Hypergravity Load Modulates Acetaminophen Nephrotoxicity via Endoplasmic Reticulum Stress in Association with Hepatic microRNA-122 Expression.

Hypergravity conditions may subject the kidney to intrinsic stress and lead to hemodynamic kidney dysfunction. However, the mechanisms underlying this phenomenon remain unclear. Accumulation of unfolded proteins in the endoplasmic reticulum (i.e., ER stress) is often observed in kidney diseases. Therefore, this study investigated whether hypergravity stress alters acetaminophen-induced renal toxicity in vivo, as well as the molecular mechanisms involved in this process. C57BL/6 mice were submitted to one or three loads of +9 Gx hypergravity for 1 h with or without acetaminophen (APAP) treatment. The protein levels of cell survival markers, including pAKT and pCREB, were decreased in the kidney after acetaminophen treatment with a single hypergravity load. Additionally, the combined treatment increased kidney injury markers, serum creatinine, and Bax, Bcl2, and Kim-1 transcript levels and enhanced ER stress-related markers were further. Moreover, multiple hypergravity loads enabled mice to overcome kidney injury, as indicated by decreases in serum creatinine content and ER stress marker levels, along with increased cell viability indices. Similarly, multiple hypergravity loads plus APAP elevated miR-122 levels in the kidney, which likely originated from the liver, as the levels of primary miR-122 increased only in the liver and not the kidney. Importantly, this phenomenon may contribute to overcoming hypergravity-induced kidney injury. Taken together, our results demonstrate that APAP-exposed mice submitted to a single load of hypergravity exhibited more pronounced kidney dysfunction due to increased ER stress, which may be overcome by repetitive hypergravity loads presumably due to increased production of miR-122 in the liver. Thus, our study provides novel insights into the mechanisms by which hypergravity stress plus APAP medication induce kidney injury, which may be overcome by repeated hypergravity exposure.

Binge Alcohol Intake After Hypergravity Stress Sustainably Decreases AMPK and Transcription Factors Necessary for Hepatocyte Survival.

BACKGROUND: Binge alcohol consumption elicits mitochondrial dysfunction in hepatocytes. An understanding of the effect of ethanol (EtOH) exposure after hypergravity stress on liver function may assist in the implementation of pathophysiological countermeasures for aerospace missions. This study investigated whether a combination of hypergravity stress and binge alcohol intake has a detrimental effect on AMP-activated protein kinase (AMPK) and other molecules necessary for hepatocyte survival. METHODS: The mice were orally administered a single dose of EtOH (5 g/kg body weight, 20% EtOH) immediately after a load to +9 Gz hypergravity for 1 hour using a small animal centrifuge and sacrificed 24 hours after treatment. For the multiple-dose model, 3 consecutive daily treatments were carried out. Immunoblottings were carried out on liver homogenates. RESULTS: Binge alcohol intake in mice immediately after a 1-hour exposure to a +9 Gz hypergravity load repressed hepatic Akt and PARP-1 levels at 24 hours posttreatment. Moreover, it sustainably diminished the level of AMPKα, a key regulator of energy metabolism, as compared to each individual treatment. Similarly, the combination of alcohol and hypergravity suppressed the levels of STAT3, FOXO1/3, C/EBPß, and CREB, transcription factors necessary for cell survival. Similar changes were not detected after 3 consecutive daily combinatorial treatments, indicating that repetitive training with hypergravity loads provides hepatoprotective effects in a binge alcohol model. CONCLUSIONS: These results show that binge alcohol exposure in mice immediately following a +9 Gz hypergravity stress persistently decreased AMPKα and other key molecules required for hepatocyte survival, and these changes may be reversed by repetitive hypergravity loads.

Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure.

This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS.

Hypergravity upregulates renal inducible nitric oxide synthase expression and nitric oxide production.

Exposure to hypergravity severely decreases renal blood flow, potentially causing renal dysfunction. Nitric oxide (NO), which is endogenously synthesized by inducible NO synthase (iNOS), plays an important role in the regulation of renal function. The purpose of this study was to examine the effect of hypergravity exposure on the production of NO in kidneys. To determine whether hypergravity induces renal hypoxia and alters renal iNOS expression and NO production, mice were exposed to short-term hypergravity at +3Gz for 1 h. The time course of iNOS mRNA expression, hypoxia-inducible factor (HIF)-1α expression, and NO production was examined. Renal HIF-1α levels were significantly elevated immediately after centrifugation, and this increase was sustained for 3 h post-exposure. iNOS mRNA levels were also significantly increased immediately after exposure and were maintained during the reoxygenation period. Immunohistochemical staining for iNOS revealed that the cortical tubular epithelium exhibited moderate to strong cytoplasmic iNOS immunoreactivity immediately after hypergravity exposure and during the reoxygenation period. The time course of NO production was similar to that of iNOS expression. Our results suggest that both hypoxia and reoxygenation might be involved in the upregulation of HIF-1α in the kidneys of mice exposed to hypergravity. Significant increases in renocortical iNOS expression immediately after centrifugation and during the reoxygenation period suggest that iNOS expression induced by hypergravity exposure might play a protective role against hypoxia/reoxygenation injury in the renal cortex. Further investigations are necessary to clarify the role of iNOS and NO in kidneys exposed to hypergravity.

A load of mice to hypergravity causes AMPKα repression with liver injury, which is overcome by preconditioning loads via Nrf2.

An understanding of the effects of hypergravity on energy homeostasis is necessary in managing proper physiological countermeasures for aerospace missions. This study investigated whether a single or multiple load(s) of mice to hypergravity has an effect on molecules associated with energy metabolism. In the liver, AMPKα level and its signaling were repressed 6 h after a load to +9 Gz hypergravity for 1 h, and then gradually returned toward normal. AMPKα level was restored after 3 loads to +9 Gz, suggestive of preconditioning adaptation. In cDNA microarray analyses, 221 genes were differentially expressed by +9 Gz, and the down-regulated genes included Nrf2 targets. Nrf2 gene knockout abrogated the recovery of AMPKα elicited by 3 loads to +9 Gz, indicating that Nrf2 plays a role in the adaptive increase of AMPKα. In addition, +9 Gz stress decreased STAT3, FOXO1/3 and CREB levels, which was attenuated during the resting time. Similarly, apoptotic markers were enhanced in the liver, indicating that the liver may be vulnerable to hypergravity stress. Preconditioning loads prevented hepatocyte apoptosis. Overall, a load of mice to +9 Gz hypergravity causes AMPKα repression with liver injury, which may be overcome by multiple loads to hypergravity as mediated by Nrf2.