Investigation of early biomarkers for the diagnosis of acute kidney injury following ischaemia reperfusion injury in anaesthetised dogs

Acute kidney injury (AKI) is a syndrome associated with high morbidity and mortality due to an abrupt decline in renal function. One of the most common causes of AKI in dogs is ischaemia reperfusion injury (IRI), as may occur during severe hypotension during anaesthesia. Favourable outcomes following AKI are more likely if therapeutic interventions are instigated early, within the first few hours of injury. Unfortunately, conventional diagnostic tests for AKI rely on markers of renal functional deterioration such as serum creatinine, that may change significantly until several days after the initial injury. Several protein biomarkers have been shown to be useful for the early diagnosis of AKI in animals and people. Multiplex assays for the simultaneous measurement of multiple renal biomarkers in canine samples have recently become available, representing a potentially efficient and cost-effective tool for investigators; however, there is limited data to support their reliable use in dogs. This thesis aimed to validate one of these multiplex assays for use with canine urine and blood, and then to document changes in urinary biomarker concentration alongside conventional diagnostic tests for AKI following ischaemia reperfusion injury in anaesthetised dogs. An experimental model of IRI AKI was utilised in six male anaesthetised greyhounds by inducing 1-hour of renal ischaemia (severe hypotension induced by acute haemorrhage) and 2-hours of reperfusion (intravenous fluid resuscitation). Urine and blood collected before, and after IRI, was used to compare the performance of two commercial multiplex assays to validated enzyme-linked immunoassays (ELISA) for the measurement of five biomarkers: clusterin, cystatin C (CysC), kidney injury molecule-1, monocyte chemoattractant protein 1, and neutrophil gelatinase-associated lipocalin (NGAL). Subsequently, extensive analytical validation was performed on one of the multiplex assays for canine urine and serum. Additionally, reference intervals for the urinary biomarkers were established using a group of 110 healthy dogs. Finally, the experimental model was used to describe temporal changes in three urinary biomarkers, NGAL, CysC, and gamma-glutamyl transpeptidase (GGT), during 3-hours of IRI in six greyhounds. Biomarker changes were compared to renal histological lesions and conventional tests for AKI. Ability of different biomarkers to detect AKI varied depending on the measurement platform used; only NGAL was significantly elevated following IRI with all assays investigated. The multiplex assay performed with acceptable accuracy and precision for the measurement of NGAL only, in both canine urine and serum. Clinically relevant urine concentrations of bilirubin, haemoglobin, and synthetic colloid solutions led to interference with measurement of all or some of the biomarkers. All biomarkers were stable in urine stored at 20 – 22oC for 2 hours, 4oC for 12 hours, or -20oC for 6 months. Despite serum creatinine remaining within a breed-specific reference interval in all dogs, urinary NGAL, CysC, and GGT concentrations, and urine protein-creatinine ratio were consistently elevated within 1-hour of IRI. The most likely origin for these changes is injured renal tubular cells, as histology revealed changes consistent with structural damage to proximal tubular cells. The findings suggest that measurement of urinary NGAL, cystatin C, and GGT concentrations, and UPC, may allow for earlier identification and treatment of AKI in anaesthetised dogs. While the multiplex assay investigated was reliable only for the measurement of NGAL in canine urine and serum, urinary GGT concentration and UPC may be of particular use as they can be measured rapidly and reliably on standard biochemistry analysers. The results of these studies provide vital information to allow further investigation into the usefulness of these biomarkers to detect peri-operative AKI in a clinical setting, and for further research investigating the aetiology and prevention of perioperative IRI AKI in dogs