OBJECTIVE: To observe the effects of acupuncture on podocyte autophagy and long non-coding RNA SOX2 overlapping transcript (LncRNA SOX2OT)/mammalian target of rapamycin C1 (mTORC1)/Unc-51-like kinase 1 (ULK1) pathway in rats with diabetic kidney disease (DKD), and to explore the mechanism by which acupuncture reduces urinary protein. METHODS: A total of 40 SPF-grade male Sprague-Dawley rats were randomly divided into a control group (n=10) and a modeling group (n=30). The DKD model was established by feeding a high-fat, high-sugar diet combined with intraperitoneal injection of streptozotocin (STZ) in the modeling group. Twenty rats with successful DKD model were randomly divided into a model group (n=10) and an acupuncture group (n=10). The acupuncture group received "spleen and stomach-regulating" acupuncture at bilateral "Zusanli" (ST36), "Fenglong" (ST40), "Yinlingquan" (SP9), and "Zhongwan" (CV12), 30 min per session, once daily, five times per week, for four weeks. The general condition, fasting blood glucose (FBG), 2-hour postprandial glucose (2hPG), serum creatinine (SCr), blood urea nitrogen (BUN), 24-hour urinary protein quantification, and urine albumin-to-creatinine ratio (UACR) were compared before and after the intervention. After intervention, urinary podocyte injury marker SPON2 was measured by ELISA. Podocyte autophagosomes and glomerular basement membrane ultrastructure in renal tissue were observed via transmission electron microscopy. Podocyte apoptosis was assessed by TUNEL staining. The protein expression of microtubule-associated protein 1 light chain 3Ⅱ (LC3-Ⅱ), mTORC1, ULK1, Beclin-1, and p62 in renal tissue was detected by Western blot. LncRNA SOX2OT expression in renal tissue was measured by real-time PCR. RESULTS: After the intervention, compared with the control group, the model group exhibited increased food and water intake, increased urine output, weight loss, and loose stools; compared with the model group, the food and water intake, urine volume, and loose stools were improved in the acupuncture group. Compared with the control group, FBG, 2hPG, SCr, BUN, 24-hour urinary protein quantification, UACR, and urinary SPON2 were all higher in the model group (P<0.01); compared with the model group, the FBG, 2hPG, SCr, BUN, 24-hour urinary protein quantification, UACR, and urinary SPON2 were all lower in the acupuncture group (P<0.01). Compared with the control group, the model group showed reduced podocyte autophagosomes and thickened glomerular basement membrane; compared with the model group, the acupuncture group had increased podocyte autophagosomes and less thickened basement membrane. Compared with the control group, the podocyte apoptosis index (AI) was higher in the model group (P<0.01); compared with the model group, the AI was lower in the acupuncture group (P<0.01). Compared with the control group, the expression of ULK1, Beclin-1, and LC3-Ⅱ proteins was lower, and the expression of mTORC1 and p62 proteins was higher in the model group (P<0.01). Compared with the model group, the expression of ULK1, Beclin-1, and LC3-Ⅱ proteins was higher, and the expression of mTORC1 and p62 proteins was lower in the acupuncture group (P<0.01). Compared with the control group, the LncRNA SOX2OT expression was lower in the model group (P<0.01). Compared with the model group, LncRNA SOX2OT expression was higher in the acupuncture group (P<0.01). CONCLUSION The "spleen and stomach-regulating" acupuncture method could improve renal function in DKD rats, reduce blood glucose and urinary protein excretion, alleviate podocyte injury, and enhance podocyte autophagy. The mechanism may be related to modulation of the renal LncRNA SOX2OT/mTORC1/ULK1 pathway.
Animals ; Podocytes/cytology* ; Diabetic Nephropathies/physiopathology* ; Rats, Sprague-Dawley ; Male ; Rats ; Mechanistic Target of Rapamycin Complex 1/genetics* ; Autophagy ; Acupuncture Therapy ; Autophagy-Related Protein-1 Homolog/genetics* ; RNA, Long Noncoding/metabolism* ; Humans ; Signal Transduction

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

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

Pinch-3 protein is an important constituent of cell membranes, which directly affects the cell morphology and mechanical properties. We observed and compared the change of morphology and cell traction force of glomerular podocytes before and after Pinch-3 gene inhibition by gene interference technology in this experiment. We found that a number of pores appeared on the cell surface, and the cell projected area were increased at the same time, with an approximate average about an increase of 40% after Pinch-3 gene inhibition. The results showed that the cell traction force of glomerular podocytes was significantly reduced, with an approximate average decrease of 40%, the maximum value of the cell traction force was reduced and the distribution of cell traction force became dispersive. All this suggested that after Pinch-3 gene inhibition, some pores created on the cell surface influenced the physical properties of glomerular podocytes and then affected the cell projected area and influenced the formation and distribution of cell traction force of the glomerular podocytes as well.
Adaptor Proteins, Signal Transducing ; genetics ; physiology ; Biomechanical Phenomena ; Cell Movement ; Genetic Engineering ; Humans ; Kidney Glomerulus ; cytology ; LIM Domain Proteins ; genetics ; physiology ; Mechanotransduction, Cellular ; physiology ; Membrane Proteins ; genetics ; physiology ; Podocytes ; cytology ; physiology ; Stress, Mechanical

Animals ; Apoptosis ; Diabetic Nephropathies ; metabolism ; pathology ; Glucose ; metabolism ; Membrane Potential, Mitochondrial ; Mice ; Mice, Transgenic ; Mitochondria ; pathology ; Podocytes ; cytology

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 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

Animals ; Epithelial Cells ; cytology ; drug effects ; Humans ; Kidney Tubules ; cytology ; drug effects ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Podocytes ; drug effects

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; physiology ; Animals ; Cell Differentiation ; Cell Line ; Cytoskeletal Proteins ; physiology ; Cytoskeleton ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Podocytes ; cytology ; RNA, Small Interfering ; Transfection ; WT1 Proteins ; metabolism