Exploratory study investigating the ability of novel biomarkers of kidney injury to detect acute kidney injury in a clinical canine model

Benjamin W Chen

Acute kidney injury (AKI) is a consequence of intra-operative hypotension (mean arterial pressure <60mmHg), one of the most common complications observed during veterinary anaesthesia. Acute kidney injury occurs after prolonged periods of hypotension and associated hypoperfusion due to reduced oxygen and nutrient delivery and the build-up of cytotoxic waste products. Acute kidney injury has a high mortality rate in both people and domestic pets, often leading to chronic impairment of kidney function. One of the most important factors in the prognosis of AKI is early diagnosis and treatment in the initiation phase of AKI. Current clinical guidelines for the diagnosis of AKI in veterinary medicine rely on serum creatinine (sCr) and urine production. Serum creatinine has many shortcomings in the diagnosis of early kidney injury, resulting in increased research interest in earlier biomarkers of kidney injury. Many biomarkers have been evaluated experimentally in animals and clinically in humans. However, there remains limited application of these biomarkers in clinical veterinary medicine. The first study in this thesis explored changes in urinary biomarkers associated with intra-operative hypotension in healthy anaesthetised dogs undergoing elective desexing. Due to the high frequency of hypotension reported in anaesthetised healthy animals, this model is considered a clinically relevant model of AKI. Observations during the first study prompted a second smaller study to explore the effects of sedation and anaesthesia on sCr. The primary aim of the first study was to determine whether renal biomarkers, validated for measurement in canine urine, would increase in concentration during a clinical model of AKI in a similar manner to that observed previously in experimental studies. A secondary aim was to determine if a relationship existed between urinary biomarkers of AKI and the lowest mean arterial blood pressure (MAP) during anaesthesia. For the first study, paired urine and blood were collected at three time points: prior to anaesthesia (T0), within 24 hours of anaesthesia (T1), and within ten days of anaesthesia, i.e., at suture removal (T10). During surgery, systemic blood pressure was measured directly via a dorsal pedal arterial catheter. Urinary concentrations of neutrophil gelatinase-associated lipocalin (uNGAL), kidney injury molecule 1 (uKIM1), clusterin (uClusterin), and monocyte chemoattractant protein 1 (uMCP-1) were measured using a magnetic bead-based multiplex assay previously validated for canine urine. In addition, urinary cystatin C concentration (uCystatinC) was measured using a particle enhanced turbidimetric immunoassay (PETIA) and urinary gamma glutamyl transpeptidase concentration (uGGT) was measured using an enzymatic colorimetric assay. Serum Cr was measured at each sample time as the “gold standard” of renal function. Urinary biomarkers were standardised against urinary creatinine concentration to account for variation in urine output and concentration. For analysis, the proportional change in each biomarker was calculated between T1 and T0 (T1/T0) and T10 and T0 (T10/T0) to account for the interindividual variability in baseline (T0) biomarker concentration. The lowest MAP in each dog was identified. To analyse the effect of magnitude and duration, MAP was categorised as < 50, <60, <70 and < 80 mmHg and the time (minutes) within each category was calculated. The total proportion of dogs with increases in each biomarker were calculated. Regression analysis was performed to determine if the proportional change in each biomarker at T1/T0 and T10/T0 could be explained by MAP or the duration of MAP within each category. Significance set at p< 0.05 adjusted for multiple factors when required. Increases in creatinine normalised biomarker concentrations at T1/T0 were observed for uGGT/Cr (82.2% of dogs), uMCP-1/Cr (87% of dogs), uClusterin/Cr (87% of dogs), uCystatin C/Cr (40.6% of dogs), uKIM-1/Cr (41% of dogs) and uNGAL/Cr (35% of dogs). The proportional increase was still present at T10/T0 but in a markedly lower percentage of dogs. A strong correlation (R2=0.61) existed between variance in uGGT/Cr at T1/T0 with duration MAP <50mmHg. The proportional increases in the other biomarkers were not associated with MAP at T1/T0 or T10/T0. These findings support the usefulness of uGGT for the diagnosis of AKI associated with hypotension. Although not associated with MAP, the proportional increases in other biomarkers may be associated with other unidentified factors causing kidney injury beyond hypotension. In addition to the primary outcomes of the first study, it was observed that sCr at T0 suggested that sedation and induction agents had a potential impact on measurement. Thus, it was decided to investigate this possibility further in a second study. The second study evaluated how anaesthetic drugs (morphine, acepromazine, medetomidine, and propofol) altered the sCr levels in healthy dogs. Blood samples from 101 dogs were used. Blood was either collected prior to administration of medication (PRE) (n=21), following sedation (SED) (n=28), or immediately after the start of general anaesthesia (IND) (n=52). The sCr in each group was compared using ANOVA with post-hoc analysis when required. Significance was defined as adjusted p < 0.05. There was a significantly lower sCr in the SED group compared to the PRE group (p=0.04). Serum Cr was also significantly lower in the IND group compared to the PRE group (p<0.01). There was no significant difference between the IND and SED groups (p=0.98). Although this study had many limitations, the results suggest that administration of premedication and/or induction agents may lower sCr. This may hinder the ability to diagnose AKI using sCr. In summary, the first study demonstrated that only uGGT had a good correlation with blood pressure and, thus, is a potential early biomarker for AKI in anaesthetised dogs with hypotension. The cause of the increase in other urinary biomarkers could not be determined; thus, significance is unknown. Thus, although the novel biomarkers have been used successfully in experimental animal AKI models, additional research needs to be conducted before these biomarkers can be justified in the clinical setting. The second study's findings highlight a need for caution when interpreting sCr from sedated or anaesthetised dogs. The results of both studies confirm the limitations of sCr as an indicator of AKI and the need for more sensitive and specific markers of AKI.

Exploratory study investigating the ability of novel biomarkers of kidney injury to detect acute kidney injury in a clinical canine model

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