Biomarkers of urinary tract disease: potential utilities

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.

Biomarkers indicating active kidney injury are likely more sensitive than markers indicating decreased function (i.e. surrogates for GFR); therefore 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.

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.

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.

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 (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).

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.