Metabolic stress promotes renal tubular inflammation by triggering the unfolded protein response

Abstract

The renal epithelium contributes to the development of inflammation during ischemic injury. Ischemia induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Ischemic tissues generate distress signals and inflammation that activates fibrogenesis and may promote adaptive immunity. Interestingly, the UPR may activate inflammation pathways. Our aim was to test whether the UPR is activated during metabolic stress and mediates a tubular inflammatory response. Glucose deprivation, not hypoxia and amino acids deprivation, activated the UPR in human renal cortical tubular cells in culture. This stress activated NF-κB and promoted the transcription of proinflammatory cytokines and chemokines, including IL-6, IL-8, TNF-α, RANTES and MCP-1. The protein kinase RNA (PKR)-like ER kinase signaling pathway was not required for the induction of inflammation but amplified cytokine. Inositol-requiring enzyme 1 activated NF-κB signaling and was required for the transcription of proinflammatory cytokines and chemokines following metabolic stress. Moreover, acute ischemia activated ER stress and inflammation in rat kidneys. Finally, the ER stress marker GRP78 and NF-κB p65/RelA were coexpressed in human kidney transplants biopsies performed before implantation, suggesting that ER stress activates tubular inflammation in human renal allografts. In conclusion, this study establishes a link between ischemic stress, the activation of the UPR and the generation of a tubular inflammatory response.

Figure 1

Glucose deprivation, not hypoxia and amino acids shortage promote ER stress. (a) GRP78 and HIF-1α expression during various metabolic stresses. Levels of GRP78 (top) and HIF-1α (bottom) expression are shown from HRCCs incubated in glucose- or amino acids-deficient culture media or challenged with hypoxia for 24 h. A representative immunoblot of three independent experiments is shown. (b) Metabolic stress promotes chaperones genes transcription. HRCCs were cultured in glucose-deprived culture medium for the indicated periods. GRP78 and GRP94 transcripts levels, measured by qRT-PCR, are presented as mean±S.E.M. relative to levels before metabolic stress over three independent experiments. ^*P<0.05. (c) Metabolic stress promotes time-dependent GRP78 expression. HRCCs were cultured in glucose-deficient medium for the indicated periods and GRP78 protein expression was determined by immunoblotting. A representative immunoblot of three independent experiments is shown

Figure 2

Metabolic stress activates the PERK and IRE1 pathways. (a) eIF2α is phosphorylated during metabolic stress. Phospho-eIF2α expression was evaluated by immunoblotting in HRCCs cultured in glucose-deficient medium for the indicated periods. A representative immunoblot of three independent experiments is shown. Density refers to peIF2α/total eIF2α. (b) ATF4 expression is upregulated during metabolic stress. ATF4 protein expression was evaluated by immunoblotting in HRCCs cultured in glucose-deficient medium for the indicated periods. A representative immunoblot of three independent experiments is shown. (c) CHOP and GADD34 transcripts expression is upregulated under metabolic stress. HRCCs were cultured in glucose-deficient medium for the indicated periods. Transcripts levels, measured by qRT-PCR, are presented as the mean±S.E.M. relative to the levels before metabolic stress for three independent experiments. ^*P<0.05; ^**P<0.01. (d) Metabolic stress promotes XBP1 splicing. HRCCs were subjected to a time course of tunicamycin (2 μg/ml) exposure or cultured in glucose-deficient medium for the indicated periods. XBP1 mRNA splicing was examined by PCR and followed by migration in polyacrylamide gels. See the Material and Methods section for more details

Figure 3

Metabolic stress generates a tubular inflammatory response and activates NF-κB signaling. (a) Heat Map of cytokines expression profiles. HRCCs were cultured in glucose-deficient medium or incubated with tunicamycin (2 μg/ml) or TNF-α (10 ng/ml). IL-6, IL-8, TNF-α, MCP-1, RANTES, CX3CL1 and IL-1β transcripts levels were measured by qRT-PCR. Expression levels shown are representative of the 2^−ΔΔC_t obtained from eight independent replicates at each time point. Time resolved supervised hierarchical clustering of transcripts expression was performed. The brightness of the green and red represents the degree to which expression was lower or higher, respectively, in the HRCCs relative to cells before treatment. (b) C/EBPβ is transiently upregulated during metabolic stress. HRCCs were cultured in glucose-deficient medium. C/EBPβ transcripts levels were measured by qRT-PCR and are presented as the mean±S.E.M. relative to levels in cells before treatment for three independent experiments. (c) IκBα is degraded and NF-κB p65/RelA expression is increased during metabolic stress. Protein expression of IκBα (left) and NF-κB p65/RelA (right) in HRCCs cultured in glucose-deficient medium or incubated with 10 ng/ml TNF-α or 2 μg/ml tunicamycin for the indicated periods was evaluated. A representative immunoblot of three independent experiments is shown. (d) NF-κB p65/RelA translocates in the nucleus during metabolic stress. NF-κB p65/RelA nuclear translocation was evaluated by immunofluorescence analysis. HRCCs were cultured in glucose-deficient medium or incubated with 10 ng/ml TNF-α or 2 μg/ml tunicamycin for 2 h. Cells were counterstained with DAPI to visualize the nuclei. White bar=10 μm. A representative picture of three independent experiments is shown

Figure 4

PERK is not required for triggering tubular inflammation during metabolic stress. (a) RNA interference directed against PERK transcripts. HRCCs were transfected with siRNA targeting PERK transcripts or control non-targeting (scramble) siRNAs. At 24 h post transfection, cells were incubated with glucose-free medium and the PERK protein level was measured at different periods of time. A representative analysis of PERK by immunoblotting is shown (n=3). (b) PERK inhibition does not reduce IκBα degradation during metabolic stress. IκBα protein levels are shown in HRCCs transfected with either PERK or scramble siRNA or control cells. At 24 h post transfection, cells were incubated for 1 h with glucose-deficient medium or incubated with 10 ng/ml TNF-α for 15 min. An immunoblot representative of three independent experiments is shown. (c) Salubrinal amplifies PERK signaling during metabolic stress. HRCCs were incubated with glucose-deficient medium for various periods of time with or without 20 μM salubrinal. CHOP transcripts were measured by qRT-PCR after different periods of time. Transcripts expression levels are presented as the mean±S.E.M. relative to the levels in non-treated cells and are representative of three independent experiments. ^*P<0.05. (d) Salubrinal amplifies tubular inflammation during metabolic stress. HRCCs were incubated with glucose-free medium for various periods of time with 20 μM salubrinal. IL-6, IL-8, TNF-α, RANTES and MCP-1 transcripts were measured by qRT-PCR after different periods of time. Transcripts expression levels are presented as the mean±S.E.M. relative to levels in control cells and are representative of three independent experiments. ^*P<0.05

Figure 5

IRE1 mediates tubular inflammation during metabolic stress. (a) RNA interference directed against IRE1 transcripts. HRCCs were transfected with siRNA targeting IRE1 transcripts or control non-targeting (scramble) siRNAs. At 24 h post transfection, cells were incubated with glucose-free medium and the IRE1 protein level was measured at different periods of time. A representative analysis of IRE1 by immunoblotting is shown (n=3). (b) IRE1 inhibition inhibits NF-κB activation during metabolic stress. Left: IκBα protein levels are shown in HRCCs transfected with either IRE1 or scramble siRNA. At 24 h after transfection, cells were incubated for 1 h with glucose-free medium. An immunoblot representative of three independent experiments is shown. Right: NF-κB p65 protein levels are shown in HRCCs transfected with either IRE1 or scramble siRNA. At 24 h after transfection, cells were incubated for 1 h with glucose-free medium. An immunoblot representative of three independent experiments is shown. Bottom: NF-κB p65/RelA nuclear translocation was evaluated by immunofluorescence analysis. HRCCs transfected with either IRE1 or scramble siRNA were cultured for 2 h in glucose-deficient medium. Cells were counterstained with DAPI (blue) to visualize the nuclei. White bar=10 μm. Nuclear NF-κB p65/RelA (red) staining is quantified by dividing the number of positive cells by the total number of cells at magnification × 10. (c) IRE1 inhibition reduces tubular inflammation during metabolic stress. HRCCs were transfected with siRNA targeting IRE1 transcripts or control non-targeting (scramble) siRNAs. At 24 h post transfection, cells were incubated with glucose-free medium and IL-8, IL-6, TNF-α and RANTES transcripts were measured by qRT-PCR at different periods of time. Transcripts expression levels are presented as a mean±S.E.M. relative to levels in cells transfected with control siRNA and are representative of three independent experiments. ^*P<0.05

Figure 6

Cold ischemia in rat kidneys activates ER stress and generates an inflammatory response. (a) Cold ischemia promotes endoplasmic reticulum stress in rat kidneys. Rats were nephrectomized and kidneys were rinsed and incubated in an IGL1 solution for up to 24 h. Left: GRP78 protein levels in rat kidneys after various times of cold ischemia were measured. A representative immunoblot is shown. Right: expression levels of the HERP, P4HB and CHOP transcripts in rat kidneys after 0, 4, 16 or 24 h of cold ischemia were measured by qRT-PCR. Expression levels are presented as the mean±S.E.M. relative to the levels in rat kidneys before cold ischemia, n=4 per group. (b) Cold ischemia promotes IκBα degradation in rat kidneys. IκBα protein levels in rat kidneys after 4 h of cold ischemia were measured. A representative immunoblot and densitometric analysis are shown. (c) Cold ischemia promotes inflammation in rat kidneys. IL-6, TNF-α, MCP-1 and CXCL1 transcripts expression levels in rat kidneys after various times of cold ischemia were measured by qRT-PCR. Expression levels are presented as the mean±S.E.M. relative to the levels in rat kidneys before cold ischemia, n=4 per group. ^*P<0.05

Figure 7

ER stress and NF-κB activation are associated in renal allograft biopsies. (a) Tubular expression of GRP78 is upregulated following cold ischemia in human kidney allografts. Left: a representative image of GRP78 immunohistochemistry in a preimplantation biopsy with a perinuclear staining (arrows) and a control biopsy. Right: quantification of tubular GRP78 staining in preimplantation biopsies compared with controls (protocol biopsies performed qt 3 months after transplantation). n=18 for preimplantation biopsies and n=15 for the control group. ^**P<0.01. (b) NF-κB signaling is activated following cold ischemia in human kidney allografts. Left: a representative image of NF-κB p65/RelA immunohistochemistry in preimplantation biopsy with nuclear staining (arrows) and control biopsy. Right: quantification of tubular NF-κB p65/RelA staining in preimplantation biopsies compared with controls (protocol biopsies performed at 3 months after transplantation). n=18 in preimplantation biopsies and n=15 in the control group. ^*P<0.05