HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

FGF21 and autophagy coordinately counteract kidney disease progression during aging and obesity.

Chronic kidney disease (CKD) has reached epidemic proportions worldwide, partly due to the increasing population of elderly and obesity. Macroautophagy/autophagy counteracts CKD progression, whereas autophagy is stagnated owing to lysosomal overburden during aging and obesity, which promotes CKD progression. Therefore, for preventing CKD progression during aging and obesity, it is important to elucidate the compensation mechanisms of autophagy stagnation. We recently showed that FGF21 (fibroblast growth factor 21), which is a prolongevity and metabolic hormone, is induced by autophagy deficiency in kidney proximal tubular epithelial cells (PTECs); however, its pathophysiological role remains uncertain. Here, we investigated the interplay between FGF21 and autophagy and the direct contribution of endogenous FGF21 in the kidney during aging and obesity using PTEC-specific fgf21- and/or atg5-deficient mice at 24 months (aged) or under high-fat diet (obese) conditions. PTEC-specific FGF21 deficiency in young mice increased autophagic flux due to increased demand of autophagy, whereas fgf21-deficient aged or obese mice exacerbated autophagy stagnation due to severer lysosomal overburden caused by aberrant autophagy. FGF21 was robustly induced by autophagy deficiency, and aged or obese PTEC-specific fgf21- and atg5-double deficient mice deteriorated renal histology compared with atg5-deficient mice. Mitochondrial function was severely disturbed concomitant with exacerbated oxidative stress and downregulated TFAM (transcription factor A, mitochondrial) in double-deficient mice. These results indicate that FGF21 is robustly induced by autophagy disturbance and protects against CKD progression during aging and obesity by alleviating autophagy stagnation and maintaining mitochondrial homeostasis, which will pave the way to a novel treatment for CKD.

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.

Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.

Autophagy and kidney aging.

Autophagy is a highly conserved intracellular degradation system in eukaryotes that maintains cellular and tissue homeostasis. Upon autophagy induction, cytoplasmic components are engulfed by a double-membrane organelle called the autophagosome that fuses with a lysosome to degrade its contents. In recent years, it has become clear that autophagy becomes dysregulated with aging, which leads to age-related diseases. Kidney function is particularly prone to age-related decline, and aging is the most significant risk factor for chronic kidney disease. This review first discuss the relationship between autophagy and kidney aging. Second, we describe how age-related dysregulation of autophagy occurs. Finally, we discuss the potential of autophagy-targeting drugs to ameliorate human kidney aging and the approaches necessary to discover such agents.

TFEB-mediated lysosomal exocytosis alleviates high-fat diet-induced lipotoxicity in the kidney.

Obesity is a major risk factor for end-stage kidney disease. We previously found that lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in obesity-related kidney disease, in both humans and experimental animal models. However, the regulatory factors involved in countering renal lipotoxicity are largely unknown. Here, we found that palmitic acid strongly promoted dephosphorylation and nuclear translocation of transcription factor EB (TFEB) by inhibiting the mechanistic target of rapamycin kinase complex 1 pathway in a Rag GTPase-dependent manner, though these effects gradually diminished after extended treatment. We then investigated the role of TFEB in the pathogenesis of obesity-related kidney disease. Proximal tubular epithelial cell-specific (PTEC-specific) Tfeb-deficient mice fed a high-fat diet (HFD) exhibited greater phospholipid accumulation in enlarged lysosomes, which manifested as multilamellar bodies (MLBs). Activated TFEB mediated lysosomal exocytosis of phospholipids, which helped reduce MLB accumulation in PTECs. Furthermore, HFD-fed, PTEC-specific Tfeb-deficient mice showed autophagic stagnation and exacerbated injury upon renal ischemia/reperfusion. Finally, higher body mass index was associated with increased vacuolation and decreased nuclear TFEB in the proximal tubules of patients with chronic kidney disease. These results indicate a critical role of TFEB-mediated lysosomal exocytosis in counteracting renal lipotoxicity.

Age-associated decline of MondoA drives cellular senescence through impaired autophagy and mitochondrial homeostasis.

Accumulation of senescent cells affects organismal aging and the prevalence of age-associated disease. Emerging evidence suggests that activation of autophagy protects against age-associated diseases and promotes longevity, but the roles and regulatory mechanisms of autophagy in cellular senescence are not well understood. Here, we identify the transcription factor, MondoA, as a regulator of cellular senescence, autophagy, and mitochondrial homeostasis. MondoA protects against cellular senescence by activating autophagy partly through the suppression of an autophagy-negative regulator, Rubicon. In addition, we identify peroxiredoxin 3 (Prdx3) as another downstream regulator of MondoA essential for mitochondrial homeostasis and autophagy. Rubicon and Prdx3 work independently to regulate senescence. Furthermore, we find that MondoA knockout mice have exacerbated senescence during ischemic acute kidney injury (AKI), and a decrease of MondoA in the nucleus is correlated with human aging and ischemic AKI. Our results suggest that decline of MondoA worsens senescence and age-associated disease.

Therapeutic potential of Beclin1 for transition from AKI to CKD: autophagy-dependent and autophagy-independent functions.

Acute kidney injury (AKI) increases the risk of chronic kidney disease (CKD), but the mechanisms of CKD development after AKI remain unclear. Recent studies have elucidated that autophagy protects against AKI, but the role of autophagy during the AKI-to-CKD transition is controversial. Beclin1 is a key molecule for autophagy as well as endocytosis and phagocytosis. Shi et al. demonstrate that Beclin1 activates autophagy and is a promising therapeutic target for AKI-to-CKD transition.

Elevated blood favipiravir levels are inversely associated with ferritin levels and induce the elevation of uric acid levels in COVID-19 treatment: A retrospective single-center study.

INTRODUCTION: Measurement of blood Favipiravir (FPV) levels and accumulation of data in COVID-19 patients are critical for assessing FPV efficacy and safety. We performed a retrospective study based on measurements of blood levels of FPV and related factors in COVID-19 patients admitted to our hospital. Furthermore, we also investigated the association between blood FPV levels and uric acid level alterations before and after FPV administration. METHODS: We enrolled 27 COVID-19 patients who had received FPV treatment at Hokushin General Hospital from April 1 to December 31, 2020. Age, gender, COVID-19 severity, presence of comorbidities, and laboratory data for each subject were investigated to identify factors that correlate with blood FPV levels. Uric acid levels were measured before and after FPV administration and a difference between the levels (i.e., a change of uric acid level) was evaluated. RESULTS: When a significant univariate variable was input by the stepwise method and a combination of variables that maintained statistical superiority was searched, serum ferritin was the only factor that independently affected blood FPV level. Furthermore, in the high-FPV group (20 µg/mL or more), a significant increase in uric acid levels was observed after FPV administration. The increment value was significantly larger than that in the low-FPV group (less than 20 µg/mL). CONCLUSIONS: Ferritin level was an important independent factor inversely affecting blood FPV level. Furthermore, a high blood FPV level induced the elevation of uric acid levels in COVID-19 treatment.

THOC4 regulates energy homeostasis by stabilizing TFEB mRNA during prolonged starvation.

TFEB, a basic helix-loop-helix transcription factor, is a master regulator of autophagy, lysosome biogenesis and lipid catabolism. Compared to posttranslational regulation of TFEB, the regulation of TFEB mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to TFEB mRNA and stabilized it after transcription by maintaining poly(A) tail length. We further found that this mode of regulation was conserved in Caenorhabditiselegans and was essential for TFEB-mediated lipid breakdown, which becomes over-represented during prolonged starvation. Taken together, our findings reveal the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function of TFEB in mediating autophagy and lipid metabolism.

Rubicon in Metabolic Diseases and Ageing.

Autophagy is a conserved cellular degradation system that maintains intracellular homeostasis. Cytoplasmic components are engulfed into double-membrane vesicles called autophagosomes, which fuse with lysosomes, and resulting in the degradation of sequestered materials. Recently, a close association between autophagy and the pathogenesis of metabolic diseases and ageing has become apparent: autophagy is dysregulated during metabolic diseases and ageing; dysregulation of autophagy is intimately associated with the pathophysiology. Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein) has been identified as a Beclin-1 associated protein. Notably, Rubicon is one of few negative regulators of autophagy whereas many autophagy-related genes are positive regulators of autophagy. Rubicon also has autophagy-independent functions including phagocytosis and endocytosis. In this mini-review, we focus on the various roles of Rubicon in different organs in the settings of metabolic diseases and ageing, and discuss its potential role as a promising therapeutic target.

Eicosapentaenoic acid attenuates renal lipotoxicity by restoring autophagic flux.

Recently, we identified a novel mechanism of lipotoxicity in the kidney proximal tubular cells (PTECs); lipid overload stimulates macroautophagy/autophagy for the renovation of plasma and organelle membranes to maintain the integrity of the PTECs. However, this autophagic activation places a burden on the lysosomal system, leading to a downstream suppression of autophagy, which manifests as phospholipid accumulation and inadequate acidification in lysosomes. Here, we investigated whether pharmacological correction by eicosapentaenoic acid (EPA) supplementation could restore autophagic flux and alleviate renal lipotoxicity. EPA supplementation to high-fat diet (HFD)-fed mice reduced several hallmarks of lipotoxicity in the PTECs, such as phospholipid accumulation in the lysosome, mitochondrial dysfunction, inflammation, and fibrosis. In addition to improving the metabolic syndrome, EPA alleviated renal lipotoxicity via several mechanisms. EPA supplementation to HFD-fed mice or the isolated PTECs cultured in palmitic acid (PA) restored lysosomal function with significant improvements in the autophagic flux. The PA-induced redistribution of phospholipids from cellular membranes into lysosomes and the HFD-induced accumulation of SQSTM1/p62 (sequestosome 1), an autophagy substrate, during the temporal and genetic ablation of autophagy were significantly reduced by EPA, indicating that EPA attenuated the HFD-mediated increases in autophagy demand. Moreover, a fatty acid pulse-chase assay revealed that EPA promoted lipid droplet (LD) formation and transfer from LDs to the mitochondria for beta-oxidation. Noteworthy, the efficacy of EPA on lipotoxicity is autophagy-dependent and cell-intrinsic. In conclusion, EPA counteracts lipotoxicity in the proximal tubule by alleviating autophagic numbness, making it potentially suitable as a novel treatment for obesity-related kidney diseases.Abbreviations: 4-HNE: 4-hydroxy-2-nonenal; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; ATG: autophagy-related; ATP: adenosine triphosphate; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; cKO: conditional knockout; CML: N-carboxymethyllysine; COL1A1: collagen type I alpha 1 chain; COX: cytochrome c oxidase; CTRL: control; DGAT: diacylglycerol O-acyltransferase; EPA: eicosapentaenoic acid; FA: fatty acid; FFA: free fatty acid; GFP: green fluorescent protein; HFD: high-fat diet; iKO: inducible knockout; IRI: ischemia-reperfusion injury; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor-related protein 2; MAP1LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleic acid; PAS: periodic-acid Schiff; PPAR: peroxisome proliferator activated receptor; PPARGC1/PGC1: peroxisome proliferator activated receptor, gamma, coactivator 1; PTEC: proximal tubular epithelial cell; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SDH: succinate dehydrogenase complex; SFC/MS/MS: supercritical fluid chromatography triple quadrupole mass spectrometry; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Urinary mulberry bodies as a potential biomarker for early diagnosis and efficacy assessment of enzyme replacement therapy in Fabry nephropathy.

BACKGROUND: The inability of enzyme replacement therapy (ERT) to prevent progression of Fabry nephropathy (FN) in the presence of >1 g/day proteinuria underscores the necessity of identifying effective biomarkers for early diagnosis of FN preceding proteinuria. Here we attempted to identify biomarkers for early detection of FN. METHODS: Fifty-one Fabry disease (FD) patients were enrolled. Urinary mulberry bodies (uMBs) were immunostained for globotriaosylceramide (Gb3) and renal cell markers to determine their origin. The association between semiquantitative uMB excretion and the histological severity of podocyte vacuolation was investigated in seven patients using the vacuolated podocyte:glomerular average area ratio. The association between the semiquantitative estimate of uMB excretion and duration of ERT was analyzed. A longitudinal study was conducted to assess the effect of ERT on uMB excretion. RESULTS: Thirty-two patients (63%) had uMBs, while only 31% showed proteinuria. The uMBs were positive for Gb3, lysosomal-associated membrane protein 1 and podocalyxin, suggesting they were derived from lysosomes with Gb3 accumulation in podocytes. We observed more severe podocyte vacuolation with increased uMB excretion (P = 0.03 for trend); however, the same was not observed with increased proteinuria. The percentage of patients with substantial uMB excretion increased with shorter ERT duration (P = 0.018). Eighteen-month-long ERT reduced uMB excretion (P = 0.03) without affecting proteinuria. CONCLUSIONS: uMB excretion, implying ongoing podocyte injury, preceded proteinuria in most patients. Semiquantitative uMB estimates can serve as novel biomarkers for early FN diagnosis and for monitoring the efficacy of FD-specific therapies.

Increased vascular permeability and severe renal tubular damage after ischemia-reperfusion injury in mice lacking adiponectin or T-cadherin.

Adiponectin (APN) is a circulating protein specifically produced by adipocytes. Native APN specifically binds to T-cadherin, a glycosylphosphatidylinositol-anchored protein, mediating the exosome-stimulating effects of APN in endothelial, muscle, and mesenchymal stem cells. It was previously reported that APN has beneficial effects on kidney diseases, but the role of T-cadherin has not been clarified yet. Here, our immunofluorescence study indicated the existence of both T-cadherin and APN protein in pericytes, subsets of tissue-resident mesenchymal stem/progenitor cells positive for platelet-derived growth factor receptor ß (PDGFRß), surrounding peritubular capillaries. In an acute renal ischemia-reperfusion (I/R) model, T-cadherin-knockout (Tcad-KO) mice, similar to APN-KO mice, exhibited the more progressive phenotype of renal tubular damage and increased vascular permeability than wild-type mice. In addition, in response to I/R-injury, the renal PDGFRß-positive cell area increased in wild-type mice, but opposingly decreased in both Tcad-KO and APN-KO mice, suggesting severe pericyte loss. Mouse primary pericytes also expressed T-cadherin. APN promoted exosome secretion in a T-cadherin-dependent manner. Such exosome production from pericytes may play an important role in maintaining the capillary network and APN-mediated inhibition of renal tubular injury. In summary, our study suggested that APN protected the kidney in an acute renal injury model by binding to T-cadherin.NEW & NOTEWORTHY In the kidney, T-cadherin-associated adiponectin protein existed on peritubular capillary pericytes. In an acute renal ischemia-reperfusion model, deficiency of adiponectin or T-cadherin exhibited the more progressive phenotype of renal tubular damage and increased vascular permeability, accompanied by severe pericyte loss. In vitro, adiponectin promoted exosome secretion from mouse primary pericytes in a T-cadherin-dependent manner. Adiponectin plays an important role in maintaining the capillary network and amelioration of renal tubular injury by binding to T-cadherin.

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.

Autophagy protects kidney from phosphate-induced mitochondrial injury.

Hyperphosphatemia is a common complication in patients with advanced chronic kidney disease (CKD) as well as an increased risk of cardiovascular mortality; however, the molecular mechanisms of phosphate-mediated kidney injury are largely unknown. Autophagy is a lysosomal degradation system, which plays protective roles against kidney diseases. Here, we studied the role of autophagy in kidney proximal tubular cells (PTECs) during phosphate overload. Temporal cessation of autophagy in drug-induced PTEC-specific autophagy-deficient mice that were fed high phosphate diet induced mild cytosolic swelling and an accumulation of SQSTM1/p62-and ubiquitin-positive protein aggregates in PTECs, indicating that phosphate overload requires enhanced autophagic activity for the degradation of increasing substrate. Morphological and biochemical analysis demonstrated that high phosphate activates mitophagy in PTECs in response to oxidative stress. PTEC-specific autophagy-deficient mice receiving heminephrectomy and autophagy-deficient cultured PTECs exhibited mitochondrial dysfunction, increased reactive oxygen species production, and reduced ATP production in response to phosphate overload, suggesting that high phosphate-induced autophagy counteracts mitochondrial injury and maintains cellular bioenergetics in PTECs. Thus, potentiating autophagic activity could be a therapeutic option for suppressing CKD progression during phosphate overload.

Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells.

Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue.

Proximal Tubule Autophagy Differs in Type 1 and 2 Diabetes.

BACKGROUND: Evidence of a protective role of autophagy in kidney diseases has sparked interest in autophagy as a potential therapeutic strategy. However, understanding how the autophagic process is altered in each disorder is critically important in working toward therapeutic applications. METHODS: Using cultured kidney proximal tubule epithelial cells (PTECs) and diabetic mouse models, we investigated how autophagic activity differs in type 1 versus type 2 diabetic nephropathy. We explored nutrient signals regulating starvation-induced autophagy in PTECs and used autophagy-monitoring mice and PTEC-specific autophagy-deficient knockout mice to examine differences in autophagy status and autophagy's role in PTECs in streptozotocin (STZ)-treated type 1 and db/db type 2 diabetic nephropathy. We also examined the effects of rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]) on vulnerability to ischemia-reperfusion injury. RESULTS: Administering insulin or amino acids, but not glucose, suppressed autophagy by activating mTOR signaling. In db/db mice, autophagy induction was suppressed even under starvation; in STZ-treated mice, autophagy was enhanced even under fed conditions but stagnated under starvation due to lysosomal stress. Using knockout mice with diabetes, we found that, in STZ-treated mice, activated autophagy counteracts mitochondrial damage and fibrosis in the kidneys, whereas in db/db mice, autophagic suppression jeopardizes kidney even in the autophagy-competent state. Rapamycin-induced pharmacologic autophagy produced opposite effects on ischemia-reperfusion injury in STZ-treated and db/db mice. CONCLUSIONS: Autophagic activity in PTECs is mainly regulated by insulin. Consequently, autophagic activity differs in types 1 and 2 diabetic nephropathy, which should be considered when developing strategies to treat diabetic nephropathy by modulating autophagy.

Suppression of autophagic activity by Rubicon is a signature of aging.

Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging.

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries.

Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.