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

Abstract

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.

Keywords: Pathology Section; hypergravity; inducible nitric oxide synthase; kidney; nitric oxide.

Figure 1. Effect of exposure to hypergravity on (A) renal HIF-1α expression, (B) iNOS mRNA expression, and (C) NO production

A. Two peaks are observed at 0 and 3 h post-exposure. A 3.3-fold increase in HIF-1α protein is observed immediately after exposure (P < 0.001), and this increase persists until 3 h post-exposure (P = 0.008). B. iNOS mRNA expression is significantly elevated immediately after exposure to hypergravity (P = 0.022), and it increases further at 3 h post-exposure (P = 0.006). C. Effect of exposure to hypergravity on renal NO production. A nitrate/nitrite assay shows similar trends in the levels of iNOS expression, both of which are significantly increased from 0 to 6 h post-exposure. The nitrate/nitrite level reaches a peak at 1 h post-exposure. *P < 0.05; *P < 0.01; ***P < 0.001.

Figure 2. Immunostaining for iNOS in the renal cortex (A to G) and medulla (H to O)

A. Renal cortex of the control mice. No iNOS expression is observed in the glomeruli or tubules in the cortex, whereas the peritubular capillaries (arrows) and inflammatory cells (arrowheads) located in the interstitium show weak to moderate iNOS immunoreactivity and serve as positive internal controls. (B to F) Renal cortex of the centrifuged mice. A significant time-dependent change in iNOS expression is noted in the renal tubular epithelial cells. Significantly increased levels of cytoplasmic iNOS expression persist between 0 and 12 h post-exposure. B. The 0-h interval group. iNOS expression is evident immediately after centrifugation. C. The 1-h interval group. D. The 3-h interval group. E. At 6 h post-exposure, maximum iNOS immunoreactivity is noted. F. The 12-h interval group. G. At 24 h post-exposure, iNOS expression disappears. H. Renal medulla of the control group. iNOS expression is absent in the medulla of the control kidney, except for the interstitial peritubular capillaries (arrows) and inflammatory cells (arrowheads) showing weak to moderate iNOS expression. I. to N. In contrast to the cortex, the medulla shows no significant time-dependent alteration in iNOS immunoreactivity. All the experimental groups shows weak cytoplasmic iNOS immunoreactivity in the tubular epithelium. O. Normal liver parenchyma is used as a positive control. There is a sharp demarcation of bile canaliculi by iNOS, whereas the cytoplasm of hepatocytes shows no iNOS expression.es shows no iNOS expression.