Podocytes exhibit a unique cytoskeletal architecture that is fundamentally linked to their function in maintaining the kidney filtration barrier. The cytoskeleton regulates podocyte shape, structure, ...stability, slit diaphragm insertion, adhesion, plasticity, and dynamic response to environmental stimuli. Genetic mutations demonstrate that even slight impairment of the podocyte cytoskeletal apparatus results in proteinuria and glomerular disease. Moreover, mechanisms underpinning all acquired glomerular pathologies converge on disruption of the cytoskeleton, suggesting that this subcellular structure could be targeted for therapeutic purposes. This review summarizes our current understanding of the function of the cytoskeleton in podocytes and the associated implications for pathophysiology.
Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been ...evaluated. Here, we identified the presence of death receptor–mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury.
Radio relics in galaxy clusters are associated with powerful shocks that (re)accelerate relativistic electrons. It is widely believed that the acceleration proceeds via diffusive shock acceleration. ...In the framework of thermal leakage, the ratio of the energy in relativistic electrons to the energy in relativistic protons should be smaller than K
e/p ∼ 10−2. The relativistic protons interact with the thermal gas to produce γ-rays in hadronic interactions. Combining observations of radio relics with upper limits from γ-ray observatories can constrain the ratio K
e/p. In this work, we selected 10 galaxy clusters that contain double radio relics, and derive new upper limits from the stacking of γ-ray observations by Fermi. We modelled the propagation of shocks using a semi-analytical model, where we assumed a simple geometry for shocks and that cosmic ray protons are trapped in the intracluster medium. Our analysis shows that diffusive shock acceleration has difficulties in matching simultaneously the observed radio emission and the constraints imposed by Fermi, unless the magnetic field in relics is unrealistically large ( ≫ 10 μG). In all investigated cases (also including realistic variations of our basic model and the effect of re-acceleration), the mean emission of the sample is of the order of the stacking limit by Fermi, or larger. These findings put tension on the commonly adopted model for the powering of radio relics, and imply that the relative acceleration efficiency of electrons and protons is at odds with predictions of diffusive shock acceleration, requiring K
e/p ≥ 10 − 10−2.
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.
Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5ʹ-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1
Direct reprogramming by forced expression of transcription factors can convert one cell type into another. Thus, desired cell types can be generated bypassing pluripotency. However, direct ...reprogramming towards renal cells remains an unmet challenge. Here, we identify renal cell fate-inducing factors on the basis of their tissue specificity and evolutionarily conserved expression, and demonstrate that combined expression of Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells (iRECs). iRECs exhibit epithelial features, a global gene expression profile resembling their native counterparts, functional properties of differentiated renal tubule cells and sensitivity to nephrotoxic substances. Furthermore, iRECs integrate into kidney organoids and form tubules in decellularized kidneys. Our approach demonstrates that reprogramming factors can be identified by targeted in silico analysis. Renal tubular epithelial cells generated ex vivo by forced expression of transcription factors may facilitate disease modelling, drug and nephrotoxicity testing, and regenerative approaches.
Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms ...disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.
•aPC protects from myocardial and renal IRIs by restricting mTORC1-mediated activation of the Nlrp3 inflammasome.•Nlrp3 inflammasome suppression by aPC is independent of its anticoagulant effect, depends on PAR-1, and can be mimicked by parmodulin-2.
The mTOR pathway has a central role in the regulation of cell metabolism, growth and proliferation. Studies involving selective gene targeting of mTOR complexes (mTORC1 and mTORC2) in renal cell ...populations and/or pharmacologic mTOR inhibition have revealed important roles of mTOR in podocyte homeostasis and tubular transport. Important advances have also been made in understanding the role of mTOR in renal injury, polycystic kidney disease and glomerular diseases, including diabetic nephropathy. Novel insights into the roles of mTORC1 and mTORC2 in the regulation of immune cell homeostasis and function are helping to improve understanding of the complex effects of mTOR targeting on immune responses, including those that impact both de novo renal disease and renal allograft outcomes. Extensive experience in clinical renal transplantation has resulted in successful conversion of patients from calcineurin inhibitors to mTOR inhibitors at various times post-transplantation, with excellent long-term graft function. Widespread use of this practice has, however, been limited owing to mTOR-inhibitor- related toxicities. Unique attributes of mTOR inhibitors include reduced rates of squamous cell carcinoma and cytomegalovirus infection compared to other regimens. As understanding of the mechanisms by which mTORC1 and mTORC2 drive the pathogenesis of renal disease progresses, clinical studies of mTOR pathway targeting will enable testing of evolving hypotheses.
The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats ...has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments in vivo.
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Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction.
garland cell nephrocytes are podocyte-like cells and thus provide a potential
model in which to study the pathogenesis ...of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two
slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, we found this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes required Cubilin and reflected size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, we screened
orthologs of human monogenic causes of nephrotic syndrome and observed conservation of the central pathogenetic alterations. We focused on the coenzyme Q
(CoQ
) biosynthesis gene
, the silencing of which disrupted slit diaphragm morphology. Restoration of CoQ
synthesis by vanillic acid partially rescued the phenotypic and functional alterations induced by
-RNAi. Notably,
colocalized with mitochondria, and
silencing increased the formation of reactive oxygen species (ROS). Silencing of
, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ
, phenocopied the effect of
-RNAi. Moreover, the ROS scavenger glutathione partially rescued the effects of
-RNAi. In conclusion,
garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of
-nephropathy.
Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we ...examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.
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