Activation of lysosomal iron triggers ferroptosis in cancer.

Iron catalyses the oxidation of lipids in biological membranes and promotes a form of cell death referred to as ferroptosis1-3. Identifying where this chemistry takes place in the cell can inform the design of drugs capable of inducing or inhibiting ferroptosis in various disease-relevant settings. Whereas genetic approaches have revealed underlying mechanisms of lipid peroxide detoxification1,4,5, small molecules can provide unparalleled spatiotemporal control of the chemistry at work6. Here, we show that the ferroptosis inhibitor liproxstatin-1 (Lip-1) exerts a protective activity by inactivating iron in lysosomes. Based on this, we designed the bifunctional compound fentomycin that targets phospholipids at the plasma membrane and activates iron in lysosomes upon endocytosis, promoting oxidative degradation of phospholipids and ferroptosis. Fentomycin effectively kills primary sarcoma and pancreatic ductal adenocarcinoma cells. It acts as a lipolysis-targeting chimera (LIPTAC), preferentially targeting iron-rich CD44high cell-subpopulations7,8 associated with the metastatic disease and drug resistance9,10. Furthermore, we demonstrate that fentomycin also depletes CD44high cells in vivo and reduces intranodal tumour growth in an immunocompetent murine model of breast cancer metastasis. These data demonstrate that lysosomal iron triggers ferroptosis and that lysosomal iron redox chemistry can be exploited for therapeutic benefits.

Integrated chemical and genetic screens unveil FSP1 mechanisms of ferroptosis regulation.

Ferroptosis, marked by iron-dependent lipid peroxidation, may present an Achilles heel for the treatment of cancers. Ferroptosis suppressor protein-1 (FSP1), as the second ferroptosis mainstay, efficiently prevents lipid peroxidation via NAD(P)H-dependent reduction of quinones. Because its molecular mechanisms have remained obscure, we studied numerous FSP1 mutations present in cancer or identified by untargeted random mutagenesis. This mutational analysis elucidates the FAD/NAD(P)H-binding site and proton-transfer function of FSP1, which emerged to be evolutionarily conserved among different NADH quinone reductases. Using random mutagenesis screens, we uncover the mechanism of action of next-generation FSP1 inhibitors. Our studies identify the binding pocket of the first FSP1 inhibitor, iFSP1, and introduce the first species-independent FSP1 inhibitor, targeting the NAD(P)H-binding pocket. Conclusively, our study provides new insights into the molecular functions of FSP1 and enables the rational design of FSP1 inhibitors targeting cancer cells.

Changes in Metabolomic Profiles Induced by Switching from an Erythropoiesis-Stimulating Agent to a Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor in Hemodialysis Patients: A Pilot Study.

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a new class of medications for managing renal anemia in patients with chronic kidney disease (CKD). In addition to their erythropoietic activity, HIF-PHIs exhibit multifaceted effects on iron and glucose metabolism, mitochondrial metabolism, and angiogenesis through the regulation of a wide range of HIF-responsive gene expressions. However, the systemic biological effects of HIF-PHIs in CKD patients have not been fully explored. In this prospective, single-center study, we comprehensively investigated changes in plasma metabolomic profiles following the switch from an erythropoiesis-stimulating agent (ESA) to an HIF-PHI, daprodustat, in 10 maintenance hemodialysis patients. Plasma metabolites were measured before and three months after the switch from an ESA to an HIF-PHI. Among 106 individual markers detected in plasma, significant changes were found in four compounds (erythrulose, n-butyrylglycine, threonine, and leucine), and notable but non-significant changes were found in another five compounds (inositol, phosphoric acid, lyxose, arabinose, and hydroxylamine). Pathway analysis indicated decreased levels of plasma metabolites, particularly those involved in phosphatidylinositol signaling, ascorbate and aldarate metabolism, and inositol phosphate metabolism. Our results provide detailed insights into the systemic biological effects of HIF-PHIs in hemodialysis patients and are expected to contribute to an evaluation of the potential side effects that may result from long-term use of this class of drugs.

Phase separation of FSP1 promotes ferroptosis.

Ferroptosis is evolving as a highly promising approach to combat difficult-to-treat tumour entities including therapy-refractory and dedifferentiating cancers1-3. Recently, ferroptosis suppressor protein-1 (FSP1), along with extramitochondrial ubiquinone or exogenous vitamin K and NAD(P)H/H+ as an electron donor, has been identified as the second ferroptosis-suppressing system, which efficiently prevents lipid peroxidation independently of the cyst(e)ine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) axis4-6. To develop FSP1 inhibitors as next-generation therapeutic ferroptosis inducers, here we performed a small molecule library screen and identified the compound class of 3-phenylquinazolinones (represented by icFSP1) as potent FSP1 inhibitors. We show that icFSP1, unlike iFSP1, the first described on-target FSP1 inhibitor5, does not competitively inhibit FSP1 enzyme activity, but instead triggers subcellular relocalization of FSP1 from the membrane and FSP1 condensation before ferroptosis induction, in synergism with GPX4 inhibition. icFSP1-induced FSP1 condensates show droplet-like properties consistent with phase separation, an emerging and widespread mechanism to modulate biological activity7. N-terminal myristoylation, distinct amino acid residues and intrinsically disordered, low-complexity regions in FSP1 were identified to be essential for FSP1-dependent phase separation in cells and in vitro. We further demonstrate that icFSP1 impairs tumour growth and induces FSP1 condensates in tumours in vivo. Hence, our results suggest that icFSP1 exhibits a unique mechanism of action and synergizes with ferroptosis-inducing agents to potentiate the ferroptotic cell death response, thus providing a rationale for targeting FSP1-dependent phase separation as an efficient anti-cancer therapy.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Nutritional and Metabolic Control of Ferroptosis.

Ferroptosis is a type of regulated cell death characterized by an excessive lipid peroxidation of cellular membranes caused by the disruption of the antioxidant defense system and/or an imbalanced cellular metabolism. Ferroptosis differentiates from other forms of regulated cell death in that several metabolic pathways and nutritional aspects, including endogenous antioxidants (such as coenzyme Q10, vitamin E, and di/tetrahydrobiopterin), iron handling, energy sensing, selenium utilization, amino acids, and fatty acids, directly regulate the cells' sensitivity to lipid peroxidation and ferroptosis. As hallmarks of ferroptosis have been documented in a variety of diseases, including neurodegeneration, acute organ injury, and therapy-resistant tumors, the modulation of ferroptosis using pharmacological tools or by metabolic reprogramming holds great potential for the treatment of ferroptosis-associated diseases and cancer therapy. Hence, this review focuses on the regulation of ferroptosis by metabolic and nutritional cues and discusses the potential of nutritional interventions for therapy by targeting ferroptosis.

Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines.

Sorafenib, a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma, has been repeatedly reported to induce ferroptosis by possibly involving inhibition of the cystine/glutamate antiporter, known as system xc-. Using a combination of well-defined genetically engineered tumor cell lines and canonical small molecule ferroptosis inhibitors, we now provide unequivocal evidence that sorafenib does not induce ferroptosis in a series of tumor cell lines unlike the cognate system xc- inhibitors sulfasalazine and erastin. We further show that only a subset of tumor cells dies by ferroptosis upon sulfasalazine and erastin treatment, implying that certain cell lines appear to be resistant to system xc- inhibition, while others undergo ferroptosis-independent cell death. From these findings, we conclude that sorafenib does not qualify as a bona fide ferroptosis inducer and that ferroptosis induced by system xc- inhibitors can only be achieved in a fraction of tumor cell lines despite robust expression of SLC7A11, the substrate-specific subunit of system xc-.

Genetic Background and Clinicopathologic Features of Adult-onset Nephronophthisis.

INTRODUCTION: Recently, nephronophthisis (NPH) has been considered a monogenic cause of end-stage renal disease (ESRD) in adults. However, adult-onset NPH is difficult to accurately diagnose and has not been reported in a cohort study. In this study, we assessed the genetic background and clinicopathologic features of adult NPH. METHODS: We investigated 18 sporadic adult patients who were suspected as having NPH by renal biopsy. We analyzed 69 genes that cause hereditary cystic kidney disease and compared clinicopathologic findings between patients with and without pathogenic mutations in NPH-causing genes. RESULTS: Seven of 18 patients had pathogenic NPH-causing mutations in NPHP1, NPHP3, NPHP4, or CEP164. Compared with patients without pathogenic mutations, those with pathogenic mutations were significantly younger but did not significantly differ in the classic NPH pathologic findings, such as tubular cysts. On the other hand, the number of tubules with thick tubular basement membrane (TBM) duplication, which was defined as >10-µm thickness, was significantly higher in patients with genetically proven adult NPH than in those without pathogenic mutations. α-Smooth muscle actin (α-SMA)-positive myofibroblasts were detected inside thick TBM duplication. CONCLUSIONS: In adult patients with NPH, thick TBM duplication was the specific finding. Our analysis also suggested that older patients tended to have no pathogenic mutations, even when they were suspected to have NPH by renal biopsy. These findings could be the novel clinical clue for the diagnosis of NPH in adult patients.

Fibromuscular dysplasia with recurrence after "long-term" following percutaneous transcatheter renal angioplasty: two case reports with a review of 26 patients.

BACKGROUND: Fibromuscular dysplasia (FMD) often causes renal artery stenosis with renovascular hypertension. Recent clinical outcomes encourage percutaneous transluminal renal angioplasty (PTRA) to treat FMD; however, the necessary follow-up period remains unclear. Moreover, previous studies have not revealed the difference in the period until recurrence between two major types of FMD-multifocal and focal. CASE PRESENTATION: We describe two patients with multifocal FMD who developed hypertension during their teenage years and had recurrence of FMD > 10 years after PTRA. We further examined the types of FMD and age of onset in 26 patients who underwent PTRA. The period until recurrence of multifocal FMD was longer than that of focal FMD. Moreover, patients with early-onset multifocal FMD are likely to have a delayed recurrence after PTRA compared to other types. CONCLUSIONS: Our report suggests that patients with multifocal FMD, especially those with onset at an early age, may need long-term follow-up for at least ≥ 10 years.

A Novel Mutation in LMX1B (p.Pro219Ala) Causes Focal Segmental Glomerulosclerosis with Alport Syndrome-like Phenotype.

A 69-year-old woman presented with mild renal dysfunction, proteinuria, and sensorineural hearing loss. A renal biopsy showed focal segmental glomerulosclerosis with thinning of the glomerular basement membrane. There was a positive family history of end-stage kidney disease and hearing loss. Although Alport syndrome was suspected from these features, a genetic test using next-generation sequencer identified a novel missense mutation in LMX1B, c.655C>G: p. (Pro219Ala). In silico analyses predicted the pathogenicity of the mutation. Thus, the present case was diagnosed as LMX1B-associated nephropathy presenting with Alport syndrome-like phenotype, expanding the disease spectrum of LMX1B nephropathy.

Concurrent analogous organ damage in the brain, eyes, and kidneys in malignant hypertension: reversible encephalopathy, serous retinal detachment, and proteinuria.

Malignant hypertension, a form of hypertensive emergency, causes acute damage in vital organs such as the brain, eyes, and kidneys. We aimed to examine the concurrency of acute hypertensive damage across the target organs to elucidate the underlying analogous pathophysiology. This single-center retrospective study evaluated the characteristics of organ damage, short-term clinical course, and interorgan relationships in patients with malignant hypertension treated between 2008 and 2019. Baseline characteristics of 20 patients who met our inclusion criteria were mean age 48 ± 13 years and blood pressure 222 ± 18/142 ± 16 mmHg; the median estimated glomerular filtration rate and urinary protein level were 49 mL/min/1.73 m2 (interquartile range [IQR] 27-79) and 1.9 g/g creatinine (IQR 0.2-4.0), respectively. Posterior reversible encephalopathy syndrome (PRES) was found in 60% of patients with major involvement and a wide variety of distribution patterns in the brainstem. In the fundus, serous retinal detachment was found in 60% of patients. Patients with PRES and serous retinal detachment showed higher levels of urinary protein than those without symptoms (P = 0.007 and 0.02, respectively), and proteinuria >1 g/g creatinine highly complicated both PRES and serous retinal detachment (91%). Matrix analysis also showed that the three symptoms were highly associated with each other. These results demonstrate the close relationship and concurrency of hypertensive acute organ damage in the brain, eyes, and kidneys. A common analogous mechanism, such as hyperperfusion-induced capillary leakage in each organ, implies an underlying pathophysiology of PRES, serous retinal detachment, and proteinuria.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5.

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

Uric Acid Elevation by Favipiravir, an Antiviral Drug.

In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation.

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischaemic insult.

Kidney enlargement effect of angioplasty for nonatherosclerotic renovascular disease: reversibility of ischemic kidney.

Renal artery stenosis causes kidney ischemia, reducing the size of the affected kidney, which eventually results in atrophy. Although renal atrophy is considered irreversible, resolution of the ischemia occasionally restores kidney size when the cause is renal artery stenosis. Angioplasty is effective in patients with nonatherosclerotic renovascular diseases (non-ARVDs). Nevertheless, renal enlargement after angioplasty has not been fully examined. We conducted a retrospective study to examine this phenomenon in non-ARVD patients. Ten patients with a <100-mm pole-to-pole length of the poststenotic kidney were treated with angioplasty. Data were collected up to 12 months after angioplasty. The mean age was 28 years; the estimated glomerular filtration rate was 92 ± 7 mL/min/1.73 m2 (mean ± SEM); blood pressure was 150/99 mmHg; 80% were women; and fibromuscular dysplasia was present in 90% of the patients. All patients had hypertension. The lengths of the poststenotic and contralateral kidney before angioplasty were 91 ± 1 and 111 ± 3 mm, respectively. After angioplasty, the length of the poststenotic kidney gradually increased during the 3 months after treatment (+5.4 mm) and that of the contralateral kidney decreased over the same time course (-3.7 mm). Enlargement was also found in the moderate atrophy subgroup (length < 92 mm), and it was greater in the <30 years old group. In a noteworthy case, renal size in the poststenotic kidney recovered from 87 to 102 mm after angioplasty. Our findings demonstrated that reduced renal size can be reversed after optimal angioplasty in non-ARVD patients, especially young patients, suggesting reversibility of the ischemic kidney.

Contribution of Rare Variants of the SLC22A12 Gene to the Missing Heritability of Serum Urate Levels.

Gout is a common arthritis caused by monosodium urate crystals. The heritability of serum urate levels is estimated to be 30-70%; however, common genetic variants account for only 7.9% of the variance in serum urate levels. This discrepancy is an example of "missing heritability." The "missing heritability" suggests that variants associated with uric acid levels are yet to be found. By using genomic sequences of the ToMMo cohort, we identified rare variants of the SLC22A12 gene that affect the urate transport activity of URAT1. URAT1 is a transporter protein encoded by the SLC22A12 gene. We grouped the participants with variants affecting urate uptake by URAT1 and analyzed the variance of serum urate levels. The results showed that the heritability explained by the SLC22A12 variants of men and women exceeds 10%, suggesting that rare variants underlie a substantial portion of the "missing heritability" of serum urate levels.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease.

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.