SGLT2 Inhibition in Patients With Type 2 Diabetes Mellitus Post-Nephrectomy: A Single-Center Case Series.

BACKGROUND: Nephrectomy is the mainstay of treatment for many kidney cancers, but has been correlated with increased incidence of acute kidney injury (AKI) and chronic kidney disease (CKD). Recently, sodium-glucose cotransporter-2 (SGLT2) inhibition has been shown to decrease the incidence of end-stage kidney disease and death in people with type 2 diabetes mellitus (T2D). However, at present, there has been no description of the use of SGLT2 inhibition in patients with T2D and solitary kidney despite the high risk of CKD progression. OBJECTIVE: To characterize the use of SGLT2 inhibition and kidney function in a series of patients with T2D with prior nephrectomy for renal cell carcinoma (RCC). DESIGN: Retrospective case series. SETTING: University hospital outpatient onco-nephrology clinic. PATIENTS: Patients post-nephrectomy for RCC with T2D who were prescribed an SGLT2 inhibitor. MEASUREMENTS: Serum creatinine, albumin to creatinine ratio (ACR), HgA1c, and blood pressure measurements. METHODS: Patients post-nephrectomy with incident use of SGLT2 inhibitor were identified from an existing registry of patients followed in the Onco-Nephrology Clinic at our institution from May 2019 to March 2021. Demographics, medication use, time since nephrectomy, cancer diagnosis, serum creatinine, ACR measurements, and blood pressure measurements were extracted from electronic medical records. RESULTS: Five patients were identified who had initiated SGLT2 inhibition post-nephrectomy. All patients were male, had T2D, and a prior history of hypertension. Renal cell carcinoma was the clinical indication for nephrectomy in all patients. None of patients were prescribed diuretics, and all were receiving renin-angiotensin system (RAS) inhibition therapies. The time from nephrectomy to SGLT2 inhibitor initiation ranged from 5 to 74 months. Baseline mean estimated glomerular filtration rate (eGFR) values were 49 mL/min/1.73 m2 (95% confidence interval [CI]: 31.5-66.5), and mean ACRs were 8.7 mg/mmol (95% CI: 0.6-16.9). After 6 months of SGLT2 inhibition, the mean eGFR and ACR values were 58 mL/min/1.73 m2 (95% CI: 29.7-86.2) and 23.8 mg/mmol (95% CI: 0-60), respectively. After 16 to 18 months of follow-up (4 patients), the mean eGFR was 56 mL/min/1.73 m2 (95% CI: 37.3-74.7), and mean ACR was 10.5 (95% CI: 0-30.5), similar to baseline values before SGTL2i therapy initiation. At baseline, mean systolic blood pressure was 128 mm Hg (95% CI: 118.3-140.9) and remained similar after 12 months of treatment (mean 131 mm Hg [95% CI: 112.3-149.7]). There were no adverse events related to AKI, electrolyte disturbances, ketoacidosis, or genitourinary infections during the 18-month follow-up period. LIMITATIONS: Small sample size, lack of a comparison group, and the variable timing of clinical data collection, including eGFR levels following initiation of SGLT2 inhibition. CONCLUSIONS: SGLT2 inhibition is becoming a standard component of nephrology care to reduce kidney function decline, cardiovascular risk, and mortality. To our knowledge, our report is the first to provide longitudinal data on SGLT2 inhibitor usage in patients with T2D and solitary kidneys post-nephrectomy. Larger prospective studies are needed to determine the efficacy and safety of SGLT2 inhibition strategies for kidney protection in patients post-nephrectomy.

CONTEXTE: La néphrectomie est le traitement de référence pour de nombreux cancers rénaux, mais elle est corrélée à une incidence accrue d'insuffisance rénale aiguë (IRA) et d'insuffisance rénale chronique (IRC). On a récemment montré que l'inhibition du cotransporteur sodium-glucose de type 2 (SGLT2) réduisait l'incidence de l'insuffisance rénale terminale et la mortalité chez les personnes atteintes de diabète de type 2 (DB2). À l'heure actuelle, malgré le risque élevé de progression vers l'IRC, il n'existe aucune description de l'utilisation des inhibiteurs du SGLT2 chez les patients DB2 ayant un seul rein. OBJECTIF: Caractériser la fonction rénale et l'utilisation des inhibiteurs du SGLT2 chez une série de patients atteints de DB2 ayant subi une néphrectomie pour traiter un carcinome rénal (CR). TYPE D'ÉTUDE: Série de cas rétrospective. CADRE: Clinique externe d'onconéphrologie d'un hôpital universitaire. SUJETS: Patients atteints de DB2 ayant subi une néphrectomie pour un CR et à qui on a prescrit un inhibiteur du SGLT2. MESURES: Créatinine sérique, rapport albumine/créatinine (RAC), HgA1c et mesures de la pression artérielle. MÉTHODOLOGIE: Les patients ayant subi une néphrectomie et ayant utilisé un inhibiteur du SGLT2 ont été identifiés dans le registre des patients suivis à la clinique d'onconéphrologie de notre établissement entre mai 2019 et mars 2021. Les données suivantes ont été extraites des dossiers médicaux : données démographiques, consommation de médicaments, temps écoulé depuis la néphrectomie, diagnostic du cancer, taux de créatinine sérique, mesures du RAC et de la pression artérielle. RÉSULTATS: Cinq patients avaient amorcé l'inhibition du SGLT2 après la néphrectomie. Tous les sujets étaient des hommes atteints de diabète de type 2 et présentant des antécédents d'hypertension. Le CR était dans tous les cas l'indication clinique pour la néphrectomie. Aucun des patients n'avait reçu une prescription de diurétiques et tous suivaient un traitement avec un inhibiteur du système rénine-angiotensine (SRA). Le délai entre la néphrectomie et l'amorce de l'inhibition du SGLT2 variait entre cinq et soixante-quatorze mois. Le DFGe initial moyen s'établissait à 49 ml/min/1,73 m2 (IC 95 % : 31,5-66,5) et le rapport albumine/créatinine moyen (RAC) à 8,7 mg/mmol (IC 95 % : 0,6-16,9). Après six mois d'inhibition du SGLT2, les valeurs moyennes de DFGe et de RAC s'établissaient respectivement à 58 ml/min/1,73 m2 (IC 95 % : 29,7-86,2) et à 23,8 mg/mmol (IC 95 % : 0-60). Après 16-18 mois de suivi (quatre patients), le DFGe moyen était de 56 ml/min/1,73 m2 (IC 95 % : 37,3-74,7) et le RAC moyen de 10,5 mg/mmol (IC 95 % : 0-30,5); des valeurs semblables aux valeurs mesurées avant le début du traitement par inhibiteur du SGTL2. La pression artérielle systolique (PAS) moyenne initiale était de 128 mmHg (IC 95 % : 118,3-140,9) et elle est demeurée quasi inchangée après douze mois de traitement (moyenne de 131 mmHg [IC 95 % : 112,3-149,7]). Aucun événement indésirable lié à l'insuffisance rénale aiguë, à des perturbations électrolytiques, à une acidocétose ou à des infections génito-urinaires n'a été observé au cours des 18 mois de suivi. LIMITES: Échantillon de petite taille, absence d'un groupe de comparaison et synchronisation variable de la collecte des données cliniques, notamment du DFGe, après le début de l'inhibition du SGLT2. CONCLUSION: L'inhibition du SGLT2 devient une partie intégrante des soins néphrologiques visant à réduire le déclin de la fonction rénale, les risques cardiovasculaires et la mortalité. À notre connaissance, notre rapport est le premier à fournir des données longitudinales sur l'utilisation des inhibiteurs du SGLT2 chez les patients atteints de diabète de type 2 ayant subi une néphrectomie. Des études prospectives de plus grande envergure sont nécessaires pour examiner l'efficacité et l'innocuité des stratégies d'inhibition du SGLT2 visant la protection rénale des patients post-néphrectomie.

Transcriptome Analysis of Kidney Grafts Subjected to Normothermic Ex Vivo Perfusion Demonstrates an Enrichment of Mitochondrial Metabolism Genes.

Normothermic ex vivo kidney perfusion (NEVKP) has demonstrated superior outcomes for donation-after-cardiovascular death grafts compared with static cold storage (SCS). To determine the mechanisms responsible for this, we performed an unbiased genome-wide microarray analysis. METHODS: Kidneys from 30-kg Yorkshire pigs were subjected to 30 min of warm ischemia followed by 8 h of NEVKP or SCS, or no storage, before autotransplantation. mRNA expression was analyzed on renal biopsies on postoperative day 3. Gene set enrichment analysis was performed using hallmark gene sets, Gene Ontology, and pathway analysis. RESULTS: The gene expression profile of NEVKP-stored grafts closely resembled no storage kidneys. Gene set enrichment analysis demonstrated enrichment of fatty acid metabolism and oxidative phosphorylation following NEVKP, whereas SCS-enriched gene sets were related to mitosis, cell cycle checkpoint, and reactive oxygen species (q < 0.05). Pathway analysis demonstrated enrichment of lipid oxidation/metabolism, the Krebs cycle, and pyruvate metabolism in NEVKP compared with SCS (q < 0.05). Comparison of our findings with external data sets of renal ischemia-reperfusion injury revealed that SCS-stored grafts demonstrated similar gene expression profiles to ischemia-reperfusion injury, whereas the profile of NEVKP-stored grafts resembled recovered kidneys. CONCLUSIONS: Increased transcripts of key mitochondrial metabolic pathways following NEVKP storage may account for improved donation-after-cardiovascular death graft function, compared with SCS, which promoted expression of genes typically perturbed during IRI.

The neurorepellent, Slit2, prevents macrophage lipid loading by inhibiting CD36-dependent binding and internalization of oxidized low-density lipoprotein.

Atherosclerosis is characterized by retention of modified lipoproteins, especially oxidized low density lipoprotein (oxLDL) within the sub-endothelial space of affected blood vessels. Recruited monocyte-derived and tissue-resident macrophages subsequently ingest oxLDL by binding and internalizing oxLDL via scavenger receptors, particularly CD36. The secreted neurorepellent, Slit2, acting through its transmembrane receptor, Roundabout-1 (Robo-1), was previously shown to inhibit recruitment of monocytes into nascent atherosclerotic lesions. The effects of Slit2 on oxLDL uptake by macrophages have not been explored. We report here that Slit2 inhibits uptake of oxLDL by human and murine macrophages, and the resulting formation of foam cells, in a Rac1-dependent and CD36-dependent manner. Exposure of macrophages to Slit2 prevented binding of oxLDL to the surface of cells. Using super-resolution microscopy, we observed that exposure of macrophages to Slit2 induced profound cytoskeletal remodeling with formation of a thick ring of cortical actin within which clusters of CD36 could not aggregate, thereby attenuating binding of oxLDL to the surface of cells. By inhibiting recruitment of monocytes into early atherosclerotic lesions, and the subsequent binding and internalization of oxLDL by macrophages, Slit2 could represent a potent new tool to combat individual steps that collectively result in progression of atherosclerosis.

Improving Sexual Function in People With Chronic Kidney Disease: A Narrative Review of an Unmet Need in Nephrology Research.

PURPOSE OF REVIEW: Sexual dysfunction occurs commonly in people with chronic kidney disease (CKD) and has been recognized as a research priority. We sought to evaluate the current state of the literature addressing sexual dysfunction in people with CKD and identify barriers and strategies to improve our management of this important symptom. SOURCES OF INFORMATION: OVID Medline and Google Scholar were searched for English, peer-reviewed studies using keywords and terms related to "Chronic Kidney Disease," "sexuality," and "sexual dysfunction OR function." METHODS: In this narrative review, we describe definitions of sexual dysfunction and contributors exacerbated by CKD, barriers to researching sexual dysfunction in people with CKD, and possible avenues for future research. KEY FINDINGS: Sexual dysfunction is common in people with CKD and results from a combination of kidney disease itself, as well as its associated physical (ie, comorbidities) and nonphysical factors. Barriers to the study of sexual dysfunction in CKD include inconsistent disease definitions, stigma, variable efficacy and safety of established therapies, and evolving gender roles in sexual function. Potential avenues for future research to improve the sexual function in people with CKD may include evaluating the safety and efficacy of established therapies in people with CKD using a variety of observational and interventional study designs, engaging people with CKD and multidisciplinary team members in research, and using implementation science methods to translate what is known about sexual function into clinical practice. Concerted efforts are required to break down barriers and improve sexual function in people with CKD. Patients have identified this as an important research priority, and national networks need to direct efforts to reduce symptom burden. LIMITATIONS: This narrative review was limited by a paucity of high-quality studies examining sexual dysfunction specifically in people with kidney disease.

CONTEXTE MOTIVANT LA REVUE: Les dysfonctions sexuelles sont fréquentes chez les personnes atteintes d'insuffisance rénale chronique (IRC) et ont été reconnues comme une priorité de recherche. Cette revue avait pour objectif d'évaluer la documentation actuelle traitant du dysfonctionnement sexuel chez les personnes atteintes d'IRC et de répertorier les obstacles à la recherche dans ce domaine et les stratégies qui permettent d'améliorer la gestion de cet important symptôme. SOURCES: Les bases de données OVID Medline et Google Scholar ont été consultées à la recherche des études rédigées en anglais et évaluées par des pairs à l'aide de mots-clés et de termes liés à Chronic Kidney Disease (insuffisance rénale chronique), sexuality (sexualité) et sexual dysfunction OR function (dysfonction OU fonction sexuelle). MÉTHODOLOGIE: Cette revue narrative présente les définitions de la dysfonction sexuelle et les contributeurs exacerbés par l'IRC. Elle décrit également les obstacles limitant la recherche sur le dysfonctionnement sexuel chez les personnes atteintes d'IRC et cerne de possibles pistes pour les recherches futures. PRINCIPAUX RÉSULTATS: Le dysfonctionnement sexuel est fréquent chez les personnes atteintes d'IRC et résulte d'une combinaison de la néphropathie elle-même et de facteurs physiques (c.-à-d. les maladies concomitantes) et non physiques qui y sont associés. Les obstacles limitant l'étude de la dysfonction sexuelle en contexte d'IRC comprennent: 1) l'incohérence dans les différentes définitions de maladie; 2) la stigmatization; 3) l'efficacité et l'innocuité variables des thérapies existantes; et 4) le caractère évolutif des rôles des genres dans la fonction sexuelle. Les possibles axes de recherche pour les études futures pourraient inclure: 1) l'utilization d'une variété de modèles d'études observationnelles et interventionnelles pour évaluer l'innocuité et l'efficacité des thérapies existantes dans cette population de patients; 2) la participation de patients et de membres de l'équipe multidisciplinaire à la recherche, et 3) l'utilization de méthodes scientifiques de mise en œuvre afin de traduire les connaissances dans la pratique clinique. Des efforts concertés sont nécessaires pour éliminer les obstacles à la recherche et améliorer la fonction sexuelle des personnes atteintes d'IRC. Il s'agit d'une importante priorité de recherche pour les patients, et les réseaux nationaux se doivent d'orienter leurs efforts vers la réduction du fardeau des symptômes. LIMITES: Cette revue narrative était limitée par le manque d'études de haute qualité examinant spécifiquement la dysfonction sexuelle chez les personnes atteintes de néphropathies.

Hyperfiltration, urinary albumin excretion, and ambulatory blood pressure in adolescents with Type 1 diabetes mellitus.

Adolescents with Type 1 diabetes mellitus (T1DM) are at risk for hyperfiltration and elevated urinary albumin-to-creatinine ratio (ACR), which are early indicators of diabetic nephropathy. Adolescents with T1DM also develop early changes in blood pressure, cardiovascular structure, and function. Our aims were to define the relationships between hyperfiltration, ACR, and 24-h ambulatory blood pressure over time in adolescents with T1DM. Normotensive, normoalbuminuric adolescents ( n = 98) with T1DM underwent baseline and 2-yr 24-h ambulatory blood pressure monitoring, glomerular filtration rate (eGFR) estimated by cystatin C (Larsson equation), and ACR measurements. Linear regression models adjusted for diabetes duration, sex, and HbA1c were used to determine associations. Hyperfiltration (eGFR ≥ 133 ml/min) was present in 31% at baseline and 21% at 2-yr follow-up. Hyperfiltration was associated with greater odds of rapid GFR decline (>3 ml·min-1·yr-1) [OR: 5.33, 95%; CI: 1.87-15.17; P = 0.002] over 2 yr. Natural log of ACR at baseline was associated with greater odds of hyperfiltration (OR: 1.71, 95% CI: 1.00-2.92; P = 0.049) and 2-yr follow-up (OR: 2.14, 95%; CI: 1.09-4.19; P = 0.03). One SD increase in eGFR, but not ln ACR, at 2-yr follow-up conferred greater odds of nighttime nondipping pattern (OR: 1.96, 95% CI: 1.06-3.63; P = 0.03). Hyperfiltration was prevalent at baseline and at 2-yr follow-up, predicted rapid decline in GFR, and was related to ACR. Elevated GFR at 2-yr follow-up was associated with nighttime nondipping pattern. More work is needed to better understand early relationships between renal hemodynamic and systemic hemodynamic changes in adolescents with T1DM to reduce future cardiorenal complications.

Influence of sex on hyperfiltration in patients with uncomplicated type 1 diabetes.

The aim of this analysis was to examine sex-based differences in renal segmental resistances in healthy controls (HCs) and patients with type 1 diabetes (T1D). We hypothesized that hyperfiltration-an early hemodynamic abnormality associated with diabetic nephropathy-would disproportionately affect women with T1D, thereby attenuating protection against the development of renal complications. Glomerular hemodynamic parameters were evaluated in HC (n = 30) and in normotensive, normoalbuminuric patients with T1D and either baseline normofiltration [n = 36, T1D-N, glomerular filtration rate (GFR) 90-134 ml·min-1·1.73 m2] or hyperfiltration (n = 32, T1D-H, GFR ≥ 135 ml·min-1·1.73 m2) during euglycemic conditions (4-6 mmol/l). Gomez's equations were used to derive efferent (RE) and afferent (RA) arteriolar resistances, glomerular hydrostatic pressure (PGLO) from inulin (GFR) and paraaminohippurate [effective renal plasma flow (ERPF)] clearances, plasma protein and estimated ultrafiltration coefficients (KFG). Female patients with T1D with hyperfiltration (T1D-H) had higher RE (1,985 ± 487 vs. 1,381 ± 296 dyne·sec-1·cm-5, P < 0.001) and filtration fraction (FF, 0.20 ± 0.047 vs. 0.16 ± 0.03 P < 0.05) and lower ERPF (876 ± 245 vs. 1,111 ± 298 134 ml·min-1·1.73 m2P < 0.05) compared with male T1D-H patients. Overall, T1D-H patients had higher PGLO and lower RA vs. HC subjects, although there were no sex-based differences. In conclusion, female T1D-H patients had higher RE and FF and lower ERPF than their male counterparts with no associated sex differences in RA Prospective intervention studies should consider sex as a modifier of renal hemodynamic responses to renal protective therapies.

The Gomez' equations and renal hemodynamic function in kidney disease research.

Diabetic kidney disease (DKD) remains the leading cause of end-stage renal disease. A major challenge in preventing DKD is the difficulty in identifying high-risk patients at an early, pre-clinical stage. Albuminuria and eGFR as measures of renal function in DKD research and clinical practice are limited by regression of one-third of patients with microalbuminuria to normoalbuminuria and eGFR is biased and imprecise in the normal-elevated range. Moreover, existing methods that are used to assess renal function do not give detailed insight into the location of the renal hemodynamic effects of pharmacological agents at the segmental level. To gain additional information about the intrarenal circulation in-vivo in humans, mathematical equations were developed by Gomez et al in the 1950s. These equations used measurements of GFR, renal blood flow (RBF), effective renal plasma flow (ERPF), renal vascular resistance (RVR), hematocrit and serum protein to calculate afferent and efferent arteriolar resistances, glomerular hydrostatic pressure and filtration pressure. Although indirect and based on physiological assumptions, these techniques have the potential to improve researchers' ability to identify early pre-clinical changes in renal hemodynamic function in patients with a variety of conditions including DKD, thereby offering tremendous potential in mechanistic human research studies. In this review, we focus on the application of Gomez' equations and summarize the potential and limitations of this technique in DKD research. We also summarize illustrative data derived from Gomez' equations in patients with type 1 (T1D) and type 2 diabetes (T2D) and hypertension.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

Sodium-glucose cotransporter-2 inhibition and the potential for renal protection in diabetic nephropathy.

PURPOSE OF REVIEW: Renal hyperfiltration has been used as a surrogate marker for increased intraglomerular pressure in patients with diabetes mellitus. Previous human investigation examining the pathogenesis of hyperfiltration has focused on the role of neurohormones such as the renin-angiotensin-aldosterone system (RAAS). Unfortunately, RAAS blockade does not completely attenuate hyperfiltration or diabetic kidney injury. More recent work has therefore investigated the contribution of renal tubular factors, including the sodium-glucose cotransporter, to the hyperfiltration state, which is the topic of this review. RECENT FINDINGS: Novel sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce proximal tubular sodium reabsorption, thereby increasing distal sodium delivery to the macula densa, causing tubuloglomerular feedback, afferent vasoconstriction and decreased hyperfiltration in animals. In humans, SGLT2 inhibition was recently shown to reduce hyperfiltration in normotensive, normoalbuminuric patients with type 1 diabetes. In clinical trials of type 2 diabetes, SGLT2 is associated with significant renal effects, including modest, acute declines in estimated glomerular filtration rate followed by the maintenance of stable renal function, and reduced albuminuria. SUMMARY: Existing data are supportive of a potential renal-protective role for SGLT2 inhibition in patients with diabetes. Dedicated renal outcome trials are ongoing and have the potential to change the clinical practice.

Glycosuria-mediated urinary uric acid excretion in patients with uncomplicated type 1 diabetes mellitus.

Plasma uric acid (PUA) is associated with metabolic, cardiovascular, and renal abnormalities in patients with type 2 diabetes but is less well understood in type 1 diabetes (T1D). Our aim was to compare PUA levels and fractional uric acid excretion (FEUA) in patients with T1D vs. healthy controls (HC) during euglycemia and hyperglycemia. PUA, FEUA, blood pressure (BP), glomerular filtration rate (GFR-inulin), and effective renal plasma flow (ERPF-paraaminohippurate) were evaluated in patients with T1D (n = 66) during clamped euglycemia (glucose 4-6 mmol/l) and hyperglycemia (9-11 mmol/l), and in HC (n = 41) during euglycemia. To separate the effects of hyperglycemia vs. increased glycosuria, parameters were evaluated during clamped euglycemia in a subset of T1D patients before and after sodium glucose cotransporter 2 (SGLT2) inhibition for 8 wk. PUA was lower in T1D vs. HC (228 ± 62 vs. 305 ± 75 µmol/l, P < 0.0001). In T1D, hyperglycemia further decreased PUA (228 ± 62 to 199 ± 65 µmol/l, P < 0.0001), which was accompanied by an increase in FEUA (7.3 ± 3.8 to 11.6 ± 6.7, P < 0.0001). In T1D, PUA levels correlated positively with SBP (P = 0.029) and negatively with ERPF (P = 0.031) and GFR (P = 0.028). After induction of glycosuria with SGLT2 inhibition while maintaining clamped euglycemia, PUA decreased (P < 0.0001) and FEUA increased (P < 0.0001). PUA is lower in T1D vs. HC and positively correlates with SBP and negatively with GFR and ERPF in T1D. Glycosuria rather than hyperglycemia increases uricosuria in T1D. Future studies examining the effect of uric acid-lowering therapies should account for the impact of ambient glycemia, which causes an important uricosuric effect.

Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1ß transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors.

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia.

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma.

The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.

Bacteria challenge in smoke-exposed mice exacerbates inflammation and skews the inflammatory profile.

RATIONALE: The pathogenesis of chronic obstructive pulmonary disease is associated with acute episodes of bacterial exacerbations. The most commonly isolated bacteria during episodes of exacerbation is nontypeable Haemophilus influenzae (NTHI). OBJECTIVES: In this study, we investigated the in vivo consequences of cigarette smoke exposure on the inflammatory response to an NTHI challenge. METHODS: C57BL/6 and BALB/c mice were exposed to cigarette smoke for 8 weeks and subsequently challenged intranasally with NTHI. MEASUREMENTS AND MAIN RESULTS: We observed increased pulmonary inflammation and lung damage in cigarette smoke-exposed NTHI-challenged mice as compared with control NTHI-challenged mice. Furthermore, although NTHI challenge in control mice was marked by increases in tumor necrosis factor-alpha, IL-6, MIP-2, and KC/GROalpha, NTHI challenge in cigarette smoke-exposed mice led to a prominent up-regulation of a different subset of inflammatory mediators, most notably MCP-1, -3, and -5, IP-10, and MIP-1gamma. This skewed inflammatory mediator expression was also observed after ex vivo NTHI stimulation of alveolar macrophages, signifying their importance to this altered response. Importantly, corticosteroids attenuated inflammation after NTHI challenge in both cigarette smoke-exposed and control mice; however, this was associated with significantly increased bacterial burden. CONCLUSIONS: Collectively, these data suggest that cigarette smoke exacerbates the inflammatory response to a bacterial challenge via skewed inflammatory mediator expression.

Cigarette smoke exposure attenuates cytokine production by mouse alveolar macrophages.

Alveolar macrophages (aMs) play a central role in respiratory host defense by sensing microbial antigens and initiating immune-inflammatory responses early in the course of an infection. The purpose of this study was to investigate the effect of cigarette smoke exposure on aMs after stimulation of innate pattern recognition receptors (PRRs) in a murine model. To accomplish this, C57BL/6 mice were exposed for 8 weeks using two models of cigarette smoke exposure, nose-only or whole-body exposure, and aMs isolated from the bronchoalveolar lavage. After stimulation of aMs with pI:C, a mimic of viral replication, and bacterial cell-wall constituent LPS, aMs from cigarette smoke-exposed mice produced significantly attenuated levels of the inflammatory cytokines TNF-alpha and IL-6, and the chemokine RANTES. This attenuation was specific to the aM compartment, and not related to changes in aM viability or expression of Toll-like receptor (TLR)3 or TLR4 between groups. Furthermore, aMs from smoke-exposed mice had decreased cytokine RNA as compared with aMs from sham-exposed mice. Mechanistically, this was associated with decreased nuclear translocation of the proinflammatory transcription factor NF-kappaB, and increased activator protein-1 nuclear translocation, in aMs from smoke-exposed mice. Attenuated cytokine production was reversible after smoking cessation. Cigarette smoke exposure also attenuated TNF-alpha production after stimulation with nucleotide-oligomerization domain-like receptor agonists, showing that the effect applies more broadly to other PRR pathways. Our data demonstrate that cigarette smoke exposure attenuates aM responses after innate stimulation, including pathways typically associated with bacterial and viral infections.