Patent Application
20120202201
This application claims priority pursuant to 35 U.S.C. §119 of European patent application 11 153 273.5, filed on 3 Feb. 2011.
A field of the invention concerns devices and methods for analysis of kidneys from bodily fluid samples
Juan et al. in Urology 835-841 (2010) describe the identification of microRNAs which are characteristic for kidney cancer in comparison to microRNAs from normal kidney.
Mitchell et al., PNAS 10513-10518 (2008) describe circulating microRNAs as markers for cancer, especially of miR-141 as a characteristic for prostate cancer.
Ali et al., J. Am. Soc. Nephrol. 1292-1298 (2007) describe the analysis of creatinine values related to acute kidney injury and acute-on-chronic renal failure.
Bagger et al., Pediatr. Nephrol. 1655-1658 (2007) describe serum creatinine levels and urine output as measures that reflect renal function.
US 2010/0173288 A1 describes that generally disease states can be analysed by measurement of all detectable microRNAs in blood serum for a patient, and describe a kit and biochip, respectively, containing essentially all mature microRNAs known from human serum.
WO 2010/145035 A 1 relates to markers for renal diseases and mentions miR-210 as a marker for renal cancer.
US 2011/0003704 A 1 describes the analysis of microvesicles for diagnosis of diseases and mentions a very large number of microRNAs, without specifically correlating a specific microRNA to acute kidney failure.
Li et al., Nephrology 599-608 (2010) describes that it has been observed in relation to renal carcinoma that miR210 is regulated by hypoxia and may have implications for tumour pathogenesis.
An embodiment of the invention is a method for analysis of kidney status in a sample of body fluid. In the method, the concentration of an analyte is determined. The analyte is selected to be miR-210 and the kidney status is acute kidney injury. Preferably, the concentration of at least one of the analytes of the group containing miR-16 and miR-320 is additionally determined. Preferably, the step of determining the concentration of the analyte contacts the sample of body fluid with a nucleic acid sequence which comprises a section that is reverse complementary to the analyte and detecting the concentration of nucleic acid hybrids formed of the nucleic acid sequence comprising the reverse complementary nucleic acid section.
An embodiment of the invention is a device containing a nucleic acid sequence containing the sequence of miR-210 and/or a nucleic acid section that is reverse complementary to miR-210 for use as an indicator substance which is specific for acute kidney injury. The device is used in methods of analysis of the invention. Preferably, the device additionally contains a nucleic acid sequence containing a section that is identical to miR-16 and/or to miR-210, and/or a nucleotide section which is reverse complementary to miR-16 and/or reverse complementary to miR-210 for use as an indicator substance which is specific for kidney status
Embodiments of the invention provide for an analytical method, and for a kit of parts suitable for performing the method, each suitable for evaluating the risk of patients to develop AKI, and suitable for evaluating the risk of mortality, and the prospects or long-term survival and renal recovery, respectively, in AKI patients.
Preferred embodiments of the invention provide a method for analysis of the status of kidney function in a sample of body fluid obtained from a patient who has not been diagnosed with acute kidney injury (AKI, also referred to as acute kidney failure) or in a sample of body fluid obtained from a patient who has been diagnosed with AKI, which analysis is suitable for and adapted to evaluating the prospects for long-term survival, e.g. for 28 days from the diagnosis of AKI, and/or for renal recovery in an AKI patient, which method for analysis comprises or consists of determining the concentration of the microRNA-210 (miR-210) comprising or consisting of the nucleic acid sequence SEQ ID NO 1: CUGUGCGUGUGACAGCGGCUGA, preferably in combination with determining the concentration of one or both of miR-320 comprising or consisting of the nucleic acid sequence SEQ ID NO 2: AAAAGCUGGGUUGAGAGGGCGA and miR-16 comprising or consisting of the nucleic acid sequence SEQ ID NO 3: UAGCAGCACGUAAAUAUUGGCG in a sample, preferably in a serum sample obtained from the patient, which patient can be diagnosed to suffer from AKI, e.g. by analysis of creatinine levels.
The present invention relates to an analytical method, and to a kit of parts, which is adapted to the analytical method, for analyzing a patient sample in order to determine the prospects of a patient to develop acute kidney injury (AKI), and to determine the prospects of patients suffering from AKI for long term survival, e.g. for 28 days from the diagnosis of AKI. The invention provides for an analyte, and for the use of the analyte, which in respect to AKI is essentially independent from a diagnosed sepsis or shock. The concentration of the analyte of the invention, preferably in combination with the concentration of one or both of two additional analytes in a sample of human body fluid, e.g. in blood serum obtained from patients, especially from patients suffering from AKI, is a marker of the propensity to develop AKI, and for survival, e.g. of 28-day survival, including renal recovery.
The invention is based on the finding that the concentration of the analyte miR-210, preferably in combination with one or both of miR-320 and miR-16, is significantly correlated with the propensity of a patient to initially develop AKI, and with the prospects of survival, e.g. for 28 days, and for renal recovery of AKI patients. In greater detail, it has been found that a serum concentration of miR-210 that is elevated in comparison to median serum levels is indicative of a propensity of a person to develop AKI, and of a reduced prospect for long-term survival of AKI patients, but is generally indicative of an increased mortality. The median serum level can e.g. be the concentration of the respective microRNA found in healthy persons, especially of miR-210s.
Concentrations of miR-320 which are decreased below median serum concentrations of healthy persons are also indicative of an increased propensity to initially develop AKI, indicative of an increased mortality, and indicative of a reduced prospect of long-term survival in AKI patients, and concentrations of miR-16 decreased below a median concentration in healthy persons are also indicative of an increased mortality.
In a first embodiment, the analytical method for determining the concentration of miR-210, optionally in combination with determining the concentration of miR-320 and/or of miR-16, allows the evaluation of the risk of a patient to develop AKI, and/or of the propensity of a patient to suffer from or develop AKI, as the concentration of the analytes of the invention is a predictive indicator for AKI, which indicator is independent from other known indicators for AKI, e.g. from diagnosis of sepsis or shock, and essentially independent from diagnosis of creatinine levels and/or from urine output.
Generally, the method for analysis can be performed by detecting the concentration of the microRNAs which are used as the analyte, i.e. miR-210, preferably additionally miR-320 and/or miR-16 in a sample of body fluid for determining the concentration of the analyte miRNA. The step of determining the concentration of the analyte in a first embodiment generally comprises the step of contacting a sample of body fluid with a nucleic acid sequence which comprises or consists of a section that is reverse complementary to the analyte microRNA (miR), and detecting the concentration or number of hybrids formed by the reverse complementary nucleic acid section with sample components. For detecting the concentration or number of analyte-specific hybrids, it is preferred that the nucleic acid sequence comprising or consisting of a nucleic acid section which is reverse complementary to the analyte miR is immobilized on a substrate, e.g. the nucleic acid sequence can be contained in a panel of nucleic acid sequences which are immobilized and spaced apart on a carrier substrate, such as panels or nucleic acid arrays in the form of a chip.
In a further embodiment, the step of determining the concentration of the one or more analyte miR can be performed by reverse transcription and PCR (RT-PCR) using an oligonucleotide primer which is specific for the analyte miR, followed by reverse transcription and amplification of at least a section of the analyte miR by PCR. For detection, hybridizing probes which are labelled with a chromophor and a spaced apart quencher, the probe having an analyte-specific nucleic acid sequence can be used, e.g. as known for the TaqMan PCR.
It is generally preferred that the analytical method is performed using an internal control for the reactions of determining the concentration of the analyte miR, e.g. by adding a known miR as a control to the sample and analysing the concentration of the added control miR by parallel detection with specificity for the control miR. The miR which is added to the sample as an internal control for the analytical method can be a known concentration or copy number of the analyte miR, e.g. a known copy number of miR-210, miR-320 and/or of miR-16, and/or an miR of human or non-human origin, e.g. a synthetic or bacterial microRNA. Preferably, the added internal control miR is produced by bacterial transcription from a DNA sequence encoding the miR, or is produced by enzymatic synthesis, e.g. by PCR, or by chemical synthesis.
The specific amplification of the added internal control miRNA is preferably used for normalization of the determined initial or original serum concentration or copy number of analyte miRNA, e.g. miR-210, miR-320 and/or of miR-16. The initial serum concentration of analyte miRNA was determined by applying a standard curve for the relation of measured amplification of added analyte miRNA of known concentration to the measured number of amplification products obtained in RT-PCR for analyte miRNA. In detail, a standard curve was generated by adding a known copy number (concentration) of analyte miRNA, e.g. of miR-16, miR-210 and miR-320, e.g. obtainable from Ambion Co. at 0.2 fmol/μL analyte miRNA, performing RT-PCR with primers specific for analyte miRNA, and measuring the copy number of specific amplification products. In addition to adding analyte miRNA of known concentration, internal control miRNA, e.g. miR-54 of C. elegans of known copy number and primers specific for internal control miRNA were added. The original serum concentration of analyte miRNA could be calculated by applying the relation of the copy number of analyte-specific amplification products to the original concentration of analyte miRNA, which relation is determined in the standard curve. Normalization could be obtained by dividing the determined concentration of original concentration of analyte miRNA by the concentration of added internal control miRNA.
Accordingly, the method for analysis preferably comprises the calculation of the concentration of the analyte miRNA by means of a standard curve that contains the pre-determined correlation between the concentration of detected analyte miRNA-specific amplification products obtained in RT-PCR to an original (initially known) concentration of analyte miRNA in serum.
In order to account for variability of RNA isolation from serum, the method preferably contains the step of adding a known concentration of a known internal control miRNA to the serum, isolating RNA from the serum, carrying out RT-PCR on the RNA with specific primer pairs added for each of the analyte miRNA and for the control miRNA, respectively, and in addition to calculating the original concentration of analyte miRNA by means of the standard curve, the method also contains the step of calculating the original concentration of added control miRNA by means of a pre-determined standard curve which contains the pre-determined correlation of the detected concentration of amplification products of the control miRNA to the original concentration of control miRNA, and forming the quotient of the calculated original concentration of analyte miRNA and the calculated concentration of control miRNA. Accordingly, it is generally preferred to determine the concentration of analyte miRNA in the serum sample as a quotient to a control miRNA. For example, if control miR-54 of C. elegans was added to 5 fmol/μL serum, for miR-210 as the analyte miRNA, the quotient of (calculated initial concentration of miR-210)/(calculated concentration of control miR-54 (C. elegans)) above 4.00098×10−5 in AKI patients were found to be indicative of a worse prognosis for survival than a lower quotient. The control miRNA preferably is an added miRNA, preferably of a non-human nucleotide sequence, or an miRNA which is determined or known to be present at constant concentrations in human serum.
Preferably, the method of analysis, as well as the determination of the standard curve for analyte miRNA and control RNA, e.g. non-coding RNA, preferably a miRNA, respectively, are carried out using at least 2 different dilutions of the isolated RNA, e.g. 2 to 6 or 3 to 4 dilutions, each e.g. diluted by a factor of 5 to a factor of 10.
In addition to or in the alternative to normalization of the analyte miRNA to an added control RNA, e.g. a control miRNA, miR-210 can be normalized to an intrinsic housekeeping RNA, e.g. to a housekeeping small RNA, which preferably is a miRNA or a snoRNA, e.g. the human equivalent to the murine snoRNA202 (SEQ ID NO 5). In these embodiments, concentrations of the analyte miRNA miR-210 are calculated in relation to the concentration of the housekeeping RNA, which is e.g. a snoRNA.
In a further embodiment, the invention relates to the use of nucleic acid sequences containing or consisting of a section that comprises or consists of the nucleic acid sequence of at least one of the analyte microRNAs, and/or to the use of nucleic acid sequences containing or consisting of a section that comprises or consists of a nucleic acid sequence is reverse complementary to at least one of the analyte microRNAs, e.g. to nucleic acid sequences containing or consisting of a nucleotide sequence of miR-210, and/or to nucleic acid sequences containing or consisting of a nucleotide sequence which is reverse complementary to miR-210, optionally additionally to nucleic acid sequences containing or consisting of miR-320, and/or miR-16 and/or nucleic acid sequences containing or consisting of a section that is reverse complementary to miR-320, and/or miR-16, as an indicator substance which is specific for kidney function, especially for use as an indicator substance that is specific for the propensity to suffer from or to develop AKI, and/or as an indicator substance, the concentration of which in a sample of body fluid is indicative of the propensity for an increased mortality for AKI patients.
Optionally, the method includes the step of determining the average concentration of miR-210, optionally additionally of miR-320 and/or of miR-16, in relation to the concentration of a housekeeping microRNA. The concentration of a housekeeping microRNA can be determined by the methods as described for the concentration of miR-210.
Preferred embodiments of the invention will now be discussed with respect to the drawings and experiments.
In the analysis of 77 AKI patients and of 30 healthy age-matched individuals which served as control (CTL), micro-RNAs were quantitatively measured by RT-PCR from serum samples. It was found that in AKI patients, concentrations of miR-210 were higher (p<0.0001) while concentrations of miR-16 (p<0.0001) and miR-320 (p<0.0001) were lower in comparison to healthy individuals. This result shows that a concentration of miR-210 higher than the median concentration in a healthy individual is indicative of AKI or of a significant propensity to suffer from or to develop AKI, whereas concentrations of miR-16 and/or of miR-320 below the median concentration of healthy individuals is also indicative of AKI, or of a significant propensity to suffer from or to develop AKI.
Multivariate Cox regression and Kaplan-Meier curve analyses revealed that the concentration of miR-210 can be used as an independent analyte indicative of the propensity to suffer from or to develop AKI, or for the presence of AKI, as an increased concentration of miR-210 above the median concentration of this analyte in healthy individuals in an independent indicator which is correlated with a reduced 28-day survival (p=0.05 and p=0.03, respectively). Detailed demographic, clinical and laboratory characteristics of a total of 77 patients are shown in Table 1, wherein
APACHE II=acute physiology and chronic health evaluation 11, BMI=body mass index, CRP=C-reactive protein, eGFR=estimated glomerular filtration rate; ICU=intensive care unit; MAP=mean arterial blood pressure; n=number of patients; RRT=renal replacement therapy; SOFA=sequential organ failure assessment:
These data show that no association between the microRNAs analysed and the APACHE II score or the SOFA score can be found. Further, no significant differences in concentrations of the microRNAs indicated in patients with or without sepsis (miR-16: p=0.3, miR-210: p=0.4; miR-320: p=0.6), surgery (miR-16: p=0.4; miR-210: p=0.7; miR-320: p=0.8), or shock (miR-16: p=0.5; miR-210: p=0.5; miR-320: p=0.4) were found. Similarly, the concentrations of the microRNAs analyzed did not differ between patients of the different intensive care units as assessed by ANOVA (miR-16: p=0.4; miR-210: p=0.3; miR-320: p=0.08). The concentrations of the microRNAs analysed did not correlate with serum creatinine levels at the start of renal replacement therapy (RRT) (miR-16: r=−0.14 p=0.2; miR-210: r=−0.3 p=0.8; miR-320: r=−0.4, p=0.8).
The analytical results for the AKI patients and healthy controls (CTL) of the determined concentration of miR-16 in serum is shown in
Normalization of measurement results for the analyte microRNAs using the detected concentration of the miR-54 (SEQ ID NO 4: UACCCGUAAUCUUCAUAAUCCGAG) was done according to Fichtlscherer et al (“Circulating microRNAs in patients with coronary artery disease”, Circ. Res. 677-684 (2010)). In short, the concentration of analyte miR, e.g. of miR-210, miR-320, and of miR-16 was calculated from the number of specific amplification products per reaction volume to obtain a calculated initial copy number of each analyte miR per initial sample volume, which initial copy number of analyte miR per initial sample volume was divided by the initial copy number concentration of added control miR, e.g. divided by the calculated concentration of control miR that was obtained from an added concentration of 5 fmol/μL serum, thus accounting for losses during RNA isolation.
Using this normalization of control miR-54 of C. elegans added to 5 fmol/μL initial sample volume, isolation of total RNA, RT-PCR using primer pairs specific for the analyte miR, e.g. for miR-210 and a primer pair specific for the control miR-54, the mean value of the concentration of miR-210 normalized by dividing the calculated initial concentration of miR-210 in the sample by the concentration of added control miR, added to a serum concentration of 5 fmol/μL serum, was determined to a value of 4.00098×10−5. Serum concentrations of miR-210 above a ratio of 4.00098×10−5 (initial concentration of miR-210 calculated from RT-PCR products)/(initial concentration calculated for control miR-54) in patients suffering from AKI indicate a worse prognosis for a period of 28 days, which was the observation period.
Standard curves of the correlation of the concentration of specific RT-PCR amplification products to the original concentration of the respective microRNA in the RNA were pre-determined with at least 4 dilutions of isolated total RNA containing the microRNA at a known concentration, each dilution differing by a factor of 10. Preferably, the known microRNA was added to a known concentration in the serum, and total RNA was isolated from serum, diluted to at least 2 dilution steps, and subjected to RT-PCR using a primer pair specific for the microRNA that was originally added to the serum.
Generally, the analytical method can include the step of measuring the concentration of a known housekeeping microRNA present in serum, and dividing the concentration of the analyte microRNA by the concentration of the housekeeping microRNA for normalization.
The results shown in
The only significant association that was found were baseline concentrations of miR-16 (r=0.3, p=0.02), miR-210 (r=0.4, pp<0.001), and of miR-320 (r=0.4, pp<0.001) correlating with lactate levels, and baseline concentrations of miR-210 additionally was correlated with heart-rate (r=0.04, p<0.0001) baseline concentrations of miR-320 was correlated with heart-rate (r=0.4, p<0.0001), and noradrenalin dose (r=0.3, p=0.04).
Following the initial analysis, AKI patients underwent renal replacement therapy, and patients were grouped 4 weeks after the therapy in survivors and non-survivors. It was found that generally both these patient groups were comparable with respect to baseline demographics and to the proportion of RIFLE categories as shown in table 1. It was found that the group of survivors and the group of non-survivors only differed with regard to the proportion of patients with metabolic acidosis (p=0.005), and SOFA score (p=0.05). It was found that patients suffering from sepsis have a higher SOFA and higher APACHE II scores, which were deemed to result from more severe cardiovascular and respiratory impairment compared to the non-septic patients.
The analytical results for the AKI patients for survivors and non-survivors are shown in
An initial analysis by the univariate cox proportional hazards analysis was done to determine a possible relationship between circulating concentrations of microRNAs at initiation of RRT and mortality. It was found that in the initial AKI patients, concentrations of miR-16, age, gender, and body mass index were not significantly associated with survival, as shown in the following Table 2.
From the data of Table 2, it was found that the variables major surgery, sepsis, APACHE II score, and SOFA score as well as concentrations of miR-210, and miR-320 display a prognostic significance at a 10% level. In multivariate analysis, it was found that only the SOFA score (p=0.005), and miR-210 (p=0.05) are independent predictors of survival. In ROC curve analysis miR-210 yielded an area under the ROC curve of 0.74 (standard error of the mean: 0.06; 95% confidence interval: 0.53 to 0.78; p=0.03), while the concentration of miR-320 showed a similar level of significance (area under the ROC curve 0.7; standard error of the mean: 0.07; 95% confidence interval: 0.53 to 0.78; p=0.03). The concentrations of miR-16 did not reach statistical significance (area under the ROC curve 0.55, standard error of the mean: 0.07, 95% confidence interval: 0.41 to 0.69, p=0.4). For comparison, the area under the curve of the SOFA score gave a value of 0.7 (standard error of the mean: 0.06, 95% confidence interval: 0.57 to 0.82, p=0.005).
As an animal model, renal ischemia/reperfusion-injury (IRI) was induced in C57B1/6 mice by bilateral clamping of both renal pedicals. The animals were anesthetized with isoflurane and generally handled according to animal experiment guidelines. After median laparatomy, renal pedicals were bluntly dissected and a non-traumatic vascular clamp was applied for 27 min. At 4 weeks after induction of bilateral IRI animals were sacrificed and RNA was isolated from whole kidneys. Sham animals were used as controls. For in vivo real-time polymerase chain reaction (RT-PCR) analysis, RNA was extracted from renal tissue with Trizol reagent (Invitrogen). After reverse transcription of total RNA, qRT-PCR was performed with the Taqman MicroRNA assay (Applied Biosystems, ABI) according to the manufacturer's recommendations to detect the level of miR-210. miR-210 was normalized to the housekeeping small RNA snoRNA202 (SEQ ID NO 5). For comparison to control animals, miR-210 concentrations were calculated in relation to the concentration of the housekeeping snoRNA-202.
The result is shown in
While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.
Various features of the invention are set forth in the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11153273.5 | Feb 2011 | EP | regional |
