DIRECT RED 80 – Hoechst 33342 Chemical

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

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 interstitial ﬁbrosis (IF) in the kidney from cats in diﬀerent 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, per ﬁeld 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) (r 5 .7939, p 5 .0098) and nonpo- larized PSR (PSRn) (r 5 .7781, p 5 .0080). There was a positive correlation of serum creatinine (sCr) at diﬀerent stages of CKD with PSRp (r 5 .7939, p 5 .0098), PSRn (r 5 .8667, p 5 .0027) and MT (r 5 .7818, p 5 .0117). Correlations between the percentage of quantiﬁed area was also positive from PSRp to PSRn (r 5 .9030, p 5 .0009) and PSRp to MT (r 5 .7939, p 5 .0098). The PSRN was also correlated with MT (r 5 .9273, p 5 .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 diﬀerent stages. 1| INTRODUCTION 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 conﬁgures, in this sense, as one of thepossible 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 reﬂect the structural loss (Bartges, 2012).Correlating senility with the development of interstitial ﬁbrosis in kidneys from senile cats requires research eﬀorts in a molecular level, to demonstrate their direct contribution to the tubular cell death, inﬂammation and ﬁbrosis process, that are not yet fully clariﬁed in cats (Brown, Elliott, Schmiedt, & Brown, 2016; Lawler et al. 2006). Tradi- tionally, trichrome stains have been used to detect collagen ﬁber in tissue sections from kidneys with interstitial ﬁbrosis. However, there is athat can, under certain circumstances, lead to a substantial underesti- mation of the collagen content in the analyzed sample (Kiernan, 2002; Whittaker, Kloner, Boughner, & Pickering, 1994).Polarized light microscopy is a powerful tool to study the ﬁbers orientation in tissues containing ﬁbrillar collagen (Changoor et al., 2011). The use of picrosirius red stain under polarized light micros- copy for detection and analysis of collagen was ﬁrst described by a Brazilian group (Junqueira, Bignolas, & Brentani, 1979; Vidal, Mello, & Pimentel, 1982). Despite the speciﬁcity conferred by the combina- tion of color and polarized light, the method is not as fully explored 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 polarization ﬁlter. 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 diﬀerent stages of chronic kidney disease in cats. Nowadays there is no eﬀective treatment to control or slow the progression of ﬁbrotic process in cats and, consequently, delay the disease progression. Several eﬀorts have been made to provide a better understanding of the renal interstitial ﬁbrosis and control its development process; although ﬁbrotic 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| MATERIAL AND METHODS The experimental group consisted of 10 cats of both sexes, being 6 males and 4 females, aged between 5 and 15 years (7.9 6 1.2 years) and evaluated in the Veterinary Hospital from Universidade Estadual do Cear´a, Cear´a, 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 CKD 5 20%), 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 where n: sample size; N: population size; p: proportion of favorable results in the population; q: proportion of unfavorable results in the population; Za/2: Critical value for the conﬁdence level (95% 5 1.96); E: absolute precision (5%).This study was approved by the Ethics Committee for Animal Use of Universidade Estadual do Ceara´, according to the principles of the Bra- zilian College of Animal Experimentation (Memo no. 11585871-7/10). All data were anonymously analyzed and a written consent was signed by the animals’ owners.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 (2 mm) were stained by Masson’s Trichrome (MT) and Picrosirius red (PSR) modiﬁed technique by Constantine and Mowry (1968).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 interstitial ﬁbrosis degree. Lesions were classiﬁed by severity score from 0 to 4, where 0 5 no interstitial ﬁbrosis; 1 5 mild (interstitial ﬁbrosis within 25% of the area from the image capturing ﬁeld); 2 5 moderate (interstitial ﬁbrosis from 26 to 50% ﬁeld); 3 5 severe (interstitial therefore, the total score quantiﬁcation of ﬁbrosis is per- formed by the following equation:Total score5 ðn:11n:21n:31n:4Þ20where n represents 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 interstitial ﬁbrosis according to the described by Yabuki et al. (2010) and S´anchez-Lara, Elliott, Syme, Brown, and Haylor (2015). Analyses were performed on 20 random ﬁelds (1003) under a trinocu- lar microscope (Nikon® Eclipse Ni with a Nikon® DS RI1 camera attached).The quantitative analysis of cortical renal interstitial ﬁbrosis per ﬁeld area was performed according to a modiﬁed technique by Encarnacion et al. (2004). The morphometric study of tissue sections stained by MTstained by PSR were analyzed separately under polarized and nonpo- larized light.Random ﬁelds (n 5 10) 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 (n 5 10) were captured from the kidney of each animal (1003).Captured images (TIFF format; 1608 3 1608 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 analyzed ﬁeld.The total area of each ROI was calculated and expressed as a per- centage of the total image area. The percentage of the total area quan- tiﬁed in 10 random ﬁelds for each kidney from each animal was obtained according to the mathematical formula: (R areas 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 percentage of the total area quan- tiﬁed in 10 random ﬁelds for each kidney of each animal was obtained according to the mathematical formula: (R areas quantiﬁed%/10). All measurements derived from the analysis were automatically transferred to a Microsoft Excel spreadsheet and statistical analysis was performed.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 captured ﬁeld area (Figure 1).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 captured ﬁeld (Figure 1).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 the Spearman’s correlation coeﬃcient (q), considering p < .05. 3| RESULTS 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 classiﬁed at this stage, with sCr 5 1.2 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(sCr 5 2.9–5.0 mg/dL) 2 cats were also ranked, with sCr 5 3.0 and4.0 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).Interstitial ﬁbrosis scores were assigned to kidneys at diﬀerent 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.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 quantiﬁcation values of each animal is shown in Table 1. There was a signiﬁcant correlation between semiquantitative quantiﬁcation by interstitial ﬁbrosis scores with quantiﬁcation by deposition area of interstitial collagen by PSRp (r 5 .7939, p 5 .0098), and PSRn(r 5 .7781, p 5 .0080) (Figures 3 and 4, respectively).Correlations of sCr quantiﬁcation 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 (r 5 .7939, p 5 .0098), PSRn (r 5 .8667,p 5 .0027) and MT (r 5 .7818, p 5 .0117). There was a positive corre- lation between PSRp and PSRn (r 5 .9030, p 5 .0009) and PSRP and TM (r 5 .7939, p 5 .0098). The PSRn was also correlated with MT (r 5 .9273, p 5 .0001). 4| DISCUSSION Interstitial ﬁbrosis 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 staging 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 interstitial ﬁbrosis were initiated, correlating the stage with histopathological ﬁndings. The most common morphologic diagnosis in cats with CKD is chronic tubulointerstitial nephritis and interstitial ﬁbrosis (Chakrabarti et al., 2012; Di Bartola, Rutgers, Zack, & Tarr, 1987; Lawler et al., 2006). Our histological ﬁndings showed tubulointerstitial nephritis and degrees of ﬁbrosis with collagen deposition accompanying the stages according to IRIS.In stage 1, interstitial ﬁbrosis was considered from absent to mild and, in the ﬁnal stages as 3 and 4, scores ranged from severe, in most ﬁelds analyzed, to very severe, present even in a few ﬁelds observed. These ﬁndings corroborate the description by Polzin (2013) and McLeland, Cianciolo, Duncan, and Quimby (2015) where the severity of interstitial ﬁbrosis was signiﬁcantly 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 suﬀered 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 quantiﬁcation per total area of collagen deposition in interstitial ﬁbrosis regions, in sections stained by PSRp (r 5 .7939, p 5 .0098) and PSRn (r 5 .8667, p 5 .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 of ﬁbrosis 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 increase ﬁber 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 (r 5 .7818, p 5 .0117), although the best correlation has been in PSRn sections (r 5 .8667, p 5 .0027). This is justiﬁed by the fact that PSR, under polarization, deﬁned the deposited collagen more precisely in the stained area. The PSR staining technique used in this study involves diﬀerentiation in two colors, where the ﬁbrotic interstitium is clearly marked in red and the other structures in a pale yellow. Trichrome staining has been used for visualization of 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 collagen ﬁbers, 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 quantiﬁcation of ﬁbrosis in the area, since the diﬀerentiation of ﬁbrosis colored blue with a background dyed stained red often becomes a challenge to distinguish pure colors observed (Street et al., 2014).PSR staining has been used as a speciﬁc method for collagen detection in tissue sections (Junqueira et al., 1979). In renal tissue, PSR has been used to quantify ﬁbrotic lesions and tubulointerstitial ﬁbrosis (Moreso et al., 1994). Sirius red F3BA was ﬁrst used as a histological technique in 1964 as a substitute for fuchsin in the Van Gieson Method (Sweat, Puchtler, & Rosenthal, 1964). The tynctorial aﬃnity of Sirius red prepared with picric acid allows very thin ﬁbers to appear bright yellow when viewed under polarization (Puchtler, Waldrop, & 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 III ﬁbrils increases its natural birefringence. When viewed under contrast polarization, collagen is bright on a dark background, thus promoting a better quantiﬁcation (Junqueira, Cossermelli, & Bren- tani, 1978). The positive correlation between the PSRp and PSRn (r 5 .9030, p 5 .0009) demonstrated that the polarization conﬁrmed the demarca- tion of collagen area, visualized in nonpolarized sections. Polarization favored the best collagen deposition visualization in areas correspond- ing to interstitial ﬁbrosis, thus allowing quantiﬁcation 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 with ﬁbrotic pro- gress are a major challenge since eﬀective antiﬁbrotic 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 diﬀerent 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 number of DIRECT RED 80 samples.