Introduction

Cardiorenal syndrome (CRS) is a complex disease that involves the cardiovascular system, the kidneys, and numerous neurohormonal systems [1]. It leads to dysfunction in one organ that impacts another [2]. This syndrome is classified into five subtypes, including acute and chronic CRS, acute and chronic renocardiac syndrome, and secondary CRS [3]. In dogs, the prevalence of azotemia with heart disease is between 7.4 and 24.1% [4]. Myxomatous mitral valve degeneration (MMVD) is the most common acquired cardiac disease in dogs, with its incidence increasing significantly with age [5]. The affected dog suffers from a reduction of cardiac output and triggering of compensatory mechanisms including the renin angiotensin aldosterone system (RAAS) and chronic stimulation of the sympathetic nervous system. This ultimately leads to congestive heart failure (CHF) [6,7]. Numerous investigations have demonstrated that both clinical and subclinical stages of MMVD are significant risk factors for the progression of kidney disease [8–12]. The American College of Veterinary Internal Medicine (ACVIM) consensus guideline for MMVD in dogs recommends treating affected dogs with pimobendan, an angiotensin converting enzyme inhibitor (ACEi), diuretics, and sildenafil [13]. Indeed, some of these medications may aggravate pre-existing renal damage [14–16]. Therefore, monitoring for renal damage during the treatment of heart disease is crucial. Routinely, serum creatinine and serum symmetric dimethylarginine (SDMA) are used as clinical renal markers in chronic kidney disease (CKD). However, they have some limitations, including low sensitivity for renal damage and the potential to be affected by concurrent diseases [17]. Proteomic techniques have emerged as powerful tools for identifying potential clinical biomarkers for various diseases in dogs, including MMVD and CKD [18–23]. Additionally, peptide mass fingerprints (PMFs), a proteomics approach that characterizes serum peptides by comparing the pattern of mass spectra have been used in exploratory research to distinguish peptide pattern differences in canine diseases, including MMVD associated with pulmonary hypertension (PH) and oral cancers [21,24], although these applications remain investigational and are not part of current diagnostic guidelines. Despite these advancements, the application of PMFs and comprehensive peptidomic profiling in CRS remains limited. Due to the complex interaction between the heart and kidneys in CRS, peptidomic analysis may help identify biomarkers and clarify the molecular mechanisms of kidney involvement. This could support earlier diagnosis and assist in designing more effective treatment plans. This investigation aimed to identify untargeted peptidomic signatures associated with cardiorenal syndrome type 2 (CRS type 2) secondary to myxomatous mitral valve disease (MMVD) using two complementary analytical approaches. First, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to compare peptide mass fingerprints (PMFs) among healthy dogs, dogs with non-azotemic MMVD, and dogs with azotemic MMVD representing MMVD-associated CRS type 2, with dogs with chronic kidney disease (CKD; IRIS stage 2) included primarily as a renal disease comparison group. PMF profiles were correlated with clinicopathologic data, echocardiography, and ultrasonography findings. Second, nano–liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) was performed to analyze peptide amino acid sequences based on mass-to-charge ratio (m/z) and to identify overexpressed proteins associated with MMVD-related cardiorenal pathophysiology, thereby providing insight into molecular association underlying the progression of CRS type 2 in dogs with chronic mitral valve disease.

We hypothesized that specific alterations in serum PMFs and peptide profiles detectable by peptidomic analysis may serve as sensitive biomarkers for detection and risk stratification of renal dysfunction in dogs with MMVD-associated CRS type 2, independent of changes induced by standard pharmacological treatments (e.g., pimobendan, ACE inhibitors, and diuretics).

Materials and methods

Results

Sample description data

The demographic characteristics of the 64 dogs enrolled in this study are presented in Table 1. The study population consisted of five groups: 15 clinically healthy individuals (Healthy), 10 dogs with MMVD at stage B1 (MMVD B1), 15 dogs with MMVD stage C without azotemia (MMVD C WOAZ), 13 dogs with MMVD stage C with azotemia (MMVD C WAZ), and 11 dogs with CKD at IRIS stage 2 (CKD stage 2). Age comparisons revealed that healthy dogs were significantly younger than dogs in all disease groups with a statistically significant difference (p < 0.01). In contrast, no significant differences were detected in body weight among the five groups (p = 0.98). The sex distribution within each group was approximately balanced between male and female dogs. No medication was administered to dogs in the Healthy, MMVD B1, and CKD stage 2 groups prior to sample collection. Dogs with MMVD stage C received standard cardiovascular therapy, which included one or more of the following: pimobendan, furosemide, spironolactone, ACEi, and sildenafil (S1 Table).

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Table 1. The demographic characteristics of the 64 dogs enrolled in this study.

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Hematologic and blood chemistry values for the Healthy, MMVD B1, and MMVD C WOAZ groups (S2 Table) were within normal reference ranges. Dogs in the MMVD C WAZ and CKD stage 2 groups showed mild azotemia, consistent with IRIS stage 2 classification. Because anemia, hypoalbuminemia, and uremia can influence serum peptide profiles through effects on protein metabolism, oxidative stress, and inflammatory activity, these variables were evaluated. No enrolled dogs demonstrated clinically significant anemia, marked hypoalbuminemia, or severe uremia based on complete blood count and serum biochemistry results. Thoracic radiographs revealed normal findings in the Healthy, MVD B1, and CKD stage 2 groups. However, cardiomegaly, defined as a VHS > 10.5, was observed in both MMVD C WOAZ and MMVD C WAZ groups, with or without concurrent pulmonary edema (S3 Table).

Thoracic radiographic and echocardiographic indices for the five study groups Healthy, MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD stage 2 are summarized in S3 Table. Radiographically, both VHS and VLAS increased significantly in the MMVD groups, particularly in MMVD C WOAZ and MMVD C WAZ, reflecting cardiomegaly and left atrial enlargement (both P < 0.001). Healthy and CKD stage 2 dogs exhibited VHS and VLAS values within normal ranges, consistent with the absence of primary cardiac disease.

Echocardiographic assessment revealed pronounced chamber enlargement in the clinical MMVD groups. LVIDd, LVIDs, normalized LVIDdN, and LA dimensions were significantly increased in MMVD C WOAZ and MMVD C WAZ compared with Healthy, MMVD B1, and CKD stage 2 groups (all P < 0.001). Systolic function, assessed by FS%, did not differ significantly across groups. E-wave and A-wave were elevated in the clinical MMVD groups (P < 0.05), while MR Vmax and TR Vmax were present only in MMVD C dogs, confirming hemodynamically significant regurgitation. CKD stage 2 dogs showed no evidence of structural cardiac enlargement and clustered closely with healthy controls in most echocardiographic parameters. Dogs in the MMVD stage B1 group demonstrated mitral regurgitation without evidence of cardiomegaly. Both the MMVD C WOAZ and MMVD C WAZ groups exhibited left atrial and left ventricular enlargement, consistent with a history of pulmonary edema in some individuals (Fig 1).

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Fig 1. Echocardiographs of the enrolled dogs.

Right parasternal shot-axis view of the left atrium level of MMVD stage B1 dogs (A) and MMVD stage C dogs (B), M-mode study of left ventricle at the papillary muscle of MMVD stage B1 (C) and MMVD stage C dogs (D).Right parasternal long axis view with a Colour Doppler study of the left atrium of MMVD stage C dogs (E).

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Abdominal ultrasonography of MMVD C WAZ and CKD stage 2 groups demonstrated mildly decreased renal perfusion, irregular renal contours, and increased echogenicity of both the cortical and medullary regions, as shown in Fig 2. These sonographic abnormalities were not observed in healthy controls, MMVD B1, MMVD C WOAZ groups.

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Fig 2. Ultrasonographic results of the enrolled dogs.

Sagittal view of the kidney of dog in the healthy (A) and CKD IRIS stage 2 group (B). Colour Doppler image of the kidney showing intrarenal vascular perfusion. Red and blue signals represent blood flow direction relative to the transducer. Patchy and reduced Doppler signal intensity indicates diminished cortical perfusion, a pattern consistent with early CKD-related vascular changes (C).

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Indirect systolic blood pressure did not differ significantly among groups (P = 0.599) and remained within the normotensive range in all dogs. Median systolic blood pressure values ranged from 129 to 131 mmHg across the five groups (S3 Table)

Urinalysis parameters across the five study groups Healthy, MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD stage 2 are summarized in S4 Table. Urine colour and clarity showed no significant differences among groups (P = 0.28 and P = 0.89, respectively). Most samples were yellow to pale yellow with clear to slightly cloudy appearance. Chemical dipstick findings, including protein, glucose, ketones, bilirubin, hemoglobin, urobilinogen, nitrite, and leukocytes, were largely unremarkable across all groups, with no statistically significant differences detected (P > 0.05). A notable difference was observed in urine concentrating ability.

Urine specific gravity (USG) decreased significantly in all disease groups compared with healthy dogs (P < 0.001). Healthy dogs demonstrated a median USG of 1.041, while MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD stage 2 groups showed progressive reductions in USG, with MMVD C WAZ exhibiting the lowest values. Urine pH did not differ significantly (P = 0.062), and crystal type distribution was comparable among groups. Sediment examination (RBCs and WBCs per high-power field) also showed no significant differences. Urine creatinine and urine protein concentrations did not vary significantly (P = 0.961 and P = 0.862, respectively). In contrast, UPCR differed significantly among groups (P < 0.001). Healthy and MMVD B1 dogs had UPCR values within the expected non-proteinuric range (median 0.09–0.10). Dogs in MMVD C WOAZ, MMVD C WAZ, and CKD stage 2 groups exhibited modest but significantly higher UPCR values (median 0.15), though values remained below the threshold for clinically relevant proteinuria (S4 Table).

Analysis of serum peptides by MALDI-TOF MS

In the 2D PLS-DA plot, the spectral data from the four replicates of each sample exhibited a distinct clustering pattern among the five groups using SIMCA®. Notably, the control group was clearly separated from the others, while the MMVD B1, MMVD C WAZ, MMVD C WOAZ, and CKD stage 2 groups clustered closely together, indicating a higher degree of similarity in their peptide profiles (Fig 3A). To ensure consistency within groups and validate the related peptide profiles, 32 replicates of pooled samples were analyzed, confirming homogeneity within each group (Fig 3B). The distinct PMFs ranged from 1 to 6 kDa of pooled samples among groups (Fig 3C).

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Fig 3. Partial least squares discriminant analysis (PLS-DA) and serum peptide mass fingerprints of dogs with myxomatous mitral valve disease and chronic kidney disease.

Two-dimensional scatterplot of the results of PLS-DA of individual (A) and pooled (B) serum samples from the Healthy, myxomatous mitral valve disease stage B1 (MMVD B1), MMVD stage C without azotemia (MMVD C WOAZ), MMVD stage C with azotemia (MMVD C WAZ), and CKD IRIS stage 2 groups (CKD stage 2), analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). (C) Peptide mass fingerprints (PMFs) of serum peptides in the mass range of 1,000–6,000 Da.

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Peptide and protein identification by nanoLC-MS/MS

A total of 19,405 proteins from all pooled samples were analyzed using in-solution digestion followed by nanoLC-MS/MS (details in S5 Table). The distribution and overlap of individual proteins across the five experimental groups Healthy, MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD Stage 2 were visualized using a Venn diagram (Fig 4A). A group of 510 common proteins was identified, suggesting they may represent a specific subset with shared characteristics or biological functions relevant to both the MMVD C WAZ and CKD stage 2 conditions (S6 Table). One hundred proteins were found to be statistically significant based on an ANOVA p < 0.05 (S7 Table). This significance suggests these proteins play a crucial role in the disease’s pathophysiology. Crucially, six common proteins were identified as being present in both MMVD C WAZ and CKD stage 2 groups and were among the 100 statistically significant proteins. These six proteins are peroxisomal membrane protein PEX14 (PEX14), glutaredoxin-2 (GLRX2), creatine kinase S-type (CKMT2), SECIS binding protein 2 (SECISBP2), LRRCT domain-containing protein, and histone H4 transcription factor (HINFP). These findings are further detailed in Fig 4B. and Table 2.

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Table 2. Significant differences in peptide alterations between the MMVD C WAZ and CKD stage 2 groups based on biological process involvement, and molecular function, using nanoLC-MS/MS data with FDR, and -log10 P-value < .05.

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Fig 4. Comparative serum proteomic profiles across Healthy, myxomatous mitral valve disease (MMVD), and chronic kidney disease IRIS stage 2 groups (CKD stage 2).

(A) Five-way Venn diagram and bar chart of total protein identification counts. The Venn diagram illustrates the number of unique and overlapping proteins identified in five different groups: Healthy, MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD stage 2. Numbers within overlapping regions indicate common proteins shared between those specific groups. The number 510 highlights the proteins found exclusively in common between the CKD stage 2 and MMVD C WAZ groups. (B) Two-way Venn diagram and bar chart of significant proteins shared between MMVD C WAZ and CKD stage 2 groups. The Venn diagram compares this list of 510 proteins (labeled “MMVD C WAZ and CKD”) with a list of proteins filtered using ANOVA at 0.05. The central overlap of 6 indicates the number of proteins that are both common to MMVD C WAZ and CKD stage 2 that meet the specific statistical criterion for significance.

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Molecular mechanism and interaction analysis

To better understand the molecular mechanisms associated with the proteins that changed across the five groups, particularly the proteins common to the MMVD C WAZ and CKD stage 2 conditions, researchers conducted several integrated analyses: Protein-protein and protein-drug interaction network analysis and pathway enrichment analysis. This focus was specifically placed on the six upregulated proteins that were identified as both statistically significant (by ANOVA) and common to MMVD C WAZ and CKD stage 2 (by Jvenn diagram). The functions of candidate peptides, including their involvement in disease pathophysiology, pathogenesis, and mechanisms of action were explored to identify prognostic biomarkers and potency assays for dogs that received treatment for cardiac diseases. The analysis also investigated the association between these six proteins and common cardiogenic drugs used for treatment, including pimobendan, furosemide, spironolactone, ACEi, and sildenafil. The protein-drug interaction network results (Fig 5) revealed distinct interaction patterns (accessed on December 5, 2025) using Stitch EMBL. Several upregulated proteins demonstrated multiple connections within the network, indicating interactions with all five tested drugs. This interaction for GLRX2 and CKMT2, which exhibited a strong relationship level in the protein interaction network (with an edge confidence >0.7), was present for SECISBP2 (with an edge confidence <0.4) with all drugs and functional partners, such as creatinine, creatinine phosphate, MgADP, MgATP, and reduced glutathione (Fig 5). The upregulated protein PEX14 was associated only with pimobendan (Fig 5A), while HINFP showed no interaction with any of the tested drugs (Fig 5A–5E).

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Fig 5. Protein–drug interaction network associated with oxidative stress and peroxisomal regulation in canine cardiorenal syndrome.

Red circles: peroxisomal membrane protein PEX14 (PEX14), glutaredoxin-2 (GLRX2), creatine kinase S-type (CKMT2), SECIS binding protein 2 (SECISBP2), LRRCT domain-containing protein, and histone H4 transcription factor (HINFP). Yellow boxes: pimobendan, spironolactone, furosemide, benazepril, sildenafil.

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Pathway enrichment analysis: Cardio-renal syndrome links

The KEGG pathway enrichment analysis using ShinyGO 0.77 (accessed on December 7, 2025), which was performed on the six significant proteins whose interactions with cardiogenic drugs are visualized in the protein-protein interaction networks (Fig 5A–5E), strongly suggests that the observed protein alterations and drug interactions are linked to the pathophysiology of CRS (Table 3). Crucially, the analysis highlights several pathways that appeared consistently across multiple drug interactions, underscoring their systemic role in both cardiac and kidney dysfunction. Peroxisome and arginine and proline metabolism pathways were associated with all five drugs (pimobendan, furosemide, spironolactone, benazepril, and sildenafil), pointing toward fundamental metabolic components, such as altered nitrogen metabolism, relevant to both CKD and cardiovascular health. Furthermore, the analysis reveals pathways highly characteristic of CRS, including the RAAS and renin secretion, which were linked to interactions involving furosemide (Fig 5B), spironolactone (Fig 5C), and benazepril (Fig 5D). The presence of diabetic cardiomyopathy (associated with Fig 5B–5D) and the aldosterone regulated sodium reabsorption pathway (Fig 5C) emphasizes the critical cardiovascular and renal regulatory components, respectively. Finally, the cGMP-PKG signaling pathway (Fig 5E) highlights an impact on vascular tone. In summation, the consistent enrichment of pathways governing the RAAS, renal regulation, cellular metabolism, and cardiovascular health provide robust evidence that the significant proteins and their drug interactions are intricately linked to the multiorgan dysfunction characteristic of cardiorenal syndrome. The biological processes and molecular functions of the protein alterations and all five drug interactions were demonstrated in S8 and S9 Tables.

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Table 3. Protein-drug pathway enrichment analysis performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG).

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Discussion

This study investigated the serum peptide profiles in healthy dogs, as well as in those diagnosed with MMVD stage B1 (MMVD B1), MMVD stage C (with and without azotemia) (MMVD C WOAZ and MMVD C WAZ), and CKD classified as IRIS stage 2 (CKD stage 2). We did not include MMVD stage B2 dogs due to the heterogeneous nature of this transitional stage, variability in cardiomegaly severity, and the frequent initiation of pimobendan therapy at this stage. Including B2 dogs would likely increase within group variability and reduce discriminatory power in peptide-based clustering. Future studies specifically targeting the B1 to B2 transition would be valuable for evaluating early peptide changes preceding overt cardiac remodelling.

The medication distribution across groups reflects expected clinical management for each disease stage and provides important context for interpreting the proteomic findings. As anticipated, dogs with MMVD C both azotemic and non-azotemic were uniformly treated with pimobendan and diuretics, consistent with ACVIM consensus guidelines for the diagnosis and treatment of myxomatous mitral valve disease in dogs [13]. The concurrent use of ACEi and spironolactone in many MMVD C dogs reflects attempts to mitigate RAAS activation, reduce afterload, and manage volume overload [35]. Importantly, Healthy, MMVD B1, and CKD stage 2 dogs were medication-naïve with respect to cardiovascular drugs. This distinction helps ensure that observed peptide alterations in these groups are attributable to underlying disease processes rather than pharmacologic effects. In contrast, the MMVD C groups experienced substantial exposure to multiple cardiogenic medications, each of which may influence neurohormonal signaling, oxidative stress, renal hemodynamics, or mitochondrial metabolism [35,36]. These drug effects are therefore relevant contributors to the complex proteomic signatures observed in the MMVD C WOAZ and MMVD C WAZ groups. The differences in sildenafil and spironolactone usage between MMVD C WOAZ and MMVD C WAZ may also reflect variations in pulmonary hypertension severity or volume status, which could further modulate peptide expression [20,21]. However, the uniform administration of pimobendan and furosemide in both MMVD C subgroups ensures that treatment effects were generally consistent within disease stages.

Hematologic and biochemical values were generally stable across groups, indicating that most dogs did not exhibit systemic inflammation, severe anemia, or metabolic disturbances that might confound peptide expression patterns. The minor reductions in RBC count, hemoglobin, and hematocrit observed in the MMVD C WAZ and CKD stage 2 groups likely reflect early renal-associated anemia or hemodilution secondary to heart failure, but the magnitude of these changes was small and clinically insignificant. The biochemical results clearly differentiated azotemic from non-azotemic groups. The significant increases in creatinine and BUN in MMVD C WAZ and CKD stage 2 confirm renal involvement in these groups and validate their classification as cardiorenal or renal disease states. Importantly, albumin concentrations remained largely within normal limits, suggesting that neither protein-losing nephropathy nor marked malnutrition influenced peptide profiles. The absence of abnormalities in liver enzymes, glucose, leukogram parameters, and most red cell indices supports that systemic inflammatory or metabolic factors were not major contributors to the proteomic patterns identified. Thus, peptide alterations observed in MMVD C WAZ and CKD stage 2 likely stem from disease specific cardiorenal mechanisms such as decreased renal clearance, oxidative stress, neurohormonal activation, and tissue remodeling rather than generalized illness or unrelated systemic factors [37,38]

Radiographic and echocardiographic findings aligned well with expected pathophysiology across disease groups. The significant increases in VHS and VLAS in MMVD C WOAZ and MMVD C WAZ are consistent with progressive cardiac remodelling associated with chronic mitral regurgitation. These radiographic indicators confirmed left atrial and ventricular enlargement, which was further supported by echocardiographic increases in LVIDd, LVIDs, LA size, and LVIDdN. The absence of cardiac enlargement in both the healthy and CKD stage 2 groups indicates that renal disease alone did not contribute to measurable cardiac structural change in this cohort. This distinction reinforces that the peptide alterations identified in CKD stage 2 dogs are not attributable to occult cardiac disease. E- and -A wave elevations and the presence of MR Vmax and TR Vmax exclusively in the MMVD C groups confirm advanced diastolic loading conditions and valvular insufficiency [39]. These functional changes likely contribute to systemic neurohormonal activation, including RAAS stimulation [40], which is central in the development of cardiorenal interactions. In the present study, systolic function indices such as FS% did not differ significantly among groups. This observation is consistent with the pathophysiology of chronic mitral regurgitation in MMVD, in which cardiac dysfunction is primarily driven by chronic volume overload rather than intrinsic myocardial systolic failure. As a result, systolic indices such as FS% often remain within normal or even supranormal ranges due to compensatory hyperdynamic ventricular contraction and reduced effective afterload created by regurgitant flow into the left atrium [41]. Importantly, both preload and afterload conditions appeared relatively stable in the present cohort. The intravascular volume status of the enrolled dogs appeared physiologically stable, as total protein concentrations across all groups remained within normal reference ranges and showed no evidence of hemoconcentration suggestive of dehydration. In addition, systemic hypertension was excluded, with systolic blood pressure measurements remaining within the normal range. Because FS% is a load-dependent parameter influenced by changes in preload and afterload [42], the preservation of this index in MMVD stage C dogs in this study may reflect the expected hemodynamic characteristics of chronic mitral regurgitation rather than the absence of heart failure. These findings support the subsequent proteomic results by validating that MMVD C WOAZ and MMVD C WAZ groups represent distinct pathophysiological states involving cardiac remodelling, circulatory congestion, and increased neurohormonal burden factors that plausibly drive the peptide alterations identified through MALDI-TOF MS and nanoLC-MS/MS.

Urinalysis findings highlighted meaningful differences in renal function across groups, particularly decreased urine concentrating ability and mild UPCR elevations in dogs with advanced MMVD and CKD. While urine colour, clarity, and dipstick chemistry were not discriminatory, USG and UPCR showed potential as informative renal indicators in the context of cardiorenal involvement. USG was significantly reduced in all MMVD and CKD groups, reflecting impaired concentrating capacity likely related to early tubular dysfunction, reduced renal perfusion, or chronic hemodynamic alterations [43]. Importantly, dogs in the MMVD C WOAZ and MMVD C WAZ groups were receiving furosemide, which physiologically increases diuresis and further lowers USG, compounding the effect of cardiac disease. The MMVD C WAZ group demonstrated the lowest USG values, consistent with azotemia and ultrasonographic evidence of reduced renal perfusion, supporting established CRS pathophysiologic mechanisms [44]. Although overt proteinuria was not present, UPCR was significantly higher in MMVD C WOAZ, MMVD C WAZ, and CKD stage 2 dogs, suggesting the possibility of subtle glomerular or tubular injury mediated by neurohormonal activation and hemodynamic stress [45]. Stable urine creatinine and urine protein values further suggest that UPCR may serve as a more sensitive early marker of renal compromise. Normal sediment and dipstick findings indicate that urinary tract inflammation or infection did not influence these patterns. Overall, the urinalysis data align closely with the proteomic findings, providing functional evidence of early renal impairment in MMVD C WAZ and CKD stage 2 dogs. The combination of reduced USG exacerbated by furosemide therapy in MMVD C groups and modest UPCR elevations supports the conclusion that subclinical renal changes in CRS are detectable both biochemically and at the peptide expression level.

Indirect systolic blood pressure measurements remained within the normotensive range in all enrolled dogs. Dogs with systemic hypertension (systolic blood pressure >140 mmHg) were excluded according to IRIS guidelines [14], minimizing the potential confounding effects of hypertension on renal or cardiovascular function. Hypertension is a well-recognized contributor to both renal injury and cardiac remodeling, particularly through increased afterload and glomerular hypertension. Therefore, the absence of systemic hypertension in this cohort suggests that the observed cardiac and renal alterations were primarily attributable to underlying MMVD or CKD rather than pressure-mediated vascular injury. These findings strengthen the interpretation that the proteomic alterations detected in this study are linked to cardiorenal pathophysiology rather than secondary effects of hypertension.

Peptide profiling was performed using MALDI-TOF MS and nanoLC-MS/MS. A key finding of this investigation was the identification of differentially expressed serum peptides potentially involved in the pathophysiological mechanisms underlying CRS, particularly in the MMVD C WAZ and CKD stage 2 groups and may aid in classification between groups and understanding of disease pathophysiology. MMVD staging was based on radiographic and echocardiographic criteria [13], while MMVD C WAZ and CKD stage 2 groups were classified using a combination of clinical parameters including BUN, serum creatinine levels, renal ultrasonographic findings (blood flow and morphology), systemic blood pressure, and urinalysis [25]. MMVD is the most common acquired cardiac disease in small-breed dogs, with the prevalence of this condition increasing with age [46]. Specifically, the prevalence of MMVD was found to be 23.9% in dogs aged 6–10 years and this rose to 30.3% in those aged 10–19 years [13]. However, in this study, the healthy group had a significantly younger average age compared to the other groups, since old animals are more likely to have other comorbidities, such as CKD or neoplasia. Nevertheless, all dogs in this study were of advanced age (>7 years) [47], and both male and female dogs were proportionately represented across all groups, suggesting that sex differences were unlikely to influence the study’s findings. Consistent with previous proteomic studies in dogs [18,21], our group sizes (10–15 dogs) reflect practical constraints and case availability rather than a powered a priori statistical calculation. Although this sample size is appropriate for exploratory mass-spectrometry based biomarker studies, larger, prospectively powered cohorts will be needed to validate these candidate peptides. Both clinical and subclinical stages of MMVD have been identified as significant risk factors for the development and progression of kidney disease [8–12]. Consequently, close monitoring of renal function is essential during the management of cardiac disease. While conventional renal biomarkers such as serum creatinine and SDMA are routinely used, they have limitations in sensitivity and may be influenced by concurrent conditions [17].

The analysis using MALDI-TOF MS revealed a distinct PMFs separation of the healthy control group from all disease affected groups (MMVD and CKD) in the 2D PLS-DA plot. This finding confirms that chronic cardiac and renal pathologies induce significant and measurable changes in the serum peptidome, highlighting the potential of MALDI-TOF MS to serve as a rapid and reliable auxiliary screening tool for the presence of these systemic diseases in dogs. This result aligns with earlier MALDI-TOF MS studies in veterinary cardiology, which similarly observed discrete clustering and distinct peptide barcodes between healthy and diseased cohorts [21]. However, the observed close clustering among the MMVD subgroups and the CKD stage 2 group suggests a considerable overlap in their low molecular weight peptidomic signatures, indicating that the peptides detected may represent shared systemic pathophysiological processes such as chronic inflammation, compromised hemostasis, or common degradation pathways related to multiple organ dysfunction (e.g., cardiorenal syndrome) [48]. This approach, coupled with the rigorous sample preparation and analysis confirmed by the internal homogeneity of the pooled samples, provides a strong foundation for future validation studies necessary to translate these promising PMFs or peptide barcodes into precise and clinically actionable biomarkers for the differential diagnosis and monitoring of canine MMVD and concurrent CKD.

All peptides and proteins were identified from each of the pooled serum samples by using nanoLC-MS/MS, successfully delineating distinct molecular signatures across the five experimental groups: Healthy, MMVD B1, MMVD C WOAZ, MMVD C WAZ, and CKD Stage 2. The identification of 100 statistically significant proteins based on ANOVA (p < 0.05) points to a core set of proteins intimately involved in the pathophysiology of these chronic conditions. A critical finding is the presence of a large subset of 510 common proteins and specifically, six shared statistically significant proteins (PEX14, GLRX2, CKMT2, SECISBP2, LRRCT domain-containing protein, and HINFP) between the MMVD C WAZ and CKD Stage 2 groups. The identification of six shared statistically significant proteins between the MMVD C WAZ and CKD Stage 2 groups provides a molecular basis for understanding the progression of cardiorenal dysfunction. These proteins serve as potential prognostic biomarkers and may act as indicators of treatment efficacy in dogs receiving pimobendan, furosemide, spironolactone, benazepril, and sildenafil [13]. The drug-protein interaction network revealed that these significant proteins are deeply embedded in pathways that appear consistently across multiple drug interactions, highlighting their systemic role in cardiac and kidney health.

In our study, peroxisome and arginine and proline metabolism were found to be associated with all five drugs used in this study (pimobendan, furosemide, spironolactone, benazepril, and sildenafil). The systemic nature of CRS is increasingly understood as a convergence of metabolic failure and oxidative stress. Central to this process is the role of peroxisomes. Peroxisomes are essential for the β-oxidation of very-long-chain fatty acids, substrates that mitochondria cannot efficiently metabolize [49]. Impaired peroxisomal function disrupts this lipid oxidation pathway, a metabolic hallmark documented in mammalian heart failure models and dogs with failing myocardium [50]. Furthermore, peroxisomes regulate cellular redox balance by producing and scavenging reactive oxygen and nitrogen species (ROS) [51,52]. The clinical importance of these metabolic functions is underscored by the protective role of peroxisomal enzymes in cardiovascular and renal [53,54]. Specifically, the overexpression of catalase (CAT), a primary peroxisomal antioxidant, facilitates significant cardioprotection by reducing oxidized lipid-induced cytotoxicity and preventing pathological mechanical alterations in vascular smooth muscle cells. Furthermore, CAT overexpression in renal tubular cells provides essential renoprotection, as demonstrated in various mouse models where it mitigates oxidative stress to prevent the development of hypertension, albuminuria, and tubulointerstitial fibrosis [55].

Building upon this metabolic foundation, our proteomic analysis identified a panel of significant proteins, including PEX14, GLRX2, CKMT2, and SECISBP2 that collectively point toward a unifying mechanism of peroxisomal and mitochondrial failure. The identification of PEX14 as a shared, statistically significant protein in both MMVD with azotemia and CKD stage 2 groups is critical. PEX14 acts as the primary docking site for peroxisomal protein import [56]. Specifically, dysregulation of PEX14 disrupts the import of antioxidant enzymes like CAT, which normally provides cardioprotection and renoprotection by mitigating oxidative stress, preventing albuminuria, and reducing tubulointerstitial fibrosis. Notably, while our analysis uniquely associated PEX14 with pimobendan using the Stitch (EMBL) database, its broader role suggests that the lack of efficient protein import promotes hydrogen peroxide (H_₂O_₂) mediated injury and dysregulated lipid metabolism, triggering pro-fibrotic signaling in both the heart and kidneys [57,58]. Complementing this peroxisomal failure is the significant detection of GLRX2 and CKMT2, which reflect severe mitochondrial and energetic compromise [59,60]. As a mitochondrial redox enzyme, GLRX2 is vital for protecting mitochondria from oxidative damage [59,61]. Its insufficiency leads to the accumulation of S-glutathionylation on metabolic enzymes, reducing ATP synthesis and establishing a self-perpetuating cycle of systemic injury characteristic of CRS [36,38,62]. Concurrently, the sarcomeric isoform CKMT2 is essential for ATP regeneration through the phosphocreatine shuttle [58]. Vulnerability to ROS leads to the oxidation of CKMT2, suppressing its enzymatic activity and causing mitochondrial overload and inefficient contractile performance [63,64]. This energetic failure, common to both advanced MMVD and CKD, facilitates the leakage of peptide fragments into the bloodstream, a pattern observed in the proteomic profiles of our disease cohorts. Finally, the identification of SECISBP2 underscores a systemic failure in cellular redox capacity [65]. SECISBP2 is the key determinant for synthesizing antioxidant selenoproteins, such as glutathione peroxidases and thioredoxin reductases [66]. The oxidative stress and inflammation hallmarks of heart disease and CKD likely impair SECISBP2 activity, suppressing downstream antioxidant defenses [67,68]. In the myocardium, this leads to mitochondrial injury and sarcomeric destabilization, while in the kidney, it contributes to tubular oxidative stress and inflammation [69]. Together, the alterations in PEX14, GLRX2, CKMT2, and SECISBP2 demonstrate that peroxisomal and mitochondrial dysfunctions are convergent drivers of metabolic derangement and structural injury in the broader context of canine cardiorenal syndrome.

Arginine and proline metabolism represents a central metabolic hub where imbalances directly contribute to the pathogenesis of both cardiovascular and renal diseases. Reduced nitric oxide (NO) synthesis from L-arginine often caused by endothelial dysfunction promotes atherosclerotic progression and the deterioration of renal function [70]. Furthermore, impaired proline catabolism via proline dehydrogenase reduces mitochondrial energy supply and ATP synthesis, establishing a link between metabolic shifting and myocardial remodeling [71]. Within the kidney, these pathways govern redox balance and apoptosis regulation, where L-proline levels specifically influence cellular survival and protect against fibrotic tissue damage [72]. Ultimately, the integration of these amino acid pathways underscores their role as both biomarkers of disease progression and therapeutic targets for ameliorating cardiorenal syndrome [70]. The identification of shared alterations in PEX14 and the arginine and proline metabolism pathway suggests an integrated peroxisomal metabolic axis in the pathogenesis of canine cardiorenal syndrome. PEX14 is a critical regulator of peroxisomal biogenesis and protein docking; its dysregulation, potentially influenced by phosphorylation changes, impairs organelle functions such as fatty acid β-oxidation and ROS scavenging [49,56]. This peroxisomal failure likely disrupts the biosynthesis of protective metabolites like proline and putrescine, which are essential for osmotic regulation, energy supply, and mitigating oxidative injury in failing cardiac and renal tissues [73]. Consequently, the convergence of PEX14 mediated peroxisomal dysfunction and impaired amino acid catabolism establishes a unifying mechanism for the energetic and structural remodeling observed in cardiorenal deterioration. The convergence of GLRX2 and the arginine-proline metabolic axis reveals a sophisticated redox-shielding mechanism that is likely compromised in canine cardiorenal disease. GLRX2 serves as a critical mitochondrial sensor, utilizing its iron-sulfur center to release active monomers during oxidative stress, which then catalyze protein deglutathionylation to maintain enzymatic integrity [74]. This antioxidant response is bolstered by L-arginine, which activates the Nrf2-Keap1 pathway to upregulate essential glutathione-dependent enzymes, and by proline metabolism, which facilitates a proadaptive stress resistance by enhancing catalase and glutaredoxin activities. In the context of the cardiorenal syndrome, the failure of this integrated system potentially driven by the previously discussed peroxisomal PEX14 dysfunction impairs the mitochondrial matrix’s buffering capacity [75]. When GLRX2-mediated deglutathionylation and the Nrf2-driven antioxidant supply fail, the resulting unchecked oxidative stress and energetic decline accelerate the structural remodeling and fibrosis observed in both heart and kidney tissues [62]. The association between the arginine-proline metabolism pathway and the creatine kinase system (CKMT2/CKMT1) highlights a critical metabolic axis for cellular energy buffering that is likely disrupted in cardiorenal disease. Arginine serves as a direct biosynthetic precursor for creatine, and our findings suggest that intracellular arginine pools dictate the capacity for creatine synthesis, which is essential for the phosphocreatine shuttle governed by mitochondrial CKMT2 [60]. In the context of the cardiorenal syndrome, the energy failure observed in failing myocardium and renal tubules may stem from a breakdown in this axis; specifically, impaired arginine availability limits the synthesis of creatine, thereby compromising the ATP-buffering capacity of CKMT2 [76]. While this metabolic shift is driven by mitochondrial and cytosolic demand, it may be further exacerbated by the previously discussed peroxisomal dysfunction, as peroxisomes regulate the upstream redox environment and amino acid catabolism necessary to maintain these arginine-driven energetic pathways. Although direct reports linking SECISBP2 specifically to arginine or proline metabolism are scarce, these pathways are functionally interdependent through the thiol-redox system [66]. This protein represents a critical regulatory layer that ensures the functional expression of the antioxidant systems previously discussed, such as the glutathione-dependent pathways linked to GLRX2.

Moreover, the identification of altered abundance in PEX14, GLRX2, CKMT2, and SECISBP2 provides a high-resolution molecular snapshot of the cellular stress encountered in dogs with advanced MMVD and CKD. We propose that these proteins serve as systemic markers of the strain caused by sustained RAAS activation and the resultant oxidative burden, which persists despite standard pharmacological intervention. The standard therapy consisting of pimobendan, furosemide, spironolactone, and benazepril, often supplemented with sildenafil aims to control congestion and modulate neurohormonal signaling. However, the persistence of these protein markers suggests a deep-seated disruption of organellar redox homeostasis. Furosemide acts at the sodium potassium chloride cotransporter 2 transporter to promote natriuresis, but its use simultaneously triggers renin release, leading to increased angiotensin II and aldosterone production [77]. This signaling stimulates nicotinamide adenine dinucleotide phosphate oxidase activity and mitochondrial ROS generation [78]. While benazepril and spironolactone attempt to mitigate this by limiting Angiotensin II conversion and blocking mineralocorticoid receptors, respectively, the chronic RAAS signaling continues to drive excessive ROS via mitochondrial pathways, contributing to myocardial remodeling and renal tubular injury [79].

Our proteomic findings suggest that this oxidative environment leads to peroxisomal and mitochondrial failure. The dysregulation of PEX14 is critical, as it impairs the import of vital antioxidant enzymes like catalase, thereby increasing H_₂O_₂-mediated injury in both the heart and kidneys [80]. Concurrently, changes in GLRX2 and CKMT2 reflect a compensatory but insufficient response to energetic compromise and mitochondrial redox imbalance [74,81]. The identified organellar dysfunction characterized by peroxisomal and mitochondrial failure are intrinsically linked to the dysregulation of arginine and proline metabolism, a central metabolic hub where biochemical supply and demand converge to accelerate cardiorenal deterioration. Arginine serves as the essential precursor for NO, a critical signaling molecule for vasodilation and renoprotection; however, the chronic oxidative stress environment of CRS reduces NO bioavailability, a deficit that pharmacological interventions like sildenafil attempt to restore by enhancing the cGMP–PKG signaling axis [82]. Ultimately, the convergence of RAAS driven oxidative stress and the relative suppression of this protective cGMP–PKG axis facilitates a toxic metabolic environment that reinforces peroxisomal and mitochondrial dysfunction. This environment shifts systemic metabolic pathways, particularly arginine and proline metabolism, toward maladaptive states: the depletion of arginine pools limits vasodilatory capacity, while dysregulated proline metabolism promotes collagen synthesis and fibrotic remodeling. These metabolic shifts do not occur in isolation but act as a self-perpetuating cycle, where impaired organellar energy production and amino acid imbalances contribute to the parallel cardiac and renal structural deterioration observed in canine cardiorenal syndrome.

This study has several limitations that should be considered when interpreting the results. First limitation is that systolic function was evaluated primarily using FS%, which is a load-dependent parameter influenced by changes in preload and afterload. Therefore, FS% may not fully reflect intrinsic myocardial contractility in this disease context. Future studies may benefit from incorporating less load-dependent indices, such as tissue Doppler imaging, speckle-tracking echocardiography–derived myocardial strain, or the Tei index, which may provide a more sensitive assessment of ventricular function in canine cardiorenal syndrome. Second, the sample size (10–15 dogs per group) was relatively small, reflecting the exploratory nature of the study and case availability. While sufficient for identifying candidate proteins, larger prospective cohorts are needed for validation. Third, the group classification relied on clinical parameters and international guidelines [13,14]; however, individual biological variability and subclinical comorbidities may still influence peptidomic results. The lack of significant changes in Stage B1 and non-azotemic MMVD groups suggests that the identified mitochondrial and peroxisomal dysfunctions are specific to the more severe, systemic phase of cardiorenal syndrome. Fourth, the use of pooled samples for nanoLC-MS/MS was necessary to identify core protein differences between disease states, but it limits the assessment of individual variation within groups. Fifth, since this study focused specifically on Type 2 cardiorenal syndrome with a small sample size, these peptidomic signatures may not apply to all CRS types, such as Type 4. Additionally, although these profiles were observed in azotemic cases, they were absent in MMVD cases without azotemia (WOAZ), suggesting that these markers reflect later stages of the cardiorenal continuum rather than early subclinical detection. These signatures provide valuable insights into the molecular remodeling associated with Type 2 CRS, but their generalizability remains to be established. Furthermore, we acknowledge the limitation that our data demonstrate an association between current therapeutic protocols and the maintenance of certain metabolic pathways, rather than causal links. As this study involved client-owned animals requiring standard-of-care, we cannot definitively rule out whether some peptide fragments are exacerbated by the treatments themselves (e.g., drug-induced RAAS activation) in the absence of a treatment-naïve control group. This clarification is included to ensure a balanced interpretation of the molecular changes observed under standard medical therapy. The absence of these datasets limits the generalizability of our findings across the entire cardiorenal syndrome spectrum, and future research incorporating treatment-naïve cases and varying CRS subtypes is warranted to validate these proteomic signatures. To mitigate this, our study design utilized MALDI-TOF MS and nLC-MS/MS as complementary steps in a discovery pipeline. MALDI-TOF MS was employed as a high-throughput screening tool to evaluate the global peptide “fingerprint” and to verify the homogeneity of individual samples within each clinical group. This ensured that the subsequent nLC-MS/MS analysis was performed on representative pooled samples. While the PMF peaks provide a rapid means of group classification based on mass distribution, our primary objective for the nLC-MS/MS analysis was to perform a deep-dive into the peptidomic landscape to identify specific biomarkers and their associated pathways (e.g., mitochondrial and peroxisomal failure). Although we did not perform a direct one-to-one assignment of MALDI peaks to peptide sequences, the nLC-MS/MS results provide the necessary biological identity to the peptidomic alterations observed during the initial screening. Finally, MALDI-TOF MS remains an investigational tool, and the resulting peptide mass fingerprints, as well as the low-molecular-weight peptides identified, can be difficult to confirm using conventional laboratory assays, requiring specialized equipment for future clinical application.

Conclusion

Our study demonstrates that canine cardiovascular-renal dysfunction originating from cardiovascular disease (CvRDh) is characterized by distinct serum peptide and protein alterations reflecting a systemic failure of cellular redox and energetic homeostasis. The identification of PEX14, GLRX2, CKMT2, and SECISBP2 as key proteomic signatures highlights a common association of peroxisomal and mitochondrial dysfunction, driven by chronic RAAS activation and oxidative stress. These molecular changes persist despite standard medical therapy, indicating that current therapies might not fully address the fundamental organellar metabolic failure. Our results suggest that proteomic profiling and PMFs have significant potential as additional tools for tracking the progression and risk assessment of advanced kidney disease in dogs with heart failure. These peptidomic characteristics could not be applicable to other CRS types, though, because this study only examined Type 2 cardiorenal syndrome with a small sample size. Furthermore, these profiles were lacking in MMVD cases without azotemia (WOAZ), although being seen in azotemic cases, indicating that these markers represent later stages of the cardiorenal continuum. These signatures provide valuable insights into the molecular remodeling associated with Type 2 CRS, potentially leading to more targeted and effective treatment plans for affected dogs. Future studies should investigate these Type 2 CRS groups to further validate the role of mitochondrial and peroxisomal biomarkers across all CRS subtypes.

Supporting information

Acknowledgments

The authors wish to express their sincere gratitude to the Faculty of Veterinary Science, Mahidol University (Thailand), and the dedicated staff at Prasu Arthorn Veterinary Teaching Hospital, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand, for their valuable support and assistance throughout this study. Special thanks are also extended to all the dog owners who generously participated in the research.