We evaluated the effects of culturing mouse MTAL cells under conditions that suppressed steady-state cytosolic Cl − on chloride channels fused into bilayers from basolateral vesicles of cultured MTAL cells. We used two agents to suppress Cl − entry: 10 −6 M PGE 2 and 10 −4 M bumetanide. Basolateral Cl − channels from control cultured MTAL cells exhibited the signature characteristics of mmClC-Ka channels: increased open-time probability ( P o ) either by raising cytosolic-face [Cl − ] or, at 2 m M cytosolic Cl − , by adding (ATP + PKA), and first-order conductance kinetics. Either 10 −6 M PGE 2 or 10 −4 M bumetanide in culture media reduced steady-state MTAL cytosolic Cl − . Chloride channels from these cells exhibited characteristics unique to CTAL mcClC-Ka channels, namely: no augmentation of P o either by raising cytosolic Cl − or with cytosolic (ATP + PKA), and multi-ion occupancy. Semi-quantitative RT-PCR and real-time quantitative PCR showed that culturing MTAL cells with 10 −6 M PGE 2 or 10 −4 M bumetanide reduced mRNA levels encoding mmClC-Ka but not mRNA levels encoding mcClC-Ka. However, when MTAL cells were cultured under control conditions, and then pre-incubated for 60 minutes with 10 −4 M bumetanide, cytosolic Cl − fell acutely but Cl − channels exhibited characteristics of mmClC-Ka channels. Thus PGE 2 and bumetanide, both of which lower steady-state MTAL cytosolic Cl − concentrations, inhibit either the transcriptional and/or the translational processes for mmClC-Ka synthesis.

Cultured mouse MTAL cells contain more mRNA encoding the Cl − channel mcClC-Ka, which mediates CTAL Cl − absorption, than mRNA encoding the Cl − channel mmClC-Ka, which mediates MTAL Cl − absorption. mmClC-Ka and mcClC-Ka have three functional differences: 1) mmClC-Ka open time probability, P o , increases with increasing cytosolic Cl − , but variations in cytosolic Cl − do not affect P o in mcClC-Ka; 2) mmClC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on P o in mcClC-Ka; and 3) mmClC-Ka channels have single-ion occupancy, while mcClC-Ka channels have multi-ion occupancy. Using basolateral vesicles from MTAL cells fused into bilayers, we evaluated the effects of 1 m M cytosolic phenylglyoxal (PGO), which binds covalently to lysine or arginine, on Cl − channels. With PGO pretreatment, Cl − channels were uniformly not gated either with increases in cytosolic-face Cl − or with (ATP + PKA) at 2 m m cytosolic-face Cl − ; and they exhibited multi-ion occupancy kinetics typical for mcClC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl − access to arginine or lysine residues on mmClC-Ka by PGO results in Cl − channels having the functional characteristics of mcClC-Ka channels.

This paper describes the kinetics of Cl- flux through mcClC-Ka Cl- channels from basolateral membranes of mouse CTAL cells. We have cloned two separate but highly homologous Cl- channels, mmClC-Ka from cultured mouse MTAL cells and mcClC-Ka from cultured mouse CTAL cells. The mmClC-Ka and mcClC-Ka channels appear to mediate net Cl- absorption in the MTAL and CTAL, respectively. The kinetics of Cl- permeation through mmClC-Ka channels exhibit traditional criteria for a first-order process, including saturation kinetics. Thus mmClC-Ka channels operate functionally as if the channels were occupied by a single Cl- ion at any given time. In the present studies, we examined conductance-concentration relations in mcClC-Ka channels, and compared both mole-fraction effects and ion selectivity characteristics in mmClC-Ka and mcClC-Ka channels. In mcClC-Ka channels, we observed both self-block at high external Cl- concentrations and, at constant ionic strength, an anomalous mole-fraction effect using external solutions containing varying F-/Cl- concentrations. Neither effect was obtained in mmClC-Ka channels. These data are consistent with the possibility that Cl- permeation through mcClC-Ka channels involved multi-ion occupancy channels that expressed single-file behavior.

The purpose of this chapter is to consider the pathophysiology of polyuric syndromes, a term used to encompass those disorders which limit the kidney’s ability to form a hypertonic urine, i. e., to excrete solutes in a minimal volume of water. To the extent that this homeostatic mechanism is impaired, polydipsia is required to avoid the development of body fluid hyperosmolality and its potentially lethal sequelae. Depending on the severity of the concentrating defect, the maintenance of a normal body fluid osmolality may require only a modest increase in the amount of solute-free water ingested, e. g., in hypercalcemic or hypokalemic states, or the daily intake of 50–85% of total body water, e. g., in infants with familial nephrogenic diabetes insipidus.

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl− channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites—threonine 185, threonine 187 and serine 270—which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P o in basolateral CTAL Cl− channels.

This series of "Physiology in Medicine" will deal with oxidative stress produced by free radicals. It is fair to say that the field was opened in 1969 by Joe M. McCord, the author of the first article in this series, working in collaboration with Irwin Fridovich at Duke University. McCord and Fridovich discovered an enzyme entitled superoxide dismutase (SOD) that catalyzed the conversion of the reactive oxygen species O2− to hydrogen peroxide according to the following reaction: From the above reaction, one can see that the reactive oxygen species O2− can be converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase. Now if one has sufficient quantities of the enzyme catalase on hand, the H2O2 can be broken down to water plus oxygen. In practical terms, reactive oxygen species include two compounds that are free radicals, O2− and OH·, both of which are characterized by having a single unpaired electron; and H2O2, which is not a free radical but which can, in the absence of catalase, lead to the formation of the free radical OH·. The consequences of the formation of these free radicals, or of H2O2 formation in excess of the rate at which it can be converted to oxygen and water, are precisely the issues to be considered in this series of articles. Stated briefly, it now seems clear that oxidative stress, particularly when free radicals are generated by leukocytes in inflammatory reactions, can lead to the formation of the noxious free radicals O2− and OH· as well as H2O2 which, as I noted above, can under certain circumstances lead to the formation of OH·. In turn, these reactive oxygen species harm tissues in a variety of ways, including DNA damage, impairment of mitochondrial respiration, and direct parenchymal impairment. Table 1indicates the articles to appear in this series and their focus. The series begins with an article by McCord, who provides a global overview of the evolution of free radicals and oxidative stress. Among other things, McCord stresses the remarkable interplay between O2, obviously a gas necessary for life, and the pejorative consequences of free radicals. Next, B. M. Babior considers the role of oxidative stress associated with the activation of phagocytes in inflammatory responses.Table 1Free Radicals and Oxidative StressAuthorTitleJoe M. McCord, PhDOverview: The evolution of free radicals and oxidative stressProfessor of Medicine, Biochemistry & MicrobiologyWebb-Waring InstituteUniversity of Colorado School of MedicineBernard M. Babior, MD, PhDPhagocytes and oxidative stressHead, Department of Molecular & Experimental MedicineDivision of BiochemistryScripps Research InstitutePaul Kubes, PhDNitric oxide and intestinal inflammationAssociate Professor of Physiology and BiophysicsUniversity of Calgary Faculty of MedicineD. Neil Granger, PhDOxidative stress and cardiac diseaseProfessor and HeadDepartment of PhysiologyLSU Medical Center/ShreveportCarroll E. Cross, MDOxidants, nitrosants, and the lungProfessor of Medicine and PhysiologyDivision of Pulmonary and Clinical Care MedicineUniversity of California School of Medicine/DavisNorman Delanty, MDOxidative injury in diseases of the central nervous systemDepartment of NeurologyUniversity of Pennsylvania School of MedicineKarl A. Nath, MB, ChBOxidative stress and acute renal failureProfessor of MedicineMayo Medical School Open table in a new tab The remaining articles focus on a series of particular examples of oxidative stress, the ways in which oxidative stress damages various parenchymal organs, and diseases in which oxidative stress plays a key developmental role: P. Kubes considers diseases of the gastrointestinal tract, D. N. Granger focuses on cardiac disease, C. E. Cross discusses the relations of oxidative stress and pulmonary disease, N. Delanty deals with oxidative stress and diseases of the central nervous system, and finally, K. A. Nath considers the role of oxidative stress in acute renal failure. It is, as McCord points out in the first article of the PIM series in this issue of AJM, a remarkable paradox that oxygen is a mixed blessing. It is obvious that the gas O2 is necessary for life but under pernicious circumstances, oxygen can be transformed into the reactive oxygen species O2− and OH·, which have pernicious consequences. The same can be said for H2O2, the product of the free radical scavenger superoxide dismutase, when catalase is unavailable.

Extracellular fluid volume is determined by sodium and its accompanying anions. There are control mechanisms which regulate sodium balance in the body. These include high and low pressure baroreceptors, intrarenal baroreceptors, renal autoregulation, tubuloglomerular feedback, aldosterone, and numerous other physical and hormonal factors. Sodium transport by the nephron involves active and passive processes which occur in several different nephron segments. Mechanisms of cotransport, Na(+)-H+ exchange, antiporters and ion-specific channels are all utilized by the nephron to maintain sodium balance. These regulatory factors and transport mechanisms for sodium in the kidney will he discussed in detail.

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Department of Internal medicine, University of Arkansas College of Medicine Little Rock 72205, USA.

Cl- channels fused from basolateral mTAL membranes into planar bilayers have distinctive functional characteristics which, when taken together, are unique among Cl- channels. The properties of these 50 to 60 pS channels can account for the characteristics of basolateral Cl- conductances in microperfused mTAL segments and thus may mediate net basolateral Cl- absorption in the intact mTAL. In the present studies, we solubilized basolateral membranes from rabbit mTAL. Since basolateral mTAL Cl- channels contain arginine- and lysine-rich domains, we exposed these solubilized membranes to sequential cation- and anion-exchange chromatography. The bound and unbound eluates from cation- and anion-exchange chromatography were reconstituted into proteoliposomes which, when fused into bilayers, yielded Cl- channels whose properties were virtually identical to those described above for native basolateral mTAL channels fused into bilayers. As judged by valinomycin-sensitive conductive 36Cl- uptake, proteoliposomes reconstituted from the unbound eluates after anion-exchange chromatography were enriched at least 30-fold in Cl- channel activity and had about 30% of the total Cl- channel activity solubilized in native vesicles.

Cet article de synthese fait le point sur les mecanismes d'absorption des sels dans la branche grele ascendante de l'anse de Henle dans la corticale et la medullaire renale. Transport du NaCl − cotransporteurs; proprietes biochimiques et electrophysiologiques. Absorption transepitheliale. Regulation par l'Adh et la PGE 2 . Force de dilution

This paper considers the quantitative interplay of various factors in modulating diluting power of in vitro medullary and cortical thick ascending limbs of Henle (MTAL and CTAL, respectively) segments from mouse and rabbit. Experimentally, the measured diluting power of the in vitro rabbit CTAL is greater than that of the rabbit MTAL, although the inherent rate of net Cl- absorption at high perfusion rates is considerably greater in the rabbit MTAL than in the rabbit CTAL. Similar results apply when comparing the rabbit CTAL to the mouse MTAL exposed to antidiuretic hormone (ADH). Our calculations show that, in the rabbit CTAL, the measured static head luminal salt concentration can be accounted for quantitatively by the measured rate of net salt absorption at a high perfusion rate together with the passive permeability coefficients for Na+ and Cl-. Moreover, with perfusion rates of 10% of single-nephron glomerular filtration rate, the transport properties of the CTAL predict that, at the end of the CTAL, the static head luminal Cl- concentration occurs if the initial perfusate contains either 50 or 150 mM Cl-. Thus one can argue that, in vivo the CTAL may be the cardinal determinant of the TAL contribution to diluting power and to external salt balance. The relatively blunted diluting power of in vitro MTAL segments can be accounted for quantitatively by assuming that luminal dilution, and the attendant osmotic gradient from lumen to cell, suppresses the inherent rate of transcellular Cl- transport. Our calculations also show that prostaglandin E2 and peritubular osmolality increases blunt tubular diluting power. Thus in vivo, the MTAL segment may be the cardinal determinant of TAL contribution to concentrating power and to intrarenal salt balance.

We evaluated the effects of osmotic gradients on 22Na+ influx in vesicles prepared from rat outer renal medulla. 22Na+ influx driven in a coflow mode by an inwardly directed 100 mM KCl gradient was measured at 20 and 60 s; 1 mM bumetanide inhibited approximately 30% of 22Na+ influx. The bumetanide-sensitive 22Na+ influx was reduced by approximately 65% when either K+ or Cl- was omitted from the aqueous phases. We found that an osmotic gradient for vesicle shrinkage, that is, 600 mM urea in the extravesicular medium, enhanced the bumetanide-sensitive 22Na+ influx twofold. Conversely, an osmotic gradient for vesicle swelling, that is, with vesicles but not extravesicular media loaded with 600 mM urea, produced a 50% suppression of bumetanide-sensitive 22Na+ influx. Moreover, 600 mM extravesicular urea, an osmotic gradient for vesicle shrinkage, also reduced uptake of the nonspecific marker [14C]mannitol. These effects of osmotic gradients were not due to alterations in ionic driving forces, since bumetanide-sensitive 22Na+ influx driven in a counterflow mode by loading the vesicles with 100 mM NaCl also was activated or suppressed by osmotic gradients for vesicle shrinkage or swelling, respectively. We conclude that osmotic gradients, and/or vesicle volume changes, modulate bumetanide-sensitive Na+:K+:2Cl- activity.

The purpose of this paper is to communicate recent work from our laboratory pertaining to the mechanism of the ADH-induced increase in salt absorption in the medullary thick ascending limb of Henle’s loop (mTALH) of mice. The specialized transport properties of the thick ascending limb of Henle’s loop enable this segment of the nephron to dissociate salt and water absorption. Sodium chloride absorption by the cortical and medullary thick ascending limb is responsible for urinary dilution, since these segments are water-impermeable, as well as urinary concentration by creating a hypertonic medullary interstitium. The primary site of action of the “loop” diuretics, the TALH absorbs roughly 15