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The primary cilium and lipophagy translate mechanical forces to direct metabolic adaptation of kidney epithelial cells

An Author Correction to this article was published on 27 September 2022

This article has been updated

Abstract

Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through β-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.

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Fig. 1: Fluid flow induces mitochondrial biogenesis in KECs.
Fig. 2: Fluid flow induces metabolic adaptation and ATP production in KECs.
Fig. 3: Fluid flow stimulates lipophagy in KECs.
Fig. 4: Fatty acids traffic from LDs to the mitochondria in KECs in an Atg5-dependent manner.
Fig. 5: Inhibition of autophagy impairs metabolism of flow-induced KECs.
Fig. 6: Inhibition of ciliogenesis impairs flow-induced mitochondrial biogenesis and activity and flow-induced lipophagy.
Fig. 7: The loss of primary cilia inhibits energy-dependent processes under shear stress.
Fig. 8: Inhibition of urinary fluid flow impairs lipid metabolism.

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Data availability

Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (https://doi.org/10.1093/nar/gkz1019, PMID 31691833) with the identifier MTBLS1852. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This article is dedicated to the memory of Beth Levine. We are grateful to A.M. Cuervo (Albert Einstein College) for sharing KECs and Ift88HM KECs. We acknowledge support from Necker Institute Imaging and the Cell Sorting Facility, the Fondation Imagine, and the Animal Histology and Morphology Core Facility (SFR Necker INSERM US24, CNRS UMS 3633). We thank C. Nguyen and J. Megret for assistance in performing in vivo and flow cytometry experiments, respectively. The work was supported by institutional funding from INSERM, CNRS and the University Paris–Descartes, and grants from Europe (E17245KK, Innovative training network DRIVE) and the Agence Nationale pour la Recherche (ANR) (R18004KK and R16167KK to P.C. and R18176KK to N.D.). The project received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 765912.

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Authors and Affiliations

Authors

Contributions

C.M., L.P.-M., F.R. and N.D. performed most of the experiments with the exception of the in vivo experiments performed by M.B. and N.D. Metabolomics and some qPCR experiments were performed, respectively, by I.N. and C.L. CRISPR clones were generated by N.K. C.M., L.P.-M., F.R., F.T., P.C. and N.D. planned the experiments. C.M., L.P.-M., F.R., E.M., M.P., G.F., F.T., P.C. and N.D. interpreted data. N.D. and P.C. conceived the study and wrote the manuscript.

Corresponding authors

Correspondence to Patrice Codogno or Nicolas Dupont.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Effect of fluid flow on TFEB activation and mitophagy in KECs.

(a) Images of Wild-type (WT) and Ift88HM KECs cells subjected to flow for 6 h, labeled with anti-TFEB antibody (green) and Hoechst 33342 (blue) to stain nuclei. Scale bars 5 μm. Representative images from n = 3 independent experiments. (b) Quantification of TFEB-positive nuclei is shown. Data are means ± SEM. The statistical significance was calculated by a two-tailed t-test (c) Wild-type (WT) and Ift88HM KECs were subjected or not (d0) to flow for 1 day (d1). Cells were then harvested, and p62 mRNA levels were quantified by RT-QPCR, normalized with respect to β-actin, and presented as fold increases, Data are means ± SEM, n = 3 independents experiments The statistical significance was calculated by a two-tailed t-test (d,e) KECs were subjected or not (d0, d4 static) to flow for 4 days (d4 shear) and Ppargc1a (d), Tim23 (e) mRNA levels were quantified by RT-QPCR, normalized with respect to β-actin, and presented as fold increases, Data in d, e) are means ± SEM, n = 3 independents experiments. The statistical significance for d) and e) was calculated by a two-tailed t-test f) KECs were maintained in static conditions for 4 days (d4 static) or not (d0 static), and the levels of TIM23 were visualized by western blot. Representative blot from n = 3 independent experiments. (g) KECs were subjected to flow or not (d0) for 1 day (d1) in the presence or absence of cycloheximide (CHX), and the levels of mitochondrial proteins (TOM20, TIM23) were visualized by western blot. Representative blot from n = 3 independent experiments. (hj) KECs were transfected with scramble (Scr) control siRNA or siRNAs against ATG16L1, Atg5, and Beclin. Cells were then subjected or not (d0) to flow for 1 day (d1), and the levels of TIM23 were visualized by western blot. Representative blot from n = 3 independent experiments. (i) TIM23/Actin ratios were determined by densitometry. Data are presented as the ratios of SiAtg to scramble values, n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test (j) Reduced Levels of ATG16L1, Atg5, and Beclin were visualized by western blot. Representative blot from n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test. Data and unprocessed blots are available as source data.

Source data

Extended Data Fig. 2 Effect of fluid flow on mitochondrial biogenesis and activity in cells.

(a) KECs and IMCD3 were subjected to flow for 4 days. Megalin and Aqp2 mRNA expression was then visualized by real-time RT–qPCR and normalized with respect to β-actin, and presented as fold increases. Data are means ± SEM. n = 3 independents experiments The statistical significance was calculated by a two-tailed t-test (b) KECs and IMCD3 were subjected or not (d0) to flow for 1 to 4 days (d1, d2, d4), and the levels of mitochondrial proteins (Tom20, Tim23, MTCO1) were visualized by western blot analysis. Representative blot from n = 3 independent experiments. (c) Tom20/Actin and Tim23/Actin ratios were determined by densitometry, Data are means ± SEM, n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test (d) KECs and IMCD3 were subjected or not (d0) to flow for 4 days (d4), and Ppargc1a mRNA expression was visualized by real-time RT–qPCR. Data are means ± SEM, n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test (e) KECs and IMCD3 were subjected to flow or not (D0) for 4 days (D4), harvested by trypsinization, and mitochondrial DNA was quantified by qPCR analysis. Data are represented as ratios between D4 and D0, Data are means ± SEM, n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test (f) KECs and IMCD3 were subjected to flow (shear) or not (static) for 4 days and then harvested by trypsinization. The oxygen consumption rate (OCR, Seahorse XF Mito Stress Test) was measured using the Seahorse extracellular flux assay; shown are f) Seahorse trace representative of n = 3 independent experiments and g) a histogram representative of n = 3 independent experiments, data are means ± SEM of 4 biological independent OCR results. The statistical significance was calculated by a two-tailed t-test. Data and unprocessed blots are available as source data.

Source data

Extended Data Fig. 3 Effect of fluid flow on triglyceride levels and effects of chloroquine on the co-localization between lipid droplet (LD) and LC3 in Atg5-deficient KECs.

(a) KECs were subjected (d4 shear) or not (d4 static) to flow for 4 days, with or without 3-MA. Cells were then harvested and triglycerides measured, Data are means ± SEM n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test (b, c) KECs were transfected with scrambled control siRNA (SiScr) or with an siRNA against Atg5. (b) Images of cells subjected to flow for 1 day, or not, and with or without chloroquine (CQ); fixed; and labeled with Lysotracker Red (red), lipidTox (green) and Hoechst 33342 (blue) to stain lysosomes, LDs and nuclei respectively. Scale bars 5 μm. Representative images from n = 3 independent experiments. (c) Relative Lysotracker-LD co-localization was quantified by Pearson’s coefficient analysis. Data are means ± SEM. Individual data points correspond to single images analyzed from n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test. Data are available as source data.

Source data

Extended Data Fig. 4 Effect of the Atglistatin on lipolysis upon shear stress in KECs.

(a) KEC cells were incubated for 16 h with Red C12, and subjected to flow for 1 day with or without Atglistatin (ATGLi). Cells were labeled with Mitotracker (green) to stain mitochondria and LipidTox to stain LDs (red) and analyzed by immunofluorescence. Scale bars 5 μm. Representative images from n = 3 independent experiments. (b) LD number and total LD area/cell quantified using Icy software from images in a). Data are means ± SEM; n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test. (c, d) Relative cellular localization of Red C12 (with droplet for c)) or with mitochondria for d)) was quantified by Pearson’s coefficient analysis. Data are means ± SEM. Individual data points correspond to single images analyzed from n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test. Data are available as source data.

Source data

Extended Data Fig. 5 Seahorse analysis of control and Atg5-deficient KECs.

(a, b) KECs were transfected twice with scrambled control siRNA (SiScr) or with an siRNA against Atg5. Cells were then subjected (shear) or not (static) to flow for 4 days and harvested by trypsinization. The OCR was measured using the Seahorse XF Mitochondria Stress Test extracellular flux assay; shown are a) Seahorse trace representative of n = 3 independent experiments and b) a histogram representative of n = 3 independent experiments, data are means ± SEM of 4 biological independent OCR results. The statistical significance of b) was calculated by a two-tailed t-test. Data are available as source data.

Source data

Extended Data Fig. 6 Loss of ciliogenesis impairs flow-induced mitochondrial biogenesis in Ift88-knockout KECs.

(a, b) WT and Ift88-/- KECs were subjected to flow, or not, for 4 days, and Ppargc1a and Tim23 mRNA levels were quantified by RT–qPCR, normalized to β-actin, and presented as fold increases. Data in a, b) are means ± SEM, n = 3 independents experiments. The statistical significance of a, b) was calculated by a two-tailed t-test. (ce) Wild-type (WT) and Ift88-/- KECs were subjected to flow, or not, for 4 days, and levels of mitochondrial proteins TOM20, TIM23, and were visualized by western blot analysis. Representative blot from n = 3 independent experiments. (d, e) The TOM20/Actin and TIM23/Actin ratios were determined by densitometry. Data are means ± SEM, n = 3 independents experiments. The statistical significance of d) and e) was calculated by a two-tailed t-test. (f, g) Images of WT and Ift88-/- KECs subjected to flow for 1 day, or not, fixed, and labeled with anti-LC3 antibody (green), LipidTox to stain LDs (red), and Hoechst 33342 to stain nuclei (blue). Scale bars 5 μm. Representative images from n = 3 independent experiments. (g) Relative LC3-LD co-localization was quantified by Pearson’s coefficient analysis. Data are means ± SEM. Individual data points correspond to single images analyzed from n = 3 independent experiments. The statistical significance was calculated by a two-tailed t-test. Data and unprocessed blots are available as source data.

Source data

Extended Data Fig. 7 Effect of Ppargc1a and Atg5 knockdown on autophagy and PGC1α expression, respectively, and effect of fluid flow on the expression of genes encoding glucose transporters.

(a, b) KECs were transfected with a scrambled control siRNA (SiScr) or with an siRNA against Ppargc1a. Cells were then subjected or not (d0) to flow for 4 days (d4) with or without chloroquine (CQ), and the levels of LC3-II were visualized by western blot. Representative blot from n = 3 independent experiments. LC3-II/Actin (b) ratios were determined by densitometry. Data are means ± SEM, n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test. (c) KECs were transfected with a scrambled control siRNA (SiScr) or with an siRNA against Atg5. Cells were then subjected or not (d0) to flow for 4 day (d4). Ppargc1a mRNA levels were quantified by real-time RT–qPCR, normalized with respect to β-actin, and presented as fold increases. Data are means ± SEM, n = 9 independents experiments. The statistical significance was calculated by a two-tailed t-test. (df) WT and Ift88HM KECs were subjected or not (d0) to flow for 4 days (d4) and Slc5a1 (d), Slc2a2 (e), Slc5a2 (f) mRNA levels were quantified by real-time RT–qPCR, normalized with respect to β-actin, and presented as fold increases, Data in df) are means ± SEM, n = 9 independents experiments for d) and e); n = 3 independents experiments for f). The statistical significance of df) was calculated by a two-tailed t-test. (g, h) KECs were transfected with a scrambled control siRNA (SiScr) or with an siRNA against Atg5. Cells were then subjected to flow, or not, for 4 days and then incubated with fluorescently labeled 2-deoxyglucose (2-NDBG) for 45 min. Cells were harvested by trypsinization, and the amount of 2-NDBG taken into the cells was analyzed by FACS. Shown are g) a representative histogram of n = 3 independent experiments and h) a plot of median fluorescence intensity of 2-NDBG dye. Data are means ± SEM, n = 3 independents experiments. The statistical significance was calculated by a two-tailed t-test. (i) Effect of fluid flow on the expression of Hnf4α mRNA. KECs were subjected or not (d0) to flow for 4 days (d4) and Hnf4α mRNA levels were quantified by real-time RT–qPCR, normalized with respect to β-actin, and presented as fold increases; Data are means ± SEM. n = 5 independents experiments. The statistical significance was calculated by a two-tailed t-test. Data and unprocessed blots are available as source data.

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Extended Data Fig. 8 Inhibition of urinary fluid flow impairs expression of enzymes involved in lipid metabolism.

(a-h) Mice subjected to UUO or a sham operation and fasted during the same period of time were euthanized 24 h after surgery. (a) Images of kidney sections subjected to immunohistochemistry for LDs (red) and LTL (green). Scale bars, 10 μm. Representative images from n = 5 (sham) and =6 (UUO) different mice (bh) Quantitative RT–qPCR measurements of b) Ppara, c) Ppargc1a, d) Mcad, e) Cpt1b f) Tim23 g) Nrf1 h) Nrf2 in UUO and sham-operated mice. Data were normalized with respect to Gapdh and are presented as fold increases. Data are means ± SEM, n = 5 and 6 for sham and UUO respectively. The statistical significance of bh) was calculated by a two-tailed t-test. (i) Model of how the primary cilium and lipophagy control metabolic fitness in proximal tubule kidney epithelial cells in response to fluid flow-induced shear stress. Shear stress induces primary cilia-dependent OXPHOS stimulation to ensure ATP production and support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis, and cytoskeleton remodeling. This metabolic adaptation is dependent on AMPK and is characterized by the stimulation of two pathways: the induction of lipophagy to produce fatty acids and the increase of mitochondrial biogenesis. The statistical significance was calculated by a two-tailed t-test. Data are available as source data.

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Supplementary information

Supplementary Information

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Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1: List of SiRNAs. Supplementary Table 2: List of primers used for qPCR. Supplementary Table 3: List of primers used for RT–qPCR.

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Miceli, C., Roccio, F., Penalva-Mousset, L. et al. The primary cilium and lipophagy translate mechanical forces to direct metabolic adaptation of kidney epithelial cells. Nat Cell Biol 22, 1091–1102 (2020). https://doi.org/10.1038/s41556-020-0566-0

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