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

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 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

This study was purposed to investigate the relationship between the catalysis of Bence Jones protein (BJP) in urine of patients with multiple myeloma(MM) and toxicity on the renal proximal tubular cells in vitro, and to explore the potential mechanism for the toxicity of BJP to renal impairment in patients with MM. The Michaelis-Menten constant (K(m)) and catalytic constant (k(cat)) of the amidase activity of BJP was calculated by Hanes equation. The LLC-PK1 cells were cultured with different concentration of BJP for 24 h, then proliferation of the cells were determined by MTT method and apoptosis were determined by flow cytometry. The results showed that the BJP from the MM patients with renal impairment significantly inhibited cell proliferation, as compared with that from MM patients without renal impairment. The BJP with higher k(cat) had higher toxicity to LLC-PK1 cells. BJP could induce apoptosis and necrosis of LLC-PK1 cells when reached a certain concentration and this effect enhanced with increase of BJP concentration. It is concluded that the catalysis of BJP and its toxicity to renal tubular epithelial cells has a positive correlation, and toxic effect of BJP on renal tubular epithelial cells results from inhibiting proliferation and inducing apoptosis and necrosis of the cells, which may be one of renal impairment mechanisms in MM patients.
Animals ; Bence Jones Protein ; metabolism ; toxicity ; Catalysis ; Coculture Techniques ; Epithelial Cells ; metabolism ; pathology ; Humans ; Kidney ; metabolism ; pathology ; Kidney Tubules ; cytology ; LLC-PK1 Cells ; Multiple Myeloma ; metabolism ; pathology ; Swine

BACKGROUND: In this study, the putative interactions between apoptosis and heat shock proteins disturbed as a result of ATP depletion were investigated as a hypoxia model. METHODS: The direct cellular damages were assessed by the release of LDH from the cytoplasm of the human tubular epithelial cells (HK-2 cells) following ATP depletion. The Bcl-2/Bax mRNA expression ratio, used as an index to assess to what extent apoptosis contributed to tubular cell damage, and the expressions of HSP 90, 72 and 27 in relation to the Bcl-2/Bax ratio in the ischemic model, as parameters of their functional contributions to tubule cell damage, were also studied. Heat preconditioning (HS) was performed at 43 degrees in a temperature-regulated water bath for 1 h. RESULTS: The release of LDH due to ATP depletion was not significantly increased in HK-2 cells compared to the control, but was slightly increased in heat preconditioned cells compared to non heat preconditioned cells, but the difference was not statistically significant (6.33 +/- 0.57 U/L vs. 8.67 +/- 2.52 U/L, p> 0.05). The Bcl-2/ Bax mRNA expression ratio increased progressively from the control to the heat preconditioned and ATP depleted cells (control; 100%, ATP depletion; 154 +/- 6%, heat preconditioning; 212 +/- 6%, heat preconditioning and ATP depletion; 421 +/- 8%). No contribution of heat preconditioning and ATP depletion was observed on the expressions of HSP90 and HSP27. However, HSP72 expression was prominent by ATP depletion, especially after heat preconditioning. CONCLUSION: There may be a possibility that the preservation of cytolytic damage and an increase in the Bcl-2/Bax mRNA expression ratio is related to the increase of HSP72 in ATP depletion as a hypoxia model.
Adenosine Triphosphate/*deficiency ; Anoxia/metabolism ; Epithelium/*metabolism ; Heat-Shock Proteins/*metabolism ; Humans ; Kidney Tubules/cytology/*metabolism ; L-Lactate Dehydrogenase/metabolism ; RNA, Messenger/metabolism ; Research Support, Non-U.S. Gov't

Animals ; Drug Carriers ; Drug Delivery Systems ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; Maleates ; metabolism ; Molecular Weight ; Oligodeoxyribonucleotides, Antisense ; metabolism ; Polyvinyls ; metabolism ; Prodrugs ; metabolism ; Proteins ; metabolism

OBJECTIVETo study whether aristololactam I (AL-I) can enter renal proximal tubular epithelial cells and the situation of intracellular distribution and accumulation.

METHODCultured human renal proximal tubular epithelial cell line (HK-2) was used as the subject. Intracellular fluorescence from AL-I and its distribution are examined by fluorescence microscopy after a treatment with different concentration of AL-I, the intracellular accumulation of AL-I was also investigated by incubated cells in AL-I -free medium for 48 h after washing-out the media containing AL-I.

RESULTAfter treatment of AL-I (concentration from 5 microg x mL(-1) to 20 microg x mL(-1)), glaucous fluorescence could be observed inside renal proximal tubular epithelial cells at 0.5 h, and the fluorescence distributed only in cytoplasm while not be observed in nuclei. Moreover, the fluorescence of AL-I could be kept in cytoplasm for more than 48 h after washing out the media containing AL-I .

CONCLUSIONAL-I is able to enter renal proximal tubular epithelial cells in short time and accumulate in cytoplasm, but not enter nuclei. This property may contribute to the cytotoxic mechanism of renal injury induced by AL-I, which may partially explain the persistent renal toxicity of AAs and its metabolites in the development of aristolochic acid nephropathy.

Animals ; Aristolochic Acids ; metabolism ; toxicity ; Cell Line ; Cell Nucleus ; drug effects ; metabolism ; Cytoplasm ; drug effects ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; pathology ; Humans ; Kidney Diseases ; metabolism ; pathology ; Kidney Tubules, Proximal ; cytology ; pathology ; Microscopy, Fluorescence

OBJECTIVETo explore the different expression of proteins between human clear-cell renal cell carcinoma (ccRCC) cell line RLC-310 and normal renal proximal tubule epithelial cell line HK-2, and to search new differentially expressed proteins of RCC.

METHODSRLC-310 and HK-2 cells were cultured in vitro. The total proteins were separated by ProteomeLab PF 2D protein fractionation system and the differential expression protein fractions of the two cell lines were analyzed and identified by capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). RT-PCR and Western blot were used to confirm the representative differential expression at mRNA and protein levels.

RESULTSOne hundred and ninty-six differentially expressed proteins were identified. These differentially expressed proteins involved in many aspects, including cell proliferation and anti-apoptosis, energy metabolism, mitochondria reduction and oxidation, oxidative stress and resistance, cell signaling, invasion and adhesion, cytoskeleton and motion, neovascularization, etc. Except for previously reported RCC associated proteins: annexin A2, fatty acid-binding protein, vimentin, fibronectin, and so on, Septin-9 was firstly found highly expressed in RLC-310 cells when compared with that in the HK-2 cells. Moreover, the overexpression of Septin-9 was confirmed by RT-PCR and Western blot analysis at both mRNA and protein levels (P < 0.05).

CONCLUSIONSThe human ccRCC cell line RLC-310 cells display differential protein profiles compared with those of the normal renal cell line HK-2 cells. The identified differential expression proteins are involved in many aspects of RCC development. It is worth further study and elucidate the molecular mechanisms of RCC. The representative differential protein Septin-9 deserves further study its role in the angiogenesis of ccRCC.

Carcinoma, Renal Cell ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Epithelial Cells ; cytology ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Kidney Tubules, Proximal ; cytology ; Proteomics ; methods ; RNA, Messenger ; metabolism ; Septins ; genetics ; 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