The Relationship between Caspase-3, -6 Expression and Morphological Changes in Kidneys of Cows

Introduction. The organism of high-yielding dairy cows is susceptible to metabolic imbalances. One of the consequences of milk production stress is the development of acidotic syndrome, which negatively effects the kidneys [1]. Ketosis, which cows are particularly susceptible to during lactation, can also lead to various nephropathies [2]. The pathological processes developed in the kidneys inhibit organ functioning, have a systemic nature and affect the overall condition of an animal. In the scientific community the role of apoptosis in the pathogenesis of various kidney diseases has long been actively discussed [3]. For example, it has been established that programmed death of tubular epithelial cells, along with necrosis, are the basic signs of acute renal failure [4]. In addition to nephrocyte damage, renal vascular damage and endothelial cell apoptosis are important factors in the development of renal dysfunction. The loss of endothelial cells by apoptosis is deemed to play a significant role in changing the microvascular network observed during renal damage [5]. Apoptosis has also been described in renal interstitial cells following ischemic renal injury [6].

Caspases are enzymes belonging to a group of proteases that are involved in cell apoptosis. 12 caspases have been described for humans and rodents, however for cattle, the exact number of enzymes from this group has to be determined yet. Caspases are divided into 3 groups: executioners, initiators and inflammatory caspases. Caspase-3 represents the executive caspase group, and caspase-6 — the initiator caspase group. Studies on the role of caspases in the development of various pathologies in the organs of cattle are extremely few. For example, the role of caspase-3 in the apoptosis of mammary gland cells in cows fed with concentrated feed has been described [7]. Data on the role of caspases in the development of renal pathologies in cattle are not available in the literature. However, caspases are known to be involved in inflammatory responses, apoptosis, vasoconstriction, and tubular necrosis in various kidney diseases in humans and rodents [8]. Furthermore, the important role of caspases in renal damage makes them a favourable target for therapy. For example, on the example of polycystic kidney disease in rats, it was demonstrated that caspase inhibition can reduce apoptosis in renal tubules and slow disease progression [9].

Therefore, the aim of the present study is to describe the relationship between caspase-3 and caspase-6 expression and morphological changes in the kidneys of high-yielding cows with pathologies.

Materials and Methods. The work was carried out at the Ural Federal Agrarian Scientific Research Center, Ural Branch of the Russian Academy of Sciences (Ekaterinburg) in the period from 2023 to 2025. To study the morphological changes in the kidneys of cows, the material was collected postmortem from animals aged 7–8 years old, that had 4–6 lactations (n=4). For histological examination, tissue specimens of no more than 0.5 cm thick were fixed in 10% Neutral Buffered Formalin (NBF) in a proportion of 1:20. After fixation, the specimens were rinsed in running water, dehydrated, then soaked in paraffin, and paraffin blocks were prepared. Paraffin sections of 3–4 µm thickness were made. Sections were deparaffinized and stained using hematoxylin and eosin and Mallory’s trichrome staining techniques.

Immunohistochemical staining of the samples with antibodies to caspase-3 and caspase-6 was performed to assess the intensity of apoptosis in the organ. For immunohistochemical analysis, sections were placed on the adhesive slides (Superfrost), then placed on a slide drying bench (+60°C) until the paraffin was visibly melted. Immunohistochemical analysis was performed in compliance with the following protocol:

1. The sections were placed in a Tris-EDTA buffer solution heated in a water-bath up to 96°C for 12 min for heat-induced epitope retrieval (HIER, pH 6; “PrimeBioMed”, Russia).
2. After 12 minute incubation in a water-bath, the sections were left in the buffer solution to cool to indoor temperature and then rinsed with distilled water.
3. The sections were incubated in an H₂O₂ solution for 10 min, then placed in distilled water for 2 min.
4. The sections were dried and outlined with a hydrophobic pen.
5. The sections were soaked in TBST buffer solution for 1 min.
6. Primary antibodies were applied to the sections, which were then placed in the humidified incubator for 1 hour at 30°C.
7. Sections were rinsed three times with TBST buffer solution.
8. Secondary antibodies were applied to the sections, which were then placed in the humidified incubator for 30 minutes at 30°C.
9. Sections were rinsed three times with TBST buffer solution.
10. DAB chromogen was applied to the sections, which were incubated until darkening, approximately during 30–60 seconds.
11. Sections were rinsed in distilled water and then counterstained with hematoxylin for 10 seconds.
12. Sections were rinsed in distilled water, decolorized, and placed in balsam.

Antibodies to caspase-3 (PAA626Bo01, Cloud-Clone Corp., China) and caspase-6 (PAC340Bo02, Cloud-Clone Corp., China) were used as primary antibodies at a 1:100 dilution. Antibodies to rabbit IgG (SAA544Rb19, Cloud-Clone Corp., China) were used as secondary antibodies with DAB (Bio-Rad Laboratories, Inc., USA) as a horseradish peroxidase substrate in accordance with the manufacturer’s instructions.

Histological description of the specimens was made, and the intensity of immunohistochemical staining was visually assessed.

Research Results. Examination of the cows’ renal specimens has revealed significant changes in the organ. The dilation in Shumlyansky Bowman capsule of renal corpuscles was seen in the renal cortex. Endothelial proliferation was visible in the Juxtaglomerular apparatus, indicating the development of intracapillary glomerulonephritis (Fig. 1.1). Cystic cavities of varying sizes were found throughout the cortex, indicating renal failure (Fig. 1.2). Protein casts were deposited in the lumen of the proximal tubules, indicating a protein metabolism disorder. Nephrocytes with degenerative changes, and a number of cells exhibiting structural abnormalities were observed. The renal tubules demonstrated narrowing of the lumen, swelling of the nephrocytes, and changes in their structure (Figs. 1.1 and 1.2).

Mallory’s trichrome staining has revealed increased connective tissue patterns in the Shumlyansky Bowman capsule and between the proximal convoluted tubules, indicating productive inflammation (Fig. 1.3). Foci of nephrosclerosis were found in renal interstitium between the tubules (Fig. 1.4). In the area of blood vessels, particularly near the perivascular zone of arcuate arteries, the pronounced sclerotic processes were observed — the extensive areas of connective tissue proliferation, stained in various shades of blue (Fig. 1.5). Within the vessels themselves, the active processes of disorganization and proliferation of vascular wall elements, and sharp narrowing of the arterial lumen were observed (Fig. 1.6).

Along with the morphological changes, expression of the proapoptotic proteins caspase-3 and caspase-6 was observed in the kidneys of the cows studied. Moderate expression of caspase-3 was revealed in the nephrocytes of the cortical tubules (Fig. 2.1), as well as in the cells of the parietal layer of the Shumlyansky-Bowman capsule of deformed glomeruli (Fig. 2.2). A large number of caspase-3-positive cells were also detected in the interstitium around the cystic cavities (Fig. 2.3). Nephrocytes in both the renal cortex and medulla tubules were intensely colored in brown, which indicates a high degree of caspase-6 expression in them (Fig. 2.4 and 2.5). At the same time, renal corpuscles that retained their structure were not expressing caspase-6, unlike the deformed glomeruli (Fig. 2.6). It is worth noting that not all the tubules had a caspase-positive reaction, which may be due to the possibility of cell death not only by apoptosis, but also by necrosis, and is discussed in the literature [4].

Discussion and Conclusion. Morphological examination of kidney tissues in cows aged from 7- to 8-year-old reveals the development of destructive processes in the organ, which are likely related not only to the age but also to the high metabolic load endured by the organism during lactation. Changes affect not only functioning of the organ parenchyma but also the stroma and microvasculature. The formation of cystic cavities, the presence of deformed glomeruli, and abnormalities in the structure of the Juxtaglomerular apparatus indicate the development of renal failure. The deposition of protein casts in the renal tubules and the death of nephrocytes indicate the development of degenerative processes. Foci of nephrosclerosis and significant microcirculatory disorders indicate the presence of inflammatory reactions and fibrotic changes in the organ.

The described changes are accompanied by the expression of apoptotic factors—caspase-3 and caspase-6. Caspase-6 is predominantly expressed by renal tubular cells, which, judging by morphological description, undergo degenerative processes caused by the protein metabolism disorder. Thus, the elevated caspase-6 expression is a marker of renal cell damage and pre-apoptotic state triggering the caspase cascade. Caspase-3 expression is observed not only in renal parenchyma functioning, but also in the stromal elements of the organ —in the interstitium and vascular wall cells. Thus, due to the executive role of caspase-3, the conclusion about extensive apoptotic processes occurring in various tissues of the organ can be drawn. The obtained data provide a profound basis for further in-depth study of pro-apoptotic factors in the context of their effect on the development of various pathologies in cattle.