, 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, the mortality rate among human and animal patients remains high. One of the speculated reasons for the high mortality is the late recognition of the disease and consequently the narrow window of opportunity for therapy. Therefore, early recognition of the disease, before overt renal failure is evident, is crucial to allow timely and thus potentially more effective therapy. The need for early diagnosis is further emphasized in veterinary medicine, because renal replacement therapies are not readily available.
Limitations of serum creatinine concentration
Despite the diagnostic advancements made in other medical fields, serum creatinine (sCr) is still being used as the marker for kidney function despite its multiple shortcomings. These include: 1) High variability among dog breeds, resulting in a wide reference range. Consequently, sCr is not expected to rise above the reference range in most dog breeds until ~75% of nephrons become non-functional. 2) sCr is affected by extra-renal factors, particularly muscle mass and hydration status, therefore lacks specificity. 3) sCr is a functional marker thus does not increase in kidney injury that is not accompanied by a concurrent decrease in kidney function. 4) There is a nonlinear relationship between sCr and glomerular filtration rate (GFR). At early stages of the disease, substantial changes in GFR are reflected in only mild changes in sCr, most of which occur within the reference range and are overlooked 5) sCr does not represent the severity of the dysfunction until a steady-state has been reached. Consequently, substantial changes in GFR at the early stages of kidney injury are associated with relatively small changes in sCr.
The above limitations of sCr are reflected by the findings of several studies demonstrating that small, and even transient, increases in sCr in human patients are detrimental. In one study, as little as 0.5 mg/dL (45 μmol/l) ncrease in sCr concentration was associated with greater in-hospital mortality (Chertow et al., 2005). In another study, a transient increase in sCr for 1–3 days was also associated with increased odds ratio for in-hospital mortality (Uchino et al., 2010). Furthermore, even a small and transient increase in sCr in patients that were discharged from the hospital was associated with the need for chronic dialysis over the ensuing 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 accurately assess kidney function, to promote timely intervention, and to determine the prognosis.
Renal biomarkers ---- characteristics
In recent years, increasing research efforts have been undertaken to identify sensitive and specific renal biomarkers. A biomarker should meet multiple requirements to be considered an ideal marker, including: 1) be detectable in urine and/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 regarding the etiology and location (i.e., glomeruli and/or tubules) of the injury, 4 reflect the severity of the injury, 5) indicate kidney injury and/or repair processes, 6) predict the likelihood of recovery.
It is yet to be determined when and if biomarkers can meet all of these requirements but it is unlikely that a single biomarker will provide all this information. It is more likely that an array of biomarkers will be needed, each providing one piece of the puzzle. Because a full panel of biomarkers might be cost- prohibitive, a subset of biomarkers may have to be selected, each providing unique and specific information that complements the others.
Potential utilities of biomarkers
Early diagnosis of kidney injury
Renal biomarkers have several potential advantages, early diagnosis being a major one (Yerramilli et al., 2016). It has been shown that kidney injury can be identified using sensitive biomarkers days before any increase in sCr is documented. Thus, measurement of biomarkers may direct the clinician's attention to ongoing kidney damage before there is measurable reduction in kidney function (Palm et al., 2016). For example, if a nephrotoxic drug is being administered, it would be more rational to monitor the patient using biomarkers capable of indicating kidney injury, rather than markers that can only indicate decreased kidney function. Once kidney injury is identified, the drug can be discontinued before there is a decrease in kidney function. This is expected to lead to a better outcome, since once kidney function decreases, recovery is expected to be slow and the patient might die before recovery has occurred.
Screening patients at risk for acute kidney injury
Biomarkers indicating active kidney injury are likely more sensitive than markers indicating decreased function (i.e. surrogates for GFR); therefor the former should be used to screen patients at high risk for kidney injury. It has been shown that the prevalence of acute kidney injury (AKI) in hospitalized dogs is relatively high when using human criteria for AKI (Thoen and Kerl, 2011). Yet, the human criteria and the International Renal Interest Society (IRIS) guidelines rely on changes in sCr concentration (or urine production) to define AKI, and therefore probably underestimate the true prevalence of AKI in hospitalized dogs and cats. The diagnosis of IRIS Grade 1 AKI is currently a diagnostic challenge. Early recognition of kidney injury in hospitalized patients might allow identification, and potentially elimination, of the cause and might promote early intervention before the injury progresses to measurable decrease in kidney function, thereby improving the likelihood of a favorable outcome. A current limitation of this approach is the availability of commercial assays for assessment of candidate biomarkers, but the availability of useful assays is expected to increase in future years.
Differentiating upper and lower urinary tract infection
Neutrophil gelatinase-associated lipocalin (NGAL) concentration was shown to increase in a subset of dogs with apparently lower urinary tract disease, potentially indicating concurrent upper urinary tract involvement (Segev et al., 2013). Yet, an increase in urinary NGAL concentration might result from local inflammation within the lower urinary system, and not necessarily kidney damage, since NGAL originates also from neutrophils recruited as part of the local inflammatory process (Decavele et al., 2011). Recent 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 based on current concepts. It is possible that in the absence of sensitive and specific tools to aid differentiating upper and lower UTI, some patients with apparent cystitis in fact sustain pyelonephritis, and are consequently being undertreated. If kidney-specific biomarkers become available, differentiation between upper and lower urinary tract infection would be easier once a UTI is documented, and sequential changes in the biomarker concentrations could guide treatment.
Markers of chronic kidney disease progression
The traditional concept is that AKI and chronic kidney disease (CKD) represent two distinct processes of kidney damage: AKI represents rapidly progressing active damage due to various causes, whereas CKD represent slowly progressive damage. Lately it has been suggested that AKI and CKD might not be separate entities, as they are influenced by various similar conditions, share common risk factors and ultimately impact each other (AKI is a risk factor for CKD and vice versa) (Cowgill et al., 2016). Results of recent studies indicate that active kidney damage is present in a subset of dogs with apparently stable (based on sCr) CKD. This finding might account, at least in part, for the wide variation in progression rate of CKD among dogs and cats (Jacob et al., 2002; Ross et al., 2006). The presence of active kidney injury, demonstrated by increased concentrations of biomarkers, likely predicts risk for rapid progression of the disease, whereas absence of active kidney injury, probably predicts a more slowly progressive kidney disease (Chen et al., 2019; Kim et al., 2019) although prospective studies critically evaluating the sensitivity and specificity of biomarkers to predict progression in CKD patients are lacking. The documentation of ongoing active kidney damage in patients with CKD might suggest that the pathophysiology of AKI and CKD share more characteristics than currently recognized, and the main difference between them being the rate of disease progression (Cowgill et al., 2016).
Identification of markers of kidney damage in dogs and cats with CKD might also facilitate the diagnosis of IRIS CKD Stage 1, which is currently challenging with available tools (Ko et al., 2021). Early identification of the disease could allow early intervention aimed to preserve kidney function. Moreover, assessment of biomarkers indicating active kidney damage in animals with CKD would facilitate the investigation of novel therapeutic interventions, allowing recruitment of animals most likely to progress without the active intervention. Active injury biomarkers could be monitored sequentially after the application of these therapies, in the same way alanine aminotransferase is used to assess efficacy of interventions during management of liver diseases. The currently available markers of kidney damage preclude such assessment, as short-term interventions are unlikely to alter kidney function, despite either benefit or harmful effects.
Candidate biomarkers
Symmetric dimethylated arginine
Symmetric dimethylated arginine (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 and therefore its reference range is more uniform (Hall et al., 2014a; Hall et al., 2014b; Hall et al., 2015). Studies in dogs and cats reported earlier detection of CKD using 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), synthesized by the liver and functions as a carrier protein for retinol (Vitamin A1). When unbound, it is freely filtered and then metabolized and reabsorbed by the proximal renal tubules, thus has been suggested as a marker of proximal tubular dysfunction. Several veterinary studies have evaluated the utility of RBP as an early marker of AKI. In a study of X-linked hereditary nephropathy, increased urinary RBP to creatinine ratio correlated with sCr and GFR. Moreover, RBP continued to increase as the disease progressed, while other biomarkers peaked and reached a plateau early in the course of disease (Nabity et al., 2012). Urinary RBP concentrations were assessed in dogs with pyometra as markers of proximal tubular function and were found to be were significantly increased compared with healthy controls (Maddens et al., 2011). In a study characterizing kidney damage during naturally occurring canine heatstroke, urinary RBP was also increased, often before any increase in sCr was documented (Segev et al., 2015).
Neutrophil gelatinase-associated lipocalin
Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most studied biomarkers thus far in veterinary medicine (Cianciolo et al., 2016; Hokamp et al., 2016; Lee et al., 2012). It is a 25 kDa protein, originally discovered in the granules of neutrophils, and is released in response to bacterial infection (Mårtensson et al., 2012). In a prospective study evaluating NGAL as a marker of kidney injury in dogs, urinary NGAL to creatinine ratio (UNCR) was higher in dogs with AKI compared with other urinary tract diseases (CKD, UTI). Receiver operator characteristics analysis, performed to assess UNCR as a predictor of AKI had an area under the curve of 0.94 (Segev et al., 2013). The median UNCR of non-azotemic IRIS AKI Grade I dogs was significantly higher than in the other groups, but no different from azotemic AKI dogs, indicating that UNCR increases before sCr, and thus may be used as an early marker of the disease. In experimentally induced AKI, UNCR preceded the increase in sCr by approximately 7 days (Palm et al., 2016). The utility of NGAL as an AKI marker of AKI in general and AKI Grade 1 in specific has been demonstrated in additional studies (Defauw et al., 2020; Monari et al., 2020).
Despite being a very sensitive and early marker of kidney injury, NGAL's specificity is questionable, as it originates from multiple tissues as well as from neutrophils and therefore may increase during inflammation and other disease processes accompanied by recruitment of neutrophil.
Urinary cystatin B, urinary clusterin and heat shock proteins, are some of the more promising biomarkers currently under investigation and show 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 of biomarker research is the specificity of the investigated biomarkers to the kidney. This concern is increasing when there is concurrent damage to other body organs, which is very common in the AKI setting. The currently used biomarkers are present in multiple body organs and thus might originate from these organs when injured, decreasing their specificity to the kidney. Measuring these biomarkers in the urine increases, at least to some extent, their specificity to the urinary system, as high molecular weight biomarkers are restricted from urinary space by the glomerular barrier. Yet, some of the investigated biomarkers are small enough to cross the glomerular barrier, and in some cases of AKI or CKD, glomerular integrity is hampered, allowing translocation of high molecular weight solute to the urinary space. Kidney specific assays (i.e., assays that detect only the kidney isoform) might overcome this limitation.
Conclusion
The development and availability of sensitive and specific kidney biomarkers in coming years will likely change some of the current approaches to diagnosis and therapy in veterinary nephrology. However, data regarding use of these agents in veterinary medicine is scarce at present and further research is warranted. Each of the biomarkers investigated so far has advantages and weaknesses and it is most likely that an array of biomarkers will be required to provide all the information required.
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