A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model ...combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our ‘omics’ approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.
Scribble (Scrib) is a scaffold protein with multifunctional roles in PCP, tight junction and Hippo signaling. This study shows that Scrib is expressed in stromal cells around the implantation chamber ...following implantation. Stromal cells transform into epithelial-like cells to form the avascular primary decidual zone (PDZ) around the implantation chamber (crypt). The PDZ creates a permeability barrier around the crypt restricting immune cells and harmful agents from maternal circulation to protect embryonic health. The mechanism underlying PDZ formation is not yet known. We found that uterine deletion of Scrib by a Pgr-Cre driver leads to defective PDZ formation and implantation chamber (crypt) formation, compromising pregnancy success. Interestingly, epithelial-specific Scrib deletion by a lactoferrin-Cre (Ltf-Cre) driver does not adversely affect PDZ formation and pregnancy success. These findings provide evidence for a previously unknown function of stromal Scrib in PDZ formation, potentially involving ZO-1 and Hippo signaling.
Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the ...accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.
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•CHAPS forms smaller micelles allowing full permeabilization of aged human organs•SHANEL enables centimeters deep molecular labeling and clearing of whole human organs•SHANEL renders intact adult human brain and kidney transparent•Deep learning and light-sheet microscopy with SHANEL allows human organ mapping
Zhao et al. present an approach for intact human organ mapping that uses a new tissue permeabilization method to clear and deeply label whole organs followed by light-sheet microscopy imaging and a deep learning-based pipeline for 3D reconstruction and data analysis.
Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and ...glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.
The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into ...specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair.
Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source.
Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome.
Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.
Perspectives in membranous nephropathy Tomas, Nicola M.; Huber, Tobias B.; Hoxha, Elion
Cell and tissue research,
08/2021, Letnik:
385, Številka:
2
Journal Article
Recenzirano
Odprti dostop
The identification of the phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 domain-containing protein 7A (THSD7A) as podocyte antigens in adult patients with membranous nephropathy (MN) ...has strongly impacted both experimental and clinical research on this disease. Evidence has been furnished that podocyte-directed autoantibodies can cause MN, and novel PLA2R- and THSD7A-specific animal models have been developed. Today, measurement of serum autoantibody levels and staining of kidney biopsies for the target antigens guides MN diagnosis and treatment worldwide. Additionally, anti-PLA2R antibodies have been proven to be valuable prognostic biomarkers in MN. Despite these impressive advances, a variety of questions regarding the disease pathomechanisms, clinical use of antibody measurement, and future treatments remain unanswered. In this review, we will outline recent advances made in the field of MN and discuss open questions and perspectives with a focus on novel antigen identification, mechanisms of podocyte injury, clinical use of antibody measurement to guide diagnosis and treatment, and the potential of innovative, pathogenesis-based treatment strategies.
Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly ...controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.
mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the ...V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, we show here that the V-ATPase is required for Rheb-dependent epithelial growth. We further uncover a positive feedback loop for the control of apical protein uptake that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, we provide evidence that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, our findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions.
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•The V-ATPase is required for mTOR-dependent tissue growth in Drosophila•V-ATPase/mTOR signaling controls apical endocytosis•Megalin activity and apical surface area is regulated by V-ATPase/mTOR signaling•Long-term mTOR inhibition causes Megalin downregulation and proteinuria in the kidney
Epithelial growth requires the endocytic uptake of proteins, which are broken down into amino acids. Amino acids recruit the growth-regulating mTORC1 complex to the lysosomal surface, thereby causing its activation. Gleixner et al. now find that in epithelial cells apical protein uptake is controlled by the lysosomal V-ATPase/mTORC1 complex in a feedforward manner. The multiligand receptor Megalin, which internalizes proteins on the apical surface, is crucial for this regulatory pathway.
Focal segmental glomerulosclerosis (FSGS) represents a glomerular scar formation downstream of various different mechanisms leading to podocytopathy and podocyte loss. Recently, significant advances ...were made in understanding genetic factors, podocyte intrinsic mechanisms, and adaptive mechanisms causing FSGS. However, while most cases of nephrotic FSGS are being treated with immunosuppressants, the underlying immune dysregulation, involved immune cells, and soluble factors are only incompletely understood. Thus, we here summarize the current knowledge of proposed immune effector cells, secreted soluble factors, and podocyte response in immune-mediated (primary) FSGS.
Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases ...and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy.
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