Determining the mechanisms underlying augmented renal drug clearance in the critically ill: use of exogenous marker compounds

This single-centre observational study was undertaken in a tertiary-level, university-affiliated ICU. Ethical approval was obtained from our institution’s Human Research Ethics Committee (HREC/09/QRBW/15), with written informed consent obtained from either the patient or the nominated substitute decision-maker.

Study participants had to be aged ≤60 years, have an expected ICU length of stay >24 hours, evidence of a systemic inflammatory response syndrome [10] in the 24 hours prior to marker administration, a plasma CR concentration <120 μmol/L and no history of chronic kidney disease (CKD) or renal replacement therapy. Patients were excluded from receiving any study markers if (1) either invasive haemodynamic monitoring or an indwelling urinary catheter (IDC) was not employed as part of standard management; (2) they were <18 years of age; (3) they were pregnant; (4) rhabdomyolysis was clinically suspected or the plasma creatinine kinase level was >5,000 IU/L; (5) they were in the ‘risk’ category or greater for acute kidney injury, as defined by the risk, injury, failure, loss, and end-stage (RIFLE) kidney disease criteria [11]; (6) there was a documented allergy and/or contraindication to one or more of the renal markers; (7) one or more markers were being employed clinically; or (8) the treating clinician considered the patient unsuitable for enrolment. Recruitment was carried out by convenience sampling.

Blood samples were then taken via the intra-arterial cannula at the following time points after marker administration: 5, 10, 15, 30, 60 and 120 minutes and 4, 6, 12 and 24 hours. Following collection, all blood samples were immediately placed on ice and centrifuged within 60 minutes at 3,000 rpm for 10 minutes. Plasma was then aliquoted off and stored for analysis at −80°C. All urine was collected via the IDC over the same 24-hour period, with a 10-ml sample stored at −80°C for later assays. The remaining urine was forwarded to the local hospital laboratory for biochemical analysis.

All marker administration was performed under medical supervision, with the patients receiving continuous cardiovascular and respiratory monitoring. All physiological parameters, fluid balance and any therapeutic interventions performed were recorded concurrently.

(S)-pindolol, (R)-pindolol, PAH and fluconazole in plasma and urine were measured by in-house liquid chromatography-tandem mass spectrometry methods validated in accordance with US Food and Drug Administration guidelines for bioanalysis. Separations were tailored for each analyte: a reverse-phase column was used for fluconazole; a hydrophilic interaction liquid chromatography column was used for PAH; and a chiral column was used for pindolol. Sinistrin was measured by using a commercially available enzyme-linked immunosorbent assay kit (FIT-GFR; BioPAL, Worcester, MA, USA). CR was measured in plasma and urine by using an isotope dilution mass spectrometry traceable assay through the institutional pathology laboratory. For comparison, estimated GFR (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula [12].

CL_CR was determined from the urinary CR concentration, 24-hour urine volume and plasma CR concentrations obtained from routine clinical testing. If multiple plasma values were available over the 24-hour study period, the mean was used in subsequent analysis. Plasma concentrations of all renal markers were plotted on a concentration/time graph, with the slope of the terminal portion calculated by linear regression. Suitability for use in further analysis was assessed by visual inspection. Backward extrapolation to time zero was performed for those markers administered IV (sinistrin and PAH). The area under the plasma concentration/time curve (AUC_0-∞) was determined using the linear trapezoidal rule and extrapolated to infinity by adding the product of the last measured plasma concentration multiplied by the terminal slope. As per previous recommendations, plasma concentrations to at least 120 minutes were required for accurate calculation of sinistrin AUC_0-∞ [13]. Plasma CL for each marker was then calculated as dose/AUC_0-∞.

The total amount recovered in urine (Ae) of PAH, fluconazole and (S)- and (R)-pindolol was calculated by multiplying the urinary concentration by the 24-hour urine volume. Renal clearance (CL_R) was then calculated as Ae/AUC_0-∞, and non-renal clearance (CL_NR) as the difference of CL − CL_R. Sinistrin CL was taken as representing the GFR, and PAH CL_R was taken as effective renal plasma flow (ERPF). The filtration fraction was calculated as GFR/ERPF. Glomerular filtration of the unbound marker was calculated as f _u × GFR, where f _u is the unbound fraction in plasma. Tubular anion secretion was determined from the net tubular secretion of PAH as CL_R − (f _u × GFR). Tubular cation secretion was determined from the net tubular secretion of (S)- and (R)-pindolol as CL_R − (f _u × GFR). Tubular reabsorption was estimated from the net reabsorption of fluconazole as (f _u × GFR) − CL_R [8]. The f _u of PAH was taken as 0.83 [9]; for (S)- and (R)-pindolol, it was 0.45 [14]; and for fluconazole, it was 0.83 [9].

Continuous data are presented as the mean (95% confidence interval (CI)). Categorical data are presented as counts (%). For bivariate correlation between continuous variables, we used a Pearson correlation coefficient (r). A paired Student’s t-test was used to compare intra-patient data. Bland-Altman analysis was employed to explore the agreement between sinistrin CL, CL_CR and CKD-EPI eGFR values. A double-sided P-value <0.05 was considered as statistically significant, and SPSS version 22 software (IBM, Armonk, NY, USA) was used for all analyses.

Twenty adult patients were recruited into the study. Demographic, illness severity, therapeutic and physiological data are provided in Table 1. As presented, the subjects were mostly male, young and primarily admitted after trauma. As per the inclusion criteria, plasma CR concentrations were low; the mean was 64.5 (53.2 to 75.7) μmol/L. Twenty-four-hour CL_CR measures were elevated (168 (139 to 197) ml/min), whereas in comparison CKD-EPI eGFR estimates were significantly lower (128 (113 to 144) ml/min, P <0.01).

Sinistrin and PAH were administered IV to all 20 patients, with no adverse effects observed. Suitable sinistrin plasma concentration/time profiles could be plotted in 15 cases, which are presented in Figure 1. PAH concentrations were assessable in 19 cases, and these are also illustrated graphically in Figure 1. Both sinistrin CL and PAH CL_R were elevated, although moderate inter-patient variation in these parameters was observed (Table 2). The mean filtration fraction was 34.0% (25.4% to 42.7%). Of note, sinistrin CL was higher than CL_CR, although this was not statistically significant (P =0.10). These measures were highly correlated, however (r =0.70, P <0.01). Bland-Altman plots comparing sinistrin CL with CL_CR, as well as with CKD-EPI eGFR, are presented in Figure 2. Graphical correlations between sinistin CL, CL_CR, PAH CL_R and filtration fraction are presented in Figure 3.

rac-Pindolol was administered enterally to 17 patients and fluconazole to 19, without any observed adverse events. Suitable concentration/time profiles could be plotted in 14 cases for (S)- and (R)-pindolol and in 18 cases for fluconazole. These are presented in Figures 4 and 5, respectively. Ae, AUC, CL, CL_R and CL_NR values for each marker are presented in Table 2.

GFR, ERPF, net tubular anion secretion, net tubular cation secretion and net tubular reabsorption were calculated as outlined above. These data, in comparison to those previously reported in healthy volunteers, are presented in Table 3.