Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

Discrete localization of phospholipase Cß3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.

Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCß3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCß3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCß3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCß3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.

Characterization and Involvement of Exosomes Originating from Chikungunya Virus-Infected Epithelial Cells in the Transmission of Infectious Viral Elements.

The Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that affects the world's popula-tion with chikungunya disease. Adaptation of the viral life cycle to their host cells' environment is a key step for establishing their infection and pathogenesis. Recently, the accumulating evidence advocates a principal role of extracellular vesicles (EVs), including exosomes, in both the infection and pathogenesis of infectious diseases. However, the participation of exosomes in CHIKV infec-tion and transmission is not well clarified. Here, we demonstrated that the CHIKV RNA and pro-teins were captured in exosomes, which were released by viral-infected epithelial cells. A viral genomic element in the isolated exosomes was infectious to naïve mammalian epithelial cells. The assay of particle size distribution and transmission electron microscopy (TEM) revealed CHIKV-derived exosomes with a size range from 50 to 250 nm. Treatments with RNase A, Triton X-100, and immunoglobulin G antibodies from CHIKV-positive patient plasma indicated that in-fectious viral elements are encompassed inside the exosomes. Interestingly, our viral plaque for-mation also exhibited that infectious viral elements might be securely transmitted to neighboring cells by a secreted exosomal pathway. Taken together, our recent findings emphasize the evidence for a complementary means of CHIKV infection and suggest the role of exosome-mediated CHIKV transmission.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

In situ localization of diacylglycerol lipase α and ß producing an endocannabinoid 2-arachidonoylglycerol and of cannabinoid receptor 1 in the primary oocytes of postnatal mice.

In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and ß are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/ß and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and ß were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLß-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Expression and localization of VIAAT in distal uriniferous tubular epithelium of mouse.

Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells.

Selective localization of diacylglycerol kinase (DGK)ζ in the terminal tubule cells in the submandibular glands of early postnatal mice.

The present immunohistochemical study was attempted to localize in the submandibular glands of mice at various postnatal stages a diacylglycerol kinase (DGK) isoform termed DGKζ which is characterized by a nuclear localization signal and a nuclear export signal. This attempt was based on following facts: the continuous postnatal differentiation of glandular cells in the rodent submandibular gland, the regulatory role of DGK in the activity of protein kinase C (PKC) through attenuation of diacylglycerol (DAG), and the possible involvement of PKC in various cellular activities including the saliva secretion as well as the cell differentiation. As a result, a selective localization of immunoreactivity for DGKζ was detected in terminal tubule (TT) cells which comprise a majority of the newborn acinar structure and differentiate into the intercalated duct cells and/or the acinar cells. The immunoreactivity was deposited in portions of the cytoplasm lateral and basal to the nucleus, but not in the nuclei themselves. Although the immunoreactive TT cells remained until later stages in female specimen than in male, they eventually disappeared in both sexes by young adult stages. The present finding suggests that the regulatory involvement of DGKζ in PKC functions via control of DAG is exerted in the differentiation of the TT cells. In addition, another possible involvement of DGKζ in the regulation of secretion of the TT cells as well as its functional significance of its nuclear localization in the submandibular ganglion cells was also discussed.

Specific IgG antibody subclasses to Angiostrongylus cantonensis in patients with angiostrongyliasis.

Total IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM specific antibodies against Angiostrongylus cantonensis somatic antigen were determined by enzyme-linked immunosorbent assay (ELISA) in sera from proven human angiostrongyliasis (PA) cases, clinically suspected angiostrongyliasis cases with eosinophilic meningitis (EM) and healthy control (HC). The specific IgA antibody in each of the patient groups was significantly higher than those of the HC group (p < 0.05). The mean ELISA value of the specific IgM in the PA group was not significantly different from that of the HC group (p > 0.05). However, the mean specific IgM ELISA value in the EM group was significantly higher than that of the HC group (p < 0.05). The levels of the specific IgG and IgG subclasses in both patient groups were significantly higher than in the healthy control (HC) group (p < 0.001). Major differences were evident in the distribution of the IgG subclass antibodies between the patient groups. The IgG1 antibody demonstrated the highest sensitivity and specificity while the IgM and IgA responses were generally poor in both patient groups. The levels of the specific IgG antibody subclasses possibly explain immune responses to the parasite.