Innovations and Emerging Therapies to Combat Renal Cell Damage: NAD⁺ As a Drug Target

Abstract

Significance: Acute kidney injury (AKI) is a common and life-threatening complication in hospitalized and critically ill patients. It is defined by an abrupt deterioration in renal function, clinically manifested by increased serum creatinine levels, decreased urine output, or both. To execute all its functions, namely excretion of waste products, fluid/electrolyte balance, and hormone synthesis, the kidney requires incredible amounts of energy in the form of adenosine triphosphate.

Recent Advances: Adequate mitochondrial functioning and nicotinamide adenine dinucleotide (NAD⁺) homeostasis are essential to meet these high energetic demands. NAD⁺ is a ubiquitous essential coenzyme to many cellular functions. NAD⁺ as an electron acceptor mediates metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis, serves as a cosubstrate of aging molecules (i.e., sirtuins), participates in DNA repair mechanisms, and mediates mitochondrial biogenesis.

Critical Issues: In many forms of AKI and chronic kidney disease, renal function deterioration has been associated with mitochondrial dysfunction and NAD⁺ depletion. Based on this, therapies aiming to restore mitochondrial function and increase NAD⁺ availability have gained special attention in the last two decades.

Future Directions: Experimental and clinical studies have shown that by restoring mitochondrial homeostasis and increasing renal tubulo-epithelial cells, NAD⁺ availability, AKI incidence, and chronic long-term complications are significantly decreased. This review covers some general epidemiological and pathophysiological concepts; describes the role of mitochondrial homeostasis and NAD⁺ metabolism; and analyzes the underlying rationale and role of NAD⁺ aiming therapies as promising preventive and therapeutic strategies for AKI. Antioxid. Redox Signal. 35, 1449–1466.

Introduction

Acute kidney injury (AKI) is a loose collection of syndromes whose defining characteristic is an abrupt loss of glomerular function (57). It can be caused by diverse mechanisms from urinary tract obstruction to sepsis, to nephrotoxin exposure—frequently multiple etiologies are involved (91). AKI is common and life-threatening, complicating more than 10% of hospital admissions and up to 50% of those who require critical care (1, 46). It increases the risk of death by three- to sevenfold when compared with those who never develop AKI (1). In addition, about 50% of patients developing moderate to severe AKI [i.e., Kidney Disease: Improving Global Outcomes (KDIGO) (58) stage 2 or 3] do not recover renal function by 30 days (86, 97, 98). Despite the high morbidity and mortality associated with AKI, therapies remain reactive and nonspecific and little impact has been made in decreasing AKI incidence and its associated long-term complications. Thus, there is a critical unmet need to find therapeutics that can predict and mitigate kidney damage, and/or improve recovery across a range of different AKI etiologies.

The pathogenesis of AKI is complex and multifactorial (40). Among the multiple pathogenic mechanisms that may lead to AKI, energy metabolism pathway alterations seem to play a key role in the development of many forms of AKI. Energy metabolic alterations can be attributed primarily to mitochondrial dysfunction and nicotinamide adenine dinucleotide (NAD⁺) deficiency. NAD⁺ is a ubiquitous coenzyme that participates in different mitochondrial and nonmitochondrial energy producing pathways, serves as a cosubstrate of aging mediating enzymes, and participates in DNA repair mechanisms. NAD⁺ is crucial to maintain mitochondrial homeostasis and adenosine triphosphate (ATP) production either through oxidative and nonoxidative mechanisms. Thus, its importance in renal physiology as it is essential to preserve an adequate kidney functioning and prevent cellular injury. Indeed, renal tubular epithelial cells (TECs) deficient in NAD⁺ cannot defend themselves against AKI and cannot repair damage once injury has occurred. Moreover, studies in which therapies aiming to improve availability of NAD⁺ have been used, and have shown that the replenishment of NAD⁺ in TECs results in decreased AKI incidence and the less probability of development of long-term complications such as chronic kidney disease (CKD). This review covers some general concepts of AKI emphasizing on its relevance as a continuously growing public health problem; it covers some pathophysiologic concepts focusing on the role of mitochondrial dysfunction and NAD⁺ deficiency in the development of AKI; and finally, it analyzes the existing evidence of NAD⁺ as a rising and promising therapeutic target in AKI.

AKI: A Public Health Problem

The modern definition of AKI was based on the RIFLE (Risk, Injury, Failure, Loss, and End-stage renal disease) criteria (6). These criteria are centered on alterations in the glomerular filtration rate (GFR), estimated imperfectly by changes in serum creatinine and, more imperfectly still, by changes in urine volume over time. The inclusion of urine output, however, provides considerable discrimination to the severity of kidney dysfunction and often is more time-sensitive compared with serum creatinine alone (53, 59). Serum creatinine is very insensitive to kidney damage and while urine output is less specific, it is more sensitive to many forms of AKI. In 2012, the KDIGO AKI Clinical Practice Guidelines (58) harmonized the adult and pediatric criteria for AKI and have become the standard definition (Table 1). The discovery of specific biomarkers for kidney damage will likely enrich the definition and diagnosis of AKI in the near future (79, 91).

Table 1.

Kidney Disease: Improving Global Outcomes Criteria for Acute Kidney Injury

Stage	sCr	UO
1	1.5–1.9 × baseline	<0.5 mL/kg/h for 6–12 h
	OR
	≥0.3 mg/dL (>26.5 μM) increase
2	2.0–2.9 × baseline	<0.5 mL/kg/h for 12 h
3	3 × baseline	<0.3 mL/kg/h for ≥24 h
	OR	OR
	Increase in sCr ≥4.0 mg/dL (353.6 μM)	Anuria for ≥12 h
	OR
	Initiation of RRT
	OR
	In patients <18 years, decrease in eGFR to <35 mL/min per 1.73 m2

AKI, acute kidney injury; GFR, glomerular filtration rate; KDIGO, Kidney Disease: Improving Global Outcomes; RRT, renal replacement therapy; sCr, serum creatinine; UO, urine output.

The incidence of AKI in Europe is estimated to be as high as 39 per 1000 population with similar rates in Australia (91). In the United States, estimates have been lower, but still quite high—about 18 per 1000. Thus, rates of AKI are approaching 6 million cases a year in the United States and more than four times this in all of Europe. As for the rest of the world, rates among hospitalized patients are quite similar to those reported in Europe meaning that there could be as many as 300 million new cases of AKI in the world in 2020! These numbers underscore the massive public health crisis that AKI represents and the urgent need to find effective treatment.

Early attempts to address AKI pharmacologically focused almost exclusively on altering renal hemodynamics. This was due, in part, to a mistaken notion that the pathogenesis of AKI was mainly ischemic and by the equally tragic approach ignoring that AKI is a collection of syndromes with multiple etiologies rather than a single disease with a unique cause and a singular therapeutic approach. Therefore, limited progress has been made and therapies still remain reactive and nonspecific with a shallow impact in morbidity and mortality. Understanding AKI as a global entity, in which multiple and different pathogenic mechanisms take part simultaneously, is crucial for the better comprehension of this syndrome, and furthermore, will allow broadening the perspective in the development of newer effective preventive and therapeutic strategies. Common causes of AKI are listed in Table 2. Moreover, understanding that although clinical manifestations of AKI might be similar among patients, the underlying pathophysiology may widely differ between them. Thus, interventions developed for one particular type/etiology of AKI may fail when studied in heterogenous groups of patients.

Table 2.

Common Causes of Acute Kidney Injury

Sepsisa

Drugs

Cardiac surgery

Acute decompensated heart failure

Noncardiac surgery

Trauma/burns

Oncologic disease

Hepatorenal syndrome

Environmental toxins, plants, insects, animals

^a AKI is often the sepsis-defining organ injury in severe infections (e.g., pneumonia)—thus, patients do not need to have sepsis first.

Pathogenesis of AKI

Understanding of AKI has advanced greatly since the beginning of the 21st century. We now understand that AKI is not a single disease, but a collection of syndromes that can be provoked by diverse pathogenic mechanisms, which often coincide (83). Nevertheless, most of the current knowledge has been extrapolated from animal models, in vitro studies, and postmortem observations in humans (83). Therefore, these observations should be interpreted with caution since the response to various kidney insults (e.g., sepsis, nephrotoxins, and ischemia) or therapies may vary widely from that in humans.

AKI is a “silent disease” in which clinical manifestations may not become evident until severe stages of the disease are reached. Because of this, prevention, early diagnosis, and intervention are almost impossible. Moreover, establishing the exact time of injury and main cause is hindered by the silent course of AKI, unlike in preclinical experimental models in which these variables are mostly controlled, and time and cause of injury are primarily determined by the characteristic features of the implemented AKI model. Conversely, AKI in the clinical setting is a multifactorial entity in which multiple pathogenic mechanisms coincide, increasing the complexity to understand and build a framework that encompasses the different elements that play a role during the pathogenesis of acute renal injury. These and many other limitations frustrate the translation of promising effective diagnostic and therapeutic strategies to the bedside.

The leading causes of AKI, namely sepsis, major surgery, heart failure, and hypovolemia, are associated to greater or lesser extent with decreased renal perfusion as a consequence of shock and extensive tubule-epithelial cell death (113). Therefore, it was compelling to explain most forms of AKI as a manifestation of decreased global renal blood flow and acute tubular necrosis (94). However, in the last two decades, the paradigm that encompasses these elements as main pathogenic events of AKI has been challenged. It is now clear that several mechanisms other than renal hypoperfusion play a role in AKI, and the extension of TEC death is not as vast as initially contemplated (62, 67, 92, 105). In multiple preclinical and clinical studies, it has been demonstrated that renal injury and AKI might occur in the absence of overt signs of hypoperfusion and/or clinical signs of hemodynamic instability (63, 74, 86). Murugan et al. (74) demonstrated in a large clinical cohort study that one-third of patients admitted to the hospital with a diagnosis of community-acquired pneumonia developed AKI without presenting clinical signs of hypoperfusion, hemodynamic instability, or requiring admission to an intensive care unit. These clinical observations have been supported and documented in preclinical models of AKI too. For instance, in animal models in which sepsis was induced to provoke AKI, animals that had normal or even increased global renal blood flow also developed AKI (10, 25–27, 61, 63, 90). In addition, histopathologic studies made in postmortem human biopsies of patients who suffered AKI have also rebutted the presumption that rapid renal function deterioration is associated with extensive acute tubular necrosis (62, 67, 105). Conversely to what was initially considered, heterogenous, focal, and patchy tubular injuries, with minimal tubule-epithelial cell death (<5%), apical vacuolization, and minor focal mesangial expansion, are the consistent findings in these specimens (62, 67, 105).

These observations clearly challenge the traditional notion of ischemic injury and significant cell death underlying AKI. Moreover, the evident dissociation that exists between the described structural alterations and the renal functional decline strongly suggests that other factors must be at play. Several heterogenous theories and mechanisms have been proposed to explain the still puzzling pathogenesis of AKI such as energy metabolism impairment, cellular metabolic reprogramming, impaired inflammatory response, and microcirculatory dysfunction (40). Although some of these seem to be disparate, many forms of AKI seem to share many of these mechanisms of injury such as energy metabolism impairment caused by mitochondrial dysfunction and NAD⁺ depletion and cellular metabolic reprogramming (41, 83).

Mitochondria and AKI

Interest in mitochondrial dysfunction, once limited to cancer, heart, and brain pathology, has exploded in the field of AKI. The kidney is one of the highest energy-demanding organs in the body, second only to the heart in abundance of mitochondria (8, 80, 120). Mitochondria are a complex network of organelles, which functions include energy synthesis, nucleotide and heme synthesis, regulation of reactive oxygen species (ROS), calcium homeostasis, and thermogenesis and regulation of mechanisms of programmed cell death (i.e., apoptosis) among others. In the kidney, mitochondria play a crucial role in terms of providing and maintaining adequate pools of energy allowing the kidney to execute all its functions, namely waste product excretion, secretion and reabsorption of nutrients, hormone synthesis, and maintenance of fluid/electrolyte equilibrium and acid-base balance. Therefore, the high susceptibility of the kidney to any sort of energy metabolic impairment and mitochondrial dysfunction. Mitochondrial dysfunction may lead to TEC injury and organ function deterioration in several ways either due to ATP deficiency or dysregulation in cell death mechanisms. ATP deficiency can occur either due to decreased synthesis due to impaired energy metabolic pathways or impaired utilization of substrates that fuel these pathways. Therefore, an adequate mitochondrial integrity, bioenergetics, and recycling (mitophagy) are essential to meet the high-energy demands of the kidney. On the contrary, healthy mitochondria are not only crucial for adequate cell functioning and survival but also to initiate repair and healing mechanisms once injury has been caused (18, 19, 23, 40, 41, 51, 67, 100, 119).

In terms of energy consumption, renal functions could be assorted into active or passive processes. For instance, glomerular blood ultrafiltration is one of the most important energy passive processes that occur in the kidney. In the glomeruli, the plasma gets filtered through the glomerular basement membrane into the tubular collecting system to produce urine. The energy requirements of this process are very low, as it is mainly regulated by endocrine/hemodynamic changes of the afferent and efferent arterioles. Conversely, functions such as tubular reabsorption and secretion of nutrients require higher amounts of energy constituting active processes. Approximately 80% of solute transport, nutrient reabsorption, and ultrafiltrate osmotic gradient regulation occur in the proximal TECs and the medullary thick ascending limb of Henle's loop. Therefore, the major pool of renal mitochondria and the highest oxygen consumption rates and oxidative phosphorylation (OXPHOS) are registered in these two segments of the renal tubular system (11, 121).

TECs primarily rely on oxidative metabolic pathways to keep an adequate flow of energy supply to be able to meet the high energetic demands (11, 49, 103, 121). These oxygen-dependent pathways mostly take place in the mitochondria, where oxygen will act as the last electron acceptor of the electron transport chain (ETC). Mitochondria transform energy in the form of ATP, through oxidation of nutrients, including glucose, fatty acids, and amino acids, in a two-prong process known as OXPHOS. First, the sequential cycling of nutrient substrates through the tricarboxylic acid cycle in the mitochondrial matrix delivers reducing equivalents (i.e., electrons) to the mitochondrial ETC in the inner mitochondrial membrane. Then, nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide (FADH₂) hand over electrons to complex I and II, respectively. This delivery of electrons to the ETC constitutes the rate-limiting step of fatty acid oxidation (FAO) (111). Then, cycling of electrons in the ETC promotes the extrusion of hydrogen ions (H⁺) out of the matrix into the intermembrane space, generating an electrochemical gradient of H⁺ across the mitochondrial membrane, which holds the energy derived from nutrients into potential energy. Finally, H⁺ re-enters the mitochondrial matrix through complex V in the ETC in favor of the electrochemical gradient, releasing the potential energy that ATP synthetase then uses to assemble ATP molecules (Fig. 1).

FIG. 1.

FAO, ETC, and glycolysis. The kidney has a high-energy demand. After cardiac myocytes, TECs contain the second-most abundant mitochondria population. For an adequate functioning, TECs require high amounts of ATP. Fatty acid oxidation (FAO) is the preferred energy source of the kidney. Long-chain FA enter the TECs with the help of CD36. Once in the cytoplasm, acyl-CoA synthetase activates FA into FA-acyl-CoA. As the outer mitochondrial membrane is impermeable to FA-acyl-CoA, CPT-1 is necessary for the transesterification of FA-acyl-CoA into acylcarnitine allowing it to cross the outer membrane to get into the inter membrane space. Acylcarnitine diffuses into the inner matrix of the mitochondria using the CACT transporter. Inside the mitochondrial matrix, the acylcarnitine is transformed back to FA-Acyl-CoA by CPT-2. β-oxidation of FA-Acyl-CoA will fuel the TCA cycle, which then will result in the production of high-energy electrons stored in NADH and FADH₂ cofactors to be carried and transferred to the ETC to act as electron donors (44). Pyruvate, a breakdown product of glycolysis can also be converted to acetyl-coA via pyruvate dehydrogenase present in the mitochondrial matrix. In the ETC, NADH and FADH₂ transfer electrons to complex I and complex II, respectively. Then a flux of electrons occurs along the ETC traveling the complex IV where they bind O2, the ultimate electron acceptor, via the NADPH oxidase. As a final step, protons generated by this process flow back into the mitochondrial matrix through ATP synthase converting ADP into ATP. ADP, adenosine-diphosphate; ATP, adenosine-triphosphate; CACT, carnitine-acylcarnitine translocase; CPT-1, carnitine palmytoiltransferase-1; CPT-2, carnitine palmytoiltransferase-2; ETC, electron transport chain; FA, fatty acids; FADH₂, flavin adenine dinucleotide; FAO, fatty acid oxidation; NADH, nicotinamide adenine dinucleotide hydrogen; TECs, tubular epithelial cells; TCA, tricarboxylic acid.

Oxidative pathways are very productive when it comes to producing energy compared with glycolytic and anaerobic mechanisms. The high amounts of ATP that are produced through the oxidation of fuel substrates are mostly intended to be used by Na⁺/K⁺ ATPase pumps along the basolateral membrane of TECs. Na⁺/K⁺ ATPase pumps constitute the main ion transporters in the tubular collecting system. Among the several functions of the Na⁺/K⁺ ATPase pumps, the most important include creating electrochemical gradients between the extracellular and intracellular matrix to facilitate diffusion of ions and reabsorption of nutrients into the blood and secretion of solutes and waste products into the ultrafiltrate that will result in the formation of urine. To generate an osmotic gradient across the cellular membrane, the Na⁺/K⁺ ATPase pump fueled by a molecule of ATP pushes 3 Na⁺ ions out and pulls 2 K⁺ ions in (103). In addition, by regulating Na⁺ and water concentrations, it adjusts the osmolarity of the urine according to the physiological needs of the organisms to maintain an adequate fluid and electrolyte balance (8).

As aforementioned, oxidative pathways are not the only source that TECs could appeal to synthesize ATP. TECs have also the ability to synthesize ATP in the absence of oxygen. In anaerobic glycolysis, pyruvate is converted to lactate instead of feeding the tricarboxylic acid cycle, inducing NAD⁺ regeneration (19). From these anaerobic pathways, only two molecules of ATP result, a small profit when compared with the more than 30 units that can be produced in the presence of oxygen (41, 49, 55). Although less efficient in terms of units of ATP generated, these anaerobic pathways play a key role during several renal physiologic and pathologic mechanisms. These anaerobic pathways allow renal cells, especially TECs, to maintain a minimum of vital energetic requirements in case of stress and injury even when this low-energy state might manifest with a downregulation of cellular functions. For instance, in sepsis-associated AKI where tissue perfusion and oxygen utilization are impaired, TECs switch to a nonoxidative metabolism in an attempt to promote cell survival at the expense of TEC function (41). However, the kidney does not only appeal to glycolysis during pathologic conditions. The renal medulla, unlike the cortex, constantly bears with low concentrations of oxygen. Most of the oxygen delivered to the kidney is utilized before reaching the medullary vasculature, making the medulla highly susceptible to hypoxic injury. Because of this constant hypoxemic state, glycolysis becomes the most suitable alternative to synthesize ATP (28, 32, 69, 77, 109, 120).

When mitochondrial function is impaired, many essential cellular mechanisms fail. This is what occurs in most forms of AKI, where mitochondrial homeostasis is busted and renal function deteriorates. A decreased mitochondria population, structural alterations (e.g., mitochondrial swelling structural disruption of the cristae), and impaired mitochondrial biogenesis are commonly observed in AKI (81, 112). Tran et al. (109) found that in sepsis-induced AKI, TEC mitochondria presented structural alterations characterized by the accumulation of acylglycerols. Accumulation of acylglycerols has a direct effect on prostaglandin E2 (PGE2) synthesis and production. During sepsis, systemic vasodilation and decreased vascular resistance are seen. As a response to these mechanisms, the sympathetic autonomic nervous system, activation of the renin/angiotensin/aldosterone system, and secretion of vasopressin cause intrarenal vasoconstriction to compensate for the decrease in renal perfusion pressure. Concomitantly, the absence of PGE2 and vasodilator counteracting effects due to mitochondrial dysfunction will exacerbate medullary ischemia by promoting renal vasculature vasoconstriction (109). Some studies have also shown that metabolic impairment contributes to the endothelial injury and microcirculatory dysfunction seen in sepsis-AKI (5, 111). Despite the increasing understanding, therapies targeting mitochondrial injury in AKI are still limited. Guo, et al. (43) found that in cisplatin-induced AKI in the mouse and in biopsy samples of human AKI kidney tissue, micro-RNA (miR)-709 was significantly upregulated in proximal tubular cells (PTCs). The expression of miR-709 in PTCs also correlated with the severity of AKI. In cultured mouse PTCs, overexpression of miR-709 induced mitochondrial dysfunction, whereas inhibition of miR-709 ameliorated cisplatin-induced mitochondrial dysfunction and cell injury. The investigators also showed that the mitochondrial transcriptional factor A (TFAM) is a target gene of miR-709, and genetic restoration of TFAM attenuated mitochondrial dysfunction and cell injury in vitro. These findings reveal a pathogenic role of miR-709 in acute tubular injury and suggest a novel target for the treatment of AKI. These findings support the concept that mitochondrial homeostasis is fundamental for the proper functioning of the kidney (19, 32). Moreover, in cases where injury has already occurred, re-establishing and preserving a healthy and functional mitochondrial population are needed to achieve adequate regeneration and healing outcomes.

Mitochondrial biogenesis

Mitochondrial biogenesis is a tightly regulated adaptive process by which the cell is able to produce new and functional mitochondria (4, 65, 104). Mitochondrial biogenesis is primarily regulated by peroxisome proliferator-activated receptor γ -coactivator-1α (PGC-1α). PGC-1α is a transcription factor that, in response to mitochondrial dysfunction, upregulates mitochondrial gene expression (8). Therefore, in cells with high mitochondrial populations, such as renal TECs, PGC-1α activity is more abundant (110). PGC-1α activity is regulated by the mammalian target of rapamycin complex 1 (mTORC1), adenosine monophosphate-activated protein kinase (AMPK), and sirtuins, more specifically sirtuin 1 (Sirt 1) and sirtuin 6 (Sirt 6). mTORC1 is a serine/threonine kinase complex that mediates glycolytic metabolism. It induces PGC-1α activity and mitochondrial biogenesis through the activation of the transcriptional repressor yin and yang 1 (YY1) (21). On the contrary, AMPK and sirtuins, key regulators of oxidative metabolism, also have the ability to upregulate the activity of PGC-1α (41).

In some forms of AKI, especially in those associated with ischemic injury and sepsis, it has been observed that PGC-1α expression and activity are downregulated. It seems that this decrease in function may be attributed to the increased inflammatory cytokines (38, 111). Although it has not been demonstrated that PGC-1α deficiency causes damage, it seems that when associated with other metabolic factors it may induce some predisposition to cell injury (109, 110). Conversely, increased expression and activity of PGC-1α seem to be protective. For instance, in TECs in which PGC-1α activity was augmented, AKI severity was markedly decreased. Furthermore, these cells had a higher capacity of regeneration after injury (109). PGC-1α also participates in the production of NAD⁺ by mediating the de novo synthesis pathway. Although under normal circumstances PGC-1α plays an important role in this pathway, its absence may be overcome by repletion of NAD by administering nicotinamide. When NAD⁺ is repleted, risk and severity of AKI are decreased, even with low levels of PGC-1α (85). Based on this, AKI might be the result of direct or indirect correlations among PGC-1α shortfall, impaired adaptive mitochondrial biogenesis, and NAD⁺ metabolic alterations. However, it seems important to highlight that NAD⁺ seems to play a more significant role in AKI, especially when all the other mechanisms are compromised.

Metabolic Reprogramming in AKI

In various forms of illness, such as cancer, cells undergo metabolic reprogramming as an adaptive response to stress or injury. Metabolic reprogramming is a process in which the cell switches from using a high-effective oxidative metabolism (i.e., OXPHOS) to less-effective glycolytic metabolism pathways. Furthermore, this metabolic reprogramming is tightly correlated with the inflammatory response. This metabolic switch is also known as the “Warburg” effect (41). Experimental data suggest that a similar reprioritization of energy metabolism pathways may occur in AKI especially when associated with sepsis or ischemia (102, 117). A decrease in fuel substrates and oxygen utilization primarily caused by macro- and microvascular dysfunction in AKI “obliges” TECs to appeal to less-efficient energy production mechanisms in an attempt to survive. Although functionally speaking, the cell enters a state of “hibernation,” where only life-essential functions are being executed, and thus, a decline in organ function will be seen, one of the mechanisms the cell possesses to bear with stress and injury.

Metabolic reprogramming in TECs occurs in a biphasic manner: an early proinflammatory response phase, primarily mediated by aerobic glycolysis, followed by a late anti-inflammatory phase, in which OXPHOS predominance is re-established (Fig. 2) (36). Although for sake of understanding and clarity these two phases are often described as individual and independent processes, it is important to state that these two phases share significant overlap (41). In the early phase, TECs, which are normally in an oxidative metabolic state (i.e., OXPHOS), reprogram their metabolic line and deviate it to glycolysis (41). The Akt/mTORC1/hypoxia-inducible factor-1 alpha (HIF-1α) complex drives the induction of aerobic glycolysis by upregulating the gene expression of glycolytic enzymes such as lactate dehydrogenase, pyruvate kinase M2, and pyruvate dehydrogenase kinase (Fig. 2). The limited amounts of ATP produced through glycolysis are intended to maintain TEC viability by preserving critical cellular components (e.g., Na⁺/K⁺ ATPase pump) that will allow the cell to survive (114). In addition, decreased usage of oxidative metabolism is also accompanied by diminished production of ROS and less oxidative stress. However, with limited ATP supplies, “nonvital” functions such as protein synthesis and solute and ion transportation (e.g., decreased synthesis and function of ion transporters) are downregulated (8, 42, 50, 68, 83, 93). The usage of glycolysis also results in increased lactate levels due to the upregulated conversion of pyruvate into lactate promoted by HIF-1α (Fig. 2) (16, 17). When the AMP:ATP ratio starts to increase, the activity of AMPK is upregulated resulting in TEC metabolic switch back to oxidative metabolism. AMPK is an essential mediator for cellular metabolism by sensing energy catabolism playing a key role in multiple functions in the cell. AMPK promotes PGC-1α activity therefore stimulating mitochondrial biogenesis. Furthermore, it stimulates the synthesis of antioxidant reagents, activates autophagy and mitophagy, and more important re-establishes the utilization of OXPHOS and finally inhibits mTORC limiting glycolysis.

FIG. 2.

Metabolic reprogramming. In the early metabolic response to AKI, renal tubular epithelial cells undergo a proinflammatory phase (acute anabolic phase) metabolism in which the Akt/mTORC1/HIF-1α complex drives the induction of aerobic glycolysis by increasing the expression of glycolytic enzymes (e.g., LDH, PKM2, and PDHK). HIF-1α promotes the conversion of pyruvate to lactate and along with PDHK inhibits the conversion of lactate into acetyl-CoA hindering the induction of the Krebs cycle and decreasing OXPHOS. In the late anti-inflammatory (adaptive catabolic phase), OXPHOS metabolic pathways are re-established. This is driven by AMPK activation, Sirt1, and Sirt6. AMPK activates Sirt1 and Sirt6. Sirt6 will block the activity of HIF-1α switching back from aerobic glycolysis to OXPHOS and is induced by the decrease in ATP levels. AMPK activates PGC-1α and with CPT-1 will stimulate FA oxidation and oxidative metabolism. Furthermore, PGC-1α along with AMPK will induce mitochondrial biogenesis (31, 32, 34). AKI, acute kidney injury; AMPK, adenosine monophosphate-activated protein kinase; HIF-1α, hypoxia-inducible factor-1 alpha; LDH, lactate dehydrogenase; mTORC1, mammalian target of rapamycin complex 1; OXPHOS, oxidative phosphorylation; PDHK, pyruvate dehydrogenase kinase; PGC-1α, PPAR γ coactivator-1α; PKM2, pyruvate kinase M2; PPAR, peroxisome proliferator-activated receptor.

This metabolic reprogramming results in energy expenditure optimization (41, 83, 117) , refinement of fuel substrate utilization, and counteraction of proapoptotic triggers (41, 48, 100, 105). These changes explain to a certain extent the cellular structural and functional dissociation observed in AKI, in which organ function decline is not directly associated with cell death, supporting the premise that TECs prioritize energy expenditure and cell survival at the expense of cell function (41, 83, 117).

In preclinical and clinical studies of sepsis-induced AKI, it has been demonstrated that the switch from OXPHOS to glycolysis may be associated with deleterious effects on mortality. Also, it has been demonstrated that in sepsis-induced AKI, AMPK activation and stimulation of OXPHOS at early time points are associated with improved overall rates of survival (33, 52). In addition, Gomez et al. have shown some preliminary data in septic diabetic patients, in whom previous exposure to metformin, an AMPK activator, had lower mortality rates at 90 days when compared with other treatments (unpublished data). However, a dissociation between renal structure and function is constantly observed. In many of these studies although survival rates improve with the activation of AMPK, this is not directly associated with an improvement in renal function, suggesting that other mechanisms must be at play (33, 78, 123).

NAD⁺ and AKI

In the last two decades, NAD⁺ has gained special attention, as increased body of evidence has demonstrated that NAD⁺ deficiency is associated with increased risk to develop acute and CKDs (3, 14, 118, 122). NAD⁺ is a ubiquitous enzyme that plays a key role in several cellular metabolic functions, aging mechanisms, and adaptive responses to cellular stress or injury among others (35). NAD⁺ along with its reduced form (i.e., NADH) serves as an electron acceptor and as a donor in the ETC in the mitochondria, and constitutes the ATP synthesis rate-limiting substrates of FAO and glycolysis. In addition, NAD⁺ also participates as a substrate in many other nonredox essential cellular functions (e.g., sirtuins, poly-adenosine diphosphate-ribose polymerases [PARPs], and ectonucleotidases), mediates oxidative stress response, and regulates calcium signaling (34, 35, 85). Therefore, it is not surprising that any type of NAD⁺ deficiency, either due to decreased synthesis or increased consumption, may lead to mitochondrial function deterioration resulting in subsequent renal injury due to impaired ATP synthesis (82, 110).

NAD⁺ intracellular concentrations, and therefore availability, are determined by a tightly regulated balance between consumption, salvage, and de novo synthesis. Alterations at any of these levels may lead to NAD⁺ deficiency, which has been associated with the development of a wide range of renal diseases (44). NAD⁺ can be synthesized de novo or recycled through several pathways in which a sort of host intrinsic and/or dietary precursors are necessary to execute these processes. Tryptophan, one of the essential amino acids, is the main precursor in the de novo NAD⁺ synthesis pathway. Tryptophan enters the cell and is converted to quinolinate, which is then converted into nicotinic acid mononucleotide (NAMN) by the action of quinolinate phosphoribosyl transferase (QPRT), the bottleneck enzyme of this process. Finally, NAMN is going to produce NAD⁺. QPRT deficiency results in NAD⁺ defective synthesis and depletion, which as discussed later in this chapter are associated with increased susceptibility to develop AKI (56, 85). Another important regulator in the de novo pathway is the mitochondrial biogenesis regulator PGC-1α. Tran and his group have described the PGC-1α ability to induce the de novo pathway, and how PGC-1α malfunctioning may result in impaired synthesis of NAD⁺ (82, 110).

NAD⁺ deficiency may result from different mechanisms such as dietary deficiency, genetic alterations of precursor's transporters, or cellular injury (44, 60). NAD⁺ depletion and impaired homeostasis may also manifest as diseases, especially affecting high-energy-consuming organs such as the brain, heart, and kidney (33, 35). In ischemic models of renal injury, it has been shown that ischemia induces the activation of NAD⁺ consuming enzymes such as PARPs and CD38 (30, 82). Also, in AKI, an accelerated consumption of NAD⁺ results due to the increased gene expression PARP (71). Furthermore, these same studies have demonstrated that impaired activity of the PAPR either by pharmacologically inhibition or in PARP1-knockout mice resulted in less severe findings of AKI (71, 89, 125).

Vitamin B3 and its different associated forms play a key role in the regulation of NAD⁺. For instance, nicotinamide one of the forms of vitamin B3 serves as a precursor of NAD⁺ in the salvage pathway (Fig. 3) (85). In experimental and clinical studies, administration of high concentrations of oral nicotinamide, much higher than the recommended nutritional dose, induces the salvage pathway resulting in increased levels of NAD⁺ regardless if other alternative pathways are compromised. Furthermore, in a recent study, Simic et al. (99) demonstrated that administration of nicotinamide riboside (NR) and pterostilbene to AKI patients resulted in increased total NAD⁺ concentrations, with no significant adverse events secondary to the administrations of the vitamin B3 precursors. Furthermore, some studies described it as the “most potent” therapy when it comes to replenishing NAD⁺ reserves (13, 108), making NR a safe and viable therapy with high feasibility of implementation (34).

FIG. 3.

NAD⁺ metabolism. NAD⁺ and its reduced form (NADH) are essential cofactors for most metabolic pathways, especially the oxidative metabolism. NAD⁺ and NADH redox cycling are necessary to produce energy in the form of ATP from fuel substrates such as glucose and FA through glycolysis and OXPHOS, respectively. NAD⁺ and NADH gain access to the mitochondria through the malate and aspartate shuttle and the glycerol-3-phosphate shuttle. NAD⁺ needs to be in constant regeneration and production to supply the energetic needs of the cell. Several NAD⁺ synthesis pathways have been described. De novo pathway: Dietary tryptophan is converted in quinolinate and then in NAMN by the QPRT. Preiss/Handler pathway: Nicotinic acid is converted to NM by NAPRT. Salvage pathway is the most important pathway by which NAD⁺ is produced. NR and NAM are converted into NMN that will be converted into NAD⁺ (94). 2-ADP ribose, 2-adenosine diphosphate ribose; ATP, adenosine triphosphate; ER, endoplasmic reticulum; G-3-P, glycerol 3 phosphate shuttle; LPS, lipopolysaccharide; M/A shuttle, malate/aspartate shuttle; NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide; NAMN, nicotinic acid mononucleotide; NAPRT, nicotinate phosphoribosyltransferase; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; QPRT, quinolinate phosphoribosyl transferase.

Other NAD⁺ roles have been extensively described especially those associated with aging. NAD⁺ serves as a cofactor to the sirtuin pathway and its deficiency is associated with shorter longevity and increased risk of degenerative diseases (9, 15, 35, 85). In addition, NAD⁺-dependent activation of Sirt-1 leads to Sirt1-mediated deacetylation and activation of PGC-1α stimulating mitochondrial biogenesis. NAD⁺ also plays a key role during the regenerative and repair mechanisms after injury, and cumulating evidence is showing that preserved NAD⁺ homeostasis is protective against damage and injury (89).

NAD⁺ and NADPH oxidases

ROS play critical roles in cellular physiology by regulating multiple biological processes and are involved in the pathological process of different types of AKI. As one of the major sources of ROS, NADPH oxidases (NOXs) produce many kinds of free radicals. ROS levels increase early in the pathological process and are predominantly localized in tubules with no capillary blood flow. NOX4 is the major renal isoform and highly expressed in the kidney, mostly in the highly metabolic proximal tubular compartment with lower levels in glomerulus (39). Furthermore, NOX4 expression is increased in a renal ischemic/reperfusion (I/R) injury model, a classical model mimicking human ischemic acute tubular necrosis that causes kidney injury and leads to programmed cell death (7, 73, 75, 124). Although evidence is clear that oxidative stress is the primary factor in AKI, NOX-derived superoxide is central to I/R- and cisplatin-induced AKI (72, 95), the functional role of NOX4 in AKI is still controversial.

Other Pathogenic Mechanisms in AKI

Inflammatory response

The inflammatory response is the host's main defense mechanism against infection. Without a coordinated and regulated inflammatory response, the host would not be able to fight and survive the invasion of external pathogens. However, in some cases, the host response to infection can be excessive or dysregulated causing more harm instead of benefit. In AKI, the inflammatory response plays a key role. It is the main mechanism by which the host eliminates the insult and initiates regeneration and healing processes. From a metabolic and phenotypical standpoint, the inflammatory response in sepsis-induced AKI can be described as biphasic: a proinflammatory phase, primarily mediated by glycolysis, followed by an anti-inflammatory phase mediated by oxidative metabolism.

The inflammatory response is initiated when an infectious organism penetrates the physical barriers and invades the host. A series of molecules, present in most external pathogens, known as pathogen-associated molecular patterns (PAMPs) are recognized by immune cells (31, 51). These inflammatory cells express in their membranes and cytoplasm a series of receptors that bind in a nonspecific manner these patterns. These receptors are known as pattern recognition receptors and can be classified as membrane toll-like receptors (TLRs) or intracytoplasmic NOD-like receptors (31). Binding of PAMPs to these pattern recognition receptors triggers a downstream cascade of signals that will end activating the nuclear factor-kappa B (NF-κB), a transcription factor which main function is to upregulate the gene expression of inflammatory cytokines and recruit immune cells to the site of injury (76). However, these pattern recognition receptors are not unique to inflammatory cells. Pattern recognition receptors are also expressed by TECs (i.e., TLR-2 and TLR-4). The expression of these inflammatory mediator receptors outside the immune system might contribute and exacerbate the tubular inflammatory damage observed in AKI (40). Furthermore, tubular expression of TLRs increases the susceptibility of TECs to inflammatory injury. During sepsis-induced AKI, TECs are exposed to PAMPs on the apical and basolateral poles, suffering a “double hit” insult resulting in increased synthesis inflammatory mediators, production of ROS, oxidative stress, and cell injury (Fig. 4) (40, 83).

FIG. 4.

Inflammatory response and microcirculatory dysfunction in AKI. PAMPs and DAMPs are inflammatory mediators derived from bacteria and host immune cells, respectively. These inflammatory mediators bind to PRRs expressed on the surface of innate immune cells, endothelial cells, and renal TECs initiating a downstream cascade of signals that will increase the synthesis of proinflammatory cytokines, ROS, oxidative stress, and endothelial activation by nitric oxide and iNOS upregulation. During inflammation, DAMPs and PAMPs are filtered in the glomeruli. Once in the tubule these are going to bind the TLRs present in the apical membrane of the TECs. In addition, some evidence suggests that TECs are also exposed to the inflammatory mediators that are present in the peritubular circulation, although the mechanisms of how this occurs are not still clear. Moreover, the inflammatory response can also injure the TECs by increasing the oxidative stress and producing ROS. Microcirculatory dysfunction is the result of a series of events that lead to an impaired delivery of oxygen and nutrients to the tissue. Endothelial activation provoked by the inflammatory response results in a cascade of events that lead to shedding of the glycocalyx, increased leukocyte migration, and endothelial permeability. In addition, microcirculatory dysfunction is characterized by a heterogeneous flow, reduced number of capillaries with continuous flow with an associated increase of capillaries with sluggish flow, or no flow. Sluggish flow and no flow, result of the increased expression of adhesion molecules on the inflammatory and endothelial cells, facilitate the migration of neutrophils and macrophage to the interstitial space. Furthermore, the areas with sluggish flow have increased production of ROS and oxidative stress. Manifested by TEC apical vacuolization (31, 32, 34). DAMP, damage-associated molecular patterns; iNOS, inducible nitric oxide synthase; PAMP, pathogen-associated molecular pattern; PRRs, pattern recognition receptors; ROS, reactive oxygen species; TLRs, toll-like receptors.

Once the insult has been eliminated and the cell is switching back to oxidative metabolism and mitochondrial homeostasis is being restored, the anti-inflammatory phase of the immune response is initiated. This anti-inflammatory phase is characterized by the activation of regeneration and healing processes. However, persistent injury and organ dysfunction are associated when mitochondrial dysfunction persists (24, 37, 47, 54).

Microcirculatory dysfunction

Although the “global-ischemia” hypothesis of AKI has been discredited, many investigators have found evidence that alterations at the microcirculatory level might play a role in the development of organ injury (84). Evidence has shown that conditions such as sepsis, trauma, and major surgery are characterized by regional changes in microcirculatory flow. Importantly, in many instances, microcirculatory alterations are not correlated with changes at a macrohemodynamic level (22, 23, 45, 96, 107, 115). Multiple mechanisms have been proposed that may lead to microcirculatory dysfunction and explain somehow the dissociation of macro- and microcirculation. Increased renal blood flow with associated decline in renal function has been attributed to a simultaneous afferent arteriolar vasoconstriction, efferent arteriolar vasodilation, and intrarenal shunting (12, 64, 66). Moreover, endothelial injury and sympathetic autonomic nervous system response to the systemic vasodilation and decreased vascular resistance that occurs in sepsis-AKI may also explain these changes. The secretion of catecholamines, activation of the renin/angiotensin system, and vasopressin may lead to a redistribution of the renal blood flow from the already hypoperfused medulla to the cortex (61, 84). This ischemic response is worsened by the decreased synthesis of prostaglandins associated with mitochondrial dysfunction due to ATP deficiency (20, 104, 110). Furthermore, renal endothelial injury impairs the activity of nitric oxide synthase, impairing the vasoconstriction counteracting effect of this molecule (106). Other mechanisms such as shedding of the glycocalyx and activation of the coagulation cascade also participate in this process resulting in increased leukocyte and platelet rolling and adhesion, reduction in blood flow velocity, and microthrombus formation (Fig. 4) (23, 83, 116). As a consequence of these pathophysiologic changes, TECs may be exposed to an increased inflammatory burden as slow moving leukocytes and platelets release damage-associated molecular patterns and PAMPs in the vicinity of TECs. More importantly, microcirculatory dysfunction can lead to altered regional blood flow distribution potentially resulting in patchy areas of ischemia and loss of autoregulation, aggravating TEC injury and dysfunction (2, 29, 84, 88). Loss of glomerular and tubular function mainly manifests as an increase in creatinine levels and decrease in urine output. These changes are usually attributed to macrohemodynamic alterations, but several studies have also demonstrated that these can also be a consequence of impaired microcirculatory hemodynamics (84). Under normal physiologic states, the GFR is tightly controlled by a series of renal regulatory mechanisms, whose main objective is to maintain a constant GFR over a wide range of blood pressures. This regulation is possible by dilation and contraction of the renal afferent and efferent arterioles. However, in AKI, these regulatory mechanisms fail and GFR control is impaired. Vasoconstriction of the afferent arteriole with associated dilation of the efferent arteriole decreases the glomerular hydrostatic pressure and decreases urine output. In addition, constriction of the afferent arteriole results in glomerular shunting and a heterogeneous redistribution of flow (12, 70, 84, 86, 101).

Cellular Regeneration Versus Fibrosis

A more in-depth and a better understanding of AKI recovery and repair is now available (43, 122–125). This increased understanding of the pathophysiological mechanisms of AKI has allowed researchers to propose novel therapeutic targets for renal diseases, especially AKI. Furthermore, with the available evidence it is clearer, that the regeneration and repair processes play a major role during AKI, and that a coordinated and regulated response of the elements that participate in these mechanisms is crucial to achieve complete recovery of renal function following injury. AKI is associated with an increased risk of progression to CKD. Following removal of the insult, renal injury can progress to either complete and adequate cellular regeneration and functional recovery, or development of fibrosis and subsequent evolution to CKD. Progression to any of these two pathways is determined by several factors such as type and severity of injury, cellular signaling mechanisms, age, previous episodes of AKI, and renal reserve among others. For example, patients with lower renal reserve (e.g., CKD, congenital heart diseases, or previous episodes of AKI) are more prone to progress to fibrosis and ESRD. However, despite improved understanding of this biology and advances in identifying novel biomarkers that better forecast the development of AKI, predicting whether repair programs after injury will result in renal recovery or fibrosis remains challenging in the absence of suitable biomarkers.

The mechanisms that mediate kidney repair and regeneration are very complex and tightly regulated, and in many ways differ from that of other organs. Mitochondrial homeostasis, monocytes/macrophages, and regulators of the cell cycle play a major role in this process. Coordination among these elements is required to achieve a successful recovery and restoration of renal function. Much of our current understanding of the pathobiology of AKI has been extrapolated from studies done in nonhuman model systems such as the zebrafish model of AKI. Zebrafish renal development, physiology, and pathophysiologic mechanisms of AKI resemble the ones observed in mammalian models of AKI, making them a good model to study AKI (28a, 122a). From these studies it is known that under normal conditions, TECs are in the G₀ phase of the cell cycle (stable cells). Following injury, TECs undergo metabolic reprogramming by which various embryologic pathways are reactivated inducing a process of dedifferentiation into a less differentiated progenitor state that will allow them to re-enter the cell cycle and give rise to newer functional cells. Through this mechanism, TECs are able to restore the tubulo-epithelial cell population (50a, 101a). Enhancing of TEC proliferation has been proposed as a possible target for therapy, and studies have shown promising results that could eventually be translated to the clinical setting. For example, Brilli Skvarca et al. (11a) demonstrated that treatment with 4-(phenylthio) butanoic acid (PTBA), a histone deacetylase inhibitor, an enhancer of renal recovery, resulted in increased TEC proliferation and reduced renal fibrosis by inducing reactivation of the Pax2a gene (renal progenitor gene). Furthermore, this resulted in less expression of renal tubular injury biomarkers such as kidney injury molecule-1 (KIM-1), and less leukocyte infiltration especially macrophages. Evidence has also demonstrated that this process is regulated by inflammatory mediators such as TLR-4 and IL-22 (3a, 60a). In preclinical models, downregulation of IL-22 activity is associated with impaired regeneration and progression to fibrosis.

Clinical Strategies for AKI

Translating this new understanding of AKI pathobiology into the clinical realm will require careful characterization of the AKI phenotype both in terms of etiology and also time-course. In many of the most common forms of AKI (e.g., sepsis, nephrotoxic), patients present with AKI already present. Therefore, prophylactic strategies are not possible. Furthermore, the course of injury may be protracted limiting the chances to stimulate repair. Interventions that, for example, force cells into cell cycle will need to be carefully timed so that they do not force cells that are injured since such therapy would be detrimental. Whether the increasing use of biomarkers (55a, 59a) to characterize the clinical course of AKI will allow for the rationale use of therapies remains to be seen. However, there is strong evidence that even in conditions such as sepsis, recovery of the kidney can happen, and when it does prognosis improves dramatically.

NAD⁺ As a Drug Target for AKI

The bulk of renal NAD⁺ research reported to date has used models of cellular tubule biology. Cellular stress associated with different AKI syndromes triggers accelerated consumption of NAD⁺. Emerging evidence suggests that interindividual differences, both at risk of AKI and further disease outcomes following AKI, may relate to their differing ability to compensate the stress-associated consumption of tubular NAD⁺ with increased NAD⁺ biosynthesis (92, 94).

A shortfall of NAD⁺ has been identified in the pathobiology of several diseases, including AKI. In mice with AKI, the bottleneck enzyme in the de novo biosynthesis pathway, QPRT, was shown to maintain renal NAD⁺ and mediate resistance to AKI (Fig. 2) (7). When QPRT is deficient or depleted, an increased risk to develop AKI is observed (7). However, the deleterious effects of QPRT and subsequent NAD⁺ can be restrained by the stimulation of other NAD⁺ synthesis pathways by administering nicotinamide, and NAD⁺ precursor (7, 88). Mehr and colleagues also conducted a small clinical trial with oral nicotinamide, one of the physiological precursors of NAD⁺ biosynthesis, in patients undergoing cardiac surgery and found a dose-related increase in NAD⁺ metabolites and lower rates of AKI (7, 85). Metabolomics suggested an elevated urinary quinolinate/tryptophan ratio (uQ/T) as an indicator of reduced QPRT. Furthermore, this same group showed that increased uQ/T may work as a biomarker to predict AKI in patients. This is important as it directly identifies patients who are most likely to respond to this specific treatment—that is, enrichment and subclassification. Niacinamide, a precursor of vitamin B3, has also been proposed as potential therapy to replenish NAD⁺. In a recent study published by Parikh and colleagues (87), patients who developed COVID-19 AKI who received therapy supplementation with niacinamide presented lower rates of renal replacement therapy and death. Furthermore, an improvement in recovery was also observed.

Different approaches to replenishing NAD⁺ levels in the aged kidney or after experimental injuries mimicking AKI have been tested in preclinical rodent models (Fig. 5). For example, supplementation with nicotinamide mononucleotide (NMN), another NAD⁺ precursor, restored renal Sirt1 activity and NAD⁺ content in 20-month-old mice and further increased both in 3-month-old mice. Moreover, supplementation with NMN significantly protected mice in both age groups from cisplatin-induced AKI (42a). This study shows that the intraperitoneal injection of NMN boosts NAD⁺ levels in both young and aged kidneys, which was also associated with increased Sirt1 activity, and protection of the kidneys from both cisplatin- and ischemia/reperfusion-induced AKI, indicating that NMN rescued AKI by activating Sirt1. Importantly, the protective effect of NMN supplementation was substantially blunted in Sirt1-deficient mice, indicating that the protective effect of NMN occurs via Sirt1 activity (42a).

FIG. 5.

NAD⁺ as a drug target for AKI. Shown are different NAD⁺ replenishment approaches reported in preclinical models of AKI. ACMS, α-amino-β-carboxymuconate-6-semialdehyde; ACMSD, α-amino-β-carboxymuconate-6-semialdehyde decarboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMS, α-amino-muconate-6-semialdehyde; CD38, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1; NADH, nicotinamide adenine dinucleotide hydrogen; NAMPT, nicotinamide phosphoribosyl transferase; NAPRT, nicotinate phosphoribosyl transferase; NMNAT1/3, nicotinamide/nicotinic acid mononucleotide adenylyl transferase 1, 2 and 3; NQO1, NAD(P)H dehydrogenase quinone 1; NRK1/2, nicotinamide riboside kinase 1 and 2; P, phosphate; PARP-1, poly (ADP-ribose) polymerase 1; Sirt-1/3, sirtuin-1 and 3; UC-MSC, umbilical cord-derived mesenchymal stromal cells.

In the framework of the NAD⁺ biosynthesis, a novel approach to increase NAD⁺ level has been the inhibition of α-amino-β-carboxymuconate-6-semialdehyde decarboxylase (ACMSD). ACMSD is an enzyme synthesized in the kidney and in the liver that belongs to the de-novo NAD⁺ biosynthesis pathway. ACMSD inhibition resulted in increased cellular NAD⁺ levels and promoted mitochondrial homeostasis and function, enhancing sirtuins activity and protecting against AKI (and liver injury) (88). Katsyuba and colleagues have also characterized several novel inhibitors of ACMSD, including TES-1025, which shows preferential distribution to the kidney in mouse PK studies and demonstrates protective efficacy in the preventive treatment of ischemia/reperfusion- and cisplatin-induced AKI murine models (56). Physiological expression of ACMSD is largely restricted to the kidney and liver, and the recent study of Liu L. et al. (65b) identified the liver as the main site at which NAD⁺ is converted from tryptophan, by the de-novo NAD⁺ biosynthesis pathway, with the NAD⁺ produced released into the bloodstream to reach other tissues. These ACMSD inhibitors may thus be a promising therapeutic potential to prevent AKI (88).

The pharmacological inhibition of poly (adenosine-diphosphate-ribose) polymerase 1 (PARP-1), one of the principal NAD⁺ consuming enzymes, was reported to protect the kidney in various preclinical models of AKI, ischemia/reperfusion-, cisplatin-induced, or septic-induced AKI rodent models, rescuing NAD⁺ levels and improving mitochondrial fitness (15a, 65a, 73, 93). Another consuming NAD⁺ enzyme, CD38, was also reported to protect rodents from septic-induced AKI. In a mouse model with lipopolysaccharide (LPS)-induced AKI, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes, as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection (98a).

An alternative approach to increase the NAD⁺ level reported to protect the kidney from cisplatin-induced injury in a mouse model was treatment with a NAD(P)H dehydrogenase quinone 1 (NQO1) activator, β-lapachone. The intracellular NAD⁺/NADH ratio in renal tissues was significantly increased in wild-type mice cotreated with cisplatin and β-lapachone compared with the ratio in mice treated with cisplatin alone. Inflammatory cytokines and biochemical markers for renal damage were significantly attenuated by β-lapachone cotreatment compared with those in the cisplatin-alone group.

Finally, other “indirect” approaches have been reported to improve mitochondrial homeostasis and sirtuin activity by regulation of expression of nicotinamide phosphoribosyl transferase (NAMPT), one of the enzymes belonging to the NAD⁺ “salvage pathway,” rescuing the metabolite nicotinamide important in NMN for the regeneration of NAD⁺. One approach is based on AICAR, an AMPK activator, demonstrating protective effect in ischemia/reperfusion- and cisplatin-induced rat models of AKI (33, 64a, 77). The second is a cell-based approach exploiting the paracrine effect of human umbilical cord-derived mesenchymal stromal cells (UC-MSC), able to remodulate the transcription induction of several genes involved in mitochondrial functions and energy supply of tubular renal cells. The transplantation of UC-MSC in a cisplatin-induced AKI mouse model induced a global metabolic reprogramming of damaged tubular cells, with an increase of NAD⁺ content driven by NAMPT expression and Sirt-3 activity (83a).

Future Directions and Conclusions

The development of newly targeted protective and therapeutic strategies for AKI is urgently needed. It is now clear that targeting healing and regenerative processes by restoring mitochondrial homeostasis and NAD⁺ is a viable strategy to prevent and/or reduce severity of AKI, and furthermore to avoid maladaptive repair processes and progression to fibrosis and CKD. Although important advances have been made in translating experimental findings into the clinical setting, larger studies are needed to confirm the viability of these therapies. In addition, the exact mechanisms by which mitochondrial dysfunction and NAD⁺ deficiency provoke AKI are still unclear, and more experimental studies are needed to better understand the pathogenesis, and also to propose alternative therapeutic targets.

Abbreviations Used

2-ADP ribose

2-adenosine diphosphate ribose

ACMS

α-amino-β-carboxymuconate-6-semialdehyde

ACMSD

α-amino-β-carboxymuconate-6-semialdehyde decarboxylase

ADP

adenosine-diphosphate

AICAR

5-aminoimidazole-4-carboxamide ribonucleotide

AKI

acute kidney injury

AMPK

adenosine monophosphate-activated protein kinase

AMS

α-amino-muconate-6-semialdehyde

ATP

adenosine triphosphate

CACT

carnitine-acylcarnitine translocase

CKD

chronic kidney disease

CPT-1

carnitine palmytoiltransferase-1

CPT-2

carnitine palmytoiltransferase-2

DAMP

damage-associated molecular pattern

ER

endoplasmic reticulum

ETC

electron transport chain

FA

fatty acids

FADH₂

flavin adenine dinucleotide

FAO

fatty acid oxidation

GFR

glomerular filtration rate

HIF-1α

hypoxia-inducible factor-1 alpha

I/R

ischemic/reperfusion

iNOS

inducible nitric oxide synthase

KDIGO

Kidney Disease: Improving Global Outcomes

KIM-1

kidney injury molecule-1

LDH

lactate dehydrogenase

LPS

lipopolysaccharide

M/A shuttle

malate/aspartate shuttle

miR

micro-RNA

mTORC1

mammalian target of rapamycin complex 1

NAD⁺

nicotinamide adenine dinucleotide

NADH

nicotinamide adenine dinucleotide hydrogen

NAM

nicotinamide

NAMN

nicotinic acid mononucleotide

NAMPT

nicotinamide phosphoribosyl transferase

NAPRT

nicotinate phosphoribosyl transferase

NF-κB

nuclear factor-kappa B

NMN

nicotinamide mononucleotide

NMNAT1/3

nicotinamide/nicotinic acid mononucleotide adenylyl transferase 1, 2 and 3

NOXs

NADPH oxidases

NQO1

NAD(P)H dehydrogenase quinone 1

NR

nicotinamide riboside

NRK1/2

nicotinamide riboside kinase 1 and 2

OXPHOS

oxidative phosphorylation

PAMP

pathogen-associated molecular pattern

PARP-1

poly (ADP-ribose) polymerase 1

PDHK

pyruvate dehydrogenase kinase

PGC-1α

peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PGE2

prostaglandin E2

PKM2

pyruvate kinase M2

PPAR

peroxisome proliferator-activated receptor

PRRs

pattern recognition receptors

PTBA

4-(phenylthio) butanoic acid

PTCs

proximal tubular cells

QPRT

quinolinate phosphoribosyl transferase

ROS

reactive oxygen species

Sirt 1

sirtuin 1

Sirt 6

sirtuin 6

TCA

tricarboxylic acid cycle

TECs

tubular epithelial cells

TFAM

transcriptional factor A, mitochondrial

TLR

toll-like receptor

UC-MSC

umbilical cord-derived mesenchymal stromal cells

uQ/T

urinary quinolinate/tryptophan ratio

YY1

yin and yang 1

Authors' Contributions

All authors contributed to the work. The concept for the article was developed by J.A.K., and C.L.M-C. drafted the text with revisions from the remaining authors.

Author Disclosure Statement

J.A.K. has received consulting fees from TES Pharma and Astellas; H.G. received grant support from TES Pharma; and F.D.F., N.G., and R.P. are employees of TES Pharma.

Funding Information

This work was supported, in part, by the National Institutes of Health (NIH) Grant 1K08GM117310-01and the University of Pittsburgh School of Medicine Dean's Faculty Advancement Award.