The renin-angiotensin-aldosterone system (RAAS) is influential in chronic kidney disease (CKD) pathophysiology, and RAAS activation is a crucial factor in CKD progression in humans (1). RAAS is thought to be a linear and maladaptive system that activates the angiotensin-converting enzyme (ACE)-angiotensin II (Ang II)-angiotensin II type I receptor (AT1R) axis (2). In addition to inducing potent vasoconstriction, conserving sodium and water, and releasing aldosterone, this axis also has pro-inflammatory, pro-oxidative, and pro-fibrosis effects (3). Treatment with ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB) to control RAAS for proteinuria and/or hypertension is widely accepted as the first-line therapy for CKD in human/veterinary medicine (4–6).

Angiotensin-converting enzyme 2 (ACE2), a homolog of ACE, was discovered in 2000 (7). Similar to ACE, ACE2 is a membrane-bound zinc metalloproteinase expressed in various organs such as the cardiovascular system and kidneys (7). However, unlike ACE, ACE2 is a mono-carboxypeptidase and has a catalytic ectodomain that generates angiotensin (1–7) directly from Ang II (8) angiotensin (1–7) mainly binds to the endogenous Mas receptor (MasR) and thus the biological effects related to angiotensin (1–7), namely vasodilation, anti-oxidation, anti-inflammation, and anti-fibrosis, are opposite to those of Ang II in humans and experimental animals (9). In cats, combining ARB plasma with recombinant human ACE2 also results in an increase in angiotensin (1–7) (10). This suggests that the ACE2-angiotensin (1–7)-MasR axis is a counterbalance to the ACE-Ang II-AT1R axis during RAAS (11). Furthermore, a decrease in renal ACE2 expression has been reported to be associated with renal injury and related complications, including albuminuria in kidney injury animal models (12, 13). Thus, it is likely that ACE2 plays a protective role in kidney disease (14, 15).

The ectodomain of ACE2 can be cleaved by enzymes, including disintegrin and metalloproteinase-17 (ADAM-17) (16, 17), and then the protein ultimately is shed into urine as urinary ACE2 (uACE2). Research indicates that renal tubular ACE2 levels in diabetic kidney disease mice correlate with uACE2 levels but not with plasma or serum ACE2 concentration (18). Moreover, injecting recombinant ACE2 markedly increase serum ACE2 but not urinary ACE2 (19), uACE2 reflects ACE2 release from proximal tubules (20, 21) and increases in both humans and mice with CKD and diabetic kidney disease (DKD) (21, 22). In addition, albuminuria and elevated biomarkers related to tubulointerstitial injury have been associated with increased uACE2 in individuals with CKD (23).

CKD commonly affects cats, especially the elder ones (24). Specifically, the histopathological expression of renal ACE2 and its relationship with renal damage have been described in CKD cats (25). However, to the best of our knowledge, the role of uACE2 in CKD cats remains unknown. Thus, our aims are several folds, including: (1) to carry out a comparative analysis against healthy controls of uACE2 concentrations and activities in cats across the different stages of CKD; (2) to investigate the relationship between uACE2 concentrations, activities, and clinicopathological information obtained from feline CKD patients; (3) to evaluate the predictive ability of uACE2 concentrations and activities in relation to CKD progression.

Although the relationship between uACE2 concentration and activity in the context of renal disease has been reported for human medicine and using experimental models, to the best of our knowledge this is the first study to simultaneously measure the concentration and activity of uACE2 in cats. Moreover, a comparison of these two measurements between CKD subjects and healthy subjects and among CKD subgroups was carried out. This was expanded into an investigation of their correlation with clinicopathological features. In the present study, UACCR was significantly higher in CKD cats compared to healthy controls. This result is similar to a previous study investigating urinary ACE2 concentration in humans (21). Additionally, the higher UACCR with advanced CKD was consistent with human findings, and, specifically, a decline in renal tubular function has previously been recognized as an important factor that is associated with increased ACE2 shedding (23).

In a previous study in humans and mice with chronic renal disease, both uACE2 protein concentration and activity were consistently increased (22), which implies that most of the uACE2 protein retains enzymatic activity and that total UAA increases as more ACE2 protein is shed into the urine. However, in the current study, it was observed that cats afflicted with CKD exhibited a notably reduced UAA when compared to healthy controls. This finding stands different from the results obtained from mice and humans (22). Interestingly, a previous study found that shed ACE2 may be cleaved, and ultimately yielded two different molecular forms of ACE2 in urine (27). These researchers further suggested that species-specific cleavage sites may exist (18, 28). Consistently, another study has also revealed that different molecular forms of the protein exist in healthy humans and CKD patients (21). The presence of different cleavage sites not only results in different molecular forms of ACE2 in urine but also implies that these different molecular forms may have different activities. It is important to note that Arg⁵⁷⁷ and Lys⁵⁹⁶ have been identified as the cleavage sites that produce shorter ACE2 fragments in mouse proximal tubular cells (29), and these amino acids are also conserved in the human (NCBI sequence ID: NP_001358344.1) and canine ACE2 (NCBI sequence ID: NP_001158732.1); however, Arg⁵⁷⁷ is replaced with Thr⁵⁷⁷ in feline ACE2 (NCBI sequence ID: XP_023104564.1). Therefore, it is possible that there are different molecular forms of uACE2 present in healthy cats and in CKD cats, and that the modified uACE2 may lose its activity after being cleaved previously in CKD cats. Further studies are needed to confirm the presence of these different ACE2 molecular forms in cats and to measure separately the activity of the various molecular forms of ACE2 in CKD and healthy feline urine.

In the present study, both UACCR and UAA demonstrated independent associations with renal indexes, with UACCR exhibiting a positive correlation with BUN and UAA showing a significant but weak negative correlation with plasma creatinine. The findings of UACCR align with previous human studies (23), though there was no analogous study regarding UAA. As above, the variability of UAA in cats differs from that observed in humans and experimental animals, Therefore, investigation into the role of UAA in cats with CKD is warranted for future research endeavors.

USG, indicative of urine concentration ability and a component of renal function, is also affected by the size of molecules within urine (30), it was found to be significantly, independently, and negatively correlated with UACCR in cats in the present study. Both an increase in uACE2 and a decrease in renal ACE2 have been reported to be associated with tubular injury in renal disease, which in turn may affect renal function (23, 31). The mechanism linking USG and ACE2 remains unclear and requires further investigation through additional studies.

Our findings show that both UACCR and UAA in cats are not significantly associated with UPC. Indeed, previous investigations exploring the histopathological change in renal injury in cats have indicated that renal injury tends to more prominent affect the tubulointerstitium but not the glomerulus (32). Furthermore, A recent study revealed no association between renal ACE2 expression and renal damage in feline CKD based on histopathology (25). In small animal clinical practice, persistent renal proteinuria usually is due to glomerular injury when dogs have renal disease, whereas this seldom occurs in cats with renal disease (33). However, to eliminate the impact of ACEI/ARB, we also excluded cases with proteinuria, as they can be prescribed with ACEI/ARB, from our study. While this refinement ensures that the results specifically apply to cats with lower urinary protein levels, it may definitely underpower the association between UACCR, UAA, and UPC.

In our study, a positive correlation was found between urinary pH and UAA in cats with CKD. Previous research suggests that ACE2 substrate hydrolysis rates are pH-dependent, but the relationship between urinary ACE2 activity and pH may not be linear (34). Additionally, species-specific variations in the effect of pH on urinary ACE2 activity were noted (34). However, further investigation is required to elucidate the actual mechanism by which pH affects UAA alterations.

There were several limitations to our study. Firstly, we have a relatively small sample size for cats (n = 67). These smaller sample sizes may decrease statistical power and affect the interpretation of our results. Secondly, because clinical renal tissue is extremely difficult to obtain, renal ACE2 concentration and activity could not be determined simultaneously in our study, thus the relationship between renal and uACE2 could not be established. In addition, without a histopathological diagnosis, we could not further investigate the relationship between specific renal injury and uACE2. Thirdly, as using ACEi or ARB could affect the expression of ACE2, we have to exclude the cases with more severe proteinuria and hypertension simultaneously. Consequently, the association between ACE2 and various degrees of proteinuria or hypertension, which may indicate poor progression, cannot be evaluated. Also, the predictive ability of uACE2 to feline renal progression was unevaluable due to the exclusion of these severe cases. Fourthly, the absence of age-matched controls in our study prevents the simultaneous evaluation of the influence of age on both uACE2 concentration and activity. Fifthly, although linear regression and Spearman tests revealed no significant correlation between freeze duration and ACE2 activity (data not shown), it is important to note that we were unable to investigate the potential degradation of activity in the same sample over time. This limitation underscores the need for further exploration in future studies. Finally, in the present study, we did not evaluate other well recognized factors of RAAS, including Ang I, ACE, and Ang II, as well as other emerging RAAS peptides, such as Ang (1–9), Ang (1–7); these could have benefited the interpretation of our results.

In conclusion, it appears that the regulation of uACE2 concentration and activity undergoes alterations in varying directions as renal function declines, particularly in cases with advanced deterioration, among feline individuals afflicted with CKD.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal studies were approved by Institutional Animal Care and Use Committee (IACUC) National Taiwan University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

T-CK: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. W-LH: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing, Formal analysis. V-CW: Conceptualization, Methodology, Supervision, Writing – review & editing. T-RJ: Methodology, Supervision, Writing – review & editing. P-ST: Methodology, Supervision, Writing – review & editing. Y-JL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.