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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Objectives To determine the prognostic potential of Sig24 for identifying prostate cancer patients at risk of developing metastases or experiencing PCSM following radical prostatectomy. Subjects and methods Sig24 scores were calculated from previously collected gene expression microarray data from the Cleveland Clinic and Mayo Clinic (I and II). The performance of Sig24 was determined using time-dependent c-index analysis, Cox proportional hazards regression and Kaplan-Meier survival analysis. Results Higher Sig24 scores were significantly associated with higher pathologic Gleason scores (GS) in all three cohorts. Analysis of the Mayo Clinic II cohort, which included time to event information, indicated that patients with high Sig24 scores also had an increased risk of developing metastasis (HR: 3.78, 95% CI: 1.96-7.29, p < 0.001) or experiencing PCSM (HR: 6.54, 95% CI: 2.16-19.83, p < 0.001). Conclusions The findings of this study demonstrate the applicability of Sig24 for the prognosis of metastasis or PCSM following radical prostatectomy. Future studies investigating the combination of Sig24 with available prognostic tests may provide new approaches to improve risk stratification for patients with prostate cancer. This article is protected by copyright. All rights reserved.

Purpose Liquid biopsies that noninvasively detect molecular correlates of aggressive prostate cancer (PCa) could be used to triage patients, reducing the burdens of unnecessary invasive prostate biopsy and enabling early detection of high-risk disease. DNA hypermethylation is among the earliest and most frequent aberrations in PCa. We investigated the accuracy of a six-gene DNA methylation panel (Epigenetic Cancer of the Prostate Test in Urine [epiCaPture]) at detecting PCa, high-grade (Gleason score greater than or equal to 8) and high-risk (D’Amico and Cancer of the Prostate Risk Assessment] PCa from urine. Patients and Methods Prognostic utility of epiCaPture genes was first validated in two independent prostate tissue cohorts. epiCaPture was assessed in a multicenter prospective study of 463 men undergoing prostate biopsy. epiCaPture was performed by quantitative methylation-specific polymerase chain reaction in DNA isolated from prebiopsy urine sediments and evaluated by receiver operating characteristic and decision curves (clinical benefit). The epiCaPture score was developed and validated on a two thirds training set to one third test set. Results Higher methylation of epiCaPture genes was significantly associated with increasing aggressiveness in PCa tissues. In urine, area under the receiver operating characteristic curve was 0.64, 0.86, and 0.83 for detecting PCa, high-grade PCa, and high-risk PCa, respectively. Decision curves revealed a net benefit across relevant threshold probabilities. Independent analysis of two epiCaPture genes in the same clinical cohort provided analytical validation. Parallel epiCaPture analysis in urine and matched biopsy cores showed added value of a liquid biopsy. Conclusion epiCaPture is a urine DNA methylation test for high-risk PCa. Its tumor specificity out-performs that of prostate-specific antigen (greater than 3 ng/mL). Used as an adjunct to prostate-specific antigen, epiCaPture could aid patient stratification to determine need for biopsy.

The COVID-19 pandemic has led to extensive morbidity and mortality throughout the world. Clinical features that drive SARS-CoV-2 pathogenesis in humans include inflammation and thrombosis, but the mechanistic details underlying these processes remain to be determined. In this study, we demonstrate endothelial disruption and vascular thrombosis in histopathologic sections of lungs from both humans and rhesus macaques infected with SARS-CoV-2. To define key molecular pathways associated with SARS-CoV-2 pathogenesis in macaques, we performed transcriptomic analyses of bronchoalveolar lavage and peripheral blood and proteomic analyses of serum. We observed macrophage infiltrates in lung and upregulation of macrophage, complement, platelet activation, thrombosis, and proinflammatory markers, including C-reactive protein, MX1, IL-6, IL-1, IL-8, TNFα, and NF-κB. These results suggest a model in which critical interactions between inflammatory and thrombosis pathways lead to SARS-CoV-2-induced vascular disease. Our findings suggest potential therapeutic targets for COVID-19., Graphical Abstract, Highlights • SARS-CoV-2 infection leads to macrophage infiltrates in the lung of infected macaques • SARS-CoV-2 upregulates proinflammatory cytokines and ISGs in macaques • SARS-CoV-2 upregulates complement and coagulation cascade in macaques • SARS-CoV-2 infection leads to endothelial damage and thrombosis in macaques, Aid et al. show that SARS-CoV-2 causes endothelial disruption and vascular thrombosis in both human and rhesus macaques lungs by inducing an upregulation of proinflammatory cytokines. Using an approach that combines histopathology and multiomics in macaques, they show the progression to vascular disease over time, which involves complement, macrophage, cytokine, and thrombosis cascades.

Background: The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Methods: Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. Results: RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Conclusions: Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection.

The hormonal milieu influences immune tolerance and the immune response against viruses and cancer, but the direct effect of androgens on cellular immunity remains largely uncharacterized. We therefore sought to evaluate the effect of androgens on murine and human T cells in vivo and in vitro. We found that murine androgen deprivation in vivo elicited RNA expression patterns conducive to IFN signaling and T-cell differentiation. Interrogation of mechanism showed that testosterone regulates T-helper 1 (Th1) differentiation by inhibiting IL-12-induced Stat4 phosphorylation: in murine models, we determined that androgen receptor binds a conserved region within the phosphatase, Ptpn1, and consequent up-regulation of Ptpn1 then inhibits IL-12 signaling in CD4 T cells. The clinical relevance of this mechanism, whereby the androgen milieu modulates CD4 T-cell differentiation, was ascertained as we found that androgen deprivation reduced expression of Ptpn1 in CD4 cells from patients undergoing androgen deprivation therapy for prostate cancer. Our findings, which demonstrate a clinically relevant mechanism by which androgens inhibit Th1 differentiation of CD4 T cells, provide rationale for targeting androgens to enhance CD4-mediated immune responses in cancer or, conversely, for modulating androgens to mitigate CD4 responses in disorders of autoimmunity.

Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar:Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.

RNA-binding proteins (RBPs) are recognized as key posttranscriptional regulators that not only modulate the spatiotemporal expression of genes during organism development but also regulate disease pathogenesis. Very limited information exists on the potential role of RBPs in modulating kidney fibrosis, which is a major hallmark of chronic kidney disease. Here, we report a novel mechanism in kidney fibrosis involving a RBP, Musashi homologue 1 (Msi1), which is expressed in tubular epithelial cells. Using two mechanistically distinct mouse models of kidney fibrosis, we show that Msi1 protein levels are significantly down-regulated in the kidneys following fibrosis. We found that Msi1 functions by negatively regulating the translation of its target mRNAs, p21 and Numb, whose protein levels are markedly increased in kidney fibrosis. Also, Msi1 overexpression and knockdown in kidney epithelial cells cause p21- and Numb-mediated cell cycle arrest. Furthermore, we observed that Numb looses its characteristic membrane localization in fibrotic kidneys and therefore is likely unable to inhibit Notch resulting in tubular cell death. Oleic acid is a known inhibitor of Msi1 and injecting oleic acid followed by unilateral ureteral obstruction surgery in mice resulted in enhanced fibrosis compared with the control group, indicating that inhibiting Msi1 activity renders the mice more susceptible to fibrosis. Given that deregulated fatty acid metabolism plays a key role in kidney fibrosis, these results demonstrate a novel connection between fatty acid and Msi1, an RNA-binding protein, in kidney fibrosis.

Background: Patients with locally advanced or recurrent prostate cancer typically undergo androgen deprivation therapy (ADT), but the benefits are often short-lived and the responses variable. ADT failure results in castration-resistant prostate cancer (CRPC), which inevitably leads to metastasis. We hypothesized that differences in tumor transcriptional programs may reflect differential responses to ADT and subsequent metastasis. Results: We performed whole transcriptome analysis of 20 patient-matched Pre-ADT biopsies and 20 Post-ADT prostatectomy specimens, and identified two subgroups of patients (high impact and low impact groups) that exhibited distinct transcriptional changes in response to ADT. We found that all patients lost the AR-dependent subtype (PCS2) transcriptional signatures. The high impact group maintained the more aggressive subtype (PCS1) signal, while the low impact group more resembled an AR-suppressed (PCS3) subtype. Computational analyses identified transcription factor coordinated groups (TFCGs) enriched in the high impact group network. Leveraging a large public dataset of over 800 metastatic and primary samples, we identified 33 TFCGs in common between the high impact group and metastatic lesions, including SOX4/FOXA2/GATA4, and a TFCG containing JUN, JUNB, JUND, FOS, FOSB, and FOSL1. The majority of metastatic TFCGs were subsets of larger TFCGs in the high impact group network, suggesting a refinement of critical TFCGs in prostate cancer progression. Conclusions: We have identified TFCGs associated with pronounced initial transcriptional response to ADT, aggressive signatures, and metastasis. Our findings suggest multiple new hypotheses that could lead to novel combination therapies to prevent the development of CRPC following ADT.

Patients with recurrent, aggressive prostate cancer typically undergo androgen-deprivation therapy (ADT), but the benefits are often short-lived, and responses are variable. Failure to respond to ADT invariably leads to metastatic disease, and ultimately death. To investigate differential responses to ADT, we performed whole-transcriptome analysis of 20 patient-matched pre-ADT biopsies and post-ADT prostatectomy specimens, and observed that all patients lost transcriptional signatures indicative of the androgen receptor (AR)-dependent subtype, after treatment. We also identified two subgroups of patients with either a strong or weak transcriptional response to ADT. The strong responders maintained the more aggressive subtype signal, while the weak responders lost expression of these genes and more resembled an AR-suppressed, basal subtype. We generated a strong responder transcriptional network using the PANDA program and integrated expression data from our cohort, protein-protein interaction, and DNA binding motif data. We also leveraged the expression data from a large public dataset of over 800 metastatic and primary samples to construct a metastatic lesion transcriptional network. We identified 20 common transcription factor coordinated groups (TFCGs) associated with both the strong responders and metastatic lesions, including GLI3/GLI2, SOX4/FOXA2/GATA4, ERF/ETV5/ETV3/ELF4, and a TFCG containing JUN, JUNB, JUND, FOS, FOSB, and FOSL1. Many TFCGs in the metastatic network were subsets of larger groups in the strong responders network, implicating these transcription factor associations as potentially critical for both the differential ADT response and metastatic disease progression. Citation Format: Nitya V. Sharma, Kathryn L. Pellegrini, Veronique Ouellet, Felipe O. Giuste, Selvi Ramalingam, Kenneth Watanabe, Eloise Adam-Granger, Lucresse Fossouo, Sungyong You, Michael R. Freeman, Paula Vertino, Karen Conneely, Adeboye O. Osunkoya, Dominique Trudel, Anne-Marie Mes-Masson, John A. Petros, Fred Saad, Carlos S. Moreno. Transcription factor relationships associated with androgen-deprivation therapy response and metastatic progression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2269.

Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250 mg/kg i.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (>2-fold, p

A large number of men are diagnosed with prostate cancer each year, but many will not experience morbidity or mortality as a result of their cancers. Therefore, biomarkers for prostate cancer are necessary to carefully select patients for initial diagnostic biopsy or to facilitate care decisions for men who have already been diagnosed with prostate cancer. RNA-based approaches to biomarker discovery allow the investigation of non-coding RNAs, gene fusion transcripts, splice variants, and multi-gene expression panels in tissue, urine, or blood as opportunities to improve care decisions. This review focuses on RNA biomarkers that are available as commercial assays, and therefore already available for potential clinical use, as well as providing an overview of newer RNA biomarkers that are in earlier stages of clinical development.

Background. Myoblast transplantation has long been studied as a potential therapy for Duchenne muscular dystrophy as the incorporation of donor myoblasts into host muscle allows the production of functional dystrophin protein. However, the clinical feasibility of this approach is limited by the poor survival of the donor cells in the weeks after transplantation. It has recently been determined that the intramuscular transplantation of large numbers of cells can lead to the formation of ischemic necrosis in the center of these cell masses. For this reason, the relationship between donor cell survival and the number of cells transplanted was investigated. Methods. Myoblasts were prepared from the hind limb muscles of male C57BL/10Sn mice and transplanted into the tibialis anterior muscles of female mdx mice at one of the following amounts: 103, 104, 105, or 106 cells. The survival of the transplanted cells was analyzed using a Y chromosome-specific qPCR. Results. It was found that donor cell survival was improved 1 week after transplantation when fewer myoblasts were transplanted, including the observation of donor cell proliferation after the transplantation of 103 myoblasts. However, concentration effects and long-term survival complicate the interpretation of these results. Conclusions. These results indicate that early donor myoblast survival was dependent on the number of cells transplanted and the volume of liquid used to deliver them into the muscle. We believe that this finding has implications for the design and interpretation of future experimentation relating to intramuscular cell therapies.

The high incidence of acute and chronic kidney injury due to various environmental factors such as heavy metals or chemicals has been a major problem in developing countries. However, the diagnosis of kidney injury in these areas can be more challenging due to the lack of highly sensitive and specific techniques that can be applied in point-of-care settings. To address this, we have developed a technique called ‘micro-urine nanoparticle detection (μUNPD)’, that allows the detection of trace amounts of molecular markers in urine. Specifically, this technique utilizes an automated on-chip assay followed by detection with a hand-held device for the read-out. Using the μUNPD technology, the kidney injury markers KIM-1 and Cystatin C were detected down to concentrations of 0.1 ng/ml and 20 ng/ml respectively, which meets the cut-off range required to identify patients with acute or chronic kidney injury. Thus, we show that the μUNPD technology enables point of care and non-invasive detection of kidney injury, and has potential for applications in diagnosing kidney injury with high sensitivity in resource-limited settings.