OBJECTIVETo observe the effect of 11R-VIVIT on lipopolysaccharide (LPS)-induced expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes.

METHODSA LPS-induced proteinuria mouse model and in vitro cultured podocytes treated with LPS were both divided into control group, LPS group and LPS+11 R-VIVIT group. The mRNA and protein expressions of uPAR in mouse kidney tissues and the podocytes were measured by real-time qPCR, laser scanning confocal microscopy and Western blotting.

RESULTSCompared with LPS group, LPS+11 R-VIVIT group showed a significantly lowered urine albumin/creatinine ratio (P<0.001) and markedly reduced mRNA and protein expressions of uPAR (PuPAR mRNA<0.001; PuPAR=0.001).

CONCLUSION11R-VIVIT can ameliorate proteinuria probably by decreasing the expression of uPAR in podocytes.

Animals ; Disease Models, Animal ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Mice, Inbred C57BL ; Oligopeptides ; pharmacology ; Podocytes ; drug effects ; metabolism ; Proteinuria ; drug therapy ; metabolism ; Receptors, Urokinase Plasminogen Activator ; metabolism

OBJECTIVETo explore the effect of resveratrol on transforming growth factor-beta1 (TGF-beta1) induced transdifferentiation of podocytes.

METHODSMouse podocytes in vitro cultured under differentiating conditions for 10 days were divided into the normal group, the model group, the high dose resveratrol group, and the low dose resveratrol group. The podocytes in the high and low dose resveratrol groups were intervened with 5 micromol/L and 2 micromol/L resveratrol respectively for 30 min. Those in the model group and the two resveratrol treated groups were continually incubated with 5 ng/mL TGF-beta1 for 72 h. Those in the normal group were routinely cultured. The protein expression of podocyte phenotypic protein molecules such as E-cadherin, P-cadherin, zonula occludens-1 (ZO-1), NEPH1, and alpha-smooth muscle-actin (alpha-SMA) were detected by immunocytochemistry, flow cytometry (FCM), and Western blot. A simple albumin influx assay was used to evaluate the filtration barrier function of podocyte monolayer.

RESULTSCompared with the normal control group, E-cadherin (+) percentage rate, the protein expression of P-cadherin, ZO-1, and NEPH1 significantly decreased in the model group (P < 0.05), but the expression of alpha-SMA and albumin permeability across podocyte monolayers increased significantly (P < 0.05). Compared with the model group, E-cadherin (+) percentage rate significantly increased (P < 0.05) and albumin permeability across podocyte monolayers decreased significantly (P < 0.05) in the high and low dose resveratrol groups. In the low dose resveratrol group, the expression of P-cadherin and NEPH1 significantly increased (P < 0.05). In the high dose resveratrol group, the expression of P-cadherin, ZO-1, and NEPH1 increased significantly, and the expression of alpha-SMA decreased significantly (P < 0.05). The correlations between resveratrol concentrations and the expression of E-cadherin (+), P-cadherin, and NEPH1 were significantly positive (r(E-cadherin (+)) = 0.772, r(P-cadherin) = 0.756, r(NEPH1) = 0.809, P < 0.05).

CONCLUSIONThe role of resveratrol in inhibiting TGF-beta1 induced phenotype abnormality might be an important mechanism for preserving the integrality of glomerular filtration barrier and decreasing proteinuria.

Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Kidney Tubules ; cytology ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; Stilbenes ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo observe the effect of amiloride on the proteinuria of the 5/6 nephrectomy rats.

METHODSTo establish the 5/6 nephrectomy rats model and divide the experiment into 3 groups, sham operated group(Sham), 5/6 nephrectomy model group(NTX) and 5/6 nephrectomy with amiloride-treated group (NTX+amiloride, n=15). The concentration of protein and mRNA of uPAR and the change of podocytes motility were detected by coomassiebluestaining, immunofluorence method and real-time PCR.

RESULTSAt second week, compared with Control group, the 24 h urine protein of NTX group was significantly increased (47.50 ± 28.05 mg vs 14.28 ± 3.8 mg, P = 0.023). There was no statistical significance in 24-hour urine protein between NTX+amiloride group and NTX group (51.56 ± 21.03 mg vs 47.50 ± 28.05 mg, P = 0.748). The same situation was also observed at the time point of 12 week, comparing with NTX group, 24-hour urine protein decreased in Sham group (188.31 ± 29.82 mg vs 21.32 ± 8.59 mg, P = 0.000) and NTX+amiloride group (188.31 ± 29.82 mg vs 121.37 ± 31.14 mg, P=0.000), with statistical significance when comparing with Sham group, the expression of uPAR mRNA in NTX group was significantly increased (9.74 ± 1.44 vs 1.01 ± 0.13, P = 0.000). In contrast, the expression of uPAR mRNA in NTX rats treated with amiloride was significantly lower than in NTX group (9.74 ± 1.44 vs 5.01 ± 1.36, P = 0.000).

CONCLUSIONAmiloride can reduce the proteinuria of the 5/6 nephrectomy rats model of transient proteinuria by inhibiting the induction of uPAR expression.

Amiloride ; pharmacology ; Animals ; Cell Movement ; Disease Models, Animal ; Nephrectomy ; Podocytes ; drug effects ; metabolism ; Proteinuria ; drug therapy ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Urokinase Plasminogen Activator ; 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

AIMTo investigate the effect and mechanism of ascorbic acid on podocyte, last barrier of glomerular filtration, in diabetic rats.

METHODSDiabetic rats induced by streptozotocin injection intraperitoneally were treated by ascorbic acid for 5 weeks. The levels of blood glucose (BG), HbA1c, urinary albumin excretion rate (UAER) and superoxide diamutase (SOD), catalase (CAT) and malondialdehyde (MDA) in renal cortex were measured. The podocyte ultrastructure was observed while the expression of desmin protein, a marker of podocyte injury, was examined.

RESULTSCompared with control group, BG and HbA1c were increased markedly in diabetic group. The activities of SOD and CAT were decreased and the concentrations of MDA were increased significantly in diabetic renal cortex. There were the increased proteinic expression of desmin, foot process effacement in podocytes and UAER markedly in diabetic rats. Compared with diabetic rats, foot process effacement and the changes of UAER were ameliorated markedly while the activities of SOD were increased, the levels of MDA and proteinic expression of desmin were decreased markedly although BG, HbA1c and the activities of CAT were no significant difference in the diabetic rats by ascorbic acid treatment.

CONCLUSIONThe findings suggest that there are marked injury in podocyte, last barrier of glomerular filtration, in diabetic rats and administration of ascorbic acid can protect podocyte by increasing antioxidative capacity and ameliorating the renal oxidative stress.

Animals ; Ascorbic Acid ; pharmacology ; Catalase ; metabolism ; Desmin ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; physiopathology ; Diabetic Nephropathies ; metabolism ; pathology ; physiopathology ; Male ; Oxidative Stress ; drug effects ; Podocytes ; drug effects ; metabolism ; ultrastructure ; Random Allocation ; Rats ; Rats, Wistar ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the effect of arctiin on mouse podocyte epithelial-mesenchymal transition (EMT) induced by advanced oxidation protein products (AOPP).

METHODSMouse podocytes were stimulated by 200 µg/ml AOPP for 24 h in the presence of 50, 100, 200, and 400 µmol/L arctiin. The expressions of α-smooth muscle actin, Grp78 and CHOP were detected using Western blotting.

RESULTThe expressions of α-SMA, Grp78 and CHOP were inhibited by arctiin, showing a dose-dependent effect within a given range of arctiin concentration.

CONCLUSIONAOPP causes endoplasmic reticulum stress to induce EMT of mouse podocytes, and arctiin can decrease EMT by alleviating the stress. This finding sheds light on a new scope of research of renal fibrosis.

Actins ; metabolism ; Advanced Oxidation Protein Products ; adverse effects ; Animals ; Cell Line ; Endoplasmic Reticulum Stress ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Furans ; pharmacology ; Glucosides ; pharmacology ; Heat-Shock Proteins ; metabolism ; Mice ; Podocytes ; metabolism ; pathology ; Transcription Factor CHOP ; metabolism

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

OBJECTIVETo investigate the effect of Qufeng Tongluo Recipe (QTR) on the expression of desmin and CD2-associated protein (CD2AP) in adriamycin-induced nephropathy rats.

METHODSThe adriamycin-induced nephropathy rat model was induced by a disposable intravenous injection of adriamycin. The model was successfully established after 3 weeks. Rats were then randomly divided into the blank control group (Group A, n =12), the model control group (Group B, n = 8), the small, medium, large dose QTR group (Group C, n = 8; Group D, n = 8; Group E, n = 8), and the positive control group (Group F, n = 8). From the fourth week normal saline was given to rats in Group A and Group B, QTR 1.0 g/mL, 2.1 g/mL, and 4.2 g/mL was respectively administered to those in Group C, D, and E. Prednisone 25 mg/kg was given to rats in Group F. All medication was performed by gastrogavage at 10 mL/kg, once daily, for 28 successive days. 24-h urinary protein excretion and sera biochemical indices were determined during medication. At the end of the experiment, ultrastructure was observed, mRNA expression of desmin, mRNA and protein of CD2AP were detected by Real-time PCR and Western blot.

RESULTS(1) Compared with Group B, 24-h urinary protein excretion significantly decreased in Group C, D, E, and F (P < 0.05). (2) Compared with Group B, Alb in Group C, D, and E increased (P < 0.05) and TC significantly decreased (P < 0.05). TG significantly increased in Group F (P < 0.05). (3) Results of electron microscope showed, compared with Group B, the morphology of foot cells was improved to various degrees in Groups D, E, and F, especially the foot process structure and the number of foot processes were significantly improved, which was more obviously shown in Group D and Group E. (4) mRNA expression of desmin, mRNA and protein of CD2AP increased in adriamycin-induced nephropathy rats (P < 0.05). After intervention, when compared with Group B, mRNA expression of desmin and CD2AP were significantly lower in Group C, D, E, and F (P < 0.05). (5) Compared with Group A, expression of desmin and CD2AP significantly increased (P < 0.05). Compared with Group B, the expression of desmin protein were obviously lower in Group C, D, E, and F, and the protein expression of desmin obviously decreased in Group D, E, and F (P < 0.05). The protein expression of desmin and CD2AP gradually decreased in Group C, D, and E (P < 0.05). Compared with Group F, the expression of CD2AP protein obviously increased in Group C and D (P < 0.05); the expression of CD2AP protein obviously decreased in Group E (P < 0.05); the expression of desmin protein was higher in Group C, D, and E (P < 0.05).

CONCLUSIONQTR's therapeutic effect on adriamycin-induced nephropathy rats might be achieved through altered expression of desmin and CD2AP.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Cytoskeletal Proteins ; metabolism ; Desmin ; metabolism ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Kidney Diseases ; chemically induced ; drug therapy ; metabolism ; Male ; Phytotherapy ; Podocytes ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved.

METHODSThe expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting.

RESULTSHigh glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes.

CONCLUSIONCTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Diabetic Nephropathies ; Glucose ; adverse effects ; MAP Kinase Signaling System ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Sialoglycoproteins ; metabolism

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