VHL-deficiency leads to reductive stress in renal cells

Abstract

Heritable renal cancer syndromes (RCS) are associated with numerous chromosomal alterations including inactivating mutations in von Hippel-Lindau (VHL) gene. Here we identify a novel aspect of the phenotype in VHL-deficient human renal cells. We call it reductive stress as it is characterised by increased NADH/NAD⁺ ratio that is associated with impaired cellular respiration, impaired CAC activity, upregulation of reductive carboxylation of glutamine and accumulation of lipid droplets in VHL-deficient cells. Reductive stress was mitigated by glucose depletion and supplementation with pyruvate or resazurin, a redox-reactive agent. This study demonstrates for the first time that reductive stress is a part of the phenotype associated with VHL-deficiency in renal cells and indicates that the reversal of reductive stress can augment respiratory activity and CAC activity, suggesting a strategy for altering the metabolic profile of VHL-deficient tumours.

Keywords: HIF; Reductive stress; Renal cell carcinoma; Respiration; VHL; VHL syndrome.

Graphical abstract

Fig. 1

Effect of hypoxia (8 h) on HIF1a protein expression (A), HRE associated transcriptional activity (B) and LDHA gene expression (C) in VHL-deficient cells. The asterisks refer to a statistically significant difference with respect to WT normoxia group unless indicated otherwise. Effect of VHL-deficiency and carbon source availability (4 h) on extracellular lactate levels (D). Asterisks refer to a statistically significant difference with respect to the same genotype on complete medium unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

(A) Effect of VHL-deficiency and carbon source availability on ATP/ADP ratio. (B) Effect of VHL-deficiency on culture growth dynamics in the complete medium. Proliferation in glucose- or glutamine-limited medium was severely stunted (limited to 10% confluence). The asterisks refer to a statistically significant difference with respect to the same genotype on complete medium unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t-test with Welch’s correction (A) or paired t-test (C). *P < 0.05, ***P < 0.001.

Fig. 3

Effect of VHL-deficiency and carbon source availability on respiratory activity based on CO₂ (A) and O₂ (B) measurement in gas phase. Results are normalised to cell number based on cyt-c absorbance. Fraction of glucose passing through the pentose phosphate pathway (PPP) based on the ratio of lactate m+1/m+2 isotopes (C). Acute effect of glucose-depletion on the respiration of WT and VHL KO cells (D). To estimate the acute effect of glucose depletion on respiration the mean values of gas flux between complete (before) and glucose-depleted medium (after) were compared using paired t-test at 4 time points (25–40 min) after changing the medium. The asterisks refer to a statistically significant difference with respect to the same genotype on complete medium unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t-test with Welch’s correction (A–C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4

Effect of VHL-deficiency on respiratory quotient (A), lipid content (B) and representative images (C), and reductive carboxylation (D) in complete medium. Representative images of HKC-8 cells stained with Bodipy (stain for neutral lipids) and Hoechst 33258 (stain for nucleic acid) (C). Scale bar equals 100 um. The asterisks refer to a statistically significant difference with respect to WT group unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t-test with Welch’s correction. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

Contribution of ¹³C₂- [1,2]-glucose (A) or ¹³C₅-[U]-glutamine (B) to labelled metabolites in complete medium. Thickness of line relates to a percentage of each labelled metabolite. Contribution of labelled citrate to m+2 succinate in ¹³C₂- [1,2]-glucose (C) supplemented complete medium. Contribution from ¹³C₂- [1,2]-glucose to citrate (indicated by m+2 isotopomer) on complete medium (D). Contribution from ¹³C₅-[U]-glutamine to succinate (indicated by m+4 isotopomer) in complete medium (E). The asterisks refer to a statistically significant difference with respect to WT group unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with an unpaired t-test with Welch’s correction. *P < 0.05, ****P < 0.0001.

Fig. 6

Effect of VHL-deficiency and carbon source availability on m+4 labelled citrate (A), fumarate (B), malate (C), m+3 labelled pyruvate (D), aspartate concentration (E), succinate/fumarate m+4 isotope ratio (F). The asterisks refer to a statistically significant difference with respect to the same genotype on complete medium unless indicated otherwise. Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t-test with Welch’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7

NADH/NAD⁺ ratio (A–B) in response to glucose depletion (4 h), pyruvate (3 mM, 4 h), resazurin (50 uM, 1h), or DNP (25 uM, 1h) in WT and VHL KO HKC-8 cells. O₂ and CO₂ fluxes in response to pyruvate (3 mM) (C–D), uncoupler dinitrophenol (DNP) (25 uM) (E–F) or resazurin (50 uM) (G–H) in WT and VHL KO HKC-8 cells. Results are normalised relative to the control group (C–H). Number on the bar indicates sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test (A, B, G, H) or unpaired t-test with Welch’s correction (C–F). *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 8

Effect of VHL-deficiency and pyruvate supplementation on metabolite pool sizes. Number on the bar indicates sample size. Values are expressed as mean ± SEM of normalised metabolite ion counts. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test. **P < 0.01, ***P < 0.001, ****P < 0.0001.