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Cells Jun 2021Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells... (Review)
Review
Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.
Topics: Genetic Diseases, Inborn; Humans; Kidney Diseases; Kidney Tubules, Proximal; Models, Biological; Podocytes; Urine
PubMed: 34204173
DOI: 10.3390/cells10061413 -
American Journal of Physiology. Renal... Aug 2022The podocyte is a key cell in maintaining renal filtration barrier integrity. Several recent studies have analyzed the genome and transcriptome in the podocyte at deep...
The podocyte is a key cell in maintaining renal filtration barrier integrity. Several recent studies have analyzed the genome and transcriptome in the podocyte at deep resolution. This avenue of "podocyte-ome" research was enabled by a variety of techniques, including ) single-cell transcriptomics, ) FACS with and without genetically encoded markers, and ) deep proteomics. However, data across various omics techniques and studies are currently not well integrated with each other. Here, we aimed to establish a common, simplified knowledge base for the mouse podocyte-ome by integrating bulk RNA sequencing, bulk proteomics of FACS-sorted podocytes, and single-cell transcriptomics. Three publicly available datasets of each omics technique from different laboratories were bioinformatically integrated and visualized. Our approach not only revealed conserved processes of podocytes but also sheds light on the benefits and limitations of the used technologies. We identified that high expression of glycan glycosylphosphatidylinositol anchor synthesis and turnover, as well as retinol metabolism, were relatively understudied features of podocytes. In addition, actin-binding molecules were organized in a podocyte-specific manner, as evidenced by differential expression in podocytes compared with other glomerular cells. We compiled a Web-based "PodIent" application that illustrates the features of the integrated dataset. This enables user-driven exploratory analysis by querying genes of interest for podocyte identity in absolute and relative quantification while also linking to functional annotation using keywords, Gene Ontology terms, and gene set enrichments. This consensus draft is a first step toward common molecular omics knowledge of kidney cells. Podocytes are key components of glomerular filtration and are affected in various kidney diseases. Here, we present an integrated, robust definition of molecular identity across proteomic, single-cell transcriptomics, and bulk transcriptomic studies on native mouse podocytes. We created the "PodIdent" app, a novel knowledge base promoting access to the presence and expression of specific proteins for podocytes.
Topics: Animals; Consensus; Kidney; Kidney Diseases; Kidney Glomerulus; Mice; Podocytes; Proteomics
PubMed: 35796460
DOI: 10.1152/ajprenal.00058.2022 -
Kidney International May 2024The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting... (Review)
Review
The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting on May 23, providing young scientists with a platform to present and discuss their research findings. Throughout the main conference, 29 speakers across 9 sessions shared their insights on podocyte biology, glomerular medicine, novel technologic advancements, and translational approaches. Additionally, the event featured 3 keynote lectures addressing engineered chimeric antigen receptor T cell- and mRNA-based therapies and the use of biobanks for enhanced disease comprehension. Furthermore, 4 brief oral abstract sessions allowed scientists to present their findings to a broad audience. The program also included a panel discussion addressing the challenges of conducting human research within the American Black community. Remarkably, after a 5-year hiatus from in-person conferences, the 14th International Podocyte Conference successfully convened scientists from around the globe, fostering the presentation and discussion of crucial research findings, as summarized in this review. Furthermore, to ensure continuous and sustainable education, research, translation, and trial medicine related to podocyte and glomerular diseases for the benefit of patients, the International Society of Glomerular Disease was officially launched during the conference.
Topics: Humans; Podocytes; Kidney Glomerulus; Kidney Diseases; Biology
PubMed: 38447880
DOI: 10.1016/j.kint.2024.01.042 -
Cells Jan 2021Fabry disease is a lysosomal storage disease with an X-linked heritage caused by absent or decreased activity of lysosomal enzymes named alpha-galactosidase A (α-gal...
Fabry disease is a lysosomal storage disease with an X-linked heritage caused by absent or decreased activity of lysosomal enzymes named alpha-galactosidase A (α-gal A). Among the various manifestations of Fabry disease, Fabry nephropathy significantly affects patients' morbidity and mortality. The cellular mechanisms of kidney damage have not been elusively described. Necroptosis is one of the programmed necrotic cell death pathways and is known to play many important roles in kidney injury. We investigated whether RIPK3, a protein phosphokinase with an important role in necroptosis, played a crucial role in the pathogenesis of Fabry nephropathy both in vitro and in vivo. The cell viability of podocytes decreased after lyso-Gb3 treatment in a dose-dependent manner, with increasing RIPK3 expression. Increased reactive oxygen species (ROS) generation after lyso-Gb3 treatment, which was alleviated by GSK'872 (a RIPK3 inhibitor), suggested a role of oxidative stress via a RIPK3-dependent pathway. Cytoskeleton rearrangement induced by lyso-Gb3 was normalized by the RIPK3 inhibitor. When mice were injected with lyso-Gb3, increased urine albuminuria, decreased podocyte counts in the glomeruli, and effaced foot processes were observed. Our results showed that lyso-Gb3 initiated albuminuria, a clinical manifestation of Fabry nephropathy, by podocyte loss and subsequent foot process effacement. These findings suggest a novel pathway in Fabry nephropathy.
Topics: Animals; Cell Death; Cytoskeleton; Female; Glycolipids; Injections, Intraperitoneal; Mice, Inbred C57BL; Models, Biological; Podocytes; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Sphingolipids; Mice
PubMed: 33513913
DOI: 10.3390/cells10020245 -
Experimental Biology and Medicine... May 2023Studies over the last decade have markedly broadened our understanding of store-operated Ca channels (SOCs) and their roles in kidney diseases and podocyte dysfunction.... (Review)
Review
Studies over the last decade have markedly broadened our understanding of store-operated Ca channels (SOCs) and their roles in kidney diseases and podocyte dysfunction. Podocytes are terminally differentiated glomerular visceral epithelial cells which are tightly attached to the glomerular capillary basement membrane. Podocytes and their unique foot processes (pedicels) constitute the outer layer of the glomerular filtration membrane and the final barrier preventing filtration of albumin and other plasma proteins. Diabetic nephropathy and other renal diseases are associated with podocyte injury and proteinuria. Recent evidence demonstrates a pivotal role of store-operated Ca entry (SOCE) in maintaining structural and functional integrity of podocytes. This article reviews the current knowledge of SOCE and its contributions to podocyte physiology. Recent studies of the contributions of SOC dysfunction to podocyte injury in both cell culture and animal models are discussed, including work in our laboratory. Several downstream signaling pathways mediating SOC function in podocytes also are examined. Understanding the pivotal roles of SOC in podocyte health and disease is essential, as SOCE-activated signaling pathways are potential treatment targets for podocyte injury-related kidney diseases.
Topics: Animals; Podocytes; Signal Transduction; Models, Animal; Diabetic Nephropathies; Cell Culture Techniques
PubMed: 36533574
DOI: 10.1177/15353702221139187 -
Cells May 2023Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2...
Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.
Topics: Rats; Animals; Phosphorylation; Caseins; Podocytes; Serine; Neurons
PubMed: 37296607
DOI: 10.3390/cells12111487 -
Scientific Reports Nov 2022Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors...
Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.
Topics: Humans; Mice; Animals; Podocytes; Focal Adhesions; src-Family Kinases; Epidermal Growth Factor; Rho Guanine Nucleotide Exchange Factors; Proteinuria; Mice, Knockout
PubMed: 36333327
DOI: 10.1038/s41598-022-21634-1 -
American Journal of Physiology. Renal... Sep 2021Low birth weight is a risk factor for chronic kidney disease, whereas adult podocyte depletion is a key event in the pathogenesis of glomerulosclerosis. However, whether...
Low birth weight is a risk factor for chronic kidney disease, whereas adult podocyte depletion is a key event in the pathogenesis of glomerulosclerosis. However, whether low birth weight due to poor maternal nutrition is associated with low podocyte endowment and glomerulosclerosis in later life is not known. Female Sprague-Dawley rats were fed a normal-protein diet (NPD; 20%) or low-protein diet (LPD; 8%), to induce low birth weight, from 3 wk before mating until (PN21), when kidneys from some male offspring were taken for quantitation of podocyte number and density in whole glomeruli using immunolabeling, tissue clearing, and confocal microscopy. The remaining offspring were fed a normal- or high-fat diet until 6 mo to induce catch-up growth and excessive weight gain, respectively. At PN21, podocyte number per glomerulus was 15% lower in low birth weight (LPD) than normal birth weight (NPD) offspring, with this deficit greater in outer glomeruli. Surprisingly, podocyte number in LPD offspring increased in outer glomeruli between PN21 and 6 mo, although an overall 9% podocyte deficit persisted. Postnatal fat feeding to LPD offspring did not alter podometric indexes or result in glomerular pathology at 6 mo, whereas fat feeding in NPD offspring was associated with far greater body and fat mass as well as podocyte loss, reduced podocyte density, albuminuria, and glomerulosclerosis. This is the first report that maternal diet can influence podocyte endowment. Our findings provide new insights into the impact of low birth weight, podocyte endowment, and postnatal weight on podometrics and kidney health in adulthood. The present study shows, for the first time, that low birth weight as a result of maternal nutrition is associated with low podocyte endowment. However, a mild podocyte deficit at birth did not result in glomerular pathology in adulthood. In contrast, postnatal podocyte loss in combination with excessive body weight led to albuminuria and glomerulosclerosis. Taken together, these findings provide new insights into the associations between birth weight, podocyte indexes, postnatal weight, and glomerular pathology.
Topics: Animals; Birth Weight; Body Size; Female; Kidney; Kidney Diseases; Kidney Glomerulus; Podocytes; Pregnancy; Prenatal Exposure Delayed Effects; Rats, Sprague-Dawley; Rats
PubMed: 34308670
DOI: 10.1152/ajprenal.00029.2021 -
Medical Science Monitor Basic Research Dec 2020BACKGROUND Palmitate, a common saturated free fatty acid, is increased in patients with diabetic nephropathy (DN). Excessive palmitate in kidney is known to cause...
BACKGROUND Palmitate, a common saturated free fatty acid, is increased in patients with diabetic nephropathy (DN). Excessive palmitate in kidney is known to cause proteinuria and fibrosis. Several studies have demonstrated that paclitaxel has anti-fibrotic and anti-inflammatory effects on kidney disease. However, whether paclitaxel can relieve podocyte injury is unclear. MATERIAL AND METHODS Immortalized mouse podocytes were used as an in vitro system. Palmitate was used to induce podocyte injury. Podocytes were divided into 4 groups: bovine serum albumin, palmitate, palmitate+1 nM paclitaxel, and palmitate+5 nM paclitaxel. The effects of paclitaxel on palmitate-induced podocyte injury were analyzed by western blot and real-time PCR. Intracellular reactive oxygen species (ROS) generation and podocyte cytoskeletons were analyzed using CM-H2DCF-DA and phalloidin staining. RESULTS Paclitaxel restored downregulated expression of nephrin and synaptopodin and upregulated VEGF expression after injury induced by palmitate. Remarkably, palmitate-induced actin cytoskeleton rearrangement in podocytes was repaired by paclitaxel. Four endoplasmic reticulum stress markers, ATF-6alpha, Bip, CHOP, and spliced xBP1, were significantly increased in palmitate-treated podocytes compared with control podocytes. Such increases were decreased by paclitaxel treatment. Palmitate-induced ROS generation was ameliorated by paclitaxel. Elevated Nox4 expression was also improved by paclitaxel. Paclitaxel alleviated the expression levels of the antioxidant molecules, Nrf-2, HO-1, SOD-1, and SOD-2. The paclitaxel effects were accompanied by inhibition of the inflammatory cytokines, MCP-1, TNF-alpha, TNF-R2, and TLR4, as well as attenuation of the apoptosis markers, Bax, Bcl-2, and Caspase-3. Furthermore, paclitaxel suppressed the palmitate-induced fibrosis molecules, fibronectin and TGF-ß1. CONCLUSIONS This study suggests that paclitaxel could be a therapeutic agent for treating palmitate-induced podocyte injury in DN.
Topics: Animals; Antioxidants; Apoptosis; Cell Death; Cell Line; Cell Survival; Endoplasmic Reticulum Stress; Fibrosis; Inflammation; Mice; Paclitaxel; Palmitates; Podocytes; Reactive Oxygen Species
PubMed: 33323915
DOI: 10.12659/MSMBR.928265 -
Journal of the American Society of... Jul 2023Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration...
SIGNIFICANCE STATEMENT
Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies.
BACKGROUND
Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood.
METHODS
To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7.
RESULTS
Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins.
CONCLUSIONS
Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.
Topics: Animals; Mice; Humans; Kidney Glomerulus; Podocytes; Endosomes; Drosophila; Kidney; Mammals
PubMed: 37022133
DOI: 10.1681/ASN.0000000000000126