Hélène Simonnet

HIF and reactive oxygen species regulate oxidative phosphorylation in cancer.

A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

The binding of beta-2-microglobulin to renal brush-border membrane: affinity measurement, inhibition by serum albumin

In the kidney, filtered proteins are rapidly reabsorbed so that the final excretion is less than 0.1% of the filtered amount for low molecular weight proteins such as beta 2-microglobulin and a few percent for albumin. In order to investigate the affinity of proteins for luminal membranes, rat renal brush-border membranes were incubated with 125I-labelled human beta 2-microglobulin and the initial binding rate determined by the filtration method. Scatchard plot analysis of binding rate revealed two types of binding sites: one with Km = 0.25.10(-6) M and Vmax = 0.1 nmol/min per mg protein and another with Km = 1.10(-5) M and Vmax = 1.3 nmol/min per mg protein. The lower affinity type is likely to represent non-specific binding the physiological role of which is to be discussed. The higher affinity sites seem to play the major role in binding rate. beta 2-Microglobulin initial binding is reversible, and inhibited by bovine serum albumin. Comparison of the time course of bound beta 2-microglobulin removal by unlabelled beta 2-microglobulin and by albumin suggests that these two proteins have a different internalization mechanism.

Protein endocytosis by a kidney tubule suspension: metabolic requirements

Endocytosis in the renal tubular cell is a permanent process serving the role of saving nitrogen from plasma peptides that are continuously cleared away by kidney glomerulus. Since small proteins appear in urine after strenuous exercise, it was hypothesized that renal ischemia impairs the tubular endocytic reabsorption of proteins. The aim of this paper is to describe a simple in vitro model of renal endocytosis and to use it in studies of endocytic metabolic requirements. The results show that rabbit renal proximal tubules in suspension are able to take up 125I-lysozyme, as well as RITC-lactalbumin. The fluorescent protein was taken up only by the ends of the everted tubule fragments, and accumulated into intracellular vesicles, demonstrating the luminal pathway of endocytosis. The amount of 125I-lysozyme taken up was equivalent to that taken up by isolated perfused tubules (Nielsen et al. (1986) Am. J. Physiol. 251, F822-F830). Anoxia decreased 12-fold the intracellular accumulation of 125I-lysozyme; however, the time-course of inhibition shows that only the late steps of endocytic accumulation are energy-dependent. Substrate deprivation studies suggest a specific role of glucose to sustain endocytosis. Lastly, renal uptake of 125I-lysozyme was shown to be strongly depressed by chloroquine, an alkalinizing agent of endosomes and lysosomes. We conclude that (1) renal tubules in suspension are a satisfactory model for endocytic studies in kidney; (2) suppressing oxygen and substrate supplies to kidney impairs endocytic tubular reabsorption of proteins.

Rapid binding of β2-microglobulin to renal brush-border membranes

Abstract 125I-labelled human β 2 - microglobulin binding to rat renal brush-border membranes was assessed by an in vitro assay under near physiological incubation conditions (i.e. low content of albumin). Binding rate was 55 pmol/min per mg protein in the presence of 200 nM of β 2 - microglobulin and degradation rate was negligible versus binding rate. The bidning rate was in reasonable agreement with the in vivo reabsorption rate, supporting the hypothesis of proteins binding to the luminal membrane during the process of reabsorption. Mild solubilizing treatment (Triton 0.1%) of brush border after β 2 - microglobulin binding yielded the labelled molecule associated with a high-molecular-weight component. Aminopeptidase activity and binding ability were to a certain extent co-purified during the course of the brush-border preparation, suggesting that most of the β 2 - microglobulin binding sites were localized in the brush-border membranes.

Effect of Acidosis, Alkalosis and Monofluoroacetate Administration on Citrate and ATP Content of Rat Renal Medulla and Papilla

1. --Renal distribution of citrate showed that there is an increase in citrate content from cortex to medulla and a decrease from medulla to papilla. Alkalosis produced an increase in citrate content and acidosis a decrease in renal citrate content, in each of the studied renal area. Monofluoroacetate produced no significant change in citrate content of medulla or papilla; it did not interfere with the acido-basic related changes in cortex citrate content, but its effect was additive. 2. --Renal distribution of ATP significantly decreased from cortex to medulla and from medulla to papilla. Acid or basic diet had no influence on intratissular ATP content. Fluoroacetate decreased renal ATP content.

Low glucose microenvironment of normal kidney cells stabilizes a subset of messengers involved in angiogenesis.

As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose‐sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well‐differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia‐inducible factor 1A (HIF1A), angiopoietin receptor (TIE‐2), and VEGF receptor 2 (FLK1) were similarly glucose‐sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.