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.
Autophagy is a highly regulated lysosomal protein degradation pathway that removes protein aggregates and damaged or excess organelles to maintain intracellular homeostasis and cell integrity. ...Dysregulation of autophagy is involved in the pathogenesis of a variety of metabolic and age-related diseases. Growing evidence suggests that autophagy is implicated in cell injury during renal diseases, both in the tubulointerstitial compartment and in glomeruli. Nevertheless, the impact of autophagy on renal disease progression and aging is still not fully understood. This review summarizes the recent advances in understanding the role of autophagy for kidney disease and aging.
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.
SARS-CoV-2 uses the receptor ACE2 for cell entry, and podocytes express ACE2.1 Glomerular changes and nephritis-like histology have been described in postmortem samples from patients with COVID-19.2 ...Other zoonoses, such as some hantaviruses, cause nephrotic syndrome, which in turn induces cardiopulmonary syndrome.3,4 Complications of nephrotic syndrome are known to be similar to capillary leak syndrome, and preventive therapies are available.5 We recommend that patients with COVID-19 who have nephritis be carefully monitored for the following conditions: pulmonary interstitial oedema, due to severe fluid overload similar to nephrotic syndrome; immune incompetence, due to renal loss of immunoglobulins; circulatory insufficiency, due to hypalbuminaemia; poor drug response because of impaired plasma protein binding; and thromboembolic events due to antithrombin deficiency. In summary, the respiratory tract is the gateway for SARS-CoV-2 infection, but we postulate that COVID-19-associated nephritis, which can be easily screened for through a simple and inexpensive urine sample analysis, might help predict complications. If validated, we believe this tool could allow early anticipation of later need for ICU admission, improved allocation of patients for special therapies (eg, in clinical trials), and initiation of preventive strategies focused on capillary leak syndrome, including treatment that could save lives.
The architectural design of our kidneys is amazingly complex, and culminates in the 3D structure of the glomerular filter. During filtration, plasma passes through a sieve consisting of a fenestrated ...endothelium and a broad basement membrane before it reaches the most unique part, the slit diaphragm, a specialized type of intercellular junction that connects neighbouring podocyte foot processes. When podocytes become stressed, irrespective of the causative stimulus, they undergo foot process effacement and loss of slit diaphragms--two key steps leading to proteinuria. Thus, proteinuria is the unifying denominator of a broad spectrum of podocytopathies. With the rising prevalence of chronic kidney disease and the fact that glomerular diseases account for the majority of patients with end-stage renal disease, further investigation and elucidation of this unique structure is of paramount importance. This Review recounts how perception of the slit diaphragm has changed over time as a result of intense research, from its first anatomical description as a thin intercellular connection, to an appreciation of its role as a dynamic signalling hub. These observations led to the introduction of novel concepts in podocyte biology, which could pave the way to development of highly desired, specific therapeutic strategies for glomerular diseases.
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.
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
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.
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.
Biological and biomedical research using
Drosophila melanogaster
as a model organism has gained recognition through several Nobel prizes within the last 100 years.
Drosophila
exhibits several ...advantages when compared to other in vivo models such as mice and rats, as its life cycle is very short, animal maintenance is easy and inexpensive and a huge variety of transgenic strains and tools are publicly available. Moreover, more than 70% of human disease-causing genes are highly conserved in the fruit fly. Here, we explain the use of
Drosophila
in nephrology research and describe two kidney tissues, Malpighian tubules and the nephrocytes. The latter are the homologous cells to mammalian glomerular podocytes and helped to provide insights into a variety of signaling pathways due to the high morphological similarities and the conserved molecular make-up between nephrocytes and podocytes. In recent years, nephrocytes have also been used to study inter-organ communication as links between nephrocytes and the heart, the immune system and the muscles have been described. In addition, other tissues such as the eye and the reproductive system can be used to study the functional role of proteins being part of the kidney filtration barrier.
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