JOURNAL OF VIROLOGY, Oct. 2011, p. 9811–9823
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 19
Pathogenic Old World Hantaviruses Infect Renal Glomerular and
Tubular Cells and Induce Disassembling of Cell-to-Cell Contacts?
Ellen Krautkra ¨mer,1* Stephan Grouls,1Nadine Stein,1Jochen Reiser,2and Martin Zeier1
Department of Nephrology, University of Heidelberg, Heidelberg, Germany,1and Division of Nephrology and Hypertension,
Leonard Miller School of Medicine, University of Miami, Miami, Florida2
Received 22 March 2011/Accepted 7 July 2011
Viral hemorrhagic fevers are characterized by enhanced permeability. One of the most affected target organs
of hantavirus-induced hemorrhagic fever with renal syndrome is the kidney, and an infection often results in
acute renal failure. To study the underlying cellular effects leading to kidney dysfunction, we infected human
renal cell types in vitro that are critical for the barrier functions of the kidney, and we examined kidney biopsy
specimens obtained from hantavirus-infected patients. We analyzed the infection and pathogenic effects in
tubular epithelial and glomerular endothelial renal cells and in podocytes. Both epithelial and endothelial cells
and podocytes were susceptible to hantavirus infection in vitro. The infection disturbed the structure and
integrity of cell-to-cell contacts, as demonstrated by redistribution and reduction of the tight junction protein
ZO-1 and the decrease in the transepithelial resistance in infected epithelial monolayers. An analysis of renal
biopsy specimens from hantavirus-infected patients revealed that the expression and the localization of the
tight junction protein ZO-1 were altered compared to renal biopsy specimens from noninfected individuals.
Both tubular and glomerular cells were affected by the infection. Furthermore, the decrease in glomerular ZO-1
correlates with disease severity induced by glomerular dysfunction. The finding that different renal cell types
are susceptible to hantaviral infection and the fact that infection results in the breakdown of cell-to-cell
contacts provide useful insights in hantaviral pathogenesis.
Hantaviruses are emerging rodent-borne viruses that cause
hemorrhagic fever in humans. Typical symptoms of viral hem-
orrhagic fevers are enhanced vascular permeability, thrombo-
cytopenia, and plasma leakage (23). The permeability of epi-
thelial and endothelial monolayers is regulated by multiprotein
complexes comprising specific transmembrane and cytosolic
proteins. Two junctional regions are formed at the site of
cell-to-cell contacts: tight and adherens junctions. In podocytes
a multiprotein complex called a glomerular slit diaphragm, a
podocyte-specific variant of adherens and tight junctions, is
responsible for the glomerular barrier function (19, 60, 63).
Tight junctions selectively regulate the permeability for ions
and solutes through the paracellular route. The permeability of
the tight junction is regulated by intra- and extracellular stimuli
(10, 27, 64, 74, 76, 81). The endothelium and epithelium rep-
resent a barrier for pathogens and, during infection, the mono-
layer integrity is often compromised due to effects of viral
replication and immune defense. However, the epithelial and
endothelial function is often influenced without observing his-
tomorphologically visible disruption or massive damage of the
monolayer in the affected tissue (5, 13, 15, 35, 57, 73, 85). The
pathomechanisms that contribute to the alteration in a barrier
function, leading to an organ dysfunction in a hantaviral infec-
tion, are not completely understood.
Hantavirus pathology is characterized by capillary leakage
and organ failure. Its severity varies from mild disease to fatal
outcome and may be influenced by intraindividual risk factors.
Gender and genetic predispositions, such as tumor necrosis
factor alpha (TNF-?) polymorphism or HLA-B8-DR3 haplo-
type, have been discussed as key determinants of disease se-
verity (33, 37, 48). Furthermore, the course of the disease
differs within strains, and the organ spectrum of illness de-
pends on the tropism of the virus. The infection with New
World hantaviruses causes hantaviral pulmonary syndrome
(HPS). The target organ that is mainly affected in its function
by the infection with a pathogenic Old World hantavirus is the
kidney, leading to the hemorrhagic fever with renal syndrome
(HFRS) (14, 42, 46, 85). Pulmonary findings in Old World
hantavirus and renal involvement in New World hantavirus
infection may be observed as well (44, 58, 86). Furthermore,
both viruses can affect several other organs (28, 39). Neverthe-
less, the acute renal failure with frequent massive proteinuria
is a symptom hallmark of Old World hantavirus infection (14,
32, 45, 53, 66, 67). Differences in organ tropism may account
for the pronounced renal involvement in Old World hantavirus
infection. However, the detection of hantavirus in the tissue of
infected patients is difficult: in renal biopsy specimens from
hantavirus-infected patients, the viral antigen was only detect-
able in the cytoplasm of tubular epithelial cells (25, 28, 29, 36,
78). Studies in a hantavirus infection model with macaques
detected viral RNA in tubular epithelial cells and rarely in the
glomerulus (71). Experimental infection of deer mice with Sin
Nombre virus revealed the expression of N protein in the
glomerulus (41). The detected viral antigens colocalize in the
areas with tubular damage (36, 71). The renal function de-
pends on the integrity of the tubular epithelium and the glo-
merular apparatus. Histopathological damage of the glomeru-
lar endothelium is not observed, despite enhanced glomerular
permeability, as revealed by often severe nonselective protein-
* Corresponding author. Mailing address: Department of Nephrol-
ogy, University of Heidelberg, Im Neuenheimer Feld 162, 69120
Heidelberg, Germany. Phone: 49 6221 91120. Fax: 49 6221 9112209.
?Published ahead of print on 20 July 2011.
uria (1, 51, 71). The molecular mechanism, whereby the tubu-
lar reabsorption and glomerular filtration function is disturbed,
and to what extent both participate in the outcome of the
clinical picture of hantavirus infection is not known. Infectious
diseases may be associated with a disruption of barrier function
due to direct or immune mediated effects on the intercellular
integrity. The massive proteinuria observed in hantavirus-in-
fected patients indicates that glomerular and tubular cells are
affected (1, 47, 68). However, the susceptibility of human renal
cells and the possible effects of hantaviral infection on the
barrier function have thus far not been elucidated. In the
present study, we examined the infection of human renal cells
in vitro and analyzed the integrity of cell-to-cell contacts of
infected cells in vitro and in renal biopsy specimens from han-
tavirus-infected patients hospitalized in our department.
MATERIALS AND METHODS
Cells and tissues. Human renal proximal epithelial cells (HREpC) were ob-
tained from Promocell (Heidelberg, Germany) and maintained in renal epithe-
lial cell growth medium 2 (Promocell). Human renal glomerular endothelial cells
(HRGEnC) were obtained from ScienCell (Carlsbad, CA) and maintained in
endothelial cell medium ECM (ScienCell). Only HREpC and HRGEnC from
passages 2 to 6 were used.
The human podocyte cell line was derived from human normal podocytes
conditionally transformed with a temperature-sensitive mutant of the simian
virus 40 (SV40) large T antigen. Growing at the permissive temperature of 33°C
allows the cells to proliferate. Thermoswitching to the nonpermissive tempera-
ture of 37°C to inactivate the SV40 T antigen results in a growth arrest and
promotes the cell to differentiate. Cells were grown for a period of 14 days at
37°C to ensure differentiation (62).
Frozen archival renal biopsy specimens of seven patients with acute hantavirus
infection (confirmed by positive IgM serology for Puumala virus antigen) and, as
controls, four samples with normal morphology from nephrectomies were used.
Biopsy specimens from hantavirus patients were taken between day 5 and 12
after onset of symptoms. This study was approved by the Ethics Committee of the
University Hospital of Heidelberg, and it adhered to the Declaration of Helsinki.
Written informed consent was obtained from all patients.
Virus and infection. The stocks of hantaviral Hantaan virus, strain 76-118
(HTNV) or Puumala virus, strain Vranica (PUUV), were propagated on Vero
E6 cells. Virus inocula, HTNV or PUUV, at a multiplicity of infection (MOI) of
0.01 were added to HREpC, HRGEnC, or differentiated podocytes. After incu-
bation for 1 h at 37°C, the unbound virus was removed by a triple washing, and
the cells were incubated for the indicated time points at 37°C. The infection was
monitored by using immunofluorescence or the Western blot analysis of hanta-
viral N protein expression with mouse monoclonal anti-nucleocapsid protein
(Progen, Heidelberg, Germany) or rabbit polyclonal anti-nucleocapsid protein
antibody. An equal loading was verified by the detection of tubulin on the same
membrane. For reinfection, Vero E6 cells were inoculated with cell-free super-
natants of infected renal cells and monitored for infection for 6 days postinfec-
tion (dpi) (HTNV) or 14 dpi (PUUV).
Immunofluorescence and Western blot analysis. For immunofluorescence,
acetone-fixed cells or frozen sections of renal biopsy specimens were stained with
primary and appropriate fluorescently labeled secondary antibodies. The follow-
ing antibodies were used: mouse or rabbit anti-ZO-1 (Invitrogen, Karlsruhe,
Germany), mouse anti-CD31 (Dako, Hamburg, Germany), goat anti-synaptopo-
din P-19 (Santa Cruz, Heidelberg, Germany), and mouse anti-cytokeratin 18
(Millipore, Schwalbach/Ts, Germany). Integrin was detected with mouse anti-
integrin ?V?3(clone LM609; Millipore). To confirm the specificity of anti-
integrin ?V?3antibody LM609, fixed cells were incubated with anti-integrin ?V?3
antibody that was pretreated with recombinant human integrin ?V?3(R&D
Systems, Wiesbaden-Nordenstadt, Germany). Recombinant protein was added
to a final concentration of 0.04 ?g/?l to integrin ?V?3antibody LM609 (final
concentration, 0.01 ?g/?l). Images were taken using a Nikon DXM1200C cam-
era attached to a Nikon Eclipse 80i upright microscope (Nikon, Du ¨sseldorf,
Germany). The quantification of ZO-1 expression was performed on slides that
were all immunolabeled with the mixture of antibodies on the same day. Images
on these slides were captured using a constant exposure time. The fluorescence
intensity of the selected areas in 32 glomeruli of seven patients and of 18
FIG. 1. Expression of marker proteins and the hantaviral receptor integrin ?V?3on renal cell types. (A) Human primary cells HREpC,
HRGEnC, and human podocytes were stained with antibodies against marker proteins for renal cell types and with anti-integrin ?V?3antibody.
(B) Lysates of renal cell types were analyzed for the expression of integrin ?3by Western blot analysis. (C) Flow cytometric analysis of cell surface
protein expression of integrin ?V?3and the endothelial marker CD31.
9812 KRAUTKRA ¨MER ET AL.J. VIROL.
This study was supported by a grant from the Deutsche Forschungs-
gemeinschaft (KR 3711/2-1) to E.K.
We thank Heike Ziebart, Vanessa Bollinger, and Charlotte Holler
for excellent technical assistance and Claudia Halfen and Michelle
Froese for critical reading of the manuscript.
No financial conflicts of interest exist.
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