PATHOGENESIS OF INTRINSIC ACUTE KIDNEY INJURY

Abstract

Purpose of review:

This review focuses on the pathogenesis of intrinsic AKI, emphasizing recent advances that hold therapeutic promise.

Recent findings:

Enhanced endothelin and reduced endothelium-derived nitric oxide release in AKI can be blocked using endothelin receptor antagonists or nitric oxide supplementation. Vasodilatory agents such as theophylline and caffeine may prevent AKI. Free labile iron is a potent factor in the generation of reactive oxygen species and tubule damage in AKI. Apoptosis via induction of p53 is an important mechanism of cell death in AKI, which can be blocked using small interfering RNA. The AKI-driven reduction in nicotinamide adenine dinucleotide can be countered using oral supplements. Surviving tubule cells regenerate after AKI, by upregulating genes encoding growth factors, such as hepatocyte growth factor (HGF). Pro-angiogenic agents (statins and erythropoietin) that can mobilize endothelial progenitor cells after AKI are currently being tested. The inflammatory response in AKI, including activation of C5a, can be therapeutically targeted. Contemporary single cell profiling technologies have identified novel genes with altered expression, new signaling pathways, and drug targets in AKI.

Summary:

Recent advances in the pathogenesis of intrinsic AKI have provided a better understanding of the clinical continuum and the rational deployment of promising therapeutics.

Introduction:

Acute kidney injury (AKI) comprises a complex continuum of molecular, structural, and functional alterations [1, 2]. Structural (intrinsic) forms of AKI are caused by systemic illnesses, prolonged ischemia, nephrotoxins, sepsis, and severe forms of primary glomerular diseases. Intrinsic AKI afflicts one-third of critically ill infants and children and is associated with adverse outcomes in the short-(death, complications) and long-term (chronic kidney disease) (CKD). This review will focus on the pathogenesis of intrinsic AKI, with emphasis on recent advances that hold therapeutic promise [3].

Morphology:

Except in primary glomerular diseases, kidney biopsy findings in intrinsic human AKI are paradoxically bland even when the reduction in glomerular filtration rate (GFR) is severe [4, 5]. There is effacement of proximal tubule brush border, cytoplasmic vacuolization, focal proximal tubular dilatation, distal tubular casts, patchy tubule cell death, and areas of cellular regeneration. Peritubular capillaries in the outer medulla display a striking vascular congestion. The molecular mechanisms underlying these morphologic changes are uncovering novel pathogenetic pathways, as outlined below.

Alterations in Hemodynamics:

In intrinsic AKI, total kidney blood flow is reduced to about 50% of normal due to persistent renal vasoconstriction. More importantly, there is marked congestion of the outer medullary zone that worsens the relative regional hypoxia, leading to cell death. Mechanisms underlying these hemodynamic alterations relate primarily to microcirculatory and endothelial damage [6], resulting in enhanced release of the vasoconstrictor endothelin and reduced release of vasodilatory endothelium-derived nitric oxide (NO). Plasma levels of endothelin are increased (and NO levels decreased) in humans with intrinsic AKI, and both represent therapeutic opportunities. In a meta-analysis of 15 adult human studies exploring NO supplementation, inhaled NO gas was associated with a reduced risk of AKI after cardiac surgery with no adverse effects [7]. Further prospective studies are in progress. Newer endothelin receptor blockers have been approved for use in hypertension and CKD and may hold promise in AKI. However, human trials of several other vasodilators have failed to demonstrate improvement in GFR in established AKI [8].

Alterations in Tubule Dynamics:

The finding of proximal tubular dilatation and distal tubular casts in human AKI indicate obstruction to tubular fluid flow. The intraluminal casts contain Tamm-Horsfall protein, the secretion of which is increased after AKI. However, obstruction alone cannot account for the profound dysfunction, and human studies using forced diuresis with mannitol or furosemide did not improve the recovery rate of patients with established AKI [9].

A role for activation of tubulo-glomerular feedback has been proposed in AKI. The increased delivery of sodium chloride to the macula densa due to proximal tubule injury induces afferent arteriolar constriction, mediated by A1 adenosine receptor activation, thereby decreasing GFR. Thus, the use of adenosine receptor antagonists such as vasodilatory theophylline and caffeine for AKI prevention is a subject of renewed clinical investigation, especially in neonates [10].

Alterations in Tubule Cell Metabolism:

A profound reduction in intracellular ATP content is a hallmark of AKI that occurs very early after injury, which triggers production of reactive oxygen species (ROS) that leads to the depletion of endogenous antioxidants with resultant dysfunction of proteins and lipids, DNA damage, and cell death. Scavengers of ROS (such as superoxide dismutase and N-acetylcysteine) protect against ischemic AKI in animal models, but human studies have been inconclusive. Free labile iron derived from red cells and injured tubule cells is another potent factor in the generation of ROS and tubule damage via activation of ferroptosis, and approaches to minimize the nephrotoxic effects of iron are under investigation [11, 12].

Alterations in tubule cell death:

A subset of tubule cells displays patchy cell death resulting from at least five mechanisms: apoptosis, necroptosis, ferroptosis, necrosis and autophagy [13]. Apoptosis is most pertinent to clinical AKI. Several apoptotic pathways, including the intrinsic, extrinsic, and regulatory factors, are activated in AKI. In particular, the proapoptotic transcription factor p53 is rapidly induced, and activates both the intrinsic and extrinsic pathways. Administration of a p53 small interfering RNA decreases serum creatinine and tubule cell death in animal models. A phase 2 trial evaluated a single intravenous dose of a p53 small interfering RNA after cardiac surgery in adults [14]. AKI incidence was reduced to 37% for the treatment group versus 50% for placebo-treated patients. AKI severity and duration were also improved in the treatment group, and no safety issues were identified. Results of larger phase 3 studies are awaited.

Necroptosis is a programmed cell death characterized by caspase-independent regulated necrosis resulting in plasma membrane rupture and subsequent release of damage-associated molecular patterns. Several novel therapeutic agents that inhibit necroptosis have already shown efficacy in experimental studies, and human translation appears to be promising [13].

Ferroptosis, a distinct form of non-apoptotic iron-dependent cell death now well described in AKI, is characterized primarily by intracellular accumulation of labile, catalytic iron, with subsequent lipid peroxidation, membrane rupture, and cell death [15–17]. Targeted inhibition of ferroptosis via widely available lipophilic antioxidants and iron chelators are achieving promising preliminary results.

Autophagy is a physiologic process by which intracellular damaged macromolecules and organelles are degraded and recycled for the synthesis of new cellular components [18]. Similar to apoptosis, autophagy in the early phases of AKI exerts protective effects by inhibiting inflammatory responses, whereas abnormal or prolonged autophagy leads to cell death. Common pathways exist between autophagy and apoptosis during the pathogenesis of AKI, which may provide unique targets for therapeutic interventions.

Alterations in tubule cell proliferation:

Surviving tubule cells possess a remarkable ability to regenerate and proliferate after AKI. Morphologically, repair is heralded by the appearance of de-differentiated epithelial cells that express vimentin, a marker for multipotent mesenchymal cells. In the next phase, the cells upregulate genes encoding growth factors, such as insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and α-melanocyte stimulating hormone (α-MSH), with resultant proliferation. In the final phase, cells express differentiation factors, and undergo re-differentiation until the normal epithelium is restored. Thus, during recovery, tubule cells recapitulate processes normally encountered during normal kidney development. HGF is nephro-protective in animal models of AKI, and recent studies have explored the effects of BB3/ANG3777, a small molecule with strong HGF-like activity, which, when administered at 24 hours after kidney ischemia in rats, improved survival and kidney function. BB3/ANG3777 is currently being tested in human AKI [19]. Human trials with recombinant IGF-1 have not demonstrated a beneficial effect. α–MSH is an anti-inflammatory and antiapoptotic cytokine that protects from AKI in animal models. However, α-MSH therapy was not effective in human AKI trials.

Extra-renal mesenchymal stem cells (MSCs) may have additional autocrine, paracrine, and growth factor-like effects on kidney regeneration. MSCs are drawn to kidney tubules that upregulate stromal cell-derived factor 1 after AKI. Exogenously administered MSCs enhance recovery from ischemic AKI in animals. In a cohort of adult patients with AKI after cardiac surgery, administration of allogeneic MSCs did not decrease the time to recovery of kidney function. MSC-derived extracellular vesicles have the advantages of lower immunogenicity and tumorigenicity; their clinical use is currently being explored [20].

Alterations in the Microvasculature:

Morphologically, endothelial cell swelling and death, with detachment of viable cells, have been observed in AKI. Circulating endothelial cells have been demonstrated in humans with septic shock. The use of pro-angiogenic agents that can mobilize endothelial progenitor cells has therefore been explored. These agents include statins and erythropoietin (EPO). Statins ameliorate experimental AKI via inhibition of vascular superoxide generation and restoration of endothelial derived NO synthase activity. In a retrospective review of adults undergoing open cardiac surgery, preoperative statin exposure was a protective factor against all stages of postoperative AKI [21]. Larger prospective randomized controlled trials of statins in AKI are under way. EPO is a potent stimulator of erythroid progenitor cells. In animal models of ischemic AKI, EPO has protective effects via amelioration of microvascular injury as well as anti-apoptotic and anti-inflammatory mechanisms. However, in a meta-analysis of controlled trials using EPO in adult patients at high risk for AKI, no reduction of AKI incidence could be demonstrated. Furthermore, in a recent study of extremely low gestational age neonates, EPO did not protect from AKI [22].

Alterations in the Inflammatory Response:

Major components of the inflammatory response in AKI include leukocyte recruitment and production of inflammatory mediators by tubule and endothelial cells. The latter include proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and the chemotactic cytokines (MCP-1, IL-8, RANTES). Human studies have shown that the plasma levels of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 are elevated in AKI and predict mortality.

Morphologically, neutrophils are the earliest leukocytes to accumulate in the kidney after AKI, in the peritubular capillaries of the outer medulla, where they adhere to endothelial cells and cause capillary plugging and congestion. Neutrophil depletion provides partial functional protection in some but not all animal models of AKI. Macrophages are the next to accumulate, largely in response to upregulation of MCP-1 in tubule cells and induction of its cognate receptor CCR2 on macrophages. Both resident macrophages as well as activated infiltrating macrophages play a role after experimental AKI. Classically activated macrophages, which predominate in early ischemic injury, produce proinflammatory cytokines. Alternatively activated macrophages modulate the inflammatory response and promote tissue repair and are prevalent during the recovery and repair phase of AKI. Dendritic cells (DCs), like macrophages, have pro- and anti-inflammatory functions and work closely with other components of the immune system to respond to kidney injury. DCs help regulate immune effector cells, and present antigenic material to T cells. The role of DCs in AKI is not fully resolved.

Intrinsic mechanisms that inhibit the inflammatory response exist. Tamm-Horsfall Protein (THP, uromodulin), secreted by the loop of Henle, inhibits proximal tubule production of proinflammatory cytokines and chemokines. THP is significantly upregulated within 48 hours of ischemia in experimental models. Genetic deletion of THP in mice resulted in more severe inflammation, increased cast formation, reduced kidney function, and diffuse tubular necrosis in the outer medulla. There is now evidence for a negative association between urinary THP and the development of human AKI [23]. Experimental administration of exogenous THP after AKI mitigates subsequent injury and hastens recovery.

Activation of the complement system in AKI, with resultant amplification of the inflammatory response, has received recent attention [24]. A predominant role for C5a in ischemic AKI has been identified during formation of the final membrane attack complex. C5a is a powerful chemoattractant that recruits inflammatory cells. The C5a receptor is normally expressed in the kidney, in proximal tubule epithelial cells and interstitial macrophages, and is upregulated after AKI. Inhibition of C5a generation using monoclonal antibodies protects against kidney dysfunction induced by ischemia in experimental models. Pre-treatment with orally active small molecule C5a receptor antagonists also substantially reduced the histologic and functional impairment induced by experimental AKI. In addition, the anti-C5 monoclonal antibody (eculizumab) ameliorates experimental ischemic AKI and is widely used in the AKI of atypical hemolytic uremic syndrome in humans.

Alterations in Gene Expression:

Attempts at unraveling the molecular basis of the myriad pathways activated by AKI have been facilitated by advances in functional genomics and transcriptome profiling. When combined with bioinformatics, these technologies have identified novel genes with altered expression, new signal transduction pathways that are activated, and even new drug targets and biomarkers in AKI [1, 2, 4, 5, 25–28]. A few clinically relevant examples are provided here.

Surprising recent data has challenged the dogma that the fibrotic response of the kidney to injury is a late phenomenon. Single-cell profiling of AKI in mice [25] and humans [26] has revealed very early activation of profibrotic transcriptional signatures. Experimental data suggest that this early fibrotic response can be blunted. In murine AKI due to ischemia-reperfusion, intraperitoneal administration of a peptide (pUR4) that inhibits fibronectin polymerization (an early event in the fibrotic cascade) soon after injury dramatically attenuated the early fibrotic response [29]. The pUR4 peptide was devoid of any adverse effects, rendering translational application to human AKI a possibility.

One of the most consistent findings from single-cell profiling in mice [25] and humans [26] is the rapid induction of an embryonic phenotype in injured proximal tubule cells, which exhibit re-expression of genes normally present only in the developing kidney (e.g., Sox4, Cd24a). This switch to the embryonic state is likely critical for regeneration of tubule cells lost during AKI. The identified candidates and their downstream effectors that accelerate repair represent new future therapies.

Genome wide association studies (GWAS) can identify potentially pathogenetic genomic sequences that are enriched in AKI cases compared to controls. A GWAS analysis of AKI cases [30] has yielded single-nucleotide polymorphisms (SNPs) involving the transcription factor interferon regulatory factor 2 (IRF2), and additional SNPs close to the transcription factor T-box 1 (TBX1). The identification of SNPs near IRF2 suggests a potential role for the immune system in AKI, a concept with already strong biologic plausibility. TBX1 is expressed during kidney development, and this finding supports the concept that genetic programs involved in nephrogenesis are reactivated after injury in post-natal life.

Alterations in Metabolomics:

Metabolomic studies in a mouse model of ischemic AKI have identified a deficiency in urinary and intra-renal nicotinamide adenine dinucleotide (NAD), an essential component of energy generation via glycolysis and the Kreb’s cycle [31]. In a phase I study of oral NAM supplementation (which generates NAD via a salvage pathway) in adults undergoing cardiac surgery, the rise in serum creatinine was prevented compared to placebo [31]. Additional translational studies are under way [32].

Summary and Conclusion:

Recent advances in the pathogenesis of intrinsic AKI provide a better understanding of the clinical continuum and the rational deployment of promising therapeutics. The clinical course of intrinsic AKI can be divided into four phases: initiation, extension, maintenance, and recovery. In the initiation phase, initial exposure to the kidney insult occurs, kidney vasoconstriction and parenchymal injury are evolving, and kidney function begins to deteriorate. Intracellular ATP depletion and generation of reactive oxygen molecules are initiated. Interventional approaches during this phase might include vasodilators, antioxidants, and iron chelators. In the extension phase, blood flow returns to the cortex, but medullary blood flow remains reduced, resulting in tubule cell death, desquamation, and luminal obstruction. Injured endothelial and epithelial cells amplify the inflammation, and endothelial denudation potentiates the vasoconstriction. The GFR continues to decline. Anti-inflammatory agents and anti-apoptotic measures are likely to be efficacious during this phase. During the maintenance phase, parenchymal injury is established, and the GFR is maintained at its nadir even though kidney blood flow begins to normalize. Cell injury and regeneration occur simultaneously. Measures to accelerate regeneration may be effective during this phase. These include growth factors, stem cells, and kidney support therapies. The recovery phase is characterized functionally by an improvement in GFR and structurally by reestablishment of tubule integrity, with fully differentiated and polarized epithelial cells. Re-expression of kidney developmental genes in injured tubule cells is likely critical for this process. However, the repair process may be incomplete or maladaptive, and AKI can progress to CKD.

Key Points:

• The pathogenetic mechanisms in AKI include vasoconstriction, microcirculatory changes, oxidant injury, tubule cell death, and an inflammatory response, all of which can be therapeutically targeted

• Injured tubule cells have a remarkable ability to regenerate and repair, which can be pharmacologically enhanced

• Contemporary single cell profiling technologies in AKI have revealed a rapid induction of an embryonic phenotype in injured proximal tubule cells, and an early activation of profibrotic transcriptional signatures

• Emerging technologies continue to identify novel genes with altered expression, new signaling pathways, and drug targets in AKI

ABBREVIATIONS

AKI

Acute Kidney Injury

ATP

Adenosine Triphosphate

CKD

Chronic Kidney Disease

EPO

Erythropoietin

GFR

Glomerular Filtration Rate

GWAS

Genome wide association studies

HGF

Hepatocyte Growth Factor

IGF-1

Insulin-like Growth Factor

MSC

Mesenchymal Stem Cell

MSH

Melanocyte Stimulating Hormone

NAD

Nicotinamide Adenine Dinucleotide

NO

Nitric Oxide

ROS

Reactive Oxygen Species

THP

Tamm-Horsfall Protein