Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c+ dendritic cells

doi:10.1038/s41419-022-05191-z

. 2022 Aug 27;13(8):739.

doi: 10.1038/s41419-022-05191-z.

Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c⁺ dendritic cells

Kurt T K Giuliani^{1

2

3}, Anca Grivei^{1

2}, Purba Nag^{1

2}, Xiangju Wang^{1

2}, Melissa Rist^{1

2}, Katrina Kildey^{1

2}, Becker Law^{1

2

4}, Monica S Ng^{1

2

3

5

6}, Ray Wilkinson^{1

2

3

4}, Jacobus Ungerer^{1

3}, Josephine M Forbes^{3

7}, Helen Healy^#^{1

2

3}, Andrew J Kassianos^#^{8

9

10

11}

Affiliations

¹ Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Brisbane, QLD, Australia.
² Kidney Health Service, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia.
³ Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia.
⁴ Institute of Health and Biomedical Innovation/School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia.
⁵ Institute of Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia.
⁶ Department of Nephrology, Princess Alexandra Hospital, Brisbane, QLD, Australia.
⁷ Mater Research Institute, University of Queensland, Brisbane, QLD, Australia.
⁸ Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
⁹ Kidney Health Service, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
¹⁰ Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
¹¹ Institute of Health and Biomedical Innovation/School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.

^# Contributed equally.

PMID: 36030251
PMCID: PMC9420140
DOI: 10.1038/s41419-022-05191-z

Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c⁺ dendritic cells

Kurt T K Giuliani et al. Cell Death Dis. 2022.

. 2022 Aug 27;13(8):739.

doi: 10.1038/s41419-022-05191-z.

Authors

Affiliations

¹ Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Brisbane, QLD, Australia.
² Kidney Health Service, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia.
³ Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia.
⁴ Institute of Health and Biomedical Innovation/School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia.
⁵ Institute of Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia.
⁶ Department of Nephrology, Princess Alexandra Hospital, Brisbane, QLD, Australia.
⁷ Mater Research Institute, University of Queensland, Brisbane, QLD, Australia.
⁸ Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
⁹ Kidney Health Service, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
¹⁰ Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.
¹¹ Institute of Health and Biomedical Innovation/School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia. andrew.kassianos@qimrberghofer.edu.au.

^# Contributed equally.

PMID: 36030251
PMCID: PMC9420140
DOI: 10.1038/s41419-022-05191-z

Abstract

Inflammasomes are multiprotein platforms responsible for the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Mouse studies have identified inflammasome activation within dendritic cells (DC) as pivotal for driving tubulointerstitial fibrosis and inflammation, the hallmarks of chronic kidney disease (CKD). However, translation of this work to human CKD remains limited. Here, we examined the complex tubular cell death pathways mediating inflammasome activation in human kidney DC and, thus, CKD progression. Ex vivo patient-derived proximal tubular epithelial cells (PTEC) cultured under hypoxic (1% O₂) conditions modelling the CKD microenvironment showed characteristics of ferroptotic cell death, including mitochondrial dysfunction, reductions in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and increases in lipid peroxidation by-product 4-hydroxynonenal (4-HNE) compared with normoxic PTEC. The addition of ferroptosis inhibitor, ferrostatin-1, significantly reduced hypoxic PTEC death. Human CD1c⁺ DC activated in the presence of hypoxic PTEC displayed significantly increased production of inflammasome-dependent cytokines IL-1β and IL-18. Treatment of co-cultures with VX-765 (caspase-1/4 inhibitor) and MCC950 (NLRP3 inflammasome inhibitor) significantly attenuated IL-1β/IL-18 levels, supporting an NLRP3 inflammasome-dependent DC response. In line with these in vitro findings, in situ immunolabelling of human fibrotic kidney tissue revealed a significant accumulation of tubulointerstitial CD1c⁺ DC containing active inflammasome (ASC) specks adjacent to ferroptotic PTEC. These data establish ferroptosis as the primary pattern of PTEC necrosis under the hypoxic conditions of CKD. Moreover, this study identifies NLRP3 inflammasome signalling driven by complex tubulointerstitial PTEC-DC interactions as a key checkpoint for therapeutic targeting in human CKD.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Hypoxia induces pathways of mitochondrial oxidative damage/dysfunction and DNA damage in human primary PTEC.**
A Left panel: fold changes (relative to normoxia) in mitochondrial superoxide levels (% MitoSOX⁺ cells) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± standard error mean (SEM). Symbols represent individual donor PTEC; n = 5. *P < 0.05, paired t test. Right panel: representative MitoSOX staining (black unfilled) compared with unstained control (grey filled) for PTEC cultured under normoxic and hypoxic conditions. Mitochondrial superoxide levels (% MitoSOX⁺ cells) are presented for each histogram, with fold change (FC) value relative to normoxic PTEC also shown. B Left panel: Fold changes (relative to normoxia) in mitochondrial membrane potential [measured as ratio of ΔMFI JC-1 red/ΔMFI JC-1 green; with delta median fluorescence intensity (ΔMFI) representing MFI test – MFI unstained control] for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 4. *P < 0.05, paired t test. Right panel: representative JC-1 dot plots of PTEC cultured under normoxic and hypoxic conditions. Mitochondrial membrane potential (ΔΨ_mt) values are presented for each histogram, with fold change (FC) value relative to normoxic PTEC also shown. C Left panel: fold changes (relative to normoxia) in γ-H2AX protein levels (as a ratio of loading control β-tubulin) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 4. *P < 0.05, paired t test. Right panel: western blot for γ-H2AX for PTEC cultured under normoxic and hypoxic conditions (15 µg total protein per lane). Representative images from one of four donor PTEC are presented; full and uncropped western blot available as Supplementary Material. D, E Fold changes (relative to normoxia) in cell viability (MTT assay) (D) and HMGB1 (ELISA) (E) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 7 for viability data and n = 3 for HMGB1 data. **P < 0.01, paired t test.

**Fig. 2. Hypoxia induces ferroptosis in human primary PTEC.**
A Left panel: fold changes (relative to normoxia) in cellular necrosis (% Annexin-V⁺ PI⁺ cells) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 4. *P < 0.05, paired t test. Right panel: representative donor Annexin-V/PI dot plots of PTEC cultured under normoxic and hypoxic conditions. The percentage of Annexin-V⁺ PI⁺ necrotic cells for each dot plot are presented, with fold change (FC) value relative to normoxic PTEC also shown. B Left panel: fold changes (relative to normoxia) in GPX4 protein levels (as a ratio of loading control β-tubulin) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 4. **P < 0.01, paired t test. Right panel: GPX4 western blot for PTEC cultured under normoxic and hypoxic conditions (15 µg total protein per lane). Representative images from one of four donor PTEC are presented; full and uncropped western blot available as Supplementary Material. C Left panel: fold changes (relative to normoxia) in 4-HNE levels (measured as mean corrected total cellular fluorescence (CTCF) of >60 cells per condition) for PTEC cultured under normoxic and hypoxic conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 6. *P < 0.05, paired t test. Right panel: immunofluorescent labelling of representative PTEC cultured under normoxic and hypoxic conditions and stained for 4-HNE (green), β-tubulin (red) and DAPI (blue). 4-HNE positivity is highlighted with white arrows. Scale bars represent 20 µm. D Left panel: representative donor bar graph of cellular necrosis for PTEC cultured under normoxic (N) and hypoxic (H) conditions in the absence (-Fer-1; DMSO vehicle control) or presence of ferrostatin-1 (+Fer-1). Right panel: fold changes (relative to hypoxia/-Fer-1) in cellular necrosis (% Annexin-V⁺ PI⁺ cells) for hypoxic PTEC cultured in the absence or presence of ferrostatin-1. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC; n = 7. *P < 0.05, paired t test.

**Fig. 3. Co-localisation of human CD1c⁺ DC with ferroptotic PTEC in fibrotic kidney tissue.**
Representative serial section immunohistochemical (IHC) staining of control/non-fibrotic (left panel) and fibrotic (middle panel) kidney tissue probed for (A) aquaporin-1, (B) GPX4, (C) 4-HNE and (D) CD1c. Regions of CD1c⁺ DC co-localisation with aqupaporin-1⁺ PTEC displaying evidence of ferroptotic cell death (↓ GPX4 and ↑ 4-HNE) are highlighted with black arrows. Scale bars represent 60 µm. Quantitative analysis (positive pixel intensity/µm² total area) of IHC staining in control and fibrotic tissue is presented (right panels). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from four randomly selected areas for each tissue sample. *P < 0.05, ***P < 0.001, Welch’s t test.

**Fig. 4. CD1c⁺ DC activated in the presence of hypoxic PTEC secrete significantly increased levels of inflammasome cytokines IL-1β/IL-18.**
A Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c⁺ DC with pre-conditioned normoxic (N; 21% O₂) or hypoxic (H; 1% O₂) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; n = 4. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. IL-12p70, IL-17, interferon (IFN)-α and IFN-γ were not detectable in any culture conditions (data not shown). B, C Secreted IL-1β (B) and IL-18 (C) protein levels (measured by LEGENDplex™ assay; pg/ml) following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c⁺ DC with pre-conditioned normoxic (white bars) or hypoxic (grey bars) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; N.D. not detectable. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 4. ****P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test. D Flow cytometric detection of intracellular IL-1β protein following 4-h co-culture of magnetic bead-enriched CD1c⁺ DC with pre-conditioned hypoxic PTEC in the presence of poly I:C. Co-cultures were treated prior to the 4-h culture period with either brefeldin A or VX-765 to enable intracellular IL-1β accumulation/detection. Representative flow cytometric histograms of IL-1β staining (black unfilled) compared with unstained control (grey filled) for PTEC (gated on live, single, CD45⁻ cells) and CD1c⁺ DC (gated on live, single, CD45⁺ lineage⁻ HLA-DR⁺ CD1c⁺ cells) are presented. One representative of three individual donor PTEC experiments is shown. E Fold changes (relative to –Fer-1) in secreted IL-1β levels (measured by ELISA; pg/ml) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c⁺ DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (-Fer-1) or presence of ferrostatin-1 (+Fer-1). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. ns not significant, paired t test.

**Fig. 5. Hypoxic PTEC trigger CD1c⁺ DC-derived IL-1β/IL-18 via activation of the NLRP3 inflammasome.**
A *NLRP3* mRNA expression relative to housekeeping gene β-2-microglobulin (*B2M)* in flow cytometry sorted CD1c⁺ DC freshly isolated (0 h) or following 24-h co-culture (DC & PTEC) with pre-conditioned normoxic (white bar) or hypoxic (grey bar) PTEC in the presence of poly I:C. DC alone cultures are included as controls. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. *P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. B Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c⁺ DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody); n = 6. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. C, D Fold changes (relative to NIL inhibitor) in secreted IL-1β (C) and IL-18 (D) protein levels (measured by LEGENDplex™ assay) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c⁺ DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 6. *P < 0.05; **P < 0.01; ****P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test.

**Fig. 6. Significantly elevated tubulointerstitial CD1c⁺ DC with ASC specks in fibrotic kidney tissue.**
A, B Immunofluorescent labelling of frozen sections from control/non-fibrotic (A) and fibrotic kidney tissue (B) stained for ASC (green), CD1c (red), aquaporin-1 (AQP-1) (white) and DAPI (blue). Scale bars represent 50 µm for small frames (left panels) and 20 µm for large frames (right panels). ASC-speck-positive CD1c⁺ DC are highlighted with white arrows. C Quantification (mean cells/mm²) of ASC-speck-negative CD1c⁺ DC (white bars) and ASC-speck-positive CD1c⁺ DC (grey bars) in control/non-fibrotic kidney tissue (n = 4) and fibrotic kidney tissue (n = 4). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from five randomly selected areas for each tissue sample. *P < 0.05, **P < 0.01, Welch’s t test.

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References

1. Xie Y, Bowe B, Mokdad AH, Xian H, Yan Y, Li T, et al. Analysis of the Global Burden of Disease study highlights the global, regional, and national trends of chronic kidney disease epidemiology from 1990 to 2016. Kidney Int. 2018;94:567–81. doi: 10.1016/j.kint.2018.04.011. - DOI - PubMed
1. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992;20:1–17. doi: 10.1016/S0272-6386(12)80312-X. - DOI - PubMed
1. Bábíčková J, Klinkhammer BM, Buhl EM, Djudjaj S, Hoss M, Heymann F, et al. Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries. Kidney Int. 2017;91:70–85. doi: 10.1016/j.kint.2016.07.038. - DOI - PubMed
1. Haase VH. Hypoxia-inducible factor signaling in the development of kidney fibrosis. Fibrogenes Tissue Repair. 2012;5:S16. doi: 10.1186/1755-1536-5-S1-S16. - DOI - PMC - PubMed
1. Friederich-Persson M, Thorn E, Hansell P, Nangaku M, Levin M, Palm F. Kidney hypoxia, attributable to increased oxygen consumption, induces nephropathy independently of hyperglycemia and oxidative stress. Hypertension. 2013;62:914–9. doi: 10.1161/HYPERTENSIONAHA.113.01425. - DOI - PMC - PubMed

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[1] Xie Y, Bowe B, Mokdad AH, Xian H, Yan Y, Li T, et al. Analysis of the Global Burden of Disease study highlights the global, regional, and national trends of chronic kidney disease epidemiology from 1990 to 2016. Kidney Int. 2018;94:567–81. doi: 10.1016/j.kint.2018.04.011. - DOI - PubMed

[2] Xie Y, Bowe B, Mokdad AH, Xian H, Yan Y, Li T, et al. Analysis of the Global Burden of Disease study highlights the global, regional, and national trends of chronic kidney disease epidemiology from 1990 to 2016. Kidney Int. 2018;94:567–81. doi: 10.1016/j.kint.2018.04.011. - DOI - PubMed

[3] Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992;20:1–17. doi: 10.1016/S0272-6386(12)80312-X. - DOI - PubMed

[4] Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992;20:1–17. doi: 10.1016/S0272-6386(12)80312-X. - DOI - PubMed

[5] Bábíčková J, Klinkhammer BM, Buhl EM, Djudjaj S, Hoss M, Heymann F, et al. Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries. Kidney Int. 2017;91:70–85. doi: 10.1016/j.kint.2016.07.038. - DOI - PubMed

[6] Bábíčková J, Klinkhammer BM, Buhl EM, Djudjaj S, Hoss M, Heymann F, et al. Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries. Kidney Int. 2017;91:70–85. doi: 10.1016/j.kint.2016.07.038. - DOI - PubMed

[7] Haase VH. Hypoxia-inducible factor signaling in the development of kidney fibrosis. Fibrogenes Tissue Repair. 2012;5:S16. doi: 10.1186/1755-1536-5-S1-S16. - DOI - PMC - PubMed

[8] Haase VH. Hypoxia-inducible factor signaling in the development of kidney fibrosis. Fibrogenes Tissue Repair. 2012;5:S16. doi: 10.1186/1755-1536-5-S1-S16. - DOI - PMC - PubMed

[9] Friederich-Persson M, Thorn E, Hansell P, Nangaku M, Levin M, Palm F. Kidney hypoxia, attributable to increased oxygen consumption, induces nephropathy independently of hyperglycemia and oxidative stress. Hypertension. 2013;62:914–9. doi: 10.1161/HYPERTENSIONAHA.113.01425. - DOI - PMC - PubMed

[10] Friederich-Persson M, Thorn E, Hansell P, Nangaku M, Levin M, Palm F. Kidney hypoxia, attributable to increased oxygen consumption, induces nephropathy independently of hyperglycemia and oxidative stress. Hypertension. 2013;62:914–9. doi: 10.1161/HYPERTENSIONAHA.113.01425. - DOI - PMC - PubMed

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Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c⁺ dendritic cells

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Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c⁺ dendritic cells

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