R. J. Turner

Bio: R. J. Turner is an academic researcher from National Institutes of Health. The author has contributed to research in topics: Kidney & Transfection. The author has an hindex of 11, co-authored 11 publications receiving 1090 citations.

The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Abstract: Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na–Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ± 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ± 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 ± 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

Osmoregulatory changes in myo-inositol transport by renal cells

Abstract: Renal medullary cells contain high concentrations of myo-inositol, sorbitol, betaine, and glycerophosphocholine, whose levels vary with urinary osmolality. Accumulation of these "compatible" organic osmolytes is believed to help the cells osmoregulate in response to the high extracellular osmolality that occurs as part of the urinary concentrating mechanism. MDCK cells (a line from dog kidney) were previously shown to accumulate myo-inositol in response to increased medium osmolality. We demonstrate here that this accumulation requires the presence of myo-inositol in the medium, implying that the myo-inositol is not synthesized by the cells but rather is transported into them from the extracellular solution. The MDCK cells contain sodium-dependent myo-inositol transporters. Relative to isotonic controls, sodium-dependent myo-inositol uptake is higher in cells exposed to increased osmolality either acutely (1-7 days) or chronically (greater than 1 year). Transport is further enhanced when the cells are cultured in myo-inositol-free medium. The transport has both high- and low-affinity components. The observed changes in transport involve changes in maximal velocity of the high-affinity component but not in its Km. We conclude that renal cells can osmoregulate by changing the number (or, less likely, the transport turnover rate) of functioning sodium-dependent myo-inositol transporters.

A1 adenosine-receptor antagonists activate chloride efflux from cystic fibrosis cells.

Abstract: A1 adenosine-receptor-antagonist drugs such as 8-cyclopentyl-1,3-dipropylxanthine (CPX) and xanthine amine congener (XAC) are found to activate the efflux of 36Cl- from CFPAC cells. These cells are a pancreatic adenocarcinoma cell line derived from a cystic fibrosis (CF) patient homozygous for the common mutation, deletion of Phe-508. The active concentrations for these compounds are in the low nanomolar range, consistent with action on A1 adenosine receptors. In addition, drug action can be blocked by exogenous agonists such as 2-chloroadenosine and also can be antagonized by removal of endogenous agonists by treatment with adenosine deaminase. Cells lacking the CF genotype and phenotype, such as HT-29 and T84 colon carcinoma cell lines, appear to be resistant to activation of chloride efflux by either drug. CFPAC cells transfected with the CF transmembrane regulator gene, CFTR, are also resistant to activation by CPX. We conclude that, since these antagonists are of relatively low toxicity and appear to act somewhat selectively, they might be considered as promising therapeutic candidates for CF.

The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is a homodimer.

Abstract: The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer.

Osmoregulation of betaine transport in mammalian renal medullary cells.

Abstract: Escherichia coli and Staphylococcus typhimurium are known to accumulate betaine by increased transport when extracellular osmolality rises. In the present studies a similar process is demonstrated in mammalian cells. Renal medullary cells contain high concentrations of "compatible" organic osmolytes such as betaine, myo-inositol, sorbitol, and glycerophosphorylcholine. The organic osmolytes occur as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Dog kidney cells in culture (MDCK) were previously shown to accumulate betaine in response to increased extracellular osmolality. We demonstrate here that this accumulation requires the presence of betaine in the medium, and this apparently is a result of uptake of extracellular betaine, rather than synthesis by the cells. MDCK cells have low- and high-affinity sodium-dependent betaine transporters with Km for betaine of approximately 6 and approximately 0.1 mM, respectively. Relative to isotonic controls, sodium-dependent betaine uptake is approximately sevenfold greater in cells chronically exposed (greater than 1 yr) to hypertonic medium (615 mosmol/kg). This is due to an increase in the maximal velocity of sodium-dependent betaine uptake with no apparent change in Km. Cells acutely exposed (1-7 days) to hypertonic medium show increased sodium-dependent betaine uptake, which is maximal after 1 day, then decreases as betaine and other osmolytes accumulate in the cells. Thus the response by which renal cells accumulate betaine following hypertonicity apparently includes an increase in the number (or, less likely, the transport turnover rate) of functioning sodium-dependent betaine transporters.

Functional Significance of Cell Volume Regulatory Mechanisms

Abstract: Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Volkl, Siegfried Waldegger, Erich Gulbins, and Dieter Haussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. ...

Pathophysiology and management of pulmonary infections in cystic fibrosis.

Abstract: This comprehensive State of the Art review summarizes the current published knowledge base regarding the pathophysiology and microbiology of pulmonary disease in cystic fibrosis (CF). The molecular basis of CF lung disease including the impact of defective cystic fibrosis transmembrane regulator (CFTR) protein function on airway physiology, mucociliary clearance, and establishment of Pseudomonas aeruginosa infection is described. An extensive review of the microbiology of CF lung disease with particular reference to infection with P. aeruginosa is provided. Other pathogens commonly associated with CF lung disease including Staphylococcal aureus, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and atypical mycobacteria are also described. Clinical presentation and assessment of CF lung disease including diagnostic microbiology and other measures of pulmonary health are reviewed. Current recommendations for management of CF lung disease are provided. An extensive review of antipseudomonal therapies in the settings of treatment for early P. aeruginosa infection, maintenance for patients with chronic P. aeruginosa infection, and treatment of exacerbation in pulmonary symptoms, as well as antibiotic therapies for other CF respiratory pathogens, are included. In addition, the article discusses infection control policies, therapies to optimize airway clearance and reduce inflammation, and potential future therapies.

Physiology of cell volume regulation in vertebrates.

Abstract: The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adap...

Aquaporins in the Kidney: From Molecules to Medicine

Abstract: The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.

Gene expression in response to abscisic acid and osmotic stress.

Abstract: Abscisic acid (ABA) was discovered in the 1950s to be a phytohormone affecting leaf abscision and bud dormancy. It was soon characterized as a sesquiterpene derived from mevalonate although certain steps of its biosynthesis in plants are still unknown (Li and Walton, 1987; Zeevaart and Creelman, 1988). Continuing work on ABA has shown that it mediates various developmental and physiological processes that affect the agronomic performance of crop plants (Austin et al., 1982; Ramagopal, 1987). These proc? esses include embryo maturation and germination as well as the response of vegetative tissues to osmotic stress (Singh et al., 1987; Zeevaart and Creelman, 1988). ABA levels increase in tissues subjected to osmotic stress by desiccation, salt, or cold (Henson, 1984; Mohapatra et al., 1988). Under these conditions, specific genes are ex? pressed that can also be induced in unstressed tissues by the application of exogenous ABA (Singh et al., 1987; Gomez et al., 1988; Mundy and Chua, 1988). Some of these genes are also expressed during the normal embryogenic program when seeds desiccate and embryos be? come dormant (Dure et al., 1981). Although different sets of ABA-responsive genes exhibit different patterns of de? velopmental and tissue-specific expression, some of them appear to be part of a general reaction to osmotic stress. This system is a normal part of the embryogenic program but is inducible in vegetative tissues at other times in the plant life cycle. Several ABA-responsive genes have now been isolated (Baker et al., 1988; Gomez et al., 1988; Marcotte et al., 1988; Mundy and Chua, 1988; Vilardell et al., 1990; Yamaguchi-Shinozaki et al., 1990). A major goal of the research discussed below is to understand the role these genes play in osmotic stress and desiccation tolerance.