Biomarker of renal dysfunction

Abstract

The present invention relates to a method for determining predisposition of a subject to developing renal dysfunction induced by physical trauma, hypotension, sepsis and/or septic shock syndrome, wherein the method comprises the steps of:a. determining the level of an anti-inflammatory cytokine present in a sample taken from the subject prior to physical trauma, prior to a hypotensive event, prior to sepsis, and/or prior to septic shock syndrome; b. determining if the subject is predisposed to developing renal dysfunction following physical trauma, hypotension, sepsis and/or septic shock syndrome on the basis of the level of an anti-inflammatory cytokine determined in step a).

Claims

1. A method of treating a subject at increased risk of renal dysfunction induced by a planned physical trauma, wherein the method comprises the step of: a) obtaining a urine sample from a subject prior to the planned physical trauma, wherein the planned physical trauma is elective surgery; b) detecting the anti-inflammatory cytokine present in the urine sample obtained in step a), wherein the anti-inflammatory cytokine is selected from the group consisting of IL-1ra, TNFsr1 and TNFsr2 and any combination thereof; c) detecting the anti-inflammatory cytokine in a pre-event normal control, wherein the pre-event normal control is a normal level of anti-inflammatory cytokine presented by a control group or a predetermined normal cytokine level; d) calculating the level of anti-inflammatory cytokine detected in step b) relative to the level of anti-inflammatory cytokine detected in step c); e) determining that the subject has a greater than normal chance of developing renal dysfunction induced by planned physical trauma when the level of anti-inflammatory cytokine determined in step b) is lower than the level of anti-inflammatory cytokine detected in step c); and f) applying therapeutic measures to treat or obviate renal dysfunction to the subject, wherein the therapeutic measures are selected from maintaining a supra-normal blood pressure; ensuring adequate tissue oxygen delivery; administration of steroids; renal replacement therapy; dialysis; or any combination thereof.

Description

(1) The present invention will now be described, by way of example, with reference to the accompanying figures, in which:

(2) FIG. 1 shows receiver/operator characteristics (ROC) curve for pre-operative urinary IL1ra showing sensitivity and specificity to distinguish patients with and without renal dysfunction as defined by MDRD eGFR drop at day 5 post-surgery of more than 25% from the pre-surgery value.

(3) FIG. 2 shows a table demonstrating the number of patients on each day of the study discussed below who had normal renal function (NF), or renal dysfunction (RD) as defined by greater than 25% MDRD drop from base line on that day, or for whom renal function data was not know (NK).

EXPERIMENTAL METHODS

(4) Four hundred and eight consecutive patients undergoing elective cardiac surgery were studied. The patients were recruited within the Cardiac Surgical Unit of the Royal Victoria Hospital Belfast in Northern Ireland (n=304) and Papworth Hospital NHS Foundation Trust in Cambridge, England (n=104). Local ethical committee approvals were received and written informed patient consent was obtained. Exclusion criteria included pre-operative dialysis dependent renal failure, significant renal disease (eGFR<40 ml min.sup.1) or diabetes mellitus. Patients on pre-operative angiotensin conversion enzyme (ACE) inhibitor therapy were not excluded from this study. The anaesthetic technique in both centers was based on the use of propofol and fentanyl. Isoflurane was used in most patients either as an adjunct anaesthetic agent or to control blood pressure. Pancuronium was used to provide muscle relaxation. Post-operative analgesia was with morphine infusion.

(5) In the 408 elective cardiac surgery patients pre-operative urinary IL1ra was measured by obtaining a urine sample before anaesthesia. The pre-operative urinary level was compared for each cytokine in patients grouped according to presence or absence of renal dysfunction defined as a drop from baseline estimated glomerular filtration rate (eGFR) of greater than 25% (as calculated by the method of modification of diet in renal disease (MDRD)) occurring within (1) the first 24 and (2) 48 postoperative hours (early renal dysfunction), (3) on the fifth postoperative day (late renal dysfunction) or (4) at any time throughout the 5 day post-operative period (early and late combined)).

(6) Cytokines Analysis (ELISA)

(7) Cytokines are measured by R&D systems Quantikine solid phase ELISA technique. This system consists of a conjugate, standard, assay diluent, calibrator diluent, wash buffer concentrate, colour reagent A, colour reagent B, and a stop solution. Reagents should be at the room temperature before beginning the assay. The microplate-consists of 96 wells. This microplate is coated with capture antibody. To each well assay diluent is added. Standards in duplicate and the samples are added to the plate and incubated for 2 h at room temperature. Any analyte present in the sample is bound by the capture antibody (immobilized antibody). After the incubation, the plate is aspirated and washed four times with the supplied wash buffer to washout any unbound materials. After washing, horseradish protease (HRP) labelled detection antibody (conjugate) is added to the plate and further incubated at room temperature. Once again, after the incubation the plate is aspirated and washed 4 times. Any unbound detection antibody is washed away. In the next step prepared substrate solution tetramethylbenzidine (TMB) is added to the wells and a blue colour develops in proportion to the amount of analyte present in the sample. After 20 minutes incubation the colour develops (blue) proportional to the cytokine concentration. For analysis colour development is stopped turning the colour in the wells to yellow. The absorbance of the colour at 450 nm is measured which is read in the microplate reader.

(8) Urinary samples for measurement of urinary IL-1ra were obtained shortly before the anaesthesia that preceded surgery.

(9) Measurement of Renal Dysfunction

(10) In 1989 Kopple et al as part of the Modification of Diet in Renal Disease study group published their findings investigating the impact of nutritional status on chronic renal insufficiency in 95 patients. The effects on progression of renal disease of a control diet of only mild dietary protein restriction were compared with 3 study diets of varying degrees of protein restriction and reduced phosphorus intake. The authors found that malnutrition and lower energy intake characterised patients with the lower GFR levels.

(11) There were some gender differences with men demonstrating a correlation between GFR and arm muscle area and percentage standard body weight especially at the onset of the experimental diets. In women, GFR correlated with dietary energy intake [Kopple et al., Kidney Int Suppl 1989; 27]. A logical development of this study from the MDRD group was the idea that analysis of patients' age weight, gender and ethnicity together with serum creatinine would allow GFR to be estimated. This assumption recognised that serum creatinine concentration alone does not adequately reflect renal function but should be considered along with the factors identified as influencing renal function in Kopple's study.

(12) To develop the prediction equation 1628 patients were enrolled in the baseline period, of which 1070 were randomly selected as the training sample whereas the remaining 558 patients constituted the validation sample. The authors then used stepwise regression to the training sample to develop the equation which was then tested and compared with the Cockcroft and Gault formula and creatinine clearance measurements in the validation sample.

(13) It was found that several measured variables were associated with a lower GFR. These included higher serum creatinine, higher serum urea and lower serum albumin levels concentrations. Independent variables associated with lower GFR included older age group, female gender and non-black ethnicity (P<0.001 for all factors).

(14) The multiple regression models explained 90.3% of the variance in the logarithm of GFR in the validation sample. Measured creatinine clearance overestimated GFR by 19%, and creatinine clearance predicted by the Cockcroft-Gault formula overestimated GFR by 16%. After adjustment for this overestimation, the percentage of variance of the logarithm of GFR predicted by measured creatinine clearance or the Cockcroft-Gault formula was 86.6% and 84.2%, respectively.

(15) MDRD study group estimated GFR is calculated from the following formula: X=32788creatinine-1.154age0.203constant where the constant is 1 for white males, 0.724 for females, and 1.21 for African Americans.

(16) MDRD estimated GFR in the present study was calculated from the above formula. According to the above formula MDRD GFR was calculated at preop day 0 and at post operative days 1, 2 and 5. For each separate post op day as well as all post op days together patients can be divided into normal and abnormal renal function groups where normal and abnormal were defined by those who sustained falls in MDRD GFR of less than or greater than 25% of baseline respectively.

(17) In summary then for post surgery cytokine values were compared between the normal and abnormal groups where normality and abnormality were defined according to the 4 definitions mentioned below.

(18) Definition 1: Abnormality is defined as having day 1 MDRD>25% drop from baseline. (Early renal dysfunction).

(19) Definition 2. Abnormality is defined as having day 2 MDRD>25% drop from baseline. (Early renal dysfunction).

(20) Definition 3. Abnormality is defined as having day 5 MDRD>25% drop from baseline. (Late renal dysfunction).

(21) Definition 4. Abnormality is defined as having at least one MDRD value>25% drop from baseline during days 1, 2, and 5. (General renal dysfunction).

(22) Statistical Methods

(23) Quantitative variables were summarized as mean and standard deviation except for those with heavily skewed distributions for which median and interquartile range were used. Comparisons between groups were obtained using the independent samples z-test or Mann-Whitney U test for quantitative variables, and the chi-squared test or Fisher's exact test for categorical variables. The ability of cytokine measurements to distinguish patients with and without renal dysfunction in the preoperative period was examined using the receiver-operator characteristic (ROC) curve. The area under this curve provides a measure of discriminatory ability; a value of 1 indicates perfect discrimination while a value of 0.5 indicates no more discriminatory ability than would be expected by chance.

(24) Results

(25) 1. Numbers of Patients in Renal Dysfunction and their Characteristics

(26) The numbers of patients who developed renal dysfunction on postoperative days 1, 2 and 5 are illustrated in FIG. 2. Table 1 shows the characteristics of patients categorized according to presence or absence of renal dysfunction defined by >25% drop from baseline eGFR pre-surgery at any time during the 5 day postoperative period. Renal dysfunction was more likely in patients who had increased weight, male gender or who had not been given an intra-operative dopamine infusion (Table 1). A requirement for postoperative resternotomy for bleeding/cardiac tapenade or post-operative adrenaline infusion was linked with renal dysfunction. Development of renal dysfunction significantly prolonged length of stay in the intensive care/high dependency unit (Table 1).

(27) TABLE-US-00001 TABLE 1 The table shows pre- and peri-operative characteristics of patients with and without renal dysfunction as defined by >25% drop from baseline eGFR at any time during the postoperative period (mean +/ SD or n(%)). Patients with MDRD Patients with MDRD eGFR drop 25% at all eGFR drop >25% at any P times (N = 349) time (N = 49) value Baseline MDRD eGFR 73.8 +/ 15.3 75.9 +/ 24.5 0.57 (ml/min/ 1.73 m.sup.2) Age (years) 62.6 +/ 10.5 65.6 +/ 10.3 0.06 Weight (kg) 77.1 +/ 13.6 82.8 +/ 15.7 0.02* Height (in) 1.69 +/ 0.10 1.71 +/ 0.08 0.06 BMI (kg/m.sup.2) 27.0 +/ 4.0 28.2 +/ 4.5 0.08 Male Gender 237/349 (68%) 42/49 (86%) 0.01* Operation time (min) 252 +/ 51 259 +/ 91 0.59 CPB time # (min) 95 +/ 34 99 +/ 41 0.52 Cross clamp time (min) 62 +/ 28 62 +/ 25 0.81 Beta blocker 226/349 (65%) 33/49 (67%) 0.72 Calcium Antagonist 83/348 (24%) 14/49 (29%) 0.47 Nitrate 162/349 (46%) 20/49 (41%) 0.46 K channel blocker 119/349 (34%) 13/49 (27%) 0.29 ACEI 152/349 (72%) 24/49 (49%) 0.47 ATII receptor blocker 13/349 (4%) 4/49 (8%) 0.14 Intra-operative 223/349 (64%) 30/49 (61%) 0.72 phenylephrine Any intra-operative 159/348 (46%) 18/49 (37%) 0.24 intotrope Intra-operative dopamine 118/348 (34%) 7/49 (14%) 0.006* Intra-operative 36/347 (10%) 6/49 (12%) 0.69 noradrenaline Intra-operative adrenaline 17/349 (5%) 3/49 (6%) 0.72 Intra-operative milrinone 25/349 (7%) 3/49 (6%) 1.00 Intra-operative IABP 3/348 (1%) 0/49 (0%) 1.00 Any post-operative 156/349 (45%) 20/49 (41%) 0.61 inotrope Post-operative dopamine 145/348 (42%) 16/49 (33%) 0.23 Post-operative 26/349 (7%) 7/49 (14%) 0.16 noradrenaline Post-operative adrenaline 17/349 (5%) 6/49 (12%) 0.05* Post-operative milrinone 28/349 (8%) 2/49 (4%) 0.56 Intra-operative IABP 1/349 (0%) 0/49 (0%) 1.00 Intra-operative PRBC 76/348 (25%)) 13/49 (27%) 0.46 Intra-operative FFP 5/349 (1%) 2/49 (4%) 0.21 Intra-operative platelets 11/349 (3%) 3/49 (6%) 0.40 Post-operative PRBCs 150/348 (43%) 26/49 (53%) 0.19 Post-operative FFP 41/349 (12%) 5/49 (10%) 0.75 Post-operative platelets 47/349 (13%) 5/49 (10%) 0.53 Intra-operative 2/349 (1%) 0/49 (0%) 1.00 hypotension >5 minutes Post-operative 2/349 (1%) 2/49 (4%) 0.08 hypotension >5 minutes Re-sternotomy for 12/349 (3%) 5/49 (10%) 0.05* bleeding Incubation time (hr) 20 (14-24) 19 (11-24) 0.16 CSICU discharge in 48 315/347 (91%) 37/49 (76%) 0.001** hrs *P < 0.05 **P < 0.01; # in 362 patients on CPB; in 359 patients on cross clamp Median (interquartile range) Abbrevations used in this table: standard deviation (SD); fresh frozen plasma (FFP) packed red blood cells (PRBCs); intra-aortic balloon pump (IABP); cardiac surgical intensive care unit (CSICU); cardiopulmonary bypass (CPB); angiotensin converting enzyme inhibitor (ACEI); angiotensin-2 (AG-II).
2. Cytokine Results

(28) Cytokine results are illustrated in Table 2. Pre-operative urinary IL1ra was lower in those patients who developed late (day 5) renal dysfunction (Table 2). The relative value of lower than normal pre-operative urinary IL1ra in predicting day 5 (late) renal dysfunction is demonstrated in the relevant receiver operator characteristic (ROC) curve shown in FIG. 1.

(29) 3. Discussion.

(30) The main findings of this study are:

(31) If patients are grouped into those with and without renal dysfunction in the fifth post-operative day, the abnormal renal function group had significantly lower pre-operative urinary IL1ra than the normal renal function group.

(32) TABLE-US-00002 TABLE 2 Table showing 24 hours pre-operative urinary cytokine concentrations (median and interquartile range) IQR) in patients categorised according to MDRD estimated 25% eGFR percentage drop from pre-operative baseline at any time post-operatively (Any MDRD >25% drop) as well as on post-operative day 1 (MDRD D1 >25% drop), day 2 (MDRD D2 >25% drop) and day 5 (MDRD D5 >25% drop). Any MDRD >25% drop No Yes Cytokine (pg ml.sup.1) n Median (IQR) n Median (IQR) P value IL1ra (pre- 342 2934 (1476-5827) 46 1907 (767-5965) 0.07 operative) TNFsr2 (pre- 344 2048 (1085-3438) 45 2052 (1244-3135) 0.97 operative) MDRD D1 >25% drop No Yes Cytokine (pg ml.sup.1) n Median (IQR) n Median (IQR) P value IL1ra (pre- 381 2879 (1448-5769) 13 2455 (810-5164) 0.40 operative) TNFsr2 (pre- 382 2059 (1092-3437) 13 1944 (809-2967) 0.50 operative) MDRD D2 >25% drop No Yes Cytokine (pg ml.sup.1) n Median (IQR) n Median (IQR) P value IL1ra (pre- 358 2908 (1443-5687) 35 2455 (1108-8741) 0.73 operative) TNFsr2 (pre- 360 2048 (1077-3423) 34 2151 (1399-3284) 0.44 operative) MDRD D5 >25% drop No Yes Cytokine (pg ml.sup.1) n Median (IQR) n Median (IQR) P value IL1ra (pre- 374 2926 (1462-5890) 16 1552 (586-3695) 0.01* operative) TNFsr2 (pre- 375 2049 (1086-3438) 16 1924 (1119-2465) 0.30 operative) (*P < 0.05)