Klotho: A multifaceted protector in sepsis-induced organ damage and a potential therapeutic target

Abstract

Sepsis is a life-threatening organ dysfunction associated with a robust systemic inflammatory and immune response to infection. Its pathological consequences lead to multiple organ deficits. Klotho was initially introduced as an antiaging molecule. Its deficiency significantly reduces lifespan, and its overexpression protects against organ injury. It reduces oxidative stress and apoptosis and has anti-inflammatory and antifibrotic properties. In this review, we discuss the underlying mechanisms of sepsis-related klotho down-regulation and the protective role of klotho in sepsis. In developing sepsis-induced multiple organ damage, klotho can modulate multiple downstream signals including nuclear factor-kappa β, mitogen activated protein kinase, and apoptosis. Multiple studies show klotho's protective effects in sepsis through activation of nuclear factor erythroid-related factor 2, Forkhead transcription factor O, and restoration of internal antioxidant activity. The proposed protective action of klotho is a promising therapeutic strategy for managing sepsis and ameliorating its related organ damage.

Key Words: Sepsis; Aging; Klotho; Cell signaling; Inflammation; Oxidation

Core Tip: Klotho, originally identified as an anti-aging protein, plays a critical role in mitigating sepsis-induced organ dysfunction by reducing oxidative stress, inflammation, and apoptosis. This review highlights klotho's potential as a therapeutic target, exploring its ability to modulate key signaling pathways like nuclear factor-kappa β, mitogen activated protein kinase, and nuclear factor erythroid-related factor 2. Reduced klotho levels correlate with increased severity of sepsis, while its supplementation shows promise in improving survival rates and alleviating organ damage. Understanding the mechanisms of klotho's cytoprotective effects may pave the way for novel treatments in managing sepsis-related organ failure.

INTRODUCTION

Sepsis is defined as “life-threatening organ dysfunction caused by a dysregulated host response to infection”[1]. Sepsis is a devastating clinical problem and the most prevalent cause of end-stage organ dysfunction and remains one of the largest contributors to health loss worldwide[2,3]. In 2020, it was estimated to affect 48.9 million people of whom 11 million died representing 20% of all global deaths[4]. Sepsis occurs in people of all ages, however, sepsis has been described as an illness of the elderly[5]. Earlier research showed over 60% of septic cases were patients over 65 years of age[3,6]. In addition to the high incidence, sepsis-related mortality has been reported to be almost 80% in patients above 80 years of age admitted to the intensive care unit[7,8].

The influence of old age on sepsis risk and outcome is evidenced clinically, and they have greater comorbidities and are more vulnerable to severe organ dysfunction[7,9]. When the integrity of the anti-inflammatory mechanism during sepsis is affected by aging, it results in a dysregulated innate immune system with excessive, long-lasting inflammatory response, and uncontrolled oxidative stress conditions. Such a phenomenon is defined as “inflamm-aging”[10,11]. Also, it was observed in different animal studies of sepsis that aged animals had a lower survival rate and exhibited excessive multi-organ dysfunction, as well as exaggerated inflammation[12,13].

Klotho is a protein that suppresses oxidative stress and inflammation[14]. In animal models, klotho was first proposed to induce premature-aging syndromes and shortened life span[15], whereas overexpression appeared to extend life span by 20%–30%[16]. Among a nationally representative sample of American adults, they found that low serum klotho concentration below 666 pg/mL was associated with a 31% higher risk of mortality[17]. Also, a prospective cohort study of patients with chronic kidney disease showed that serum klotho concentration of less than 760 pg/mL was associated with a higher mortality rate[18]. Given that sepsis and low klotho protein expression are independent predictors of mortality in sepsis, deficiency of klotho before or during the onset of sepsis appears to worsen the prognosis. The purpose of this review is to gain a deeper understanding of the protective role of klotho in sepsis (Figure 1).

Figure 1 The role of klotho administration after sepsis. FGF-R: Fibroblast growth factor-receptors; FoxO: Forkhead transcription factor O; HO-1: Heme oxygenase-1; ILs: Interleukins; MAPK: Mitogen activated protein kinase; Mn-SOD: Manganese-superoxide dismutase; NADPH: Nicotinamide adenine dinucleotide phosphate; NF-κB: nuclear factor-kappa β; Nrf-2: Nuclear factor erythroid related factor 2; PAR: Pathogen-associated receptor; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor-α; TWEAK: Tumor necrosis factor-related weak inducer of apoptosis.

KLOTHO PROTEIN: AN OVERVIEW

In 1997, Kuro-o et al[15] identified “klotho", the aging suppressor gene whose mutation manifests in a syndrome mimicking advanced aging. The name was taken from the mythological Greek figure responsible for spinning the thread of life[15]. Klotho predominantly originates in renal distal and proximal convoluted tubules, parathyroid glands, and the choroid plexus. Recent findings also show its presence in cardiovascular tissues, liver, urine, blood, and cerebrospinal fluid[19-23]. The klotho gene family comprises three subtypes: (1) Α-klotho, the most prevalent subtype; (2) β-klotho; and (3) γ-klotho[20]. Klotho functions as a protective factor against aging by promoting antioxidants[24], anti-inflammatory effects[25], and protects against cellular apoptosis, fibrosis, and senescence[26,27]. Low expression levels can lead to accelerated aging, a syndrome resembling human aging that includes skin and muscle atrophy, osteoporosis, cognitive impairment, and atherosclerosis—together, increased oxidative stress production and upregulation of inflammatory cytokines increased the risk of multisystem dysfunction[23,26-28]. Klotho exhibits alternatively spliced transcripts that yield either a secreted soluble form known as soluble klotho or a full-length single-pass transmembrane protein referred to as membrane klotho. The latter functions as a coreceptor regulator for fibroblast growth factor (FGF)-23[29,30].

Interestingly, klotho depletion contributes to acute kidney injury[14] and cardiac damage[31], and is found in individuals with chronic kidney disease and diabetes[32,33]. Also, a low klotho level is involved in a variety of conditions, including coronavirus disease 2019[34], cancer[29], and sepsis[35]. The klotho level was used as a multiorgan damage and mortality marker in experimental and clinical stressful conditions[28,36,37].

SEPSIS AND KLOTHO EXPRESSION

Klotho deficiency results in the development of renal, cardiovascular, and brain injuries and death in septic animals and is associated with endothelial dysfunction, oxidative stress activation, inflammatory responses, impaired immunity, and apoptosis[25,28,38]. Klotho protein exerts its function as a humoral factor and influences the pathophysiological process by modulating different intracellular signal pathways[39]. Research suggests a complex relationship between klotho expression and sepsis. Klotho deficiency aggravates sepsis-related multiple organ dysfunction[28]. The severity of septic shock influences the low expression of klotho and correlates with disease severity. Mild sepsis was associated with slight klotho reduction[40]. Klotho levels start to decline within 4 hours following the onset of systemic hyperinflammation.

Furthermore, research indicates that administering recombinant klotho 1 hour or 4 hours after lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) surgery is linked to reduced organ damage[28]. The administration of klotho is currently in the preclinical stage and has not yet received approval for clinical use. If the clinical application of klotho begins successfully, the dosage will be determined based on the preclinical findings, with early administration within the first 24 hours.

Low klotho mRNA expression was reported in an experimental model of gram-negative sepsis induced by LPS injection[25]. In addition, the CLP model in klotho knockout mice demonstrated significantly higher mortality, higher concentrations of cytokines, and high oxidative stress[28]. It has been reported that renal klotho expression was significantly reduced in patients and animals with sepsis[13,38]. Klotho deficiency has been reported to increase the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, aggravating sepsis-induced multiple organ dysfunction syndrome[23,25,28]. In contrast, recombinant klotho ameliorates sepsis-induced multiple organ dysfunction[13,23,41]. Also, its administration increases the survival rate and decreases lung injury, neutrophil influx, and inflammatory factors[42].

Physiological and experimental lower levels of klotho and sepsis

Reduction of klotho protein expression physiologically (by natural aging) or by using genetic models or pharmacological agents (experimental) was shown to elicit multiple detrimental effects in various previous studies. Table 1 below summarizes these studies[22,25,28,38,43-46].

Table 1 Effect of klotho downregulation on sepsis models.

Klotho regulator	Sepsis model	Mechanism	Effect	Ref.
Aged mice	LPS	Klotho downregulation/upregulation of NF-κB	Cardiovascular dysfunction. Higher myocardial levels of cytokines TNF-α, IL-6, and IL-1β	Hui et al[25]
Aged mice	LPS	Exacerbated inflammation associated with aging/klotho downregulation	More severe and persistent cardiac dysfunction. Higher myocardial IL-6, ICAM-1, and vascular cell adhesion molecule levels	Li et al[22]
Aged mice	CLP	Serum TNF-α and IL-6 were elevated. Caspase-3 tissue expression. The bacterial count increased	The survival rate was reduced. Apoptosis activation in the thymus and spleen. Impairment of innate and adaptive immunity	Inoue et al[43]
Genetically deficient Kl/+ mice	CLP	Klotho downregulation/upregulation of NF-κB	Low survival rate. Increase in the production of TNF-α, IL-1β, IL-6. Increase in thiobarbituric acid reactive substances and lactate with a reduction in reduced glutathione. The autonomic response in sepsis was much more harmful through impairment of the vasopressor effect and baroreflex sensitivity	Jorge et al[28]
Klotho-siRNA fibroblasts. In-vitro study	LPS	Upregulation of superoxide production/downregulation of glutathione antioxidant. Upregulation of high mobility group box-1. Increase in NF-κB activation	Reduction in wound healing. Apoptotic cell death induction. Imbalance in intracellular zinc and calcium amounts. Increased secretion of TNF-α, IL-6, and IL-Iβ, concurrently with suppression of anti-inflammatory cytokine IL-10	Mytych et al[44]
Haploinsufficient mice (Kl+/–)	LPS	Down-regulation in renal and brain klotho gene expression. Elevation in plasma plasma-neutrophil gelatinase-associated lipocalin mRNA level. High mRNA levels of E-selectin, ICAM-1, and retinoic acid-inducible gene-I	Renal and brain damage were observed. Endothelial activation promotes immune cell infiltration into the tissue, which increases tissue damage	Jou-Valencia et al[38]
Klotho knockout mice	CLP	Reduction in klotho level/Hyper-cytokinemia. Impairment of bacterial clearance	Decrease in survival rate when compared to the wild-type. Activation of innate immune cells. Elevation of apoptosis in lymphocytes. Increasing serum concentrations of TNF-α, IL-6, and monocyte chemoattractant protein	Inoue et al[45]
Adult mice	CLP	Reduction in klotho level	Increase in serum creatine and blood urea nitrogen. Damage to renal tissue. Autophagy activation	Chen et al[46]

CLP: Cecal ligation and puncture; ICAM-1: Intracellular adhesion molecules; IL: Interleukin; LPS: Lipopolysaccharide; NF-κB: Nuclear factor-kappa β; TNF-α: Tumor necrosis factor-α.

Pharmacological overexpression of klotho

As shown in the previous section, the reduction in klotho level had detrimental effects on sepsis, thus attempts were made to use different pharmacological techniques to rescue animals with sepsis and its associated organ damage. Table 2 summarizes some of these studies and their findings[13,14,22,23,25,31,35,38,41].

Table 2 Summary of the protective effects of different klotho regulators in different sepsis models.

Klotho regulator	Sepsis model	Mechanism	Effect	Ref.
Treated mice with klotho protein (0.02 mg/kg) for 4 days	LPS	Klotho administration/attenuates the reactive oxygen species/p38-mitogen activated protein kinase signaling pathway	Reversal of myocardial injury. Reduction in atrial and brain natriuretic peptide (atrial natriuretic peptide and BNP). Reduction in apoptosis	Yan et al[31]
Recombinant klotho protein	CLP	Restoration of endogenous klotho expression	Alleviated CLP-induced acute renal injury by reducing serum creatinine and BUN levels	Chen et al[13]
Recombinant klotho protein	LPS	Klotho administration/suppression of NF-κB activation	Cardiac function was improved. Lower myocardial levels of chemokines and cytokines TNF-α, IL-6, and IL-1β	Hui et al[25]
Recombinant klotho protein	LPS	Anti-inflammatory effect of klotho administration/suppression of IL-6 and adhesion molecules	Return to the baseline levels for myocardial ICAM-1, VCAM-1, and IL-6. Recovery from cardiac dysfunction	Li et al[22]
Recombinant IL-37	LPS	Anti-inflammatory effect of IL-37/klotho upregulation/suppression of IL-6 and adhesion molecules	Improvement of cardiac functions	Li et al[22]
Pretreatment-recombinant α-klotho protein	LPS	Antiapoptotic mechanism through caspase-3 reduction. Anti-inflammatory by shifting the balance towards an anti-inflammatory environment. Antioxidant activities	Amelioration of septic cardiorenal injury. Reduction in serum levels of troponin, NGAL, BNP, and creatinine. Reduction in the renal cytokines IL-6, IL-1, and TNF-α levels, with marked elevation in IL-10. Reduction in malondialdehyde and nitric oxide production	Liu et al[41]
Recombinant human-klotho protein	CLP. LPS in-vitro study in the human HK2 epithelial cell line	Klotho exerts its protective effects by upregulating nuclear factor erythroid related factor 2 to suppress the ferroptosis signaling pathway	Alleviated kidney injury and increased HK2 cell viability. Administration of klotho upregulates klotho expression in blood, renal tissue, and HK2 cells. Low levels of the inflammatory factors TNF-α and IL-6 and oxidative stress responses. Improvement in mitochondrial number, structure, and function in HK2 cells	Zhou et al[14]
Recombinant α-klotho protein	In-vitro LPS administration to HPAEpiCs. CLP in-vivo	Prevents activation of the Bcl-2/Bax/caspase-3 signaling pathway	Klotho increased mouse survival and decreased IL-1β, IL-6, and TNF-α levels. Reducing the percentage of apoptotic cells in lung tissue and HPAEpiCs exposed to LPS	Li et al[42]
Treatment with human Wharton’s Jelly-Derived Mesenchymal Stem Cells	CLP	Klotho protein expression was high. Reduction in NF-κB tissue expression. Expression of Bax was reduced, whereas Bcl-X was increased	Survival was improved. Glomerular filtration rate and renal and liver functions were improved. Reduction in apoptosis. Levels of the proinflammatory cytokines IL-1α, IL-6, interferon-γ, and TNF-α were reduced. Endothelial function was improved	Cóndor et al[23]
Resveratrol and Recombinant murine α-klotho protein	CLP	Increased tissue expression of klotho protein in both treated groups. The expression of Bax and caspase-3 was reduced, whereas that of Bcl-2 was increased. The reno-protective effect exerted by klotho is through the antiapoptotic effect	Resveratrol has the same effect as exogenous administration of the klotho protein. Improvement in renal function and structure, decrease in serum creatinine and BUN	Chen et al[35]
Recombinant klotho protein	LPS	Reduced level of myeloid peroxidase. Attenuation of renal and brain E-selectin, VCAM-1, ICAM-1 mRNA, endothelial adhesion molecule expression, and neutrophil infiltration	Renal and brain inflammation were reduced. Low plasma BUN and plasma NGAL levels. Decreased renal and brain levels of IL-6, IL-1β, IL-8 and TNF-α	Jou-Valencia et al[38]

BNP: Brain natriuretic peptide; BUN: Blood urea nitrogen; CLP: Cecal ligation and puncture; HK2: Renal tubular; HPAEpiCs: Human pulmonary alveolar epithelial cells; ICAM-1: Intracellular adhesion molecule-1; IL: Interleukin; LPS: Lipopolysaccharide; NF-κB: Nuclear factor-kappa β; NGAL: Neutrophil gelatinase-associated lipocalin; TNF-α: Tumor necrosis factor-α; VCAM-1: Vascular cell adhesion molecule.

MOLECULAR PATHWAYS INVOLVED IN KLOTHO ACTION

Nuclear factor-kappa β

Sepsis is associated with increased production of inflammatory cytokines, including TNF-α, IL-6, IL-1 β, and TNF-related weak inducer of apoptosis (TWEAK)[47]. From a pathophysiological view, impaired heart, kidney, or multiple organ functions during sepsis are consequences of inflammation and metabolic disturbance[2]. TWEAK is a TNF-superfamily member that activates the FGF-14 receptor to trigger multiple signaling cascades. FGF-14 levels are relatively low in normal tissue but increase rapidly following tissue injury and inflammatory conditions[48]. Upon TWEAK engagement, trimerization of FGF-14 promotes intracellular signal transduction through canonical and non-canonical nuclear factor-kappa β (NF-κB) pathways and protein kinase cascades including extracellular signal-regulated kinases, c-Jun N-terminal kinase, p38-mitogen activated protein kinase (MAPK), and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)[49]. Following sepsis, TWEAK-dependent canonical NF-κB activation and TNF-α production induce multiple responses including cell proliferation, expression of other inflammatory cytokines, and downregulation of protective factors such as klotho[25,28,50]. Targeting NF-κB by administering klotho improved cardiac, renal, and liver functions and reduced TNF-α, IL-6, and IL-1β[23,25].

Reactive oxygen species /p38-MAPK

The antiaging functions of klotho are closely associated with its antioxidative stress effect, inhibiting reactive oxygen species (ROS) production and promoting ROS clearance[51]. The antioxidants superoxide dismutase (SOD) and catalase are reduced with aging. This reduction is associated with activating nicotinamide adenine dinucleotide phosphate and increasing production of hydrogen peroxide[52]. Previous findings showed that increased oxidative stress inhibits klotho expression while low klotho expression in tissues aggravates oxidative damage[28,31]. Studies revealed that overexpression of klotho alleviated oxidative stress-mediated cell injuries[38,41], including heart[31], kidney[41], and endothelial cells[23].

The previous findings indicated that oxidative stress arises after sepsis exposure, which harms intracellular organs[53].

It is reported that MAPKs have an important role in regulating oxidative stress-related signaling and participate in the pathogenesis of sepsis[54]. MAPKs are involved in regulating a variety of physiological processes in cells, including proliferation, differentiation, and apoptosis[54]. Klotho treatment inhibited P38-MAPK activation post-sepsis exposure[31]. Klotho prevents oxidative injury and apoptosis through activation of the Forkhead transcription factor O (FoxO) family of transcription factors by inhibition of its phosphorylation and stimulation of manganese SOD (Mn-SOD)[55,56]. The antioxidant effect of klotho protein is mediated by modulating antioxidant signaling pathways involving Mn-SOD, PI3K/Akt/FoxO, and nuclear factor erythroid-related factor 2 (Nrf-2) pathways[57].

Mitochondria-dependent apoptosis

Previous reports showed that excessive intracellular ROS can cause oxidative damage to membrane proteins and mitochondrial DNA, leading to structural changes and mitochondrial impairments[14,58]. Moreover, ROS-induced mitochondrial dysfunction, including oxidative injury, could lead to an aggravated imbalance between ROS generation and elimination, causing ROS overload and ultimately triggering the initiation of apoptosis[59]. The activation of apoptosis is characterized by the decline of Bcl and the presence of Bax and caspase-3 activity[23,35,40]. An imbalance between Bax and Bcl-2 plays an important role in the mitochondria-mediated caspase cascade, which was confirmed by an increased Bax/Bcl-2 ratio, release of cytochrome-c from the mitochondria to the cytoplasm, cleavage of caspase-3, and subsequent induction of apoptosis[60]. Klotho increased Bcl and reduced the activity of caspase-3[23,35]. Accumulating evidence indicated that klotho exerts an antiapoptotic effect by suppressing endothelial apoptosis via MAPK and mitochondria-dependent apoptosis pathways as klotho administration inhibited caspase-3 activation[42,61].

FGF-23

The biological effects of FGF-23 are strictly dependent on the cognate coreceptor klotho, which is fundamental for FGF-23 exerting its action at the organ level[29]. Regarding inflammation, FGF-23 has been associated directly with high levels of C-reactive protein and IL-6 in patients with sepsis[37,49]. In addition, klotho was inversely related to IL-6[62]. There is information from biological knowledge in experimental models showing binding between the cytokines and circulating FGF-23 and klotho in proinflammatory conditions. In CLP mice[63], injection of IL-1β as an inflammatory cytokine doubles the serum concentration of intact FGF-23. Contrary to FGF-23, α-klotho protein expression is reduced in response to inflammatory stimuli[25,38].

Nrf-2

Nrf-2, the core transcription factor of the endogenous cellular antioxidant stress system, translocates to the nucleus and binds to specific gene loci, thereby attenuating the cellular oxidative stress response and protecting cellular function[64]. In addition, different studies confirmed that increasing Nrf-2 levels were associated with klotho elevation, effectively reducing the related organ damage[65,66]. Klotho protein administration inhibits ferroptosis by activating the Nrf-2 signaling pathway[14]. Klotho overexpression protects cardiac function by regulating the Wnt signaling pathway and activating the AKT/Nrf-2/heme oxygenase-1 pathway to slow cellular senescence and antioxidant activity[67,68]. Studies have shown that klotho promotes angiogenesis and maintains endothelial cell integrity by regulating apoptosis and autophagy of endothelial cells[23,38].

The therapeutic role of klotho

The fall in klotho level after sepsis is of pathophysiological and clinical importance[36,37]. In different sepsis studies, the administration of recombinant klotho protein resulted in considerable improvement in cardiac, liver, and renal functions and structure, with a diminution of oxidation, inflammation, and apoptosis[23,35,38]. Moreover, klotho gene delivery might be a prophylactic agent against sepsis-induced multiple organ injuries.

When a single dose of klotho protein was intraperitoneally injected into septic rats 30-60 minutes after CLP or LPS, it considerably preserved endogenous klotho levels and suppressed the increase in creatinine and blood urea nitrogen[13,25]. Thus, the earlier klotho administration, the better the outcome. Young adult mice expressing more significant plasma klotho levels than adult mice were more resistant to myocardial inflammation[22]. If these results are translated into a clinical setting, klotho would be promising as a prophylactic and therapeutic agent for patients with a high risk of sepsis-induced multiple organ injuries.

Clinical application of klotho

Clinical sepsis studies noted that the serum concentration of α-klotho was over-expressed, and FGF-23 was downregulated as a counter-regulatory mechanism in response to acute inflammation[40,62]. The acute increase of serum α-klotho during septic shock may be an attempt to modulate proinflammatory pathways, which then decrease as sepsis resolves[40]. This finding contrasts with other clinical settings that showed lower klotho levels in septic patients[37,38,69]. These findings are summarized in Table 3[37,38,40,62,69].

Table 3 Summary of clinical data showing the change in klotho expression in septic patients.

Sepsis model	Mechanism	Effect	Ref.
Sepsis patients. Collection of warm postmortem biopsies from patients who died of sepsis and renal failure in the ICU	Reduction in renal klotho level. Increased mRNA levels of kidney damage markers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1	Correlated to renal damage	Jou-Valencia et al[38]
Sepsis patients	Reduced serum klotho level	An early predictor of AKI to allow immediate interventions	Pei et al[69]
A prospective cohort of ICU patients with sepsis and previously normal renal function	Low klotho and high erythropoietin plasma levels are cofactors for activating FGF-23	The klotho level is an early predictor of the development of AKI, mortality, and long-term CKD progression in sepsis patients	Toro et al[37]
Patients with CKD are admitted to the ICU with acute inflammation/sepsis	At the peak of infection, suppressing the active form of FGF-23 and activating α-klotho	Counter-regulatory response to acute inflammation	Dounousi et al[62]
Patients admitted to the ICU with septic shock within the previous 72 hours	Increasing serum level of α-klotho	Higher mortality rate	Abdelmalik et al[40]

AKI: Acute kidney injury; CKD: Chronic kidney disease; FGF: Fibroblast growth factor; ICU: Intensive care unit.

CONCLUSION

Data from animal or clinical studies show decreased klotho levels in the kidney, heart, and plasma following sepsis. These findings suggest that sepsis is a klotho-deficient state, and this deficiency might have a role in organ dysfunction. The importance of klotho are as follows: First, klotho is a potential early biomarker for diagnosing acute organ injury; Second, early administration of exogenous klotho protein, or enhancement of endogenous klotho by resveratrol or IL-37, may prevent sepsis-induced organ damage; Third, administration of klotho protein and upregulation of endogenous klotho expression after sepsis may accelerate recovery; and Fourth, even if klotho does not change the course of damage, treating klotho deficiency could decrease the risk of progression to a severe prognosis. Elucidating the molecular mechanisms of how klotho functions as a cytoprotective protein will involve its cell regeneration, antioxidant, anti-inflammatory, and antiapoptotic effects.