Established AKI cases
Cases were ascertained from a single center prospective non-concurrent observational cohort of inpatients who were identified as having suffered acute kidney injury (AKI) during a hospital admission. The institutional review board of the University of California, San Diego (UCSD) reviewed and approved the study as well as the consent document. Primary providers referred potentially eligible patients to investigators and study coordinators. Patients were eligible for enrollment if they were greater than 18 years of age and met the serum creatinine criteria for acute kidney injury as set out by the Acute Kidney Injury Network (AKIN) [13]: an abrupt rise by ≥0.3 mg/dl within a 48-hour period. Patients were excluded if they received chronic renal replacement therapy (hemodialysis or peritoneal dialysis) within the 6 months prior to admission; had ever been in receipt of a kidney transplant; pregnant or breast feeding; currently incarcerated or otherwise institutionalized (nursing home, rehabilitation); were placed under hospice/comfort care; or did not have reasonable expectation of survival past the present hospitalization. If eligible, the patient or authorized representative was presented the study and consent for participation was obtained. All relevant patient data were derived from the electronic and paper medical records, as well as direct interview of the patient or surrogate. Basic demographic information and co-morbid conditions were recorded on enrollment. Etiology of AKI was determined by chart review of provider diagnosis, and verification by study personnel; diagnosis followed the categories set by the Project to Improve Care of Acute Renal Disease (PICARD) study investigators [1]. Daily assessment included medication review, physical exam (as recorded in medical record or assessed by study personnel when missing), vital signs, intake and output, and labs. Clinical data elements were collected daily and the need for and utilization pattern of renal replacement therapy was also monitored and recorded. Blood and urine samples were collected at time of entry, daily for 7 days maximum, and at hospital discharge. Twenty-four hour urine collections were performed at study entry and hospital discharge for creatinine/urea clearance (in approximation of glomerular filtration rate - GFR) as well as urinary kallikrein activity. One sample of whole blood was collected for genomic DNA preparation (and genetic analysis of the KLK1 locus) at the time of study entry. At the time of discharge, follow-up appointments with either the primary care physician or a nephrologist (not all patients were seen by a nephrologist during the hospitalization), extent of renal recovery, and dialysis dependence (if needed during hospitalization) were ascertained. Recovery: pre-defined study endpoints were 12 months of follow-up, start of maintenance hemodialysis or peritoneal dialysis, receipt of a kidney transplant, or death; recovery of renal function was the primary outcome, defined as a return to within 10% of baseline eGFR or lowest eGFR prior to AKI event. Recovery was assessed at 6 months of follow-up.
Healthy (non-hospital) controls
In addition, we obtained data for a control group of 38 healthy adult subjects. Each healthy control was selected from only one member from each of 38 twin pair sets. Twin pairs were recruited by a population-based twin registry in southern California [14], and by newspaper advertisement. These twins are of European, African-American, and Asian biogeographic ancestry. Ethnicity was established by self-identification. Self-reported zygosity was confirmed by extensive SNP genotyping. There was no clinical evidence for kidney disease or any other cardiovascular disorder in any of the controls. Untimed (spot) urine collections were performed, and one sample of EDTA-anticoagulated whole blood was collected for genetic analysis of the KLK1 locus at the time of study entry.
Replication: Incipient (early) AKI cases in an intensive care unit (ICU), with ICU control subjects
An additional sample of controls was ascertained for replication of findings, in an ICU setting. In brief, the replication sample consisted of n = 44 subjects ("ICU controls") who did not develop AKI during a 7-day observation period after hospital ICU admission, as well as n = 11 subjects (ICU cases) who did develop AKI, as defined above. Patients were screened at ICU admission for potential study participation at three academic medical centers (University of California San Diego, University of Alabama, and Université de Montréal) between July 2006 and December 2008. Patients were eligible for enrollment if they were age 18 or older and had a life expectancy of at least 48 hours. Controls were excluded if they had AKI according to the AKIN criteria [13], were admitted to the ICU > 48 hours prior to screening, transferred from another ICU, had a serum creatinine > 2 mg/dl ≤ 3 days before ICU admission, were prisoners, received dialysis within the 12 months prior to admission, had a functioning kidney transplant, were on anticoagulants or warfarin within the last 7 days, suffered from decompensated cirrhosis, had CKD stage 5, were anemic (hemoglobin < 9.0 mg/dl or hematocrit < 27%), or were already enrolled in another research project.
The study was approved by the Institutional Review Boards at each institution, and written informed consent was obtained from all participants or their health care surrogates. Following informed consent, data on past medical history were collected once, and clinical, laboratory and process-of-care elements were collected daily. Each institution's local laboratory measured serum creatinine values. AKI was defined as an increase in serum creatinine level of more than 0.3 mg/dL or more than 50% from a reference creatinine within 48 hours (AKIN criteria)[13]. Patients without AKI within the first 4 days had continued blood and urine samples twice daily, for a total study observation period of 7 days.
Biochemical assays
Urine was assayed for kallikrein by an alkaline amidolytic activity assay as previously described (4), using the chromogenic substrate S-2266: [D] Val-Leu-Arg-paranitroanilide (Kabi Pharmacia; Franklin, OH, USA) [5, 15]. The activity of kallikrein per liter of urine (units per liter, U/L) is calculated from the formula: U/L = (9.55·A), where A = absorbance, after a 30-min assay incubation of the paranitroanilide product in a spectrophotometer at 405 nm [5, 15]. Inclusion or exclusion of the kallikrein inhibitor aprotinin (Trasylol, Miles Inc., West Haven, CT, USA) indicated that a fraction of human urinary S-2266 amidolytic activity in the absence of aprotinin was non-kallikrein (likely urokinase) [15]; thus, aprotinin (20 kallikrein inhibitory U/mL) was systematically included in the assay blank, to assure specificity for kallikrein measurement. The inter-assay coefficient of variation was 18.1%, and activities from n = 20 samples measured on two separate occasions correlated highly (Spearman R = 0.92, P< 0.01). In n = 87 subjects, activity correlated (Spearman R = 0.82, P< 0.0001) when results were compared for kallikrein excretion normalized to time versus creatinine excretion. Specificity of the S-2266 amidolytic assay for glandular (KLK1, including renal, pancreas, and salivary) kallikrein activity in urine arises from two features: first, the substrate S-2266 is cleaved only by a particular subset of serine proteases, including KLK1; and second, the inclusion of aprotinin in the assay blank, which specifically inhibits serine proteases including KLK1; nonetheless, only an immunoassay specific for lysyl-bradykinin (the kinin product of KLK1) generation could provide absolute assurance of specificity for KLK1.
Clinical chemistries (serum or urine, electrolytes or creatinine) were measured by spectrophotometric autoanalyzer. Urine aldosterone concentration was determined by enzyme immunoassay (Alpco Diagnostics, Salem, NH, USA). Urine catecholamine concentration was determined by commercial ELISA kit (Labor Diagnostika Nord GmBH & Co. KG, Nordhorn, Germany). Urine values were normalized to creatinine concentration in the same sample.
DNA extraction and bisulfite treatment of CpG sites
A sample of EDTA-anticoagulated whole blood was obtained from participants and stored at -70°C prior to DNA extraction. Timed and spot urine samples were obtained and frozen at -70°C before assay. Blood DNA was prepared from blood leukocytes with a commercial kit (QIAamp® DNA Mini Kit; Qiagen, USA). Urine DNA was prepared from the urine pellet with spun columns (urine DNA isolation kit; Norgen, Canada). Both DNAs were subjected to sodium metabisulfite (Na2S2O5) treatment (Imprint™ DNA Modification Kit; Sigma, USA), and then eluted in 20 μL elution buffer. Bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected.
KLK1promoter CpG methylation analysis
Pyrosequencing for allele discrimination (Pyrosequencing; Qiagen, USA) provides real-time extension-based DNA analysis that can evaluate multiple CpG sites [16]. CpG methylation analysis at the 5'/upstream/proximal promoter region of human kallikrein serine protease 1 (KLK1) gene was performed. The KLK1 gene was analyzed by a single PCR amplicon spanning 4 CpGs in a 263 bp region, with two biotinylated sequencing (extension) primers (Figure 1). The consecutive 4 CpGs were located -203 to -135 bp from the transcription initiation site: sequentially at -203, -196, -154, and -135 bp (Figure 1).
As a control, global CpG methylation analysis was completed using PyroMark LINE-1 reagents (Pyrosequencing; Qiagen, USA). We thus determined the methylation status of three CpG sites in LINE-1 repetitive (LTR-like) elements, wherein methylation levels of CpG sites represent global methylation status across the genome, because of the repetitive nature of LINE-1 elements [17, 18].
PCR amplification was performed using 10X PCR buffer, 3.0 mM MgCl2, 200 μM of each dNTP, 0.2 μM each of forward and reverse primers, HotStar DNA polymerase (Qiagen, USA) 1.25 U, and ~10 ng of bisulfite-converted DNA per 50 μl reaction. PCR cycling conditions were: 94°C 15 min; 45 cycles of 94°C 30 s, 56°C 30 s, 72°C 30 s; 72°C 5 min; and then products were held at 4°C. The PCR was performed with one of the PCR primers biotinylated to convert the PCR product to single-stranded DNA template. PCR products (each 10 μl) were sequenced by Pyrosequencing PSQ96 HS System (Pyrosequencing, Qiagen, USA). The methylation status of each locus was analyzed individually as a T/C SNP using Pyro-QCpG™ software (Pyrosequencing).
AKI case samples were evaluated at study entry (baseline, time of diagnosis). After bisulfite modification and PCR amplification, KLK1 blood DNA promoter methylation data from 13 AKI patients and 30 controls were obtained. KLK1 urine DNA promoter methylation data from 9 AKI and 22 controls were available. LINE-1 blood DNA methylation data from 14 AKI patients and 32 controls were available for evaluation. LINE-1 urine DNA methylation data from 15 AKI patients and 32 controls were obtained.
Statistical analyses
Results are expressed as the mean value ± one standard error of the mean (SEM) for continuous variables. For comparisons of two groups, unpaired two-sided t-tests or one-way ANOVA (enabling adjustment for covariates of age, sex, and ethnicity) were performed. Non-parametric Wilcoxon Rank Sum test was used to confirm parametric tests in the face of relatively small sample sizes. Proportions were evaluated by Fisher's exact test (2 × 2 tables) or chi-square test (3 × 2 tables). Statistical analyses were performed in R2.10.1 <http://www.r-project.org/ > or SPSS-17 (Statistical Package for the Social Sciences; Chicago, IL, USA). A P value of < 0.05 was considered significant. Multiple linear regression was performed with default criteria of entry (p < 0.05) and exit (p > 0.10) from the multivariate regression model, using stepwise or forward options. Recovery from AKI was pre-defined as return (within 6 months follow-up) to within 10% of baseline eGFR, or lowest eGFR prior to AKI event.