As a major active component extracted from traditional Chinese herb Tripterygium wilfordii Hook F, triptolide exhibits multiple pharmacological effects. Autophagy is an evolutionary conserved intracellular catabolic process involved in cytoplasmic materials degradation. Autophagic dysfunction contributes to the pathologies of many human diseases, which makes it a promising therapeutic target. Recent studies have shown that triptolide exerts neuroprotection, anti-tumor activities, organ toxicity, and podocyte protection by modulating autophagy. This article highlights the current information on triptolide-modulated autophagy, analyzes the possible pathways involved, and describes the crosstalk between autophagy and apoptosis modulated by triptolide, in hope of providing implications for the roles of autophagy in pharmacological effects of triptolide and expanding its novel usage as an autophagy modulator.
Animals ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Humans ; Neoplasms ; drug therapy ; pathology ; Neuroprotective Agents ; pharmacology ; Phenanthrenes ; pharmacology ; Podocytes ; drug effects

The aim of this paper was to explore the effects of triptolide( TP),the effective component of Tripterygium wilfordii on improving podocyte epithelial-mesenchymal transition( EMT) induced by high glucose( HG),based on the regulative mechanisms of Nod-like receptor protein 3( NLRP 3) inflammasome in the kidney of diabetic kidney disease( DKD). The immortalized podocytes of mice in vitro were divided into the normal( N) group,the HG( HG) group,the low dose of TP( L-TP) group,the high dose of TP( HTP) group and the mannitol( MNT) group,and treated by the different measures,respectively. More specifically,the podocytes in each group were separately treated by D-glucose( DG,5 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) + TP( 5 μg·L~(-1))or HG( 30 mmol·L~(-1)) + TP( 10 μg·L~(-1)) or DG( 5 mmol·L~(-1)) + MNT( 24. 5 mmol·L~(-1)). After the treatment of HG or TP at 24,48 and 72 h,firstly,the activation of podocyte proliferation was investigated. Secondly,the protein expression levels of the epithelial markers in podocytes such as nephrin and ZO-1,the mesenchymal markers such as collagen Ⅰ and fibronectin( FN) were detected,respectively. Finally,the protein expression levels of NLRP3 and apoptosis-associated speck-like protein( ASC) as the key signaling molecules of NLRP3 inflammasome activation,as well as the downstream effector proteins including caspase-1,interleutin( IL)-1β and IL-18 were examined,severally. The results indicated that,for the cultured podocytes in vitro,HG could cause the low protein expression levels of nephrin and ZO-1,induce the high protein expression levels of collagen Ⅰ and FN and trigger podocyte EMT. Also HG could cause the high protein expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 and induce NLRP3 inflammasome activation. On the other hand,the co-treatment of TP( L-TP or H-TP) and HG for podocytes could recover the protein expression levels of nephrin and ZO-1,inhibit the protein expression levels of collagen Ⅰ and FN and ameliorate podocyte EMT. Also the co-treatment of TP( L-TP or H-TP) and HG could down-regulate the protein expression levels of NLRP3 and ASC,inhibit NLRP3 inflammasome activation and reduce the protein expression levels of the downstream effector molecules including caspase-1,IL-1β and IL-18. On the whole,HG could activate NLRP3 inflammasome and induce podocyte EMT in vitro. TP at the appropriate dose range could inhibit NLRP3 inflammasome activation and ameliorate podocyte EMT,which may be one of the critical molecular mechanisms of TP protecting againstpodocyte inflammatory injury in DKD.
Animals ; Caspase 1/metabolism* ; Cells, Cultured ; Diabetic Nephropathies ; Diterpenes/pharmacology* ; Epithelial-Mesenchymal Transition ; Epoxy Compounds/pharmacology* ; Glucose ; Inflammasomes/metabolism* ; Interleukin-18/metabolism* ; Interleukin-1beta/metabolism* ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Phenanthrenes/pharmacology* ; Podocytes/drug effects*

To explore the effects and molecular mechanisms of triptolide（TP）on improving podocyte epithelial-mesenchymal transition（EMT）induced by high dose of D-glucose（HG）, the immortalized podocytes of mice were divided into the normal group（N）, the high dose of D-glucose group（HG）, the low dose of TP group（L-TP）, the high dose of TP group（H-TP）and the mannitol group（MNT）, and treated by the different measures respectively. More specifically, the podocytes in each group were separately treated by D-glucose（DG, 5 mmol·L⁻¹）or HG（25 mmol·L⁻¹）or HG（25 mmol·L⁻¹）+ TP（3 μg·L⁻¹）or HG（25 mmol·L⁻¹）+ TP（10 μg·L⁻¹）or DG（5 mmol·L⁻¹）+ MNT（24.5 mmol·L⁻¹）. After the intervention for 24, 48 and 72 hours, firstly, the activation of podocyte proliferation was investigated. Secondly, the protein expression levels of the epithelial markers in podocytes such as nephrin and podocin, the mesenchymal markers such as desmin and collagen Ⅰ and the EMT-related mediators such as snail were detected respectively. Finally, the protein expression levels of Wnt3α and β-catenin as the key signaling molecules in Wnt3α/β-catenin pathway were examined severally. The results indicated that, HG could cause the low protein expression levels of nephrin and podocin and the high protein expression levels of desmin, collagen Ⅰ and snail in podocytes, and inducing podocyte EMT. On the other hand, HG could cause the high protein expression levels of Wnt3α and β-catenin in podocytes, and activating Wnt3α/β-catenin signaling pathway. In addition, L-TP had no effect on the activation of podocyte proliferation, the co-treatment of L-TP and HG could significantly recover the protein expression levels of nephrin and podocin, inhibit the protein expression levels of desmin, collagen I and snail in podocytes, thus, further improving podocyte EMT. And that, the co-treatment of L-TP and HG could obviously decrease the high protein expression levels of Wnt3α and β-catenin induced by HG in podocytes, and inhibit Wnt3α/β-catenin signaling pathway activation. On the whole, HG can induce podocyte EMT by activating Wnt3α/β-catenin signaling pathway; L-TP can ameliorate podocyte EMT through inhibiting Wnt3α/β-catenin signaling pathway activation, which may be one of the effects and molecular mechanisms .
Animals ; Cells, Cultured ; Diterpenes ; pharmacology ; Epithelial-Mesenchymal Transition ; Epoxy Compounds ; pharmacology ; Glucose ; Mice ; Phenanthrenes ; pharmacology ; Podocytes ; drug effects ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; beta Catenin ; metabolism

All-trans retinoic acid (ATRA) is a vitamin A derivative and plays an important role in the regulation of cell aggregation, differentiation, apoptosis, proliferation, and inflammatory response. In recent years, some progress has been made in the role of ATRA in renal diseases, especially its protective effect on podocytes. This article reviews the research advances in podocyte injury, characteristics of ATRA, podocyte differentiation and regeneration induced by ATRA, and the protective effect of ATRA against proliferation, deposition of fibers, and apoptosis.
Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cytoprotection ; Humans ; Podocytes ; drug effects ; physiology ; Tretinoin ; pharmacology

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.
AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Angiotensin II/*pharmacology ; Angiotensin II Type 1 Receptor Blockers/pharmacology ; Animals ; Blotting, Western ; Cell Line ; Cell Nucleus/metabolism ; Crk-Associated Substrate Protein/*metabolism ; Cytoplasm/metabolism ; Focal Adhesion Kinase 1/metabolism ; Losartan/pharmacology ; Metformin/pharmacology ; Mice ; Microscopy, Confocal ; Podocytes/cytology/drug effects/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Ribonucleotides/pharmacology ; Signal Transduction/*drug effects

OBJECTIVETo observe the effect of Wenyang Huoxue Lishui Recipe (WHLR) containing serum on the expression of cathepsin L (CatL) in puromycin aminonucleoside-induced injury of mouse glomerular podocytes.

METHODSMouse podocyte cells (MPCs) in vitro cultured were divided into the normal control group, the model group, the dexamethasone (DEX) group, 10% WHLR containing serum group, 20% WHLR containing serum group, the vehicle serum control group. MPCs in the normal control group were cultured at 37 degrees C culture solution for 24 h. 45 mg/L puromycin was acted on MPCs in the model group for 24 h. On the basis of puromycin intervention, 1 limol/L DEX was co-incubated in MPCs of the DEX group for 24 h; 10% or 20% WHLR containing serum was co-incubated in MPCs of the 10% WHLR containing serum group and 20% WHLR containing serum group for 24 h. The vehicle serum control group was also set up by incubating with WHLR containing serum alone for 24 h. The expression of CatL and its substrate Synaptopodin in podocytes were detected by cell immunofluorescence staining. FITC-conjugated phalloidin was used to stain F-actin. A cortical F-actin score index (CFS index) was designed to quantify the degree of cytoskeletal reorganization in cultured podocytes.

RESULTSCompared with the normal control group, the expression of synaptopodin significantly decreased and the expression of CatL significantly-increased in the model group. F-actin arranged in disorder, gradually forming pericellular F-actin ring. CFS index was obviously elevated (P < 0.01). Compared with the model group, the epression of synaptopodin increased, the expression of CatL decreased, and CFS index also decreased in the DEX group, 10% WHLR containing serum group, and 20% WHLR containing serum group (P < 0.05, P < 0.01). Compared with the DEX group, the expression of synaptopodin decreased in 10% WHLR containing serum group, CFS index also decreased in 20% WHLR containing serum group (P < 0.05).

CONCLUSIONSWHLR could up-regulate the expression of synaptopodin, down-regulate the expression of CatL, and alleviate cytoskeletal reorganization of F-actin. It was helpful to stabilize the cytoskeleton of F-actin and improve the merging of podocytes.

Actins ; metabolism ; Animals ; Cathepsin L ; metabolism ; Cells, Cultured ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Kidney Glomerulus ; cytology ; Mice ; Microfilament Proteins ; metabolism ; Podocytes ; drug effects ; pathology ; Puromycin Aminonucleoside ; adverse effects ; Up-Regulation

The present study was designed to determine the mechanism underlying the treatment of nephrotic syndrome using astragaloside by observing the effects of astragaloside on the expression of nephrin and podocin proteins and genes in kidneys of rats with adriamycin nephropathy. The rats were injected with adriamycin and, after successful model establishment, randomly divided into a model group, a Methylprednisolone (MP) group, and an astragaloside group. The 24-h complete urine samples were collected. Biochemical indicators were monitored, and kidney tissues were collected for pathological analysis using light microscopy and electron microscopy. The mRNA expression of nephrin and podocin was measured in the kidney tissues using the real-time qPCR, and the protein expression levels of nephrin and podocin were detected using Western blot analysis. At the end of 12 weeks of drug intervention, the urinary protein level was lower in the MP and astragaloside groups than that in the model group (P = 0.008 and P = 0.01, respectively). Serum albumin was higher in the MP and astragaloside groups than in the model group (P < 0.001 and P = 0.012, respectively). Podocytes in the MP group were nearly normal, and fusion of podocytes in the astragaloside group was significantly less than that in the control group. The nephrin and podocin mRNA and protein expression levels in the intervention groups were higher (P < 0.05) than that in the model group. Due to the increased expression of podocyte-related nephrin and podocin proteins, astragaloside maintained slit diaphragm integrity and decreased the level of proteinuria in rats with adriamycin nephropathy.
Animals ; Astragalus Plant ; chemistry ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Glucosides ; administration & dosage ; Humans ; Kidney ; drug effects ; metabolism ; Kidney Diseases ; chemically induced ; drug therapy ; Male ; Podocytes ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

PURPOSE: The aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes. MATERIALS AND METHODS: Human podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting. RESULTS: ZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 microM) montelukast treatment (p<0.01; n=3). CONCLUSION: Our results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.
Acetates/*pharmacology ; Blotting, Western ; Dose-Response Relationship, Drug ; Humans ; Interleukin-13/*pharmacology ; Leukotriene Antagonists/*pharmacology ; Microscopy, Confocal ; Podocytes/*drug effects/metabolism ; Proteinuria/pathology ; Quinolines/*pharmacology ; Tight Junctions ; Zonula Occludens-1 Protein/*metabolism

PURPOSE: The aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes. MATERIALS AND METHODS: Human podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting. RESULTS: ZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 microM) montelukast treatment (p<0.01; n=3). CONCLUSION: Our results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.
Acetates/*pharmacology ; Blotting, Western ; Dose-Response Relationship, Drug ; Humans ; Interleukin-13/*pharmacology ; Leukotriene Antagonists/*pharmacology ; Microscopy, Confocal ; Podocytes/*drug effects/metabolism ; Proteinuria/pathology ; Quinolines/*pharmacology ; Tight Junctions ; Zonula Occludens-1 Protein/*metabolism

OBJECTIVETo investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN).

METHODSThirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSThe PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group.

CONCLUSIONThe PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

Animals ; Creatinine ; blood ; Female ; Kidney ; drug effects ; metabolism ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; Male ; Podocytes ; drug effects ; metabolism ; Proteinuria ; pathology ; Puromycin Aminonucleoside ; adverse effects ; RANK Ligand ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor Activator of Nuclear Factor-kappa B ; metabolism