Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury.

Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 °C to 22 °C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 °C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury.

VHL-deficiency leads to reductive stress in renal cells.

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.

Equilibrium or disequilibrium? A dual-wavelength investigation of photosystem I donors.

Oxidation of photosystem I (PSI) donors under far-red light (FRL), slow re-reduction by stromal reductants and fast re-reduction in the dark subsequent to illumination by white light (WL) were recorded in leaves of several C(3) plants at 810 and 950 nm. During the re-reduction from stromal reductants the mutual interdependence of the two signals followed the theoretical relationship calculated assuming redox equilibrium between plastocyanin (PC) and P700, with the equilibrium constant of 40 +/- 10 (Delta E (m) = 86-99 mV) in most of the measured 24 leaves of nine plant species. The presence of non-oxidizable PC of up to 13% of the whole pool, indicating partial control of electron transport by PC diffusion, was transiently detected during a saturation pulse of white light superimposed on FRL or on low WL. Nevertheless, non-oxidizable PC was absent in the steady state during fast light-saturated photosynthesis. It is concluded that in leaves during steady state photosynthesis the electron transport rate is not critically limited by PC diffusion, but the high-potential electron carriers PC and P700 remain close to the redox equilibrium.

Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves.

The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC), and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal were measured immediately before and after FRL was turned on or off. The same derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied to fit the model to the measurements. Typical best-fit pool sizes were 1.0-1.5 micromol m(-2) of P700, 3 PC/P700 and 1 Cyt f/P700, apparent equilibrium constants were 15 between P700 and PC and 3 between PC and Cyt f. Cyclic electron flow (CET) was calculated from the slope of the signal after FRL was turned off. CET activated as soon as electrons accumulated on the PSI acceptor side. The quantum yield of CET was close to unity. Consequently, all PSI in the leaf were able to perform in cycle, questioning the model of compartmentation of photosynthetic functions between the stroma and grana thylakoids. The induction of CET was very fast, showing that it was directly redox-controlled. After longer dark exposures CET dominated, because linear e- transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase.

Photosystem II cycle and alternative electron flow in leaves.

Sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.) were grown in the laboratory and leaves were taken from field-grown birch trees (Betula pendula Roth). Chlorophyll fluorescence, CO2 uptake and O2 evolution were measured and electron transport rates were calculated, J(C) from the CO2 uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, J(O) from the O2 evolution rate, and J(F) from Chl fluorescence parameters. Mesophyll diffusion resistance, r(md), used for the calculation of J(C), was determined such that the in vivo Rubisco kinetic curve with respect to the carboxylation site CO2 concentration became a rectangular hyperbola with Km(CO2) of 10 microM at 22.5 degrees C. In sunflower, in the absence of external O2, J(O) = 1.07 J(C) when absorbed photon flux density (PAD) was varied, showing that the O2-independent components of the alternative electron flow to acceptors other than CO2 made up 7% of J(C). Under saturating light, J(F), however, was 20-30% faster than J(C), and J(F)-J(C) depended little on CO2 and O2 concentrations. The inter-relationship between J(F)-J(C) and non-photochemical quenching (NPQ) was variable, dependent on the CO2 concentration. We conclude that the relatively fast electron flow J(F)-J(C) appearing at light saturation of photosynthesis contains a minor component coupled with proton translocation, serving for nitrite, oxaloacetate and oxygen reduction, and a major component that is mostly cyclic electron transport around PSII. The rate of the PSII cycle is sufficient to release the excess excitation pressure on PSII significantly. Although the O2-dependent Mehler-type alternative electron flow appeared to be under the detection threshold, its importance is discussed considering the documented enhancement of photosynthesis by oxygen.