Journal of Clinical Virology 75 (2016) 16–20

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Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Human kidney damage in fatal dengue hemorrhagic fever results of

glomeruli injury mainly induced by IL17
Carla Pagliari a,b , Juarez Antônio Simões Quaresma c,d , Luciane Kanashiro-Galo a ,
Leda Viegas de Carvalho e , Webster Oliveira Vitoria b , Wellington Luiz Ferreira da Silva a ,
Ricardo Penny e , Barbara Cristina Baldez Vasconcelos c,f ,
Pedro Fernando da Costa Vasconcelos c,f,∗ , Maria Irma Seixas Duarte a
a
Faculdade de Medicina da Universidade de São Paulo, Lab. da Disciplina de Patologia de Moléstias Transmissíveis, Departamento de Patologia, São Paulo,
Brazil
b
Programa de Pós-Graduação em Ciências da Saúde, Instituto de Assistência Médica ao Servidor Público Estadual, SP, Brazil
c
Universidade do Estado do Pará, Belém, Pará, Brazil
d
Núcleo de Medicina Tropical, UFPA, Belém, Pará, Brazil
e
Hospital Guilherme Álvaro, Seção de Anatomia Patológica, Santos, SP, Brazil
f
Instituto Evandro Chagas, Ananindeua, Pará, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Acute kidney injury is an unusual complication during dengue infection. The objective of this study
Received 22 October 2015 was to better identify the characteristics of glomerular changes focusing on in situ immune cells and
Received in revised form cytokines. An immunohistochemical assay was performed on 20 kidney specimens from fatal human
14 December 2015
cases of dengue hemorrhagic fever (DHF). It was observed a lymphomononuclear infiltrate, neutrophils
Accepted 20 December 2015
and nuclear fragmentation in the glomeruli, hydropic degeneration, nuclear retraction, eosinophilic
tubules and intense acute congestion. Sickle erythrocytes were frequent in glomeruli and inflamma-
Keywords:
tory infiltrate. The glomeruli presented endothelial swelling and mesangial proliferation. Lymphocytes
Dengue
Kidney CD4+ predominated over CD8+ T cells, B cells and natural killer cells. There were also an expressive
Immunohistochemistry number of macrophagic CD68+ cells. S100, Foxp3 and CD123 cells were not identified. Cells expressing
Cells IL17 and IL18+ cytokines predominated in the renal tissues, while IL4, IL6, IL10, IL13, TNF-alpha and
Cytokines IFN-gamma were rarely visualized. The high number of cells expressing IL17 and IL18+ could reflect the
acute inflammatory response and possibly contribute to the local lesion. CD8+ T cells could play a role
in the cytotoxic response. DHF is a multifactorial disease of capillary leakage associated with a “Tsunami
of cytokines expression”. The large numbers of cells expressing IL17 seems to play a role favoring the
increased permeability.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Dengue is the most important viral mosquito-borne diseases
affecting humans in tropical and subtropical regions worldwide.
The presentation of the infection ranges from asymptomatic infec-
tion to severe fatal Dengue Hemorrhagic Fever (DHF), which is
characterized by hemorrhagic manifestations, thrombocytopenia,
plasma leakage and or shock [18,11,9,24,7].
∗ Corresponding author at: Instituto Evandro Chagas, Rodovia Br-316, km-07, s/n,
67030-000 Ananindeua, Brazil.
E-mail addresses: pedrovasconcelos@iec.pa.gov.br,
pedro.vasconcelos@globo.com (P.F. da Costa Vasconcelos).
During fatal DHF, viral antigens have been detected in many tis-
sues and organs including liver, lung, lymph nodes, skin and kidney.
An immunohistochemistry Immunohistochemical studies revealed
that viral antigens in the kidney are present as granular deposits in
the lining cells of the tubules [13,17].
Acute kidney injury is an unusual complication during severe
dengue and is associated to hypotension, rhabdomyolysis or
hemolysis. However, cases of dengue with acute kidney injury
without such characteristics have also been reported. In those
cases, the injury is explained as been probably mediated by a direct
effect of dengue virus-DENV [21,2].
Most studies concerning renal involvement during the course
of dengue have reported considerable rates of mortality [20]. The
analysis of renal biopsies demonstrate glomerular changes such as

http://dx.doi.org/10.1016/j.jcv.2015.12.005
1386-6532/© 2015 Elsevier B.V. All rights reserved.
 C. Pagliari et al. / Journal of Clinical Virology 75 (2016) 16–20 17

Fig. 1. Histological aspects of kidney injury in cases of dengue hemorrhagic fever. (A) Glomerular congestion with some elongated and angled red blood cells. Some tubule
cells have pyknotic nuclei (×400); (B) discrete endothelial proliferation. Acute tubular necrosis (nuclear pyknosis) (×400); (C) chronic tubulointerstitial nephritis. Focal acute
tubular necrosis (×200); (D) intense congestion, some tubule cells with pyknotic nuclei, possible tubular necrosis (×200). (E) Glomerular congestion. Some white blood cells
in capillary loops; (F) infiltrated tubulointerstitial nephritis, possible chronic pyelonephritis, mononuclear cell infiltration, mild tubular dilation, hyaline casts, congestion of
peritubular capillaries (×200).

hypertrophy and hyperplasia of mesangial and endothelial cells, Table 1

List of antibodies applied in the immunohistochemistry protocol, with mark, code
monocyte-like cells in some glomerular capillary lumens and focal
and work dilution.
thickening of the glomerular basement membrane. In other cases,
immune complex deposition is a common histologic feature [8]. Antibody Mark/code Work dilution
Vascular alterations have been also observed and seem to be Monoclonal anti-CD4 Dako/M0834 1:1000
related to the imbalance of the host immune response, as a conse- Monoclonal anti-CD8 Dako/M7103 1:20
quence of the pattern of cytokines and cells present at the sites of Monoclonal anti-CD20 Dako/M755 1:400
Monoclonal anti-CD57 Neomarkers/MS-136-P 1:200
lesions [5].
Monoclonal anti-CD68 Dako/M814 1:200
Although the liver and lungs are considered important target Polyclonal anti-S100 Dako/Z311 1:1000
organs of DENV in severe disease, other organs are also injured and Monoclonal anti-Foxp3 Ebioscience/14-4776 1:50
matter of studies, such as spleen, heart and kidneys [31,30,3]. Monoclonal anti-CD123 Ebioscience/14-1239-82 1:50
Polyclonal anti-IL4 RD systems/AF204NA 1:40
Considering the importance of severe dengue (DHF) in the con-
Polyclonal anti-IL6 RD systems/AF206NA 1:20
text of public health worldwide, the objective of this study was to Polyclonal anti-IL10 RD systems/AF217NA 1:40
better identify the immune and histopathology characteristics of Polyclonal anti-IL13 RD systems/AF213NA 1:500
glomerular, tubular and interstitial renal changes focusing on the Polyclonal anti-IL17 RD systems/AF317NA 1:500
phenotype of immune cells at that site and their implications to the Polyclonal anti-IL18 Santa cruz/SC 6178 1:100
Polyclonal anti-TNF alpha RD systems/AF210NA 1:20
immunopathogenesis of dengue. The results of our study suggest
Monoclonal anti-IFN-gamma RD systems/MAB285 1:30
that glomeruli injury is induced by interleukin 17 (IL17).

2. Materials and methods
2.1. Samples
Twenty formalin-fixed paraffin-embedded kidney specimens of
fatal dengue cases were selected from the archives of the Depart-
ment of Pathology of Hospital Guilherme Álvaro, municipality of
Santos, São Paulo State, Brazil. Specimens were obtained during
viscerotomy of human patients who died with DHF during the year
of 2010, following the ethical standards of the institutional review
committee. The patients’ ages ranged from 4 to 62 years old, nine
were male and 11 female.
Diagnosis was confirmed by correlation of clinical data, serologic
diagnosis (IgM antibodies and NS1 antigens detection by ELISA)
and histological examination by hematoxylin-eosin staining and
detection of viral antigens through immunohistochemical assay
[28,25]. The control group (n = 5) included fprmalin-fixed paraffin-
embedded kidney specimens from patients who died without any
infectious disease or renal damage, confirmed by histological eval-
uation.
2.2. Immunohistochemistry analysis
Sections were made from formalin-fixed, paraffin-embedded
kidney samples and re-hydrated by a series of increasing ethanol
gradient. Endogenous peroxidase activity was blocked by incubat-
ing sections in 3% H2 O2 solution at room temperature for 20 min.
For antigen retrieval, sections were incubated in retrieval solution
(Dako, S2367) pH 9.0 for 20 min at 95 ◦ C. Immunohistochemical
staining was performed by incubation with specific primary anti-
body (Table 1). The tissue samples were incubated with the specific
secondary antibody and a streptavidin-biotin peroxidase system,
according to the manufacturer’s instructions (LSAB system, code
K0690, DakoCytomation, Carpinteria, CA, USA). Each reaction was
visualized with 3,3 -diaminobenzidine-tetrahydrochloride (DAB)
(Sigma–Aldrich Chemical Co., St. Louis, MO, USA D5637). All
18 C. Pagliari et al. / Journal of Clinical Virology 75 (2016) 16–20
Fig. 2. immunohistochemistry phenotype of cells and cytokines in human kidney from patients with dengue hemorrhagic fever. The main findings in glomeruli were
characterized by CD4+ T cells, macrophages (CD68+ cells) and the cytokines IL17, IL18 and IL6. The cytokines IL18 and IL6 were also frequently visualized in the tubules.
(Streptavidin-Biotin peroxidase method, ×200).
specimens were counterstained with Mayer’s Hematoxylin and
mounted for light microscope. Positive and negative controls fol-
lowed each immunohistochemistry reaction.
2.3. Quantitative analysis
The immunostained cells in the glomeruli were quantified by
counting the number of positive cells in 20 randomized glomeruli
in each specimen. It was considered the mean number of positive
cells/glomeruli.
2.4. Ethical aspects
The study was approved by the Ethics Committee of the Medical
School, University of São Paulo process 253/12.
3. Results
3.1. Necropsy findings
The most frequent gross findings observed at necropsy included
pleural effusion (50%), hepatosplenomegaly (20%), ascites (30%),
sickle cell traits (15%), bleeding in the lungs (40%), gastrointestinal
tract (15%) and peritoneum (20%).
3.2. Histological findings
The most frequent histological findings in kidney were charac-
terized by glomerular congestion with some elongated and angled
red blood cells, some tubular cells with pyknotic nuclei, conges-
tion of glomerular and peritubular capillaries, foci of mild chronic
inflammation with plasma cells, acute tubular necrosis, focal tubu-
lar atrophy and fibrosis, infiltrated tubulointerstitial nephritis,
mononuclear cell infiltration, mild tubular dilation, hyaline casts,
congestion of peritubular capillaries and discrete endothelial pro-
liferation (Fig. 1).
The presence of sickle erythrocytes was also frequently
observed in the glomeruli and in the inflammatory infiltrate. Also,
glomeruli presented endothelial edema and mesangial prolifera-
tion.
3.3. Immunohistochemitry findings
The immunohistochemical assay of kidney tissues showed the
presence of inflammatory cells with a clear predominance of CD4+
T cells over CD8+ T cells. B cells and natural killer cells were also
present in the renal tubules. An excessive number of macrophages
CD68+, but absence of S100+ cells was observed in the tissue. Treg
cells, immunostained by Foxp3 and plasmacytoid dendritic cells
(CD123) were also absent.
Considering the pattern of cytokines, there was a predominance
of cells expressing IL17 and IL18, followed by IL6. The cytokines IL4,
IL10, IL13, TNF-␣ and IFN-␥ were rarely visualized. Both IL18 and
IL6 were also frequently expressed in the tubules. Fig. 2 demon-
strates by immunohistochemical staining the most representative
expressed cytokines in the kidney; Fig. 3A presents the quantifi-
cation of the immunostained cells, and Fig. 3B expresses different
cytokines in the renal tissue.
The control group showed a low number of cells in the kidney
tissues. Moreover Treg and S100+ dendritic cells were not detected
in that group. No Cytokines were immunostained in the cells
examined. The histological findings demonstrated renal glomeruli
preserved to both distal and proximal tubules.
4. Discussion
DHF presents as severe disease. Hemorrhagic phenomena,
thrombocytopenia, and plasma leakage to natural cavities are
important clinic signs, indicating important hemodynamic dys-
function. In many cases, due the disease severity, the outcome is
fatal.
It has been hypothesized that immunological mechanisms (i.e.,
cytokine storm) occur during severe dengue disease. Some in situ
findings demonstrate the DENV interactions with target cells such
as dendritic cells, hepatocytes and endothelial cells, and the conse-
 C. Pagliari et al. / Journal of Clinical Virology 75 (2016) 16–20
Fig. 3. Quantification of cells and cytokines in the glomeruli. Mean values considering 20 glomeruli. Black column—group of DHF renal lesions; white column—control group.
quent production of immune mediators that modulate the adaptive
humoral and cellular immune response [6,10].
The glomerulonephritis that can follows DHF is commonly
characterized by mesangial proliferation and immune com-
plex deposition [23]. Experimentally, glomerular enlargement,
increased endocapillary and mesangial cellularity, glomerular IgM
and immune-complex deposition and proliferative lesions in the
glomeruli were observed [4,1].
During the course of experimental infection by DENV type 2, the
induction of diffuse proliferative glomerular lesions with enlarged
volume and accompanied by increased endocapillarity and mesan-
gial cellularity were demonstrated [19].
Recently the histopathologic changes and the ultrastructure
lesions of multiple organs during severe dengue were described. In
the kidney, parenchyma and circulatory damages, as well as acute
tubular necrosis of proximal convoluted tubules with loss of the
basement membrane were reported [27], with detection of DENV
antigens in macrophages and monocytes. The authors speculated
that the kidney is probably not a target organ for the virus but that
the injuries observed were result of the host immune response at
that site [27].
In the present work using renal tissues obtained from 20 fatal
dengue cases, we observed in the glomeruli and in the inflam-
matory infiltrate the presence of sickle erythrocytes, hydropic
degeneration, nuclear retraction (possible early stage of acute tubu-
lar necrosis), eosinophilic tubules, and intense acute congestion.
The glomeruli also showed endothelial edema and mesangial pro-
19
liferation, besides hemorrhage. It is also noteworthy the presence
of multifocal interstitial nephritis.
Some chemical elements of immune response are related to dis-
ease severity such as IL-6, IL-8, TNF-␣, IFN-␥, and the complement
system. The increased vascular permeability during severe dengue
(i.e., DHF) seems to be mediated by IL-2, TNF-␣ and IFN-␥ [12,22].
The CD4+ and CD8+ T cells, and also natural killer cells are com-
mon source of IFN-␥. Although IL-18 induces the production of
IFN-␥, in the present study this factor was rarely visualized in the
glomeruli or interstitium. The cytokine IL-18 could reflect the acute
inflammatory response and contribute to the local lesion.
In contrast to what is observed in liver lesions in severe dengue,
we could not observe the occurrence of apoptosis in renal lesions.
However, the elevated number of CD8+ T cells, could indicate these
cells play a role in the cytotoxic response in such lesions.
In addition, the CD4+ and CD8+ T cells could also play a role as
a source of IL-17, the predominant expressed cytokine, whose pro-
duction is induced in part by IL6 [26]. On the other hand, the DHF
is described as a multi-factorial disease, where the capillary leak-
age commonly observed is associated with a “Tsunami of cytokines
expression”. The predominance of IL17 might partly explain this
leakage and the increased vascular permeability.
The IL17 belongs to a family composed by six members, from
IL17A to IL17F. IL17A, also named CTLA8, is produced by CD4+
and CD8+ T cells, ␥␦ T lymphocytes, mast cells, neutrophils, ker-
atinocytes and iNKT cells [14]. This cytokine plays some roles in
20 C. Pagliari et al. / Journal of Clinical Virology 75 (2016) 16–20
immune response, such as inducing the production of IL1␤, IL6, IL8
and neutrophils activation [15].
CD4+ T lymphocytes predominate in the renal lesions of severe
hemorrhagic dengue, although CD8+, CD20+ and NK cells are also
present in considerable numbers. Similar to the published data
on liver lesions, the IL18 seems to contribute to the formation of
renal lesions in fatal dengue. By the way, the presence of large
numbers of cells expressing IL17 seems either to play a role enhanc-
ing the increased permeability or, it is just an indication of the
local immune response. IL17 is an important activating cytokine of
endothelium. Apparently endothelial cells express some receptors
to IL17, produce neutrophil chemo attractants and express adhe-
sion molecules related to leukocyte transmigration. Finally, IL17
promotes the expression of E-selectin, VCAM-1 and ICAM–1 as well
[29].
It was previously demonstrated that acute infection by DENV-
2 induces high levels of IL17 in severe fatal dengue patients [16].
The present results obtained from renal tissues of fatal cases may
contribute to the better understanding of immune mechanisms in
DHF cases.
In summary, in this study, the CD4+ predominated over CD8+ T
cells, B cells and natural killer cells; the high number of IL17 and
IL-18+ cells could reflect the acute inflammatory response and con-
tribute to the local lesion; and the presence of large numbers of cells
expressing IL17 seems to indicate this cytokine plays a role favoring
the increase of vascular permeability.
Conflict of interest
None.
Funding
This study was partially supported by the National Institute
of Science and Technology for Viral Hemorrhagic Fevers—INCT-
FHV INCT-FHV (MCT/CNPq/FNDCT/CAPES/FAPESPA No. 15/2008)
process 573739/2008-0, CNPq process 501549/2003-0, and CNPq
process 301641/2010-2.
WOV—received a trainee grant from Instituto de Assistência
Médica ao Servidor Público Estadual—CNPq (National Counsel of
Technological and Scientific Development); BCBV—is a trainee of
the INCT-FHV.
Acknowledgment
The authors are grateful to Prof. Washington Luis Conrado dos
Santos from Fundação Oswaldo Cruz, Salvador, Bahia, for the assis-
tance in the histological analysis.
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