Thampi George

Angiotensin II inhibits HCO 3 absorption via a cytochrome P-450-dependent pathway in MTAL

The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10-8 M in the bath) inhibits [Formula: see text] absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 μM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 μM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10-8 M), the major P-450 metabolite in the MTAL, inhibited [Formula: see text] absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10-8 M) to the bath had no effect on [Formula: see text]absorption. The inhibition of [Formula: see text]absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 μM) or herbimycin A (1 μM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso- N-acetylpenicillamine (SNAP; 10 μM) or the NO synthase (NOS) substratel-arginine (1 mM) to the bath stimulated [Formula: see text] absorption by 35%, suggesting that NO directly regulates MTAL[Formula: see text] absorption. Addition of 10-11 to 10-10 M ANG II to the bath did not affect [Formula: see text] absorption. We conclude that ANG II inhibits [Formula: see text]absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of [Formula: see text]absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.

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.

The basolateral NHE1 Na+/H+ exchanger regulates transepithelial HCO3 - absorption through actin cytoskeleton remodeling in renal thick ascending limb

In the renal medullary thick ascending limb (MTAL), inhibiting the basolateral NHE1 Na+/H+ exchanger with amiloride or nerve growth factor (NGF) results secondarily in inhibition of the apical NHE3 Na+/H+ exchanger, thereby decreasing transepithelial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption. MTALs from rats were studied by in vitro microperfusion to identify the mechanism underlying cross-talk between the two exchangers. The basolateral addition of 10 μm amiloride or 0.7 nm NGF decreased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in tubules bathed with amiloride or a Na+-free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption by vasopressin or stimulation by hyposmolality, factors that regulate \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption through primary effects on apical Na+/H+ exchange. Treatment of MTALs with amiloride or NGF for 15 min decreased polymerized actin with no change in total cell actin, as assessed both by fluorescence microscopy and by actin Triton X-100 solubility. Jasplakinolide prevented amiloride-induced actin remodeling. Vasopressin, which inhibits \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption by an amount similar to that observed with amiloride and NGF but does not act via NHE1, did not affect cellular F-actin content. These results indicate that basolateral NHE1 regulates apical NHE3 and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the MTAL by controlling the organization of the actin cytoskeleton.

Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb.

Background: Bacterial molecules act through Toll-like receptors to impair renal tubule function. Results: Activation of ERK and inhibition of bicarbonate absorption by LPS requires both TLR4 and TLR2 in thick ascending limb. Conclusion: LPS-induced cell signaling may depend on interaction between TLR4 and TLR2. Significance: Interaction of TLR4 with TLR2 could provide new mechanisms for control and therapeutic targeting of LPS-induced immune responses. Previously we demonstrated that basolateral LPS inhibits HCO3− absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO3− absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2−/− mice. In contrast, inhibition of HCO3− absorption by lumen LPS was preserved in TLR2−/− MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2−/− mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2−/− MTALs. Inhibition of HCO3− absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO3− absorption by TLR2-specific ligands was preserved in MTALs from TLR4−/− mice. These results indicate that the effect of basolateral LPS to inhibit HCO3− absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.

Nerve Growth Factor Inhibits HCO3 − Absorption in Renal Thick Ascending Limb through Inhibition of Basolateral Membrane Na+/H+ Exchange

Nerve growth factor (NGF) inhibits transepithelial HCO3 − absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitroin Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pH i recovery after lumen or bath Na+addition. NGF (0.7 nm in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3 − absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525–12529). To determine whether a similar mechanism could explain inhibition of HCO3 − absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mm Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3 − absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3 − absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3 − absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3 − absorption.

Nerve Growth Factor Inhibits Na+/H+ Exchange and Absorption through Parallel Phosphatidylinositol 3-Kinase-mTOR and ERK Pathways in Thick Ascending Limb

In the medullary thick ascending limb, inhibiting the basolateral NHE1 Na+/H+ exchanger with nerve growth factor (NGF) induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and transepithelial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption. The inhibition by NGF is mediated 50% through activation of extracellular signal-regulated kinase (ERK). Here we examined the signaling pathway responsible for the remainder of the NGF-induced inhibition. Inhibition of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption was reduced 45% by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 and 50% by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), a downstream effector of PI3K. The combination of a PI3K inhibitor plus rapamycin did not cause a further reduction in the inhibition by NGF. In contrast, the combination of a PI3K inhibitor plus the MEK/ERK inhibitor U0126 completely eliminated inhibition by NGF. Rapamycin decreased NGF-induced inhibition of basolateral NHE1 by 45%. NGF induced a 2-fold increase in phosphorylation of Akt, a PI3K target linked to mTOR activation, and a 2.2-fold increase in the activity of p70 S6 kinase, a downstream effector of mTOR. p70 S6 kinase activation was blocked by wortmannin and rapamycin, consistent with PI3K, mTOR, and p70 S6 kinase in a linear pathway. Rapamycin-sensitive inhibition of NHE1 by NGF was associated with an increased level of phosphorylated mTOR in the basolateral membrane domain. These findings indicate that NGF inhibits \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the medullary thick ascending limb through the parallel activation of PI3K-mTOR and ERK signaling pathways, which converge to inhibit NHE1. The results identify a role for mTOR in the regulation of Na+/H+ exchange activity and implicate NHE1 as a possible downstream effector contributing to mTORs effects on cell growth, proliferation, survival, and tumorigenesis.In the medullary thick ascending limb, inhibiting the basolateral NHE1 Na(+)/H(+) exchanger with nerve growth factor (NGF) induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and transepithelial HCO(3)(-) absorption. The inhibition by NGF is mediated 50% through activation of extracellular signal-regulated kinase (ERK). Here we examined the signaling pathway responsible for the remainder of the NGF-induced inhibition. Inhibition of HCO(3)(-) absorption was reduced 45% by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 and 50% by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), a downstream effector of PI3K. The combination of a PI3K inhibitor plus rapamycin did not cause a further reduction in the inhibition by NGF. In contrast, the combination of a PI3K inhibitor plus the MEK/ERK inhibitor U0126 completely eliminated inhibition by NGF. Rapamycin decreased NGF-induced inhibition of basolateral NHE1 by 45%. NGF induced a 2-fold increase in phosphorylation of Akt, a PI3K target linked to mTOR activation, and a 2.2-fold increase in the activity of p70 S6 kinase, a downstream effector of mTOR. p70 S6 kinase activation was blocked by wortmannin and rapamycin, consistent with PI3K, mTOR, and p70 S6 kinase in a linear pathway. Rapamycin-sensitive inhibition of NHE1 by NGF was associated with an increased level of phosphorylated mTOR in the basolateral membrane domain. These findings indicate that NGF inhibits HCO(3)(-) absorption in the medullary thick ascending limb through the parallel activation of PI3K-mTOR and ERK signaling pathways, which converge to inhibit NHE1. The results identify a role for mTOR in the regulation of Na(+)/H(+) exchange activity and implicate NHE1 as a possible downstream effector contributing to mTORs effects on cell growth, proliferation, survival, and tumorigenesis.

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.

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.

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.

A two-hit mechanism for sepsis-induced impairment of renal tubule function.

Renal insufficiency is a common and severe complication of sepsis, and the development of kidney dysfunction increases morbidity and mortality in septic patients. Sepsis is associated with a variety of defects in renal tubule function, but the underlying mechanisms are incompletely understood. We used a cecal ligation and puncture (CLP) model to examine mechanisms by which sepsis influences the transport function of the medullary thick ascending limb (MTAL). MTALs from sham and CLP mice were studied in vitro 18 h after surgery. The results show that sepsis impairs the ability of the MTAL to absorb HCO(3)(-) through two distinct mechanisms. First, sepsis induces an adaptive decrease in the intrinsic capacity of the tubules to absorb HCO(3)(-). This effect is associated with an increase in ERK phosphorylation in MTAL cells and is prevented by pretreatment of CLP mice with a MEK/ERK inhibitor. The CLP-induced reduction in intrinsic HCO(3)(-) absorption rate appears to involve loss of function of basolateral Na(+)/H(+) exchange. Second, sepsis enhances the ability of LPS to inhibit HCO(3)(-) absorption, mediated through upregulation of Toll-like receptor 4 (TLR4)-ERK signaling in the basolateral membrane. The two inhibitory mechanisms are additive and thus can function in a two-hit capacity to impair renal tubule function in sepsis. Both effects depend on ERK and are eliminated by interventions that prevent ERK activation. Thus the TLR4 and ERK signaling pathways represent potential therapeutic targets to treat or prevent sepsis-induced renal tubule dysfunction.