Comparison of Plasma and Urine Biomarker Performance in Acute Kidney Injury

Background

New renal biomarkers measured in urine promise to increase specificity for risk stratification and early diagnosis of acute kidney injury (AKI) but concomitantly may be altered by urine concentration effects and chronic renal insufficiency. This study therefore directly compared the performance of AKI biomarkers in urine and plasma.

Methods

This single-center, prospective cohort study included 110 unselected adults undergoing cardiac surgery with cardiopulmonary bypass between 2009 and 2010. Plasma and/or urine concentrations of creatinine, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), liver fatty acid-binding protein (L-FABP), kidney injury molecule 1 (KIM1), and albumin as well as 15 additional biomarkers in plasma and urine were measured during the perioperative period. The primary outcome was AKI defined by AKIN serum creatinine criteria within 72 hours after surgery.

Results

Biomarkers in plasma showed markedly better discriminative performance for preoperative risk stratification and early postoperative (within 24h after surgery) detection of AKI than urine biomarkers. Discriminative power of urine biomarkers improved when concentrations were normalized to urinary creatinine, but urine biomarkers had still lower AUC values than plasma biomarkers. Best diagnostic performance 4h after surgery had plasma NGAL (AUC 0.83), cystatin C (0.76), MIG (0.74), and L-FAPB (0.73). Combinations of multiple biomarkers did not improve their diagnostic power. Preoperative clinical scoring systems (EuroSCORE and Cleveland Clinic Foundation Score) predicted the risk for AKI (AUC 0.76 and 0.71) and were not inferior to biomarkers. Preexisting chronic kidney disease limited the diagnostic performance of both plasma and urine biomarkers.

Conclusions

In our cohort plasma biomarkers had higher discriminative power for risk stratification and early diagnosis of AKI than urine biomarkers. For preoperative risk stratification of AKI clinical models showed similar discriminative performance to biomarkers. The discriminative performance of both plasma and urine biomarkers was reduced by preexisting chronic kidney disease.     

Clinical Impact of Kidney Function on Presepsin Levels

Objective

Presepsin is highlighted as a diagnostic and prognostic marker of sepsis. Little information is available regarding the accurate association between presepsin levels and the degree of kidney function. We analyzed presepsin levels in patients with a glomerular filtration rate (GFR) in the categories G1 to G5, evaluated via inulin renal clearance test, and receiving hemodialysis (HD).

Methods

Patients who were not receiving HD were included if they had undergone inulin renal clearance measurements for the accurate measurement of GFR (measured GFR), and patients who were receiving hemodialysis (HD) were included if they had anuria. Exclusion criteria were infection, cancer, liver disease, autoimmune disorders, or steroid or immunosuppressant use. GFR category was defined as follows; G1: GFR ≥ 90 ml/min/1.73m², G2: GFR = 60 to 90 ml/min/1.73m², G3: GFR = 30 to 60 ml/min/1.73m², G4: GFR = 15 to 30 ml/min/1.73m², G5: GFR ≤ 15 ml/min/1.73m².

Results

Seventy-one patients were included. The median (IQR) presepsin values of patients in each GFR category were as follows: G1 + G2: 69.8 (60.8–85.9) pg/ml; G3: 107.0 (68.7–150.0) pg/ml; G4: 171.0 (117.0–200.0) pg/ml; G5: 251.0 (213.0–297.5) pg/ml; and HD: 1160.0 (1070.0–1400.0) pg/ml. The log-transformed presepsin values, excluding patients receiving HD, inversely correlated with the measured GFR (Pearson’s correlation coefficient = -0.687, P < 0.001). The multivariate analysis revealed that measured GFR and hemoglobin levels significantly correlated with elevated presepsin levels.

Conclusion

Presepsin levels were markedly high in patients receiving HD, similar to values seen in patients with severe sepsis or septic shock. In patients who were not receiving HD, presepsin levels increased as GFR decreased. Thus, the evaluation of presepsin levels in patients with chronic kidney disease requires further consideration, and a different cutoff value is needed for diagnosing sepsis in such patients.     

Measurement of Glomerular Filtration Rate by Inulin Clearance without Urine Collection

A continuous infusion method for measuring inulin clearance (and hence glomerular filtration rate) which does not require urine collection has been re-evaluated and found to give satisfactory results. In two non-oedematous and anephric patients the plasma level of inulin became virtually constant four hours after a single intravenous dose, and hence this is the minimum time required for the infusion with this method.     

Using an Isolated Rat Kidney Model to Identify Kidney Origin Proteins in Urine

The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.     

The Influence of Angiotensin Infusion on the Urine Composition in Individual Kidney Function Tests

Thirty-two patients with hypertension were given a pressor dose of angiotensin in the course of individual kidney function tests. In eight patients with unilateral renal artery stenosis the differences in urine sodium and creatinine concentration between the affected and the nonaffected kidney did not become greater with angiotensin infusion. In four patients with unilateral pyelonephritis and falsely positive individual kidney function tests, these tests became normal following angiotensin infusion. It is concluded that angiotensin infusion is not a useful means of improving the results of individual kidney function tests.     

Vitamin K Intake and Plasma Desphospho-Uncarboxylated Matrix Gla-Protein Levels in Kidney Transplant Recipients

Vitamin K is essential for activation of γ-carboxyglutamate (Gla)-proteins including the vascular calcification inhibitor matrix Gla-protein (MGP). Insufficient vitamin K intake leads to production of uncarboxylated, mostly inactive proteins and contributes to an increased cardiovascular risk. In kidney transplant recipients, cardiovascular risk is high but vitamin K intake and status have not been defined. We investigated dietary vitamin K intake, vascular vitamin K status and its determinants in kidney transplant recipients. We estimated vitamin K intake in a cohort of kidney transplant recipients (n = 60) with stable renal function (creatinine clearance 61 [42–77] (median [interquartile range]) ml/min), who were 75 [35–188] months after transplantation, using three-day food records and food frequency questionnaires. Vascular vitamin K status was assessed by measuring plasma desphospho-uncarboxylated MGP (dp-ucMGP). Total vitamin K intake was below the recommended level in 50% of patients. Lower vitamin K intake was associated with less consumption of green vegetables (33 vs 40 g/d, p = 0.06) and increased dp-ucMGP levels (621 vs 852 pmol/L, p<0.05). Accordingly, dp-ucMGP levels were elevated (>500 pmol/L) in 80% of patients. Multivariate regression identified creatinine clearance, coumarin use, body mass index, high sensitivity-CRP and sodium excretion as independent determinants of dp-ucMGP levels. In a considerable part of the kidney transplant population, vitamin K intake is too low for maximal carboxylation of vascular MGP. The high dp-ucMGP levels may result in an increased risk for arterial calcification. Whether increasing vitamin K intake may have health benefits for kidney transplant recipients should be addressed by future studies.     

Relation of Plasma Homocysteine to Plasma Amyloid Beta Levels

Background Elevated plasma homocysteine and amyloid β (Aβ) have been associated with Alzheimer’s disease (AD). We investigated the cross-sectional association between these biomarkers. Methods We used linear regression to relate plasma homocysteine and Aβ adjusting for age, gender, creatinine, APOE-ε4, and ethnic group in 327 persons aged 78 ± 6.6 years. Results Plasma homocysteine correlated with age, serum creatinine, plasma Aβ40 and Aβ42, and was inversely correlated with serum vitamin B12, and folate. Aβ42, but not Aβ40, was related to later development of dementia. Homocysteine was related to higher Aβ40 levels (coefficient = 2.0; P < 0.0001) and this association was attenuated after adjustment for creatinine (coefficient = 1.0; P < 0.0001). The crude association between homocysteine and Aβ42 was weaker (coefficient = 0.5; P = 0.01) and became non-significant after adjustment for creatinine (coefficient = 0.4; P = 0.06). These associations were unrelated to ethnicity, the presence of APOE-ε4 or dementia. Analyses by quartiles of homocysteine showed that these association were driven primarily by the fourth quartile. Conclusions Plasma homocysteine is directly related to Aβ40. The association with Aβ42 is not significant. These results seem to indicate that homocysteine is related to aging but not specifically to AD. Special issue dedicated to John P. Blass.     

Evaluation of Exome Sequencing to Estimate Tumor Burden in Plasma

Accurate estimation of systemic tumor load from the blood of cancer patients has enormous potential. One avenue is to measure the presence of cell-free circulating tumor DNA in plasma. Various approaches have been investigated, predominantly covering hotspot mutations or customized, patient-specific assays. Therefore, we investigated the utility of using exome sequencing to monitor circulating tumor DNA levels through the detection of single nucleotide variants in plasma. Two technologies, claiming to offer efficient library preparation from nanogram levels of DNA, were evaluated. This allowed us to estimate the proportion of starting molecules measurable by sequence capture (<5%). As cell-free DNA is highly fragmented, we designed and provide software for efficient identification of PCR duplicates in single-end libraries with a varying size distribution. On average, this improved sequence coverage by 38% in comparison to standard tools. By exploiting the redundant information in PCR-duplicates the background noise was reduced to ∼1/35000. By applying our optimized analysis pipeline to a simulation analysis, we determined the current sensitivity limit to ∼1/2400, starting with 30 ng of cell-free DNA. Subsequently, circulating tumor DNA levels were assessed in seven breast- and one prostate cancer patient. One patient carried detectable levels of circulating tumor DNA, as verified by break-point specific PCR. These results demonstrate exome sequencing on cell-free DNA to be a powerful tool for disease monitoring of metastatic cancers. To enable a broad implementation in the diagnostic settings, the efficiency limitations of sequence capture and the inherent noise levels of the Illumina sequencing technology must be further improved.     

PIMM as an Efficient Tool to Estimate Gas Phase Formation Enthalpies of Energetic Molecules

Some new approaches to computer-aided design of new energetic materials require highly efficient techniques to estimate relevant properties. To date, evaluation of the formation enthalpies makes a significant contribution to the computational cost when calculated with standard quantum mechanical techniques. In a quest for a more efficient procedure, the PIMM model is evaluated, as implemented in the 1991 version. According to present results, this method, while being roughly one order of magnitude more efficient than MNDO schemes for large molecules, is comparable to PM3 and far superior to AM1 with regard to the accuracy of calculated gas-phase enthalpies of formation.     

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice

Background

Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia.

Methods

Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression.

Results

A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups.

Conclusions

These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.     

A Specific Response to Toxic Cadmium Levels in Red Kidney Bean Embryos

Cadmium salts are highly toxic to excised embryos of Phaseolus vulgaris cv. Red Kidney, inhibiting growth at concentrations in excess of 3 mg/L Inhibition is accompanied by specific toxic responses, namely inhibition of greening and induction of acute curvature in the embryonic axis. Comparisons among some 24 elements at 10 mg/l for cadmium-like effects showed silver to affect curvature, but not greening, while cobalt and nickel were the converse.     

Altered Levels of Acetylcholinesterase in Alzheimer Plasma

Background

Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal Findings

Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were ∼20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G₁+G₂ forms and not G₄ tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer''s disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G₁+G₂ forms, the subset of AChE species which are increased in Alzheimer''s brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer''s plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion

Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.     

Thrombin-Anti-Thrombin Levels and Patency of Arterio-Venous Fistula in Patients Undergoing Haemodialysis Compared to Healthy Volunteers: A Prospective Analysis

Background

Patients on haemodialysis (HD) are at an increased risk of sustaining thrombotic events especially to their vascular access which is essential for maintenance of HD.

Objectives

To assess whether 1) markers of coagulation, fibrinolysis or endothelial activation are increased in patients on HD compared to controls and 2) if measurement of any of these factors could help to identify patients at increased risk of arteriovenous (AVF) access occlusion.

Patients/Methods

Venous blood samples were taken from 70 patients immediately before a session of HD and from 78 resting healthy volunteers. Thrombin-antithrombin (TAT), D-dimer, von Willebrand factor (vWF), plasminogen activator inhibitor-1 antigen (PAI-1) and soluble p-selectin were measured by ELISA. C-reactive protein (hsCRP) was measured by an immunonephelometric kinetic assay. Determination of the patency of the AVF was based upon international standards and was prospectively followed up for a minimum of four years or until the AVF was non-functioning.

Results

A total of 70 patients were studied with a median follow-up of 740 days (range 72-1788 days). TAT, D-dimer, vWF, p-selectin and hsCRP were elevated in patients on HD compared with controls. At one year follow-up, primary patency was 66% (46 patients). In multivariate analysis TAT was inversely associated with primary assisted patency (r= -0.250, p= 0.044) and secondary patency (r = -0.267, p= 0.031).

Conclusions

The novel finding of this study is that in patients on haemodialysis, TAT levels were increased and inversely correlated with primary assisted patency and secondary patency. Further evaluation is required into the possible role of TAT as a biomarker of AVF occlusion.     

冠心病患者血浆同型半胱氨酸和内皮功能的研究(英文)

目的:探讨冠心病患者血浆中同型半胱氨酸、内皮素、循环内皮细胞水平与冠状动脉粥样硬化性心脏病发生过程的关系。方法:对62例冠心病患者组和50例健康对照组分别采用高效液相色谱分析法和放射免疫分析法测定血浆中同型半胱氨酸水平和内皮素水平,同时测定血浆中循环内皮细胞的数量。结果:冠心病组同型半胱氨酸(21.27±5.73)μmol/L、内皮素(84.30±13.68)ng/L、循环内皮细胞(12.08±5.85)和cells/0.9μl明显高于对照组同型半胱氨酸(9.12±4.87)μmol/L、内皮素(47.65±12.71)ng/L、循环内皮细胞(2.35±1.02)cells/0.9μl,P均小于0.001。同型半胱氨酸与内皮素、循环内皮细胞呈正相关(r=0.601,P0.01;r=0.645,P0.01),且冠心病组血浆循环内皮细胞和内皮素呈正相关(r=0.850,P0.01)。结论:同型半胱氨酸及内皮素对冠状动脉粥样硬化性心脏病的发生发展有促进作用,对其检测有临床实用价值。     

Validation and Assessment of Three Methods to Estimate 24-h Urinary Sodium Excretion from Spot Urine Samples in Chinese Adults

24-h urinary sodium excretion is the gold standard for evaluating dietary sodium intake, but it is often not feasible in large epidemiological studies due to high participant burden and cost. Three methods—Kawasaki, INTERSALT, and Tanaka—have been proposed to estimate 24-h urinary sodium excretion from a spot urine sample, but these methods have not been validated in the general Chinese population. This aim of this study was to assess the validity of three methods for estimating 24-h urinary sodium excretion using spot urine samples against measured 24-h urinary sodium excretion in a Chinese sample population. Data are from a substudy of the Prospective Urban Rural Epidemiology (PURE) study that enrolled 120 participants aged 35 to 70 years and collected their morning fasting urine and 24-h urine specimens. Bias calculations (estimated values minus measured values) and Bland-Altman plots were used to assess the validity of the three estimation methods. 116 participants were included in the final analysis. Mean bias for the Kawasaki method was -740 mg/day (95% CI: -1219, 262 mg/day), and was the lowest among the three methods. Mean bias for the Tanaka method was -2305 mg/day (95% CI: -2735, 1875 mg/day). Mean bias for the INTERSALT method was -2797 mg/day (95% CI: -3245, 2349 mg/day), and was the highest of the three methods. Bland-Altman plots indicated that all three methods underestimated 24-h urinary sodium excretion. The Kawasaki, INTERSALT and Tanaka methods for estimation of 24-h urinary sodium excretion using spot urines all underestimated true 24-h urinary sodium excretion in this sample of Chinese adults. Among the three methods, the Kawasaki method was least biased, but was still relatively inaccurate. A more accurate method is needed to estimate the 24-h urinary sodium excretion from spot urine for assessment of dietary sodium intake in China.