We used in situ hybridization and immunocytochemistry with polyclonal antibodies against the mouse bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (mBSC2) to determine the location of this cotransporter in rat brain. Northern blots and in situ hybridization showed the presence of cotransporter mRNA in the brain, with an especially high level of expression in the choroid plexus (CP). Affinity-purified anti-BSC2 antibody identified proteins of 145-155 kDa on Western blot analysis and immunoprecipitation of brain and CP membrane protein. Indirect immunofluorescence demonstrated that BSC2 protein is located on the apical surface of the CP and is heterogeneously distributed in cell bodies and dendrites of neurons in the central and peripheral nervous system. The apical localization of BSC2 in the CP was confirmed by 86Rb+ uptakes in primary cultures of CP cells grown on permeable filters and confocal immunofluorescence microscopy. The apical localization of the cotransporter in CP epithelium suggests a role for the cotransporter in cerebrospinal fluid K+ homeostasis. In neurons, the cotransporter may help regulate intracellular Cl- concentration and thereby affect neuronal response to gamma-aminobutyric acid.

A thiazide sensitive Na-Cl cotransporter, rTSC1, has recently been cloned from a rat kidney cortex cDNA library. The molecular regulation and nephron localization of this protein is unknown. The purpose of this study was to examine the nephron distribution and subcellular localization of the rTSC1 protein in the rat kidney. In situ hybridization showed rTSC1 transcripts were localized to short, convoluted tubule segments in the kidney cortex. Polyclonal antibodies raised against a 110 amino acid segment from the amino terminus of rTSC1 recognized three major bands of 135, 140 and 155 kDa on Western blotting of membrane protein from cortex but not outer medulla of the rat kidney. Immunofluorescence studies using the antibody alone and in double labeling experiments with antibodies against the H+ ATPase and calbindin D28, showed intense labeling of apical membranes in the distal nephron beginning in the initial distal convoluted tubule and terminating within the connecting tubule. The intensity of labeling diminished from proximal to distal sites along the distal tubule. Ultrastructural studies by immunoelectron microscopy showed the cotransporter protein to be localized predominately on apical microvilli of the distal convoluted tubule cells. These results are consistent with rTSC1 encoding the apical thiazide sensitive Na-Cl cotransporter in the distal tubule.

We studied the effects of inhibition of apical NaCl entry on the structural correlates for electrolyte transport in the distal convoluted tubule (DCT) of rats. Thiazide diuretics were used to block NaCl entry specifically in the DCT. Metolazone or hydrochlorothiazide (HCTZ) were applied for three days subcutaneously via osmotic minipumps. The renal epithelial structure of control and treated rats was studied by light and electron microscopy. Distribution of the thiazide-sensitive NaCl cotransporter (rTSC1), calbindin D28K and Ca(2+)-Mg(2+)-ATPase was examined by immunohistochemistry, and the content of rTSC1 transcripts by Northern blot and in situ hybridization. In treated rats the DCT epithelium had lost the structural characteristics of electrolyte transporting epithelia and the cells were in different stages of apoptosis. In damaged cells calbindin D28K and Ca(2+)-Mg(2+)-ATPase were strongly decreased; the rTSC1 was shifted from the luminal membrane to the basal cell half and was found additionally in small membrane vesicles in intercellular and peritubular spaces. Transcripts of rTSC1 were drastically reduced in homogenates of kidney cortex and almost absent in damaged DCT cells. All other tubular segments were unaffected by the treatment. Focal inflammatory infiltrates were found to be specifically surrounding DCT profiles. Thus, inhibition by thiazides of apical NaCl entry into DCT cells is associated with apoptosis of DCT cells and focal peritubular inflammation.

A bumetanide-sensitive Na-K-Cl cotransporter (rBSC1) was recently cloned from a rat renal outer medulla (OM) cDNA library and shown to be expressed predominantly in the kidney. The purpose of the present study was to examine the nephron distribution of cotransporter transcripts and protein in rat kidney. In situ hybridization showed an intense signal only in the outer medulla and extending along cortical medullary rays consistent with expression of rBSC1 transcripts in medullary (MTAL) and cortical (CTAL) thick ascending limbs. Polyclonal antibodies raised in rabbits against a unique 67 amino acid segment from the carboxyl terminus of rBSC1 identified a broad major band of 130 to 160 (midpoint of 150) kDa and at least two minor bands of 50 to 70 kD on Western blotting of homogenates from cortex (C) and outer medulla (OM), but not inner medulla (IM), of rat kidney. Thus the Na-K-Cl cotransporter protein detected by the polyclonal rBSC1 antibody in rat kidney was similar in size to the major approximately 150 kD bumetanide binding protein detected by others in mouse and dog kidneys. Immunofluorescence studies using the anti-rBSC1 polyclonal antibody on rat kidney sections showed an intense signal limited to apical surfaces of MTAL and CTAL segments. Colocalization with anti-Tamm-Horsfall antibody which is present in all TABA cells except macula densa cells confirmed the absence of anti-rBSC1 fluorescence in the macula densa cells. These results are consistent with rBSC1 encoding the, or the major isoform of the, apical Na-K-Cl cotransporter in the thick ascending limb. The Na-K-Cl cotransporter functionally detected in macula densa cells may be encoded by a different BSC isoform.