The Re Designation Of Acute Renal Failure Biology

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The re-designation of Acute Renal Failure by Acute Kidney Injury is not reflected in the current diagnosis and stratification of patients. Despite the change in nomenclature AKI diagnosis is limited to the use of functional surrogates of change in GFR: plasma creatinine and urine output. Plasma creatinine delays AKI diagnosis by 1-3 days 13, 205, 398. Urine output is possibly earlier, but is unreliable. Biomarkers reflecting injury promise earlier diagnosis. However, their performance in heterogenous critically ill patients is influenced by the duration from insult until time of measurement.16, 17 In the EARLYARF trial,30 triaging to intervention by tubular enzymes urinary AP and GGT appeared to be compromised by many false negatives. These arose because sampling missed this combination of biomarkers short duration temporal "window of opportunity" for diagnosis.30

Most studies have assessed biomarker performance in comparison to the current 'gold standard' of AKI diagnosis, change in plasma creatinine. Creatinine change measures functional change, whereas injury biomarkers measure structural injury to the tubular cells. From a biological point of view comparing one with the other is compromised. This, coupled with other limitations of creatinine, including imprecise measure of GFR, delayed increase following GFR drop, and variation with age, gender, and muscle mass, further compromises the assessment of biomarker performance. These limitations may result in underestimation of biomarker performance even when they perfectly reflect tubular injury.218, 227 Assessment of the new biomarkers beyond comparisons with creatinine change, such as against hard outcomes including mortality or dialysis requirement as a reference standard is more valid.

We aimed to evaluate the temporal profile of AKI biomarkers in high-risk critically ill patients, who presented early in the course of postulated insult to the kidney. We aimed to test the hypothesis that detection nearer the likely time of insult to the kidney will improve the predictive ability of the biomarkers and hence their utility, by measuring the biomarkers within 4 hours of ED admission and at multiple time points in the ICU. We also aimed to investigate the performance of these biomarkers to predict mortality or requirement for dialysis.


The EDAKI study was a prospective observational study investigating the utility of plasma and urinary biomarkers from admission to the ED throughout ICU stay. Details of the inclusion and exclusion criteria are described in Chapter 2. Briefly, critically ill patients with cardiac arrest, sustained or prolonged hypotension, or probable ruptured AAA were included in the study. Plasma and urinary samples were collected in the ED, ICU admission, and 4, 8, 16 hours post ICU admission, and at 2, 4 and 7-days. Plasma samples were assayed for creatinine, NGAL, CysC, and urine samples were assayed for AP, GGT, -GST, -GST, NGAL, CysC and albumin. Details of measurement of these biomarkers are described in Chapter 2. For each patient, postulated time of insult was adjudicated using time of ambulance call (for cardiac arrest), or when symptoms of severe shock such as mental obtundation, or documented hypotension (for hypotension), or onset of abdominal pain (for suspected ruptured AAA).

Baseline creatinine was determined from a chart review, according to the hierarchy: (1) pre-admission creatinine 7-365 days prior to hospital admission, (2) creatinine at 30-day follow up, (3) hospital final creatinine, (4) on admission creatinine. AKI was defined based on plasma creatinine criteria: either an increase of more than 26.5 mol/l within 48 hours, or a relative increase of more than 50% above baseline within 7 days of admission.8, 14, 15 Cut-points for plasma NGAL (150 ng/ml) and plasma CysC (0.98ng/ml) were determined from previous published data.205, 261 For urinary biomarkers, cut points were determined from the EARLYARF data.17 These were determined using the same sensitivity of AKI (KDIGO)15 for prediction of mortality or dialysis of 61.2% (These cut-points were: NGAL 140 g/l, CysC 0.96 mg/l, GGT 130 U/L, AP 11.3 U/L, -GST 5.2 g/l, and -GST 14.9 g/l, Pickering & Endre, unpublished result).

ResultsDemographic and clinical characteristics

Patients were recruited between the 24th March 2010 and 29th February 2012. One hundred and nine patients were recruited in the ED. Fifteen patients were excluded because they were not admitted to the ICU. In the ICU, another 17 patients were excluded: three did not fit the inclusion criteria, four were not followed up in the ICU, one with inter-hospital transfer of more than 4 hours, one with drug overdose, six were not expected to survive 24 hours, and two refused consent (Error: Reference source not found). In total, 77 patients were included in the analysis. Plasma and urine samples were collected on ED arrival, ICU admission, and at 4, 8, 16 hours post ICU admission, and at 2, 4 and 7-days. Not all patients had samples collected at every time point (Error: Reference source not found).

Forty-five patients (58%) had AKI. The demographic and clinical characteristics of these patients are presented in Error: Reference source not found. Of the 77 patients, 49 (64%) were admitted due to cardiac arrest, 22 (29%) due to sustained hypotension, 5 (7%) due to ruptured AAA, and one due to profound hypotension. All 5 patients with profound hypotension and ruptured AAA had AKI, whilst 77% with sustained hypotension and 45% with cardiac arrest had AKI. There was no difference in baseline creatinine between those without AKI (No-AKI) and those with AKI. Forty-three patients had available pre-admission baseline creatinine. The others were determined from 30-day follow up (n=18), hospital final (n=15), or on admission (n=3). There were no differences in the demographic profile or baseline co-morbidities between No-AKI and AKI patients. Patients with AKI had greater illness severity, higher APACHE II and SOFA scores, compared to those without AKI (p<0.0001).


We hyposthesized that earlier measurement of biomarkers following insult is better diagnosed AKI or predicted mortality. However, we showed that earlier measurement of biomarkers in the ED did not improve its diagnostic or predictive performance in patients with significant kidney hypoperfusion following cardiac arrest, ruptured AAA or hypotension. These patients presented early following insult at a median of 1.1 (0.8 - 1.4). AKI occurred in 60% of patients, and 40% died or needed dialysis. Urinary NGAL, and albumin predicted mortality or dialysis, and improved a risk prediction model.

Biomarker performance in diagnosis of AKI and prediction of mortality or dialysisDiagnosis of AKI

The EDAKI study was designed to investigate the hypothesis that measuring injury biomarkers as early as possible following insult (e.g in the ED for an acute admission) would improve their diagnostic performance. This was hypothesised following observation of increased urinary GGT within 4 to 12 hours following insult after cardiopulmonary bypass in a sub-cohort of the EARLYARF trial.30 We showed that urinary GGT concentration was high during the first 12 hours following insult. However, ED urinary GGT failed to diagnose AKI, whilst GGT measured within 4 to 12 hours following insult within the ICU did diagnose AKI. As a result, we suggest that a 4-hour period was required to detect differences in urinary GGT in AKI compared to those without AKI. However, all patients in the study had reduced GFR secondary to reduced kidney perfusion, and increased urinary GGT by 2 to 3 times in the ED, regardless whether they hadAKI or not.

Earlier measurement of other biomarkers did not improve their performance. Plasma NGAL and CysC were diagnostic of AKI when measured early following insult. However, urinary NGAL, CysC, GGT and -GST were only diagnostic of AKI later in the ICU. This is of considerable interest, however small sample volumes limiting repeated analysis and missing ED urinary samples may have influenced this finding. We also showed that lower concentration of ED and ICU urinary -GST was diagnostic of AKI. It may be possible that they were released very rapidly following insult and its concentration was exhausted by the time it is measured in the ED, 1.1 (0.8 - 1.4) hours after insult.

Our study included patients who presented early in the ED following insult, which allowed evaluation of the biomarker temporal profile early following insult. However, the results cannot be generalized to other more heterogenous critically ill patients with varying time of insult. Furthermore, limitations in using plasma creatinine in AKI diagnosis limit the utility of this finding. Hence a similar analysis is needed for assessment of biomarker performance in prediction of more definitive clinical outcomes, such as mortality or dialysis need.

Prediction of the composite outcome of mortality or dialysis

Due to the inherent limitations in using plasma creatinine as a 'gold standard', the performance of these biomarkers is best assessed against a definite clinical outcome, which include mortality or dialysis need. Initiation of dialysis is dependent on clinician, usually based on creatinine level, urine output, or presence of acidosis. Hence, mortality would be the best outcome to use in a larger study. A composite outcome of mortality or dialysis was selected due to small number of patients and outcome in this study. We showed that of all biomarkers, urinary NGAL and albumin were independently predictive of mortality or dialysis. A ten-fold increase in urinary NGAL or albumin increased the risk of mortality or dialysis by about 300%. Siew et al. showed that urinary NGAL and Interleukin-18 (IL-18) were independently predictive of mortality or dialysis in a study of 451 critically ill patients [Adjusted OR of 1.86 (1.31 to 2.64) for IL-18, and 1.53 (1.07 to 2.19)].221 Greater concentrations of albumin is associated with an increased risk of multiple organ failure.399 Albuminuria is also a predictor of mortality in critically ill patients.400, 401 In a study involving 431 ICU patients, the urinary albumin to creatinine ratio measured on ICU admission, and 4 to 6 hours later are higher in non-survivors compared to survivors, and their change is an independent predictor of ICU mortality.401 Recent studies had reported that high urinary albumin was associated with increased risk of AKI in adult402 and paediatric403 cardiac surgical patients.

Both urinary NGAL and albumin improved clinical predictor model containing APACHE II score and AKI in prediction of mortality or dialysis. However, combining both urinary NGAL and albumin did not increase the IDI by urinary NGAL alone. In addition, plasma CysC, and urinary -GST showed an improved the risk prediction model, however to a lesser extent compared to urinary NGAL. Of interest is how much the improvement of risk assessment is clinically important. This requires consensus definition and acceptance amongst clinician. An 11% improvement for urinary NGAL in risk assessment of patients who died or needed dialysis may be sufficient, given the high-risk of AKI in this group of critically ill patients.

Severity of injury: preformed versus induced/filtered biomarkersPreformed Biomarkers: Tubular enzymes

Urinary GGT, -GST and -GST were elevated in the ED. Their mean concentrations were 2 to 100 times higher than the cut-points in the EARLYARF trial30 across the whole cohort of patients regardless whether they had AKI or not. The outer strata of medulla contain the S3 portion of the proximal tubule, and medullary thick ascending limb of the distal tubules.77-79 This area is poorly oxygenated, with a partial pressure of about 10 to 20 mmHg. This is because oxygen diffuses from arterial to venous vena riecta, reducing oxygen tension in the outer medulla.77, 404 The demand for active tubular reabsorption of sodium to maintain the osmotic gradient further renders this area relatively hypoxia. In the presence of significant kidney hypoperfusion, preferential shunting of blood away from the already hypoxic outer medulla may result in ischaemia, and depletion of ATP which leads to tubular cell injury.78

Ischaemic injury resulted in the shedding and excretion of proximal tubular brush border enzymes and epithelial tubule cells.79, 80 GGT and -GST are proximal tubular brush border enzymes, whereas -GST is located in distal tubules.274, 405 A modest increase of N-acetyl-β-D-glucosaminidase (NAG) has been described following kidney hypoperfusion after cardiac surgery.319, 406 Similar to cardiac surgery patients, our study comprised of those exposed to significant loss of kidney perfusion. Reduced kidney perfusion following cardiac arrest, severe hypotension, or ruptured AAA may result in further hypoxia to the outer medulla, ischaemia, ATP depletion, tubular injury, and shedding of these tubular enzymes. As they are preformed,13 in the EARLYARF trial these tubular enzymes, GGT and AP were released early following insult, and rapidly reduce once most of the brush border has been shed (within 4 to 12 hours).17 It is possible that they are more sensitive to injury; hence their release occurs with milder injury, such as a brief episode of ischaemia. Loss of brush border membranes and tubular cells may result in loss of polarity, and luminal obstructions, which may further contribute to further GFR reduction.79, 80 This also results in translocation of Na+/K+ ATPase from basolateral to luminal membrane, resulting in impaired sodium reabsorption.79, 81

A pilot study showed that these enzymes were useful in diagnosis of AKI,275 however this finding was not repeated in the larger study.17, 30 The enzymes were not diagnostic of AKI or predictive of dialysis, 30-day17, 30 or 1-year mortality.407 This may be due to their short duration of shedding or release; hence their elevation in many patients may have passed by the time patients present in hospital. However, the current study demonstrates that these enzymes were not diagnostic of AKI or predictive of mortality or dialysis even with early presentation following insult. As transient functional impairment (Transient-AKI) is associated with higher risk of mortality,63 the significance of an increase tubular enzymes remain to be tested in a larger patient cohort. Since their assays are cheap and rapidly performed, their utility in detecting milder form of injury remains a further area of potential exploration.

Induced or Filtered Biomarkers

Induced biomarkers such as NGAL, and freely filtered biomarkers such as CysC increase following injury to the proximal and distal tubules. Urinary NGAL is expressed in the distal tubules and collecting duct. In AKI, NGAL expression in the distal tubules is upregulated.231, 233 Some is absorbed into the circulation and filtered later at the glomerulus.231, 233 Reabsorption of filtered NGAL along with other low molecular weight proteins is impaired in the presence of proximal tubular injury, further increasing its urinary excretion.235, 236 Urinary CysC is freely filtered at the glomerulus, and similarly reabsorbed by the proximal tubule. In the presence of proximal tubule injury, reabsorption of CysC is similarly impaired, leading to increased urinary excretion.186 These biomarkers are released early following insult, and their concentration remains high for a longer duration.17, 269 Their utility in diagnosis of AKI and prediction of mortality has been documented in many studies across different population groups.19, 221, 238, 258, 408

Combination of preformed and induced or filtered biomarkers

All except one patient (ID058) had increase in urinary concentration of at least one of the 3 measured tubular enzymes (GGT, -GST or -GST) above the pre-specified cut-points. Sixty-six patients had a concurrent increase of an induced or a filtered biomarker of injury (plasma or urinary NGAL, or urinary CysC above the pre-specified cut-points). We propose that the presence of tubular enzymuria alone indicates a milder form of injury, while the presence of tubular enzymuria, plus induced or filtered biomarkers represents more severe form of injury. Since all of our patients were exposed to significant kidney hypoperfusion, the differences in the observed severity of injury may result from differences in duration of decrease of kidney perfusion, or the effect of compensatory mechanism or intervention. We postulate that a shorter duration of kidney hypoperfusion may result in of tubular enzymes only, whereas a much longer duration of hypoperfusion may result in increase both tubular enzymes and induced or filtered biomarkers (Error: Reference source not found). This is supported by longer duration until return of spontaneous circulation (ROSC) in the sub-cohort of cardiac arrest patients (n=49) with increases in both tubular enzymes and induced or filtered biomarkers, compared to those with only increases tubular enzymes (23  13 min versus 14  7 min, p=0.04, Error: Reference source not found). Using these results, we arbitrarily divided our cohort according to severity of structural injury. Patients with both increased tubular enzymes, and induced or filtered biomarkers is classified as severe structural injury, whereas those with only increased tubular enzymes is classified as moderate structural injury.

These concepts are illustrated by temporal profile of biomarkers in 2 examples of AKI patients with (A) severe injury, (ID079), and (B) moderate injury, following cardiac arrest are shown in Error: Reference source not found. In (A) the ED concentrations of all measured urinary biomarkers except CysC were above the pre-specified cut-points as previously defined. Plasma and urinary NGAL, and CysC increased progressively with sustained increases, whereas urinary GGT, -GST and -GST reduced exponentially within the first 24 hours. In (B) the ED urinary GGT, -GST and -GST were above the pre-specified cut-points, while the others were below cut-points. Here, again urinary GGT, -GST and -GST reduced exponentially within the first 24 hours.

Cross tabulation of severity of functional loss by KDIGO definition, and structural injury by the pre-specified cut points is shown in Table 4.9. Table 4.10 showed proportion of patients who died or needed dialysis in each group. Mortality or dialysis increased with increasing severity of functional AKI (p=0.001). Mortality or dialysis also increased progressively with increasing severity of structural classification, but this was not statistically significant (p=0.25).

We showed that injury biomarkers are excreted early following significant kidney hypoperfusion. However, the release of tubular enzymes occurs in short period of time, whereas excretion of induced or filtered biomarkers is more sustained. Induced or filtered biomarkers concentration correlate with severity of injury better than preformed biomarkers. However, preformed biomarkers facilitates in identifying those with tubular cell injury.

Study strengths and limitations

Unique aspects of this study were that it (1) examined critically ill patients at high-risk of developing AKI secondary to significant kidney hypoperfusion, and (2) captured patients very soon after insult. In 14 patients with hypotension secondary to septic shock, the onset of sepsis may have decreased filtration prior to the hypotensive event recorded as the time of insult. A lower than expected recruitment rate meant the final study numbers, after 2-years of recruitment, were lower than expected (100). This reduced slightly the power of the study to detect differences in biomarker performance between the ED and ICU. Recruitment was interrupted for a period of approximately six weeks due to a major earthquake in Christchurch. Patient recruitment was stopped after 2 years due to time constraint for completion of this thesis and lack of further funds. Several samples especially the early ones were missed especially during the training phase of the early part of recruitment and due to the priority given to active resuscitation of patients. Some later samples were also missed due to an interruption following Canterbury Health Laboratory (CHL) industrial action that took place over a period of 6 months during the recruitment phase.

4.5 Conclusion

Fifty-eight percent of patients with significant kidney hypoperfusion secondary to cardiac arrest, hypotension or ruptured AAA had AKI. Earlier measurement of biomarkers in the ED did not improve their AKI diagnostic performance. Urinary NGAL and albumin were independently predictive of mortality or dialysis. Urinary NGAL modestly improved the risk prediction model by 11% in predicting mortality or dialysis. Induced or filtered biomarkers correlate with severity of injury better than preformed biomarkers. However, preformed biomarkers facilitate detection of tubular cell injury.

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