Kidney damage biomarkers detect acute kidney injury but only functional markers predict mortality after paraquat ingestion

Acute kidney injury (AKI) is common following paraquat ingestion. The diagnostic performance of injury biomarkers was investigated in serial blood and urine samples from patients from 5 Sri Lankan hospitals. Functional AKI was diagnosed using serum creatinine (sCr) or serum cystatin C (sCysC). The 95th centile in healthy subjects defined the urinary biomarker cutoffs for diagnosing structural AKI. 50 poisoned patients provided 2 or more specimens, 76% developed functional AKI [AKIN stage 1 (n=12), 2 (n=7) or 3 (n=19)]; 19/26 patients with AKIN stage 2/3 also had functional AKI by sCysC criteria (≥50% increase). Urinary cystatin C (uCysC), clusterin (uClu) and NGAL (uNGAL) increased within 24h of ingestion compared with NoAKI patients and healthy controls. Each biomarker demonstrated moderate diagnostic utility [AUC-ROC: uCysC 0.79, uNGAL 0.79, uClu 0.68] for diagnosis of functional AKI at 16h. Death occurred only in subjects with functional AKI. Structural biomarker-based definitions detected more AKI than did sCr or sCysC, but did not independently predict death. Renal injury biomarkers did not add clinical value to patients who died rapidly due to multi-organ failure. Use of injury biomarkers within 16-24h may guide early intervention for reno-protection in less severe paraquat poisoning.NHMRC project grant 101177

Urinary microRNAs as non-invasive biomarkers for toxic acute kidney injury in humans

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell’s viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell’s viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p  0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers

MicroRNAs in Human Nephrotoxicity

Acute kidney injury (AKI) is common following poisoning and envenoming. A major challenge in the management of AKI is the modest sensitivity of current biomarkers for early diagnosis. Better biomarkers of toxic AKI may help in earlier diagnosis and allow early and appropriate treatment. MicroRNAs are a class of RNAs which have recently come into prominence as promising diagnostic and prognostic biomarkers. They are present in biological fluids and these circulating microRNAs have been attracting considerable attention as novel biomarkers for organ injury. This thesis will explore the potential of microRNA biomarkers for early diagnosis of nephrotoxicity and determine their ability to predict and quantify organ damage, and provide insights into mechanisms of nephrotoxicity in Russell’s viper envenoming, oxalic acid, glyphosate and paraquat poisonings. This thesis identified a lack of human studies on the most important causes of nephrotoxic injury, such as pesticides, chemicals, snake envenomation, and medicines other than aminoglycosides and cisplatin. A high sensitivity discovery platform was used to identify signature microRNAs to distinguish patients with AKI and without AKI following individual poisons, i.e. oxalic acid, glyphosate and paraquat and Russell’s viper envenoming. These were validated in a larger cohort. Urinary microRNA signatures provided a stronger signal for AKI in oxalic acid poisoning compared to serum microRNA. Kidney injury had the greatest impact on urinary microRNA while poisoning itself was better reflected in serum microRNA. This result led to the analysis of urinary microRNAs in four different causes of toxic-AKI. Several microRNAs including miR-30a-5p, miR-30b, miR-191 and miR-204, were promising biomarkers for the early detection of toxic AKI. The target mRNAs of these microRNA are largely involved in renal cell death/necrosis, oxidative stress, apoptosis and mitochondrial dysfunction. These urinary microRNAs are potential alternatives to traditional urinary proteins or serum creatinine markers. This thesis demonstrated microRNA hold great promise as AKI diagnostic markers

A modified low-cost colorimetric method for paracetamol (acetaminophen) measurement in plasma

Background: Despite a significant increase in the number of patients with paracetamol poisoning in the developing world, plasma paracetamol assays are not widely available. The purpose of this study was to assess a low-cost modified colorimetric paracetamol assay that has the potential to be performed in small laboratories with restricted resources. Methods: The paracetamol assay used in this study was based on the Glynn and Kendal colorimetric method with a few modifications to decrease the production of nitrous gas and thereby reduce infrastructure costs. Preliminary validation studies were performed using spiked aqueous samples with known concentrations of paracetamol. Subsequently, the results from the colorimetric method for 114 stored clinical samples from patients with paracetamol poisoning were compared with those from the current gold-standard high-performance liquid chromatography method. A prospective survey, assessing the clinical use of the paracetamol assay, was performed on all patients with paracetamol poisoning attending the Peradeniya General Hospital, Sri Lanka, over a 10-month period. Results: The recovery study showed an excellent correlation (r² > 0.998) for paracetamol concentrations from 25 to 400 mg/L. The final yellow color was stable for at least 10 min at room temperature. There was also excellent correlation with the high-performance liquid chromatography method (r² = 0.9758). In the clinical cohort study, use of the antidote N-acetylcysteine was avoided in over a third of patients who had the plasma paracetamol concentration measured. The cost of consumables used per assay was $0.50 (US). Conclusions: This colorimetric paracetamol assay is reliable and accurate and can be performed rapidly, easily, and economically. Use of this assay in resource-poor clinical settings has the potential to have a significant clinical and economic impact on the management of paracetamol poisoning

Method of stabilizing blood for the determination of methemoglobin

Methemoglobin (MetHb) is a significant clinical problem for some poisonings. Its measurement is a problem as both formation and reduction of MetHb can occur even after sampling with time. The objective of this study was to discover a method to stabilize the blood samples for the determination of MetHb. First, hemolysates were prepared by diluting the MetHb blood samples with phosphate buffers under different pH values. The samples were stored at 4–8°C and a day-to-day variability in the amount of MetHb was determined using the method described by Evelyn and Malloy. The results show that there is a significantchange in the amount of MetHb stored in both KH₂PO₄/Na₂HPO₄ and KH₂PO₄/Na₂HPO₄.2H₂O buffer solutions at pH of 6.7 and 6.9. Buffer solution containing phosphate composition of KH₂PO₄/Na₂HPO₄·2H₂O (pH=7.0) gives relatively stable values for MetHb during the storage and the amount of MetHb samples in the buffer solution retain constant up to 9 days. Therefore, stabilized MetHb blood samples can be prepared using KH₂PO₄/Na₂HPO₄·2H₂O buffer solution (pH=7) with non-ionic detergent and the samples can be stored for several days at 4–8°C

MicroRNAs in toxic acute kidney injury: Systematic scoping review of the current status

Abstract Acute kidney injury induced by nephrotoxic agents is common, increasing in incidence and associated with considerable morbidity and mortality in developing countries. MicroRNAs are stable biomarkers that can be detected in extracellular fluids. This systematic scoping review aims to describe published research on urinary and circulating microRNAs in toxic acute kidney injury in both animal and human studies. We conducted a literature search, using EMBASE and Medline, for articles on urinary and circulating microRNA in nephrotoxic injuries to February 2020. A total of 21 publications studied acute kidney injury from 12 different toxic agents. Cisplatin was the most common nephrotoxic agent (n = 10), followed by antibiotics (n = 4). There were no randomized controlled trials. An increase in urinary miR‐218 predicted acute kidney injury in six different studies, suggesting it is a promising biomarker for nephrotoxin‐induced acute kidney injury. There were many factors that prevented a more comprehensive synthesis of microRNA performance including highly variable models, no consistent protocols for RNA isolation, cDNA synthesis and PCR amplification, and variability in normalization methods using reference controls. In conclusion, while microRNAs are promising biomarkers to study nephrotoxic acute kidney injury, the replication of most positive findings is not assessable due to deficient reporting of negative outcomes. A very narrow range of poisons have been studied, and more human data are required. In particular, further studies are needed on the most important causes of nephrotoxic injury, such as pesticides, chemicals, snake envenoming, and medicines other than aminoglycosides and cisplatin