Inflammation in AKI: Current Understanding, Key Questions, and Knowledge Gaps

SPECIAL ARTICLE www.jasn.org

Inﬂammation in AKI: Current Understanding, Key Questions, and Knowledge Gaps

† ‡ Hamid Rabb,* Matthew D. Grifﬁn, Dianne B. McKay, Sundararaman Swaminathan,§ | Peter Pickkers, Mitchell H. Rosner,§ John A. Kellum,¶ and Claudio Ronco,** the Acute Dialysis Quality Initiative Consensus XIII Work Group

*Division of Nephrology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland; †Regenerative Medicine Institute, School of Medicine, College of Medicine Nursing and Health Sciences, National University of Ireland, Galway, Ireland; ‡Division of Nephrology and Hypertension, Department of Medicine, University of California, San Diego, La Jolla, California; §Division of Nephrology, University of Virginia School of Medicine, Charlottesville, Virginia; |Department of Intensive Care Medicine, Radboud University Medical Center, Nijmegen, The Netherlands; ¶Center for Critical Care Nephrology and Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; and **Department of Nephrology Dialysis and Transplantation, Saint Bortolo Hospital and the International Renal Research Institute, Vicenza, Italy

ABSTRACT Inflammation is a complex biologic response that is essential for eliminating METHODS microbial pathogens and repairing tissue after injury. AKI associates with intrarenal and systemic inflammation; thus, improved understanding of the cellular and The 13th ADQI Consensus Conference molecular mechanisms underlying the inflammatory response has high potential on Therapeutic Targets of Human AKI for identifying effective therapies to prevent or ameliorate AKI. In the past decade, held in Charlottesville, Virginia in April much knowledge has been generated about the fundamental mechanisms of of 2014 (www.adqi.net) was attended by inflammation. Experimental work in small animal models has revealed many details an international group of experts and of the inflammatory response that occurs within the kidney after typical causes of focused on an objective scientific review AKI, including insights into the molecular signals released by dying cells, the role of of the current literature, developing a pattern recognition receptors, the diverse subtypes of resident and recruited consensus of opinion, with evidence immune cells, and the phased transition from destructive to reparative inflamma- where possible, to distill current litera- tion. Although this expansion of the basic knowledge base has increased the number ture and articulate a research agenda to of mechanistically relevant targets of intervention, progress in developing therapies address important unanswered ques- that improve AKI outcomes by modulation of inflammation remains slow. In this tions. Similar to other ADQI meetings, article, we summarize the most important recent developments in understanding amodified Delphi approach was fol- the inflammatory mechanisms of AKI, highlight key limitations of the commonly used lowed. Details of the methods can be animal models and clinical trial designs that may prevent successful clinical found in the summary by Okusa et al.1 application, and suggest priority approaches for research toward clinical translation in the introduction to the series. in this area. Basic Concepts of Inflammation J Am Soc Nephrol 27: 371–379, 2016. doi: 10.1681/ASN.2015030261 Inflammation is a complex response that is needed to eradicate harmful pathogens and mediate tissue repair after injury. Inflammation is a complex biologic re- identifying effective therapies to prevent sponse that is essential for eliminating or ameliorate AKI. The Acute Dialysis microbial pathogens and repairing tissue Quality Initiative Consensus (ADQI) Published online ahead of print. Publication date after diverse forms of injury. AKI is Conference XIII convened to discuss available at www.jasn.org. known to be associated with intrarenal mechanisms of AKI and identify poten- fl Correspondence: Dr. Hamid Rabb, Johns Hopkins and systemic in ammation. Improved tial therapeutic targets. This paper sum- Hospital, Ross 965, 720 Rutland Avenue, Baltimore, understanding of the cellular and mo- marizes the work of the group focus on MD 21205. Email: [email protected] fl fl lecular mechanisms underlying the in- in ammation and in ammatory mech- Copyright © 2016 by the American Society of flammatory response is important for anisms of injury. Nephrology

J Am Soc Nephrol 27: 371–379, 2016 ISSN : 1046-6673/2702-371 371 SPECIAL ARTICLE www.jasn.org

However, excess and unresolved inflam- TGF-b, IL-10, heme oxygenase 1, resolvins, with the nonimmunologic cells of the mation can promote autoimmune dis- and protectin D1) mediators by res- kidney after acute injury and the result- orders, fibrosis, and tissue damage.2 ident and recruited cell populations are ing coordinated set of inflammatory Classically, release of cytokines and important determinants of the injury responses have been the subject of inten- recruitment of neutrophils and macro- and repair phases.6 Under ideal condi- sive investigation. As outlined in the phages to the site of injury are consid- tions, a fine balance between inflamma- opening section, a detailed understand- ered the hallmark features of the early tory and anti-inflammatory factors ing of the cellular and molecular inflammatory response followed by cells ensures robust tissue repair and a return programs that dictate the course of in- of adaptive immunity at later stages. of homeostatic conditions. However, trarenal inflammation is considered to More recent data, however, suggest that AKI often results in an abnormal repair be one of the most promising pathways T cells also participate in early inflamma- process as a result of prolonged hypoxia toward developing new therapies for tory responses in AKI.3 The recruitment and sustained secretion of profibrotic cy- preventing AKI, lessening the severity of immune effector cells is facilitated by tokine (e.g., IL-13 and TGF-b1), leading of initial parenchymal damage, or en- the upregulation of adhesion molecules to post-AKI fibrosis and chronic renal hancing subsequent repair and regener- on various cell types within the kidney.4 dysfunction.6,7 ation of the kidney.6,12 Coupled to this is Subsequently, the balance between pro- As depicted in Figure 1, recent scien- the emerging concept that mechanisms and anti-inflammatory mediators signif- tific discoveries regarding the triggers, of intrarenal inflammation during AKI icantly affects the extent of tissue injury responders, and consequences of local- also exert potentially harmful effects on and repair after an acute event. Inflam- ized inflammatory responses have laid distantorgansandtissuesthroughre- mation during ischemia-reperfusion the groundwork for a more rational ap- lease of soluble mediators or re-entry (IR) has many similarities in its immune proach to identifying targets of interven- of activated leukocytes into the blood- signature to inflammation occurring in tion for AKI. However, in the clinical stream.4,13 The current knowledge response to a microbial pathogen.5 setting, far more so than in the experi- base, although exquisite in some of its Cellular damage and its associated mental setting, there is a broad range of details, is highly skewed toward experi- molecular products are thought to be key primary and secondary modifiers of the mental work carried out in rodents, and triggers for inflammation after acute archetypal genetically and epigenetically translation to human AKI is in its infancy. tissue injury.5 Within the kidney, renal predefined inflammatory responses that Furthermore, it primarily documents tubular epithelial cells are extremely sus- remain understudied. In the subsequent events occurring in previously healthy, ceptible to intrinsic oxidative stress, par- sections of this article, we first outline in nonaged kidneys suffering single episodes ticularly during the reperfusion phase of greater detail some of the important of injury and typically, addresses the role IR.2,5,6 Necrotic cells release damage– recent insights into the cellular and mo- of one target at a time— limitations that associated molecular patterns, such as lecular mechanisms of intrarenal are discussed in greater detail in subse- high–mobility group box 1, histones, inflammation during AKI. Then, we dis- quent sections. heat shock proteins, fibronectin, and cuss the current status limitations of pre- Figure 2 illustrates an idealized model biglycan into the extracellular spaces, clinical (animal model) investigation of AKI–associated renal inflammation which subsequently, activate pattern rec- and clinical trial design in this area. that has emerged from the recent pre- ognition receptors, such as toll-like re- Finally, we identify key challenges for fu- clinical literature. It provides a poten- ceptors (TLRs), and nucleotide–binding ture successful translation of basic ex- tially valuable conceptual framework oligomerization domain–like receptors, perimental work on inflammation in on which to evaluate future approaches such as the nucleotide–binding oligo- AKI and suggest how these may be ad- for staging and therapeutically targeting merization domain–,LRR–,andpyrin dressed in a more cohesive manner by episodes of AKI in human subjects. domain–containing 3 inflammasome, the research community. An initial phase of cell damage/cell expressed in epithelial and endothelial death occurs that varies in its extent and cells, dendritic cells (DCs), monocytes/ The Renal Immune System and the complexity in accordance with the specific macrophages, and lymphocytes.6–9 Acti- Phases of the Intrarenal Inflammatory nature of the insult. This phase lasts vated renal parenchyma cells and DCs Response to Acute Injury minutes to hours; may involve epithelial, also secrete chemokines, including During the past decade, a remarkable endothelial, and other renal parenchymal CXCL1, CXCL8, CCL2, and CCL5, that amount of new knowledge has been cells in some or all zones of the affected promote acute neutrophil– and monocyte/ garnered regarding the intrinsic immu- kidneys; and generates a broad range of macrophage–dependent inflamma- nologic cells of the healthy adult kidney triggering factors for acute inflammatory tory responses in AKI.6,10 Time- as well as the blood-borne counterparts response.4,7,14 Recent progress in identify- dependent changes in the expression of that transit through the renal paren- ing and elucidating the mechanisms of ac- proinflammatory (e.g.,TNF-a,IFN-g, chyma during health and disease.6,11 tion of proinflammatory cell death/cell IL-6, IL-1b, IL-23, IL-17, C3, C5a, and Not surprisingly, the interactions of damage–associated factors has been re- C5b) and anti-inflammatory (e.g., IL-4, these resident and infiltrating leukocytes markable and is now revealing many

372 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 371–379, 2016 www.jasn.org SPECIAL ARTICLE

recognition that many of the same triggers and cellular mediators that are re- sponsible for initial organ damage are also key players in a subsequent phase of repair and regeneration.7,23,24 In the experimental setting, this phase has been shown to be essential for optimal recovery of renal function after AKI and result from programmed transitions in the phenotype of immune effector cells (especially mononuclear phagocytes) involving specific alternative intracellular signaling pathways and soluble mediators.6,11,23–25 The recent progress in understanding these inherent restorative mechanisms has led to a new emphasis on harnessing them to actively promote repair and regeneration after established acute organ injury, including AKI.7,26,27 However, interventions that target earlier phases of inflammation during AKI fl Figure 1. Interactions between injury, in ammatory mediators and outcome during AKI. may bring unintended negative conse- (Upper panel) Varying types of insult instigate activation of the innate and adaptive immune quences by disrupting the role played system within the kidney. Renal dysfunction during AKI may result from the initial receptor– by the same mediators in the transition mediated triggers as well the subsequent cellular responses and various secondary se- fl quelae. (Lower panel) In the clinical setting, the nature and outcome of the inflammatory from proin ammatory to prorepair 23 response in AKI are dictated by not only an archetypal genome–encoded response pro- mechanisms (Figure 2). gram but also, primary and secondary modifiers (including therapeutic interventions), which have received less attention in experimental studies and must be taken into con- Targets for Prevention and sideration in the design of clinical trials of inflammation-modifying therapies. DAMP, Treatment of Renal Inflammation damage–associated molecular pattern; PAMP, pathogen–associated molecular pattern; Associated with AKI PRRs, pattern recognition receptors. As discussed above, experimental models of AKI have shown common inflamma- novel potential therapeutic targets for pre- the production of archetypal proinflam- tory triggers involving epithelial cells, vention or very early treatment of AKI.7,14 matory mediators (cytokines, chemokines, endothelial cells, resident inflammatory Overlapping with this initial phase and enzymes, free radical species, and lipid me- cells, and infiltrating leukocytes. These lasting hours to days is a phase of acute diators) with high cytotoxic potential, inflammatory triggers initiate localized inflammation that is mediated primarily whichtendtoamplifyandextendcell and systemic responses through recep- by immune cells. Among the important damage and cell death.4,6 In this manner, tors and cells of the innate and adaptive immediate responders is the abundant the phase of acute inflammatory response immune systems.6 The innate immune network of resident mononuclear phago- may be seen as consolidating and worsen- system is a phylogenically conserved early cyticcells(oftensubdividedintorenalDCs ing organ dysfunction after injury. Thus, defense system activated by families of and macrophages), which is juxtaposed improved understanding of its cellular and membrane-bound and cytoplasmic re- between the tubular epithelium and the molecular components from preclinical ceptors that are exquisitely sensitive to peritubular capillaries within the intersti- models represents another significant molecules released from pathogens or tial space.6,11 However, responses of means to identify targets of intervention dead/dying cells. Several families of innate almost equal rapidity occur from the for limiting organ damage in the early immune receptors are constitutively ex- bloodstream involving endothelial adher- stages of AKI. It has become recognized, pressed in the kidney, and experimental ence and activation of neutrophils and however, that the acute inflammatory re- data have clearly shown activation of these monocytes as well as intraparenchymal sponse incorporates counter-regulatory receptors to be associated with some of the infiltration by neutrophils, monocytes, ef- components, such as recruited regula- earliest events of AKI. In preclinical studies, fector memory T cells, and other less tory T cells (Tregs) and induced anti– blockade of several of the innate immune frequent myeloid and lymphoid effec- inflammatory cytokines (e.g., IL-10), which receptors (e.g.,TLR2,28 nucleotide–binding tors.6,15–19 The initial activation–induced are also amenable to manipulation.20–22 oligomerization domain–,LRR–,andpyrin responses of these resident and recruited Among the most striking advances domain–containing 3, and nucleotide– immune cells are typically dominated by in knowledge of the past decade is the binding oligomerization domain 229,30)

J Am Soc Nephrol 27: 371–379, 2016 Inﬂammation in AKI 373 SPECIAL ARTICLE www.jasn.org

healthy cells, thereby transducing death signals to the nearby healthy cells and propagating the local tissue injury.38 Ex- perimental data from many laboratories show that parenchymal cell injury can be ameliorated by blockade of membrane– bound or intracytoplasmic innate immune receptors. Rodent models show that these receptors are rapidly activated after renal artery occlusion, leading to a cascade of intracellular signaling events that ultimately cause apoptosis and ne- crosis of the triggered cells and the production of proinflammatory molecules. Many laboratories have shown in rodent models that interfering with the triggering of pattern recognition receptors prevents the expansion of renal tubular cell Figure 2. Types of inflammatory responses during AKI. An idealized schematic is shown of injury.6,8,28 the phases of inflammation that occur within the healthy kidney after acute insults on the Other concepts that require better basis of current understanding from preclinical (mostly rodent) models. Initial cell damage definition to develop clinically relevant fl and death trigger, over minutes to hours, a primary acute in ammatory response involving therapeutics involve the soluble media- fi resident and in ltrating leukocytes. This phase, if appropriately regulated, evolves over tors of cellular injury, which may be several days into a phase of active repair and regeneration, which is dependent on regu- produced locally (within the injured latory and reprogrammed leukocyte subsets. As indicated in the lower panel, molecular and cellular details of the early and later phases of the inflammatory process provide specific kidney) or systemically. Cytokines and therapeutic target opportunities, but strategies that involve blocking/inhibiting elements of chemokines produced locally recruit a the acute inflammatory response may, paradoxically, introduce a risk of disrupting the cascade of inflammatory effector cells natural transition to the repair phase if improperly timed. into injured tissue, and experimental blockade of several cytokines and chemokines can abrogate AKI.39 Cytokines has been shown to prevent experimental expected in the future. Multiple inflam- and chemokines lead to upregulation of AKI in rodent models. Blockade of TLR2, matory and parenchymal cell types are adhesion molecules on endothelial cells, however, has recently been tested in hu- involved in AKI, and in fact, an impor- such as intercellular adhesion molecule- mansandisinphase2trialsintheUnited tant epithelial-endothelial axis exists 1, P-selectin, and E-selectin, and these States and Europe (OPN-305; Opsona that promotes the infiltration of inflam- adhesion molecules are essential for the Therapeutics).31 matory cells after initial injury.38 Be- recruitment of leukocytes into areas of Virtually all immune cells have been causetherearesomanydifferentcell injury.6 Thus, there has been much in- implicated in AKI, and some are thought types involved in the inflammatory re- terest in adhesion molecule blockade in to be deleterious (e.g., neutrophils,10,16 sponse and because different inflamma- AKI as a way to interrupt the epithelial- monocytes/macrophages,25,32 DCs,33,34 tory cells contribute to injury and repair, endothelial axis of injury.38,39 NKT cells,17,35 NK cells,19 and B it is very important to be mindful of the Molecules generated within injured cells18,36), whereas others are likely pro- phenotype and kinetics of the inflam- cells have also been recently identified as tective (e.g., Tregs20,21). Others, such as matory response in designing effective potential targets to abrogate inflamma- macrophages, play different roles depend- anti–inflammatory approaches to AKI tion.40 For example, the cysteine prote- ing on the type and time at which they (Figure 2). ases (calpains and caspases) play a role in arrive in the injured tissue.25 M1 macro- Experimental data suggest that it is hypoxia-induced injury in proximal tu- phages contribute to inflammation also very important to consider paren- bular cells, and mitochondrial mediators and tissue injury in the injury phase, chymal cells in the design of anti- are gaining interest as potential anti– whereas M2 macrophages exert anti- inflammatory approaches, because they inflammatory targets in several cell types. inflammatory functions in postischemic can play an essential role in the earliest Inhibitors of mitochondrial permeability kidneys and facilitate renal tubular regen- phases of AKI. Injured parenchymal transition pore opening, quinone analogs, eration during the recovery phase.37 At- cells, such as renal tubular epithelial cells superoxide dismutase mimetics, Szeto– tempts have been made to enhance Tregs and endothelial cells, release damage– Schiller peptides, and mitochondrial using IL-2 complexes in mice,22 and clin- associated molecular patterns that trig- division inhibitors have already shown ical translation of this strategy might be ger innate immune receptors on nearby promise in diverse models of AKI, and

374 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 371–379, 2016 www.jasn.org SPECIAL ARTICLE there have already been limited clinical injury.45 There are fundamental structural For example, CLP-induced sepsis lacks the trials of mitochondrial-targeted thera- and functional differences between the hyperdynamic phase seen in human sepsis pies.40 Finally, microRNAs generated in mouse and human kidney that may influ- and is associated with early lymphocyte ap- injured renal tubular cells are also gaining ence susceptibility to AKI as well as the optosis, which is usually a feature of the interest as potential targets.41,42 degree and nature of inflammatory re- recovery phase of human sepsis.50,51 Fur- In summary, a large and rapidly ex- sponses. (1)Expressionofsomemolecu- thermore, CLP does not reproducibly panding array of inflammatory cell types lar markers that determine susceptibility result in kidney injury. The absence of and molecular mediators has been im- to ischemic injury and inflammation dif- sepsis-associated comorbidities that are plicated in the pathogenesis of AKI, and fers substantially between mouse and hu- common in human patients and known data from rodent models have clearly man kidneys. As an example, tissue factor, to affect the immune system (e.g.,old shown that deletion, blockade, or en- expressed by the renal tubules in mice, is age, diabetes mellitus, and CKD) is also a hancement of many of these can improve not strongly expressed by the human kid- limiting factor that needs to be considered experimental outcomes. Before rational ney.46,47 (2) Human blood is neutrophil in translating findings derived from the targets are developed, however, a better predominant, whereas mouse blood is current models.52 It has been reported understanding is needed of the putative lymphocyte predominant. (3) Mouse se- that the CD-1 outbred strain of mice may cell types involved in injury and repair, rum has factors that inhibit TNF-a be more susceptible to sepsis-induced AKI the dynamic interactions between pa- expression and production, and thus, it and as a result of greater genetic heteroge- renchymal and immune cells, and the limits systemic proinflammatory re- neity, constitute a more clinically relevant signals that amplify injurious responses sponse.47 In this regard, greater use of pig model of human disease. The baboon and repair. It is imperative that future models of AKI might be advantageous, be- model of sepsis, induced by injecting live studies define these important events to cause they may more closely resemble gram–negative (Escherichia coli) bacteria, allow pharmacologic development of the human anatomy, physiology, immune could also be considered as a useful rationale targets to prevent or treat AKI. system, and susceptibility to ischemic model, because its immune system more AKI.48,49 As an example, renal medullary closely resembles that of humans, and Limitations and Pitfalls of Animal thicknessinpigsisclosetothatofahuman sepsis in this species has been shown to Models to Study Inflammation in AKI kidney. Thus, pig models may be particu- be associated with a hyperdynamic state.53 Rodent models of IR, sepsis, and toxin– larly useful in examining therapeutic tar- An ideal CLP model should include treat- induced kidney injury are the commonly gets identifiedinrodentsasameanstoward ment with fluid and antibiotics to more used approaches to study the pathophys- selecting the most promising candidates closely mimic the intensive care unit iology of AKI and examine novel thera- for translation into human trials. The (ICU) scenario.50 However, supportive peutic approaches. The risk of severe well recognized limitations to the wider treatment with fluids and antibiotics is AKI is substantially increased by the pres- use of pig models of AKI include the cost, quite variable across laboratories and ence of preexisting CKD, diabetes mellitus, specialized expertise, and facilities that are many times, inadequate. hypertension, and proteinuria. Develop- necessary for them to be established. ing animal models of renal ischemia su- Toxin-Induced Models of AKI perimposed on diabetes, hypertension, Animal Models of Sepsis-Induced AKI The advantages of the most commonly and/or CKD would likely advance our un- An ideal animal model of sepsis would used nephrotoxic model of AKI, cisplatin- derstanding of AKI pathogenesis.43,44 Al- mimic hemodynamic, inflammatory, induced nephrotoxicity, include its tech- though current models have substantially pathophysiologic, and clinical features nicalsimplicity,itsclinicalrelevancetoone contributed to our current understanding, as well the course and outcome of human specific form of AKI in human patients, there are several important limitations to sepsis. Commonly used models include and its amenability to analyzing individual their use in the study of inflammation and injection of bacterial toxin, such as LPS, immunologic cell types and mediators. therapeutic targeting of inflammation in injection of bacteria, and perforation of There are also significant disadvantages, human AKI. the colon to induce fecal peritonitis. however, and these include variable re- There are several limitations to the use producibility of kidney injury and the Renal IR Injury of rodent LPS injection (endotoxemia) as fact that the inflammatory mechanisms There are remarkable sex- and strain- a model of human sepsis. In the first place, involved differ substantially from those of related differences in immune responses mice are less sensitive to LPS than humans. more common clinical causes of AKI. that affect susceptibility to AKI. For Furthermore, although mice exhibit earlier As an example, CD11c+ DCs have been example, C57BL/6 (B6) mice have a andhighercytokineresponsethanhumans, shown to enhance renal inflammation T–helper subset 1–predominant im- certain pathogenic features of LPS toxicity, and exacerbate renal injury in renal IR mune system and are highly susceptible including kidney injury, are ameliorated by in the mouse, whereas in cisplatin- to AKI, whereas BALB/C mice have a simple volume replacement.50 The cecal li- induced AKI, depletion of CD11c+ DCs T–helper subset 2–predominant system gation and puncture (CLP) model also has is reported to induce greater neutrophil and are more resistant to ischemic tissue substantial differences from human sepsis. accumulation and worsen renal injury.54

J Am Soc Nephrol 27: 371–379, 2016 Inﬂammation in AKI 375 SPECIAL ARTICLE www.jasn.org

It is important to be cautious when treatment. For example, measurement clinical end points, such as patient sur- interpreting pathophysiologic findings of known markers of AKI could facilitate vival, length of hospital stay, and RRT- and therapeutic targets derived from early detection and enrollment, thus im- free days. Especially for relatively small the existing animal models of AKI. Ro- proving the ability to administer a trial phase 2 trials with compounds that show dent cisplatin AKI models may not intervention within the window of ther- promising data in animal studies, a com- exactly recapitulate human disease; un- apeutic opportunity in patients with bination of study drug–induced effects like patients treated with cisplatin who subclinical AKI. Regarding chosen end on these end points may facilitate the de- undergo repeat dosing with very low points, it has become clear that, with sign of larger trials.61 short–term mortality, rodent models are more sophisticated RRTs, short-term Currently, a cutoff level of 25% or usually short term and require high-dose (e.g., 28 days) survival is not an adequate 50% decrease in GFR is the most com- cisplatin, which has a high short–term outcome measure to determine whether monly used outcome measure in clinical mortality. Development of animal mod- an investigational compound is effective trials of intervention for AKI, and this els that manifest comorbidity and risk (i.e., renoprotective) in this specific degree of GFR decrease is accepted by the factors commonly known to affect the group of patients. Current Food and FDA and the EMEA as a clinical end immune response and increase the risk Drug Administration (FDA) require- point. An alternative end point with of human AKI is imperative to increase ments for phase 3 AKI trials are clinical relevance, however, is require- the clinical applicability of such observa- patient–centered clinical outcomes, ment for initiation of RRT. A therapeutic tions. In addition to the more wide- such as death, need for acute dialysis, compound that results in prevention of spread confirmation of key findings in myocardial infarction, stroke, and all– renal damage, more swift recovery of large animal models, the application of cause nonelective hospitalization, which renal function, or protection of the other novel approaches, such as the use usually require a larger number of trial kidneys from additional damage is likely of humanized mice, in which immuno- participants. Therefore, additional dis- to result in a lower proportion of treated deficient mice are reconstituted with hu- cussion with the FDA is required to clar- patients requiring RRT. However, only man immune system, hold promise for ify the importance of and qualification patients that suffer the most severe forms experimentally testing the many target- pathway for short-term changes in renal of AKI are likely to be treated with RRT, ing strategies that have arisen from ro- function and other alternative out- implying that many more patients are dent model experiments. Development comes. For example, future studies needed to conduct a trial for which the of national and international human might examine the effect of short-term primary end point is RRT-free days. AKI tissue repositories will be also of great increases in serum creatinine on long– Importantly, in studies conducted in value for more precisely documenting the term clinical outcomes as an alternative several thousands of patients, these issues course and progression of tissue injury to a hard clinical outcome.57 It is now will not influence interpretation. How- and inflammation in human AKI. known that AKI acquired in the ICU ever, in phase 2 trials involving several has important long–term sequelae, in- dozens to a few hundred patients, out- Study Design Considerations and cluding an increased risk for ESRD.58 come analyses on the basis of GFR as a Challenges for Clinical Trials in AKI However, both the number of patients continuous variable rather than a 25% or Optimally designed clinical trials will be and the duration of follow-up required 50% decrease in GFR would increase essential for determining the therapeutic to address end points, such as ESRD, sensitivity and specificity. Decline in GFR value of interventions that target inflam- make it unlikely that pharmacologic may significantly affect how drugs are matory response in the setting of AKI. interventional trials with sufficient sta- metabolized and how patients can man- Unfortunately, the vast majority of com- tistical power to show effects on these age fluid load. The need for dialysis pleted clinical trials related to prevention long-term consequences of ICU- requires well defined criteria, because or treatment of AKI have been negative. acquired AKI will be conducted in the in some cases, patientsmaynot be offered This may well be related to the above– near future. Therefore, the primary aim dialysis because of the burden of comor- described complexity, multifactorial na- of clinical research in AKI should be to bidity and perceived futility, dialysis may ture, and dynamic progression of AKI as determine whether a new intervention be refused by patients or family members well as our inability to determine the actually ameliorates inflammation– on other grounds, or patients may die phase that the patient is in and the extent associated kidney tissue damage during before dialysis is initiated. Furthermore, of tissue injury that has already oc- the patient’s clinical stay. For this reason, because the event rate is critical for power curred.55,56 However, study design issues measurement of markers of renal injury analyses for clinical trials and often on may also have played a significant role in (e.g., urinary excretion of markers of tu- the basis of historical data, in addition to the negative outcomes of clinical trials. bular damage or cell arrest59 and func- inclusion and exclusion criteria during In the case of both patient inclusion and tional assessment, such as endogenous trial planning, adaptive trial design could chosen end points, refinements to cur- creatinine clearance or iohexol clear- offer a useful strategy for phase 2 clinical rent trial approaches may improve the ance60) may provide more refined evi- trials.56,57 Furthermore, in view of the sensitivity to detect beneficial effects of dence of efficacy compared with hard long–term clinical consequences of

376 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 371–379, 2016 www.jasn.org SPECIAL ARTICLE

ICU-acquired AKI,58,62 more attention (3) The current lack of an effective in- (6) Data and biologic samples from com- should be given to relatively small dete- tervention to treat AKI is partly the pleted negative sepsis trials could riorations in renal function, mainly by result of insensitive trial designs. be reanalyzed for evidence of positive clinicians and trialists. It is recognized, effects on renal inflammation and re- of course, that obtaining reliable mark- nal injury. ers and accurate assessment of renal FUTURE GUIDANCE function in the setting of AKI remains (7) In addition to the design of trials in- challenging. New techniques, such as (1) Knowledge acquired in idealized ro- volving novel inflammation–modifying contrast-enhanced ultrasonography, dent models must be critically evalu- agents, emerging knowledge of the which enables the quantification of renal ated in more clinically relevant animal mediators of inflammation in AKI tissue perfusion, could be of distinct models and directly investigated in should prompt new clinical trials of value in future trials. Bedside assessment human subjects with AKI using new older drugs that may be successfully of regional renal blood flow early in the imaging and biospecimen analysis repurposed for AKI prevention or course of AKI could be a valuable diag- technologies. Because there is limited therapy. fl nostic and prognostic tool,63 although knowledge on human in ammatory validation studies in patients in the response during AKI, it is imperative (8) Future studies should also try to fl – ICU to determine if this technique can to identifying animal models that identify novel in ammation driven fl facilitate the early diagnosis of AKI and best represent human in ammatory biomarkers to assist in identifying guide the use of therapeutic interven- response. and tracking drug reactions in clini- tions that target microcirculatory perfu- cal trials. (2) Greater emphasis could be placed sion have yet to be performed. on using current knowledge of As described in this article, many inflammation to develop and test potential targets related to inflammation ACKNOWLEDGMENTS multitargeted approaches aimed at have been identified, and for several of identifying synergistic combinations them, drugs have already been devel- A complete list of participants in the Acute of interventions in AKI. oped. In addition, there are older drugs Dialysis Quality Initiative Consensus XIII that may have new applications in the (3) Research focused on identifying and Work Group is provided in the Supplemental prevention or treatment of AKI. In that measuring inflammatory phases in Appendix. regard, it is important to realize that, for the setting of human AKI is needed to many clinical trials in sepsis that did not appropriately target inflammation- show an effect on overall mortality, a modifying therapies and identify op- DISCLOSURES fi bene cial effect on the kidney may have timal times to start and stop such The following authors report no conflicts of 64 been missed. We would advocate therapies. Additionally, great empha- interest: H.R., M.D.G., D.B.M., S.S., M.H.R., and strongly, therefore, for the determina- sis should be given to comparative C.R. P.P. is the principal investigator of the Safety, – fi Tolerability, Efficacy and QoL Study of Human tion of kidney speci c end points, espe- analysis regarding the nature and ki- recAP in the Treatment of Patients with SA-AKI cially in sepsis trials, because sepsis netics of inflammatory response in represents a major cause of AKI. Trial (NCT02182440) that investigates the effects of humans and different experimental recombinant alkaline phosphatase (AP) in patients models of AKI. with sepsis-associated AKI and has received re- imbursement for travel costs and consultancy fees KEY CONCLUSIONS (4) International collaboration to develop from AM-Pharma (Amsterdam, The Netherlands), a shared resource of tissue samples the producer of recombinant AP. J.A.K. has received (1) Recent preclinical animal experiments from human subjects with AKI would consulting fees from Abbott Laboratories (Chicago, have more accurately documented the be of high value for selecting the most IL), Alere (San Diego, CA), Alung (Pittsburgh, PA), phases of inflammation after AKI promising inflammation–related ther- AM-Pharma (Amsterdam, The Netherlands), Astute along with many novel cellular and apeutic targets. Medical (San Diego, CA), Atox Bio (Ness Ziona, molecular details. However, the ap- Israel), Baxter (Chicago, IL), Cytosorbents (Princeton, plicability of this knowledge to human (5) Trial designs in AKI should combine NJ), Grifols (Barcelona, Spain), Roche (Basel, Switzerland) and Spectral Diagnostics (Toronto, AKI remains poorly understood. renal injury, renal function, and clinical end points. Especially for phase 2 Canada), J.A.K. has also received research grants from Alere (San Diego, CA), Astute Medical (San (2) Preclinical experiments have typically clinical trials, the focus to detect ben- fi fi Diego, CA), Atox Bio (Ness Ziona, Israel), Bard focused on de ning the roles of single e cial renal effects should shift from (Covington, GA), Baxter (Chicago, IL), Cytosorbents mechanistic pathways, cell types, or clinical end points, including mortal- (Princeton, NJ), Grifols (Barcelona, Spain), and molecular mediators in young, ge- ity, to renal damage and renal function Spectral Diagnostics (Toronto, Canada) and has netically homogenous animals under end points, such as kidney perfusion licensed technologies through the University of idealized circumstances. and actual GFR. Pittsburgh (Pittsburgh, PA) to Astute Medical

J Am Soc Nephrol 27: 371–379, 2016 Inﬂammation in AKI 377 SPECIAL ARTICLE www.jasn.org

(San Diego, CA), Cytosorbents (Princeton, NJ), and 16. Awad AS, Rouse M, Huang L, Vergis AL, 29. Shigeoka AA, Mueller JL, Kambo A, Spectral Diagnostics (Toronto, Canada). Reutershan J, Cathro HP, Linden J, Okusa Mathison JC, King AJ, Hall WF, Correia JS, MD: Compartmentalization of neutrophils in Ulevitch RJ, Hoffman HM, McKay DB: the kidney and lung following acute ischemic An inflammasome-independent role for REFERENCES kidney injury. Kidney Int 75: 689–698, 2009 epithelial-expressed Nlrp3 in renal ische- 17. Li L, Huang L, Sung SS, Lobo PI, Brown MG, mia-reperfusion injury. J Immunol 185: – 1. Okusa MD, Rosner MH, Kellum JA, Ronco C: Gregg RK, Engelhard VH, Okusa MD: NKT cell 6277 6285, 2010 Acute Dialysis Quality Initiative XIII Workgroup: activation mediates neutrophil IFN-gamma 30. Shigeoka AA, Kambo A, Mathison JC, King Therapeutic Targets of Human AKI: Harmoniz- production and renal ischemia-reperfusion AJ, Hall WF, da Silva Correia J, Ulevitch RJ, – ing Human and Animal AKI. J Am Soc Nephrol injury. JImmunol178: 5899 5911, 2007 McKay DB: Nod1 and nod2 are expressed in 27: 371–379, 2016 18. Renner B, Strassheim D, Amura CR, Kulik L, human and murine renal tubular epithelial 2. Medzhitov R: Inflammation 2010: New adven- Ljubanovic D, Glogowska MJ, Takahashi K, cells and participate in renal ischemia re- Carroll MC, Holers VM, Thurman JM: B cell perfusion injury. J Immunol 184: 2297–2304, tures of an old flame. Cell 140: 771–776, 2010 3. Linfert D, Chowdhry T, Rabb H: Lymphocytes subsets contribute to renal injury and renal 2010 protection after ischemia/reperfusion. JIm- 31. Reilly M, Miller RM, Thomson MH, Patris V, and ischemia-reperfusion injury. Transplant munol 185: 4393–4400, 2010 Ryle P, McLoughlin L, Mutch P, Gilboy P, Rev (Orlando) 23: 1–10, 2009 19. Zhang ZX, Wang S, Huang X, Min WP, Sun H, Miller C, Broekema M, Keogh B, McCormack 4. Ratliff BB, Rabadi MM, Vasko R, Yasuda K, Liu W, Garcia B, Jevnikar AM: NK cells induce W, van de Wetering de Rooij J: Randomized, Goligorsky MS: Messengers without borders: apoptosis in tubular epithelial cells and double-blind, placebo-controlled, dose- Mediators of systemic inflammatory response contribute to renal ischemia-reperfusion in- escalating phase I, healthy subjects study of in AKI. J Am Soc Nephrol 24: 529–536, 2013 jury. J Immunol 181: 7489–7498, 2008 intravenous OPN-305, a humanized anti- 5. Chen GY, Nuñez G: Sterile inflammation: 20. Gandolfo MT, Jang HR, Bagnasco SM, Ko TLR2 antibody. Clin Pharmacol Ther 94: 593– Sensing and reacting to damage. Nat Rev GJ, Agreda P, Satpute SR, Crow MT, King LS, 600, 2013 Immunol 10: 826–837, 2010 Rabb H: Foxp3+ regulatory T cells partici- 32. Day YJ, Huang L, Ye H, Linden J, Okusa MD: 6. Kurts C, Panzer U, Anders HJ, Rees AJ: The pate in repair of ischemic acute kidney injury. Renal ischemia-reperfusion injury and aden- immune system and kidney disease: Basic Kidney Int 76: 717–729, 2009 osine 2A receptor-mediated tissue pro- concepts and clinical implications. Nat Rev 21. Kinsey GR, Sharma R, Huang L, Li L, Vergis tection: Role of macrophages. Am J Physiol Immunol 13: 738–753, 2013 AL, Ye H, Ju ST, Okusa MD: Regulatory T Renal Physiol 288: F722–F731, 2005 7. Anders HJ, Schaefer L: Beyond tissue injury- cells suppress innate immunity in kidney 33. Bajwa A, Huang L, Ye H, Dondeti K, Song S, damage-associated molecular patterns, toll- ischemia-reperfusion injury. J Am Soc Nephrol Rosin DL, Lynch KR, Lobo PI, Li L, Okusa MD: like receptors, and inflammasomes also drive 20: 1744–1753, 2009 Dendritic cell sphingosine 1-phosphate regeneration and fibrosis. J Am Soc Nephrol 22. Kim MG, Koo TY, Yan JJ, Lee E, Han KH, receptor-3 regulates Th1-Th2 polarity in kid- 25: 1387–1400, 2014 Jeong JC, Ro H, Kim BS, Jo SK, Oh KH, Surh ney ischemia-reperfusion injury. J Immunol 8. Kim HJ, Lee DW, Ravichandran K, O Keys D, CD, Ahn C, Yang J: IL-2/anti-IL-2 complex 189: 2584–2596, 2012 Akcay A, Nguyen Q, He Z, Jani A, Ljubanovic attenuates renal ischemia-reperfusion injury 34. Li L, Huang L, Ye H, Song SP, Bajwa A, Lee SJ, fl D, Edelstein CL: NLRP3 in ammasome through expansion of regulatory T cells. JAm Moser EK, Jaworska K, Kinsey GR, Day YJ, knockout mice are protected against ischemic Soc Nephrol 24: 1529–1536, 2013 Linden J, Lobo PI, Rosin DL, Okusa MD: but not cisplatin-induced acute kidney injury. J 23. Zhang MZ, Yao B, Yang S, Jiang L, Wang S, Dendritic cells tolerized with adenosine A₂AR – Pharmacol Exp Ther 346: 465 472, 2013 Fan X, Yin H, Wong K, Miyazawa T, Chen J, agonist attenuate acute kidney injury. JClin 9. Vallés PG, Lorenzo AG, Bocanegra V, Vallés Chang I, Singh A, Harris RC: CSF-1 signaling Invest 122: 3931–3942, 2012 R: Acute kidney injury: What part do toll-like mediates recovery from acute kidney injury. J 35. Yang SH, Lee JP, Jang HR, Cha RH, Han SS, receptors play? Int J Nephrol Renovasc Dis 7: Clin Invest 122: 4519–4532, 2012 Jeon US, Kim DK, Song J, Lee DS, Kim YS: – 241 251, 2014 24. Kulkarni OP, Hartter I, Mulay SR, Hagemann Sulfatide-reactive natural killer T cells abro- 10. Bolisetty S, Agarwal A: Neutrophils in acute J, Darisipudi MN, Kumar Vr S, Romoli S, gate ischemia-reperfusion injury. JAmSoc kidney injury: Not neutral any more. Kidney Thomasova D, Ryu M, Kobold S, Anders HJ: Nephrol 22: 1305–1314, 2011 – Int 75: 674 676, 2009 Toll-like receptor 4-induced IL-22 acceler- 36. Jang HR, Gandolfo MT, Ko GJ, Satpute SR, fi 11. Nelson PJ, Rees AJ, Grif n MD, Hughes J, ates kidney regeneration. J Am Soc Nephrol Racusen L, Rabb H: B cells limit repair after fi Kurts C, Duf eld J: The renal mononuclear 25: 978–989, 2014 ischemic acute kidney injury. JAmSoc phagocytic system. JAmSocNephrol23: 25. Lee S, Huen S, Nishio H, Nishio S, Lee HK, Nephrol 21: 654–665, 2010 194 –203, 2012 Choi BS, Ruhrberg C, Cantley LG: Distinct 37. Jang HR, Rabb H: Immune cells in experi- 12. Bajwa A, Kinsey GR, Okusa MD: Immune macrophage phenotypes contribute to kid- mental acute kidney injury. Nat Rev Nephrol mechanisms and novel pharmacological ney injury and repair. JAmSocNephrol22: 11: 88–101, 2015 therapies of acute kidney injury. Curr Drug 317–326, 2011 38. Molitoris BA: Therapeutic translation in acute Targets 10: 1196–1204, 2009 26. Bonventre JV, Yang L: Cellular pathophysi- kidney injury: The epithelial/endothelial axis. 13.GramsME,RabbH:Thedistantorganeffectsof ology of ischemic acute kidney injury. JClin JClinInvest124: 2355–2363, 2014 acute kidney injury. Kidney Int 81: 942–948, 2012 Invest 121: 4210–4221, 2011 39. Yalavarthy R, Edelstein CL: Therapeutic and 14. Linkermann A, Chen G, Dong G, Kunzendorf 27. Kinsey GR, Li L, Okusa MD: Inflammation in predictive targets of AKI. Clin Nephrol 70: U, Krautwald S, Dong Z: Regulated cell death acute kidney injury. Nephron, Exp Nephrol 453–463, 2008 in AKI. JAmSocNephrol25: 2689–2701, 109: e102–e107, 2008 40. Tábara LC, Poveda J, Martin-Cleary C, Selgas 2014 28. Shigeoka AA, Holscher TD, King AJ, Hall FW, R, Ortiz A, Sanchez-Niño MD: Mitochondria- 15. Carlin LM, Stamatiades EG, Auffray C, Hanna Kiosses WB, Tobias PS, Mackman N, McKay targeted therapies for acute kidney injury. RN, Glover L, Vizcay-Barrena G, Hedrick CC, DB: TLR2 is constitutively expressed within Expert Rev Mol Med 16: e13, 2014 Cook HT, Diebold S, Geissmann F: Nr4a1- the kidney and participates in ischemic renal 41. Lorenzen JM, Kaucsar T, Schauerte C, dependent Ly6C(low) monocytes monitor injury through both MyD88-dependent and Schmitt R, Rong S, Hübner A, Scherf K, endothelial cells and orchestrate their dis- -independent pathways. JImmunol178: Fiedler J, Martino F, Kumarswamy R, Kölling posal. Cell 153: 362–375, 2013 6252–6258, 2007 M, Sörensen I, Hinz H, Heineke J, van Rooij

378 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 371–379, 2016 www.jasn.org SPECIAL ARTICLE

E, Haller H, Thum T: MicroRNA-24 antagonism puncture model. Am J Pathol 165: 1433– 58. Gammelager H, Christiansen CF, Johansen prevents renal ischemia reperfusion injury. J 1446, 2004 MB, Tønnesen E, Jespersen B, Sørensen HT: Am Soc Nephrol 25: 2717–2729, 2014 52. Leelahavanichkul A, Huang Y, Hu X, Zhou H, Five-year risk of end-stage renal disease 42. Lee CG, Kim JG, Kim HJ, Kwon HK, Cho IJ, Tsuji T, Chen R, Kopp JB, Schnermann J, among intensive care patients surviving Choi DW, Lee WH, Kim WD, Hwang SJ, Choi Yuen PS, Star RA: Chronic kidney disease dialysis-requiring acute kidney injury: A na- S, Kim SG: Discovery of an integrative net- worsens sepsis and sepsis-induced acute tionwide cohort study. Crit Care 17: R145, work of microRNAs and transcriptomics kidney injury by releasing High Mobility 2013 changes for acute kidney injury. Kidney Int Group Box Protein-1. Kidney Int 80: 1198– 59. Bihorac A, Chawla LS, Shaw AD, Al-Khafaji A, 86: 943–953, 2014 1211, 2011 Davison DL, Demuth GE, Fitzgerald R, Gong 43. Anders H, Schlöndorff D: Murine models of 53. Kinasewitz GT, Chang AC, Peer GT, Hinshaw MN, Graham DD, Gunnerson K, Heung M, renal disease: Possibilities and problems in LB, Taylor FB Jr.: Peritonitis in the baboon: A Jortani S, Kleerup E, Koyner JL, Krell K, studies using mutant mice. Exp Nephrol 8: primate model which stimulates human Letourneau J, Lissauer M, Miner J, Nguyen 181–193, 2000 sepsis. Shock 13: 100– 109, 2000 HB, Ortega LM, Self WH, Sellman R, Shi J, 44. Becker GJ, Hewitson TD: Animal models of 54. Tadagavadi RK, Reeves WB: Renal dendritic Straseski J, Szalados JE, Wilber ST, Walker chronic kidney disease: Useful but not per- cells ameliorate nephrotoxic acute kidney MG, Wilson J, Wunderink R, Zimmerman J, fect. Nephrol Dial Transplant 28: 2432– injury. JAmSocNephrol21: 53–63, 2010 Kellum JA: Validation of cell-cycle arrest 2438, 2013 55. Jo SK, Rosner MH, Okusa MD: Pharmacologic biomarkers for acute kidney injury using 45. Kennedy SE, Erlich JH: Murine renal ischae- treatment of acute kidney injury: Why drugs clinical adjudication. Am J Respir Crit Care mia-reperfusion injury. Nephrology (Carlton) haven’t worked and what is on the horizon. Clin Med 189: 932–939, 2014 13: 390–396, 2008 J Am Soc Nephrol 2: 356–365, 2007 60.MacedoE,MehtaRL:Measuringrenalfunc- 46. Luther T, Flössel C, Mackman N, Bierhaus A, 56. Molitoris BA, Okusa MD, Palevsky PM, tion in critically ill patients: Tools and strate- Kasper M, Albrecht S, Sage EH, Iruela-Arispe Chawla LS, Kaufman JS, Devarajan P, Toto gies for assessing glomerular filtration rate. L, Grossmann H, Ströhlein A, Zhang Y, RM, Hsu CY, Greene TH, Faubel SG, Kellum CurrOpinCritCare19: 560–566, 2013 Nawroth PP, Carmeliet P, Loskutoff DJ, JA, Wald R, Chertow GM, Levin A, Waikar SS, 61. Pickkers P, Heemskerk S, Schouten J, Laterre Müller M: Tissue factor expression during Murray PT, Parikh CR, Shaw AD, Go AS, PF, Vincent JL, Beishuizen A, Jorens PG, human and mouse development. Am J Chinchilli VM, Liu KD, Cheung AK, Weisbord Spapen H, Bulitta M, Peters WH, van der Pathol 149: 101–113, 1996 SD, Mehta RL, Stokes JB, Thompson AM, Hoeven JG: Alkaline phosphatase for treat- 47. Mestas J, Hughes CC: Of mice and not men: Thompson BT, Westenfelder CS, Tumlin JA, ment of sepsis-induced acute kidney injury: A Differences between mouse and human im- Warnock DG, Shah SV, Xie Y, Duggan EG, prospective randomized double-blind placebo- munology. J Immunol 172: 2731–2738, 2004 Kimmel PL, Star RA: Design of clinical trials in controlled trial. Crit Care 16: R14, 2012 48. Giraud S, Favreau F, Chatauret N, Thuillier R, AKI: A report from an NIDDK workshop. Tri- 62. Liotta M, Olsson D, Sartipy U, Holzmann MJ: Maiga S, Hauet T: Contribution of large pig als of patients with sepsis and in selected Minimal changes in postoperative creatinine for renal ischemia-reperfusion and trans- hospital settings. Clin J Am Soc Nephrol 7: values and early and late mortality and car- plantation studies: The preclinical model. J 856–860, 2012 diovascular events after coronary artery by- Biomed Biotechnol 2011: 532127, 2011 57. Okusa MD, Molitoris BA, Palevsky PM, pass grafting. Am J Cardiol 113: 70–75, 2014 49. Lieberthal W, Nigam SK: Acute renal failure. Chinchilli VM, Liu KD, Cheung AK, Weisbord 63. Kalantarinia K, Okusa MD: Ultrasound con- II. Experimental models of acute renal failure: SD, Faubel S, Kellum JA, Wald R, Chertow trast agents in the study of kidney function in Imperfect but indispensable. Am J Physiol GM, Levin A, Waikar SS, Murray PT, Parikh health and disease. Drug Discov Today Dis Renal Physiol 278: F1–F12, 2000 CR, Shaw AD, Go AS, Chawla LS, Kaufman Mech 4: 153–158, 2007 50. Doi K, Leelahavanichkul A, Yuen PS, Star RA: JS, Devarajan P, Toto RM, Hsu CY, Greene 64. Marshall JC: Why have clinical trials in sepsis Animal models of sepsis and sepsis-induced TH, Mehta RL, Stokes JB, Thompson AM, failed? Trends Mol Med 20: 195– 203, 2014 kidney injury. JClinInvest119: 2868–2878, Thompson BT, Westenfelder CS, Tumlin JA, 2009 Warnock DG, Shah SV, Xie Y, Duggan EG, 51. Ganopolsky JG, Castellino FJ: A protein C Kimmel PL, Star RA: Design of clinical trials in fi fl de ciency exacerbates in ammatory and acute kidney injury: A report from an NIDDK This article contains supplemental material online hypotensive responses in mice during poly- workshop–prevention trials. Clin J Am Soc at http://jasn.asnjournals.org/lookup/suppl/doi:10. microbial sepsis in a cecal ligation and Nephrol 7: 851–855, 2012 1681/ASN.2015030261/-/DCSupplemental.

J Am Soc Nephrol 27: 371–379, 2016 Inﬂammation in AKI 379