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

Abstract

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 HCO₃⁻ 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 HCO₃⁻ absorption by basolateral LPS. Adding LPS to the bath decreased HCO₃⁻ absorption by 30% in rat and mouse MTALs perfused in vitro. The inhibition of HCO₃⁻ 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 HCO₃⁻ 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 HCO₃⁻ absorption despite the presence of physiological stimuli that activate ERK in the MTAL. We conclude that basolateral LPS inhibits HCO₃⁻ 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.

renal insufficiency is a common and severe complication of sepsis.¹ The development of kidney dysfunction predicts a poor outcome, leads to prolonged hospitalization, and doubles the risk for mortality in septic patients (14, 45, 49, 59, 66, 70). Sepsis and endotoxemia induce a variety of defects in renal tubule function in association with alterations in fluid and electrolyte homeostasis that contribute to sepsis pathogenesis (19, 45, 70). These include a urinary concentrating defect (35, 57), increased fractional excretion of sodium and glucose (57, 68, 69, 75), and hypotension (11, 69, 70), as well as the development of systemic metabolic acidosis that contributes to multiple organ dysfunction (6, 11, 46, 70) and is an independent risk factor for mortality in septic patients (24, 48). The immunopathological mechanisms that underlie renal tubule dysfunction during sepsis are poorly understood.

Recently, we demonstrated that Gram-negative bacterial lipopolysaccharide (LPS) and Gram-positive bacterial molecules (lipoteichoic acid; peptidoglycan) can act directly through Toll-like receptors (TLR) to impair the transport function of renal tubules, identifying a new pathophysiological mechanism that can contribute to renal tubule dysfunction during sepsis (31, 32). In particular, absorption of HCO₃⁻ by the medullary thick ascending limb (MTAL) is inhibited by LPS, the dominant cell wall molecule of Gram-negative bacteria (31). The inhibition by LPS is mediated through activation of its cell-surface receptor TLR4. Our studies also revealed a novel sidedness to LPS receptor signaling, whereby LPS inhibits HCO₃⁻ absorption in the MTAL through the activation of different TLR4-dependent signaling pathways in the basolateral and apical membranes (31). The direct action of LPS to inhibit renal tubule HCO₃⁻ absorption may exacerbate and/or impair the ability of the kidneys to correct systemic metabolic acidosis during sepsis (31, 32). Understanding the mechanisms that underlie LPS-induced transport inhibition in the MTAL may lead to the identification of potential therapeutic targets to treat or prevent renal tubule dysfunction during sepsis. At present, the specific intracellular signals that are triggered by LPS in the MTAL and the transport proteins that are targeted downstream of LPS signaling to result in inhibition of HCO₃⁻ absorption are undefined.

The MTAL participates in acid-base homeostasis by reabsorbing most of the filtered HCO₃⁻ not reabsorbed by the proximal tubule (2, 26). Absorption of HCO₃⁻ by the MTAL depends on H⁺ secretion mediated by the apical membrane Na⁺/H⁺ exchanger NHE3 (2, 3, 7, 33, 82). The regulation of MTAL HCO₃⁻ absorption by a variety of physiological factors is achieved through regulation of this exchanger (26, 29, 33, 47, 81, 82). We have shown that the basolateral Na⁺/H⁺ exchanger NHE1 also is an important determinant of the HCO₃⁻ absorption rate in the MTAL. Inhibition of NHE1 with amiloride or nerve growth factor, or by NHE1 knockout, results secondarily in inhibition of apical NHE3, thereby decreasing HCO₃⁻ absorption (28, 34, 77, 78). The rate of HCO₃⁻ absorption in the MTAL thus depends on a regulatory interaction between the basolateral and apical membrane Na⁺/H⁺ exchangers, whereby basolateral NHE1 enhances the activity of apical NHE3 (28, 30, 34, 77, 78). Studies in a variety of cell systems, including immune cells, endothelial cells, and intestinal epithelial cell lines, have shown that Na⁺/H⁺ exchange activity attributable to NHE1 plays a role in LPS-induced inflammatory responses (22, 40, 52, 55, 56, 64, 65) and that NHE1 is a target for LPS-induced regulation (9, 40, 52, 56, 64, 65). These findings raise the possibility that LPS could inhibit HCO₃⁻ absorption in the MTAL through a primary effect on basolateral NHE1. Whether NHE3, the apical exchanger responsible for absorption of NaCl and/or NaHCO₃ by epithelia of the kidney and intestinal tract, is modulated directly by LPS or is a downstream target of TLR signaling in renal cells has not been determined. Infection with enteropathogenic Eschericia coli for several hours decreased NHE3 activity in an intestinal epithelial cell line (38), indicating that bacterial-epithelial cell interactions can influence the activity of this exchanger.

The signal transduction pathways activated through TLRs in renal tubules are poorly defined. Activation of extracellular signal-regulated kinase (ERK) is a prominent intracellular signaling event involved in LPS-induced immune responses. Stimulation of ERK by LPS through TLR4 is important for a variety of responses in immune cells, including activation of transcription factors that promote the expression of genes encoding cytokines and other inflammatory mediators, regulation of cell proliferation and differentiation, and protection against apoptosis (4, 5, 15, 17, 21, 36, 42, 51, 53, 63). With respect to the kidney, LPS was reported to increase ERK phosphorylation and activity in a mouse renal epithelial cell line (72), and activation of ERK through TLR4 played a role in mediating the effect of uropathogenic E. coli to stimulate chemokine secretion in cultured collecting duct cells (10). We have shown that the ERK pathway plays an important role in the regulation of Na⁺/H⁺ exchange activity and HCO₃⁻ absorption in the MTAL. Activation of ERK mediates inhibition of HCO₃⁻ absorption by aldosterone and nerve growth factor (79, 83). Moreover, ERK-dependent inhibition of HCO₃⁻ absorption in the MTAL involves specific targeting of the ERK pathway to inhibit either basolateral NHE1 or apical NHE3 depending on the physiological stimulus (29, 34, 79, 83). Recent studies using selective kinase inhibitors suggest that ERK may be involved in mediating inhibition of HCO₃⁻ absorption by basolateral LPS in the MTAL (31). However, whether LPS directly activates ERK in renal tubules and whether the ERK pathway functions as a signaling intermediate to couple TLR4 to inhibition of ion transport in the MTAL remains to be determined.

The purpose of the present study was to determine the signal transduction and transport mechanisms responsible for inhibition of HCO₃⁻ absorption by basolateral LPS in the MTAL. We focus on mechanisms underlying inhibition by basolateral LPS because this pathway is upregulated during sepsis (80). The results show that basolateral LPS inhibits HCO₃⁻ absorption through the activation of a TLR4/myeloid differentiation factor 88 (MyD88)/ERK pathway that is coupled to inhibition of NHE3. These studies identify NHE3 as a downstream target of LPS-induced TLR4 signaling in the MTAL and show that the ERK pathway plays a role in linking TLRs to the inhibition of renal tubule ion transport.

METHODS

Animals.

Male Sprague-Dawley rats (50–100 g body wt) were purchased from Taconic (Germantown, NY). Mice deficient in TLR4 (C57BL/10ScNJ; TLR4^−/−), MyD88 [B6.129P2(SJL)-Myd88^tm1.1Defr/J; MyD88^−/−], and TIR (Toll/interleukin-1 receptor) domain-containing adapter-inducing interferon-β (Trif) (C57BL/6J-Ticam1^Lps2/J; Trif^−/−), and wild-type control mice (C57BL/10SnJ for TLR4^−/−; C57BL/6J for MyD88^−/− and Trif^−/−) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were 6 to 8 wk old. The animals were maintained under pathogen-free conditions in microisolator cages and received standard rodent chow (NIH 31 diet, Ziegler) and water up to the time of experiments. All protocols in this study were approved by the IACUC of The University of Texas Medical Branch.

Tubule perfusion and measurement of net HCO₃⁻ absorption.

MTALs were isolated and perfused in vitro as previously described (25, 31, 79). Tubules were dissected from the inner stripe of the outer medulla at 10°C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipets for perfusion at 37°C. The tubules were perfused and bathed in control solution that contained (in mM) 146 Na⁺, 4 K⁺, 122 Cl⁻, 25 HCO₃⁻, 2.0 Ca²⁺, 1.5 Mg²⁺, 2.0 phosphate, 1.2 SO₄²⁻, 1.0 citrate, 2.0 lactate, and 5.5 glucose (equilibrated with 95% O₂-5% CO₂, pH 7.45 at 37°C). Solutions containing LPS (ultra pure E. coli K12, InvivoGen) and other experimental agents were prepared as described (31, 34, 77, 83). Experimental agents were added to the bath solution as described in results. In one series of HCO₃⁻ transport experiments (see Fig. 5B), Na⁺ in the bath solution was replaced with N-methyl-d-glucammonium (NMDG⁺).

Fig. 5.

Inhibition of HCO₃⁻ absorption by bath LPS does not involve basolateral Na⁺/H⁺ exchange. MTALs from rats were studied with 10 μM amiloride in the bath (A) or in a Na⁺-free bath (B), conditions that inhibit basolateral Na⁺/H⁺ exchange (28, 34, 77, 78). LPS (500 ng/ml) was then added to and removed from the bath solution. In B, Na⁺ in the bath was replaced with NMDG⁺; the lumen was perfused with control solution containing 146 mM Na⁺ (28, 77). J_HCO3⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

The protocol for study of transepithelial HCO₃⁻ absorption was as described (25, 34, 83). Tubules were equilibrated for 20–30 min at 37°C in the initial perfusion, and bath solutions and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.5–1.9 nl·min⁻¹·mm⁻¹. One to three 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5–10 min after an experimental agent was added to or removed from the bath solution. The absolute rate of HCO₃⁻ absorption (J_HCO3⁻, pmol·min⁻¹·mm⁻¹) was calculated from the luminal flow rate and the difference between total CO₂ concentrations measured in perfused and collected fluids (25). An average HCO₃⁻ absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in Figs. 1, 4, 5, and 7. Mean values ± SE (n = number of tubules) are presented in the text.

Fig. 1.

Inhibitors of extracellular signal-regulated kinase (ERK) activation eliminate inhibition of HCO₃⁻ absorption by bath lipopolysaccharide (LPS). Medullary thick ascending limbs (MTAL) from Sprague-Dawley rats and C57BL/6J mice were isolated and perfused in vitro. Tubules were studied in control solution (A and C) or bathed with 15 μM U0126 or 15 μM PD98059 (B and D), and then LPS (500 ng/ml) was added to and removed from the bath solution. Absolute rates of HCO₃⁻ absorption (J_HCO3⁻) were measured as described in methods. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P values are for paired t-test. NS, not significant. Mean values are given in results.

Fig. 4.

Role of myeloid differentiation factor 88 (MyD88) and TIR (Toll/interleukin-1 receptor) domain-containing adapter-inducing interferon-β (Trif) in responses to LPS. A: MTALs from wild-type, MyD88^−/−, and Trif^−/− mice were perfused in vitro under control conditions, and then LPS (500 ng/ml) was added to and removed from the bath solution. J_HCO3⁻, data points, lines, and P values are as in Fig. 1. NS, not significant. B: MTALs from wild-type, MyD88^−/−, and Trif^−/− mice were incubated for 15 min at 37°C in vitro in the absence (control) and presence of LPS. The tubules were stained with p-ERK antibody and analyzed by confocal immunofluorescence as in Fig. 2A. The effect of LPS to increase ERK phosphorylation was eliminated in MTALs from MyD88^−/− mice. Images are representative of at least six tubules of each type. C: intensity of p-ERK staining was quantified for experiments in B as described in methods and is presented as a percentage of control level measured in the same experiment. *P < 0.05 vs. control. Control fluorescence intensity did not differ in the three groups.

Fig. 7.

Inhibition of HCO₃⁻ absorption by bath LPS is additive to inhibition by aldosterone. MTALs from rats were bathed with aldosterone (1 nM) and then LPS (500 ng/ml) was added to and removed from the bath solution. J_HCO3⁻, data points, lines, and P value are as in Fig. 1.

Measurement of pH_i and apical Na⁺/H⁺ exchange activity.

pH_i was measured in isolated, perfused MTALs by use of the pH-sensitive dye BCECF and a computer-controlled spectrofluorometer (CM-X, SPEX Industries) coupled to the perfusion apparatus, as previously described (77, 81). MTALs were perfused and bathed in Na⁺-free, HEPES-buffered solution that contained (in mM) 145 NMDG⁺, 4 K⁺, 147 Cl⁻, 2.0 Ca²⁺, 1.5 Mg²⁺, 1.0 phosphate, 1.0 SO₄²⁻, 1.0 citrate, 2.0 lactate, 5.5 glucose, and 5 HEPES (equilibrated with 100% O₂; titrated to pH 7.4). The lumen solution also contained furosemide to block Na⁺-K⁺-2Cl⁻ cotransport activity, and the bath contained ethylisopropyl amiloride (50 μM) to eliminate any contribution of basolateral Na⁺/H⁺ exchange to the Na⁺-induced changes in pH_i. Apical Na⁺/H⁺ exchange activity was determined by measurement of the initial rate of pH_i increase after addition of 145 mM Na⁺ to the lumen solution (Na⁺ replaced NMDG⁺) (81, 82). Interruption of pH_i recovery at various points along the recovery curve permits determination of the Na⁺/H⁺ exchange rate over a range of pH_i values, with appropriate corrections for a variable background acid loading rate (81). The Na⁺-dependent pH_i recovery was inhibited ≥90% by lumen ethylisopropyl amiloride (50 μM) under all experimental conditions. Apical Na⁺/H⁺ exchange rates (J_Na⁺/H⁺, pmol·min⁻¹·mm⁻¹) were calculated as (dpH_i/dt) × β_i × V, where dpH_i/dt (pH units/min) is the initial slope of the record of pH_i vs time, β_i is the intrinsic intracellular buffering power (mM/pH unit), and V is cell volume per unit tubule length (nl/mm), measured as previously described (77, 81, 82). Experimental agents were added to the bath solution as described in results.

Immunoblotting.

Immunoblot analysis of phosphorylated ERK1/2 was carried out using a previously described inner stripe tissue preparation (30, 83). Thin strips of tissue were microdissected at 10°C from the inner stripe of the outer medulla of rat kidney. This preparation is highly enriched in MTALs and exhibits regulatory changes in signaling proteins that accurately reflect changes observed in the MTAL (27, 30, 79, 83). After dissection, the tissue strips were divided into samples of equal amount and incubated for various times at 37°C in the absence and presence of LPS using the same control solution as in HCO₃⁻ transport experiments. After incubation, the tissue samples were homogenized in ice-cold PBS and solubilized for 2 h at 4°C in RIPA buffer with protease inhibitors. Samples of equal protein content (50 μg/lane) were separated by SDS-PAGE using 9% gels and transferred to PVDF membranes as described (30, 34). Membranes were blocked with 5% BSA in Tris-buffered saline (TBS)/Tween and incubated overnight at 4°C with antiphospho-ERK1/2-Thr202/Tyr204 (1:2,500) or anti-ERK1/2 (1:5,000) antibody (Cell Signaling Technology). After washing in TBS, horseradish peroxidase-conjugated anti-rabbit secondary antibody was applied, and immunoreactive bands were detected by chemiluminescence (Luminol Reagent, Santa Cruz, CA). Protein bands were quantified by densitometry (MetaMorph).

Confocal immunofluorescence microscopy.

MTALs were studied by confocal microscopy as previously described (30, 78). Rat and mouse MTALs were microdissected and mounted on Cell-Tak-coated coverslips at 10°C. The tubules were then incubated in the absence and presence of LPS for 15 min at 37°C in a flowing bath using the same control solution as in HCO₃⁻ transport experiments. After incubation, the tubules were washed with PBS and fixed and permeabilized in acetone at −20°C for 10 min. The tubules were incubated in Image-iT FX signal enhancer (Invitrogen) for 30 min at room temperature, washed, and blocked in 10% goat serum in PBS for 1 h at room temperature. The tubules were then incubated overnight at 4°C with a 1:200 dilution of antiphospho-ERK1/2 antibody, washed, and then incubated for 1 h at room temperature in Alexa 488-conjugated goat anti-rabbit IgG antibody (1:100; Invitrogen) in blocking buffer. Fluorescence staining was examined using a Zeiss laser-scanning confocal microscope (LSM510 UV META) as described (30, 31, 78). Tubules were imaged longitudinally, and Z-axis optical sections (0.4 μm) were obtained through a plane at the center of the tubule, which provides a cross-sectional view of cells in the lateral tubule walls. For individual experiments, two to four tubules from the same kidney for each experimental condition, or from wild-type and null-mutant mice, were fixed and stained identically and imaged in a single session at identical settings of illumination, gain, and exposure time. Fluorescence intensity of antiphospho-ERK1/2 (p-ERK1/2) staining was quantified as previously described (30). Two-dimensional image analysis was performed using MetaMorph software in which a box (4.2 × 1.4 μm) was positioned on the cytoplasm in the midregion of the cell, and pixel intensity per unit area was determined after background subtraction. Three different cells were analyzed per optical section, and three optical sections were analyzed per tubule, one section at the center of the tubule and two sections positioned 0.12 μm above and below the center section. The measurements were averaged to obtain a value for each tubule. Fluorescence intensity for experimental groups was expressed as a percentage of the control value measured in the same experiment. Mean values (n = number of tubules) were used for statistical analysis.

Analysis.

Results are presented as means ± SE. Differences between means were evaluated using Student's t-test for paired or unpaired data, or analysis of variance with Newman-Keuls multiple range test, as appropriate. P < 0.05 was considered statistically significant.

RESULTS

Inhibitors of ERK activation eliminate inhibition of HCO₃⁻ absorption by bath LPS.

Under control conditions, adding LPS (500 ng/ml) to the bath decreased HCO₃⁻ absorption by 32% (from 15.1 ± 0.6 to 10.3 ± 0.7 pmol·min⁻¹·mm⁻¹) in MTALs from rats (Fig. 1A) and by 29% (from 15.7 ± 0.6 to 11.1 ± 0.8 pmol·min⁻¹·mm⁻¹) in MTALs from mice (Fig. 1C). The inhibition by LPS is rapid (<15 min), sustained for up to 60 min, and reversible. To determine whether the ERK pathway is involved in mediating the inhibition by LPS, we examined the effects of U0126 and PD98059, two selective inhibitors of MEK1/2 that block activation of ERK1/2 in the MTAL (30, 79, 83). The inhibition of HCO₃⁻ absorption by bath LPS was eliminated completely in rat and mouse MTALs bathed with U0126 or PD98059 (Fig. 1, B and D). These results extend our previous observations (31) and support an essential role for the ERK pathway in mediating the inhibition of HCO₃⁻ absorption by basolateral LPS.

LPS activates ERK in the MTAL.

To determine whether LPS activates ERK in the MTAL, we examined the effects of LPS on ERK phosphorylation. MTALs dissected from rats were incubated in the absence and presence of LPS for 15 min, stained with p-ERK antibody, and then analyzed by confocal immunofluorescence (Fig. 2, A and B). Stimulation with LPS increased p-ERK staining 1.5 ± 0.1-fold. The LPS-induced increase in ERK phosphorylation was eliminated by PD98059. Treatment with LPS for 15 min caused a similar increase in ERK phosphorylation in MTALs from mice (see below).

Fig. 2.

LPS increases ERK phosphorylation in the MTAL. A: MTALs dissected from rats were incubated in vitro at 37°C in control solution, LPS (500 ng/ml), PD98059 (15 μM), or PD98059 + LPS for 15 min and then fixed and stained with antiphospho-ERK1/2-Thr202/Tyr204 (p-ERK) antibody. The tubules were analyzed by confocal immunofluorescence as described in methods. Images are Z-axis sections (0.4 μm) taken through a plane at the center of the tubule showing a cross-sectional view of cells in the lateral tubule walls (30, 31, 78). LPS increased p-ERK labeling, and this increase was eliminated by PD98059. Images are representative of at least eight tubules of each type. Scale bar = 5 μm. B: intensity of p-ERK staining for experiments in A was quantified as described in methods and is presented as a percentage of the control level. Bars are means ± SE. *P < 0.05 vs. control (ANOVA). C: time course of ERK activation. Inner stripe tissue from rats was incubated in vitro at 37°C in the absence and presence of LPS for the indicated times, and cell lysates were then immunoblotted with p-ERK antibody for analysis of ERK phosphorylation and anti-ERK antibody for total ERK level. Blots are representative of 3 independent experiments. D: p-ERK levels normalized for total ERK were determined for experiments in C by densitometry. p-ERK/ERK ratios are presented as a percentage of the control value (0 min) measured in the same experiment. Bars are means ± SE. *P < 0.05 vs. 0 min.

To determine the time course of LPS-induced ERK activation, inner stripe tissue was incubated in vitro in the absence and presence of LPS for various times, and then ERK phosphorylation was analyzed by immunoblotting (Fig. 2, C and D). Phosphorylation of ERK was increased within 15 min of exposure to LPS and remained elevated for at least 60 min. ERK phosphorylation was increased 1.5 ± 0.1-fold at 15 min, 1.6 ± 0.1-fold at 30 min, and 1.5 ± 0.1-fold at 60 min relative to control tissue not treated with LPS (n = 3; P < 0.05). Taken together with the preceding immunofluorescence experiments, these results indicate that LPS induces rapid and sustained activation of ERK in the MTAL and support a role for the ERK pathway in mediating inhibition of HCO₃⁻ absorption.

LPS-induced ERK activation is mediated through TLR4.

Previously we demonstrated that basolateral LPS inhibits HCO₃⁻ absorption in the MTAL through activation of its cell-surface receptor TLR4 (31). To determine whether TLR4 mediates the activation of ERK, the effects of LPS on ERK phosphorylation were examined in MTALs from wild-type and TLR4^−/− mice (Fig. 3). Similar to results in MTALs from rats, treatment with LPS for 15 min increased ERK phosphorylation 1.6 ± 0.1-fold in MTALs from wild-type mice. In contrast, LPS had no effect on phosphorylation of ERK in MTALs from TLR4^−/− mice. These results indicate that activation of ERK by LPS in the MTAL is mediated through TLR4. The inability of LPS to activate ERK in MTALs from the TLR4^−/− mice can explain our previous finding that basolateral LPS fails to inhibit HCO₃⁻ absorption in the TLR4^−/− MTALs (31).

Fig. 3.

LPS-induced ERK activation is mediated through Toll-like recptor 4 (TLR4). A: MTALs dissected from wild-type and TLR4^−/− mice were incubated in vitro for 15 min at 37°C in the absence (control) and presence of LPS. The tubules were then fixed and stained with p-ERK antibody and analyzed by confocal immunofluorescence as in Fig. 2A. LPS increased ERK phosphorylation in MTALs from wild-type mice but had no effect in MTALs from TLR4^−/− mice. Images are representative of at least six tubules of each type. B: intensity of p-ERK staining was quantified for experiments in A as described in methods and is presented as a percentage of control level measured in the same experiment. *P < 0.05 vs. wild-type control.

Role of MyD88 and Trif in responses to LPS.

Binding of LPS to the TLR4 complex generates intracellular signals through two distinct pathways mediated by recruitment of the adaptor molecules MyD88 and Trif (42, 43, 58). To determine the role of these adaptors in the inhibition of HCO₃⁻ absorption by bath LPS, MTALs from wild-type, MyD88^−/−, and Trif^−/− mice were perfused in vitro. As shown in Fig. 4A, adding LPS to the bath decreased HCO₃⁻ absorption by 28% (from 14.3 ± 0.7 to 10.3 ± 0.8 pmol·min⁻¹·mm⁻¹) in MTALs from wild-type mice and by 23% (from 15.3 ± 0.6 to 11.8 ± 0.5 pmol·min⁻¹·mm⁻¹) in MTALs from Trif^−/− mice. In contrast, bath LPS had no effect on HCO₃⁻ absorption in MTALs from MyD88^−/− mice. These results demonstrate that inhibition of HCO₃⁻ absorption by basolateral LPS is dependent on MyD88.

Further experiments were carried out to determine the role of MyD88 and Trif in LPS-induced ERK activation. The effects of LPS on ERK phosphorylation were studied in MTALs from wild-type, MyD88^−/−, and Trif^−/− mice by confocal immunofluorescence. As shown in Fig. 4, B and C, LPS increased ERK phosphorylation 1.5 ± 0.1-fold in MTALs from wild-type mice and 1.6 ± 0.1-fold in MTALs from Trif^−/− mice but had no effect on phosphorylation of ERK in MTALs from MyD88^−/− mice. Taken together, these results demonstrate that the TLR4-dependent effects of basolateral LPS to stimulate ERK and inhibit HCO₃⁻ absorption are mediated through MyD88.

Inhibition by bath LPS does not involve basolateral Na⁺/H⁺ exchange.

We have shown previously that activation of ERK inhibits HCO₃⁻ absorption in the MTAL through two distinct mechanisms: 1) primary inhibition of basolateral NHE1, which results secondarily in inhibition of apical NHE3 (77, 83); and 2) direct coupling of the ERK pathway to inhibition of NHE3 (29, 79). To test whether NHE1 is involved in LPS-induced inhibition of HCO₃⁻ absorption, the effects of bath LPS were examined in the presence of 10 μM bath amiloride and in the absence of bath Na⁺, two conditions that inhibit basolateral Na⁺/H⁺ exchange and prevent inhibition of HCO₃⁻ absorption mediated through NHE1 (28, 34, 77, 78). The results in Fig. 5 show that bath LPS reduced HCO₃⁻ absorption by 30 ± 1% in tubules bathed with amiloride and by 29 ± 2% in tubules studied in a Na⁺-free bath, decreases similar to those observed in MTALs studied in control conditions (Fig. 1). Thus the inhibition of HCO₃⁻ absorption by basolateral LPS is not mediated through basolateral Na⁺/H⁺ exchange.

Bath LPS inhibits apical Na ⁺/H ⁺ exchange through an ERK-dependent pathway.

Further studies were carried out to determine whether bath LPS decreases HCO₃⁻ absorption through primary inhibition of the apical Na⁺/H⁺ exchanger NHE3. MTALs were studied under control conditions and with LPS or U0126 + LPS in the bath solution for 15–20 min. As shown in Fig. 6, bath LPS decreased apical Na⁺/H⁺ exchange activity over the range of pH_i values studied. The exchanger exhibited a sigmoidal dependence on pH_i, as reported previously (77, 81, 82). Kinetic analysis showed that the inhibition of apical Na⁺/H⁺ exchange by LPS was due to a 30% decrease in maximal velocity (V_max, 82 ± 4 pmol·min⁻¹·mm⁻¹, control vs. 57 ± 3 pmol·min⁻¹·mm⁻¹, bath LPS; P < 0.05), with no change in apparent affinity for intracellular H⁺ ${\mathrm{(K}}_{\mathrm{app}}^{\prime }$, 7.39 pH units, control vs. 7.41 pH units, bath LPS). The effect of bath LPS to decrease apical Na⁺/H⁺ exchange activity was eliminated in tubules bathed with U0126 (Fig. 6, A and B). These results indicate that basolateral LPS inhibits apical NHE3 in the MTAL through an ERK-dependent signaling pathway.

Fig. 6.

Bath LPS inhibits apical Na⁺/H⁺ exchange through an ERK-dependent pathway. A: MTALs from rats were studied under control conditions and with 500 ng/ml LPS or 15 μM U0126 + LPS in the bath for 15–20 min. Apical Na⁺/H⁺ exchange rates (J_Na⁺/H⁺) were determined from initial rates of pH_i increase measured after addition of Na⁺ to the tubule lumen (see methods). Data points are from 16 control tubules, 19 tubules with LPS, and 10 tubules with U0126 + LPS. Solid (control) and dashed (bath LPS) lines are from least-squares fits to the Hill equation (81, 82); kinetic parameters (V_max and apparent affinity for intracellular H⁺) are given in results. B: data from A were grouped over the indicated pH_i intervals to obtain mean exchange rates (± SE). *P < 0.05 vs. other conditions in each interval. U0126 alone has no effect on apical Na⁺/H⁺ exchange activity (79).

Inhibition of HCO₃⁻ absorption by bath LPS is additive to inhibition by aldosterone.

In previous studies, we demonstrated that aldosterone (1 nM) decreases HCO₃⁻ absorption by 29% in the MTAL through ERK-dependent inhibition of NHE3 (29, 79). These findings suggest that aldosterone and bath LPS inhibit HCO₃⁻ absorption through a common mechanism and raise the question of whether LPS can influence MTAL function in the presence of physiological stimuli that increase ERK activity. To address this, we asked whether bath LPS would inhibit HCO₃⁻ absorption in the presence of aldosterone. In tubules bathed with 1 nM aldosterone, adding LPS to the bath decreased HCO₃⁻ absorption by 30%, from 12.1 ± 0.3 to 8.5 ± 0.7 pmol·min⁻¹·mm⁻¹ (Fig. 7). Thus LPS inhibits HCO₃⁻ absorption by a similar amount in the absence or presence of aldosterone. These results suggest that the mechanisms by which LPS and aldosterone inhibit NHE3 through ERK are different and show that the ability of basolateral LPS to impair MTAL function is maintained despite the presence of other, physiological stimuli that activate the ERK pathway.

DISCUSSION

Impaired renal function accentuates the pathogenesis and lethality of sepsis through the loss of metabolic, fluid, and electrolyte homeostasis (19, 45, 70). Recently, we demonstrated that Gram-negative LPS inhibits HCO₃⁻ absorption in the MTAL through activation of TLR4, establishing that bacterial molecules can act directly to alter the transport function of renal tubules by interacting with cell-surface receptors and activating intracellular signaling pathways (31). Understanding the mechanisms involved in the inhibition of HCO₃⁻ absorption by LPS will provide insights into cellular pathways and signaling molecules that link innate immune receptors to renal tubule transport processes and that can be activated to impair renal tubule function during sepsis and other inflammatory conditions. In the present study, we show that basolateral LPS decreases HCO₃⁻ absorption in the MTAL by inhibiting apical NHE3 through activation of the MEK/ERK signaling pathway (Fig. 8). Activation of ERK by LPS is mediated through TLR4 and the adaptor protein MyD88 and decreases NHE3 activity through a decrease in V_max. These results identify NHE3 as a downstream target of TLR4 signaling in renal epithelial cells and show that bacterial molecules can impair the absorptive functions of renal tubules through inhibition of this exchanger.

Fig. 8.

Model for inhibition of HCO₃⁻ absorption by basolateral LPS in the MTAL. Basolateral LPS decreases HCO₃⁻ absorption by inhibiting Na⁺/H⁺ exchanger 3 NHE3 through activation of the MEK/ERK signaling pathway. The LPS-induced ERK activation is mediated through TLR4 and the adaptor molecule MyD88. Arrows do not necessarily imply direct relationships; regulatory steps may involve additional signaling components.

Although TLR4 on renal cells plays a role in mediating inflammatory kidney injury in a variety of infectious and noninfectious conditions (12, 18, 20, 44, 50, 61, 67, 74, 84–87), mechanisms of TLR4 signaling in segments of the nephron are poorly understood. In response to LPS, the intracellular adaptor molecules MyD88 and Trif are recruited to the TLR4 complex and mediate the induction of distinct intracellular signaling pathways and inflammatory responses (39, 42, 43, 58). A prominent feature of TLR signaling is the induced expression of immune and inflammatory mediators through the activation of mitogen-activated protein kinases (4, 15, 36, 43). Activation of ERK by LPS in a variety of cell types leads to the activation of transcription factors that regulate the induction of genes encoding cytokines and other proteins involved in the inflammatory response, including tumor necrosis factor-α, IL-1β, IL-10, and Cox-2 (4, 5, 17, 21, 36, 43, 63). LPS stimulates ERK1/2 through TLR4-mediated phosphorylation and activation of its upstream activator MEK1/2 (4, 5, 17, 21, 76). In macrophages, LPS activates ERK through both MyD88-dependent and MyD88-independent (Trif-dependent) pathways (39, 41, 53, 63). The results of the present study show that LPS increases ERK1/2 phosphorylation in the MTAL. Both the activation of ERK and the inhibition of HCO₃⁻ absorption by basolateral LPS are mediated through TLR4, are blocked by selective MEK1/2 inhibitors, and are eliminated in MyD88-deficient MTALs but preserved in MTALs that lack Trif.² Taken together, these results support the conclusion that basolateral LPS inhibits HCO₃⁻ absorption in the MTAL through the activation of a TLR4/MyD88/MEK1/2/ERK1/2 signaling pathway and identify MEK/ERK as a key downstream pathway that links TLR4 to inhibition of MTAL transport. In addition, we reported recently that the ability of basolateral LPS to inhibit HCO₃⁻ absorption through ERK is upregulated in MTALs from septic mice (80). These findings suggest that the ERK pathway may represent a potential therapeutic target to treat or prevent sepsis-induced renal tubule dysfunction. Important areas for future work will be to identify the signaling components that connect the TLR4/MyD88 receptor complex to activation of MEK1/2 in the MTAL and to understand how the LPS-induced ERK pathway may be upregulated during sepsis.

The Na⁺/H⁺ exchanger isoform NHE3 is expressed selectively in the apical membrane of renal and intestinal epithelial cells, where it mediates the reabsorption of NaCl and/or NaHCO₃ (2, 8, 16, 26, 60, 71). In the MTAL, NHE3 mediates the luminal H⁺ secretion required for HCO₃⁻ absorption, as evidenced by functional studies (26, 33), immunocytochemical localization (3, 7), inhibitor sensitivity (28, 77, 82), acute regulatory responses (27, 81, 82), and chronic adaptations (33, 47). The present study shows that LPS acts via ERK to inhibit NHE3, resulting in a decrease in HCO₃⁻ absorption. LPS inhibits NHE3 when exchanger activity is studied independently of the activity of other transporters and in the absence of a change in the driving force for the exchanger, indicating that the LPS-induced ERK pathway is coupled directly to inhibition of NHE3. LPS decreases NHE3 activity through a decrease in V_max, with no change in the intracellular H⁺ affinity. Thus LPS-induced ERK activation leads to a decrease in the intrinsic activity of individual transporters within the membrane and/or alters the subcellular distribution of NHE3 to decrease the number of transporters in active apical membrane domains. The molecular mechanisms that underlie acute regulation of NHE3 by ERK are undefined. NHE3 is a component of macromolecular complexes in the plasma membrane, where its activity is regulated through phosphorylation of its carboxy-terminal cytoplasmic domain by protein kinases such as PKA, and through interactions with a variety of accessory proteins and signaling molecules, including calcineurin homologous protein, megalin, the cytoskeleton-binding protein ezrin, and sodium-hydrogen exchanger regulatory factor (NHERF)-1/2 (1, 8, 16, 60). Whether these function as targets for mediating inhibition of NHE3 by ERK, or a downstream effector of ERK, is not known. Whether apical NHE3 activity is regulated in the MTAL by membrane trafficking, and whether this process may be influenced by ERK, also remains to be determined.

The ubiquitously expressed Na⁺/H⁺ exchanger isoform NHE1 has been identified as a target for modulation by LPS in several cell types (9, 40, 52, 56, 65). Moreover, we have shown that ERK can inhibit HCO₃⁻ absorption in the MTAL through inhibition of basolateral NHE1 (34, 77, 83). The results of the present study show, however, that NHE1 plays no role in mediating the inhibition of HCO₃⁻ absorption by basolateral LPS. Thus either NHE1 activity is not affected by basolateral LPS signaling in the MTAL or changes in NHE1 activity induced by basolateral LPS are not involved in mediating inhibition of HCO₃⁻ absorption.

In previous studies we demonstrated that ERK-dependent regulation of HCO₃⁻ absorption in the MTAL involves ERK signal specificity. Nerve growth factor (NGF) and aldosterone both inhibit HCO₃⁻ absorption through activation of ERK, but the inhibition occurs through different mechanisms. NGF decreases HCO₃⁻ absorption through direct coupling of the ERK pathway to inhibition of basolateral NHE1, which results secondarily in inhibition of apical NHE3 (34, 77, 83). In contrast, aldosterone decreases HCO₃⁻ absorption through direct coupling of the ERK pathway to inhibition of NHE3, independent of NHE1 (29, 79). Thus, in the MTAL, the ERK pathway can be targeted primarily to inhibit basolateral NHE1 or apical NHE3, depending on the physiological stimulus. The results of the present study suggest an additional level of specificity in ERK-dependent regulation of Na⁺/H⁺ exchange in the MTAL. Basolateral LPS inhibits HCO₃⁻ absorption through mechanisms that appear to be identical to those observed with aldosterone, namely, direct coupling of the ERK pathway to inhibition of NHE3 through a decrease in V_max. Our results show, however, that the inhibition of HCO₃⁻ absorption by basolateral LPS is fully maintained in the presence of a maximal inhibitory concentration of aldosterone. The additivity of the LPS and aldosterone inhibitory effects suggests that the molecular mechanisms mediating ERK-dependent inhibition of NHE3 by the two stimuli may differ. The potential mechanisms by which ERK signals arising from different stimuli may differentially regulate NHE3 are undefined and may be complex (54). Possible contributing factors could include the targeting of different downstream effectors of ERK to different regulatory components of the NHE3 macromolecular complex and the regulation of different ERK pools through the activation of different upstream kinases. The latter mechanism is of interest in view of recent findings that TLR-dependent activation of ERK is mediated through a specific a mitogen-activated protein kinase kinase kinase (MAP3K) Tpl2, which activates ERK1/2 through phosphorylation and activation of MEK1/2 (4, 5, 17, 21, 76). Tpl2 is required for LPS-induced ERK activation in various cell types and functions as a specific downstream mediator of ERK activation in TLR signaling (4, 17, 21, 23, 42, 76). Thus Tpl2 enables TLR-induced ERK activation that is distinct from ERK activation mediated through other members of the MAP3K family, such as the mitogen-responsive MAP3K Raf-1 (5, 13, 23, 37, 76). Whether Tpl2 mediates TLR-dependent activation of ERK in renal tubules and may play a role in the specificity of ERK signaling in the MTAL will be important areas for future research. Our finding that LPS and aldosterone appear to inhibit NHE3 through different ERK-dependent mechanisms has important implications for the pathogenesis of sepsis because it reveals mechanisms of TLR signal specificity that enable bacterial molecules to impair renal tubule transport despite the presence of other, physiological stimuli that converge on the same signaling pathways. Understanding the molecular events that underlie this TLR signal specificity will be important for selective therapeutic targeting of pathogen-induced inflammatory pathways in renal tubules. The Tpl2-ERK pathway recently has been identified as a potential therapeutic target for treatment of a number of autoimmune and inflammatory diseases (23, 37).

In addition to the acute effect of the ERK pathway to inhibit NHE3 in the MTAL, ERK has been shown to play a role in the long-term (24 h) stimulation of NHE3 by acid media in renal proximal tubule cells (73). This chronic stimulation likely involves an effect of acid-induced ERK activation to mediate increased transcription of endothelin-1, which then functions as an autocrine or paracrine factor to upregulate NHE3 (62). These findings illustrate further the complexity of ERK-dependent regulation of NHE3, which may involve both direct and indirect mechanisms. As noted earlier, the acute effect of LPS to inhibit HCO₃⁻ absorption through ERK is enhanced in MTALs from septic mice that exhibit systemic metabolic acidosis (80). These findings underscore the importance of understanding the distinct molecular components responsible for specificity of ERK signaling in response to different stimuli, and how these pathways interact, to permit effective therapeutic targeting of ERK signals leading to pathogen-induced renal tubule dysfunction.

Fine control of NHE3 in renal tubules is essential for regulation of sodium and volume balance, acid-base balance, and blood pressure (2, 8, 26, 60, 71). In the present study, we demonstrate that interaction of LPS with TLR4 induces cell signals that inhibit NHE3 in the MTAL. The decrease in NHE3 activity results in a decrease in HCO₃⁻ absorptive capacity that could impair the ability of the kidneys to correct systemic metabolic acidosis that contributes to sepsis pathogenesis (6, 11, 24, 46, 48, 70). These studies identify NHE3 as a target of TLR4 signaling in the MTAL and suggest that activation of TLRs by bacterial molecules and other microbial pathogens can disrupt renal tubule absorptive processes important for volume and acid-base homeostasis through inhibition of this exchanger. TLR4 is expressed constitutively throughout the nephron, including segments of the proximal tubule, thick ascending limb, and collecting duct (10, 18, 20, 31, 44, 50, 84–87), and TLR4 has been shown to play a role in mediating inflammatory kidney injury in a variety of conditions, including sepsis, ischemia-reperfusion, diabetes, and nephrotoxic injury (12, 18, 44, 50, 67, 74, 84–87). Our studies raise the possibility that effects of LPS or endogenous ligands to activate TLR4 signaling and impair NHE3 function could contribute to renal transport defects observed in these pathological conditions.

In addition to our finding that LPS decreases NHE3 activity acutely in the MTAL through TLR4-ERK signaling, LPS may induce long-term NHE3 regulation. Intraperitoneal injection of LPS to induce endotoxemia decreased NHE3 protein expression after 6–12 h in whole kidneys of mice (75) and in inner stripe of outer medulla of kidneys from rats (57). The precise systemic factors responsible for downregulation of NHE3 in the LPS-treated animals and whether renal NHE3 expression is decreased in more clinically relevant models of sepsis (14) remain to be determined. However, we found that the capacity of the MTAL to absorb HCO₃⁻ is reduced in a mouse model of sepsis induced by cecal ligation and puncture (80), an effect that could be explained by decreased NHE3 expression. Thus, during Gram-negative sepsis, LPS may act through a combination of short-term and long-term mechanisms to reduce NHE3 activity in renal tubules: acute inhibition through the induction of TLR4 signaling pathways coupled directly to inhibition of NHE3, and chronic inhibition through the induction of undefined molecular signals that reduce NHE3 protein abundance.

In summary, the results of the present study demonstrate that basolateral LPS inhibits apical NHE3 in the MTAL through the activation of a TLR4/MyD88/MEK/ERK signaling pathway. The LPS-induced inhibition of NHE3 results in a decrease in transepithelial HCO₃⁻ absorption and is due to a decrease in V_max of the exchanger. The ability of LPS to inhibit HCO₃⁻ absorption through ERK is maintained despite the presence of other, physiological stimuli that activate the ERK pathway in MTAL cells. These studies identify NHE3 as a direct downstream target of TLR4 signaling in the MTAL and suggest that bacterial molecules can impair renal absorptive functions important for extracellular fluid volume and acid-base regulation through the induction of TLR-mediated intracellular signals that modulate NHE3 activity. Our results identify ERK as a key signaling intermediate that links the TLR4/MyD88 receptor complex to downstream modulation of ion transport proteins and raise the possibility that molecular components of the TLR4-ERK signaling pathway may be therapeutic targets for treatment of sepsis-induced renal tubule dysfunction.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-038217.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: B. A. Watts, III, E. R. Sherwood, and D. W. Good conception and design of research; B. A. Watts, III and T. George performed experiments; B. A. Watts, III, T. George, and D. W. Good analyzed data; B. A. Watts, III, E. R. Sherwood, and D. W. Good interpreted results of experiments; B. A. Watts, III and D. W. Good prepared figures; B. A. Watts, III and D. W. Good edited and revised manuscript; B. A. Watts, III and D. W. Good approved final version of manuscript; D. W. Good drafted manuscript.