Evaluation of plasma microRNA-122, high-mobility group box 1 and keratin-18 concentrations to stratify acute gallstone disease: a pilot observational cohort study in an emergency general surgery unit.

OBJECTIVE: To obtain pilot data to evaluate the discriminatory power of biomarkers microRNA-122 (miR-122), high-mobility group box 1 (HMGB1), full-length keratin-18 (flk-18) and caspase-cleaved keratin-18 (cck-18) in plasma to identify potential biliary complications that may require acute intervention. DESIGN: An observational biomarker cohort pilot study. SETTING: In a Scottish University teaching hospital for 12 months beginning on 3 September 2014. PARTICIPANTS: Blood samples were collected from adults (≥16 years old) referred with acute biliary-type symptoms who have presented to hospital within 24 hours prior were recruited. Patients unable or refused to give informed consent or were transferred from a hospital outside the National Health Service regional trust were excluded. PRIMARY OUTCOME MEASURES: To evaluate whether circulating miR-122, HMGB1, flk-18 and cck-18 can discriminate between people with and without gallstone disease and uncomplicated from complicated gallstone disease during the first 24 hours of hospital admission. RESULTS: 300 patients were screened of which 285 patients were included. Plasma miR-122, cck-18 and flk-18 concentrations were increased in patients with gallstones compared with those without (miR-122: median: 2.89×104 copies/mL vs 0.90×104 copies/mL (p<0.001); cck-18: 121.2 U/L vs 103.5 U/L (p=0.031); flk-18: 252.4 U/L vs 145.1 U/L (p<0.001)). Uncomplicated gallstone disease was associated with higher miR-122 and cck-18 concentrations than complicated disease (miR-122: 5.72×104 copies/mL vs 2.26×104 copies/mL (p=0.023); cck-18: 139.7 U/L vs 113.6 U/L (p=0.047)). There was no significant difference in HMGB1 concentration between patients with and without gallstones (p=0.559). Separation between groups for all biomarkers was modest. CONCLUSION: miR-122 and keratin-18 plasma concentrations are elevated in patients with gallstones. However, this result is confounded by the association between biomarker concentrations, age and gender. In this pilot study, miR-122 and keratin-18 were not sufficiently discriminatory to be progressed as clinically useful biomarkers in this context.

Urinary Biomarkers of Aminoglycoside-Induced Nephrotoxicity in Cystic Fibrosis: Kidney Injury Molecule-1 and Neutrophil Gelatinase-Associated Lipocalin.

Aminoglycosides are commonly used for the treatment of pulmonary exacerbations in patients with cystic fibrosis (CF). However, they are potentially nephrotoxic. This prospective observational cohort study aimed to investigate the potential validity of two urinary renal biomarkers, Kidney Injury Molecule-1 (KIM-1) and Neutrophil Gelatinase-associated Lipocalin (NGAL), in identifying aminoglycoside-induced nephrotoxicity in children with CF. Children and young adults up to 20 years of age with a confirmed diagnosis of CF were recruited from ten United Kingdom hospitals. Participants provided urine samples for measurement of KIM-1 and NGAL concentrations, at baseline, at regular outpatient appointments, and before, during and after exposure to clinically-indicated treatment with the aminoglycoside tobramycin. 37/158 patients recruited (23.4%) received at least one course of IV tobramycin during the study. The median peak fold-change during tobramycin exposure for KIM-1 was 2.28 (IQR 2.69) and 4.02 (IQR 7.29) for NGAL, in the absence of serum creatinine changes. Baseline KIM-1 was positively associated with cumulative courses of IV aminoglycosides (R2 = 0.11; ß = 0.03; p < 0.0001). KIM-1, in particular, may be a useful, non-invasive, biomarker of acute and chronic proximal tubular injury associated with exposure to aminoglycosides in patients with CF, but its clinical utility needs to be further evaluated in prospective studies.

HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis.

Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration, and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a nonautonomous fashion, consistent with its role as a DAMP. Liver-specific HMGB1 deficiency reduced HCC development in 3 mouse models of chronic injury but not in a model lacking chronic liver injury. As with CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature, and hepatocarcinogenesis in CLD.

HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

Risk stratification after paracetamol overdose using mechanistic biomarkers: results from two prospective cohort studies.

BACKGROUND: Paracetamol overdose is common but patient stratification is suboptimal. We investigated the usefulness of new biomarkers that have either enhanced liver specificity (microRNA-122 [miR-122]) or provide mechanistic insights (keratin-18 [K18], high mobility group box-1 [HMGB1], and glutamate dehydrogenase [GLDH]). The use of these biomarkers could help stratify patients for their risk of liver injury at hospital presentation. METHODS: Using data from two prospective cohort studies, we assessed the potential for biomarkers to stratify patients who overdose with paracetamol. We completed two independent prospective studies: a derivation study (MAPP) in eight UK hospitals and a validation study (BIOPAR) in ten UK hospitals. Patients in both cohorts were adults (≥18 years in England, ≥16 years in Scotland), were diagnosed with paracetamol overdose, and gave written informed consent. Patients who needed intravenous acetylcysteine treatment for paracetamol overdose had circulating biomarkers measured at hospital presentation. The primary endpoint was acute liver injury indicating need for continued acetylcysteine treatment beyond the standard course (alanine aminotransferase [ALT] activity >100 U/L). Receiver operating characteristic (ROC) curves, category-free net reclassification index (cfNRI), and integrated discrimination index (IDI) were applied to assess endpoint prediction. FINDINGS: Between June 2, 2010, and May 29, 2014, 1187 patients who required acetylcysteine treatment for paracetamol overdose were recruited (985 in the MAPP cohort; 202 in the BIOPAR cohort). In the derivation and validation cohorts, acute liver injury was predicted at hospital presentation by miR-122 (derivation cohort ROC-area under the curve [AUC] 0·97 [95% CI 0·95-0·98]), HMGB1 (0·95 [0·93-0·98]), and full-length K18 (0·95 [0·92-0·97]). Results were similar in the validation cohort (miR-122 AUC 0·97 [95% CI 0·95-0·99], HMGB1 0·98 [0·96-0·99], and full-length K18 0·93 [0·86-0·99]). A combined model of miR-122, HMGB1, and K18 predicted acute liver injury better than ALT alone (cfNRI 1·95 [95% CI 1·87-2·03], p<0·0001 in the MAPP cohort; 1·54 [1·08-2·00], p<0·0001 in the BIOPAR cohort). INTERPRETATION: Personalised treatment pathways could be developed by use of miR-122, HMGB1, and full-length K18 at hospital presentation for patient stratification. This prospective study supports their use for hepatic safety assessment of new medicines. FUNDING: Edinburgh and Lothians Health Foundation, UK Medical Research Council.

Dynamic and accurate assessment of acetaminophen-induced hepatotoxicity by integrated photoacoustic imaging and mechanistic biomarkers in vivo.

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

A Short-Term Biological Indicator for Long-Term Kidney Damage after Radionuclide Therapy in Mice.

Folate receptor (FR)-targeted radionuclide therapy using folate radioconjugates is of interest due to the expression of the FR in a variety of tumor types. The high renal accumulation of radiofolates presents, however, a risk of radionephropathy. A potential option to address this challenge would be to use radioprotectants, such as amifostine. Methods for early detection of kidney damage that-in this case-cannot be predicted based on dose estimations, would facilitate the development of novel therapies. The aim of this study was, therefore, to assess potentially changing levels of plasma and urine biomarkers and to determine DNA damage at an early stage after radiofolate application. The identification of an early indicator for renal damage in mice would be useful since histological changes become apparent only several months after treatment. Mice were injected with different quantities of 177Lu-folate (10 MBq, 20 MBq and 30 MBq), resulting in mean absorbed kidney doses of ~23 Gy, ~46 Gy and ~69 Gy, respectively, followed by euthanasia two weeks (>85% of the mean renal radiation dose absorbed) or three months later. Whereas all investigated biomarkers remained unchanged, the number of γ-H2AX-positive nuclei in the renal cortex showed an evident dose-dependent increase as compared to control values two weeks after treatment. Comparison with the extent of kidney injury determined by histological changes five to eight months after administration of the same 177Lu-folate activities suggested that the quantitative assessment of double-strand breaks can be used as a biological indicator for long-term radiation effects in the kidneys. This method may, thus, enable faster assessment of radiopharmaceuticals and protective measures by preventing logistically challenging long-term investigations to detect kidney damage.

Characterization of Drug-Specific Signaling Between Primary Human Hepatocytes and Immune Cells.

It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO). Hepatocyte toxicity, cytokine release and activation of oxidative stress pathways were measured. Supernatants were transferred to monocyte-derived dendritic cells and cell phenotype and function were assessed. High-mobility group box 1 protein (HMGB1) and lactate dehydrogenase release as well as adenosine triphosphate depletion occurred in a drug-, time-, and concentration-dependent manner with SMX-NO and flucloxacillin, whereas isoniazid and amoxicillin were nontoxic. Furthermore, drug-induced activation of nuclear factor (erythroid-derived 2)-like 2 marker genes was observed when hepatocytes were exposed to test drugs. The disulfide isoform of HMGB1 stimulated dendritic cell cytokine release and enhanced the priming of naive T-cells. Incubation of dendritic cells with supernatant from drug-treated hepatocytes resulted in 2 distinct cytokine profiles. SMX-NO/flucloxacillin stimulated secretion of TNF-α, IL-6, IL-1α, and IL-1-ß. Isoniazid which did not induce significant hepatocyte toxicity, compared with SMX-NO and flucloxacillin, stimulated the release of a panel of cytokines including the above and IFN-γ, IL-12, IL-17A, IP-10, and IL-10. Collectively, our study identifies drug-specific signaling pathways between hepatocytes and immune cells that could influence whether drug exposure will result in an immune response and tissue injury.

Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.

OBJECTIVE: Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression. DESIGN AND RESULTS: Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl4-injected OpnHep transgenic yet it was less in wild type and almost absent in Opn-/- mice. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) protected mice from CCl4-induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K-pAkt1/2/3 pathway to upregulate collagen-I. CONCLUSIONS: During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.

Redox-dependent regulation of hepatocyte absent in melanoma 2 inflammasome activation in sterile liver injury in mice.

Sterile liver inflammation, such as liver ischemia-reperfusion, hemorrhagic shock after trauma, and drug-induced liver injury, is initiated and regulated by endogenous mediators including DNA and reactive oxygen species. Here, we identify a mechanism for redox-mediated regulation of absent in melanoma 2 (AIM2) inflammasome activation in hepatocytes after redox stress in mice, which occurs through interaction with cytosolic high mobility group box 1 (HMGB1). We show that in liver during hemorrhagic shock in mice and in hepatocytes after hypoxia with reoxygenation, cytosolic HMGB1 associates with AIM2 and is required for activation of caspase-1 in response to cytosolic DNA. Activation of caspase-1 through AIM2 leads to subsequent hepatoprotective responses such as autophagy. HMGB1 binds to AIM2 at a non-DNA-binding site on the hematopoietic interferon-inducible nuclear antigen domain of AIM2 to facilitate inflammasome and caspase-1 activation in hepatocytes. Furthermore, binding of HMGB1 to AIM2 is stronger with fully reduced all-thiol HMGB1 than with partially oxidized disulfide-HMGB1, and binding strength corresponds to caspase-1 activation. These data suggest that HMGB1 redox status regulates AIM2 inflammasome activation. CONCLUSION: These findings suggest a novel and important mechanism for regulation of AIM2 inflammasome activation in hepatocytes during redox stress and may suggest broader implications for how this and other inflammasomes are activated and how their activation is regulated during cell stress, as well as the mechanisms of inflammasome regulation in nonimmune cell types. (Hepatology 2017;65:253-268).

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice.

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP-induced acute liver injury (APAP-ALI) and justifies development of anti-inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N-acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP-ALI. The anti-HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti-HMGB1 polyclonal antibody treatment improves survival in a model of APAP-ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti-HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP-ALI. The mouse anti-HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody-Fc backbones. Effector function-deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP-ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP-induced serum elevations of alanine aminotransferase and microRNA-122 and completely abrogated markers of APAP-induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C-X-C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. CONCLUSION: This is the first report describing the generation of a partly humanized HMGB1-neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP-ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1-specific therapy as a means to treat APAP-ALI and other inflammatory conditions. (Hepatology 2016;64:1699-1710).

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

BACKGROUND AND AIMS: Colorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss. RESULTS: Elevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated. METHODS: Hmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice. CONCLUSIONS: HMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

Detection of Drug-Induced Acute Kidney Injury in Humans Using Urinary KIM-1, miR-21, -200c, and -423.

Drug-induced acute kidney injury (AKI) is often encountered in hospitalized patients. Although serum creatinine (SCr) is still routinely used for assessing AKI, it is known to be insensitive and nonspecific. Therefore, our objective was to evaluate kidney injury molecule 1 (KIM-1) in conjunction with microRNA (miR)-21, -200c, and -423 as urinary biomarkers for drug-induced AKI in humans. In a cross-sectional cohort of patients (n = 135) with acetaminophen (APAP) overdose, all 4 biomarkers were significantly (P < .004) higher not only in APAP-overdosed (OD) patients with AKI (based on SCr increase) but also in APAP-OD patients without clinical diagnosis of AKI compared with healthy volunteers. In a longitudinal cohort of patients with malignant mesothelioma receiving intraoperative cisplatin (Cp) therapy (n = 108) the 4 biomarkers increased significantly (P < .0014) over time after Cp administration, but could not be used to distinguish patients with or without AKI. Evidence for human proximal tubular epithelial cells (HPTECs) being the source of miRNAs in urine was obtained first, by in situ hybridization based confirmation of increase in miR-21 expression in the kidney sections of AKI patients and second, by increased levels of miR-21, -200c, and -423 in the medium of cultured HPTECs treated with Cp and 4-aminophenol (APAP degradation product). Target prediction analysis revealed 1102 mRNA targets of miR-21, -200c, and -423 that are associated with pathways perturbed in diverse pathological kidney conditions. In summary, we report noninvasive detection of AKI in humans by combining the sensitivity of KIM-1 along with mechanistic potentials of miR-21, -200c, and -423.