Renal Biomarkers

Adapted from the IRIS education article by Gilad Segev, Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel.

Despite advances in the management of kidney diseases, including the introduction of renal replacement therapies such as dialysis, the death rate among human and animal patients remains high. One suspected reason is that the disease is recognized late, which leaves only a narrow window of opportunity for treatment. Early recognition of kidney disease, before overt kidney failure is evident, is therefore crucial to allow timely and potentially more effective treatment. The need for early diagnosis is even greater in veterinary medicine, because renal replacement therapies are not readily available.

Limitations of Serum Creatinine

Despite diagnostic advances in other fields of medicine, serum creatinine (sCr) is still used as the marker of kidney function, even though it has many shortcomings:

  1. It varies widely between dog breeds, which results in a wide reference range. As a consequence, sCr is not expected to rise above the reference range in most dog breeds until about 75% of nephrons have stopped working.
  2. It is affected by factors outside the kidney, particularly muscle mass and hydration status, and so it lacks specificity.
  3. It is a functional marker, so it does not increase when kidney injury occurs without a concurrent decrease in kidney function.
  4. Its relationship with glomerular filtration rate (GFR) is not linear. In the early stages of disease, substantial changes in GFR cause only small changes in sCr, most of which occur within the reference range and are overlooked.
  5. It does not reflect the severity of kidney dysfunction until a steady state has been reached. As a result, substantial changes in GFR early in kidney injury are associated with relatively small changes in sCr.

These limitations are reflected in several studies showing that small, and even transient, increases in sCr are harmful in human patients. In one study, an increase in sCr of as little as 0.5 mg/dL (45 µmol/L) was associated with higher in-hospital mortality (Chertow et al., 2005). In another, a transient increase in sCr lasting 1 to 3 days was also associated with increased odds of death in hospital (Uchino et al., 2010). Furthermore, even a small, transient increase in sCr in patients discharged from hospital was associated with the need for chronic dialysis over the following three years (Wald et al., 2009). These studies imply that relying on sCr as the only marker of kidney function does not provide all the information needed to assess kidney function accurately, to prompt timely intervention, and to determine the prognosis.

Characteristics of an Ideal Biomarker

In recent years, increasing research effort has gone into identifying sensitive and specific kidney biomarkers. To be considered ideal, a biomarker should:

  1. Be detectable in urine or blood, using methods that are readily available and cost-effective
  2. Be highly predictive of kidney injury, with high sensitivity and specificity
  3. Provide information on the cause and location of the injury, such as the glomeruli or the tubules
  4. Reflect the severity of the injury
  5. Indicate kidney injury or repair processes
  6. Predict the likelihood of recovery

It is not yet known whether or when biomarkers will meet all of these requirements, but it is unlikely that a single biomarker will provide all of this information. More likely, a panel of biomarkers will be needed, each providing one piece of the puzzle. Because a full panel might be too expensive, a subset of biomarkers may have to be chosen, each providing unique information that complements the others.

Potential Uses of Biomarkers

Early Diagnosis of Kidney Injury

Early diagnosis is one of the main potential advantages of kidney biomarkers (Yerramilli et al., 2016). Sensitive biomarkers have been shown to identify kidney injury days before any increase in sCr is documented. Measuring biomarkers may therefore alert the clinician to ongoing kidney damage before there is any measurable reduction in kidney function (Palm et al., 2016). For example, when a drug that can damage the kidneys is being given, it would be more rational to monitor the patient with biomarkers that indicate kidney injury than with markers that can only indicate decreased kidney function. Once kidney injury is identified, the drug can be stopped before kidney function decreases. This is expected to lead to a better outcome, because once kidney function has decreased, recovery is expected to be slow, and the patient might die before recovery occurs.

Screening Patients at Risk of Acute Kidney Injury

Biomarkers of active kidney injury are likely to be more sensitive than markers of decreased function, which are surrogates for GFR, so the former should be used to screen patients at high risk of kidney injury. The prevalence of acute kidney injury (AKI) in hospitalized dogs has been shown to be relatively high when human criteria for AKI are used (Thoen and Kerl, 2011). However, both the human criteria and the International Renal Interest Society (IRIS) guidelines rely on changes in sCr (or urine production) to define AKI, and they therefore probably underestimate the true prevalence of AKI in hospitalized dogs and cats. Diagnosing IRIS Grade I AKI is currently a challenge. Early recognition of kidney injury in hospitalized patients might allow the cause to be identified and potentially removed, and could prompt early intervention before the injury progresses to a measurable decrease in kidney function, improving the chance of a good outcome. A current limitation of this approach is the limited availability of commercial assays for candidate biomarkers, although more useful assays are expected to become available.

Differentiating Upper From Lower Urinary Tract Infection

The concentration of neutrophil gelatinase-associated lipocalin (NGAL) was shown to increase in a subset of dogs with apparently lower urinary tract disease, potentially indicating that the upper urinary tract was also involved (Segev et al., 2013). However, an increase in urinary NGAL might result from local inflammation in the lower urinary tract rather than kidney damage, because NGAL also comes from neutrophils recruited as part of the local inflammatory process (Decavele et al., 2011). Unpublished data suggest that other, kidney-specific biomarkers are also increased in dogs with apparently lower urinary tract infection (UTI), indicating that some of these dogs have subclinical pyelonephritis and ongoing kidney injury that would go unnoticed with current approaches. Without sensitive and specific tools to distinguish upper from lower UTI, some patients with apparent cystitis may actually have pyelonephritis and be undertreated. If kidney-specific biomarkers become available, distinguishing upper from lower urinary tract infection would become easier once a UTI is documented, and serial changes in biomarker concentrations could guide treatment.

Markers of Chronic Kidney Disease Progression

The traditional view is that AKI and chronic kidney disease (CKD) are two distinct processes of kidney damage: AKI is rapidly progressing active damage from various causes, whereas CKD is slowly progressive damage. More recently, it has been suggested that AKI and CKD may not be separate entities, because they are influenced by many of the same conditions, share risk factors, and ultimately affect each other, with AKI being a risk factor for CKD and vice versa (Cowgill et al., 2016). Recent studies indicate that active kidney damage is present in a subset of dogs with CKD that appears stable on the basis of sCr. This finding might explain, at least in part, the wide variation in how quickly CKD progresses in dogs and cats (Jacob et al., 2002; Ross et al., 2006). Active kidney injury, shown by increased biomarker concentrations, probably predicts a risk of rapid progression, whereas its absence probably predicts more slowly progressive disease (Chen et al., 2019; Kim et al., 2019), although prospective studies that critically evaluate how well biomarkers predict progression in CKD patients are lacking. The documentation of ongoing active kidney damage in patients with CKD suggests that AKI and CKD may share more features than currently recognized, and that the main difference between them may be the rate of disease progression (Cowgill et al., 2016).

Identifying markers of kidney damage in dogs and cats with CKD might also help diagnose IRIS CKD Stage 1, which is currently challenging with the available tools (Ko et al., 2021). Early identification could allow early intervention to preserve kidney function. Moreover, measuring biomarkers of active kidney damage in animals with CKD would help in the investigation of new treatments, by allowing the recruitment of the animals most likely to progress without intervention. Active injury biomarkers could then be monitored after these treatments are applied, in the same way that alanine aminotransferase (ALT) is used to assess treatment in liver disease. The markers currently available do not allow this, because short-term interventions are unlikely to change kidney function, whether they are beneficial or harmful.

Candidate Biomarkers

Symmetric Dimethylarginine (SDMA)

Symmetric dimethylarginine (SDMA) is a methylated form of the amino acid arginine. Like creatinine, SDMA is a filtration marker, but it is not influenced by muscle mass, so its reference range is more uniform (Hall et al., 2014a; Hall et al., 2014b; Hall et al., 2015). Studies in dogs and cats have reported earlier detection of CKD with SDMA than with sCr (Nabity et al., 2015; Yerramilli et al., 2016).

Retinol-Binding Protein (RBP)

Retinol-binding protein is a low-molecular-weight protein (21 kDa) made by the liver that carries retinol (vitamin A1). When not bound, it is freely filtered and then reabsorbed and broken down by the proximal renal tubules, so it has been suggested as a marker of proximal tubular dysfunction. Several veterinary studies have evaluated RBP as an early marker of AKI. In dogs with X-linked hereditary nephropathy, an increased urinary RBP-to-creatinine ratio correlated with sCr and GFR, and RBP continued to increase as the disease progressed, while other biomarkers peaked and plateaued early in the course of disease (Nabity et al., 2012). Urinary RBP concentrations were significantly higher in dogs with pyometra than in healthy controls (Maddens et al., 2011). In a study of kidney damage in dogs with naturally occurring heatstroke, urinary RBP was also increased, often before any increase in sCr (Segev et al., 2015).

Neutrophil Gelatinase-Associated Lipocalin (NGAL)

NGAL is one of the most studied biomarkers in veterinary medicine so far (Cianciolo et al., 2016; Hokamp et al., 2016; Lee et al., 2012). It is a 25 kDa protein, first discovered in the granules of neutrophils, that is released in response to bacterial infection (Mårtensson et al., 2012). In a prospective study of NGAL as a marker of kidney injury in dogs, the urinary NGAL-to-creatinine ratio (UNCR) was higher in dogs with AKI than in dogs with other urinary tract diseases such as CKD and UTI. In a receiver operating characteristic analysis, UNCR as a predictor of AKI had an area under the curve of 0.94 (Segev et al., 2013). The median UNCR in non-azotemic dogs with IRIS AKI Grade I was significantly higher than in the other groups but no different from that of dogs with azotemic AKI, indicating that UNCR rises before sCr and so may be used as an early marker of the disease. In experimentally induced AKI, UNCR rose approximately 7 days before sCr (Palm et al., 2016). The usefulness of NGAL as a marker of AKI in general, and of AKI Grade I in particular, has been demonstrated in further studies (Defauw et al., 2020; Monari et al., 2020).

Although NGAL is a very sensitive and early marker of kidney injury, its specificity is questionable, because it comes from multiple tissues as well as from neutrophils, so it may increase during inflammation and other disease processes that recruit neutrophils.

Other Promising Biomarkers

Urinary cystatin B, urinary clusterin, and heat shock proteins are among the more promising biomarkers under investigation, with encouraging results (Bruchim et al., 2017; Chen et al., 2019; Hezzell et al., 2020; Kavkovsky et al., 2020; Yerramilli et al., 2016).

Kidney Specificity

One of the main concerns in biomarker research is whether the biomarkers being investigated are specific to the kidney. This concern increases when other organs are damaged at the same time, which is very common in AKI. The biomarkers currently used are present in several organs and may come from those organs when they are injured, which reduces their specificity to the kidney. Measuring these biomarkers in urine increases their specificity to the urinary system, at least to some extent, because the glomerular barrier keeps high-molecular-weight biomarkers out of the urine. However, some of the biomarkers being investigated are small enough to cross the glomerular barrier, and in some cases of AKI or CKD the glomerular barrier is damaged, allowing high-molecular-weight substances into the urine. Kidney-specific assays, which detect only the kidney form of a molecule, might overcome this limitation.

Conclusion

The development and availability of sensitive and specific kidney biomarkers in the coming years will probably change some current approaches to diagnosis and treatment in veterinary nephrology. However, data on the use of these markers in veterinary medicine are limited at present, and further research is needed. Each biomarker investigated so far has strengths and weaknesses, and a panel of biomarkers will most likely be required to provide all the information needed.

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