Nerve Growth Factor Inhibits Na+/H+ Exchange and HCO3- Absorption through Parallel Phosphatidylinositol 3-Kinase-mTOR and ERK Pathways in Thick Ascending Limb

Abstract

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.The Na ϩ /H ϩ exchanger isoform NHE1 2 is expressed ubiquitously in the plasma membrane of nonpolarized cells and in the basolateral membrane of epithelial cells, where it plays essential roles in basic cell functions such as the maintenance of intracellular pH (pH i ) and cell volume (1-3). NHE1 is involved in other important cellular processes, including proliferation, survival, adhesion, migration, and tumor formation (2-7). These specialized functions involve regulation of NHE1 by a variety of receptor-mediated signaling networks as well as physical interactions of NHE1 with the actin cytoskeleton (2, 3, 5). By comparison, the role of NHE1 in epithelial function remains poorly understood. In particular, the contributions of NHE1 to transcellular acid-base transport and the possible mechanisms involved are largely undefined.The medullary thick ascending limb (MTAL) of the mammalian kidney participates in acid-base regulation by reabsorbing most of the filtered HCO 3Ϫ not reabsorbed by the proximal tubule (8, 9). Absorption of HCO 3 Ϫ by the MTAL depends on H ϩ secretion mediated by the apical membrane NHE3 Na ϩ /H ϩ exchanger (8, 10 -13) and basolateral HCO ...

Inhibition of the TNF-α^|^ndash;Induced Serine Phosphorylation of IRS-1 at 636/639 by AICAR

“…It has been reported that the serine phosphorylation of IRS-1 at 636/639 is also regulated by the Ser/Thr kinase mammalian target of rapamycin (mTOR) (25), which is activated by TNF-. This cascade seems to exist both dependent and independent of the MEK/ ERK or JNK pathways (26,27). Therefore, AICAR may inhibit the serine phosphorylation of IRS-1 at 636/639 and 307 through the inhibition of mTOR signaling.…”

Toll-like receptor 4 (TLR-4) regulates renal ion transport

“…Interestingly, they observed that the effect of apical LPS on transport was blocked by rapamycin, which inhibits the mTOR/PI3-kinase pathway, whereas the effect of basolateral LPS was blocked by an inhibitor of the ERK pathway. While the authors had previously shown that these pathways inhibit bicarbonate reabsorption and NHE1 and NHE3 activity in thick ascending limbs (7,11), these novel data indicate polarized signaling of the TLR-4 receptor in renal epithelial cells, an important finding in TLR biology not previously described in other cells. In addition, the authors determined that blocking basolateral Na/H exchange with amiloride prevented the inhibitory effect of apical but not basolateral LPS.…”

Acute kidney injury: what part do toll-like receptors play?

“…48 Although the underlying signaling mechanisms are different, HCO 3 − reabsorption through the activation of an extracellular-signal-regulated kinase (ERK)-dependent pathway is decreased in the presence of basolateral LPS. 48,49 In addition, basolateral LPS inhibits HCO 3 − reabsorption in the MTAL through the activation of a TLR4/MyD88/mitogenactivated protein kinase (MEK)/ERK pathway is coupled to the inhibition of sodium-hydrogen exchanger (NHE) 3. NHE3 has been identified as a target of TLR4 signaling in the MTAL.…”

“…The TLR2 is expressed selectively in the basolateral membrane of MTAL cells, in contrast to TLR4, which is expressed in both basolateral and apical membrane domains. 49,52 The MTAL HCO 3 − reabsorption is inhibited by bacterial lipopeptides and gram-positive bacterial cell wall structures (lipoteichoic acid and peptidoglycan) recognized by TLR2. Moreover, the inhibition by gram-positive bacterial components is additive to inhibition by basolateral LPS, due to the TLR2 and TLR4 agonists activating different cell signaling pathways.…”