Clinical Application of Renal Biomarkers

Kidney disease in dogs and cats is common; estimated prevalence ranges from 7% to 20%.¹ The International Renal Interest Society (IRIS) has established guidelines for clinical staging and grading of kidney disease with criteria for acute kidney injury (AKI) and chronic kidney disease (CKD). These guidelines have helped veterinarians identify cases in which there is kidney injury or disease that might be mitigated by therapeutic intervention. Despite the clinical significance of kidney disease, however, early detection of kidney injury remains challenging.

The most accurate assessment of renal function is considered to be measurement of glomerular filtration rate (GFR).^2,3 However, this procedure is labor-intensive and time-consuming, making it an impractical test in daily clinical practice. Indirect assessment of renal function using serum creatinine and blood urea nitrogen (BUN) is easy to perform, and these tests are widely available. However, both analytes have well-established limitations, resulting in a lack of sensitivity and specificity for detection of kidney disease.⁴ These values typically do not increase until approximately 75% of the functional renal mass is lost.^5,6

Identifying novel biomarkers for kidney injury and disease is appealing and would provide additional tools for diagnosing and monitoring affected dogs and cats. Ideal biomarkers for the detection of kidney disease would be specific (i.e., not affected by comorbidities), strongly correlated with GFR, and more sensitive than serum creatinine for detecting disease. In addition, ideal biomarkers could be used to determine severity and location of the injury as well as to monitor disease progression or response to therapy, and testing would be readily available from a reference laboratory or as an in-clinic assay. A few promising candidates have been found to be useful for evaluating kidney disease in clinical and research settings (FIGURE 1). This review describes the current evidence with regard to widely available renal biomarkers for dogs and cats and their potential utility in clinical practice.

Figure 1. Overview of renal biomarkers in dogs and cats. Asterisks indicate those for which measurement is widely available.
FGF-23=fibroblast growth factor 23; GFR=glomerular filtration rate; GGT=γ-glutamyl transpeptidase; KIM-1=kidney injury molecule 1; NAG=N-acetyl-β-D-glucosaminidase; NGAL=neutrophil gelatinase-associated lipocalin; PIIINP=procollagen type III amino-terminal propeptide; RBP=retinol-binding protein; SDMA=symmetric dimethylarginine; TGF-β1=transforming growth factor β1; THP=Tamm-Horsfall protein; UPC=urine protein:creatinine ratio.

Renal Biomarkers in Blood

The 2 most commonly used serum biomarkers, with widely available testing, are creatinine and symmetric dimethylarginine (SDMA).

Creatinine

The most widely used indirect marker of renal function (GFR) is serum creatinine. Although this review focuses primarily on new renal biomarkers, we need to consider several essential factors with regard to the clinical utility of serum creatinine to help clinicians interpret results.

It is widely accepted that serum creatinine correlates inversely and exponentially with GFR, and creatinine above the reference interval correlates with the loss of approximately 50% to 60% of renal function.^6,7 The poor sensitivity of serum creatinine stems partly from its high variability between individuals, leading to wide population-based reference intervals.^8-10 The low variability observed within an individual dog or cat over weeks to months means that small increases in serum creatinine, even within the reference interval, may reflect significant decreases in renal function.⁴ Therefore, the sensitivity of serum creatinine for detecting kidney disease can probably be improved by evaluating serial measurements in an individual patient.

Considerable variation in serum creatinine measurements between instruments and laboratories leads to markedly different results, which can affect clinical interpretation of trending results for an individual patient. Studies have shown that serum creatinine results from a single sample measured on different instruments or at different laboratories can range from 0.9 to 2.3 mg/dL.^11,12 Therefore, to minimize this significant analytical variation, serial determination of serum creatinine in a given patient should be performed consistently on the same analyzer or at the same laboratory.

Another significant inherent limitation of serum creatinine measurement is its dependence on muscle mass. Many patients with kidney disease are geriatric or cachectic; decreased muscle mass can lead to decreased serum creatinine, which could lead to overestimation of renal function. That is, decreases in GFR that would result in increased creatinine may be offset by muscle wasting in animals with kidney disease.^4,13 Therefore, interpretation of serial creatinine measurements must account for changes in muscle mass, and creatinine may be an unreliable indicator of declining renal function in patients with concurrent muscle wasting.

SDMA

SMDA is a methylated amino acid (arginine) that is primarily excreted by the kidneys and does not seem to be reabsorbed by the tubules for reuse.¹⁴ A high-throughput immunoassay has been developed and validated, allowing SDMA to be a standard analyte on chemistry panels offered by IDEXX Laboratories, Inc. (idexx.com); the derived reference is ≤14 µg/dL for dogs and cats.¹⁵ SDMA is an endogenous surrogate marker of GFR in dogs and cats; its exponential relationship with GFR is similar to that of serum creatinine,^16,17 and it has been incorporated into the IRIS staging system. Longitudinal clinical studies have indicated that SDMA can potentially be used to detect earlier decline in renal function in some patients and as an early marker of CKD.^16,18-20 However, its usefulness for monitoring CKD progression has not been well established. Intra-individual and analytical variations of SDMA are higher than those of serum creatinine, potentially making it harder to track true changes in renal function over time. In dogs, a critical difference between measurements of approximately 6 µg/dL is needed to indicate a clinically significant change in serial SDMA concentrations.²¹

Unlike serum creatinine, SDMA does not seem to be influenced by muscle mass and has been shown to not correlate with lean body mass in adult dogs and geriatric cats.^17,22 However, recent studies have provided preliminary evidence that SDMA concentrations may be influenced by diseases such as diabetes mellitus, neoplasia (lymphoma), and nephrolithiasis.²³ In addition, there have been reports of animals with elevated SDMA considered to have normal renal function.^23,24 These reports highlight the need for further research to explore the effects of nonrenal disease on SDMA concentrations.

Renal Biomarkers in Urine

Use of urine biomarkers for early detection of AKI is a valuable area of focus in the veterinary profession. These biomarkers may help practitioners identify opportunities to intervene during earlier stages of renal damage, when they may have an opportunity to prevent additional damage, especially for patients that are receiving nephrotoxic medications or are exposed to other risk factors for AKI (e.g., sepsis, toxin exposure, recent surgery). Urine biomarkers include renal proteinuria, neutrophil gelatinase-associated lipocalin (NGAL), and γ-glutamyl transpeptidase (GGT).

Renal Proteinuria

Persistent renal proteinuria has been associated with a greater risk for renal disease progression and death in dogs and cats.^25-27 In small animal practice, persistent renal-origin proteinuria is often overlooked as an early marker of kidney disease. It is often not recognized until azotemia has developed, leading to a missed opportunity for timely therapeutic intervention. Treatments that attenuate proteinuria have been associated with slowed kidney disease progression and improved survival outcomes in dogs.^25,28-30

Proteinuria describes any type of protein in the urine. It results from 2 major mechanisms: loss of selective glomerular filtration (resulting in increased plasma protein in the filtrate) or impaired tubular resorption of the filtered protein. The first-line screening test for detecting proteinuria is the urine dipstick colorimetric test. If proteinuria is persistent, the urine protein:creatinine ratio (UPC) should be quantified; values of >0.4 in cats and >0.5 in dogs are considered proteinuric. Historically, a UPC >2 was thought to be associated more with glomerular injury;^31,32 however, more recent evidence suggests that the magnitude of proteinuria cannot always be used to localize renal proteinuria to glomerular or tubular damage.³³

Recent advances in determining the patterns of proteinuria in dogs and cats with renal injury include urine electrophoresis (SDS-PAGE [sodium dodecyl-sulfate polyacrylamide gel electrophoresis]). Primary tubular damage generates proteins of low molecular weight, and primary glomerular damage generates proteins of intermediate to high molecular weight. However, the predominant patterns seen in proteinuric dogs are mixed, indicating that injury to both glomeruli and renal tubules is common.^32,34 Further studies are needed to determine if urine electrophoresis can predict disease outcomes, document effectiveness of therapeutic interventions, or help clinicians determine which patients would benefit from a more invasive comprehensive renal biopsy.

NGAL

NGAL is an emerging urine biomarker for in-clinic detection of glomerular and tubulointerstitial injury. In healthy animals, this low–molecular-weight protein passes freely through the glomerular membrane and is almost completely reabsorbed by the kidneys’ proximal tubules.^4,33 There is growing evidence for use of NGAL as a biomarker for AKI. Recently, the usefulness of this biomarker for identifying early renal injury (compared with serum creatinine) in dogs and cats receiving nephrotoxic medications (e.g., aminoglycosides) has been explored.³⁵ One study showed that in dogs given gentamicin, NGAL increased significantly several days before creatinine.³⁵ This finding may enable closer monitoring of drug-induced kidney injury in our patients, allowing for informed decisions regarding current therapies. The main limitation of using NGAL as a biomarker for renal injury is that increases can result from extrarenal causes (i.e., lower urinary tract disease).³² Because urinary NGAL concentrations can be markedly increased in pyuric samples, the clinical practitioner should interpret results of urinary NGAL with caution for any patient affected by concurrent lower urinary tract disease.

GGT

GGT is a brush border enzyme in the proximal renal tubular epithelial cells that can be released into the urine with the onset of tubular damage. Because of its large molecular size, plasma GGT is not freely filtered through the glomerular membrane; therefore, increased concentrations of GGT in the urine can reliably reflect tubular injury, as long as the glomerular barrier is intact.³⁶ This tubular enzyme can be routinely measured in urine by using the same analyzers used to measure GGT in canine and feline serum or plasma. Urine GGT is generally present at low levels in healthy animals.³⁶ Defined reference ranges for urine GGT are in progress; multiple studies cite different values for healthy adult dogs.^36,37 Although practitioners may not routinely measure urine GGT, doing so could be considered a prudent practice for patients at increased risk for tubular injury (e.g., those receiving nephrotoxic medications). Serial GGT measurements may enable recognition of early AKI and provide a potential mechanism for monitoring response to therapy for AKI.

Future Perspectives

Current Markers

With regard to SDMA, further research is needed to establish if this marker is more sensitive for monitoring and predicting disease outcomes and to explore the influence of other nonrenal factors on SDMA concentration. In addition, if SDMA does allow for earlier diagnosis of CKD or AKI, further studies are needed to determine how to best manage those patients. With regard to UPC, although measurement of this ratio is an essential diagnostic step for evaluating renal proteinuria, its low specificity for determining the source and severity of kidney injury leads to the need for more specific urine markers of glomerular and tubular damage.

Future Markers

Several renal biomarkers of interest in veterinary research show promise as possible future clinically applicable tools for identifying and monitoring kidney injury and disease (FIGURE 1).  Several markers on the horizon of veterinary research have shown to be specific indicators of glomerular damage, including immunoglobulins (A, G, and M), C-reactive protein, thromboxane B₂, and transferrin. Similarly, numerous markers of tubular damage are being explored, including RBP (retinol-binding protein), cystatins B and C, THP (Tamm-Horsfall protein), NAG (N-acetyl-β-D-glycosaminidase), KIM-1 (kidney injury molecule 1), clusterin, and F₂-isoprostanes. In addition, a few markers for renal parenchymal fibrosis (TGF-β1 [transforming growth factor β1] and PIIINP [procollagen III aminoterminal peptide]) and for altered renal metabolism (FGF-23 [fibroblast growth factor 23]) have been identified. Measurement of these biomarkers may become more widely available for in-clinic diagnosis and localization of early renal injury. More studies need to be performed before measurement of these biomarkers can confidently be developed for this purpose, but the clinical practitioner should keep them in mind in the years to come.

Conclusion

Significant advances have been made for evaluating a variety of new biomarkers in dogs and cats with kidney disease. However, the number of commercially available tests for kidney function and injury is limited, and further investigation is needed. This article highlights several new biomarkers that will probably aid in the early detection, monitoring, and assessment of prognosis in dogs and cats with kidney disease. It is unlikely that a single biomarker will be able to provide a complete global picture of kidney function or injury in an individual animal. It is more likely that we will need a panel of markers to give us a comprehensive evaluation of renal health.