Unique Transcriptional Programs Identify Subtypes of AKI

BASIC RESEARCH www.jasn.org

† ‡ | Katherine Xu,* Paul Rosenstiel, Neal Paragas, Christian Hinze,§ Xiaobo Gao, Tian Huai Shen,* Max Werth,* Catherine Forster,¶ Rong Deng,* Efrat Bruck,* Roger W. Boles,* Alexandra Tornato,* † Tejashree Gopal,* Madison Jones,* Justin Konig,* Jacob Stauber,* Vivette D’Agati, †† ‡‡ Hediye Erdjument-Bromage,** Subodh Saggi, Gebhard Wagener, Kai M. Schmidt-Ott,§ || Nicholas Tatonetti,§§ Paul Tempst, Juan A. Oliver,* Paolo Guarnieri,§§ and Jonathan Barasch*

Departments of *Medicine, Division of Nephrology, †Pathology, ‡‡Anesthesiology, and §§Systems Biology, Columbia University Medical Center, New York, New York; ‡Department of Medicine, Division of Nephrology, University of Washington, Seattle, Washington; §Max Delbrück Center for Molecular Medicine, Berlin, Germany; |Department of Medicine, Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York; ¶Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; **Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York; ††Department of Medicine, State University of New York Downstate Medical Center, Brooklyn, New York; ||Memorial Sloan Kettering Cancer Center, New York, New York BASIC RESEARCH ABSTRACT Two metrics, a rise in serum creatinine concentration and a decrease in urine output, are considered tantamount to the injury of the kidney tubule and the epithelial cells thereof (AKI). Yet neither criterion emphasizes the etiology or the pathogenetic heterogeneity of acute decreases in kidney excretory function. In fact, whether decreased excretory function due to contraction of the extracellular fluid volume (vAKI) or due to intrinsic kidney injury (iAKI) actually share pathogenesis and should be aggregated in the same diagnostic group remains an open question. To examine this possibility, we created mouse models of iAKI and vAKI that induced a similar increase in serum creatinine concentration. Using laser microdissection to isolate specific domains of the kidney, followed by RNA sequencing, we found that thousands of genes responded specifically to iAKI or to vAKI, but very few responded to both stimuli. In fact, the activated gene sets comprised different, functionally unrelated signal transduction pathways and were expressed in different regions of the kidney. Moreover, we identifieddistinctivegeneexpressionpatternsinhumanurineas potential biomarkers of either iAKI or vAKI, but not both. Hence, iAKI and vAKI are biologically unrelated, suggesting that molecular analysis should clarify our current definitions of acute changes in kidney excretory function.

J Am Soc Nephrol 28: 1729–1740, 2017. doi: https://doi.org/10.1681/ASN.2016090974

The critical function of the kidney, conserved from kidney’s excretory function decreases indepen- planaria1 to mammals,2,3 is to regulate the extracel- dently of the ECFV. Adding further complexity, lular ﬂuid volume (ECFV). When Na+ and water ’ are scarce and ECFV decreases, the kidney s excre- Received September 12, 2016. Accepted November 12, 2016. tory function also decreases, ensuring the conser- K.X. and P.R. contributed equally to this work. vation of Na+ and water and the maintenance of ECFV. However, the same homeostatic neuronal Published online ahead of print. Publication date available at and hormonal pathways (e.g., sympathetic and www.jasn.org. angiotensin-aldosterone systems) that regulate ef- Correspondence: Dr. Paolo Guarnieri, Department of Systems fectors of volume retention (epithelial sodium Biology, Irving Cancer Research Center, 1130 St. Nicholas Ave- nue, New York, NY 10032 or Dr. Jonathan Barasch, Department channel, Na/KATPase, and osmolytes) are appro- of Medicine/Nephrology, Nephrology Div. 4 Stem, Department priated by diseases such as congestive heart failure of Medicine, 622 W 168th Street, New York, NY 10032. E-mail: and cirrhosis in the absence of volume depletion. [email protected] or [email protected] Consequently, in patients with such diseases, the Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 1729–1740, 2017 ISSN : 1046-6673/2806-1729 1729 BASIC RESEARCH www.jasn.org mechanisms of injury that affect the cells of the nephron, such patterning of gene expression in the kidneys of mice subjected as severe ischemia, bacterial endotoxins, pancreatic enzymes, to either severe ECFV depletion (vAKI) or transient renal is- and nephrotoxic drugs may also decrease the kidney’s excre- chemia (iAKI). Because the elevation of sCr currently defines tory function. AKI stage, which is thought to reflect the intensity of kidney Poor or absent kidney excretory function due to extrarenal injury,14–16 we chose conditions to match sCr. In addition, causes (i.e., ECFV depletion or diseases such as heart failure) because whole kidney analyses could render the genetic signa- has traditionally been labeled “prerenal” renal failure or tures of different nephron segments undetectable, we chose “hemodynamic” renal failure, emphasizing the notion that laser capture microdissection to assay different microana- cells of the nephron were likely uninjured, consequently dis- tomic kidney regions. tinguishing “prerenal” conditions from direct or “intrinsic” Our results indicate that equivalent acute decreases of nephron injury. Although conceptually straightforward and of kidney excretory function (e.g., rises in sCr) due to intrinsic critical importance to guide therapeutic interventions, the kidney injury activate sharply different genetic programs than separation of patients with acute decreases in kidney excretory those activated by homeostatic responses to volume depletion, functions into categories of “prerenal” or “intrinsic” kidney with very limited overlap. Further, we translated these data to failure has bedeviled clinicians for decades.4,5 The discovery of humans where we found that the same genes were activated in biomarkers specific for intrinsic kidney injury6,7 has facili- clinically distinct groups of patients. Thus, the term “AKI” as tated diagnosis, but the usefulness of these markers remains based on acute increases in sCr is likely to identify patients to be established in patients subject to different stressors that with fundamentally different processes, and although it might reduce excretory function. be useful for epidemiologic and actuarial purposes, it obfus- Recent epidemiologic studies found that patients with acute cates etiology and potentially confounds therapeutic decision- decreases in kidney excretory function of similar magnitude, as making. determined by the concentration of circulating waste products (serum creatinine, sCr), had poor prognoses, regardless of the stimulus.8 Further, because acute increases in sCr of equal RESULTS magnitude had equal clinical significance and presaged a similar clinical course,5 it followed that any acute decrease in kid- Patterning of the Genetic Responses to vAKI and iAKI ney excretory function reflected some degree of kidney injury. The concentration of sCr increased to the same extent (no In this view, even modest elevations in sCr due to nonrenal significant differences) in the two models of AKI, the first diseases are but an initial phase of a continuous pathogenesis due to volume depletion (vAKI; 1.9-fold increase in sCr; resulting in kidney cell damage.9–11 In fact, even increases in P,0.01) and the second due to transient kidney ischemia sCr due to ECFV depletion have been proposed to represent a (iAKI; 1.5-fold increase in sCr; P,0.03; Supplemental Figure 1). forme fruste of intrinsic kidney disease11–13 in that both might The isolation of microanatomic domains of the kidneys by activate the same pathways of injury, but with different timing laser capture was first validated in kidneys by both RNA se- or with varying degrees of intensity. Although experimental quencing and quantitative real-time PCR, which confirmed the data supporting this hypothesis is scant, it has gained wide- appropriate enrichment of known nephron segment–specific spread backing from several influential renal community markers (Supplemental Figure 2). associations such as the Acute Dialysis Quality Initiative An unsupervised hierarchic clustering analysis (Supple- (ADQI),14 the Acute Kidney Injury Network (AKIN),15 and mental Figure 3) revealed that transcriptional profiles were the Kidney Disease Improving Global Outcomes (KDIGO).16 grouped by anatomic domains, and were separated according These groups all recommend that patients with acute decreases to the etiology of the rise in sCr. iAKI induced 12-fold in kidney excretory function be diagnosed principally on the more differentially expressed genes (DEGs) than vAKI basis of the sCr level and urine output (RIFLE and AKIN), a (q-value,0.01). Remarkably, the majority of expressed genes concept encapsulated in the diagnostic acronym AKI. were different and nonoverlapping between vAKI and iAKI. In Although some guidelines suggest that ECFV depletion is an addition, these genes had distinct expression patterns: iAKI important “risk factor for AKI”16 and may influence sCr mea- DEGs localized predominately to the outer stripe of the outer surements,15 the biologic relationship between ECFV deple- medulla (OSOM), whereas vAKI DEGs localized predomi- tion and AKI remains indeterminate. nately to the inner stripe of the outer medulla (ISOM; Figure Although grouping all patients with acute rises in sCr into 1). When we applied statistical cut offs, 92.3% (1158) of the 2 the single clinical entity of “AKI” has provided quantifiable genes upregulated in iAKI (.2-fold; P value ,10 5)werenot data across many different clinical scenarios, it is at variance expressed in the vAKI model (Supplemental Table 1A). Sim- with the striking lack of correlation between the degree of sCr ilarly, 51.7% (103 genes) of the upregulated genes in vAKI 2 elevation and kidney pathology in biopsies of critical care and (.2-fold; P value ,10 5) were not expressed in the iAKI other patients,4,17,18 implying that there may be different cel- model (Supplemental Table 1B). lular mechanisms that mediate decreases in kidney excretory To examine whether the transcriptional profiles of the two function. To directly test this hypothesis, we examined the types of AKI suggested distinct functional responses, we

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Figure 1. Limited overlap of gene expression between iAKI and vAKI. Differential gene expression was most prominent in the OSOM in the iAKI model and in the ISOM in the vAKI model. The heatmaps portray only the significant DEGs (q-value,0.01). Gene expression was z-score–transformed on a per-gene basis and then hierarchically clustered. performed gene-set enrichment analyses (false discovery rate outer edge of the OSOM, a region known to be most sensitive ,25%).19 As shown in Figure 2A, this analysis indicated that to ischemic damage.22 In contrast, vAKI kidneys had no de- iAKI yielded many more significantly induced gene sets than tectable histologic cellular derangements, including apical vAKI, including classic injury/repair (Hippo, ErbB, MAPK) megalin and actin in the proximal tubule (data not shown). and inflammatory (JAK/STAT, NOD-Like, NFkB, TLR, and Moreover, the patterning and number of apoptotic cells dif- Chemokine) pathways, probably reflecting the fact that kidney fered. Consistent with the histologic analysis, iAKI kidneys injury and repair overlap in time. In addition, the signaling demonstrated focal clusters of TUNEL+ cells in the kidney pathway impact analysis20 (q=0.01), which provides direc- cortex and OSOM (104 TUNEL+ per 630 mm2 kidney region), tionality to gene interactions, identified Wnt and PPAR path- whereas vAKI kidneys had scant positive cells (1 TUNEL+ cell way activation in iAKI (Figure 2B). In marked contrast, none per 630 mm2 kidney region) and only scattered TUNEL+ cells of these pathways were modulated by vAKI. Instead, vAKI in the papilla. Hence, two stimuli that induce equivalent re- induced metabolic (TCA, gluconeogenesis, oxidative phos- ductions in kidney excretory function displayed markedly dif- phorylation, respiratory electron transport), transport (metal ferent genetic and pathologic responses. transport), and osmo-regulatory (sulfur amino acid, glycine- serine-threonine pathways) gene sets.21 Differential Expression of Specific Genes In sum, the patterning of gene expression (the number and In iAKI kidneys, we confirmed the upregulation of genes location of DEGS) differed sharply in vAKI and iAKI. This known to be associated with intrinsic injury, specifically: indicated that cellular responses to the two stimuli were un- Spp1 (OPN), Cxcl1 (GRO-a), and Lcn2 (NGAL) with P val- 2 related, as supported by histologic and TUNEL analyses of the ues ,10 21; Clu (clusterin), Havcr (KIM-1), and Timp1 2 kidneys (Supplemental Figure 1). Ischemic kidneys consis- (TIMP1) with P values ,10 10;andS100a8/9 (calprotectin) 2 2 tently demonstrated regions of coagulative necrosis at the with P values ,10 7 to ,10 9 (Supplemental Table 2). They

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Figure 2. Different patterning of iAKI and vAKI pathways. (A) Functional analyses using gene set enrichment analysis (GSEA) against KEGG, Reactome, Biocarta, and PID Pathway databases. Each row (thin lines) demonstrates a pathway found in one or more of the queried databases. Significant pathway enrichment is represented in a binary manner (enrichment=red; de-enrichment=blue; unchanged=white; false discovery rate ,25%). (B) Functional analysis using GSEA was supplemented with signaling pathway impact analysis leveraging the topologic information available from canonical signaling pathways. Each horizontal division (boxes) contains an aggregate of gene sets ascribable to a known signaling pathway analyzed by one or more of the queried databases (i.e., KEGG, Reactome, Biocarta, PID). Each shaded row within the division represents an individual gene set analyzed by a single database; most signaling pathways were analyzed by multiple gene sets and databases. Pathway activation (red) or inhibition (green) was determined by signaling pathway impact analysis; the depth of shading of red or green reflects the degree of GSEA enrichment or de-enrichment. were upregulated on average approximately 180-fold. As an some tubules (Figure 3, Supplemental Table 2), rather than example of a well studied iAKI-specificgene,Lcn2 was in- by the OSOM where iAKI changes in gene expression were 2 tensely expressed (214-fold, P value ,10 21)andlikemany generally found. other known iAKI genes, it localized to the OSOM and In vAKI kidneys, upregulated genes were particularly local- 2 ISOM (Figure 3, Supplemental Table 2). More interest- ized to cortex and ISOM; e.g., Pappa2 (6.35-fold, P,10 8)and 2 ingly, we found nearly 1000 novel genes that were markedly Stc1 (10.72-fold, P,10 15). They were upregulated from upregulated in iAKI. Most localized in the OSOM and they 2-fold to 128, with a mean of 10.8-fold (see Supplemental were upregulated from 2- to 3067-fold (mean=29-fold). Table 1B). Because vAKI not only decreased kidney excretory 2 Particularly notable were Krt20 (CK20; 1643-fold, P,10 12), function but also increased serum osmolality (see Supplemen- 2 Tactstd2 (TROP2; 4.26-fold, P,10 9), and Gc (VDBP; 8.26- tal Material), which is known to significantly alter kidney gene 2 fold, P,10 6) (Figure 3, Supplemental Table 1A). transcription,25 we also examined a model of vAKI in the ab- Surprisingly, a separate group of genes previously suggested sence of hyper-osmolarity (see Concise Methods). This model as iAKI biomarkers (b2M, Timp2, Netrin, Igfbp7, Tnfsf10, elevated sCr 1.5-fold and induced the same genes as the stan- 2 Hgf) were unchanged or even downregulated, on average dard vAKI model; e.g., Pappa2 (38-fold, P,10 3), Stc1 (3- 2 2 0.48-fold (see references, Gauer et al. and Mar et al.). Down- fold, P,10 3), Ddit4l (2.8-fold, P,10 2), Tuba4a (2-fold, 2 regulation of the expression of these genes was specific for iAKI P=0.05), and Impa1 (4.7-fold, P,10 2). In contrast, neither because these genes were not modulated by vAKI (Figure 1). model of vAKI upregulated iAKI genes Lcn2 (NGAL) and Some of these genes were expressed by the glomerulus and by Havcr (KIM1) (p=NS).

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due to vAKI or iAKI. However, because the former rapidly re- sponds to fluid resuscitation, but the latter generally has a longer time to resolution, the patient’s evolution facilitates the diagnosis. Hence, to first establish the prevalence of iAKI and vAKI in patients, we reviewed the creatinine kinetics in a cohort of patients from our previous emergency department studies.26,27 Remark- ably, the majority of changes in sCr were very transient; e.g., analysis of .2000 emergency department patients26,27 showed that volume administration normalized sCr in 72% of patients within 48 hours of presentation. To determine whether transient changes in sCr are common, we developed an algorithm to examine the electronic health records of 3.8 million patients at New York Pres- byterian-Columbia University Medical Center. We detected 61,726 events of “AKI,” defined as a deflection of sCr $0.3 mg/dl above baseline, but like in our emergency department studies, we again found that most sCr elevations were transient: 33% of events resolved within 1 day, 60% within 2 days, and 73% within 3 days of the initial rise in sCr. Hence, the evolution of sCr in the majority of patients entering the hospital indicates that vAKI is likely to be among the most common causes of acute decrease in kidney excretory function.4,8,28 Accordingly, although the major focus of clinical research in AKI concerns intrinsic forms of AKI, genes expressed in vAKI may be helpful in clinical practice as well. We first selected secreted gene products from the Secreted Protein Database29 or from the Max Planck Unified Proteome Database30 of human urine. This informatics pipeline identified 267 secreted iAKI (Supplemental Table 3A) and 30 secreted vAKI (Supplemental Table 3B) candidate urinary bio- Figure 3. Detection of novel biomarkers in mouse. Immunofluo- markers. Of these, we tested 40 proteins that originated from rescence (CK20) and in situ hybridization (Tacstd2, Lcn2) demon- different regions of the nephron using urine collected from strate specificity for iAKI. Note that CK20 (red) was expressed in iAKI or vAKI patients that were previously adjudicated by proximal tubules (megalin=green) and in intercalated cells (IC) (inset: strict criteria26 (see Concise Methods). In agreement with CK20=red; AQP2=white). Note that Tacstd2 was expressed in distal our studies with mouse models, CHI3L1 (Chi3l1), TROP2 nephron segments. Lcn2 RNA was expressed by thick ascending (Tacstd2), TPA (Plat), and CK20 (Krt20)(Figure4A)were limbs of Henle (TALH) as well as ICs of the collecting ducts in iAKI. Timp2 and Igfbp7 RNA are shown for comparison. Note the ex- prominently expressed in the urine of patients who achieved pression of these genes in scattered glomerular (G) and tubular cells. the diagnosis of intrinsic kidney injury (i.e., iAKI) during their hospitalization. Further, these proteins were minimally or inconsistently expressed in patients diagnosed with volume- It has been proposed that vAKI is a forme fruste of iAKIand that reversible vAKI. For comparison, we also assayed standard bio- vAKI can rapidly progress to iAKI.11–13 On the other hand, it may markers of iAKI such as NGAL,6 VDBP,31 TIMP2/IGFBP732 be that vAKI is an appropriate kidney homeostatic response to (Figure 4B) which demonstrated a range of specificity for iAKI. ECFV depletion, which does not cause tubular cell damage. In this Of the genes we detected in vAKI mouse kidneys, we found case, correction of the ECFV depletion should quickly reverse the that full-length PAPPA2 (Figure 4A) was detected in the urine of activated genetic program, whereas tubular damage would be ex- patients with vAKI (9 of 13) and in the urine of some normal pected to require a longer period of repair. To test this, we restored patients (3 of 13), but not in the urine of patients with iAKI. ad libitum water access to mice with vAKI (see Concise Methods) Interestingly, iAKI urine contained PAPPA2 immuno-reactive and found that differentially expressed vAKI genes normalized fragments rather than the full-length protein suggesting that within 24 hours (Supplemental Figure 4). Hence, genes overex- iAKI urine contained proteases that cleave PAPPA2, and that pressed in vAKI were sensitive to transient changes in ECFV. the molecular weight of this biomarker in the setting of an elevated sCr might distinguish between iAKI and vAKI. Toexamine Translational Application of iAKI and vAKI Genes this, we mixed urine of vAKI patients and iAKI patients and found Because our results establish a clear-cut difference between iAKI that PAPPA2 was degraded, particularly when urine pH was and vAKI, we examined whether the data could be translated to acidic (pH 5.5)33 (Figure 4C). A preliminary proteomic analysis humans. In clinical medicine, it may be initially difficult to de- of the iAKI urine samples identified proteases known to be termine whether an acute decrease in kidney excretory function is active in tubular damage; e.g., cathepsin D,34 cathepsin B,35

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Figure 4. Detection of novel biomarkers in human. (A) Secreted proteins (CHI3L1, TROP2, TPA, CK20) were elevated in most human iAKI urine but not in most vAKI urine. Conversely, secreted protein PAPPA2 was elevated in many vAKI urines, and some normal urines, but it is degraded in iAKI patients. Blue bars denote canonical molecular weights of the analyte. (B) Secreted proteins (NGAL, VDBP, TIMP2, IGFBP7) are shown for comparison and demonstrate different degrees of speciﬁcity of iAKI patients, as listed. Blue bars denote canonical molecular weights of the analyte. (C) Proteolysis of vAKI urinary PAPPA2 by iAKI urine was rescued by protease inhibitors, pepstatin A, or combinations of inhibitors. PAPPA2 was also degraded by cathepsin D. aminopeptidase N,36 MMP-9,37 and MMP-8.38 Further, when including ECFV depletion, extrarenal diseases, and damage to cathepsin D was added to vAKI urine samples, PAPPA2 was di- cells of the nephron. It may be the case that all causes of de- gested, whereas the addition of pepstatin A to the urine coincu- fective renal excretion activate a common molecular pathway. bation preserved PAPPA2 immunoreactivity. These data show On the other hand, if genetic readouts speciﬁc to different that full-length urinary PAPPA2 reports vAKI or normal urine, forms of defective renal excretion were evident at the time of but degraded protein reports intrinsic kidney injury. patient presentation, then a “precision medicine” approach to AKI could be implemented, akin to the efforts currently un- derway in the study of CKD. DISCUSSION By isolating different domains of the kidney, we demonstrated that thousands of genes were unique to either iAKI or The acute failure of the kidney to excrete salt, water, urea, vAKI. Although these data were obtained with laser microdis- creatinine, and other wastes results from a variety of processes section, which analyzes RNA from a variety of cells within the

1734 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1729–1740, 2017 www.jasn.org BASIC RESEARCH captured domain, they were in agreement with an emerging more severe (acute shock) models of vAKI might cause iAKI map of gene expression in specific kidneycells. For example, we (e.g., hemorrhagic or cardiogenic shock), our two models were adapted the method of Gay et al.39 to create a novel mouse to consistent with human presentations of vAKI and iAKI.61–63 isolate newly-synthesized collecting duct RNA and found In this light, perhaps the conversion of vAKI to iAKI may 86.3% of iAKI genes (.2-fold; P value ,0.05) were not generally require a “second hit” (a well known phenomenon expressed in the vAKI model, and 95.9% of vAKI genes in nephrotoxicity64–66 and sepsis67). (.2-fold; P value ,0.05) were not expressed in the iAKI model The distinct patterning of vAKI and iAKI genes may have a (T. Shen, unpublished data) consistent with laser capture mi- number of clinical applications. First, because vAKI genes rap- croscopy. Our findings were also in agreement with transcrip- idly reversed with volume resuscitation, the proteins encoded tomic analyses by Star and colleagues40 and with measurements by these genes may serve as novel biomarkers of volume dis- of tubular cell energetics (which were preserved in vAKI but not orders, perhaps useful to guide rehydration (or cardiotonic) in iAKI models).41 Hence, it is apparent that vAKI is not an therapy and limit volume overload. Second, simple ratios of attenuated form of iAKI; rather, each activated a distinct genetic iAKI and vAKI genes might confirm the presence of tubular program despite similar levels of sCr. damage and clarify the meaning of an elevated sCr. Lastly, the iAKIinducedchanges ininflammatory, epithelialgrowth, distinct patterning of genes may provide an explanation for the and cell repair genes. These include the Hippo signaling dissociation of iAKI biomarkers from sCr at lower levels of pathway,42,43 particularly Yap1, an antiapoptotic transcrip- RIFLE,AKIN,andKDIGOscores.Notonlydothelimitations tion cofactor which increased 1.7-fold (P,105), whereas its of sCr reduce its capacity to judge the performance of an iAKI inactivator, Lats2,44 demonstrated a 0.65-fold decrease biomarker,68,69 but vAKI-induced elevated sCr should be (P,104), implicating the Hippo pathway in epithelial re- compared with vAKI-induced genes rather than iAKI- generation.45 In addition, iAKI activated Wnt7a (80.5-fold, induced genes. Pooling molecularly and spatially distinct P,104), a gene which is essential for tubular repair and programs on the basis of sCr reduces the utility of iAKI regeneration,46 butthatcandrivetransformationto biomarkers, because many of these proteins are not even chronic damage when its expression is sustained.47,48 Other expressed when sCr rises due to vAKI.26,70,71 For example, classic inflammatory and repair pathways, such as MAPK, Lcn2 (siderocalin-NGAL) was rapidly stimulated by injuri- JAK/STAT, NFkB, TLR, and chemokine, were also markedly ous stimuli,27,72–76 but it was poorly responsive to even activated in iAKI, in agreement with landmark studies.49–52 prolonged volume derangements27,70,72 (including cirrho- Yet,atthesamesCr,noneoftheiAKIpathwayswereacti- sis71,76 and diuretics72) despite elevation of sCr in all of vated in vAKI. Perhaps the absence of injury in vAKI was these cases. Consequently, we suggest that elevated sCr due to protective mechanisms such as the renal protective even at the same stage can reflect very different clinical, factors known to be modulated in iAKI (e.g., prostaglan- cellular, and molecular responses, but simultaneous mea- dins, NO, HIF).4,53–57 As an example, PAPPA2 is a metal- surement of both iAKI and vAKI markers could provide loproteinase secreted by the thick ascending limb of Henle, discriminatory power. which targets the IGFBP system, permitting IGF-mediated In sum, the kidney’s response to environmental challenges cell survival and growth.58–60 However, PAPPA2 is degraded is fine-tuned, producing different genetic readouts in different in iAKI through proteolysis, thereby removing a potential cells in different parts of the nephron. A transient elevation of protective mechanism that is found in salt-sensitive volume sCr is the most prevalent renal abnormality in clinical medi- stress.58 In sum, iAKI and vAKI models sharply diverged in cine and if these rapid fluctuations are volume-sensitive ele- clinical phenotype (Supplemental Figure 1) as well as in vations (as suggested by our emergency department series26), transcriptional (Figure 1, Supplemental Tables 1–3) and new tools specific to vAKI (in addition to those for iAKI) could in specific pathways including cell survival and cell repair provide quick prospective diagnoses and treatment plans for a (Figure 2). broad range of patients. The stark distinctions between iAKI and vAKI raise the question of whether these entities represent continuous stages of injury that might ultimately converge. As our vAKI mice CONCISE METHODS were subjected to severe and prolonged volume derangements and could not have survived further volume depletion, we Clinical Samples argue that the volume stimulus was of sufficient severity to Patient samples and data collection were approved by the Institutional induce kidney injury, if capable. Hence, vAKI and iAKI phe- Review Board of Columbia University with written informed consent notypes may be difficult to interconvert, given that the vAKI of the patients. Emergency room urine samples were selected at ran- mice do not demonstrate iAKI histology, iAKI genes, or iAKI dom from our multicenter prospective cohort study,26,27 using our molecular pathways. In fact, the vAKI gene set surprisingly published criteria for iAKI, vAKI, and control including clinical his- resembled the control gene set more closely than the iAKI tory, time to resolution of elevated sCr (vAKI,72 hours; iAKI$7 gene set (see Figure 1, Supplemental Figure 3). Although there days), and rapid responses to volume challenges, all aimed at identi- is no doubt that additional more prolonged (.3 days) or even fying only “gold standard” patients (Supplemental Material).

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Analysis of Large Clinical Dataset (1:200; FL-1321; Vector Laboratories). Fluorescent secondary We examined the electronic health records of 3.8 million patients at antibodies, Alexa Fluor 594-AffiniPure F(ab9)2 Fragment Donkey New York Presbyterian-Columbia University Data Warehouse. Of Anti-Rabbit IgG and Alexa Fluor 647-AffiniPure F(ab9)2 Fragment these patients, 569,519 had atleast one creatinine available.Wedefined Donkey Anti-Goat IgG (1:1000; Jackson Immunoresearch Laborato- baseline creatinine during each of their hospitalizations if at least three ries), were used for CK20 and AQP2 identification, respectively. All sCr were available (83,601 patients) and demonstrated stable sCr slides were costained with 49,6-diamidino-2-phenylindole to identify (change#0.2 mg/dl; median number of sCr measurements defining nuclei. baseline per hospitalization was four). The baseline creatinine was fi de ned as the mean of the longest sequence of stable creatinine val- Laser Capture Microdissection ues. An AKI event was defined as a deflection from baseline of $0.3 Kidneys were embedded in O.C.T. Compound and immediately snap mg/dl evident in 61,726 events in 27,464 patients. We defined the frozen in dry ice and kept at 280°C until time of sectioning. Sections of time to resolution as the number of hours between the peak creati- 8–10 mm(20mm for glomeruli) were collected on nuclease-free glass nine value during the event and the time of the first drop $0.3 mg/dl slides covered with a thin membrane (Zeiss Microscopy, Thornwood, from this peak. NY), fixed in 70% ethanol for 30 seconds, stained with 1% cresyl violet acetate solution, and dehydrated in 70% and 100% ethanol followed by Mouse Husbandry air-drying for 30 minutes. Regions of interest were identified morpho- Female wild-type C57Bl/6 mice, aged 10–12 weeks (Jackson Labs, Bar logically and 15–20 cross sections (for cortex, OSOM; ISOM) or ap- Harbor, ME), were used according to protocols approved by the Co- proximately 1500 cross sections (glomerulus) were microdissected lumbia Institutional Animal Care and Use Committee. (PALM MicroBeam; Zeiss Microscopy, Thornwood, NY). We collected RNA from vAKI (n=5), iAKI (n=3), and control (n=3) (50 independent Renal Volume Depletion Model samples in total). Domain-specific RNA was validated by RT-qPCR We tested different durations of water deprivation72 which reduced (Supplemental Figure 2; Supplemental Table 4). food intake (6.260.3 versus 1.260.4 g on day 3; P,0.001), until we found a significant rise in sCr at 72 hours. Consequently, water was RNA Extraction and RNA Sequencing withheld from mice for 72 hours. Body weight and food intake were Total RNA was isolated using Ambion RNAqueous Micro Kit (Life measured daily. Food intake was determined by weighing chow pel- Technologies, Carlsbad, CA). RNA concentration and integrity for lets and spillage. Kidneys were harvested and blood was collected at each sample were assessed on RNA 6000 Chips using an Agilent 2100 72 hours or mice were rehydrated for an additional 24 hours. The Bioanalyzer (Agilent Technologies, Santa Clara, CA). Poly-A pull- furosemide model utilized a single dose of 50 mg/kg followed by down was used to enrich mRNAs (200 ng to 1 mg per sample, sample briefer water and food deprivation (48 hours).77,78 RIN was .8.0) and then libraries were prepared using single-end 100 bp reads for each sample with Illumina TruSeq RNA prep kits (Illu- Renal Ischemia Reperfusion Injury Model mina, San Diego, CA). Libraries were sequenced using Illumina Hi- To compare the vAKI model with acute renal vascular ischemia, we Seq2000 at Columbia Genome Center. Batch effects were analyzed evaluated a range of ischemic doses until we matched the elevated using PC analysis (Supplemental Figure 5, Supplemental Material). sCr in the volume depletion model. We found that brief bilateral renal artery ischemia raised sCr at the 24 hours point, similar to Data Analysis the volume depletion model. Consequently, mice were anesthetized Illumina RTA was used to perform base calling, and CASAVA (version with isoflurane and placed on a warming table to maintain a rectal 1.8.2) wasused forconvertingbase callfiles (.BCL) toFASTQ format and temperature of 37°C. Left and right renal pedicles were clamped using for performing sequence adaptor trimming. Reads were then mapped to microvascular clamps (Fine Science Tools, Foster City, CA) for 10 min- the mouse reference genome (mm9) using Tophat79 (version 2.0.4) utes. After the clamps were removed, reperfusion of the kidneys was allowing four mismatches (–read-mismatches=4) and a maximum of visually confirmed. The kidneys and blood were harvested at 24 hours. ten multiple hits (–max-multihits=10). The relative expression was cal- culated using cufflinks80 (version 2.0.2) with default settings. Gene ex- Clinical Measurements pression levels were normalized by library size and gene length into 81 sCR, sodium, and blood urea nitrogen were measured using Creati- FPKMs and log2 transformed. Counts tables were generated with nine and EC8+ cartridges read by an i-STAT Handheld (Abbott Point HTSeq (http://www-huber.embl.de/users/anders/HTSeq) version of Care, Princeton, NJ). 0.6.1. Transcripts with zero counts across all samples were removed and mathematical artifacts (e.g., negative infinites) were replaced with Immunohistochemistry “NA.” Statistical analysis was performed in R version 3.1.0 and addi- Kidneys were fixed (4% PFA/0.1M PB at 4°C overnight), transferred to tional Bioconductor packages were part of release 2.14. 30% sucrose/0.1M PB (4°C overnight), and embedded in O.C.T. Compound (Tissue-Tek). Frozen sections of 20 mmwereusedfor Data Availability immunofluorescence staining with rabbit anti-CK20 (1:200; The FASTQ dataset can be accessed from NCBI’s GEO Acces- ab118574; Abcam), goat anti-AQP2 (1:400; sc-9880; Santa Cruz Bio- sion GSE81741 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? technology), and Fluorescein-labeled Lotus Tetragonolobus Lectin acc=GSE81741).

1736 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1729–1740, 2017 www.jasn.org BASIC RESEARCH

Generation of Heatmaps Pittsburgh, PA), and proteins detected using anti-human antibodies: Genes that were differentially expressed in iAKI versus control and polyclonal human CHI3L1 (1:1000; AF2599; R&D Systems), poly- vAKI versus control were included. The expression data shown is the clonal human TROP2 (1:1000; AF650; R&D Systems), monoclonal variance-stabilized data generated using the DESeq package from human TPA (PLAT) (1:1000; ab157469; Abcam), monoclonal human Bioconductor according to the DESeq vignette, (http://bioconductor. CK20 (1:1000; ab118574; Abcam), polyclonal human PAPPA2 org/packages/release/bioc/html/DESeq.html, http://bioconductor.org/ (1:1000; AF1668; R&D Systems), monoclonal human NGAL packages/release/bioc/vignettes/DESeq/inst/doc/DESeq.pdf). Hierar- (1:1,000; BPD-HYB-211–01–02; Enzo Lifesciences), monoclonal hu- chic clustering used Pearson correlation distance plus single link- man VDBP (1:1000; MAB3778; R&D), polyclonal human IGFBP7 age. The variance-stabilized expression values were visualized with (1:1000; AF1334; R&D Systems), and polyclonal human TIMP2 heatmap.2 (gplots package, http://cran.r-project.org/web/packages/ (1:1000; AF971; R&D Systems). gplots/index.html). Probe Synthesis for In Situ Hybridization Identification of Genes and Pathways Mouse kidney mRNA was reverse transcribed using SuperScript III DEGs were identified using edgeR package82 version 3.6. We used the First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen), and tar- Benjamini & Hochberg83 procedure for controlling false discovery get genes were amplified using the following primers: Timp2, rate of the multiple tests and accepted as significant a q-value,0.01. forward: 59-gatcagagccaaagcagtgag-39 and T7 embedded reverse: 59- Pathway enrichment analysis was performed using the commercially ggattaccTAATACGACTCACTATAGGGttctctgtgacccagtccatc-39; available version of signaling pathway impact analysis, PathwayGuide IGFBP7, forward: 59-ctctcctcttcctcctcttcg-39 and T7 embedded reverse: (Advaita Corporation http://www.advaitabio.com/)20 against 59-ggattaccTAATACGACTCACTATAGGG tgacctcacagctcaagaaca-39; KEGG84 and Reactome,85 whereas gene-set enrichment analyses19 Ngal, forward: 59-aaaaacagaaggcagctttacg-39 and T7 embedded reverse: were performed against MSigDB canonical pathways from the cu- 59-ggattaccTAATACGACTCACTATAGGGaaagatggagtggcagacaga-39; rated gene sets (C2) v4.0. Trop2, forward: 59-GCAATGGGCTCACAGGTATT-39,andT7-embedded reverse: 59-GGCCAGTGAATTGTAATACGACTCACTATAGG- Identification of Biomarkers GAGGCGGTTTGTATTTGCCCGACTTCC-39. The PCR products Candidate biomarkers were filtered according to the Max Plank Uni- were used as templates for in vitro transcription. Probes were syn- fied Proteome (1542 proteins assessed March 20, 2014), Secreted thesized by T7 RNA polymerase (Roche) and digoxigenin-labeled ProteinDB,29 or by prediction (signal peptide and without a trans- RNAs were subsequently purified by PureLink RNA Mini Kit membrane domain—according to Ensembl!).86,87 We identified the (Life Tech). proteins that were exclusive to condition, demonstrated $2-fold change compared with control, and were expressed with an In Situ Hybridization for Frozen Sections . FPKM 1. Kidneys fixed in 4% PFA were sectioned (8 mm), air-dried for 1–3 hours, then refixed in 4% PFA for 10 minutes and treated with pro- Protein Identification by Nano-Liquid Chromatography teinase K (1 mg/ml), acetylated and prehybridized, and hybridizations Coupled to Tandem Mass Spectrometry Analysis were at 68°C–72°C overnight in 50% formamide, 53 SSC, 53 Den- fl Urinary protein preparations were resolved brie ybySDS-PAGE, hardts, 250 mg/ml baker’s yeast RNA (Sigma), and 500 mg/ml herring stained with Coomassie Blue, and separated protein bands excised sperm DNA (Sigma). Washes were at 72°C in 53 SSC for 5–10 minutes, 88 for in situ trypsin digestion of polypeptides. Peptides were eluted then at 72°C in 0.23SSC for 1 hour. Sections were stained overnight with m with 3 l of 40% acetonitrile containing 0.1% formic acid and anti-digoxigenin antibody (1:5000 dilution; Boehringer-Ingelheim, m diluted to 20 l with 0.1% formic acid for nano-liquid chroma- Mannheim, Germany) and alkaline phosphatase activity detected 89 tography coupled to tandem mass spectrometry as described. If with BCIP, NBT (Boehringer-Ingelheim), and 0.25 mg/ml levami- not analyzed immediately, the peptide pools are stable when kept sole. Sections were dehydrated and mounted in Permount (Fisher in organic buffer (40% acetonitrile/0.1%formic acid) and stored Scientific). at 280°C.

Post–Liquid Chromatography-tandem Mass Spectrometry Analysis ACKNOWLEDGMENTS Database searches (parameters in Supplemental Material) were car- ried out using Mascot version 2.5.090 with the human segment of J.B. was supported by R21 DK091729, R01DK073462, R01DK092684, Uniprot protein database (20,210 sequences; European Bioinformat- and by a March of Dimes Research Grant. K.X. was supported by F31 ics Institute, Swiss Institute of Bioinformatics, and Protein Informa- DK105799. tion Resource).

Western Blot DISCLOSURES Urine (8.3 ml) was loaded on 4%–15% SDS-polyacrylamide gel (Bio- Columbia University has issued patents and patent applications for the use of Rad Laboratories), blotted using nitrocellulose (GE Healthcare, biomarkers.

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1740 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1729–1740, 2017