Podocytes maintain high basal levels of autophagy independent of mTOR signaling

While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases

Role of dietary amino acid balance in diet restriction-mediated lifespan extension, renoprotection, and muscle weakness in aged mice.

Extending healthy lifespan is an emerging issue in an aging society. This study was designed to identify a dietary method of extending lifespan, promoting renoprotection, and preventing muscle weakness in aged mice, with a focus on the importance of the balance between dietary essential (EAAs) and nonessential amino acids (NEAAs) on the dietary restriction (DR)-induced antiaging effect. Groups of aged mice were fed ad libitum, a simple DR, or a DR with recovering NEAAs or EAAs. Simple DR significantly extended lifespan and ameliorated age-related kidney injury; however, the beneficial effects of DR were canceled by recovering dietary EAA but not NEAA. Simple DR prevented the age-dependent decrease in slow-twitch muscle fiber function but reduced absolute fast-twitch muscle fiber function. DR-induced fast-twitch muscle fiber dysfunction was improved by recovering either dietary NEAAs or EAAs. In the ad libitum-fed and the DR plus EAA groups, the renal content of methionine, an EAA, was significantly higher, accompanied by lower renal production of hydrogen sulfide (H2 S), an endogenous antioxidant. Finally, removal of methionine from the dietary EAA supplement diminished the adverse effects of dietary EAA on lifespan and kidney injury in the diet-restricted aged mice, which were accompanied by a recovery in H2 S production capacity and lower oxidative stress. These data imply that a dietary approach could combat kidney aging and prolong lifespan, while preventing muscle weakness, and suggest that renal methionine metabolism and the trans-sulfuration pathway could be therapeutic targets for preventing kidney aging and subsequently promoting healthy aging

Protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes.

To examine the cell-protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes, we analyzed the renal phenotype of tamoxifen (TM)-inducible podocyte-specific Atg5-deficient (iPodo-Atg5-/-) mice with experimental endothelial dysfunction. In both control and iPodo-Atg5-/- mice, high fat diet (HFD) feeding induced glomerular endothelial damage characterized by decreased urinary nitric oxide (NO) excretion, collapsed endothelial fenestrae, and reduced endothelial glycocalyx. HFD-fed control mice showed slight albuminuria and nearly normal podocyte morphology. In contrast, HFD-fed iPodo-Atg5-/- mice developed massive albuminuria accompanied by severe podocyte injury that was observed predominantly in podocytes adjacent to damaged endothelial cells by scanning electron microscopy. Although podocyte-specific autophagy deficiency did not affect endothelial NO synthase deficiency-associated albuminuria, it markedly exacerbated albuminuria and severe podocyte morphological damage when the damage was induced by intravenous neuraminidase injection to remove glycocalyx from the endothelial surface. Furthermore, endoplasmic reticulum stress was accelerated in podocytes of iPodo-Atg5-/- mice stimulated with neuraminidase, and treatment with molecular chaperone tauroursodeoxycholic acid improved neuraminidase-induced severe albuminuria and podocyte injury. In conclusion, podocyte autophagy plays a renoprotective role against diabetes-related structural endothelial damage, providing an additional insight into the pathogenesis of massive proteinuria in diabetic nephropathy

Ketone bodies : A double-edged sword for mammalian life span.

Accumulating evidence suggests health benefits of ketone bodies, and especially for longevity. However, the precise role of endogenous ketogenesis in mammalian life span, and the safety and efficacy of the long-term exogenous supplementation of ketone bodies remain unclear. In the present study, we show that a deficiency in endogenous ketogenesis, induced by whole-body Hmgcs2 deletion, shortens life span in mice, and that this is prevented by daily ketone body supplementation using a diet containing 1,3-butanediol, a precursor of β-hydroxybutyrate. Furthermore, feeding the 1,3-butanediol-containing diet from early in life increases midlife mortality in normal mice, but in aged mice it extends life span and prevents the high mortality associated with atherosclerosis in ApoE-deficient mice. By contrast, an ad libitum low-carbohydrate ketogenic diet markedly increases mortality. In conclusion, endogenous ketogenesis affects mammalian survival, and ketone body supplementation may represent a double-edged sword with respect to survival, depending on the method of administration and health status

AIF1L regulates actomyosin contractility and filopodial extensions in human podocytes

<div><p>Podocytes are highly-specialized epithelial cells essentially required for the generation and the maintenance of the kidney filtration barrier. This elementary function is directly based on an elaborated cytoskeletal apparatus establishing a complex network of primary and secondary processes. Here, we identify the actin-bundling protein allograft-inflammatory-inhibitor 1 like (AIF1L) as a selectively expressed podocyte protein <i>in vivo</i>. We describe the distinct subcellular localization of AIF1L to actin stress fibers, focal adhesion complexes and the nuclear compartment of podocytes <i>in vitro</i>. Genetic deletion of <i>AIF1L</i> in immortalized human podocytes resulted in an increased formation of filopodial extensions and decreased actomyosin contractility. By the use of SILAC based quantitative proteomics analysis we describe the podocyte specific AIF1L interactome and identify several components of the actomyosin machinery such as MYL9 and UNC45A as potential AIF1L interaction partners. Together, these findings indicate an involvement of AIF1L in the stabilization of podocyte morphology by titrating actomyosin contractility and membrane dynamics.</p></div

AIF1L prevents formation of filopodial extensions in podocytes.

<p>(a-c) Immunofluorescence studies revealed the presence of numerous thin filopodial extensions evolving prom pre-existing lamellipodial structures in both AIF1L knockouts (dashed boxes indicate areas of higher magnification; white arrows indicate filopodial extensions). (d-e) Quantification of filopodia showed higher numbers per cell in conditions of AIF1L loss; also, measurements of filopodia demonstrated overall increased length in respective AIF1L knockout clones (n = 53 WT, 53 KO-A and 42 KO-B podocytes out of 3 independent experiments were analyzed for filopodia number; filopodia from those cells were measured for length, n = 277 WT, 1175 KO-A and 778 KO-B filopodia; **** p<0.0001). (f-h) Seeding of podocytes on thin fibrillar collagen gels for 3 hours resulted in an overall multi-polar morphology with several membrane protrusions. Numerous filopodia extended from those areas of membrane protrusion in AIF1L knockout cells. (black arrows indicate filopodial extensions; dashed boxes indicate areas of magnification).</p

Cisplatin Nephrotoxicity Is Critically Mediated by the Availability of BECLIN1

Cisplatin nephrotoxicity is a critical limitation of solid cancer treatment. Until now, the complex interplay of various pathophysiological mechanisms leading to proximal tubular cell apoptosis after cisplatin exposure has not been fully understood. In our study, we assessed the role of the autophagy-related protein BECLIN1 (ATG6) in cisplatin-induced acute renal injury (AKI)—a candidate protein involved in autophagy and with putative impact on apoptosis by harboring a B-cell lymphoma 2 (BCL2) interaction site of unknown significance. By using mice with heterozygous deletion of Becn1, we demonstrate that reduced intracellular content of BECLIN1 does not impact renal function or autophagy within 12 months. However, these mice were significantly sensitized towards cisplatin-induced AKI, and by using Becn1+/−;Sglt2-Cre;Tomato/EGFP mice with subsequent primary cell analysis, we confirmed that nephrotoxicity depends on proximal tubular BECLIN1 content. Mechanistically, BECLIN1 did not impact autophagy or primarily the apoptotic pathway. In fact, a lack of BECLIN1 sensitized mice towards cisplatin-induced ER stress. Accordingly, the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blunted cisplatin-induced cell death in Becn1 heterozygosity. In conclusion, our data first highlight a novel role of BECLIN1 in protecting against cellular ER stress independent from autophagy. These novel findings open new therapeutic avenues to intervene in this important intracellular stress response pathway with a promising impact on future AKI management

AIF1L knockout clones exhibit an impaired actomyosin contractility reserve.

<p>(a-c) Western blot experiments and quantification by densitometry demonstrated decreased levels of MYL9 as well as MYH9 in respective AIF1L knockout clones, whereas UNC45A or MYPT were not affected (n = 6 WT and KO WB intensities out of 3 independent experiments; *** p<0.001; **** p<0.0001). (d-g) Treatment of wild type cells and AIF1L knockout clones with the myosin-II inhibitor blebbistatin resulted in a more rapid dissolution of FA complexes in conditions of AIF1L loss, indicating a decreased actomyosin contractility reserve (n = 29 WT, 27 KO-A and 29 KO-B podocytes out of 3 independent experiments were analyzed; * p<0.05; **** p<0.0001). (h-j) Washout experiments for blebbistatin showed that in conditions of reconstituting actomyosin contractility podocytes show a high generation rate of filopodial protrusions (note that these structures are associated with FA sites at the filopodial basis indicated by white arrows; white dashed boxes indicate areas of magnification;(n = 20 podocytes per condition were analyzed; n.s.–non significant, **** p<0.0001)).</p

AIF1L localizes to filamentous actin, adhesion sites and cellular protrusions.

<p>(a) Expression of GFP-AIF1L in wild type podocytes localized to focal adhesion sites, as revealed by co-staining with the FA marker PAXILLIN (PXN—dashed boxes indicate sites of magnification, white arrows indicate FA tips or nascent FAs with less intense co-labeling of AIF1L and PAXILLIN). (b) In co-stainings with the filamentous actin marker phalloidin and the nuclear dye DAPI AIF1L localized to stress fibers and to the nuclear compartment (dashed boxes indicate sites of magnification; white asterisk indicates localization of AIF1L to the nucleus). (c) Filopodial extensions and the leading edge zone showed also an accumulation of AIF1L (white arrows indicate filopodial extensions; white asterisks indicate the leading edge zone; pictures in c were gamma and intensity adjusted to increase filopodia visualization).</p