Podocyte Development Research Articles

Molecular Regulation of Podocyte Development by Transcription Factor 21

Molecular Regulation of Podocyte Development by Transcription Factor 21

Kidney Organoid Modeling of WT1 Mutations Reveals Key Regulatory Paths Underlying Podocyte Development.

Wilms tumor-1(WT1) is a crucial transcription factor that regulates podocyte development. However, the epigenomic mechanism underlying the function of WT1 during podocyte development has yet to be fully elucidated. Here, single-cell chromatin accessibilityand gene expressionmaps of foetal kidneys and kidney organoids are generated. Functional implications of WT1-targeted genes, which are crucial for the development of podocytes and the maintenance of their structure, including BMPER/PAX2/MAGI2 that regulates WNT signaling pathway, MYH9 that maintains actin filament organization and NPHS1 that modulates cell junction assembly are identified. To further illustrate the functional importance of WT1-mediated transcriptional regulation during podocyte development, cultured and implanted patient-derived kidney organoids derived from the Induced Pluripotent Stem Cell (iPSCs) of a patient with a heterozygous missense mutation in WT1 are generated. Results from single-cell RNA sequencing (scRNA-seq)and functional assays confirm that the WT1 mutation leads to delays in podocyte development and causes damage to cell structures, due to its failure to activate the targeting genes MAGI2, MYH9, and NPHS1. Notably, correcting the mutation in the patient iPSCs using CRISPR-Cas9 gene editing rescues the podocyte phenotype. Collectively, this work elucidates the WT1-related epigenomic landscape with respect to human podocyte development and identifies the disease-causing role of a WT1 mutation.

Spatially Anchored Molecular Neighborhoods in Lupus Nephritis


 
 
 Background: 
 Lupus nephritis (LN) affects 1 in 3 individuals with systemic lupus erythematosus. Suboptimal treatment precipitates irreversible kidney damage, leading to end stage renal disease. Kidney damage in lupus is characterized by immune cells injuring epithelial, endothelial, and stromal cells. We characterized molecular neighborhoods composed of immune cells interacting with resident cells of the kidney through spatial transcriptomics (ST). 
 Methods: 
 Visium ST experiments were conducted in 8 healthy controls (233 glomeruli) and 3 LN samples (48 glomeruli). Cell type labels from the Kidney Precision Medicine Project single cell RNA-seq atlas were transferred to deconvolute ST spots into specific cell types. Using histology and NPHS2 expression, glomeruli were selected as functional tissue units. Data were normalized, dimensionally reduced, and clustered with Seurat v4. Spatially anchored gene signatures of LN were identified. Glomeruli were re-clustered according to cell composition, to identify associated neighborhoods by fisher’s exact test. We characterized the cell composition, differentially expressed genes (DEGs), and pathways of relevant neighborhoods. 
 Results: 
 Between LN and control glomeruli, we identified HSPA8, PLEK, COL1A2 DEGs, associated with hypoxia, fibrosis, and immune response. We identified glomerular neighborhoods consistent with immune complex deposition, endothelial dysfunction (e.g. wire loop lesions), and mesangial cell expansion enriched in LN (p<0.05). Immune complex-mediated injury neighborhoods were characterized by interferon signaling, endothelial cell migration, and vascular genesis, consistent with DEGs BST2, CXCL12, and ENG. Endothelial dysfunction neighborhoods present cellular adhesion, immune cell signaling, and hypoxic pathways. DEGs included ITGB2, HLA-DPB1, and EGR1. Pathways enriched in mesangial expansion neighborhoods included matrix adhesion, podocyte development, and ERK1 and ERK2 cascade, aligned with ITGB3, NPHS1, and APOE DEGs. 
 Conclusion/ Clinical Impact and Implications: 
 Neighborhood characterization provides insight into cell-cell interactions that drive kidney disease progression. Future directions will change how kidney biopsy specimens drive treatment by delineating specific cell-cell interactions, linking molecular and histopathological signatures, and defining genes associated with therapeutic resistance. 
 
 

Wilms' tumor gene 1: lessons from the interface between kidney development and cancer.

In 1990, mutations of the Wilms' tumor-1 gene (WT1), encoding a transcription factor in the embryonic kidney, were found in 10-15% of Wilms' tumors; germline WT1 mutations were associated with hereditary syndromes involving glomerular and reproductive tract dysplasia. For more than three decades, these discoveries prompted investigators to explore the embryonic role of WT1 and the mechanisms by which loss of WT1 leads to malignant transformation. Here, we discuss how alternative splicing of WT1 generates isoforms that act in a context-specific manner to activate or repress target gene transcription. WT1 also regulates posttranscriptional regulation, alters the epigenetic landscape, and activates miRNA expression. WT1 functions at multiple stages of kidney development, including the transition from resting stem cells to committed nephron progenitor, which it primes to respond to WNT9b signals from the ureteric bud. WT1 then drives nephrogenesis by activating WNT4 expression and directing the development of glomerular podocytes. We review the WT1 mutations that account for Denys-Drash syndrome, Frasier syndrome, and WAGR syndrome. Although the WT1 story began with Wilms' tumors, an understanding of the pathways that link aberrant kidney development to malignant transformation still has some important gaps. Loss of WT1 in nephrogenic rests may leave these premalignant clones with inadequate DNA repair enzymes and may disturb the epigenetic landscape. Yet none of these observations provide a complete picture of Wilms' tumor pathogenesis. It appears that the WT1 odyssey is unfinished and still holds a great deal of untilled ground to be explored.

ГЕНЕТИЧЕСКИЕ ПРИЧИНЫ СТЕРОИДРЕЗИСТЕНТНОГО НЕФРОТИЧЕСКОГО СИНДРОМА

Nephrotic syndrome (NS) is a severe glomerulopathy characterized by severe proteinuria, hypoproteinemia, hypoalbuminemia, hyperlipidemia and widespread edema. There’ve been over sixty genes identified to date that are responsible for the development, structural integrity and functioning of podocytes. Pathogenic nucleotide variants of these genes lead to the podocytopathy in steroid-resistant nephrotic syndrome in 30% of cases. Depending on the protein damaged, the severity of the course, the extrarenal manifestations as well as the therapeutic options may vary. The molecular genetic research has therefore resulted in the fore in recent decades when pediatric patients with steroid-resistant NS are diagnosed.

Patient-derived podocyte spheroids reveal new insights into the etiopathogenesis of Alport syndrome

Alport syndrome (AS) is a rare disease characterized by defective glomerular basement membranes, caused by mutations in COL4A3, COL4A4, and COL4A5, which synthesize collagen type IV. Patients present with progressive proteinuria, hematuria and podocyte loss. There is currently no cure for Alport syndrome, and this is mainly due to its complex and variable pathogenesis, as well as the lack of models that can faithfully mimic the human phenotype. Here we have developed a novel human culture model of Alport syndrome and used it to study the effects of different mutations on podocyte development and biology. First, we established a differentiation protocol that allowed us to generate podocyte spheroids from patient-derived human induced pluripotent stem cells (hiPSCs). We have then carried out discovery proteomics and demonstrated that a total of 178 proteins were differentially expressed between Alport (AS1 and AS3) and control (LT) podocytes. GO analysis indicated alterations in several metabolic pathways, such as oxidative phosphorylation, RNA maturation, chromatin condensation, and proliferation. Although functional assays showed no changes in lactate production and mitochondrial potential compared to healthy controls, immunofluorescence and electron microscopy analysis showed key morphological changes related to the phenotypical maturation of Alport podocytes. Moreover, the studied mutations led to persistent proliferation, increased reactive oxygen species (ROS) production and the concomitant expression of peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) in podocytes. These data on patient-derived podocytes provide evidence that collagen mutations, in addition to playing a central role in the defective development of the glomerular filtration barrier, cause significant alterations in podocyte development and metabolism very early in development, even before the formation of the filtering apparatus. In conclusion, our study provides a new methodological platform for the differentiation of podocytes and to study human podocytopathies in a personalized manner, and reveals new insights into the etiopathogenesis and pathobiology of Alport syndrome.

Modeling Podocyte Ontogeny and Podocytopathies with the Zebrafish.

Podocytes are exquisitely fashioned kidney cells that serve an essential role in the process of blood filtration. Congenital malformation or damage to podocytes has dire consequences and initiates a cascade of pathological changes leading to renal disease states known as podocytopathies. In addition, animal models have been integral to discovering the molecular pathways that direct the development of podocytes. In this review, we explore how researchers have used the zebrafish to illuminate new insights about the processes of podocyte ontogeny, model podocytopathies, and create opportunities to discover future therapies.

Osr1 Maintains Renal Progenitors and Regulates Podocyte Development by Promoting wnt2ba via the Antagonism of hand2.

Knowledge about the genetic pathways that control nephron development is essential for better understanding the basis of congenital malformations of the kidney. The transcription factors Osr1 and Hand2 are known to exert antagonistic influences to balance kidney specification. Here, we performed a forward genetic screen to identify nephrogenesis regulators, where whole genome sequencing identified an osr1 lesion in the novel oceanside (ocn) mutant. The characterization of the mutant revealed that osr1 is needed to specify not renal progenitors but rather their maintenance. Additionally, osr1 promotes the expression of wnt2ba in the intermediate mesoderm (IM) and later the podocyte lineage. wnt2ba deficiency reduced podocytes, where overexpression of wnt2ba was sufficient to rescue podocytes and osr1 deficiency. Antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression. These studies reveal new insights about the roles of Osr1 in promoting renal progenitor survival and lineage choice.

23-OR: Loss of the Transcription Factor Tcf21 in Adult Podocytes Leads to Susceptibility in Diabetic Kidney Disease

Background: Diabetic kidney disease (DKD) is the most common cause of dialysis induction in the United States. Tcf21 is a basic helix-loop-helix transcription factor that is essential for the development of the lungs, heart, and kidneys. Previously, we reported that Tcf21 regulates podocyte development. However, the precise role of Tcf21 in mature individuals and DKD remains unclear. Method: We generated mice that lacked Tcf21 only in podocytes after birth (iKO) using a doxycycline induction system. We also constructed a diabetes mouse model using streptozotocin injections. In addition, we used cultured human podocytes to investigate the effects of Tcf21 overexpression and the factors that regulate Tcf21 expression. Results: When the Tcf21 gene was deleted specifically in podocytes in 3-week-old mice, urinary protein was not observed. In contrast, when diabetes was induced in these mice, massive urinary protein and strong glomerulosclerosis was present in 60% of iKO mice. Ultrastructural analysis revealed glomerular basement membrane thickening and podocyte foot process effacement, suggesting that iKO mice are susceptible to DKD. In addition, RNA-seq analysis of Tcf21-overexpressing podocytes showed accumulation of genes involved in the interferon pathway, extracellular matrix production, senescence, and autophagy in gene ontology analysis. Analysis of individual genes showed upregulation of interferon-related genes such as IFI6 and IFI27, increased expression of PAI-1, which is one of the senescence-associated secreted phenotypes, and decreased expression of HMGA2, whose SNIP is known to correlate with susceptibility to DKD. In addition, Tcf21 expression was suppressed by 58% and 27% after TGF-β and high glucose plus insulin stimulation, respectively. Conclusion: Our results suggest that Tcf21 protects against DKD in mature individuals. Elucidation of the mechanism by which Tcf21 prevents DKD may lead to the identification of novel therapeutic targets. Disclosure N.Teramoto: None. Y.Maezawa: None. T.Minamizuka: None. M.Koshizaka: None. K.Yokote: None.

COUP-TFII in Kidneys, from Embryos to Sick Adults.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear hormone receptor of unknown ligands. This molecule has two interesting features: (1) it is a developmental gene, and (2) it is a potential hormone receptor. Here, we describe the possible roles of COUP-TFII in the organogenesis of the kidneys and protection from adult renal diseases, primarily in mouse models. COUP-TFII is highly expressed in embryos, including primordial kidneys, and is essential for the formation of metanephric mesenchyme and the survival of renal precursor cells. Although the expression levels of COUP-TFII are low and its functions are unknown in healthy adults, it serves as a reno-protectant molecule against acute kidney injury. These are good examples of how developmental genes exhibit novel functions in the etiology of adult diseases. We also discuss the ongoing research on the roles of COUP-TFII in podocyte development and diabetic kidney disease. In addition, the identification of potential ligands suggests that COUP-TFII might be a novel therapeutic target for renal diseases in the future.

The Atypical Cyclin-Dependent Kinase 5 (Cdk5) Guards Podocytes from Apoptosis in Glomerular Disease While Being Dispensable for Podocyte Development.

Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and—in contrast to neurons—does not impact on glomerular development or maintenance.

Autonomous Calcium Signaling in Human and Zebrafish Podocytes Controls Kidney Filtration Barrier Morphogenesis.

Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. These findings establish podocyte cell-autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.

Studies on the Role of the Transcription Factor Tcf21 in the Transdifferentiation of Parietal Epithelial Cells into Podocyte-Like Cells

Background/Aims: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. Methods: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. Results: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin‑1, β-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. Conclusion: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage.

Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.

POS-381 Podocyte B7-1 Mediates Cell Injury and Glomerulosclerosis through Communicating with β-catenin Pathway

Podocyte injury is a hallmark of glomerular diseases, however, the intrinsic mechanisms are not elucidated. Previous studies have shown that B7-1 may be an inducer to podocyte injury. However, the intrinsic role of B7-1 in podocyte injury is controversial. In this study, we have identified that podocyte B7-1 definitely contributes to cell injury and glomerulosclerosis, and the underlying mechanisms are intimately correlated with activation of β-catenin pathway. Our studies provide the importance evidence to the role of B7-1 in podocyte injury and development of glomerulosclerosis. The expression of B7-1 were assessed in various CKD models, human biopsies and isolated glomeruli. Podocyte-specific B7-1 transgenic mice (podo-B7-Tg) were constructed. Podocyte, glomerular injury and β-catenin signaling were assessed in podo-B7-Tg mice at 2, 3 and 6-month age, and advanced oxidative protein products (AOPPs)-administrated mice. In ADR and 5/6 nephrectomized mice, B7-1 interference plasmids (B7-shR) were injected through hydrodynamic-based gene delivery approach. In cultured podocyte cell line (MPC5), ectopic expression of B7-1 was by transient transfection and lentivirus-mediated transduction. The podocyte injury and β-catenin signaling were assessed. The binding of NF-κB-p65 and B7-1 was tested by ChIP assay, and testified by luciferase reporter assessment. The protein binding with B7-1 was assessed by mass spectrum and defined by co-immunoprecipitation. The binding of β-catenin and B7-1 was also determined by ChIP assay. The expression of B7-1 was upregulated in various CKD models (ADR, db/db, 5/6 nephrectomyin and human biopsies), and increased predominantly in podocytes. In vivo, there is a slightly but significantly increase in urinary albumin secretion and podocyte injury in podo-B7-Tg mice at age of 3-month, and further elevated at 6-month-age. Compared with wild-type mice, administration of AOPPs in podo-B7-Tg mice further enhanced the expression of B7-1, which resulting in the acceleration of renal fibrosis, and the further elevated activation of β-catenin pathway. Knockdown of B7-1 could restore podocyte integrity and retard renal fibrosis in ADR and 5/6 nephrectomized mice. The activation of β-catenin pathway was also apparently abolished by B7-1 interference. In cultured cells, exogenous expression of B7-1 induced podocyte injury and β-catenin pathway activation. The transcriptional activity of B7-1 was regulated by NF-κB-p65 through directly triggering the gene promoter. Moreover, B7-1 was identified to bind with HSP90AB1 and LRP5, forming a complex to trigger the activation of β-catenin pathway. Interestingly, there were also several TCF/LEF binding consensus sites in the promoter region of B7-1. Ectopic expression of β-catenin also induced the mRNA and protein expression of B7-1. Podocyte B7-1 which depends on the regulation of NF-κB pathway, plays an important role in maintaining cellular homeostasis. Additionally, B7-1 binds to HSP90AB1 and LRP5 to form a complex, further inducing the activation of β-catenin. While β-catenin can also promote B7-1 transcription through TCF/LEF transcription factors. Our studies provide a new theory in the pathogenesis of glomerulosclerosis, and give an important clue to the therapeutic strategy to podocyte injury through targeting the two-way networks of B7-1 and β-catenin.

Planar cell polarity pathway in kidney development, function and disease.

Planar cell polarity (PCP) refers to the coordinated orientation of cells in the tissue plane. Originally discovered and studied in Drosophila melanogaster, PCP is now widely recognized in vertebrates, where it is implicated in organogenesis. Specific sets of PCP genes have been identified. The proteins encoded by these genes become asymmetrically distributed to opposite sides of cells within a tissue plane and guide many processes that include changes in cell shape and polarity, collective cell movements or the uniform distribution of cell appendages. A unifying characteristic of these processes is that they often involve rearrangement of actomyosin. Mutations in PCP genes can cause malformations in organs of many animals, including humans. In the past decade, strong evidence has accumulated for a role of the PCP pathway in kidney development including outgrowth and branching morphogenesis of ureteric bud and podocyte development. Defective PCP signalling has been implicated in the pathogenesis of developmental kidney disorders of the congenital anomalies of the kidney and urinary tract spectrum. Understanding the origins, molecular constituents and cellular targets of PCP provides insights into the involvement of PCP molecules in normal kidney development and how dysfunction of PCP components may lead to kidney disease.

Sustained activation of C3aR in a human podocyte line impairs the morphological maturation of the cells.

The C3a receptor (C3aR) has been reported to be involved in various physiological and pathological processes, including the regulation of cellular structure development. Expression of C3aR has been reported in podocytes; however, data concerning the role of C3aR in podocyte morphology is scarce. The aim of the present study was to examine the effect of C3aR activation on the architectural development of podocytes. An immortal human podocyte line (HPC) was transfected with a C3a expression lentivirus vector or recombinant C3a. SB290157 was used to block the activation of C3aR. The expression of C3a in HPC cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and ELISAs. Phase contrast and fluorescence microscopy were used to observe the morphology of the podocytes. The adhesive ability of HPC cells was analyzed using an attachment assay. RT-qPCR, cyto-immunofluorescence and western blotting were used to determine the expression levels of the adhesion-associated genes. The expression levels of carboxypeptidases in HPC cells was also detected by RT-qPCR. Compared with the untransfected and control virus-transfected HPC cells, the C3a-overexpressing cells (HPC-C3a) failed to expand their cell bodies and develop an arborized appearance in the process of maturation, which the control cells exhibited. In addition, HPC-C3a cells presented with decreased adhesive capacity, altered focal adhesion (FA) plaques and decreased expression of FA-associated genes. These effects were blocked by a C3aR antagonist; however, the addition of purified C3a could not completely mimic the effects of C3a overexpression. Furthermore, HPC cells expressed carboxypeptidases, which have been reported to be able to inactivate C3a. In summary, the results demonstrated that sustained C3aR activation impaired the morphological maturation of HPC cells, which may be associated with the altered expression of FA-associated genes and impaired FA. Since chronic complement activation has been reported in renal diseases, which indicate sustained C3aR activation in renal cells, including podocytes and podocyte progenitors, the possible role of C3aR in the dysregulation of podocyte architecture and podocyte regeneration requires further research.

Transcription Factor 21 Regulates Nephron Progenitor Cells Self-Renewal/Induction and Podocyte Development

Transcription Factor 21 Regulates Nephron Progenitor Cells Self-Renewal/Induction and Podocyte Development

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

Berberine protects against diabetic kidney disease via promoting PGC-1α-regulated mitochondrial energy homeostasis.

Background and PurposeDisordered lipid metabolism and disturbed mitochondrial bioenergetics play pivotal roles in the initiation and development of diabetic kidney disease (DKD). Berberine is a plant alkaloid, used in Chinese herbal medicine. It has multiple therapeutic actions on diabetes mellitus and its complications, including regulation of glucose and lipid metabolism, improvement of insulin sensitivity, and alleviation of oxidative damage. Here, we investigated the reno‐protective effects of berberine.Experimental ApproachWe used samples from DKD patients and experiments with models of DKD (db/db mice) and cultured podocytes, to characterize energy metabolism profiles using metabolomics. Molecular targets and mechanisms involved in the regulation of mitochondrial function and bioenergetics by berberine were investigated, along with its effects on metabolic alterations in DKD mice.Key ResultsMetabolomic analysis suggested altered mitochondrial fuel usage and generalized mitochondrial dysfunction in patients with DKD. In db/db mice, berberine treatment reversed the disordered metabolism, podocyte damage and glomerulosclerosis. Lipid accumulation, excessive generation of mitochondrial ROS, mitochondrial dysfunction, and deficient fatty acid oxidation in DKD mouse models and in cultured podocytes were suppressed by berberine. These protective effects of berberine were accompanied by activation of the peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) signalling pathway, which promoted mitochondrial energy homeostasis and fatty acid oxidation in podocytes.Conclusion and ImplicationsPGC‐1α‐mediated mitochondrial bioenergetics could play a key role in lipid disorder‐induced podocyte damage and development of DKD in mice. Restoration of PGC‐1α activity and the energy homeostasis by berberine might be a potential therapeutic strategy against DKD.