Polarization microscopy as a tool for quantitative evaluation of collagen using picrosirius red in different stages of CKD in cats

R E S E A R C H A R T I C L E

Polarization microscopy as a tool for quantitative evaluation of

collagen using picrosirius red in di

_ff

erent stages of CKD in cats

G. B. Morais

_|

_{D. A. Viana}

_|

_{F. M. O. Silva}

_|

_{F. A. F. Xavier J}

_unior

_|

K. M. Farias

_|

_{C. D}

_’

_{O Pessoa}

_|

_{J. A. M. Silveira}

_|

_{A. P. N. N. Alves}

_|

M. R. L. Mota

_|

_{F. D. O. Silva}

_|

_{C. M. S. Sampaio}

_|

_{J. M. G. Verdugo}

_|

J. S. A. M. Evangelista

1_{Faculdade de Veterinaria, Universidade}

Estadual do Ceara, Ceara, Brazil

2_{Departamento de Fisiologia e Farmacologia,}

Universidade Federal do Ceara, Ceara, Brazil

3_{Faculdade de Farmacia Odontologia e}

Enfermagem, Universidade Federal do Ceara, Ceara, Brazil

4_{Departamento de Geologia, Universidade}

Federal do Ceara, Ceara, Brazil

5_{Centro de Ci^encias da Sa}

ude, Universidade Estadual do Ceara, Ceara, Brazil

6_{Instituto Cavanilles Universidad de}

Valencia, Valencia, Brazil

Correspondence

G. B. Morais, Programa de Pos-graduaç~ao em Ci^encias Veterinarias, Faculdade de Veterinaria, Universidade Estadual do Ceara, Av. Dr. Silas Munguba, 1700, Campus Itaperi 60740-903, Fortaleza, Ceara, Brazil.

E-mail: [email protected]

Abstract

Chronic kidney disease (CKD) is a relevant disease in feline clinic. The tubulointerstitial damage,

with collagen deposition and_fibrosis, is an important result of this process. The aim of this study

was to quantify and correlate the deposition of collagen and severity of interstitialfibrosis (IF) in

the kidney from cats in di_fferent stages of CKD. Kidney fragments from 10 adult cats with CKD

were analyzed and stained by Masson’s trichrome (MT) and Picrosirius red (PSR) for circular

polar-ized microscopy. Random quantitative analysis was performed on MT sections to classify the

degree of IF, perfield area, with and without circular polarization. Statistics correlations were

per-formed by Spearman_’s (q;p<.05). There was a significant correlation of IF quantification with the

area of interstitial collagen deposition by polarized PSR (PSRp) (r5.7939,p5.0098) and

nonpo-larized PSR (PSRn) (r5.7781,p5.0080). There was a positive correlation of serum creatinine

(sCr) at different stages of CKD with PSRp (r5.7939,p5.0098), PSRn (r5.8667,p5.0027)

and MT (r5.7818,p5.0117). Correlations between the percentage of quanti_fied area was also

positive from PSRp to PSRn (r5.9030,p5.0009) and PSRp to MT (r5.7939,p5.0098). The

PSRN was also correlated with MT (r5.9273,p5.0001). The correlation with IF and sCr follows

the disease evolution and the quantification of collagen by PSR is an excellent tool for analyzing

the disease severity at di_fferent stages.

K E Y W O R D S

chronic kidney disease, feline,fibrosis, picrosirius red, kidney

1

_{I N T R O D U C T I O N}

Chronic kidney disease (CKD) is one of the main illnesses that

culmi-nate in death in cats (Egenvall et al., 2009). Progressive kidney diseases

lead to a_fibrosis process that deprives the body of its function and

promotes consequent decline in kidney function (Eddy, 2000; Hakim &

Lazarus, 1989; Prunotto et al., 2011). The process is irreversible,

lead-ing inevitably to end-stage renal disease (ESRD) (Chakrabarti, Syme,

Brown, & Elliott, 2012; Hewitson, 2012; Yabuki et al., 2010).

Chronic kidney disease is common in older cats (Bartges, 2012;

Marino et al., 2014.). The age con_figures, in this sense, as one of the

possible factors involved in the establishment of CKD in this species

(Brown, Elliott, Schmiedt, & Brown, 2016). Senility leads to a decrease

in the number of nephrons and consequent decrease in organ function

as a whole. It is worth noting that the degree of functional impairment

does not always re_flect the structural loss (Bartges, 2012).

Correlating senility with the development of interstitialfibrosis in

kidneys from senile cats requires research efforts in a molecular level,

to demonstrate their direct contribution to the tubular cell death,

in_flammation and_fibrosis process, that are not yet fully clari_fied in cats

(Brown, Elliott, Schmiedt, & Brown, 2016; Lawler et al. 2006).

Tradi-tionally, trichrome stains have been used to detect collagen_fiber in

tis-sue sections from kidneys with interstitial_fibrosis. However, there is a

lack of selective precision, failing to reveal collagen with very_fine_fibers Review Editor: Prof. Alberto Diaspro

as it might be (Rich & Whittaker, 2005; Whittaker & Canham, 1991).

One explanation for this under-utilization is the inability to visualize

all types of collagen when the polarized light is linearly used, since

fibers or their portions appear dark when in parallel alignment to the transmission axis of any polarizationfilter. In contrast, the use of

cir-cularly polarized light eliminates this problem and enables the

visual-ization of each fiber portion (Rich & Whittaker, 2005; Whittaker

et al., 1994).

Fibrotic lesions are considered relevant and present in di_fferent

stages of chronic kidney disease in cats. Nowadays there is no e_ffective

treatment to control or slow the progression of_fibrotic process in cats

and, consequently, delay the disease progression. Several e_fforts have

been made to provide a better understanding of the renal interstitial

fibrosis and control its development process; althoughfibrotic lesions in feline CKD have been described, there are no studies quantifying

the collagen deposition under polarized light and correlating these

lesions with the disease stages. The aim of this study was to quantify

and correlate the deposition of collagen and severity of interstitial

fibrosis in kidneys from cats at different stages of CKD.

2

M A T E R I A L A N D M E T H O D S

2.1

_{Experimental animals}

The experimental group consisted of 10 cats of both sexes, being 6

males and 4 females, aged between 5 and 15 years (7.96_{1.2 years)}

and evaluated in the Veterinary Hospital from Universidade Estadual

do Ceara, Ceara, Brazil from 2014 to 2015. The inclusion criteria were:

history of kidney disease through clinical, laboratory, and imaging

eval-uation, with death during that period (not necessarily due to kidney

dis-ease), not presenting any evidence of acute kidney disease, neoplasia,

and ureter/urethral obstruction.

Cats selected in this study were treated animals that died during

the study period, having a clinical history compatible with CKD and

which have different causes of death, such as pancreatitis, poisoning,

among others, which were sent for necropsy.

The calculation was performed using the sample calculation for

qualitative variable (incidence of CKD520%), considering a_finite

pop-ulation of animals, evaluated during a period of time, that had

consist-ent changes with CKD. The sample size was calculated as follows:

n5 N :ðp: qÞ: Z/=2

N2₁

ð Þ:E21_{ðp: qÞ: Z} /=2

All data were anonymously analyzed and a written consent was signed

by the animals’owners.

2.3

Sample preparation

Both kidneys were collected and measured after the animals’death. A

small representative sample from the renal cortex of the right kidney,

in full cross section of about 1.5 cm2_{, was collected,fixed in 10%}

buf-fered formalin and processed by standard histological processing

tech-niques. Sections (2mm) were stained by Masson’s Trichrome (MT) and

Picrosirius red (PSR) modified technique by Constantine and Mowry

(1968).

2.4

Quantitative analysis of interstitial

_fi

brosis scores

A random quantitative analysis by semi-quantitative scoring system

was performed in 20 random fields by an experienced pathologist

(Viana, D A), according to the methods previously established by Raij,

Azar, & Keane (1984). Sections stained by MT were analyzed to classify

the interstitialfibrosis degree. Lesions were classified by severity score

from 0 to 4, where 0 5no interstitial fibrosis; 15 mild (interstitial

fibrosis within 25% of the area from the image capturing field); 25moderate (interstitialfibrosis from 26 to 50%field); 35severe

(interstitial therefore, the total score quantification of fibrosis is

per-formed by the following equation:

Total score5 n:1

1n:21n:31n:4

ð Þ

20

wherenrepresents the number of_fields that received the respective

score for each slide.

This quantification method is supported by quantitation studies for

scores (Gibson-Corley, Olivier, & Meyerholz et al., 2013) and quantifi

-cation of interstitialfibrosis according to the described by Yabuki et al.

(2010) and Sanchez-Lara, Elliott, Syme, Brown, and Haylor (2015).

Analyses were performed on 20 randomfields (1003) under a

trinocu-lar microscope (Nikon® Eclipse Ni with a Nikon® DS RI1 camera

attached).

2.5

Digital and quantitative analysis by image area

The quantitative analysis of cortical renal interstitial fibrosis perfield

area was performed according to a modified technique by Encarnacion

et al. (2004). The morphometric study of tissue sections stained by MT

stained by PSR were analyzed separately under polarized and

nonpo-larized light.

Randomfields (n510) from the cortical area of each slide stained

by PSR (1003) were captured, with and without circular polarization

(Nikon Eclipse Cipol microscope with an attached Nikon DS-Ri2 digital

camera). Digital images of histological sections stained by MT were

captured in a standardized way (1003) through a trinocular microscope

(Nikon®_{Eclipse Ni with an attached Nikon}®_{DS RI1camera). Random}

fields (n510) were captured from the kidney of each animal (1003). Captured images (TIFF format; 160831608 pixels, corresponding

to 11 MB), were used to select regions of interest (ROI), corresponding

to tubulointerstitial_fibrosis, excluding tubular, vascular, and glomerular

epithelium. An initial arithmetic subtraction of the background lighting

was performed and images were readjusted to a RGB Stack Model

Software. Then these images were adjusted by the threshold tool, and

areas related to interstitial _fibrosis were delimited, with subsequent

quantification of total collagen per area in each analyzedfield.

The total area of each ROI was calculated and expressed as a

per-centage of the total image area. The perper-centage of the total area

quan-ti_fied in 10 random _fields for each kidney from each animal was

obtained according to the mathematical formula: (Rareas quantified%/

10). All measurements derived from the analysis were automatically

transferred to a Microsoft Excel spreadsheet and subjected to

statisti-cal analysis.

The total area of each ROI was calculated and expressed as a

per-centage of the total image area. The perper-centage of the total area

quan-tified in 10 randomfields for each kidney of each animal was obtained

according to the mathematical formula: (Rareas quanti_fied%/10). All

measurements derived from the analysis were automatically

trans-ferred to a Microsoft Excel spreadsheet and statistical analysis was

performed.

2.6

Picrosirius polarized (PSRp)

The aim was to distinguish the birefringent (bright) from the

birefrin-gent (dark) materials. Captured color images were binarized (black and

white). Birefringent regions, which correspond to positive staining,

were converted to white. A threshold was set in black areas were

excluded and the remaining volume of the white area was quantified in

relation to the capturedfield area (Figure 1).

2.7

Picrosirius nonpolarized (PSRn)

The aim was to distinguish positively stained material (red) from

unstained one (yellow) and tubular regions (lumen/empty space; white)

of the staining within the ROI. Captured color images were binarized

(black and white) where the stained material was positively constituted

of darker regions. Therefore, the areas to be quantified were adjusted

and the percentage quantification of the delimited area was applied, in

relation to the total area of the capturedfield (Figure 1).

2.8

Statistical analysis

GraphPad PRISM® _{v6.0 (GraphPad Software, CA, USA) and Action}®

v2.7 software were used. Statistical analysis was performed using the

Spearman’s rank correlation coefficient and results were expressed by

3

R E S U L T S

3.1

Staging of chronic kidney disease (CKD) by the

international renal interest society (IRIS)

All cats from this study were staged for CKD by serum creatinine (sCr)

according to the IRIS staging 1 to 4. In stage 1, animals have sCr values

below 1.6 mg/dL, associated with image changes or clinical history

sug-gestive of chronicity for kidney disease. One animal was classi_fied at

this stage, with sCr51.2 mg/dL and ultrasonographic image indicating

loss of corticomedullary definition. Animals belonging to stage 2

(sCr 5 _{1.6–2.8 mg/dL) had sCr} 5 _{1.8 and 2.1 mg/dL. In stage 3}

(sCr52.9_–5.0 mg/dL) 2 cats were also ranked, with sCr53.0 and

4.0 mg/dL and ultrasound images showing irregularities of renal cortex

surface. In stage 4, sCr levels exceed 5.0 mg/dL; 5 cats were classified

at this stage, with sCr values ranging from 6.0 to 14.0 mg/dL (Table 1).

3.2

Quantitative analysis of interstitial

_fi

brosis and

correlations

Interstitial_fibrosis scores were assigned to kidneys at di_fferent stages

of CKD (Figure 2). The degree of interstitial_fibrosis followed the stage

in the samples analyzed, being possible to observe that the _fibrosis

area, and consequently the deposition of collagen, is larger in more

advanced stages.

5 3 4.0 285.0 18.591 6.839 22.140 1

6 3 6.0 290.0 17.201 9.758 14.885 1 1 1

7 2 2.1 100.0 8.086 3.810 5.916 1

8 2 1.8 117.5 6.830 2.853 3.337 1

9 3 3.0 255.0 11.948 6.197 8.217 1 1

10 1 1.2 100.0 10.435 3.564 11.706 1

Quantitative analysis by interstitial _fibrosis scores in sections

stained by MT was correlated with the percentage of the interstitial

collagen deposition total area, highlighted by PSRp and PSRn. Total

quanti_fication values of each animal is shown in Table 1. There was a

signi_ficant correlation between semiquantitative quanti_fication by

interstitial _fibrosis scores with quanti_fication by deposition area of

interstitial collagen by PSRp (r 5 .7939, p 5 .0098), and PSRn

(r5.7781,p5.0080) (Figures 3 and 4, respectively).

Correlations of sCr quanti_fication with total area of collagen

depo-sition in sections stained by PSR under and without polarization were

described in the correlation matrix (Figure 3). There was a positive

cor-relation of sCr with PSRp (r5.7939,p 5.0098), PSRn (r5.8667,

p5.0027) and MT (r5.7818,p5.0117). There was a positive

corre-lation between PSRp and PSRn (r5.9030,p5.0009) and PSRP and

TM (r5.7939,p5 .0098). The PSRn was also correlated with MT

(r5.9273,p5.0001).

4

_{D I S C U S S I O N}

Interstitialfibrosis has been positively correlated with serum creatinine

in cats diagnosed with CKD and staged based on these values

accord-ing to IRIS (Chakrabarti et al., 2012; Yabuki et al., 2010). The stagaccord-ing

based on the sCr according to IRIS is as an important tool for

establish-ing the diagnosis, treatment, and prognosis of CKD in cats (Boyd,

Lang-ston, Thompson, Zivin, & Imanishi, 2008; Iris, 2015; Polzin, 2013). Cats

evaluated on this study had their sCr measured to be classified in

stages as recommended by IRIS. With this step completed, analysis and

assignment scores for interstitialfibrosis were initiated, correlating the

stage with histopathological_findings.

The most common morphologic diagnosis in cats with CKD is

chronic tubulointerstitial nephritis and interstitialfibrosis (Chakrabarti

et al., 2012; Di Bartola, Rutgers, Zack, & Tarr, 1987; Lawler et al.,

2006). Our histologicalfindings showed tubulointerstitial nephritis and

degrees offibrosis with collagen deposition accompanying the stages

according to IRIS.

In stage 1, interstitial_fibrosis was considered from absent to mild

and, in thefinal stages as 3 and 4, scores ranged from severe, in most

fields analyzed, to very severe, present even in a fewfields observed. These _findings corroborate the description by Polzin (2013) and

McLeland, Cianciolo, Duncan, and Quimby (2015) where the severity

of interstitial _fibrosis was signi_ficantly higher in advanced stages of

CKD when compared to early stages. Interstitial fibrosis leads to an

increased production of extracellular matrix and collagen deposition in

areas that have su_ffered injury and subsequent repair (Farris et al.,

2011; Hewitson, 2009; Hewitson, Darby, Bisucci, Jones, & Becker,

1998; Lawson, Elliott, Wheeler-Jones, Syme, & Jepson, 2015; S

anchez-Lara et al. 2015; Wynn, 2010).

There was a positive correlation between sCr and quanti_fication

per total area of collagen deposition in interstitial fibrosis regions, in

sections stained by PSRp (r5.7939,p5.0098) and PSRn (r5.8667,

p5.0027). This indicates that the degree of tissue damage, resulting in

repair (fibrosis), follows the stages, and severity of the disease. Similar

results were also demonstrated by Encarnacion et al. (2004) when

cor-relating the quantification offibrosis area in digital images in CKD from

transplanted human biopsies.

The correlation of sCr with PSR also showed good accuracy of the

method to detect deposition and consequent amount of collagen in

areas where_fibers are thin. Constantine and Mowry (1968) reported

that sections stained by PSR under polarization allowed to distinguish

and highlight thin_fibers from collagen deposition in_fibrotic regions, as

well as to increasefiber refrangibility in polarization. Thus, polarization

of sections stained by PSR enhanced the detection of collagen_fibers

not visualized when the PSR is not polarized, increasing the

visualiza-tion definition of areas with collagen deposition.sCr values correlated

with MT was also positive (r5.7818,p5.0117), although the best

correlation has been in PSRn sections (r5.8667,p5.0027). This is

justi_fied by the fact that PSR, under polarization, de_fined the deposited

collagen more precisely in the stained area. The PSR staining technique

used in this study involves differentiation in two colors, where the

interstitial _fibrosis in routine histological analysis to highlight the

fibrotic area in different colors from other structures (Montes & Jun-queira, 1991).

Although MT usually intensely stain collagen fibers, other areas

like renal tubules and structures containing collagen, such as reticular

fibers and the basal membrane, are not selectively stained, not allowing the differentiation of these structures during quantification, particularly

when there are very thin collagenfibers, which can have different

col-ors in the same tissue section (Constantine & Mowry, 1968; Montes &

Junqueira, 1991; Street et al., 2014; Taboga & Vidal, 2003). Another

aspect to be emphasized is that MT might result in a limited method

for the quanti_fication of_fibrosis in the area, since the di_fferentiation of

fibrosis colored blue with a background dyed stained red often

Valentine, 1973; Wolman, 1975). The intensity of birefringence by

Sir-ius red is superior when compared to other staining techniques to

col-lagen, since its molecule binding within the upper groove of collagen

types I and IIIfibrils increases its natural birefringence. When viewed

under contrast polarization, collagen is bright on a dark background,

thus promoting a better quanti_fication (Junqueira, Cossermelli, &

Bren-tani, 1978).

The positive correlation between the PSRp and PSRn (r5.9030,

p5_{.0009) demonstrated that the polarization confirmed the}

demarca-tion of collagen area, visualized in nonpolarized secdemarca-tions. Polarizademarca-tion

favored the best collagen deposition visualization in areas

correspond-ing to interstitial _fibrosis, thus allowing quanti_fication per area with

better detailing. The quantitation per area allows to correlate the

severity of tubulointerstitial damage and repair with the degree of

injury and, consequently, with the severity and stage of the disease.

It is noteworthy that, in clinical practice, diseases withfibrotic

pro-gress are a major challenge since e_ffective anti_fibrotic agents are not

yet widely studied and also little used in cats (Razzaque & Taguchi,

2002).

In the face of our results it is possible to conclude that correlation

between interstitial fibrosis and sCr follows the disease evolution.

Quantification of collagen can be an excellent tool for analyzing the

severity of interstitial tubule damage at di_fferent stages of CKD and

PSR is a good option in this process. Our results validate the use of

PSR in the nonpolarized and polarized light microscopy for this purpose

and, additionally, emphasize the importance of evaluating a larger

num-ber of samples.

A C K N O W L E D G M E N T S

We thank Fundaç~ao Nucleo de Tecnologia Industrial do Cear a

(NUTEC), Comiss~ao de Aperfeiçoamento de Pessoal do Nível

Supe-rior (CAPES), Conselho Nacional de Pesquisa (CNPq) and Fundaç~ao

Cearense de Apoio ao Desenvolvimento Científico e Tecnologico

(FUNCAP) for providing all resources necessary for this study

development.

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