GDF-15 Suppresses Puromycin Aminonucleoside-Induced Podocyte Injury by Reducing Endoplasmic Reticulum Stress and Glomerular Inflammation.

GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its association with kidney disease progression, while other studies identified GDF15 to have protective effects. To investigate the potential protective role of GDF15 on podocytes, we first performed in vitro studies using a Gdf15-deficient podocyte cell line. The lack of GDF15 intensified puromycin aminonucleoside (PAN)-triggered endoplasmic reticulum stress and induced cell death in cultivated podocytes. This was evidenced by elevated expressions of Xbp1 and ER-associated chaperones, alongside AnnexinV/PI staining and LDH release. Additionally, we subjected mice to nephrotoxic PAN treatment. Our observations revealed a noteworthy increase in both GDF15 expression and secretion subsequent to PAN administration. Gdf15 knockout mice displayed a moderate loss of WT1+ cells (podocytes) in the glomeruli compared to wild-type controls. However, this finding could not be substantiated through digital evaluation. The parameters of kidney function, including serum BUN, creatinine, and albumin-creatinine ratio (ACR), were increased in Gdf15 knockout mice as compared to wild-type mice upon PAN treatment. This was associated with an increase in the number of glomerular macrophages, neutrophils, inflammatory cytokines, and chemokines in Gdf15-deficient mice. In summary, our findings unveil a novel renoprotective effect of GDF15 during kidney injury and inflammation by promoting podocyte survival and regulating endoplasmic reticulum stress in podocytes, and, subsequently, the infiltration of inflammatory cells via paracrine effects on surrounding glomerular cells.

[Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology].

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

Characteristics of sodium and water retention in rats with nephrotic syndrome induced by puromycin aminonucleoside.

INTRODUCTION: Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated. METHODS: The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na+ channel (ENaC), Na+/H+ exchanger 3 (NHE3), Na+-K+-2Cl- cotransporter 2 (NKCC2) and Na+-Cl- cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected. RESULTS: The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group. CONCLUSIONS: The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.

Nicorandil protects podocytes via modulation of antioxidative capacity in acute puromycin aminonucleoside-induced nephrosis in rats.

Nephrotic syndrome, characterized by proteinuria and hypoalbuminemia, results from the dysregulation of glomerular podocytes and is a significant cause of end-stage kidney disease. Patients with idiopathic nephrotic syndrome are generally treated with immunosuppressive agents; however, these agents produce various adverse effects. Previously, we reported the renoprotective effects of a stimulator of the mitochondrial ATP-dependent K+ channel (MitKATP), nicorandil, in a remnant kidney model. Nonetheless, the cellular targets of these effects remain unknown. Here, we examined the effect of nicorandil on puromycin aminonucleoside-induced nephrosis (PAN) rats, a well-established model of podocyte injury and human nephrotic syndrome. PAN was induced using a single intraperitoneal injection. Nicorandil was administered orally at 30 mg/kg/day. We found that proteinuria and hypoalbuminemia in PAN rats were significantly ameliorated following nicorandil treatment. Immunostaining and ultrastructural analysis under electron microscopy demonstrated that podocyte injury in PAN rats showed a significant partial attenuation following nicorandil treatment. Nicorandil ameliorated the increase in the oxidative stress markers nitrotyrosine and 8-hydroxy-2-deoxyguanosine in glomeruli. Conversely, nicorandil prevented the decrease in levels of the antioxidant enzyme manganese superoxide dismutase in PAN rats. We found that mitochondrial Ca2+ uniporter levels in glomeruli were higher in PAN rats than in control rats, and this increase was significantly attenuated by nicorandil. We conclude that stimulation of MitKATP by nicorandil reduces proteinuria by attenuating podocyte injury in PAN nephrosis, which restores mitochondrial antioxidative capacity, possibly through mitochondrial Ca2+ uniporter modulation. These data indicate that MitKATP may represent a novel target for podocyte injury and nephrotic syndrome.NEW & NOTEWORTHY Our findings suggest that the mitochondrial Ca2+ uniporter may be an upstream regulator of manganese superoxide dismutase and indicate a biochemical basis for the interaction between the ATP-sensitive K+ channel and Ca2+ signaling. We believe that our study makes a significant contribution to the literature because our results indicate that the ATP-sensitive K+ channel may be a potential therapeutic target for podocyte injury and nephrotic syndrome.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology / 中国中药杂志

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

The role of PTEN in puromycin aminonucleoside-induced podocyte injury.

Podocytes are specialized cells of the glomerulus that play important structural and functional roles in maintaining the filtration barrier. Loss and injury of podocytes are leading factors of glomerular disease and kidney failure. Recent studies found that phosphatase and tensin homolog (PTEN) may play a critical role in maintaining the normal structure and function in podocytes. However, we still understand very little about how PTEN is regulated under podocyte injury conditions. In this study, We therefore investigated whether PTEN could play a role in podocyte injury induced by puromycin aminonucleoside (PAN), and whether dexamethasone (DEX) alleviates podocyte injury by PTEN/PI3K/Akt signaling. Our results showed that PI3K/Akt pathway was activated in podocytes exposed to PAN conditions, accompanied by down-regulation of the PTEN and microtubule-associated light chain 3 (LC3) expression.podocyte-specific knockout of PTEN significantly promoted podocyte injury, The potential renoprotection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and the inhibition of apoptosis.These novel findings also suggest that targeting PTEN might be a novel and promising therapeutic strategy against podocyte injury.

Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes.

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.

Synthesis of a Peptidoyl RNA Hairpin via a Combination of Solid-Phase and Template-Directed Chain Assembly.

Peptidoyl RNAs are the products of ribosome-free, single-nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting from a synthetic and a bioorganic point of view. A synthesis of a stabilized version of peptidoyl RNA, with an amide bond between the C-terminus of a peptide and a 3'-amino-2',3'-dideoxynucleoside in the RNA chain was developed. The preferred synthetic route used an N-Teoc-protected aminonucleoside support and involved a solution-phase coupling of the amino-terminal oligonucleotide to a dipeptido dinucleotide. Exploratory UV-melting and NMR analysis of the hairpin 5'-UUGGCGAAAGCdC-LeuLeu-AA-3' indicated that the peptide-linked RNA segments do not fold in a cooperative fashion. The synthetic access to doubly RNA-linked peptides on a scale sufficient for structural biology opens the door to the exploration of their structural and biochemical properties.

Sinkihwan-gamibang ameliorates puromycin aminonucleoside-induced nephrotic syndrome.

Nephrotic syndrome (NS) is a kidney disease characterized by hypertriglyceridemia, massive proteinuria, hypo-albuminemia and peripheral edema. Sinkihwan-gamibang (SKHGMB) was recorded in a traditional Chinese medical book named "Bangyakhappyeon ()" and its three prescriptions Sinkihwan, Geumgwe-sinkihwan, and Jesaeng-sinkihwan belong to Gamibang. This study confirmed the effect of SKHGMB on renal dysfunction in an NS model induced by puromycin aminonucleoside (PAN). The experimental NS model was induced in male Sprague Dawley (SD) rats through injection of PAN (50 mg·kg-1)via the femoral vein. SKHGMB not only reduced the size of the kidneys increased due to PAN-induced NS, but also decreased proteinuria and ascites. In addition, SKHGMB significantly ameliorated creatinine clearance, creatinine, and blood urea nitrogen. SKHGMB relieved glomeruli dilation and tubules fibrosis in the glomeruli of the NS model. SKHGMB inhibited the protein and mRNA levels of the NLRP3 inflammasome including NLRP3, ASC, and pro-caspase-1 in NS rats. SKHGMB reduced the protein and mRNA levels of fibrosis regulators in NS rats. The results indicated that SKHGMB exerts protective effects against renal dysfunction by inhibiting of renal inflammation and fibrosis in NS rats.

Hirudin attenuates puromycin aminonucleoside-induced glomerular podocyte injury by inhibiting MAPK-mediated endoplasmic reticulum stress.

Damage to podocytes is an important determinant of renal pathology. The puromycin aminonucleoside (PAN) mice nephropathy model is commonly used in the study of renal disease with podocyte injury. Hirudin has a broad nephroprotective effect and has been shown to treat renal interstitial fibrosis in previous studies. Mice were given PAN by gavage to prepare animal models, and MPC5 cells were incubated with PAN in vitro. Twenty-four hours urine was collected for analysis of urinary protein levels. Renal pathological changes were observed by hematoxylin and eosin staining. Immunofluorescence detection of nephrin in kidney tissues and cells. Apoptosis was analyzed with over TUNEL. Cytoskeleton, endoplasmic reticulum stress (ERS), p38 MAPK signaling, and apoptosis-related proteins were assessed by western blot analysis. The data suggested that hirudin attenuated reduced renal injury and increased urine protein in PAN mice. Hirudin also attenuated cytoskeletal protein (synaptopodin, nephrin, and podocin) disruption, ERS activation, and apoptosis in PAN mice and PAN-induced podocytes. In addition, hirudin inhibited the expression of p38 MAPK signaling key proteins upregulated by PAN, thereby suppressing ERS. The p38 MAPK agonist was able to partially antagonize the inhibition of p38 MAPK signaling by hirudin in PAN-induced podocytes, thereby reactivating the ERS inhibited by hirudin, promoting cytoskeletal protein degradation and increasing the level of apoptosis. In conclusion, hirudin could decrease podocyte injury by inhibiting p38 MAPK signaling-mediated ERS, resulting in the protection of the kidney from PAN damage. These findings may provide an experimental basis for hirudin treatment of podocyte injury diseases.

The Nephrotoxin Puromycin Aminonucleoside Induces Injury in Kidney Organoids Differentiated from Induced Pluripotent Stem Cells.

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.

Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics.

Lymphatic vessels are highly responsive to changes in the interstitial environment. Previously, we showed renal lymphatics express the Na-K-2Cl cotransporter. Since interstitial sodium retention is a hallmark of proteinuric injury, we examined whether renal sodium affects NKCC1 expression and the dynamic pumping function of renal lymphatic vessels. Puromycin aminonucleoside (PAN)-injected rats served as a model of proteinuric kidney injury. Sodium 23Na/1H-MRI was used to measure renal sodium and water content in live animals. Renal lymph, which reflects the interstitial composition, was collected, and the sodium analyzed. The contractile dynamics of isolated renal lymphatic vessels were studied in a perfusion chamber. Cultured lymphatic endothelial cells (LECs) were used to assess direct sodium effects on NKCC1. MRI showed elevation in renal sodium and water in PAN. In addition, renal lymph contained higher sodium, although the plasma sodium showed no difference between PAN and controls. High sodium decreased contractility of renal collecting lymphatic vessels. In LECs, high sodium reduced phosphorylated NKCC1 and SPAK, an upstream activating kinase of NKCC1, and eNOS, a downstream effector of lymphatic contractility. The NKCC1 inhibitor furosemide showed a weaker effect on ejection fraction in isolated renal lymphatics of PAN vs controls. High sodium within the renal interstitium following proteinuric injury is associated with impaired renal lymphatic pumping that may, in part, involve the SPAK-NKCC1-eNOS pathway, which may contribute to sodium retention and reduce lymphatic responsiveness to furosemide. We propose that this lymphatic vessel dysfunction is a novel mechanism of impaired interstitial clearance and edema in proteinuric kidney disease.

Sinkihwan-gamibang ameliorates puromycin aminonucleoside-induced nephrotic syndrome / 中国天然药物

Nephrotic syndrome (NS) is a kidney disease characterized by hypertriglyceridemia, massive proteinuria, hypo-albuminemia and peripheral edema. Sinkihwan-gamibang (SKHGMB) was recorded in a traditional Chinese medical book named "Bangyakhappyeon ()" and its three prescriptions Sinkihwan, Geumgwe-sinkihwan, and Jesaeng-sinkihwan belong to Gamibang. This study confirmed the effect of SKHGMB on renal dysfunction in an NS model induced by puromycin aminonucleoside (PAN). The experimental NS model was induced in male Sprague Dawley (SD) rats through injection of PAN (50 mg·kg-1)via the femoral vein. SKHGMB not only reduced the size of the kidneys increased due to PAN-induced NS, but also decreased proteinuria and ascites. In addition, SKHGMB significantly ameliorated creatinine clearance, creatinine, and blood urea nitrogen. SKHGMB relieved glomeruli dilation and tubules fibrosis in the glomeruli of the NS model. SKHGMB inhibited the protein and mRNA levels of the NLRP3 inflammasome including NLRP3, ASC, and pro-caspase-1 in NS rats. SKHGMB reduced the protein and mRNA levels of fibrosis regulators in NS rats. The results indicated that SKHGMB exerts protective effects against renal dysfunction by inhibiting of renal inflammation and fibrosis in NS rats.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

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.

Deiodinase-3 is a thyrostat to regulate podocyte homeostasis.

BACKGROUND: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. METHODS: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. FINDINGS: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of Dio3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvß3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. INTERPRETATION: The study provided a mechanistic insight into how D3-αvß3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves' disease, explained a plausible link between thyroid disorder and NS. FUNDING: This work was supported by American Thyroid Association (ATA-2018-050.R1).

Effect of tacrolimus on the expression of Park7 in glomerular podocytes injured by puromycin aminonucleoside. / ä»–å ‹èŽ«å¸å¯¹å˜Œå‘¤éœ‰ç´ æŸä¼¤çš„è‚¾å°çƒè¶³ç»†èƒžé‡ç»„äººå¸•é‡‘æ£®è›‹ç™½7表达的影响.

OBJECTIVES: To study the effect of puromycin aminonucleoside (PAN) on the apoptosis of mouse podocyte clone 5 (MPC-5) and the expression of recombinant human Parkinson's disease 7 (Park7) and to study the protective mechanism of tacrolimus (FK506) against MPC-5 injury. METHODS: MPC-5 cells were cultured in vitro and then divided into three groups: blank control (control), PAN, and FK506. The cells in the PAN group were added with PAN (with a concentration of 50 mg/L) to establish a model of MPC-5 injury, and those in the FK506 group were added with PAN (with a concentration of 50 mg/L) and FK506 (with a concentration of 5 mg/L). An inverted microscope was used to observe the morphology and structure of MPC-5 cells at 12, 24, and 48 hours after treatment. Flow cytometry was used to measure cell apoptosis rate. Quantitative real-time PCR was used to measure the mRNA expression of Park7. Western blot and immunofluorescent staining were used to measure the protein expression of Park7. RESULTS: The control group had a large number of foot processes of the cell body at all time points, with tight connections between cells and a normal morphology. Compared with the control group, the PAN group had a significantly smaller cell volume at all time points, with loose connections between cells and the presence of ruptured cells. Compared with the PAN group, the FK506 group had an increased cell volume at all time points, with tighter connections between cells and a better morphology. The PAN group had a significantly higher apoptosis rate than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the apoptosis rate at all time points (P<0.01). The PAN group had a significantly higher mRNA expression level of Park7 than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the mRNA expression level of Park7 at all time points (P<0.01). Western blot showed that the PAN group had a significantly higher protein expression level of Park7 than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the protein expression level of Park7 at all time points (P<0.01). Immunofluorescent staining showed that in the PAN group, there was a significantly lower expression of Park7 protein in cell membrane and cytoplasm, with a dense cluster distribution and increased fluorescence intensity. Compared with the PAN group, the FK506 group had a significant improvement in the distribution of Park7 protein. CONCLUSIONS: PAN can act on MPC-5 cells and cause morphological and structural damage and apoptosis of MPC-5 cells, as well as upregulated mRNA and protein expression of Park7. FK506 can downregulate the mRNA and protein expression of Park7 in the model of MPC-5 injury, maintain cellular homeostasis, reduce proteinuria, and delay glomerulosclerosis.

PPARα agonist exerts protective effects in podocyte injury via inhibition of the ANGPTL3 pathway.

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.

Inhibition of microRNA-130b Alleviated Podocyte Injury Induced by Puromycin Aminonucleoside in vitro.

OBJECTIVE: To investigate the effect of microRNA-130b (miR-130b) on podocyte injury induced by puromycin aminonucleoside (PAN) and its possible mechanisms. METHODS: The immortalized podocytes (HPC) were treated by 25, 50, or 100 µg/mL PAN, then real-time polymerase chain reaction (PCR) was used to detect the expression of miR-130b. The HPC were transfected with miR-130b inhibitor or normal control (NC) inhibitor, and then the cells were stimulated with 100 µg/mL PAN for 24h. Western blot was used to detect the protein expression of synaptopodin and nephrin. Phalloidin dying was used to detect the changes in the cytoskeleton. Flow cytometry was used to measure podocyte apoptosis. Luciferase reporter gene assays were performed to explore the interaction between miR-130b and PGC1α. RESULTS: PAN significantly upregulated the expression of miR-130b. The western blot showed that inhibition of miR-130b increased the protein expression of synaptopodin and nephrin compared to the negative control inhibitor group. The phalloidin dying showed that inhibition of miR-130b alleviated cytoskeletal remodeling of podocytes induced by PAN. Flow-cytometric analysis showed that apoptosis was decreased after miR-130b silencing. The miR-130b mimic could significantly down-regulate the protein expression of PGC1α, and the dual luciferase reporter assay showed that miR-130b induced a decrease in PGC1α 3'-UTR luciferase activity compared to the control mimic group, but there was no significant difference between the control mimic group and the mut·PGC1α 3'-UTR group. CONCLUSION: miR-130b ameliorates podocyte injury induced by PAN through inhibiting the expression of PGC1α.

Dysfunction of the Klotho-miR-30s/TRPC6 axis confers podocyte injury.

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.

Effects of tacrolimus on autophagy protein LC3 in puromycin-damaged mouse podocytes.

OBJECTIVE: To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury. METHODS: An in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha (LC3) expression were compared. RESULTS: Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group. CONCLUSIONS: Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.