Apoptosis in kidney tissue of senior and geriatric cats with chronic kidney disease

Abstract

Apoptosis, an important pathological event associated with kidney disease progression, is expected to be a therapeutic target in chronic kidney disease (CKD). However, its role in naturally occurring CKD in aged cats remains unclear. Therefore, here, we investigated kidney tissues from aged cats (≥10 years) with or without azotemic CKD to evaluate apoptotic events using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The positive TUNEL signals of the renal cells were quantified and statistically analyzed for correlation with the severity of plasma creatinine (pCre) concentration, renal lesions (glomerulosclerosis, interstitial cell infiltration, peritubular capillaries, and interstitial fibrosis), and oxidative damage of the kidney tissue. Oxidative damage was evaluated using immunohistochemistry for 8-hydroxy-2’-deoxyguanosine (OHdG) and 4-hydroxynonenal (HNE). In the TUNEL assay, regardless of azotemia, positive nuclear signals were observed in the tubular epithelial and intraluminal cells, interstitial infiltrating cells, and glomerular cells. Quantitative TUNEL scores showed no significant differences between the azotemic and non-azotemic groups in any compartment of the kidney tissues. In the azotemic group, TUNEL scores did not correlate with pCre or renal lesion severity. However, the scores showed a significant positive correlation with the scores of 8-OHdG and 4-HNE. These findings suggest that apoptosis associated with oxidative damage in renal tissue is an initial pathological event that leads to CKD, rather than a change following CKD progression, in aged cats. Inhibiting apoptosis by antioxidant treatment may be a key strategy to prevent the development of CKD.

INTRODUCTION

Apoptosis is programmed cell death regulated by complex signaling pathways and is involved in the reduction of functional kidney tissue mass through the loss of renal epithelial cells, thereby resulting in the progression of chronic kidney disease (CKD) [26, 30]. Apoptosis of tubular epithelial cells (TECs) is one of the critical pathological events in tubulointerstitial damage (TID) progression, which is the final common pathway in feline and human CKD [1, 24, 33]. In an experimental rat model of TID induced by unilateral ureteral obstruction, the prevention of tubular apoptosis suppressed the degree of tubulointerstitial fibrosis (TIF) and tissue inflammation [7]. Studies using 5/6 nephrectomy rats have demonstrated that the degree of apoptosis in the glomeruli and tubules was significantly higher than that in sham-operated rats and that tubular apoptosis is associated with tubular atrophy and TIF [32, 35].

Apoptosis in cells is induced by various factors, and oxidative stress is an important endogenous pathway for the activation of apoptosis [21, 29]. Oxidative stress is a state in which the equilibrium between the production of reactive oxygen species (ROS) and antioxidant capacity is disrupted. Excessive production of ROS directly or indirectly induces renal tissue apoptosis, such as in kidneys damaged by cadmium toxicity [4, 34], cisplatin toxicity [15, 17], and ischemia-reperfusion injury [9, 28]. Therefore, antioxidant therapy is currently expected to be a therapeutic strategy for subsequent CKD in humans [13, 25].

CKD is defined as a long-term persistent structural damage and functional decline of the kidney, and its prevalence is extremely high in aged cats [1]. In our recent study, it was suggested that renal tissue oxidative stress could be involved in the development and/or early progression of naturally occurring CKD in aged cats by immunohistochemical analysis of the oxidative stress markers, 8-hydroxy-2’-deoxyguanosine (OHdG) and 4-hydroxynonenal (HNE) [12]. If oxidative damage in the kidney induces apoptosis of renal epithelial cells, suppressing oxidative damage would be an effective therapeutic strategy to prevent the apoptosis-induced reduction of functional renal tissue mass in feline CKD. However, whether such oxidative stress is associated with renal tissue apoptosis in feline CKD is unclear because apoptotic events in the kidneys of feline patients with CKD have not been investigated previously. Therefore, this study aimed to investigate the apoptosis in renal tissues from aged cats using a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and evaluate the association of apoptotic events with the progression and severity of CKD and oxidative damage in renal tissues.

MATERIALS AND METHODS

Samples

Formalin-fixed paraffin-embedded (FFPE) kidney tissue samples were obtained from 20 cats over 10 years of age. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Veterinary Clinical Research Committee of Kagoshima University, Japan (accession number: KVH190002). Based on medical records, 13 cats with persistently high plasma creatinine (pCre) concentrations (≥140 µmol/L) with low urine specific gravity (<1.035) were classified into the azotemic CKD group. Seven cats with normal pCre concentrations (<140 µmol/L) were classified into the non-azotemic group. The signalment and pCre values for the cases are shown in Supplementary Table 1. Cats diagnosed with acute kidney injury, neoplastic kidney disease, polycystic kidney disease, or glomerulonephritis were excluded. In addition, cases exposed to nephrotoxins such as nonsteroidal anti-inflammatory drugs, anticancer drugs, or lily, as well as cases with systemic and infectious diseases that induce renal tubular damage, such as feline infectious peritonitis, were not included.

Tissue preparation

Serial or mirror sections of kidney FFPE blocks, 3 µm thick, were prepared and stained with hematoxylin-eosin, periodic acid-Schiff, and Masson’s trichrome stains for histopathological analysis. The TUNEL assay kit for in situ apoptosis detection was applied to the prepared FFPE sections (FUJIFILM Wako Pure Chemical, Osaka, Japan), and the assay was performed according to the manufacturer’s protocol as follows: (i) deparaffinization and rehydration; (ii) protein digestion using protease solution at 37°C for 5 min; (iii) washing with phosphate-buffered saline (PBS); (iv) labeling of 3′-terminals of DNA with TdT reaction solution at 37°C for 10 min; (v) washing in PBS; (vi) inactivation of intrinsic peroxidase with 3% H₂O₂ for 5 min; (vii) washing with PBS; (viii) labeling with peroxidase-conjugated antibody at 37°C for 10 min; (ix) washing with PBS; and (x) detection of signals using 3,3′-diaminobenzidine (DAB) (DAB buffer tablet; Merck, Darmstadt, Germany). Counterstaining was performed using Mayer’s hematoxylin. Sections incubated with DNase I and those without TdT were used as positive and negative control sections, respectively.

Immunohistochemistry (IHC) of 8-OHdG as a marker of DNA oxidative damage, 4-HNE as a terminal product of lipid peroxidation, CD204 as a macrophage marker, and CD34 as an endothelial marker were also applied to the prepared FFPE sections. Polymer-based IHC was used to detect 8-OHdG and CD204. Alkaline phosphatase- and peroxidase-conjugated secondary antibodies were used for 8-OHdG and CD204, respectively. Labeled streptavidin-biotin-based IHC was used to detect 4-HNE and CD34.

IHC for 8-OHdG and CD204 was performed as follows: (i) deparaffinization and rehydration; (ii) microwave heating using 10 mM citrate buffer (pH 6) at 700 W for 10 min; (iii) for CD204, treatment with 3% H₂O₂ for 30 min; (iv) washing with PBS; (v) blocking with 0.25% casein/PBS for 60 min; (vi) incubation with anti-8-OHdG mouse monoclonal antibody (1:800; MOG-020P; Japan Institute for the Control of Aging, Shizuoka, Japan) or anti-CD204 mouse monoclonal antibody (1:400; clone SRA-E5; Cosmo Bio, Tokyo, Japan) at 4°C overnight; (vii) washing with PBS; (viii) incubation with alkaline phosphatase polymer-conjugated universal antibody (Simple Stain AP [MULTI]; Nichirei Biosciences, Tokyo, Japan) for 8-OHdG or peroxidase-polymer-conjugated universal antibody (Simple Stain MAX-PO [MULTI]; Nichirei Biosciences) for CD204 for 30 min; (ix) washing with PBS; and (x) detection of immunoreactivity using Fast Red II Substrate Kit (Nichirei Biosciences) for 15 min for 8-OHdG, and DAB for 5 min for CD204. The IHC of 4-HNE and CD34 was performed with following reagents: anti-4HNE rabbit polyclonal antibody (1:50; 4°C overnight; ab46545; Abcam, Cambridge, UK), anti-CD34 goat polyclonal antibody (1:100; 4°C overnight; sc-7045; Santa Cruz Biotechnology, Dallas, TX, USA), biotinylated goat anti-rabbit IgG antibody (1:200; 30 min; Vector Laboratories, Newark, CA, USA), biotinylated rabbit anti-goat IgG antibody (1:200; 30 min; Vector Laboratories), and peroxidase-conjugated streptavidin (ready-to-use; 30 min; Vector Laboratories). Sections were incubated with avidin and biotin blocking solutions (ready-to-use; 15 min each; Vector Laboratories) before incubation with the primary antibody. The cross-reactivity of the antibodies used in cats has been demonstrated previously [12, 19, 22]. For negative control sections, normal goat IgG (Medical & Biological Laboratories, Tokyo, Japan), normal rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA), and normal mouse IgG (Santa Cruz Biotechnology) were used instead of primary antibodies.

Quantitative analysis

Positive signals for TUNEL, 8-OHdG, and 4-HNE were quantified separately in the renal cortex and medulla. To assess the number of positive nuclei for TUNEL and 8-OHdG, digital images were taken at ×400 (10 non-duplicate images/section), and the percentage of positive nuclei in the TECs was calculated. The number of positive nuclei for TUNEL and 8-OHdG per glomerulus were also calculated. In 4-HNE, the number of 4-HNE-positive tubules per observation field (×200) was calculated in the renal cortex. To assess positive medullary signals, a semi-quantitative method described in a previous study was applied [18]. Briefly, the renal medulla was observed at ×200, and each observation field was graded from 0 to +4 (0, no signals; +1, up to 25% tubules exhibiting positive signals; +2, 26–50% tubules positive; +3, 51–75% tubules positive; and +4, 76–100% tubules positive). The score was calculated by multiplying the number of fields by the grade coefficient. For example, if out of the 20 fields examined, five exhibited grade 0, six grade +1, four grade +2, three grade +3, and two grade +4 changes, the final score for medullary 4-HNE was calculated as follows: [(0 × 5/20) + (1 × 6/20) + (2 × 4/20) + (3 × 3/20) + (4 × 2/20)] × 100=155.

CD34-positive peritubular capillaries (PTCs) were assessed using point-counting method [19]. Digital images of the renal cortex were captured at ×200, and grid lines were drawn on the images using Adobe Photoshop Elements 9 (Adobe Systems, San Jose, CA, USA). After excluding circles containing glomeruli or large vessels from the analysis, circles with CD34-positive PTCs were counted. The extent of glomerulosclerosis, interstitial mononuclear cell infiltration, and interstitial fibrosis was evaluated using a semi-quantitative method as described previously [18].

Statistical analysis

The Shapiro–Wilk test was used to confirm normal distribution. The statistical association between the number of cases with immunosignals and the group was analyzed using the Fisher’s exact test. Pearson’s or Spearman’s rank correlation coefficient was used to evaluate the correlation between the parameters. Mann–Whitney U test was used to compare the two groups. Statistical significance was defined as P<0.05. The analysis was performed using EZR ver. 1.55 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [8].

RESULTS

Histopathology

TIF lesions, observed as blue fibrotic tissue when stained with Masson’s trichrome, were observed to varying degrees in the kidneys of both non-azotemic and azotemic cats (Fig. 1). The interstitial mononuclear cell infiltrate comprised macrophages, lymphocytes, and plasma cells. Dilated tubules with tubular atrophy and/or flattened epithelium were also observed in these TIF lesions. In addition, varying degrees of glomerulosclerosis, characterized by an increase in mesangial matrices and thickening of the glomerular basement membrane, were observed in most cases.

Fig. 1.

Histopathological findings in kidneys from aged cats. (a) The cortex of a non-azotemic cat. (b) The cortex of an azotemic cat with chronic kidney disease. Tubulointerstitial lesions with fibrosis are observed in both cats. Masson’s trichrome stain. Scale bars, 50 µm.

TUNEL assay and IHC

TUNEL-positive nuclei were detected from the proximal tubules to the collecting ducts in all 20 cats, regardless of whether they were in the non-azotemic or azotemic group (Fig. 2a and 2b). Positive TUNEL signals were also detected in the glomeruli of two cats each in the azotemic and non-azotemic groups, and signals were observed in the nuclei of glomerular cells, especially in podocytes (Fig. 2c). Signals were also sporadically detected in the nuclei of interstitial-infiltrated cells in all cases (Fig. 2d). Furthermore, positive nuclei were detected in tubule intraluminal cells in the cortex of two out of seven non-azotemic cats and nine out of 13 azotemic cats. In the medulla, positive nuclei of tubule intraluminal cells were detected in three out of seven non-azotemic cats and nine out of 13 azotemic cats. The number of cases with TUNEL-positive signals is summarized in Table 1. No significant differences were detected between the non-azotemic and azotemic groups (Table 1).

Fig. 2.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive signals in kidneys from azotemic cats with chronic kidney disease. (a) Cortex. (b) Medulla. (c) A glomerulus. (d) Interstitial infiltrated cells. Arrows show TUNEL-positive apoptotic cells. TUNEL assay. Scale bars, 50 µm.

Table 1. Number of cases with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive or -negative signals in each renal site.

The 8-OHdG-positive nuclei were detected in the TECs of all cats, regardless of azotemic or non-azotemic status. In the glomeruli, 8-OHdG-positive signals were detected in four non-azotemic cats and seven azotemic cats. Cells with 8-OHdG-positive nuclei were different from those with TUNEL-positive nuclei (Fig. 3). The 4-HNE-positive signals were detected in four out of seven non-azotemic cats and 12 out of 13 azotemic cats, and the signals were restricted to the distal nephrons, which included the distal tubules, loops of Henle, and collecting ducts. No signals for 4-HNE were detected in the glomeruli. CD204-positive signals were observed in interstitial infiltrating cells and tubule intraluminal cells, which were different from those observed in TUNEL-positive cells (Fig. 4).

Fig. 3.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and 8-hydroxy-2’-deoxyguanosine (OHdG) immunohistochemistry in the mirror sections. (a and c) TUNEL assay. (b and d) 8-OHdG immunohistochemistry. (a and b) The cortex of a non-azotemic cat. (c and d) The glomeruli of an azotemic cat with chronic kidney disease. The cells labeled with black arrows indicate positive signals for TUNEL but not for 8-OHdG. The cells labeled using black arrowheads indicate positive-signals for 8-OHdG, but not for TUNEL. Scale bars, 50 µm.

Fig. 4.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and CD204 immunohistochemistry in the mirror sections from an azotemic cat with chronic kidney disease. (a) TUNEL assay. (b) CD204 immunohistochemistry. Tubular intraluminal cells (black arrow) indicate positive signals for TUNEL, but not for CD204. Scale bars, 50 µm.

Quantitative analysis

Glomerulosclerosis and interstitial cell infiltration scores in the azotemic CKD group were significantly higher than those in the non-azotemic group (Fig. 5). The CD34-positive PTCs score in the azotemic CKD group was lower than that in the non-azotemic group (Fig. 5). Interstitial fibrosis scores did not differ between the two groups (Fig. 5). TUNEL-positive TECs scores were also compared between the azotemic and non-azotemic groups, and no significant differences were detected in the renal cortex and medulla (Fig. 6). The values of clinicopathological and pathological parameters (pCre, glomerulosclerosis, interstitial cell infiltration, CD34-positive PTCs, interstitial fibrosis, 8-OHdG, and 4-HNE) were compared between the groups with and without TUNEL-positive glomeruli, and there were no significant differences between any of the parameters (Fig. 7). The correlation between the TUNEL scores and each parameter was also analyzed (Table 2). In the azotemic group, the TUNEL score significantly correlated with the 8-OHdG and 4-HNE scores for the cortex or medulla. In the non-azotemic group, the TUNEL score negatively correlated with the interstitial cell infiltration in the cortex.

Fig. 5.

Differences in histopathological parameters between the non-azotemic and azotemic chronic kidney disease groups. (a) Glomerulosclerosis. (b) Interstitial cell infiltration. (c) CD34-positive peritubular capillaries. (d) Interstitial fibrosis. Mann–Whitney U test. *: P<0.05, **: P<0.01.

Fig. 6.

Differences in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) scores between the non-azotemic and azotemic chronic kidney disease groups. (a) TUNEL score for tubular epithelial cells (TECs) in cortex. (b) TUNEL score for TECs in medulla. No significant differences are detected in TUNEL scores. Mann–Whitney U test.

Fig. 7.

Differences in clinicopathological and pathological parameters between the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive and TUNEL-negative groups in the glomerular cell nuclei. (a) Creatinine. (b) Glomerulosclerosis. (c) Interstitial cell infiltration. (d) CD34-positive peritubular capillaries. (e) Interstitial fibrosis. (f) Cortical 8-hydroxy-2’-deoxyguanosine (OHdG). (g) Medullary 8-OHdG. (h) Glomerular 8-OHdG. (i) Cortical 4-hydroxynonenal (HNE). (j) Medullary 4-HNE. No significant differences are detected in any parameters. Mann–Whitney U test.

Table 2. Correlation between tubular terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling scores and data from clinicopathological and histomorphometrical analyses in the azotemic chronic kidney disease and non-azotemic groups.

	Azotemic CKD		Non-azotemic
	Cortical TUNEL	Medullary TUNEL	Cortical TUNEL	Medullary TUNEL
pCre	NS	NS	NS	NS
Glomerulosclerosis	NS	NS	NS	NS
Interstitial cell infiltration	NS	NS	–0.759*	NS
CD34-positive PTCs	NS	NS	NS	NS
Interstitial fibrosis	NS	NS	NS	NS
Cortical 8-OHdG	0.582*	NS	NS	0.778*
Cortical 4-HNE	0.637*	NS	NS	NS
Medullary 8-OHdG	NS	0.643*	NS	NS
Medullary 4-HNE	0.593*	NS	NS	NS

Data: correlation coefficients. CKD, chronic kidney disease; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; pCre, plasma creatinine concentration; PTCs, peritubular capillaries; 8-OHdG, 8-hydrox-2’-deoxyguanosine; 4-HNE, 4-hydroxynonenal; NS, not significant. *, P<0.05 (Pearson correlation coefficient or Spearman’s rank correlation coefficient).

DISCUSSION

In this study, clear TUNEL-positive apoptotic cells were observed in the TECs of all cases, and quantitative analysis revealed no significant differences in TUNEL-positive signal levels between the azotemic CKD and non-azotemic groups. This finding suggests that apoptosis of TECs is not a pathological event in the later stage of CKD.

Tissue apoptosis is subsequently induced by tissue oxidative stress via complex pathways [21, 29]; therefore, we performed IHC of 8-OHdG and 4-HNE to evaluate the correlation of apoptosis with oxidative damage in the present study. Consequently, tubular TUNEL scores significantly correlated with tubular 8-OHdG and 4-HNE levels. Moreover, analysis using mirror sections revealed that TUNEL-positive apoptosis and 8-OHdG-positive nuclear oxidative damage did not occur simultaneously. Our recent preliminary study suggested that oxidative damage occurs in the kidneys of aged cats with CKD, regardless of whether they are azotemic or non-azotemic, and is an important pathological event during early-stage CKD in aged cats [12], and the present study additionally suggested that apoptotic events in TECs involve oxidative stress-related induction and early progression of CKD in aged cats.

TUNEL-positive signals were observed in glomerular cells in several cases in both non-azotemic and azotemic cats, but the severity of glomerulosclerosis did not differ between the groups with and without glomerular apoptosis. Glomerulosclerosis is initially induced by the dysfunction of the glomerular filtration barrier due to podocyte damage [2, 27], and apoptosis of mesangial cells contributes to the progression of glomerulosclerosis [5]. Several studies have indicated that ROS is closely associated with glomerular cell apoptosis [3, 14, 16, 36]. The contribution of glomerular cell apoptosis to the progression of glomerulosclerosis in aged cats was unclear in the present study and further investigation is required.

TUNEL-positive signals were also observed in tubule intraluminal cells. In our previous study, macrophage infiltration in renal tissues was investigated in canine and feline CKD, and it was demonstrated that intraluminal macrophages were more prominent in feline CKD than in canine CKD [22]. Therefore, we initially suspected that TUNEL-positive intraluminal cells were macrophages. To test this hypothesis, IHC of CD204 and TUNEL were analyzed using mirror sections. However, no cells that were double-positive for TUNEL and CD204 were observed in any case, suggesting that TUNEL-positive intraluminal cells were apoptotic epithelial cells that dropped from the tubular epithelium to the lumen, and this phenomenon might be a pathological event in tubular damage in feline CKD. A study on human diabetic nephropathy suggested that a decrease in the cell adhesion molecule-1 protein causes apoptosis of renal TECs and is implicated in the development and progression of tubular damage and TID [10]. TID is a typical histopathological change in feline CKD [1, 33], and our results suggest that shedding of the apoptotic tubular epithelium into the tubular lumen may be a pathological event in the development and early progression of naturally occurring CKD in aged cats.

In addition, podocytes that shed from the glomeruli may be included in the intraluminal apoptotic cells of the tubules, as TUNEL-positive signals were also observed in podocytes. Apoptosis and podocyte sloughing have been considered the primary mechanisms underlying podocyte loss in glomerular diseases [2, 27, 31, 38], and this event may contribute to the progression of glomerular damage in feline CKD. However, apoptotic podocytes were observed in only two cases each in the azotemic and non-azotemic groups, and it was not considered a common pathological mechanism in feline CKD.

Renal TIF is a crucial and common pathological event in CKD, and its severity is closely associated with a decline in renal function in humans with CKD [6, 37]. This association has also been strongly suggested in feline CKD [1, 33], and the present study also revealed TIF lesions of varying degrees in the kidneys of aged cats. To clarify the association between renal tubular apoptosis and the severity of TIF, we analyzed the correlation between TUNEL and TIF scores in the azotemic CKD group and found no statistical correlation. Furthermore, no statistical correlation was observed between the tubular TUNEL score and glomerulosclerosis and interstitial cell infiltration scores in the present analysis. Therefore, in naturally occurring CKD in aged cats, it has been suggested that renal tubular apoptosis is not a pathological event that follows tubulointerstitial or glomerular damage. However, this does not mean that apoptosis is not involved in the pathological mechanism underlying feline CKD. As one of the complicated mechanisms underlying TIF progression, the transformation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) is a well-known process after TEC damage, and our previous study showed that EMT occurred in the kidney tissues of cats with CKD [33]. Oxidative damage-related apoptosis in TECs, as suggested in the present study, might be a triggering event that induces EMT in feline CKD.

CD34-positive PTCs were estimated to evaluate renal capillary rarefaction, which is a pathological event that occurs before the progression of CKD. In humans and experimental animals, a decrease in PTCs leads to insufficient oxygen supply to TECs, which induces TIF [11, 20]. However, the incidence of renal capillary rarefaction in cats with CKD remains controversial. Our previous study suggested that the occurrence of this event in CKD is different between dogs and cats [19]. Briefly, CD34-positive PTCs were well preserved in the kidneys of cats with CKD, but were significantly reduced in the kidneys of dogs with CKD. In contrast, a recent study demonstrated renal capillary rarefaction in feline CKD using CD31 immunohistochemistry [23]. In the present study, renal capillary rarefaction was re-evaluated, and a significant reduction in CD34-positive PTCs was observed in azotemic CKD in aged cats. Therefore, it was suggested that renal capillary rarefaction is a possible pathological event in feline CKD if the focus is on cats aged >10 years. However, no correlation between renal tissue apoptosis and renal capillary rarefaction was observed in the present study.

To summarize, this study investigated renal apoptotic events in aged cats using the TUNEL method, and suggested that apoptosis in kidney tissue could be an initial pathological event that leads to CKD, rather than a change following CKD progression. Further, the degree of the apoptotic event significantly correlated with the severity of oxidative damage in TECs. Therefore, oxidative damage-related apoptosis of TECs is considered to be involved in the early pathogenesis of naturally occurring CKD in aged cats. In human CKD, various antioxidants have been reported to help in prevention and treatment by inhibiting oxidative stress and apoptosis [13, 25], and based on the findings of the present study, we suspect that the inhibition of apoptosis by antioxidants would be an important preventive strategy against CKD in aged cats.

The present study has some limitations. First, the sample size was small, and the number of cases in each stage of CKD was not uniform because cats with stable late stages were difficult to obtain for autopsy (Supplementary Table 1). Second, analysis of oxidative stress markers in blood and urine was not possible. Third, the irregular number of cats with different CKD stages, particularly the small number of cats in the later stages of CKD, may have affected the results of the study. Therefore, although this study suggests that apoptosis is related to the early pathogenesis of CKD in aged cats, it is difficult to define this theory on the basis of the results of this study alone. Further studies are needed to evaluate the role of renal tissue apoptosis in aged cats.

CONFLICT OF INTEREST

The authors declare no conflicts of interest in relation to the research, authorship, or publication of this article.