Monophosphoryl lipid A pretreatment suppresses sepsis- and LPS-induced proinflammatory cytokine production in the medullary thick ascending limb.

Sepsis is the leading cause of acute kidney injury in critically ill patients. Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of septic kidney injury; however, the sites and mechanisms of renal TNF-α production during sepsis remain to be defined. In the present study, we showed that TNF-α expression is increased in medullary thick ascending limbs (MTALs) of mice with sepsis induced by cecal ligation and puncture. Treatment with lipopolysaccharide (LPS) for 3 h in vitro also increased MTAL TNF-α production. Sepsis and LPS increased MTAL TNF-α expression through activation of the myeloid differentiation factor 88 (MyD88)-IL-1 receptor-associated kinase 1-ERK signaling pathway. Pretreatment with monophosphoryl lipid A (MPLA), a nontoxic immunomodulator that protects against bacterial infection, eliminated the sepsis- and LPS-induced increases in MTAL TNF-α production. The suppressive effect of MPLA on TNF-α was mediated through activation of a phosphatidylinositol 3-kinase-dependent pathway that inhibits MyD88-dependent ERK activation. This likely involves MPLA-phosphatidylinositol 3-kinase-mediated induction of Tollip, which negatively regulates the MyD88-ERK pathway by inhibiting activation of IL-1 receptor-associated kinase 1. These regulatory mechanisms are similar to those previously shown to mediate the effect of MPLA to prevent sepsis-induced inhibition of MTAL [Formula: see text] absorption. These results identify the MTAL as a site of local TNF-α production in the kidney during sepsis and identify molecular mechanisms that can be targeted to attenuate renal TNF-α expression. The ability of MPLA pretreatment to suppress MyD88-dependent ERK signaling in the MTAL during sepsis has the dual beneficial effects of protecting tubule transport functions and attenuating harmful proinflammatory responses.

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

LPS inhibits HCO3- 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-ß (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 HCO3- 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.

Monophosphoryl lipid A prevents impairment of medullary thick ascending limb [Formula: see text] absorption and improves plasma [Formula: see text] concentration in septic mice.

Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway.

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4-/- or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF)-/- mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.

High-mobility group box 1 inhibits HCO3- absorption in the medullary thick ascending limb through RAGE-Rho-ROCK-mediated inhibition of basolateral Na+/H+ exchange.

High-mobility group box 1 (HMGB1) is a nuclear protein released extracellularly in response to infection or injury, where it activates immune responses and contributes to the pathogenesis of kidney dysfunction in sepsis and sterile inflammatory disorders. Recently, we demonstrated that HMGB1 inhibits HCO3 (-) absorption in perfused rat medullary thick ascending limbs (MTAL) through a basolateral receptor for advanced glycation end products (RAGE)-dependent pathway that is additive to Toll-like receptor 4 (TLR4)-ERK-mediated inhibition by LPS (Good DW, George T, Watts BA III. Am J Physiol Renal Physiol 309: F720-F730, 2015). Here, we examined signaling and transport mechanisms that mediate inhibition by HMGB1. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by the Rho-associated kinase (ROCK) inhibitor Y27632 and by a specific inhibitor of Rho, the major upstream activator of ROCK. HMGB1 increased RhoA and ROCK1 activity. HMGB1-induced ROCK1 activation was eliminated by the RAGE antagonist FPS-ZM1 and by inhibition of Rho. The Rho and ROCK inhibitors had no effect on inhibition of HCO3 (-) absorption by bath LPS. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by bath amiloride, 0 Na(+) bath, and the F-actin stabilizer jasplakinolide, three conditions that selectively prevent inhibition of MTAL HCO3 (-) absorption mediated through NHE1. HMGB1 decreased basolateral Na(+)/H(+) exchange activity through activation of ROCK. We conclude that HMGB1 inhibits HCO3 (-) absorption in the MTAL through a RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of NHE1. The HMGB1-RAGE-RhoA-ROCK1 pathway thus represents a potential target to attenuate MTAL dysfunction during sepsis and other inflammatory disorders. HMGB1 and LPS inhibit HCO3 (-) absorption through different receptor signaling and transport mechanisms, which enables these pathogenic mediators to act directly and independently to impair MTAL function.

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.

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.

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

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) 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 HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) 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 HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) 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.

Basolateral LPS inhibits NHE3 and HCOFormula 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-ß (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.

High sodium intake increases HCO(3)- absorption in medullary thick ascending limb through adaptations in basolateral and apical Na+/H+ exchangers.

A high sodium intake increases the capacity of the medullary thick ascending limb (MTAL) to absorb HCO(3)(-). Here, we examined the role of the apical NHE3 and basolateral NHE1 Na(+)/H(+) exchangers in this adaptation. MTALs from rats drinking H(2)O or 0.28 M NaCl for 5-7 days were perfused in vitro. High sodium intake increased HCO(3)(-) absorption rate by 60%. The increased HCO(3)(-) absorptive capacity was mediated by an increase in apical NHE3 activity. Inhibiting basolateral NHE1 with bath amiloride eliminated 60% of the adaptive increase in HCO(3)(-) absorption. Thus the majority of the increase in NHE3 activity was dependent on NHE1. A high sodium intake increased basolateral Na(+)/H(+) exchange activity by 89% in association with an increase in NHE1 expression. High sodium intake increased apical Na(+)/H(+) exchange activity by 30% under conditions in which basolateral Na(+)/H(+) exchange was inhibited but did not change NHE3 abundance. These results suggest that high sodium intake increases HCO(3)(-) absorptive capacity in the MTAL through 1) an adaptive increase in basolateral NHE1 activity that results secondarily in an increase in apical NHE3 activity; and 2) an adaptive increase in NHE3 activity, independent of NHE1 activity. These studies support a role for NHE1 in the long-term regulation of renal tubule function and suggest that the regulatory interaction whereby NHE1 enhances the activity of NHE3 in the MTAL plays a role in the chronic regulation of HCO(3)(-) absorption. The adaptive increases in Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL may play a role in enabling the kidneys to regulate acid-base balance during changes in sodium and volume balance.

Toll-like receptor 2 mediates inhibition of HCO(3)(-) 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.

Nerve growth factor inhibits Na+/H+ exchange and formula 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 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 mTOR's effects on cell growth, proliferation, survival, and tumorigenesis.

Aldosterone inhibits apical NHE3 and HCO3- absorption via a nongenomic ERK-dependent pathway in medullary thick ascending limb.

Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na(+)/H(+) exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO(3)(-) absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO(3)(-) absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U-0126 and PD-98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid (

Nongenomic regulation by aldosterone of the epithelial NHE3 Na(+)/H(+) exchanger.

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na(+)/H(+) exchangers (NHE). In the MTAL, apical NHE3 mediates H(+) secretion necessary for HCO(3)(-) absorption; basolateral NHE1 influences HCO(3)(-) absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO(3)(-) absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na(+)/H(+) exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na(+)/H(+) exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V(max). Inhibition of HCO(3)(-) absorption by aldosterone was not affected by 0.1 mM lumen Zn(2+) or 1 mM lumen DIDS, arguing against the involvement of an apical H(+) conductance or apical K(+)-HCO(3)(-) cotransport. These results demonstrate that aldosterone inhibits HCO(3)(-) absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger.

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 HCO3- 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 microM amiloride or 0.7 nM NGF decreased HCO3- absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal HCO3- absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on HCO3- absorption in tubules bathed with amiloride or a Na(+)-free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of HCO3- absorption by vasopressin or stimulation by hyposmolality, factors that regulate HCO3- 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 HCO3- 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 HCO3- absorption in the MTAL by controlling the organization of the actin cytoskeleton.

Transepithelial HCO3- absorption is defective in renal thick ascending limbs from Na+/H+ exchanger NHE1 null mutant mice.

In the medullary thick ascending limb (MTAL) of rat kidney, inhibiting basolateral Na(+)/H(+) exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na(+)/H(+) exchange, thereby decreasing transepithelial HCO(3)(-) absorption. To assess the possible role of the Na(+)/H(+) exchanger NHE1 in this regulatory process, MTALs from wild-type and NHE1 knockout (NHE1(-/-)) mice were studied using in vitro microperfusion. The rate of HCO(3)(-) absorption was decreased 60% in NHE1(-/-) MTALs (15.4 +/- 0.5 pmol.min(-1).mm(-1) wild-type vs. 6.0 +/- 0.5 pmol.min(-1).mm(-1) NHE1(-/-)). Transepithelial voltage, an index of the NaCl absorption rate, did not differ in wild-type and NHE1(-/-) MTALs. Basolateral addition of 10 microM amiloride or 0.7 nM NGF decreased HCO(3)(-) absorption by 45-49% in wild-type MTALs but had no effect on HCO(3)(-) absorption in NHE1(-/-) MTALs. Inhibition of HCO(3)(-) absorption by vasopressin and stimulation by hyposmolality, both of which regulate MTAL HCO(3)(-) absorption through primary effects on apical Na(+)/H(+) exchange, were similar in wild-type and NHE1(-/-) MTALs. Thus the regulatory defect in NHE1(-/-) MTALs is specific for factors (bath amiloride and NGF) shown previously to inhibit HCO(3)(-) absorption through primary effects on basolateral Na(+)/H(+) exchange. These findings demonstrate a novel role for NHE1 in transepithelial HCO(3)(-) absorption in the MTAL, in which basolateral NHE1 controls the activity of apical NHE3. Paradoxically, a reduction in NHE1-mediated H(+) extrusion across the basolateral membrane leads to a decrease in apical Na(+)/H(+) exchange activity that reduces HCO(3)(-) absorption.

An apical K(+)-dependent HCO(3)- transport pathway opposes transepithelial HCO(3)- absorption in rat medullary thick ascending limb.

Absorption of HCO(3)(-) in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na(+)/H(+) exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO(3)(-)/OH(-)/H(+) transport pathways in the rat MTAL and to determine their role in transepithelial HCO(3)(-) absorption. MTALs were perfused in vitro in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2). Lumen addition of either 120 mM Cl(-) or 50 mM Na(+) (50 microM EIPA present) had no effect on intracellular pH (pH(i)). Lumen Cl(-) addition also had no effect on pH(i) in the presence of 145 mM Na(+) or in the nominal absence of HCO(3)(-)/CO(2). Thus there was no evidence for apical Cl(-)/HCO(3)(-) (OH(-)) exchange, Na(+)-dependent Cl(-)/HCO(3)(-) exchange, or Na(+)-HCO(3)(-) cotransport. In contrast, in tubules studied in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2) and 120 mM K(+), removal of luminal K(+) induced a rapid and pronounced decrease in pH(i) (DeltapH(i) = 0.56 +/- 0.06 pH U). pH(i) recovered following lumen K(+) readdition. The initial rate of net base efflux induced by lumen K(+) removal was decreased 85% at the same pH(i) in the nominal absence of HCO(3)(-)/CO(2), indicating a dependence on HCO(3)(-)/CO(2) and arguing against apical K(+)/H(+) exchange. A combination of the apical K(+) channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K(+)-induced pH(i) changes, arguing against electrically coupled K(+) and HCO(3)(-) conductances. The effect of lumen K(+) on pH(i) was inhibited by 1 mM H(2)DIDS. In addition, lumen addition of DIDS increased transepithelial HCO(3)(-) absorption from 10.7 +/- 0.7 to 14.9 +/- 0.7 pmol x min(-1) x mm(-1) (P < 0.001) and increased pH(i) slightly in MTAL studied in physiological solutions (25 mM HCO(3)(-) and 4 mM K(+)). Lumen DIDS stimulated HCO(3)(-) absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K(+)-dependent HCO(3)(-) transport pathway that mediates coupled transfer of K(+) and HCO(3)(-) from cell to lumen in the MTAL. This mechanism, possibly an apical K(+)-HCO(3)(-) cotransporter, functions in parallel with apical Na(+)/H(+) exchange and opposes transepithelial HCO(3)(-) absorption.

Aldosterone potentiates 1,25-dihydroxyvitamin D3 action in renal thick ascending limb via a nongenomic, ERK-dependent pathway.

Recently, we demonstrated that aldosterone inhibits HCO3- absorption in the rat medullary thick ascending limb (MTAL) via a nongenomic pathway blocked by inhibitors of extracellular signal-regulated kinase (ERK) activation. Here we examined the effects on the MTAL of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which regulates cell functions through nongenomic mechanisms in nonrenal systems. Addition of 1 nM 1,25(OH)2D3 to the bath decreased HCO3- absorption by 24%, from 15.0 +/- 0.3 to 11.4 +/- 0.5 pmol. min-1. mm-1 (P < 0.001). This inhibition was maximal within 60 min and was eliminated by pretreatment with actinomycin D, cycloheximide, or inhibitors of protein kinase C. In MTAL bathed with 1 nM aldosterone [added 15-20 min before 1,25(OH)2D3], the absolute (5.6 +/- 0.3 vs. 3.6 +/- 0.3 pmol. min-1. mm-1) and fractional (49 +/- 2 vs. 24 +/- 2%) decreases in HCO3- absorption induced by 1,25(OH)2D3 were significantly greater than those in the absence of aldosterone (P < 0.05). The effect of aldosterone to potentiate inhibition by 1,25(OH)2D3 was not affected by spironolactone but was eliminated by the MAPK kinase/ERK inhibitor U-0126. U-0126 did not affect inhibition of HCO3- absorption by 1,25(OH)2D3 alone. Aldosterone induced rapid activation of ERK via a transcription-independent pathway. We conclude that 1) 1,25(OH)2D3 inhibits HCO3- absorption in the MTAL via a genomic pathway involving protein kinase C, which may contribute to 1,25(OH)2D3-induced regulation of urinary net acid and/or Ca2+ excretion and 2) aldosterone potentiates inhibition by 1,25(OH)2D3 through an ERK-dependent, nongenomic pathway. These results identify a novel regulatory interaction whereby aldosterone acts via nongenomic mechanisms to enhance the genomic response to 1,25(OH)2D3. Aldosterone may influence a broad range of biological processes, including epithelial transport, by modifying the response of target tissues to 1,25(OH)2D3 stimulation.