The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Jan 28, 2015
Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomer... more Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all ...
A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and cha... more A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Olfactomedin-related proteins are members of a gene family with conserved C-terminus motifs. We i... more Olfactomedin-related proteins are members of a gene family with conserved C-terminus motifs. We isolated one member of this gene family, BMZ olfactomedin-related protein, from rat glomerulus during searching for glomerulus-specific genes. The BMZ olfactomedin-related protein was uniquely immunolocalized in podocytes in the kidney. By double label immunofluorescence microscopy and immunoelectron microscopy, localization of the protein was demonstrated at the Golgi apparatus in the podocyte. To examine a role of C-terminus amino acid sequence, SDEL, of this protein in the retention at the Golgi apparatus, BMZ olfactomedin-related protein with wild-type SDEL sequence or mutated KDEL or PEET sequence at the C-terminus was expressed in CHO cells by transfection with plasmid expression vectors. BMZ olfactomedin-related protein with SDEL C-terminus was accumulated to the perinuclear region and was co-localized with giantin (a marker of Golgi apparatus) in these cells. On the other hand, BM...
Background/Aims: A chemokine fractalkine (FKN/CX3CL1) is induced primarily by endothelial cells a... more Background/Aims: A chemokine fractalkine (FKN/CX3CL1) is induced primarily by endothelial cells and accumulates inflammatory cells via its receptor CX3CR1. Since glomerular preferential expression of FKN/CX3CL1 gene was reported in normal human kidney, we presumed FKN/CX3CL1 might play some roles in glomerular physiology. The purpose of this study is to examine the expression and localization of FKN/CX3CL1 in normal and proteinuric glomeruli. Methods: Normal and proteinuric rat kidneys were studied. The gene and protein expressions of FKN/CX3CL1 and CX3CR1 were examined by real-time RT-PCR, in situ hybridization and immunohistochemistry, Western blotting. Results: By real-time RT-PCR, glomerular preferential expression of FKN/CX3CL1 was confirmed, whereas CX3CR1 was detected in glomeruli and cortices. The localization of FKN/CX3CL1 gene and protein were demonstrated in glomerular cells including podocytes. In nephrotic puromycin aminonucleoside (PAN) nephrosis glomeruli, increased e...
To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a... more To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes -IFI27, CRHBP, FLJ10154 and SEMA5B- were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD -DDX5, HSPC138, and MGC10940- were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA. Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus. Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.
Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under n... more Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.
Abstract A plasma membrane Ca 2+-pump ATPase preparation purified from porcine aorta was incubate... more Abstract A plasma membrane Ca 2+-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several ...
High affinity Ca2(+)-activated, Mg2(+)-dependent ATP-ase [Ca2+ + Mg2+)-ATPase) activities were ch... more High affinity Ca2(+)-activated, Mg2(+)-dependent ATP-ase [Ca2+ + Mg2+)-ATPase) activities were characterized in the membrane fractions of the porcine aorta. The (Ca2+ + Mg2+)-ATPase activity, similar to those found in the plasma membranes of the erythrocyte and the heart, ie, Ca2(+)-pumping ATPase activity, was not found in the membrane fractions isolated by the conventional method. The activity, however, became apparent in a plasma membrane-enriched fraction obtained from the microsomes treated with digitonin. The enzyme activity was stimulated by a purified C-kinase and by cyclic GMP or a purified cyclic GMP-dependent protein kinase (G-kinase). In addition to the Ca2(+)-pumping ATPase which requires millimolar concentration of Mg2+ for its activity, another high affinity (Ca2+ + Mg2+)-ATPase was detected that required micromolar concentration of Mg2+ for its full activation. Cell fractionation studies suggested its localization to plasma membranes, but the biochemical characteristics of the enzyme indicated that the enzyme could not be a biochemical expression of the plasma membrane Ca2+ pump.
Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in ... more Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Jan 28, 2015
Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomer... more Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all ...
A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and cha... more A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Olfactomedin-related proteins are members of a gene family with conserved C-terminus motifs. We i... more Olfactomedin-related proteins are members of a gene family with conserved C-terminus motifs. We isolated one member of this gene family, BMZ olfactomedin-related protein, from rat glomerulus during searching for glomerulus-specific genes. The BMZ olfactomedin-related protein was uniquely immunolocalized in podocytes in the kidney. By double label immunofluorescence microscopy and immunoelectron microscopy, localization of the protein was demonstrated at the Golgi apparatus in the podocyte. To examine a role of C-terminus amino acid sequence, SDEL, of this protein in the retention at the Golgi apparatus, BMZ olfactomedin-related protein with wild-type SDEL sequence or mutated KDEL or PEET sequence at the C-terminus was expressed in CHO cells by transfection with plasmid expression vectors. BMZ olfactomedin-related protein with SDEL C-terminus was accumulated to the perinuclear region and was co-localized with giantin (a marker of Golgi apparatus) in these cells. On the other hand, BM...
Background/Aims: A chemokine fractalkine (FKN/CX3CL1) is induced primarily by endothelial cells a... more Background/Aims: A chemokine fractalkine (FKN/CX3CL1) is induced primarily by endothelial cells and accumulates inflammatory cells via its receptor CX3CR1. Since glomerular preferential expression of FKN/CX3CL1 gene was reported in normal human kidney, we presumed FKN/CX3CL1 might play some roles in glomerular physiology. The purpose of this study is to examine the expression and localization of FKN/CX3CL1 in normal and proteinuric glomeruli. Methods: Normal and proteinuric rat kidneys were studied. The gene and protein expressions of FKN/CX3CL1 and CX3CR1 were examined by real-time RT-PCR, in situ hybridization and immunohistochemistry, Western blotting. Results: By real-time RT-PCR, glomerular preferential expression of FKN/CX3CL1 was confirmed, whereas CX3CR1 was detected in glomeruli and cortices. The localization of FKN/CX3CL1 gene and protein were demonstrated in glomerular cells including podocytes. In nephrotic puromycin aminonucleoside (PAN) nephrosis glomeruli, increased e...
To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a... more To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes -IFI27, CRHBP, FLJ10154 and SEMA5B- were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD -DDX5, HSPC138, and MGC10940- were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA. Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus. Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.
Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under n... more Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.
Abstract A plasma membrane Ca 2+-pump ATPase preparation purified from porcine aorta was incubate... more Abstract A plasma membrane Ca 2+-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several ...
High affinity Ca2(+)-activated, Mg2(+)-dependent ATP-ase [Ca2+ + Mg2+)-ATPase) activities were ch... more High affinity Ca2(+)-activated, Mg2(+)-dependent ATP-ase [Ca2+ + Mg2+)-ATPase) activities were characterized in the membrane fractions of the porcine aorta. The (Ca2+ + Mg2+)-ATPase activity, similar to those found in the plasma membranes of the erythrocyte and the heart, ie, Ca2(+)-pumping ATPase activity, was not found in the membrane fractions isolated by the conventional method. The activity, however, became apparent in a plasma membrane-enriched fraction obtained from the microsomes treated with digitonin. The enzyme activity was stimulated by a purified C-kinase and by cyclic GMP or a purified cyclic GMP-dependent protein kinase (G-kinase). In addition to the Ca2(+)-pumping ATPase which requires millimolar concentration of Mg2+ for its activity, another high affinity (Ca2+ + Mg2+)-ATPase was detected that required micromolar concentration of Mg2+ for its full activation. Cell fractionation studies suggested its localization to plasma membranes, but the biochemical characteristics of the enzyme indicated that the enzyme could not be a biochemical expression of the plasma membrane Ca2+ pump.
Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in ... more Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions.
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Papers by Yutaka Yoshida