Mouse Medullary Thick Ascending Limb Research Articles

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1.

LPS inhibits absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-β (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

High-mobility group box 1 inhibits HCO(3)(-) absorption in medullary thick ascending limb through a basolateral receptor for advanced glycation end products pathway.

High-mobility group box 1 (HMGB1) is a damage-associated molecule implicated in mediating kidney dysfunction in sepsis and sterile inflammatory disorders. HMGB1 is a nuclear protein released extracellularly in response to infection or injury, where it interacts with Toll-like receptor 4 (TLR4) and other receptors to mediate inflammation. Previously, we demonstrated that LPS inhibits HCO(3)(-) absorption in the medullary thick ascending limb (MTAL) through a basolateral TLR4-ERK pathway (Watts BA III, George T, Sherwood ER, Good DW. Am J Physiol Cell Physiol 301: C1296-C1306, 2011). Here, we examined whether HMGB1 could inhibit HCO(3)(-) absorption through the same pathway. Adding HMGB1 to the bath decreased HCO(3)(-) absorption by 24% in isolated, perfused rat and mouse MTALs. In contrast to LPS, inhibition by HMGB1 was preserved in MTALs from TLR4(-/-) mice and was unaffected by ERK inhibitors. Inhibition by HMGB1 was eliminated by the receptor for advanced glycation end products (RAGE) antagonist FPS-ZM1 and by neutralizing anti-RAGE antibody. Confocal immunofluorescence showed expression of RAGE in the basolateral membrane domain. Inhibition of HCO(3)(-) absorption by HMGB1 through RAGE was additive to inhibition by LPS through TLR4 and to inhibition by Gram-positive bacterial molecules through TLR2. Bath amiloride, which selectively prevents inhibition of MTAL HCO(3)(-) absorption mediated through Na⁺/H⁺ exchanger 1 (NHE1), eliminated inhibition by HMGB1. We conclude that HMGB1 inhibits MTAL HCO(3)(-) absorption through a RAGE-dependent pathway distinct from TLR4-mediated inhibition by LPS. These studies provide new evidence that HMGB1-RAGE signaling acts directly to impair the transport function of renal tubules. They reveal a novel paradigm for sepsis-induced renal tubule dysfunction, whereby exogenous pathogen-associated molecules and endogenous damage-associated molecules act directly and independently to inhibit MTAL HCO(3)(-) absorption through different receptor signaling pathways.

Lumen LPS inhibits HCO3−absorption in the medullary thick ascending limb through TLR4-PI3K-Akt-mTOR-dependent inhibition of basolateral Na+/H+exchange

Sepsis and endotoxemia induce defects in renal tubule function, but the mechanisms are poorly understood. Recently, we demonstrated that lipopolysaccharide (LPS) inhibits HCO3(-) absorption in the medullary thick ascending limb (MTAL) through activation of different Toll-like receptor 4 (TLR4) signaling pathways in the basolateral and apical membranes. Basolateral LPS inhibits HCO3(-) absorption through ERK-dependent inhibition of the apical Na(+)/H(+) exchanger NHE3. Here, we examined the mechanisms of inhibition by lumen LPS. Adding LPS to the lumen decreased HCO3(-) absorption by 29% in rat and mouse MTALs perfused in vitro. Inhibitors of phosphoinositide 3-kinase (PI3K) or its effectors Akt and mammalian target of rapamycin (mTOR) eliminated inhibition of HCO3(-) absorption by lumen LPS but had no effect on inhibition by bath LPS. Exposure to LPS for 15 min induced increases in phosphorylation of Akt and mTOR in microdissected MTALs that were blocked by wortmannin, consistent with activation of Akt and mTOR downstream of PI3K. The effects of lumen LPS to activate Akt and inhibit HCO3(-) absorption were eliminated in MTALs from TLR4(-/-) and MyD88(-/-) mice but preserved in tubules lacking Trif or CD14. Inhibition of HCO3(-) absorption by lumen LPS was eliminated under conditions that inhibit basolateral Na(+)/H(+) exchange and prevent inhibition of HCO3(-) absorption mediated through NHE1. Lumen LPS decreased basolateral Na(+)/H(+) exchange activity through PI3K. We conclude that lumen LPS inhibits HCO3(-) absorption in the MTAL through TLR4/MyD88-dependent activation of a PI3K-Akt-mTOR pathway coupled to inhibition of NHE1. Molecular components of the TLR4-PI3K-mTOR pathway represent potential therapeutic targets for sepsis-induced renal tubule dysfunction.

Basolateral LPS inhibits NHE3 and HCO3−absorption through TLR4/MyD88-dependent ERK activation in medullary thick ascending limb

Sepsis is associated with defects in renal tubule function, but the underlying mechanisms are incompletely understood. Recently, we demonstrated that Gram-negative bacterial lipopolysaccharide (LPS) inhibits HCO(3)(-) absorption in the medullary thick ascending limb (MTAL) through activation of Toll-like receptor 4 (TLR4). Here, we examined the mechanisms responsible for inhibition of HCO(3)(-) absorption by basolateral LPS. Adding LPS to the bath decreased HCO(3)(-) absorption by 30% in rat and mouse MTALs perfused in vitro. The inhibition of HCO(3)(-) absorption was eliminated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/ERK inhibitors U0126 and PD98059. LPS induced a rapid (<15 min) and sustained (up to 60 min) increase in ERK phosphorylation in microdissected MTALs that was blocked by PD98059. The effects of basolateral LPS to activate ERK and inhibit HCO(3)(-) absorption were eliminated in MTALs from TLR4(-/-) and myeloid differentiation factor 88 (MyD88)(-/-) mice but were preserved in MTALs from TIR (Toll/interleukin-1 receptor) domain-containing adapter-inducing interferon-β (Trif)(-/-) mice. Basolateral LPS decreased apical Na(+)/H(+) exchanger 3 NHE3 activity through a decrease in maximal velocity (V(max)). The inhibition of NHE3 by LPS was eliminated by MEK/ERK inhibitors. LPS inhibited HCO(3)(-) absorption despite the presence of physiological stimuli that activate ERK in the MTAL. We conclude that basolateral LPS inhibits HCO(3)(-) absorption in the MTAL through activation of a TLR4/MyD88/MEK/ERK pathway coupled to inhibition of NHE3. These studies identify NHE3 as a target of TLR4 signaling in the MTAL and show that bacterial molecules can impair the absorptive functions of renal tubules through inhibition of this exchanger. The ERK pathway links TLR4 to downstream modulation of ion transport proteins and represents a potential target for treatment of sepsis-induced renal tubule dysfunction.

Toll-like receptor 2 mediates inhibition of HCO3−absorption by bacterial lipoprotein in medullary thick ascending limb

Bacterial infection and sepsis are associated with renal tubule dysfunction and dysregulation of systemic electrolyte balance but the underlying mechanisms are incompletely understood. Recently, we demonstrated that HCO(3)(-) absorption by the medullary thick ascending limb (MTAL) is inhibited by gram-negative bacterial LPS through activation of Toll-like receptor 4 (TLR4). Here, we examined whether MTAL transport is altered by activation of TLR2, the receptor predominantly responsible for recognizing gram-positive bacteria. Confocal immunofluorescence showed expression of TLR2 in the basolateral membrane domain of rat and mouse MTALs. The functional role of TLR2 was examined in perfused MTALs using Pam(3)CSK(4), a bacterial lipoprotein analog that specifically activates TLR2. Adding Pam(3)CSK(4) to the bath decreased HCO(3)(-) absorption by 25%. The inhibition by Pam(3)CSK(4) was eliminated in MTALs from TLR2(-/-) mice. HCO(3)(-) absorption was also inhibited by the TLR2 agonists lipoteichoic acid and peptidoglycan, two cell wall components of gram-positive bacteria. The MEK/ERK inhibitor U0126 eliminated inhibition of HCO(3)(-) absorption by bath LPS but had no effect on inhibition by Pam(3)CSK(4). The inhibition by Pam(3)CSK(4) was eliminated by the protein kinase C inhibitors chelerythrine Cl and bisindolylmaleimide. Moreover, the inhibition by Pam(3)CSK(4), lipoteichoic acid, and peptidoglycan was additive to inhibition by LPS. Thus, agonists of basolateral TLR2 and TLR4 inhibit HCO(3)(-) absorption independently through distinct signaling pathways. We conclude that bacterial components act directly through TLRs to modify the transport function of renal tubules. During polymicrobial sepsis, gram-positive bacterial molecules acting through TLR2 and gram-negative LPS acting through TLR4 can function through parallel signaling pathways to impair MTAL transport. The inhibition of luminal acidification may impair the ability of the kidneys to correct systemic acidosis that contributes to sepsis pathogenesis.

Lipopolysaccharide directly alters renal tubule transport through distinct TLR4-dependent pathways in basolateral and apical membranes

Bacterial infection of the kidney is associated with renal tubule dysfunction and dysregulation of systemic electrolyte balance. Whether bacterial molecules directly affect renal tubule transport is unknown. We examined the effects of LPS on HCO3(-) absorption in the isolated rat and mouse medullary thick ascending limb (MTAL). LPS decreased HCO3(-) absorption when added to bath or lumen. The MEK/ERK inhibitor U0126 eliminated inhibition by bath LPS but had no effect on inhibition by lumen LPS. Conversely, the mammalian target of rapamycin (mTOR) inhibitor rapamycin eliminated inhibition by lumen LPS but had no effect on inhibition by bath LPS. Inhibiting basolateral Na(+)/H(+) exchange with amiloride eliminated inhibition of HCO3(-) absorption by lumen but not bath LPS. Confocal immunofluorescence showed expression of TLR4 in basolateral and apical membrane domains. Inhibition of HCO3(-) absorption by bath and lumen LPS was eliminated in MTALs from TLR4(-/-) mice. Thus LPS inhibits HCO3(-) absorption through distinct TLR4-dependent pathways in basolateral and apical membranes. These results establish that bacterial molecules can directly impair the transport function of renal tubules, identifying a new mechanism contributing to tubule dysfunction during bacterial infection. The LPS-induced reduction in luminal acidification may contribute to Gram-negative pathogenicity by promoting bacterial adherence and growth and impairing correction of infection-induced systemic acid-base disorders.

Cl- channels in basolateral TAL membranes. XVI. MTAL and CTAL cells each contain the mRNAs encoding mmClC-Ka and mcClC-Ka

Our prior data indicate that two separate but homologous basolateral chloride (Cl-) channels, mmClC-Ka and mcClC-Ka, are the principal mediators of net Cl- absorption in mouse medullary thick ascending limb (MTAL) and cortical thick ascending limb (CTAL) cells, respectively. In the present studies, we evaluated the possibility that there might be translational or post-translational suppression of mmClC-Ka and mcClC-Ka activity in CTAL and MTAL cells, respectively. Polymerase chain reaction (PCR) fragments were prepared that were highly specific for either mmClC-Ka or mcClC-Ka, the cDNAs encoding mmClC-Ka and mcClC-Ka, respectively. Using reverse transcription (RT)-PCR with these highly specific products, mRNAs specific for non-homologous channel sequences in either mmClC-Ka or mcClC-Ka were present in both MTAL and CTAL cells. Both mouse MTAL and CTAL cells contain the mRNAs encoding mmClC-Ka and mcClC-Ka. There may be translational or post-translational suppression of mmClC-Ka activity in CTAL cells, and of mcClC-Ka activity in MTAL cells.

Effect of luminal atrial natriuretic peptide on chloride reabsorption in mouse cortical thick ascending limb: inhibition by endothelin.

Insofar as neutral endopeptidase inhibition has afforded evidence for a tubular luminal action of atrial natriuretic peptide (ANP), the present study was undertaken to investigate a possible effect of the peptide on chloride reabsorption (JCl) in thick ascending limb (TAL). Luminal addition of ANP to in vitro microperfused cortical TAL (CTAL) significantly decreased JCl with a threshold and a maximum concentration of 10(-12) M and 10(-9) M, respectively. A similar effect of 10(-9) M ANP was observed in medullary TAL (MTAL). The effect of luminal ANP was significantly reduced by HS-142-1, a specific inhibitor of guanylyl cyclase receptor, and by H-8, a protein kinase G inhibitor, but was not affected by the protein kinase C inhibitor bisindolylmaleimide I. Unexpectedly, the effect of ANP was not additive with that of endothelin (ET), a peptide that was previously shown to decrease JCl in TAL through a calcium-independent, protein kinase C-mediated pathway. Indeed, ET-1 (10(-8) M in the lumen) significantly decreased JCl and prevented a further effect of ANP on the same tubule. Similarly, the decrease of JCl induced by simultaneous addition of ET and ANP was not higher than that obtained with each agent alone. Conversely, the inhibitory effect of ANP was enhanced in the presence of cyclic guanosine monophosphate (cGMP; 10(-6) M in the lumen). ET-1 significantly attenuated the ANP-stimulated generation of cGMP in microdissected CTAL and failed to prevent a further decrease of JCl promoted by a permeant cGMP analogue. It is concluded that luminal ANP decreased Cl reabsorption in mouse CTAL and MTAL. This effect was abrogated by ET-1 as a result of the inhibition of ANP-stimulated cGMP generation.

Cl- channels in basolateral TAL membranes: XIII. Heterogeneity between basolateral MTAL and CTAL Cl- channels

Antidiuretic hormone (ADH) or adenosine 3', 5'-cyclic phosphate (cAMP) analogues augment net NaCl absorption in microperfused mouse medullary thick ascending limb (MTAL) segments but not in cortical thick ascending limb (CTAL) segments. This ADH-dependent MTAL effect is due to increased apical Na+/K+/2Cl- admittance and apical K+ recycling accompanied by a rise in calculated intracellular Cl- concentrations and by a threefold rise in basolateral Cl- conductance. rbClC-Ka, a 75.2 member of the ClC family of Cl- channels, mediates net Cl- absorption in the MTAL. The gating characteristics of rbClC-Ka channels from their intracellular surfaces are, to our knowledge, unique among Cl- channels. The channels are activated by small increases in intracellular Cl- (K1/2 = 10 mM Cl-). Adenosine triphosphate plus the catalytic subunit of protein kinase A (ATP + PKA) gate rbClC-Ka when cytosolic Cl- concentrations are 25 mM. Thus, in mouse MTAL segments, ADH-dependent rises in cytosolic Cl- are primarily responsible for basolateral Cl- conductance increases. These experiments compared the properties of Cl- channels fused into bilayers from basolaterally enriched vesicles from cultured mouse CTAL cells with rbClC-Ka channels. The key findings were that anti-rbClC-Ka, antibody that recognizes and blocks rbClC-Ka, recognized and blocked basolateral Cl- channels in CTAL cells, that the extracellular faces of the CTAL channels were, like rbClC-Ka, substrate gated with a K1/2 of approximately 170 mM Cl-, and that, unlike rbClC-Ka channels, cytosolic faces of basolateral CTAL Cl- channels were not gated by either increasing cytosolic Cl- concentrations or cytosolic (ATP + PKA). This failure of activation of basolateral CTAL Cl- channels was confirmed using excised patch clamp studies. Finally, on Western blots, anti-rbClC-Ka recognized a 74 kDa band on basolateral CTAL vesicles. Basolateral CTAL Cl- channels probably share a high degree of structural homology and possibly molecular mass with rbClC-Ka channels. However, significant differences between rbClC-Ka channels and CTAL Cl- channels account for the inability of increasing either cytosolic Cl- or (PKA + ATP) to raise Po in CTAL basolateral Cl- channels.

Expression of Cl−/ HCO 3 − exchanger in the basolateral membrane of mouse medullary thick ascending limb

Although a basolateral Cl-/HCO3- exchanger (AE) has been implicated in the arginine vasopressin (AVP)-dependent hypertonic regulatory increase in the medullary thick ascending limb (MTAL), there are conflicting data regarding whether this exchanger is indeed present in this tubule segment. In this study, mouse MTAL was examined whether Cl-/HCO3- exchange activity was present in the basolateral membrane and whether mRNAs from the known AE genes are expressed. Cl-/HCO3- exchange activity was examined in isolated perfused MTAL tubules under isotonic conditions and in the absence of arginine vasopressin. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to monitor intracellular pH. Removal of basolateral Cl- induced reversible cell alkalization that was independent of external Na+ and completely inhibited by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The rate and extent of cell alkalinization were significantly greater in the presence than absence of external CO2/HCO3-. A voltage clamp did not inhibit cell alkalinization induced by basolateral Cl- removal. Consistently, addition of basolateral Cl- induced reversible cell acidification in MTAL depleted of intracellular Cl-. Furthermore, mRNA encoding two members (AE2 and AE3) of the AE gene family were demonstrated in microdissected mouse MTAL tubules by reverse transcription-polymerase chain reaction. It is concluded that AE is present in the basolateral membrane of mouse MTAL.

Cl- channels in basolateral renal medullary membranes. XII. Anti-rbClC-Ka antibody blocks MTAL Cl- channels.

Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.

Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb.

Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.

Calcium and Magnesium: Low Passive Permeability and Tubular Secretion in the Mouse Medullary Thick Ascending Limb of Henle's Loop (MTAL)

Recent studies from our laboratory have shown that in the mouse and rat nephron Ca2+ and Mg2+ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca2+ and Mg2+ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PDte.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca2+ and Mg2+ net fluxes in the mTAL are not driven by the transepithelial PDte. At zero voltage, a small but significant net secretion of Ca2+ into the tubular lumen was observed. With a high lumen-positive PDte generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca2+ and Mg2+ reabsorption was noted; instead significant and sustained Ca2+ and Mg2+ net secretion occurred. When a lumen-positive PDte was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca2+ net secretion and a lesser Mg2+ net secretion, not modified by ADH, were observed. Replacement of Na+ by K+ in the lumen perfusate induced, in the absence of PDte changes, important but reversible net secretions of Ca2+ and Mg2+. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca2+ and Mg2+ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca2+ and Mg2+, the secretory processes would appear to be of cellular origin.

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Cl- channels in basolateral renal medullary vesicles. IX. Channels from mouse MTAL cell patches and medullary vesicles.

The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.

Vasopressin regulates apical and basolateral Na(+)-H+ antiporters in mouse medullary thick ascending limbs

We assessed in isolated perfused mouse medullary thick ascending limb (MTAL) segments Na(+)-H+ antiporter activity in both apical and basolateral membranes and the effects of arginine vasopressin (AVP) on the activities of these antiporters under isotonic conditions using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to monitor intracellular pH (pHi). When the apical Na(+)-H+ antiporter was inhibited in the absence of AVP with removal of luminal Na+ plus addition of 0.5 mM amiloride, a small but significant increase in pHi was observed after luminal NH4Cl-induced acidification of MTAL cells to pHi less than 6.7. This increase in pHi was dependent on basolateral Na+ and inhibited with 0.5 mM basolateral amiloride, consistent with the function of a basolateral Na(+)-H+ antiporter. Basolateral AVP (100 microU/ml) enhanced the rate of pHi recovery due to the basolateral Na(+)-H+ antiporter by more than twofold. In contrast, AVP decreased the apical Na(+)-H+ antiporter activity by 50%. In the absence of AVP, addition of 0.5 mM amiloride to the luminal perfusate reduced steady-state pHi by 0.40 +/- 0.07 units, whereas exposure of the basolateral membrane to the same concentration of amiloride had no effect on pHi (delta pHi = 0.01 +/- 0.01 units). AVP reduced the magnitude of cell acidification on exposure of apical membranes to amiloride (delta pHi = 0.16 +/- 0.03) but increased the pHi response to basolateral amiloride (delta pHi = 0.09 +/- 0.00). Thus Na(+)-H+ antiporters are present on both apical and basolateral membranes of the mouse MTAL in the absence of AVP. AVP stimulates the basolateral, while inhibiting the apical, Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation by verapamil of hormonal action on the Henle's loop of mice

In order to know the role of cytosolic calcium in the modulation of the hormone action on sodium chloride transport across the thick ascending limbs of Henle's loop, we examined whether verapamil, a blocker of cellular calcium entry, can modulate the effects of arginine vasopressin (AVP) or glucagon in stimulating transepithelial voltage (Vt) and cyclic AMP generation in the mouse medullary thick ascending limb (MAL). The pretreatment of the renal tubule with 5 X 10(-5)M verapamil reduced the Vt stimulated with 200 microU/mliter AVP from 1.7 +/- 0.3 mV to 0.4 +/- 0.4 mV (N = 7, P less than 0.05). The changes in Vt were well correlated with those of unidirectional Cl flux from the lumen to the bath. However, verapamil did not influence the Vt stimulated with 10(-3) M dibutyryl cyclic AMP. The pretreatment of the MAL with 10(-5) M verapamil also inhibited the cyclic AMP generation in the MAL from 72.1 +/- 17.9 to 50.6 +/- 13.6 fmoles/mm/7 min (N = 7, P less than 0.05) as well as in the medullary collecting tubule from 147.6 +/- 46.6 to 121.2 +/- 41.6 fmoles/mm/7 min (N = 4, P less than 0.05). The effect of verapamil in inhibiting the AVP-stimulated cAMP was dose-dependent: the cAMP generation was inhibited by 28.9 +/- 6.8 and 61.1 +/- 9.3% with 10(-5) M and 10(-4) M verapamil, respectively. When verapamil was added to the medium simultaneously with AVP, the generation of cyclic AMP was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)