Biomarkers for evaluation of renal function

Introduction

Chronic kidney disease (CKD) is very common in dogs and cats, particularly among the geriatric population, and consequently diagnosing and managing the condition is a routine aspect of small animal clinical practice (1). The kidneys possess a significant functional reserve, therefore in the initial period clinical signs of CKD are often absent. Polyuria and polydipsia are present in many affected animals in the early stages, but can be mild and are often overlooked by pet owners. In veterinary medicine, diagnosing CKD can be particularly challenging in the initial stages, despite the fact that this is when medical management is likely most effective. Early diagnosis enables timely intervention, which is aimed at preserving functional renal parenchyma, slowing disease progression, delaying the onset of clinical signs, and enhancing both quality of life and longevity. Conversely, when the disease is identified at an advanced stage, treatment is primarily symptomatic, as a substantial portion of functional kidney tissue has already been lost.

How is CKD best identified?

Measurement of glomerular filtration rate (GFR) is regarded as the gold standard for assessing renal function. However, the methods used can be technically demanding (depending on the specific technique employed), and the results are often challenging to interpret, making GFR measurement impractical for routine clinical use (2). Renal biopsy is similarly considered as gold standard for diagnosing and characterizing kidney disease, but it is an invasive and costly procedure, typically reserved for specific indications, such as evaluation of glomerular disease. As a result, the diagnosis of CKD primarily relies on the interpretation of laboratory tests and imaging modalities. Common diagnostic tools for the detection and monitoring of kidney disease include surrogate markers of GFR (e.g., serum creatinine [sCr], urea (or BUN), and symmetric dimethyl-arginine (SDMA)), urinalysis, and diagnostic imaging (2, 3). A key limitation of these markers is their low sensitivity, particularly in the early stages of the disease, as well as their low specificity (Figure 1) (4). The non-linear relationship between filtration markers and GFR implies that substantial changes in GFR in early CKD lead to only small changes in the concentrations of these markers (Figure 2) – and these subtle changes often occur within the “normal” reference range and are consequently overlooked. This is likely the origin of the common misconception that a reduction of at least 75% in kidney function is required for detection using surrogate markers of GFR (5). In reality, any decrease in GFR is expected to result in an increased concentration of filtration markers; however, due to the limitations mentioned above, such changes frequently go undetected, particularly when the reference interval of the marker is broad.

Another limitation of the currently used markers is their inability to distinguish between the underlying causes of their elevation, such as hemodynamic reasons (i.e., pre-renal), post-renal, or intrinsic renal factors (e.g., acute kidney injury or chronic kidney disease) (6). Therefore, it is crucial to recognize that these functional markers reflect a single point in time for kidney function, and cannot identify the cause of the increase or determine trends without serial measurements.

Biomarkers to assess kidney function 

Urine concentration ability

Urine specific gravity (USG) is frequently used in veterinary practice as a supplementary tool for assessing renal function, and is considered a relatively sensitive marker for the early diagnosis of CKD. Urine concentration ability is often impaired before measurable reductions in kidney function become evident, so decreased kidney function may be suspected in animals presenting with polyuria and polydipsia (Figure 3). However, USG is nonspecific, and a renal cause should only be suspected after ruling out other differential diagnoses; furthermore, it is important to note that a single sample of unconcentrated urine (i.e., USG <1.030 in dogs or <1.035 in cats) does not confirm a loss of urine concentration ability (7). Persistence of this finding should always be assessed in conjunction with daily water intake. Furthermore, USG is not a highly sensitive marker for CKD, as urine concentration ability is typically lost only after significant reductions in kidney function have occurred. Notably, some animals, particularly cats, may retain their urine concentration ability at the early stages of the disease, even when functional markers exceed the upper limit of the reference range. Therefore, in cases of persistent azotemia with preserved urine concentration ability, CKD should be considered right after excluding other causes of azotemia. In the advanced stages of CKD, however, all affected animals are expected to lose their ability to concentrate urine.

Serum creatinine

Creatinine is primarily derived from the breakdown of creatine and creatine phosphate in muscle tissue (8,9). It is released into the circulation at a relatively constant rate, proportional to muscle mass, and is cleared by glomerular filtration, making it a reliable biomarker for GFR. However, its concentration varies widely due to differences in muscle mass, being lower in small-breed dogs and higher in large-breed dogs, resulting in a broad reference range. Therefore, utilization of breed-specific reference intervals has been suggested (8). Additionally, creatinine reference ranges vary between laboratories, further complicating its interpretation, and so there are several limitations to the sensitivity and specificity of sCr in detecting renal dysfunction (10) (Table 1). As noted earlier, GFR may decline significantly in the early stages of CKD, with only minimal increases in sCr levels, often remaining within the reference range. Consequently, sCr has limited utility for the early diagnosis of CKD unless careful attention is given to subtle changes within the reference range. To address this, it is beneficial to establish the baseline sCr concentration for an individual animal when healthy, and to use consistent methodologies and the same laboratory for all subsequent measurements. This approach enables comparisons of future values to the patient’s baseline, rather than relying on broad and variable reference intervals, making serial measurements a more effective diagnostic tool. In contrast, during the later stages of CKD, even minor declines in GFR result in significant and rapid increases in sCr concentrations. 

 

Table 1. Internal and external factors affecting serum creatinine (sCr) concentration (10).

 

When interpreting sCr values, it is very important to incorporate the patient’s body size: small breeds typically have low baseline sCr (<1.0 mg/dL/88.4 μmol/L), medium-sized dogs tend to have sCr concentrations around 1.0 mg/dL, and large breeds usually have sCr concentrations exceeding this level, though rarely surpassing 1.4 mg/dL (124 μmol/L). Therefore, a toy breed dog with a creatinine concentration of 1.3 mg/dL (115 μmol/L) may fall within the “general” reference range, but is atypical for the breed, and should be considered abnormal.

SDMA

A byproduct of intracellular protein metabolism, SDMA is primarily cleared by glomerular filtration. Unlike creatinine, its concentration is not influenced by sex or bodyweight in domestic animals (8), so as a filtration marker it offers the advantage of being unaffected by muscle mass, enabling earlier detection of CKD in certain cases, and hence has been incorporated into the IRIS (International Renal Interest Society) CKD guidelines for the diagnosis and staging of CKD (11). Studies have suggested that evaluating SDMA may facilitate earlier diagnosis of CKD (12,13), and both SDMA and creatinine can be used independently to assess kidney function. However, when there is uncertainty about whether a single marker accurately reflects renal function, it is advisable to consider a panel of biomarkers. This approach is particularly useful in animals with very high or low muscle mass, where sCr interpretation can be challenging. Persistent SDMA elevations above 14 μg/dL indicate reduced kidney function, even when sCr remains within the reference interval, making it a valuable marker for identifying animals in IRIS CKD stage 1 (11). In cats, an elevated serum SDMA concentration as a single-point measurement or screening test has shown moderate effectiveness in confirming CKD, but further monitoring is recommended for accurate diagnosis and staging (13).

Urine protein to creatinine ratio (UPC)

Persistent renal proteinuria may be an early indication of kidney disease, so when detected, pre-renal, renal, and post-renal causes should be evaluated. Renal proteinuria is most commonly of glomerular origin; however, tubular proteinuria (and, to a lesser extent, interstitial proteinuria) should also be considered. If renal proteinuria is confirmed, it should be quantified using the urine protein to creatinine ratio (UPC). Normal UPC values are <0.2 for both dogs and cats. UPC values between 0.2-0.5 in dogs and 0.2-0.4 in cats are classified as borderline proteinuria. Persistent renal proteinuria with a UPC >0.4 in cats or >0.5 in dogs is considered abnormal. High-magnitude persistent proteinuria (UPC >2.0) is most often glomerular in origin, and warrants further diagnostic investigation. Glomerular diseases can be broadly categorized as immune complex-mediated or non-immune complex-mediated. A diagnostic workup should aim to identify the underlying cause (e.g., an infectious agent), as its recognition and elimination are high-priority therapeutic goals, with the potential to achieve complete remission. A good-quality renal biopsy, assessed by a nephropathologist using specialized stains and electron microscopy, provides critical information for characterizing the pathological pattern and guiding treatment decisions (e.g., immunosuppression). In cases of persistent renal proteinuria, a diagnosis of CKD stage 1 should be made, even if kidney function remains within normal limits. Changes in the magnitude of proteinuria should always be interpreted in the context of kidney function, as proteinuria may decrease in the late stages of CKD, due to a reduction in the number of functional nephrons.

Overall assessment of kidney function

There is no single biomarker with sufficiently high sensitivity and specificity to accurately detect the full spectrum of kidney diseases and their associated complexities. Clinicians are advised to integrate all available diagnostic information for a comprehensive assessment of kidney function. In cases of uncertainty, additional diagnostic modalities, such as imaging techniques and renal biopsy, should be considered when appropriate (Figure 4). Any isolated increase in functional biomarkers, or the presence of persistent renal proteinuria, should alert clinicians to investigate for presence of kidney disease. Even if these values normalize on subsequent measurements, periodic monitoring is recommended to detect any changes in kidney function promptly (14). When faced with uncertainty between normal kidney function and early-stage CKD, clinicians should schedule regular follow-ups every few months to monitor trends, and intervene when necessary.

Monitoring animals with kidney disease

Biomarkers of kidney function are valuable not only for diagnosing kidney disease but also for monitoring its progression. Differentiating between stable and progressive CKD has significant clinical implications for diagnostic evaluation, therapeutic planning, and prognostic assessments. A common approach to assess CKD progression involves serial measurements of sCr or SDMA over time and calculating the slopes of these biomarkers. Although evidence supports the validity of slopes derived from inverse functional biomarkers (such as sCr or SDMA) as indicators of progressive changes in GFR and CKD, there is no consensus on what slope values define stable kidney function (i.e., stable CKD) versus progressive CKD (15).

Novel and emerging biomarkers 

In recent years, additional biomarkers have emerged as valuable tools for assessing kidney function and monitoring CKD. A key limitation of traditional markers is their inability to detect renal damage that does not coincide with changes in kidney function, so substantial tubular damage may go undetected if kidney function remains unaffected. Biomarkers of tubular damage were initially studied for the early detection of acute kidney injury (16), but recent evidence suggests that they may also be useful for the diagnosis and monitoring of CKD.

Cystatin B

Cystatin B is an intracellular protein belonging to the cysteine protease inhibitor family. It is found ubiquitously in many cell types, but because of its intracellular location, only trace concentrations are detected in serum and urine of healthy subjects. The presence of Cystatin B in urine is indicative of active injury to renal tubular epithelial cells, often due to apoptosis or necrosis (16). Studies in dogs and cats have demonstrated that urinary Cystatin B is also increased in animals with stable CKD, as defined by functional markers, suggesting that ongoing tubular damage may occur even when CKD is deemed stable. This highlights its potential utility in detecting injury before functional markers exhibit abnormalities. However, the sensitivity of Cystatin B for diagnosing CKD stage 1 has yet to be fully determined. In a recent study, urinary Cystatin B was shown to differentiate between stable and progressive CKD in dogs with CKD stage 1 (17), suggesting that it may become a valuable component of CKD monitoring protocols. An increase in Cystatin B levels compared to previous measurements should alert clinicians to possible ongoing underlying causes that could accelerate disease progression.

FGF-23

FGF-23 is a phosphaturic hormone first identified in humans with genetic phosphate-wasting disorders. In healthy animals it is primarily secreted by osteocytes and osteoblasts in response to hyperphosphatemia and elevated plasma calcitriol levels. Within the kidneys, FGF-23 inhibits calcitriol production by suppressing the activity of the vitamin D synthesis enzyme (25-hydroxyvitamin D-1α-hydroxylase) and promotes phosphaturia by downregulating sodium-phosphorus type II cotransporters in the proximal tubules. In the parathyroid gland, it reduces parathyroid hormone (PTH) production and secretion (18). FGF-23 levels rise as renal function declines in human patients (19), and similar increases have been observed in cats with CKD. Azotemic cats with hyperphosphatemia exhibit higher FGF-23 concentrations compared to normophosphatemic cats within the same IRIS stage. This biomarker has potential utility in managing phosphate levels in cats with CKD. Note that FGF-23 measurement is not recommended in hyperphosphatemic cats, as it is expected to be elevated in these cases, but elevated FGF-23 levels in normophosphatemic cats with CKD may indicate the need for further phosphorus restriction (19).

Conclusion

Diagnosing and managing CKD is a routine aspect of small animal clinical practice, but because the kidneys have significant functional reserve, clinical signs are often absent in the early stages. Urine concentration ability is often diminished before other parameters used to detect CKD, so any animal presenting with polyuria and polydipsia should have its USG measured; however, this is a non-specific test, and reduced USG can also occur with other conditions. Both SDMA and creatinine can be used independently to assess kidney function, but it is often advisable to consider a panel of biomarkers should kidney damage be suspected, and it is essential to differentiate between stable and progressive CKD, as this has significant clinical implications for diagnostic evaluation, treatment options and prognosis. Serial testing is to be recommended in order to optimize the sensitivity of these parameters. Recently, novel biomarkers are beginning to be identified that may help earlier and more accurate diagnosis of CKD in the future. 

References

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