Onset and Evolution of Southern Annular Mode-Like Changes at Centennial Timescale.

The Southern Westerly Winds (SWW) are the surface expression of geostrophic winds that encircle the southern mid-latitudes. In conjunction with the Southern Ocean, they establish a coupled system that not only controls climate in the southern third of the world, but is also closely connected to the position of the Intertropical Convergence Zone and CO2 degassing from the deep ocean. Paradoxically, little is known about their behavior since the last ice age and relationships with mid-latitude glacier history and tropical climate variability. Here we present a lake sediment record from Chilean Patagonia (51°S) that reveals fluctuations of the low-level SWW at mid-latitudes, including strong westerlies during the Antarctic Cold Reversal, anomalously low intensity during the early Holocene, which was unfavorable for glacier growth, and strong SWW since ~7.5 ka. We detect nine positive Southern Annular Mode-like events at centennial timescale since ~5.8 ka that alternate with cold/wet intervals favorable for glacier expansions (Neoglaciations) in southern Patagonia. The correspondence of key features of mid-latitude atmospheric circulation with shifts in tropical climate since ~10 ka suggests that coherent climatic shifts in these regions have driven climate change in vast sectors of the Southern Hemisphere at centennial and millennial timescales.

An assessment of cinacalcet HCl effects on bone histology in dialysis patients with secondary hyperparathyroidism.

AIMS: Cinacalcet lowers plasma parathyroid hormone (PTH) levels in patients with secondary hyperparathyroidism (sHPT), but the bone histologic response has not been described. This prospective, double-blind, placebo-controlled trial assessed the effects of cinacalcet on bone histology and serum markers of bone metabolism in dialysis patients with sHPT. METHODS: Patients with intact PTH (iPTH) > or = 300 pg/ml were randomly assigned 2:1 to receive cinacalcet or placebo with concurrent vitamin D and/or phosphate binder therapy. Cinacalcet (30 - 180 mg/day) was used to achieve iPTH levels < or = 200 pg/ml. Bone biopsies were performed before and after one year of treatment. RESULTS: Baseline and end-of-study data were available from 32 patients (19 cinacalcet, 13 placebo). Baseline bone turnover was elevated in 27, reduced in 3 and normal in 2 patients. Serum bone-specific alkaline phosphatase (BSAP) and N-telopeptide (NTx) were elevated. Cinacalcet treatment decreased PTH and diminished activation frequency, bone formation rate/bone surface, and fibrosis surface/bone surface. Adynamic bone was observed in three patients receiving cinacalcet; in two of these, PTH levels were persistently low (< 100 pg/ml). The histomorphometric parameter changes in bone corresponded to PTH, BSAP and NTx reductions. Bone mineralization parameters remained normal. CONCLUSIONS: Treatment with cinacalcet lowered PTH and reduced bone turnover and tissue fibrosis among most dialysis patients with biochemical evidence of sHPT.

Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier.

Na(v)1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na(v)1.2 and its beta2 subunit, but not Na(v)1.6 or beta1, are clustered in developing central nervous system nodes and that clustering of Na(v)1.2 and Na(v)1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na(v)1.2 alpha and beta2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na(v)1.6 or beta1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na(v)1.2 and Na(v)1.6 channels is differentially controlled and suggest that myelination induces Na(v)1.6 clustering.

Effect of ICAM-1 blockade on lung inflammation and physiology during acute viral bronchiolitis in rats.

Viral respiratory infections cause acute bronchiolitis and physiologic dysfunction in human infants and in animals. It is possible that the pulmonary dysfunction is a consequence of the inflammatory cells that are recruited during viral illness. We hypothesized that blockade of intercellular adhesion molecule-1 (ICAM-1), a major cell adhesion molecule, would impede the ingress of leukocytes during viral infection and attenuate virus-induced pulmonary dysfunction. Adult male rats were inoculated with parainfluenza type 1 (Sendai) virus or sterile vehicle, and treated with blocking or nonblocking MAb specific for rat ICAM-1. Respiratory system resistance, oxygenation (PaO2), methacholine responsiveness, and bronchoalveolar lavage (BAL) leukocyte counts were measured in anesthetized, paralyzed, ventilated rats. Treatment with the blocking ICAM-1 antibody reduced virus-induced increases in BAL neutrophils and lymphocytes by 70% (p < 0.001), but did not affect BAL monocytes/macrophages. Peripheral blood leukocyte counts were elevated in anti-ICAM-1 blocking antibody-treated rats (p = 0.0003). Although virus-induced increases in resistance and decreases in PaO2 were not affected by anti-ICAM-1 treatment, there was a small but significant attenuation of virus-induced methacholine hyperresponsiveness (p = 0.02). We conclude that ICAM-1 has an important role in neutrophil and lymphocyte infiltration during respiratory viral illness, and that virus-induced changes in pulmonary physiology are not related directly to the numbers of neutrophils and lymphocytes that migrate to the air spaces during infection.

Glomerulonefritis y vasculitis asociados con exposición al silice: presentación de dos casos y revisión de la literatura / Glomerulonephritis and vasculitis associated with silica exposition: presentation of two cases and review of the literature

La asociación entre silicosis y enfermedades sistémicas se ha reportado desde hace décadas. En este artículo reportamos dos pacientes con antecedentes de severa exposición al sílice, uno de ellos con diagnóstico de silicosis hecho por biopsia ganglionar, que desarrollaron glomerulonegritis rapidamente progresiva con inmunufluorescencia negativa uno, y vasculitis de tipo poliarteritis nodosa (PAN)con severo compromiso renal el otro, ambos varios años después de la exposición al sílice. La "nefropatía silicótica" es un síndrome clínico que incluye anormalidades de la función renal que van desde formas leves hasta insuficiencia renal rapidamente progresiva, desproporcionadas a los hallazgos histológicos. Se han reportado diversas formas de glomerulonefritis, vasculitis y colagenopatías asociadas con exposición al sílice. en la patogénesis estarían involucrados un efecto tóxico directo a nivel renal y un estímulo recurrente del sistema inmune, aunque aún queda por dilucidar el mecanismo exacto y comprobar la existencia real de esta asociación.

Glomerulonefritis y vasculitis asociados con exposición al silice: presentación de dos casos y revisión de la literatura / Glomerulonephritis and vasculitis associated with silica exposition: presentation of two cases and review of the literature

La asociación entre silicosis y enfermedades sistémicas se ha reportado desde hace décadas. En este artículo reportamos dos pacientes con antecedentes de severa exposición al sílice, uno de ellos con diagnóstico de silicosis hecho por biopsia ganglionar, que desarrollaron glomerulonegritis rapidamente progresiva con inmunufluorescencia negativa uno, y vasculitis de tipo poliarteritis nodosa (PAN)con severo compromiso renal el otro, ambos varios años después de la exposición al sílice. La "nefropatía silicótica" es un síndrome clínico que incluye anormalidades de la función renal que van desde formas leves hasta insuficiencia renal rapidamente progresiva, desproporcionadas a los hallazgos histológicos. Se han reportado diversas formas de glomerulonefritis, vasculitis y colagenopatías asociadas con exposición al sílice. en la patogénesis estarían involucrados un efecto tóxico directo a nivel renal y un estímulo recurrente del sistema inmune, aunque aún queda por dilucidar el mecanismo exacto y comprobar la existencia real de esta asociación. (AU)

Serial segmental bronchoalveolar lavage in individual rats.

Bronchoalveolar lavage (BAL) is a well-characterized technique for analysis of cellular constituents of the airways and air spaces, but whole lung lavage requires that the animal be euthanized. We describe a technique of segmental BAL in rats that allows serial measurements of inflammation. A tracheal tube was placed, under direct visualization, in lightly anesthetized animals, and a catheter was passed through the tracheal tube and advanced to a wedge position. Five 0.1-ml volumes of buffer solution were instilled and then withdrawn with gentle suction. In normal rats, the percentages of neutrophils, eosinophils, and mononuclear cells had a high level of agreement in the segmental samples compared with those obtained subsequently by whole lung lavage. In rats with acute pulmonary inflammation, the differential leukocyte counts from segmental samples exhibited patterns of change that differed from those of whole lung lavage; however, most segmental samples were obtained from the left lung base so that regional variability could be minimized in serial studies. Lung mechanics and airway inflammation were not affected by repeated segmental BALs done 2 wk apart.

Prevention of chronic postbronchiolitis airway sequelae with IFN-gamma treatment in rats.

After viral bronchiolitis at an early age, Brown Norway (BN) rats develop chronic airway dysfunction consisting of inflammation, remodeling, episodic reversible obstruction, and hyperresponsiveness. We hypothesized that supplementation of interferon gamma (IFN-gamma) during viral illness would alter the inflammatory response and attenuate the postviral sequelae. Weanling rats were treated daily with aerosolized interferon gamma (IFN-gamma), from 2 d prior through 7 d after inoculation, and compared with saline-treated infected rats and with noninfected control rats. The IFN-gamma treatment had no significant effect on viral titers, growth retardation, or total bronchoalveolar leukocytes, but there was a slight decrease in lung interleukin-4 (IL-4) mRNA (p = 0.015) during the first week. Despite having minimal effects on the acute illness, the IFN-gamma had marked effects on postviral sequelae, the IFN-gamma group having less bronchiolar inflammation (p = 0.025) and fibrosis (p = 0.01), and lacking abnormalities in pulmonary resistance (p = 0.028) and dynamic compliance (p = 0.006) compared with the untreated postviral group. We conclude that IFN-gamma modulated the inflammatory response to viral illness, such that acute airway injury did not evolve into chronic airway dysfunction. If similar processes contribute to the development of human asthma, it may be possible to interrupt the progression of airway dysfunction with an early immunomodulatory intervention.

Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats.

Recent data suggest that sex hormones affect the thiazide-sensitive NaCl cotransporter (TSC) density or binding capacity (Chen, Z., D.A. Vaughn, and D.D. Fanestil. 1994. J. Am. Soc. Nephrol. 5:1112-1119). Thus, we determined the effect of ovariectomy (OVX) and estrogen replacement on the ultrastructural localization of TSC in rat kidney using immunocytochemistry. Kidneys of intact female (CON) and OVX rats fed ad libitum for 6 and 9 wk or pair-fed for 9 wk were processed for transmission electron microscopy. Immunogold localization of rat TSC (rTSC1) demonstrated intense label in the apical plasma membrane of CON distal convoluted tubule (DCT). In OVX DCT, rTSC1 label and apical plasma membrane microprojections were decreased. Western blots of renal membrane protein from pair-fed CON and OVX revealed bands at 129-135 kD, but the OVX signal was reduced. Morphometric analyses demonstrated that injecting 10 microg/ kg body weight 17beta-estradiol subcutaneously 4x/wk in OVX rats restored DCT apical microprojections and label density for rTSC1. Thus, in OVX rats (a) rTSC1 immunoreactive renal membrane protein is reduced; (b) apical plasma membrane complexity and immunogold label for rTSC1 in DCT is decreased; and (c) estradiol replacement restores DCT ultrastructure and rTSC1 label to normal. We conclude that estrogen enhances the density of rTSC1 in the DCT, and may alter renal Na transport by this mechanism.

Attenuation of virus-induced airway dysfunction in rats treated with imiquimod.

Viral respiratory infections cause acute airway abnormalities consisting of inflammation and physiological dysfunction in both animals and humans. It is likely that inflammatory cell products, such as cytokines, contribute substantially to viral-induced airway dysfunction. We hypothesized that imiquimod, an immune response enhancing agent that induces interferon-alpha, would attenuate the development of airway dysfunction during acute viral illness in rats. Adult Brown Norway rats were inoculated with parainfluenza type 1 (Sendai) virus or sterile vehicle, and treated with either imiquimod or water. Respiratory system resistance (Rrs), arterial oxygen tension (Pa,O2), lung viral titres and bronchoalveolar lavage (BAL) leucocyte counts were measured in anaesthetized, paralysed, ventilated rats. Virus-infected, water-treated rats had a significant decrease in Pa,O2 and had significant increases in leucocyte count and Rrs when compared to both the virus-infected, imiquimod-treated, (Pa,O2, p = 0.03; leucocyte count, p = 0.02; and Rrs, p = 0.009) and noninfected, water-treated rats (Pa,O2, p = 0.007; leucocyte count, p = 0.001; and Rrs, p = 0.01). In addition, imiquimod suppressed BAL eosinophils in both virus-infected (p = 0.02) and noninfected (p = 0.001) groups, and lowered overall virus titres (p = 0.03). Thus, both virus-induced airway inflammation and physiological dysfunction were attenuated significantly by imiquimod treatment in this animal model. By further delineating mechanisms by which infections induce airway dysfunction in animal models, more specific pharmacological interventions can be developed for the treatment of virus-induced asthma.

Effects of dexamethasone on acute virus-induced airway dysfunction in adult rats.

Respiratory viral infections have been associated with airway obstruction and hyperresponsiveness, and exacerbations of asthma. Although virus-induced asthma is thought to be precipitated by airway inflammation, the clinical efficacy and rationale for using antiinflammatory treatment during such exacerbations remains controversial. The purpose of this study was to use a well characterized animal model of respiratory viral illness to test the hypothesis that the inflammatory response to viral infection is responsible for the development of airway dysfunction. Adult rats were inoculated with either Sendai virus or sterile vehicle and treated with daily injections of dexamethasone or saline. At postinoculation d 4, 5, or 6, rats were evaluated for airway obstruction, hyperresponsivenes, inflammation, and lung viral titers. Saline-treated infected rats had significant airway obstruction (increased resistance, decreased dynamic compliance), hyperresponsiveness (i.v. methacholine), and inflammation (increased bronchoalveolar lavage leukocytes) compared with noninfected controls. In contrast, dexamethasone-treated infected rats had no increase in bronchoalveolar lavage leukocytes and significantly smaller changes in airway physiology, but had increased lung viral titers compared with saline-treated infected rats. We conclude that glucocorticoid suppression of the inflammatory response to respiratory viral infection largely prevents virus-associated airway dysfunction.

Induction of sodium channel clustering by oligodendrocytes.

As oligodendrocytes wrap axons of the central nervous system (CNS) with insulating myelin sheaths, sodium channels that are initially continuously distributed along axons become segregated into regularly spaced gaps in the myelin called nodes of Ranvier. It is not known whether the regular spacing of nodes results from regularly spaced glial contacts or is instead intrinsically specified by the axonal cytoskeleton. Contact with Schwann cells induces clustering of sodium channels along the axons of peripheral neurons in vitro and in vivo. Similarly, it has been suggested that astrocyte contact induces clustering of sodium channels along CNS axons. Here we show that oligodendrocytes are necessary for clustering of sodium channels in vitro and in vivo. The induction, but not the maintenance, of sodium-channel clustering along the axons of highly purified rat retinal ganglion cells in culture depends on a protein secreted by oligodendrocytes. Surprisingly, the oligodendrocyte-induced clusters are regularly spaced at the predicted interval in the absence of glial-axonal contact. Mutant rats that are deficient in oligodendrocytes develop few axonal sodium channel clusters in vivo. These results demonstrate a crucial role for oligodendrocytes in inducing clustering of sodium channels.

Fluorescence depolarization as an early measure of T lymphocyte stimulation.

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl alpha-D-mannopyranoside (alphaMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

Expression of the sodium-chloride cotransporter in osteoblast-like cells: effect of thiazide diuretics.

The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.

Expression of the Na(+)-K(+)-2Cl- cotransporter BSC2 in the nervous system.

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.

Chronic, episodic, reversible airway obstruction after viral bronchiolitis in rats.

Viral bronchiolitis in human infants has been associated with persistent airway abnormalities, but not proven as a cause. Previously we observed some adult rats had airway obstruction and hyperresponsiveness following bronchiolitis at an early age. The purpose of this study was to determine, via serial measurements of lung mechanics, whether the postbronchiolitis airway obstruction was episodic or continuous, and to determine the magnitude and duration of glucocorticoid effects. Rats were either virus- (n = 14) or sham-inoculated (n = 8) at 3 wks of age. Lung mechanics were measured 6 times in each rat at postinoculation Weeks 11-18. Half the rats in each group were treated with dexamethasone for 3 d at Week 15. The virus group had higher lung resistance (p = 0.03) and lower dynamic compliance (p = 0.005) than control rats, with airway obstruction occurring in an episodic pattern. Dexamethasone treatment had a transient effect in postbronchiolitis rats; lung resistance normalized in Week 15 (p = 0.006), then returned to pretreatment levels by Weeks 16-18. We conclude that viral bronchiolitis in rats can result in a chronic syndrome of intermittent, reversible airway obstruction which has multiple parallels with human asthma over a prolonged time period.

The Na-(K)-Cl cotransporter family in the mammalian kidney: molecular identification and function(s).

A new solute carrier gene family, SLC12, was recently described based on the molecular identification of three electroneutral Na-(K)-Cl cotransport proteins. In mammals, these proteins are encoded by three distinct but related genes: SLC12A1, SLC12A2, SLC12A3, which are located on different chromosomes. Although the expression patterns of these three cotransport proteins differ significantly, all of them are expressed in the mammalian kidney and participate in several important aspects of renal function. This review summarizes the information learned from the molecular identification of these cotransporters, evaluates the patterns of expression within the kidney, and discusses the roles that these cotransporters play in renal physiology and pathophysiology.

Thiazide treatment of rats provokes apoptosis in distal tubule cells.

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.

Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium, and the glomerular afferent arteriole.

Na-K-Cl cotransport plays an important role in the kidney in NaCl reabsorption in the thick ascending limb of Henle and a less well defined role in the inner medullary collecting duct (IMCD). Two Na-K-Cl cotransporters encoded by different genes have been identified in the mammalian kidney: BSC1/NKCC2 which localizes to the apical thick ascending limb of Henle and BSC2/NKCC1 which was isolated from a mouse IMCD cell line (mIMCD-3) but its localization has not been determined. In this study we generated a polyclonal antibody (anti-mBSC2) against the mouse BSC2/NKCC1 protein in order to characterize and localize this protein in mouse kidney. Western blot analysis with affinity-purified anti-mBSC2 showed a protein doublet of 140 and 150 kD which was most abundant in the renal papilla but also seen in cortex and outer medulla. The 140-150-kD bands were not seen with preimmune serum or with anti-mBSC2 preabsorbed with specific antigen. Immunolocalization confirmed expression of mBSC2 protein on the basolateral surface of terminal IMCD segments and demonstrated expression in the papillary surface epithelium. Immunofluorescence also revealed the unexpected presence of the BSC2 protein at the juxtaglomerular afferent arteriole, in a juxtaglomerular structure probably representing the extraglomerular mesangium, and throughout the glomerular mesangium.

Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney.

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.