High prevalence of renal amyloidosis in cats in an overcrowded colony

Abstract

Multiple cats in an overcrowded colony died of acute kidney injury with renal amyloidosis. To investigate the etiology of these deaths, we performed histological analysis of the kidneys of 27 colony cats and 29 noncolony cats (8 with acute kidney injury, 9 with chronic kidney disease, and 12 nonazotemic). Congo red-positive amyloid deposition was observed prominently in the glomeruli and cortical and medullary interstitium and was identified as AA amyloid using permanence. The prevalence and severity of renal amyloidosis were significantly higher in colony cats than in noncolony cats. Feline immunodeficiency virus showed no association with feline leukemia virus infection. Based on our results, adverse environmental conditions may accelerate the incidence of feline renal amyloidosis.

Amyloidosis is a disease characterized by extracellular deposition of amyloid proteins in organs and rarely occurs in dogs and cats. Amyloid proteins are easily deposited in the kidneys; however, the deposition sites differ between dogs and cats. In dogs, amyloid proteins tend to deposit in the glomeruli, and renal biopsy can be used to diagnose renal amyloidosis. In contrast, in cats, amyloids are more likely to deposit in the medullary interstitium [2, 3], and cases are rarely diagnosed by renal biopsy, which involves examination of the cortical tissue. Therefore, feline renal amyloidosis is not well-understood.

Recently, multiple cat deaths due to acute kidney injury (AKI) occurred in an overcrowded colony. Histopathological examination of the kidneys revealed amyloidosis. This disease is well-known to occur in the Abyssinian cat breed and is specifically associated with familial renal amyloidosis. Previous studies reported that in Abyssinian families, AA amyloids are deposited throughout the body and renal amyloidosis is particularly prominent, resulting in death from renal failure at a young age [2]. Various clinical conditions such as chronic inflammation, infections, and tumors have been reported to be associated with the incidence of secondary amyloidosis. In a study of 91 dogs diagnosed with renal amyloidosis, chronic inflammatory disease was present in 80% of cases and tumors in 20% of cases [11]. These inflammatory diseases included urinary tract infections, arthritis, pancreatitis, tumors, and immune-mediated diseases [11]. Additionally, inflammation predisposes dogs with dermatomyositis to amyloidosis [5]. Glomerular and interstitial amyloidosis has been detected in cats naturally infected with feline immunodeficiency virus (FIV) [10]. In the present study, we histologically surveyed kidney tissues from cats and cats outside the colony and evaluated the prevalence and severity of renal amyloidosis in the colony.

Kidney tissues from 27 cats in the colony were collected at a private veterinary hospital during autopsy or postmortem using Tru-cut needle puncture. The clients associated with the colony provided consent for autopsy and tissue sampling after the cat’s death. Kidney tissues from 29 cats outside the colony were obtained by autopsy at Kagoshima University, Japan. A total of 56 cats were classified as follows: group 1, comprising 27 cats in the colony; group 2, eight cats that died of AKI outside the colony; group 3, nine aged cats with naturally occurring chronic kidney disease (CKD); and group 4, 12 nonazotemic cats. An attending veterinarian interviewed the cat colony owners.

Kidney tissue samples obtained from Kagoshima University were fixed in 10% neutral-buffered formalin, whereas those obtained from a private veterinary hospital were fixed in masked formalin (maskedformA0.5, Japan TANNER, Osaka, Japan). After fixation, the tissues were embedded in paraffin, and 3 μm thick sections were cut and stained with hematoxylin and eosin, periodic Schiff, and Masson’s trichrome stains for general histopathological observation. The sections were also cut to 8 µm and stained with alkaline Congo red stain for amyloid detection. AA and AL amyloids were distinguished by permanganate pretreatment in combination with Congo red staining. Sections stained with Congo red were observed using bright-field optical microscopy and polarized light microscopy.

The degree of Congo red staining was evaluated using a previously described quantitative method [8, 14]. Briefly, each glomerulus was graded from 0 to +4 (0, no staining; +1, up to 25% positive area; +2, 26–50% positive area; +3, 51–75% positive area; and +4, 76–100% positive area). The score was calculated by multiplying the number of glomeruli by the grade coefficient. For example, of the 50 glomeruli examined, 8 showed grade 0, 7 showed grade +1, 20 showed grade +2, 10 showed grade+3, and 5 showed grade +4; the final score was calculated as follows: [(0 × 8/50) + (1 × 7/50) + (2 × 20/50) + (3 × 10/50) + (4 × 5/50)] × 100=194. To assess interstitial staining in the cortex and medulla, non-crossed observation fields (×400) were graded from 0 to +4, and the score was calculated in the same manner. Multiple comparisons among the four groups were performed using nonparametric Steel–Dwass test. The score for negative cases was assigned as 1/2 of the lowest value of the positive cases. The association between amyloid deposition and the group classification was evaluated using Fisher’s exact test. A significant association was defined as P<0.05. EZR version 4.1.2 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) was used for all analyses.

The number of cats that died in the colony exceeded 30 over four years and five months (November 2017–April 2022). The owner claimed that the cats were kept indoors and did not have access to the outside. The temperature was controlled with an air conditioner throughout the year. However, fleas, ticks, and parasites were prevalent among the cats that received clinical therapy. The cats were not kept in cages and were free to roam around the house, and bowls of food, water, and litter trays were set multiple times but were shared among the cats. The body condition score was low overall, with no cats exceeding a score of 4/9 (Supplementary Table 1).

The signals and clinical information of the cat colonies (group 1) are shown in Supplementary Table 1. Serologic tests for FIV and feline leukemia virus (FeLV) were performed in 18 of 27 cases using a kit (Thinka FIV/FeLV, ARKRAY, Kouka, Japan), among which four cases were positive for FIV and none were positive for FeLV. Only three cats were vaccinated. Most patients had various underlying or preexisting diseases, including upper respiratory infections, gingivostomatitis, bites, and CKD. In patients diagnosed with severe renal amyloidosis, increased echogenicity and parenchyma enlargement were observed bilaterally on ultrasonography (Fig. 1).

Fig. 1.

Echographic finding of the kidney from a colony cat with severe renal amyloidosis. Increased echogenicity and parenchyma enlargement, particularly in the cortex, are observed.

General histopathological observations revealed various renal lesions such as glomerulosclerosis, tubular atrophy or dilatation, tubular necrosis, interstitial cell infiltration, and interstitial fibrosis. Although the findings varied between cases, many cats in the colony showed prominent tubular necrosis and infiltration of inflammatory cells. Similar findings were observed in patients with AKI. Glomerulosclerosis, interstitial cell infiltration, and interstitial fibrosis were observed in all CKD cases, with interstitial fibrosis particularly notable in many cases. Similar but milder findings to those observed in CKD cases were found in nonazotemic patients. No patients with overt glomerulonephritis were included in this analysis.

Congo red staining revealed amyloid deposition in the glomeruli and cortical or medullary interstitium in many cases. The deposits were observed as orange-red substances under bright-field light microscopy and green birefringent substances under polarized light microscopy (Figs. 2 and 3). Colorization and birefringence of amyloid deposition were not detected in sections treated with permanganate in any cases of amyloid deposition. Similar substances exhibiting an orange-red color under bright-field light microscopy, green birefringent substances under polarized light microscopy, and discoloration with permanganate treatment were identified to varying degrees in the small arteries in all cases.

Fig. 2.

Congo red-stained kidney sections from cats in the colony. A–C: Exemplar cat with severe renal amyloidosis. Amyloid deposits exhibited an orange-red color under bright field microscopy (A and B) and green birefringence under polarized light microscopy (C). In this case, apparent amyloid deposition was observed in the glomeruli (A and C) and medullary interstitium (B). D: Another cat with severe renal amyloidosis. Amyloid deposition was more evident in the cortical interstitium than in the glomeruli. Scale bars: 50 µm.

Fig. 3.

Congo red-stained kidney sections from cats outside the colony. A and B: Cats with acute kidney injury (AKI) (cortex). C: Cat with chronic kidney disease (CKD) (medulla). D: Nonazotemic cat (medulla). In each case, varying amounts of amyloid deposition were observed, which was particularly severe in AKI cases. Scale bars: 50 µm.

In the colony (group 1), amyloid deposition was observed in 26 of 27 cases. Glomerular, cortical, and medial interstitial depositions were found in 18, 19, and 26 of 27 cases, respectively. Outside the colony, amyloid deposition was observed in of 3 of 8 cats with AKI (group 2), 2 of 9 cats with CKD (group 3), and 2 of 12 nonazotemic cats (group 4). Glomerular, cortical interstitial, and medial interstitial deposits were observed in 2, 3, and 3 of 8 cases, respectively. Glomerular and cortical interstitial depositions were not detected in cats with CKD, but medial interstitial deposition was observed in 2 of 9 cases. Glomerular, cortical, and medial interstitial depositions were observed in 1, 0, and 2 of 12 cases, respectively. Deposition at the glomerular and medullary interstitial sites significantly differed between groups (Table 1).

Table 1. Number of cats showed amyloid depositions in each renal site.

	Glomeruli		Cortical interstitium		Medullary interstitium
	Positive	Negative	Positive	Negative	Positive	Negative
Group 1 (colony)	18	9	19	8	26	1
Group 2 (acute kidney injury)	2	6	3	5	3	5
Group 3 (chronic kidney disease)	0	9	0	9	2	7
Group 4 (nonazotemic)	1	11	0	12	2	10
P value	<0.01		<0.01		<0.01

The results of quantitative analysis are shown in Fig. 4. The glomerular and cortical interstitial scores in the colony were significantly higher than those in CKD and nonazotemic cats (Fig. 4a and 4b). The medullary interstitial score in the colony was significantly higher than that in AKI, CKD, and nonazotemic cats (Fig. 4c). No significant differences were detected among AKI, CKD, and nonazotemic cats at any site.

Fig. 4.

Quantitative results of Congo-red positive staining. A: Glomeruli, B: Cortical interstitium, C: Medullary interstitium. The data are shown in box plots. Steel-Dwass test.

Alkaline Congo red staining was used to detect amyloid proteins. Substances that showed both an orange-red color under bright-field light microscopy and green birefringence under polarized light microscopy were considered amyloid deposits. Permanganate treatment is widely used to distinguish between AA and AL amyloids [13], with AA amyloid proteins uniquely showing negative staining for Congo red after treatment. In the present study, amyloid deposition was detected in the kidneys in many cases and was demonstrated to be an AA amyloid following permanganate treatment. Congo red-positive material was found in the small arteries in all cases. However, those in the small arteries should not be dismissed as true amyloid deposits. Although these deposits were theoretically considered to be AA amyloids, as they presented with green birefringence under polarized light microscopy and discoloration with permanganate treatment, immunohistochemical analysis was required to confirm that they were true amyloid deposits. However, to the best of our knowledge, antibodies specific to feline AA amyloids are not commercially available.

Amyloid proteins are more likely to be deposited in the medullary interstitium of feline kidneys [2, 3]. Cases in the present colony showed prominent medullary interstitial amyloid deposition, which was significantly more severe than that in cases outside the colony. In addition, cortical interstitial amyloid deposition and glomerular amyloid deposition were consistently found in colony cases and were significantly more severe than in cases of CKD and nonazotemia outside the colony. This result indicates extremely intense amyloidosis in these colony cases, which is a clinically significant finding given the rarity of this condition among feline kidney diseases.

An association between infectious viral diseases and feline amyloidosis has long been suspected. Feline infectious peritonitis has been identified as a possible cause of amyloidosis [6, 12], however, its significance in the pathogenesis of amyloidosis remains unclear. Recently, an increasing number of studies has focused on FIV-induced renal amyloidosis in cats. In a survey of renal diseases in cats naturally infected with FIV, renal amyloid deposition was detected in the glomeruli and interstitium in 38% (8/21) of cases [10]. Another study of the kidneys from 34 cats naturally infected with FIV (13 asymptomatic, 21 immunodeficiency syndrome), 30 cats seronegative for FIV, 20 cats experimentally infected with FIV, and 5 cats that were specific pathogen-free showed that renal amyloidosis was present in 35% (12/34), 3.3% (1/30), 0% (0/20), and 0% (0/5) of cases, respectively [1]. Amyloid deposition was absent in cats experimentally infected with FIV in isolation units, and these cats showed no susceptibility to secondary infections, indicating that FIV infection alone is insufficient to induce the development of amyloidosis; this result was observed even in cases caused by short-term infection (<24 months) [1]. Similarly, a retrospective case-control study of feline renal amyloidosis in shelters in Italy did not reveal a direct relationship between FIV and renal amyloidosis. The study showed that the prevalence of AA amyloidosis surveyed in three shelters and renal amyloidosis was 57.1% (16/28), 73.0% (19/26), and 52.0% (13/25) of cats in each shelter, with no association observed between FIV, FeLV, and feline coronavirus [4]. In the present study, serological tests for FIV and FeLV were performed in 18 cases in a colony, and 22.2% (4/18) of these cases tested positive for FIV.

The high prevalence of renal amyloidosis in cats housed in colonies may be associated with environmental factors. Increased serum amyloid A levels are the principal cause of AA amyloidosis, and chronic inflammatory responses induced by various diseases may spread because of unfavorable environmental conditions in the colony. In addition, chronic stress from a poor environment induces a decline in immune function, promoting further inflammation and abnormal accumulation of amyloid proteins. Because the deposited amyloid was AA, the spread of infectious or inflammatory diseases in the colony was suspected to be the cause of the high incidence of renal amyloidosis. Additionally, many cats in this colony were assumed to have persistent inflammation, either primary or secondary to an underlying disease, which may have led to the production of serum amyloid A in the liver and secondary AA amyloidosis.

Another theory is that transmission of the disease through feces may be responsible for the high prevalence of renal amyloidosis in colonies. In laboratory mice, intravenous injection of an amyloid-enhancing factor extracted from the spleen induced systemic AA amyloidosis, and oral administration of this material accelerated disease onset [7]. Oral transmission has also been suggested as a cause of systemic amyloidosis in captive cheetahs, which are well-known to have a high prevalence of AA amyloidosis [9]. A previous study suggested disease transmission rapidly occurs through feces, as feces from affected cheetahs contained AA amyloid fibrils distinct from those found in the liver. The increased transmissibility of these fibrils was attributed to differences in their molecular weight and shape [15]. In the current study, interviews with the colony owner revealed that litter trays were shared rather than assigned to individual cats. Although we did not perform biochemical analysis of feces, the possibility of amyloidosis transmission between cats housed in the colony was suspected.

In the present study, renal amyloid deposition was not prominent in CKD and nonazotemic cats. However, two AKI cases showed glomerular and interstitial amyloid deposition similar to that observed in colony cases. The environmental conditions in both cases were unclear from the medical records, and whole-body autopsies were not performed. One cat had hypertrophic cardiomyopathy and was serologically positive for FIV but the cause of renal amyloidosis in these cases could not be determined.

There were some limitations to this study. Clinical and clinicopathological data, such as SAA, proteinuria, blood pressure, and comorbidities, were lacking. Additionally, we did not perform pathological analysis of other organs, such as the spleen and liver. Finally, kidney samples were not evaluated macroscopically.

In conclusion, we investigated the prevalence of renal amyloidosis in cats housed in overcrowded colony. Many cats in this colony died of renal amyloidosis. The cause of the disease in this colony is unclear but may be associated with unfavorable environmental conditions. Clinical veterinarians should consider AA amyloidosis as a potential cause of AKI if renal failure occurs frequently in households with multiple cat breeds or in shelters. In addition, improving the environment in which cats live is essential for preventing the renal amyloidosis. In particular, proper hygiene management, infection prevention measures, and optimization of living conditions, including stress reduction, are crucial.

CONFLICTS OF INTERESTS

The authors declare no conflicts of interest.