Project description:The aim of the study was to gain mechanistic insights of the protective effect of forskolin on thapsigargin-induced podocyte death by assessing, analyzing and comparing the respective gene expression profiles. Overall design: Gene expression profiles of vehicle (DMSO)-, thapsi-, and thapsi+fors-treated podocytes were analyzed. 3 replicates per condition. Vehicle (DMSO)-treated cells were served as control/reference samples.
Project description:Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R.
Project description:Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.
Project description:Investigation of DNAse Hypersensitivity changes in podocytes cultured under normal or high glucose conditions transfected with a miR-93 mimic or a nontargeting mimic. Examination of the changes in hypersensitivity induced by high glucose culture conditions compared to normal glucose conditions to mimic the diabetic millieu. Further, to see if miR-93 overexpression can reverse these changes.
Project description:Primary membranous nephropathy is usually caused by antibodies against the podocyte antigen membrane M-type phospholipase A2 receptor (PLA2R). The treatment of membranous nephropathy is not fully satisfactory. The calcineurin inhibitor tacrolimus is used to treat membranous nephropathy, but recurrence upon drug withdrawal is common. TNF superfamily members are key mediators of kidney injury. We have now identified key TNF receptor superfamily members in podocytes and explored the regulation of PLA2R expression and the impact of tacrolimus. Data mining of single cell transcriptomics and glomerular transcriptomics data identified TNFRSF12a/Fn14 as the highest expressed TNF receptor superfamily gene in human membranous nephropathy, and this was confirmed by immunohistochemistry that also identified NF?B activation in membranous nephropathy podocytes. Additionally, glomerular transcriptomics identified PLA2R1 expression as being increased in membranous nephropathy in the parenteral administration of the Fn14 ligand TWEAK increased podocyte PLA2R expression in mice. Furthermore, in cultured human podocytes, TWEAK increased the expression of PLA2R as well as the expression of other genes recently identified by GWAS as linked to membranous nephropathy: NFKB1 and IRF4. Interestingly, IRF4 encodes the FK506-binding protein 52 (FKBP52), a protein associated with tacrolimus. Tacrolimus prevented the increased expression of PLA2R, NFKB1 and IRF4 induced by TWEAK in cultured podocytes. In conclusion, TWEAK upregulates the expression of PLA2R and of other genes linked to membranous nephropathy in podocytes, and this is prevented by tacrolimus. An impact of tacrolimus on the expression of PLA2R and other genes in podocytes may underlie its efficacy in treating the disease as well as the frequent recurrence of nephrotic syndrome upon tacrolimus withdrawal.
Project description:Aim:Our study is aimed at investigating whether Lipopolysaccharide- (LPS-) treated podocytes could polarize naive CD4+ T cells into different subsets in vitro. Materials and Methods:Podocytes and mouse bone marrow-derived dendritic cells (BMDCs) were first cultured with 25??g/ml LPS for 6 hours, respectively. Then, naive CD4+ T cells were cocultured with the LPS-treated podocytes or BMDCs at a ratio of 1?:?1 or 1?:?1?:?1. After 48 hours, we collected the suspended cells and supernatant from all groups to measure T helper (Th)17 cells, regulatory T (Treg) cells, and cytokine concentration. Results:We observed the expression of CD80 and major histocompatibility complex class II molecule (MHC II) in podocytes but did not found the upregulation of them after treating podocytes with LPS. LPS-treated podocytes could induce naive CD4+ T cells to Th17 cells and Treg cells with a higher ratio of Th17/Treg than BMDCs. Possible interaction between podocytes and BMDCs may exist in the induction process of Th17 cells and Treg cells. Conclusion:Our study proved that CD80 and MHC II were constitutively expressed in podocytes but not upregulated by LPS. LPS-treated podocytes could polarize naive CD4+ T cells into Th17 and Treg cells and affect the Th17/Treg balance and may incline to cause a Th17 response.
Project description:It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN). Here we used shRNA transfection to knockdown the insulin receptor (IR) gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.
Project description:Glomerular podocytes are highly differentiated epithelial cells that are key components of the kidney filtration units. Podocyte damage or loss is the hallmark of nephritic diseases characterized by severe proteinuria. Recent studies implicate that hormones including glucocorticoids (ligand for glucocorticoid receptor) and vitamin D3 (ligand for vitamin D receptor) protect or promote repair of podocytes from injury. In order to elucidate the mechanisms underlying hormone-mediated podocyte-protecting activity from injury, we carried out microarray gene expression studies to identify the target genes and corresponding pathways in response to these hormones during podocyte differentiation. We used immortalized human cultured podocytes (HPCs) as a model system and carried out in vitro differentiation assays followed by dexamethasone (Dex) or vitamin D3 (VD3) treatment. Upon the induction of differentiation, multiple functional categories including cell cycle, organelle dynamics, mitochondrion, apoptosis and cytoskeleton organization were among the most significantly affected. Interestingly, while Dex and VD3 are capable of protecting podocytes from injury, they only share limited target genes and affected pathways. Compared to VD3 treatment, Dex had a broader and greater impact on gene expression profiles. In-depth analyses of Dex altered genes indicate that Dex crosstalks with a broad spectrum of signaling pathways, of which inflammatory responses, cell migration, angiogenesis, NF-?B and TGF? pathways are predominantly altered. Together, our study provides new information and identifies several new avenues for future investigation of hormone signaling in podocytes.
Project description:Progressive kidney diseases affect approximately 500 million people worldwide. Podocytes are terminally differentiated cells of the kidney filter, the loss of which leads to disease progression and kidney failure. To date, there are no therapies to promote podocyte survival. Drug repurposing may therefore help accelerate the development of cures in an area of tremendous unmet need. In a newly developed high-throughput screening assay of podocyte viability, we identified the BRAFV600E inhibitor GDC-0879 and the adenylate cyclase agonist forskolin as podocyte-survival-promoting compounds. GDC-0879 protects podocytes from injury through paradoxical activation of the MEK/ERK pathway. Forskolin promotes podocyte survival by attenuating protein biosynthesis. Importantly, GDC-0879 and forskolin are shown to promote podocyte survival against an array of cellular stressors. This work reveals new therapeutic targets for much needed podocyte-protective therapies and provides insights into the use of GDC-0879-like molecules for the treatment of progressive kidney diseases.
Project description:Studies have shown that podocytes and renal tubular epithelial cells from patients with HIV-associated nephropathy (HIVAN) express HIV-1 transcripts, suggesting that productive infection of renal epithelial cells precipitates development of HIVAN. However, podocytes and renal tubular epithelial cells do not express CD4 receptors, and it is unclear how these cells become productively infected <i>in vivo</i> We investigated the mechanisms underlying the infection by HIV-1 of podocytes cultured from the urine of children with HIVAN. We observed low-level productive infection on exposure of these cells to primary cell-free HIV-1 supernatants. However, envelope-defective recombinant HIV-1 did not infect the renal epithelial cell lines. Moreover, treatment of podocytes to inhibit endocytic transport or dynamin activity or remove cell surface heparan sulfate proteoglycans reduced infection efficiency. Transfection of CD4- 293T cells with a cDNA expression library developed from a podocyte cell line derived from a child with HIVAN led to the identification of TNF-<i>?</i> as a possible mediator of HIV-1 infection. Overexpression of transmembrane TNF-<i>?</i> in cultured CD4- renal tubular epithelial cells, 293T cells, and HeLa cells enabled the infection of these cells; exposure to soluble TNF-<i>?</i> did not. Immunohistochemistry showed TNF-<i>?</i> expression in podocytes of renal sections from children with HIVAN. Furthermore, we found that TNF-<i>?</i> enhanced NF-<i>?</i>B activation and integration of HIV-1 into the podocyte DNA. Finally, inhibition of dynamin activity blocked TNF-<i>?-</i>mediated infection. These data establish a role for transmembrane TNF-<i>?</i> in facilitating the viral entry and integration of HIV-1 into the DNA of renal epithelial cells.