Toll-like receptor 2 mediates inhibition of HCO(3)(-) absorption by bacterial lipoprotein in medullary thick ascending limb

Abstract

Bacterial infection and sepsis are associated with renal tubule dysfunction and dysregulation of systemic electrolyte balance but the underlying mechanisms are incompletely understood. Recently, we demonstrated that HCO(3)(-) absorption by the medullary thick ascending limb (MTAL) is inhibited by gram-negative bacterial LPS through activation of Toll-like receptor 4 (TLR4). Here, we examined whether MTAL transport is altered by activation of TLR2, the receptor predominantly responsible for recognizing gram-positive bacteria. Confocal immunofluorescence showed expression of TLR2 in the basolateral membrane domain of rat and mouse MTALs. The functional role of TLR2 was examined in perfused MTALs using Pam(3)CSK(4), a bacterial lipoprotein analog that specifically activates TLR2. Adding Pam(3)CSK(4) to the bath decreased HCO(3)(-) absorption by 25%. The inhibition by Pam(3)CSK(4) was eliminated in MTALs from TLR2(-/-) mice. HCO(3)(-) absorption was also inhibited by the TLR2 agonists lipoteichoic acid and peptidoglycan, two cell wall components of gram-positive bacteria. The MEK/ERK inhibitor U0126 eliminated inhibition of HCO(3)(-) absorption by bath LPS but had no effect on inhibition by Pam(3)CSK(4). The inhibition by Pam(3)CSK(4) was eliminated by the protein kinase C inhibitors chelerythrine Cl and bisindolylmaleimide. Moreover, the inhibition by Pam(3)CSK(4), lipoteichoic acid, and peptidoglycan was additive to inhibition by LPS. Thus, agonists of basolateral TLR2 and TLR4 inhibit HCO(3)(-) absorption independently through distinct signaling pathways. We conclude that bacterial components act directly through TLRs to modify the transport function of renal tubules. During polymicrobial sepsis, gram-positive bacterial molecules acting through TLR2 and gram-negative LPS acting through TLR4 can function through parallel signaling pathways to impair MTAL transport. The inhibition of luminal acidification may impair the ability of the kidneys to correct systemic acidosis that contributes to sepsis pathogenesis.

Fig. 1.

Toll-like receptor 2 (TLR2) is expressed in the basolateral membrane domain of the medullary thick ascending limb (MTAL). MTALs dissected from rats (A–C) and from wild-type (D) and TLR2^−/− (E) mice were stained with anti-TLR2 antibody and analyzed by confocal immunofluorescence as described in methods. B: merged differential interference contrast image of the tubule in A to establish cell boundaries for the TLR2 localization. Images are z-axis sections (0.4 μm) taken through a plane at the center of the tubule, which shows a cross-sectional view of cells in the lateral tubule walls (33, 81). TLR2 staining was observed selectively in the basolateral membrane domain. The TLR2 staining was absent in the presence of blocking peptide and in MTALs from TLR2^−/− mice. Images are representative of at least 4 tubules of each type. Scale bar = 5 μm.

Fig. 2.

Pam₃CSK₄ inhibits HCO₃⁻ absorption in the MTAL. MTALs from rats were isolated and perfused in vitro in control solution, and then Pam₃CSK₄ (1 μg/ml) was added to and removed from the bath solution. Absolute rates of HCO₃⁻ absorption (JHCO₃⁻) were measured as described in methods. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P value is for paired t-test. Mean values are given in results.

Fig. 3.

Inhibition of HCO₃⁻ absorption by Pam₃CSK₄ is eliminated in MTALs from TLR2^−/− mice. MTALs from wild-type control (A) and TLR2^−/− (B) mice were perfused in vitro in control solution, and then Pam₃CSK₄ was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 2. NS, not significant. Mean values are given in results.

Fig. 4.

Lipoteichoic acid and peptidoglycan inhibit HCO₃⁻ absorption in the MTAL. MTALs from rats were perfused in vitro in control solution, and then lipoteichoic acid (1 μg/ml; A) or peptidoglycan (10 μg/ml; B) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 2. Mean values are given in results.

Fig. 5.

LPS and Pam₃CSK₄ inhibit HCO₃⁻ absorption through distinct signaling pathways. MTALs from rats were perfused in the absence (control) or presence of U0126 (15 μM) in the bath, and then LPS (500 ng/ml; A) or Pam₃CSK₄ (1 μg/ml; B) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 2.

Fig. 6.

Inhibition of HCO₃⁻ absorption by Pam₃CSK₄ is blocked by inhibitors of PKC. MTALs were bathed with the PI3K inhibitors LY294002 (20 μM) or wortmannin (100 nM; A), or with the PKC inhibitors chelerythrine Cl (100 nM) or bisindolylmaleimide (100 nM; B), and then Pam₃CSK₄ (1 μg/ml) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 2. Mean values are given in results.

Fig. 7.

Inhibition of HCO₃⁻ absorption by TLR2 agonists is additive to inhibition by LPS. A and B: MTALs were bathed with LPS (500 ng/ml), and then Pam₃CSK₄, lipoteichoic acid, or peptidoglycan was added to and removed from the bath solution. C: MTALs were bathed with Pam₃CSK₄, and then LPS was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 2. Mean values are given in results.