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*

Initially, when periaqueductal gray (PAG) is electrically stimulated, analgesia is induced, and this phenomenon is called stimulation-produced analgesia. Nucleus raphe magnus (NRM) as well as PAG are known to be the potent analgesic centers. NRM could modulate the nociceptive response of spinal cord neurons through spinally projecting fibers. However, as well as the above analgesic effects have been confined to the somatic pain, it was variable according to species, and the analgesic effect by NRM stimulation on the visceral pain was not yet clarified. In this study the analgesic effect by NRM stimulation on the visceral pain was examined through recording the activities of the dorsal horn neurons with renal input and renal pain, as a type of visceral pain. The renal pain was induced by ureteral occlusion or renal arterial occlusion, which in turn activated the renal mechanoreceptor or chemoreceptor. These cells had concomitant somatic input. In order to compare the effects of NRM stimulation on the renal pain with somatic pain, the somatic stimulation such as squeezing was conducted on the peripheral receptive field. The main results are summarized as follows: 1) After an electrical stimulation of NRM, spontaneous activities of dorsal horn neurons with renal input were reduced to 73.3 +/- 9.7% of the control value. 2) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by a brush, a type of non-noxious stimuli, did not change significantly. But the activities by a squeeze, a type of noxious stimuli, the activities were reduced to 63.2 +/- 7.2% of the control value. 3) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by occlusion of ureter or renal artery were reduced to 46.7 +/- 8.8% and 49.0 +/- 8.0% of the control value respectively. 4) The inhibitory effect of NRM on the dorsal horn neurons with renal input did not show any difference between renal A delta fiber and C fiber group. 5) By the electrical stimulation of NRM, the activities evoked by ureteral occlusion showed more reduction in the high threshold cell group than in the wide dynamic range cell group. These results suggest that activation of NRM can alleviate the renal pain as well as the somatic pain by modulating the dorsal horn neurons activities.
Afferent Pathways/cytology/physiology ; Animal ; Cats ; Electric Stimulation ; Female ; Kidney/innervation/*physiopathology ; Male ; Nervous System/cytology ; Nervous System Physiology ; Neurons/physiology ; *Pain Threshold ; Raphe Nuclei/*physiology ; Spinal Cord/cytology/physiology ; Support, Non-U.S. Gov't

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

To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD68. Nine cases of nonspecific tubulointerstitial change and 4 cases of nonallograft tubulointerstitial nephritis were used as a control. Inflammatory infiltration was present in all cases studied. T cells predominated in the interstitium of chronic rejection and were followed by macrophages and B cells. The degree of interstitial infiltration of frozen section was not accordant with that of paraffin sections. Allografts with nonspecific tubulointerstitial changes or tubulointerstitial nephritis of native kidneys showed similar distribution pattern in terms of type and degree. However, the degree of infiltrate did not give any statistical significance among groups. The CD4/CD8 ratios in interstitial infiltrates were less than 1.0 in 6 cases and was not accordant with those of peripheral blood. Proportion of nudelta T cells increased over 10% in 2 cases in tissue and in 3 cases in peripheral blood. In 3 cases of chronic rejection in which both tissue and blood results were available, there was no concordance of CD4/CD8 or nudeltaT/CD3 between them. Tubular expression of HLA DR was, however, present only in 4 cases of chronic rejection. In conclusion, T lymphocytes were predominant regardless of diagnosis or disease activity. T lymphocyte subset did not give any suggestion as to the diagnosis or disease activity in chronic rejection. Furthermore nudelta T cells had only limited value. Lymphocytic subsets in peripheral blood would not be predictors of tissue destruction in chronic rejection.
Flow Cytometry ; Graft Rejection/*immunology ; Human ; Kidney/cytology/*immunology ; Kidney Transplantation/*immunology ; Leukocytes, Mononuclear/*immunology ; Phenotype ; Receptors, Antigen, T-Cell, gamma-delta/immunology ; Support, Non-U.S. Gov't

To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD68. Nine cases of nonspecific tubulointerstitial change and 4 cases of nonallograft tubulointerstitial nephritis were used as a control. Inflammatory infiltration was present in all cases studied. T cells predominated in the interstitium of chronic rejection and were followed by macrophages and B cells. The degree of interstitial infiltration of frozen section was not accordant with that of paraffin sections. Allografts with nonspecific tubulointerstitial changes or tubulointerstitial nephritis of native kidneys showed similar distribution pattern in terms of type and degree. However, the degree of infiltrate did not give any statistical significance among groups. The CD4/CD8 ratios in interstitial infiltrates were less than 1.0 in 6 cases and was not accordant with those of peripheral blood. Proportion of nudelta T cells increased over 10% in 2 cases in tissue and in 3 cases in peripheral blood. In 3 cases of chronic rejection in which both tissue and blood results were available, there was no concordance of CD4/CD8 or nudeltaT/CD3 between them. Tubular expression of HLA DR was, however, present only in 4 cases of chronic rejection. In conclusion, T lymphocytes were predominant regardless of diagnosis or disease activity. T lymphocyte subset did not give any suggestion as to the diagnosis or disease activity in chronic rejection. Furthermore nudelta T cells had only limited value. Lymphocytic subsets in peripheral blood would not be predictors of tissue destruction in chronic rejection.
Flow Cytometry ; Graft Rejection/*immunology ; Human ; Kidney/cytology/*immunology ; Kidney Transplantation/*immunology ; Leukocytes, Mononuclear/*immunology ; Phenotype ; Receptors, Antigen, T-Cell, gamma-delta/immunology ; Support, Non-U.S. Gov't

The prevalence of renal disease continues to increase worldwide. When normal kidney is injured, the damaged renal tissue undergoes pathological and physiological events that lead to acute and chronic kidney diseases, which frequently progress to end stage renal failure. Current treatment of these renal pathologies includes dialysis, which is incapable of restoring full renal function. To address this issue, cell-based therapy has become a potential therapeutic option to treat renal pathologies. Recent development in cell therapy has demonstrated promising therapeutic outcomes, in terms of restoration of renal structure and function impaired by renal disease. This review focuses on the cell therapy approaches for the treatment of kidney diseases, including various cell sources used, as well recent advances made in preclinical and clinical studies.
Cell- and Tissue-Based Therapy/*methods ; Fetal Stem Cells/transplantation ; Humans ; Kidney/cytology ; Kidney Diseases/*therapy ; Pluripotent Stem Cells/transplantation ; Stem Cell Transplantation/methods

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

The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.
Animals ; Basement Membrane/drug effects/metabolism ; Cattle ; Cells, Cultured ; Diabetic Nephropathies/metabolism ; Endothelial Cells/cytology/*drug effects/*metabolism ; Gene Expression/drug effects ; Glucose/*pharmacology ; Heparan Sulfate Proteoglycan/genetics/*metabolism ; Kidney Glomerulus/*cytology ; Sulfur Radioisotopes/diagnostic use ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S.

PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.
Antioxidants/metabolism ; Apoptosis/*drug effects ; Cation Transport Proteins/*metabolism ; Cell Cycle/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Epithelial Cells/*cytology/drug effects/*metabolism ; Glucose/*pharmacology ; Glutathione/metabolism ; Humans ; Kidney Tubules/*cytology ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Sodium-Hydrogen Antiporter/*metabolism

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.
Animals ; Apoptosis/*physiology ; Caspases/biosynthesis ; Cell Line ; DNA Fragmentation ; Endothelial Cells/drug effects ; Enzyme Inhibitors/*pharmacology ; Kidney/cytology ; Phosphotransferases (Alcohol Group Acceptor)/*antagonists & inhibitors/physiology ; Propylene Glycols/*pharmacology ; Research Support, Non-U.S. Gov't ; Sphingosine/pharmacology/*physiology ; Swine ; Up-Regulation