ABSTRACT
Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.
Subject(s)
Nephrotic Syndrome , Podocytes , Apoptosis , Cells, Cultured , Humans , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacology , Nephrotic Syndrome/chemically induced , Nephrotic Syndrome/drug therapy , Nephrotic Syndrome/metabolism , Podocytes/metabolism , Proteinuria/metabolism , Puromycin/pharmacology , Puromycin Aminonucleoside/metabolism , Puromycin Aminonucleoside/pharmacology , Rituximab/metabolism , Rituximab/pharmacologyABSTRACT
Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.
Subject(s)
Acute Kidney Injury , Induced Pluripotent Stem Cells , Acute Kidney Injury/chemically induced , Acute Kidney Injury/metabolism , Humans , Kidney , Organoids/metabolism , Puromycin Aminonucleoside/metabolism , Puromycin Aminonucleoside/pharmacologyABSTRACT
In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.
Subject(s)
Adenomatous Polyposis Coli/pathology , Albuminuria/pathology , Nephrotic Syndrome/pathology , Podocytes/pathology , Transcytosis/physiology , Adenomatous Polyposis Coli/metabolism , Albuminuria/metabolism , Animals , Disease Models, Animal , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Male , Mice , Mice, Inbred C57BL , Nephrotic Syndrome/chemically induced , Nephrotic Syndrome/metabolism , Podocytes/metabolism , Puromycin/pharmacology , Puromycin Aminonucleoside/pharmacologyABSTRACT
Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to exert protective effects on podocytes, whereas angiopoietin-like 3 (ANGPTL3) has been shown to exert significant pathogenic effects on these cells. This study aimed to investigate the link between the protective effects of PPARα activation and the pathogenic effects of ANGPTL3 in podocytes. Both PPARα and ANGPTL3 were expressed in cultured podocytes. PPARα mRNA and protein levels decreased whereas ANGPTL3 mRNA and protein levels increased in a time-dependent manner in podocytes treated with puromycin aminonucleoside (PAN). Gemfibrozil, a pharmacological agonist of PPARα, increased PPARα levels and activity in podocytes. The drug also decreased ANGPTL3 levels by potentially weakening ANGPTL3 promoter activity in both normal and PAN-treated podocytes. Furthermore, gemfibrozil significantly decreased PAN-induced apoptosis and F-actin rearrangement. Primary podocytes from Angptl3-knockout mice were cultured. There was no significant difference between Angptl3-/- podocytes treated with or without gemfibrozil in the lamellipodia numbers after PAN treatment. The results suggested that the protective effects of gemfibrozil on podocytes were not exerted following knockout of the Angptl3 gene. This study identified a novel mechanism of the PPARα agonist gemfibrozil that exerts its protective effects by inhibiting PAN-induced apoptosis and cytoskeleton rearrangements through inhibition of ANGPTL3 expression.
Subject(s)
Actin Cytoskeleton/drug effects , Angiopoietin-like Proteins/physiology , Gemfibrozil/pharmacology , PPAR alpha/agonists , Podocytes/drug effects , Pseudopodia/drug effects , Puromycin Aminonucleoside/pharmacology , Angiopoietin-Like Protein 3 , Animals , Apoptosis , Hypolipidemic Agents/pharmacology , Mice , Mice, Knockout , Podocytes/metabolism , Podocytes/pathology , Protective Factors , Pseudopodia/metabolismABSTRACT
Klotho deficiency was observed in virtually all kinds of kidney disease and is thought to play a critical role in podocyte injury. However, the underline mechanisms involved in podocyte injury remain unknown. miRNAs have diverse regulatory roles, and miR-30 family members were essential for podocyte homeostasis. Our study revealed that Klotho and miR-30s were downregulated in PAN-treated podocytes. The ectopic expression of Klotho ameliorates PAN induced podocyte apoptosis through upregulating miR-30a and downregulating Ppp3ca, Ppp3cb, Ppp3r1, and Nfact3 expression, which are the known targets of miR-30s. We also found that Klotho regulates TRPC6 via miR-30a to activate calcium/calcineurin signaling. Further, glucocorticoid (Dexamethasone, DEX) was found to sustain Klotho and miR-30a levels during PAN treatment in vitro. Eventually, in rats, PAN treatment substantially downregulated Klotho and miR-30a levels, lead to podocyte injury and increased proteinuria. The transfer of exogenous Klotho to podocytes of PAN-treated rats could increase miR-30a expression, reduce TRPC6 expression, and also ameliorated podocyte injury and proteinuria. In conclusion, Klotho, acting on miR-30s, which directly regulates its target genes, contributes to podocyte apoptosis induced by PAN. It is a novel mechanism underlying PAN-induced podocyte injury.
Subject(s)
Glucuronidase/metabolism , Kidney Diseases/metabolism , MicroRNAs/metabolism , Podocytes/metabolism , TRPC6 Cation Channel/metabolism , Animals , Calcium Signaling , Cells, Cultured , Dexamethasone/pharmacology , Disease Models, Animal , Down-Regulation , Humans , Kidney Diseases/genetics , Kidney Diseases/pathology , Klotho Proteins , Male , MicroRNAs/genetics , Podocytes/drug effects , Podocytes/pathology , Puromycin Aminonucleoside/pharmacology , Rats , Rats, WistarABSTRACT
BACKGROUND: Focal segmental glomerulosclerosis (FSGS) causes renal fibrosis and may lead to kidney failure. FSGS and its common complication, proteinuria, are challenging to treat. Corticosteroids are ineffective in many patients with FSGS, and alternative treatments often yield suboptimal responses. Repository corticotropin injection (RCI; Acthar® Gel), a naturally sourced complex mixture of purified adrenocorticotropic hormone analogs and other pituitary peptides, may have beneficial effects on idiopathic FSGS via melanocortin receptor activation. METHODS: Two studies in a preclinical (female Sprague-Dawley rats) puromycin aminonucleoside FSGS model assessed the effect of RCI on renal function and morphology: an 8-week comparison of a single RCI dose with methylprednisolone (N = 27), and a 12-week chronic RCI dose range study (N = 34). Primary outcomes were proteinuria and renal pathology improvements for measures of renal fibrosis, tubular damage, glomerular injury, and total kidney injury score. Impact of RCI treatment was also determined by assessing urinary biomarkers for renal injury, podocyte expression of podoplanin (a biomarker for injury), podocyte effacement by electron microscopy, and histological staining for fibrosis biomarkers. RESULTS: Compared with saline treatment, RCI 30 IU/kg significantly reduced proteinuria, with a 38% reduction in peak mean urine protein levels on day 28 in the 8-week model, and RCI 10 IU/kg, 30 IU/kg, and 60 IU/kg reduced peak mean urine protein in the 12-week model by 18, 47, and 44%, respectively. RCI also showed significant dose-dependent improvements in fibrosis, interstitial inflammation, tubular injury, and glomerular changes. Total kidney injury score (calculated from histopathological evaluations) demonstrated statistically significant improvements with RCI 30 IU/kg in the 8-week study and RCI 60 IU/kg in the 12-week study. RCI treatment improved levels of urinary biomarkers of kidney injury (KIM-1 and OPN), expression of podoplanin, and podocyte morphology. RCI also reduced levels of desmin and fibrosis-associated collagen deposition staining. Methylprednisolone did not improve renal function or pathology in this model. CONCLUSIONS: These results provide evidence supporting the improvement of FSGS with RCI, which was superior to corticosteroid treatment in this experimental model. To the authors' knowledge, this is the first evidence that a drug for the treatment of FSGS supports podocyte recovery after repeated injury.
Subject(s)
Adrenal Cortex Hormones/therapeutic use , Adrenocorticotropic Hormone/administration & dosage , Glomerulosclerosis, Focal Segmental/drug therapy , Kidney/pathology , Animals , Biomarkers/urine , Disease Models, Animal , Female , Fibrosis , Glomerulosclerosis, Focal Segmental/pathology , Glomerulosclerosis, Focal Segmental/physiopathology , Injections , Kidney/drug effects , Kidney/metabolism , Membrane Glycoproteins/metabolism , Podocytes/pathology , Proteinuria/prevention & control , Puromycin Aminonucleoside/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Smad3 signaling and transgelin expression are often activated during puromycin aminonucleoside (PAN)-induced podocyte injury. Here, we investigated whether the Smad3 inhibitor SIS3 can ameliorate damage to injured podocytes. A model of PAN-induced podocyte injury was constructed using the MPC5 cell line. The effects of SIS3 on the expression of the podocyte cytoskeletal proteins transgelin, p15INK4B , phosphor-smad3, phosphor-JAK/stat3, the apoptotic marker cleaved caspase 3, and c-myc were investigated using western blot. The distribution of F-actin in PAN-induced podocyte injury was observed under an immunofluorescence microscope. PAN-induced podocyte injury altered the distribution of F-actin and transgelin, and colocalization of these two proteins was observed. Transgelin expression and Smad3 phosphorylation were increased in the MPC5 cell line with prolonged PAN treatment. In addition, c-myc expression, p15INK4B , and JAK phosphorylation were all increased after treatment with PAN. Treatment with the Smad3 inhibitor SIS3 reversed these phenomena and protected against PAN-induced podocyte injury. Moreover, stimulating podocytes directly with TGFß-1 also led to enhanced expression of transgelin or phosphor-JAK/stat3, and this could be inhibited by SIS3. In conclusion, transgelin expression was induced through the Smad3 signaling pathway during PAN-induced podocyte injury, and the resulting abnormal distribution of F-actin and the enhanced expression of transgelin could be reversed by blockade of this pathway.
Subject(s)
Isoquinolines/pharmacology , Podocytes/drug effects , Puromycin Aminonucleoside/antagonists & inhibitors , Pyridines/pharmacology , Pyrroles/pharmacology , Smad3 Protein/antagonists & inhibitors , Animals , Cells, Cultured , Mice , Podocytes/metabolism , Podocytes/pathology , Puromycin Aminonucleoside/pharmacology , Smad3 Protein/metabolismABSTRACT
Objective: The aim of our study is to investigate the relationship between podocyte autophagy and apoptosis induced by Puromycin Aminonucleoside (PAN) and to clarify its mechanism.Methods: Podocytes were cultured in vitro. The apoptosis rates of each group were detected using flow cytometry. The expression of LC3-II protein and changes in distribution were detected through laser scanning confocal microscope, and the western blot protocol was employed for detection of protein expression of LC3-II. The autophagosomes were detected by transmission electron microscopy.Results: In this study, We found that autophagosome increased followed by apoptosis after podocyte injury. Furthermore, we conformed that the activation of autophagy could inhibit the apoptosis to alleviate the injury of podocyte at an early stage.Conclusions: Autophagy occurred earlier before apoptosis and autophagy mediated podocyte apoptosis induced by PAN. These findings indicate that autophagy may become a novel therapeutic target for the treatment of podocyte injury and proteinuria in the future.
Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Podocytes/pathology , Puromycin Aminonucleoside/pharmacology , Animals , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagosomes/ultrastructure , Blotting, Western , Cell Shape/drug effects , Cells, Cultured , Mice , Podocytes/drug effects , Podocytes/ultrastructure , Time FactorsABSTRACT
BACKGROUND: Oxidative stress is a major factor responsible for minimal-change nephrotic syndrome (MCNS), which occurs most commonly in children. However, the influence of oxidative stress localized to mitochondria remains unclear. We examined the effect of a mitochondrion-targeting antioxidant, MitoTEMPO, in rats with puromycin aminonucleoside (PAN)-induced MCNS to clarify the degree to which mitochondrial oxidative stress affects MCNS. MATERIALS AND METHODS: Thirty Wistar rats were divided into three groups: normal saline group (n = 7), PAN group (n = 12), and PAN + MitoTEMPO group (n = 11). Rats in the PAN and PAN + MitoTEMPO groups received PAN on day 1, and those in the PAN + MitoTEMPO group received MitoTEMPO on days 0 to 9. Whole-day urine samples were collected on days 3 and 9, and samples of glomeruli and blood were taken for measurement of lipid peroxidation products. We also estimated the mitochondrial damage score in podocytes in all 3 groups using electron microscopy. RESULTS: Urinary protein excretion on day 9 and the levels of lipid peroxidation products in urine, glomeruli, and blood were significantly lower in the PANã+ãMitoTEMPO group than in the PAN group (p = 0.0019, p = 0.011, p = 0.039, p = 0.030). The mitochondrial damage score in podocytes was significantly lower in the PANã+ãMitoTEMPO group than in the PAN group (p <0.0001). CONCLUSIONS: This mitochondrion-targeting agent was shown to reduce oxidative stress and mitochondrial damage in a MCNS model. A radical scavenger targeting mitochondria could be a promising drug for treatment of MCNS.
Subject(s)
Antioxidants/pharmacology , Drug Delivery Systems , Mitochondria , Nephrosis, Lipoid , Organophosphorus Compounds/pharmacology , Piperidines/pharmacology , Proteinuria , Puromycin Aminonucleoside/adverse effects , Animals , Lipid Peroxidation/drug effects , Male , Mitochondria/metabolism , Mitochondria/pathology , Nephrosis, Lipoid/chemically induced , Nephrosis, Lipoid/drug therapy , Nephrosis, Lipoid/pathology , Nephrosis, Lipoid/urine , Oxidative Stress/drug effects , Podocytes/metabolism , Podocytes/pathology , Proteinuria/chemically induced , Proteinuria/drug therapy , Proteinuria/pathology , Proteinuria/urine , Puromycin Aminonucleoside/pharmacology , Rats , Rats, WistarABSTRACT
BACKGROUND: Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear. METHODS: To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. RESULTS: Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridge-like prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. CONCLUSIONS: The three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.
Subject(s)
Glomerular Basement Membrane/pathology , Imaging, Three-Dimensional , Nephrosis/pathology , Podocytes/pathology , Puromycin Aminonucleoside/pharmacology , Tomography, X-Ray Computed/methods , Animals , Cells, Cultured , Disease Models, Animal , Injections, Intraperitoneal , Male , Microscopy, Electron, Scanning/methods , Nephrosis/chemically induced , Podocytes/cytology , Podocytes/drug effects , Random Allocation , Rats , Rats, Wistar , Reference ValuesABSTRACT
Triptriolide (T11) is a natural diterpene diepoxide that derived from Chinese traditional herb medicine (TCHM) Tripterygium wilfordii Hook.F (TWHF). From a structural point of view, T11 is very similar to triptolide (T9), one of the most effectively compounds in TWHF that have already been systematically investigated in the past decades. However, the basic functions and medicinal properties of T11 have not yet been well investigated mainly due to its low abundance in its plant organ. The present study aimed to investigate the protective effects of T11 on puromycin aminonucleoside (PAN) induced apoptotic mouse podocytes and the underlying mechanism. The results showed that T11 had no significant toxicity in podocytes in high dosage, and showed prominent protective effects on PAN induced podocytes injury. Further studies indicated that T11 might exert its protective effects by inhibiting of apoptosis and restoring of survival in PAN induced podocytes.
Subject(s)
Apoptosis/drug effects , Diterpenes/pharmacology , Phenanthrenes/pharmacology , Podocytes/drug effects , Animals , Drugs, Chinese Herbal/pharmacology , Epoxy Compounds/pharmacology , Mice , Puromycin Aminonucleoside/pharmacology , Tripterygium/chemistryABSTRACT
Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.
Subject(s)
Doxorubicin/adverse effects , Gene Expression Regulation/drug effects , Kidney Diseases/metabolism , Podocytes/metabolism , Puromycin Aminonucleoside/adverse effects , Signal Transduction/drug effects , Transcriptome/drug effects , Animals , Doxorubicin/pharmacology , Humans , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Mice , Phosphorylation/drug effects , Podocytes/pathology , Puromycin Aminonucleoside/pharmacologyABSTRACT
MicroRNAs are a group of small non-coding RNAs that play key roles in almost every aspect of mammalian cell. In kidney, microRNAs are required for maintaining normal function of renal cells, disruption of which contributes to pathogenesis of renal diseases. In this study, we investigated the potential role of miRNAs as key regulators of podocyte survival by using a primary cell culture model from non-human primates (NHPs). Through microRNA profile comparison in glomeruli from mouse, rat and NHP, miR-27b was found to be among a list of glomeruli-enriched miRNA conserved across species. In NHP primary podocyte culture, significant downregulation of miR-27b was observed during treatment of puromycin aminonucleoside (PAN), a classic nephrotoxin. Overexpression of miR-27b enhanced PAN-induced apoptosis and cytoskeleton destruction in podocytes while its inhibition had a protective effect. Target identification analysis identified Adora2b as a potential direct target of miR-27b. Ectopic expression of miR-27b suppressed both Adora2b mRNA and protein expression, whereas inhibition of miR-27b increased the transcript and protein expression levels of Adora2B. Dual luciferase assay further confirmed Adora2b as a direct target of miR-27b. Furthermore, knockdown of Adora2b by siRNAs enhanced PAN-induced apoptosis, similar to the phenotypes we had observed with miR-27b overexpression. In addition, stimulating the adenosine signaling by an Adora2b agonist, NECA, improved podocyte survival upon PAN treatment. Taken together, our data identified a novel role of miR-27b-adora2b axis in primary podocyte survival upon injury and suggested a critical role of adenosine signaling pathway in podocyte protection.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell Survival/drug effects , MicroRNAs/genetics , Podocytes/drug effects , Puromycin Aminonucleoside/pharmacology , Receptor, Adenosine A2B/genetics , Animals , Antagomirs/genetics , Antagomirs/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Macaca fascicularis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , MicroRNAs/agonists , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , Podocytes/cytology , Podocytes/metabolism , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2B/metabolism , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Signal TransductionABSTRACT
BACKGROUND/AIMS: The purpose of this study was to investigate the impact of rapamycin (RAP) on autophagy in podocytes and the therapeutic effects of RAP on idiopathic membranous nephropathy (IMN). METHODS: We established an in vitro model of IMN by preconditioning mouse podocytes with puromycin aminonucleoside (PAN). A Cell Counting Kit-8 was used to detect the proliferation of each group of podocytes. Podocyte apoptosis was analyzed by flow cytometry via annexin V/propidium iodide dual staining. Subsequently, we observed the number of autophagosomes by transmission electron microscopy. Western blotting was used to detect the levels of LC3, mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, and p-P70S6K in each group. RESULTS: The number of podocytes in the PAN + 100 ng/mL RAP group, PAN + 200 ng/mL RAP group, and PAN + 300 ng/mL RAP group was significantly increased (P < 0.01). The apoptotic rate of podocytes was significantly different between the PAN group and the PAN + RAP group (P < 0.001). There were fewer autophagic corpuscles in the PAN group and more autophagosomes were observed in the PAN + RAP group. LC3 protein expression was down-regulated in the PAN group, while its expression was up-regulated in the PAN + RAP group. In the PAN group, the levels of phosphorylated mTOR, 4EBP1, and P70S6K were increased, while in the PAN + RAP group, protein phosphorylation was reduced. CONCLUSIONS: RAP can effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway, and activate podocyte autophagy, consequently reducing podocyte apoptosis. Therefore, RAP could be used for the treatment of idiopathic membranous nephropathy.
Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Signal Transduction/drug effects , Sirolimus/pharmacology , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Cell Proliferation/drug effects , Eukaryotic Initiation Factors , Mice , Microtubule-Associated Proteins/metabolism , Phosphoproteins/metabolism , Podocytes/cytology , Podocytes/drug effects , Podocytes/metabolism , Puromycin Aminonucleoside/pharmacology , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oxidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron microscope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matrix. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.
Subject(s)
Gene Expression Regulation/drug effects , Kidney Glomerulus/metabolism , Nephrosis/chemically induced , Nephrosis/metabolism , Puromycin Aminonucleoside/adverse effects , STAT3 Transcription Factor/metabolism , Animals , Kidney Glomerulus/ultrastructure , Male , Nephrosis/pathology , Phosphorylation/drug effects , Puromycin Aminonucleoside/pharmacology , Rats , Rats, WistarABSTRACT
Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a downstream mediator of PI3K, which is essential for maintaining the functional integrity of podocytes. However, little is known about the role of SGK3 in podocyte function. Herein, we demonstrated that SGK3 contributes to the maintenance of podocyte integrity. Conditionally immortalized mouse podocyte cells (MPCs) were treated with puromycin aminonucleoside (PAN). PAN treatment inhibited the activity of SGK3 and the expression of podocin. Short hairpin RNA (shRNA)-mediated knockdown of SGK3 also reduced podocin expression in the absence of PAN. Adriamycin (ADR)-treated mice developed proteinuria and had decreased renal glomerular SGK3 expression in comparison to control mice. Consistent with a role for SGK3 in the ADR effect, SGK3 knockout (KO) mice had markedly reduced kidney podocin expression and significantly elevated proteinuria compared with wild-type mice. Electron microscopy revealed that SGK3 KO mice displayed partial effacement of podocyte foot processes. Further, a SGK3 target protein, glycogen synthase kinase-3 (GSK3), was discovered to be dramatically activated in PAN and SGK3 shRNA-treated MPCs and in SGK3 KO mice. Taken together, these data strongly suggest that SGK3 plays a significant role in regulating podocyte function, likely by controlling the expression and activity of GSK3.-Peng, L.-Q., Zhao, H., Liu, S., Yuan, Y.-P., Yuan, C.-Y., Mwamunyi, M.-J., Pearce, D., Yao, L.-J. Lack of serum- and glucocorticoid-inducible kinase 3 leads to podocyte dysfunction.
Subject(s)
Podocytes/enzymology , Protein Serine-Threonine Kinases/deficiency , Animals , Cell Line, Transformed , Gene Expression Regulation, Enzymologic/drug effects , Gene Knockdown Techniques , Intracellular Signaling Peptides and Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Podocytes/pathology , Protein Serine-Threonine Kinases/metabolism , Puromycin Aminonucleoside/adverse effects , Puromycin Aminonucleoside/pharmacologyABSTRACT
Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target CTGF was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Mechanotransduction, Cellular , Phosphoproteins/metabolism , Podocytes/metabolism , Transcription Factors/metabolism , Acyltransferases , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Fluorescent Antibody Technique , HEK293 Cells , Humans , Kidney Glomerulus/metabolism , Male , Mice , Phosphoproteins/genetics , Podocytes/cytology , Podocytes/drug effects , Proteinuria/genetics , Proteinuria/metabolism , Proteomics , Puromycin Aminonucleoside/pharmacology , Rats , Stress, Mechanical , Transcription Factors/genetics , YAP-Signaling ProteinsABSTRACT
Glucocorticoids (GCs) are commonly used in the treatment of nephrotic syndrome. However, high doses and long periods of GC therapy can result in severe side effects. The present study aimed to selectively deliver albuminmethylprednisolone (MP) nanoparticles towards glomerular podocytes, which highly express the specific neonatal Fc receptor (FcRn) of albumin. Bovine serum albumin (BSA) was labeled with a fluorescent dye and linked with modified MP via an amide bond. The outcome nanoparticle named BSA633MP showed a uniform size with a diameter of approximately 10 nm and contained 12 drug molecules on average. The nanoconjugates were found to be stable at pH 7.4 and acidsensitive at pH 4.0, with approximately 72% release of the MP drug after 48 h of incubation. The nanoparticle demonstrated a 36fold uptake in receptorspecific cellular delivery in the FcRnexpressing human podocytes compared to the uptake in the non-FcRn-expressing control cells. Colocalization further confirmed that uptake of the nanoconjugates involved receptormediated endocytosis followed by lysosome associated transportation. In vitro cellular experiments indicated that the BSA633MP ameliorated puromycin aminonucleosideinduced podocyte apoptosis. Moreover, in vivo fluorescence molecular imaging showed that BSA633-MP was mainly accumulated in the liver and kidney after intravenous dosing for 24 h. Collectively, this study may provide an approach for the effective and safe therapy of nephrotic syndrome.
Subject(s)
Drug Carriers , Histocompatibility Antigens Class I/metabolism , Methylprednisolone , Nanoparticles/chemistry , Podocytes/metabolism , Receptors, Fc/metabolism , Serum Albumin, Bovine , Animals , Apoptosis/drug effects , Cattle , Cell Line, Transformed , Drug Carriers/chemistry , Drug Carriers/pharmacology , Humans , Methylprednisolone/chemistry , Methylprednisolone/pharmacology , Puromycin Aminonucleoside/adverse effects , Puromycin Aminonucleoside/pharmacology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/pharmacologyABSTRACT
PURPOSE: The purpose of this study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSC) on podocytes of puromycin amino nuclear glucoside (PAN) -induced nephrosis in mice. METHODS: Mice were randomly divided into Control, PAN and BMSC groups. Mice were injected with PAN (0.5 mg/g weight) via the tail vein. The 24-h urinary protein was obtained after modelling, and urinary protein excretion was determined. The blood and kidney specimens were isolated after the tenth day of modelling. Blood samples were collected for measuring serum creatinine (SCr) and blood urea nitrogen (BUN). A sample of kidney was taken for observing pathological changes through hematoxylin-eosin staining and electron microscopy, and the rest of the kidney was used for detecting the protein and mRNA expression of nephrin, CD2AP, synaptopodin, TRPC6 by real-time quantitative PCR, Western-blot and immunohistochemistry. RESULTS: After PAN injection, podocyte foot process fusion was detected by electron microscopy, and the 24 h urinary protein excretion increased compared with control mice on days 3, 7 and 10 post-PAN injection (P.
Subject(s)
Bone Marrow Cells/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Nephrosis/therapy , Podocytes/metabolism , Allografts , Animals , Bone Marrow Cells/pathology , Mesenchymal Stem Cells/pathology , Mice, Inbred BALB C , Nephrosis/chemically induced , Nephrosis/metabolism , Nephrosis/pathology , Podocytes/pathology , Puromycin Aminonucleoside/adverse effects , Puromycin Aminonucleoside/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Therapeutic options for treating glomerulopathies, the main cause of chronic kidney disease, are limited. Podocyte dedifferentiation is a major event in the pathogenesis of glomerulopathies. The goal of the present study was, therefore, to develop an assay to monitor podocyte differentiation suitable for compound screening. EXPERIMENTAL APPROACH: We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under the control of the promoter of nephrin, a marker of podocyte differentiation. Mean CFP fluorescence intensity per glomerulus (MFG) was determined by summation of all glomerular voxels from confocal z-stacks in the absence and presence of pharmaceutical compounds. KEY RESULTS: In untreated cultured glomeruli, MFG remained fairly stable during the first 5 days, when foot processes were already effaced, and the level of many podocyte-specific proteins was only mildly affected, as revealed by proteomics. Between day 6 and 9, MFG decreased to almost zero. The decrease in MFG was paralleled by a decrease in CFP and nephrin expression, as determined by RT-PCR, western blots and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration-dependently induced a complete loss of MFG. Dexamethasone (25 µM) and pioglitazone (10 µM) markedly attenuated the effect of 0.6 µg·mL-1 PAN on MFG. CONCLUSION AND IMPLICATIONS: In summary, we established a novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes in situ. Our assay is suitable for compound screening to identify drugs for the treatment of glomerulopathies.