Sepsis-Induced Acute Kidney Injury

524  Section 15 / Infectious Diseases and Sepsis

CHAPTER 90 

Sepsis-Induced Acute Kidney Injury Hernando Gómez and John A. Kellum

OBJECTIVES This chapter will: 1. Review the epidemiology of sepsis-induced acute kidney injury. 2. Discuss the evidence supporting novel mechanisms leading to acute kidney injury in the setting of sepsis. 3. Review novel approaches to the diagnosis of sepsis-induced acute kidney injury. 4. Review current and potential therapies in the context of novel mechanisms of disease.

Sepsis is the most common cause of acute kidney injury (AKI) in critically ill patients,1 affecting 40% to 50% of cases.2 Importantly, the development of AKI in the setting of sepsis increases the risk of in-hospital death sixfold to eightfold,2,3 and among survivors, the risk of progression to chronic kidney disease.4 Despite this, the mechanisms by which sepsis causes AKI are not well understood. Therefore current therapy remains reactive rather than preventive and nonspecific. Given that the leading clinical conditions associated with AKI, namely, sepsis, major surgery, heart failure, and hypovolemia,2 are associated with hypoperfusion, it is tempting to attribute all AKI to ischemia. However, an increasing body of evidence suggests that at

least in a portion of patients, AKI can occur in the absence of overt signs of hypoperfusion. Langenberg et al. showed, for example, that AKI developed in septic animals despite normal or increased renal blood flow.5 In a human study, Prowle et al. were able to demonstrate that decreased renal blood flow (RBF) was not a universal finding, even in patients with well-established sepsis-induced AKI.6 Furthermore, in a large-scale study, including more than 1800 patients with community-acquired pneumonia, Murugan et al. found that a fifth to a quarter of patients with nonsevere pneumonia who were never admitted to an ICU and whom never displayed overt signs of shock or hypoperfusion still developed AKI.7 Complementary to the insights from clinical and in vivo studies, in vitro experiments in which hemodynamics are no longer relevant have shown that incubation of human renal tubular epithelial cells with plasma from septic patients induces damage of tubular epithelial cells evidenced by the increased release of tubular enzymes, elevated permeability, and the decreased expression of key molecules for tubular functional integrity.8 Taken together these data provide evidence that, at least in some patients, renal injury cannot be explained solely on the basis of the classic paradigm of hypoperfusion and that other mechanisms must come into play. Recent studies in septic animals and postmortem observations in septic humans have provided evidence of what sepsis-induced AKI actually looks like. Despite representing

Chapter 90 / Sepsis-Induced Acute Kidney Injury  525 the latest stages of the disease, these kidneys were characterized by a strikingly bland histology with focal areas of tubular injury, which was also entirely discordant with the profound functional impairment seen premortem. In addition, and contrary to prior understanding, necrosis and apoptosis were largely absent,9,10 which not only argues in favor of the notion that sepsis-induced AKI is not equivalent to acute tubular necrosis (ATN) but also supports the hypothesis that at least in the early stages, this phenotype may represent a concerted, organized, common underlying adaptive mechanism.10 A consistent observation in these studies, regardless of species, disease stage, severity, or organ examined, appears to be the presence of three main alterations: inflammation,11,12 diffuse microcirculatory flow abnormalities,13 and cellular bioenergetic adaptive responses to injury.10,14 The study and understanding of these three domains may provide a roadmap to unravel the mechanisms by which sepsis causes AKI and perhaps organ injury in general and may facilitate the development of more targeted therapies. In this chapter we provide an overview of the current clinical classification system and the epidemiology of sepsis-induced AKI and then focus on the roles the above-mentioned mechanisms may play in the genesis of sepsis-induced AKI and on the discussion of potential therapeutic implications.

DEFINITION OF ACUTE KIDNEY INJURY IN THE CLINICAL SETTING The definition of AKI has undergone important transformations in recent years. Traditionally it was based on the assessment of renal function, and in particular, on the assessment of changes in glomerular filtration rate (GFR). Although practical at the bedside, this approach is limited by the fact that functional changes do not necessarily reflect structural alterations.4 An additional limitation is the interpretation of GFR through the quantification of creatinine. Although creatinine levels correlate well with GFR in steady-state conditions, AKI usually occurs in the setting of rapidly changing hemodynamic, microcirculatory, metabolic, and local conditions. Finally, the assessment of renal dysfunction based on glomerular function does not take into account the presence of tubular dysfunction, which has been recognized as an important pathophysiologic event. Despite these limitations, the standardization of two measures of glomerular function has provided the scientific community with a tool, in a common language, to assess the occurrence of AKI. These measures are serum creatinine and urine output. Using these tools, AKI has been defined as any of the following: • Increase in serum creatinine by ≥0.3 mg/dL (≥26.5 µmol/L) within 48 hours • Increase in serum creatinine to ≥1.5 times baseline, presumed or known to have occurred in the previous 7 days • Urinary volume < 0.5 mL/kg/hr for 6 hours Several classification systems have been developed, each with virtues and caveats, including the Risk, Injury, Failure, Loss, End-stage kidney disease (RIFLE) criteria, the Acute Kidney Injury Network (AKIN) criteria, and the pRIFLE (a modification for pediatrics). More recently, in an attempt to harmonize RIFLE, AKIN, and pRIFLE, the Kidney Disease: Improving Global Outcomes (KDIGO) has proposed a unified version of these classification systems, which is shown in Table 90.1.15

TABLE 90.1 KDIGO Criteria and Staging for Acute Kidney Injury STAGE

SERUM CREATININE

URINE OUTPUT

1

1.5–1.9 times baseline creatinine OR ≥0.3 mg/dL (≥26.5 µmol/L) increase 2.0–2.9 times baseline creatinine 3.0 times baseline creatinine OR Increase in serum creatinine ≥ 4.0 mg/dL (353.6 µmol/L) OR Initiation of renal-replacement therapy OR In patients < 18 years, decrease in eGFR to <35 mL/ min/1.73 m2

<0.5 mL/kg/hr for 6–12 hr

2 3

<0.5 mL/kg/hr for ≥12 hr <0.3 mL/kg/hr for ≥24 hr; OR Anuria for >12 hr

KDIGO, Kidney Disease: Improving Global Outcomes.

EPIDEMIOLOGY OF SEPSIS-INDUCED ACUTE KIDNEY INJURY Sepsis is the leading cause of AKI in acutely ill patients.2,16 Acute kidney injury occurs in as many as 40% to 50% of septic critically ill patients, which increases the risk of death sixfold to eightfold1–3,17 and the risk of advancing to renal fibrosis and chronic kidney disease.4 Importantly, a large proportion of patients who usually are considered to be less severely compromised and thus at lower risk still develop AKI. Murugan et al. showed in a large cohort of patients admitted to the emergency department with nonsevere community-acquired pneumonia that 34% of these patients developed AKI, many of whom never required admission to an ICU.7 This suggests that AKI is related not only to shock states or critical illness and that patients with non–life-threatening infections also may be at high risk of developing renal dysfunction and its short- and long-term consequences.

NOVEL CONCEPTS IN THE PATHOPHYSIOLOGY OF SEPSIS-INDUCED ACUTE KIDNEY INJURY Recent evidence suggests that the origin of most cases of AKI is multifaceted and that several concurrent mechanisms may be at play. These mechanisms include inflammation; profound, heterogeneous distortion of microvascular flow at the peritubular and glomerular levels; and tubular epithelial cell injury and impairment. Given that these three major events occur early in the course of sepsis and that cell death seldom occurs, early sepsis-induced AKI may be the clinical and biochemical manifestation of tubular cell responses to injury. Evidence from animal studies suggests that such response is, at least in part, adaptive, in that it is driven by metabolic reprogramming and by downregulation and reprioritization of energy expenditure to avoid energy imbalance favoring individual cell survival

526  Section 15 / Infectious Diseases and Sepsis processes (e.g., maintenance of membrane potential and cell cycle arrest) at the expense of organ function (i.e., tubular absorption and secretion of solutes).

shunts has been proposed to result in areas of hypoperfusion and hypoxia.23,24 In relation to this, the endothelium also provides an essential system of retrograde communication that allows the microcirculation to fine tune and couple blood flow distribution to metabolic demand, which is, in essence, the concept of regional autoregulation. Tyml et al. have shown that lipopolysaccharide (LPS)-induced endothelial injury results in loss of such retrograde communication rate between microvessels 500 microns apart,25 suggesting that sepsis may impair not only the response to vasoactive mediators but also the capacity of peripheral microvascular beds to autoregulate. Similarly, endothelial dysfunction results in increased vascular permeability and worsening interstitial edema,26,27 with two important consequences. First, edema increases the diffusion distance oxygen has to travel to reach target cells,28 further creating areas at risk for hypoxia. Second, given that the kidney is an encapsulated organ, tissue edema contributes to increased venous output pressures, aggravating congestion, and perpetuating microvascular perfusion alterations.29,30 Endothelial cells are also important determinants of vascular tone and play an important role in the responsiveness to vasoactive mediators.31 Injury to the arterial and arteriolar endothelium has shown consistently to result in impaired responsiveness to vasoactive substances, which may explain the loss of vasomotor tone during sepsis. Nitric oxide (NO) also has been shown to have a potential role in the genesis of microvascular dysfunction and in the pathophysiology of AKI. Although sepsis is characterized

Renal Microcirculation During Sepsis-Induced Acute Kidney Injury Sepsis causes profound alterations in microvascular blood flow distribution.13,18 Such alterations are characterized by an increase in the heterogeneity of regional blood flow distribution, a decrease in the proportion of capillaries with “nutritive” (or continuous) blood flow, and an increase in the proportion of capillaries with intermittent or no flow.13,19 The renal microcirculation is disturbed in a similar fashion, as has been described recently in different models of sepsisinduced AKI,12,20,21 even in the setting of normal or even increased RBF.22 Multiple mechanisms seem to frame this characteristic microcirculatory derangement, including endothelial dysfunction, impaired red blood cell deformability, thinning and damage of the glycocalyx layer, increased leukocyte activation and recruitment, and activation of the coagulation cascade with fibrin deposition19 (Fig. 90.1). Importantly, these alterations in microcirculatory flow and endothelial function are thought to contribute directly to the development of organ dysfunction through multiple mechanisms. Uncoupling of microcirculatory blood flow distribution from metabolic demand with the creation of microvascular

al heli

ot

End

Altered endothelial cell–cell communication

Endothelial activation/ dysfunction

Peritubular capillary cell

Glycocalyx dysfunction

↑ iNOS

O2– ↑ NO

ONOO–

↓ aPC

Oxidative stress

DAMPs - Cytokines Neutrophil Vascular lumen

Tissue factor

Adhesion

Rolling

↓ NO ↑ Transmigration ↓ eNOS

Coagulation cascade activation Platelets

H2O

Altered r flow scula microva

Endothelial leak and edema

Endothelial activation/ dysfunction

Selectins

DAMPs - Cytokines

Integrins

Leukocyte

ICAM/VCAM-1

Activated leukocyte

FIGURE 90.1  Sepsis induces profound alterations in microcirculatory blood flow, including renal peritubular blood flow. Multiple mechanisms are thought to contribute to microcirculatory dysfunction. Damage- and pathogen-associated molecular patterns (DAMPs and PAMPs), oxidative stress, and altered nitric oxide production induce endothelial dysfunction. One of the key components of this is denudation of the glycocalyx from the luminal membrane of endothelial cells with several consequences: alteration of the coloidosmotic gradient between the lumen of the capillaries and the protein-rich area protected by the glycocalyx layer, resulting in increased leak of plasma and fluid into the interstitium; exposure of endothelial receptors expressed in the context of inflammatory injury that promote adhesion and rolling of platelets and neutrophils, which contribute to further inflammation. The inflammatory injury also results in an inducible nitric oxide synthase (iNOS)-dependent decrease in endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. eNOS-derived NO is key in protecting the endothelium by several mechanisms, including direct vasodilatation, by inhibiting platelet aggregation and leukocyte activation. Platelet adhesion and activation of the coagulation cascade in the setting of endothelial dysfunction further impair flow.

Chapter 90 / Sepsis-Induced Acute Kidney Injury  527 by global increased NO production,32 the expression of one of the most important catalyzers of its production, inducible NO synthase (iNOS), is rather heterogeneous.32 Accordingly, it is possible that the heterogeneous expression of iNOS may result in heterogeneous regional concentrations of NO, which could result in the presence of vascular beds deprived of NO even in the setting of elevated systemic levels.33 This is important because it is reminiscent of the characteristic heterogeneous pattern of microvascular dysfunction described in sepsis and may relate pathophysiologically with areas of shunting and hypoxia.33 Importantly, selective inhibition of iNOS not only can restore the renal microcirculatory derangements during sepsis but also is associated with decreased functional manifestations of renal injury, suggesting that microcirculatory abnormalities may be in the mechanistic pathway of sepsis-induced AKI.20 However, the interactions between NO, microvascular dysfunction, and AKI are far from straightforward; sepsis also is known to result in iNOS-dependent decrease in endothelial-derived NO synthase (eNOS) activity, which will also alter microvascular flow homeostasis.34,35 Endothelial synthase-derived NO is key in protecting the endothelium by several mechanisms, including direct vasodilatation, by inhibiting platelet aggregation and leukocyte activation. During sepsis, inflammation, and oxidative stress, the uncoupled eNOS36 not only induces endothelial cell dysfunction but also results in denudation of the glycocalyx. The glycocalyx is a layer of organized glycosaminoglycan branches that protrudes from the surface of the endothelial cell membrane into the capillary lumen. This glycosaminoglycan layer has important biomechanical functions, including maintenance of adequate capillary flow, preservation of oncotic and hydrostatic pressure gradient balance to limit filtration, and impeding platelet and leukocyte adhesion.37 Damage and loss of the glycocalyx layer is thought to result in altered red blood cell flow, capillary leak, and exposure of endothelial adhesion molecules, which leads to increased adhesion of platelets and leukocytes. All of these mechanisms contribute to the microvascular dysfunction phenotype characteristic of sepsis to further oxidative stress and inflammation. Finally, sluggish peritubular flow also may result in amplification of the inflammatory signal. As demonstrated by Goddard et al.38 in myocardial capillaries during a porcine model of endotoxemia, leukocytes decrease their velocities and increase their transit time in these areas of sluggish flow. In addition, there is evidence of upregulation of inflammatory molecules, such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 139,40 in these peritubular capillaries that would contribute to leukocyte activation and prolonged leukocyte transit. This prolonged transit may translate directly into a greater time of exposure of the endothelium and neighboring tubular epithelial cells to activated, cytokine-secreting leukocytes, and to other pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) that ultimately amplify the inflammatory signal and induce focal oxidative stress and tubular injury. The tubular epithelial cells exposed to this amplified signal then act as primary targets for this alarm and trigger a response in adjacent segments of the proximal tubule evidenced by the induction of oxidative stress and vacuolization (Fig. 90.2). The lack of apoptosis and necrosis suggests this is an organized, adaptive response that ultimately signals other tubular cells to shut down in a paracrine fashion. Importantly, this provides an explanation for why only a few heterogeneous groups of tubular epithelial cells demonstrate the bland, yet typical histopathology changes.

Inflammation Propagates Renal Damage During Sepsis A strong association between cytokine levels (interleukin [IL]-6, IL-10, and macrophage migration inhibitory factor) and the development of sepsis-induced AKI7,41 supports the hypothesis that systemic inflammation is an important mediator of this process. During sepsis, although the inflammatory response is fundamental to clear the infection and later promote tissue recovery, it also can result in tissue damage and organ dysfunction.42 In addition to leukocytes, dendritic cells, and resident macrophages, tubular epithelial cells are capable of recognizing and responding to PAMPs through pattern-recognition receptors, including toll-like receptors (TLR), C-type lectin receptors, retinoic acid inducible gene 1-like receptors, and nucleotide-binding oligomerization domain-like receptors,43 which result in the upregulation of inflammatory gene transcription and initiation of innate immunity. This response also is stimulated by endogenous substances released by injured cells and tissues known as DAMPs, which include DNA, RNA, histones, high-mobility group box 1 (HMGB1) and S100 proteins and are recognized by these same receptors.44 Proinflammatory mediators activate endothelial cells and induce upregulation of adhesion molecules such as E-selectin, which has been demonstrated to play a major role in leukocyte recruitment into the kidney during the late stages of sepsis-induced AKI.45 Although not seen in all models of sepsis-induced AKI,46 elimination of neutrophils or blocking adhesion molecules that are required for neutrophil recruitment into the kidney completely abolished sepsisinduced AKI in a cecal ligation and puncture (CLP)–induced sepsis model.45 This observation can be explained by the fact that leukocytes leaving peritubular capillaries have a close proximity to tubular epithelial cells and can activate directly tubular epithelial and dendritic cells by releasing proinflammatory mediators and DAMPs. The cycle then is perpetuated by the release of mediators such as leukotriene B4 and platelet-activating factor, which increase vascular permeability and upregulate the expression of adhesion molecules that promote further inflammation.47–49 In addition, DAMPs, PAMPs, and proinflammatory cytokines that are filtered readily through the glomerulus can activate these tubular epithelial cells from within the tubule (Fig. 90.2).50 It has been shown recently that mammalian tubular epithelial cells (including human) express TLR2 and TLR4 and that these cells are capable of recognizing inflammatory mediators such as LPS in a TLR4-dependent manner.51–54 Furthermore, Krüger et al.51 demonstrated that damaged human tubules stain positively for the TLR4 ligand and HMGB1 and that in vitro stimulation of human tubular epithelial cells with HMGB1 results in proinflammatory responses through TLR4,51 suggesting that such mediators can act in an autocrine and paracrine fashion and may contribute to further tubular cell damage. The recognition that tubular epithelial cells actually are equipped with machinery to recognize the inflammatory signal supports the hypothesis that their response may be organized and not random. In support of this, Kalakeche et al.52 have shown that TLR4-dependent LPS recognition in the tubular epithelial cells occurs in the S1 segment of the proximal tubule, that assembly of LPS with TLR4 in the tubular epithelial cell produces internalization of LPS through fluid-filled endocytosis, and that this triggers an organized oxidative outburst in epithelial cells of the adjacent tubular segments (S2 and S3) but not in the S1 segment (see Fig. 90.2). These

528  Section 15 / Infectious Diseases and Sepsis Circulating DAMPs/PAMPs and inflammatory mediators Peritubular capillary Microcirculatory dysfunction

Proximal tubule segments Filtered DAMPs/PAMPs and inflammatory mediators S1

TLR-4

Increased time of exposure to Sluggish activated peritubular = leukocytes + flow decrease O2 availability

TNFα

S3

Paracrine signalling

Vascular lumen

Tubular lumen

S2

Distal nephron

Activated leukocyte DAMPs/PAMPs Other inflammatory mediators (i.e., cytokines)

Activated endothelium Expression of ICAM, VCSM Dendritic cell Alarm signal

FIGURE 90.2  The proximal tubular epithelial cells are exposed to inflammatory injury from the peritubular capillaries and within the tubule, after damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) filter through the glomerulus and are recognized by pattern recognition receptors, including TLR2 and TLR4. The response of the tubular epithelial cell and the proximal tubule to inflammation seems to be organized because after TLR4-mediated recognition of LPS by cells of the S1 segment of the proximal tubule trigger a response characterized by oxidative stress and apical blebbing of cells in the S2 and S3 segments, but not in the S1 segment. Kalakeche et al.52 have suggested that the S1 segment cells may communicate in a paracrine fashion with cells down the proximal tubule by secreting TNF into the tubular lumen. This is an important mechanism that may explain how the proximal tubule shuts down the nephron when sensing danger.

findings have led Kalakeche et al.52 to suggest that the S1 segment of the proximal tubule may act as a sensor of danger that activates a series of events resulting in oxidative stress within distal tubular segments (S2, S3) and that potentially could explain tubular dysfunction in the setting of sepsis.

(Adaptive) Responses of Tubular Epithelial Cells to Inflammation With the exception of T lymphocytes and intestinal epithelia, and despite multiple triggering stimuli,55 significant necrosis or apoptosis does not occur during sepsis,9,10 which suggests that during the acute phase, regardless of the consequences at the organ level, the cellular response is successful at preventing death. This denotes a possible underlying adaptive mechanism10,47,56 and an opportunity to understand

the response of the tubular epithelial cells to sepsis. Accordingly, the tubular epithelial cell response to injury may be characterized at least in part by processes that limit proapoptotic triggers, by (1) reprogramming metabolism to optimize and prioritize energy consumption and to maintain energy homeostasis, (2) maintaining cellular organelle function through quality control processes (general autophagy and mitophagy), and (3) limiting cell cycling and DNA replication.

Metabolic Reprogramming and Reprioritization of Energy Consumption Energy balance dysregulation and mitochondrial injury are two major triggers of apoptosis and, consequently, two of the most highly regulated cellular defense mechanisms to injury.57 Although still controversial, sepsis seems to be

Chapter 90 / Sepsis-Induced Acute Kidney Injury  529 Phase

Acute anabolic phase: mTORC1-

Adaptive catabolic phase: AMPK-SirT AMP: ATP LKB1

mTORC1 NAD+/NADH

mTOR Raptor Metabolic control

AMPKα β γ

ACC

SirT1, 6 HIF-1α

Cpt1 PGC-1α

LDH

PKM2

Inflammatory phenotype

Repair phenotype

Mitophagy

Aerobic glycolysis– ‘Warburg’

Proinflammatory

Biogenesis

PDH

↑ Lactate

Metabolic phenotype

PDHK

Fatty acid oxidation – OXPHOS

T-CD4+ and macrophages

Th17 and M1

Cytokine profile

MCP-1, TNFα, IL-1, 6

Increased fat deposition Pro-fibrotic Progression to CKD

Antiinflammatory

T-CD4+ and macrophages

Treg and M2

Cytokine profile

IL-10, IL-4

Repair and re-differentiation to functional epithelium Restoration of epithelial function

FIGURE 90.3  The study of activated inflammatory cells has demonstrated that the Warburg effect is not exclusive of cancer cells. Inflammation induces a metabolic reprogramming that results in a shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis as main process for energy derivation. In monocytes this early shift towards aerobic glycolysis has been attributed to the activation of the hypoxia inducible factor-1α (HIF-1α), which results in increased expression of cytoplasm glucose transporters, enhanced activity of glycolytic enzymes, expression of pyruvate kinase isoform M2, which slows down conversion of phosphoenolpyruvate to pyruvate, and of pyruvate dehydrogenase kinase, which blocks pyruvate dehydrogenase limiting entrance of pyruvate to the Krebs cycle. At a later stage, the shift back to OXPHOS in inflammatory cells has been attributed to the NAD dependent deacetylaces Sirt1 and Sirt6, which turn off aerobic glycolysis by inhibiting HIF-1a signaling. These mechanisms, as demonstrated in the figure, may be operational in tubular epithelial cells that denote a biphasic metabolic response to sepsis, that will determine the innate inflammatory response, and possibly, the repair phenotypes during convalescence.

associated with maintenance of adenosine triphosphate (ATP) levels in the kidney,58 albeit with a decrease in production,59,60 suggesting a significant decrease in ATP utilization. Furthermore, analogous to the evolutionarily conserved defense response to hypoxia, in which nonvital functions are limited to avoid overtaxing energy expenditure,57 sterile inflammation by administration of LPS has been shown to induce downregulation of renal tubular cell ion transporters,61 which account for more than 70% of ATP cellular consumption.62 Furthermore, there is evidence that experimental sepsis induces similar effects. Gupta et al.63 showed that, in the presence of LPS, proximal tubules of mice have a delayed uptake of low-molecular-weight dextran, a sign of reduced endocytic capacity. Good et al.64 have shown in an LPS-induced rodent sepsis model that LPS inhibits NHE1 (Na+/H+ exchanger 1) and thus blocks bicarbonate reabsorption in the medullary thick ascending limb of the loop of Henle. Finally, Hsiao et al. have shown that sodium transport (tubular sodium reabsorption) is decreased as

early as 9 hours after induction of sepsis by cecal ligation and puncture.65 Prioritization of energy expenditure during sepsis seems to be part of a more complex cellular metabolic reprogramming strategy, which is key to the survival of the cell in the acute phases of the septic syndrome and may determine the repair phenotype during the convalescent phase (Fig. 90.3). Immune cells are known to use a strategy of metabolic reprogramming comparable to the Warburg effect used by tumor cells (i.e., when cancer cells switch their metabolism from oxidative phosphorylation to glycolysis to derive energy and substrates) to fit their energy and substrate requirements in tune with changing environmental conditions.66 A phasic switch between glycolysis and oxidative phosphorylation (OXPHOS) has been shown to frame the biochemical phenotype of this metabolic reprogramming in immune cells. Modifications to this phasic regulation have been shown to affect outcome in animal models of sepsis. For example, Yang et al. demonstrated that

530  Section 15 / Infectious Diseases and Sepsis knockdown or inhibition of pyruvate kinase isoenzyme 2 (PKM2), an enzyme required for the Warburg effect to occur in monocytes during inflammation, improved the survival of rodents exposed to endotoxin or CLP-induced sepsis. Although the evidence is significantly less robust in TEC, Escobar et al. have shown that activation of a master regulator of energy balance that promotes OXPHOS over glycolysis before the induction of sepsis by CLP protects from AKI in a rodent model,67 suggesting that metabolic reprogramming also may be important in the response of the tubular epithelial cells. Along these same lines Opal et al. have shown that pharmacologic activation of SirT1 (silent information regulator T1), a downstream AMPK effector that regulates metabolism, by a small molecule activator or STAC administered at the time of CLP improved survival in rodents with CLP-induced sepsis.68 These findings were in agreement with those by Vachharajani et al., who found that specific inhibition of SirT1 by EX-527 at the time of CLP increased mortality in a similar model.69 Finally, Kang et al. showed that human and mouse fibrotic kidneys had lower expression of enzymes and regulators involved in fatty acid oxidation (FAO) than controls and that inhibition of FAO in primary human cells resulted in phenotypical changes consistent with fibrosis, including lipid deposition, dedifferentiation, and ATP depletion. These authors also demonstrated that restoring FAO by activating key regulators such as the peroxisome proliferator-activated receptor α and the PPAR-γ co-activator-1a (PPARGC1A) protected mice from tubulointerstitial fibrosis after induction of fibrosis with folic acid.70 Han et al. demonstrated that animals lacking LKB1, a key regulator of proliferation, polarity, cell metabolism, and upstream regulator of AMPK, progressed to the fibrotic phenotype71 described by Kang et al. Importantly they were able to restore FAO and rescue animals from the fibrotic phenotype by using fenofibrate, the same PGC-1α agonist that rescued animals in the study by Kang et al. Activation of AMPK with A769662 resulted in the same effects, again suggesting that different mediators of this energy regulatory pathway have key roles in regulating progression to the mechanisms that lead to CKD. Taken together this evidence suggests that during sepsis, the response of the tubular epithelial cell may be characterized by an organized reprogramming of metabolism, which promotes hierarchic downregulation of major energy sinks such as ion transport, while fueling only those processes necessary to cell survival (i.e., maintenance of membrane potential)72 and adjusts energy and substrate acquisition between glycolysis and OXPHOS to adapt to changing environmental conditions (see Fig. 90.3). This evidence also suggests that reprogramming of metabolism as a defense strategy goes beyond the acute phases of the septic syndrome, defining the repair phenotype and thus influencing progression to chronic organ dysfunction. This is a highly conserved mechanism across species that seems to frame the core strategy of cellular response to threatening circumstances. It also provides the conceptual ground to suggest that cellular metabolic reprogramming is a pillar of the tubular epithelial response to sepsis.

Mitochondrial Quality Control Processes: Mitophagy and Biogenesis Mitochondria play a key role in cellular homeostasis in the setting of physiologic and pathologic stress. Mitochondria are common targets of inflammatory injury, causing mitochondrial dysfunction, increased production of ROS, and thus harm to the host cell. However, the cell can defend

itself from injured mitochondria by triggering quality control processes that attempt to repair dysfunctional mitochondria (fusion and fission), eliminate those beyond the possibility of repair (mitophagy), and reconstitute the pool of healthy mitochondria (biogenesis). Mitophagy is an evolutionarily conserved quality control mechanism by which eukaryotic cells remove and digest dysfunctional mitochondria from the cytoplasm.73,74 During sepsis, TLR-mediated inflammation,75 oxidative stress,76,77 and alterations in the electron transport chain that uncouple respiration and depolarize the mitochondrial membrane are potent triggers of mitophagy.74 This early mitochondrial uncoupling characterized by an increment in O2 consumption (VO2) is not to be confused with the adaptive response it triggers, which is framed by the activation of mitophagy and is characterized by a decrement in VO2 and conservation of energy. In the kidney, mitophagy is activated as early as 3 hours after CLP-induced sepsis,65 suggesting it is part of the early response of tubular epithelial cells to injury. Importantly, insufficient activation of mitophagy has been associated with a worse outcome in critically ill patients and has been postulated to contribute to cell and organ dysfunction.78 On the other hand, stimulation of autophagy has been shown to be effective at protecting cells65 and organ function78 in the setting of experimental inflammatory insults. Furthermore, in the setting of experimental sepsis induced by CLP, decreased autophagy has been associated with increased blood urea nitrogen and creatinine levels and a decline in proximal tubular sodium transport.65 As a protective response, mitophagy offers several advantages, namely, removal of dysfunctional mitochondria, with subsequent decrement in ROS/RNS production, energy conservation, limiting oxidative stress damage, and importantly, intercepting proapoptotic signals at the mitochondrial level impeding triggering of apoptosis.74,79–81 Indeed, Carchman et al. have shown that inhibition of mitophagy results in a robust apoptotic signal in hepatocytes of animals subjected to CLP.59 It is unknown, however, what mitophagyinduced maintenance of renal function really means. The adaptive response, framed by metabolic downregulation, would most likely decrease tubular and renal function and not promote it, just as hibernation promotes the loss of function. Indeed, increased or preserved renal function in the setting of stress may result in harm in the long run. However, animal and human data associate acute stimulation of autophagy with preserved renal function and its faulty activation or decline with worse outcome. It is possible that the interplay of autophagy and tubular cell function varies with time and that persistence of the initial protective response ultimately may be deleterious in the subacute or chronic phases. Mitochondrial biogenesis is a cellular quality control process by which the cell, by coordinating mitochondrial and nuclear gene expression, reconstitutes functional mitochondria. Several environmental stressors, including exercise, oxidative stress, and inflammation, can induce mitochondrial biogenesis. Importantly, recent evidence has suggested that mitophagy and biogenesis are coupled through several mechanisms, including redox pathways and TLR9-dependent mechanisms,82 and that biogenesis is key to the defense of the tubular epithelial cell to inflammation. Tran et al. demonstrated that animals lacking PGC-1α, which regulates the activation of biogenesis, were more susceptible to developing AKI after exposure to endotoxin than wild type animals. Activation of mitochondrial biogenesis is a natural response of tubular epithelial cells as demonstrated by Bartz et al. These authors showed that sepsis induced by injection of infected peritoneal clots with Staphylococcus aureus naturally induces

Chapter 90 / Sepsis-Induced Acute Kidney Injury  531 activation of mitochondrial biogenesis.83 This has been demonstrated in other organs, such as the liver, in which Carchman et al. showed that mitochondrial biogenesis was activated in hepatocytes of rodents exposed to CLP and endotoxin in a TLR4-dependent mechanism,82 suggesting biogenesis may be a global cellular defense strategy. Finally, Piantadosi et al., using the same model as Bartz et al., further demonstrated that exogenous activation of mitochondrial biogenesis using inhaled carbon monoxide at 250 ppm significantly increased survival, suggesting biogenesis as a potential therapeutic target.84

Cell Cycle Arrest A growing body of evidence indicates that mitochondria are involved intimately in the regulation of the cell cycle.74 The ability of mitochondria to move within the cell, change shape, and coalesce in different ways has emerged recently as an important feature, which may influence the cell cycle.85 Briefly, the cell cycle is the progression of cells through a number of steps in preparation for mitosis (G0, G1, S, G2, M). This preparation portrays several checkpoints in which the cell seems to evaluate whether it is prepared to advance to the next phase. Of particular interest to renal tubular injury in sepsis and the involvement of mitochondrial regulation is the G1-S checkpoint. Only at and during this stage, mitochondria have been shown to coalesce into a single, tubular network of mitochondria. This mesh seems to act as syncytia, with electrical coupling and unusual hyperpolarization,86 which fits well with prior studies showing an increase in O2 consumption during the G1-S transition of the cell cycle.87 This also relates to the finding that a reduction in ATP production induced by specific ETC mutations produces cell cycle arrest at the G1-S checkpoint.88 Together, these data indicate that the formation of this giant tubular network is necessary to meet the energy requirement needed to synthesize all the components for adequate cell division. It also suggests that the G1-S border is an important checkpoint of the cycle, whereby the inability to meet such energy requirements induces cell cycle arrest presumably to prevent a potentially lethal energy imbalance.85 Yang et al.89 recently showed in a rodent model of CLP-induced sepsis that G1-S cell cycle arrest was associated with kidney injury and that recovery of renal function paralleled cell cycle progression 48 hours after CLP. These findings have become even more clinically relevant as tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7), two markers involved in G1-S cycle arrest, have been identified as the most sensitive and specific markers to predict risk of development of AKI in critically ill patients.90–92 We speculate that the renal cell cycle arrest in the epithelial tubular cell may provide an advantage by avoiding replication because (1) it conserves energy and prevents triggering apoptosis or necrosis and (2) limiting replication diminishes the probability of DNA damage, reducing not only energy consumption employed in DNA repair but also the chances of triggering apoptosis (Fig. 90.4).

Glomerular Hemodynamics During Sepsis-Induced Acute Kidney Injury One of the most impressive characteristics of sepsis-induced AKI is the evident disconnect between the very minor structural changes in the architecture of the kidney and

the astounding functional decline. Clinically, such functional decline is quantified currently in terms of GFR surrogates, such as creatinine and urine output. Despite that, as we have discussed earlier, it is recognized that tubular injury may be at least as important in explaining the functional alterations during sepsis-induced AKI, it is also clear that there are significant changes in the physiology of the glomerulus that merit attention. Sepsis-induced AKI is, by definition, characterized by a decline in GFR. GFR depends on the generation of a sufficient net filtration pressure (NFP) within the glomerular capillaries that will drive filtration. Following the principles of the Starling law of the capillary, NFP will be determined by the forces pushing fluid into the Bowman’s capsule (glomerular capillary hydrostatic pressure, PC), and forces opposing this movement (glomerular capillary oncotic pressure, πC, and hydrostatic pressure in Bowman’s space, PB). Glomerular capillary hydrostatic pressure is dependent on changes in mean arterial pressure (MAP), as well as on the vascular tone of the afferent and efferent arterioles. In this way, although GFR is maintained throughout a wide range of mean arterial pressures by virtue of its autoregulatory capacity (i.e., autoregulation), a decline in MAP below this autoregulatory margin will result in a decrease in NFP, PC, and thus GFR. However, despite that during sepsis, RBF is maintained and in some cases increased, GFR still drops, and AKI still occurs. Decreases such as these in GFR may be explained by three nonmutually exclusive mechanisms: a decline in MAP, vasoconstriction of the afferent arteriole, and predominant vasodilatation of the efferent arteriole. This latter mechanism seems to be operative during sepsis, leading to a decrease in GFR by decreasing PC. Some authors have postulated that this acute decline of GFR may be protective because with less filtration (1) the renal tubule will be exposed to less inflammation by virtue of a decrease in exposure to filtered DAMPs and PAMPs and (2) because the tubule will require less energy expenditure in reabsorbing sodium and chloride, thereby maintaining energetic balance. This hypothesis also links tubular injury to the alterations in GFR. Sepsis-induced tubular injury has been associated with a decrease in tubular reabsorption of sodium and chloride.65 Increased delivery of NaCl to the macula densa triggers the vasoconstriction of the afferent arteriole by a mechanism known as tubuloglomerular feedback (TGF),93 thus decreasing GFR further. In addition, activation of the renal sympathetic nervous system can account further for increasing the tone of the afferent arteriole.94 The recruitment of alternative pathways that bypass the glomerulus (i.e., from afferent to efferent arterioles) may be yet another mechanism explaining the drop in GFR seen in patients with sepsis.95,96 Although these mechanisms appeal to reason, several details are still unknown, for example, if tubular injury precedes (and thus drives) the decline in GFR in the setting of sepsis. Finally, increments in venous pressure may be particularly important to renal function, because the kidney is contained inside a nondistensible capsule. Thus promoting renal venous congestion may contribute to the genesis, but, most likely, maintenance of AKI during sepsis.97

Potential for Diagnostic and Therapeutic Targets No specific therapeutic measures are available to prevent or treat sepsis-induced AKI. A potential reason for this may be that often therapy is started too late in the disease process. The development of new biomarkers, which also provide insights in the pathophysiology of the disease,

532  Section 15 / Infectious Diseases and Sepsis Exogenous DAMP Exogenous PAMP Endogenous Cytokine

Healthy RTECs G3

G1

Early sepsis G3 OR Arrest / repair

M G2 G4

Maladaption / apoptosis/ fibrosis G1

A

G3 DAMPs and PAMPs

S

IGFBP7

TIMP-2

p27 Cyc D CDK 4/6 G1 cell-cycle arrest

p21

p53 Cyc E CDK 2 Protection/repair

B

makes it possible to detect kidneys at risk for injury and thus enable earlier initiation of interventions.90–92 The knowledge that inflammation, microvascular dysfunction, and adaptive responses of tubular cells are involved in the development of sepsis-induced AKI provides new diagnostic and therapeutic avenues. Because these mechanisms are closely interlinked with each other, modulating one of these components simultaneously alters other components. As increased levels of proinflammatory mediators (e.g., IL-6) are associated with the development of AKI,41 it is tempting to speculate that eliminating these mediators or endotoxin can prevent sepsis-induced AKI. Experimentally it has been shown that removal of such mediators by hemadsorption completely protects against AKI in a CLP-model of sepsis.8,98,99 A clinical study demonstrated that reducing endotoxin by polymyxin-B hemoperfusion reduced RIFLE scores and urine tubular enzymes.8 However, clinical studies have reported conflicting results. The EUPHAS study, an Italian randomized, nonblinded trial, reported in 64 patients with severe sepsis or septic shock improvement in the primary outcomes of hemodynamics and organ dysfunction and a decrease in 28-day mortality as a secondary outcome.100 However, a prospective, multicenter French study that randomized

S

M G2

FIGURE 90.4  Alterations of the renal tubular epithelial cell cycle. During sepsis, differentiated tubular epithelial cells (which are characterized by having low turnover rates with majority being in G0 phase) exposed to damage-associated molecular patterns (DAMPs) and pathogenassociated molecular patterns (PAMPs) activate signaling pathways that either increase proliferation or arrest the cell cycle at G1 phase presumably in preparation for cell division (A). It has been hypothesized that injury and repair may be part of the same process and that appropriate coordination results in either arrest and repair, or maladaptive repair with apoptosis, and the classic features of chronic kidney disease. Insulin-like growth factor binding protein -7 (IGFBP7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) are implicated in regulation of G1 cell cycle arrest and have been identified recently as the most reliable biomarkers to predict the development of AKI in the septic and critically ill population. This has resulted in the hypothesis that release of these biomarkers may be protective of the renal tubular cell in the setting of sepsis-induced AKI (B).

243 patients with abdominal infections and septic shock showed that treatment with polymyxin B hemoperfusion had an insignificant increment in 28-day mortality and no improvement in organ failure.101 Despite that the mechanism by which polymyxin B hemoperfusion may provide an advantage is removal of endotoxin, none of the studies conducted so far has targeted therapy to specific levels of lipopolysaccharide. The EUPHRATES trial is an ongoing multicenter, placebo-controlled study that will attempt to overcome this limitation by enrolling septic patients in 50 ICUs in the United States and Canada, with endotoxin activity assay of 0.6 or greater, and targeting 28-day mortality as its primary outcome.102 Along the same lines, alkaline phosphatase (AP) is an endogenous enzyme that exerts detoxifying effects through dephosphorylation of endotoxins and proinflammatory extracellular ATP and is reduced during systemic inflammation. Heemskerk et al.103 demonstrated that administration of AP was associated with a decreased expression of iNOS synthase in proximal tubule cells isolated from urine and that this related to an attenuated urinary excretion of glutathione S-transferase A1-1, a proximal tubule injury marker. In a small, randomized trial, Pickkers et al. showed

Chapter 90 / Sepsis-Induced Acute Kidney Injury  533 that the administration of exogenous AP in septic patients improved endogenous creatinine clearance and reduced the requirement and duration of renal replacement therapy.104 Modulating TNF-α signaling may be yet another therapeutic option, because a polymorphism in the promoter region of the TNFA gene is associated with markers of kidney disease severity and distant organ dysfunction.105 To improve microcirculatory perfusion, vasodilators in the setting of sepsis are currently under investigation, including nitroglycerin,18,106 NO administration, and modulation of NO production.33,35 Furthermore, drugs with pleiotropic effects on the vasculature, such as statins107 and erythropoietin,108 have the potential to prevent kidney injury by enhancing eNOS expression and decreasing vascular permeability. However, it is important to consider that regional microcirculatory autoregulation is possible only if sufficient perfusion pressure is attained; thus early resuscitation goals still must focus on achieving a mean arterial pressure sufficient enough to ensure perfusion. Asfar et al. have shown that such a goal must be a mean arterial pressure of 65 and 70 mm Hg and that higher levels of MAP result in improved outcomes (decreased need for RRT) only in the subpopulation of patients with chronic hypertension.109 However, it is important to explore these treatment options, bearing in mind that these mechanisms are part of the natural host response to sepsis and that, although known perpetrators of injury, they are also necessary for bacterial clearance, tissue protection and repair, and ultimately survival. Accordingly, the reader must not expect a single treatment modality to emerge as a magic bullet to prevention and/or treatment sepsis-induced AKI.

CONCLUSION Close examination of the histology of various organs of patients dying from sepsis has dramatically changed the way we think of sepsis-induced organ dysfunction. The recognition that, in the case of the kidney, sepsis-induced AKI cannot be explained entirely by the traditional concept of acute tubular necrosis and that sepsis does not cause overt apoptosis and necrosis in failing organs has challenged the notion that ischemia is the only mechanism explaining organ dysfunction. Importantly, it also has prompted many to suggest that the response to the septic environment may early on be adaptive in nature. In this review, we have now put forth a conceptual model that cellular energy regulation is fundamental to the adaptive response and that such regulation is driven at least in part by metabolic downregula-

tion and reprioritization of energy utilization and by mitochondrial quality control processes such as mitophagy. Further work is warranted to better understand the role, timing, and reach of these multiple mechanisms in the pathogenesis of sepsis-induced AKI and if this can be translated into novel diagnostic and therapeutic interventions to improve outcome in this patient population.

Key Points 1. Sepsis-induced acute kidney injury is not equivalent to acute tubular necrosis. 2. Sepsis-induced acute kidney injury is the result of the intersection of multiple mechanisms that include, but are not limited to, hypoperfusion. 3. The early renal and tubular epithelial cell response to sepsis framed by active metabolic reprogramming, cell cycle arrest, mitophagy, and biogenesis is important because it may not only represent an adaptive strategy to stress but also may play a key role in repair processes beyond the early stages of disease.

Key References 1. Hoste EAJ, Bagshaw SM, Bellomo R, et al. Epidemiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study. Intensive Care Med. 2015;41(8):1411-1423. doi:10.1007/s00134-015-3934-7. 7. Murugan R, Karajala-Subramanyam V, Lee M, et al. Acute kidney injury in non-severe pneumonia is associated with an increased immune response and lower survival. Kidney Int. 2010;77(6):527-535. doi:10.1038/ki.2009.502. 46. Singbartl K, Bishop JV, Wen X, et al. Differential effects of kidney-lung cross-talk during acute kidney injury and bacterial pneumonia. Kidney Int. 2011;80(6):633-644. doi:10.1038/ ki.2011.201. 47. Gomez H, Ince C, De Backer D, et al. A unified theory of sepsis-induced acute kidney injury: inflammation, microcirculatory dysfunction, bioenergetics, and the tubular cell adaptation to injury. Shock. 2014;41(1):3-11. doi:10.1097/ SHK.0000000000000052. 99. Peng Z-Y, Wang H-Z, Carter MJ, et al. Acute removal of common sepsis mediators does not explain the effects of extracorporeal blood purification in experimental sepsis. Kidney Int. 2012;81(4):363-369. doi:10.1038/ki.2011.320. A complete reference list can be found online at ExpertConsult.com.

Chapter 90 / Sepsis-Induced Acute Kidney Injury  533.e1

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