Exploring Endogenous Tryptamines: Overlooked Agents Against Fibrosis in Chronic Disease? A Narrative Review

Abstract

Long regarded as illicit substances with no clinical value, N-dimethylated tryptamines—such as N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and bufotenine—have been found to produce naturally in a wide variety of species, including humans. Known for their psychoactive effects through serotonin receptors (5-HTRs), N-dimethylated tryptamines are currently being reinvestigated clinically for their long-term benefits in mental disorders. Endogenous tryptamine is methylated by indolethylamine-N-methyltransferase (INMT), which can then serve as an agonist to pro-survival pathways, such as sigma non-opioid intracellular receptor 1 (SIGMAR1) signaling. Fibrogenic diseases, like metabolic-associated fatty liver disease (MAFLD), steatohepatitis (MASH), and chronic kidney disease (CKD) have shown changes in INMT and SIGMAR1 activity in the progression of disease pathogenesis. At the cellular level, endothelial cells and fibroblasts have been found to express INMT in various tissues; however, little is known about tryptamines in endothelial injury and fibrosis. In this review, I will give an overview of the biochemistry, molecular biology, and current evidence of INMT’s role in hepatic fibrogenesis. I will also discuss current pre-clinical and clinical findings of N-methylated tryptamines and highlight new and upcoming therapeutic strategies that may be adapted for mitigating fibrogenic diseases. Finally, I will mention recent findings for mutualistic gut bacteria influencing endogenous tryptamine signaling and metabolism.

1. Introduction

The enactment of the U.S. Controlled Substances Act of 1971 marked a pivotal shift in international drug regulation and domestic healthcare oversight, influencing not only the pharmaceutical landscape but also hindering therapeutic research in psychedelics and disproportionately affecting marginalized communities through its enforcement [1,2,3,4,5,6]. Among some of the first substances deemed to have no medical utility, to be unsafe for treatments under medical supervision, and to have a high potential for abuse are the potent, short-lived psychedelic N,N-methylated tryptamines: N,N-dimethyltryptamine (DMT) as well as bufotenine (5-HO-DMT) and later, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) in 2011 [1,7,8]. N-methylated tryptamines have since been found to be a component of indigenous South American ethnobotanical ceremonies pre-dating Western colonization [9]. Today, N-methylated tryptamines are often misused outside of medical and scientific research, particularly by younger people and gender and sexual minorities [10,11,12]. However, their clinical application in treating depression, anxiety, substance dependency, and post-traumatic stress disorder (PTSD) has been gaining traction [13,14,15,16].

In the modern era, DMT was first synthesized in 1931 by Richard Manske and characterized in nature in 1946 by Oswaldo Gonçalves de Lima from the root bark of Mimosa hostilis [17,18]. The psychoactive properties and metabolism of DMT were made akin to lysergic acid diethylamide (LSD-25) and mescaline when characterized in humans in 1956 by Stephen Szára [19]. Since then, N,N-methylated tryptamines have had research focus on their effects on the brain, substance misuse, and psychotherapeutics [20,21,22]. Recent robust in vitro and in vivo experiments have found that these substances can induce cellular proliferation, repair and redox mechanisms, as well as anti-inflammatory and antifibrogenic responses [15,23,24,25,26]. The benefits of N,N-methylated tryptamines are continuing to be reassessed and uncovered; however, their endogenous role remains in controversy [27].

In 1961, Axelrod discovered the enzyme indolethylamine-N-methyltransferase (INMT) in rabbit lung [28]. In 1965, concentrations of DMT (blood: 8.0 to 55 ng/mL; urine: 43 μg/day), 5-HO-DMT (blood: 1.0 to 40 ng/mL; urine: 63 μg/day), and 5-MeO-tryptamine (blood: 20 to 80 ng/mL; urine: 37 μg/day) were discovered in normal human blood and urine [29]. In 1972, Lewis Mandel et al. discovered human INMT in lung tissue, and the enzyme was found to monomethylate and dimethylate tryptamine with the universal one-carbon methyl donor S-adenosylmethionine (SAM) [30]. In 1988, an enzyme Thioether S-Methyltransferase (Temt) was discovered in mouse lungs and livers, which was determined to be chiefly responsible for methylating dimethylselenide (DMSe) with SAM to form trimethylselenonium (TMSe⁺) for selenium metabolite detoxification and elimination [31,32,33]. Although it would not be discovered until 2015 in a study investigating poor selenium status, single nucleotide polymorphism (SNP) influences on TMSe⁺ production in rural Argentinian and Bangladeshi women revealed TEMT activity by INMT [34,35]. INMT has recently been confirmed to be consequential in the methylation of volatile DMSe to form the urine-excreted trimethylselenonium (TMSe⁺) through site-directed mutagenic experiments [36]. In 1995, Strassman initiated the investigation of endogenous DMT and Warner et al. cloned and characterized mouse lung TEMT [37]. In 1998, mouse Inmt—identified as Temt—was determined to be a marker for renal proximal tubules, which are key drivers in renal fibrosis [38,39]. In 1999, Thompson et al. cloned and characterized the human INMT gene from skeletal and placental tissues [40]. In 2005, the crystal structure of human INMT in complex with S-adenosylhomocysteine (SAH) was solved (PDB: 2A14; rcsb.org/structure/2a14). In 2014, a selective noncompetitive inhibitor (N,N-Dimethylaminopropyltryptamine) to rabbit INMT, and predicted by in silico modeling to inhibit human INMT, was synthesized [41]. In 2023, E. coli were engineered to produce DMT, 5-HO-DMT, and 5-MeO-DMT in high quantities by expressing human INMT and supplemented media with tryptophan or glucose [42]. It is clear that illicit N-methylated tryptamines are endogenously made, but current research endeavors continue to stress the potential role for INMT in regulating neurochemistry, mood, consciousness, and sensory perception [3,43,44,45,46,47].

Recently, variations in INMT expression and activity have been identified in acute and chronic diseases in the liver, kidneys, and heart, particularly in early fibrogenic states [48,49,50,51]. Fibrosis is a severe feature of both chronic diseases and acute injuries, often originating from or reinforced by endothelial injury and dysfunction at the onset of pathogenesis [52,53,54,55,56]. Endothelial injury in chronic conditions like MAFLD, MASH, and CKD, as well as in acute injuries such as ischemia, disrupt the delicate balance of vascular function and act as major contributors in tissue fibrogenesis [57,58,59,60,61]. In MAFLD and MASH, for example, lipotoxic stress initiates disruption in hepatocyte homeostasis, causing oxidative distress and damage which then can trigger immune infiltration and significant tissue damage [62,63]. The major mediators in liver injury response are liver sinusoidal endothelial cells (LSECs), which are considered early initiators and reinforcers for hepatic stellate cell (HSC) activation [62,63,64]. HSCs are retinoid storing cells, responsible for tissue fibrosis following their transdifferentiation to myofibroblasts, which interestingly have recent evidence of also playing a role in renal fibrosis—known as renal stellate cells (RSCs) in the cortex—in a similar manner [62,65,66,67]. Reactive oxygen species (ROS) as well as lipid peroxidation byproducts from damaged hepatocytes provoke endothelial dysfunction and HSC activation, creating a pro-inflammatory and pro-fibrotic environment [65,68,69]. Similarly, in CKD, endothelial cells in the renal microvasculature are exposed to hemodynamic stress, hyperglycemia in diabetes, circulatory-derived oxidative stress and uremic toxins [70,71,72,73]. Comprising the glomerular filtration barrier—a vital cellular architecture in regulating renal filtration function—are endothelial cells, a basement membrane, and podocytes [74,75]. The recent feature of RSCs in renal fibrosis may also pose a significant avenue in mitigating cortical fibrogenesis [67]. Exposure to these insults increases the risk for endothelial cell activation and podocyte injury, leading to a compromised vascular barrier, increased permeability, recruitment of immune cells, and tissue injury [76]. The resulting transforming growth factor beta (TGFβ)-mediated endothelial and podocyte injury contributes to significant renal fibrosis [53,77,78,79].

Ischemic injury, whether in the liver or kidneys, causes rapid endothelial cell damage due to the lack of oxygen and nutrient supply [80,81]. The resulting hypoxia induces hypoxia inducible factor-1alpha (HIF-1α) stabilization, which, often in feedback synergy with TGFβ, and/or endothelin-1 (ET-1), promotes extracellular collagen deposition, fibroblast, HSC activation, vascular remodeling, and inflammation [82,83,84,85,86]. Notably, hypoxia has also been shown to be responsible for a shift toward N-methylated tryptamines biosynthesis, and in vitro studies found DMT to be protective against hypoxia [87,88]. Hypoxia also impairs endothelial nitric oxide production, leading to increased vascular tone and further endothelial dysfunction [89]. In the liver, fibrosis is often irreversible following severe or chronic insults like ischemia or MASH, and the lack of any current therapeutics available for liver fibrosis leaves patients with a greater risk for developing organ failure and cancer [62]. Collectively, these diseases create a cycle where endothelial injury fuels inflammation, TGFβ signaling, and hypoxia-driven fibroblast and stellate cell activation, leading to sustained fibrosis [63,89]. This endothelial-initiated inflammation and fibrogenic outcome is critical in the pathogenesis of chronic liver and kidney diseases, underscoring the need to target endothelial dysfunction in therapeutic strategies to mitigate fibrosis and prevent disease acceleration [62,89,90].

A key role for endogenous N-methylated tryptamines was identified in 2009 as the agonist to the sigma non-opioid intracellular receptor 1 (SIGMAR1) signaling pathway [91,92,93,94]. SIGMAR1 is a ubiquitous, versatile intracellular receptor involved with inter-organelle crosstalk, specifically the mitochondria and endoplasmic reticulum (ER), and plays a role vascular function [95,96,97,98,99]. Pre-clinical research on SIGMAR1 is currently receiving attention across a spectrum of liver, kidney, and heart diseases for its pro-survival effects against insults [96,100,101,102,103]. In neurobiology, colocalization of SIGMAR1 and INMT in motoneurons is suspected, suggesting that there may be subcellular spatial regulation in favor of agonizing SIGMAR1 via INMT [104]. Indeed, some researchers have suggested the benefits of N-methylated tryptamines for multiple diseases [105]. Advances in our understanding of the role of INMT in human biology will undoubtedly improve our understating of tryptamine and selenium metabolism, thus prompting new therapeutic strategies for diseases.

2. Tryptamine Metabolism and Biochemistry

Serving as the skeleton to a major class of psychedelics, a tryptamine is an indole heterocycle with an ethylamine group at the C3 position (Figure 1A). Tryptamine is also the metabolite formed from the decarboxylation of tryptophan via aromatic L-amino acid decarboxylase (AADC; an alternatively expressed enzyme encoded by the dopa decarboxylase (DDC) gene), which is primarily expressed in liver, kidneys, spleen, brain, and heart and is regulated by hepatocyte nuclear factor 1 (HNF1α) for nonneuronal tissue expression [106,107,108]. Using SAM, INMT methylates tryptamine to form N-methyltryptamine (NMT) and a second methylation can occur by exchanging the spent SAH for new SAM to form DMT (Figure 1B) [43,109]. Chu et al. 2014 reported the allosteric activity of DMT on INMT, which may be a result of end-product inhibition [41]. Other reports have suggested the production of N,N,N-trimethyltryptamine (TMT) and 5-hydroxy-TMT (5-HO-TMT) by INMT, but there is not much evidence exploring their biological role [42,110]. Interestingly, plants and fungi have been shown to produce N,N,N-trimethyltryptamine and the psilocin (4-HO-DMT) derivative aeruginascin (4-HO-TMT), respectively [111,112].

INMT may also methylate serotonin (5-HO-tryptamine; 5-HT) to form the mono- and dimethylated ethylamine derivatives 5-HO-NMT and bufotenine. 5-HT arises from the C5 hydroxylation of tryptophan by tryptophan hydroxylase-1 (TPH-1) prior to decarboxylation by AADC [109,113]. Although not well established, it is suggested that melatonin—a 5-HT derivative canonically produced in a stepwise manner of N-acetylation of the ethylamine by serotonin N-acetyltransferase (SNAT) and then O-methylation of the C5 hydroxyl by N-acetylserotonin O-methyltransferase (ASMT)—is produced in an alternative manner with ASMT preceding SNAT N-acetylation [114]. Localized in the retina and pineal gland, ASMT production of 5-MeO-tryptamine may be possible (Figure 1B), but it is not yet known if 5-MeO-tryptamine may be trafficked for methylation in tissues or cells expressing high levels of INMT [114,115]. The presence of 5-MeO-tryptamine in blood, however, might serve as a source for 5-MeO-DMT via INMT [29,116]. It is worth mentioning here that DMT, 5-HO-DMT, and 5-MeO-DMT have a slight lipophilic character (logP ~2) and have been found in vesicles, suggesting some feasibility in being trafficked or localized to lipid bilayers [43,110,117]. A 1974 study in 5-MeO-DMT biosynthesis suggests that INMT may have the ability to methylate the C5 hydroxyl, but no recent studies have confirmed this [109,118]. Alternatively, a recent study using rat KOs of INMT have shown that the N-methylated tryptamines NMT and DMT are still biosynthesized, which suggests that there could be an unknown redundant or compensatory enzyme for the endogenous production of N-dimethylated tryptamines [119]. Nevertheless, genetic limitations between other mammals could be another reason for the differences in methylation capacity and biological function, since INMT in humans share ~57% sequence homology and are oppositely oriented in the genome compared to mice, along with possible alternative splicing (Gene [INMT]. Bethesda (MD): National Library of Medicine (USA), National Center for Biotechnology Information; 2004 (accessed on 3 October 2024). Available online: https://www.ncbi.nlm.nih.gov/gene/11185) [120]. Recent evidence in prostate cancer has also uncovered SMYD3 in the promoter region of INMT and Inmt, but DNA methylation patterns in INMT may influence variations in expression too, as seen in head and neck squamous cell carcinoma [121,122]. Most interestingly, the knockdown of INMT in vitro amplifies the anticancer activity of selenium compounds, further suggesting INMT’s role in selenium metabolism [121].

In silico experiments exploring DMT in metabolism have provided thermodynamic insight to methylation by INMT as well as catabolic pathways, such as via monoamine oxidases A and B (MAO-A, and -B) [123,124]. A recent study has illustrated the N-methylation(s) of the C3 ethylamine in tryptamine and has determined that the second methylation to form DMT (δG^‡ = 142.42 kJ/mol) has a much higher activation barrier than the formation of NMT (δG^‡ = 81.42 kJ/mol), suggesting that DMT formation is thermodynamically disfavored, potentially explaining the low physiological abundance in the brain [123]. Another recent in silico study noted the preferential oxidation of DMT by the MAO-A flavoenzyme with FADH (δG^‡ = 70.3 kJ/mol), demonstrating a lower activation barrier compared to tryptamine (δG^‡ = 78.2 kJ/mol) by δG^‡ = 8 kJ/mol, suggesting a more thermodynamically favorable oxidation and again highlighting a rationale for the low abundance of DMT [124]. Indeed, early rat experiments have shown that tryptamine exists for matter of seconds in vivo [125]. Nonetheless, a recent study implementing in silico techniques to investigate INMT methylation of DMSe to TMSe (Figure 1C) reinforced previous evidence for the preferential binding of DMSe over tryptamines in INMT [34,35,36]. While INMT may show versatile activity between both tryptamine and selenium metabolism, the argument can be made that INMT’s contribution to selenium turnover is important for maintaining cellular viability, such as that conducted by the selenoprotein GPX4, in staving off ferroptosis by quenching lipid peroxides arising from antioxidant plasmalogens [126,127,128,129,130]. Conversely, one could also suggest that tryptamine derivatives in hypoxic or fibrotic environments may serve as short-lived antioxidants where the lipophilic character supports localized quenching of ROS, but this has yet to be shown [24,117,131].

The physiological relevance of N-methylated tryptamines has been difficult to determine for many reasons. Its social perception has narrowly associated N-methylated tryptamines with neurobiology [132]. N-methylated tryptamine synthesis may be inconsequential to health and disease since they arise from a minor pathway compared to the classical kynurenine, serotonin/melatonin, and indole metabolic pathways [133]. Improvement in the robustness of high-throughput experiments and the public availability of data have brought tryptophan metabolism, INMT, and endogenous N-methylated tryptamines into the spotlight again [15,26,51,87,134].

Human tryptophan metabolism predominantly occurs when the essential amino acid is liberated by digestive peptidases and absorbed by the intestinal lumen via SLC6A14 and SLC6A19 neutral amino acid transporters and shuttled to portal circulation via SLC16A10, SLC7A5-SLC3A2, and SLC7A8-SLC3A2 [135,136]. Next, entering the blood, tryptophan is then trafficked to the liver by albumin (75–90%) where 90–95% of diet-derived hepatic tryptophan is directed to the kynurenine pathway and remaining tryptophan is directed to minor metabolic pathways: serotonin/melatonin (1–2%) and indole (5%), which primarily occur in the gut (Figure 1A,B) [137,138]. The kynurenine pathway supplies the crucial bioenergetic metabolite nicotinamide adenine dinucleotide (NAD), as well as kynurenine, kynurenic acid, xanthurenic acid, and cinnabarinic acid which play significant roles in immunoregulation and the cellular redox state [139]. In fact, gut microbiota generating indole acetic acid, like tryptamine, dampen hepatic inflammation in MAFLD [140].

Albeit physiologically low in abundance, N-methylated tryptamines also have an impact on other aspects of metabolism like serotonin, such as lipid metabolism [93,141,142]. Mediated by metabolites from the kynurenine pathway and gut microbiota, agonism of the aryl-hydrocarbon receptor (AhR) has been shown to inhibit de novo lipogenesis [139,140]. Mice given 5-HO-DMT demonstrated reduced cyclooxygenase lipid products and particularly reduced 20-hydroxy-5, 8, 11, 14-eicosatetraenoic acid (20-HETE) levels which play an important role in endothelial dysfunction, as seen in lipidomics performed on their paws [93]. Looking to bypass untoward side effects of 5-HO-DMT, a recent study in mice was able to demonstrate liposomal encapsulation of bufotenine for delivery while maintaining the efficacy of 5-HO-DMT in dampening lipid metabolism and inflammation [142]. The untoward psychotoxic effects of methylated tryptamines are a significant challenge to effectively target SIGMAR1. Strategies to deliver methylated tryptamines are growing as mentioned, but many would require a lipophilic scaffold or delivery system that bypasses 5-HTR signaling at the plasma membrane and maintains intracellular efficacy. Further exploration in drug design and delivery is required to optimize the targeting efficiency of these SIGMAR1 agonists. Consistent with N-methylated tryptamines’ influence on lipid metabolism, studies investigating alterations to SIGMAR1 activity have demonstrated changes to lipid metabolism [143,144]. While further studies are required to explore and deepen our understanding of the impact of N-methylated tryptamines, the evidence of protective effects could be investigated further for therapeutic exploitation.

2.1. Redox Biochemistry

N-dimethylated tryptamines have been described for their effect on 5-HTRs (

Table 1. Receptor interactions among N-dimethylated tryptamines.

Receptor	DMT	5-HO-DMT	5-MeO-DMT
5-HTR1A	Agonist	Agonist	Agonist
5-HTR1B	Partial Agonist	Partial Agonist	Partial Agonist
5-HTR1D	Weak Agonist	Unknown	Weak Agonist
5-HTR2A	Agonist	Agonist	Agonist
5-HTR2B	Agonist	Agonist	Agonist
5-HTR2C	Agonist	Agonist	Agonist
5-HTR7	Agonist	Unknown	Agonist
SIGMAR1	Agonist	Agonist	Agonist
TAAR1	Agonist	Unknown	Agonist
TAAR6	Predicted Agonist	Unknown	Predicted Agonist

) [1,22,43,145,146,147,148,149]. 5-HTRs have been long been implicated in peripheral tissues, where serotonin signaling can mediate whole-body lipid metabolism, inflammation, and play a role in fibrogenic diseases [150,151,152,153,154]. N-methylated tryptamines may also owe their positive outcomes to 5-HTR signaling. Particularly of therapeutic interest are 5-HTRs 1B, 2A, and 2B in HSCs, which have been associated with pro-fibrotic outcomes [155,156,157]. Indeed, 5-HTR signaling is vital in immunoregulation, reviewed elsewhere [158,159]. It is not yet established if endogenous N-methylated tryptamines may compensate or interfere with 5-HTR signaling.

Studies on tryptophan derivatives and serotonin have suggested potent radical scavenging properties as a result of favorable free radical attacking substituents and the tryptamine skeleton [131,160]. Consistent with other reports, serotonin also interacts with lipids to protect lipids from oxidation, suggesting that N-methylated tryptamines may have the same function [117,161]. Since serotonin has shown radical scavenging activity at the C5 hydroxyl, substituents to the tryptamine skeleton may drastically influence radical scavenging properties as well as stability of the radical from further propagation. The C2 position of the tryptamine backbone is susceptible to oxidation, suggesting that tryptamine derivatives could also act as antioxidants [131]. Given the lipophilic character, N-methylated tryptamines could also be short lived due to their rapid spontaneous reaction with hydroperoxides, lipoperoxides, superoxide, and other ROS. Tryptophan derivatives have been shown to spontaneously degrade as a result of oxidation and UV exposure into products such as 2-indolone, supporting further investigations of the antioxidant capacity of N-methylated tryptamines in biological systems [131]. Future electron paramagnetic resonance experiments could help in elucidating this.

2.2. Molecular Biology

Besides their known role in 5-HTR signaling, some N-methylated tryptamines have been investigated for their potential as endogenous ligands to trace amine-associated receptors (TAARs) (Table 1) [45,162,163,164]. While mostly studied in the context of the brain and olfaction, TAAR1 has been shown to be agonized by DMT and it is predicted to also agonize TAAR6. In humans, there are nine TAAR genes, with six functional genes and three pseudogenes [163]. TAARs exist outside of the brain, but no conclusive function in peripheral tissues has been determined as of yet [163]. More research is necessary to determine the likelihood of N-methylated tryptamines in TAAR signaling. In the periphery, TAAR1 mRNA is found in the liver, kidney, and heart, suggesting that there may be roles in N-methylated tryptamine signaling through TAAR1 [162,165]. However, recent evidence in the tryptamine perfused rat kidney suggests that vascular responses are likely mediated through 5-HTR_1A [166].

Recent evidence in hepatic portal vein hypertension has shown that 5-HTR_1A agonism confers reduction in hypertension, suggesting an influence of 5-HTR_1A agonism on endothelial dysfunction [167]. 5-HTR_1A has not yet been described extensively in endothelial cells, so further research is needed to confirm if endothelial 5-HTR_1A is responsible for these observed benefits, similar to the effects of endothelin signaling via endothelin receptor A (ETAR) [68,168,169,170,171]. Canonical endothelial dysfunction, however, is well known to be mediated by ET-1 and has also been implicated in MAFLD, MASH, CKD, and CVD [52,53,54,56,77,172,173,174,175]. ET-1 is a potent vasoconstrictor peptide primarily produced by endothelial cells. ET-1 exerts its effects by binding to endothelin receptor A or -B (ET_AR or ET_BR, respectively), which are G-protein coupled receptors expressed on endothelial cells, smooth muscle cells, HSCs, podocytes, fibroblasts, and other various cell types [53,54,56,170,173,176]. Upon ET-1 binding, ET_AR, for example, activates G proteins, leading to the stimulation of phospholipase C and phosphatidylinositol hydrolysis to form IP3 and subsequently promote the release of calcium, triggering vasoconstriction [176,177]. Endothelial dysfunction arises from insults and pathogenic crosstalk through ET-1/ET_AR or ET_BR between endothelial and other cells in their local milieu or from circulation [176,178]. In the kidney, this has been documented in models of focal segmental glomerulosclerosis and DKD, demonstrating a promise for ameliorating oxidative stress and fibrosis when targeted [77,179,180,181,182]. Similarly in the liver, endothelin signaling is a contributor to HSC activation for fibrosis, and ET_AR blocking has been shown to reduce hepatic fibrosis in rats and mice [173,178,183,184]. As a consequence to endothelin signaling, TGFβ release can be upregulated and commit to a viscous positive feedback loop by TGFβ subsequently upregulating transcription of endothelin pathway proteins [177,182,185,186]. The ET-1/ET_AR is an attractive target in mitigating endothelial dysfunction and resulting fibrosis but because of its ubiquity and systemic consequences in human health, there remain therapeutic challenges.

TGFβ signaling is a well-studied fibrogenic pathway that is ubiquitously present throughout the body and exists among most cell types. Briefly, TGFβ is a secreted latent protein at the cell membrane that, upon mechanobiological changes or extracellular sources of TGFβ, stimulate release and signaling via TGFβ receptors, induce SMAD phosphorylation, nuclear translocation, and transcription of extracellular matrix (ECM) proteins, like collagen [187,188]. Endothelial dysfunction promotes inflammation and TGFβ signaling in the local milieu and can contribute to circulating TGFβ [189,190,191,192,193]. ECs, HSCs, and likely RSCs, are susceptible to a complex system of pro-fibrogenic, pro-inflammatory signals that drive responses in acute insults chronic disease. Other overlapping disease factors may instigate endothelial dysfunction, such as xanthine oxidoreductase, as seen in fatty liver, CKD, and CVD [70]. Indeed, pioneering research in ET-1 HSC activation identified in vitro superoxide generated by xanthine oxidoreductase significantly impacted endothelin receptor expression [170]. Now, it has been reported that antagonizing ET_AR ameliorates liver fibrosis, as seen in a mouse model using nanoparticle delivery systems [194]. However, while great strides have been made in targeting serotonin, TAAR, endothelin, and TGFβ receptors, new interest in SIGMAR1 agonism may provide benefits without serious influence on mood or undesired effects.

SIGMAR1 was first identified as an opioid receptor but was soon considered an orphan receptor due to low specificity with opioid peptides [91]. It was not until Fontanilla et al. discovered DMT as a ligand for SIGMAR1 in 2009 with a K_d = 14.75 μM [91]. Since then, it has been suggested that 5-HO-DMT and 5-MeO-DMT act on SIGMAR1 in a similar manner; however, it has not yet been fully elucidated if 5-HO-DMT and 5-MeO-DMT have greater effect on SIGMAR1 agonism in reducing fibrogenic and pro-inflammatory genes (Table 1) [15,25,88,93,195]. Moreover, SIGMAR1 can induce antioxidant and anti-fibrogenic effects by prompting antioxidant pathways, anti-autophagic, and endocytic recycling. SIGMAR1 has been implicated in activating antioxidant response elements to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) [196]. Autophagy can be downregulated through SIGMAR1-inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) interactions that inhibit the endonuclease activity of IRE1α [197,198]. Especially important is the upregulation of endocytic receptors to reduce ECM deposition, like LRP1, which has been recently suggested to have enhanced expression by SIGMAR1 [199,200]. Interestingly, the N-methylated ethylamine moieties of a tryptamine resemble other endogenous agonists to SIGMAR1 signaling, such as the essential nutrient choline [201]. Choline is a one-carbon and lipid metabolite partly relied on from diet that contributes to, among many pathways, one-carbon metabolism as a methyl donor to form SAM [202]. Choline has recently been found to agonize SIGMAR1, deepening its role beyond metabolism and in maintaining mitochondrial and ER dynamics [95,99,201,203]. Choline metabolism, like tryptamine, is partly contributed to by microbiota, like Enterobacteriaceae or Ruminococcaceae, which impacts health and disease [137,138,140,204,205,206,207,208].

2.3. Tryptamine Metabolism and the Gut Microbiome

Gut bacteria deeply impact human health and metabolism, especially in contributing to immunoregulatory mechanisms via kynurenine metabolites [137,140,204,209,210]. Microbiota-derived tryptamine arises from decarboxylation of tryptophan (Figure 1A) by bacteria like Ruminococcus gnavus and has been associated with liver fibrosis in MAFLD [209,211,212,213,214]. Like choline, gut-derived tryptophan metabolites have conflicting evidence to their benefit or harm [212,215,216]. A recent article found R. gnavus-derived tryptamine to impair insulin sensitivity in mice and monkeys via TAAR1 [217]. In contrast, the enrichment of R. gnavus also has been shown to be lower in CVD when complicated with NAFLD, suggesting that comorbidities may influence the microbiome composition [218]. Additionally, Enterobacteriaceae have a positive association with MAFLD severity and progression where shifts in tryptophan and choline metabolism can impact disease pathogenesis [211,216]. Similarly, Enterobacteriaceae has been implicated in CKD, enriching as disease progresses and correlating with low eGFR [219,220]. Targeting Enterobacteriaceae through tryptophan metabolism by using D-tryptophan, however, has shown effectiveness in reducing colitis and may be a potential therapeutic [221,222].

Intriguingly, microbial-derived short-chain fatty acids were recently shown to impact SIGMAR1 [102]. Indeed, a SIGMAR1 KO also showed evidence for gut dysbiosis, suggesting that there may be a microbial relationship to SIGMAR1 activity [223]. It remains to be seen if microbiota-derived tryptamines have any contribution to endogenous N-methylated tryptamine levels, but further experiments may help to determine if SIGMAR1 agonism is mediated through host-microbe circumstances.

3. INMT in Models of Human Diseases

In vivo murine studies, particularly ones in fibrogenic diseases, have demonstrated that Inmt is expressed in many healthy tissues and have found an alteration of Inmt mRNA expression as a result of insult or disease [48,50,224]. Both male and female mouse kidneys have shown a high expression of Inmt in cortical segments of healthy proximal tubule cells, which can be further utilized in in situ RNA-sequencing to identify spatial cellular changes in whole-tissue models of acute kidney injury (AKI) and repair [225]. Indeed, proximal tubules are early contributors to fibrogenic signaling in the injured kidney [39].

In utero experiments have also shown a change in Inmt expression. In 2012, a time-course mouse study using a hyperhomocysteinemia model with cystathionine β-synthase (Cbs)-deficient mice found both wild-type and Cbs-deficient in uterus interimplantation sites had increased Inmt mRNA but several folds more in Cbs-deficient mice in early pregnancy [226]. The study did not further investigate if Inmt activity is consequential for implantation and in utero embryonic development; however, the generation of the Inmt knockout (KO) rat and mouse (mousephenotype.org/data/genes/MGI:102963) has not reported embryonic lethality, suggesting that Inmt may have functionality beyond implantation, pregnancy, and embryonic viability [119,227].

3.1. INMT in Renal Diseases

Originally found as a marker for proximal tubule cells in the kidney, INMT has been implicated in many forms of renal diseases, such as nephrolithiasis, diabetic kidney disease (DKD), AKI, and renal fibrosis [38,50,228,229,230]. In search of a genetic susceptibility to nephrolithiasis, a Japanese 2012 genome-wide association study with 5892 participants identified a risk SNP, rs1000597, in INMT [228]. A model of DKD, a prominent subgroup of CKD, downregulates proximal tubule Inmt expression in db/db mice but is rescued by treatments of the angiotensin receptor blocker irbesartan and the sodium-glucose cotransporter 2 inhibitor (SGLT2i) dapagliflozin, suggesting Inmt may be important in injury or disease resolution [50]. In line with this evidence, publicly available transcriptomic data from DKD-induced mouse models identified the downregulation of Inmt [231].

In a model of AKI, Inmt expression in male C57BL/6 mice subjected to ischemia–reperfusion injury (IRI) was downregulated in pro-fibrotic, maladaptive proximal tubule cells [232]. In another IRI experiment aiming to characterize the transition of AKI-to-CKD, C57BL/6CN male mice were found to have Inmt expression downregulated in proximal tubules as a result of IRI [233]. Interestingly, studies conducted in 2018 and 2019 demonstrated that administering DMT to prevent renal injury in a rat model of clamp-induced IRI significantly mitigated IRI with an intramuscular dose of 2.95 mL/kg body weight of a 2.45 mg/mL DMT solution, administered once 15 min before ischemia and once 15 min before reperfusion [229,230]. Inmt was not assessed in this study, but the study may suggest that the role of Inmt may be in mitigating cell and tissue insults via agonizing SIGMAR [91,229,230]. Ultimately, other studies in rats have found that Inmt is expressed in healthy brain, lung, liver, and kidney tissues [92,234].

Public databases of transcriptomic data suggest human INMT has been found to be expressed in liver, lung, heart, and kidney tissues (Human Protein Atlas: proteinatlas.org/ENSG00000241644-INMT/tissue+cell+type, and The Kidney Precision Medicine Project: https://www.kpmp.org (accessed on 27 September 2024) [235,236]. When breaking down INMT expression by single-cell RNAseq in these organs: fibroblast subpopulations, endothelial, smooth muscle, mesangial cells, macrophages, and HSCs are expressors [235,236]. The KPMP database includes single-nucleus RNAseq which associated INMT expression with repairing fibroblasts and degenerative endothelial cells, and although consistent with a protective effect, more research is necessary to conclude a role for INMT in fibrosis [236].

Recent studies in fibrogenic MAFLD, MASH, CVD, and CKD have implicated changes in SIGMAR1 activity as well as INMT [48,100]. Interestingly INMT variants, which impact the methylation ability of INMT, have been associated with a risk nephrolithiasis, but a recent study has shown amelioration through SIGMAR1 agonism [96]. Fukumoto et al. has also shown the preferential activity of INMT in TMSe production, which has recently been found to be compensated by thiopurine S-methyltransferase [36,237]. Ultimately, INMT may be important as a redundant enzyme between tryptamine and selenium metabolism where insults may determine the preferential activity of INMT.

3.2. INMT in Hepatic Diseases

Identified in hepatic endothelial cells in 2018, INMT became a fibrotic-gene candidate in human MAFLD in 2021 [48,238]. A recent article investigating a mouse model of liver fibrogenesis and regression using thioacetamide found downregulated INMT which rapidly increased to normal expression levels in the regressive period [239]. A 2016 study demonstrated hepatic Inmt is downregulated in mice treated with 30 mg/kg diclofenac, a common analgesic [240]. A recent rat model of primary sclerosing cholangitis identified hepatic tryptophan and monoamine metabolism in regulating liver fibrosis, suggesting tryptamines may play a role in liver fibrosis [241]. In Gata4 KO mice, a transcription factor regulating liver fibrosis, hepatocyte Inmt was found to be upregulated but present in low protein abundance; however, recent evidence by Arroyo et al. demonstrated Gata4 as responsible for HSC deactivation [242,243]. Future studies in the Gata4 KO, with a focus on INMT, will help in delineating the activity of INMT in HSCs activation. HSCs activation and subsequent maladaptive ECM deposition is induced by a myriad of insults; however, ET-1 is implicated as a major driver of fibrosis in MASH [52,172,177,183]. Opposing HSC activation and fibrogenic signaling has been shown by LRP1 mitigating rise in TGFβ in vitro [244]. Interestingly, studies have shown that activated HSCs have ER-localized SIGMAR1 translocated to the plasma membrane, which are suggested to be more susceptible to agonization from agents in the local milieu [245]. Studies have shown in mice that the absence of functional LRP1 accelerates the progression of MAFLD and MASH, further underscoring the benefits to LRP1 endocytic signaling against disease [246]. While it has yet to be shown, the sum of evidence here indicates that the SIGMAR1 agonizing products of INMT may promote endocytic recycling via increased LRP1 levels to mitigate ECM deposition.

Hepatic autophagy through ER-stress induced IRE1α activation, on the other hand, has been implicated with pro-fibrotic effects [247]. While not yet investigated, methylated tryptamines could potentiate the SIGMAR1-dependent inhibition of IRE1α endonuclease activity, thus promoting antifibrogenic effects in the liver. Further direct investigations are necessary to confirm.

A rat model investigating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) AhR agonism and toxicity found that hepatic Inmt was downregulated following TCDD administration, irrespective of TCDD susceptibility [248]. Responsible for pro-inflammatory gene expression as a transcription factor, AhR is agonized by several tryptophan metabolites from the Kynurenine pathway. The decrease in hepatic Inmt may reflect upstream regulation by pro-inflammatory signals, since methylated tryptamines have been shown to induce anti-inflammatory effects [25,195]. An obese Zucker rat model investigating the anti-steatotic effect of soy protein found Inmt to be upregulated compared to a casein diet by mRNA RNAseq and verified by qPCR [249]. Metformin, a diabetic drug improving liver health, has been used in MAFLD to identify INMT expression as a target [250]. While INMT has not been directly explored in the liver, it is expressed across disease profiles and largely associated with positive outcomes. It has yet to be demonstrated but experiments specifically incorporating the Inmt KO into mouse models of liver and kidney injury may uncover the role of INMT N-methylated tryptamines.

A 24h in vitro FACS flow cytometry experiment investigating the influence of DMT on LX-2 HSCs demonstrated an increase in MHC-II expression (4.6% with 100 μM DMT alone and 11.6% at 100 μM DMT with 500IU interferon gamma (IFNγ)), which is consistent with other reports using IFNγ to downregulate collagens involved in fibrosis (Unpublished data) [251,252]. INMT in HSCs has yet to be fully described, but SIGMAR1 in activated HSCs has been shown to be highly expressed and can be blocked by novel targeted delivery of silencing RNA (siRNA) nanoparticles [253]. While the Han et al. study demonstrated antagonism of SIGMAR1 leads to a reduction in collagen proteins, it remains to be determined if agonism may also influence fibrogenesis. Considering that ferroptosis is a large contributor to MASH and fibrosis, the role INMT in mediating selenium metabolism and SIGMAR1-dependent expression of antioxidant genes may be a significant factor in dampening ferroptotic fate [254,255]. Selenoproteins, like GPX4, are expressed in all forms of life and play a role in quenching ROS and other free-radical substances. The intake of dietary selenium is required for selenoprotein synthesis at very low concentrations at roughly 55 μg/day, thus underscoring the vital importance of sufficient selenium levels to maintain homeostasis. INMT balances selenium levels through the production of the major urine metabolite TMSe; however, there are no studies investigating the flux of TMSe produced by INMT in fibrotic diseases. Given the lipophilic character and susceptibility to oxidation at the indolic C-2 position, methylated tryptamines may also serve to spontaneously quench propagative lipoperoxides that otherwise promote ferroptosis [126]. Indeed, ferroptosis contributes to HSC activation and liver fibrogenesis [256]. More thorough experiments implementing ⁷⁶Se, ¹³C-SAM and ¹³C-tryptophan could help in determining the role of INMT in fibrogenic MAFLD by investigating the contribution of INMT to selenium and N-methylated tryptamine metabolism. This will be vital in understanding INMT activity in HSCs. Expanding an isotope tracing experiment to incorporate human subjects with MAFLD would confirm the preferential flux of selenium or N-methylated tryptamine metabolites in disease.

It is noteworthy to mention that Alzheimer’s disease shares many molecular characteristics of chronic liver and renal pathologies, such as fibrosis, and a recent study found that DMT ameliorates Alzheimer’s disease through SIGMAR1 [257]. Associated with fibrosis, and often with comorbidities, Alzheimer mouse models have similarly linked benefits from choline to reduced pathology [258,259,260,261,262,263]. Indeed, pathologies such as diabetes and Alzheimer’s are relevant to liver and kidney diseases due to the presence of underlying endothelial dysfunction, which in turn promote fibrogenic and ROS-stimulating pathways in these tissues. There could be hope yet for revisiting a clinical application of N-methylated tryptamine in chronic, fibrotic diseases [261].

Altogether, INMT in diseases, such as liver, kidney, and heart fibrosis, Alzheimer’s disease and diabetes, may play a critical role as an antifibrogenic mediator through the production of its metabolites. Future studies must delineate the exact metabolic contribution of INMT to either methylation of tryptamines or selenium metabolism, with a focus on validated models of fibrosis.

3.3. INMT in Cancer

Cancer is notorious in modifying metabolic pathways to support proliferation, evade immune defensive measures, alter the local microenvironment and syphon metabolites from neighboring cells. Across most cancers, INMT is downregulated [122,224]. INMT downregulation in hepatocellular carcinoma (HCC) is a prognostic indicator for poor outcomes and is interestingly met with an increase in SIGMAR1 [224,264]. Indeed, SIGMAR1 has been shown to be protective against ferroptosis in HCC, perhaps as a hijacked mechanism to promote cancer success [254,264]. INMT has been used to develop an HCC-specific profile of selenium metabolism [265]. Selenoproteomic and metabolomic patient studies implementing isotope-tracing ⁷⁶Se could further our understanding of INMT in progressive-to-terminal liver disease [266]. INMT has also been downregulated in prostate cancer, having an antiproliferative, pro-apoptotic effect when overexpressed [267]. Head and neck squamous cell carcinoma also demonstrates downregulation of INMT expression, influenced by hypomethylation changes in the promoter [122]. Lung adenocarcinoma has also demonstrated a low expression of INMT and is associated with poor prognosis; however, INMT improves immunotherapy response [268]. Perhaps higher expressing INMT in cancer promotes a resistance to cell death via SIGMAR1 in this context [264]. More research is required to determine the metabolic function of INMT across cancer as it often induces global shifts in metabolism.

4. Conclusions

Evidence presented here suggests that endogenously N-dimethylated tryptamines DMT, 5-HO-DMT, and 5-MeO-DMT likely have a biological function outside of neuroregulatory mechanisms. Studies have highlighted the versatile roles of INMT in mediating both tryptamine and selenium metabolic pathways. INMT’s contribution to either tryptamine or selenium metabolism remains to be delineated, but recent data in INMT KO rats suggest that N-dimethylated tryptamines may have an alternative metabolic route [119]. Selenoproteomic and metabolomic patient studies implementing isotope-tracing ⁷⁶Se, ¹³C-SAM and ¹³C-tryptophan could help in determining the role of INMT in MAFLD, MASH, and CKD. Likewise, these studies could define a role for N-methylated tryptamines outside of neuroscience and usher advances in drug targeting and repurposing.

The changes in hepatic, renal, and cardiac INMT expression are consistent with upregulation in injury resolution and downregulation in maladaptive and degenerative tissues. Further studies are required to confirm these phenomena across tissues and cell types.

Benefits of SIGMAR1 agonism in fibrotic, chronic diseases have received growing attention; however, intracellular localization increases the complexity of accurate drug targeting with N-dimethylated tryptamines due to partial 5-HTR agonism at the cell membrane. Studies have highlighted the ubiquity and role of SIGMAR1 in regulating mitochondrial integrity, bioenergetics, and endoplasmic reticulum stress [95,98,99]. SIGMAR1 is an attractive target in mitigating renal injury and fibrosis, growing in popularity. Endothelial agonism of SIGMAR1 may aid in reducing both local and systemic insults (Figure 2). Nonetheless, future research in agonizing SIGMAR1 in liver diseases may be therapeutically promising through means of N-methylated tryptamines or their derivatives.