Animals ; Calpain ; metabolism ; Epithelial Cells ; metabolism ; Kidney ; cytology ; Kidney Tubules ; cytology ; metabolism ; Rats ; Reperfusion Injury ; metabolism

OBJECTIVETo investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines.

METHODSImmunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines.

RESULTSCoordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01).

CONCLUSIONSSimilar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

Adult ; Animals ; Cell Line ; Gene Expression ; Humans ; Kidney ; metabolism ; Kidney Tubules, Collecting ; metabolism ; Kidney Tubules, Distal ; metabolism ; Kidney Tubules, Proximal ; cytology ; Polycystic Kidney, Autosomal Dominant ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Swine ; TRPP Cation Channels ; metabolism

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

Animals ; Epithelial-Mesenchymal Transition ; Fibrosis ; Kidney ; pathology ; Kidney Tubules ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; pathology

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.
Animal ; Biological Transport/drug effects ; Dexamethasone/pharmacology* ; Dose-Response Relationship, Drug ; Drug Interactions ; Glycerophospholipids/analysis ; Isoenzymes/drug effects ; Kidney Cortex/cytology ; Kidney Tubules, Collecting/drug effects* ; Kidney Tubules, Collecting/cytology ; Mice ; Mice, Transgenic ; Oleic Acid/pharmacology ; Phospholipase D/drug effects*

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/ non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl-/HCO3- exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.
Animals ; Anion Exchange Protein 1, Erythrocyte/*metabolism ; Callithrix/*metabolism ; Gene Expression Profiling/veterinary ; *Gene Expression Regulation ; Immunohistochemistry/veterinary ; Kidney Tubules/cytology/physiology/ultrastructure ; Kidney Tubules, Collecting/cytology/*metabolism/ultrastructure ; Male ; Microscopy, Electron, Transmission/veterinary

Phosphorylation of beta-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition (EMT). Introducing mimic peptide of tyrosine residue 654 domain of beta-catenin into cells may influence phosphorylation of beta-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of beta-catenin (PM) with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells (HK-2). The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of beta-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.
Carrier Proteins/*metabolism ; Epithelial Cells/cytology ; Epithelial Cells/*metabolism ; Fibroblasts/cytology ; Fibroblasts/metabolism ; Kidney Tubules/*cytology ; Peptides/metabolism ; Permeability ; Phosphorylation ; Tyrosine/*metabolism ; beta Catenin/*metabolism

The effects of oxygen partial pressure on cryopreservation of the cells with organ preservation solution were explored. Hypoxic UW solution was made by purging the UW solution with argon. The pig proximal tubule epithelial cells (LLC-PK1 cells) were cryopreserved in hypoxic UW solution (Ar-UW group) or standard UW solution (UW group) at 4 degrees C for 48 h. Trypan blue staining and LDH detection were performed to evaluate the injury of the cells. The results showed that the oxygen partial pressure in Ar-UW group was significantly declined from 242+/-6 mmHg to 83+/-10 mmHg. After cryopreservation at 4 degrees C for 48 h, LDH leakage rate and Trypan blue-stained rate in Ar-UW group were (11.3+/-3.4)% and (10.5+/-4.7)%, respectively, which were significantly lower than in UW group [(49.5+/-6.9)% and (47.6+/-9.3)% respectively, both P<0.01]. It was concluded that lower oxygen partial pressure of UW solution was more beneficial to the cryopreservation of LLC.
Adenosine ; Allopurinol ; Cell Hypoxia ; Cell Line ; Cryopreservation ; Cryoprotective Agents ; Epithelial Cells/*cytology ; Glutathione ; Insulin ; Kidney Tubules, Proximal/cytology ; Organ Preservation Solutions ; Oxygen/pharmacology ; Raffinose ; Swine ; Tissue Preservation/methods

OBJECTIVETo investigate the mechanisms of decorin inhibiting epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor beta1 (TGF-beta1) in renal tubular epithelial cells.

METHODHK-2 cells in vitro were divided into 4 groups: (1) negative control group; (2) decorin group, added with decorin 100 ng/ml ; (3) TGF-beta1 group, added with TGF-beta1 10 ng/ml; (4) decorin and TGF-beta1 group, added with decorin 100 ng/ml and TGF-beta1 10 ng/ml. The protein level of phosphor-ERK, phosphor-PI3K, phosphor-Smad(3) and beta-catenin was detected by Western blotting method. The snail mRNA level was tested by real time-PCR, while the lymphoid enhancer factor-1 (LEF-1) mRNA level was measured by RT-PCR.

RESULTSThe snail (2.59 +/- 0.70:1.02 +/- 0.13) and LEF-1 mRNA (1.85 +/- 0.08:0.30 +/- 0.11) were significantly up-regulated, meanwhile the protein level of phosphor-ERK (1.11 +/- 0.09:0.47 +/- 0.07), phosphor-PI3K (14.79 +/- 1.02:2.48 +/- 0.06), phosphor-Smad(3) (0.95 +/- 0.02:0.08 +/- 0.01) and beta-catenin (1.46 +/- 0.20:0.49 +/- 0.05) were significantly increased in TGF-beta1 group compared to control group, while there were no statistically significant difference in all figures between control group and decorin group. The phosphor-ERK protein level (0.58 +/- 0.08) and the snail mRNA level (1.24 +/- 0.03) were significantly down-regulated in TGF-beta1 and decorin group compared to TGF-beta1 group, however there were no statistically significant differences in the level of phosphor-PI3K (15.84 +/- 1.64), phosphor-Smad(3) (0.90 +/- 0.04) and beta-catenin (1.42 +/- 0.09) between these two groups.

CONCLUSIONDecorin inhibited EMT induced by TGF-beta1 which may be through blocking the ERK signal transduction pathway.

Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Decorin ; pharmacology ; Epithelial Cells ; cytology ; Fibronectins ; Humans ; Kidney Tubules ; cytology ; pathology ; Proteoglycans ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore the toxic effects of lead acetate on the apoptosis and ultrastructure of human renal tubular epithelial cells (HK-2).

METHODSAfter HK-2 cells were exposed to 5, 10 and 20 µmol/L lead acetate for 24 h, the morphological changes of HK-2 cells were observed by Hochest 33342-PI staining, and the ultrastructure changes of HK-2 cells were examined under a electron microscope, LDH activity and MDA content in supernatant of HK-2 cellular culture were detected by spectrophotometer, DNA damage of HK-2 was determined by DNA ladder and the apoptotic rates of HK-2 cells were measured by flow cytometry.

RESULTSThe morphological changes of apoptotic HK-2 cells in exposure group were observed by Hochest 33342-PI staining. The cytoplasm vacuoles, karyopycnosis, nuclear membrane vague and apoptotic bodies in HK-2 cells of exposure group were found under electron microscopy. LDH activity and MDA contents in exposure group increased significantly, as compared to control group (P < 0.01). The results of DNA Ladder showed that DNA damage of HK-2 cells in exposure group appeared. The apoptotic rates of HK-2 cells exposed to 5, 10, 20 µmol/L lead acetate were 14.16% ± 2.94%, 19.45% ± 2.73%, 25.01% ± 3.97%, respectively, which were significantly higher than that (5.81% ± 2.18%) in control group (P < 0.05).

CONCLUSIONLead acetate could remarkably induce the apoptosis of HK-2 cells and affect the kidney.

Apoptosis ; drug effects ; Cell Line ; Epithelial Cells ; cytology ; drug effects ; ultrastructure ; Humans ; Kidney Tubules, Proximal ; cytology ; drug effects ; ultrastructure ; Organometallic Compounds ; toxicity