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

Abstract

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 $\mathrm{H}\mathrm{C}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$ 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 $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ 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-β (TRIF)^−/− mice. The effect of MPLA to prevent LPS inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ 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 $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ 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.

sepsis is the most common cause of acute kidney injury in critically ill patients, and the development of kidney dysfunction prolongs hospitalization and is associated with exceptionally high mortality rates in patients with sepsis (5, 16, 65, 76). Sepsis and endotoxemia induce a variety of defects in renal tubule function in association with alterations in metabolic, fluid, and electrolyte homeostasis that potentiate sepsis pathogenesis (4, 17, 27, 34, 51, 55, 60–62, 66, 71). Significant among these alterations is the development of metabolic acidosis, which contributes to multiple organ dysfunction (6, 29, 36, 38) and is a marker of poor outcome and increased mortality in septic patients (20, 29, 30, 39, 49). The pathophysiological mechanisms that underlie renal tubule dysfunction during sepsis remain poorly understood, and there is a continuing need to identify new and effective therapeutic interventions (16, 54, 76). Recent evidence suggests that maladaptive responses of the renal tubule epithelial cells to inflammatory stimuli play an important role in kidney dysfunction during sepsis (76). These studies underscore the need for improved understanding of the cellular and molecular mechanisms of sepsis-induced kidney dysfunction at the level of individual nephron segments and for the identification of preventive and therapeutic approaches that protect renal tubule cells against the deleterious effects of infectious stimuli.

Prior exposure of cells or organisms to a sublethal dose of LPS results in an adaptive response that confers protection against a subsequent LPS challenge. This phenomenon, known as endotoxin tolerance, has been studied extensively in immune cells and is well described in both experimental animals and humans (7, 9, 11, 43). Pretreatment with LPS can induce a complex state of immune reprogramming that is characterized functionally by reduced responsiveness to LPS, attenuated proinflammatory responses, increased production of anti-inflammatory mediators, and improved resistance to bacterial infection and septic shock (7, 9, 11). The mechanistic basis for endotoxin tolerance involves upregulation of negative regulatory molecules that suppress Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88) signaling in tolerant cells (7, 9, 11, 18, 47). However, despite these potentially beneficial properties, the toxicity of LPS has precluded its clinical use as a therapeutic or prophylactic agent.

Monophosphoryl lipid A (MPLA) is a chemically modified derivative of LPS that retains the beneficial immunomodulatory properties of LPS without the inherent toxicity (9, 10). MPLA is produced by hydrolysis of diphosphoryl lipid A from native LPS, resulting in structural alterations that reduce the systemic toxicity of MPLA by >99% compared with native lipid A or LPS (9, 10). The ability of MPLA to augment adaptive immune responses with minimal proinflammatory side effects led to its current use as a vaccine adjuvant in humans (9, 10). In addition to its immunoadjuvant activity, MPLA enhances the innate host response to infection and acts as a protective agent against bacterial sepsis (9, 56, 58). Pretreatment with MPLA induces resistance to endotoxemia in experimental animals and humans and improves survival rates following infection with gram-negative or gram-positive bacteria and sepsis induced by cecal ligation and puncture (2, 3, 9, 12, 56, 58, 59). The immunomodulatory effects of MPLA are mediated through stimulation of TLR4 and involve a nonspecific increase in bacterial clearance, enhanced neutrophil recruitment, and attenuated production of proinflammatory cytokines (9, 10, 47, 56–59, 63). MPLA-induced protection against infectious agents is associated with tissue-specific alterations in immune responses in multiple organ systems, including myeloid cells, lung, liver, and endothelium (9, 47, 56–59, 63). The ability of MPLA to induce protection against LPS in other cell systems raises the possibility that MPLA could alter the responses of renal tubule epithelial cells to LPS stimulation. Whether treatment with MPLA may influence renal tubule function has not been investigated.

The medullary thick ascending limb (MTAL) participates in the renal regulation of acid-base balance by reabsorbing most of the filtered $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ not reabsorbed by the proximal tubule (22, 31). We have demonstrated previously that the MTAL is a target for impaired function in response to LPS and sepsis. In particular, basolateral LPS inhibits $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL through the activation of a TLR4-MyD88-ERK signaling pathway that is upregulated in MTALs from septic mice (25, 70, 71). The direct action of LPS to decrease $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption would contribute to sepsis pathogenesis by impairing the ability of the kidneys to attenuate metabolic acidosis that contributes to morbidity and mortality (20, 25, 30, 39, 71). The effect of LPS to impair MTAL transport thus provides a well-defined system to test whether treatment with MPLA can protect against LPS-induced renal tubule dysfunction. Whether renal tubule epithelial cells are capable of endotoxin tolerance and its possible functional significance are unclear.

The purpose of the present study was to determine whether MPLA can induce protection against LPS in the MTAL and to identify cellular mechanisms involved. The results show that pretreatment with MPLA prevents the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL. This effect is mediated through MPLA stimulation of a TLR4-Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway that prevents LPS-induced ERK activation. These studies demonstrate that MPLA acts directly on the MTAL to protect against LPS and support nontoxic TLR4-based immunomodulators such as MPLA as potential therapeutic agents to prevent or treat renal tubule dysfunction during bacterial infection.

METHODS

Animals.

Male Sprague-Dawley rats (50–90 g body wt) were purchased from Taconic (Germantown, NY). Mice deficient in TLR4 (C57BL/10ScNJ; TLR4^−/−), TLR2 (B6.129-Tlr2^tm1Kir/J; TLR2^−/−), or TRIF (C57BL/6J-Ticam1^Lps2/J; TRIF^−/−) and wild-type control mice (C57BL/10SnJ for TLR4^−/−; C57BL/6J for TLR2^−/− and TRIF^−/−) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were 6–12 wk old. The animals were maintained under pathogen-free conditions in microisolator cages and received standard rodent chow (NIH 31 diet; Zeigler) and water up to the time of experiments. All protocols in this study were approved by the Institutional Animal Care and Use Committee of The University of Texas Medical Branch.

Tubule perfusion and measurement of net $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption.

MTALs were isolated and perfused in vitro as previously described (21, 25). 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 $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, 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). Experimental agents were added to the lumen and bath solutions as described in results. Monophosphoryl lipid A derived from Salmonella enterica serotype Minnesota Re 595 (Sigma-Aldrich) was prepared as a stock solution by gentle vortexing in DMSO and diluted into bath and lumen solutions to a final concentration of 1 µg/ml. Solutions containing LPS (500 ng/ml, ultrapure Escherichia coli K12; InvivoGen) and other experimental agents were prepared as described (24, 25, 68–70). MPLA was studied at 1 µg/ml because 1) this concentration is within the range used to study the immunomodulatory effects of MPLA in classic immune and nonimmune cells and in animal models of bacterial tolerance (12, 47, 56–59, 63) and 2) it induces highly reproducible protection against LPS in the MTAL, with no effect on basal $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rate (see results).

The protocol for study of transepithelial $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption was as described (21, 25, 70). Tubules were equilibrated 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). In all protocols, the solutions were identical in the initial and recovery periods. The initial period was 2 h in all experiments. Except for experiments in Fig. 8, MPLA was removed from the bath solution when LPS was added (experimental period) to prevent possible competition for binding to basolateral TLR4 (see results). 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 $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, 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 (21). An average $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ 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 the figures. Mean values ± SE (n = number of tubules) are presented in the text.

Fig. 8.

LPS can inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the presence of bath MPLA. MTALs from rats were pretreated with MPLA (bath and lumen) plus LY294002 for 2 h, and then LPS was added to and removed from the bath solution. MPLA was present in the bath solution throughout the experiment. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, data points, lines, and P value is as in Fig. 1. Mean values are given in results.

Confocal immunofluorescence microscopy.

MTALs were studied by confocal microscopy as previously described (24, 67, 69, 70). In brief, MTALs were dissected and mounted on Cell-Tak-coated coverslips at 10°C. The tubules were then incubated at 37°C in a flowing bath using the same solutions as in $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ transport experiments. The specific protocols used for incubation are given in results. Following incubation, the tubules were washed with PBS, fixed and permeabilized in cold acetone, and held 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% normal donkey serum in PBS for 1 h at room temperature. The tubules were then incubated overnight at 4°C with anti-phospho-Akt-Ser473 (Ab 193H12; 1:200) or anti-phospho-ERK1/2-Thr202/Tyr204 (1:200) antibodies (Cell Signaling Technology), washed, and then incubated for 1 h at room temperature in Alexa 488-conjugated donkey anti-rabbit IgG antibody (Invitrogen) in blocking buffer. Fluorescence staining was examined using a Zeiss laser-scanning confocal microscope (LSM510 UV META), as described (26, 67, 70). 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 phospho-Akt or phospho-ERK staining was quantified as previously described (26, 69, 70). Two-dimensional image analysis was performed using MetaMorph software, in which a box (1.4 × 4.2 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.

Immunoblotting.

Immunoblot analyses were performed on inner stripe of the outer medulla dissected from rat kidneys as previously described (26, 69, 70). This tissue preparation is highly enriched in MTALs and exhibits regulated changes in signaling proteins that accurately reflect changes observed in the MTAL (23, 24, 68, 70, 72). The tissue was divided into samples of equal amount and incubated in vitro at 37°C in the same solutions used for $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ transport experiments. The specific protocols used for incubation are given in results. Following incubation, samples of equal protein content (50 µg/lane) were subjected to immunoblotting using anti-phospho-Akt-Ser473 (Ab D9E; 1:2,000) or anti-Akt (1:2,000) antibodies (Cell Signaling Technology), as described (24, 69). Immunoreactive protein bands were detected by chemiluminescence (Luminol Reagent, Santa Cruz. CA) and quantified by densitometry (MetaMorph). Different anti-phospho-Akt antibodies were used in the immunoblot and confocal immunofluorescence experiments to verify activation of Akt by MPLA using two independent probes.

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 Tukey's test for multiple comparisons, as appropriate. P < 0.05 was considered statistically significant.

RESULTS

MPLA prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS in the MTAL.

Basolateral LPS inhibits $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL (25, 70). To determine whether treatment with MPLA could attenuate inhibition by LPS, MTALs from rats were studied in vitro in control solution or in the presence of MPLA for 2 h, and then LPS was added to the bath solution. MPLA was removed from the bath when LPS was added to prevent possible competition for binding to basolateral TLR4 (see methods). Under control conditions, adding LPS to the bath decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 24% (from 14.4 ± 0.7 to 10.9 ± 0.5 pmol·min⁻¹·mm⁻¹; Fig. 1A). In contrast, in MTALs pretreated with MPLA in bath and lumen, adding LPS to the bath had no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (14.5 ± 0.3 pmol·min⁻¹·mm⁻¹, MPLA, vs. 14.5 ± 0.3 pmol·min⁻¹·mm⁻¹, MPLA + LPS; Fig. 1B). Pretreatment with MPLA in the bath alone also eliminated inhibition by LPS (13.6 ± 0.5 pmol·min⁻¹·mm⁻¹, MPLA, vs. 13.5 ± 0.4 pmol·min⁻¹·mm⁻¹, MPLA + LPS; Fig. 1C). However, in MTALs pretreated with MPLA in the lumen alone, LPS decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 17% (from 15.0 ± 0.6 to 12.5 ± 0.6 pmol·min⁻¹·mm⁻¹; Fig. 1D). These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL and that this protective effect is mediated through MPLA acting from the basolateral surface.

Fig. 1.

Monophosphoryl lipid A (MPLA) pretreatment prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS in medullary thick ascending limb (MTAL). MTALs from rats were isolated and perfused in vitro. Tubules were studied in control solution (A) or with MPLA (1 µg/ml) in bath and lumen (B), bath alone (C), or lumen alone (D) for 2 h, and then LPS was added to and removed from the bath solution. In B and C, MPLA was removed from the bath when LPS was added (see methods). Absolute rates of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$) 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.

MPLA alone has no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption.

The effect of MPLA on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption was assessed using two protocols. In the first protocol, we compared $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rates measured in MTALs perfused and bathed in vitro in either control solution or MPLA for 2 h. As shown in Fig. 2A, the $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rate for tubules treated with MPLA did not differ from that measured in controls (14.7 ± 0.3 pmol·min⁻¹·mm⁻¹, control, vs. 14.6 ± 0.3 pmol·min⁻¹·mm⁻¹, MPLA). Similar results were obtained for MTALs studied with MPLA in bath and lumen or in bath alone. In the second protocol, we examined directly the effect of MPLA addition on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in individual MTALs. As shown in Fig. 2B, adding MPLA to bath and lumen or to bath alone had no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (14.0 ± 0.5 pmol·min⁻¹·mm⁻¹, control, vs. 13.9 ± 0.6 pmol·min⁻¹·mm⁻¹, MPLA). MPLA had no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption for up to 50 min in these experiments. Thus MPLA prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS but, by itself, has no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. These findings are consistent with the ability of MPLA to induce protection against endotoxin without inherent immunotoxic effects (3, 9, 10).

Fig. 2.

MPLA alone has no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. A: MTALs from rats were isolated and perfused in vitro for 2 h in control solution or with MPLA in bath and lumen or bath alone. Data points are values for single tubules. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, absolute rate of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. P value compares control vs. MPLA (unpaired t-test). Number of tubules is 7 for control and 11 for MPLA. Mean values are given in results. B: MTALs from rats were isolated and perfused in vitro in control solution, and then MPLA was added to and removed from the bath and lumen or bath solutions. Treatment with MPLA was for up to 50 min. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, data points, lines, and P value is as in Fig. 1. Mean values are given in results.

MPLA prevents LPS-induced inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through a PI3K-Akt pathway.

Further studies were performed to determine the mechanism by which MPLA protects against LPS in the MTAL. Studies in immune cells suggest that anti-inflammatory effects of MPLA depend on PI3K signaling (47, 57). To determine the role of PI3K in the ability of MPLA to prevent LPS-induced inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption, we examined the effect of LY294002, a selective inhibitor of PI3K that blocks PI3K activation and PI3K-mediated regulation in the MTAL (23, 24, 69). In MTALs pretreated with bath and lumen MPLA plus LY294002, adding LPS to the bath decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 26% (from 14.1 ± 0.5 to 10.4 ± 0.5 pmol·min⁻¹·mm⁻¹; Fig. 3A). This inhibition was fully reversible and is similar to that induced by LPS under control conditions (Fig. 1A). LPS induced a similar inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in MTALs pretreated with bath MPLA plus LY294002 (not shown). To define further the PI3K pathway involved in MPLA's protective effect, we examined the role of Akt, a direct target of PI3K (45). As shown in Fig. 3B, in MTALs pretreated with MPLA plus the selective Akt Inhibitor VIII (42, 69), bath LPS decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 24% (from 13.3 ± 0.3 to 10.1 ± 0.4 pmol·min⁻¹·mm⁻¹). Thus inhibiting PI3K or its downstream effector Akt restored the ability of basolateral LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in MTALs pretreated with MPLA. These results support the view that MPLA prevents the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through the activation of a PI3K-Akt signaling pathway. In addition, these results show that the protective effect of MPLA against LPS inhibition is mediated specifically through the activation of an intracellular signaling pathway and is not due to a nonspecific metabolic or toxic effect of MPLA on the MTAL cells or to an irreversible interaction of MPLA with TLR4 or other components of the basolateral membrane.

Fig. 3.

Inhibitors of PI3K and Akt eliminate the ability of MPLA to prevent inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS. MTALs were pretreated with MPLA plus bath LY294002 (20 µM; A) or bath Akt Inhibitor VIII (5 µM; B) for 2 h, and then LPS was added to and removed from the bath solution. MPLA was removed from the bath when LPS was added. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

MPLA induces PI3K-dependent activation of Akt in the MTAL.

To determine directly whether MPLA activates PI3K-Akt signaling in the MTAL, we examined the effect of MPLA on Akt phosphorylation. MTALs dissected from rats were incubated in vitro in the absence (control) and presence of the PI3K inhibitor LY294002 for 15 min, then treated with MPLA for 2 h. The tubules were stained with anti-phospho-Akt (p-Akt) antibody and analyzed by confocal immunofluorescence microscopy. As shown in Fig. 4, A and B, stimulation with MPLA increased p-Akt staining 1.6 ± 0.1-fold. The MPLA-induced increase in Akt phosphorylation was eliminated by LY294002 (Fig. 4A). MPLA caused a similar activation of Akt in MTALs from mice (see below). The effect of MPLA on Akt was examined further by immunoblot analysis of the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs. These experiments used an anti-p-Akt antibody different from that used in the confocal imaging experiments (see methods). The results in Fig. 4, C and D, show that treatment with MPLA for 2 h increased Akt phosphorylation without a change in total Akt level. These studies demonstrate that MPLA activates Akt in the MTAL and that this activation depends on PI3K. Together with the experiments in Fig. 3, these results support the view that MPLA prevents LPS-induced inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through the activation of a PI3K-Akt signaling pathway.

Fig. 4.

MPLA increases Akt phosphorylation through PI3K in the MTAL. A: MTALs from rats were incubated in vitro at 37°C in the absence (control) and presence of LY294002 for 15 min, then treated with MPLA for 2 h. The tubules were fixed and stained with anti-phospho-Akt-Ser473 (p-Akt) antibody and 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. MPLA increased p-Akt labeling, and this increase was eliminated by LY294002. Images are representative of at least six tubules of each type. Scale bar = 5 µm. B: the intensity of p-Akt staining for experiments in A was quantified as described in methods and is presented as a percentage of control level measured in the same experiment. Bars are means ± SE. *P < 0.05 vs. control. C: inner stripe tissue was incubated in vitro at 37°C in the absence (control) and presence of MPLA for 2 h. Cell lysates were immunoblotted with p-Akt antibody to analyze Akt phosphorylation and with anti-Akt antibody for total Akt level. Blots show three independent experiments. D: p-Akt level normalized for total Akt was determined for experiments in C by densitometry. The p-Akt-to-Akt ratio is presented as a percentage of control value measured in the same experiment. Bars are means ± SE. *P < 0.05 vs. control.

MPLA-induced Akt activation is mediated through TLR4 and does not require TLR2.

The immunomodulatory functions of MPLA are mediated through TLR4 (9, 10, 47, 56). To determine whether TLR4 mediates MPLA-induced PI3K-Akt signaling in the MTAL, the effect of MPLA on Akt phosphorylation was examined in tubules from wild-type (WT) and TLR4^−/− mice. As shown in Fig. 5, A and B, treatment with MPLA for 2 h increased Akt phosphorylation by 1.6 ± 0.2-fold in MTALs from WT mice but had no effect on Akt phosphorylation in MTALs from TLR4-deficient mice. These results indicate that the PI3K-dependent activation of Akt by MPLA in the MTAL is mediated through TLR4.

Fig. 5.

MPLA-induced activation of Akt is mediated through TLR4 but does not depend on TLR2. A: MTALs dissected from wild-type (WT) and TLR4^−/− mice were incubated in vitro at 37°C in the absence (control) and presence of MPLA for 2 h. The tubules were then fixed and stained with p-Akt antibody and analyzed by confocal immunofluorescence as in Fig. 4A. MPLA increased Akt phosphorylation in MTALs from WT mice but had no effect in MTALs from TLR4^−/− mice. Images are representative of seven to eight tubules of each type. B: the intensity of p-Akt 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. control. C: MTALs from WT and TLR2^−/− mice were incubated in vitro at 37°C in the absence (control) and presence of MPLA for 2 h, then analyzed for p-Akt staining as in A. MPLA caused a similar increase in Akt phosphorylation in WT and TLR2^−/− MTALs. Images are representative of six to eight tubules of each type. D: the intensity of p-Akt staining was quantified for experiments in C as described in methods and is presented as a percentage of control level measured in the same experiment. *P < 0.05 vs. control.

The effects of basolateral LPS to activate ERK and inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL require a novel interaction between TLR4 and TLR2 in the basolateral membrane (26). To determine whether the TLR4-mediated activation of PI3K-Akt by MPLA also requires TLR2, we examined the effect of MPLA on Akt phosphorylation in MTALs from TLR2^−/− mice. As shown in Fig. 5, C and D, MPLA increased Akt phosphorylation by 1.6 ± 0.1-fold in TLR2^−/− MTALs, an increase similar to that observed in MTALs from WT controls. Thus the TLR4-mediated activation of Akt by MPLA does not depend on TLR2. These results are in direct contrast to the TLR4-mediated activation of ERK by basolateral LPS, which requires TLR4 interaction with TLR2 (26). This difference in the requirement of TLR2 for TLR4 signaling may account, in part, for the effects of MPLA and LPS to activate different TLR4-dependent signaling pathways at the basolateral membrane.

MPLA activates Akt and prevents LPS-induced inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through TRIF.

Stimulation of TLR4 generates cell signals through two distinct pathways mediated by recruitment of the cytosolic adaptor proteins MyD88 and TRIF (8, 10, 33, 52). We have shown that the TLR4-dependent activation of ERK by basolateral LPS is mediated through MyD88 (70). Studies in immune cells indicate that TLR4-dependent immunomodulatory effects of MPLA are mediated preferentially through TRIF (9, 10, 47). To determine the role of TRIF in MPLA signaling in the MTAL, we examined the effect of MPLA on Akt phosphorylation in tubules from WT and TRIF^−/− mice. As shown in Fig. 6, A and B, treatment with MPLA for 2 h increased Akt phosphorylation 1.7 ± 0.1-fold in MTALs from WT mice but had no effect on phosphorylation of Akt in MTALs from TRIF^−/− mice. These results indicate that activation of PI3K-Akt signaling by MPLA in the MTAL is mediated through a TRIF-dependent pathway.

Fig. 6.

MPLA activates Akt and prevents LPS inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through TRIF. A: MTALs from WT and TRIF^−/− mice were incubated in vitro at 37°C in the absence (control) and presence of MPLA for 2 h. The tubules were then stained with p-Akt antibody and analyzed by confocal immunofluorescence as in Fig. 4A. The effect of MPLA to increase Akt phosphorylation was eliminated in MTALs from TRIF^−/− mice. Images are representative of at least six tubules of each type. B: the intensity of p-Akt staining was quantified for experiments in A as described in methods and is expressed as a percentage of control level measured in the same experiment. *P < 0.05 vs. control. C and D: MTALs from WT (C) and TRIF^−/− (D) mice were perfused in vitro with MPLA in bath and lumen for 2 h, and then LPS was added to and removed from the bath solution. MPLA was removed from the bath when LPS was added. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Further studies were performed to examine the role of TRIF in mediating the effect of MPLA to prevent LPS-induced inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. MTALs from WT and TRIF^−/− mice were perfused and bathed in vitro with MPLA for 2 h, and then LPS was added to the bath solution (Fig. 6, C and D). In previous studies, we demonstrated that bath LPS decreases $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 28% in MTALs from WT mice (26, 70). As shown in Fig. 6C, the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption was eliminated in WT MTALs pretreated with MPLA (13.8 ± 0.3 pmol·min⁻¹·mm⁻¹, MPLA, vs. 13.9 ± 0.2 pmol·min⁻¹·mm⁻¹, MPLA + LPS). In contrast, bath LPS decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 28% (from 14.6 ± 0.6 to 10.5 ± 0.6 pmol·min⁻¹·mm⁻¹) in MTALs from TRIF^−/− mice pretreated with MPLA (Fig. 6D). This inhibition is similar to that induced by bath LPS in TRIF^−/− MTALs in the absence of MPLA (70). Similar results were obtained for MTALs pretreated with MPLA in bath alone (not shown). Thus the effect of MPLA to eliminate LPS-induced inhibition is mediated through TRIF. Taken together, our results indicate that MPLA prevents the inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by bath LPS through TLR4- and TRIF-dependent activation of PI3K-Akt.

MPLA prevents LPS-induced ERK activation through PI3K.

Basolateral LPS inhibits $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL through the activation of ERK (70). Therefore we tested whether MPLA prevents LPS-induced transport inhibition by blocking ERK activation. MTALs were preincubated for 2 h in control solution, MPLA, or LY294002 + MPLA, and then LPS was added for an additional 15 min. As in $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ transport experiments, MPLA was removed from the incubation solution when LPS was added. The tubules were stained with anti-p-ERK antibody and analyzed by confocal immunofluorescence as described (70). As shown in Fig. 7, A and B, LPS increased ERK phosphorylation 1.5 ± 0.1-fold under control conditions, and this increase was eliminated in MTALs pretreated with MPLA (Fig. 7A, b vs. c). The effect of MPLA to prevent LPS-induced ERK activation was abolished by LY294002 (Fig. 7Ad). Thus inhibiting PI3K restored the ability of LPS to activate ERK in MPLA-treated MTALs. Treatment with MPLA alone had no effect on ERK phosphorylation (Fig. 7C). These results show that pretreatment with MPLA blocks activation of ERK by LPS in the MTAL and that this effect of MPLA is mediated through PI3K. Thus our results support a mechanism in which MPLA prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS by stimulating a TLR4-TRIF-PI3K-Akt pathway that blocks LPS-induced ERK activation. In addition, they establish that the signaling pathways activated by MPLA and LPS through basolateral TLR4 in the MTAL are distinct, with MPLA activating PI3K-Akt and LPS activating ERK (25, 70).

Fig. 7.

MPLA prevents LPS-induced activation of ERK through PI3K. A: MTALs were preincubated in vitro at 37°C for 2 h in control solution (a and b), MPLA (c), or LY294002 plus MPLA (d), and then LPS was added for 15 min. MPLA was removed from the incubation solution when LPS was added (c and d). The tubules were stained with anti-phospho-ERK1/2-Thr202/Tyr204 (p-ERK) antibody and analyzed by confocal immunofluorescence microscopy as described in methods. Pretreatment with MPLA prevented LPS-induced ERK phosphorylation (b vs. c), and this effect of MPLA was blocked by LY294002 (d). Images are representative of eight tubules of each type. LY294002 alone did not affect p-ERK level (not shown). B: the intensity of p-ERK staining for experiments in A was quantified as described in methods and is presented as a percentage of the control-control (a) level. Bars are means ± SE. *P < 0.05 vs. a or c (ANOVA). Cont, control; LY, LY294002. C: MTALs were incubated in vitro in the absence (control) and presence of MPLA for 2 h, then analyzed for p-ERK staining by confocal immunofluorescence as in A. Images are representative of eight tubules of each type. MPLA had no effect on ERK phosphorylation. Similar results were obtained for MPLA treatment of 15 min (not shown).

Bath LPS can inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the presence of bath MPLA.

In the preceding experiments, MPLA was removed from the bath solution when LPS was added to prevent possible competition for binding to basolateral TLR4. To explore this possibility more directly, further experiments were performed to examine the functional interaction between bath MPLA and LPS. MTALs were pretreated with MPLA + LY294002, a condition in which bath LPS inhibits $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption because MPLA-induced PI3K signaling is blocked (Fig. 3). However, in contrast to the previous experiment in which MPLA was removed from the bath when LPS was added, the effect of bath LPS on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption was examined in the continued presence of bath MPLA. As shown in Fig. 8, adding LPS to the bath decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 29% (from 13.8 ± 1.0 to 9.8 ± 1.0 pmol·min⁻¹·mm⁻¹) with MPLA present in the bath solution. This inhibition is similar to that observed under the same conditions when bath MPLA was removed (Fig. 3), indicating that the ability of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption was not affected by the physical presence of MPLA in the bath solution. Thus these experiments provide no evidence that MPLA competes with LPS for binding to basolateral TLR4. They further support an essential role for PI3K signaling in mediating the effect of MPLA to prevent LPS-induced transport inhibition.

MPLA does not prevent inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone.

To test the selectivity of MPLA's effects in the MTAL, we examined whether pretreatment with MPLA would prevent inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone. Aldosterone inhibits $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL through an ERK-dependent pathway distinct from the ERK pathway that mediates inhibition by basolateral LPS (68, 70). As shown in Fig. 9, adding aldosterone to the bath decreased $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by 30% (from 13.1 ± 0.4 to 9.2 ± 0.6 pmol·min⁻¹·mm⁻¹) in MTALs pretreated with MPLA. This inhibition is similar to that induced by aldosterone in the absence of MPLA (68). Thus pretreatment with MPLA did not prevent inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone. These results further support MPLA as a nontoxic immunomodulator, in that the MPLA-induced signaling pathway is targeted selectively to block activation of ERK by LPS but does not prevent ERK-dependent regulation by the physiological stimulus aldosterone.

Fig. 9.

MPLA pretreatment does not prevent inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone. MTALs were perfused in vitro with MPLA in bath and lumen for 2 h, and then aldosterone (1 nM) was added to and removed from the bath solution. MPLA was removed from the bath when aldosterone was added. J$\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$, data points, lines, and P value is as in Fig. 1. Mean values are given in results.

DISCUSSION

Kidney dysfunction is a common and severe complication of sepsis that greatly increases morbidity and mortality, and there is a critical need to develop novel treatment strategies (5, 16, 54, 65, 76). MPLA is a low-toxicity TLR4-based immunomodulator that protects cells and organ systems against LPS and improves host resistance to bacterial infections (2, 3, 9, 10, 12, 56, 58). In the current study, we tested whether the beneficial immunomodulatory effects of MPLA against LPS would extend to the renal tubule. Our results show that pretreatment with MPLA prevents the effect of basolateral LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the isolated, perfused MTAL. This protective effect of MPLA is mediated directly through the activation of a basolateral TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced activation of ERK through TLR4 and MyD88 (Fig. 10). Importantly, MPLA itself has no effect on the $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rate, consistent with MPLA's well-established low toxicity profile. These results provide new evidence that renal tubule epithelial cells are capable of immune reprogramming that conveys resistance to LPS and identify TLR4-based lipid A analogs as potential novel therapeutic agents to treat or prevent renal tubule dysfunction in response to bacterial infections. Immunomodulatory strategies that enhance renal tubule resistance to bacterial infection may be beneficial in a variety of clinical scenarios in which patients are at increased risk of developing infectious complications, including those that have suffered major burns or trauma, patients that have undergone major surgery or received immunosuppressive therapies, and patients that have survived the acute phase of sepsis (9, 56).

Fig. 10.

Model of cell mechanisms by which MPLA prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS in the MTAL. MPLA activates a basolateral TLR4-TRIF-PI3K-Akt signaling pathway that prevents LPS-induced activation of ERK through TLR4 and MyD88. This, in turn, prevents ERK-mediated inhibition of NHE3 and $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (70). ^§The precise molecular mechanism underlying the inhibitory signaling interaction is undetermined but may involve MPLA-induced Akt-dependent activation of intracellular molecules that negatively regulate the TLR4-MyD88 pathway (see text for details). Arrows do not necessarily imply direct relationships; regulatory steps may involve additional signaling components.

Although the ability of MPLA to promote resistance to infection is well described, the cellular mechanisms underlying MPLA's protective effects are incompletely understood (9). The extent to which MPLA's ability to convey resistance to LPS extends to nonimmune cells also remains uncertain. Evaluation of the mechanisms of MPLA's immunomodulatory effects using in vivo models is complicated by its interactions with cells of the innate immune system, which can elicit multiple systemic effects, such as changes in myeloid cell recruitment or production of inflammatory mediators, that can secondarily modify tissue responses to infection (32, 56, 58). Thus it is difficult to determine from studies performed in vivo whether MPLA affects cellular responses directly or whether its effects are mediated through changes in the activity of other immunoregulatory factors. The isolated, perfused MTAL provides an ideal system to examine whether MPLA directly affects the function of native renal tubules and to determine cellular mechanisms involved. The results of the present study demonstrate that MPLA prevents inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS directly through the activation of a PI3K-Akt signaling pathway. This conclusion is supported by several observations, including the following: 1) inhibition of PI3K or Akt restores the ability of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in MPLA-treated MTALs, 2) MPLA directly stimulates Akt through PI3K in the MTAL, 3) the effects of MPLA both to stimulate PI3K-Akt and to prevent LPS-induced transport inhibition are eliminated in MTALs from TRIF^−/− mice, and 4) the effect of MPLA to block LPS-induced ERK activation depends on PI3K. The protective effects of MPLA persist after MPLA has been removed from the extracellular fluid, indicating an adaptive change in MTAL signaling. Thus the induction of a refractory state to LPS depends on the direct activation of a signal transduction pathway by MPLA in MTAL cells, independent of interactions of MPLA with innate immune cells or changes in other immunomodulatory factors within the host environment. These findings reflect the phenomenon of in vitro endotoxin tolerance in immune cells, which is induced by repeated LPS stimulation and mediated directly through changes in intracellular signaling pathways (7, 11, 18, 41, 73). Our finding that PI3K mediates the immunoregulatory effect of MPLA in the MTAL is consistent with prior studies demonstrating that PI3K signaling contributes to the development of LPS tolerance (1, 14, 19, 28, 37, 40, 41, 46, 50, 53, 74) and plays a role in the ability of MPLA to suppress production of proinflammatory mediators in immune cells (47, 57).

Our results provide new insights into the complexity of TLR4 signaling in renal tubule cells. In the MTAL, MPLA and LPS both act through basolateral TLR4, but the two agonists stimulate different intracellular signaling pathways and induce different functional responses. In general, TLR4 signaling involves two distinct downstream pathways mediated through recruitment of the adaptor proteins MyD88 and TRIF (8, 10, 33, 52). Our results show that the different cellular effects of MPLA and LPS in the MTAL are dependent on different TLR4 adaptors. LPS induces MyD88-dependent stimulation of ERK, which mediates inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (70). MPLA induces TRIF-dependent stimulation of PI3K-Akt, which blocks LPS-induced ERK activation and prevents LPS inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (Fig. 10). MPLA alone has no effect on ERK phosphorylation or $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption, providing further evidence that the MPLA- and LPS-activated TLR4 pathways are distinct. Our finding that MPLA induces resistance to LPS in the MTAL through TRIF is consistent with results in other systems indicating that the immunoadjuvant properties of MPLA and its ability to enhance host responses to infection are mediated preferentially through TRIF-biased signaling (9, 10, 47) and that the TRIF pathway plays a role in the development of endotoxin tolerance (8, 9). The effects of MPLA and LPS to activate different TLR4 pathways in the MTAL may reflect the ability of the TLR4 receptor complex to distinguish subtle structural differences between LPS components, resulting in the preferential activation of MyD88- or TRIF-dependent signaling and different cellular responses (9, 13, 15). Our results with MPLA provide novel insight into an immunomodulatory mechanism within renal tubule cells in which the TLR4-induced MyD88 and TRIF pathways play opposing roles in determining the tubule response to LPS; in particular, PI3K signals generated through TRIF prevent LPS-induced MTAL dysfunction by downregulating inhibitory signals generated through MyD88. In addition, they support TLR4 agonists that preferentially activate TRIF-dependent signaling as a new approach to augment renal tubule defenses against bacterial pathogens. It will be important in future studies to assess whether the protective effect of MPLA against LPS through TLR4-TRIF signaling in the MTAL extends to other nephron segments. The possibility that MPLA may act through TRIF-PI3K signaling to modulate the production of immune mediators in the kidney during infection also will be important for future investigation.

Little direct information is available regarding how MPLA and LPS interact functionally as agonists of TLR4. Recent studies in neutrophils indicate that MPLA competes with LPS for TLR4 binding (57). However, we found in the MTAL that basolateral LPS can induce full inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the presence of MPLA when MPLA-induced PI3K activation is blocked (Fig. 8). This experiment confirms the essential role of PI3K signaling in MPLA's protective effect and shows that the physical presence of MPLA does not prevent access of LPS to the basolateral TLR4 receptor. Thus, although MPLA and LPS both induce cell signals in the MTAL through basolateral TLR4, our experiments provide no evidence that the two agonists compete for TLR4 binding.¹ One possible explanation for this finding is that MPLA and LPS preferentially bind and activate different TLR4 subpopulations in MTAL cells. In this regard, we have shown that the effects of LPS to activate MyD88-dependent ERK signaling and inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL require a novel interaction between TLR4 and TLR2 in the basolateral membrane (26). In contrast, the effect of MPLA to activate TRIF-dependent PI3K signaling is mediated through TLR4 but does not require TLR2. Thus the basolateral membrane of the MTAL may present TLR4 in two different molecular states: one in which TLR4 associates physically with TLR2 and preferentially mediates signaling by LPS (26) and a second in which TLR4 functions independently of TLR2 and preferentially mediates signaling by MPLA. The presence of two distinct TLR4-based recognition systems could explain both the effect of basolateral LPS and MPLA to activate different downstream TLR4 signaling pathways (LPS activating MyD88-ERK; MPLA activating TRIF-PI3K) and the apparent lack of competition between LPS and MPLA for basolateral TLR4 binding. Consistent with this possibility, previous studies reported that different structural forms of LPS can induce the formation of different TLR4 receptor clusters in membrane microdomains, where the recruitment and interaction of TLR4 with different coreceptors and accessory proteins determine the cellular response (64). Further work is needed to evaluate this possibility in the MTAL, as well as the relative affinities of MPLA and LPS for basolateral TLR4. Our results show, however, that the MPLA-induced protection against LPS in the MTAL depends specifically on the activation of a TLR4-TRIF-PI3K signaling pathway and does not involve a competitive effect of MPLA to prevent LPS TLR4 binding.

The effect of MPLA to stimulate PI3K-Akt signaling prevents LPS inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by blocking LPS-induced ERK activation. This, in turn, prevents ERK-mediated inhibition of the apical Na⁺/H⁺ exchanger NHE3 that decreases $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption (70). These findings are consistent with studies in other systems demonstrating that the LPS-tolerant phenotype is characterized by an impaired ability of LPS to activate ERK (18, 73) and that activation of PI3K-Akt signaling suppresses LPS-induced ERK activation and cytokine production in immune cells (1, 18, 19, 28). The molecular mechanisms underlying the inhibitory interaction between the MPLA-induced PI3K-Akt and LPS-induced ERK pathways in the MTAL remain to be determined. Our results show, however, that MPLA does not prevent ERK-mediated inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone. This finding indicates that the MPLA-induced PI3K pathway does not function as a nonspecific inhibitor of ERK signaling but instead is targeted selectively to block ERK activation by LPS. LPS activates ERK through TLR4-MyD88-mediated recruitment and activation of downstream mediators IL-1 receptor-associated kinase (IRAK)-4/1, TNF receptor-associated factor 6 (TRAF6), and transforming growth factor-β-activated kinase 1 (TAK1; 33, 41, 44). It is now recognized that a molecular hallmark of the LPS-tolerant state is cellular reprogramming that involves the upregulation of intracellular molecules that negatively regulate the TLR4-MyD88 signaling pathway (7, 9, 11, 18, 41). A major regulator of endotoxin tolerance in human and mouse models is the inactive kinase IRAK-M, which is induced by LPS challenge and downregulates TLR4 signaling by suppressing IRAK4/1-MyD88 interactions (7, 35, 41, 44, 53, 75). Other negative regulators of TLR4-MyD88 signaling include Toll-interacting protein (Tollip), suppressor of cytokine signaling-1 (SOCS-1), and GSK-3, which are implicated in suppressing recruitment and activation of IRAK1 (8, 18, 41, 46, 48, 53, 77). These different regulatory proteins undergo early activation by LPS in tolerant cells and can be modulated through interactions with PI3K-Akt signaling (1, 7, 14, 40, 41, 46, 50). These findings raise the possibility that the induction of IRAK-M or other molecules that downregulate TLR4-MyD88 signaling could explain the effect of the MPLA-induced PI3K pathway to selectively block activation of ERK by LPS in the MTAL. Studies to define the role of these intracellular molecules in mediating the protective effect of MPLA against LPS in the MTAL will provide new insights into the molecular regulation of TLR4 signaling in renal epithelial cells and may aid in identifying targets to preserve renal tubule function during sepsis and other inflammatory disorders.

An important finding of our study is that treatment with MPLA alone has no effect on MTAL $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. This result is significant on a mechanistic level because we have shown that activation of PI3K-Akt signaling by other regulatory factors decreases $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through inhibition of apical and basolateral membrane Na⁺/H⁺ exchangers (24, 69). In contrast, the PI3K-Akt signaling pathway activated by MPLA is not coupled directly to a change in $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rate but prevents ERK-mediated inhibition by LPS. These findings may be the result of specific targeting of MPLA signaling in the MTAL, whereby PI3K-Akt signals generated by MPLA through TLR4 and TRIF selectively activate intracellular molecules that downregulate LPS signaling through TLR4 and MyD88, as detailed above. The absence of an effect of MPLA on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption also is significant on a therapeutic level because it shows that MPLA treatment itself does not adversely affect the basic transport function of the MTAL and that the ability of MPLA to prevent inhibition by LPS is not due to a nonspecific cytotoxic or metabolic effect on the MTAL cells. The absence of toxicity and specificity of MPLA signaling are established further in the MTAL by the finding that pretreatment with MPLA does not prevent ERK-mediated inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by aldosterone (68). This result shows that MPLA is able to protect against LPS without impairing the regulation of MTAL transport by physiological stimuli such as aldosterone that act through the ERK pathway. Thus our studies show that MPLA offers unique properties of nontoxicity and efficacy that enhance its potential for use as a clinical immunomodulator to prevent renal tubule dysfunction in response to infectious stimuli.

Our studies with MPLA provide new evidence that renal tubules are capable of developing resistance to LPS and identify cellular mechanisms involved. In addition, they demonstrate the functional importance of MPLA to preserve MTAL $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. It is possible, however, that the ability of MPLA to protect against LPS in the MTAL may be influenced in vivo by the timing or duration of MPLA exposure or by interactions with the innate immune system in the host environment. Thus, to establish the pathophysiological significance of these findings, it will be important to demonstrate that treatment with MPLA protects MTAL function during bacterial infection in vivo. Using a clinically relevant cecal ligation and puncture (CLP) model in mice, we have shown that sepsis impairs $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL through two distinct mechanisms: 1) it induces an adaptive decrease in the intrinsic capacity of the MTAL to absorb $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ and 2) it enhances the ability of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption through upregulation of the basolateral TLR4-ERK signaling pathway (71). The CLP model thus provides an ideal system to test whether the protective effects of MPLA extend to the MTAL in vivo. The results of the current study demonstrating that MPLA acts directly on the MTAL to induce LPS resistance, independent of interactions with the innate immune system, and identifying the intracellular signaling mechanisms involved provide a critical basis for the design and interpretation of the in vivo studies. Two observations support the view that MPLA will attenuate the effects of sepsis to impair MTAL $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. First, the effects of CLP both to decrease the basal $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption rate and to enhance inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption by LPS depend on ERK activation (71). The current study shows that MPLA protects against LPS and preserves MTAL $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption specifically by downregulating ERK activation. Second, in preliminary studies using a MPLA pretreatment protocol shown to increase survival in CLP mice (56), we found that MPLA increased Akt phosphorylation in MTALs of septic mice (B. Watts and D. Good, unpublished observation). This finding is significant in view of our current results showing that MPLA suppresses ERK activation and prevents LPS inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL through activation of Akt. Based on these observations, studies are in progress to determine whether pretreatment of CLP mice with MPLA to activate PI3K-Akt signaling in the MTAL results in downregulation of ERK that prevents sepsis-induced impairment of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption. An effect of MPLA to preserve renal tubule $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption would have clinical benefit by enhancing the ability of the kidneys to attenuate metabolic acidosis that contributes to increased mortality in septic patients (30, 39, 49). As noted above, a prophylactic treatment strategy that protects renal tubule function against bacterial infection without excessive inflammation has significant importance for clinical scenarios in which patients are at increased risk of developing secondary or nosocomial infections.

In summary, the results of the present study demonstrate that pretreatment with the immunomodulator MPLA prevents the effect of LPS to inhibit $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption in the MTAL. This effect is mediated directly through MPLA stimulation of a TLR4-TRIF-dependent PI3K-Akt pathway, which blocks LPS-induced ERK activation through TLR4 and MyD88. Importantly, MPLA itself has no effect on $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ absorption, and pretreatment with MPLA does not prevent ERK-mediated inhibition of $\mathrm{H}\mathrm{C}{\mathrm{O}}_3^{-}$ by aldosterone, indicating that the low toxicity profile of MPLA extends to the MTAL and that the MPLA-induced signaling pathway is targeted selectively to prevent TLR4-MyD88-mediated ERK activation by LPS. These results provide new evidence that renal tubule epithelial cells are capable of immune reprogramming that conveys resistance to LPS and identify detoxified TLR4-based immunomodulators such as MPLA as potential novel therapeutic agents to treat or prevent renal tubule dysfunction in response to bacterial infection.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 38217.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.A.W., E.R.S., and D.W.G. conceived and designed research; B.A.W. and T.G. performed experiments; B.A.W. and D.W.G. analyzed data; B.A.W. and D.W.G. interpreted results of experiments; B.A.W. and D.W.G. prepared figures; D.W.G. drafted manuscript; B.A.W. and D.W.G. edited and revised manuscript; B.A.W., E.R.S., and D.W.G. approved final version of manuscript.