Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response.

Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.

Dynamic changes of podocytes caused by fibroblast growth factor 2 in culture.

Fibroblast growth factor 2 (FGF2) augments podocyte injury, which induces glomerulosclerosis, although the mechanisms remain obscure. In this study, we investigated the effects of FGF2 on cultured podocytes with interdigitating cell processes in rats. After 48 h incubation with FGF2 dynamic changes in the shape of primary processes and cell bodies of podocytes resulted in the loss of interdigitation, which was clearly shown by time-lapse photography. FGF2 reduced the gene expressions of constituents of the slit diaphragm, inflections of intercellular junctions positive for nephrin, and the width of the intercellular space. Immunostaining for the proliferation marker Ki-67 was rarely seen and weakly stained in the control without FGF2, whereas intensely stained cells were frequently found in the presence of FGF2. Binucleation and cell division were also observed, although no significant increase in cell number was shown. An in vitro scratch assay revealed that FGF2 enhanced migration of podocytes. These findings show that FGF2 makes podocytes to transition from the quiescent state into the cell cycle and change their morphology due to enhanced motility, and that the culture system in this study is useful for analyzing the pathological changes of podocytes in vivo.

Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice.

A main feature of Fabry disease is nephropathy, with polyuria an early manifestation; however, the mechanism that underlies polyuria and affected tubules is unknown. To increase globotriaosylceramide (Gb3) levels, we previously crossbred asymptomatic Glatm mice with transgenic mice that expressed human Gb3 synthase (A4GALT) and generated the GlatmTg(CAG-A4GALT) symptomatic Fabry model mice. Additional analyses revealed that these mice exhibit polyuria and renal dysfunction without remarkable glomerular damage. In the present study, we investigated the mechanism of polyuria and renal dysfunction in these mice. Gb3 accumulation was mostly detected in the medulla; medullary thick ascending limbs (mTALs) were the most vacuolated tubules. mTAL cells contained lamellar bodies and had lost their characteristic structure ( i.e., extensive infolding and numerous elongated mitochondria). Decreased expression of the major molecules-Na+-K+-ATPase, uromodulin, and Na+-K+-2Cl- cotransporter-that are involved in Na+ reabsorption in mTALs and the associated loss of urine-concentrating ability resulted in progressive water- and salt-loss phenotypes. GlatmTg(CAG-A4GALT) mice exhibited fibrosis around mTALs and renal dysfunction. These and other features were consistent with pathologic findings in patients with Fabry disease. Results demonstrate that mTAL dysfunction causes polyuria and renal impairment and contributes to the pathophysiology of Fabry nephropathy.-Maruyama, H., Taguchi, A., Nishikawa, Y., Guili, C., Mikame, M., Nameta, M., Yamaguchi, Y., Ueno, M., Imai, N., Ito, Y., Nakagawa, T., Narita, I., Ishii, S. Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice.

Induction of interdigitating cell processes in podocyte culture.

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.

Disruption of the mitochondria-associated ER membrane (MAM) plays a central role in palmitic acid-induced insulin resistance.

The mitochondria-associated ER membrane (MAM) is a specialized subdomain of ER that physically connects with mitochondria. Although disruption of inter-organellar crosstalk via the MAM impairs cellular homeostasis, its pathological significance in insulin resistance in type 2 diabetes mellitus remains unclear. Here, we reveal the importance of reduced MAM formation in the induction of fatty acid-evoked insulin resistance in hepatocytes. Palmitic acid (PA) repressed insulin-stimulated Akt phosphorylation in HepG2 cells within 12h. Treatment with an inhibitor of the ER stress response failed to restore PA-mediated suppression of Akt activation. Mitochondrial reactive oxygen species (ROS) production did not increase in PA-treated cells. Even short-term exposure (3h) to PA reduced the calcium flux from ER to mitochondria, followed by a significant decrease in MAM contact area, suggesting that PA suppressed the functional interaction between ER and mitochondria. Forced expression of mitofusin-2, a critical component of the MAM, partially restored MAM contact area and ameliorated the PA-elicited suppression of insulin sensitivity with Ser473 phosphorylation of Akt selectively improved. These results suggest that loss of proximity between ER and mitochondria, but not perturbation of homeostasis in the two organelles individually, plays crucial roles in PA-evoked Akt inactivation in hepatic insulin resistance.

Severity and Frequency of Proximal Tubule Injury Determines Renal Prognosis.

AKI increases the risk of developing CKD, but the mechanisms linking AKI to CKD remain unclear. Because proximal tubule injury is the mainstay of AKI, we postulated that proximal tubule injury triggers features of CKD. We generated a novel mouse model to induce proximal tubule-specific adjustable injury by inducing the expression of diphtheria toxin (DT) receptor with variable prevalence in proximal tubules. Administration of high-dose DT in mice expressing the DT receptor consistently caused severe proximal tubule-specific injury associated with interstitial fibrosis and reduction of erythropoietin production. Mild proximal tubule injury from a single injection of low-dose DT triggered reversible fibrosis, whereas repeated mild injuries caused sustained interstitial fibrosis, inflammation, glomerulosclerosis, and atubular glomeruli. DT-induced proximal tubule-specific injury also triggered distal tubule injury. Furthermore, injured tubular cells cocultured with fibroblasts stimulated induction of extracellular matrix and inflammatory genes. These results support the existence of proximal-distal tubule crosstalk and crosstalk between tubular cells and fibroblasts. Overall, our data provide evidence that proximal tubule injury triggers several features of CKD and that the severity and frequency of proximal tubule injury determines the progression to CKD.

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality.

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (DstGt) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt23Rbrc. Motor abnormalities in homozygous dt23Rbrc/dt23Rbrc mice are not as severe as homozygous DstGt/DstGt mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt23Rbrc/dt23Rbrc mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt23Rbrc/dt23Rbrc mice than in homozygous DstGt/DstGt mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.

Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies.

Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at -25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20-100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at -80 °C, but were severely damaged at -25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at -25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs.

Decreased urinary calbindin 1 levels in proteinuric rats and humans with distal nephron segment injuries.

BACKGROUND: Several proteins have been proposed as new urinary biomarkers of kidney injuries, but they are not always capable of identifying the kidney nephron segment that has been injured. Since calbindin 1 protein is exclusively localized in the kidney distal nephron segment, it is presumed that its expression is altered during distal nephron segment injuries, resulting in changes in its urinary excretion. METHODS: Calbindin 1 expression in normal rat kidneys was compared with that in the kidneys of rats that had suffered distal nephron segment injuries (unilateral ureteral obstruction [UUO] or anti-glomerular basement membrane glomerulonephritis [anti-GBM GN]) using immunohistochemical examinations and real-time polymerase chain reaction. The urinary calbindin 1 protein concentration of normal rats was also compared with that of anti-GBM GN rats and of cisplatin nephropathy rats using Western blotting. We also compared the kidney and urinary calbindin 1 protein concentrations of normal human subjects with those of proteinuric patients [immunoglobulin (Ig)A nephropathy; IgAN] with distal nephron segment injuries. RESULTS: Calbindin 1 mRNA expression in the renal cortices and calbindin 1 protein expression in the kidney distal nephron segments were decreased in the UUO and anti-GBM GN rat kidneys. The urinary calbindin 1 protein levels of the anti-GBM GN rats were also markedly decreased, whereas those of the cisplatin nephropathy rats were slightly decreased. The human IgAN patients displayed decreased renal calbindin 1 protein expression in their dilated distal tubules, and some patients displayed decreased urinary calbindin 1 levels. CONCLUSION: Since it has been demonstrated that decreased urinary calbindin 1 levels are indicative of decreased calbindin 1 kidney expression due to distal nephron segment injuries, calbindin 1 might be a useful urinary biomarker for identifying distal nephron segment injuries.

Heparin increasing podocyte-specific gene expressions.

AIM: Heparin, a highly sulfated glycosaminoglycan, has been shown to have a renoprotective effect on renal diseases, but its mechanisms remain to be elucidated. In this study, we examined the effect of heparin on podocytes by using primary cultured podocytes positive for podocyte-specific markers including podocin and podocalyxin. METHODS: Podocytes were cultured from highly purified glomeruli isolated by the method with renal perfusion with magnetic beads and digestion of collagenase. Podocyte-specific gene expressions and proteins were examined by real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence microscopy. RESULTS: Real-time PCR showed that addition of heparin to the culture media significantly upregulated most of the podocyte-specific genes in a dose-dependent and time-dependent manner. Western blotting showed a marked increase in protein levels of nephrin and podocin. Podocin localization at cell-cell contact sites became conspicuous in the presence of heparin. The effect of heparin was observed even in culture media deprived of bovine foetal serum. Heparan sulfate, less sulfated than heparin, and hyaluronan did not show such effects, but sulfated dextran did markedly. CONCLUSION: Heparin acts on cultured podocytes to increase podocyte-specific gene expressions. A high degree of sulfation is crucial for the effect of heparin.

A symptomatic Fabry disease mouse model generated by inducing globotriaosylceramide synthesis.

Fabry disease is a lysosomal storage disorder in which neutral glycosphingolipids, predominantly Gb3 (globotriaosylceramide), accumulate due to deficient α-Gal A (α-galactosidase A) activity. The GLAko (α-Gal A-knockout) mouse has been used as a model for Fabry disease, but it does not have any symptomatic abnormalities. In the present study, we generated a symptomatic mouse model (G3Stg/GLAko) by cross-breeding GLAko mice with transgenic mice expressing human Gb3 synthase. G3Stg/GLAko mice had high Gb3 levels in major organs, and their serum Gb3 level at 5-25 weeks of age was 6-10-fold higher than that in GLAko mice of the same age. G3Stg/GLAko mice showed progressive renal impairment, with albuminuria at 3 weeks of age, decreased urine osmolality at 5 weeks, polyuria at 10 weeks and increased blood urea nitrogen at 15 weeks. The urine volume and urinary albumin concentration were significantly reduced in the G3Stg/GLAko mice when human recombinant α-Gal A was administered intravenously. These data suggest that Gb3 accumulation is a primary pathogenic factor in the symptomatic phenotype of G3Stg/GLAko mice, and that this mouse line is suitable for studying the pathogenesis of Fabry disease and for preclinical studies of candidate therapies.

Profiling and annotation of human kidney glomerulus proteome.

BACKGROUND: The comprehensive analysis of human kidney glomerulus we previously performed using highly purified glomeruli, provided a dataset of 6,686 unique proteins representing 2,966 distinct genes. This dataset, however, contained considerable redundancy resulting from identification criteria under which all the proteins matched with the same set of peptides and its subset were reported as identified proteins. In this study we reanalyzed the raw data using the Mascot search engine and highly stringent criteria in order to select proteins with the highest scores matching peptides with scores exceeding the "Identity Threshold" and one or more unique peptides. This enabled us to exclude proteins with lower scores which only matched the same set of peptides or its subset. This approach provided a high-confidence, non-redundant dataset of identified proteins for extensive profiling, annotation, and comparison with other proteome datasets that can provide biologically relevant knowledge of glomerulus proteome. RESULTS: Protein identification using the Mascot search engine under highly stringent, computational strategy generated a non-redundant dataset of 1,817 proteins representing 1,478 genes. These proteins were represented by 2-D protein array specifying observed molecular weight and isoelectric point range of identified proteins to demonstrate differences in the observed and calculated physicochemical properties. Characteristics of glomerulus proteome could be illustrated by GO analysis and protein classification. The depth of proteomic analysis was well documented via comparison of the dynamic range of identified proteins with other proteomic analyses of human glomerulus, as well as a high coverage of biologically important pathways. Comparison of glomerulus proteome with human plasma and urine proteomes, provided by comprehensive analysis, suggested the extent and characteristics of proteins contaminated from plasma and excreted into urine, respectively. Among the latter proteins, several were demonstrated to be highly or specifically localized in the glomerulus by cross-reference analysis with the Human Protein Atlas database, and could be biomarker candidates for glomerular injury. Furthermore, comparison of ortholog proteins identified in human and mouse glomeruli suggest some biologically significant differences in glomerulus proteomes between the two species. CONCLUSIONS: A high-confidence, non-redundant dataset of proteins created by comprehensive proteomic analysis could provide a more extensive understanding of human glomerulus proteome and could be useful as a resource for the discovery of biomarkers and disease-relevant proteins.

Glomerular expression of connexin 40 and connexin 43 in rat experimental glomerulonephritis.

BACKGROUND: Gap junctional intercellular communication is thought to play an important role in the maintenance of cell differentiation and homeostasis. Gap junctions connect glomerular mesangial cells to each other. In this study, we examined the glomerular expression of connexins (Cxs) 40 and 43 at both the protein and transcript levels in anti-Thy1.1 glomerulonephritis (GN). METHODS: Anti-Thy1.1 GN was induced by intravenous injection of anti-Thy1.1 monoclonal antibody 1-22-3. Cx protein expression was examined by immunofluorescence, immunoelectron microscopy, and Western blotting. Changes in mRNA levels were detected by real-time reverse transcriptase-polymerase chain reaction. RESULTS: Cx40 was detected in mesangial cells in normal rat glomeruli; its expression was reduced on days 3 and 7 and recovered to normal on day 14 following GN induction. Cx43 was detected in mesangial cells and podocytes in normal rat glomeruli, and its expression did not change during the disease course of GN. Expression of Cx40 and Cx43 was also detected in extraglomerular mesangial cells; this expression did not change during the disease course. Opposing patterns of expression between Cx40 and smooth muscle actin (SMA) were observed with double-immunofluorescence labeling. SMA is a differentiation marker of mesangial cells; it is often expressed during proliferation but not under physiological conditions. CONCLUSION: These results suggest that Cx40 expression in mesangial cells is related to mesangial cell regeneration. Thus, Cx expression regulation could be a therapeutic target for glomerular diseases.

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. METHODS: Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. RESULTS: The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 µg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. CONCLUSIONS: The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

A single-nucleotide substitution of CjTKPR1 determines pollen production in the gymnosperm plant Cryptomeria japonica.

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution ("T" to "C" at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.

Novel expression of claudin-5 in glomerular podocytes.

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.

Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney.

BACKGROUND: Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney. METHODS: Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies. RESULTS: Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-1 was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501. CONCLUSION: This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.

Expression and localization of insulin-like growth factor binding proteins in normal and proteinuric kidney glomeruli.

AIM: Insulin-like growth factor I (IGF-I) acts on target cells in an endocrine and/or local manner through the IGF-I receptor (IGF-IR), and its actions are modulated by multiple IGF binding proteins (IGFBP). To elucidate the roles of local IGFBP in kidney glomeruli, the expression and localization of their genes were examined and compared with normal and proteinuric kidney glomeruli. METHODS: A cDNA microarray database (MAd-761) was constructed using human kidney glomeruli and cortices. The gene expression levels of IGF-I, IGF-1R and IGFBP (1-10) were examined in glomeruli and cortices by polymerase chain reaction (PCR) and in situ hybridization (ISH), and the expression levels of IGFBP that were abundantly found in the glomerulus were compared between normal and proteinuric kidneys in rats and humans. RESULTS: IGFBP-2, -7 and -8 were demonstrated to be abundantly and preferentially expressed in the glomerulus. In PCR, the expression levels of the IGFBP-2, -7, -8 and -10 genes in glomeruli were shown to have more than doubled compared with their levels in the cortices. In ISH, the IGFBP-2, -7, -8 and -10 genes were found to be localized in glomerular cells including podocytes, and their increased expression was observed in inflammatory glomeruli. IGF-I gene expression was localized in glomerular podocytes, whereas the IGF-IR gene was expressed in glomerular podocytes and cortical tubular cells. In nephrotic rats, the expression of the IGFBP-10 gene was increased in glomerular podocytes; however, the expression levels of IGFBP-2, -7 and -8 did not change. CONCLUSION: IGFBP-2, -7, -8 and -10 are produced by normal and injured glomerular podocytes and may regulate local IGF-I actions in podocytes and/or cortical tubular cells in the kidney.

Gap junction with MLO-A5 osteoblast-like cell line induces ALP and BSP transcription of 3T3-L1 pre-adipocyte like cell line via Hspb1 while retaining adipogenic differentiation ability.

In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.

SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.