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Journal of Visualized Experiments : JoVE May 2023Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus....
Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes. Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients' mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.
Topics: Humans; Podocytes; Biopsy; Kidney Glomerulus; Epithelial Cells; Cell Differentiation
PubMed: 37306454
DOI: 10.3791/65364 -
International Journal of Molecular... Dec 2023Diabetes nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) worldwide, and podocyte injury is the central contributor to the progression of DN....
Diabetes nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) worldwide, and podocyte injury is the central contributor to the progression of DN. Despite the emerging evidence that has established the importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of DN, abnormal protein O-GlcNAcylation is also augmented. Currently, the mechanism associating these two hyperglycemia-induced disorders remains poorly understood. This study intended to elucidate whether ER stress drives hyper-protein O-GlcNAcylation to cause podocyte injury in DN. We used both type 1 and type 2 DN models to confirm the occurrence of ER stress and excessive protein O-GlcNAcylation, and then podocyte purification was also conducted for further investigation. Nephroseq V5 data were mined and in vitro studies were applied to reveal the involvement of ER stress and hyper-O-GlcNAcylation in podocyte injury. Our results indicated that ER stress was induced in both type 1 and type 2 DN, and the human RNA-seq data from Nephroseq V5 showed that O-GlcNAcylation-related genes were significantly upregulated in the DN patients. We further demonstrated that ER stress occurred prior to hyper-O-GlcNAc modification and that pharmacologically inhibited protein O-GlcNAcylation can help decrease the podocyte apoptosis induced by hyperglycemia. Together, these discoveries will aid in uncovering the activation of the ER stress-O-GlcNAcylation axis in podocyte injury under DN, which will help open up new therapeutic approaches for preventing DN progression.
Topics: Humans; Podocytes; Diabetic Nephropathies; Endoplasmic Reticulum Stress; Proteins; Hyperglycemia; Diabetes Mellitus, Type 2
PubMed: 38139429
DOI: 10.3390/ijms242417603 -
Kidney International Nov 2022Podocyte loss and resultant nephron loss are common processes in the development of glomerulosclerosis and chronic kidney disease. While the cortical distribution of...
Podocyte loss and resultant nephron loss are common processes in the development of glomerulosclerosis and chronic kidney disease. While the cortical distribution of glomerulosclerosis is known to be non-uniform, the relationship between the numbers of non-sclerotic glomeruli (NSG), podometrics and zonal differences in podometrics remain incompletely understood. To help define this, we studied autopsy kidneys from 50 adults with median age 68 years and median eGFR 73.5 mL/min/1.73m without apparent glomerular disease in a cross-sectional analysis. The number of NSG per kidney was estimated using the physical dissector/fractionator combination, while podometrics were estimated using model-based stereology. The number of NSG per kidney was directly correlated with podocyte number per tuft and podocyte density. Each additional 100,000 NSG per kidney was associated with 26 more podocytes per glomerulus and 16 podocytes per 10 μm increase in podocyte density. These associations were independent of clinical factors and cortical zone. While podocyte number per glomerulus was similar in the three zones, superficial glomeruli were the smallest and had the highest podocyte density but smallest podocytes. Increasing age and hypertension were associated with lower podocyte number, with age mostly affecting superficial glomeruli, and hypertension mostly affecting juxtamedullary glomeruli. Thus, in this first study to report a direct correlation between the number of NSG and podometrics, we suggest that podocyte number is decreasing in NSG of individuals losing nephrons. However, another possible interpretation may be that more nephrons might protect against further podocyte loss.
Topics: Adult; Humans; Aged; Cross-Sectional Studies; Kidney Glomerulus; Podocytes; Kidney; Hypertension
PubMed: 36175177
DOI: 10.1016/j.kint.2022.07.028 -
Archives of Biochemistry and Biophysics Oct 2020Podocytes are unique, highly specialized, terminally differentiated cells that form an essential, integral part of the glomerular filter. These cells limit the outside... (Review)
Review
Podocytes are unique, highly specialized, terminally differentiated cells that form an essential, integral part of the glomerular filter. These cells limit the outside border of the glomerular basement membrane, forming a tight barrier that prevents significant protein loss from the capillary space. The slit diaphragm formed by podocytes is crucial for maintaining glomerular integrity and function. They are the target of injury in many glomerular diseases, including hypertension and diabetes mellitus. Accumulating studies have revealed that AMP-activated protein kinase (AMPK), an essential cellular energy sensor, might play a fundamental role in regulating podocyte metabolism and function. AMPK participates in insulin signaling, therefore controls glucose uptake and podocytes insulin sensitivity. It is also involved in insulin-dependent cytoskeleton reorganization in podocytes, mediating glomerular albumin permeability. AMPK plays an important role in the regulation of autophagy/apoptosis processes, which influence podocytes viability. The present review aimed to highlight the molecular mechanisms associated with AMPK that are involved in the regulation of podocyte function in health and disease states.
Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Diabetic Nephropathies; Humans; Insulin; Insulin Resistance; Kidney Glomerulus; Podocytes; Signal Transduction
PubMed: 32781053
DOI: 10.1016/j.abb.2020.108541 -
Cells May 2024Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate... (Review)
Review
Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.
Topics: Podocytes; Sphingolipids; Humans; Animals; Lipid Metabolism; Kidney Diseases; Membrane Microdomains
PubMed: 38891023
DOI: 10.3390/cells13110890 -
Clinical Science (London, England :... Jun 2020Chronic kidney disease (CKD) substantially reduces quality of life and leads to premature death for thousands of people each year. Dialysis and kidney organ transplants... (Review)
Review
Chronic kidney disease (CKD) substantially reduces quality of life and leads to premature death for thousands of people each year. Dialysis and kidney organ transplants remain prevalent therapeutic avenues but carry significant medical, economic and social burden. Podocytes are responsible for blood filtration selectivity in the kidney, where they extend a network of foot processes (FPs) from their cell bodies which surround endothelial cells and interdigitate with those on neighbouring podocytes to form narrow slit diaphragms (SDs). During aging, some podocytes are lost naturally but accelerated podocyte loss is a hallmark of CKD. Insights into the origin of degenerative podocyte loss will help answer important questions about kidney function and lead to substantial health benefits. Here, approaches that uncover insights into podocyte mechanobiology are reviewed, both those that interrogate the biophysical properties of podocytes and how the external physical environment affects podocyte behaviour, and also those that interrogate the biophysical effects that podocytes exert on their surroundings.
Topics: Animals; Biomechanical Phenomena; Biophysics; Health; Humans; Kidney Diseases; Podocytes; Stress, Mechanical
PubMed: 32501496
DOI: 10.1042/CS20190764 -
Journal of Diabetes Research 2020Diabetic nephropathy (DN) is not only an important microvascular complication of diabetes but also the main cause of end-stage renal disease. Studies have shown that the... (Review)
Review
Diabetic nephropathy (DN) is not only an important microvascular complication of diabetes but also the main cause of end-stage renal disease. Studies have shown that the occurrence and development of DN are closely related to morphological and functional changes in podocytes. A series of morphological changes after podocyte injury in DN mainly include podocyte hypertrophy, podocyte epithelial-mesenchymal transdifferentiation, podocyte detachment, and podocyte apoptosis; functional changes mainly involve podocyte autophagy. More and more studies have shown that multiple signaling pathways play important roles in the progression of podocyte injury in DN. Here, we review research progress on the pathological mechanism of morphological and functional changes in podocytes associated with DN, to provide a new target for delaying the occurrence and development of this disorder.
Topics: Animals; Apoptosis; Autophagy; Cell Transdifferentiation; Diabetic Nephropathies; Disease Progression; Humans; Podocytes; Research; Signal Transduction
PubMed: 32695831
DOI: 10.1155/2020/7504798 -
Frontiers in Immunology 2022Idiopathic membranous nephropathy is the main cause of chronic kidney disease (CKD). Studies have shown sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly...
Idiopathic membranous nephropathy is the main cause of chronic kidney disease (CKD). Studies have shown sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly delay renal outcomes in patients with CKD, but the exact mechanism remains unknown. In this study, we investigated the mechanism by which the SGLT2 inhibitor canagliflozin attenuates podocyte injury by reversing the imbalance in Helper T cell 1 (Th1)/Helper T cell 2 (Th2) in peripheral blood of rats with membranous nephropathy (MN). MN rats were gavaged with canagliflozin (10 mg/kg/d) and losartan (10 mg/kg/d), respectively, for eight weeks. Compared with the MN group, the urinary ratio of total protein and the creatinine levels of the canagliflozin group decreased significantly. Canagliflozin improved the glomerulus pathological damage, increased the expression levels of podocyte marker proteins. The protective effect of canagliflozin on kidneys was more obvious than that of losartan. Treatment with canagliflozin increased the proportion of Th1 cells by 2.3 times, decreased the proportion of Th2 cells by 68.5%, and significantly restrained the synthesis of immunoglobulin G1 in B-cells and glomerulus subepithelial immune complex deposition. Co-culture of B-cells derived from MN rats with podocytes triggered the activation of phosphorylation of mTOR and ULK1 of podocytes, inhibited podocyte autophagy and resulted in podocyte injury. B-cells derived from canagliflozin treatment rats reversed these effects above. In conclusion, canagliflozin exerts a protective effect on kidneys by reversing the imbalance in Th1/Th2 cells in MN rats and restoring the autophagy of podocytes inhibited by the abnormal immunoglobulin G secretion from B-cells.
Topics: Rats; Animals; Podocytes; Glomerulonephritis, Membranous; Canagliflozin; Losartan; Autophagy; Renal Insufficiency, Chronic
PubMed: 36531996
DOI: 10.3389/fimmu.2022.993869 -
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