The Role of Autophagy in Kidney Inflammatory Injury via the NF-κB Route Induced by LPS

Abstract

Acute kidney injury (AKI) is a systemic inflammatory response syndrome associated with poor clinical outcomes. No treatments effective for AKI are currently available. Thus, there is an urgent need of development of treatments effective for AKI. Autophagy, an intracellular proteolytic system, is induced in renal cells during AKI. However, whether autophagy is protective or injurious for AKI needs to be clearly clarified. We addressed this question by pharmacological inhibition of autophagy using a mouse model of lipopolysaccharide (LPS) induced-AKI. We found that autophagy was induced in renal cortex of mice during LPS-induced AKI as reflected by a dose-and time-dependent increased accumulation of light chain 3-II (LC3-II), the common marker of autophagy, compared to that of control group; 2) the occurrence of intensive, punctate and increased immunohistochemical staining image of LC3-II in renal cortex; 3) the significant increase in the expression levels of Beclin-1, another key marker of autophagy; 4) the significantly increased levels of plasma urea and serum creatinine and 5) the significant increase in autophagagosome area ratio. We observed that 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy, blocked autophagy flux, alleviated AKI and protected against LPS-induced AKI. LPS triggered kidney inflammation by activation of the canonical NF-κB pathway. This route can be modulated by autophagy. Activation of the canonical NF-κB pathway was reduced in 3-MA+LPS as compared to that in LPS-treated group of mice. Mice pretreated with 3-MA before exposure to LPS showed a reduction in p65 phosphorylation, resulting in the accumulation of ubiquitinated IκB. In conclusion, impairment of autophagy ameliorates LPS-induced inflammation and decreases kidney injury. The accumulation of ubiquitinated IκB may be responsible for this effect.

Keywords: 3-methyladenine; IκB; LPS-induced kidney injury; autophagy; inflammation.

Figure 1

Analysis of the expression of microtubule-associated protein LC3-II in kidney homogenate by Western blot. A. The expression levels of LC3-II proteins in kidney homogenates of male mice intraperitoneally injected (I.P.) with lipopolysaccharide (LPS) at the indicated doses (n=6 for each dose). The assay was repeated three times. Left panel, the representative Western blots for LC3-II; Right panel, quantification of LC3-II by densitometry (n=3); *P<0.05 vs 0 mg/kgLPS group; B. The expression levels of LC3-II proteins in kidney homogenates of male mice intraperitoneally injected with lipopolysaccharide (LPS) at 10 mg/kg for the indicated time points (n=6, for each time points). Left panel, the representative Western blots for LC3II; Right panel, protein quantification of LC3-II by densitometry (n=3); *P<0.05 vs. LPS stimulation at 0 h.

Figure 2

Detection of LC3-II expressions for the occurrence of LPS-induced autophagy in renal cortex and medulla via immunohistochemical staining. A. Immuohistochemical staining of LC3-II in renal cortex and medulla of mice intraperitoneally injected without (upper panels) and with (lower panels) lipopolysaccharide (LPS) for 24 h: compared to those in the cortex (cortex-con) and medulla (medulla-con) of the controlled mice, the immunohistochemical staining intensity of LC3-II was significantly increased in cortex (cortex-LPS) but only slight increase in medulla (medulla-LPS) of the LPS-stimulated mice. B. Analysis of the average optical intensity (AOD) and integral optical density (IOD) of the immunohistochemical staining intensity of LC3-II in cortex and medulla of mice stimulated without or with LPS. The immunohistochemical staining intensity of LC3-II in renal cortex was significantly increased in LPS-stimulated mice (Cortex-LPS), *P<0.05 vs. Cortex-CON (n=8); while the immunohistochemical staining intensity of LC3-II in renal cortex of LPS-stimulated mice was also significantly higher than in renal medulla (Medulla-LPS), # P<0.05 vs. Medulla-LPS (n=8).

Figure 3

Inhibition of autophagy by 3-MA dramatically attenuated LPS-induced AKI in mice. The mice were pretreated i.p. without or with 3-MA (15 mg/kg in 0.1 mL of 0.9 % normal saline)(n=8) for 1h, followed by exposure to LPS (10 mg/kg in 0.1 mL of 0.9 % normal saline)(n=8) for 24h. The homogenates of one side of kidney were used to examined the expression levels of Beclin-1(A) and LC3-II (B). The other side of kidney was used for immunohistochemical staining for LC3-II to examine its expression in renal cortex (C). A. Left panel, representative Western blots for Beclin-1; Right panel, quantification of Beclin-1 protein by densitometry (n=3); B. Left panel, the representative Western blots for LC3-II; Right panel, quantification of LC3-II protein by densitometry (n=3); C. left panel, immunohistochemical staining for LC3-II protein in renal cortex; right panel, quantitative analysis of the optical intensity of the immunohistochemical staining image of LC3-II protein. *P<0.05 vs. CON; # P<0.05 vs. LPS.

Figure 4

Pharmacological inhibition of autophagy improved kidney injury. The mice were pretreated i.p. without or with or 3-MA at 15 mg/kg (in 0.1 mL of 0.9 % normal saline)(n=8) for 1h, followed by exposure to LPS (10 mg/kg in 0.1 mL of 0.9 % normal saline, n=8) for 24h. Blood samples were collected from mice in these groups. The levels of the serum creatinine and plasma urea were measured. A. Levels of plasma urea; B. Levels of serum creatinine; C. TEM visualization of the injury in renal cortex of mice, the red arrows point to autophygosomes; D. Quantitative analysis of the number per area of autophygosomes; *P<0.05 vs. CON; #P<0.05 vs. LPS.

Figure 5

Autophagy activated NF-κB pathway. A. Measurement of the expression level of IL-1β gene To characterize which the intracellular proinflammatory pathways are involved in LPS-induced autophagy, both canonical and non-canonical routes of NF-κB were studied with Western blot. B. LPS stimulated the classic canonical route via phosphorylation of p65; C. but did not cause any effects non-canonical route as indicated by without changes in p52 phosphorylation : * P<0.05 vs. CON; #P<0.05 vs. LPS.

Figure 6

Accumulation of ubiquitylated IκB. A. The total IκB levels were measured in kidney tissue homogenates of mice in four groups. Mice pretreated with 3MA (15 mg/kg) followed by challenge with by LPS (10 mg/kg) displayed significantly increased levels of IκB. After being co-immunoprecipitated with either antibody for p65 or with FK1, the LPS+3MA-treated mice displayed significantly higher levels of p65 (B) and FK1(C), demonstrating the binding of both ubiquitin and p65 to IκB in treated animals. *P<0.05 vs. CON; #P<0.05 vs. LPS.