24, 36, and 48 h (p < 0.05).
related to the immune complex, and we showed that
purified HBV directly affected HMCs.
There were some limitations to our study. We did
not explore the mechanisms through which HBV
increased HMC proliferation and expression of
extracellular matrix proteins. HBV X Protein (HBx) acts an
indirect transcriptional transactivitor to regulate cell
proliferation, transdifferentiation and apoptosis .
Some studies have shown that HBx can induce
mesangial cell proliferation through the upregulation
of interleukin-1 and interleukin-6 . However, in
these previous studies, HBx was transfected into
mesangial cells artificially; therefore, it remains
unknown whether HBV can induce HMC proliferation
through a natural infection. Further studies will be
required to clarify this.
In conclusion, our findings support the hypothesis
that HBV exerts direct effects on HMCs, and will
hopefully enrich our understanding of the pathogenesis
DZ and JD carried out all experiments in this study, collated the information,
performed the literature search and drafted the manuscript. CY performed
the EdU assays. LW performed the ELISA experiments. WL, the corresponding
author, designed the research project, performed the literature search and
prepared the manuscript. All authors read and approved the final
This work was supported by Starting Foundation of Beijing Friendship
Hospital, Capital Medical University (201007), Beijing Municipal Science and
Technology Commission Funds (D131100004713001).
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