R E S E A R C H A R T I C L E
Morphological characterization of ckd in cats: Insights of
fibrogenesis to be recognized
G. B. Morais
1|
D. A. Viana
2|
J. M. Verdugo
3|
M. G. Rosell
o
4|
J. O. Porcel
4|
D. D. Rocha
5|
F. A. F. Xavier J
unior
1
|
K. D. S. M. Barbosa
1|
F. M. O. Silva
1|
G. A. C. Brito
6|
C. M. S. Sampaio
7|
J. S. A. M. Evangelista
11Comparative Morphology Laboratory,
Universidade Estadual do Ceara, Ceara, Brazil
2Laboratory of Veterinary Pathology and
Legal Medicine Universidade Estadual do Ceara, Ceara, Brazil
3Institute Cavanilles of Evolutionary
Biodiversity, Universitad de Valencia, Valencia, Spain
4College of Veterinary Medicine,
Universitad Cardenal Herrera, Spain
5Laboratory of Experimental Oncology,
Universidade Federal do Ceara, Ceara, Brazil
6Laboratory of Morphology and Image
Processing, Universidade Federal do Ceara, Ceara, Brazil
7Health Sciences Center, Universidade
Federal do Ceara, Ceara, Brazil Correspondence
G. B. Morais, Programa de Pos-graduaç~ao em Ci^encias Veterinarias, Faculdade de Veterinaria, Universidade Estadual do Ceara, Fortaleza, Ceara, Brazil. Av. Dr. Silas Munguba, 1700—Campus Itaperi—60740-903—Fortaleza, Ceara, Brazil.
Email: glaycianebm@yahoo.com.br
Review Editor: Prof. Alberto Diaspro
Abstract
Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis and its
pathoge-nesis is associated with the activity of mesenchymal cells (fibroblasts), being essentially
characterized by a process of excessive accumulation resulting from the deposition of extracellular
matrix components. The aim of this study was to characterize the morphological presentation of
chronic and fibrotic lesions in the glomerular, tubular, interstitial, and vascular compartments in
feline CKD, as well as the possible participation of myofibroblasts in renal fibrotic processes in this
species. Cat kidneys were collected and processed according to the conventional techniques for
light microscopy, circular polarization, immunohistochemistry, and electron microscopy. Fibrotic
alterations were present in all compartments analyzed. The main findings in the glomerular
com-partment were different degrees of glomerular sclerosis, synechia formation, Bowman’s capsule
calcification, in addition to glomerular basement membrane thickening and pericapsular fibrosis.
The tubulointerstitial compartment had intense tubular degeneration and the immunostaining in
tubular cells for mesenchymal cell markers demonstrated the possibility of mesenchymal epithelial
transition and consequent involvement of myofibroblasts in the development of interstitial tubule
damage. Infiltration of inflammatory cells, added to vessel thickening and fibrosis, demonstrated
the severity and role of inflammation in the development and perpetuation of damage. Thus, we
may conclude that fibrotic lesions play a relevant role in feline CKD and the mechanism of
perpet-uation of these lesions need further elucidation regarding the origin and participation of
myofibroblasts and consequent mesenchymal epithelial transition in this species.
K E Y W O R D S
collagen, feline fibrosis, myofibroblasts, renal histopathology
1
|I N T R O D U C T I O N
Chronic kidney disease (CKD) is relevant and frequent in the clinical
rou-tine of small animals, being defined as changes present in one or both
kid-neys for more than 3 months (Bartges, 2012; Egenvall et al., 2009).
Kidney disease can occur due to direct injury to the kidney and, in several
cases, this initial harm will trigger the repair or fibrogenesis process to
contain the damage (Liu, 2006; Mutsaers, Stribos, Glorieux, Vanholder, &
Olinga, 2015).
Repairing in damaged tissue after chronic or repetitive injury
basi-cally involves two distinct steps: a regenerative phase, where damaged
cells are replaced by cells of the same type, leaving no evidence of
damage; and a healing phase or fibrosis where the connective tissue
replaces the normal parenchymal tissue (Hutchison, Fligny, & Duffield,
2013; Wynn, 2007).
Fibrosis is a response characterized by the sustained production of
growth factors, proteolytic enzymes, angiogenic factors and cytokines,
leading to progressive and excessive production, deposition and
contraction of extracellular matrix components (ECM) (Hutchison et al.,
2013).
Renal fibrosis is characterized by glomerulosclerosis and
tubulointer-stitial fibrosis. The pathogenesis of renal fibrosis is associated with the
activity of mesenchymal cells (fibroblasts) and is essentially characterized
by an excessive accumulation process resulting from the deposition of
ECM components (Bonventre, 2014; Hewitson, 2009; Liu, 2006).
Renal interstitial fibroblast has been the focus of studies on
tubulo-interstitial fibrosis. When activated they are called myofibroblasts and
synthesizeasmooth muscle actin (a-SMA), marking present in various
forms of progressive renal disease in humans, as well as components of
ECM such as fibronectin and collagen (Badid, Mounier, Costa, &
Des-mouliere, 2000; Jinde et al., 2001; Hewitson and Becker, 1995;
Hewit-son, 2009; Qi et al., 2006).
Lesions affecting nephrons which progress to fibrosis are
subdi-vided into those that start in the glomerulus and those that begin in
the tubules. Glomerular diseases constitute the overwhelming majority
of diseases progressing to CKD in humans (Kaissling, Lehir, & Kriz,
2013). However, primary glomerular diseases are considered rare in
cats and tubulointerstitial fibrosis seems to be the most common final
result in feline CKD (Ichii et al., 2011; Lulich, Osborne, O’brien, &
Pol-zin, 1992).
Interstitial fibrosis is a typical finding resulting from
tubulointersti-tial damage (TID), whose CKD severity is related to this damage in
humans and cats with similar developmental characteristics in both
species (Goumenos et al., 2001; Hewitson and Becker, 1995; Yabuki
et al., 2010). Studies have sought to elucidate the complex mechanism
underlying TID in cats that involves the induction and proliferation of
myofibroblasts and consequent fibrotic process (Chakrabarti, Syme,
Brown, & Elliott, 2013; Yabuki et al., 2010).
Several studies have recently shown efforts to demonstrate and
characterize CKD in cats (Brown, Elliott, Schmiedt, & Brown, 2016;
Chakrabarti et al., 2013). In addition to this effort, we propose to
char-acterize the morphological presentation of chronic and fibrotic lesions
in the glomerular, tubular, interstitial and vascular compartments in
feline CKD, as well as the possible participation of myofibroblasts in
renal fibrotic processes in cats.
2
|M E T O D O L O G Y
2.1
|Experimental animals
Adult cats (Felis catus) of different breeds and both genders were
selected in the State University of Ceara (Brazil) and Cardenal Herrera
University (Spain) from 2014 to 2016. Animals were evaluated in the
clinical and hospital routine in both institutions, being forwarded to the
Necropsy Sector, presenting renal lesions characterized as chronic in
the Between.
2.2
|Ethical aspects
This study was approved under the official memo no. 11585871-7/10
by the Ethics Committee for Animal Use of the State University of
Ceara according to the principles of the Brazilian College of Animal
Experimentation. All data were anonymously analyzed and an informed
consent was signed and obtained by all the animals’owners.
2.3
|Sample preparation
Both kidneys were macroscopically evaluated, measured and sectioned
longitudinally during the necroscopic examination. Several fragments
were removed from cortical and spinal areas with1.5 cm2per animal.
Some fragments were fixed in 10% buffered formalin solution for
clas-sical histological analysis, immunohistochemistry and confocal
micros-copy for 24 h at room temperature and other selected fragments were
fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde
for electron microscopy for5 days at 48C.
2.4.
|Light microscopy
Sections fixed in buffered formalin were washed in running water for
approximately 1h and the conventional histological processing
tech-nique was performed with resin (Paraplast®). 2lm sections were stained
by hematoxylin-eosin (H-E), periodic acid-Schiff (PAS), Masson’s
tri-chrome, periodic acid silver methenamine staining (PAMS) and
picrossir-ius red using the counter-staining technique with hematoxylin. Images
were obtained through a Nikon® Eclipse Ni trinocular microscope with
Nikon® DS RI1 coupled camera and NIS Elements version 4.2 software.
2.5.
|Circular polarization microscopy
For polarization microscopy, sections stained by picrossirius red
according to the modified technique of Constantine and Mowry
(1968), which did not use counter staining with hematoxylin, were
sub-jected to circular polarization by the Nikon Eclipse CiPol microscope
with Nikon® DS coupled digital camera - Ri2 and use of the NIS
Elements version 4.2 software.
2.6.
|Immunohistochemistry and confocal microscopy
Immunostaining was performed on 2-lm sections, which were briefly
subjected to heating at 968C for 30 min in citrate buffer (10 mmol L21,
pH 6.0) to enhance antigen retrieval. Endogenous peroxidase activity
was blocked with peroxide hydrogen (Dako, USA) for 30 min. Sections
were then incubated for 1 h at room temperature with the primary
antibodies against a-SMA (1:100; Abcam, UK) and anti-vimentin
(1:200; clone V9, Dako, USA). Negative controls included buffer alone
or equivalent concentrations of an irrelevant mouse monoclonal
anti-body of normal IgG. Specific labeling was detected with EnVisionTM/
HRP Detection Kit (Dako, USA) fora-SMA and vimentin. Slides were
washed in PBS and revealed with DAB liquid (Dab1cromogen
substra-te1buffer) (Dako, USA) for 1min. Sections were counterstained in
Mayer’s hematoxylin.
For confocal microscopy, previous steps of slides preparation were
followed by the application ofa-SMA primary antibody. Then, a rabbit
secondary IgG conjugated with Alexa Fluor 6476 was used to detect
a-SMA. Additionally, counterstaining was conducted for the nucleus
with 40-6-diamino-2-phenylindole dilactate (DAPI) 7 and F-actin with
Alexa Fluor 488-phalloidin6. Slides were examined under a confocal
laser scanning microscope (LSM 710). The excitation lines used were
405, 488, and 633 nm, for nuclei (blue) anda-SMA (red), respectively.
The immunoreaction specificity was accessed by omission of the
pri-mary antibody.
2.7.
|Electron microscopy
Kidney tissue fragments were fixed with 4% paraformaldehyde and
2.5% glutaraldehyde. After fixation, samples were washed five times
0.1M PB (53), followed by the addition of 2% osmium solution (in 0.1
MPB) and kept at room temperature in a dark chamber for 1–2 h. Then
samples were washed in cold distilled water for up to 10 min. The
dehydration process was started with a rinsing in 30% alcohol solution
followed by increasing concentrations of ethylic alcohol. After,
frag-ments were washed with 2% uranyl acetate in 70%ethanol solution.
Another step of dehydration was initiated with 70 and 96% ethanol
solution, after which samples were washed with propylene oxide
solu-tion at room temperature and taken for immersion in Araldite resin
(Electron Microscopy Sciences, Fort Washington, PA). For the resin
polymerization samples remained in the oven at 708C for 3 days. Then,
fragments in resin were taken to the ultramicrotome to obtain 1-lm
sections stained by Toluidine blue to select the area to be analyzed.
Thin sections were cut at silver-grey interference color (60–70 nm) and
placed on copper mesh grids. Grids were examined with a FEI—G5
transmission electron microscope, and digital photomicrographs were
obtained.
3
|R E S U L T S
Our results were divided into fibrotic alterations that are present in the
glomerular, tubular, interstitial and vascular compartment of the kidney.
These alterations were chosen since they are commonly found and
well characterized in human kidneys with chronic kidney disease in
resemblance to cats (Table 1).
With the aid of light microscopy, our findings demonstrated
differ-ent degrees of glomerular sclerosis in H–E staining (Figure 1b–e) and
Masson’s trichrome (Figure 1g–j). These lesions were characterized by
the deposition of fibrotic material in the space where the mesangial
cells were located (Figure 1j). Regarding the changes that comprised
the glomerular capsule, our findings were thickening (Figure 1m) and
pericapsular fibrosis (Figure 1n). Another relevant finding in the
ana-lyzed cases was the formation of crescents or synechia where
glomeru-lar tuft adhesion occurs to Bowman0s capsule (Figure 1m). Bowman’s capsule calcification was observed in some kidneys (Figure 1o). Some
sections had thickening of the basal glomerular membrane, confirmed
in PAMS (Figure 1l).
In the tubular compartment, hydropic degeneration was a frequent
finding. Tubules with flattened and vacuolated cytoplasm (Figure 2e)
and irregularity of the basal tubular membrane were observed in PAMS
(Figure 2b). Some tubules with calcification were also observed (Figure
2a). Some tubular cells showed positive immunoblotting for vimentin
(Figure 3f) and tubules epithelial detachment was observed with
intense labeling for vimentin (Figure 3e). It was possible to observe the
integrity of tubules bordered by degenerating tubules with tubular
lumen completely filled by lipid content and foci of lipid deposition
T A B L E 1 Histopathological findings in chronic human and feline kidney disease in the glomerular, tubular, interstitial and vascular segments
Morphological changes Human Feline
Glomerulus Crescents or synechia Vizjak et al., 2003 Chakrabarti et al., 2012
Esclerosis Vizjak et al., 2003; Nanayakkara et al.,
2012; Wijkstr€om et al., 2013; Lopez-Marín et al. 2014
Chakrabarti et al., 2012; McLeland et al., 2015; Brown et al., 2016.
Mesangial proliferation Vizjak et al., 2003;
Wijkstr€om et al., 2013
McLeland et al., 2015
Glomerular tuft collapse Lopez-Marín et al., 2014 Brown et al., 2016
Thickening Bowman’s Capsule Wijkstr€om et al., 2013 McLeland et al., 2015;
Brown et al., 2016
Tubules Hydropic degeneration Lopez-Marín et al., 2014 McLeland et al., 2015; Brown et al., 2016.
Integrity of tubular membrane Wijkstr€om et al., 2013 Brown et al., 2016
Interstice Interstitial fibrosis Jinde et al., 2001; Vizjak et al., 2003; Nanayakkara et al., 2012; Wijkstr€om et al., 2013; Lopez-Marín et al., 2014
Chakrabarti et al., 2012; McLeland et al., 2015; Brown et al., 2016.
Mononuclear inflammatory infiltrate
Vizjak et al., 2003; Nanayakkara et al., 2012; Wijkstr€om et al., 2013; Lopez-Marín et al., 2014
Chakrabarti et al., 2012; McLeland et al., 2015; Brown et al., 2016
Vascular Intimate layer proliferation Lopez-Marín et al., 2014 McLeland et al., 2015
surrounded by inflammatory cells in the interstice near the tubules
(Fig-ure 2e,f).
At electron microscopy it was possible to observed intense
degen-eration and flattening of the epithelium with accumulation in the tubular
lumen of lipid material from the proximal tubular cells (Figure 2h).
Proxi-mal tubular cells had loose chromatin and elongated nuclei. In some
tubules, tubular cells had more evident nucleoli and a very
heterogene-ous nuclear shape in the same tubular segment (Figure 2e). Tubular
degeneration with evident interstitial tubular damage was evidenced in
confocal microscopy where elongated cells, with cytoplasm
immunola-belled bya-SMA, surrounded these tubules (Figure 4a,b).
In the interstitial compartment the most frequent findings were
interstitial fibrosis and inflammatory infiltrate. Interstitial fibrosis was
considered moderate to severe with abundant deposit of extracellular
matrix and collagen (Figure 5a), evidenced in the Masson’s trichrome
and picrossirius red staining (Figure 5b,c, respectively). This deposit
was visualized on circular polarization microscopy where collagen fibers
were refractive (Figure 5d). Collagen deposition has been shown to be
heterogeneous and is predominantly distributed in the renal cortical
region, showing glomerular pericapsular fibrosis, perivascular fibrosis
and interstitial fibrosis. In electron microscopy, areas of interstitial
fibrosis had deposition of collagen with the fibers arranged in circular
beams with striated appearance (Figure 5e,f).
In the immunomodulated sections fora-SMA in confocal
micros-copy, evident interstitial marking of elongated cells surrounding tubules
and the glomerular capsule suggested the presence of myofibroblasts
in these regions (Figure 4c,d). Markings in the glomerular tuft and
vessels were evident and considered a positive internal control of the
reaction (Figure 4a,d). In the immunohistochemistry sections for
a-SMA, interstitial cells with elongated cytoplasm and nucleus
immersed in extracellular matrix from regions of interstitial fibrosis near
tubules had positive marking and appearance similar to that observed
in confocal microscopy (Figure 3a–d). The interstitium was also strongly
marked by vimentin, mainly in areas where there was intense
inflamma-tory infiltrate and fibrosis (Figure 3e).
The inflammatory infiltrate was predominantly mononuclear
(Fig-ure 6d–f) with diffuse distribution, in most cases, in the cortical (Figure
6a,b) and medullary (Figure 6c) regions. Some kidneys with discrete
fibrosis deposition had a discrete and localized infiltrate.
Vascular sclerosis was a consistent finding regarding the vascular
compartment. Both the proliferation of the middle layer and the
forma-tion of concentric collagen bundles around blood vessels were
identi-fied (Figure 7).
4
|D I S C U S S I O N
Dysfunction or direct injury in any glomerular component such as
podocytes, mesangium, endothelium and glomerular basement
mem-brane (GBM) generates a repair response in an attempt to control
dam-age, resulting in sclerosis or glomerulosclerosis. In glomerulosclerosis,
there are changes in the synthesis and deposition of collagen in the
glomerular matrix, as well as direct lesions in the GBM, which modify
the filtration properties and, consequently, the glomerular function as a
whole (Kaissling et al., 2013; Kriz and Le Hir, 2005). As it progresses,
F I G U R E 1 Photomicrographs of cat kidneys with chronic kidney disease. Cortical region from cats showing different stages of glomerulosclerosis, where (a) represents a glomerulus with degree of zero sclerosis and (b–e), respectively, degrees ranging from mild to very severe, HE3400. (f–j) correspond to the same classification previously quoted in HE stained by Masson’s trichrome. Figure (k) shows a glomerulus with severe sclerosis stained with picrossírius red and hematoxylin,3400. In (l), thickening of the glomerular basement membrane can be observed by staining periodic acid methanamine silver,3400. (m) and (n) show the glomerular tuft collapsing evidenced by the arrow, called the synechia, and the thickening of the glomerular capsule by interstitial fibrosis surrounding Bowman’s capsule, HE
3400. Figure (o) shows glomerular calcification, HE340 [Color figure can be viewed at wileyonlinelibrary.com]
glomerular sclerosis shows different degrees of fibrotic material
deposi-tion, demonstrated and confirmed by Masson’s trichrome staining, as
well as rarefaction of the glomerular cellular constituents as visualized
in our study.
Usually the development or progression of renal disease begins
with the separation of the glomerular tuft, generally occurring adhesion
of the tuft to the Bowman’s capsule (parietal epithelium) characterizing
the formation of a crescent or glomerular synechia (Kwoh, Shannon,
Miner, & Shaw, 2006; Reidy and Kaskel, 2007; Kaissling et al., 2013).
This finding was very relevant in our study since some glomeruli
presenting mild degrees of glomerular sclerosis had this change. The
extent of synechia leads to the collapse and sclerosis of the capillary
tuft associated with filtration that is diverted in places where adhesion
occurs, thus instigating the process of established sclerosis (Kriz et al.,
1994; Kwoh et al., 2006).
According to some authors, glomerular lesions are the primary
cause in most renal pathologies that trigger tubular lesions, especially
in dogs and humans (Ahmad, 2015; Cianciolo et al., 2013; Klosterman
et al., 2011; Kopp, 2013; Schneider et al., 2013; Sumnu, Gursu, &
Ozturk, 2015). Most cats with CKD do not present histological
F I G U R E 3 Photomicrographs of cat kidney with chronic renal disease immunostaining fora-SMA and vimentin. In (a), the renal interstitium we observed elongated cells with cytoplasm presenting positive marking toa-SMA immersed in abundant fibrotic matrix,31000. (b–d) demonstrate the different intensities of interstitial staining bya-SMA, where in (b and d) the markings are intense and in discrete (c) in the peritubular compartment,3100,3200,3200, respectively. In (e), the interstitial labeling by vimentin was evident in the cells that make up the inflammatory infiltrate and in (f) some tubules had cells immunostained by vimentin in the cortical region,3200, respectively [Color fig-ure can be viewed at wileyonlinelibrary.com]
evidence of primary glomerular disease. It should be noted that
glomer-ulonephritis by deposition of immunocomplexes and amyloidosis,
con-sidered as primary causes of CKD in cats, may result in chronic
tubulointerstitial lesions (Asproni, Abramo, Millanta, Lorenzi, & Poli,
2013; Brown et al., 2016; Glick, Horn, & Holscher, 1978; Nash, Wright,
Spencer, Thompson, & Fisher, 1979).
Extensive research into the elucidation of glomerular disease role
as a primary trigger of CKD in cats needs to be performed. Tubular
lesions are more relevant in this species since they are closely related
to direct cell damage, hypoxia or ischemia, causing interstitial fibrosis,
thus being characterized as a consistent finding in kidneys of cats with
CKD (Schmiedt, Brainard, Hinson, Brown, & Brown, 2015).
The process of interstitial fibrosis per se perpetuates renal lesions.
Ischemia resulting from interstitial expansion by collagen deposition
leads to poor perfusion and consequent hypoxia in compartments and
surrounding cells. Tubules have an intense metabolic activity, being
more vulnerable to hemodynamic insults (Brown et al., 2016).
During a tubular degeneration, epithelial cells degenerate and
dis-appear long before the dissolution of basal tubular membrane (BTM).
Thus, tubular cells remain isolated from the interstitium until complete
degeneration of BTM, whose removal occur much later (Kaissling et al.,
2013). Injured tubular cells lead to local inflammation in order to
pro-mote repair through fibrosis.
Tubular damages found in our study demonstrated degrees of
severity ranging from lesions of hydropic degeneration and steatosis in
the tubular cells to complete denudation of the tubular basement
mem-brane and detachment of the tubular epithelium with consequent
tubu-lointerstitial fibrosis passing through tubular calcification. According to
F I G U R E 5 Photomicrographs of cat kidneys with chronic kidney disease demonstrating interstitial fibrosis. The deposition of fibrotic material in HE and Masson’s trichrome, respectively,3100 is observed in (a and b). (c) shows a section stained by the picrossirius red and in (d) under circular polarization, where the refraction of collagen fibers is observed in contrast to the dark field,3100. In ultrafine sections of transmission electron microscopy, (e) shows the organization in circular bundles of collagen deposition (2lm bar) and (f) can be observed in greater increase the striation of the collagen fibers (200 nm bar) [Color figure can be viewed at wileyonlinelibrary.com]
Chakrabarti, Syme, & Elliott (2012), cat kidneys with tubular
mineraliza-tion and inflammatory infiltramineraliza-tion had a higher correlamineraliza-tion with severe
interstitial fibrosis than kidneys where mineralization was not present.
The author also associated tubular mineralization with high rates of
glo-merular sclerosis.
Some animals had moderate to severe degrees of TID with tubular
rupture and extravasation of the lipid content of proximal tubular cells,
observed in semi-thin sections stained by toluidine blue and electron
microscopy, obliterating the tubular light or even lodging as pellets in the
interstitium surrounded by inflammatory infiltrate. This finding
corrobo-rates with McLeland, Cianciolo, Duncan, & Quimby (2015) and Brown
et al. (2016) when describing the presence of chronic granulomatous
inflammation adjacent to foci of interstitial lipid deposition in
spontane-ous CKD in cats with severe TID, resulting from multiple ischemic injuries.
In our study, cells immunolabelled bya-SMA and vimentin were
observed, circumferential to the tubules that had epithelium with
F I G U R E 6 Photomicrographs of the characterization of inflammatory infiltrates in cat kidneys with chronic renal disease. (a and b) show the distribution and intensity of inflammatory infiltration in the renal cortical region, picrossirius red stained with hematoxylin and Masson’s Trichrome (3100) and (c) in the medular region, HE3100. In (d), the predominance of mononuclear cells such as plasma cells and
alterations and loss of glomerular basement membrane integrity,
sug-gesting the presence of myofibroblasts. The massive accumulation of
myofibroblasts around degenerate tubules is a hallmark of chronic renal
injury (Kaissling et al., 2013). Elongated cells immunolabelled bya-SMA
and vimentin were also encapsulated in an abundant interstitial matrix
reaffirming the possible involvement of myofibroblasts in interstitial
fibrosis secondary to TID.
In a study conducted by Yabuki et al. (2010) the induction and
proliferation of myofibroblasts in feline CKD using a-SMA and
vimentin as markers of these cells was demonstrated. They may
orig-inate from resident peritubular fibroblasts or through the process of
epithelial-mesenchymal transition (EMT) and express a-SMA that
characterize them as contractile smooth muscle cells (Sandbo and
Dulin, 2011).
F I G U R E 7 Photomicrographs of cat kidneys with chronic kidney disease demonstrating vascular sclerosis. In (a and b) it is possible to observe the proliferation of the middle layer, HE and Masson’s trichrome,340 and320, respectively. The formation of concentric bundles of perivascular collagen is observed in (e) (3400) and (f), picrosirius red stained by hematoxylin3400 and transmission electron microscopy 2lm bar. (c and d) reinforce the deposit of collagen by picrossirius red under circular polarization,3100 [Color figure can be viewed at wileyonlinelibrary.com]
Some tubular cells were positively labeled for vimentin and
nega-tively labeled for a-SMA. Vimentin is a cytoskeletal protein not
expressed in epithelial cells. Tubular expression of vimentin was
signifi-cantly correlated with the degree of interstitial fibrosis in humans (Jinde
et al., 2001) and in cats, thus demonstrating the capacity of
transforma-tion in myofibroblasts in interstitial fibrosis and TID as a factor of
severity in feline CKD (Yabuki et al., 2010; Chakrabarti et al., 2012).
This tubular positive immunostaining previously cited is correlated
with the mesenchymal epithelial transition (MET) and the participation
of proximal tubular cells in the interstitial fibrosis process through
notypic modification and activation of genes of the mesenchymal
phe-notype, in detriment to genes of the epithelial phephe-notype, is
questioned and neglected (Grgic, Duffield, & Humphreys, 2012). For
some researchers MET is a reality in kidneys that undergo persistent
chronic injury under the action of inflammatory cytokines that
stimu-late this process, such as the transforming growth factor beta-1
(TGF-b1) (Moll et al., 2013; Zhou, Ma, Lin, & Qin, 2014).
Fibrosis results from persistent inflammatory triggers that produce
inflammation, tissue destruction and repair processes simultaneously
(Hutchison et al., 2013). Severe mononuclear infiltration associated
with areas of fibrosis, tubular damage and interstitial lipid deposition
shows the inflammation triggered in kidneys areas analyzed in our
study. The recruitment of inflammatory cells, mainly macrophages, is
related to the release of proinflammatory factors and profibrotic
cyto-kines, such as TGF-b, which perpetuate the process (Brown et al.,
2016; Chakrabarti et al., 2012; Hutchison et al., 2013; Lech and
Anders, 2013). This mediator, among others, allows local mesenchymal
cells to transform cells expressinga-SMA and producing ECM,
charac-teristics of myofibroblasts.
When inflammation is chronic myofibroblasts continuously
synthe-size ECM, leading to the formation of fibrotic scarring in the kidney.
Although some cell types have the potential to produce and secrete
ECM components such as epithelial and endothelial cells and
leuko-cytes, it is the myofibroblasts that produce the pathogenic fibrous
col-lagen observed in all forms of fibrosis (Hinz et al., 2012; Liu, 2006).
The presence of vascular sclerosis with proliferation of the middle
layer and the formation of concentric bundles of collagen around blood
vessels was observed in our study. Atherosclerosis is an aggravating
factor in CKD, which can lead to renal ischemia and subsequent
tubulo-interstitial damage (Nangaku, 2006).
Thus, we may conclude that fibrotic lesions play a relevant role in
glomerular, tubular, interstitial and vascular compartments in CKD in
cats. The characterization of fibrotic lesions present in samples
ana-lyzed in our study corroborate with the literature and reinforce the
need for further elucidation of processes such as
epithelial-mesenchymal transition and its components, such as the origin and
par-ticipation of myofibroblasts, which lead to the progression of CKD and
fibrosis in cats.
A C K N O W LE D G M E N T S
We thank Fundaç~ao Nucleo de Tecnologia Industrial do Ceara
(NUTEC), Comiss~ao de Aperfeiçoamento de Pessoal do Nível Superior
(CAPES), Conselho Nacional de Pesquisa (CNPq) and Fundaç~ao
Cear-ense de Apoio ao Desenvolvimento Científico e Tecnologico
(FUN-CAP), Universidad de Valencia and Universidad Cardenal Herrera for
providing all resources necessary for this study development.
CO N F L I CT S O F I N T E RE S T
The authors of this article declare that there is no potential conflicts of
interest including employment, consultancies, stock ownership,
honora-ria, paid expert testimony and patent applications/registrations related
to the current manuscript. This manuscript is submitted on behalf of all
authors.
O RC I D
G. B. Morais http://orcid.org/0000-0002-3627-7939
F. A. F. Xavier Junior http://orcid.org/0000-0002-2635-1306
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How to cite this article:Morais GB, Viana DA, Verdugo JM, et al. Morphological characterization of ckd in cats: Insights of
fibrogenesis to be recognized.Microsc Res Tech. 2017;00:1–12.
https://doi.org/10.1002/jemt.22955
