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 vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi 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.     

Hyposmolality stimulates Na(+)/H(+) exchange and HCO(3)(-) absorption in thick ascending limb via PI 3-kinase

The signal transduction mechanisms that mediateosmotic regulation of Na⁺/H⁺ exchange are notunderstood. Recently we demonstrated that hyposmolality increasesHCO₃ absorption in the renal medullary thickascending limb (MTAL) through stimulation of the apical membraneNa⁺/H⁺ exchanger NHE3. To investigate themechanism of this stimulation, MTALs from rats were isolated andperfused in vitro with 25 mM HCO₃-containingsolutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitorswortmannin (100 nM) and LY-294002 (20 µM) blocked completely thestimulation of HCO₃ absorption by hyposmolality. Intissue strips dissected from the inner stripe of the outer medulla, theregion of the kidney highly enriched in MTALs, hyposmolality increasedPI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-inducedPI 3-K activation. Further studies examined the interaction betweenhyposmolality and vasopressin, which inhibits HCO₃absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO₃ absorption, due to aneffect of cAMP to inhibit hyposmolality- induced activation of PI 3-K.In contrast to their effects to block stimulation by hyposmolality, PI3-K inhibitors and vasopressin have no effect on inhibition of apicalNa⁺/H⁺ exchange (NHE3) andHCO₃ absorption by hyperosmolality. These resultsindicate that hyposmolality increases NHE3 activity andHCO₃ absorption in the MTAL through activation of aPI 3-K-dependent pathway that is inhibited by vasopressin and cAMP.Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediatedthrough different signal transduction mechanisms.

     

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.     

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.     

More actors in ammonia absorption by the thick ascending limb

This review will briefly summarize current knowledge on the basolateral ammonia transport mechanisms in the thick ascending limb (TAL) of the loop of Henle. This segment transports ammonia against a concentration gradient and is responsible for the accumulation of ammonia in the medullary interstitium, which, in turn, favors ammonia secretion across the collecting duct. Experimental data indicate that the sodium/hydrogen ion exchanger isoform 4 (NHE4; Scl9a4) is a sodium/ammonia exchanger and plays a major role in this process. Disruption of murine NHE4 leads to metabolic acidosis with inappropriate urinary ammonia excretion and decreases the ability of the TAL to absorb ammonia and to build the corticopapillary ammonia gradient. However, NHE4 does not account for the entirety of ammonia absorption by the TAL, indicating that, at least, one more transporter is involved.     

CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes

These experiments evaluated salt transport processes in isolated cortical thick limbs of Henle (cTALH) obtained from mouse kidney. When the external solutions consisted of Krebs-Ringer bicarbonate (KRB), pH 7.4, and a 95% O2-5% CO2 gas phase, the spontaneous transepithelial voltage (Ve, mV, lumen-to-bath) was approximately mV; the net rate of Cl- absorption (JnetCl) was approximately 3,600 pmols s-1 cm-2; the net rate of osmotic solute absorption Jnetosm was twice JnetCl; and the net rate of total CO2 transport (JnetCO2) was indistinguishable from zero. Thus, net Cl- absorption was accompanied by the net absorption of a monovalent cation, presumably Na+, and net HCO3- absorption was negligible. This salt transport process was stimulated by (CO2 + HCO3- ): omission of CO2 from the gas phase and HCO3- from external solutions reduced JnetCl, Jnetosm, and Ve by 50%. Furthermore, 10(-4) M luminal furosemide abolished JnetCl and Ve entirely. The lipophilic carbonic anhydrase inhibitor ethoxzolamide (10(-4) M, either luminal or peritubular) inhibited (CO2 + HCO3-)-stimulated JnetCl, Jnetosm, and Ve by approximately 50%; however, when the combination (CO2 + HCO3-) was absent, ethoxzolamide had no detectable effect on salt transport. Ve was reduced or abolished entirely by omission of either Na+ or Cl- from external solutions, by peritubular K+ removal, by 10(-3) M peritubular ouabain, and by 10(-4) M luminal SITS. However, Ve was unaffected by 10(-3) M peritubular SITS, or by the hydrophilic carbonic anhydrase inhibitor acetazolamide (2.2 x 10(-4) M, lumen plus bath). We interpret these data to indicate that (CO2 + HCO3-)-stimulated NaCl absorption in the cTALH involved two synchronous apical membrane antiport processes: one exchanging luminal Na+ for cellular H+; and the other exchanging luminal Cl- for cellular HCO3- or OH-, operating in parallel with a (CO2+ HCO3-)-independent apical membrane NaCl cotransport mechanism.     

Signal transduction in a compliant thick ascending limb

In several previous studies, we used a mathematical model of the thick ascending limb (TAL) to investigate nonlinearities in the tubuloglomerular feedback (TGF) loop. That model, which represents the TAL as a rigid tube, predicts that TGF signal transduction by the TAL is a generator of nonlinearities: if a sinusoidal oscillation is added to constant intratubular fluid flow, the time interval required for an element of tubular fluid to traverse the TAL, as a function of time, is oscillatory and periodic but not sinusoidal. As a consequence, NaCl concentration in tubular fluid alongside the macula densa will be nonsinusoidal and thus contain harmonics of the original sinusoidal frequency. We hypothesized that the complexity found in power spectra based on in vivo time series of key TGF variables arises in part from those harmonics and that nonlinearities in TGF-mediated oscillations may result in increased NaCl delivery to the distal nephron. To investigate the possibility that a more realistic model of the TAL would damp the harmonics, we have conducted new studies in a model TAL that has compliant walls and thus a tubular radius that depends on transmural pressure. These studies predict that compliant TAL walls do not damp, but instead intensify, the harmonics. In addition, our results predict that mean TAL flow strongly influences the shape of the NaCl concentration waveform at the macula densa. This is a consequence of the inverse relationship between flow speed and transit time, which produces asymmetry between up- and downslopes of the oscillation, and the nonlinearity of TAL NaCl absorption at low flow rates, which broadens the trough of the oscillation relative to the peak. The dependence of waveform shape on mean TAL flow may be the source of the variable degree of distortion, relative to a sine wave, seen in experimental recordings of TGF-mediated oscillations.     

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-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.     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.

     

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.     

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.     

Feedback-mediated dynamics in a model of a compliant thick ascending limb

The tubuloglomerular feedback (TGF) system in the kidney, which is a key regulator of filtration rate, has been shown in physiologic experiments in rats to mediate oscillations in tubular fluid pressure and flow, and in NaCl concentration in the tubular fluid of the thick ascending limb (TAL). In this study, we developed a mathematical model of the TGF system that represents NaCl transport along a TAL with compliant walls. The model was used to investigate the dynamic behaviors of the TGF system. A bifurcation analysis of the TGF model equations was performed by deriving and finding roots of the characteristic equation, which arises from a linearization of the model equations. Numerical simulations of the full model equations were conducted to assist in the interpretation of the bifurcation analysis. These techniques revealed a complex parameter region that allows a variety of qualitatively different model solutions: a regime having one stable, time-independent steady-state solution; regimes having one stable oscillatory solution only; and regimes having multiple possible stable oscillatory solutions. Model results suggest that the compliance of the TAL walls increases the tendency of the model TGF system to oscillate.     

肾脏髓袢升支粗段钾离子通道的研究进展

肾脏髓袢升支粗段(thick ascending limb of the Henle'loop,TAL)具有重要的生理功能,它可以重吸收约20%～30%的Na+、K+、Cl-等物质,且在尿液稀释和浓缩机制中发挥着重要作用。而TAL上的钾离子通道在产生细胞膜电位、与Na+-K+-2Cl-及Na+-K+-ATPase相关的K+再循环、调整细胞容积中起重要作用。因此,对TAL中钾离子通道的研究具有较为重要的生理意义。本文对有关TAL中钾离子通道的类型、功能、调控及其相关疾病等问题的研究进展简要综述。