Localization and rapid regulation of Na+/myo-inositol cotransporter in rat kidney.

myo-inositol, a major compatible osmolyte in renal medulla, is accumulated in several kinds of cells under hypertonic conditions via Na+/myo-inositol cotransporter (SMIT). To investigate the physiological role of the SMIT, we sought to determine its localization by in situ hybridization and its acute regulation by NaCl and furosemide administration. Northern analysis demonstrated that SMIT is strongly expressed in the medulla and at low levels in the cortex of kidney. Intraperitoneal injection of NaCl rapidly induced SMIT mRNA in both the cortex and medulla, and furosemide completely abolished this induction. In situ hybridization revealed that SMIT it predominantly present in the medullary and cortical thick ascending limbs of Henle's loop (TALH) and macula densa cells. Less intense signals were seen in the inner medullary collecting ducts (IMCD). NaCl loading increased the signals throughout the TALH, and furosemide reduced the signals. SMIT in the IMCD is less sensitive to these kinds of acute regulation. Thus, the distribution pattern of SMIT does not correspond to the corticomedullary osmotic gradient, and SMIT in the TALH and macula densa cells is regulated very rapidly. These results suggest that SMIT expression in TALH may be regulated by intracellular and/or peritubular tonicity close to the basolateral membrane, which is supposed to be proportional to the magnitude of NaCl reabsorption.

Na+/myo-inositol transport is regulated by basolateral tonicity in Madin-Darby canine kidney cells.

We investigated the effects of change in basolateral osmolality on Na(+)-dependent myo-inositol uptake in Madin-Darby canine kidney cells to test our hypothesis that the Na+/myo-inositol transporter (SMIT), an osmolyte transporter, is mainly regulated by osmolality on the basolateral surface. A significant osmotic gradient between both sides of the epithelium persisted at least 10 h after basolateral osmolality was increased. [3H]myo-inositol uptake increased in a basolateral osmolality-dependent manner. The magnitude of the increase is comparable to that for making both sides hypertonic. Apical hypertonicity also increased the uptake on the basal side, but the magnitude of the increase was significantly smaller than the basolateral or both sides hypertonicity. Betaine-gamma-amino-n-butyric acid transporter activity, measured by [3H]gamma-amino-n-butyric uptake, showed a pattern similar to SMIT activity in response to basolateral hypertonicity. The most plausible explanation for the polarized effect of hypertonicity is that the basal membrane is much more water permeable than the apical membrane. These results seem to be consistent with the localization and regulation of the SMIT in vivo.

Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells.

Madin-Darby canine kidney cells behave like the renal medulla and accumulate small organic solutes (osmolytes) in a hypertonic environment. The accumulation of osmolytes is primarily dependent on changes in gene expression of enzymes that synthesize osmolytes (sorbitol) or transporters that uptake them (myo-inositol, betaine, and taurine). The mechanism by which hypertonicity increases the transcription of these genes, however, remains unclear. Recently, it has been reported that yeast mitogen-activated protein (MAP) kinase and its activator, MAP kinase-kinase, are involved in osmosensing signal transduction and that mutants in these kinases fail to accumulate glycerol, a yeast osmolyte. No information is available in mammals regarding the role of MAP kinase in the cellular response to hypertonicity. We have examined whether MAP kinase and MAP kinase-kinase are regulated by extracellular osmolarity in Madin-Darby canine kidney cells. Both kinases were activated by hypertonic stress in a time- and osmolarity-dependent manner and reached their maximal activity within 10 min. Additionally, it was suggested that MAP kinase was activated in a protein kinase C-dependent manner. These results indicate that MAP kinase and MAP kinase-kinase(s) are regulated by extracellular osmolarity.

Induction of Na+/myo-inositol cotransporter mRNA after focal cerebral ischemia: evidence for extensive osmotic stress in remote areas.

Myo-inositol is one of the major organic osmolytes in the brain. It is accumulated into cells through an Na+/ myo-inositol cotransporter (SMIT) that is regulated by extracellular tonicity. To investigate the role of SMIT in the brain after cerebral ischemia, we examined expression of SMIT mRNA in the rat brain after middle cerebral artery occlusion, which would reflect alteration of extracellular tonicity. The expression of SMIT mRNA was markedly increased 12 h after surgery in the cortex of the affected side and lasted until the second day. Increased expression was also found in the contralateral cingulate cortex. Up-regulated expression was found predominantly in the neurons in remote areas, although nonneuronal cells adjacent to the ischemic core also expressed this mRNA. These results suggest that cerebral ischemia causes extensive osmotic stress in brain and that the neuronal cells respond to this stress by increasing SMIT expression.

Rapid and transient up-regulation of Na+/myo-inositol cotransporter transcription in the brain of acute hypernatremic rats.

The osmoregulatory system is well developed in the brain. Osmolytes contribute to maintenance of cell volume and cellular functions without changing intracellular ionic composition. Myo-inositol is regarded as one of the major osmolytes in the brain. In the present study, we investigated the changes in expressions of sodium myo-inositol cotransporter (SMIT) mRNA in the brain of acute hypernatremic rats by in-situ hybridization and Northern blot methods. Under moderate acute hypernatremic conditions, SMIT mRNA level increased markedly at 1 h and returned to almost control levels at 3 h, in accordance with plasma Na+ concentrations. Especially, distinct increases in SMIT mRNA expression were observed in the granule cells and glial cells in the cerebellum. These findings indicate that SMIT plays an important role in osmoregulation, especially in the early stages of acute hypernatremia in the brain.

Expression of Na+/myo-inositol cotransporter mRNA in the inner ear of the rat.

We have demonstrated the cellular localization of Na+/myo-inositol cotransporter (SMIT) mRNA in the rat inner ear by in situ hybridization. In the cochlea, the most intense SMIT mRNA signals were observed in fibrocytes of the spiral ligament, moderate signals were found in the spiral limbus, inner hair cells and spiral ganglion cells, while the hybridization signals were almost undetectable in the marginal cells of the stria vascularis and outer hair cells. In the vestibular system, moderate hybridization signals were found in the sensory epithelium, fibrocytes and vestibular ganglion cells. These findings suggest that SMIT plays an important role in maintenance of intracellular ionic balance and cell volume in the inner ear, especially in the fibrocytes associated with generation of the ion gradients between the endolymph and perilymph.

Expression of Na+/myo-inositol cotransporter mRNA in normal and hypertonic stress rat eyes.

We studied the localization of Na+/myo-inositol cotransporter (SMIT) mRNA in normal and hypertonic stress rat eyes by in situ hybridization histochemistry using cRNA probes. SMIT mRNA signals were observed in the iris-ciliary body, the lens epithelial cells, and the ganglion cell layer and the inner nuclear layer of the retina. There was a rapid increase on SMIT mRNA in the retina of hypertonic stress rats compared with control rats. These findings suggest that Na+/myo-inositol cotransporter gene expression is osmotically regulated in vivo to protect retinal neuronal function against hypertonic stress.

Cellular localization of Na+/MYO-inositol co-transporter mRNA in the rat brain.

The distribution of Na+/MYO-inositol co-transporter (SMIT) mRNA in the rat brain was studied by in situ hybridization histochemistry. The highest levels of SMIT mRNA were observed in the choroid plexus. Intense hybridization signals were found in the pineal gland, the area postrema, the hippocampus, the locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and the Purkinje cell and granule cell layers of the cerebellum. Low to moderate levels of labelling were detected in almost all neurones and small glia-like cells throughout the brain. These results suggest that almost all cells in the brain possess an SMIT-mediated osmotic and ionic regulatory system, and uneven densities of positive SMIT mRNA signals may reflect the differences in sensitivity of the cells to osmotic and ionic changes and also reflect differences in permeability of capillaries.

GLAST mRNA expression in the periventricular area of experimental hydrocephalus.

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.

Response to osmotic stimuli in mesangial cells: role of system A transporter.

It has been suggested that mesangial cells have an osmoregulatory mechanism like that of renal medullary cells, such as intracellular accumulation of polyols in response to hypertonicity. We examined osmoregulatory role of neutral amino acids transported by system A in cultured mesangial cells. The contents of almost all amino acids increased under hypertonic conditions to more than twice the value in isotonic cells. In hypertonic cells, the system A transport activity, measured by Na(+)-dependent 2-(methylamino)isobutyric acid (MeAIB) uptake, was 3.8-fold the uptake in isotonic cells, reaching a maximum 16 h after the switch to hypertonic medium. The response to hypertonicity was the result of an increase in maximal velocity without change in Michaelis constant and was dependent on RNA and protein synthesis. When medium osmolality decreased from hypertonic to isotonic, MeAIB uptake reverted to the isotonic level within 16 h and a large transient efflux of L-proline occurred within 10 min. These results suggest that mesangial cells respond to extracellular hypertonicity by increasing system A transport activity and neutral amino acids can function as compatible osmolytes in mesangial cells.

Differential regulation of adenine nucleotide translocators by hypertonicity in the brain.

To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up-regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT)2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up-regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of NaCl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.

Neuroprotective role of Na+/myo-inositol cotransporter against veratridine cytotoxicity.

Na+/myo-inositol cotransporter has been shown to protect cells from the perturbing effects of hypertonic stress by the accumulation of myo-inositol. Here we report a regulatory mechanism for the cotransporter. Induction of myo-inositol cotransporter mRNA was observed after exposure to veratridine, a voltage-gated sodium channel opener. The veratridine-elicited induction was inhibited when Na+ was eliminated from the bath, although calcium chelation failed to modify the gene expression. Veratridine evoked an accumulation of Na+ in the cells, which paralleled the abundance of the mRNA. These results strongly suggested that an increase in Na+ influx due to sodium channel opening affected transcription of the cotransporter gene. Activity of the myo-inositol cotransporter was also up-regulated after veratridine exposure. To clarify the possible roles of myoinositol accumulation under veratridine exposure, we next examined the neurotoxic effects of veratridine when myo-inositol uptake was blocked. Neither 30 microM veratridine nor 500 microM 2-O,C-methylene myo-inositol, a competitive inhibitor of myo-inositol, elicited apparent cytotoxicity. However, a combination of these agents markedly increased cytotoxicity in culture, suggesting that an adequate amount of myo-inositol was necessary when the cells were stimulated with veratridine.

Localization of rat CLC-K2 chloride channel mRNA in the kidney.

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+ exchanger, Na+-Cl- cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle's loop, but the signal in the cortical thick ascending limb of Henle's loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.

Na+/myo-inositol cotransport is regulated by tonicity in cultured rat mesangial cells.

Mesangial cells are considered to be faced with osmotic stress under physiological (such as extraglomerular mesangial cells) and pathophysiological (for example, diabetes mellitus) conditions. To see if mesangial cells have an osmoregulatory mechanism, like renal medullary cells, we measured the intracellular contents of organic osmolytes in isotonic and hypertonic conditions. Cultured rat mesangial cells are well tolerant of acute increase in osmolality up to 500 mOsm/kg. The myo-inositol content increased in hypertonic cells more than six-fold the value in isotonic cells. The contents of glycerophosphorylcholine and sorbitol also increased but were less than that of myo-inositol. The Na(+)-dependent myo-inositol uptake in hypertonic cells was a 12-fold uptake in isotonic cells, reaching a maximum 24 hours after the switch to a hypertonic medium. The uptake rate increased as medium osmolality increased from 300 to 500 mOsm/kg. Raffinose is the most effective solute to increase the myo-inositol uptake. NaCl, glucose and mannitol also increased the uptake rate (NaCl > glucose > mannitol). The increased uptake by hypertonicity was the result of an increase in Vmax without change in Km and was dependent on RNA and protein synthesis. These results indicate that mesangial cells respond to extracellular hypertonicity by increasing myo-inositol transport activity and accumulating myo-inositol into the cells, suggesting that myo-inositol functions as an organic osmolyte in mesangial cells.

Expression of betaine transporter mRNA: its unique localization and rapid regulation in rat kidney.

Betaine is a major compatible osmolyte in the renal medulla. It is taken up into cells via the betaine gamma-amino-n-butyric acid transporter (BGT-1). We investigated the localization of BGT-1 mRNA and its acute regulation by NaCl and furosemide administration. In situ hybridization revealed that BGT-1 mRNA is predominantly present in the outer medulla and papilla. Less intense signals were seen in the inner medulla and no signals were found in the cortex. Microscopic examination suggested that intense signals were present in the medullary thick ascending limbs of Henle's loop (MTAL) and the inner medullary collecting ducts (IMCD). A reverse transcription and polymerase chain reaction assay of individual microdissected segments along the nephron confirmed its localization. Intraperitoneal administration of NaCl rapidly increased the signal in the MTAL, and furosemide prevented the increase in BGT-1 mRNA by NaCl loading. In contrast, BGT-1 mRNA in the IMCD is less sensitive to these kinds of acute regulation. These results suggest that BGT-1 expression in the MTAL is rapidly regulated in response to the magnitude of NaCl absorption, as suggested for the expression of Na+/myo-inositol cotransporter.