High-mobility group box 1 inhibits HCO3- absorption in the medullary thick ascending limb through RAGE-Rho-ROCK-mediated inhibition of basolateral Na+/H+ exchange

Abstract

High-mobility group box 1 (HMGB1) is a nuclear protein released extracellularly in response to infection or injury, where it activates immune responses and contributes to the pathogenesis of kidney dysfunction in sepsis and sterile inflammatory disorders. Recently, we demonstrated that HMGB1 inhibits HCO3 (-) absorption in perfused rat medullary thick ascending limbs (MTAL) through a basolateral receptor for advanced glycation end products (RAGE)-dependent pathway that is additive to Toll-like receptor 4 (TLR4)-ERK-mediated inhibition by LPS (Good DW, George T, Watts BA III. Am J Physiol Renal Physiol 309: F720-F730, 2015). Here, we examined signaling and transport mechanisms that mediate inhibition by HMGB1. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by the Rho-associated kinase (ROCK) inhibitor Y27632 and by a specific inhibitor of Rho, the major upstream activator of ROCK. HMGB1 increased RhoA and ROCK1 activity. HMGB1-induced ROCK1 activation was eliminated by the RAGE antagonist FPS-ZM1 and by inhibition of Rho. The Rho and ROCK inhibitors had no effect on inhibition of HCO3 (-) absorption by bath LPS. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by bath amiloride, 0 Na(+) bath, and the F-actin stabilizer jasplakinolide, three conditions that selectively prevent inhibition of MTAL HCO3 (-) absorption mediated through NHE1. HMGB1 decreased basolateral Na(+)/H(+) exchange activity through activation of ROCK. We conclude that HMGB1 inhibits HCO3 (-) absorption in the MTAL through a RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of NHE1. The HMGB1-RAGE-RhoA-ROCK1 pathway thus represents a potential target to attenuate MTAL dysfunction during sepsis and other inflammatory disorders. HMGB1 and LPS inhibit HCO3 (-) absorption through different receptor signaling and transport mechanisms, which enables these pathogenic mediators to act directly and independently to impair MTAL function.

Keywords: HMGB1; NHE1; ROCK; kidney; sepsis.

Fig. 1.

High mobility group box 1 (HMGB1) inhibits HCO₃⁻ absorption in the medullary thick ascending limb (MTAL). MTALs from rats were isolated and perfused in vitro in control solution, and then HMGB1 (1 μg/ml) was added to and removed from the bath solution. Absolute rates of HCO₃⁻ absorption (JHCO₃⁻) were measured as described in materials and methods. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. The P value is for paired t-test. Mean values are given in results.

Fig. 2.

Rho-associated kinase (ROCK) inhibitor eliminates inhibition of HCO₃⁻ absorption by HMGB1 but not by LPS. MTALs were bathed with 10 μM Y27632, and then HMGB1 (A) or LPS (B) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. NS, not significant. Mean values are given in results.

Fig. 3.

Inhibition of HCO₃⁻ absorption by HMGB1 is mediated by Rho. A and B: MTALs were bathed with Rho Inhibitor I (0.8 μg/ml) for 120 min, and then HMGB1 (A) or LPS (B) was added to and removed from the bath solution. C: time control experiments in which MTALs were bathed with control solution for 120 min, and then HMGB1 was added to and removed from the bath solution. Protocol details are provided in materials and methods. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Fig. 4.

Inhibition of HCO₃⁻ absorption by HMGB1 is not mediated through MyD88. MTALs from MyD88-deficient (MyD88^−/−) mice were perfused in vitro under control conditions, and then HMGB1 was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Fig. 5.

HMGB1 activates ROCK1 through RAGE and Rho. A and B: inner stripe tissue was incubated in vitro at 37°C in the absence (Control) and presence of the RAGE antagonist FPS-ZM1 for 15 min (A) or Rho Inhibitor I for 120 min (B), and then treated with HMGB1 for 15 min. ROCK1 activity was determined in cell lysates by immunoprecipitation kinase assay using recombinant MYPT1 as a substrate (see materials and methods). Substrate phosphorylation was analyzed by immunoblotting using anti-phospho-MYPT1(Thr⁶⁹⁶) antibody (p-MYPT1). Immunoprecipitates were immunoblotted with anti-ROCK1 antibody for total ROCK1 levels. Negative control (−, without kinase) and positive control (+, active ROCK II) lanes are shown to verify assay specificity. Experimental and −/+ control lanes are from the same gel. Blots are representative of 4 independent experiments of each type. ROCK1 activity was determined by densitometric analysis of phosphorylated MYPT1 and expressed as a percentage of the control value measured in the same experiment. Bars are means ± SE. ^*P < 0.05 vs. Control (ANOVA). C: microdissected MTALs were incubated in vitro at 37°C under control conditions and in the presence of HMGB1 or Y27632+HMGB1. Treatment with HMGB1 was for 15 min. Tubules were treated with Y27632 for 15 min before HMGB1 addition. The tubules were fixed and stained with anti-phospho-MYPT1(Thr⁶⁹⁶) antibody (p-MYPT1) and analyzed by confocal immunofluorescence as described in materials and methods. HMGB1 increased p-MYPT1 labeling, and this increase was eliminated by Y27632. Quantification of p-MYPT1 fluorescence intensity was as described in materials and methods and is given in results. Images are representative of at least 7 tubules of each type.

Fig. 6.

HMGB1 activates RhoA. A and B: inner stripe tissue was incubated in vitro at 37°C in the absence (Control) and presence of HMGB1 for the indicated times. RhoA activity was measured in cell lysates using a rotekin-RBD pull-down assay in which levels of activated RhoA (RhoA-GTP) are measured by immunoblotting with anti-RhoA Ab (see materials and methods). Total RhoA level was determined in a portion of each lysate. Negative and positive control lanes indicate assay of cell lysates loaded with GDP (−) or GTPγS (+). Blots are representative of 3 independent experiments of each type. C: activity of RhoA was determined for experiments in A and B by densitometric analysis of RhoA-GTP and expressed as a percentage of the control value measured in the same experiment. Results for 3- and 5-min HMGB1 treatment are combined. Bars are means ± SE. ^*P < 0.05 vs. Control.

Fig. 7.

Inhibition of HCO₃⁻ absorption by HMGB1 is eliminated by inhibitors of basolateral Na⁺/H⁺ exchange. MTALs were studied with 10 μM amiloride in the bath (A) or in a Na⁺-free bath (B), conditions that inhibit basolateral Na⁺/H⁺ exchange (25, 31, 96). HMGB1 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⁺. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Fig. 8.

HMGB1 inhibits basolateral Na⁺/H⁺ exchange activity through a ROCK-dependent pathway. MTALs were perfused in vitro under control conditions and with HMGB1 or Y27632+HMGB1 in the bath solution. Treatment with HMGB1 was for 10 min. Tubules were bathed with Y27632 for 10 min before addition of HMGB1. Basolateral Na⁺/H⁺ exchange rates (JNa⁺/H⁺) were determined from initial rates of intracellular pH (pH_i) increase in response to bath Na⁺ addition (see materials and methods). Data points are for individual tubules. Mean values are given in results. Initial pH_i was 6.75 ± 0.03 and did not differ in the 3 groups. The number of tubules is 6 for Control, 6 for HMGB1, and 7 for Y27632+HMGB1. ^*P < 0.05 vs. Control or Y27632+HMGB1 (ANOVA).

Fig. 9.

Jasplakinolide prevents inhibition of HCO₃⁻ absorption by HMGB1 but not by LPS. MTALs were bathed with 0.05 μM jasplakinolide for 35–50 min, and then HMGB1 (A) or LPS (B) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Fig. 10.

ROCK inhibitor does not prevent inhibition of HCO₃⁻ absorption by bath amiloride. MTALs were bathed with Y27632, and then amiloride (10 μM) was added to and removed from the bath solution. JHCO₃⁻, data points, lines, and P values are as in Fig. 1. Mean values are given in results.

Fig. 11.

Model for inhibition of HCO₃⁻ absorption by HMGB1 in the MTAL. Extracellular HMGB1 activates a basolateral RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of the basolateral Na⁺/H⁺ exchanger NHE1. Inhibition of NHE1 results secondarily in inhibition of apical NHE3 through actin cytoskeleton remodeling, thereby decreasing HCO₃⁻ absorption (25, 31, 96, 97). Arrows do not necessarily imply direct relationships; regulatory steps may involve additional signaling components. See text for details.